CN107773762A - ADC based on PD L1 antibody coupling chemotherapeutics and its preparation method and application - Google Patents
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- CN107773762A CN107773762A CN201610792030.XA CN201610792030A CN107773762A CN 107773762 A CN107773762 A CN 107773762A CN 201610792030 A CN201610792030 A CN 201610792030A CN 107773762 A CN107773762 A CN 107773762A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to Field of Drug Discovery, and in particular to a kind of ADC based on PD L1 antibody coupling chemotherapeutics and its preparation method and application.The present invention be based on antibody coupling technology, using PD L1 antibody targeting but be not limited only to PD L1 antibody and antineoplastic maytansine（DM1）And the like anti-cancer properties, formed immune drug system；The immune drug system not only remain single PD L1 antibody targeting and independent chemotherapeutics to the strong lethal of tumour, immune drug system also makes substantial amounts of DM1 effectively be enriched to tumor locus, reduce the dosage of medicine, so as to reduce the killing of medicine normal tissue, the toxic side effect of medicine is reduced.
Description
Technical field
The invention belongs to Field of Drug Discovery, and in particular to a kind of ADC based on PD-L1 antibody coupling chemotherapeutics and its
Preparation method and application.
Background technology
Tumour is one of three big diseases of current serious threat human health.Global cancer patients in 2012 and death
All it is being continuously increased, newly-increased cases of cancer has nearly half to appear in Asia, and wherein most is in China, the newly-increased cases of cancer of China
It is in first.In 4 kinds of malignant tumours such as liver, esophagus, stomach and lung, Chinese new cases and the death toll Jun Ju worlds are first
Position.2012 annual datas of national tumour Register issue show that China increases cases of cancer about 3,500,000 newly every year, there are about simultaneously
2500000 people are therefore dead.Pernicious stomach cancer therapeutic effect is poor, and the late period rate of transform is high, and how bad prognosis is.Clinically use at present more
Conventional treatments such as operative treatment, radiotherapy, chemotherapy and immunization therapy, but chemotherapy or radiotherapy can not change the existence of patient
Phase, even for the cancer patient of chemosensitivity first, also it is easy to recur or produce resistance.And enter again after recurring
When row chemotherapy or radiotherapy, curative effect then substantially reduces.Therefore, majority recurrence can not be avoided, for the patient that cure rate is extremely low, it is low
Toxicity, more effective targeted therapy just seem very necessary.In recent years, treated for the antibody target tropism of cell surface molecule
Achieve huge progress.During antineoplaston, because antibody-targeted therapy has specific height, Small side effects, partly declines
The features such as phase is long, have become a kind of method of very promising tumor biotherapy.But monoclonal antibody is present
It is low to the gentle effect of function of tumor.
The content of the invention
In order to overcome the problems of in the prior art, it is an object of the invention to provide one kind to be based on PD-L1 antibody
It is coupled ADC of chemotherapeutics and its preparation method and application.
To achieve these goals and other related purposes, the present invention adopt the following technical scheme that：
The first aspect of the present invention, there is provided a kind of antibody-drug conjugates, including cytotoxic drug, antibody and idol
The coupling chain of len antibody and drug toxicity.
Preferably, the cytotoxic drug is selected from, but not limited to, maytansine DM1.
Preferably, the antibody is selected from, but not limited to, Programmed death ligand-1 PD-L1.
Preferably, the coupling chain is selected from, but not limited to, 3- (2- pyridines two are thio) propionyl hydrazines PDPH.
Preferably, maytansine DM1 and Programmed death ligand-1 PD-L1 molar ratio is (1~2)：1.
Preferably ,-S-S- the groups for being coupled chain PDPH are connected with maytansine DM1-SH groups.
Preferably, it is coupled chain PDPH-NH2Group and Programmed death ligand-1 PD-L1-COOH group is connected.
The second aspect of the present invention, there is provided the preparation method of afore mentioned antibodies-drug conjugates, including：
(1) maytansine DM1 (DM1-PDPH) of the synthesis with hydrazide group group：Take maytansine DM1 and the 3- (sulphur of 2- pyridines two
Generation) propionyl hydrazine PDPH, dissolves, reaction；
(2) the Programmed death ligand-1 PD-L1 of oxidation is prepared：PD-L1 is mixed with oxidation buffer solution, carries out oxygen
Change；
(3) PD-L1-DM1 is prepared：The maytansine DM1 with hydrazides group that step (1) is obtained obtains with step (2)
Oxidation Programmed death ligand-1 mixing, reaction, obtain PD-L1-DM1.
