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CN106467575A - Cysteine engineered Antibody-toxin conjugate - Google Patents

Cysteine engineered Antibody-toxin conjugate Download PDF

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Publication number
CN106467575A
CN106467575A CN201510507792.6A CN201510507792A CN106467575A CN 106467575 A CN106467575 A CN 106467575A CN 201510507792 A CN201510507792 A CN 201510507792A CN 106467575 A CN106467575 A CN 106467575A
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pab
antibody
tdc
toxin conjugate
cysteine engineered
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CN106467575B (en
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朱义
王茜
王一茜
卓识
李�杰
陈澜
余永国
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Chengdu Bailidote Biological Pharmaceutical Co ltd
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Sichuan Baili Pharmaceutical Co Ltd
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Abstract

The present invention is by 205 L-Valine of light chain of target antibody（V）It transform cysteine as（C）, and by the free sulfhydryl groups of this improved cysteine（‑SH）Be coupled small molecule high activity cytotoxin（Payload）Mc vc PAB OH connexon carry out site-directed coupling, form the excellent cysteine engineered antibody toxin conjugate of homogeneity, its toxin compares antibody ratios（DAR）For 1.6 2.0.This antibody toxin conjugate has formula：2C1‑LC‑V205C‑mc‑vc‑PAB‑payload.Meanwhile, the treatment use invention further discloses preparation of this TDC medicine, purification process, to expression EGFRvIII and overexpression EGFRwt tumor.

Description

Cysteine engineered Antibody-toxin conjugate
Technical field
The present invention relates to a kind of compound and its production and use, particularly to cysteine engineered Antibody-toxin conjugate (TDC) of a class and its production and use.
Background technology：
EGF-R ELISA EGFR (Epidermal Growth Factor Receptor) is a kind of glycoprotein, belong to one kind of ErbB receptor family, this family includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).EGFR is the receptor of epithelium growth factor (EGF) cell proliferation and signal transduction, insertion cell membrane, molecular weight 170KDa, by activate with ligand binding.After activation, EGFR is converted into dimer by monomer.EGFR is also possible to be polymerized to activate with other members of ErbB receptor family, such as ErbB2/Her2/neu.
The usual low amounts of EGFR is expressed in multiple normal tissue cells, including skin, liver etc., related to normal physiological effect.
The generation of EGFR overexpression and kinds of tumors with develop related, including head and neck cancer, bladder cancer, ovarian cancer, nonsmall-cell lung cancer, colorectal cancer, cerebral glioma, renal carcinoma, carcinoma of prostate, cancer of pancreas, breast carcinoma (Atalay et al., 2003；Herbst and Shin,2002).
Kinds of tumors also expresses the EGFR of saltant type, EGRFvIII (de2-7 EGFR) while overexpresses wild-type EGFR (EGFRwt).EGRFvIII is the EGFR saltant type (Sugawa et al., 1990) of EGFRwt 6-273 aminoacid deletion, the cell continuous activation of mediating ligand independent form.EGRFvIII is reported in kinds of tumors, including cerebral glioma, breast carcinoma, nonsmall-cell lung cancer, ovarian cancer and carcinoma of prostate (Wikstrand et al., 1997；OlapadeOlaopa et al.,2000).The appearance of EGRFvIII often indicates that tumor prognosis are bad.
EGRFvIII is specific expressed, and in tumor cell tissue, normal tissue cell does not express EGRFvIII, and this provides good target spot for targeted therapy.
Antibody-toxin conjugate (Antibody drug conjugate, ADC) be targeted therapy hot fields, two medicine Adcetris and Kadcyla in the granted listing of the U.S. show good clinical efficacy, and have more than 50 ADC medicines and carrying out clinical stage research.
With EGFRvIII as target spot, phase II clinical trials are carried out in the U.S. by the ADC medicine ABT-414 of the non-site-directed coupling of interchain disulfide bond sulfydryl, embody certain clinical effectiveness, but because it adopts non-site-directed coupling mode, its medicine homogeneity is poor, patient is caused with more serious toxic and side effects, limit its clinical application amount, directly affects its clinical therapeutic efficacy.
Content of the invention：
The Antibody-toxin conjugate (TDC) that the compound of the present invention is cysteine engineered has formula：2C1-LC-V205C-mc-vc-PAB-payload.Wherein 2C1 is that 205 L-Valine (V) of parental antibody 2A1 light chain transform the antibody after cysteine (C) as.The aminoacid sequence of the weight chain variable district of 2C1 is SEQ ID NO:1,2C1 light-chain amino acid sequence is SEQ ID NO:12.The aminoacid sequence of the parental antibody 2A1 weight chain variable district of 2C1 is SEQ ID NO:1,2A1 light-chain amino acid sequence is SEQ ID NO:14.2C1 maintains the ability (affinity) of its parental antibody 2A1 and antigen binding, wild-type egf receptor (EGFRwt) and the EFGR (de2-7 EGFR/EGRFvIII) of people that antigen is behaved.2C1 carries out Antibody-toxin conjugate (TDC) site-directed coupling by the cysteine sulfydryl of 205 transformations of its light chain with mc-vc-PAB-payload, and toxin is 1.6-2.0 than antibody ratios DAR.Antibody-toxin conjugate (TDC) medicine that the compound of the present invention is cysteine engineered is mainly used in treating the tumor of expression EGFRvIII and overexpression EGFRwt, including brain colloid carcinoma, nonsmall-cell lung cancer, head and neck cancer, colorectal cancer, bladder cancer, colorectal cancer, renal carcinoma, carcinoma of prostate, cancer of pancreas, breast carcinoma etc..
