CN107375941A - A kind of anti-human DLL4 monoclonal antibodies and aplysiatoxin derivative MMAE conjugate - Google Patents
A kind of anti-human DLL4 monoclonal antibodies and aplysiatoxin derivative MMAE conjugate Download PDFInfo
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- CN107375941A CN107375941A CN201710593411.XA CN201710593411A CN107375941A CN 107375941 A CN107375941 A CN 107375941A CN 201710593411 A CN201710593411 A CN 201710593411A CN 107375941 A CN107375941 A CN 107375941A
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- mmgz01
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- mmae
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/07—Tetrapeptides
Abstract
The invention discloses a kind of anti-human DLL4 monoclonal antibodies MMGZ01 and aplysiatoxin derivative MMAE coupling conjugate, it is related to biopharmaceutical technology.It is characterized in that：The Preparation method and use of small molecule toxins aplysiatoxin derivative MMAE and anti-human DLL4 monoclonal antibodies MMGZ01 coupling conjugate.The present invention uses anti-human DLL4 monoclonal antibodies MMGZ01, choose vcMMAE joint and drug regimen, phosphine reducing agent tricarboxylic ethyl phosphine TCEP partial reduction antibody MMGZ01 is used in coupling process, then it is coupled with vcMMAE and carries out the purifying of conjugate, pass through the optimization to technique, prepare new anti-human DLL4 monoclonal antibody drugs conjugate MvM03, described conjugate MvM03 beneficial effects are the tumor-targeting for promoting lps molecule MMAE, the toxic action to organism normal cell is reduced, reaches more preferable therapeutic effect.
Description
Technical field
The present invention is antibody coupling drug field.In particular it relates to anti-human DLL4 monoclonal antibodies MMGZ01
It is coupled small lps molecule aplysiatoxin derivative MMAE conjugate.
Background technology
DLL4 (Delta like 4) is an important part of Notch receptor in Notch signal paths, DLL4 by with
The combination of flanking cell surface Notch receptor, participate in regulation and control adjacent cells propagation, apoptosis, differentiation and stem cells hyperplasia and
The biological processes such as renewal.In addition DLL4 is overexpressed in mankind tumor tissue, and participates in tumor vascular occurrence and development, to mesh
Before untill, people successively in the tumor vessels such as kidney, carcinoma of urinary bladder, colon cancer, brain tumor and breast cancer observe DLL4 mistake
Expression.Multinomial research is found in recent years, and DLL4/Notch signal paths are thin in tumour generation, Tumor Angiongesis and Tumor Stem
Important function has been played during the tumor development such as the propagation of born of the same parents and renewal.It can be seen that DLL4 is expected to turn into research tumour hair
Life system and the novel target for developing dependent diagnostic reagent, the specific antibody developed with this and its tumor stem cell treatment method
Have a good application prospect.
Anti-tumor monoclonal antibody is coupled by chemical modification and small molecule toxins, and the existing specificity of these conjugates is known
The ability of other tumour antigen, the toxicity of lps molecule killing tumor cell is also remained, targets tumor locus, selective killing
Tumour cell.The country there is no anti-human DLL4 antibody drug conjugates Patents and document report at present, only on a small quantity by dipeptides
Joint valine-citrulline (Val-Cit, vc) and aplysiatoxin derivative the MMAE ((Monomethyl for influenceing microtubule polymerization
Auristain E) report (patent No. of joint and drug regimen applied to antibody drug coupling：CN 103145847 B；CN
103254317 B；CN 103394083 A；CN 1938046 B；CN 102936281 B).Therefore, inventor want to by
Anti-human DLL4 monoclonal antibodies MMGZ01 is coupled with a kind of toxin small-molecule drug, makes full use of the targeting of antibody and toxin small
The toxicity of molecule, curative effect is improved, reduce toxic side effect.
((Monomethyl Auristain E) is that a kind of artificial synthesized have high toxicity to aplysiatoxin derivative MMAE
Antitubulin, it is similar with aplysiatoxin can to suppress cell division, with maleimidocaproyl-
After the connection of dipeptide (valine-citrulline or vc) joint, its toxicity is 10-1000 times of adriamycin.Due to
MMAE toxicity is higher and non-selectivity, therefore it does not carry out treatment of cancer individually as medicine.In order to reduce toxicity and carry
High selectivity, by MMAE by linker and antibody coupling, using the targeting of antibody, curative effect is improved, reduces toxic side effect.