Preferably, in step (1), maytansine DM1 and 3- (2- pyridines two are thio) propionyl hydrazine PDPH is taken to be dissolved in methanol.
Preferably, in step (2), the oxidation buffer solution is sodium iodate oxidation buffer solution.
Preferably, in step (3), the reaction is carried out at room temperature.
The third aspect of the present invention, there is provided purposes of the afore mentioned antibodies-drug conjugates in anti-tumor medicine is prepared.
Preferably, the anti-tumor medicine is selected from, but not limited to, lung cancer therapy medicine.
The fourth aspect of the present invention, there is provided a kind of cancer therapeutics compositions, the active ingredient of described pharmaceutical composition
In contain afore mentioned antibodies-drug conjugates.
Preferably, in described pharmaceutical composition, afore mentioned antibodies-drug conjugates for the pharmaceutical composition active ingredient it
One or sole active ingredient.
Preferably, pharmaceutically acceptable carrier is also included in aforementioned pharmaceutical compositions.
Pharmaceutical composition of the present invention in use, afore mentioned antibodies-drug conjugates as sole active ingredient, it is or foregoing anti-
Body-drug conjugates can mix system as one of active ingredient with one or more pharmaceutically acceptable carriers or excipient
Into the pharmaceutical dosage form of different way of administration.
Preferably, the dosage form of described pharmaceutical composition be tablet, capsule, powder, granule, syrup, solution,
Oral liquid, spirit, tincture, aerosol, powder spray, injection, injection sterile powder, or suppository.Above-mentioned preparation type can
To understand according to the related definition in pharmacy (sixth version, People's Health Publisher, Cui Fude), the preparation of above-mentioned preparation can be with
Prepared according to the method for the related preparations in pharmacy (sixth version, People's Health Publisher, Cui Fude).
The dosage of described pharmaceutical composition is in the scope known to clinician.Afore mentioned antibodies-drug coupling
Thing can change as the effective dose of active component with the order of severity of mode of administration and disease.Most of large-scale lactation is moved
For thing, the taking dose of daily active ingredient afore mentioned antibodies-drug conjugates is about 0.01~1000mg.Preferably, it is grown up
The scope of clinical administration amount is 0.01-200mg/ days, more preferably 0.05-100mg/ days.
The fifth aspect of the present invention, there is provided a kind of method for treating tumour, including forgoing neoplasms medicine is applied to
Patient.
Compared with prior art, the present invention has the advantages that：
The present invention's is intended to the targeting using PD-L1 antibody based on antibody coupling technology but is not limited only to PD-L1 antibody
With the anti-cancer properties of antineoplastic maytansine (DM1) and the like, immune-drug system is formed；Immune-the drug system
Not only remain single PD-L1 antibody targeting and independent chemotherapeutics to the strong lethal of tumour ,-drug system is immunized
Substantial amounts of DM1 is effectively enriched to tumor locus, reduce the dosage of medicine, so as to reduce killing for medicine normal tissue
Wound, reduce the toxic side effect of medicine.
Brief description of the drawings
Fig. 1：DM1 compounds (DM1-PDPH) synthesis schematic diagram with hydrazides group.
Fig. 2：PD-L1-DM1 synthesis schematic diagram.
Fig. 3：DM1 standard curve.
Fig. 4：It is PD-L1-DM1 vitro cytotoxicity design sketch.
Fig. 5：It is PD-L1-DM1 antibody activity flow cytometer detection figure.
Fig. 6：It is PD-L1-DM1 antibody activity laser co-focusing detection figure.
Fig. 7：It is tumor killing effect figure in PD-L1-DM1 bodies.
Fig. 8 is PD-L1-DM1 treatment tumor effect figures.
Embodiment
First, antibody-drug conjugates (antibody-drug conjugates, ADC)
Antibody-drug conjugates (antibody-drug conjugates, ADC) are antibody-targeted therapy medicines of new generation
Thing, it is mainly used in the treatment of cancer and tumour.From structure, ADC medicines by " bullet " medicine (cell toxicity medicament), antibody and
The part of coupling chain 3 of coupled antibody and medicine is formed, and cytotoxic drug is connected into antibody egg by the method for chemical coupling
Bai Shang.Having benefited from the ADCs of stable bond subtype definitely can carry medicine to intracellular, and in peripheral blood and cell table
Seldom degrade in face；Under equal dosage, it can be maintained for a long time in body using stable type ADCs more like sustained release agent is the same
The casualty-producing concentrations of effect without discharging unnecessary toxin so that ADCs in antitumor have more efficient lethal effect and
Smaller adverse reaction.