The completely newly cysteine engineered Antibody-toxin conjugate (TDC) that the present invention records compares the ADC of non-site-directed coupling, and to have a medicine homogeneity good, and the advantages of Small side effects, preclinical study result shows that it is significantly better than non-site-directed coupling ADC.
Brief description
Fig. 1 detects 2C1-LC-V205C-mc-vc-PAB-MMAE TDC DAR for HIC-HPLC；
Fig. 2A assembles situation for SEC-HPLC detection 2C1-LC-V205C-mc-vc-PAB-MMAE TDC；
Fig. 2 B carries out the 2A1-mc-vc-PAB-MMAE ADC sample of the non-site-directed coupling of cysteine for SEC-HPLC detection by antibody native interchain disulfide bond；
Fig. 3 A is the affinity that 2C1-LC-V205C-mc-vc-PAB-MMAE TDC and 2C1 maintains 2A1 and antigen EGFRvIII；
Fig. 3 B is 2C1-LC-V205C-mc-vc-PAB-MMAE TDC, 2C1-LC-V205C-mc-vc-PAB-MMAF TDC, 2C1-LC-V205C-mc-vc-PAB-PBD TDC, 2C1-LC-V205C-mc-vc-PAB-SN38TDC, 2C1-LC-V205C-mc-vc-PAB-DOX TDC maintain 2A1 and the affinity of antigen EGFRvIII；
Fig. 4 A is the IC50 testing result of the application on human skin squamous cell carcinoma A431 to EGFRwt overexpression for the 2C1-LC-V205C-mc-vc-PAB-MMAE TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC；
Fig. 4 B is 2C1-LC-V205C-mc-vc-PAB-MMAF TDC and its corresponding 2A1-mc-vc-PAB-MMAF ADC, the IC50 testing result to the application on human skin squamous cell carcinoma A431 of EGFRwt overexpression；
Fig. 4 C is the IC50 testing result of the application on human skin squamous cell carcinoma A431 to EGFRwt overexpression for the 2C1-LC-V205C-mc-vc-PAB-PBD TDC and its corresponding 2A1-mc-vc-PAB-PBD ADC；
Fig. 4 D is the IC50 testing result of the U87-EGFRvIII to expression EGFRvIII for the 2C1-LC-V205C-mc-vc-PAB-MMAE TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC；
Fig. 4 E is the IC50 testing result of the U87-EGFRvIII to expression EGFRvIII for the 2C1-LC-V205C-mc-vc-PAB-MMAF TDC and its corresponding 2A1-mc-vc-PAB-MMAF ADC；
Fig. 4 F is the IC50 testing result of the U87-EGFRvIII to expression EGFRvIII for the 2C1-LC-V205C-mc-vc-PAB-PBD TDC and its corresponding 2A1-mc-vc-PAB-PBD ADC；
Fig. 4 G is the IC50 testing result of the U87-EGFRvIII to expression EGFRvIII for the 2C1-LC-V205C-mc-vc-PAB-SN38 TDC and its corresponding 2A1-mc-vc-PAB-SN38 ADC；
Fig. 4 H is the IC50 testing result of the U87-EGFRvIII to expression EGFRvIII for the 2C1-LC-V205C-mc-vc-PAB-Dox TDC and its corresponding 2A1-mc-vc-PAB-Dox ADC；
Fig. 5 A carries out single intravenous injection result for tumor-bearing mice Composition analyzed 1mg/kg dosage；
Fig. 5 B is tumor-bearing mice Composition analyzed 6mg/kg dosed administration result；
Fig. 6 A detects neutrophil count result for rat toxicity；
Fig. 6 B detects AST testing result for rat toxicity；
Fig. 6 C monitors body weight change situation for rat toxicity detection is daily.
The synthesis of embodiment 1 mc
The maleic anhydride 3.5g (0.036mol) of 6-aminocaprolc acid 3.9g (0.03mol) and 1.2eq is added in 30ml glacial acetic acid.Reactant liquor is in 120 DEG C of stirring reaction 4～6h.After completion of the reaction, stop heating, naturally cool to room temperature.60 DEG C of concentrating under reduced pressure remove most of acetic acid.Gained brown color viscous solution is poured into water, and adds ethyl acetate 20ml × 3 extraction, merges organic layer.Organic layer uses water, saturated common salt water washing, anhydrous sodium sulfate drying successively, filters, and filtrate reduced in volume obtains pale tan oil, adds the stirring of 50ml water, has off-white powder to separate out, filters, the target product 5.08g of 50 DEG C of drying under reduced pressure, yield 80%.mp：89-92℃.m/z：212.2[M+H]+.1HNMR(400Mz,DMSO)：13.21 (br, 1H, COOH), 6.75 (s, 2H, COCH=CHCO), 3.63 (t, 2H, J=7.2Hz, NCH2CH2), 2.42 (t, 2H, J=7.4Hz, CH2COOH), 1.52-1.68 (m, 4H, NCH2CH2CH2CH2), 1.30-1.42 (m, 2H, NCH2CH2CH2CH2).
