CN106215193A - A kind of little molecule vacuole LMP-1 antibody polypeptides and the conjugate of cyclophosphamide coupling - Google Patents
A kind of little molecule vacuole LMP-1 antibody polypeptides and the conjugate of cyclophosphamide coupling Download PDFInfo
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- CN106215193A CN106215193A CN201610754312.0A CN201610754312A CN106215193A CN 106215193 A CN106215193 A CN 106215193A CN 201610754312 A CN201610754312 A CN 201610754312A CN 106215193 A CN106215193 A CN 106215193A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
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Abstract
The invention discloses the coupling conjugate of little molecule vacuole LMP-1 antibody polypeptides and cyclophosphamide, belong to antibody coupling drug world.Cyclophosphamide and the coupling conjugate of vacuole LMP-1 antibody polypeptides；Described vacuole LMP-1 antibody polypeptides is brand-new sequence.Coupling conjugate preparation process includes: (1) prepares the cyclophosphamide of 2 8mmol/L concentration with 0.1 mol/L HCl solution；(2) 4 DEG C, drip 1%NaNO2, continuously stirred, react 20 30 minutes；(3) with the vacuole LMP-1 antibody polypeptides described in buffer solution of pH8 10；(4) described vacuole LMP-1 antibody polypeptides solution adds diazotizing cyclophosphamide, regulate pH to 9 10；(5) 48 DEG C, react 2 hours；(6) PBS 2 days, to obtain final product.Described coupling conjugate, in preparation application in tumor.Have the beneficial effects that the tumor-targeting promoting cyclophosphamide, improve curative effect, reduce toxic and side effects.
Description
Technical field
The present invention is antibody coupling drug world.In particular it relates to vacuole LMP-1 antibody polypeptides coupling resists
The conjugate of tumour medicine cyclophosphamide.
Background technology
Cyclophosphamide is broad-spectrum anti-tumor medicine, is used for treating leukemia and other tumors.It is in vitro without anti-tumor activity,
Enter internal elder generation afterwards and change into aldophosphamide through microsome function oxidase in liver.And aldehyde amide is unstable, thin in tumor
Intracellular resolves into amide chlormethine and acrylic aldehyde, and amide chlormethine has cytotoxicity to tumor cell.Cyclophosphamide is difunctional alkane
Agent and cell cycle nonspecific agent (CCNSA), may interfere with DNA and RNA function, and especially with bigger on the former impact, it is sent out with DNA
Raw cross link, suppression DNA synthesis, the S phase is acted on the most obvious.It is clinically used for malignant lymphoma, multiple myeloma, white blood
Disease, breast carcinoma, ovarian cancer, cervical cancer, carcinoma of prostate, colon cancer, bronchogenic carcinoma, pulmonary carcinoma etc., have certain curative effect.Can also be used for class
The treatment of rheumatic arthritis, primary nephrotic syndrome of children and autoimmune disease.
While it is true, cyclophosphamide has serious toxic and side effects.Common adverse effect: 1. bone marrow depression；2. phlebitis；3.
Digestive tract reaction；4. alopecia etc..Producing main reason is that of toxic and side effects, cyclophosphamide is broad-spectrum anti-tumor requirement, special
Property is poor, also has certain lethal effect to normal cell.Therefore, inventor wants to cyclophosphamide with specific
Antibody coupling, thus produce the targeting of tumor cell, improve curative effect, reduce toxic and side effects.
Vacuole LMP-1 (Vacuole membrane protein 1, Vmp1) is a newly discovered class transmembrane protein,
Guard at evolution camber, point out it may participate in the interaction between albumen and the important vital movement across species.In recent years
Research find Vmp1 be protein excretion, organelle formed and many cells growth course required, many tumors be correlated with
Cytology process plays the effect of key in (including cell membrane transport, growing multiplication, autophagy etc.), prompting Vmp1 may be swollen
Tumor generation, development have played important function.The more important thing is, Vmp1 is to connect between cell-ECM and compact siro spinning technology formation
Important component, its power in terms of regulating cell adhesive capacity, prompting Vmp1 may participate in HCC Invasion and Metastasis process.
Research finds, vacuole LMP-1 (Vacuole membrane protein 1, Vmp1) is high at tumor cell surface
Expressing, therefore, vacuole LMP-1 antibody with targeting in tumor cell surface, and itself can have the effect of anti-breast cancer.