Therefore, antibody-drug conjugates (antibody-drug conjugates, ADC) had both overcome to a certain extent
Antibody kills
While hindering insufficient, the toxic side effect of Chemotherapeutic Drugs On Normal tissue has been significantly reduced.
2nd, PD-L1
PD-L1 full name are that Programmed Death Ligand-1 (Programmed death ligand-1) are widely present in tumour
On cell.Many research shows at present, the PD-L1 molecules of a variety of human tumor great expressions and patient clinical pathological characters and pre-
After be closely related, turn into tumour detection and Index for diagnosis new biological indicator.Tumour cell is blocked using this part
B7.1's on PD-1 and T cell is mutually distinguishable, so as to escape the monitoring of immune system.
3rd, DM1
DM1 is also referred to as maytansine DM1, English name Maytansine DM1.No. CAS：139504-50-0.
4th, PDPH
PDPH refers to 3- (2- pyridines two are thio) propionyl hydrazine, and English name is 3- (2-Pyridyldithio) propionyl
hydrazide(PDPH)。
5th, PD-L1-DM1
The PD-L1-DM1 of the present invention, belong to antibody-drug conjugates, including cytotoxic drug, antibody and coupling resist
The coupling chain of body and drug toxicity.The cytotoxic drug is selected from, but not limited to, maytansine DM1.The antibody is selected from but unlimited
In Programmed death ligand-1 PD-L1.The coupling chain is selected from, but not limited to, 3- (2- pyridines two are thio) propionyl hydrazines PDPH.
Maytansine DM1 and Programmed death ligand-1 PD-L1 molar ratio is (1~2)：1.
In one embodiment, coupling chain PDPH-S-S- groups are connected with maytansine DM1-SH groups.It is coupled chain PDPH
- NH2Group and Programmed death ligand-1 PD-L1-COOH group is connected.
6th, PD-L1-DM1 preparation method
The PD-L1-DM1 preparation methods of the present invention include step：
(1) maytansine DM1 (DM1-PDPH) of the synthesis with hydrazide group group：Take maytansine DM1 and the 3- (sulphur of 2- pyridines two
Generation) propionyl hydrazine PDPH, dissolves, reaction；
(2) the Programmed death ligand-1 PD-L1 of oxidation is prepared：PD-L1 is mixed with oxidation buffer solution, carries out oxygen
Change；
(3) PD-L1-DM1 is prepared：The maytansine DM1 with hydrazides group that step (1) is obtained obtains with step (2)
Oxidation Programmed death ligand-1 mixing, reaction, obtain PD-L1-DM1.
In one embodiment, in step (1), maytansine DM1 and 3- (2- pyridines two are thio) propionyl hydrazine PDPH is taken to be dissolved in first
In alcohol.
In one embodiment, in step (2), the oxidation buffer solution is sodium iodate oxidation buffer solution.
In one embodiment, in step (3), the reaction is carried out at room temperature.
7th, PD-L1-DM1 purposes
The PD-L1-DM1 of the present invention can be used for preparing anti-tumor medicine.
In one embodiment, PD-L1-DM1 of the invention is prepared into lung cancer therapy medicine, can realize to the good of tumor tissues
Treatment.
Therefore, anti-tumor medicine can be prepared using the PD-L1-DM1 of the present invention, the treatment for tumor disease.
8th, cancer therapeutics compositions
Contain afore mentioned antibodies-drug conjugates in the active ingredient of the cancer therapeutics compositions of the present invention, such as：
PD-L1-DM1.In described pharmaceutical composition, PD-L1-DM1 be the pharmaceutical composition one of active ingredient or it is unique effectively into
Point.Pharmaceutically acceptable carrier is also included in aforementioned pharmaceutical compositions.
" pharmaceutically acceptable " composition applies to people and/or animal and (such as toxicity, stimulated without excessively bad side reaction
And allergy) have rational benefit/risk than material." pharmaceutically acceptable carrier " be for by the present invention meat
Cinnamic aldehyde sends acceptable solvent, suspending agent or the excipient pharmaceutically or on food of animal or people to.Carrier can be liquid
Or solid.
