CN109200291B - Antibody coupling drug targeting EGFR (epidermal growth factor receptor), preparation method and application thereof - Google Patents
Antibody coupling drug targeting EGFR (epidermal growth factor receptor), preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an antibody coupling drug targeting EGFR, a preparation method and application thereof. The antibody drug conjugate targeting the EGFR is named as LR004-VC-MMAE and comprises an antibody, a cytotoxic drug and a linker, wherein the antibody drug conjugate has a structure shown in a formula I, wherein the mAb is an LR004 monoclonal antibody, and n is 2-8. The novel antibody coupling drug LR004-VC-MMAE can target EGFR antigen and has strong activity of killing tumor cells. Compared with LR004, the affinity, the endocytic activity and the targeting property of the antibody are not influenced, and the biological function of the antibody is well reserved. Compared with LR004, the tumor inhibition effect of the LR004-VC-MMAE antibody coupling drug is remarkably improved, and the situation of tumor disappearance appears. Compared with LR004, the LR004-VC-MMAE antibody conjugate drug total antibody has longer half-life, slower clearance rate, lower concentration of free MMAE in plasma, short half-life and fast clearance speed, and is beneficial to reducing toxicity.
Description
Technical Field
The invention belongs to the field of biotechnology medicine. Specifically, the invention provides an antibody coupling drug capable of generating a targeted tumor killing effect, a preparation method and application thereof.
Technical Field
According to the global cancer report, 1810 ten thousands of cancer cases are predicted to be newly added in 2018, the number of deaths reaches 960 ten thousands, 18 deaths per minute are caused by cancer on average, and the global cancer burden is further increased. China is a big cancer country, and the number of new patients is on a remarkable rising trend. Among them, solid tumors such as lung cancer, breast cancer, colorectal cancer, etc. are cancers with a very high incidence rate, and the disease has progressed to a middle or advanced stage when it is usually diagnosed. At present, the main treatment means are surgical treatment, radiotherapy and pharmacotherapy, and the treatment strategy is poor in disease prognosis, low in 5-year survival rate and large in toxic and side effects, so that great pain is brought to patients. In recent years, with the continuous development of molecular biology, molecular targeted therapy becomes a new direction of tumor therapy, and brings good news to cancer patients.
Epidermal growth factor receptor (EGFR, HER-1, ErbB-1) is a tyrosine kinase receptor, one of the four members of the EGFR tyrosine kinase family, and EGFR activation is triggered by the binding of specific ligands such as Epidermal Growth Factor (EGF) and transforming growth factor-alpha (TGF-alpha). When EGFR binds to a ligand, it dimerizes and results in activation of the intracellular tyrosine kinase domain, further activating downstream signaling pathways. EGFR and its ligands are part of the cell signaling system, the signaling network of which plays an important role in the development and progression of tumors, and EGFR is overexpressed in tumors of various epithelial origins, such as head and neck cancer, lung cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, esophageal cancer, etc. EGFR is therefore an important target in tumor therapy. Currently, the targeted therapy of EGFR mainly comprises tyrosine kinase inhibitor (tinib) small molecule drugs and anti-EGFR antibody drugs. Among these, antibody drugs generally act by blocking EGFR ligand binding, resulting in inhibition of downstream effects, such as inhibition of intracellular signaling, inhibition of cell cycle progression, induction of apoptosis, and inhibition of DNA repair, angiogenesis, tumor cell motility, invasion, and metastasis. However, antibody therapy has certain limitations and is usually combined with chemotherapeutic drugs to achieve therapeutic efficacy.
An Antibody-drug conjugate (ADC) is one of the strategies for improving the curative effect of the Antibody, and the ADC takes the Antibody as a carrier, is connected with a cytotoxic drug through a linker and is delivered to a target site, so that the targeting property of the cytotoxic drug is improved, the anti-tumor activity of the Antibody is increased, the treatment window is integrally improved, and an effective weapon is provided for the accurate medical treatment of diseases. ADCs are typically composed of 3 building blocks of an antibody (antibody), a warhead molecule (warhead), and a linker (linker) that links the two. ADCs have extremely high requirements for cytotoxicity of cytotoxins, usually IC50＜10-9mol/L, so that the limited amount of cytotoxin carried by the antibody can be utilized to effectively kill the tumor cells. MMAE (monomethylauristatin E) is a linear polypeptide cytotoxic molecule that inhibits tumor growth by acting on tubulin, IC, in a variety of human cancer cell lines50Is 10-9～10-11mol/L is 200 times of vinblastine which is a traditional anticancer small molecule drug, and is widely applied to the research and development of ADC. In 2011, MMAE has been successfully used in the buduximab petrolatum (adocetris) for treating Hodgkin Lymphoma (HL) and Anaplastic Large Cell Lymphoma (ALCL) approved by FDA, and according to the phase ii clinical test report, 102 patients are administered at 1.8mg/kg for three weeks, the total remission rate of HL patients is 75% (34% total response, 40% partial response), the total remission rate of ALCL patients is 87% (53% total response), and the treatment effect is significant. The linker of the Val-Cit dipeptide is most widely used in the coupling of MMAE ADCs that are efficiently cleaved by cathepsin B to release toxin, which, because this enzyme is scarcely distributed in the blood, makes such ADCs highly stable in the blood and selectively release toxin in tumor tissues.
At present, antibody conjugated drugs (ADC) targeting targets such as CD30, CD33, CD22 and Her-2 are approved by FDA to be on the market, and since EGFR can be highly expressed in various tumors, the ADC taking EGFR as the target can be used for treating various solid tumors, the tumor treatment types of the ADC are expanded, the clinical and market prospects are wide, and the ADC can bring hopes for treating various tumors, especially for treating the solid tumors.
The LR004 antibody is an antibody that targets EGFR and is used to treat related cancers that exhibit EGFR, either alone or in combination with one or more additional therapeutic agents (patent publication No.: CN 106470697 a; WO 2016/044234 EN). The advantages of the LR004 antibody are mainly the following: with respect to the antibody itself, the LR004 sialic acid is N-acetylneuraminic acid (NANA is seen to more closely resemble humans), and the LR004 antibody does not contain detectable amounts of the effective oligosaccharide immunogen galactose- α -1, 3-galactose structure, both features of which result in a lower immune response; in terms of activity, the LR004 antibody exhibited good therapeutic efficacy in a variety of tumor cells (a431, CEO, MDA-MB-468).
