CN109939242A - Antibody prodrug conjugate and its preparation and application - Google Patents
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Abstract
The invention discloses a kind of antibody prodrug conjugate and its preparations and application.The antibody prodrug conjugate is coupled by the shielded prodrugs of antibody and functional group by the connexon that microenvironment responds, structure are as follows:Wherein Ab is antibody, and L represents the connection unit of connection antibody and drug molecule, and D represents drug molecule, and X is protected functional group on drug molecule, and Caging represents blocking group.Antibody prodrug conjugate of the invention has the tissue penetration enhanced and lower toxic side effect, can be used for treating tumour, immunological diseases or infectious diseases.Particularly, the antibody prodrug conjugate of target tumor is released prodrugs by tumor microenvironment stimulation, prodrugs do not kill normal cell, it is deep into after tumor tissues by specific activation, tumour penetration power is improved relative to existing ADC drug molecule, toxic side effect is reduced, good oncotherapy effect is shown as.
Description
Technical field
The present invention relates to biotech drug fields, and in particular to a kind of preparation side of antibody prodrug conjugate (ProADC)
Method and application thereof.
Background technique
In tumor area, chemotherapy is still one of current most important treatment means, but due to toxic side effect etc.,
Significantly limit its development.The appearance of antibody drug conjugates (ADC) provides a kind of new resolving ideas for the above problem.
By the way that drug molecule and antibody to be coupled, drug molecule is transported to tumour week using the targeting of antibody molecule by people
It encloses, to realize that tumour-specific kills.Up to the present, there have been 4 ADC drug listings in the whole world, has more than 40 ADC medicines
Object is in clinical development, and on the whole, ADC drug development has a extensive future.
But for ADC drug, due to size is too big, cell internalizing rate is slow etc., ADC drug entities are penetrated
Ability is poor, therefore function and effect are bad in treatment of solid tumors.In this case, pass through the connexon of tumor microenvironment sensitivity
Drug molecule is connected with antibody, people have devised the ADC drug of tumor microenvironment response release.What this mode obtained
ADC drug can release the small-molecule drug that size is small, diffusion is fast after reaching around tumour, thus realize better tumour
Penetration power.But the ADC drug of this tumour release still remains problem.Due to the company of most of tumor microenvironment response
Connecing son all has poor metabolic stability, along with the heterogeneity of tumor microenvironment, the ADC drug molecule meeting of many release types
There is the case where early release small-molecule drug.These small-molecule drugs can be diffused into the normal cell closed on, cause onlooker
Toxicity.
Therefore, current ADC drug there are aiming at the problem that, need one kind and can either solve drug entities penetration power again can
Reduce the new method of poisonous side effect of medicine.
Summary of the invention
To solve the above problems, the present invention is directed to develop a kind of antibody prodrug conjugate (ProADC).Pass through tumour micro-loop
Antibody and prodrugs are carried out coupling and form antibody prodrug conjugate by the connexon of border response.Antibody prodrug conjugate is targeting
After tumour, is stimulated by tumor microenvironment, release prodrugs.Prodrugs do not kill normal cell, are deep into
It can be by specific activation after tumor tissues.The present invention improves the tumour penetration power of ADC drug molecule on the whole, reduces drug
Molecule toxic side effect.
Specifically, the present invention provides a kind of antibody prodrug conjugate as shown in Equation 1 or its is pharmaceutically acceptable
Salt.
In formula 1, Ab is antibody；
L represents the optional connection unit that can be used for antibody and connect with drug molecule；
D represents drug molecule；
X is protected functional group on drug molecule；
Caging represents blocking group；
N is the integer greater than 0.
Preferably, Ab is full length antibody or antibody fragment with cancer target effect in formula 1.The usual full length antibody
Comprising two heavy chains and two light chains, pass through disulfide-bonded between heavy chain and light chain.The antibody fragment is to be tried by chemistry
The antibody for the non-overall length that agent or genetic engineering obtain.
