CN104744584B - A kind of polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate and its preparation method and application - Google Patents
A kind of polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate and its preparation method and application Download PDFInfo
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- CN104744584B CN104744584B CN201510133532.7A CN201510133532A CN104744584B CN 104744584 B CN104744584 B CN 104744584B CN 201510133532 A CN201510133532 A CN 201510133532A CN 104744584 B CN104744584 B CN 104744584B
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- polyethylene glycol
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Abstract
The invention discloses a kind of polyethylene glycol rTRAIL mutant tripolymer aplysiatoxin conjugates and its preparation method and application.A kind of polyethylene glycol rTRAIL mutant tripolymer aplysiatoxin conjugate, the amino acid sequence of rTRAIL mutant is coupled the aplysiatoxin for having mono methoxy polyethylene glycol maleimide and band connection arm as shown in SEQ ID No.1 on rTRAIL mutant tripolymer.Conjugate of the present invention has stronger tumor cell killing potential than rTRAIL mutant tripolymer aplysiatoxin conjugate, has longer half-life period than rTRAIL mutant monomethylated polyethylene glycol maleimide conjugate；Show in conjugate of the present invention, mono methoxy polyethylene glycol maleimide, rTRAIL mutant tripolymer, aplysiatoxin are not individually to play each self-applying, and stronger synergistic effect is produced between three.
Description
Technical field
The invention belongs to biotechnologys and drug field, and in particular to a kind of polyethylene glycol-rTRAIL mutant tripolymer-
Aplysiatoxin conjugate and its preparation method and application.
Background technology
Tumor necrosis factor (TNF) relevant apoptosis induction ligand (TNF related apoptosis-inducing
Ligand, TRAIL or Apo2L, TNFSF10) it is to be after the antiapoptotic factors of TNF, FasL third TNF families found later
A kind of bio-pharmaceutical that anti-cancer applications have good prospects.
TRAIL is expressed in immune system cell, including NK cells, T cell, macrophage and dendritic cells, and position
In cell membrane, soluble form can be processed into through cysteine proteinase.TRAIL is by being combined hair with its cell-membrane receptor
Wave apoptosis-induced effect.Have now been found that 5 kinds of TRAIL receptors, including：TRAIL-R1 (DR4, TNFSF10a) and TRAIL-R2
(DR5, TNFRSF10b)；TRAIL-R3 (DcR1, TNFRSF10c) and TRAIL-R4 (DcR2, TNFRSF10d)；Recycle bone protection
Plain (OPG, TNFRSF11b).DR4 and DR5 has one section of death domain (DD), is apoptosis-induced institute after TRAIL bind receptors
It is required.Its excess-three receptor can not be apoptosis-induced although can be combined with TRAIL due to lacking functional death structural domain.
TRAIL can activate mitochondria dependence and non-mitochondria to rely on two antiapoptotic signals ways after being combined with DR4 or DR5
Diameter, mediated death signal transduction enter into the cell, and Set-out slide effect object caspase-3 mRNA (caspase-3) finally causes tumour thin
Born of the same parents' apoptosis.
In recent years, it has been found that recombinate human tumor cell line apoptosis of the solvable TRAIL induction of wide spectrum, but simultaneously there is also
Some relatively low to TRAIL sensibility or tolerance tumour cells, as all people's K-1735, most of breast cancer are thin
Born of the same parents system, prostate cancer cell line LNcaP etc., although these cell surfaces more or less also express DR4 or DR5, due to cell
The reasons such as albumen height expression of the missing of key enzyme, mutation and other inhibition apoptosis, cause TRAIL to combine in interior apoptosis pathway
It can not cause the final apoptosis of cell after DR4 or DR5.
The study found that soluble type rsTRAIL (rTRAIL) can improve tumour cell pair with chemoradiotherapy plus application
The sensibility of rTRAIL, the two have synergistic effect.RTRAIL international at present is 95~281 amino acids segments,
RTRAIL monomers tend to form the preferable tripolymer of bioactivity and exist, and at the top of tripolymer, there are one zinc ions to combine
Site plays an important role to stablizing trimeric configuration.
As Chinese patent literature that Authorization Notice No. is 102936281 B of CN disclose a kind of rTRAIL mutant and its
Aplysiatoxin conjugate, the death receptor specific bond of the conjugate rTRAIL mutant and tumor cell surface, MMAE is determined
It to being transported to tumour cell, and discharges and plays a role in tumour cell, can both kill low to TRAIL sensibility or even resist
Tumour cell also reduces the toxic side effect that MMAE is administered alone generation.
The conjugate is disadvantageous in that the half-life period of rTRAIL itself is very short, partly declining in rodent
Phase is only 3 to 5 minutes and the half-life period in inhuman primate is 23 to 30 minutes.RTRAIL is even with aplysiatoxin
After connection, because the molecular weight of product increases limited (1316Da), it is difficult to the shortcomings that improving rTRAIL half-life shorts.For rTRAIL-
The shortcomings that MMAE conjugate half-life shorts, there is an urgent need for being further transformed to improve its pharmacokinetic property, improve conjugate
Druggability.
Invention content
The present invention provides a kind of polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugates, and the conjugate is not
Only there is good anti-tumor effect, also there is longer half-life period.
A kind of polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate, the amino acid sequence of rTRAIL mutant
Row are coupled as shown in SEQ ID No.1, on the rTRAIL mutant tripolymer mono methoxy polyethylene glycol maleimide
And the aplysiatoxin of band connection arm.
Mono methoxy polyethylene glycol maleimide makes conjugate of the present invention have better pharmacokinetic property, such as
Longer half-life period, higher bioavilability；And aplysiatoxin then makes conjugate of the present invention have stronger killing tumour thin
The ability of born of the same parents.And discovery is tested, conjugate of the present invention (authorizes public than rTRAIL mutant tripolymer-aplysiatoxin conjugate
Announcement number is 102936281 B of CN) there is stronger tumor cell killing potential, second more poly- than rTRAIL mutant-monomethylation two
Alcohol maleimide conjugate (Authorization Notice No. is 103408654 B of CN) has longer half-life period.Show of the invention even
Join in object, mono methoxy polyethylene glycol maleimide, rTRAIL mutant tripolymer, aplysiatoxin are not independent play respectively
Self-applying, stronger synergistic effect is produced between three.