The innovativeness and the advancement of the invention lie in that the advantages of the LR004 antibody are fully adopted, and the good targeting property, affinity and endocytosis capacity of the LR004 antibody are utilized to prepare the novel antibody coupling drug LR004-VC-MMAE, which shows stronger anti-solid tumor effect than the known ADC both in vivo and in vitro, and compared with the LR004, the activity is obviously improved. In the pharmacokinetic research, the LR004-VC-MMAE antibody coupled drug total antibody has longer half-life, slower clearance rate and lower concentration of free MMAE in plasma, which is beneficial to reducing toxicity. In conclusion, the antibody coupling drug LR004-VC-MMAE targeting EGFR shows good application prospect.
Disclosure of Invention
One of the purposes of the invention is to provide an antibody coupling drug LR004-VC-MMAE targeting EGFR and a preparation method thereof, which have high-efficiency targeting, endocytic activity and affinity activity to EGFR positive tumor tissues.
The second object of the present invention is to provide an antitumor agent containing the above ADC as an active ingredient and use thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to an antibody conjugate drug targeting EGFR, which is named as LR004-VC-MMAE and consists of an antibody, a cytotoxic drug and a linker, wherein the antibody drug conjugate has a structure shown as the following formula I:
wherein, the mAb is LR004 monoclonal antibody, n is 2-8, and the preferable n is 4.
In the present invention, the LR004 monoclone antibody is a human murine chimeric antibody that specifically binds to the N-terminal portion of human EGFR, and the antibody has a molecular weight of about 153 kDa. The monoclonal antibody can be prepared in chinese hamster ovary cells CHO using methods known in the art. The cytotoxic drug is MMAE. The linker is a Val-Cit (VC) linker, which is called MC-VC-PAB-linker. The MMAE is firstly connected with a VC joint, and after purification preparation, the maleimide group of the MMAE and the interchain disulfide bond of the LR004 antibody generate addition reaction to prepare the antibody coupling drug.
Further, the invention also provides a method for preparing the antibody conjugated drug, which comprises the following steps:
1) dissolving MC-VC-PAB-MMAE (i.e. linker-cytotoxic drug) in dimethyl sulfoxide to obtain a linker-cytotoxic drug stock solution;
2) dissolving a reducing agent in a buffer solution to prepare a reducing agent stock solution;
3) mixing the LR004 antibody with the reducing agent solution in the step 2) to perform reduction reaction for 1-2h to obtain a solution after the antibody is reduced;
4) dropwise adding the linker-cytotoxic drug stock solution obtained in the step 1) into the solution after the antibody reduction, and carrying out addition reaction for 1-2h to obtain the antibody conjugated drug.
In the method, preferably, the reducing agent in step 3) is TCEP, and the molar amount of the reducing agent is 2 to 4 times the molar amount of the LR004 antibody when the reducing agent is mixed with the LR004 antibody.
In the method, preferably, the molar amount of the linker-cytotoxic drug added in the step 4) is 4 to 8 times of the molar amount of the LR004 antibody in the mixing.
In the method, preferably, the reaction in the step 3) and the reaction in the step 4) are carried out at a stirring speed of 100-300 rpm under the protection of nitrogen, and the reaction temperature in the step 3) is 35-40 ℃, preferably 37 ℃.
In the method, preferably, the method further comprises a step of further purifying the obtained antibody conjugated drug LR004-VC-MMAE, preferably an AKTA purifier protein purification system, and collecting the peak of the required antibody conjugated drug component; after the collection, the mixture is ultrafiltered and centrifuged by using a 30kDa ultrafiltration tube, concentrated and filtered by a sterile filter membrane.
Still further, the invention also provides application of the antibody coupling drug in preparing a drug for tumor targeted therapy, wherein the tumor is an EGFR positive solid tumor, and comprises esophageal cancer, squamous cell carcinoma, lung cancer, breast cancer, pancreatic cancer, head and neck cancer, colon cancer, prostate cancer and osteosarcoma cancer.
Furthermore, the invention also provides a pharmaceutical composition for tumor targeted therapy, which comprises a pharmaceutically effective amount of the antibody conjugate drug and a pharmaceutically acceptable adjuvant.
Compared with the prior art, the invention has the beneficial effects that:
1. the LR004 monoclonal antibody is utilized, MMAE is used as a warhead molecule, Val-Cit is used as a linker, and the LR004-VC-MMAE is coupled with VC-MMAE by a chemical method of reducing disulfide bonds between antibody chains, so that the LR004-VC-MMAE which targets the EGFR and mainly treats solid tumors is constructed. Preferably, TCEP is used as a reducing agent, after reaction conditions are optimized to a certain extent, the antibody coupling drug with the average drug antibody coupling ratio (DAR) of about 4 is prepared, the components with the naked antibody and the coupling ratio of 8 are both lower than 5 percent, the monomer content is higher than 95 percent, the quality is stable and controllable, the repeatability is good, the yield is considerable, and the stability of the antibody is not influenced.
2. The novel antibody coupling drug LR004-VC-MMAE can target EGFR antigenIt also has strong activity of killing tumor cells. Compared with LR004, the affinity, the endocytic activity and the targeting property of the antibody are not influenced, and the biological function of the antibody is well reserved. Compared with LR004, the tumor inhibiting activity is obviously improved in-vitro activity evaluation, and IC50All in nM. In vivo activity evaluation, compared with LR004, the tumor inhibition effect of the LR004-VC-MMAE antibody coupling drug is remarkably improved, and the situation of tumor disappearance appears. In the pharmacokinetic research, the LR004-VC-MMAE antibody coupled drug total antibody has longer half-life period and slower clearance rate, the concentration of free MMAE in plasma is lower, the half-life period is short, the clearance speed is fast, and the toxicity is favorably reduced.
Drawings
FIG. 1 is a synthesis roadmap for MMAE, VC linker and VC-MMAE
Wherein FIG. 1a shows the synthesis route of MMAE
FIG. 1b shows the synthesis route of VC linker (MC-VC-PAB-PNP)
FIG. 1c shows the scheme for the synthesis of VC-MMAE (MC-VC-PAB-MMAE)
FIG. 2 is a graph showing the determination of EGFR expression levels in various tumor cells.
Wherein, FIG. 2a shows WesternBlot detection of EGFR expression levels in different tumor cells.
FIG. 2b is a flow cytometry assay to detect EGFR expression levels in different tumor cells.
FIG. 3 is a binding activity assay of LR004 and LR 004-VC-MMAE.
Wherein, FIG. 3a is the affinity curve of LR004-VC-MMAE and control LR004 measured by ELISA method and antigen EGFR.
FIG. 3b is a flow cytometry assay of the affinity curves of LR004 and LR004-VC-MMAE against EGFR-positively expressing cells.
FIG. 4 shows that the immune confocal method detects the endocytosis of LR004 and LR004-VC-MMAE in KYSE520 cells.