Ab is full human monoclonal antibody Trastuzumab in an application example of the inventionNeedle
To Her2 target spot, for treating the highly expressed tumour of Her2 receptor.
Preferably, L is that tumor microenvironment responds the connexon being broken in formula 1.
It is furthermore preferred that L is the connection of the connexon of tumor microenvironment pH response, the connexon of reduction response or enzyme response
Son.Wherein, the connexon of pH response includes but is not limited to brown key, carbonic acid ester bond, phosphoric acid ester bond and its derivative, restores response
Connexon includes but is not limited to the disulfide bond of glutathione (GSH) response, hydrogen peroxide (H2O2) response phenyl boric acid ester bond and its
Derivative, the connexon of enzyme response include but is not limited to the chemical bond of cathepsin B (Cathepsin B) cutting, phosphate
The chemical bond and its spread out that chemical bond, the carboxy-lesterase (Carboxylesterase) of enzyme (Phosphoesterase) cutting are cut
Biology.
In an application example of the invention, L is the connexon of tumor microenvironment pH response, and specific structure is formula 2
The sulfydryl to dissociate on maleimide structure and antibody in compound represented occurs 1,4 addition reactions and forms stable C-S
Carbonyl reaction on key and hydrazide structure and drug molecule forms the acyl palm fibre key to tumor microenvironment pH response, to will resist
Body and drug molecule connect.
Preferably, D is DNA alkylating agent, DNA intercalator, tubulin binding agent, enzyme inhibitor, immunoregulation agent in formula 1
Deng.
D is adriamycin in an application example of the invention.Acyl on compound shown in 13 carbonyls of adriamycin and formula 2
Hydrazine forms the acyl palm fibre key of tumor microenvironment pH response.
Preferably, X is amino, sulfydryl, hydroxyl or the carboxyl on selected drug molecule in formula 1.
X is the amino in drug molecule adriamycin saccharide ring in an application example of the invention.
Preferably, Caging is the trans- cyclo-octene protecting group (TCO) of tetrazine molecule removing, transition metal removing in formula 1
Allyl-based protection, transition metal removing propargyl protecting group or its analog, light removing adjacent nitre benzyl (ONB) or its
Analog, TCO removing to nitrine benzyl (PAB) or its analog.
Caging is the trans- cyclo-octene protecting group (TCO) of tetrazine molecule removing in an application example of the invention,
Wherein the tetrazine molecule is 1,2,4,5- dimethyl tetrazine.
Antibody prodrug conjugate of the invention in the preparation, usually first carries out the protection of functional group, so to drug molecule
The protected drug molecule of functional group is reacted with connexon afterwards, then is connect with antibody, and the antibody prodrug conjugate is obtained.
Below with the adriamycin prodrug TCO-Dox-Mal and its antibody of the trans- cyclo-octene protection of tumor microenvironment pH response
For prodrug conjugate Ab-Dox-TCO, illustrate the preparation flow of antibody prodrug conjugate of the invention.Those skilled in the art
It should be appreciated that each step can be modified and be supplemented according to techniques known based on following preparation thinkings, from
And prepare the various antibody prodrug conjugates of different compositions, structure and purposes.
The preparation process of antibody prodrug conjugate Ab-Dox-TCO the following steps are included:
1) in organic solvent by adriamycin (Dox) and compound 2, organic base is as catalyst, under the conditions of 10-100 DEG C
Reaction overnight, obtains compound 3；
2) compound 3 and 6- maleimide hexanoyl hydrazine trifluoroacetate (6-Maleimidocaproic Acid
Hydrazide, Trifluoroacetic Acid) in organic solvent, organic acid overnight, is obtained as catalyst, room temperature reaction
The adriamycin prodrug TCO-Dox-Mal (compound 4) of the trans- cyclo-octene protection responded to tumor microenvironment pH；
3) compound 4 and the trastuzumab monoclonal antibody (HS-Ab) restored are in PBS buffer solution system, 0~60 DEG C of condition
Lower reaction 0.5-24h obtains antibody prodrug conjugate Ab-Dox-TCO.
