CN107261137A - Two kinds of Anti-HER 2 chaetocin conjugates and preparation method thereof and antitumor application thereof - Google Patents
Two kinds of Anti-HER 2 chaetocin conjugates and preparation method thereof and antitumor application thereof Download PDFInfo
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Abstract
The present invention relates to biotechnology and drug field, specifically two kinds Anti-HER 2 chaetocin conjugate Trastuzumab vc Chaetocin (T L C) and Pertuzumab vc Chaetocin (P L C) and its synthesis preparation method and application.The antibody coupling matter is obtained by the drug molecule with linker and monoclonal antibody coupling, and structure is as follows.Present invention also offers a kind of preparation method with linker drug molecule.Antibody coupling matter prepared by the present invention has targeting to HER2 positive cells, both the killing to HER2 positive tumor cells can have been strengthened, also the toxic side effect that (+) Chaetocin is administered alone generation can be reduced, there is huge potential treatment use value to cancer caused by the tumour cell of HER2 positive expressions.
Description
Technical field
The present invention relates to biotechnology and drug field, specifically, be two kinds of Anti-HER 2s-chaetocin conjugate and
Its preparation method and anti-tumor aspect application.
Background technology
Human epidermal growth factor receptor 2 (HER2) is the Epidermal Growth Factor Receptor Family with tyrosine kinase activity
One member.The polymerization of acceptor can cause the phosphorylation of receptor tyrosine residue, and start many A signal pathways and cause carefully
Born of the same parents breed and tumour occurs, and mainly altimeter reaches in breast cancer.The overexpression of HER2 genes not only develops with tumour
And be one of neoplasm targeted therapy medicament selection mutually outside the Pass, or an important clinical treatment monitoring and prognostic indicator,
Important target spot.For the research relative maturity of the target spot, and the medicine Trastuzumab of the Overexpression it has been directed to
(Herceptin) it is Herceptin Trastuzumab.
Herceptin is Humanized monoclonal antibodies derived from a kind of recombinant DNA, selectively affects people's epidermis
The extracellular position of growth factor receptors -2 (HER2), disabling signal path suppresses the growth of tumour cell, and Trastuzumab can be with
The immunocyte of itself is stimulated to go to destroy cancer cell, the only tumour to HER2 height expression is effective.Handkerchief trastuzumab is also a kind of logical
The Humanized monoclonal antibodies of biological technique method production are crossed, by combining HER2, retardance HER2 and miscellaneous the two of other HER receptors
It is poly-, it is suppressed that the downstream signal transduction process related to tumour growth；It is different from the epitope that Herceptin is combined, to it is low,
Middle expression HER2 tumour is also effective.
More thio diketopiperazine (Epipolythiodioxopiperazines, ETPs) is that a class is main by marine fungi
The important activity secondary metabolite of generation, its architectural feature be diketopiperazine parent nucleus in contain sulphur bridge.ETPs has extensive
Bioactivity, such as antiproliferative, cell toxicant, immunosupress, antiviral and antibacterial, many of compound is even more to show
Very high anti-tumor activity.Chaetocin (+)-Chaetocin is one, ETP fields " star molecule ", ocean is found in earliest true
In bacterium Chaetronium minutum metabolite, scientist has been carried out greatly around the antitumor activity of the molecule in recent years
Quantity research, shows that the compound has istone lysine specific methyltransferase (HKMTs) inhibitory action, can strong inhibition
The growth of tumour cell.The disulfide bond that (+)-Chaetocin excellent activity has with itself also has compared with Important Relations, Ke Nengneng
Enough modes of action with similar alkylating agent modify target cellular protein, block many cell pathways and then cause death of neoplastic cells.
Antibody coupling medicine is to pass through bioactivity connector by monoclonal antibody (mAb) and potent toxicity medicine
(Linker) coupling is formed, and it has had chemicals and the respective advantage of antibody drug concurrently, passes through efficiently special antigen-antibody
Reaction, carries antineoplastic and goes directly target, antibody endocytosis enters cell, is further degraded in lysosome and discharges medicine
Reach the purpose of killing tumor cell.