FIG. 5 is in vivo imaging of LR004-VC-MMAE in tumor-bearing nude mice.
FIG. 6 is the IC of LR004-VC-MMAE and MMAE in tumor cells with different EGFR expression levels50The value is obtained.
FIG. 7 shows the study of the drug effect of LR004-VC-MMAE in A431 nude mouse graft tumor model.
FIG. 7a is a study of the efficacy of LR004-VC-MMAE in A431 nude mouse graft tumor model at different dose groups.
FIG. 7b shows LR004-VC-MMAE 15mg/kg dose at A431 (tumor volume 400-500 mm)3) The study of drug efficacy in a nude mouse transplantation tumor model of (1).
FIG. 8 shows the study of the drug effect of LR004-VC-MMAE in KYSE520 nude mouse transplantation tumor model.
FIG. 8a study of the pharmacological effects of LR004-VC-MMAE at different doses in KYSE520 nude mouse graft tumor model.
FIG. 8b is a graph showing the change in body weight of the control group and the administered group in the experiment.
15mg/kg) toxicity to each organ of nude mice.
FIG. 9 is a graph showing the time course of the LR004-VC-MMAE components in nude mice.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 Synthesis of VC-MMAE (MC-VC-PAB-MMAE):
1.1 Synthesis of MMAE
The synthetic route is shown in figure 1 a:
step a: synthesis of Boc-Dap-N-demethylephedrine
The compound Boc-Dap-OH (500mg,1.7mmol) and (1S,2R) - (. + -.) -demethylephedrine (289.3mg,1.9mmol) were dissolved in 10mL DMF and DEPC (368.9mg,2.2 mmol) and Et were added sequentially3N (228.8mg.2.2mmol), and the reaction was stirred at room temperature overnight. After the reaction is finished, adding water to stop the reaction, extracting the mixture for three times by using ethyl acetate, extracting the mixture for one time by using saturated NaCl solution, and then adding anhydrous Na2SO4Dry overnight. And (3) column chromatography separation and purification, wherein the separation conditions are as follows: ethyl acetate ═ 5: 1, obtaining 485mg of white solid. C23H36N2O5.MS(ESI)m/z:321.1H NMR(400MHz, DMSO-d6)δ7.66(d,J＝8.6Hz,1H),7.36–7.25(m,4H),7.22–7.16(m,1H), 5.37(d,J＝4.6Hz,1H),4.46(t,J＝5.1Hz,1H),3.97(dp,J＝8.5,6.6Hz,1H), 3.52(d,J＝9.7Hz,1H),3.40–3.31(m,2H),3.18(s,1H),3.09–2.95(m,1H), 2.15–2.04(m,1H),1.71(s,2H),1.61–1.48(m,2H),1.41(s,10H),1.01(dd,J＝ 8.8,6.6Hz,7H).
And step b, synthesizing Dap-N-demethyl ephedrine. The reaction product was dissolved in 6mL of DCM, and 1mL of TFA was added under ice-bath and stirred at room temperature overnight. After the reaction, DCM is added for extraction for three times, organic phases are combined and saturated NaHCO is respectively used3Extracting the solution and saturated NaCl solution once, and adding anhydrous Na2SO4Dry overnight. DCM was suction filtered and spin dried and washed twice with ether to give the product without further purification.
Step c: Fmoc-L-Val-Dil-OButAnd (4) synthesizing. Reactant Dil-OButHCl (500mg,1.7mmol) and Fmoc-L-Val-OH (780mg,2.3mmol) were dissolved in 10mL 2-Me-THF, CDMT (403.5mg,2.3mmol) and NMM (256.6mg,2.55mmol) were added sequentially and the reaction was stirred at room temperature overnight. After the reaction is finished, adding ethyl acetate for extraction for three times, combining organic phases, washing the organic phases once by saturated NaCl, and adding anhydrous Na2SO4Dry overnight. And (3) performing column chromatography separation, wherein the separation conditions are as follows: ethyl acetate ═ 6: 1 to obtain 486mg of a reaction product. C34H48N2O6.MS(ESI)m/z:581.1H NMR (400MHz,DMSO-d6)δ7.89(d,J＝7.5Hz,2H),7.77–7.69(m,2H),7.62(d,J＝ 8.8Hz,1H),7.42(t,J＝7.4Hz,2H),7.31(tdd,J＝7.5,3.2,1.2Hz,2H),4.33–4.13(m,4H),3.77(s,1H),3.25(s,3H),2.93(s,3H),2.57(d,J＝2.7Hz,1H), 2.17(dd,J＝15.6,9.3Hz,1H),2.11–1.95(m,1H),1.77(s,1H),1.40(s,9H), 1.32–1.24(m,3H),0.93–0.86(m,9H),0.70(t,J＝7.3Hz,3H).
Step d, L-Val-Dil-OButAnd (4) synthesizing. The reaction product obtained in the previous step is dissolved in 15mLTo THF as water, 200. mu.L of Et was added2NH, stirred at rt overnight. After the reaction was completed, the reaction solution was spin-dried and washed with ether for 3 times to obtain 357mg of product without further purification.
Step e, Fmoc- (N-Me) -L-Val-L-Val-Dil-OButAnd (4) synthesizing. The reaction product from the previous step was dissolved in 10mL EtOAc, and after Fmoc- (N-Me) -L-Val-OH (455mg, 1.2mmol) was added followed by DEPC (214mg,1.3mmol) and 1.6mL DIEA, the reaction was stirred at room temperature overnight. After the reaction, water was added to stop the reaction. Extracting with ethyl acetate for three times, mixing organic phases, extracting with saturated NaCl solution once, and adding anhydrous Na2SO4Dry overnight. And (3) performing column chromatography separation, wherein the separation conditions are as follows: ethyl acetate 4: 1 to obtain white solid 536 mg. C40H59N3O7.MS(ESI)m/z:694.1H NMR (400MHz,DMSO-d6)δ＝7.90(d,J＝7.5,2H),7.70–7.60(m,2H),7.46–7.38 (m,2H),7.32(td,J＝7.5,1.2,2H),4.61(s,1H),4.39(td,J＝13.1,11.8,5.9,2H), 4.30(q,J＝7.3,6.5,1H),4.23(t,J＝6.6,1H),3.76(s,1H),3.39(q,J＝7.0,1H),3.24 (d,J＝7.9,3H),2.96(d,J＝9.4,3H),2.80(d,J＝8.9,3H),2.64–2.53(m,1H),2.16 (dd,J＝15.6,9.6,1H),2.10–1.92(m,2H),1.83–1.61(m,1H),1.41(s,9H),1.33 –1.21(m,2H),1.10(t,J＝7.0,1H),0.90–0.71(m,18H).