The synthesis of embodiment 2 Mc-OSu
4.7g (22mmol) MC and 25g (22mmol) HOSu is added under nitrogen protection in 50ml acetonitrile.Separately take 4.5g (22mmol) DCC to be dissolved in 25ml acetonitrile, keep interior temperature at 0 DEG C about, be slowly dropped in reactant liquor.Room temperature reaction is overnight again after 0 DEG C of reaction 2 hours for reactant liquor.Filter, filter cake is washed with acetonitrile 10ml × 3, filtrate reduced in volume is extremely dry.Gained grease obtains light tan solid 6.4g, yield 95% in reduced pressure at room temperature 6h.(purification does not directly cast single step reaction) m/z：309.2[M+H]+.1HNMR(400Mz,CDCl3)：1～2 (m, 6H, CCH2CH2CH2C), 2.68 (t, 2H, CH2CO), 2.95 (s, 4H, COCH2CH2CO), 3.68 (t, 2H, CH2N), 6.81 (s, 2H, CH=CH).
The synthesis of embodiment 3 Fmoc-Val-OSu
Fmoc-Val 10g and HOSu 3.4g is added in 100mlTHF.Separately take DCC6g to be dissolved in 50ml acetonitrile, keep interior temperature at 0 DEG C about, be slowly dropped in reactant liquor.Reactant liquor is stirred at room temperature reaction 24 hours.Filter, filter cake is washed with THF, and filtrate reduced in volume obtains clear oil thing.Grease is not purified directly to cast single step reaction.m/z：437.4[M+H]+
The synthesis of embodiment 4.Fmoc-vc
The aqueous solution 60ml (NaHCO3 2g, 1.05eq) of Cit 4.0g (1.05eq) and sodium bicarbonate is added in 20mlTHF.Separately take 22.35mmolFmoc-Val-OSu to be dissolved in 60mlDME, then be added in reactant liquor.Reactant liquor stirring reaction 24 hours at room temperature.After completion of the reaction, add 15% aqueous citric acid solution 110ml in system, be then extracted twice with EA again, merge organic layer, be concentrated under reduced pressure to give white solid.And add methyl tertiary butyl ether(MTBE) 100ml to stir and wash in white solid, filter, filter cake obtains product 4.83g, yield 65% in 40 DEG C of drying under reduced pressure 4h.m/z：497.6(M+H)+.1HNMR(400Mz,DMSO):0.92 (6H,, 1.35～1.65 m) (4H, m), 2.10 (1H, m), 3.01 (2H, q), 3.99 (1H, t), 4.01-4.45 (2H, m), 4.45 (2H, t), 5.46 (2H, br), 6.03 (1H, t), 7.20-8.02 (8H, m), 8.25 (1H, d).
The synthesis of embodiment 5.Fmoc-vc-PABOH
Add DCM/MeOH=2/1 mixed solvent 60ml in reaction bulb, add Fmoc-vc 2g (4.2mmol) and PABOH 1.04g (2eq), stirring and dissolving partly adds EEDQ 2.0g (2eq) afterwards.Reaction system lucifuge stirring reaction 2.0d at ambient temperature.After completion of the reaction, 40 DEG C are concentrated under reduced pressure to give white solid.Collect white solid, add methyl tertiary butyl ether(MTBE) 100ml to stir and wash, filter, filter cake is washed with methyl tertiary butyl ether(MTBE), and 40 DEG C of drying under reduced pressure of gained white solid obtain 2.2g, yield about 88%.m/z：602.6(M+H)+.1HNMR(400Mz,DMSO):0.95 (6H, m), 1.45～1.69 (4H, m), 2.10 (1H, m), 3.11 (2H, m), 3.99 (1H, m), 4.30 (2H,, 4.05～-4.66 d) (2H, m), 4.55 (2H, d), 5.21 (1H, t), 5.51 (2H,), 6.11 br (1H, t), 7.09-8.10 (12H, m), 8.21 (1H, d), 10.51 (1H, br).
The synthesis of embodiment 6.vc-PABOH
Add Fmoc-vc-PABOH 490mg (0.815mmol) stirring and dissolving in 10mlNMP, add diethylamine 2ml.Stirring reaction 24h at room temperature.After completion of the reaction, in 40 DEG C of concentrating under reduced pressure, in gained grease, add 20mlDCM stirring and crystallizing, filter, filter cake is washed with DCM, and gained solid drying under reduced pressure obtains 277mg, yield 90%.m/z：380.2(M+H)+.1HNMR(400Mz,DMSO):0.89 (6H, m), 1.31～1.61 (4H, m), 1.82 (1H, m), 2.86 (1H, m), 2.89 (2H, d), 4.38 (2H, d), 4.44 (1H, m), 5.01 (1H,), br 5.35 (2H, br), 5.84 (1H, br), 7.14 (2H, d), 7.42 (2H, d), 8.08 (1H, br), 9.88 (1H, br).
The synthesis of embodiment 7.mc-vc-PABOH
Add vc-PABOH 205mg (0.54mmol) and MC-OSu 184mg (1.1eq) in 10mlNMP, finish stirring reaction 24h at room temperature.Reaction finishes, and in 40 DEG C of concentrating under reduced pressure, adds 20ml methyl tertiary butyl ether(MTBE) stirring and crystallizing in gained grease.Filter, filter cake is washed with methyl tertiary butyl ether(MTBE), obtains 310mg product, yield 100%.m/z:573.3(M+H)+.1HNMR(400Mz,DMSO):0.89(6H,m)、1.15-1.99(10H,m)、2.11(1H,m)、2.31(2H,t)、3.21(2H,m)、3.53(2H,t)、4.32(1H,t)、4.51(1H,m)、4.59(2H,br)、5.24(1H,br)、5.56(2H,br)、6.20(1H,br)、7.12(2H,s)、7.23(2H,d),7.58(2H,d)、7.94(1H,d),8.17(1H,d)、10.21(1H,br).