Cytotoxic drug is used as the bullet of ADCs medicines, undertakes killing tumor cell, plays the main work(of ADCs curative effects
Can, it is ADCs important component.Drug candidate bullet high concentration is in aplysiatoxin in clinical in recent years, the medicine such as maytansine
Thing, searching out development of the new medicine bullet to ADCs medicines and promoting has important meaning, and marine natural products is antitumor
The important sources of active material, with very high research and development value, and there is no at present on by Trastuzumab and
Pertuzumab and other oceans are toxin conjugated, to treat the report for the disease that HER2 is mediated.
The content of the invention
It is an object of the invention to provide two kinds of Anti-HER 2s-chaetocin conjugate and preparation method thereof and antitumor side
Apply in face.
There is provided two kinds of Anti-HER 2s-chaetocin conjugate (T-L-C or P-L-C), its structure for the first aspect of the present invention
Formula is as follows：
Wherein, mAb refers to Herceptin Trastuzumab or handkerchief trastuzumab Pertuzumab, through protease-sensitive type
Valine-citrulline linker (vc) is connected with ocean toxin chaetocin (+)-Chaetocin.
That is, described Anti-HER 2-chaetocin conjugate is respectively：
A kind of Anti-HER 2 with following structural formula-chaetocin conjugate Trastuzumab-vc-Chaetocin,
That is T-L-C：
Wherein, Anti-HER 2 be selectively acting in the Herceptin Trastuzumab of HER2 extracellular regions,
Trastuzumab is connected through protease-sensitive type valine-citrulline linker (vc) with chaetocin (+)-Chaetocin, formula
In n represent drug antibody coupling ratio, described drug antibody coupling ratio n scope is 1-8.
It is preferred that, scope of the medicine antibody coupling than n is 1-2, i.e., each Trastuzumab antibody in described T-L-C
1-2 (+)-Chaetocin molecules of molecule coupling labeled.
A kind of Anti-HER 2 with following structural formula-chaetocin conjugate Pertuzumab-vc-Chaetocin, i.e.,
P-L-C：
Wherein, Anti-HER 2 is HER heterodimericization inhibitor handkerchief trastuzumab Pertuzumab, and Pertuzumab is through egg
White enzyme responsive type valine-citrulline linker (vc) is connected with chaetocin (+)-Chaetocin, and the n in formula represents medicine and resisted
Body coupling ratio, described drug antibody coupling ratio n scope is 1-8.
It is preferred that, scope of the medicine antibody coupling than n is 1-2, i.e., each Pertuzumab antibody point in described P-L-C
1-2 (+)-Chaetocin molecules of son coupling.
There is provided a kind of preparation method with linker drug molecule, i.e. vc-Chaetocin, bag for the second aspect of the present invention
Include following steps (synthesis path is shown in Fig. 1)：
(A) by chaetocin (+)-Chaetocin and N, N- diisopropylethylamine mixed dissolution in N,N-dimethylformamide
In, stir 20min under room temperature under nitrogen protection；Take valine-citrulline dipeptides and N, N- diisopropyl that maleimide is amine-modified
Base ethamine is added in reaction solution, is stirred overnight at room temperature；
(B) add trifluoroacetic acid after completion of the reaction and reaction solution is quenched in acetic acid, oil pump is concentrated under reduced pressure, silica gel column chromatography purifying
Yellow-brown solid is obtained, the solid is the band linker drug molecule.
The partially synthetic reaction condition referenced patent document WO/ with linker drug molecule vc-Chaetocin
2006/060533(Conjugates of 1,8-bis-naphthalimides with an antibody)；Preparation method is obtained
To band linker drug molecule vc-Chaetocin there is no relevant report.
There is provided above-mentioned Anti-HER 2-chaetocin conjugate (T-L-C or P-L-C) system for the third aspect of the present invention
Preparation Method, comprises the following steps：
(a) that Herceptin or handkerchief trastuzumab stoste are replaced into phosphate using Sephadex G-25 desalting columns is anti-
Buffer solution is answered, super filter tube concentration adjustment antibody concentration prepares Herceptin or handkerchief trastuzumab antibody.