Based on above-mentioned theory basis and research practice, the anti-human DLL4 monoclonals will with independent intellectual property right of the invention
Antibody MMGZ01 is connected with MMAE by valine-citrulline joints, designs a kind of novel antibodies drug conjugates
MvM03.MvM03 design opens a kind of new ideas of cancer therapy.The research approach plays antibody target sexual clorminance, effectively
Toxic side effect caused by toxin pharmaceutical is reduced, commanding elevation will be occupied in preclinical study and clinical safety evaluation.Therefore,
The exploitation of antibody drug conjugates MvM03 with independent intellectual property right, the clinical application of novelty is provided for oncotherapy system
Scheme.
The content of the invention
Goal of the invention
It is an object of the invention to overcome deficiency of the prior art, there is provided a kind of Idiotype is strong, higher anti-human of purity
Antibody drug conjugates MvM03 of DLL4 monoclonal antibodies MMGZ01 and aplysiatoxin derivative MMAE couplings and preparation method thereof
And application, the present invention chooses Val-Cit and MMAE joint and drug regimen is coupled with anti-human DLL4 antibody, and it is anti-to obtain it
Body drug conjugates MvM03, promote MMAE tumor-targeting, improve curative effect, reduce toxic side effect.It is used in the present invention
Antibody patent applied for (the patent No.：CN105384818A phosphine reducing agent tricarboxylic ethyl phosphine), while in coupling process is used
TCEP (Tris- (2-carboxyethyl)-phosphine hydrochloride) carries out the reduction of antibody.Compared to
Pinatuzumab vedotin and the Patents file (patent No.：The A1. of EP 2478912) provide coupling process in made
Thio-alcohol reducing agent dithiothreitol (DTT), TCEP can be used in the range of wider PH and reaction condition it is more gentle,
Dissolubility is good, small toxicity, while TCEP does not react with other functional groups in protein.Based on this, the application is also to coupling
Technique has carried out further optimization, prepares average coupling ratio and is less than 10% for 4.5 or so, naked anti-component, content of monomer is big
In 90%, and inside and outside drug effect confirms the antibody coupling medicine of the anti-human DLL4 antibody of energy specific killing target cell.
Technical scheme
Technical program of the present invention lies in provide a kind of anti-human DLL4 monoclonal antibodies MMGZ01 and aplysiatoxin derivative
The antibody drug conjugates of MMAE couplings, it is characterised in that：Anti-human DLL4 monoclonal antibodies MMGZ01 and aplysiatoxin derivative
MMAE antibody drug conjugates.The preparation process of the conjugate includes：
Step 1：Anti-human DLL4 monoclonal antibodies MMGZ01 coagulates by Protein A posts affinitive layer purifications and fine jade glucose
After glue G25 FF column molecular sieves chromatography replaces antibody-solutions system, BCA methods measure antibody concentration, by reducing agent TCEP and antibody
With 3: 1 mixed in molar ratio, MMGZ01 is reduced, opens MMGZ01 interchain disulfide bonds, sample reacts 1 hour in 4 DEG C, with containing 1M
The DTPA PBS solutions of PH 7.0 chromatograph desalination by fine jade glucose G25 FF column molecular sieves.
Step 2：The toxin MMAE powder with two peptide linker Val-Cit is taken, i.e. it is molten to be substantially dissolved in DMSO by vc-MMAE
In liquid, vc-MMAE solution and step 1 are obtained into antibody products coupling reaction is carried out with 15: 1 mixed in molar ratio, be placed on ice
It is gently mixed, reacts 1 hour.
Step 3：, the product MMGZ01-vcMMAE that is obtained step 2 using fine jade glucose gel G25 FF desalting columns, i.e.,
MvM03, sieve chromatography purifying is carried out, remove the impurity in reaction system.
Step 4：The product MvM03 in step 3 is concentrated with super filter tube on low speed refrigerated centrifuge, produced anti-
Body drug conjugates MvM03；After BCA methods measure conjugate concentration, -20 DEG C of preservations are placed in.
Preferably, the antibody drug conjugates MvM03 prepared according to the above method possesses following characteristics：Naked anti-component is low
In 10%, content of monomer is more than 90%.
It is further preferred that the antibody drug conjugates MvM03 prepared according to the above method is applied to targeted therapy people
In DLL4 positive tumor medicines.
