CN109200291A - A kind of antibody coupling drug targeting EGFR and preparation method thereof and its purposes - Google Patents

A kind of antibody coupling drug targeting EGFR and preparation method thereof and its purposes Download PDF

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CN109200291A
CN109200291A CN201811245835.8A CN201811245835A CN109200291A CN 109200291 A CN109200291 A CN 109200291A CN 201811245835 A CN201811245835 A CN 201811245835A CN 109200291 A CN109200291 A CN 109200291A
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antibody
mmae
drug
cancer
reaction
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CN109200291B (en
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李卓荣
胡馨月
苗庆芳
王蓉
金洁
刘秀均
崔阿龙
李毅
甄永苏
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Institute of Medicinal Biotechnology of CAMS
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Abstract

The invention discloses a kind of antibody coupling drugs for targeting EGFR and preparation method thereof and its purposes.The antibody coupling drug for targeting EGFR, is named as LR004-VC-MMAE, is made of antibody, cytotoxic drug and connexon, and the antibody drug conjugates have structure shown in Formulas I, wherein mAb is LR004 monoclonal antibody, n=2~8.Novel antibodies coupling drug LR004-VC-MMAE can either targeting EGFR antigen, and have the activity of strong killing tumor cell.And compared to LR004 itself, the affinity of antibody, endocytic activity and targeting are not influenced, its biological function is preferably retained.Compared to LR004, the tumor killing effect of LR004-VC-MMAE antibody coupling drug has the raising of conspicuousness, and the case where tumor disappearance occurs.Compared to LR004, the total antibody of LR004-VC-MMAE antibody coupling drug is presented longer half-life period, slower clearance rate, and the MMAE concentration dissociated in blood plasma is lower, and half-life short, and removing speed is fast, advantageously reduces toxicity.

Description

A kind of antibody coupling drug targeting EGFR and preparation method thereof and its purposes
Technical field
The invention belongs to biotech drug fields.Specifically, the present invention provides one kind can produce target tumor Antibody coupling drug, the Its Preparation Method And Use of lethal effect.
Technical background
It is shown according to " global cancer report ", the whole world in 2018 is estimated to increase 18,100,000 cases of cancer newly, and death toll reaches 9600000,18 people of average minute clock is further aggravated because of cancer mortality, global cancer burden.China is cancer big country, neopathy Number is in significant ascendant trend.Wherein, the solid tumors such as lung cancer, breast cancer, colorectal cancer are the high cancers of disease incidence, and When usually making a definite diagnosis, the state of an illness has evolved to middle and advanced stage.Current main treatment means are operative treatment, radiotherapy and drug Chemotherapy, not only disease prognosis is poor for the therapeutic strategy, and survival rate is low within 5 years, and its toxic side effect is big, brings greatly to patient Pain.In recent years, with the continuous development of molecular biology, molecular targeted therapy becomes the new direction of oncotherapy, gives Cancer patient brings Gospel.
EGF-R ELISA (EGFR, HER-1, ErbB-1) is a kind of tyrosine kinase receptor, is EGFR tyrosine One of four members of kinase families, EGFR activation are grown by playing ligands specific such as epidermal growth factor (EGF) and conversion The combination of the factor-α (TGF- α) triggers.As EGFR and ligand binding, dimerization occurs and leads to tyrosine kinase intracellular Area's activation, further activates downstream signaling pathway.EGFR and its ligand are a part of cellular signal transduction system, signal Conducting networks play an important role during the formation and development of tumour, and EGFR was in the tumour of a variety of epithelial origins Expression, such as head and neck cancer, lung cancer, colon cancer, cancer of pancreas, breast cancer, oophoroma, the cancer of the esophagus.Therefore EGFR is oncotherapy In a critically important target spot.The targeted therapy of EGFR mainly has junket ammonia kinase inhibitor (for Buddhist nun's class) small molecule medicine at present Object and anti-egfr antibodies drug.Wherein, the mechanism of action of antibody drug generally blocks EGFR ligand binding, causes under inhibition Trip effect such as inhibits intracellular signal transduction, inhibits cell cycle progression, induces cell apoptosis and inhibit DNA to repair, blood vessel Generation, Tumor Cell Migration, invasion and transfer.But the treatment of antibody has certain limitation, usually wants and chemotherapy Drug combination can be only achieved curative effect.
Antibody-drug conjugates (Antibody-drug conjugate, ADC) are the strategies for improving antibody curative effect One, it is to be connected cytotoxic drug using antibody as carrier by connexon and sent to target area, both improved Cytotoxic drugs The targeting of object, and increase the anti-tumor activity of antibody, therapeutic window is improved on the whole, is mentioned for " the precisely medical treatment " of disease Effective weapon is supplied.ADC is generally by antibody (antibody), bullet molecule (warhead) and the connexon for connecting the above two (linker) 3 component compositions.ADC has high requirement, usual IC to cytotoxic cytotoxicity50< 10-9mol/ L could utilize the cytotoxin of limited quantity entrained by antibody effectively to kill tumour cell in this way.MMAE(monomethyl Auristatin E) it is a kind of linear polypeptide class Cytotoxic molecules, it inhibits tumour growth by acting on tubulin, In a variety of human cancer cell lines, IC50It is 10-9~10-11Mol/L is 200 times of traditional anti-cancer small-molecule drug vincaleukoblastinum, existing It has been widely used in the research and development of ADC.2011, MMAE was employed successfully in the Hodgkin lymphoma of FDA approval listing (HL) and the appropriate all esters of former times monoclonal antibody of the therapeutic agent cloth of primary cutaneous type (ALCL) (brentuximab vedotin, Adcetris in), according to II clinical trial phase, 102 patients are with 1.8mg/kg administration three weeks, patient's HL Overall response rate 75% (34% total regression, the response of 40% part), patient's ALCL Overall response rate is 87% (53% total regression), and therapeutic effect is aobvious It writes.The connexon of Val-Cit dipeptides is most widely used in the coupling of MMAE class ADC, is efficiently split by cathepsin B Solve and discharge toxin, since this enzyme is in blood almost without distribution so that such ADC have in blood it is very high steady It is qualitative and toxin is optionally discharged in tumor tissues.