But the molecular weight of vacuole LMP-1 antibody is relatively big, has antigenicity, the long-term people of being used for knows from experience generation and neutralizes reaction, reduces medicine
Effect.Therefore, the invention provides a kind of high specificity, little molecule vacuole LMP-1 antibody polypeptides that purity is higher and ring phosphinylidyne
The conjugate of amine coupling, promotes the tumor-targeting of cyclophosphamide, improves curative effect, reduces toxic and side effects.
Summary of the invention
Goal of the invention
The present invention provides a kind of high specificity, little molecule vacuole LMP-1 antibody polypeptides that purity is higher and cyclophosphamide
The conjugate of coupling, promotes the tumor-targeting of cyclophosphamide, improves curative effect, reduces toxic and side effects.
Technical scheme
Technical program of the present invention lies in the coupling providing a kind of little molecule vacuole LMP-1 antibody polypeptides with cyclophosphamide
Conjugate, it is characterised in that: cyclophosphamide and the coupling conjugate of vacuole LMP-1 antibody polypeptides；Described vacuole LMP-1 resists
Body polypeptide is brand-new sequence.The preparation of described coupling conjugate, step includes: (1) prepares 2-with 0.1mol/L HCl solution
The cyclophosphamide of 8mmol/L concentration；(2) 4 DEG C, drip 1%NaNO2, continuously stirred, react 20-30 minute；(3) pH8-10 is used
The vacuole LMP-1 antibody polypeptides described in buffer solution；(4) described vacuole LMP-1 antibody polypeptides solution adds weight
The cyclophosphamide of nitridation, regulates pH to 9-10；(5) 4-8 DEG C, react 2 hours；(6) PBS 2 days, to obtain final product.Described vacuole
LMP-1 antibody polypeptides uses chemical synthesis to prepare.Described coupling conjugate, preparation answering in tumor
With.It is preferably in preparation for treating the application in breast cancer medicines.
Beneficial effect
The little molecule vacuole LMP-1 antibody polypeptides of the present invention and the coupling conjugate of cyclophosphamide, can be used for promoting ring
The tumor-targeting of phosphamide, improves curative effect, reduces toxic and side effects.Have the beneficial effects that (1) is improved cyclophosphamide and swollen in vivo
The concentration of tumor tissue；Reduce cyclophosphamide in whole blood, the distribution of the tissues such as Yi Jixin, liver, spleen, lung, kidney, brain simultaneously；(2) notable
Property suppression tumor cell propagation；(3) toxicity of cyclophosphamide is reduced；(4) little molecule vacuole LMP-1 antibody polypeptides and ring phosphorus
The coupling conjugate of amide can specific binding with tumor cell；(5) tumor-bearing mice survival rate is improved.
Detailed description of the invention
Polypeptide is by Shanghai raw work gill synthesis.
Embodiment 1
Prepare the coupling conjugate of vacuole LMP-1 antibody polypeptides and cyclophosphamide: prepare with 0.1mol/L HCl solution
The cyclophosphamide of 2mmol/L concentration；4 DEG C, drip 1%NaNO2, continuously stirred, react 20 minutes；With the buffer solution of pH8
Described vacuole LMP-1 antibody polypeptides；Vacuole LMP-1 antibody polypeptides solution adds diazotizing cyclophosphamide, regulation
PH to 9；At 4 DEG C, react 2 hours；Use PBS 2 days again, lyophilizing, to obtain final product.
Embodiment 2
Prepare the coupling conjugate of vacuole LMP-1 antibody polypeptides and cyclophosphamide: prepare with 0.1mol/L HCl solution
The cyclophosphamide of 8mmol/L concentration；4 DEG C, drip 1%NaNO2, continuously stirred, react 30 minutes；With the buffer solution of pH10
Described vacuole LMP-1 antibody polypeptides；Vacuole LMP-1 antibody polypeptides solution adds diazotizing cyclophosphamide, regulation
PH to 10；At 8 DEG C, react 2 hours；Use PBS 2 days again, lyophilizing, to obtain final product.
Embodiment 3
Body with transplanted human breast carcinoma model inspection vacuole LMP-1 antibody polypeptides Yu the coupling conjugate of cyclophosphamide
Interior vigor.
6-8w SCID in age female mice, mice is randomly divided into 5 groups, often group 10.(1) blank group；(2) conjugate low dosage
Group；(3) dosage group in conjugate；(4) conjugate high dose group；(5) cyclophosphamide positive group.Set up transplanted human breast carcinoma mould
Type, the 7th day after inoculation breast cancer cell, tail intravenously administrable is administered respectively.Dosage regimen is: blank group adds same volume
Solvent, 0.05,0.1,0.2mmol/Kg experimental group is that conjugate sets 3 dosage:, the ring phosphinylidyne that positive group is 0.2mmol/Kg
Amine, is administered, continuous 14 days every day.After 21 days, observe mouse survival quantity, calculate survival rate.Result shows, conjugate can be effective
Ground protection tumor-bearing mice, dosage 0.05,0.1,0.2mmol/Kg time can improve the survival rate of tumor-bearing mice, survival rate is respectively
It is 40.98,68.92,79.01%.