It is further preferred that in the step 1 and step 3, described purifying includes affinity column, desalting column and surpassed
Liquid system is changed in filter.
It is further preferred that described affinity column is Protein A post affinity columns.
It is further preferred that described desalting column is fine jade glucose gel G25 FF desalting columns.
It is further preferred that the ultrafiltration concentration system uses the super filter tube of 50kD films.
Wherein, the PBS is phosphate-buffered salt liquid solution, phpsphate-buffered saline.
Wherein, the vcMMAE is synthesized by Shanghai Chemicals companies.
Wherein, the MMGZ01 molecular sequences refer to patent CN105384818A.
Wherein, the fine jade glucose gel G-25 FF desalting columns, are made by mixing by sephadex and Ago-Gel,
It is prepared again through highly cross-linked, has that stability is good, the good flow velocity of rigidity is fast, reproducible, high resolution, particle are uniform, is not had
There is the advantages that non-specific adsorption, volume is not shunk after filling, the sephadex of simultaneously beyond tradition can be substituted completely.Agar
Sugared gel represents that the cross-link dextran of different size model is represented with English alphabet G, the Arab behind G with English alphabet FF
Numeral is 10 times of the water number of gel, and G-25 absorbs water 2.5 grams when being every gram of gel expansion, and G-25 separating ranges are 1000-
5000Da, aim at protein desalination and design.
The present invention compared with prior art the advantages of have it is following some：
The present invention is obtained anti-human DLL4 monoclonal antibodies MMGZ01 and derived with aplysiatoxin by antibody drug coupling technology
The antibody drug conjugates MvM03 of thing MMAE couplings, the conjugate had both remained MMGZ01 targeting, made full use of again
MMAE suppression microtubule polymerization ability, so as to the more efficiently therapeutic effect for playing target killing tumour；
The peptide linker of Val-Cit (vc) two used in the present invention, overcomes hydrazone key joint in drug effectiveness and secure side
The defects of face, and pass through joint vc-MMAE maleimide and the anti-human DLL4 monoclonal antibodies MMGZ01 of partial reduction
Sulfydryl carries out addition reaction and obtains antibody drug conjugates MvM03.
Compared with existing coupling process, the present invention uses phosphine reducing agent three in antibody moiety reduction reaction system
Carboxyethyl phosphine TCEP (Tris- (2-carboxyethyl)-phosphine hydrochloride) goes back original antibody, and TCEP can be
Used in the range of wider PH and reaction condition be more gentle, dissolubility is good, small toxicity, at the same TCEP not with its in protein
He reacts at functional group, it can be ensured that the controllability of reaction and the uniformity of result；
The present invention has also carried out further optimization, prepared conjugate MvM03 to coupling process, and average coupling ratio is
4.42, naked anti-component is less than 10%, and content of monomer is more than 90%, and inside and outside drug effect confirms that the conjugate can effectively and specificity is killed
Hinder target cell.
Beneficial effect
The antibody drug by anti-human DLL4 monoclonal antibodies MMGZ01 and aplysiatoxin derivative MMAE couplings of the present invention
Conjugate MvM03, available for the tumor-targeting for promoting MMAE, curative effect is improved, reduces toxic side effect.Beneficial effect is (1)
Conjugate MvM03 remains the high affine vigor to DLL4；(2) combined with expressing people DLL4 cell-specific, avoid MMAE
To the lethal effect of normal cell；(3) equal conspicuousness suppresses the growth of tumour, propagation in vivo and in vitro.
Specific implementation method
Embodiment 1
Prepare and be coupled by the antibody drug of anti-human DLL4 monoclonal antibodies MMGZ01 and aplysiatoxin derivative MMAE couplings
Thing MvM03：(1) antibody moiety：MMGZ01 passes through Protein A post affinitive layer purifications, with PH 7.0 PBS to above-mentioned pure
The MMGZ01 antibody of change is chromatographed by fine jade glucose gel G25 FF column molecular sieves and replaces antibody-solutions system；Determined using BCA methods
Antibody concentration；4-12% PAGE gels electrophoresis checking purified product is that electrophoresis is pure；By reducing agent TCEP and above-mentioned antibody with
3: 1 mol ratio is sufficiently mixed, and after 4 DEG C are reacted 1 hour, passes through fine jade glucose gel with the PBS solutions of PH 7.0 of the DTPA containing 1M
G25 FF column molecular sieves are chromatographed to said mixture desalination；(2) conjugate part：Vc-MMAE powder is taken to be substantially dissolved in DMSO
In solution, vc-MMAE solution and above-mentioned antibody products are sufficiently mixed with 15: 1 mol ratio, is gently mixed, reacts 1 on ice
Hour；Using super filter tube on low speed refrigerated centrifuge by said mixture ultrafiltration desalination, produce antibody drug conjugates
MvM03。
Embodiment 2
With high performance liquid chromatography HPLC detection MMAE and anti-human DLL4 monoclonal antibodies MMGZ01 coupling situation.