Above-mentioned steps 1) described in organic solvent such as N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), two
Chloromethanes (DCM), tetrahydrofuran (THF) etc.；Organic bases such as triethylamine, ethylenediamine, piperidines, N as catalyst, N- bis- are different
Propylethylamine (DIEPA), pyridine, dimethylamino naphthyridine (DMAP) etc..
Above-mentioned steps 2) reaction organic solvent such as methanol (MeOH) used, ethyl alcohol, ether, tetrahydrofuran (THF), 1,
Six alkane of 4- epoxy etc.；Organic acids such as formic acid, acetic acid, trifluoroacetic acid (TFA) as catalyst etc..
Antibody prodrug conjugate or its pharmaceutically acceptable salt provided by the invention, and drug based on this
Combination, can be used for treating tumour, immunological diseases or infectious diseases.The connexon responded by tumor microenvironment by antibody and
Prodrugs connect, and form antibody prodrug conjugate, have the tumor tissues penetration power and lower toxic side effect of enhancing,
It is eventually exhibited as good oncotherapy effect.
Detailed description of the invention
Fig. 1 is the preparation flow figure of antibody prodrug conjugate Ab-Dox-TCO.
Fig. 2 is that SDS-PAGE characterizes antibody prodrug conjugate Ab-Dox-TCO, wherein (a) is coomassie brilliant blue staining knot
Fruit (b) is fluorescence imaging result.
Fig. 3 is antibody prodrug conjugate Ab-Dox-TCO penetration power test result under tumour slightly acidic condition.
Fig. 4 is that tumor cytotoxicity of the antibody prodrug conjugate Ab-Dox-TCO under tumour slightly acidic condition acts on test
As a result.
It is good that Fig. 5 shows that antibody prodrug conjugate Ab-Dox-TCO has on Her2MDA-MB-231 tumor model
Targeting.
Fig. 6 is therapeutic effect figure of the antibody prodrug conjugate Ab-Dox-TCO on Her2MDA-MB-231 tumor model,
Wherein A and B is respectively the gross tumor volume and mouse weight versus time curve of different experiments group mouse.
Specific embodiment
The conjunction of the adriamycin prodrug (TCO-Dox-Mal) of the trans- cyclo-octene protection of 1. tumor microenvironment pH of embodiment response
It is as follows at the synthetic route of TCO-Dox-Mal:
(1) synthesis of compound 1
Cyclo-octene -2- alcohol ((Z)-cyclooct-2-en-1-ol) (8.5g, 67mmol), benzoic acid is added in 1L quartz bottle
Methyl esters (methyl benzoate) (9.2g, 67mmol), 500mL anhydrous ether and 1000mL n-hexane.Reaction solution passes through illumination
It reacts for 24 hours, optical source wavelength 254nm, intensity 300W.During light reaction, reaction system is recycled by peristaltic pump, is passed through
Silver nitrate silicagel column (silver nitrate and silica gel quality score ratio be 25:200) carries out product enrichment, and wriggling flow rate pump is 18mL/
min.After reaction, silver nitrate silica gel is taken out, 500mL ammonium hydroxide is added and stirs 5min, is then extracted with 1000mL methylene chloride
It takes.Merge organic phase, after taking out solvent by Rotary Evaporators, is purified with column chromatography.Eluent system is ethyl acetate: stone
Oily ether=1:50 (volume ratio).Purifying obtains compound 1.1H NMR(CDCl3, 400MHz) and δ (ppm): 5.98-5.91 (m, 1H),
5.59-5.55(d,1H),4.61(s,1H),2.50-2.47(m,1H),2.07-1.81(m,4H),1.70-1.42(m,4H),
1.15-1.06(m,1H),0.8-0.72(m,1H).GC-MS/MS(m/z):calcd.for C8H14O,126.10392；found
126.10415.