With Authorization Notice No. be CN 102936281 B, CN 103408654 B Chinese invention patent it is identical, the present invention
RTRAIL mutant be derived from the 95th~281 amino acid of overall length trail protein, and the 109th asparagine (Asn) is prominent
Become cysteine (Cys).
In rTRAIL mutant tripolymer, the sulfydryl of three cysteine residues is in free state, for being coupled single first
The aplysiatoxin of oxygroup polyethylene glycol maleimide or band connection arm, after coupling, each tripolymer can combine 1-2 PEG
Molecule and 1-2 MMAE molecule, therefore in conjugate of the present invention, rTRAIL mutant tripolymer, mono methoxy polyethylene glycol horse
Come acid imide, band connection arm aplysiatoxin molar ratio be 1:(1~2):(2~1).Under different molar ratios, conjugate
Tumor cell killing potential or half-life period will be different.
Wherein, when rTRAIL mutant tripolymer, the sea hare poison of mono methoxy polyethylene glycol maleimide, band connection arm
The molar ratio 1 of element:2:When 1, the half-life period of conjugate is longest；And when the molar ratio of three is 1:1:When 2, the anti-of conjugate swells
Tumor activity is most strong.
Consider half-life period and antitumor activity the two indexs, preferably, in conjugate of the present invention, rTRAIL is prominent
Variant tripolymer, mono methoxy polyethylene glycol maleimide, band connection arm aplysiatoxin molar ratio be 1:1:2 or 1:1:
1；More preferably 1:1:2.
Preferably, the molecular weight of the mono methoxy polyethylene glycol maleimide is 3000~15000Da.As excellent
Choosing, the linking arm are the amine-modified valine-citrulline dipeptides of maleimide.Mono methoxy polyethylene glycol maleimide with
And above-mentioned linking arm realizes idol by maleimide base group reacts to form thioether bond with the sulfydryl in rTRAIL mutant
Connection.
The synthetic method of the linking arm, the synthetic method of the aplysiatoxin of band connection arm are CN referring to Authorization Notice No.
The related content that the Chinese invention patent of 102936281 B is recorded.
The present invention also provides the preparation sides of the polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate
Method includes the following steps：
(1) it by rTRAIL mutant polymer depolymerization, regroups to obtain rTRAIL mutant tripolymer；
Specially：RTRAIL mutant is dissolved in the buffer solution containing zinc ion, addition tricresyl phosphate (β-chloroethyl) ester, 30
~40 DEG C of water-bath 0.5h~3h.
The preparation process of rTRAIL mutant is referring to the Chinese invention patent that Authorization Notice No. is 102936281 B of CN.
There are more free sulfhydryl groups in rTRAIL mutant (monomer), thus between monomer can by disulfide bond formation polymer, but
The multimeric structure is unstable, and tricresyl phosphate (β-chloroethyl) ester (TCEP) can make unstable polymer depolymerization, is kept
Free state, the zinc ion in reaction system further promotes rTRAIL mutant monomer to form stable tripolymer, in stabilization
RTRAIL mutant tripolymer in, be mutated 109 cysteine sulfydryls of acquisition by separate out.
(2) by rTRAIL mutant tripolymer and mono methoxy polyethylene glycol maleimide hybrid reaction, poly- second is obtained
Glycol-rTRAIL mutant tripolymer conjugate；
Preferably, the molar ratio of rTRAIL mutant tripolymer and mono methoxy polyethylene glycol maleimide is 1:
1.5~8,20~30 DEG C of reaction temperature, reaction time 30s~1h.
(3) it by the aplysiatoxin hybrid reaction of polyethylene glycol-rTRAIL mutant tripolymer conjugate and band connection arm, obtains
To the polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate.
Preferably, mole of polyethylene glycol-rTRAIL mutant tripolymer conjugate and the aplysiatoxin of band connection arm
Than being 1:2~10,20~30 DEG C of reaction temperature, 30~60min of reaction time.
Experiment finds, when rTRAIL mutant tripolymer first with mono methoxy polyethylene glycol maleimide amine coupling again with band
When the aplysiatoxin coupling of linking arm, target conjugate can be obtained；And when TRAIL mutant tripolymer elder generation and band connection arm
When aplysiatoxin is coupled again with mono methoxy polyethylene glycol maleimide amine coupling, it is but unable to get target conjugate.This be because
There are space steric effect between three free sulfhydryl groups on rTRAIL mutant tripolymer, and mono methoxy polyethylene glycol horse
Take the aplysiatoxin that imido molecular weight is far longer than band connection arm, is coupled mono methoxy polyethylene glycol maleimide in the ban
When, it is also difficult to occupy all reaction compartments even if mono methoxy polyethylene glycol maleimide is excessively added, is always band connection
The aplysiatoxin of arm leaves certain reaction compartment；And if first the aplysiatoxin of coupling band connection arm, the sea hare of band connection arm are malicious
Element can occupy all reaction compartments, and can not further be coupled mono methoxy polyethylene glycol maleimide.
Moreover, rTRAIL mutant tripolymer is first also helped with mono methoxy polyethylene glycol maleimide and is carried
The water solubility of high rTRAIL mutant tripolymer, it is heavy caused by the aplysiatoxin coupling with band connection arm in next step to substantially reduce
Form sediment, thus from yield for also superior to rTRAIL-MMAE conjugates.Conjugate aqueous solution of the present invention has than simple
RTRAIL molecules or rTRAIL-MMAE conjugates more preferably stability, show as not precipitating after multigelation.
It therefore, can by adjusting the addition of the aplysiatoxin of mono methoxy polyethylene glycol maleimide, band connection arm
To adjust the PEG/MMAE being coupled on rTRAIL mutant tripolymer (polyethylene glycol/aplysiatoxin) ratio.