May be anti-containing other interference because stoste pH value is not the optimal conditions of coupling drug below, and in Stock solutions
The material answered, is less than 30kDa's by substitutional solution, and with the small molecule and other molecular weight in super filter tube concentration removing system
Impurity molecule.
(b) three (2- carboxyethyls) phosphines (TCEP) are added into Anti-HER 2 buffer solution, reduction reaction is carried out.
Because the reactive cysteine in Anti-HER 2 surface is present in the form of interchain disulfide bond, thus not
It is coupled in the presence of free cysteine and linker drug molecule vc-Chaetocin, so before being coupled with drug molecule, needing
Monoclonal antibody interchain disulfide bond is reduced and opens the cysteine sulfydryl for obtaining free state.
Common disulfide bond reducing agent is dithiothreitol (DTT) (DTT), because can be amine-modified with maleimide containing sulfydryl in DTT
The reactive polypeptide of valine-citrulline two, preferably three (2- carboxyethyls) phosphine (TCEP), and the stability of TCEP in aqueous here
It is all fine with dissolubility；The buffer solution is pH 7.4 PBS；The mol ratio of TCEP and Anti-HER 2 is 12:1；
The condition of the reduction reaction is preferably：37 DEG C of water-bath 1.5h.
(c) monoclonal antibody that the purifying of Sephadex G-25 desalting columns is reduced is crossed, the hair shell with linker that synthesis is obtained is added
Element (+)-Chaetocin is mixed, and carries out coupling reaction.Preferably, the linker be the amine-modified valine of maleimide-
Citrulling dipeptides.(its synthetic method bibliography：Dubowchik,G.M.,Firestone,R.A.,Padilla,L.,et
al.Cathepsin B-labile dipeptide linkers forlysosomal release of doxorubicin
from internalizing immunoconjugates:modelstudies of enzymatic drug release
and antigen-specific in vitro anticancer activity[J].Bioconjugate Chemistry,
2002,13(4):855-869.).The mol ratio of chaetocin (+)-Chaetocin and Anti-HER 2 with linker are 10:1,
The temperature of the coupling reaction is preferably ice bath reaction 1.5h.
(d) after the completion of reacting, add cysteine solution and reaction is quenched, cross desalting column purifying, super filter tube is concentrated to give institute
State Anti-HER 2-chaetocin conjugate.The cysteine of the addition is the 20mM solution of 20 times of excess molar ratios, is quenched anti-
Should be preferably 45min under ice bath；Because the molecular weight of Anti-HER 2 of the present invention-chaetocin conjugate is all higher than 140kDa, and
The molecular weight of other molecules present in reaction system is respectively less than 2kDa, using the small molecule in desalting column removing system, is used in combination
Super filter tube concentrated antibody, obtains described Anti-HER 2-chaetocin conjugate.
The fourth aspect of the present invention is there is provided above-mentioned Anti-HER 2-chaetocin conjugate (T-L-C or P-L-C) in system
Application in standby anti-tumor medicine.
It is preferred that, described tumour is stomach cancer, lung cancer, liver cancer or breast cancer.
It is preferred that, described tumour is HER2 positive tumors.Anti-HER 2-chaetocin conjugate of the present invention and not idol
The Anti-HER 2 of connection medicine is compared, and has higher affinity and obvious lethality to HER2 positive tumor cells.
It is preferred that, the HER2 positive tumor cells are stomach cancer cell SGC-7901, and lung carcinoma cell NCI-H460, liver cancer is thin
Born of the same parents SMMC-7721 and three kinds of breast cancer cells SKBR-3, AU565 and MBA-MD-231.
It is furthermore preferred that described HER2 positive tumor cells are breast cancer cell SKBR-3, AU565.
