CN115814104A

CN115814104A - Antibody drug conjugate formulations and uses thereof

Info

Publication number: CN115814104A
Application number: CN202111085625.9A
Authority: CN
Inventors: 肖莉莉; 胡朝红
Original assignee: Shanghai Miracogen Inc
Current assignee: Shanghai Miracogen Inc
Priority date: 2021-09-16
Filing date: 2021-09-16
Publication date: 2023-03-21

Abstract

The invention relates to an antibody drug conjugate preparation and application thereof, and particularly provides an anti-epidermal growth factor receptor antibody drug conjugate preparation which has good curative effect on diseases related to Epidermal Growth Factor Receptor (EGFR), and the stability of the preparation is remarkably improved compared with the prior art.

Description

Antibody drug conjugate formulations and uses thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an antibody drug conjugate preparation and application thereof.

Background

The Epidermal Growth Factor Receptor (EGFR, also known as HER1, c-ErbB 1) is a cell surface Receptor of the Epidermal Growth Factor family, a transmembrane glycoprotein consisting of 1186 amino acid residues and having a molecular weight of 170 kD. EGFR belongs to ErbB subfamily of type I tyrosine kinase receptor (ErbB 1-4), and has tyrosine kinase activity. EGFR is stably expressed in many epithelial tissues, including skin and hair follicles. Abnormal expression of epidermal growth factor receptor or activation by receptor mutation may lead to canceration. There are many solid tumors that show overexpression of epidermal growth factor receptors, such as colorectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, and the like. Growth factors such as transforming growth factor alpha and epidermal growth factor are endogenous ligands of epidermal growth factor receptors. These ligands bind with epidermal growth factor receptor, activate the activity of receptor intracellular tyrosine protein kinase, and initiate multiple downstream signal transduction pathways, thereby regulating the growth and differentiation of normal cells, increasing the invasiveness of tumor cells, promoting angiogenesis, and inhibiting apoptosis of tumor cells. The overexpression of the epidermal growth factor receptor in tumors and the important function of the epidermal growth factor receptor in the growth and differentiation of tumor cells enable the epidermal growth factor receptor to become a tumor treatment target with good prospect.

At present, two therapeutic antibodies against epidermal growth factor receptors are available on the market, one is a human-mouse chimeric antibody C225 antibody (Erbitux, erbitux or Cetuximab, imclone (now Eli Lilly) company), which has specific affinity with epidermal growth factor receptors, can block the binding of ligands such as EGF, TGF and the like with the epidermal growth factor receptors, and inhibit the phosphorylation and downstream signaling of the receptors, thereby inhibiting the growth of tumor cells, inducing apoptosis, and reducing the production of matrix metalloproteinase and vascular epithelial growth factor. In 2004, the FDA approved erbitux for colorectal cancer and 2006 approved it for head and neck cancer, with more clinical trials for other oncologic indications. Clinically, the effective rate (ORR) of erbitux and irinotecan for treating colorectal cancer is 23 percent, and the effective rate of erbitux and other therapeutic drugs for treating head and neck cancer is 13 to 30 percent. Because of the human-mouse chimeric antibody, erbitux developed an antibody response in 3.7% of patients in clinical trials.

Another therapeutic antibody against epidermal growth factor receptor is panitumumab (vegtibix, panitumumab, amgen), a fully humanized monoclonal antibody prepared using transgenic mouse technology, without murine protein sequences. The antibody targets Epidermal Growth Factor Receptor (EGFR), is approved by FDA for marketing in 2006 9 months, is used in combination with fluoropyrimidine, oxaliplatin and irinotecan, or is used to treat EGFR-positive metastatic colorectal cancer after chemotherapy. 2006. The annual FDA approved its monotherapy chemotherapy-resistant metastatic colorectal cancer (mCRC). However, panitumumab is an IgG2 subtype antibody, and CDC activity, ADCC activity, and other biological activities of IgG2 are significantly reduced as compared with IgG 1; in addition, igG2 subtype antibodies are less stable, which may be the main reason why the fully human antibody panitumumab has no significant advantage over the chimeric antibody erbitux in clinical effect. The overall survival rate (OR) for the clinical treatment of colorectal cancer reaches 8%, and the non-progression survival period is prolonged by only 3.6 months.

There is a large amount of Clinical data showing that erbitumumab and panitumumab are only effective against KRAS wild type (KRAS wild type) expressed by EGFR, but not against KRAS mutants, and therefore, the guidelines published by the american Clinical Oncology association clearly indicate that anti-EGFR monoclonal drugs are only suitable for use in KRAS wild type colon cancer patients (Allegra CJ, jessup JM, someield MR, hamilton SR, hammond EH, hayes DF, et al. American society of Clinical Oncology Clinical options: testing for KRAS gene mutations in substrates with catalytic chemical expression from monoclonal antibody therapy. J.Clin Oncol. 2009 27.

Therefore, there is a need in the art for humanized anti-epidermal growth factor receptor antibody drugs with higher biological activity, especially antibody drugs with therapeutic effect on KRAS mutants, such as antibody drug conjugates, to further improve therapeutic effect and reduce side effects.

Disclosure of Invention

The inventor of the application provides an anti-epidermal growth factor receptor antibody drug conjugate preparation through a large amount of experiments and creative work, the preparation has good curative effect on diseases related to Epidermal Growth Factor Receptor (EGFR), and compared with the prior art, the stability of the preparation is obviously improved.

To this end, in a first aspect of the invention, the invention provides an antibody drug conjugate formulation comprising:

an antibody-drug conjugate or a salt thereof, the concentration of the antibody-drug conjugate or the salt thereof being 1-20mg/mL (e.g., 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL, or 20mg/mL, or 3-5mg/mL, or 2-6mg/mL, or 1-7mg/mL, or 1-8mg/mL, or 1-9mg/mL, or 1-10 mg/mL);

a citrate buffer at a concentration of 10-50mM (e.g., 10mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50mM, or 19-21mM, or 18-22mM, or 17-23mM, or 16-24 mM), and at a pH of 6.3-6.7 (e.g., 6.3, 6.4, 6.5, 6.6, or 6.7);

trehalose at a concentration of 1-10% (e.g., 1%, 2%, 3%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 8%, 9%, or 10%, or 4-7%, or 4.5-6.5%, or 5-6%);

sodium chloride at a concentration of 0.1-2% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2%, or 0.2-0.4%);

tween 80 at a concentration of 0.01 to 0.2% (e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, or 0.2%, or 0.02 to 0.08%, or 0.03 to 0.07%, or 0.04 to 0.06%);

the antibody drug conjugate has a structure shown in a formula I,

Ab-(L-D) _p

formula I

Wherein:

ab represents an anti-epidermal growth factor receptor antibody, wherein the anti-epidermal growth factor receptor antibody comprises a heavy chain and a light chain, wherein CDR1, CDR2, CDR3 of the heavy chain variable region comprise the amino acid sequences as set forth in SEQ ID NOs: 5-7 or a mutant thereof, wherein the light chain variable region CDR1, CDR2, CDR3 comprise the sequences as shown in SEQ ID NOs: 12 to 14 or a mutant thereof;

l represents a linker which is 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB);

d represents a cytotoxic agent which is MMAE;

p represents 1 to 9 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9, or such as 1 to 7), for example 2 to 6, for example 3 to 5, particularly for example 3.9, 4.0 or 4.1.