Have on the antibody coupling drug (ADC) of the target spots such as targeting CD30, CD33, CD22 and Her-2 ratified by FDA at present City can be used for the treatment of a variety of solid tumors using EGFR as the ADC of target spot since EGFR high in kinds of tumors can be expressed, The oncotherapy type of ADC is extended, clinical and wide market can be the treatment especially solid tumor of numerous tumours Treatment zone is wished.
LR004 antibody is a kind of antibody for targeting EGFR, the antibody individually or with one or more additional therapeutic agent groups It shares in the associated cancer (Patent publication No: 106470697 A of CN for the treatment of performance EGFR； WO 2016/044234 EN). , mainly there is the following in the advantages of LR004 antibody: being directed to antibody itself, the sialic acid of LR004 is N-acetyl-neuraminate (NANA is regarded the more similar mankind), LR004 antibody is free of effective oligosaccharide immunogen galactolipin-α -1,3- gala of detectable amount Sugared structure, this two point feature make it generate lower immune response；In terms of activity, LR004 antibody is in kinds of tumor cells Good curative effect is presented in (A431, CEO, MDA-MB-468).
Novelty and the advanced above-mentioned advantage for being sufficiently to use LR004 antibody of the invention, utilizes its good target A kind of novel antibodies coupling drug LR004-VC-MMAE has been made in tropism, compatibility and endocytosis ability, equal in vivo and in vitro It shows the effect compared with the stronger solid tumor resisting of known ADC, compares LR004, activity is significantly improved.It is dynamic in medicine generation In mechanics study, longer half-life period, slower clearance rate, blood is presented in the total antibody of LR004-VC-MMAE antibody coupling drug The MMAE concentration dissociated in slurry is lower, advantageously reduces toxicity.In conclusion targeting the antibody coupling drug of EGFR LR004-VC-MMAE shows good application prospect.
Summary of the invention
An object of the present invention is to be the provision of a kind of antibody coupling drug LR004-VC- for targeting EGFR MMAE and preparation method thereof has efficient targeting, endocytic activity and affine activity to the tumor tissues of the EGFR positive.
The second object of the present invention is to provide using above-mentioned ADC as anti-tumor drug of active constituent and application thereof.
In order to achieve the above object, present invention employs following technological means:
A kind of antibody coupling drug targeting EGFR of the invention, is named as LR004-VC-MMAE, the drug It is made of antibody, cytotoxic drug and connexon, the antibody drug conjugates have the structure as shown in following formula I:
Wherein, mAb is anti-for LR004 monoclonal, n=2~8, preferred n=4.
In the present invention, the LR004 Dan Kelong antibody is human mouse chimeric antibody, specifically binds to the N-terminal of Human epidermal growth factor receptor Part, the molecular weight of the antibody are about 153kDa.The monoclonal antibody can be used method as known in the art in China It is made in hamster ovary cell CHO.The cell toxicity medicament is MMAE.The connexon is Val-Cit (VC) connector, full name MC-VC-PAB~connector.MMAE is connected with VC connector first, after purified preparation, maleimide base group again with The interchain disulfide bond of LR004 antibody occurs addition reaction and antibody coupling drug is made.
Further, the invention also provides a kind of methods for preparing the antibody coupling drug, including following step It is rapid:
1) MC-VC-PAB-MMAE (i.e. connexon-cell toxicity medicament) is dissolved in dimethyl sulfoxide, it is thin obtains connexon- Cytotoxic drug stock solution；
2) it dissolves reduce agent in buffer, configures reducing agent stock solution；
3) LR004 antibody is mixed with the reducing agent solution in the step 2) and carries out reduction reaction 1-2 h, obtain antibody Solution after reduction；
4) connexon in the step 1)-cell toxicity medicament stock solution is added drop-wise in the solution after antibody reduction, into Row addition reaction 1-2h, obtains antibody coupling drug.
In the described method, it is preferred that the preferred TCEP of reducing agent in step 3), with mole of LR004 antibody when mixing On the basis of amount, the mole of reducing agent is 2-4 times of antibody mole.
In the described method, it is preferred that added when the step 4) mixes on the basis of the mole of LR004 antibody Connexon-cell toxicity medicament mole is 4-8 times of antibody mole.
In the described method, it is preferred that step 3) and the reaction in step 4) they are 100~300rpm in speed of agitator, It is carried out under nitrogen protection, the reaction temperature in step 3) is 35~40 DEG C, preferably 37 DEG C.
In the described method, it is preferred that further include the antibody coupling drug LR004-VC-MMAE that will obtain further The step of purifying, preferably AKTA purifier protein purification system, antibody coupling medicine group swarming needed for collecting；It has collected Bi Hou carries out ultrafiltration centrifugation using 30kDa super filter tube, and concentration is filtered through sterilised membrane filter.
Further, the invention also provides the antibody coupling drugs in preparation for neoplasm targeted therapy Purposes in drug, wherein the tumour is EGFR positive entity tumor, including the cancer of the esophagus, dermoid cancer, lung Cancer, breast cancer, cancer of pancreas, head and neck cancer, colon cancer, prostate cancer, osteosarcoma cancer.
Further, the invention also provides a kind of pharmaceutical composition for neoplasm targeted therapy, contain pharmacy Upper a effective amount of antibody coupling drug of the present invention and the adjuvant pharmaceutically allowed.
