CN102258789B - Miniaturized Endoglin antibody and adriamycin conjugate and preparation method thereof - Google Patents

Abstract

The invention provides a miniaturized Endoglin antibody and adriamycin (ADM) conjugate and a preparation method thereof. The conjugate is Fab-ADM; and the molecular ratio of Fab to ADM is 1:2. The preparation method comprises the following steps of: (1) selecting N-hydroxyl succinimido-inter-(N-maleimide) benzoate (MBS) as a cross-linking agent for later use; (2) modifying adriamycin by using thehetero-bifunctional cross linker N-hydroxyl succinimido-inter-(N-maleimide) benzoate (MBS); (3) performing sulfhydrylization on an Endoglin antibody fragment Fab; and (4) preparing the conjugate. Theconjugate Fab-ADM has a strong tumor inhibition effect and can obviously prolong the survival time of lump animals.

Description

Conjugate of miniaturization Endoglin antibody and amycin and preparation method thereof

Technical field

The present invention relates to prepare antitumor drug, relate in particular to miniaturization Endoglin antibody with antitumor action and conjugate of amycin and preparation method thereof.

Background technology

Utilize antibody to the selectivity of tumour specific antigen, antitumor drug is carried out the design of active targeting, can improve antitumor drug to the selectivity of tumor cell, reduce it to the toxicity of normal organ.At present, the research of antibody drug mainly contains both direction: one is the miniaturization of antibody drug.Because antibody and conjugate thereof are macromolecular substances.The molecular mass of IgG type antibody is about 150kD, and huge antibody or conjugate molecule are difficult to arrive by capillary endothelial layer and ECS the tumor cell in solid tumor deep.Therefore, development miniaturization antibody drug is significant to improving curative effect.By the Fab fragment that the enzyme action method obtains, its molecular mass approximately is equivalent to 1/3 of complete antibody.Studies show that, the easier penetration cell external series gap of antibody fragment arrives the tumor cell in deep.Compare the less immunogenic of antibody fragment with complete antibody.Use antibody fragment, can reduce the antibody response of human body.Another research direction is exactly the high efficiency of antibody drug.Antibody connects " bullet " that tumor cell is had strong lethal effect, can reduce the consumption of medicine and to the toxicity of other organs, can also reduce medical expense.

At the beginning of 1970's, Judah doctor Folkman of Harvard University is formal the proposition just: the generation of tumor, development and transfer are all closely related with tumor-blood-vessel growth (Tumor Angiogenesis), so the therapeutic strategy of target tumor angiogenesis just seems particularly important.Studies show that, the Endoglin high expressed is in the endotheliocyte of various tumor tissues and tumor tissues marginal vessel origin, but seldom appearing at the endotheliocyte of normal structure, is one of significant molecule of tumor-blood-vessel growth, can be used as the important target spot of the anti-angiogenic treatment of tumor and targeted therapy.Amycin (Adriamycin, ADM) be a kind of antitumor antibiotics, but synthetic, the dna dependent rna of severe jamming DNA synthesize and protein synthesis, antitumor spectra is wider, kinds of tumors all there is effect, belong to cell cycle nonspecific agent (CCNSA), the tumor cell of various growth cycles is had killing action.This paper is with the Endoglin antibody fragment of Primary Study miniaturization and the antitumor action of amycin conjugate.

Summary of the invention

The object of the invention is to: a kind of have the miniaturization Endoglin antibody of antitumor action and the conjugate of amycin are provided, and this conjugate Fab-ADM not only has stronger tumor suppression effect, but the life span of significant prolongation tumor animal also.

For achieving the above object, technical scheme of the present invention is: the conjugate that miniaturization Endoglin antibody and amycin are provided, wherein, conjugate Fab-ADM is as cross-linking agent by N-hydroxy-succinamide base-meta-(N-dimaleoyl imino) benzoate (MBS), MBS at first with one contain-molecule of NH2 is that amycin (ADM) reaction and stable bond are active intermediate product, and then with one contain-molecule of S-is through pepsin digestion, the antibody passage Fab reaction of dithiothreitol, DTT (DTT) reduction obtains end-product, i.e. conjugate Fab-ADM; The molecular proportion of Fab and ADM is 1: 2.

The present invention also provides the preparation method of miniaturization Endoglin antibody and amycin conjugate: may further comprise the steps:

(1), select MBS for subsequent use as cross-linking agent;

(2), the MBS of amycin modifies:

The MBS of amycin modifies: the adjustment doxorubicin concentration is 10mg/ml, and MBS concentration is 2mg/ml, adjusts the reflection system take MBS and amycin mol ratio as 10: 1, behind room temperature reaction 30min, mixes with the monoclonal antibody of sulfhydrylation immediately.

(3), the sulfhydrylation of Endoglin antibody passage Fab

Through the monoclonal antibody molecule that saturated ammonium sulphate and propylene polydextran gel S-200HR (Sephacryl S-200HR) purification obtain, the molecular proportion when reacting with pepsin is about 20: 1, and 37 ℃, to react 3-6 hour, pH value of reaction system is 4.5; After reaction finishes, add the Tris (Tris) of 2M, the pH8.0 cessation reaction; Adjustment is 10mg/ml through F (ab ') 2 concentration of Sephacryl S-200HR gel column purification gained, add dithiothreitol, DTT (DTT) reduction, its final concentration is 50mM, room temperature reaction 30min, after the desalination immediately with step (2) in the MBS amycin coupling of modifying.