It should be noted that the concentration of trehalose, sodium chloride or tween 80 represents a mass volume concentration (w/v%), and refers to the mass (unit: g) of trehalose, sodium chloride or tween 80 per 100mL of the antibody drug conjugate preparation.

In addition, the "citric acid buffer" is prepared by citric acid and sodium citrate, and different pH values are realized by adjusting different citric acid and sodium citrate ratios. The concentration of the "citrate buffer" refers to the total concentration of citric acid and sodium citrate.

In addition, the above "antibody drug conjugate" refers to a composition containing ADC molecules of the same or different DAR values.

In particular, the invention provides compositions comprising a plurality of anti-Epidermal Growth Factor Receptor (EGFR) ADC molecules. In certain instances, each ADC in a composition described herein comprises the same number of one or more cytotoxic agents. In other cases, each ADC in the compositions described herein comprises a different number of one or more cytotoxic agents.

In the antibody drug conjugates described herein, 1, 2, 3, 4, 5, 6, 7, 8 or more cytotoxic agents may be coupled to each anti-Epidermal Growth Factor Receptor (EGFR) antibody.

The Drug Antibody Ratio (DAR) refers to the number of molecules of cytotoxic agent coupled to anti-Epidermal Growth Factor Receptor (EGFR) antibody. The number of molecules of cytotoxic agent contained in an ADC described herein is typically an integer, and when the number of molecules of cytotoxic agent contained in an ADC described herein (e.g., p in formula I) is a fraction, the fraction refers to the average number of cytotoxic agents per anti-Epidermal Growth Factor Receptor (EGFR) antibody conjugate in a composition comprising a plurality of ADC molecules.

In some embodiments, the concentration of the antibody drug conjugate or salt thereof is 2-6mg/mL.

In some embodiments, the concentration of the antibody drug conjugate or salt thereof is 4mg/mL.

In some embodiments, the concentration of the citrate buffer is 15-25mM.

In some embodiments, the concentration of the citrate buffer is 20mM.

In some embodiments, the pH of the citric acid buffer is 6.3-6.5.

In some embodiments, the pH of the citrate buffer is 6.3.

In some embodiments, the trehalose is at a concentration of 4-7%.

In some embodiments, the trehalose is at a concentration of 5.5%.

In some embodiments, the concentration of sodium chloride is 0.1-0.5%.

In some embodiments, the concentration of sodium chloride is 0.3%.

In some embodiments, the concentration of tween 80 is 0.01 to 0.1%.

In some embodiments, the concentration of tween 80 is 0.05%.

In some embodiments, FR1, FR2, FR3, FR4 regions of the heavy chain variable region of the anti-epidermal growth factor receptor antibody comprise the amino acid sequences set forth in SEQ ID NOs: 8 to 11 or a mutant thereof.

In some embodiments, FR1, FR2, FR3, FR4 regions of the anti-epidermal growth factor receptor antibody light chain variable region comprise the amino acid sequences set forth in SEQ ID NOs: 15 to 18 or a mutant thereof.

In some embodiments, the anti-epidermal growth factor receptor antibody heavy chain constant region is selected from a human IgG, igM, igA, igD, igA constant region, or a mutant of the above constant regions.

In some embodiments, the IgG is selected from IgG1, igG2, igG3, and IgG4.

In some embodiments, the light chain constant region of the anti-epidermal growth factor receptor antibody is a human lambda constant region, a kappa constant region, or a mutant of the above constant regions.

In some embodiments, the sequence of the heavy chain variable region of the anti-epidermal growth factor receptor antibody comprises the amino acid sequence set forth in SEQ ID NO:1, or a sequence comprising a sequence identical to SEQ ID NO:1, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%.

In some embodiments, the anti-epidermal growth factor receptor antibody has the sequence of the heavy chain variable region as set forth in SEQ ID NO:1 is shown.

In some embodiments, the sequence of the light chain variable region of the anti-epidermal growth factor receptor antibody comprises the amino acid sequence set forth in SEQ ID NO:2, or a sequence comprising a sequence identical to SEQ ID NO:2, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%.

In some embodiments, the light chain variable region of the anti-epidermal growth factor receptor antibody has the sequence set forth in SEQ ID NO:2, respectively.

In some embodiments, the sequence of the heavy chain constant region of the anti-epidermal growth factor receptor antibody comprises the amino acid sequence as set forth in SEQ ID NO:3, or a sequence comprising a sequence identical to SEQ ID NO:3, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%.

In some embodiments, the anti-epidermal growth factor receptor antibody has a heavy chain constant region with a sequence as set forth in SEQ ID NO:3, respectively.

In some embodiments, the sequence of the light chain constant region of the anti-epidermal growth factor receptor antibody comprises a sequence as set forth in SEQ ID NO:4, or a sequence comprising a sequence identical to SEQ ID NO:4, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%.

In some embodiments, the light chain constant region of the anti-epidermal growth factor receptor antibody has the sequence set forth in SEQ ID NO:4, respectively.

In some embodiments, the trehalose is trehalose dihydrate.

In some embodiments, the citric acid buffer is formulated from citric acid and sodium citrate.

In some embodiments, the citric acid is citric acid monohydrate.

In some embodiments, the sodium citrate is sodium citrate dihydrate.

In a second aspect of the invention, the invention provides a method of preparing the aforementioned formulation, comprising:

1) Carrying out reduction reaction on the anti-epidermal growth factor receptor antibody and a reducing agent to obtain a reduced anti-epidermal growth factor receptor antibody, wherein the anti-epidermal growth factor receptor antibody is as described above;

2) Carrying out coupling reaction on the reduced anti-epidermal growth factor receptor antibody and vcMMAE;

3) Quenching the coupling reaction, and performing buffer solution replacement on the coupling reaction product to obtain the antibody drug conjugate preparation, wherein the antibody drug conjugate preparation is as described above.