Beneficial effects of the present invention are：
1st, conjugate of the present invention has the biological function of both Anti-HER 2 and (+)-Chaetocin, both has
The killing ability of Trastuzumab or Pertuzumab antibodies on tumor cell, again there is (+)-Chaetocin to rely histone
The inhibitory action of propylhomoserin specific methyltransferase (HKMTs), and the mode of action of similar alkylating agent modify target cellular protein,
Under the synergy of the two, obvious antitumous effect is shown；
2nd, conjugate of the present invention is combined by anti-HER2 monoclonal antibody with the HER2 receptor-specifics of tumor cell surface, occurs endocytosis
(+)-Chaetocin is transported to release in tumour cell and played a role by effect.Antibody coupling matter prepared by the present invention is to HER2
Positive cell has targeting, can both strengthen the killing to HER2 positive tumor cells, can also reduce (+)-Chaetocin
The toxic side effect produced to normal cell is administered alone, has huge potential to cancer caused by the tumour cell of HER2 positive expressions
Treatment use is worth.
Brief description of the drawings
The linker drug molecule vc-Chaetocin of Fig. 1 present invention synthesis path；
Fig. 2 conjugates T-L-C HPLC analysis charts after carrying out reduction：(a)Trastuzumab；(b)T-L-C；
Fig. 3 conjugates P-L-C HPLC analysis charts after carrying out reduction：(a)Pertuzumab；(b)P-L-C；
Inhibited proliferations (48h) of each concentration (+)-Chaetocin of Fig. 4 to different tumour cells；
Each concentration Trastuzumab and T-L-C of Fig. 5 compare (72h) the inhibitory action of different cells；
Each concentration Pertuzumab and P-L-C of Fig. 6 compare (72h) the inhibitory action of different cells；
Each concentration P-L-C and T-L-C of Fig. 7 compare the inhibitory action (72h) of breast cancer cell of the same race (t inspections：*, P<
0.05；*, P<0.01；* *, P<0.001)；
Fig. 8 .P-L-C and T-L-C compares the inhibitory action (72h) of different breast cancer cells (t inspections：*, P<
0.05；*, P<0.01；* *, P<0.001).
Embodiment
The embodiment provided with reference to embodiment the present invention elaborates.
Embodiment 1：Preparations of the vc-Chaetocin with linker drug molecule
Preparation process is as follows：
(1) by chaetocin (+)-Chaetocin 15mg (0.022mmol) and the μ L of N, N- diisopropylethylamine 16.5
(0.100mmol), 2.4mL DMFs are blended in after the lower magnetic agitation 20min of room temperature under nitrogen protection, take Malaysia
Imide modified valine-citrulline dipeptides 37mg (0.050mmol) and the μ L (0.103mmol) of N, N- diisopropylethylamine 17
It is added in reaction solution, is stirred overnight at room temperature；
(2) TLC monitors reaction and finished, and reaction solution, oil pump decompression is quenched in 0.40mL 1.3M trifluoroacetic acids and 0.60mL acetic acid
Concentrated by rotary evaporation, silica gel column chromatography purifying, eluant, eluent is dichloromethane：Methanol=6:1, obtain yellow-brown solid product 6-L-C
6.8mg, yield：24.4%.1H NMR(500MHz,CD3OD)δH(ppm):8.08 (d, J=6.0Hz, 2H ,-CH=CH-),
7.57-7.30 (m, 10H, ArH), 6.76 (d, J=6.0Hz, 2H, ArH), 4.59-4.51 (m, 8H ,-CH 2O-,-CH-,-
NHCHCO-),4.23-4.16(m,2H,-CH 2-), 3.52 (t, J=7.5Hz, 2H ,-CH 2N-),3.12(s,6H,-NCH 3),
2.76-2.69(m,2H,-CH 2NHCO-),2.31-2.19(m,5H,-CH(CH3)2,-CH 2-),1.64-1.58(m,6H,-
CH 2CO-,-CH 2-),1.35-1.31(m,6H,-CH 2-),0.99-0.97(m,6H,-CH(CH 3)2).ESI-MS[(M+Na)+]m/
z:1317.3。
Synthesis path of the invention with linker drug molecule vc-Chaetocin is as shown in Figure 1.