The synthesis of embodiment 8.mc-vc-PAB-PNP
Mc-vc-PABOH 168.6mg (0.294mmol) is taken to be dissolved in 5ml anhydrous pyridine under nitrogen protection, reaction system is cooled to 0 DEG C about.Separately take PNP179mg (3eq) to be dissolved in 5mlDCM, then be slowly added in reaction system.And remove ice bath after keeping 10min in 0 DEG C about, react 3h then at being stirred at room temperature.Reaction finishes, and adds 70mlEA and 100ml 15% aqueous citric acid solution, divides and takes organic layer.Organic layer uses citric acid, water, saturated common salt water washing, then anhydrous sodium sulfate drying successively, filters, and filtrate reduced in volume obtains light yellow oil to dry, adds methyl tertiary butyl ether(MTBE) crystallize to obtain off-white powder 86mg, yield 40%.m/z:738(M+H)+.1HNMR(400Mz,CDCl3/CD3OD):0.84(6H,m)、1.11-1.84(10H,m)、2.05(1H,m)、2.15(2H,t)、3.09(2H,m)、3.32(2H,t)、4.12(1H,m)、4.38(1H,m)、5.15(2H,s)、6.61(2H,s)、6.84(1H,d),7.61(1H,d)、7.21(2H,d),7.50(2H,d)、7.61(2H,d),8.18(2H,d)、9.59(1H,br).
The synthesis of embodiment 9.mc-vc-PAB-MMAE
20mg mc-vc-PAB-PNP (1.5eq) and 3mgHOBT is added in 2mlDMF.It is stirred at room temperature in a moment, add 13mgMMAE, 0.5ml pyridine, 25ulDIEA.Reactant liquor stirring reaction 2d at room temperature.After completion of the reaction, reactant liquor is directly with preparing column purification, and lyophilizing after composition concentration needed for collection obtains about 10mg product, yield about 42%.m/z:1317.1(M+H)+.
The synthesis of embodiment 10.mc-vc-PAB-MMAF
According to the operation of embodiment 9, obtain mc-vc-PAB-MMAF about 12.5mg, yield 45.2%, m/z:1331.7(M+H)+.
The synthesis of embodiment 11 mc-vc-PAB-PBD
According to the operation of embodiment 9, obtain mc-vc-PAB-PBD about 9.5mg, yield 32.5%, m/z:1325.4(M+H)+.
The synthesis of embodiment 12 mc-vc-PAB-DOX
According to the operation of embodiment 9, obtain mc-vc-PAB-DOX about 11.2mg, yield 38.9%, m/z:1143.2(M+H)+.
The synthesis of embodiment 14 mc-vc-PAB-SN-38
After the dichloromethane dissolving that 100mg 10-O-Boc-SN-38 is dried with 10ml, add 25.6mg (1eq) DMAP, the dichloromethane solution (62mg triphosgene 2ml dichloromethane dissolves) of Deca triphosgene at 0 DEG C, drip and finish, continue at reaction 12h at 0 DEG C, dichloromethane is removed under reduced pressure, after the DMF dissolving being dried with 10ml, add 144mg mc-vc-PABOH, go to and 24h is stirred at room temperature, obtain mc-vc-PAB-SN-38 41mg through preparation liquid phase separation, two step yields total for 19.7%, m/z:992.1(M+H)+.
Embodiment 15, the expression and purification of 2C1 antibody
Using FreestyleTM293-F (Invitrogen) suspension cell expresses 2C1 antibody.Day before transfection, with 6 × 105/mL density, cell is inoculated in the 1L shaking flask of the complete medium of F17 containing 300mL (FreestyleTM F17 expresses culture medium, Gibco company), 37 DEG C, 5%CO2,120rpm cell culture table incubated overnight.Next day, carry out the transfection of antibody expressing plasmid, wherein plasmid with PEI:PEI ratio is 2:1.Transfection one day after, adds TN1 supplemented medium by 2.5% (v/v), supernatant is collected by centrifugation after continuing culture 4 days.
Collect the cell expression supernatant obtaining, through Protein A affinity column (Mabselect Sure LX, GE company), with 0.1M citric acid (pH3.0) eluting, the antibody of capture is adjusted to pH7.0 by 1/10 (v/v) with 1M Tris-HCl (pH9.0), pass through gel permeation chromatography post SEC (Superdex 200 again, GE company) remove the impurity such as polymer and endotoxin, antibodies buffer is replaced as PBS (pH7.4) simultaneously, collect UV280nm target peak sample and be concentrated into 5mg/ml through ultra-filtration centrifuge tube (30KD, Pall company).
The 2C1 antibody being obtained by the method, concentration is 5mg/ml, and target antibody monomer (POI%) is more than 98%, for follow-up test.
Embodiment 16, prepare, by being coupled 2C1 antibody and mc-vc-PAB-MMAE, the 2C1 antibody that 2C1-LC-V205C-mc-vc-PAB-MMAE TDC sample cell is expressed, through Mabselect Sure purification, add Tris solution to neutralize after low pH eluting at once, and change the Tris-HCl buffer that liquid is pH7.5.Compound mc-vc-PAB-MMAE, white powder, it is dissolved in standby in DMA.In order to remove the screen on mutation cysteine residues, need first to reduce antibody.According to 20 times of molecular proportions, the DTT aqueous solution of 1M is added in 2C1 antibody-solutions, mix latter 20 DEG C and react 4 hours.Response time to after the pH of sample is adjusted to 5.0, and remove DTT and the screen in sample by SP Sepharose F.F. cation-exchange chromatography.Subsequently according to 10 times of molecular proportions, DHAA solution is added in sample, 25 DEG C of lucifuges are reacted 3 hours, so that antibody interchain disulfide bond is reconnected.It is subsequently adding mc-vc-PAB-MMAE solution so that mc-vc-PAB-MMAE is coupled with antibody mutation cysteine, fully mix latter 25 DEG C and react 1 hour.Reaction removes, using SP Sepharose F.F. cation-exchange chromatography, the mc-vc-PAB-MMAE not being coupled upper antibody molecule after terminating, and obtains 2C1-LC-V205C-mc-vc-PAB-MMAE TDC sample.