In some embodiments, the method comprises:

(1) Replacing (preferably three times) the aforementioned anti-epidermal growth factor receptor antibody (preferably 10 mg) in a reduction buffer (preferably containing 25mM sodium borate, pH8.0,25mM NaCl,5mM EDTA), detecting the concentration of the protein, and calculating the content of the protein;

(2) Adding DTT into the product of the step (1), and reacting for 1-5 hours (preferably 2 hours) at room temperature to obtain a reduced anti-epidermal growth factor receptor antibody, wherein the amount of the DTT substance is 2.0-3.0 times, preferably 2.5 times that of the protein substance;

(3) Displacing (preferably three times) the product of step (2) into a coupling buffer (preferably comprising 50mM Tris, pH7.2,150mM NaCl,5mM EDTA) and calculating the amount of free thiol species;

(4) Adding vc-MMAE (namely MC-vc-PAB-MMAE) into the product of the step (3), and reacting for 1-5 hours (preferably 2 hours) under room temperature condition, wherein the amount of the substance of the vc-MMAE is 1.0-1.5 times, preferably 1.1 times that of the reduced anti-epidermal growth factor receptor antibody;

(5) Adding N-acetylcysteine to the product of step (4), and standing still (preferably for 5 minutes) to obtain a mixture containing the antibody drug conjugate, wherein the amount of the N-acetylcysteine is 15-25 times, preferably 20 times that of the vc-MMAE;

(6) Displacing (preferably, displacing three times) the mixed solution obtained in the step (5) into a conjugate stock solution to obtain the antibody drug conjugate preparation;

the coupling stock solution comprises:

trehalose, at a concentration of 1-10% (e.g., 1%, 2%, 3%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 8%, 9%, or 10%, or 4-7%, or 4.5-6.5%, or 5-6%);

sodium chloride at a concentration of 0.1-2% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2%, or 0.2-0.4%); and

in the antibody drug conjugate preparation, the concentration of the antibody drug conjugate is 1-20mg/mL (such as 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL or 20mg/mL, or 3-5mg/mL, or 2-6mg/mL, or 1-7mg/mL, or 1-8mg/mL, or 1-9mg/mL, or 1-10 mg/mL).

In some embodiments, the concentration of the citrate buffer is 15-25mM.

In some embodiments, the concentration of the citrate buffer is 20mM.

In some embodiments, the pH of the citric acid buffer is 6.3-6.5.

In some embodiments, the pH of the citrate buffer is 6.3.

In some embodiments, the trehalose is at a concentration of 4-7%.

In some embodiments, the trehalose is at a concentration of 5.5%.

In some embodiments, the concentration of sodium chloride is 0.1-0.5%.

In some embodiments, the concentration of sodium chloride is 0.3%.

In some embodiments, the concentration of tween 80 is 0.01 to 0.1%.

In some embodiments, the concentration of tween 80 is 0.05%.

In some embodiments, the concentration of the antibody drug conjugate is 2-6mg/mL.

In some embodiments, the concentration of the antibody drug conjugate is 4mg/mL.

In some embodiments, the trehalose is trehalose dihydrate.

In some embodiments, the citric acid is citric acid monohydrate.

In some embodiments, the sodium citrate is sodium citrate dihydrate.

In a third aspect of the invention, the invention provides an antibody drug conjugate formulation prepared by the aforementioned method.

In a fourth aspect of the invention, the invention provides a composition comprising the aforementioned formulation.

In some embodiments, the composition further comprises a known chemotherapeutic agent for treating tumors, such as doxorubicin (Adriamycin), cyclophosphamide, taxanes such as paclitaxel (Taxol), docetaxel (Taxotere), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (runder, froron, arninoxin), 5-FU plus leucovorin, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, or the like, or a combination thereof.

In some embodiments, the composition further comprises known immunotherapeutic agents for treating tumors, such as PD-1 mab (e.g., parbolzumab, nivolumab Wu Liyou mab), PD-L1 mab (e.g., atezolizumab), TIGIT mab, 4-1BB mab, VEGFR2 mab (e.g., ramucirumab, apatinib), HER2 mab (e.g., trastuzumab biosilimer, trastuzumab-dkst), and the like, or combinations thereof.

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, tacrolimus, and the like; (3) Antimetabolites such as azathioprine and 6-mercaptopurine, and the like; (4) Polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT 3; (5) alkylating agents, such as cyclophosphamide. Specifically, the immunosuppressive agent is, for example, methylprednisolone, prednisone, azathioprine, pleconate, sirolimus, sulley, cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod and the like.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

In a fifth aspect of the invention, the invention provides the use of the aforementioned formulation or the aforementioned composition for the manufacture of a medicament for the prevention and/or treatment of a disease associated with Epidermal Growth Factor Receptor (EGFR).

In a sixth aspect of the invention, the invention provides the aforementioned formulation or the aforementioned composition for use in the prevention and/or treatment of a disease associated with Epidermal Growth Factor Receptor (EGFR).

In a seventh aspect of the present invention, the present invention provides a method for preventing and/or treating a disease associated with Epidermal Growth Factor Receptor (EGFR), comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of the aforementioned formulation or the aforementioned composition.

In some embodiments, the disease associated with Epidermal Growth Factor Receptor (EGFR) is a tumor associated with EGFR, such as a tumor associated with EGFR overexpression, further such as selected from colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, renal cancer, prostate cancer, gastric cancer, pancreatic cancer, and brain glioma.

In some embodiments, the tumor is a KRAS gene mutated tumor, further, for example, KRAS gene mutated colon, rectal, lung, or pancreatic cancer.

In some embodiments, the tumor is a tumor with a mutation in the BRAF gene, further, for example, selected from the group consisting of cancer of colon, rectum, and lung with a mutation in the BRAF gene.

In an eighth aspect of the invention, the invention provides the use of the aforementioned preparation or the aforementioned composition in the preparation of an agent or a medicament for inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth, and inhibiting tumor cell proliferation.

In a ninth aspect of the invention, the invention provides the aforementioned formulation or the aforementioned composition for use in inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth, inhibiting tumor cell proliferation.

In a tenth aspect of the present invention, the present invention provides a method for inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth, inhibiting tumor cell proliferation, which comprises administering to a subject in need thereof an effective amount of the aforementioned formulation or the aforementioned composition.

In some embodiments, the tumor is selected from the group consisting of colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer, and brain glioma.