Pharmaceutically acceptable carrier is the various auxiliary materials and/or excipient pharmaceutically commonly used, including but not limited to sugar
Class (such as lactose, dextrose and saccharose), starch (such as cornstarch and potato starch), cellulose and its derivates (such as carboxymethyl
Sodium cellulosate, ethyl cellulose and methylcellulose), tragacanth gum powder, malt, gelatin, talcum, kollag is (such as tristearin
Acid and magnesium stearate), calcium sulfate, vegetable oil is polynary such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil
Alcohol (such as propane diols, glycerine, D-sorbite, mannitol and polyethylene glycol), alginic acid, emulsifying agent (such as Tween, polyoxyethylene
Castor oil), wetting agent (such as NaLS), colouring agent, flavor enhancement, tablet agent, stabilizer, antioxidant, preservative, nothing
Pyrogen water, isotonic salting liquid and phosphate buffer etc.；The carrier can improve the stability, activity and biology of formula as needed
Validity etc..
Pharmaceutical composition of the present invention in use, afore mentioned antibodies-drug conjugates as sole active ingredient, it is or foregoing anti-
Body-drug conjugates can mix system as one of active ingredient with one or more pharmaceutically acceptable carriers or excipient
Into the pharmaceutical dosage form of different way of administration.
The dosage form of described pharmaceutical composition be tablet, capsule, powder, granule, syrup, solution, oral liquid,
Spirit, tincture, aerosol, powder spray, injection, injection sterile powder, or suppository.Above-mentioned preparation type can be according to medicine
Related definition in agent (sixth version, People's Health Publisher, Cui Fude) understands that the preparation of above-mentioned preparation can be according to medicament
The method for learning the related preparations in (sixth version, People's Health Publisher, Cui Fude) is prepared.
The dosage of described pharmaceutical composition is in the scope known to clinician.Afore mentioned antibodies-drug coupling
Thing can change as the effective dose of active component with the order of severity of mode of administration and disease.Most of large-scale lactation is moved
For thing, the taking dose of daily active ingredient afore mentioned antibodies-drug conjugates is about 0.01~1000mg.Preferably, it is grown up
The scope of clinical administration amount is 0.01-200mg/ days, more preferably 0.05-100mg/ days.
9th, the method for treating tumour
Forgoing neoplasms medicine is applied to patient, can be used to treat tumour.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.The test method of unreceipted actual conditions in the following example,
Generally according to normal condition, or the condition proposed by according to each manufacturer.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can be selected between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987 and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1 carries hydrazide group DM1 (DM1-PDPH) synthesis
It is 1 by maytansine DM1 and 3- (2- pyridines two are thio) propionyl hydrazine PDPH mol ratios:1 to 2:1, weighing quality is
73.83mg maytansine DM1,23mg 3- (2- pyridines two are thio) propionyl hydrazine PDPH, after being dissolved in methanol, room temperature is placed at room temperature
2h, or 4 DEG C of placement 4h, obtain the DM1 compounds (DM1-PDPH) with hydrazides group.
PDPH-DM1 synthesis schematic diagram is as shown in Figure 1.
The preparation for the PD-L1 antibody that embodiment 2 aoxidizes
Mixed with 20mM sodium iodate oxidation buffer solution with isometric PD-L1 antibody, be placed in ice bath, lucifuge reaction 30
Minute.Excessive Potassiumiodate is removed using desalting column.
The PD-L1-DM1 of embodiment 3 preparation
By the crosslinking agent modification of embodiment 1 with the DM1 compounds (DM1-PDPH) of hydrazides group and the oxygen of embodiment 2
The PD-L1 antibody mixing of change, reacts 2 hours at room temperature.Excess is removed using PBS reactant, or using desalting column
Crosslinking agent is modified DM1 (DM1-PDPH).
PD-L1-DM1 synthesis schematic diagram is as shown in Figure 2.