Compared to the prior art, the beneficial effects of the present invention are:
1, for the present invention by using MMAE for bullet molecule using LR004 monoclonal antibody, Val-Cit is connexon, It is coupled by the chemical method and VC-MMAE that restore antibody interchain disulfide bond, constructs one kind and target EGFR and controlled with solid tumor Antibody coupling drug LR004-VC-MMAE based on treatment.It is preferred that TCEP is carried out centainly as reducing agent to its reaction condition After optimization, antibody coupling drug of the average drug antibody coupling ratio (DAR) 4 or so is prepared, naked anti-and Conjugate ratio is 8 component is below 5%, and content of monomer is greater than 95%, and stable and controllable for quality, and reproducible, yield is considerable, and not shadow Ring the stability of antibody.
2, novel antibodies coupling drug LR004-VC-MMAE, can either targeting EGFR antigen, and have strong killing The activity of tumour cell.And compared to LR004 itself, the affinity of antibody, endocytic activity and targeting are not influenced, preferably Remain its biological function.In vitro in activity rating, compared to LR004, antitumor activity is significantly improved, IC50In nM rank.In vivo in activity rating, compared to LR004, the tumor suppression of LR004-VC-MMAE antibody coupling drug is imitated Fruit has the raising of conspicuousness, and the case where tumor disappearance occurs.In pharmacokinetic, LR004-VC-MMAE antibody The total antibody of coupling drug is presented longer half-life period, slower clearance rate, and the MMAE concentration dissociated in blood plasma is lower, and half Declining, the phase is short, and removing speed is fast, advantageously reduces toxicity.
Detailed description of the invention
Fig. 1 is MMAE, the synthetic route chart of VC connexon and VC-MMAE
Wherein, Fig. 1 a is the synthetic route of MMAE
Fig. 1 b is the synthetic route of VC connexon (MC-VC-PAB-PNP)
Fig. 1 c is the synthetic route of VC-MMAE (MC-VC-PAB-MMAE)
Fig. 2 is the EGFR expression in the different tumour cells of measurement.
Wherein, Fig. 2 a is the expression that WesternBlot detects EGFR in different tumour cells.
Fig. 2 b is the expression that flow cytometry detects EGFR in different tumour cells.
Fig. 3 is the combination activity analysis of LR004 and LR004-VC-MMAE.
Wherein, Fig. 3 a is that ELISA method measures LR004-VC-MMAE and compares the affinity song of LR004 and antigen EGFR Line.
Fig. 3 b is that flow cytometry measurement LR004 and LR004-VC-MMAE is directed to the affine of EGFR positive expression cell Force curve.
Fig. 4 is endocytosis feelings of the immune detection of the focus method altogether LR004 and LR004-VC-MMAE in KYSE520 cell Condition.
Fig. 5 is LR004-VC-MMAE in the intracorporal living imaging of tumor bearing nude mice.
Fig. 6 is IC of the LR004-VC-MMAE and MMAE in the tumour cell of different EGFR expressions50Value.
Fig. 7 is drug efficacy study of the LR004-VC-MMAE in A431 Nude Mouse Model.
Fig. 7 a is drug efficacy study of the LR004-VC-MMAE of various dose group in A431 Nude Mouse Model.
Fig. 7 b be LR004-VC-MMAE 15mg/kg dosage group in A431 (gross tumor volume is 400-500 mm3) nude mice Drug efficacy study in Transplanted tumor model.
Fig. 8 is drug efficacy study of the LR004-VC-MMAE in KYSE520 Nude Mouse Model.
Drug efficacy study of the LR004-VC-MMAE of Fig. 8 a various dose in KYSE520 Nude Mouse Model.
Fig. 8 b is control group and administration group changes of weight curve graph in experiment.
15mg/kg) to the toxicity of each internal organs of nude mice.
Fig. 9 is the Drug-time curve figure of LR004-VC-MMAE each component in nude mouse.
Specific embodiment
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with describing And it is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.This field skill Art personnel should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and Form is modified or is replaced, but these modifications and replacement are fallen within the protection scope of the present invention.
The synthesis of embodiment 1VC-MMAE (MC-VC-PAB-MMAE):
The synthesis of 1.1MMAE
Synthetic route is as shown in Figure 1a:
The synthesis of step a:Boc-Dap-N- demethyl ephedrine
Compound Boc-Dap-OH (500mg, 1.7mmol) and (1S, 2R)-(±)-demethyl ephedrine (289.3mg, It 1.9mmol) is dissolved in 10mL DMF, sequentially adds DEPC (368.9mg, 2.2 mmol) and Et3N (228.8mg.2.2mmol), It is stirred to react at room temperature overnight.After completion of the reaction, add water to stop reaction, be extracted with ethyl acetate three times, with saturation NaCl solution After extraction is primary, anhydrous Na2SO4It is dried overnight.Column chromatographic isolation and purification, separation condition are petroleum ether: ethyl acetate=5:1, Obtain white solid 485mg.C23H36N2O5.MS(ESI)m/z:321.1H NMR(400MHz, DMSO-d6) δ 7.66 (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)
The synthesis of step b:Dap-N- demethyl ephedrine.Above-mentioned reaction product is dissolved in 6mL DCM, is added under ice bath Enter TFA 1mL, is stirred overnight at room temperature.After completion of the reaction, DCM is added to extract three times, merges organic phase, respectively with saturation NaHCO3Solution and saturation NaCl solution extract one time, and anhydrous Na is added2SO4It is dried overnight.DCM is filtered and be spin-dried for, ether is used It washes twice, obtains product, without being further purified.