Embodiment 2：The preparation of Anti-HER 2-chaetocin conjugate (T-L-C or P-L-C)
The preparation method of T-L-C conjugates includes：
(1) Herceptin stoste (20mg/mL) is replaced into by PBS reaction bufferings by Sephadex G-25 desalting columns
Liquid (pH 7.4), and concentration is adjusted to 10mg/mL with super filter tube concentration monoclonal antibody；
(2) 500 μ L (10mg/mL) Herceptin solution are taken, the 20mM TCEP solution 21 of 12 times of excess molar ratios is added
μ L, 1.5h is incubated at 37 DEG C；
(3) desalting column is crossed with the PBS of the DTPA containing 1mM, the monoclonal antibody being reduced is purified, to monoclonal antibody solution under condition of ice bath
The middle μ L of 5.38mM 6-L-C solution 64 for adding 10 times of excess molar ratios, stand reaction 1.5h；
(4) the μ L of 20mM cysteine solutions 35 of 20 times of excess molar ratios are added, 45min is stood, PBS crosses desalting column, are surpassed
Chimney filter is concentrated into 500 μ L, obtains antibody coupling medicine T-L-C (8.3mg/mL).
The preparation method of P-L-C conjugates includes：
(1) handkerchief trastuzumab stoste (10mg/mL) is replaced into by PBS reaction bufferings by Sephadex G-25 desalting columns
Liquid (pH 7.4), and concentration is adjusted to 8mg/mL with super filter tube concentration monoclonal antibody；
(2) 500 μ L (8mg/mL) handkerchief trastuzumab solution are taken, the μ of 20mM TCEP solution 17 of 12 times of excess molar ratios is added
L, 1.5h is incubated at 37 DEG C；
(3) desalting column is crossed with the PBS of the DTPA containing 1mM, the monoclonal antibody being reduced is purified；To monoclonal antibody solution under condition of ice bath
The middle μ L of 5.38mM 6-L-C solution 51 for adding 10 times of excess molar ratios, stand reaction 1.5h；
(4) the μ L of 20mM cysteine solutions 27 of 20 times of excess molar ratios are added, 45min is stood, PBS crosses desalting column, are surpassed
Chimney filter is concentrated into 500 μ L, obtains antibody coupling medicine P-L-C (5.2mg/mL).
The cysteine sulfydryl of Trastuzumab and Pertuzumab monoclonal antibodies and vc-Chaetocin dimaleoyl imino
Alkylated reaction occurs for group, becomes the conjugate for being coupled different number Chaetocin.
Monoclonal antibody and (+)-Chaetocin have different maximum absorption wavelengths, but (+)-Chaetocin maximum suction
Wavelength is received in 302nm or so, antibody also has absorption here, so HPLC analyses are carried out at 280nm to T-L-C and P-L-C,
Simultaneously using Trastuzumab and Pertuzumab monoclonal antibodies as reference, the change of observation retention time and peak shape.
Pure antibody after carrying out reduction, has light chain (Fig. 2 a, 3a peaks 1), heavy chain (Fig. 2 a peaks 2,3a peaks 3) two at 280nm
Main peak；It is coupled after (+)-Chaetocin, such as Fig. 2 b and Fig. 3 b, obvious change all occurs for the appearance time and peak shape of light and weight chain,
Because the structure composition of two kinds of monoclonal antibodies is different, the position number difference that medicine is connected is to the shadow in terms of antibody polarity, molecular weight
Ring also different, thus light and weight chain appearance time can difference, such as Herceptin appearance time after drug molecule is connected pushes away
Late.Show that the medicine number that antibody has been coupled on (+)-Chaetocin, but coupling is less by HPLC analyses.