Embodiment 17, prepare 2C1-LC-V205C-mc-vc-PAB-MMAF TDC sample by being coupled 2C1 antibody and mc-vc-PAB-MMAF, according to the operating procedure of embodiment 16, prepare 2C1-LC-V205C-mc-vc-PAB-MMAF TDC by being coupled 2C1 antibody and mc-vc-PAB-MMAF.
Embodiment 18, prepare 2C1-LC-V205C-mc-vc-PAB-PBD TDC sample by being coupled 2C1 antibody and mc-vc-PAB-PBD, according to the operating procedure of embodiment 16, prepare 2C1-LC-V205C-mc-vc-PAB-PBDTDC by being coupled 2C1 antibody and mc-vc-PAB-PBD.
Embodiment 19, prepare 2C1-LC-V205C-mc-vc-PAB-SN38 TDC sample by being coupled 2C1 antibody and mc-vc-PAB-SN38, according to the operating procedure of embodiment 16, prepare 2C1-LC-V205C-mc-vc-PAB-SN38 TDC by being coupled 2C1 antibody and mc-vc-PAB-SN38.
Embodiment 20, prepare 2C1-LC-V205C-mc-vc-PAB-Dox TDC sample by being coupled 2C1 antibody and mc-vc-PAB-Dox, according to the operating procedure of embodiment 16, prepare 2C1-LC-V205C-mc-vc-PAB-Dox TDC by being coupled 2C1 antibody and mc-vc-PAB-Dox.
Embodiment 21, prepare 2A1-mc-vc-PAB MMAE ADC sample by being coupled 2A1 antibody and mc-vc-PAB-Dox
The 2A1 antibody of cell expression, adds Tris solution to neutralize after Mabselect Sure purification, low pH eluting at once, and changes the Tris-HCl buffer that liquid is pH7.5.Mc-vc-PAB-MMAE, white powder, it is dissolved in standby in DMA.In order to open the interchain disulfide bond of antibody, need first to reduce antibody.According to 20 times of molecular proportions, the DTT aqueous solution of 1M is added in 2A1 antibody-solutions, mix latter 20 DEG C and react 4 hours.Response time to after the pH of sample is adjusted to 5.0, and the DTT in sample is removed by SP Sepharose F.F. cation-exchange chromatography.It is subsequently adding mc-vc-PAB-MMAE solution so that mc-vc-PAB-MMAE is coupled with the cysteine residues of antibody and the interchain disulfide bond opened, fully mix latter 25 DEG C and react 1 hour.Reaction removes, using SP Sepharose F.F. cation-exchange chromatography, the mc-vc-PAB-MMAE not being coupled upper antibody molecule after terminating.Obtain 2A1-mc-vc-PAB-MMAE ADC sample.
Embodiment 22, prepare 2A1-mc-vc-PAB MMAF ADC sample by being coupled 2A1 antibody and mc-vc-PAB-MMAF
According to the operating procedure of embodiment 21, prepare 2A1-mc-vc-PAB MMAF ADC by being coupled 2A1 antibody and mc-vc-PAB-MMAF.
Embodiment 23, prepare 2A1-mc-vc-PAB PBD ADC sample by being coupled 2A1 antibody and mc-vc-PAB-PBD
According to the operating procedure of embodiment 21, prepare 2A1-mc-vc-PAB-PBD ADC by being coupled 2A1 antibody and mc-vc-PAB-PBD.
Embodiment 24, prepare 2A1-mc-vc-PAB-SN38 ADC sample by being coupled 2A1 antibody and mc-vc-PAB-SN38
According to the operating procedure of embodiment 21, prepare 2A1-mc-vc-PAB-SN38 ADC by being coupled 2A1 antibody and mc-vc-PAB-SN38.
Embodiment 25, prepare 2A1-mc-vc-PAB Dox ADC sample by being coupled 2A1 antibody and mc-vc-PAB-Dox
According to the operating procedure of embodiment 21, prepare 2A1-mc-vc-PAB-Dox ADC by being coupled 2A1 antibody and mc-vc-PAB-Dox.
Embodiment 26, HIC-HPLC detection toxin are than antibody ratios DAR
Analyze TDC and ADC sample with high performance liquid chromatography hydrophobic chromatography method, according to corresponding calculated by peak area DAR.Concrete grammar is as follows：
Chromatographic column：HICBu‐NP5(5μm,4.6x 35mm)；
Mobile phase：A:2M ammonium sulfate, the phosphate buffer of 0.025M, pH7；B：The phosphate buffer of 0.025M, pH7；C：100% isopropanol；
Buffer A balance, buffer B and buffer C gradient elution, 25 DEG C, 214nm detects.DAR computing formula is：DAR=(0D area × 0+1D area × 1+2D area × 2)/(0D area+1D area+2D area).
Accompanying drawing 1, HIC-HPLC detection 2C1-LC-V205C-mc-vc-PAB-MMAE TDC DAR.1 it is calculated site-directed coupling DAR=1.9 with reference to the accompanying drawings, its compound homogeneity is fine.