Step c:Fmoc-L-Val-Dil-OButSynthesis.Reactant Dil-OBut.HCl (500mg, 1.7 mmol) and Fmoc-L-Val-OH (780mg, 2.3mmol) is dissolved in 10mL 2-Me-THF, sequentially add CDMT (403.5mg, 2.3mmol) with NMM (256.6mg, 2.55mmol), it is stirred to react at room temperature overnight.To after completion of the reaction, ethyl acetate be added Extraction three times, merges organic phase, washed once with saturation NaCl, anhydrous Na is added2SO4It is dried overnight.Column chromatography for separation, separation Condition is petroleum ether: ethyl acetate=6:1 obtains reaction product 486mg.C34H48N2O6.MS(ESI)m/z:581.1H NMR (400MHz,DMSO-d6) δ 7.89 (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-OButSynthesis.Upper step reaction product is dissolved in the anhydrous THF of 15mL, 200 μ L are added Et2NH is stirred overnight at room temperature.It is spin-dried for reaction solution after completion of the reaction, after washing 3 times with ether, obtains 357mg product, is not necessarily to It is further purified.
Step e:Fmoc- (N-Me)-L-Val-L-Val-Dil-OButSynthesis.Upper step reaction product is dissolved in 10mL In EtOAc, Fmoc- (N-Me)-L-Val-OH (455mg, 1.2mmol) is added, sequentially adds DEPC (214mg, 1.3mmol) After 1.6mL DIEA, reaction is stirred at room temperature overnight.After completion of the reaction, water is added to stop reaction.Ethyl acetate extraction three is added It is secondary, merge organic phase, it is primary with saturation NaCl solution extraction, anhydrous Na is added2SO4It is dried overnight.Column chromatography for separation, lightning strip Part is petroleum ether: ethyl acetate=4:1 obtains white solid 536mg.C40H59N3O7.MS(ESI)m/z:694.1H NMR δ=7.90 (400MHz, DMSO-d6) (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).
Top reaction product is dissolved in the DCM of 10mL by step f:Fmoc- (N-Me)-L-Val-L-Val-Dil-OH., The TFA of 1.5mL is added, reaction is stirred at room temperature overnight.After completion of the reaction, DCM is added to extract three times, merges organic phase, uses respectively It is saturated NaHCO3Solution and saturation NaCl solution extract one time, and anhydrous Na is added2SO4It is dried overnight.DCM is filtered and be spin-dried for, is used Ether washes twice, and obtains product 523mg.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).
The synthesis of step g:Fmoc-MMAE.By reaction step b product (300mg, 0.93mmol) and reaction step f product (464mg, 0.73mmol) is dissolved in the DCM of 15mL, sequentially adds 600 μ L of HATU (461mg, 1.2mmol) and DIEA, room temperature Under be stirred to react overnight.After completion of the reaction, be added DCM extract three times, merge organic phase, respectively with 2% citric acid solution with Saturation NaCl solution respectively extracts one time, and anhydrous Na is added2SO4It is dried overnight.DCM is filtered and is spin-dried for, ether is added and washs 3 times, Without being further purified, yellowish solid 495mg is obtained.
Step h: the reaction product of step g is dissolved in 10mL DCM, and 300 μ L Et are added2NH is stirred to react at room temperature Overnight.It is spin-dried for reaction dissolvent after reaction, is isolated and purified using preparation liquid phase.Chromatographic condition is as follows: A phase H2O+ 0.08%TFA, B phase acetonitrile+0.08%TFA.45% Gradient elution of 0-35min B phase collects sterling, and reaction product is lyophilized. 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).
The synthesis of 1.2VC connexon (MC-VC-PAB-PNP)
Synthetic route is as shown in Figure 1 b:
The synthesis of step a:Fmoc-Val-OSu.By reaction product Fmoc-L-Val-OH (10g, 29.3 mmol) and HoSu (3.7g, 32.3mmol) is dissolved in 100mL THF, and DCC (6.6 g, 32.3mmol) are added under ice bath, after addition, remove Ice bath is stirred to react overnight at room temperature.After completion of the reaction, reaction solution is moved into ice bath, is filtered after solid is precipitated completely, it will Filtrate is spin-dried for, and is threaded to foam-like.Reaction product is purer, without being further purified.
Step b: upper step reaction product (16g, 36.6mmol) is dissolved in 90mL DME, L-Cit (6.7 g, 38.5mmol) it is dissolved in NaHCO3It at salt in the 90mL water of (3.2g, 38.5mmol), is then added in reaction system, 50mL THF Hydrotropy, reaction are stirred overnight at room temperature to clarification.After completion of the reaction, 15% citric acid water isometric with THF is added under ice bath Solution is precipitated completely to reaction product white solid, and Buchner funnel is filtered to filtrate and clarified, and filter cake is drained, and vacuum oven is dry It is dry.After drying, white solid is ground, is washed with ether, filters, obtains white solid.C26H32N4O6.1H NMR (400MHz, DMSO-d6) δ 12.48 (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 (7.6g, 15.3mmol) of reaction step b and PABOH (3.76g, 30.6mmol) is molten In 140mL CH2Cl2With the CH of 70mL3OH in the mixed solvent is protected from light and EEDQ (7.5g, 30.6mmol) is added.It stirs at room temperature Reaction, is spin-dried for reaction solution after completion of the reaction, and ether washing filters.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: it above walks reaction product (8.4g, 19mmol) and is dissolved in 150mL DMF, Et is added2NH 25mL, at room temperature It is stirred to react overnight.After completion of the reaction, it is spin-dried for solvent, after ether washes three times, obtains yellowish solid 2.3g.C28H40N6O7.1H NMR (400MHz, DMSO-d6) δ 9.91 (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 product (0.5g, 1.3mmol) is dissolved in 18mL DMF, addition MC-OSu (0.45g, 1.4mmol), reaction is stirred at room temperature overnight.After completion of the reaction, be spin-dried for reaction dissolvent, after ether washes three times, filter yellow is solid Body 0.69g, without being further purified.