The HPLC of Agilent 1200 analysis antibody drug conjugates MvM03 coupling situation.Sample detection condition is as follows：
(1) mobile phase A：20mmol/L PBS (pH 7.0)+1.5mol/L ammonium sulfate；(2) Mobile phase B：20mmol/L PBS(pH
7.0)/isopropanol=7.5/2.5；(3) gradient：10-100%B；(4) elution time：20min；(5) flow velocity：0.60mL/
min；(6) sample size：10uL；(7) Detection wavelength：280nm.According to appearance number and each peak area, pro rata calculates antibody
Drug coupling ratio (DAR).
Experimental result is shown in Fig. 1, compared with there is the MMGZ01 of main peak in 9.5min, MvM03 also respectively 12.5min,
Appearance when 15min, 17min and 18min, the number of the corresponding upper MMAE small molecules of coupling is 2,4,6 and 8 respectively,
And corresponding peak area ratio is 15.80%, 23.50%, 30.47% and 16.03%, DAR values, which are finally calculated, is
4.42。
Embodiment 3
Antibody drug conjugates MvM03 and people's DLL4 antigens affinity detect：Using ELISA method, by 1 μ g/mL DLL4
Antigen is added in 96 hole elisa Plates with every μ L of hole 100,4 DEG C, and coating is overnight；PBS is three times after board-washing, by the conjugate of embodiment 1
MvM03 and MMGZ01, by blank group, concentration gradient 0,3.9,7.8,15.6,31.3,62.5,125,250,500,1000nM
It is separately added into 96 orifice plates, 37 DEG C, is incubated 2 hours；PBS after board-washing, adds coupling horseradish peroxidase three times
The sheep anti-mouse igg antibody of (horseradish peroxidase, HRP), 37 DEG C, it is incubated 1 hour；PBS is three times after board-washing,
TMB nitrite ions are added, room temperature lucifuge is reacted 20 minutes, is eventually adding terminate liquid terminating reaction, ELIASA detection OD450-
OD630。
Experimental result is shown in Fig. 2, and compared with MMGZ01, MvM03 is lower slightly to the affinity of DLL4 antigens, but still in concentration according to
Type, i.e. affinity is relied to be incremented by with the increase of drug dose, and by calculating, MMGZ01 and MvM03 EC50It is 32nM respectively
And 42.3nM, illustrating that the affinity of conjugate is not obvious reduces.
Embodiment 4
MvM03 suppresses HUVEC cell growth assays in vitro：Using MTT colorimetric methods.By the HUVEC cells of logarithmic growth, with
1.0 ×105Add in 96 well culture plates, cultivate 12 hours；Experiment is divided into 3 groups, respectively blank group, MMGZ01 groups and implementation
The antibody drug conjugates MvM03 groups that example 1 obtains；The DMEM culture mediums that same volume contains 5% hyclone are separately added into,
MMGZ01 and MvM03 (1 nM, 5nM, 10nM, 20nM, 40nM, 80nM, 160nM, 320nM, 640nM, 1280nM).Set per hole
3 multiple holes, after cultivating 72 hours, 12 μ L 5mg/mL MTT solution are added per hole, after acting on 4 mouse, DMSO is added, in vibration
10min is reacted on device, absorbance A value is determined at ELIASA 570nm and 630nm.Each experimental group is calculated to HUVEC cells
The inhibiting rate of growth, and draw curve map.
Experimental result is shown in Fig. 3, it is seen that compared with MMGZ01, the antibody drug conjugates MvM03 of embodiment 1 can effectively suppress
The growth of HUVEC cells, so as to block blood vessel to provide blood oxygen to tumor tissues, tumour is caused to become feeble and die.
Brief description of the drawings
Fig. 1 is high-efficient liquid phase chromatogram technique analysis antibody drug conjugates MvM03.