Subordinate list 1,2C1-LC-V205C-mc-vc-PAB-payload TDC, 2A1-mc-vc-PAB-payload ADC coupling efficiency DAR table
Subordinate list 1 shows, by the cysteine engineered TDC compound coupling efficiency all higher (theoretical upper values are 2.0) carrying out site-directed coupling, DAR >=1.7, product homogeneity is considerably better than the ADC compound (DAR theoretical upper values are 8.0) carrying out the non-site-directed coupling of cysteine by antibody native interchain disulfide bond.
Embodiment 27, SEC-HPLC detection TDC assemble situation
By TDC sample preservation in 37 DEG C, analyze its gathering situation with SEC-HPLC respectively within the 0th, 7,21 days, concrete grammar is as follows：
Chromatographic column：TSKgel SuperSW mAb HR(7.8mm×30cm)
Mobile phase：0.1M sodium sulfate, 0.1M, the phosphate buffer of pH6.7.
25 DEG C, 280nm detects.
Accompanying drawing 2A, SEC-HPLC detection 2C1-LC-V205C-mc-vc-PAB-MMAE TDC assembles situation, and sample is deposited 3 weeks in 37 DEG C, assembles body burden and is substantially not changed in；
Accompanying drawing 2B, SEC-HPLC detection carries out the 2A1-mc-vc-PAB-MMAE ADC sample of the non-site-directed coupling of cysteine by antibody native interchain disulfide bond, and sample is coupled after terminating and detects immediately, there are about 30% aggregation, including a large amount of high molecular weight aggregates.
Subordinate list 2,2C1-LC-V205C-mc-vc-PAB-payload TDC, 2A1-mc-vc-PAB-payload ADC subject monomers content list
Subordinate list 2 shows, is substantially less than by the ADC compound subject monomers content that antibody native interchain disulfide bond carries out the non-site-directed coupling of cysteine and passes through the cysteine engineered TDC compound carrying out site-directed coupling.
Embodiment 28, TDC maintain skeleton antibody 2C1 and original antibodies 2A1 and contrast TDC, 2C1 and 2A1 relative affinity for EGFRvIII for the affinity indirect elisa method of EGFRvIII.Comprise the following steps that：Restructuring EGFRvIII-His*6 antigen wrapper sheet；Fishskin gelatin is closed；Dilute 2A1,2C1,2C1-LC-V205C-mc-vc-PAB-MMAE TDC, 2C1-LC-V205C-mc-vc-PAB-MMAF TDC, 2C1-LC-V205C-mc-vc-PAB- PBD TDC, 2C1-LC-V205C-mc-vc-PAB-SN38TDC, 2C1-LC-V205C-mc-vc-PAB-DOX TDC respectively, maximum concentration 50ug/ml, 4 times of gradient dilutions, totally 11 concentration；Two anti-incubations of HRP labelling；TMB develops the color, and absorbs at detection 450nm.Testing result with A450 to concentration map, as shown in figure 3, the TDC after 2C1, coupling maintains the affinity similar to 2A1, EC50 value very close to；Illustrate that the site-directed coupling of the rite-directed mutagenesises of light chain V205C and 2C1 and mc-vc-PAB-payload on 2A1 does not affect the affinity of itself and EGFRvIII antigen.
Accompanying drawing 3A, 2C1-LC-V205C-mc-vc-PAB-MMAE TDC and 2C1 maintains 2A1 and the affinity of antigen EGFRvIII.
Accompanying drawing 3B, 2C1-LC-V205C-mc-vc-PAB-MMAE TDC, 2C1-LC-V205C-mc-vc-PAB-MMAF TDC, 2C1-LC-V205C-mc-vc-PAB-PBD TDC, 2C1-LC-V205C-mc-vc-PAB-SN38 TDC, 2C1-LC-V205C-mc-vc-PAB-DOX TDC maintains 2A1 and the affinity of antigen EGFRvIII, and 2C1-LC-V205C-mc-vc-PAB-payload can carry out site-directed coupling with multiple small molecular cell toxin and keep Ag-Ab affinity.
Embodiment 29, cytotoxicity Composition analyzed
Measure the cellular cytoxicity activity of TDC and ADC by following experiments process：In the tumor cell culture base of the people TDC and ADC being added separately to EGFR overexpression or EGFRVIII expression, cell culture measured cell survival rate after 72 hours.Experiment in vitro based on cell is used for measuring the apoptosis of cell survival rate, cytotoxicity and TDC of the present invention induction.
Measure the pharmacy in vitro of Antibody-toxin conjugate by cell proliferation test.CellTiterAqueousOne Solution Cell Proliferation Assay is commercially available (Promega Corp., Madison, WI).CellTiterAQueous One Solution Cell Proliferation Assay (a) is a kind of detectable of the living cells quantity being detected with colorimetry in cell proliferation and cytotoxicity experiment.This reagent contains a new tetrazole compound [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt；MTS] and a kind of electron coupling agent (phenazine ethosulfate；PES).PES has enhanced chemical stability, and this makes it can be mixed to form stable solution with MTS.This convenient " single solution " pattern, is in first generation CellTiterImprovement on the basis of AQueous Assay, CellTiterUsed in AQueous Assay, electron coupling agent PMS and MTS solution is provided separately from.MTS (Owen ' s reagent) is become a kind of coloured first product by cell biological reduction, can be directly dissolved in (following formula) in culture medium.This conversion is likely to complete in the presence of NADPH or NADH that the dehydrogenase in the cell that metabolism enlivens produces.During detection, only need to be by a small amount of CellTiterAQueous One Solution Reagent is directly added in the culture medium of cultivation plate hole, is incubated 14 hours, then reads the absorbance of 490nm with microplate reader.