Step f: the reaction product of reaction step e is dissolved in 12mL DMF, and (PNP) is added after dissolution2CO(1.1g, 3.6mmol) with DIEA 0.5mL, it is stirred at room temperature lower overnight.After completion of the reaction, solvent, silica gel column separating purification, separation are spin-dried for Condition is methylene chloride: methanol=20:1.Obtaining product is yellowish solid 0.42g.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).
The synthesis of 1.3VC-MMAE (MC-VC-PAB-MMAE)
Synthetic route is as illustrated in figure 1 c:
MC-VC-PAB-PNP (93mg, 0.125mmol) and MMAE (60mg, 0.083mmol) are dissolved in 4mL DMF, according to Secondary addition HoBt (10mg, 0.074mmol) and 0.5mL pyridine are stirred to react overnight at room temperature.Reaction product uses preparation solution Mutually isolated and purified.Chromatographic condition is as follows: A phase H2O+0.08%TFA, B phase acetonitrile+0.08%TFA.0-35min B phase 65% Gradient elution collects sterling, and reaction product is lyophilized.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).
The preparation of 2 antibody drug conjugates LR004-VC-MMAE of embodiment
The following steps are included:
1) MC-VC-PAB-MMAE (i.e. connexon-cell toxicity medicament) that embodiment 1 is prepared is dissolved in dimethyl Asia Sulfone obtains connexon-cell toxicity medicament stock solution.
2) TCEP reducing agent is dissolved in buffer PBS (DTPA containing 1mM), configures the reducing agent deposit of certain molar weight Liquid.
3) buffer of LR004 antibody is replaced before reduction, buffer solution system is PBS (DTPA containing 1mM).Replacing options are Desalting column replacement.The replacing options are as follows: desalting column loading 2.5mL, elute 3.5mL with displacement buffer, are concentrated by ultrafiltration dense Degree, ultrafiltration revolving speed 4000rpm, 4 DEG C of centrifugation 10min.Configuration obtains 5mg/mL LR004 antibody-solutions.
4) the 5mg/mL LR004 antibody-solutions in step 3) and the reducing agent TCEP solution in the step 2) are mixed It closes, 37 DEG C, speed of agitator carries out 1~2h of reduction reaction under the conditions of being 200rpm, nitrogen protection when reaction, obtains antibody reduction Solution afterwards.The mole of added TCEP reducing agent is 3 times of LR004 antibody mole；
5) connexon in the step 1)-cell toxicity medicament solution is added drop-wise in the solution after antibody reduction, stirring Revolving speed carries out 1~2h of addition reaction under the conditions of being 200rpm, and nitrogen protection when reaction obtains antibody coupling drug.When mixing with On the basis of the mole of LR004 antibody, added connexon-cell toxicity medicament solution is 6 times of LR004 antibody mole.
6) the antibody coupling drug LR004-VC-MMAE needs after reaction, obtained are further purified, using AKTA Purifier protein purification system, antibody coupling medicine group swarming needed for collecting.After collection, 30kDa super filter tube is used Ultrafiltration centrifugation is carried out, concentrating buffer solution concentration, preferably 4 DEG C of centrifuging temperature, the preferred 4000rpm of revolving speed, centrifugation time is preferred 12min.After purification, it is filtered through sterilised membrane filter and is stored in -80 DEG C, whether reached before activity rating through reagents detection endotoxin Mark.
The LR004-VC-MMAE antibody drug conjugates being prepared have the structure as shown in following formula I:
Wherein, mAb is LR004 monoclonal antibody；N=4.
The detection of expression of EGFR in the different tumour cells of embodiment 3
3.1WesternBlot detects the EGFR expression of different tumour cells
Collect the esophageal cancer cell (KYSE450) of logarithmic phase, breast cancer cell (MDA-MB-468), head & neck cancer cell (SCC-25), colon cancer cell (HCT-116), pancreatic cancer cell (AsPC-1), osteosarcoma cell (143B) and prostate cancer are thin Born of the same parents (PC-3).Cell dissociation is collected cell and is counted, and collects 1 × 107Above-mentioned cell, centrifugation abandon supernatant it is spare.RIPA is pre-chilled Protease inhibitors is added in Protein Extraction Reagent (phosphorylated protein needs while inhibitors of phosphatases is added).In Protein Extraction 0.1M PMSF mother liquor, PMSF final concentration 1mM are added before starting.Cell count, with cell quantity 1 × 1071ml cracking is added Liquid, 4 DEG C of centrifugation packing, saves, BCA method measures protein concentration.WesternBlot experiment, applied sample amount are carried out according to protein concentration For 20 holes μ g/, transferring film time 1h.Film is dyed using Ponceaux staining reagent after the completion of transferring film.With 3% after dyeing BSA-TBST is closed, and dilutes primary antibody with 3%BSA-TBST, 4 DEG C overnight, and TBST is washed film 5 times.With 5% skimmed milk power- TBST dilutes secondary antibody, goat anti-mouse IgG (H+L) HRP, room temperature jog 40min.TBST is washed film 6 times.After ECL is added on film Exposure: 10s-5min (time for exposure adjusts with different luminous intensities), develop 2min, fixing, as a result as shown in Figure 2 a.