Fmoc- (N-Me) -L-Val-L-Val-Dil-OH the top reaction product was dissolved in 10mL of DCM, 1.5mL of TFA was added and the reaction was stirred at RT overnight. After the reaction, DCM is added for extraction for three times, organic phases are combined and saturated NaHCO is respectively used3Extracting the solution and saturated NaCl solution once, and adding anhydrous Na2SO4Dry overnight. DCM was filtered off with suction and spin dried, washed twice with ether to give 523mg of product. C36H51N3O7.MS(ESI)m/z:638.1H NMR(400MHz,DMSO-d6)δ＝ 12.25(s,1H),7.90(d,J＝7.5,3H),7.63(d,J＝7.4,2H),7.42(t,J＝7.4,2H),7.33(q, J＝7.4,2H),4.62(s,1H),4.37(ddt,J＝36.6,21.5,8.2,5H),3.34(s,1H),3.26(d, J＝7.9,1H),2.96(d,J＝9.2,3H),2.80(d,J＝9.5,3H),2.56(dd,J＝16.2,8.1,1H), 2.20(dt,J＝15.8,7.9,1H),2.11–1.91(m,2H),1.77(s,1H),1.30(s,1H),1.01– 0.67(m,21H).
Step g: and (3) synthesizing Fmoc-MMAE. The reaction step b product (300mg,0.93mmol) and reaction step f product (464mg,0.73mmol) were dissolved in 15mL of DCM and HATU (461mg,1.2mmol) and DIEA 600. mu.L were added sequentially and the reaction stirred at room temperature overnight. After the reaction is finished, DCM is added for extraction for three times, organic phases are combined and are respectively extracted once by 2 percent citric acid solution and saturated NaCl solution, and anhydrous Na is added2SO4Dry overnight. DCM was filtered off with suction and spin dried, washed 3 times with diethyl ether without further purification to give 495mg of a yellowish solid.
Step h: dissolve the reaction product of step g in 10mL DCM and add 300. mu.L Et2NH, stirred at rt overnight. And (4) after the reaction is finished, the reaction solvent is dried in a spinning mode, and the preparation liquid phase is used for separation and purification. The chromatographic conditions were as follows: phase A H2O + 0.08% TFA, B-phase acetonitrile + 0.08% TFA. Eluting with 45% phase B at constant gradient for 0-35min, collecting pure product, and lyophilizing. MMAE.C39H67N5O7.1H NMR (500MHz,DMSO-d6)δ(ppm):7.45–7.21(m,5H),4.67(m,2H),4.34–4.06(m, 2H),3.89(dd,J＝9.1,2.0Hz,1H),3.76–3.66(m,1H),3.61–3.53(m,1H),3.49 –3.39(m,1H),3.36(m,J＝13.8,4.9Hz,5H),3.25–3.12(m,2H),2.88–2.82 (m,1H),2.59–2.44(m,2H),2.37–2.29(m,3H),2.24(m,J＝9.0,6.9Hz,1H), 2.20–2.02(m,2H),2.01–1.77(m,4H),1.76–1.54(m,2H),1.50–1.25(m,2H), 1.24–1.08(m,6H),1.07–0.81(m,19H).
Synthesis of 1.2VC linker (MC-VC-PAB-PNP)
The synthetic route is shown in FIG. 1 b:
step a, synthesizing Fmoc-Val-OSu. The reaction product Fmoc-L-Val-OH (10g,29.3 mmol) and HoSu (3.7g,32.3mmol) were dissolved in 100mL THF, DCC (6.6 g,32.3mmol) was added under ice-bath, after the addition was complete, the ice-bath was removed and the reaction was stirred at room temperature overnight. After the reaction is finished, the reaction solution is transferred to an ice bath, suction filtration is carried out after the solid is completely separated out, and the filtrate is dried in a spinning mode until the filtrate is foamed. The reaction product was relatively pure and no further purification was required.
Step b: the reaction product from the previous step (16g,36.6mmol) was dissolved in 90mL DME, L-Cit (6.7 g,38.5mmol) was dissolved in NaHCO3(3.2g,38.5mmol) in 90mL of water, adding into the reaction system, adding 50mL of THF for dissolving, and stirring at room temperature overnight for clarification. After the reaction is finished, adding 15% citric acid aqueous solution with the same volume as THF under ice bath until white solid of a reaction product is completely separated out, carrying out suction filtration by using a Buchner funnel until the filtrate is clear, pumping out a filter cake, and drying by using a vacuum drying oven. After drying, grinding the white solid, washing with diethyl ether, and filtering to obtain the white solid. C26H32N4O6.1H NMR(400MHz, DMSO-d6)δ12.48(s,1H),8.17(d,J＝7.4Hz,1H),7.90(d,J＝7.5Hz,2H),7.76 (t,J＝7.1Hz,2H),7.41(q,J＝7.7Hz,3H),7.33(td,J＝7.5,2.3Hz,2H),6.00(s, 1H),5.63(s,2H),4.30–4.21(m,3H),4.16(td,J＝8.1,5.1Hz,1H),3.93(dd,J＝ 9.2,7.0Hz,1H),2.98–2.89(m,2H),1.99(h,J＝6.8Hz,1H),1.77–1.66(m, 1H),1.57(dtd,J＝13.9,9.2,5.7Hz,1H),1.41(dq,J＝10.5,6.1,5.0Hz,2H), 0.88(dd,J＝13.0,6.7Hz,6H).
Step c: the reaction product of reaction step b (7.6g,15.3mmol) and PABOH (3.76g, 30.6mmol) were dissolved in 140mL CH2Cl2And 70mL of CH3To the OH-mixed solvent, EEDQ (7.5g,30.6mmol) was added in the dark. Stirring and reacting at room temperature, spin-drying the reaction solution after the reaction is finished, washing with diethyl ether, and performing suction filtration. C33H39N5O6.1H NMR(400MHz,DMSO-d6)δ＝9.99(s,1H),8.11(d, J＝7.5,1H),7.90(d,J＝7.6,2H),7.75(t,J＝8.0,2H),7.55(d,J＝8.2,2H),7.43(q, J＝8.0,7.5,3H),7.33(t,J＝7.4,2H),7.24(d,J＝8.1,2H),5.99(t,J＝6.0,1H),5.41 (s,2H),5.10(t,J＝5.8,1H),4.43(d,J＝5.5,3H),4.31(s,1H),4.25(d,J＝6.3,2H), 3.94(t,J＝7.9,1H),3.03–2.88(m,2H),2.00(d,J＝9.0,1H),1.76–1.65(m,1H), 1.60(d,J＝9.8,1H),1.48–1.36(m,2H),0.92–0.85(m,6H).
