CN106866822A - Cysteine engineered antibody toxin conjugate - Google Patents
Cysteine engineered antibody toxin conjugate Download PDFInfo
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Abstract
205 valines of light chain by target antibody of the invention（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 connexons carry out site-directed coupling, form the excellent cysteine engineered antibody toxin conjugate of homogeneity, its toxin compares antibody ratios（DAR）It is 1.6 2.0.This antibody toxin conjugate has formula：1C5‑LC‑V205C‑mc‑vc‑PAB‑payload.Meanwhile, preparation, purification process invention further discloses this TDC medicine, in the treatment use of the tumour of overexpression EGFRwt.
Description
Technical field
The present invention relates to a kind of compound and its production and use, a more particularly to class is cysteine engineered to be resisted
Body-toxin conjugated thing（TDC）And its production and use.
Technical background：
EGF-R ELISA EGFR（Epidermal Growth Factor Receptor）It is a kind of glycoprotein, belongs to
One kind of ErbB receptor family, the family includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3)
With Her 4 (ErbB-4).EGFR is epidermal growth factor（EGF）Cell breeds the acceptor with signal transduction, and insertion cell membrane divides
Son amount 170KDa, by being activated with ligand binding.After activation, EGFR is converted into dimer by monomer.EGFR is also possible to and ErbB
Other members of receptor family are polymerized to activate, such as ErbB2/Her2/neu.
The usual low amounts of EGFR is expressed in various normal tissue cells, including skin, liver etc., phase is acted on normal physiological
Close.
EGFR overexpression is related with development to the generation of kinds of tumors, including head and neck cancer, carcinoma of urinary bladder, oophoroma, non-small thin
Born of the same parents' lung cancer, colorectal cancer, glioma, kidney, prostate cancer, cancer of pancreas, breast cancer（Atalay et al., 2003;
Herbst and Shin, 2002）.
Antibody-toxin conjugate（Antibody drug conjugate, ADC）It is the hot fields of targeted therapy,
In the U.S., two medicines Adcetris and Kadcyla of granted listing show good clinical efficacy, and have more than 50
ADC medicines are carrying out clinical stage research.
The brand-new cysteine engineered Antibody-toxin conjugate that this patent is disclosed（TDC）Compared to current clinical research
The ADC of the non-site-directed coupling technology that the stage mainly uses has the advantages of medicine homogeneity is good, and toxicity is smaller, preclinical study knot
Fruit shows that its antitumor activity is significantly better than non-site-directed coupling ADC.
The content of the invention：
The cysteine engineered Antibody-toxin conjugate of compound of the invention（TDC）With formula：1C5-LC-V205C-mc-
vc-PAB-payload.Wherein 1C5 is 205 valines of parental antibody 1A5 light chains（V）It transform cysteine as（C）Afterwards anti-
Body.The amino acid sequence of the weight chain variable district of 1C5 is SEQ ID NO:1,1C5 light-chain amino acid sequence is SEQ ID NO:
12.The amino acid sequence of the parental antibody 1A5 weight chain variable districts of 1C5 is SEQ ID NO:1,1A5 light-chain amino acid sequence is
SEQ ID NO:14.1C5 maintains the ability of its parental antibody 1A5 and antigen binding（Affinity）, the wild type that antigen is behaved
EGF-R ELISA（EGFRwt）.1C5 is by its light chain 205 cysteine sulfydryls and mc-vc-PAB- of transformation
Payload carries out Antibody-toxin conjugate（TDC）Site-directed coupling, toxin is 1.6-2.0 than antibody ratios DAR.Of the inventionization
The cysteine engineered Antibody-toxin conjugate of compound（TDC）Medicine is mainly used in treating the tumour of overexpression EGFRwt, including
Head and neck cancer, colorectal cancer, brain colloid carcinoma, cancer of pancreas, non-small cell lung cancer, carcinoma of urinary bladder, colorectal cancer, kidney, prostate cancer, breast
Gland cancer etc..
Brief description of the drawings
Fig. 1 is HIC-HPLC detection 1C5-LC-V205C-mc-vc-PAB-MMAE TDC DAR figures.