3.2 flow cytometries detect the expression of different cells
LR004-VC-MMAE is chosen, concentration is 10 μ g/mL, respectively with 5 × 105Esophageal cancer cell (KYSE520, KYSE150), dermoid cancer A431, non-small cell lung cancer cell (HCC827, NCI-H1975, A549), breast cancer is thin Born of the same parents (MDA-MB-468, MCF-7), pancreatic cancer cell (AsPC-1) and primary cutaneous type cell Karpas299 are 4 1h is incubated at DEG C jointly, 2% FBS/PBS is washed 2 times, and goat anti-human igg's (Zhong Shan Golden Bridge, 1:200 dilution) of FITC label is added After washing twice with 500 μ L PBS cell liquid is resuspended in 4 DEG C of incubation 1h, PBS, with the fluorescence of flow cytomery difference cell Intensity, as a result as shown in Figure 2 b.
The affinity determination of embodiment 4LR004-VC-MMAE
4.1ELISA method detects the combination activity of LR004-VC-MMAE and antigen EGFR
Antigen EGFR (being purchased from ACRO Biosystems) is diluted to 2 μ g/mL with PBS, is laid on 96 orifice plates, every 100 μ of hole L, 4 DEG C are incubated overnight rear PBST and wash 3 times, are closed overnight with 2%BSA, absorb confining liquid, washed 3 times with PBST, by albumen LR004-VC-MMAE and control LR004 are diluted to a series of various concentrations, and 96 holes for being coated EGFR antigen are added Plate, 50 μ L of every hole, 37 DEG C of incubation 1h, PBST are washed 3 times, each 5min.The Goat anti-Human of alkali phosphatase enzyme mark is then added (Fab specificity) secondary antibody (1:1000 dilution), 37 DEG C of incubation 1h, PBST washings three times, each 5min.Tmb substrate colour developing is added About 10min surveys absorbance value in 405nm wavelength, and mapping indicates the binding curve of the two with EGFR.The result shows that LR004- VC-MMAE is in concentration dependant in conjunction with antigen EGFR, and is coupled little to the affine activity influence of antibody itself after MMAE (Fig. 3 a).
The affinity constant of 4.2Biocare method measurement LR004-VC-MMAE
The LR004-VC-MMAE of 1 μ g/mL is bound on CM5 chip respectively, by the EGFR antigen (ACRO of various concentration Biosystems it) is injected in system, and flows through chip and combined with conjugate.Chip surface uses the MgCl of 3M2It is solved From.By Biacore T200 system-computed affinity constant value.Affinity numerical value is as follows:
Table 1LR004-VC-MMAE affinity constant
4.3 flow cytometries detect LR004-VC-MMAE and combine activity
The different concentration of LR004-VC-MMAE and LR004 is chosen, respectively with 5 × 1054 DEG C of cell common incubation 1h, 2% FBS/PBS is washed 2 times, and 4 DEG C of goat anti-human igg's (Zhong Shan Golden Bridge, 1:200 dilution) that FITC label is added is incubated for 1h, PBS washing Cell liquid is resuspended with the PBS of 500 μ L afterwards twice, with its fluorescence intensity of flow cytomery.The result shows that in different EGFR In positive tumor cell, the combination activity of LR004-VC-MMAE and LR004 are suitable (such as Fig. 3 b).
Endocytic activity of the 5 confocal laser scanning microscope LR004-VC-MMAE of embodiment in cell
Single cell suspension is made in KYSE520 cell in logarithmic growth phase, cell count simultaneously adjusts its concentration, presses Every hole 1 × 104LR004-VC-MMAE is added in 96 orifice plates in a cell inoculation after 37 DEG C of culture 2h, its concentration is 5 μ eventually g/mL.4 DEG C of incubation 30min after conjugate are added in observation for conjugate situation in conjunction with cell surface antigen, collect thin Born of the same parents' sample, PBS are washed 2 times, and cell is centrifuged to glass slide with cell rejection tablet machine, and 4% paraformaldehyde fixes 10min, and PBST is washed 3 times, 0.2%TritonX-100/PBS permeabilization 10min, PBST is washed 3 times, overnight with 5% sheep blood serum closing.37 DEG C of incubations The anti-human fluorescence secondary antibody 30min of Alexa Fluor488 label, PBST are washed 3 times, and Hoechst3342 contaminates core 15min, and PBST washes 3 It is secondary, anti-fluorescence quenching is added dropwise, covered is in confocal microscopy.Cell is observed to the endocytosis situation of conjugate When, need 37 DEG C to be incubated for for 24 hours after albumen is added.With LAMP-1 antibody (be purchased from Cell Signaling Thechnology) and Donkey anti-rabbit secondary antibody (being purchased from the green skies) label lysosome that Alexa Fluro 555 is marked.Remaining operation and above process phase Together.
The result shows that LR004-VC-MMAE is in 4 DEG C of incubations, can in conjunction with EGFR receptor on cell membrane, but not into Enter cell interior.And when 37 DEG C of incubations, LR004-VC-MMAE enters cell interior by endocytosis, can in lysosome common location (as shown in Figure 4).
Embodiment 6LR004-VC-MMAE is in the intracorporal target tumor ability of nude mice
With DyLight680 antibody labeling kit (being purchased from Thermo Scientific) difference label L R004-VC- MMAE, positive control LR004 and negative control rituximab-VC-MMAE, specific method is refering to operational manual.It will KYSE520 cell inoculation is in BALB/c nude mice oxter, when knurl product to 200-300mm3When, by the above-mentioned three of label with The dosage tail vein injection mice with tumor (n=2) of 20mg/kg.Utilize the toy of XENGOEN (Caliper company of the U.S.) Living imaging instrument is observed.It respectively at different time points, is handled using Medical anesthetic agent isoflurane, is placed in 37 DEG C in advance It (is anaesthetized simultaneously) on the observation board of heat, it is intracorporal in tumor bearing nude mice to monitor drug fluorescence using the CCD camera lens for being cooled to -90 DEG C The case where distribution and target tumor.As a result, it has been found that LR004-VC-MMAE can preferably target transplanted tumor in nude mice position, when 48h It can be seen that stronger tumour enrichment degree, fluorescence intensity can be maintained 6 days (Fig. 5).