The concentration mensuration of conjugate in this experiment：Using ultraviolet spectrophotometry, the UV absorption at 280nM is determined
Value, passes through the concentration of langbobier law calculating antibody.The assay method of coupling ratio in this experiment：Coupling ratio is determined to refer to determine
The number of small-molecule drug, i.e., the mol ratio of medicine and antibody in conjugate are connected on each monoclonal antibody molecule.Ultraviolet point can be used
Two methods of light photometry and Ellman-BCA methods are determined.Ultraviolet spectrophotometry：Antibody is determined using ultraviolet specrophotometer
With the absorbance of drug molecule maximum absorption wave strong point, the standard curve at 280nm and 302nm is drawn out, then by determining
Absorbance of the antibody coupling medicine at two wavelength, the amount of antibody and drug molecule is calculated according to langbobier law, is tried to achieve
(+)-Chaetocin/ antibody ratios.Ellman-BCA methods：Ellman ' s reagents can be used for oneself in colorimetric method for determining biological sample
By sulfhydryl content, BCA methods are conventional protein concentration quantitative approach, sensitivity is high, and taken amount is few, simple to operate, the color of formation
Complex stabilities are good；Conjugate, which is opened disulfide bond by reducing agent, becomes free sulfydryl, then utilizes Ellman method meters
The content of unreacted sulfydryl is calculated, determines that conjugate concentration calculates coupling ratio in conjunction with BCA methods.The idol obtained by two methods
Connection is than having good uniformity, as a result as shown in table 1.
1 two kinds of table determines the contrast of coupling ratio methods and resultses
| Sample | T-L-C | P-L-C |
| A280(nm) | 0.664 | 0.397 |
| A302(nm) | 0.072 | 0.060 |
| DAR (UV methods) | 1.81 | 1.89 |
| A412(nm) | 0.022 | 0.011 |
| A570(nm) | 0.558 | 0.416 |
| DAR (Ellman-BCA methods) | 1.79 | 1.86 |
Embodiment 3：Cell in vitro proliferation assay biological activity test
In the present embodiment, it have studied the effect that chaetocin (+)-Chaetocin breeds to each tumor cell line；With HER2
Positive cell SKBR-3, AU565, HER2 negative cells MBA-MD-231 and normal cell HaCaT are object, are determined
The biological activity of Trastuzumab, Pertuzumab monoclonal antibody and T-L-C, P-L-C conjugate.
The present embodiment evaluates the antiproliferative effect of medicine using CCK-8 reagents.The main component of reagent is water solubility four
Azoles salt WST-8, WST-8 are generated orange-yellow water miscible formazan (formazan) after intracellular dehydrogenase biological reducing, generation
Formazans amount is linear related to living cells quantity.
The cell line of the present embodiment selection has：Stomach cancer cell SGC-7901, lung carcinoma cell NCI-H460, liver cancer cells
SMMC-7721, breast cancer cell SKBR-3, AU565, MBA-MD-231 and the Normal Immortalized keratinocyte HaCaT of people.
This experiment have studied the effect that chaetocin (+)-Chaetocin breeds to each tumor cell line first.SGC-7901、
NCI-H460, AU565 cell in 1640 culture mediums containing 10% hyclone, SMMC-7721, MBA-MD-231 cell containing
In the DMEM culture mediums of 10% hyclone, SKBR-3 cells in McCoy ' the s 5a culture mediums containing 10% hyclone, in
37 DEG C, 5%CO2Cultivated in incubator；Six kinds of cells are respectively with 6 × 103-8×103The density of individual cell per well is seeded to 96 holes
After plate, 100 μ L/ holes, culture 24h, original culture medium in 96 orifice plates is suctioned out, the chaetocin of the nutrient solution dilution of various concentrations is added
(+)-Chaetocin, 100 μ L/ holes, each concentration sets multiple holes, and sets Vehicle controls and the cell-free medium hole of respective concentration,
37 DEG C, 5%CO2Cultivated in incubator after 48h, add 10 μ L CCK-8,37 DEG C, 5%CO per hole240-60min is cultivated in incubator,
The OD values under 450nm are determined, and calculate ICs of chaetocin (+)-Chaetocin to various cells50It is worth (μM).Result of calculation such as table
Shown in 2, to inhibited proliferation such as Fig. 4 of each tumour cell.