Fig. 2 is SEC-HPLC detection 1C5-LC-V205C-mc-vc-PAB-MMAE TDC aggregation situation maps.
Fig. 3 is the 1A5- that SEC-HPLC detections carry out the non-site-directed coupling of cysteine by antibody native interchain disulfide bond
Mc-vc-PAB-MMAE ADC sample drawings.
Fig. 4 is affine with antigen EGFRwt for 1C5-LC-V205C-mc-vc-PAB-MMAE TDC and 1C5 maintains 1A5
Try hard to.
Fig. 5 be 1C5-LC-V205C-mc-vc-PAB-MMAE TDC, 1C5-LC-V205C-mc-vc-PAB-MMAF TDC,
1C5-LC-V205C-mc-vc-PAB-PBD TDC、1C5-LC-V205C-mc-vc-PAB-SN38 TDC、1C5-LC-V205C-
Mc-vc-PAB-DOX TDC maintain 1A5 to try hard to the affine of antigen EGFRwt.
Fig. 6 is 1C5-LC-V205C-mc-vc-PAB-MMAE TDC and its corresponding 1A5-mc-vc-PAB-MMAE ADC
To the IC50 testing result figures of the A431 cells of overexpression EGFR.
Fig. 7 is 1C5-LC-V205C-mc-vc- PAB-MMAF TDC and its corresponding 1A5-mc-vc-PAB-MMAF ADC
To the IC50 testing result figures of the A431 cells of overexpression EGFR.
Fig. 8 is 1C5-LC-V205C-mc-vc-PAB-PBD TDC and its corresponding 1A5-mc-vc-PAB-PBD ADC couple
The IC50 testing result figures of the A431 cells of overexpression EGFR.
Fig. 9 is 1C5-LC-V205C-mc-vc-PAB-SN38 TDC and its corresponding 1A5-mc-vc-PAB-SN38 ADC
To the IC50 testing result figures of the A431 cells of overexpression EGFR.
Figure 10 is 1C5-LC-V205C-mc-vc-PAB-Dox TDC and its corresponding 1A5-mc-vc-PAB-Dox ADC couple
The IC50 testing result figures of the A431 cells of overexpression EGFR.
Specific embodiment
The synthesis of the mc of embodiment 1
6-aminocaprolc acid 3.9g (0.03mol are added in 30ml glacial acetic acid）With the maleic anhydride 3.5g (0.036mol of 1.2eq）.
Reaction solution is in 120 DEG C of 4~6h of stirring reaction.After completion of the reaction, stop heating, naturally cool to room temperature.60 DEG C concentrated under reduced pressure to remove
Remove most of acetic acid.Gained brown color viscous fluid is poured into water, and adds the extraction of ethyl acetate 20ml × 3, merges organic layer.Have
Machine layer uses water, saturated common salt water washing, anhydrous sodium sulfate drying, filtering, filtrate decompression to be concentrated to give pale tan oil successively,
The stirring of 50ml water is added, there is off-white powder to separate out, filtered, 50 DEG C of target product 5.08g 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 the Mc-OSu of embodiment 2
4.7g (22mmol) MC and 25g are added in 50ml acetonitriles under nitrogen protection（22mmol）HOSu.Separately take 4.5g
（22mmol）DCC is dissolved in 25ml acetonitriles, keeps interior temperature at 0 DEG C or so, is slowly dropped into reaction solution.Reaction solution is in 0 DEG C
Room temperature reaction is overnight again after 2 hours for reaction.Filtering, filter cake is washed with acetonitrile 10ml × 3, and filtrate decompression is concentrated to dryness.Gained oil
Shape thing obtains the g of light tan solid 6.4, yield 95% in reduced pressure at room temperature 6h.（Do not purify and 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 the Fmoc-Val-OSu of embodiment 3
Fmoc-Val 10g and HOSu 3.4g are added in 100mlTHF.Separately take DCC6g to be dissolved in 50ml acetonitriles, keep interior temperature
At 0 DEG C or so, it is slowly dropped into reaction solution.Reaction solution is stirred at room temperature reaction 24 hours.Filtering, filter cake is washed with THF,
Filtrate decompression is concentrated to give clear oil thing.Grease is not purified directly to cast single step reaction.m/z：437.4 [M+H]+.