When the molar ratio of rTRAIL mutant tripolymer and mono methoxy polyethylene glycol maleimide is 1:When 1.5~2,
The molar ratio of polyethylene glycol-rTRAIL mutant tripolymer conjugate and the aplysiatoxin of band connection arm is 1:When 4~10, it can obtain
Molar ratio to rTRAIL mutant tripolymer, mono methoxy polyethylene glycol maleimide, aplysiatoxin is 1:1:2 poly- second
Glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate；
When the molar ratio of rTRAIL mutant tripolymer and mono methoxy polyethylene glycol maleimide is 1:2~4, poly- second
The molar ratio of glycol-rTRAIL mutant tripolymer conjugate and the aplysiatoxin of band connection arm is 1:When 2~8, it can be obtained
RTRAIL mutant tripolymer, mono methoxy polyethylene glycol maleimide, aplysiatoxin molar ratio be 1:1:1 poly- second two
Alcohol-rTRAIL mutant tripolymer-aplysiatoxin conjugate；
When the molar ratio of rTRAIL mutant tripolymer and mono methoxy polyethylene glycol maleimide is 1:4~8, poly- second
The molar ratio of glycol-rTRAIL mutant tripolymer conjugate and the aplysiatoxin of band connection arm is 1:When 2~6, it can be obtained
RTRAIL mutant tripolymer, mono methoxy polyethylene glycol maleimide, aplysiatoxin molar ratio be 1:2:1 poly- second two
Alcohol-rTRAIL mutant tripolymer-aplysiatoxin conjugate.
It is anti-in preparation that the present invention also provides the polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugates
Application in tumour medicine.
The antitumor drug has the apoptosis of tumor cells of death receptor for inducing cell surface expression.Preferably,
The antitumor drug is drugs against colon cancer, anti-leukemia drug, ovarian cancer resistance medicament, anti-gastric cancer medicament, anti-lung-cancer medicament, resists
Breast cancer medicines or medicines resistant to liver cancer.
The antitumor drug includes a effective amount of polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate,
And at least one pharmaceutically acceptable carrier, diluent or excipient.When preparation, usually active constituent and excipient are mixed
It closes, or with figuration dilution agent or Bao Ke in carrier existing for capsule or sachet.When excipient plays diluent
When, the medium of solid, semisolid or fluent material as excipient, carrier or active constituent can be used in it.Therefore, composition can
To be tablet, pill, pulvis, solution, syrup, sterilizing injecting solution etc..
Suitable excipient includes：Lactose, glucose, sucrose, sorbierite, mannitol, starch, microcrystalline cellulose, poly- second
Alkene pyrrolidone, cellulose, water etc.；Preparation may also include：Wetting agent, emulsifier, preservative (such as methyl hydroxybenzoate and third
Ester), sweetener etc..The antitumor drug can be made into unit or polynary dosage form, and each dosage form includes to generate desired treatment
Imitate and calculate rTRAIL mutant-aplysiatoxin conjugate of predetermined amount, and suitable pharmacy excipient.
The antitumor drug can be administered by conventional route, including (but being not limited to)：Intramuscular, peritonaeum
Interior, intravenous, subcutaneous, intradermal, local administration etc..
It is that the PEG-rTRAIL-MMAE conjugates of safe and effective amount are applied to people when using the drug, the wherein safety
It is a effective amount of to range preferably from 0.5~50 mg/kg weight, more preferably 1~30 mg/kg weight.Certainly, specific agent
For amount it is also contemplated that the factors such as administration route, patient health situation, these are all within the scope of skilled practitioners technical ability.
In addition, the present invention conjugate can also with other treatment drug combination, including (but being not limited to)：It is various thin
Intracellular cytokine, such as IFN, TNF, IL-2；Various tumor chemotherapeutic drugs, as 5-FU, methotrexate influence Nucleic acid
Drug；The alkylating agents drug such as mustargen, cyclophosphamide；Adriamycin, actinomycin D etc. interfere transcription that RNA is prevented to synthesize
Drug；Vincristine, camptothecin etc. influence the drug of protein synthesis and certain hormone medicines, etc..
Compared with prior art, beneficial effects of the present invention are：
(1) conjugate of the invention has both good pharmacokinetic property and tumor cell killing potential, also, this hair
Bright conjugate has stronger tumor cell killing potential than rTRAIL mutant tripolymer-aplysiatoxin conjugate, than
RTRAIL mutant-monomethylated polyethylene glycol maleimide conjugate has longer half-life period.Show of the invention even
Join in object, mono methoxy polyethylene glycol maleimide, rTRAIL mutant tripolymer, aplysiatoxin are not independent play respectively
Self-applying, stronger synergistic effect is produced between three；
(2) present invention is dashed forward rTRAIL using the space steric effect of the free sulfhydryl groups on rTRAIL mutant tripolymer
Variant tripolymer is first coupled with mono methoxy polyethylene glycol maleimide amine coupling with the aplysiatoxin of band connection arm and prepares this again
Invention conjugate, avoiding the aplysiatoxin of first coupling band connection arm causes the aplysiatoxin of band connection arm to occupy all reactions skies
Between, and the case where can not further be coupled mono methoxy polyethylene glycol maleimide；Preparation method is ingenious in design, reaction letter
It is single, two step modification reactions can be completed in same system, and adjust inventory and product of different nature can be obtained；
(3) rTRAIL mutant tripolymer is first also helped with mono methoxy polyethylene glycol maleimide and is carried by the present invention
The water solubility of high rTRAIL mutant tripolymer, it is heavy caused by the aplysiatoxin coupling with band connection arm in next step to substantially reduce
It forms sediment so that the preparation yield of conjugate of the present invention is above 60%, significantly larger than the preparation yield of rTRAIL-MMAE conjugates
(about 25%).