Step d reaction product from previous step (8.4g,19mmol) was dissolved in 150mL DMF and Et was added2NH 25mL, stirred the reaction at room temperature overnight. After the reaction, the solvent was spin-dried and washed with ether three times to obtain a yellowish solid (2.3 g). C28H40N6O7.1H NMR(400MHz,DMSO-d6)δ9.91(s,1H),8.07 (d,J＝7.6Hz,1H),7.82(d,J＝8.6Hz,1H),7.55(d,J＝8.1Hz,2H),7.23(d,J＝ 8.1Hz,2H),7.01(s,2H),5.99(t,J＝5.9Hz,1H),5.42(s,2H),5.10(t,J＝5.7Hz, 1H),4.43(d,J＝4.9Hz,2H),4.20(t,J＝7.7Hz,1H),3.00(dq,J＝26.1,6.7Hz, 2H),2.17(dq,J＝14.6,7.0Hz,2H),1.97(q,J＝6.7Hz,1H),1.71(q,J＝7.7Hz, 1H),1.64–1.57(m,1H),1.54–1.43(m,6H),1.20(q,J＝7.8Hz,2H),1.10(t,J ＝7.0Hz,1H),0.84(dd,J＝12.7,6.7Hz,6H).
Step e: reaction step d the product (0.5g,1.3mmol) was dissolved in 18mL DMF and MC-OSu (0.45g,1.4mmol) was added and the reaction stirred at room temperature overnight. After the reaction was complete, the reaction solvent was spin-dried, washed three times with ether, and suction filtered to obtain 0.69g of a yellow solid without further purification.
Step f, dissolving the reaction product of reaction step e in 12mL DMF, adding (PNP)2CO (1.1g,3.6mmol) and DIEA, 0.5mL, was stirred overnight at room temperature. After the reaction is finished, the solvent is dried by spinning, and the separation and purification are carried out by a silica gel column under the separation conditions of dichloromethane: methanol 20: 1. the product was obtained as a yellowish solid, 0.42 g. C35H43N7O11.1H NMR(500MHz,DMSO-d6)δ(ppm):10.09(s, 1H),8.32(d,J＝9.1Hz,2H),8.14(d,J＝7.1Hz,1H),7.83(d,J＝8.6Hz,1H), 7.66(d,J＝8.4Hz,2H),7.57(d,J＝9.1Hz,2H),7.41(d,J＝8.6Hz,2H),7.01(s, 2H),6.00(t,1H),5.44(s,2H),5.24(s,2H),4.39(m,J＝12.6Hz,7.1,1H),4.20(t, J＝7.2Hz,1H),3.37(m,J＝7.1Hz,2H),2.99(m,2H),2.22–2.09(m,2H),1.96 (m,J＝13.5Hz,6.7,1H),1.72(m,1H),1.65–1.56(m,1H),1.52–1.43(m,6H), 1.22–1.15(m,2H),0.87–0.82(m,6H).
1.3 Synthesis of VC-MMAE (MC-VC-PAB-MMAE)
The synthetic route is shown in FIG. 1 c:
MC-VC-PAB-PNP (93mg,0.125mmol) and MMAE (60mg,0.083mmol) were dissolved in 4mL DMF, and HoBt (10mg,0.074mmol) and 0.5mL pyridine were added sequentially and the reaction was stirred at room temperature overnight. The reaction product is separated and purified using a preparative liquid phase. The chromatographic conditions were as follows: phase A H2O + 0.08% TFA, B-phase acetonitrile + 0.08% TFA. And (3) eluting the B phase by 65% gradient at 0-35min, collecting the pure product, and freeze-drying the reaction product. C68H105N11O15.1H NMR(500MHz,DMSO-d6)δ＝10.05(s, 1H),7.88(d,J＝7.6,2H),7.73(dd,J＝10.7,7.8,2H),7.56(q,J＝13.9,10.7,2H), 7.41(dd,J＝7.6,4.1,2H),7.37–7.21(m,7H),7.16(m,2H),5.16–4.95(m,4H), 4.77-4.69(m,1H),4.69-4.59(m,1H),4.55-4.49(m,2H),4.46–4.38(m,2H), 4.37-4.17(m,5H),4.09-3.94(m,3H),3.93-3.89(m,1H),3.80-3.75(m,1H), 3.34-3.29(m,1H),3.28-3.21(m,4H),3.19(d,J＝13.3,3H),3.11(s,2H),3.03(dq, J＝13.5,6.6,2H),2.98-2.95(m,1H),2.96–2.91(m,1H),2.86(dd,J＝17.7,3.2, 3H),2.40(d,J＝15.9,2H),2.32-2.24(m,2H),2.16-2.04(m,3H),2.04-1.91(m, 3H),1.86-1.65(m,5H),1.63–1.43(m,5H),1.41-1.27(m,3H),1.09–0.94(m, 6H),0.94–0.69(m,21H).
Example 2 preparation of antibody drug conjugate LR004-VC-MMAE
The method comprises the following steps:
1) the MC-VC-PAB-MMAE (i.e., linker-cytotoxic drug) prepared in example 1 was dissolved in dimethyl sulfoxide to obtain a linker-cytotoxic drug stock solution.
2) TCEP reducing agent is dissolved in buffer PBS (containing 1mM DTPA), and a certain molar quantity of reducing agent stock solution is prepared.
3) Before reduction, the buffer solution of the LR004 antibody is replaced, and the buffer solution is PBS (containing 1mM DTPA). The replacing method is desalting column replacing. The replacement method comprises the following steps: loading 2.5mL of desalting column, eluting 3.5mL with displacement buffer solution, ultrafiltering to concentrate concentration, ultrafiltering at 4000rpm, and centrifuging at 4 deg.C for 10 min. Thus, a 5mg/mL LR004 antibody solution was prepared.
4) Mixing the 5mg/mL LR004 antibody solution in the step 3) with the reducing agent TCEP solution in the step 2), carrying out reduction reaction for 1-2h at 37 ℃ and under the condition of stirring speed of 200rpm, and carrying out nitrogen protection during the reaction to obtain a solution after the antibody is reduced. The molar amount of the added TCEP reducing agent is 3 times that of the LR004 antibody;
5) dropwise adding the connector-cytotoxic drug solution obtained in the step 1) into the solution after the antibody reduction, carrying out addition reaction for 1-2h under the condition of stirring rotation speed of 200rpm, and carrying out nitrogen protection during the reaction to obtain the antibody coupling drug. The molar amount of the LR004 antibody is taken as a reference during mixing, and the added connexon-cytotoxic drug solution is 6 times of the molar amount of the LR004 antibody.