The amount of first product detecting at 490nm is directly proportional to the viable count in culture.Because the first product of MTS is solvable in tissue culture medium (TCM), CellTiterAQueous One Solution Assay operating procedure compared with MTT or INT method is less.
The research system being detected as pharmacy in vitro using A431 (EGFR overexpressing cell) and U87-EGFRVIII (EGFR mutant stable cell lines) in the present invention.In 96 orifice plates, carry out plating cells 6000/ hole, after 24 hours, carry out antibody dosing.Drug level to A431 is 2uM-3.8nM, and twice dilutes, and the Drug level to U87-EGFRVIII is 2nM-0.4pM.MTS detection cytoactive after processing 72 hours.
Accompanying drawing 4A-4C, 2C1-LC-V205C-mc-vc-PAB-MMAE TDC, 2C1-LC-V205C-mc-vc-PAB-MMAF TDC, 2C1-LC-V205C-mc-vc-PAB-PBD TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC, the IC50 testing result of 2A1-mc-vc-PAB-MMAF ADC, the 2A1-mc-vc-PAB-PBD ADC application on human skin squamous cell carcinoma A431 to EGFRwt overexpression.TDC cellular cytoxicity activity is better than ADC.
Accompanying drawing 4D-4H、2C1-LC-V205C-mc-vc-PAB-MMAE TDC、2C1-LC-V205C-mc-vc-PAB-MMAF TDC、2C1-LC-V205C-mc-vc-PAB-PBD TDC、2C1-LC-V205C-mc-vc-PAB-SN38 TDC、2C1-LC-V205C-mc-vc-PAB-Dox TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC、2A1-mc-vc-PAB-MMAF ADC、2A1-mc-vc-PAB-PBD ADC、2A1-mc-vc-PAB-SN38ADC、The IC50 testing result of the U87-EGFRvIII to expression EGFRvIII for the 2A1-mc-vc-PAB-Dox ADC.TDC cellular cytoxicity activity is better than ADC.
Subordinate list 3, TDC, ADC are to EGFRwt overexpressing cell system A431 and EGFRvIII expression stability strain U87-EGFRVIII cytotoxicity IC50Testing result.
Subordinate list 3 result shows，2C1-LC-V205C-mc-vc-PAB-MMAE TDC、2C1-LC-V205C-mc-vc-PAB-MMAF TDC、2C1-LC-V205C-mc-vc-PAB-PBD TDC、2C1-LC-V205C-mc-vc-PAB-SN38 TDC、2C1-LC-V205C-mc-vc-PAB-Dox TDC is better than its corresponding 2A1-mc-vc-PAB-MMAE ADC to EGFRwt overexpressing cell system A431 and EGFRvIII expression stability strain U87-EGFRVIII cellular cytoxicity activity、2A1-mc-vc-PAB-MMAF ADC、2A1-mc-vc-PAB-PBD ADC、2A1-mc-vc-PAB-SN38ADC、2A1-mc-vc-PAB-Dox ADC.Embodiment 30, tumor-bearing mice Composition analyzed
U87-EGFRvIII bearing mouse model is established, to evaluate the internal drug effect of TDC and ADC coupling drug in the present invention.It is subcutaneously injected into the BALB/c nude mice both sides in 4～6 weeks Mus ages with 3 × 106 U87-EGFRvIII cells，Treat that mouse tumor mean size grows to 65～88mm3，Random packet，Every group 5，At the 0th day，2C1-LC-V205C-mc-vc-PAB-MMAE、2C1-LC-V205C-mc-vc-PAB-MMAF、2C1-LC-V205C-mc-vc-PAB-PBD、2A1-mc-vc-PAB-MMAE、2A1-mc-vc-PAB-MMAF、2A1-mc-vc-PAB-PBD carries out single intravenous injection (accompanying drawing 5A) with 1mg/kg dosage respectively，2C1-LC-V205C-mc-vc-PAB-SN38、2C1-LC-V205C-mc-vc-PAB-DOX、2A1-mc-vc-PAB-SN38、2A1-mc-vc-PAB-DOX is with 6mg/kg dosed administration (accompanying drawing 5B).Data display is tumor average volume ± SE during measurement.
Accompanying drawing 5A, 2C1-LC-V205C-mc-vc-PAB-MMAE, 2C1-LC-V205C-mc-vc-PAB-MMAF, 2C1-LC-V205C-mc-vc-PAB-PBD, 2A1-mc-vc-PAB-MMAE, 2A1-mc-vc-PAB-MMAF, 2A1-mc-vc--PAB-PBD, at the 0th day, carry out single intravenous injection with 1mg/kg dosage respectively.TDC group (2C1-LC-V205C-mc-vc-PAB-MMAE, 2C1-LC-V205C-mc-vc-PAB-MMAF, 2C1-LC-V205C-mc-vc-PAB-PBD) tumor killing effect is significantly better than ADC group (2A1-mc-vc-PAB-MMAE, 2A1-mc-vc-PAB- MMAF, 2A1-mc-vc-PAB-PBD).
Accompanying drawing 5B, 2C1-LC-V205C-mc-vc-PAB-SN38,2C1-LC-V205C-mc-vc-PAB-DOX, 2A1-mc-vc-PAB-SN38,2A1-mc-vc-PAB-DOX, at the 0th day, carry out single intravenous injection with 6mg/kg dosage respectively.TDC group (2C1-LC-V205C-mc-vc-PAB-SN38,2C1-LC-V205C-mc-vc-PAB-DOX) tumor killing effect is significantly better than ADC group (2A1-mc-vc-PAB-SN38,2A1-mc-vc-PAB-DOX).