In some embodiments, the tumor is a tumor with a mutation in the BRAF gene, further, for example, selected from the group consisting of colon cancer, rectal cancer, and lung cancer with a mutation in the BRAF gene.

Has the advantages that:

the antibody drug conjugate preparation of the invention keeps clear in appearance under different temperature conditions, the DAR value is basically kept unchanged, the contents of high polymer% (HMW%) and acidic peak% are moderate, and the preparation stability is obviously improved compared with the prior art.

Drawings

FIG. 1 HIC-HPLC chart for determining the drug/antibody ratio of antibody drug conjugates.

FIG. 2 in vitro cell activity assay of monoclonal antibody and antibody drug conjugate, wherein O represents BA03 monoclonal antibody, and A-solidup represents MYK-3 antibody drug conjugate.

FIG. 3 growth inhibitory Activity of MYK-3 on colon cancer cells HT-29, wherein O represents BA03 monoclonal antibody, and A represents MYK-3 antibody drug conjugate.

FIG. 4 growth inhibitory activity of MYK-3 on brain glioma cancer cells U87-MG, wherein O represents BA03 monoclonal antibody, and A-solidup represents MYK-3 antibody drug conjugate.

FIG. 5 growth inhibitory activity of MYK-3 on lung carcinoma cells A549, wherein O represents BA03 monoclonal antibody, and A-solidup represents MYK-3 antibody drug conjugate.

FIG. 6 growth inhibitory activity of MYK-3 on KRAS mutant colon cancer cells LoVo, where A represents the erbitux monoclonal antibody and A-tangle-solidup represents the MYK-3 antibody drug conjugate.

FIG. 7 Effect of monoclonal antibodies and antibody drug conjugates on the volume of mouse HT-29 colon carcinoma transplants. Data are presented as mean ± standard deviation; p < 0.05, P < 0.01, P < 0.001, compared to the buffer control group.

FIG. 8 Effect of monoclonal antibodies and antibody drug conjugates on body weight in a mouse HT-29 colon cancer graft tumor model.

FIG. 9 the growth inhibitory activity of MYK-3 on the transplanted tumor of KRAS mutant colon cancer cell LoVo in nude mice.

FIG. 10 comparison of the change in mass properties of MYK-3 in citrate buffer solutions of different pH.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present invention, unless otherwise indicated, any concentration range, percentage range, ratio range or numerical range should be understood to include any integer value within the range, and fractional value within the range, as appropriate.

In the present invention, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. The light chains of antibodies can be classified into two types, kappa and lambda. Heavy chains can be divided into five types, namely mu, delta, gamma, alpha or epsilon, and antibodies can be divided into five types, namely IgM, igD, igG, igA and IgE according to the difference of the heavy chains. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V) _H ) And heavy chain constant region (C) _H ) And (4) forming. The heavy chain constant region consists of 3 domains (C) _H 1、C _H 2 and C _H 3) And (4) forming. Each light chain is composed of a light chain variable region (V) _L ) And a light chain constant region (C) _L ) And (4) forming. The light chain constant region consists of a domain C _L And (4) forming. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and component C1q of the complement system. V _H And V _L Regions may also be subdivided into regions of high degeneracy, known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FRs). Each V _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. Variable region (V) of each heavy/light chain pair _H And V _L ) Antibody binding sites were formed separately. The assignment of amino acids to regions or domains follows either 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.

The monoclonal antibody variant can be obtained by a traditional genetic engineering method. The person skilled in the art is fully aware of methods for the engineering of DNA molecules using nucleic acid mutations. Alternatively, nucleic acid molecules encoding the heavy and light chain variants may be obtained by chemical synthesis.

In the present invention, algorithms for determining sequence identity (homology) and percent sequence similarity are, for example, the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1977) Nucl.acid.Res.25: 3389-3402 and Altschul et al (1990) J.mol.biol.215:403-410. BLAST and BLAST 2.0 can be used to determine percent amino acid sequence identity in the present invention using, for example, the parameters described in the literature or default parameters. Software for performing BLAST analysis is publicly available through the biotechnology information center.

In the present invention, such amino acid sequences having at least 70% sequence identity to an amino acid sequence include polypeptide sequences substantially identical to the amino acid sequence, e.g., those sequences that contain at least 70% sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity as compared to the polypeptide sequence of the present invention when the methods described herein (e.g., BLAST analysis using standard parameters) are employed.

In the present invention, a mutant of the amino acid sequence refers to a sequence which has more than 70% identity to the amino acid sequence, such as more than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, such as a sequence having 3, 2 or 1 substituted, deleted or added amino acids. Preferably, the amino acid substitution, addition or deletion does not exceed 3 amino acids. More preferably, the amino acid to be substituted, added or deleted is not more than 2 amino acids. Most preferably, the amino acid substitution, addition or deletion does not exceed 1 amino acid.

A "substituted" variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted into its same position. The substitutions may be single, in which only one amino acid in the molecule is substituted; or may be multiple, wherein two or more amino acids of the same molecule are substituted. Multiple substitutions may be at consecutive sites. Likewise, an amino acid may be substituted with multiple residues, wherein such variants include both substitutions and insertions. An "insertion" (or "addition") variant is a variant in which one or more amino acids are inserted into an amino acid at a specific position immediately adjacent to a stretch of native sequence. By immediately adjacent to an amino acid is meant attached to the alpha-carboxy or alpha-amino functionality of that amino acid. A "deletion" variant is a variant in which one or more amino acids in the native amino acid sequence are removed. Typically, a deletion variant has one or two amino acids deleted in a particular region of its molecule.

In the present invention, the MMAE has the structure:

in the invention, L-D in the formula I is MC-vc-PAB-MMAE, and the structure of the L-D is shown as the following formula:

in the present invention, anti-epidermal growth factor receptor antibody Ab is connected to L-D through a thiol group generated by reducing its own disulfide bond, as shown below:

in the present invention, the Drug Antibody Ratio (DAR) or drug loading (loading) is represented by p, i.e., formula i: ab- (L-D) _p The average number of drug moieties (i.e., cytotoxic agents) per antibody in the molecule of (a) may be an integer or a fraction. The ADC of formula I comprises a collection of antibodies coupled to a range of drug moieties. The average number of drug modules per antibody in the ADC preparation from the conjugation reaction can be verified by conventional means, such as mass spectrometry, ELISA assay, HIC and HPLC. The quantitative distribution of the ADC in p can also be determined. In some cases, a homogeneous ADC slave with p being a certain valueSeparation, purification and validation in ADCs with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.