Description of the drawings
Fig. 1 is polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin idol that ratio is coupled with different PEG/MMAE
Join the structural schematic diagram of object；
Wherein, polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate hereinafter referred to as PEG-rTRAIL-
MMAE conjugates, PEG1-rTRAIL-MMAE2Indicate the PEG-rTRAIL-MMAE conjugates that PEG/MMAE coupling ratios are 0.5,
PEG1-rTRAIL-MMAE1Indicate the PEG-rTRAIL-MMAE conjugates that PEG/MMAE coupling ratios are 1, PEG2-rTRAIL-
MMAE1Indicate the PEG-rTRAIL-MMAE conjugates that PEG/MMAE coupling ratios are 2；
Fig. 2 is the SDS-PAGE analyses after rTRAIL mutant tripolymer is reacted with PEG and MMAE；
Wherein, M is low molecular weight protein marker；Swimming lane 1 is rTRAIL mutant N109C tripolymers；Swimming lane 2 is
RTRAIL mutant N109C tripolymers first reacted with mPEG-MAL after result；Swimming lane 3 is rTRAIL mutant N109C trimerizations
Body is first reacted with mPEG-MAL, then the result with vcMMAE reactions；Swimming lane 4 be rTRAIL mutant N109C tripolymers first with
The result of vcMMAE reactions；Swimming lane 5 is first reacted with vcMMAE for rTARIL mutant N109C tripolymers, then anti-with mPEG-MAL
The result answered；Swimming lane 6 is what rTRAIL mutant N109C tripolymers first reacted 30 seconds with mPEG-MAL, then were reacted with vcMMAE
As a result；
Fig. 3 is the molecule of rTRAIL mutant N109C, rTRAIL-MMAE conjugate and PEG-rTRAIL-MMAE conjugates
Mesh analysis result；
Fig. 4 is rTRAIL114-281, rTRAIL mutant N109C, rTRAIL-MMAE conjugate and PEG-rTRAIL-MMAE
The C.D analysis result of conjugate；
Fig. 5 is respectively through MMAE, rTRAIL mutant, rTRAIL-MMAE conjugates and PEG-rTRAIL-MMAE conjugates
The cell cycle analysis result of breast cancer cell line MCF-7 after handling 8 hours；
Wherein, G1, G2, S, G2/ M is each stage in the cell cycle, %G2/ M marks are in G2The cell quantity of/M phases
Account for the percentage of total cell quantity；
Fig. 6 is physiological saline, rTRAIL114-281, MMAE, BSA-MMAE conjugate, rTRAIL-MMAE conjugates and PEG-
RTRAIL-MMAE conjugates handle the variation diagram of mouse interior tumor volume after lotus knurl nude mouse model respectively；
Wherein, " iv.q2d × 4 " indicate tail vein injection, are administered within every two days, are administered four times altogether；
Fig. 7 physiological saline, rTRAIL114-281, MMAE, BSA-MMAE conjugate, rTRAIL-MMAE conjugates and PEG-
RTRAIL-MMAE conjugates handle the variation diagram of mouse interior tumor weight after lotus knurl nude mouse model respectively；
Fig. 8 is physiological saline, rTRAIL114-281, MMAE, BSA-MMAE conjugate, rTRAIL-MMAE conjugates and PEG-
The apoptosis activity analysis result in situ of rTRAIL-MMAE conjugates；
Fig. 9 is reason brine, rTRAIL114-281, MMAE, BSA-MMAE conjugate, rTRAIL-MMAE conjugates and PEG-
The animal toxicity analysis result of rTRAIL-MMAE conjugates；
Figure 10 is reason brine, rTRAIL114-281, MMAE, BSA-MMAE conjugate, rTRAIL-MMAE conjugates and PEG-
The changes of weight figure of rTRAIL-MMAE conjugates each test group of animals after being administered respectively；
Figure 11 is that SD is big after rTRAIL mutant, rTRAIL-MMAE conjugates and PEG-rTRAIL-MMAE conjugates are administered
Blood concentration changes with time figure in mouse body；
Figure 12 is the cancer target capability study result figure of PEG-rTRAIL-MMAE conjugates；
Figure 13 is the preparation flow figure and schematic diagram of PEG-rTRAIL-MMAE conjugates.
Specific implementation mode
Invention is further described in detail with reference to the accompanying drawings and detailed description.
Embodiment 1 prepares polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate
1, the preparation (such as Fig. 1) of the PEG-rTRAIL-MMAE conjugates with different PEG/MMAE ratios
(1) PEG/MMAE ratios be 0.5 (i.e. N109C tripolymers, PEG, MMAE molar ratio be 1:1:2)
A kind of polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate (hereinafter referred to as PEG-rTRAIL-
MMAE conjugates) preparation method, include the following steps：
1. preparing rTRAIL mutant N109C；
The preparation method of rTRAIL mutant N109C is special referring to the Chinese invention that Authorization Notice No. is CN 102936281B
The 1st~6 part of specific implementation mode in profit, amino acid sequence is as shown in SEQ ID No.1.
2. taking the rTRAIL mutant N109C of 1mg to be dissolved in 1mL PBS, (pH 6.0 contains 10 μM of ZnCl2) in, it is added 10
Tricresyl phosphate (β-chloroethyl) ester (TCEP) of ten times of amounts of μ L, 30 DEG C of water-bath 0.5h；It is added while stirring with 100 μ L PBS
(mPEG-MAL, wherein PEG molecular weight are 1.5 times of excessive mono methoxy polyethylene glycol maleimides of (pH 6.0) dissolving
15000Da), 30s is reacted at 20 DEG C；
3. 10 times of excessive vcMMAE (i.e. aplysiatoxins of band connection arm, by Jiangyin Kang Nuotai are added into reaction system
Bioisystech Co., Ltd is on behalf of synthesis), 60min is reacted at 20 DEG C, excessive cysteine is added and terminates reaction；
4. the super filter tube for being 10kDa with Millipore molecular cut offs removes the small molecule in reaction system.It is obtained
Conjugate crosses the moisture film degerming in 0.22 μm of aperture, and -20 DEG C save backup；
5. using the preparation yield of conjugate obtained by Their Determination by Spectrophotometry (280nm)；It is solidifying using 12% SDS-PAGE
Gel electrophoresis method measures the relative molecular mass of gained conjugate, is used in combination Quantity One softwares to quantify the amount of each monomer, obtains
PEG/MMAE is coupled ratio in conjugate.