6) After the reaction is finished, the obtained antibody conjugated drug LR004-VC-MMAE needs to be further purified, and an AKTA purifier protein purification system is adopted to collect the component peak of the needed antibody conjugated drug. After the collection is finished, ultrafiltration centrifugation is carried out by using a 30kDa ultrafiltration tube, the concentration of the buffer solution is concentrated, the centrifugation temperature is preferably 4 ℃, the rotation speed is preferably 4000rpm, and the centrifugation time is preferably 12 min. After purification, the endotoxin is filtered by a sterile filter membrane and stored at-80 ℃, and before activity evaluation, whether the endotoxin reaches the standard is detected by a limulus reagent.
The prepared LR004-VC-MMAE antibody drug conjugate has a structure shown in the following formula I:
wherein, the mAb is LR004 monoclonal antibody; n is 4.
Example 3 detection of EGFR expression in different tumor cells
3.1WesternBlot assay for EGFR expression levels in different tumor cells
Esophageal cancer cells (KYSE450), breast cancer cells (MDA-MB-468), head and neck cancer cells (SCC-25), colon cancer cells (HCT-116), pancreatic cancer cells (AsPC-1), osteosarcoma cells (143B), and prostate cancer cells (PC-3) were collected at log phase. Digesting the cells, collecting the cells, counting the cells, and collecting 1 × 107The cells were centrifuged to remove the supernatant. Precooling RIPA protein extraction reagent, and adding protease inhibitor (phosphatase inhibitor is required to be added simultaneously for phosphorylation protein). 0.1M PMSF stock solution was added before the start of protein extraction, PMSF final concentration 1 mM. Cell count, in number of cells 1X 107Adding 1ml of lysate, centrifugally packaging at 4 ℃, preserving, and determining the protein concentration by using a BCA method. WesternBlot assay was performed according to protein concentration, loading was 20. mu.g/well, and membrane transfer time was 1 h. After the film transfer was completed, the film was stained with ponceau red staining reagent. After staining, blocking was performed with 3% BSA-TBST, primary antibody was diluted with 3% BSA-TBST, and membrane was washed 5 times with TBST overnight at 4 ℃. Diluting the secondary antibody with 5% skimmed milk powder-TBST, and goat anti-mouse IgG(H + L) HRP, gentle shaking at room temperature for 40 min. TBST membrane washing 6 times. Film exposure after ECL addition to film: 10s-5min (exposure time adjusted for different light intensities), developed for 2min, and fixed, the results are shown in FIG. 2 a.
3.2 flow cytometry detection of expression levels in different cells
Selecting LR004-VC-MMAE with concentration of 10 μ g/mL, and mixing with 5 × 105The esophageal cancer cells (KYSE520, KYSE150), squamous cell carcinoma A431, non-small cell lung cancer cells (HCC827, NCI-H1975, A549), breast cancer cells (MDA-MB-468, MCF-7), pancreatic cancer cells (AsPC-1) and anaplastic large cell lymphoma cells Karpas299 were incubated at 4 ℃ for 1H, washed 2 times with 2% FBS/PBS, added FITC-labeled goat anti-human IgG (Medtronic bridge, 1:200 dilution) and incubated at 4 ℃ for 1H, washed twice with PBS and then resuspended in 500. mu.L PBS, and the fluorescence intensity of the different cells was measured by flow cytometry, as shown in FIG. 2 b.
Example 4 affinity assay for LR004-VC-MMAE
4.1ELISA method for detecting the binding activity of LR004-VC-MMAE and antigen EGFR
Antigen EGFR (purchased from ACRO Biosystems) was diluted to 2. mu.g/mL with PBS, plated in 96-well plates, 100. mu.l per well, PBST washed 3 times after overnight incubation at 4 ℃, blocked overnight with 2% BSA, the blocking solution aspirated, washed 3 times with PBST, protein LR004-VC-MMAE and control LR004 diluted to a series of different concentrations, added to 96-well plates already coated with EGFR antigen, incubated for 1h at 37 ℃ at 50. mu.L per well, washed 3 times with PBST, 5min each. Subsequently, alkaline phosphatase-labeled goat anti-human (Fab-specific) secondary antibody (1:1000 dilution) was added, incubated at 37 ℃ for 1h, and PBST washed three times for 5min each. The TMB substrate is added for developing for about 10min, and the absorbance value is measured at the wavelength of 405nm, and the binding curve of the TMB substrate and the EGFR is plotted. The results show that LR004-VC-MMAE is concentration dependent on binding to the antigen EGFR and that the affinity activity on the antibody itself after coupling to MMAE is not much affected (fig. 3 a).
4.2 determination of the affinity constant of LR004-VC-MMAE by the Biocare method
LR004-VC-MMAE of 1. mu.g/mL was bound to CM5 chips, and EGFR antigen (ACRO Biosystems) was injected into the system at different concentrations and flowed through the chipsAnd combining with the conjugate. The chip surface adopts 3M MgCl2Dissociation is carried out. Affinity constant values were calculated by the Biacore T200 system. The affinity values are as follows:
TABLE 1LR004-VC-MMAE affinity constants
4.3 flow cytometry for detecting LR004-VC-MMAE binding activity
Selecting different concentrations of LR004-VC-MMAE and LR004, which are respectively 5 × 105The cells were incubated at 4 ℃ for 1h, washed 2 times with 2% FBS/PBS, added with FITC-labeled goat anti-human IgG (China fir gold bridge, 1:200 dilution) and incubated at 4 ℃ for 1h, washed twice with PBS, and then resuspended in 500. mu.L of PBS, and the fluorescence intensity was measured with a flow cytometer. The results showed that the binding activity of LR004-VC-MMAE to LR004 was comparable in different EGFR-positive tumor cells (see fig. 3 b).
Example 5 confocal laser microscopy of LR004-VC-MMAE endocytosis Activity in cells
Preparing single cell suspension from KYSE520 cells in logarithmic growth phase, counting cells, adjusting concentration, and measuring at 1 × 10 per well4The cells were inoculated in a 96-well plate and cultured at 37 ℃ for 2h, then LR004-VC-MMAE was added to a final concentration of 5. mu.g/mL. For observation of the binding condition of the conjugate and the cell surface antigen, the conjugate is added and incubated at 4 ℃ for 30min, a cell sample is collected and washed for 2 times by PBS, the cell is centrifuged on a glass slide by a cell flaker, 4% paraformaldehyde is fixed for 10min, PBST is washed for 3 times, 0.2% TritonX-100/PBS is permeabilized for 10min, PBST is washed for 3 times, and 5% goat serum is used for sealing overnight. Incubating Alexa Fluor 488-labeled anti-human fluorescent secondary antibody at 37 ℃ for 30min, washing by PBST for 3 times, staining nuclei by Hoechst3342 for 15min, washing by PBST for 3 times, dripping an anti-fluorescent quenching agent, covering a cover glass, and observing by a confocal microscope. When the endocytosis of the conjugate by the cells is observed, the protein is added and then the incubation is carried out for 24 hours at 37 ℃. Lysosomes were labeled with LAMP-1 antibody (purchased from Cell Signaling therapeutics) and Alexa Fluro 555-labeled donkey-anti-rabbit secondary antibody (purchased from petunia). The rest of the operation is the same as the above process.