(2) synthesis of compound 2
Compound 1 (304mg, 2.41mmol) and 15mL methylene chloride are added in 50mL single port bottle.System is cooled to 0 DEG C
Afterwards, it is separately added into dimethylamino naphthyridine (4- (N, N-Dimethylamino) pyridine) (1.16g, 9.50mmol) and to nitre
Base phenyl chloroformate (4-nitrophenylchloroformate) (0.9g, 4.46mmol).Then by reaction system holding chamber
Temperature is stirred to react overnight.After reaction, solvent is removed with Rotary Evaporators, then carries out column chromatographic purifying.Column chromatographic elution
System is ethyl acetate: petroleum ether=1:10 (volume ratio).Purifying obtains compound 2.1H NMR(CDCl3, 400MHz) and δ
(ppm):8.29-8.26(d,2H),7.41-7.38(d,2H),6.02-5.94(m,1H),5.59-5.54(d,1H),5.44(s,
1H),2.55-2.50(m,1H),2.25-2.20(dd,1H),2.11-1.69(m,4H),1.60-1.50(m,2H),1.26-
1.13(m,1H),0.88-0.8(m,1H).GC-MS/MS(m/z):calcd.for C15H17NO5,291.11067；found
291.11075.
(3) synthesis of compound 3
Compound 2 (20mg, 0.0687mmol) is dissolved in 2mL DMF solvent, N, N- diisopropyl are then respectively adding
Base ethamine (Diisopropylethylamine) (80mg, 0.62mmol) and doxorubicin hydrochloride (Doxorubicin
hydrochloride)(45mg,0.0776mmol).Reaction system is stirred overnight at room temperature, after reaction, with rotation
Turn evaporimeter removal solvent, is purified by column chromatography.Column chromatographic elution system is CH2Cl2: MeOH=50:1 (volume ratio).
Purifying obtains compound 3.1H NMR(CDCl3, 400MHz) and δ (ppm): 13.97 (s, 1H), 13.23 (s, 1H), 8.04-8.02
(d,1H),7.80-7.76(dd,1H),7.39-7.37(d,1H),5.81-5.70(m,1H),5.50-5.43(m,2H),5.28-
5.23(d,2H),5.09-5.07(d,1H),4.74(s,2H),4.16-4.11(q,1H),4.07(s,3H),3.86(s,1H),
3.68(s,1H),3.28-3.24(d,1H),3.03-2.98(d,2H),2.42(m,1H),2.34-2.30(d,1H),2.18-
2.14(d,1H),2.03-1.77(m,7H),1.61(m,3H),1.47-1.44(m,1H),1.29-1.28(d,3H),1.01(m,
1H),0.75(m,1H).HRMS(m/z):calcd.for C36H41NO13,695.25779；found[M+H]+:
696.26570,[M+NH3+H]+:713.29153,[M+Na]+:718.24760.