(2) PEG/MMAE ratios be 1 (i.e. N109C tripolymers, PEG, MMAE molar ratio be 1:1:1)
1. taking the rTRAIL mutant N109C of 1mg to be dissolved in 1mL PBS, (pH 6.0 contains 10 μM of ZnCl2) in, it is added 10
Tricresyl phosphate (β-chloroethyl) ester (TCEP) of ten times of amounts of μ L, 37 DEG C of water-bath 2h；It is added while stirring with 100 μ L PBS (pH
6.0) (mPEG-MAL, wherein PEG molecular weight are the 2 times of excessive mono methoxy polyethylene glycol maleimides dissolved
5000Da), 40min is reacted at 25 DEG C；
2. 4 times of excessive vcMMAE are added into reaction system (by Jiangyin Kang Nuotai Bioisystech Co., Ltd on behalf of conjunction
At), 40min is reacted at 25 DEG C, excessive cysteine is added and terminates reaction；
3. the super filter tube for being 10kDa with Millipore molecular cut offs removes the small molecule in reaction system.It is obtained
Conjugate crosses the moisture film degerming in 0.22 μm of aperture, and -20 DEG C save backup；
4. using the preparation yield of conjugate obtained by Their Determination by Spectrophotometry (280nm)；It is solidifying using 12% SDS-PAGE
Gel electrophoresis method measures the relative molecular mass of gained conjugate, is used in combination Quantity One softwares to quantify the amount of each monomer, obtains
PEG/MMAE is coupled ratio in conjugate.
(3) PEG/MMAE ratios be 2 (i.e. N109C tripolymers, PEG, MMAE molar ratio be 1:2:1)
1. taking the rTRAIL mutant N109C of 1mg to be dissolved in 1mL PBS, (pH 6.0 contains 10 μM of ZnCl2) in, it is added 10
Tricresyl phosphate (β-chloroethyl) ester (TCEP) of ten times of amounts of μ L, 40 DEG C of water-bath 3h；It is added while stirring with 100 μ L PBS (pH
6.0) (mPEG-MAL, wherein PEG molecular weight are the 8 times of excessive mono methoxy polyethylene glycol maleimides dissolved
3000Da), 1h is reacted at 30 DEG C；
2. 2 times of excessive vcMMAE are added into reaction system (by Jiangyin Kang Nuotai Bioisystech Co., Ltd on behalf of conjunction
At), 30min is reacted at 30 DEG C, excessive cysteine is added and terminates reaction；
3. the super filter tube for being 10kDa with Millipore molecular cut offs removes the small molecule in reaction system.It is obtained
Conjugate crosses the moisture film degerming in 0.22 μm of aperture, and -20 DEG C save backup；
4. using the preparation yield of conjugate obtained by Their Determination by Spectrophotometry (280nm)；It is solidifying using 12% SDS-PAGE
Gel electrophoresis method measures the relative molecular mass of gained conjugate, is used in combination Quantity One softwares to quantify the amount of each monomer, obtains
PEG/MMAE is coupled ratio in conjugate.
The measurement result of absorption photometry shows that the preparation yield of PEG-rTRAIL-MMAE conjugates is above 60%, far
Far above the preparation yield (about 25%) of rTRAIL-MMAE conjugates, show that rTRAIL first undergoes mPEG-MAL modifications and is coupled again
The mode of vcMMAE is remarkably improved the preparation yield of PEG-rTRAIL-MMAE conjugates.
2, influence of the reaction time to PEG/MMAE ratios
It is that 1 citing (attached drawing 2) is divided into three kinds of conditions in modification with PEG/MMAE ratios：1. first adding mPEG-MAL
Reaction 40 minutes, then vcMMAE is added to react 40 minutes；2. first plus vcMMAE reacts 40 minutes, then mPEG-MAL is added to react 40 points
Clock；3. first plus mPEG-MAL reacts 30 seconds, adds vcMMAE and react 40 minutes.Remaining reaction condition all same.Under the conditions of each
Reaction product resolving gel concentration be 12% SDS-PAGE analysis coupling result.
As a result as shown in Fig. 2：Only formerly add mPEG-MAL (swimming lane 2) again plus under conditions of vcMMAE (swimming lane 3),
Will appear the band (band above N109C bands) of PEG-rTRAIL-MMAE conjugates, and no matter mPEG-MAL and rTRAIL
The reaction time of tripolymer is 30 seconds (swimming lanes 6) or 40 minutes (swimming lane 3), and PEG-rTRAIL-MMAE conjugates can be obtained,
And yield is close.And vcMMAE (swimming lane 4) is formerly added then to be unable to get PEG- again plus under conditions of mPEG-MAL (swimming lane 5)
RTRAIL-MMAE conjugates
The above results show mPEG-MAL and vcMMAE, and there are spaces during modifying rTRAIL mutant tripolymer
Steric hindrance, the larger mPEG-MAL of first coupling molecule amount, ability leaving space give smaller vcMMAE；If first coupling molecule amount compared with
Small vcMMAE, then it " will occupy " all energy couplings mutation cysteine on free sulfhydryl groups (three) so that mPEG-
The position that MAL is not coupled.
3, the structural analysis of PEG-rTRAIL-MMAE conjugates
By taking PEG/MMAE ratios are 1 PEG-rTRAIL-MMAE conjugates as an example, molecular sieve (size-exclusion
Chromatography, SEC) chromatography analyzes PEG-rTRAIL-MMAE conjugates, dual modification is undergone with analysis
Whether rTRAIL mutant tripolymer maintains its active Trimeric structures, and analysis result is shown in Fig. 3.