The synthesis of the Fmoc-vc of embodiment 4.
Cit 4.0g are added in 20mlTHF（1.05eq）With the aqueous solution 60ml of sodium acid carbonate（NaHCO3 2g,1.05eq）.
Separately take 22.35mmolFmoc-Val-OSu to be dissolved in 60mlDME, then be added into reaction solution.Reaction solution is stirred at room temperature
Reaction 24 hours.After completion of the reaction, to 15% aqueous citric acid solution 110ml is added in system, then it is extracted twice with EA again, is closed
And organic layer, it is concentrated under reduced pressure to give white solid.And washed to adding methyl tertiary butyl ether(MTBE) 100ml to stir 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, m)、1.35 ~ 1.65 (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 the Fmoc-vc-PABOH of embodiment 5.
DCM/MeOH=2/1 mixed solvent 60ml are added in reaction bulb, Fmoc-vc 2g are added（4.2mmol）And PABOH
1.04g（2eq）, EEDQ 2.0g are added behind stirring and dissolving part（2eq）.Lucifuge stirring is anti-at ambient temperature for reaction system
Answer 2.0d.After completion of the reaction, 40 DEG C are concentrated under reduced pressure to give white solid.White solid is collected, methyl tertiary butyl ether(MTBE) 100ml is added
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, d)、 4.05~-4.66 (2H, m)、4.55 (2H,
d)、5.21 (1H, t)、5.51 (2H, br)、6.11(1H, t)、7.09 -8.10 (12H, m)、 8.21 (1H, d)、
10.51(1H, br)。
The synthesis of embodiment 6.vc-PABOH
Fmoc-vc-PABOH 490mg are added in 10mlNMP（0.815mmol）Stirring and dissolving, adds diethylamine 2ml.In
Stirring reaction 24h at room temperature.After completion of the reaction, it is concentrated under reduced pressure in 40 DEG C, 20mlDCM stirring and crystallizings are added in gained grease,
Filtering, 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
Vc-PABOH 205mg are added in 10mlNMP（0.54 mmol）With MC-OSu 184mg（1.1eq）, finish in room temperature
Lower stirring reaction 24h.Reaction is finished, concentrated under reduced pressure in 40 DEG C, and the stirring analysis of 20ml methyl tertiary butyl ether(MTBE)s is added in gained grease
It is brilliant.Filtering, filter cake is washed with methyl tertiary butyl ether(MTBE), obtains 310mg products, 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) , 8.1 7 (1H, d)、10.21 (1H, br)。
The synthesis of the mc-vc-PAB-PNP of embodiment 8.
Mc-vc-PABOH 168.6mg are taken under nitrogen protection（0.294mmol）It is dissolved in 5ml anhydrous pyridines, reaction system is cold
But to 0 DEG C or so.Separately take PNP179mg（3eq）It is dissolved in 5mlDCM, then is slowly added into reaction system.And in 0 DEG C
Left and right removes ice bath after keeping 10min, and 3h is reacted then at being stirred at room temperature.Reaction is finished, and adds 70mlEA and the lemons of 100ml 15%
Aqueous acid, divides and takes organic layer.Organic layer uses citric acid, water, saturated common salt water washing, then anhydrous sodium sulfate drying, mistake successively
Filter, filtrate decompression is concentrated to dryness and obtains light yellow oil, adds methyl tertiary butyl ether(MTBE) crystallization to obtain off-white powder 86mg, receives
Rate 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 the mc-vc-PAB-MMAE of embodiment 9.
20mg mc-vc-PAB-PNP (1.5eq are added in 2mlDMF）And 3mgHOBT.It is stirred at room temperature in a moment, adds
13mgMMAE, 0.5ml pyridine, 25ulDIEA.Reaction solution stirring reaction 2d at room temperature.After completion of the reaction, reaction solution is directly used
Post purifying is prepared, is freezed after composition concentration needed for collecting, obtain about 10mg products, yield about 42%.m/z: 1317.1 (M+H)
+。
The synthesis of the mc-vc-PAB-MMAF of embodiment 10.