In certain embodiments, less than the theoretical maximum of drug moieties are conjugated to the antibody in the conjugation reaction. In general, antibodies do not contain many free and reactive cysteine thiol groups to which a drug moiety can be attached; in fact, most cysteine thiol groups in antibodies exist as disulfide bridges. In certain embodiments, the antibody may be reduced with a reducing agent such as Dithiothreitol (DTT) or Tricarbonylethylphosphine (TCEP) under partially or fully reducing conditions to produce reactive cysteine thiol groups.

In the present invention, "treatment" refers to clinical intervention in an attempt to alter the natural course of the treated individual or cell, either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis. In some embodiments, the antibody drug conjugates of the invention are used to delay the onset of a disease or disorder, or to slow the progression of a disease or disorder. The above parameters for assessing successful treatment and improvement of a disease can be readily measured by conventional procedures familiar to physicians. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or determining Response Rate (RR).

In the present invention, "subject" refers to a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

In the present invention, "effective amount" refers to an amount effective in achieving a desired therapeutic or prophylactic effect at a necessary dose and time. The "therapeutically effective amount" of a substance/molecule of the invention may vary depending on 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 also encompasses an amount of the substance/molecule that outweighs any toxic or detrimental consequences. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic effect. Typically, but not necessarily, since a prophylactic dose is administered to a subject prior to the onset of disease or at an early stage of disease, the prophylactically effective amount will be lower than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the tumor volume; 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 tumor growth to some extent; and/or to reduce to some extent one or more symptoms associated with cancer.

For the prevention or treatment of disease, the appropriate dosage of an antibody drug conjugate of the invention (either alone or in combination with one or more other therapeutic agents such as chemotherapeutic agents) will depend 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 administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and responsiveness to the antibody drug conjugate, and the discretion of the attending physician. Suitably, the antibody drug conjugate is administered to the patient at once or through a series of treatments.

"pharmaceutically acceptable carrier" as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammal to which they are exposed at the dosages and concentrations employed. Typically, the 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 glucoseMannose, sucrose, trehalose or dextrin; 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 ^TM Polyethylene glycol (PEG) and PLURONICS ^TM 。

In some embodiments, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt, wherein the inorganic acid salt is a hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate and carbonate, sulfate or phosphate, and the organic acid salt is a formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α -ketoglutarate, α -glycerophosphate, alkylsulfonate or arylsulfonate; preferably, the alkyl sulfonate is a methyl sulfonate or an ethyl sulfonate; the aryl sulfonate is benzene sulfonate or p-toluene sulfonate.

Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid to provide a pharmaceutically acceptable anion.

In the present invention, the KRAS gene has the same meaning as that of the K-RAS gene, which is one of RAS gene family members, encodes K-RAS protein, and is involved in the generation, proliferation, migration, spread and angiogenesis of various tumors. The common mutation sites are codon 12 and codon 13 of exon 2 of K-RAS gene and codon 61 of exon 3, wherein there are 7 mutation hot spots: G12C, G12R, G12S, G V, G12D, G12A, G V/D7 mutations account for more than 90%. In one embodiment of the invention, the tumor is a tumor with KRAS gene mutation associated with EGFR overexpression.

In the present invention, the BRAF (v-RAF murine sarroma viral oncogene homologB 1) gene is a protooncogene and is one of the RAF family members. About 8% of human tumors develop BRAF mutation, and the vast majority of mutant forms of BRAF gene mutation are BRAFV600E mutation, which causes the continuous activation of downstream MEK/ERK signaling pathway and is important for the growth, proliferation, invasion and metastasis of tumors. In one embodiment of the invention, the tumor is a tumor with a mutation in the BRAF gene associated with overexpression of EGFR.

In the present invention, 20 kinds of conventional amino acids and abbreviations thereof follow conventional usage. See Immunology-A Synthesis (2 nd edition, E.S. Golub and D.R. Gren, eds., sinauer Associates, sunderland, mass. (1991)), which is incorporated herein by reference.

The antibody BA03 in the invention, namely BA03 in Chinese invention patent application CN 103772504A, is prepared by referring to example 3 in the patent application, and the sequence of each part of the antibody is as follows:

the heavy chain variable region sequence is:

QVQLQESGPGLVKPSETLSLTCTVSGFSLSNYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDYYDYEFAY WGQGTLVTVSS(SEQ ID NO：1)。

wherein the underlined portions are CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 6), CDR3 (SEQ ID NO: 7), respectively;

the underlined portions are FR1 (SEQ ID NO: 8), FR2 (SEQ ID NO: 9), FR3 (SEQ ID NO: 10) and FR4 (SEQ ID NO: 11), respectively.

The light chain variable region sequence is:

EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK(SEQ ID NO：2)。

wherein the underlined portions are CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO: 13), CDR3 (SEQ ID NO: 14), respectively;

the non-underlined portions are FR1 (SEQ ID NO: 15), FR2 (SEQ ID NO: 16), FR3 (SEQ ID NO: 17) and FR4 (SEQ ID NO: 18), respectively.

The heavy chain constant region sequence is:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：3)。

the light chain constant region sequence is:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO：4)。

the invention is further illustrated by the following examples.

Example 1: preparation method of antibody drug conjugate

10mg of BA03 antibody was taken and replaced with a reducing buffer (25 mM sodium borate, pH8.0,25mM NaCl,5mM EDTA) by using a 15mL 30KD ultrafiltration apparatus for three times in total; the final volume was about 1mL, transferred to a new Eppendorf centrifuge tube (weighed) and weighed; detecting the protein concentration and calculating the total protein amount. Adding 2.5 times of mole number of DTT into the antibody, keeping the temperature for 2 hours at room temperature, and continuously mixing uniformly; the solution was replaced into coupling buffer (50mM Tris, pH7.2,150mM NaCl,5mM EDTA) three times using a 15ml 30KD ultrafiltration unit. Taking the concentrated solution, measuring the protein concentration by A280, weighing, and calculating the total amount of the protein; taking 10 mu l of sample, and determining the number of free sulfydryl by an Ellman's method;

and calculating the molar concentration of free thiol groups by the following formula:

b-cuvette optical path length (typically 1 cm).

Free thiol moles were calculated from the free thiol molar concentration and total protein solution volume.