As shown in figure 3, rTRAIL mutant N109C goes out a cysteine residues due to comparing rTRAIL multimutations, because
This disulfide bond for foring intramolecular mispairing leads to the formation of dimer, occurs after tripolymer peak is shown as in molecular sieve result
One dimer peak；And then only there is tripolymer in the coupled product rTRAIL-MMAE of rTRAIL mutant N109C and MMAE
Peak, show reduction, coupling and etc. rear rTRAIL mutant dimerization body portion depolymerization and re-formed with the coupled product of MMAE
Tripolymer；Double modified outcome PEG-rTRAIL-MMAE also tripolymers.In addition the result also indicates that removing reaction system
In small molecule after, remaining is uniform conjugate.
4, the secondary structure composition of C.D analysis PEG-rTRAIL-MMAE conjugates
By taking the PEG-rTRAIL-MMAE conjugates that PEG/MMAE coupling ratios are 1 as an example, Chirascan Plus are utilized
CD spectrophotometer (Applied Photophysics Co., Ltd) circular dichroism spectrometers far ultraviolet section 200~
260nm scans rTRAIL114-281(the 114-281 amino acid fragments of wild type rTRAIL), rTRAIL mutant N109C and
The group of the secondary structure elements of each sample is calculated in rTRAIL-MMAE conjugates, PEG-rTRAIL-MMAE conjugates, simulation
Proportional, analysis result is shown in Table 1 and Fig. 4.
Table 1
Note：1. conjugate refers to rTRAIL-MMAE conjugates, 2. conjugate refers to PEG-rTRAIL-MMAE conjugates.
By table 1 as it can be seen that rTRAIL-MMAE conjugates and PEG-rTRAIL-MMAE conjugates and rTRAIL114-281In two level
It is similar in structure composition, such as the secondary structures composition such as spiral, parallel, folding；And N109C then depositing due to mispairing dimer
Largely changing the original secondary structures of rTRAIL.It should be the result shows that rTRAIL mutant tripolymer and MMAE be independent
Coupling, or be coupled simultaneously with PEG and MMAE, smaller to its original secondary structural change, also from another side, performance is singly repaiied
Original activity of rTRAIL mutant tripolymer is very likely maintained after decorations or double modifications.
1 PEG-rTRAIL-MMAE conjugates of example are detected to act on the cell-cycle arrest of tumour cell
MMAE (aplysiatoxin) can be by inhibiting tubulin dimerization to prevent cell division so that cell is blocked
In the G2-M phases.Therefore, rTRAIL-MMAE and PEG-rTRAIL-MMAE in theory can be by inside tumor cells
Discharge effects of the MMAE to arresting cell cycle.In order to verify the effect, using human breast carcinoma cell lines MCF-7 as cell membrane
Type, using flow cytomery each sample treated MCF-7 cell cycle stages, to evaluate rTRAIL114-281,
The cell-cycle arrest of rTRAIL-MMAE and PEG-rTRAIL-MMAE acts on.Wherein PEG-rTRAIL-MMAE is PEG/MMAE
For 1 conjugate, the results are shown in Figure 5.
As seen from Figure 5, rTRAIL114-281It is acted on as a control group without apparent cell-cycle arrest, MCF-7 cells
Cell cycle distribution belongs to normal condition.And rTRAIL-MMAE with PEG-rTRAIL-MMAE just as positive control MMAE,
Significant cell-cycle arrest ability is all shown, the MCF-7 cell overwhelming majority after processing is in the G2-M phases.The result table
Bright two kinds of conjugates, which can enter tumour cell and discharge MMAE, to play a role, and meets the original intention of conjugate design, is expected to pass through
MMAE this independently of the mode of TRAIL apoptosis pathway overcome tolerance of the tumour cell to TRAIL.
Detect the internal antitumor activity evaluation of 2 PEG-rTRAIL-MMAE conjugates of example
1, the internal antitumor activity evaluation of the PEG-rTRAIL-MMAE conjugates with different PEG/MMAE ratios
The PEG-rTRAIL-MMAE conjugates of different PEG/MMAE ratios (0.5,1,2) are injected into different human tumor cells
Nude Mouse Model evaluates the internal antitumor activity of each PEG-rTRAIL-MMAE conjugates.
Specific evaluation procedure is：When tumour is grown to 50~100mm3When size, start tail vein injection administration, every other day later
Administration, dosage be 24mg/kg (being calculated with protein content) every time, be administered four times altogether；It waits the (the 18th at the end of testing
It), humanity puts to death experimental group and nude mice of control group, removes tumour, weighs in the balance and take knurl weight, and calculate tumor suppression percentage
(tumour inhibiting rate, %), calculation is as follows：The calculation formula of tumour inhibiting rate：Tumour inhibiting rate (%)=(solvent control group knurl weight-administration group
Knurl weight)/solvent control group knurl weight * 100%.
Result of calculation is shown in Table 2.
Table 2
As can be seen from Table 2, the PEG-rTRAIL-MMAE conjugates of difference PEG/MMAE ratios (0.5,1,2) are swollen to different people
Oncocyte Nude Mouse Model all has apparent antitumous effect.
Table 3
Contrast table 2 and table 3 are it is found that when PEG/MMAE is 0.5, and PEG-rTRAIL-MMAE conjugates are to different human tumours
The internal antitumor activity of cell Nude Mouse Model is than rTRAIL-MMAE conjugates and PEG-rTRAIL conjugates
Height shows that under certain PEG/MMAE ratios (0.5), the collaboration of PEG, rTRAIL mutant tripolymer and MMAE three are made
It is best with reaching.
2, PEG-rTRAIL-MMAE conjugates are compared with the internal antitumor activity of rTRAIL-MMAE conjugates
It selects Non-small cell lung carcinoma cell line NCI-H460 as model cell, NCI-H460 is transplanted naked in Balb/c
Mouse subcutaneously builds human tumour Transplanted tumor model.