4 each PD-L1 of embodiment is coupled DM1 amount
Using ultraviolet spectrophotometry, by determining UV absorptions of the conjugate PD-L1-DM1 at 252nm and 280nm
Value, and carry out by langbobier law that Conjugate ratio is calculated；To DM1 in antibody coupling medicine PD-L1-DM1 is prepared
Concentration Testing.DM1 UV absorption detects DM1 concentration in 252nm using ultraviolet specrophotometer.Dilution proportion first, obtain
Obtained its standard curve：Y=0.0008x+0.0011.200ul is taken to be detected at 252nm again, its absorbance value is
0.08984.By standard curve obtain DM1 concentration be：0.08984=0.0008x+0.0011, x=110.925ug/ml.DM1
Molecular weight is 692.2, and the concentration of antibody is 1mg/mL, antibody molecule amount about 5*104。
Each the quantity of antibody molecule carrying DM1 molecules is：
N (DM1)/N (PD-L1)=(110.925*10-3/692.2)/(1/5*104)=8.0125.
I.e. each PD-L1 antibody molecules carry 8.0125 DM1 molecules.
The cell toxicity test of embodiment 5
To PD-L1 antibody, the toxicity of PD-L1 antibody couplings medicine (ADC, PD-L1-DM1) cell is evaluated.Cell is spread
Plate：Take the logarithm the A549 (gastric carcinoma cells) in growth period, is counted after HMEC-1 (human microvascular endothelial cell (mvec)) digestion, centrifugation,
96 orifice plate middle berth cell densities are 5 × 103/ hole, 100 μ l serum-containing mediums are added per hole, and the blanc cell hole that makes a circle in week is every
It is individual to be supplied with 100 μ l PBS, it is placed on 5%CO2, in 37 DEG C of cell culture incubator overnight.
PD-L1 antibody and PD-L1 antibody couplings DM1 (PD-L1-DM1) are taken respectively, is added in cell hole, and effect antibody is dense
Degree is all 0.02 μ g/ml, and is incubated with cell, in 5%CO2, after 37 DEG C are incubated 24h, old culture medium is discarded, is added
After PBS board-washings 2 times, 100ul is added per hole and contains 10%CCK-8 cell culture fluid in 5%CO2, 2.5h is incubated at 37 DEG C, is made
With the full-automatic ELIASAs of BIO-TEK Elx800 at 450nm wavelength, the light absorption value of sample well is read, and calculate cells survival
Rate；Cells survival rate computational methods：
Survival rate=(OD450scan-OD450nc)/(OD450pc-OD450nc) × 100%
Wherein, OD450sam：Light absorption value of the experimental group under 450nm wavelength；
OD450no：Light absorption value of the negative control group under 450nm wavelength；
OD450pc：Light absorption value of the positive controls under 450nm wavelength.
PD-L1-DM1 vitro cytotoxicity design sketch is as shown in Figure 4.
Specifically, experimental result is as shown in Figure 4 A：Compared to Control group PD-L1-DM1 medicine groups to HMEC-1 cells
Very little is influenceed, no level of signifiance, shows that its biological safety is preferable.
Experimental result is as shown in Figure 4 B：Compared to Control groups, PD-L1 antibody has obvious increasing to A549 stomach cancer cells
Rejection ability is grown, and antibody coupling medicine PD-L1-DM1 groups then have higher cell lethality, and carried compared with PD-L1 antibodyomes
It is high by about one time；
As can be seen here the antibody coupling target-oriented drug and its to oncotherapy effect better than antibody drug in itself.
The antibody activity of embodiment 6 detects
Pair it is the independent chemotherapeutics DM1 of evaluation and PD-L1 antibody coupling chemotherapeutics with the research of cell surface interaction
The important measurement index of thing (PD-L1-DM1).By the research evaluation pair with the interaction of cell surface, can deduce
Internal targeting and the effect being enriched with, and then evaluate its potential using value.Specific experiment process is as follows：
(1) plating cells：Take the logarithm growth period A549 cell dissociations, centrifugation after count, it is close in 24 orifice plate middle berth cells
Spend for 2 × 105/ hole, 500 μ l serum-containing mediums are added per hole, the blanc cell hole that makes a circle in week is each mended with 500 μ l PBS
Foot, is placed on 7%CO2, in 37 DEG C of cell culture incubator overnight.
(2) free DM1, antibody coupling medicine DM1 (PD-L1-DM1) is taken to add in cell hole respectively, with A549 cells
It is incubated, in 5%CO2, 37 DEG C of incubation 6h.