To the reduced antibody, vc-MMAE (obtained from Haoyuan chemical technology Co., ltd., shanghai, inc., cat. No. HY-15575) in a molar amount of 1.1 times that of the free thiol was added (dissolved in DMSO), and the mixture was mixed, reacted at room temperature for 2 hours, and mixed intermittently. Adding N-acetylcysteine with the mole number 20 times that of the added vc-MMAE into the reaction solution, uniformly mixing, and standing for 5 minutes. The antibody drug conjugate MYK-3 was obtained by replacing the conjugate stock solution (20 mM sodium citrate (Na-citrate), 0.3% NaCl,5% Trehalose (Trehalose), 0.05% Tween-80, pH 6.0) with a 15ml 30KD ultrafiltration unit for three times; the samples were stored at 4 ℃. It should be noted that after quenching the conjugation reaction with N-acetylcysteine, an antibody drug conjugate MYK-3 has been prepared. The subsequent step (i.e. the replacement of the conjugate stock with a 15ml 30kD ultrafiltration device) was to place the antibody drug conjugate MYK-3 in the conjugate stock for storage and ready for use.

Determination of drug/antibody ratio:

prepared Antibody Drug conjugate MYK-3 was analyzed by HIC-HPLC (Jun Ouyang, drug-To-Antibody (DAR) Ratio and Drug distribution Hydrophoc Interaction Chromatography and Reverse Phase High Performance Chromatography, lamp Dual (ed.), antibody Drug Conjugates, chapter 17, methods in Molecular biology, vol 1045, 275 p283) To determine the Drug/Antibody Ratio (Drug-loaded Antibody Ratio, DAR), see FIG. 1, with an average number DAR of 4.1 calculated from the peak area of the map.

Example 2: determination of in vitro cell Activity of antibody drug conjugate MYK-3

The cell activity detection method comprises the following steps:

1.1 after 3-4 cell passages, pouring out the culture medium, rinsing with 5mL of DPBS, digesting the cells with 3mL of trypsin, re-suspending with the culture medium, centrifuging by a centrifuge, discarding the supernatant, re-suspending with the culture medium, taking out 0.5mL of the cells, and counting by a cell counter. Plating is carried out on a 96-well cell culture plate (the DiFi cells are 10000 cells/well, the HT-29 cells are 5000 cells/well, the A549 cells are 2000 cells/well, the U87-MG cells are 3000 cells/well, and the LoVo cells are 4000 cells/well), after 24 hours of culture, monoclonal antibody BA03 and antibody drug conjugate MYK-3 with serial dilution concentration are added, heat preservation is carried out for 72 hours, 20 mul CCK8 color reagent is added into each well, OD450-650 is detected by a microplate reader at the wavelength of 450-650nm, and four-parameter fitting is carried out.

In vitro cell activity test results:

the following cell lines were purchased from Shanghai institute of cell biology, china academy of sciences.

Activity in EGFR-highly expressed DiFi cells (human colorectal cancer cells): MYK-3 has remarkably increased cell growth inhibitory activity, EC, than monoclonal antibody BA03 ₅₀ About 10 times lower (EC of BA 03) ₅₀ Is 51.9ng/ml, EC for MYK-3 ₅₀ Is 5.1 ng/ml) as shown in FIG. 2.

Activity in other tumor cells with moderate and low expression of EGFR: MYK-3 also showed significant cell growth inhibitory activity (as shown in FIG. 3, FIG. 4, FIG. 5) against cancer cells with moderate and low expression of EGFR (human colon cancer cell HT29, human lung cancer cell A549, human brain astrocytoma cell U87-MG) relative to mAb itself (EC of HT-29) ₅₀ Is 611ng/ml, EC of A549 ₅₀ Is 28.3. Mu.g/ml, EC for U87-MG ₅₀ It was 5.3. Mu.g/ml.

In addition, we also tested the activity in the intermediate EGFR expressing KRAS mutant colon cancer cells LoVo (Dunn EF, ilda M, myers RA, hintz KA, campbell DA, armstrong EA, li C and Wheeler DL. Dasatinib susceptizers KRAS tissue tumor tissue to cetriximab. Oncogene 2011 30) and found that MYK-3 showed significant tumor growth inhibitory activity on KRAS mutant colon cancer cells LoVo (as shown in fig. 6, EC 561-574 ₅₀ 3.2. Mu.g/ml), whereas BA03 alone had little inhibitory activity against this cell line.

Example 3: experiment of mouse in vivo transplantation tumor

Experimental method for mouse in vivo transplantation tumor:

HT-29 colon cancer cells are cell strains with low EGFR expression and BRAF mutation, and EGFR targeting monoclonal antibody Erbitux (Erbitux) for treating colorectal cancer which is sold on the market at present has no growth inhibition activity on the HT-29 cell strains.

HT-29 cell transplantation model: collecting tumor cells in logarithmic growth phase, counting, re-suspending in 1 XPBS, and regulating cell suspension concentration to 3 XP 10 ⁷ And/ml. Nude mice were inoculated subcutaneously on the right back with tumor cells, 3X 10, using a 1ml syringe (No. 4 needle) ⁶ 0.1 ml/mouse when the tumor volume reaches 150-200mm ³ Random block method for later useGrouping, 8 mice in each group, ensuring that the tumor volume and the weight of the mice are uniform among groups. The mean tumor volumes of the groups differed from the mean tumor volumes of all experimental animals by no more than ± 10%. The tail vein was dosed once every four days (

days

1, 5, 9, 13) for a total of 4 doses, with tumor volume and mouse body weight measured periodically. There were 8 mice per administration group.

Experimental results of mouse transplantable tumor

Mouse HT-29 colon carcinoma transplantable tumor experiments: the experiment was divided into five groups including buffer group (20 mM sodium citrate, 0.3% sodium chloride, 5% trehalose, 0.05% Tween 80, pH 6), BA03 monoclonal antibody group (5 mg/kg), MYK-3 group (1 mg/kg), MYK-3 group (5 mg/kg) and non-binding ADC group (5 mg/kg) (human IgG-vcMAE conjugate, where IgG is IgG purified from healthy human serum, prepared in the same manner as MYK-3). MYK-3 dosed mice had significantly reduced transplanted tumor volumes compared to the control group, showing a significant anti-tumor growth effect (fig. 7). On day 18, the tumor growth inhibition rate of the MYK-3 group at 5mg/Kg dose compared with the buffer solution group reaches 54%, 46% compared with the monoclonal antibody BA03 group at the same dose, and 42% compared with the unconjugated ADC group.

Mouse weight: the body weight of mice dosed with MYK-3 was unchanged compared to the control group (see FIG. 8), indicating that MYK-3 had no toxic effect of reducing body weight in mice.