According to the operation of embodiment 9, mc-vc-PAB-MMAF about 12.5mg, yield 45.2%, m/z are obtained: 1331.7(M+H)+
The synthesis of the mc-vc-PAB-PBD of embodiment 11
According to the operation of embodiment 9, mc-vc-PAB-PBD about 9.5mg, yield 32.5%, m/z are obtained: 1325.4 (M+H)+.
The synthesis of the mc-vc-PAB-DOX of embodiment 12
According to the operation of embodiment 9, mc-vc-PAB-DOX about 11.2mg, yield 38.9%, m/z are obtained: 1143.2 (M+H)+.
The synthesis of the mc-vc-PAB-SN-38 of embodiment 14
After the 10-O-Boc-SN-38 that 100mg is bought is dissolved with the dry dichloromethane of 10ml, 25.6mg is added（1eq）
DMAP, in the dichloromethane solution that triphosgene is added dropwise at 0 DEG C（62mg triphosgenes 2ml dichloromethane dissolves）, drip and finish, continue at
12h is reacted at 0 DEG C, dichloromethane is removed under reduced pressure, after being dissolved with the dry DMF of 10ml, add 144mg mc-vc-PABOH, turned
To 24h is stirred at room temperature, mc-vc-PAB-SN-38 41mg are obtained by preparing liquid phase separation, two step yields it is total be 19.7%, m/
z: 992.1(M+H)+ 。
The expression and purification of embodiment 15,1C5 antibody
Use FreestyleTM293-F（Invitrogen）Suspension cell expresses 1C5 antibody.The day before transfection, with 6 × 105
Cell is inoculated in the complete mediums of F17 containing 300mL by individual/mL density（FreestyleTM F17 express culture medium, and Gibco is public
Department）1L shaking flasks in, 37 DEG C, 5%CO2,120rpm cell culture table incubated overnight.Next day, antibody expression matter is carried out with PEI
The transfection of grain, wherein plasmid:PEI ratios are 2:1.Transfect one day after, by 2.5%（v/v）TN1 supplemented mediums are added, continues to train
Supernatant is collected by centrifugation after supporting 4 days.
The cell expression supernatant that collection is obtained, through Protein A affinity columns（Mabselect Sure LX, GE are public
Department）, with 0.1M citric acids（pH3.0）Wash-out, the antibody 1M Tris-HCl of capture（pH9.0）By 1/10（v/v）Regulation to
PH7.0, then by gel permeation chromatography post SEC（Superdex 200, GE company）The impurity such as removal polymer and endotoxin, together
When antibodies buffer is replaced as PBS（pH7.4）, UV280nm target peak samples are collected through ultra-filtration centrifuge tube（30KD, Pall are public
Department）It is concentrated into 5mg/ml.
The 1C5 antibody obtained by the method, concentration is 5mg/ml, target antibody monomer（POI%）More than 98%, after being used for
Continuous experiment.
Embodiment 16, prepare 1C5-LC-V205C-mc-vc-PAB- by being coupled 1C5 antibody and mc-vc-PAB-MMAE
MMAE TDC samples
The 1C5 antibody of cell expression, purifies by Mabselect Sure, adds Tris solution to neutralize at once after low pH wash-outs,
And change the Tris-HCl buffer solutions that liquid is pH7.5.Compound mc-vc-PAB-MMAE, white powder is dissolved in standby in DMA
With.In order to remove the screen on mutation cysteine residues, it is necessary to first reduce antibody.According to 20 times of molecular proportions by 1M's
The DTT aqueous solution is added in 1C5 antibody-solutions, and 20 DEG C are reacted 4 hours after mixing.Reaction time after by the pH of sample adjust to
5.0, and the DTT and screen in sample are removed by SP Sepharose F.F. cation-exchange chromatographies.Then according to 10
Times molecular proportion adds DHAA solution in sample, and 25 DEG C of lucifuges are reacted 3 hours, reconnects antibody interchain disulfide bond.Then
Add mc-vc-PAB-MMAE solution so that mc-vc-PAB-MMAE is coupled with antibody mutation cysteine, fully mix
25 DEG C are reacted 1 hour afterwards.Reaction is removed using SP Sepharose F.F. cation-exchange chromatographies after terminating and is not coupled upper antibody
The mc-vc-PAB-MMAE of molecule, obtains 1C5-LC-V205C-mc-vc-PAB-MMAE TDC samples.