Embodiment 4
The in-vitro multiplication rate experiment of breast cancer cell: use MTT colorimetry.By the human breast cancer cell of logarithmic growth, with
1.0×105Add in 96 well culture plates, cultivate 24h；Experiment is divided into 5 groups, is respectively blank group, positive group and low middle high dose
The coupling conjugate of the vacuole LMP-1 antibody polypeptides that obtains of embodiment 1 and cyclophosphamide；It is separately added into same volume
DMEM culture medium (HyClone, containing hyclone 20%), cyclophosphamide (1mmol/ml) and vacuole LMP-1 antibody polypeptides with
The coupling conjugate (18,36,72 μm ol/ml) of cyclophosphamide.Every hole sets five multiple holes, cultivates 48h, and every hole adds MTT, effect
After 4h, add DMSO, hatch 30min, at microplate reader 620nm, measure absorbance A value, become breast cancer cell suppression ratio by formula
=(1 experimental group light absorption value/matched group light absorption value) × 100%.The coupling conjugate of cyclophosphamide positive group and embodiment 1 is real
The suppression ratio of the breast cancer cell testing group is respectively 82.7,57.06,76.1 and 92.5% (p ＜ 0.05).Result shows, implements
The coupling conjugate of example 1 can effectively suppress growth of tumour cell, and the tumor control rate of high dose conjugate is higher than cyclophosphamide.
Embodiment 5
Body with transplanted human breast carcinoma model inspection vacuole LMP-1 antibody polypeptides Yu the coupling conjugate of cyclophosphamide
Interior distribution.
6-8w SCID in age female mice, mice is randomly divided into 3 groups, often group 6.(1) blank group；(2) conjugate group；(3)
Cyclophosphamide positive group.Setting up transplanted human breast carcinoma model, the 7th day after inoculation breast cancer cell, tail vein is given respectively
Medicine is administered.Dosage regimen is: blank group adds the solvent of same volume, and experimental group is the conjugate of 0.2mmol/Kg dosage, sun
Property group be the cyclophosphamide of 0.2mmol/Kg, every day be administered, continuous 7 days.After 14 days, after sacrifice, take whole blood respectively, with
And the tissue such as the heart, liver, spleen, lung, kidney, brain, tumor.Whole blood anticoagulant heparin, by heparin anti-coagulating centrifugation hemocyte, takes supernatant
Blood plasma 0.5ml, adds 2.67%HCLO4, centrifugation plasma protein, and supernatant heating in water bath 100 DEG C 20 minutes is to be measured；Each group
Knit through homogenate, removing protein, centrifugal after, take supernatant.The supernatant of blood plasma and tissue is added NBP respectively show in the basic conditions
Colour response, then carries out colorimetric determination under 575nm wavelength.
Calculate mice plasma and each concentration organizing cyclophosphamide.
Table 1 conjugate is blood plasma and tissue distribution concentration in tumor model animal
* P < 0.05, * * P < 0.01 is compared with positive group.
Experimental result is shown in Table 1, compares with cyclophosphamide positive group, vacuole LMP-1 antibody polypeptides and the idol of cyclophosphamide
Concentration in connection compound group in-vivo tumour tissue is significantly raised, (p ＜ 0.01), and the tissue such as the heart, liver, spleen, lung, kidney, brain, with
And the concentration of cyclophosphamide in blood plasma substantially reduces, the especially drug level in blood plasma will be 68.80 ± 22.67 μ g/ml (p
＜ 0.01), there is significant difference.
Embodiment 6
The coupling conjugate of vacuole LMP-1 antibody polypeptides and cyclophosphamide toxic action.Embodiment 1 is used to combine
Thing, measures median lethal dose(LD 50): take 60 mices and be randomly divided into 6 groups, and often group l0 is only.Each group mice elder generation fasting 12h, abdominal cavity note respectively
Penetrate embodiment 2 conjugate of various dose, dosage is respectively 125,250,500,1000,2000,5000mg/Kg, give by reagent
Fasting 3-4 hour again after thing.Hereafter Continuous Observation 2 weeks, with or without the phenomena of mortality, calculate median lethal dose(LD 50) LD50.And observe skin,
Mucosa, hair color, eyes, breathe, circulate, autonomous and central nervous system's behavior expression.