Fig. 2 is elisa assay antibody drug conjugates MvM03 and people's DLL4 binding abilities.
Fig. 3 is inhibitory action of the MTT analysis drug conjugates MvM03 to HUVEC cell growths.
Claims (11)
- A kind of 1. new anti-human DLL4 monoclonal antibodies MMGZ01 and aplysiatoxin derivative MMAE antibody drug conjugates MvM03, it is characterised in that：Joint Val-Cit toxin is carried including anti-human DLL4 monoclonal antibodies MMGZ01 molecule coupling labeleds MMAE, i.e. vc-MMAE, average each MMGZ01 molecule coupling labeleds MMAE molecular number is 4.42.
- 2. antibody drug conjugates MvM03 according to claim 1, it is characterised in that：Its preparation method includes following step Suddenly：Step 1：Anti-human DLL4 monoclonal antibodies MMGZ01 passes through Protein A posts affinitive layer purifications and fine jade glucose gel G25 After FF column molecular sieves chromatography replaces antibody-solutions system, BCA methods measure antibody concentration, by reducing agent TCEP and antibody with 3：1 Mixed in molar ratio, MMGZ01 is reduced, open MMGZ01 interchain disulfide bonds, sample reacts 1 hour in 4 DEG C, with the DTPA's containing 1M PH 7.0PBS solution chromatographs desalination by fine jade glucose G25 FF column molecular sieves.Step 2：Taking the toxin MMAE powder with two peptide linker Val-Cit, i.e. vc-MMAE is substantially dissolved in DMSO solution, Vc-MMAE solution and step 1 are obtained into antibody products coupling reaction is carried out with 15: 1 mixed in molar ratio, be placed on ice gently Stirring, react 1 hour.Step 3：, the product MMGZ01-vcMMAE, the i.e. MvM03 that are obtained step 2 using fine jade glucose gel G25FF desalting columns, Sieve chromatography purifying is carried out, removes the impurity in reaction system.Step 4：The product MvM03 in step 3 is concentrated with super filter tube on low speed refrigerated centrifuge, produces antibody medicine Thing conjugate MvM03；After BCA methods measure conjugate concentration, -20 DEG C of preservations are placed in.
- 3. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's is anti- Body drug conjugates MvM03, it is characterised in that：Described antibody drug conjugates MvM03 antibody moiety uses chemical method, Interchain disulfide bond is reduced into mercapto groups using TCEP reducing agents, so as to modify antibody structure.
- 4. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's is anti- Body drug conjugates MvM03, it is characterised in that：In the step 1, MMGZ01 and reducing agent TCEP mol ratio is 1: 3.
- 5. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's is anti- Body drug conjugates MvM03, it is characterised in that：The antibody drug conjugates MvM03 prepared by the above method possesses following Feature：Naked anti-component is less than 10%, and content of monomer is more than 90%.
- 6. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's is anti- Body drug conjugates MvM03, it is characterised in that：In the step 1 and step 3, described purifying includes affinity column, taken off Liquid system is changed in salt plug and ultrafiltration.
- 7. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's is anti- Body drug conjugates MvM03, it is characterised in that：In the step 1 and step 3, described affinity column is Protein A Post affinity column.
- 8. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's is anti- Body drug conjugates MvM03, it is characterised in that：In the step 1 and step 3, described desalting column is fine jade glucose gel G25FF desalting columns.
- 9. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's is anti- Body drug conjugates MvM03, it is characterised in that：Described anti-human DLL4 monoclonal antibodies MMGZ01 and described vcMMAE idol Connection is than being 4.42.
- 10. new anti-human DLL4 monoclonal antibodies MMGZ01 according to claim 2 and aplysiatoxin derivative MMAE's Antibody drug conjugates MvM03, it is characterised in that：The antibody drug conjugates MvM03 prepared by the above method is employed Targetted in treatment in people's DLL4 tumour medicines.
- 11. antibody drug conjugates MvM03 according to claim 4, it is characterised in that：It is used to treat breast cancer drug preparing Application in thing.
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| CN111494645A (en) * | 2020-05-20 | 2020-08-07 | 中国药科大学 | Conjugate of anti-human D LL 4 humanized antibody and maytansine alkaloid DM1, and preparation method and application thereof |
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| CN116239513A (en) * | 2023-05-05 | 2023-06-09 | 天津凯莱英制药有限公司 | Preparation method of MMAE key intermediate, preparation method of MMAE and antibody coupling drug |
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