The results show that LR004-VC-MMAE can be combined with EGFR receptor on cell membrane but does not enter the cell interior when incubated at 4 ℃. While LR004-VC-MMAE enters the interior of the cell through endocytosis and can be co-localized in lysosomes when incubated at 37 ℃ (as shown in figure 4).
Example 6 tumor targeting ability of LR004-VC-MMAE in nude mice
LR004-VC-MMAE, positive control LR004 and negative control rituximab-VC-MMAE were labeled with DyLight680 antibody labeling kit (purchased from Thermo Scientific), respectively, the specific methods refer to the protocol. KYSE520 cells were inoculated to the axilla of BALB/c nude mice when the tumor volume was 200-300mm3The three labeled drugs were administered at a dose of 20mg/kg into a tumor-bearing mouse (n-2) via tail vein injection. Observations were made using a small animal in vivo imager from XENGOEN (Caliper, usa). At different time points, the drug is treated by medical anesthetic isoflurane, placed on an observation plate preheated at 37 ℃ (anesthetized simultaneously), and the distribution of the drug fluorescence in a tumor-bearing nude mouse and the condition of targeting tumors are monitored by a CCD lens cooled to-90 ℃. As a result, the LR004-VC-MMAE can better target the transplanted tumor site of the nude mouse, a stronger tumor enrichment degree can be seen in 48 hours, and the fluorescence intensity can be maintained for 6 days (figure 5).
Example 7 cell killing Activity of LR004-VC-MMAE against different EGFR expressing tumor cells
In combination with the results of the expression level of EGFR in tumor cells obtained in example 3, the cells were evaluated for the in vitro cell killing activity of the antibody-conjugated drug LR 004-VC-MMAE. In particular to esophageal cancer cells (KYSE520, KYSE150), squamous cell carcinoma A431, non-small cell lung cancer cells (HCC827, NCI-H1975, A549), breast cancer cells (MDA-MB-468, MCF-7), pancreatic cancer cells (AsPC-1) and anaplastic large cell lymphoma cells Karpas 299. The method comprises the following specific steps:
cells in logarithmic growth phase were counted by centrifugation and resuspended at 1X 104-3×104Perwell was inoculated into 96-well plates and incubated at 37 ℃ for 2 h. Then, LR004-VC-MMAE (MMAE as positive control) was added at different concentrations, with 3 parallel wells set for each drug concentration. Incubate at 37 ℃ for 48h, each wellThe incubation was continued for 1-2h with the addition of 20. mu.L of CCK8 reagent. The color reaction was observed and the absorbance at 450nm was measured with a microplate reader. The blank group (without cells) and the negative control group (without drug treatment) were set during the experiment, and the cell survival rate was calculated according to the following formula: cell viability ═ 100% (medicated a450 value-blank a450 value)/(control a450 value-blank a450 value). IC (integrated circuit)50The value calculation uses SPSS software. The results (FIG. 6) show that LR004-VC-MMAE has close IC to MMAE50The LR004-VC-MMAE obtained by the invention not only can effectively exert partial biological functions of the LR004 antibody, but also has high killing activity of the MMAE to tumor cells, and shows good targeting property and selectivity.
Example 8 study of antitumor Effect of LR004-VC-MMAE on A431 transplantation tumor model
8.1LR004-VC-MMAE model of A431 transplanted tumor (initial tumor volume about 100 mm)3) Research on antitumor effect
Taking female BALB/c nude mice with the weight of 18-22g, inoculating EGFR positive cell A431 under the right axilla of the nude mice, and inoculating 5 x 10 cells to each nude mouse6And (4) cells. The tumor mass is about 100mm3At the same time, the groups were randomly grouped according to body weight and tumor volume, 6 individuals in each group were used, and a blank control group was set. The following dosing schedule was followed: LR004-VC-MMAE (1mg/kg, 5mg/kg, 10mg/kg and 15mg/kg), MMAE (0.3mg/kg), LR004(15 mg/kg). The administration mode is tail vein administration, 200 μ L each, and the control group is not treated, and is administered once every 4 days for 6 times. During the experiment, the body weight and the tumor volume of the nude mice are measured every 3 to 4 days. Tumor volume was calculated according to the formula V a b/2 (a: tumor long diameter, b: tumor short diameter) and tumor growth curves were plotted (fig. 7 a).
Tumor inhibition rate (mean tumor volume in control group-mean tumor volume in experimental group)/mean tumor volume in control group x 100%
The results showed that after 60 days of experiment, the tumor inhibition rates of LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg) groups were 97.7%, 98.2% and 98.9%, respectively. Compared with LR004 (tumor suppression rate of 82.6%), there was a statistically significant difference.
8.2LR004-VC-MMAE model of A431 transplantation tumor (initial tumor volume about 400-3) Research on antitumor effect
Taking female BALB/c nude mice with the weight of 18-22g, inoculating EGFR positive cell A431 under the right axilla of the nude mice, and inoculating 5 x 10 cells to each nude mouse6And (4) cells. The tumor mass is about 400-500mm long3At the same time, the patients are randomly grouped according to the body weight and the tumor volume, 6 patients are in each group, and a blank control group and an LR004-VC-MMAE 15mg/kg dose group are arranged. The rest of the operation is the same as the above 8.1 process. The results showed that the control group reached a tumor volume of about 2500mm after 40 days of the experiment3While the average tumor volume of the LR004-VC-MMAE 15mg/kg dose group is about 370mm3The tumor inhibition rate was 87.2%. It shows that LR004-VC-MMAE ADC also has good response to large tumors, and can suppress the tumors from growing any more and even reduce the tumor volume, and the tumor inhibition curve is shown in figure 7 b.