By taking PEG/MMAE ratios are 1 PEG-rTRAIL-MMAE conjugates as an example, when tumour is grown to 50~100mm3Size
When, (it is divided into two groups according to the dosage of design：The dosage of one of which is each 4mg/kg, wherein about containing 65 μ g/kg
MMAE；Another group of dosage is each 24mg/kg, wherein about containing 390 μ g/kgMMAE) start tail vein injection administration simultaneously
Tumor size is measured every other day, is administered every other day later, is administered four times altogether；Tumor size (V) measurement method is to be distinguished with vernier caliper
The radial diameter (L) and trans D (L) of tumour are measured, tumor size calculation is V=L*W2/2。
Meanwhile using physiological saline as blank control group；Made with the conjugate BSA-MMAE of bovine serum albumin(BSA) and MMAE
For non-binding control groups (dosage is each 10mg/kg, wherein about contain 390 μ g/kgMMAE), because BSA point
Son amount (66kDa) it is similar to molecular weight (67kDa) of rTRAIL mutant N109C tripolymers, therefore setting BSA-MMAE to
The effect for excluding conjugate is caused by EPR (penetrability and retention effect of enhancing) effect of large biological molecule substance；
With rTRAIL114-281(dosage is each 10mg/kg) and MMAE (dosage is every time 390 μ g/kg) are single
Solely experimental comparison group of the administration as conjugate, dosage respectively in PEG-rTRAIL-MMAE (PEG/MMAE ratios are 1)
The parts TRAIL and MMAE are equivalent；
Simultaneously be arranged rTRAIL-MMAE experimental groups (PEG/MMAE 0) (dosage be each 10mg/kg, wherein containing about
Have 390 μ g/kg MMAE), to analyze influences of the PEG to PEG-rTRAIL-MMAE conjugates.
The gross tumor volume comparison result of each experimental mice is shown in Fig. 6.
As seen from Figure 6, rTRAIL-MMAE and PEG-rTRAIL-MMAE (24mg/kg, wherein about containing 390 μ g/kg
MMAE extremely significant antitumous effect) is all shown, and the antitumor activity of PEG-rTRAIL-MMAE is better than rTRAIL-
MMAE conjugates show that PEG couplings have the antitumor activity of PEG-rTRAIL-MMAE and are obviously improved effect.
Also, PEG-rTRAIL-MMAE conjugates only need four intravenously administrables, the growth of tumour just obviously to be inhibited,
Antitumor activity be substantially better than PEG-rTRAIL conjugates disclosed in patent No. ZL201310342861.3 (daily administration, continuous 9
It).
And rTRAIL114-281It is administered alone group, MMAE is administered alone group, the antitumor activity of BSA-MMAE groups compares physiology
Brine group does not show significant difference.And PEG-rTRAIL-MMAE is in low dosage (4mg/kg, wherein about containing 65 μ g/kg
Apparent antitumous effect is still shown when MMAE).
To further confirm that the antitumous effect of each experimental group, humanity put to death each experimental mice and take out tumour weigh,
It takes pictures, the results are shown in Figure 7.As seen from Figure 7, tumor weight is consistent with the tumor size variation that Fig. 6 is shown.
3, Apoptosis situation in TUNEL technologies detection tumor tissue section
TdT-mediated dUTP-biotin nick end labeling (TUNEL) are that one kind is withered for detecting cell
Die the analysis method of (apoptosis).Apoptosis has one important to be characterized as that intracellular DNA can be fragmented into segment (DNA
Fragmentation), TUNEL can effectively detect the generation of DNA fragmentation, it is possible to convey the message of Apoptosis.Cause
This 24 hours after the 4th administration, takes out a mouse, stripping from each experimental group at random during the zoopery of part 2
From tumour, it is made as histotomy, is used in combination TUNEL methods to detect apoptosis of tumor cells situation, to confirm the antitumor effect of the sample
Fruit is produced by inducing apoptosis of tumour cell.
The results are shown in Figure 8, and other than physiological saline group, remaining each experimental group all shows the Apoptosis that degree differs
Situation, and wherein rTRAIL-MMAE and PEG-rTRAIL-MMAE (24mg/kg, wherein about containing 390 μ g/kg MMAE, PEG/
MMAE ratios are that 1) experimental group presents most Apoptosis, followed by MMAE and rTRAIL114-281.The result with the 2nd
The testing result divided is consistent, shows that conjugate generates final antitumor activity by inducing apoptosis of tumour cell.
Detect the oxicity analysis of 3 PEG-rTRAIL-MMAE conjugates of example
The model of nude mice bearing tumor in example 2 is detected after pharmacodynamic experiment, takes out the potential toxicity target organs of TRAIL
(such as liver, lung and kidney), is made histotomy, is dyed for HE.Finally utilize cell in each histotomy of histologic analysis
Lesion situation, the results are shown in Figure 9.
As seen from Figure 9, MMAE groups cause serious stethemia, considerably beyond a small amount of caused by cervical dislocation nude mice
The degree of pulmonary congestion, in addition visible pulmonary branches tracheal epithelial cell severe detachment, shows to cause nude mice when MMAE is administered alone
Serious pulmonary toxicity；And local hepatocellular injury (as shown by arrows) caused by MMAE is also shown by hepatic tissue section, this is then
Show that MMAE equally causes the hepatic injury of nude mice.And rTRAIL114-281, rTRAIL-MMAE, PEG-rTRAIL-MMAE group then
Visible toxicity is not shown, cellular morphology is similar to physiological saline group in each histotomy, shows rTRAIL114-281、
RTRAIL-MMAE and PEG-rTRAIL-MMAE does not cause side effect, i.e. MMAE and rTRAIL while playing antitumor activity
Or significantly reduce the cytotoxicity of itself after PEG-rTRAIL couplings.
In addition, as shown in Figure 10, the tumor bearing nude mice weight by monitoring each experimental group can be also found that the lotus of only MMAE groups
Tumor nude mouse weight rapidly declines upon administration, and to 20 grams hereinafter, showing that MMAE causes general toxicity, and other group of lotus knurl is naked
Mouse weight fluctuates in normal range (NR), and the result is consistent with the result of Figure 10.