(4) synthesis of compound 4
Compound 3 (69.5mg, 0.1mmol) is dissolved in 20mL dehydrated alcohol, 6- maleimide is then sequentially added
Amine hexanoyl hydrazine trifluoroacetate (6-maleimidocaprohydrazide trifluoroacetate salt) (101.8mg,
0.3mmol) and 5.6 μ L trifluoroacetic acids (TFA).Reaction system is protected from light stirring 12h at normal temperature, after reaction by reaction system
It is diluted with methanol, is purified by HPLC.HPLC purification condition is that 5-95% acetonitrile carries out gradient elution, elution flow rate
For 25mL/min.Purifying obtains compound 4 (TCO-Dox-Mal).1H NMR(CDCl3, 400MHz) δ (ppm): 13.98 (s,
1H),13.23(s,1H),10.21(s,1H),8.04-8.02(d,1H),7.80-7.76(t,1H),7.40-7.38(d,1H),
6.66(s,2H),5.81-5.70(m,1H),5.53-5.44(m,2H),2.28-2.23(d,2H),4.76-4.67(d,2H),
4.15-4.11(m,1H),4.08(s,3H),3.88(s,1H),3.69-3.67(d,1H),3.52-3.48(m,2H),3.33-
3.24(m,1H),2.98-2.90(d,1H),2.57-2.14(m,3H),2.08-1.80(m,6H),1.70-1.54(m,4H),
1.30-1.24(m,8H),1.06-0.97(m,1H),0.87-0.84(m,1H),0.79-0.71(m,1H).13C NMR(CDCl3,
500MHz)δ(ppm):213.93,187.09,186.68,170.91,162.83,161.04,156.18,155.67,155.06,
135.77,135.50,134.08,134.04,133.61,131.78,131.25,120.88,119.85,118.44,111.58,
111.38,100.72,74.13,69.68,69.58,67.30,65.55,60.42,56.69,53.43,46.83,40.61,
37.73,36.67,35.87,34.01,31.59,30.24,28.31,26.47,24.05,22.66,21.06,16.86,
14.20.HRMS(m/z):calcd.for C46H54N4O15,902.35857；found[M+NH3+H]+:920.39239.
The preparation of 2. antibody prodrug conjugate Ab-Dox-TCO of embodiment and the measurement of DAR
The preparation flow of antibody prodrug conjugate is referring to Fig. 1.10mg trastuzumab monoclonal antibody is dissolved in 10mL PBS
In solution, DTT (33.3 μ L, 3.33 μm of ol) is then added in 37 DEG C of reduction 30min.After reaction, it will restore
Trastuzumab monoclonal antibody carries out desalination with 10 desalting column of PD, is then centrifuged for being concentrated to 10mg/mL spare.It is ready to 100mM's
TCO-Dox-Mal DMSO solution takes 33.3 μ L to be added in the monoclonal antibody solution after above-mentioned 10mg/mL is restored.Then reaction system
In 4 DEG C of progress 1h.After, it carries out repeating desalination and concentration, finally obtains antibody prodrug conjugate.Wherein, antibody prodrug is coupled
The concentration of object measures (1mg/mL=1.4AU) by A280 on Nanodrop.The concentration of Dox is inhaled by 495nm in UV detector
Receiving light acquisition, (Dox absorptivity is ε 495=8030cm-1M-1).Thus obtained antibody coupling matter ultimate density is 66.67 μ
M, DAR's is 3.
Embodiment 3.SDS-PAGE characterizes antibody prodrug conjugate
The antibody for taking 16 μ L10mg/mL antibody prodrug conjugate Ab-Dox-TCO and equal volume, concentration, is separately added into 4 μ
L SDS-PAGE sample-loading buffer, then boils sample 30min at 95 DEG C, albumen is allowed sufficiently to be denaturalized.Then the 10 well-done samples of μ L are taken,
Carry out SDS-PAGE electrophoresis.Deposition condition is 12% polyacrylamide gel, 100V voltage 1h.After, gel is carried out to examine horse
The dyeing of this brilliant blue, is developed after decoloration with ChemiDoc XRS+molecular imager (Molecular Imager), such as Fig. 2 institute
Show.There is Comassie channel and fluorescence channel (exct:488nm) in the channel wherein used
The measurement of 4. antibody prodrug conjugate penetration power under the conditions of tumour is slightly sour of embodiment
First by layer overlay DMEM Agar substrate glue in 96 orifice plates (containing 2mg agar in 100mL DMEM).To matrix
After gelling is solid, plant into the highly expressed MDA-MB-231 breast cancer cell of Her2 receptor.Implantation methods are every 100 μ L of hole, are planted close
Degree is 10000, every hole cell, and cell culture condition is 5%CO at 37 DEG C2, the culture medium used is containing 10%FBS's
DMEM.After cell culture 2 weeks, in light microscopic observation 3D cell mass upgrowth situation and form, pick out that form is round and smooth, and size exists
100-500 μm of 3D cell carries out penetration power measurement.