Embodiment 17, prepare 1C5-LC-V205C-mc-vc-PAB- by being coupled 1C5 antibody and mc-vc-PAB-MMAF
MMAF TDC samples
According to the operating procedure of embodiment 16,1C5-LC-V205C- is prepared by being coupled 1C5 antibody and mc-vc-PAB-MMAF
mc-vc-PAB-MMAF TDC。
Embodiment 18, prepare 1C5-LC-V205C-mc-vc-PAB-PBD by being coupled 1C5 antibody and mc-vc-PAB-PBD
TDC samples
According to the operating procedure of embodiment 16,1C5-LC-V205C-mc- is prepared by being coupled 1C5 antibody and mc-vc-PAB-PBD
vc-PAB-PBDTDC。
Embodiment 19, prepare 1C5-LC-V205C-mc-vc-PAB- by being coupled 1C5 antibody and mc-vc-PAB-SN38
SN38 TDC samples
According to the operating procedure of embodiment 16,1C5-LC-V205C- is prepared by being coupled 1C5 antibody and mc-vc-PAB-SN38
mc-vc-PAB-SN38 TDC。
Embodiment 20, prepare 1C5-LC-V205C-mc-vc-PAB-Dox by being coupled 1C5 antibody and mc-vc-PAB-Dox
TDC samples
According to the operating procedure of embodiment 16,1C5-LC-V205C-mc- is prepared by being coupled 1C5 antibody and mc-vc-PAB-Dox
vc-PAB-Dox TDC。
Embodiment 21, prepare 1A5-mc-vc-PAB-MMAE ADC samples by being coupled 1A5 antibody and mc-vc-PAB-Dox
The 1A5 antibody of cell expression, purifies by Mabselect Sure, adds Tris solution to neutralize at once after low pH wash-outs,
And change the Tris-HCl buffer solutions that liquid is pH7.5.Mc-vc-PAB-MMAE, white powder is dissolved in standby in DMA.For
The interchain disulfide bond of antibody is opened, it is necessary to first reduce antibody.The DTT aqueous solution of 1M is added according to 20 times of molecular proportions
In 1A5 antibody-solutions, 20 DEG C are reacted 4 hours after mixing.Reaction time is adjusted to 5.0 the pH of sample after, and by SP
DTT in Sepharose F.F. cation-exchange chromatographies removal sample.It is subsequently adding mc-vc-PAB-MMAE solution so that
Mc-vc-PAB-MMAE and antibody are coupled with the cysteine residues of the interchain disulfide bond opened, after fully mixing 25 DEG C it is anti-
Answer 1 hour.Reaction is removed using SP Sepharose F.F. cation-exchange chromatographies after terminating and is not coupled upper antibody molecule
mc-vc-PAB-MMAE.Obtain 1A5-mc-vc-PAB-MMAE ADC samples.
Embodiment 22, prepare 1A5-mc-vc-PAB-MMAF ADC samples by being coupled 1A5 antibody and mc-vc-PAB-MMAF
Product.
According to the operating procedure of embodiment 21,1A5-mc-vc- is prepared by being coupled 1A5 antibody and mc-vc-PAB-MMAF
PAB –MMAF ADC。
Embodiment 23, prepare 1A5-mc-vc-PAB-PBD ADC samples by being coupled 1A5 antibody and mc-vc-PAB-PBD.
According to the operating procedure of embodiment 21,1A5-mc-vc- is prepared by being coupled 1A5 antibody and mc-vc-PAB-PBD
PAB-PBD ADC。
Embodiment 24, prepare 1A5-mc-vc-PAB-SN38 ADC samples by being coupled 1A5 antibody and mc-vc-PAB-SN38
Product.
According to the operating procedure of embodiment 21,1A5-mc-vc- is prepared by being coupled 1A5 antibody and mc-vc-PAB-SN38
PAB-SN38 ADC。
Embodiment 25, prepare 1A5-mc-vc-PAB-Dox ADC samples by being coupled 1A5 antibody and mc-vc-PAB-Dox.