Result: embodiment 1 conjugate is 2143.65mg/Kg to median lethal dose(LD 50) LD50 of mice.
Embodiment 7
The combination rate of conjugate and breast cancer cell: use ELISA method, by the human breast cancer cell of logarithmic growth, with
1.0×105Add in 96 hole ELISA Plate, 4 DEG C, be coated overnight；After PBS washes plate tri-times, by the conjugate of embodiment 1, by blank
Group, Concentraton gradient 0,18,36,72 μm ol/ml is separately added in 96 orifice plates, and the most every hole adds quantitative anti-vacuole LMP-1
Antibody competition combines, 37 DEG C, after hatching 2 hours；After PBS washes plate tri-times, add coupling horseradish peroxidase
The anti-human IgG antibodies of (horseradish peroxidase, HRP), hatches 1 hour by 37 DEG C；After PBS washes plate tri-times again, add
ABC solution, hatches 10-20 minute, finally terminates reaction with flowing water；Microscope is observed, and described embodiment 1 conjugate is thin with tumor
The cell that born of the same parents combine is brownish black, for positive cell, uses computer software to calculate the quantity of the cell that is positive.
Result: the quantity that embodiment 1 conjugate is combined with breast cancer cell, with dosage escalation, has notable compared with blank group
Sex differernce, when dosage is 72 μm ol/ml, positive cell quantity reaches 372.9 ± 54.7 (p ＜ 0.05).
SEQUENCE LISTING
<110>Suzhou Pu Luoda bio tech ltd
<120>a kind of vacuole LMP-1 antibody polypeptides and the conjugate of cyclophosphamide coupling
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213>artificial sequence
<400> 1
Asn Gly Asn Phe Thr Asp Pro Ser Ser
1 5
Claims (5)
1. one kind little molecule vacuole LMP-1 antibody polypeptides and the coupling conjugate of cyclophosphamide, it is characterised in that: ring phosphinylidyne
Amine and the coupling conjugate of vacuole LMP-1 antibody polypeptides；The sequence of described vacuole LMP-1 antibody polypeptides is SEQ ID NO:
1。
The preparation of coupling conjugate the most according to claim 1, it is characterised in that: step includes: (1) uses 0.1 mol/L
The cyclophosphamide of HCl solution preparation 2-8mmol/L concentration；(2) 4 DEG C, dripping 1%NaNO2, continuously stirred, reaction 20-30 divides
Clock；(3) with the vacuole LMP-1 antibody polypeptides described in buffer solution of pH8-10；(4) described vacuole LMP-1 antibody is many
Peptide solution adds diazotizing cyclophosphamide, regulates pH to 9-10；(5) 4-8 DEG C, react 2 hours；(6) PBS 2 days, i.e.
?.
Coupling conjugate the most according to claim 2, it is characterised in that: described vacuole LMP-1 antibody polypeptides employingization
Prepared by synthetic method.
4. according to the arbitrary described coupling conjugate of claim 1-3, it is characterised in that: in preparation in tumor
Application.
Coupling conjugate the most according to claim 4, it is characterised in that: in preparation for treating answering in breast cancer medicines
With.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110124057A (en) * | 2019-06-06 | 2019-08-16 | 天津医科大学总医院 | A kind of anti-tumor drug or pharmaceutical carrier of the cyclodextrin comprising glutamine modification |
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| CN101429542A (en) * | 2008-08-29 | 2009-05-13 | 芮屈生物技术（上海）有限公司 | Hybridization in situ detection kit for VMP1 gene, detection method and uses thereof |
| CN105039333A (en) * | 2015-08-21 | 2015-11-11 | 天津医科大学 | Liver cancer targeted peptide and application thereof |
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2016
- 2016-08-29 CN CN201610754312.0A patent/CN106215193A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429542A (en) * | 2008-08-29 | 2009-05-13 | 芮屈生物技术（上海）有限公司 | Hybridization in situ detection kit for VMP1 gene, detection method and uses thereof |
| CN105039333A (en) * | 2015-08-21 | 2015-11-11 | 天津医科大学 | Liver cancer targeted peptide and application thereof |
Non-Patent Citations (1)
| Title |
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| 刘德传等: "M T T法测定乳腺癌对化疗药物敏感性的实验研究", 《东南国防医药》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110124057A (en) * | 2019-06-06 | 2019-08-16 | 天津医科大学总医院 | A kind of anti-tumor drug or pharmaceutical carrier of the cyclodextrin comprising glutamine modification |
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