Example 9 study of antitumor Effect of LR004-VC-MMAE on KYSE520 transplantable tumor model
Taking female BALB/c nude mice with the weight of 18-22g, inoculating EGFR positive cell A431 under the right axilla of the nude mice, and inoculating 5 x 10 cells to each nude mouse6And (4) cells. The tumor mass is about 100mm3At the same time, the groups were randomly grouped according to body weight and tumor volume, 8 per group, and a blank control group was set. The following dosing schedule was followed: LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg), LR004(15mg/kg) and Rituximab-VC-MMAE (15 mg/kg). The administration mode is tail vein administration, 200 μ L each, and the control group is not treated, and is administered once every 4 days for 4 times. During the experiment, the body weight and the tumor volume of the nude mice are measured every 3 to 4 days. Tumor volume was calculated according to formula V ═ a ×, b × (b) × (2) (a: tumor major diameter, b: tumor minor diameter) and tumor growth curves were plotted. The control group, LR004(15mg/kg) and Rituximab-VC-MMAE (15mg/kg) were euthanized in 44 days, and LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg) was continuously observed until the end of the experiment for 60 days. The results showed that the negative control group, rituximab-VC-MMAE (15mg/kg), had no antitumor activity. LR004(15mg/kg) did not show significant anti-tumor activity in the tumor, and the tumor inhibition rate was highIt is only 13.8%. The LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg) group showed disappearance of tumors, wherein 6 nude mice in the LR004-VC-MMAE (5mg/kg) group showed disappearance of tumors, 7 nude mice in the LR004-VC-MMAE (10 mg/kg) group showed disappearance of tumors, and all nude mice in the LR004-VC-MMAE (15mg/kg) group showed disappearance of tumors (FIG. 8).
Example 10 pharmacokinetic profiles of LR004-VC-MMAE in nude mice
10.1ELISA method for detecting concentration of total antibody in nude mice
40 BALB/c nude mice (body weight 20-22g) received tail vein injection of LR004-VC-MMAE 15mg/kg, were sacrificed at time points 0, 0.5, 1, 2, 8, 24, 48, 72, 120, 216 hours, and whole blood was collected. 4 mice were set at each time point. The whole blood of the nude mice is collected and stored at the temperature of 80 ℃ after centrifugation. Preparing an LR004-VC-MMAE standard: 6 dilution standard points containing 0.5mL of standard diluent were prepared: 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625 ng/mL. The specific ELISA detection method was as described in application example 4.1.
10.2LC-MS/MS method for detecting concentrations of free MMAE and bound MMAE in nude mice
Free MMAE assay: mu.L of the above serum sample was added to 60. mu.L of an acetonitrile solution containing 200ng/mL of Tolbutamide, and centrifuged by vortexing. 10 μ L was injected into the LC-MS/MS system.
Total MMAE detection: mu.L of cathepsin B was added to 12. mu.L of the above serum sample and incubated at 37 ℃ for 3 hours. The reaction was stopped by the addition of 38. mu.L of a solution of Tolbutamide in acetonitrile at 200ng/mL and vortexed for centrifugation. 10 μ L was injected into the LC-MS/MS system.
LC-MS/MS System: waters HSS T31.8 μm 2.1X 50 mm; the phase A is 0.1% of formic acid + H2O; phase B was 0.1% formic acid + acetonitrile. B, 10 to 90 percent and 0 to 2 min; the flow rate is 0.5 mL/min; the column temperature was 50 ℃. MMAE measures molecular weight 718.7/152.2.
The formula for calculating the concentration of bound MMAE (total MMAE concentration-free MMAE concentration)/hydrolysis rate of cathepsin B × 100%, the curve for the traditional Chinese medicine is shown in fig. 9, and the pharmacokinetic parameters are shown in tables 2-4.
The results show that the half-life of the total antibody (conjugated antibody and unconjugated antibody) of the LR004-VC-MMAE is about 5 days, the clearance rate is low, and the anti-tumor activity can be continuously exerted. The concentration of free MMAE is low, the free MMAE can be reduced to about 2ng/mL after 24 hours, the half-life period is short, the clearance rate is high, and the toxicity can be reduced.
TABLE 2 pharmacokinetic parameters of Total antibody in LR004-VC-MMAE
TABLE 3 pharmacokinetic parameters of LR004-VC-MMAE in combination with MMAE
TABLE 4 pharmacokinetic parameters of free MMAE in LR004-VC-MMAE
Claims (11)
1. An antibody drug conjugate targeting EGFR, designated LR004-VC-MMAE, wherein said drug is comprised of an antibody, a cytotoxic drug and a linker, said antibody drug conjugate having the structure shown in formula I below:
wherein, the mAb is LR004 monoclonal antibody, and n is 2-8.
2. The antibody conjugate drug of claim 1, wherein n-4.
3. A method for preparing an antibody-conjugated drug according to claim 1 or 2, comprising the steps of:
1) dissolving MC-VC-PAB-MMAE in dimethyl sulfoxide to obtain a connexon-cytotoxic drug stock solution;
2) dissolving a reducing agent in a buffer solution to prepare a reducing agent stock solution;
3) mixing the LR004 antibody with the reducing agent solution in the step 2) to perform reduction reaction for 1-2h to obtain a solution after the antibody is reduced;
4) dropwise adding the linker-cytotoxic drug stock solution obtained in the step 1) into the solution after the antibody reduction, and carrying out addition reaction for 1-2h to obtain the antibody conjugated drug.
4. The method according to claim 3, wherein the reducing agent in step 3) is TCEP and the molar amount of the reducing agent is 2 to 4 times the molar amount of the LR004 antibody in the mixing.
5. The method as claimed in claim 3, wherein the molar amount of the linker-cytotoxic drug added in step 4) is 4-8 times the molar amount of the antibody based on the molar amount of the LR004 antibody.
6. The method according to claim 3, wherein the reaction in step 3) and step 4) is carried out at a stirring speed of 100-300 rpm under the protection of nitrogen, and the reaction temperature in step 3) is 35-40 ℃.
7. The process according to claim 6, wherein the reaction temperature in step 3) is 37 ℃.
8. The method of claim 3, further comprising the step of further purifying the resulting antibody conjugated drug LR004-VC-MMAE, collecting the desired antibody conjugated drug component peaks; after the collection, the mixture is ultrafiltered and centrifuged by using a 30kDa ultrafiltration tube, concentrated and filtered by a sterile filter membrane.
9. The process of claim 3, wherein the purification is performed using an AKTA purifier protein purification system.
10. Use of an antibody-conjugated drug according to claim 1 or 2 for the preparation of a medicament for tumor-targeted therapy, wherein said tumor is an EGFR-positive solid tumor, including esophageal cancer, squamous cell carcinoma, lung cancer, breast cancer, pancreatic cancer, head and neck cancer, colon cancer, prostate cancer, osteosarcoma cancer.
11. A pharmaceutical composition for tumor-targeted therapy, comprising a pharmaceutically effective amount of the antibody-conjugated drug of claim 1 or 2 and a pharmaceutically acceptable adjuvant.
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