Then, antibody prodrug conjugate Ab-Dox-TCO is respectively placed in normal physiological conditions pH 7.4 and simulation tumour is micro-
It is incubated in the PBS solution of environment pH 6.5 for 24 hours, drug concentration is 1mM (calculating by Dox content), incubation temperature 37 at this time
℃.Then, the antibody prodrug conjugate Ab-Dox-TCO under the conditions of two kinds is separately added into above-mentioned ready 3D cell, medicine
Object ultimate density is 10 μM, and incubation conditions are 5%CO at 37 DEG C2, it is incubated for 3h.After experiment, by 3D cell with 100 μ L PBS
It washes 3 times, imaging experiment is then carried out by confocal microscope.Imaging pattern is z-staking, scanning range 5-50
μm, excitation wavelength 488nm.
Experimental result is as shown in Figure 3, it has been found that antibody prodrug conjugate has better under tumour slightly acidic environment
Tumour penetration power, penetration power is up to 50 μm.
The measurement of 5. antibody prodrug conjugate cellkilling capacity under the conditions of tumour is slightly sour of embodiment
The highly expressed MDA-MB-231 breast cancer cell of Her2 receptor is planted first in 96 orifice plates.Implantation methods are every hole
100 μ L, planting density are 2000, every hole cell, and cell culture condition is 5%CO at 37 DEG C2, the culture medium that uses be containing
The DMEM of 10%FBS.Then, antibody prodrug conjugate Ab-Dox-TCO is respectively placed in normal physiological conditions pH7.4 and simulation
It is incubated in the PBS solution of tumor microenvironment pH 6.5 for 24 hours, drug concentration is 1mM (calculating by Dox content) at this time, is incubated for temperature
Degree is 37 DEG C.After cell culture 1 day, it is separately added into the above-mentioned antibody prodrug conjugate of various concentration, continues incubated cell.Carefully
Born of the same parents cultivate the 2nd day, are washed cell 3 times with 100 μ L PBS, and replacement fresh culture continues cell culture.Then it is added in experimental group
The prodrug activator 1,2,4,5- dimethyl tetrazine (Me of 1.5 times of equivalents2TZ), isometric PBS is then added in control group.Cell training
It supports the 4th day, is washed cell 3 times with 100 μ L PBS, replace fresh culture.MTS reagent (20 hole μ L/) is added to continue to be incubated for 1h, so
The each group of light absorption value at 490nm is recorded under microplate reader afterwards, cell viability is calculated with this.
By experimental result it was found that either under normal physiological conditions or under tumour slightly acidic condition, we
Antibody prodrug conjugate cannot all kill tumour cell.As addition prodrug activator 1,2,4,5- dimethyl tetrazine (Me2TZ after)
The activity of prodrug can effectively be restored.Particularly, our antibody prodrug conjugate is before addition under tumour slightly acidic condition
Medicine activator 1,2,4,5- dimethyl tetrazine (Me2TZ) there is optimal tumor-killing effect afterwards, as shown in Figure 4.
Targeting ability measurement of the 6. antibody prodrug conjugate of embodiment in Her2MDA-MB-231 tumor model
We have prepared the highly expressed MDA-MB-231 mouse tumor model of Her2 receptor first.Take 3-4 weeks female
BABL/C nude mice 10,2 × 10 are inoculated with respectively at oxter6A highly expressed MDA-MB-231 cell of Her2 receptor, then in nothing
It is raised under bacterium grade environment, to tumor size to 100mm3Start to test.
Antibody prodrug is coupled CY5 fluorescence on substance markers, labeling method is to be added 10 in 10mg/mL antibody prodrug conjugate
Reaction system is carried out desalination by 10 desalting column of PD after reaction by NHS-CY5, the 37 DEG C of reaction 2h of times equivalent, then from
It is spare that the heart is concentrated to 10mg/mL.