According to the operating procedure of embodiment 21,1A5-mc-vc- is prepared by being coupled 1A5 antibody and mc-vc-PAB-Dox
PAB-Dox ADC。
Embodiment 26, HIC-HPLC detects toxin than antibody ratios DAR
TDC and ADC samples are analyzed with high performance liquid chromatography hydrophobic chromatography method, according to correspondence calculated by peak area DAR.Specific method
It is as follows：
Chromatographic column：Proteomix® HICBu‐NP5 (5 μm, 4.6 x 35 mm)；
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 solution C gradient elutions, 25 DEG C, 214nm detections.DAR computing formula are：DAR=（0D
Area × 0+1D areas × 1+2D area × 2）/（0D area+1D area+2D areas）.
1 site-directed coupling DAR=1.81 is calculated with reference to the accompanying drawings, its compound homogeneity is fine.
Subordinate list 1,1C5-LC-V205C-mc-vc-PAB-payload TDC, 1A5-mc-vc-PAB-payload ADC idols
Connection efficiency DAR tables
Subordinate list 1 shows, higher by the cysteine engineered TDC compound coupling efficiencies for carrying out site-directed coupling（Theoretical highest
Be worth is 2.0）, DAR >=1.7, product homogeneity is considerably better than carries out the non-fixed point of cysteine by antibody native interchain disulfide bond
The ADC compounds of coupling（DAR theoretical upper values are 8.0）.
Embodiment 27, SEC-HPLC detection TDC aggregation situations
By TDC Sample storages in 37 DEG C, its aggregation situation is analyzed with SEC-HPLC respectively within the 0th, 14 days, specific method is as follows：
Chromatographic column：TSKgel SuperSW mAb HR（7.8mm×30cm）
Mobile phase：0.1M sodium sulphate, 0.1M, the phosphate buffer of pH6.7.
25 DEG C, 280nm detections
Accompanying drawing 2, SEC-HPLC detection 1C5-LC-V205C-mc-vc-PAB-MMAE TDC aggregation situations, sample deposits 2 in 37 DEG C
Week, aggregation content is not changed in substantially；
The detection of accompanying drawing 3, SEC-HPLC carries out the 1A5-mc- of the non-site-directed coupling of cysteine by antibody native interchain disulfide bond
Vc-PAB-MMAE ADC samples, sample is coupled after terminating and detects immediately, 48% aggregation is there are about, including a large amount of macromolecules
Amount aggregation.
Subordinate list 2,1C5-LC-V205C-mc-vc-PAB-payload TDC, 1A5-mc-vc-PAB-payload ADC mesh
Mark content of monomer list
Subordinate list 2 shown, the ADC compound subject monomers of the non-site-directed coupling of cysteine are carried out by antibody native interchain disulfide bond
Content is substantially less than by the cysteine engineered TDC compounds for carrying out site-directed coupling.
Embodiment 28, TDC maintains the affinity of skeleton antibody 1C5 and original antibodies 1A5 for EGFRvIII
The relative affinity of TDC, 1C5 and 1A5 for EGFRwt is contrasted with indirect elisa method.Comprise the following steps that：
Restructuring EGFRwt-His*6 antigen wrapper sheets；Fishskin gelatin is closed；1A5,1C5,1C5-LC-V205C-mc-vc- are diluted respectively
PAB-MMAE TDC、1C5-LC-V205C-mc-vc-PAB-MMAF TDC、1C5-LC-V205C-mc-vc-PAB-PBD TDC、
1C5-LC-V205C-mc-vc-PAB-SN38 TDC, 1C5-LC-V205C-mc-vc-PAB-DOX TDC, maximum concentration 50ug/
Ml, 4 times of gradient dilutions, totally 11 concentration；The secondary antibody of HRP marks is incubated；TMB develops the color, and is absorbed at detection 450nm.Testing result
Concentration is mapped with A450, as shown in figure 3, the TDC after 1C5, coupling maintains the affinity similar to 1A5, EC50 values connect very much
Closely；Illustrate on 1A5 the rite-directed mutagenesis and 1C5 of light chain V205C and the site-directed coupling of mc-vc-PAB-payload do not influence its with
The affinity of EGFRwt antigens.