Example 4: inhibitory Activity of MYK-3 on growth of transplanted tumor of KRAS mutant colon cancer cell LoVo in nude mouse

Model construction according to example 3 (at 2X 10) ⁶ 0.1 ml/mouse inoculated tumor cells) and a dosing method, the transplanted tumor growth inhibitory activity of MYK-3 on KRAS mutant colon cancer cells LoVo in nude mice is detected, the experiment is divided into six groups, including a dilution buffer group (20 mM sodium citrate, 0.3% sodium chloride, 5% trehalose, 0.05% Tween 80, pH6), an erbitumomab group (3 mg/kg), a MYK-3 group (three doses of 0.3mg/kg, 1mg/kg and 3 mg/kg) and a non-binding control ADC group (3 mg/kg), wherein the non-binding control ADC represents an ADC control (non-binding ADC control, anti-CD20 mAb) with the same drug loading rate in non-binding mode-vcMMAE), the conjugate being prepared in the same way as MYK-3. The results of the experiment are shown in FIG. 9.

As can be seen from the figure, MYK-3 shows a complete inhibition of tumor growth in LoVo cells at the 3mg/Kg dose, and MYK-3 at 1mg/Kg has shown a still stronger activity than the marketed drug Erbitux at the 3mg/Kg dose.

Example 5 Effect of pH on MYK-3 stability

The inventors have conducted further intensive studies on pH, starting from the antibody drug conjugate obtained in example 1. The results show that the pH of the optimal formulation for MYK-3 is 6.3. That is, the optimal formulation was formulated with 20mM citric acid buffer, 5.5% trehalose dihydrate, 0.3% sodium chloride and 0.05% PS80, pH6.3, and the concentration of antibody-drug conjugate MYK-3 was 4mg/mL.

To examine the effect of different pH of MYK-3 on its quality in a 20mM citric acid buffer solution (containing 5.5% trehalose dihydrate, 0.3% sodium chloride and 0.05% PS80), the procedure was the same as in example 1 except that the recipe (recipe F1-F5 to be investigated) of the coupling stock solution replaced using a 15ml 30KD ultrafiltration device after quenching the coupling reaction with N-acetylcysteine was different from that in example 1.

The compositions and concentrations of the formulations F1 to F5 to be examined were the same except that the pH of the citric acid buffer solutions were different. The method specifically comprises the following steps: 4mg/mL of antibody-drug conjugate MYK-3, 20mM citrate buffer, pH 5.6 (F1), 6.0 (F2), 6.3 (F3), 6.5 (F4), or 6.7 (F5), 5.5% trehalose dihydrate, 0.3% sodium chloride, and 0.05% PS80.

Specifically, the test solutions were exchanged into the recipes F1 to F5 (pH: 5.6 to 6.7) to be examined, examined for 10 days under the conditions of 40. + -. 2 ℃/75% +/-5% RH, and sampled and analyzed at set time points, and the test items included appearance, pH, protein concentration, SEC, iCIEF and HIC. If the sample is cloudy in appearance or has a more pronounced opalescence, no subsequent correlation analysis (e.g., SEC, iCIEF, and HIC) will be performed. The specific experimental design is shown in table 1.

TABLE 1 influence of pH (5.6-6.7) on the stability of MYK-3 (20 mM citrate buffer)

Note: t0 denotes the starting point, D denotes day, and √ denotes detection.

From the appearance, as the time increases, the sample is turbid when F1 is placed at 40 ℃ for 3 days, the sample is turbid when F2 is placed at 40 ℃ for 7 days, and obvious opalescence and precipitation can be observed when the temperature of F1 and F2 is reduced to room temperature and 2-8 ℃, and the lower the pH value is, the more precipitation is; however, other prescriptions remained clear throughout and no significant change was observed.

The SEC, iCIEF and HIC assay data and trends are shown in Table 2 and FIG. 10. Since the samples for the F1 prescription were cloudy after being left at 40 degrees for 3 days, the SEC, iCIEF and HIC tests were not performed again for the F1 prescription at 3 and 7 days; meanwhile, since the samples for the F2 prescription were cloudy after being left at 40 degrees for 7 days, the SEC, iCIEF, and HIC tests for the F2 prescription at 7 days were no longer performed. HIC data showed that pH was different and drug loading (DAR) values for the F3 and F4 prescriptions were essentially unchanged (see fig. 10 c); the SEC data showed that the rate of increase of the high-mer% (HMW%) of the F3, F4 and F5 prescriptions decreased with increasing pH, with the decrease being substantially comparable (as in fig. 10 a); the iCIEF data show that the% acidity peaks for the F3, F4, and F5 prescriptions increased with increasing pH, but the rising trend for the F3 prescription was significantly lower than for the F4 and F5 prescriptions (see fig. 10 b). According to the test data, the optimal formula of MYK-3 is determined to be F3 by combining the detection results of appearance, SEC, iCIEF, HIC and the like, namely the pH value of the formula is 6.3. Accordingly, the amounts of citric acid and sodium citrate in the recipe were adjusted to obtain a final formulation of 20mM citrate buffer pH 6.3.

TABLE 2 pH (5.6-6.7) Effect on MYK-3 (20 mM citrate buffer) stability Experimental data

The final formulation of the MYK-3 formulation based on the above is as follows:

although specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that, based upon the overall teachings of the disclosure, various modifications and alternatives to those details could be developed and still be encompassed by the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

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Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

20 25 30

<210> 18

<211> 9

<212> PRT

<213> Artificial

<220>

<223> light chain variable region FR4

<400> 18

Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5

Claims

1. An antibody drug conjugate formulation comprising:

an antibody drug conjugate or a salt thereof, the concentration of the antibody drug conjugate or the salt thereof being 1-20mg/mL, preferably 2-6mg/mL, more preferably 4mg/mL;

a citric acid buffer at a concentration of 10-50mM, preferably 15-25mM, more preferably 20mM, and a pH of 6.3-6.7, preferably 6.3-6.5, more preferably 6.3;

trehalose, the concentration of the trehalose is 1-10%, preferably 4-7%, more preferably 5.5%;

sodium chloride, the concentration of the sodium chloride is 0.1-2%, preferably 0.1-0.5%, more preferably 0.3%;

tween 80, wherein the concentration of the tween 80 is 0.01 to 0.2 percent, preferably 0.01 to 0.1 percent, and more preferably 0.05 percent;

the antibody drug conjugate has a structure shown in a formula I,

Ab-(L-D) _p

formula I

Wherein:

ab represents an anti-epidermal growth factor receptor antibody, wherein the anti-epidermal growth factor receptor antibody comprises a heavy chain and a light chain, wherein CDR1, CDR2, CDR3 of the variable region of the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 5-7 or a mutant thereof, wherein the light chain variable region CDR1, CDR2, CDR3 comprise the sequences as shown in SEQ ID NOs: 12 to 14 or a mutant thereof;

d represents a cytotoxic agent which is MMAE;

p represents 1 to 9, preferably 2 to 6, more preferably 3 to 5.