The antibody prodrug conjugate that CY5 is marked is injected to above-mentioned Her2MDA-MB-231 mouse according to the dosage of 50mg/kg,
Control group then injects the NHS-CY5 of equivalent.After injecting 3h, the distribution of mouse systemic drug is observed in small animal imaging instrument.
The access used in experiment is Cy5channel.
Experimental result is as shown in Figure 5, it can be seen that our antibody prodrug conjugate has extraordinary tumor tissues rich
Collect effect.
Therapeutic effect measurement of the 7. antibody prodrug conjugate of embodiment in Her2MDA-MB-231 tumor model
We have prepared the highly expressed MDA-MB-231 mouse tumor model of Her2 receptor first.Take 3-4 weeks female
BABL/C nude mice 30,2 × 10 are inoculated with respectively at oxter6A highly expressed MDA-MB-231 cell of Her2 receptor, then in nothing
It is raised under bacterium grade environment, to tumor size to 30-50mm3Start to test.
Experiment is divided into three groups, first group of injection 30mg/kg antibody prodrug conjugate, before second group of injection 30mg/kg antibody
Medicine conjugate and 3.3mg/kg prodrug activator 1,2,4,5- dimethyl tetrazine (Me2TZ), third group injects isometric PBS.
Wherein, antibody prodrug conjugate was administered at the 2nd, 6,9 and 12 day, 1,2,4,5- dimethyl tetrazine (Me of prodrug activator2TZ) exist
Subsequent one day is administered.Treatment cycle is 2 weeks, during which observes different groups of gross tumor volume size variations respectively and mouse weight becomes
Change.
Experimental result is as shown in Figure 6, it can be seen that PBS group and the antibody prodrug conjugate not activated are all without tumour
Inhibitory effect, the antibody prodrug conjugate after only activating can play good tumor-killing effect (see A in Fig. 6).By small
Mouse changes of weight is it was found that our antibody prodrug conjugate has biological safety well (see B in Fig. 6).
Claims (13)
1. a kind of antibody prodrug conjugate or its pharmaceutically acceptable salt, the structure of the antibody prodrug conjugate such as 1 institute of formula
Show:
In formula 1, Ab is antibody, and L represents the connection unit of connection antibody and drug molecule, and D represents drug molecule, and X is drug point
Protected functional group on son, Caging represent blocking group, and n is the integer greater than 0.
2. antibody prodrug conjugate as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that Ab in formula 1
For full length antibody or antibody fragment with cancer target effect.
3. antibody prodrug conjugate as claimed in claim 2 or its pharmaceutically acceptable salt, which is characterized in that Ab be for
The full human monoclonal antibody Trastuzumab of Her2 target spot.
4. antibody prodrug conjugate as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that L is in formula 1
The connexon of tumor microenvironment response fracture.
5. antibody prodrug conjugate as claimed in claim 4 or its pharmaceutically acceptable salt, which is characterized in that L is tumour
The connexon of the connexon of microenvironment pH response, the connexon of reduction response or enzyme response.
6. antibody prodrug conjugate as claimed in claim 5 or its pharmaceutically acceptable salt, which is characterized in that L is tumour
The connexon of microenvironment pH response is the sulfydryl hair to dissociate on the maleimide structure and antibody in 2 compound represented of formula
The carbonyl reaction that raw Isosorbide-5-Nitrae addition reaction is formed on C-S key and its hydrazide structure and drug molecule is formed to tumor microenvironment pH
The acyl palm fibre key of response and the connection unit constituted；
7. antibody prodrug conjugate as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that D is in formula 1
DNA alkylating agent, DNA intercalator, tubulin binding agent, enzyme inhibitor or immunoregulation agent.
8. antibody prodrug conjugate as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that X is in formula 1
Amino, sulfydryl, hydroxyl or carboxyl on drug molecule.
9. antibody prodrug conjugate as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that in formula 1
Caging is the trans- cyclo-octene protecting group of tetrazine molecule removing, the allyl-based protection of transition metal removing, transition metal take off
The propargyl protecting group removed or its analog, the adjacent nitre benzyl of light removing or its analog, TCO removing to nitrine benzyl or its
Analog.