ICs of chaetocin (+)-Chaetocin of table 2 to individual cell50It is worth (μM)
From table 2 and Fig. 4, chaetocin (+)-Chaetocin has obvious lethal effect to each tumour cell, can be with
Medicine bullet as antibody coupling medicine.
This experiment uses pure monoclonal antibody Trastuzumab, Pertuzumab and obtained two kinds of conjugates T-L-C, P-L-C
The research of Cell culture invitro proliferation function is carried out to two kinds of HER2 tumor cell lines being overexpressed, while also negative to HER2
Tumour cell and normal cell have carried out the research of Cell culture invitro proliferation function.AU565 cells are containing 10% hyclone
1640 culture mediums in, MBA-MD-231, HaCaT cell are in the DMEM culture mediums containing 10% hyclone, SKBR-3 cells
In McCoy ' the s 5a culture mediums containing 10% hyclone, in culture in 37 DEG C, 5%CO2 incubators；Four kinds of cells respectively with
6×103-8×103The density of individual cell per well is seeded to after 96 orifice plates, 100 μ L/ holes, culture 24h, is suctioned out original in 96 orifice plates
Culture medium, adds Trastuzumab, Pertuzumab, T-L-C or P-L-C (0.1 μm of hole of the nutrient solution dilution of various concentrations
The sterilised membrane filter filtration sterilization in footpath), 100 μ L/ holes, each concentration sets multiple holes, and sets the Vehicle controls of respective concentration and acellular
Cultivate datum hole, 37 DEG C, in 5%CO2 incubators after culture 72h, add 10 μ L CCK-8 per hole, 37 DEG C, train in 5%CO2 incubators
1-2h is supported, the OD values under 450nm is determined, the inhibited proliferation of each cell is compared as shown in Fig. 5,6,7,8.
From Fig. 5 and 6, after (+)-Chaetocin is coupled on antibody, it is compared with pure monoclonal antibody, T-LC and P-
L-C to HER2 height expression two kinds of breast cancer cells killing ability be all better than do not connect medicine Trastuzumab and
Pertuzumab monoclonal antibodies, and to HER2 negative cells MBA-MD-231 and normal cell HaCaT all without obvious inhibitory action,
Show conjugate specific killing HER2 positive cells.
As shown in Figure 7, inhibitory action of two kinds of difference ADC medicines to breast cancer cell of the same race is compared, P-L-C suppression is made
With T-L-C, P-L-C and T-L-C is all better than, to connect medicine number close, but activities present is variant, it may be possible to due to
Pertuzumab with Trastuzumab structures and mechanism of action are different, or Pertuzumab is after (+)-Chaetocin is connected
Endocytosis ability is better than Trastuzumab, and specific mechanism needs further research.
As shown in Figure 8, either T-L-C or P-L-C, identical to both sources (same focus, metastatic mammary gland
Cancer) breast cancer cell have obvious inhibitory action, illustrate obtained by conjugate have preferably to this class breast cancer
Inhibition；T-L-C has differences to the activities present of two kinds of breast cancer cells in each concentration section, and P-L-C is to two kinds of mammary gland
The activities present of cancer is more apparent in the difference of high concentration section.
In summary, Anti-HER 2 of the present invention-chaetocin conjugate T-L-C and P-L-C has obvious anti-
Tumor promotion, and with good targeting, small molecule drug toxicity can be transported to tumor locus, and there is provided a kind of new
Antibody coupling medicine for treating the positive metastatic breast cancer cells of HER2.
The preferred embodiment to the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (8)
1. a kind of Anti-HER 2 with following structural formula-chaetocin conjugate Trastuzumab-vc-Chaetocin, i.e.,
T-L-C：
Wherein, Anti-HER 2 is Herceptin Trastuzumab, Trastuzumab through protease-sensitive type valine-melon
Propylhomoserin linker vc is connected with chaetocin (+)-Chaetocin, and the n in formula represents drug antibody coupling ratio, and n scope is 1-8.