Accompanying drawing 4,1C5-LC-V205C-mc-vc-PAB-MMAE TDC and 1C5 maintain the affine of 1A5 and antigen EGFRwt
Power.
Accompanying drawing 5,1C5-LC-V205C-mc-vc-PAB-MMAE TDC, 1C5-LC-V205C-mc-vc-PAB-MMAF
TDC、1C5-LC-V205C-mc-vc-PAB-PBD TDC、1C5-LC-V205C-mc-vc-PAB-SN38 TDC、1C5-LC-
V205C-mc-vc-PAB-DOX TDC maintain the affinity of 1A5 and antigen EGFRwt, 1C5-LC-V205C-mc-vc-PAB-
Payload with various small molecular cell toxin can carry out site-directed coupling and keep Ag-Ab affinity.
Embodiment 29, cytotoxicity Composition analyzed
The cellular cytoxicity activity of TDC and ADC is determined by following experiments process：TDC and ADC are added separately to EGFR and cross scale
Up to or EGFRVIII expression people tumor cell culture base in, cell culture determines cell survival rate after 72 hours.Based on thin
The experiment in vitro of born of the same parents is used to determine the apoptosis of cell survival rate, cytotoxicity and TDC of the present invention induction.
The pharmacy in vitro of Antibody-toxin conjugate is determined by cell proliferation test.CellTiter 96®
AqueousOne Solution Cell Proliferation Assay are commercially available（Promega Corp., Madison,
WI）.AQueous One Solution Cell Proliferation Assay (a) of CellTiter 96 be it is a kind of with than
Color method come detect cell propagation and cytotoxicity experiment in living cells quantity detection reagent.This reagent contain one it is 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 that the solution of stabilization can be mixed to form with MTS.This
Convenient " single solution " pattern is planted, is the improvement on the basis of the AQueous Assay of first generation CellTiter 96,
The electron coupling agent PMS used in the AQueous Assay of CellTiter 96 is provided separately from MTS solution. MTS
(Owen ' s reagent) turns into a kind of coloured formazan product by cell biological reduction, can be directly dissolved in culture medium（Figure
1）.This conversion is likely to what is completed in the presence of the NADPH or NADH that the dehydrogenase being metabolized in active cell is produced.
During detection, only a small amount of AQueous One Solution Reagent of CellTiter 96 need to be directly added into culture plate
In the culture medium in hole, it is incubated 1-4 hour, the absorbance of 490nm is then read with ELIASA.
The amount that formazan products are detected at 490nm is directly proportional to the viable count in culture.Because MTS formazans are produced
Thing is solvable in tissue culture medium (TCM), the AQueous One Solution Assay and MTT or INT method phases of CellTiter 96
It is less than operating procedure.
A431 is used in the present invention（EGFRwt overexpressing cells）As the research system that pharmacy in vitro is detected.In 96 orifice plates
In, the hole of plating cells 6000/ is carried out, after 24 hours, carry out antibody dosing.Drug level to A431 is 2nM-0.4pM.Treatment
MTS detections cytoactive after 72 hours.
Accompanying drawing 6-10 and table 3,1C5-LC-V205C-mc-vc-PAB-MMAE TDC, 1C5-LC-V205C-mc-vc-
PAB-MMAF TDC、1C5-LC-V205C-mc-vc-PAB-PBD TDC、1C5-LC-V205C-mc-vc-PAB -SN38 TDC、
1C5-LC-V205C-mc-vc-PAB-Dox TDC and its corresponding 1A5-mc-vc-PAB-MMAE ADC, 1A5-mc-vc-PAB-
MMAF ADC, 1A5-mc-vc-PAB-PBD ADC, 1A5-mc-vc-PAB-SN38 ADC, 1A5-mc-vc-PAB-Dox ADC pairs
The IC50 testing results of the A431 cells of overexpression EGFR.TDC cellular cytoxicity activities are better than ADC.
The present invention is not limited to by the scope of the specific embodiment disclosed in embodiment, and these embodiments are used for illustrating this
Invention several aspects, functionally equivalent any embodiment belong to the scope of the present invention.