10. antibody prodrug conjugate as described in claim 1 or its pharmaceutically acceptable salt, which is characterized in that described anti-
L is the connexon of tumor microenvironment pH response in body prodrug conjugate, and D is adriamycin, and X is the amino in adriamycin saccharide ring,
Caging is the trans- cyclo-octene protecting group of tetrazine molecule removing.
11. the preparation method of any antibody prodrug conjugate of claim 1~10 first carries out functional group to drug molecule
Protection, then reacts the protected drug molecule of functional group with connexon, then connect with antibody, and it is even to obtain the antibody prodrug
Join object.
12. preparation method as claimed in claim 11, which comprises the following steps:
1) in organic solvent by adriamycin and compound 2, organic base reacts overnight as catalyst, under the conditions of 10-100 DEG C,
Obtain compound 3；
2) in organic solvent, organic acid is as catalyst, room temperature for compound 3 and 6- maleimide hexanoyl hydrazine trifluoroacetate
Reaction overnight, obtains compound 4, i.e. the adriamycin prodrug TCO-Dox- of the trans- cyclo-octene protection of tumor microenvironment pH response
Mal；
3) compound 4 and the trastuzumab monoclonal antibody restored react 0.5- under the conditions of PBS buffer solution system, 0~60 DEG C
For 24 hours, antibody prodrug conjugate Ab-Dox-TCO is obtained；
13. claim 1~10 any the antibody prodrug conjugate or its pharmaceutically acceptable salt are treated in preparation
Application in the drug of tumour, immunological diseases or infectious diseases.
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| CN113321610A (en) * | 2020-02-28 | 2021-08-31 | 国家纳米科学中心 | Cy5.5 prodrug for super-resolution fluorescence imaging and preparation method and application thereof |
| CN113321698A (en) * | 2020-02-28 | 2021-08-31 | 国家纳米科学中心 | Monomethyl auristatin E prodrug and preparation method and application thereof |
| CN113321692A (en) * | 2020-02-28 | 2021-08-31 | 国家纳米科学中心 | Adriamycin prodrug, preparation method and application thereof |
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| CN114849039A (en) * | 2022-05-26 | 2022-08-05 | 华中科技大学同济医学院附属协和医院 | Bionic robot system for intestinal drug delivery and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113321610A (en) * | 2020-02-28 | 2021-08-31 | 国家纳米科学中心 | Cy5.5 prodrug for super-resolution fluorescence imaging and preparation method and application thereof |
| CN113321698A (en) * | 2020-02-28 | 2021-08-31 | 国家纳米科学中心 | Monomethyl auristatin E prodrug and preparation method and application thereof |
| CN113321692A (en) * | 2020-02-28 | 2021-08-31 | 国家纳米科学中心 | Adriamycin prodrug, preparation method and application thereof |
| CN113321608A (en) * | 2020-02-28 | 2021-08-31 | 国家纳米科学中心 | Fluorescent prodrug for specifically labeling tumor cells, and preparation method and application thereof |
| CN113321698B (en) * | 2020-02-28 | 2022-08-23 | 国家纳米科学中心 | Monomethyl auristatin E prodrug and preparation method and application thereof |
| CN113321692B (en) * | 2020-02-28 | 2022-10-14 | 国家纳米科学中心 | Adriamycin prodrug, preparation method and application thereof |
| CN113633780A (en) * | 2020-09-11 | 2021-11-12 | 上海邈基生物科技有限公司 | Small molecule conjugate and application thereof |
| CN113633780B (en) * | 2020-09-11 | 2023-08-29 | 上海邈基生物科技有限公司 | Small molecule conjugate and application thereof |
| CN114849039A (en) * | 2022-05-26 | 2022-08-05 | 华中科技大学同济医学院附属协和医院 | Bionic robot system for intestinal drug delivery and preparation method and application thereof |
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