Embodiment 31, rat toxicity detection
The present invention is the cysteine engineered Antibody-toxin conjugate toleration of assessment，Adopt normal Sprague-Dawley rat with Single-dose intravenous injection TDC or ADC (first day)，Wherein 2C1-LC-V205C-mc-vc-PAB-MMAE、2C1-LC-V205C-mc-vc-PAB-MMAF、2C1-LC-V205C-mc-vc-PAB-PBD、2A1-mc-vc-PAB-MMAE、2A1-mc-vc-PAB-MMAF、2A1-mc-vc-PAB-PBD dosage is 50mg/kg，2C1-LC-V205C-mc-vc-PAB-SN38、2C1-LC-V205C-mc-vc-PAB-DOX、2A1-mc-vc-PAB-SN38、2A1-mc-vc-PAB-DOX dosage is 100mg/kg.5th day and the 12nd day collection blood sample after administration, carries out neutrophil count (accompanying drawing 6A) and AST detection (accompanying drawing 6B), and monitoring body weight change situation (accompanying drawing 6C) daily.After sampling in 12nd day, put to death rat.
Accompanying drawing 6A, TDC and ADC rat toxicity detects, neutrophil cell situation of change.At the 12nd day, ADC group neutrophil cell number was significantly higher than comparison and TDC group, and display ADC group toxicity is noticeably greater than TDC group.
Accompanying drawing 6B, TDC and ADC rat toxicity detects, the horizontal situation of change of AST in liver.At the 12nd day, in ADC group liver, AST level was significantly higher than comparison and TDC group, and ADC group shows obvious hepatic injury, and TDC group safety is considerably better than ADC group.
Accompanying drawing 6C, TDC and ADC rat toxicity detects, body weight change situation.At first 5 days, the equal continuous decrease of ADC group body weight, gradually recovers for 5 days；TDC group and matched group significant difference, show that TDC group safety is considerably better than ADC group.
The present invention is not limited to by the scope of the specific embodiments disclosing in embodiment, and these embodiments are used for illustrating several aspects of the present invention, and functionally equivalent any embodiment belongs to the scope of the present invention.In fact, in addition to shown and described herein, the various modifications of the present invention are also obvious to those skilled in the art and belong to this paper scope of the following claims.

Claims (11)

1. cysteine engineered Antibody-toxin conjugate, has below formula：2C1-LC-V205C-mc-vc-PAB-payload.
2. the cysteine engineered Antibody-toxin conjugate described in claim 1 it is characterised in that：Wherein 2C1 is 205 L-Valine of target antibody light chain（V）It transform cysteine as（C）Antibody afterwards, the aminoacid sequence of 2C1 weight chain variable district is SEQ ID NO:1, light-chain amino acid sequence is SEQ ID NO:12.
3. the cysteine engineered Antibody-toxin conjugate described in claim 2 it is characterised in that：Comprise a ripe weight chain variable district, its aminoacid sequence at least 95% and SEQ ID NO:1 is identical.
4. the cysteine engineered Antibody-toxin conjugate described in claim 2 it is characterised in that：Comprise a ripe light chain, its aminoacid sequence at least 95% and SEQ ID NO:12 is identical.
5. the cysteine engineered Antibody-toxin conjugate described in claim 1 it is characterised in that：Wherein mc-vc-PAB-OH connexon is：
.
6. the cysteine engineered Antibody-toxin conjugate described in claim 1 it is characterised in that：Wherein Payload is small molecule high activity cytotoxin, including but not limited to MMAE, MMAF, PBD, SN-38 and Dox；
MMAE, MMAF, PBD, SN-38 and Dox molecular formula is respectively：
.
7. the cysteine engineered Antibody-toxin conjugate described in claim 1 it is characterised in that：Wherein toxin compares antibody ratios（DAR）For 1.6-2.0.
8. the cysteine engineered Antibody-toxin conjugate described in claim 2 it is characterised in that：The site amino sequence that mc-vc-PAB connexon carries out site-directed coupling with antibody is：GLSSP C TKSFN, wherein, C Cysteine for the 205 L-Valine transformations of target antibody light chain.
9. the cysteine engineered Antibody-toxin conjugate described in claim 2 it is characterised in that：2C1 antibody is selected from IgG1, IgG2, IgG3, IgG4, Fab and F (ab)2.
10. the cysteine engineered Antibody-toxin conjugate described in claim 1 it is characterised in that：Its indication is the tumor of expression EGFRvIII and overexpression EGFRwt, including brain colloid carcinoma, nonsmall-cell lung cancer, head and neck cancer, colorectal cancer, bladder cancer, colorectal cancer, renal carcinoma, carcinoma of prostate, cancer of pancreas, breast carcinoma etc..
Cysteine engineered Antibody-toxin conjugate described in 11. claim 1 it is characterised in that：Its main site-directed coupling step is：First by DTT also original antibody, release the shielding on the cysteine residues of transformation on antibody, and DTT and screen are removed by cation-exchange chromatography；Then using DHAA oxidised antibody, the interchain disulfide bond of antibody is made to reconnect；The cysteine residues being eventually adding mc-vc-PAB-payload with antibody transformation are coupled, and remove the mc-vc-PAB-payload not being coupled upper antibody molecule by cation-exchange chromatography.
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