Embodiment 7LR004-VC-MMAE is directed to the cell killing activity of the tumour cell of different EGFR expression
The tumour cell EGFR expression quantity obtained in 3 in conjunction with the embodiments is as a result, carry out antibody coupling drug to these cells The cell in vitro killing activity of LR004-VC-MMAE is evaluated.Specially esophageal cancer cell (KYSE520, KYSE150), in squamous Chrotoplast cancer A431, non-small cell lung cancer cell (HCC827, NCI-H1975, A549), breast cancer cell (MDA-MB-468, MCF-7), pancreatic cancer cell (AsPC-1) and primary cutaneous type cell Karpas299.Specific step is as follows:
The cell centrifugation of logarithmic growth phase, which is resuspended, to be counted, and with 1 × 104-3×104/ hole is inoculated in 96 orifice plates, in 37 DEG C of culture 2h.Then, the LR004-VC-MMAE (MMAE is as positive control) of various concentration is added, each drug concentration is set Set 3 parallel holes.In 37 DEG C of incubation 48h, every hole is added 20 μ L CCK8 reagents and continues to cultivate 1-2h.Color reaction is observed, is used in combination Microplate reader measures the light absorption value at 450nm.Blank group (without cell) and negative control group (nothing are respectively set in experimentation Drug-treated), the survival rate of cell: cell survival rate=(dosing group A450 value-blank group A450 is calculated according to the following formula Value)/(450 values of control group A-blank group A450 value) × 100%.IC50Value, which calculates, uses SPSS software.According to result (Fig. 6) institute Show, LR004-VC-MMAE and MMAE have close IC50Value, and do not show in EGFR negative cells system Karpas299 anti- Tumor promotion, therefore the LR004-VC-MMAE of invention acquisition can not only effectively play the biology of LR004 antibody moiety Function is learned, MMAE is also had concurrently to the Efficient killing effect activity of tumour cell, in addition shows good targeting and selectivity.
Antitumor drug efficacy study of the embodiment 8LR004-VC-MMAE to A431 Transplanted tumor model
8.1LR004-VC-MMAE is to A431 Transplanted tumor model (starting tumor volume about 100mm3) antitumor drug effect grind Study carefully
Taking weight is female BAl BIc/c nude mice of 18-22g, and EGFR positive cell A431 is inoculated in armpit on the right side of nude mice Subcutaneously, every inoculation 5 × 106A cell.It is long to about 100mm to tumor mass3When, it is grouped at random according to weight and knurl product, Every group 6, if blank control group.It is handled according to following dosage regimens: LR004-VC-MMAE (1mg/kg, 5mg/kg, 10mg/ Kg and 15mg/kg), MMAE (0.3mg/kg), LR004 (15mg/kg).Administration mode is tail vein administration, and every 200 μ L are right It does not deal with, is administered once according to group within every 4 days, be administered 6 times altogether.During experiment, every 3~4 days bodies to nude mice weight and tumour Product measures.Gross tumor volume (a: tumour major diameter, b: tumour minor axis) is calculated according to formula V=a*b*b/2, it is raw to draw tumour Long curve (Fig. 7 a).
Tumour inhibiting rate=(control group mean tumor volume-experimental group mean tumor volume)/control group mean tumor volume × 100%
The results show that LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg) group is swollen after testing 60 days Tumor inhibiting rate is respectively 97.7%, 98.2% and 98.9%.With LR004 (tumour
8.2LR004-VC-MMAE is to A431 Transplanted tumor model (starting tumor volume about 400-500 mm3) antineoplastic Effect research
Taking weight is female BAl BIc/c nude mice of 18-22g, and EGFR positive cell A431 is inoculated in armpit on the right side of nude mice Subcutaneously, every inoculation 5 × 106A cell.It is long to about 400-500mm to tumor mass3When, divided at random according to weight and knurl product Group, every group 6, if blank control group and LR004-VC-MMAE 15mg/kg dosage group.Remaining operation and above-mentioned 8.1 process phase Together.The results show that control group gross tumor volume reaches about 2500mm after testing 40 days3, and LR004-VC-MMAE 15mg/kg dosage group mean tumour volume is about 370mm3, tumor control rate 87.2%.Illustrate LR004-VC-MMAE ADC Also there is good response to big tumour, and tumour can be suppressed and no longer grown and even reduce gross tumor volume, tumor suppression curve is such as Fig. 7 b.
Antitumor drug efficacy study of the embodiment 9LR004-VC-MMAE to KYSE520 Transplanted tumor model
Taking weight is female BAl BIc/c nude mice of 18-22g, and EGFR positive cell A431 is inoculated in armpit on the right side of nude mice Subcutaneously, every inoculation 5 × 106A cell.It is long to about 100mm to tumor mass3When, it is grouped at random according to weight and knurl product, Every group 8, if blank control group.According to following dosage regimens handle: LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg), LR004 (15mg/kg) and Rituximab-VC-MMAE (15mg/kg).Administration mode is tail vein administration, often Only 200 μ L, control group do not deal with, are administered once within every 4 days, are administered 4 times altogether.During experiment, every 3~4 days to nude mice weight It is measured with the volume of tumour.Gross tumor volume (a: tumour major diameter, b: tumour minor axis) is calculated according to formula V=a*b*b/2, Draw tumor growth curve.Control group, LR004 (15mg/kg) and Rituximab-VC-MMAE (15mg/kg) gave in 44 days Nude mice euthanasia, LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg), which continues observation to experiment in 60 days, to be terminated.As a result It has been shown that, negative control group rituximab-VC-MMAE (15mg/kg) is without anti-tumor activity.LR004 (15mg/kg) is in the tumour In and not shown obvious anti-tumor activity, tumor control rate be only 13.8%.LR004-VC-MMAE (5mg/kg, 10mg/kg and 15mg/kg) there is tumor disappearance situation in group, wherein and LR004-VC-MMAE (5mg/kg) organizes 6 nude mouse tumors and disappears, LR004-VC-MMAE (10 mg/kg) organizes 7 nude mouse tumors and disappears, and LR004-VC-MMAE (15mg/kg) organizes whole nude mouse tumors It disappears (Fig. 8).