(3) after being incubated 6h, DM1 groups and ADC groups (PD-L1-DM1) are gently washed 2 times with culture medium respectively, and by cell dissociation
Centrifuge in 15ml centrifuge tubes, then washed 2 times (800rpm × 5min) with PBS, concentration is 1 × 106/ ml, each 1ml, use
The secondary antibody (rabbit-anti people) that Alexa Fluor 610 are marked, and pass through the red fluorescence intensity of the flow cytometer detection antibody.
Experimental result is as shown in Figure 5：Show in figure, PD-L1-DM1 antibody activity and the PD-L1 activity of itself are almost
It is the same, it can be seen that PD-L1-DM1 antibody activity is not influenceed by being coupled.
The antibody activity of embodiment 7 detects
Pair with cell surface interaction research be evaluation PD-L1 antibody couplings chemotherapeutics (PD-L1-DM1) antibody
The important measurement index of activity.After antibody coupling, some changes may occur for its space structure, and it is evaluated by laser co-focusing
Repercussion effect pair with cell surface, and then evaluate its potential using value.Specific experiment process is as follows：
(1) plating cells：Take the logarithm growth period A549 cell dissociations, centrifugation after count, it is thin in confocal ware middle berths
Born of the same parents' density is 2 × 104/ ware, addition 1ml has blood serum medium after cell attachment, is placed on 5%CO2, 37 DEG C of cell culture
In case overnight.
(2) free antibody PD-L1, antibody coupling medicine DM1 (PD-L1-DM1) is taken to add cell hole respectively
In, it is incubated with A549 cells, in 5%CO2, 37 DEG C of incubation 6h.
(3) after being incubated 6h, DM1 groups and ADC groups (PD-L1-DM1) are gently washed 2 times with culture medium respectively, then wash 2 with PBS
Time, the secondary antibody (rabbit-anti people) marked using Alexa Fluor 610, and the green glimmering of cell surface is detected by laser co-focusing
Luminous intensity.
Experimental result is as shown in Figure 6：Further demonstrate that, PD-L1-DM1 antibody activity and PD-L1 itself activity are almost
It is the same, PD-L1-DM1 antibody activity is not influenceed by being coupled.
The PD-L1-DM1 of embodiment 8 treatment tumor effect detections
Mouse tumor model has been initially set up, has then been grouped at random, has been grouped into 3 groups, including PBS control group
(Control), PD-L1-DM1 groups, PD-L1 groups and DM1 groups.
Lung cancer in mice tumor model (A549) has been initially set up, then will be vaccinated with A549 cells (human lung carcinoma cell)
Balb/c nude mices are grouped at random, are grouped into 3 groups, including PBS control group (Control), PD-L1, PD-L1-DM1 group group.
Every group of Balb/c nude mice is 5, and the dosage of the Balb/c nude mice per injections of average every lotus knurl is 5mg/kg, is injected 3 times, is put down
Injection in every 7 days is once.Start recording and observe at any time in first day drug injection and record every group and be vaccinated with A549 cells
Lotus knurl Balb/c nude mices Survival, every 2 days measurement tumor sizes simultaneously weighing body weight is carried out to it.Gross tumor volume calculating side
Method is as follows：
Gross tumor volume calculation formula：Long × wide × wide/2
The 30th day Balb/c nude mice to whole lotus knurls is dissected after drug injection is carried out to lotus knurl Balb/c nude mices,
Its tumor tissues is taken out, and measures the quality and volume of the tumor tissues of every group of lotus knurl Balb/c nude mice, and data are carried out to it
Processing.
As a result as shown in fig. 7, after PD-L1 antibody coupling DM1 medicines (PD-L1-DM1) treatment, gross tumor volume reduces
80%.As shown in figure 8, after (PD-L1-DM1) treatment, gross tumor volume is obviously reduced.Fully show that prepared PD-L1 antibody is even
Connection DM1 medicines (PD-L1-DM1) can realize the good treatment to tumor tissues.
The experiment further proves that the antibody coupling matter (PD-L1-DM1) of this method synthesis is improved in clinical tumor
Treatment.These benefit from antibody target tropism and in the strong cytotoxicities of tumor locus enrichment and DM1 so that it is to tumour
The fragmentation effect of cell is best.ADC medicines (PD-L1-DM1) are undoubtedly compound with more novel antibodies-chemotherapy of market prospects
The pharmaceutical dosage form for the treatment of.