Claims (11)
1. cysteine engineered Antibody-toxin conjugate, with below formula：1C5-LC-V205C-mc-vc-PAB-
payload。
2. the cysteine engineered Antibody-toxin conjugate described in claim 1, wherein 1C5 is target antibody light chain 205
Valine（V）It transform cysteine as（C）Antibody afterwards, the amino acid sequence of 1C5 weight chain variable districts is SEQ ID NO:1, gently
Chain amino acid sequence is SEQ ID NO:12.
3. the cysteine engineered Antibody-toxin conjugate described in claim 2, comprising a weight chain variable district for maturation,
Its amino acid sequence at least 90% and SEQ ID NO:1 is identical.
4. the cysteine engineered Antibody-toxin conjugate described in claim 2, comprising a light chain for maturation, its amino
Acid sequence at least 90% and SEQ ID NO:12 is identical.
5. the cysteine engineered Antibody-toxin conjugate described in claim 1, wherein mc-vc-PAB-OH connexons are：
。
6. the cysteine engineered Antibody-toxin conjugate described in claim 1, 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：
。
7. the cysteine engineered Antibody-toxin conjugate described in claim 1, wherein toxin compares antibody ratios（DAR）For
1.6-2.0。
8. the cysteine engineered Antibody-toxin conjugate described in claim 2, mc-vc-PAB connexons are carried out with antibody
The site amino sequence of site-directed coupling is：GLSSPCTKSFN, wherein,CIt is the 205 valine transformations of target antibody light chain
Cysteine.
9. the cysteine engineered Antibody-toxin conjugate described in claim 2,1C5 antibody be selected from IgG1, IgG2, IgG3,
IgG4, Fab and F (ab)2。
10. the cysteine engineered Antibody-toxin conjugate described in claim 1, its indication is overexpression EGFRwt's
Tumour, including it is head and neck cancer, colorectal cancer, brain colloid carcinoma, cancer of pancreas, non-small cell lung cancer, carcinoma of urinary bladder, colorectal cancer, kidney, preceding
Row gland cancer, breast cancer etc..
Cysteine engineered Antibody-toxin conjugate described in 11. claims 1, its main site-directed coupling step is：First
Using DTT also original antibodies, the shielding on the cysteine residues transformed on antibody is released, and remove by cation-exchange chromatography
DTT and screen；Then DHAA oxidised antibodies are used, the interchain disulfide bond of antibody is reconnected；It is eventually adding mc-vc-
PAB-payload is coupled with the cysteine residues of antibody transformation, and is not coupled upper antibody by cation-exchange chromatography removal
The mc-vc-PAB-payload of molecule.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018064964A1 (en) * | 2016-10-08 | 2018-04-12 | 四川百利药业有限责任公司 | Cysteine modified antibody-drug conjugate and preparation method thereof |
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| WO2019034177A1 (en) * | 2017-08-18 | 2019-02-21 | 四川百利药业有限责任公司 | Antibody drug conjugate having two different drugs |
| CN108853514B (en) * | 2017-08-18 | 2022-07-22 | 四川百利药业有限责任公司 | Antibody drug conjugates with two different drugs |
| CN108727498A (en) * | 2018-04-24 | 2018-11-02 | 四川百利药业有限责任公司 | Cysteine engineered Antibody-toxin conjugate |
| CN108743966A (en) * | 2018-04-24 | 2018-11-06 | 四川百利药业有限责任公司 | Cysteine engineered Antibody-toxin conjugate |
| CN108743966B (en) * | 2018-04-24 | 2021-10-19 | 四川百利药业有限责任公司 | Cysteine engineered antibody-toxin conjugates |
| CN108727498B (en) * | 2018-04-24 | 2021-11-26 | 四川百利药业有限责任公司 | Cysteine engineered antibody-toxin conjugates |
| CN109485655A (en) * | 2018-12-20 | 2019-03-19 | 河北百灵威超精细材料有限公司 | A kind of preparation method of biotin maleimide |
| CN113583086A (en) * | 2021-08-02 | 2021-11-02 | 联宁（苏州）生物制药有限公司 | Synthetic method of intermediate LND1035 of antibody-coupled drug |
| CN113583086B (en) * | 2021-08-02 | 2024-01-30 | 联宁（苏州）生物制药有限公司 | Synthesis method of intermediate LND1035 of antibody coupled drug |
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