Embodiment 10LR004-VC-MMAE pharmacokinetic situations in nude mouse
10.1ELISA method detects the concentration of total antibody in nude mouse
40 BALB/c nude mices (weight 20-22g) are received into tail vein injection LR004-VC-MMAE 15mg/kg, when Between put 0,0.5,1,2,8,24,48,72,120,216 hour, put to death, take whole blood.4 mouse are arranged in each time point.It collects Nude mice whole blood is centrifuged -80 DEG C of preservations.It prepares LR004-VC-MMAE standard items: preparing 6 containing 0.5mL Standard diluents Dilution standard point: 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL.ELISA is specifically examined Survey method is the same as described in Application Example 4.1
10.2LC-MS/MS method detects the concentration of sequestered MMAE and mating type MMAE in nude mouse
Sequestered MMAE detection: the second that 60 μ L contain 200ng/mL Tolbutamide is added in above-mentioned 20 μ L of blood serum sample Nitrile solution, vortex centrifugal.10 μ L are injected into LC-MS/MS system.
Total MMAE detection: 1 μ L cathepsin B is added in the 12 above-mentioned serum samples of μ L, is incubated for 3 hours at 37 DEG C. The acetonitrile solution that 38 μ L contain 200ng/mL Tolbutamide is added and terminates reaction, vortex centrifugal.10 μ L are injected into LC-MS/ MS system.
LC-MS/MS system: 1.8 μm of 2.1 × 50mm of Waters HSS T3；Formic acid+the H that A phase is 0.1%2O；B phase For 0.1% formic acid+acetonitrile.B:10%-90%, 0-2min；Flow velocity is 0.5mL/min；Column temperature is 50 DEG C.MMAE detection point Son amount 718.7/152.2.
Mating type MMAE concentration calculation formula=(total MMAE concentration-dissociate MMAE concentration)/cathepsin B hydrolysis speed Rate × 100%, wherein Drug-time curve is as shown in figure 9, pharmacokinetic parameter is as shown in Table 2-4.
The result shows that total antibody (antibody of coupling and the antibody not being coupled) half-life period of LR004-VC-MMAE is about 5 It, clearance rate is lower, sustainable performance anti-tumor activity.Sequestered MMAE concentration is lower, for 24 hours after 2ng/mL can be reduced to left The right side, half-life short, clearance rate is high, can reduce toxicity.
The pharmacokinetic parameter of total antibody in table 2LR004-VC-MMAE
The pharmacokinetic parameter of mating type MMAE in table 3LR004-VC-MMAE
The pharmacokinetic parameter of free MMAE in table 4LR004-VC-MMAE

Claims (9)

1. a kind of antibody coupling drug for targeting EGFR, is named as LR004-VC-MMAE, which is characterized in that the drug It is made of antibody, cytotoxic drug and connexon, the antibody drug conjugates have the structure as shown in following formula I:
Wherein, mAb is LR004 monoclonal antibody, n=2~8.
2. antibody coupling drug according to claim 1, which is characterized in that n=4.
3. a kind of method for preparing antibody coupling drug of any of claims 1 or 2, which comprises the following steps:
1) MC-VC-PAB-MMAE is dissolved in dimethyl sulfoxide, obtains connexon-cell toxicity medicament stock solution；
2) it dissolves reduce agent in buffer, configures reducing agent stock solution；
3) LR004 antibody is mixed with the reducing agent solution in the step 2) and carries out reduction reaction 1-2h, obtain antibody reduction Solution afterwards；
4) connexon in the step 1)-cell toxicity medicament stock solution is added drop-wise in the solution after antibody reduction, is added At reaction 1-2h, the antibody coupling drug is obtained.
4. according to the method described in claim 3, it is characterized in that, the preferred TCEP of reducing agent in step 3), with LR004 when mixing On the basis of the mole of antibody, the mole of reducing agent is 2-4 times of antibody mole.
5. according to the method described in claim 3, it is characterized in that, with the mole of LR004 antibody when the step 4) mixes On the basis of, the mole of added connexon-cell toxicity medicament is 4-8 times of antibody mole.
6. according to the method described in claim 3, it is characterized in that, the reaction in step 3) and step 4) is in speed of agitator 100~300rpm is carried out under nitrogen protection, and the reaction temperature in step 3) is 35~40 DEG C, preferably 37 DEG C.
7. according to the method described in claim 3, it is characterized in that, further including the antibody coupling drug LR004-VC- that will be obtained The step of MMAE is further purified, preferably AKTA purifier protein purification system, antibody coupling drug component needed for collecting Peak；After collection, ultrafiltration centrifugation is carried out using 30kDa super filter tube, concentration is filtered through sterilised membrane filter.
8. purposes of the antibody coupling drug according to claim 1 or 2 in the drug that preparation is used for neoplasm targeted therapy, Wherein, the tumour is EGFR positive entity tumor, including the cancer of the esophagus, dermoid cancer, lung cancer, breast cancer, pancreas Cancer, head and neck cancer, colon cancer, prostate cancer, osteosarcoma cancer.
9. a kind of pharmaceutical composition for neoplasm targeted therapy, which is characterized in that the claim 1 containing pharmaceutical effective amount Or antibody coupling drug and the adjuvant pharmaceutically allowed described in 2.
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