It is described above, only presently preferred embodiments of the present invention, it is not any to the present invention in form and substantial limitation,
It should be pointed out that for those skilled in the art, on the premise of the inventive method is not departed from, can also make
Some improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, it is the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution for any equivalent variations that above-described embodiment is made, still fall within the scope of technical scheme
It is interior.
Claims (10)
1. a kind of antibody-drug conjugates, include the coupling of cytotoxic drug, antibody and coupled antibody and drug toxicity
Chain.
2. antibody-drug conjugates according to claim 1, it is characterised in that the cytotoxic drug is stepped on from U.S.A
Plain DM1；The antibody Procedure for selection death ligand -1PD-L1.
3. antibody-drug conjugates according to claim 2, it is characterised in that the coupling chain selects 3- (2- pyridines two
It is thio) propionyl hydrazine PDPH.
4. antibody-drug conjugates according to claim 2, it is characterised in that maytansine DM1 matches somebody with somebody with programmed death
Body -1PD-L1 molar ratio is (1~2)：1.
5. antibody-drug conjugates according to claim 3, it is characterised in that coupling chain PDPH-S-S- groups and U.S.
Step on plain DM1-SH groups connection.
6. antibody-drug conjugates according to claim 3, it is characterised in that coupling chain PDPH-NH2Group and program
Property death ligand-1PD-L1-COOH group connection.
7. the preparation method of the antibody-drug conjugates as described in any one of claim 1~6, including：
(1) maytansine DM1 (DM1-PDPH) of the synthesis with hydrazide group group：Take maytansine DM1 and 3- (2- pyridines two are thio) third
Hydrazides PDPH, dissolve, reaction；
(2) the Programmed death ligand-1 PD-L1 of oxidation is prepared：PD-L1 is mixed with oxidation buffer solution, aoxidized；
(3) PD-L1-DM1 is prepared：The oxygen that the maytansine DM1 with hydrazides group that step (1) is obtained obtains with step (2)
The Programmed death ligand-1 mixing of change, reaction, obtains PD-L1-DM1.
8. according to the method for claim 7, it is characterised in that methods described also includes any one of following or multinomial：
1) in step (1), maytansine DM1 and 3- (2- pyridines two are thio) propionyl hydrazine PDPH is taken to be dissolved in methanol；
2) in step (2), the oxidation buffer solution is sodium iodate oxidation buffer solution；
3) in step (3), the reaction is carried out at room temperature.
9. purposes of the antibody-drug conjugates as described in any one of claim 1~6 in anti-tumor medicine is prepared.
10. a kind of cancer therapeutics compositions, such as claim 1~6 times is contained in the active ingredient of described pharmaceutical composition
Antibody-drug conjugates described in one.
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| US20150174268A1 (en) * | 2012-07-18 | 2015-06-25 | Shanghai Birdie Biotech, Inc. | Compounds for targeted immunotherapy |
| WO2016111645A1 (en) * | 2015-01-09 | 2016-07-14 | Agency For Science, Technology And Research | Anti-pd-l1 antibodies |
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| CN105899539A (en) * | 2014-01-10 | 2016-08-24 | 博笛生物科技（北京）有限公司 | Compounds and compositions for immunotherapy |
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| US20150174268A1 (en) * | 2012-07-18 | 2015-06-25 | Shanghai Birdie Biotech, Inc. | Compounds for targeted immunotherapy |
| US20150018516A1 (en) * | 2013-02-07 | 2015-01-15 | Immunomedics, Inc. | Pro-drug form (p2pdox) of the highly potent 2-pyrrolinodoxorubicin conjugated to antibodies for targeted therapy of cancer |
| CN105899539A (en) * | 2014-01-10 | 2016-08-24 | 博笛生物科技（北京）有限公司 | Compounds and compositions for immunotherapy |
| CN104491868A (en) * | 2014-11-26 | 2015-04-08 | 中国人民解放军第二军医大学 | Novel chemotherapeutic drug nano ADC based on antibody conjugation and preparation method and application thereof |
| CN105777906A (en) * | 2014-12-19 | 2016-07-20 | 苏州丁孚靶点生物技术有限公司 | Anti-PD - L1 human antibody and application thereof |
| WO2016111645A1 (en) * | 2015-01-09 | 2016-07-14 | Agency For Science, Technology And Research | Anti-pd-l1 antibodies |
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| WO2022237884A1 (en) * | 2021-05-13 | 2022-11-17 | 清华大学 | Antibody drug conjugate, preparation method therefor and application thereof |
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