2. Anti-HER 2 according to claim 1-chaetocin conjugate Trastuzumab-vc-Chaetocin, it is special
Levy and be, described drug antibody coupling ratio n scope is 1-2.
3. a kind of Anti-HER 2 with following structural formula-chaetocin conjugate Pertuzumab-vc-Chaetocin, i.e. P-
L-C：
Wherein, Anti-HER 2 is handkerchief trastuzumab Pertuzumab, Pertuzumab through protease-sensitive type valine-melon ammonia
Sour linker vc is connected with chaetocin (+)-Chaetocin, and the n in formula represents drug antibody coupling ratio, and n scope is 1-8.
4. Anti-HER 2 according to claim 3-chaetocin conjugate Pertuzumab-vc-Chaetocin, it is special
Levy and be, described drug antibody coupling ratio n scope is 1-2.
5. a kind of preparation method of chaetocin (+)-Chaetocin with linker, i.e. vc-Chaetocin, including following step
Suddenly：
(A) by chaetocin (+)-Chaetocin and DIPEA mixed dissolution in DMF,
The lower stirring 20min of room temperature under nitrogen protection；Take valine-citrulline dipeptides and N, N- diisopropylethylamine that maleimide is amine-modified
It is added in reaction solution, is stirred overnight at room temperature；
(B) add trifluoroacetic acid after completion of the reaction and reaction solution is quenched in acetic acid, oil pump is concentrated under reduced pressure, silica gel column chromatography purifying is obtained
Yellow-brown solid, the solid is described band linker drug molecule.
6. a kind of preparation method of Anti-HER 2-chaetocin conjugate as described in claim 1-4 is any, including following step
Suddenly：
(a) Herceptin or handkerchief trastuzumab stoste are replaced into phosphate reaction using Sephadex G-25 desalting columns to delay
Fliud flushing, super filter tube concentration adjustment antibody concentration, prepares Herceptin or handkerchief trastuzumab；
(b) TCEP is added into monoclonal antibody buffer solution, reduction reaction is carried out；The buffer solution is pH 7.4 PBS；TCEP
Mol ratio with monoclonal antibody is 12:1；The condition of the reduction reaction is：37 DEG C of water-bath 1.5h；
(c) monoclonal antibody that the purifying of Sephadex G-25 desalting columns is reduced excessively, chaetocin (+) of the addition with linker-
Chaetocin is mixed, and carries out coupling reaction；The linker is the amine-modified valine-citrulline dipeptides of maleimide；Band is even
Chaetocin (+)-Chaetocin and monoclonal antibody that connect base mol ratio are 10:1, the temperature of the coupling reaction is reacted for ice bath
1.5h；
(d) after the completion of reacting, add cysteine solution and reaction is quenched, cross desalting column purifying, super filter tube is concentrated to give described anti-
HER2 antibody-chaetocin conjugate；The cysteine of the addition is the 20mM solution of 20 times of excess molar ratios, and reaction, which is quenched, is
45min under ice bath.
7. a kind of Anti-HER 2-chaetocin conjugate as described in claim 1-4 is any is in anti-tumor medicine is prepared
Application.
8. described Anti-HER 2-chaetocin conjugate according to claim 7 is in anti-tumor medicine is prepared
Using, it is characterised in that described tumour is HER2 positive tumors.
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| CN108404138A (en) * | 2018-03-09 | 2018-08-17 | 中国药科大学 | A kind of conjugate and its application of targeting CD24 monoclonal antibodies and two alkoxide of diethylamine azo |
| CN113975399A (en) * | 2021-11-09 | 2022-01-28 | 启东启晨生物科技有限公司 | Antibody conjugate anti-tumor drug with DNA as drug carrier |
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| CN108404138A (en) * | 2018-03-09 | 2018-08-17 | 中国药科大学 | A kind of conjugate and its application of targeting CD24 monoclonal antibodies and two alkoxide of diethylamine azo |
| CN113975399A (en) * | 2021-11-09 | 2022-01-28 | 启东启晨生物科技有限公司 | Antibody conjugate anti-tumor drug with DNA as drug carrier |
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