Detect pharmacokinetic of the 4 PEG-rTRAIL-MMAE conjugates of example in SD rat bodies
Whether enhanced compared to rTRAIL-MMAE conjugates by the modification of PEG for research PEG-rTRAIL-MMAE conjugates
Pharmacokinetic property, such as Increased Plasma Half-life, bioavilability improve etc., select the SD rats as Research of Animal Model for Study
The PEG-rTRAIL-MMAE conjugates of rTRAIL mutant, rTRAIL-MMAE conjugates and difference PEG/MMAE ratios
Pharmacokinetics.
Pass through eye socket blood taking method timing acquiring rat serum after administration (being administered once, dosage is every SD rat 1mg)
Sample prepares serum, and blood concentration of each sample in SD rat bodies is detected followed by the mode of ELISA.As a result such as Figure 11
Shown in table 4, table 5.
As seen from Figure 11, the pharmacokinetics that the blood concentration-time curve of PEG-rTRAIL-MMAE conjugates is reflected
Property is better than rTRAIL mutant and rTRAIL-MMAE conjugates.
Table 4
Note：AUCinfIndicate that 0 arrives just infinite area under the curve (mg/litre * hours), t1/2Indicate half-life period (hour),
CL indicates clearance rate (l/h/kilogram), similarly hereinafter.
By table 4 as it can be seen that the half-life period of PEG-rTRAIL-MMAE conjugates has reached 11.54 ± 6.20 hours, considerably beyond
RTRAIL-MMAE couplings in rTRAIL mutant (1.91 ± 0.94 hours) and patent of invention (ZL201210416648.8)
Object (4.26 ± 3.34 hours), also superior to the half-life period of the PEG-rTRAIL in patent of invention (ZL201310342861.3)
(276.68 ± 85.51min, about 4.61 ± 1.42 hours).As it can be seen that the coupling of MMAE also significantly extends PEG-rTRAIL-
The half-life period of MMAE conjugates.Meanwhile in terms of bioavilability and clearance rate be also PEG-rTRAIL-MMAE conjugates more
It is advantageous.The dual modification for showing rTRAIL mutant experience PEG and MMAE, moving property to the medicine of the conjugate obtained improves
Obviously, one of the principal element that its internal antitumor activity in detecting example 2 improves is eventually become.
Table 5
By table 5 as it can be seen that when the ratio higher of PEG/MMAE, the medicine of PEG-rTRAIL-MMAE conjugates moves property more preferably,
It is longer etc. including bioavilability (AUC) higher, half-life period.Also, the PEG-rTRAIL-MMAE that PEG/MMAE ratios are 5 is even
The half-life period for joining object is 9.06 ± 3.29 hours, the significantly larger than half-life period of PEG-rTRAIL conjugates；Show the coupling of MMAE
Also the medicine for substantially improving PEG-rTRAIL-MMAE conjugates moves property.
Detect the cancer target capability study of 5 PEG-rTRAIL-MMAE conjugates of example
MMAE and rTRAIL mutant N109C tri- is directly replaced using water-soluble Sulfo-Cyanine5maleimide
Aggressiveness reacts, and the PEG-rTRAIL-Cy5 with different PEG/Cy5 ratios is prepared according to method same as Example 1, to
Simulate the behavior of the PEG-rTRAIL-MMAE conjugates with different PEG/MMAE ratios in animal body.Sulfo-
The molecular weight of Cyanine5maleimide is 803, and the MMAE containing attachment is 1316.63, and the two molecular weight is closer to,
The change degree of its structure also will be closer to after modification rTRAIL.In addition, utilizing bovine serum albumin(BSA) BSA and fluorescent dye
Cy5 is coupled in the same way, is compareed as non-binding, and the shadow of EPR (increased penetrability and anelasticity) effect is excluded
It rings, as a result as shown in figure 12.
As seen from Figure 12, PEG-rTRAIL-Cy5 can specifically be targeted to tumor locus, and BSA-Cy5 is then complete always
Status cloth does not show the ability of targeting, this show PEG-rTRAIL-MMAE conjugates can active targeting deliver drug to tumour
Position.The result with the MMAE in detection example 2 due to there is no targeting that cannot inhibit tumour growth, and PEG-rTRAIL-MMAE
Then since its targeting ability can effectively inhibit the result of tumour consistent.
Claims (4)
1. a kind of polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate, the amino acid sequence of rTRAIL mutant
As shown in SEQ ID No.1, which is characterized in that coupling has mono methoxy polyethylene glycol horse on the rTRAIL mutant tripolymer
Take the aplysiatoxin of acid imide and band connection arm,
Wherein, rTRAIL mutant tripolymer, mono methoxy polyethylene glycol maleimide, band connection arm aplysiatoxin rub
You are than being 1:(1~2):(2~1),
The molecular weight of the mono methoxy polyethylene glycol maleimide is 3000~15000Da,
The linking arm is the amine-modified valine-citrulline dipeptides of maleimide,
The preparation method of the polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate, includes the following steps：
(1) it by rTRAIL mutant polymer depolymerization, regroups to obtain rTRAIL mutant tripolymer；
(2) by rTRAIL mutant tripolymer and mono methoxy polyethylene glycol maleimide hybrid reaction, polyethylene glycol-is obtained
RTRAIL mutant tripolymer conjugate；
(3) by the aplysiatoxin hybrid reaction of polyethylene glycol-rTRAIL mutant tripolymer conjugate and band connection arm, institute is obtained
State polyethylene glycol-rTRAIL mutant tripolymer-aplysiatoxin conjugate.
2. polyethylene glycol as described in claim 1-rTRAIL mutant tripolymer-aplysiatoxin conjugate prepare it is antitumor
Application in drug.
3. application as claimed in claim 2, which is characterized in that the antitumor drug has dead for inducing cell surface expression
Die the apoptosis of tumor cells of receptor.
4. application as claimed in claim 3, which is characterized in that the antitumor drug is drugs against colon cancer, anti-leukemia medicine
Object, ovarian cancer resistance medicament, anti-gastric cancer medicament, anti-lung-cancer medicament, anti-breast cancer medicines or medicines resistant to liver cancer.
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