2. The formulation of claim 1, wherein the anti-epidermal growth factor receptor antibody has one or more of the following characteristics:

1) The FR1, FR2, FR3 and FR4 regions of the heavy chain variable region of the anti-epidermal growth factor receptor antibody respectively comprise the amino acid sequences shown in SEQ ID NO: 8-11 or a mutant thereof;

2) The FR1, FR2, FR3 and FR4 regions of the variable region of the anti-epidermal growth factor receptor antibody light chain respectively comprise the amino acid sequences shown in SEQ ID NO:15 to 18 or a mutant thereof;

3) The heavy chain constant region of the anti-epidermal growth factor receptor antibody is selected from human IgG, igM, igA, igD, igA constant regions or mutants of the constant regions;

preferably, the IgG is selected from IgG1, igG2, igG3 and IgG4;

4) The light chain constant region of the anti-epidermal growth factor receptor antibody is a human lambda constant region, a kappa constant region or a mutant of the constant region.

3. The formulation of any one of claims 1-2, wherein the anti-epidermal growth factor receptor antibody has one or more of the following characteristics:

1) The sequence of the heavy chain variable region of the anti-epidermal growth factor receptor antibody comprises the amino acid sequence shown in SEQ ID NO:1, or a sequence comprising a sequence identical to SEQ ID NO:1, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%;

preferably, the sequence of the heavy chain variable region of the anti-epidermal growth factor receptor antibody is as shown in SEQ ID NO:1 is shown in the specification;

2) The sequence of the light chain variable region of the anti-epidermal growth factor receptor antibody comprises the amino acid sequence shown in SEQ ID NO:2, or a sequence comprising a sequence identical to SEQ ID NO:2, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%;

preferably, the light chain variable region of the anti-epidermal growth factor receptor antibody has the sequence shown in SEQ ID NO:2 is shown in the specification;

3) The sequence of the heavy chain constant region of the anti-epidermal growth factor receptor antibody comprises the amino acid sequence shown in SEQ ID NO:3, or a sequence comprising a sequence identical to SEQ ID NO:3, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%;

preferably, the sequence of the heavy chain constant region of the anti-epidermal growth factor receptor antibody is as shown in SEQ ID NO:3 is shown in the specification;

4) The sequence of the light chain constant region of the anti-epidermal growth factor receptor antibody comprises the amino acid sequence shown as SEQ ID NO:4, or a sequence identical to SEQ ID NO:4, preferably greater than 75%, 80%, 85%, 90%, 95%, 99%;

preferably, the light chain constant region of the anti-epidermal growth factor receptor antibody has the sequence shown in SEQ ID NO:4, respectively.

4. The formulation of any one of claims 1-3, wherein the trehalose is trehalose dihydrate;

or the citric acid buffer solution is prepared from citric acid and sodium citrate;

preferably, the citric acid is citric acid monohydrate;

preferably, the sodium citrate is sodium citrate dihydrate.

5. A method of making the formulation of any one of claims 1-4, comprising:

1) Subjecting an anti-epidermal growth factor receptor antibody to a reduction reaction with a reducing agent to obtain a reduced anti-epidermal growth factor receptor antibody, the anti-epidermal growth factor receptor antibody being as defined in any one of claims 1 to 3;

3) Quenching the conjugation reaction and subjecting the conjugation reaction product to buffer exchange to obtain the antibody drug conjugate preparation as defined in any one of claims 1 to 4.

6. The method of claim 5, wherein the method comprises:

(1) Displacing (preferably three times) an anti-epidermal growth factor receptor antibody (preferably 10 mg) as defined in any one of claims 1-3 into a reduction buffer (preferably comprising 25mM sodium borate, ph8.0,25mM nacl,5mM EDTA), detecting the concentration of the protein, calculating the content of the protein;

(4) Adding vc-MMAE into the product of the step (3), and reacting for 1-5 hours (preferably 2 hours) at room temperature, wherein the amount of the substance of the vc-MMAE is 1.0-1.5 times, preferably 1.1 times that of the reduced anti-epidermal growth factor receptor antibody;

(5) Adding N-acetylcysteine to the product of step (4), and allowing to stand (preferably for 5 minutes) to obtain a mixture containing an antibody drug conjugate as defined in claim 1, the amount of the substance of N-acetylcysteine being 15-25 times, preferably 20 times, the amount of vc-MMAE;

the coupling stock solution comprises:

sodium chloride, the concentration of the sodium chloride is 0.1-2%, preferably 0.1-0.5%, more preferably 0.3%; and

in the antibody drug conjugate preparation, the concentration of the antibody drug conjugate is 1-20mg/mL, preferably 2-6mg/mL, and more preferably 4mg/mL;

preferably, the trehalose is trehalose dihydrate;

preferably, the citric acid buffer is prepared from citric acid and sodium citrate;

preferably, the citric acid is citric acid monohydrate;

preferably, the sodium citrate is sodium citrate dihydrate.

7. An antibody drug conjugate formulation prepared by the method of any one of claims 5 to 6.

8. A composition comprising the formulation of any one of claims 1-4 or 7, optionally further comprising a pharmaceutically acceptable carrier, diluent or excipient.

9. Use of a formulation according to any one of claims 1 to 4 or 7 or a composition according to claim 8 selected from one or more of:

1) The use thereof for the preparation of a medicament for the prevention and/or treatment of diseases associated with the Epidermal Growth Factor Receptor (EGFR);

2) The application in preparing the reagent or the medicine for inhibiting the angiogenesis of the tumor, delaying the progress of the tumor, inhibiting the growth of the tumor and inhibiting the proliferation of the tumor cells.

10. The use according to claim 9, wherein the disease associated with Epidermal Growth Factor Receptor (EGFR) is a tumor associated with EGFR, such as a tumor associated with EGFR overexpression, further such as selected from the group consisting of colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer and brain glioma;

alternatively, the tumor is selected from colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer, and brain glioma;

alternatively, the tumor is a KRAS gene mutated tumor, further, for example, KRAS gene mutated colon, rectum, lung or pancreas cancer;

alternatively, the tumor is a tumor with BRAF gene mutation, and further, the tumor is selected from colon cancer, rectal cancer and lung cancer with BRAF gene mutation.