(4), the preparation of conjugate

The amycin that MBS modifies and the mol ratio of sulfhydrylation monoclonal antibody are approximately 15: 1, room temperature reaction 18h; Conjugate is crossed Sephacryl S-200HR gel column purification and ultrafiltration and concentration, measures protein concentration in absorbance 280nm place.

Description of drawings

Fig. 1 is the preparation principle figure of conjugate; A:MBS reaction principle figure (drawing from Bioconjugate Techniques (2nd Edition) Greg T.Hermanson, P287) wherein; B: amycin; C: through the antibody passage Fab of pepsin digestion, DTT reduction gained; D: antibody through the pepsin digestion schematic diagram (from Internet:http: //www.binglixue.com/zhikao/yaodian/jichu/my05.htm);

Fig. 2 is that the free ADM of mtt assay analysis and conjugate Fab-ADM are to the external increment suppression ratio of hepatoma cell strain BEL-7402;

Fig. 3 is that tumor-bearing mice is at the gross tumor volume of different monitoring points;

Fig. 4 is that tumor-bearing mice is in the survival rate of different monitoring points.

The specific embodiment

1 materials and methods

1.1 material

1.1.1 laboratory animal and cell strain

The hybridoma cell strain, the hepatoma cell strain BEL-7402 that secrete anti-Endoglin monoclonal antibody are preserved by this laboratory, and rat liver cancer H22 cell strain derives from Chinese Typical Representative culture collection center; The cleaning II level Male Kunming strain mice of body weight 18-22g is provided by Guangdong Medical Lab Animal Center.

1.1.2 medicine and reagent

Doxorubicin hydrochloride (doxorubicin hydrochloride) is Italian Pfizer Italia S.r.l. company product.Sephacryl S-200HR gel is available from GE company; The ultrafiltration concentration pipe is available from Millipore company.Special-shaped bifunctional coupling agent MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester) is available from Sigma company.Albumen Marker is available from Fermentas company.Pepsin is available from Amersco company.MTT cell proliferation and cytotoxicity detection kit are available from green skies biotechnology research institute.DMEM dry powder and FBS are available from Gibco company.Other routine biochemistry reagent are domestic analytical pure.

1.2 method

1.2.1 antibody coupling matter preparation

1.2.1.1 antibody coupling matter preparation principle

The preparation of conjugate selects MBS as cross-linking agent, the mechanism of action of MBS is seen Figure 1A, MBS at first with one contain-molecule R (being in this experiment amycin (structural formula is seen Figure 1B)) reaction and the stable bond of NH2 be active intermediate product, and then with one contain-molecule R ' (being the antibody passage Fab (Fig. 1 C) through pepsin digestion, DTT reduction in this experiment) reaction of S-obtains end-product, i.e. conjugate.After pepsin acts on complete antibody molecule, can the nearly C end-grain cutting of disulfide bond between the IgG heavy chain is disconnected, obtain 2 sections of F (ab ') with two valency antibody activities, the fraction (this moment, the major part of Fc section all was hydrolyzed into more micromolecular peptide, did not present any biologic activity) (Fig. 1 D) that also has the Fc section.F (ab ') 2 can expose disulfide bond and be combined (Fig. 1 C) with cross-linking agent after DTT reduction.

1.2.1.2 the MBS of amycin modifies:

The adjustment doxorubicin concentration is 10mg/ml, and MBS concentration is 2mg/ml, adjusts the reflection system take MBS and amycin mol ratio as 10: 1, behind room temperature reaction 30min, mixes with the monoclonal antibody of sulfhydrylation immediately;

1.2.1.3Endoglin the sulfhydrylation of antibody passage Fab

The monoclonal antibody molecule that obtains through saturated ammonium sulphate and propylene polydextran gel S-200HR (Sephacryl S-200HR) purification, molecular proportion when reacting with pepsin is about 20: 1,37 ℃, react 3-6 hour (optimum response is 3 hours), pH value of reaction system is 4.5.After reaction finishes, add the Tris of 2M, the pH8.0 cessation reaction.Adjustment is 10mg/ml through F (ab ') 2 concentration of Sephacryl S-200HR gel column purification gained, adds DTT reduction (its final concentration is 50mM), room temperature reaction 30min, the amycin coupling of modifying with MBS immediately after the desalination.

1.2.1.4 the preparation of conjugate

The amycin that MBS modifies and the mol ratio of sulfhydrylation monoclonal antibody are approximately 15: 1, room temperature reaction 18h; Conjugate is crossed Sephacryl S-200HR gel column purification and ultrafiltration and concentration, measures protein concentration in absorbance 280nm place.

1.2.1.5 the molecular proportion of Fab and ADM is calculated in the conjugate

According to the additivity of absorbance, can in same sample, measure simultaneously plural component without separating.

$A_{280}={\mathrm{\ϵ}}_{\mathrm{Fab}}^{280}{c}_{\mathrm{Fab}}+{\mathrm{\ϵ}}_{\mathrm{ADM}}^{280}{c}_{\mathrm{ADM}}$ $A_{232.5}={\mathrm{\ϵ}}_{\mathrm{Fab}}^{232.5}{c}_{\mathrm{Fab}}+{\mathrm{\ϵ}}_{\mathrm{ADM}}^{232.5}{c}_{\mathrm{ADM}}$

$c_{\mathrm{ADM}}/c_{\mathrm{Fab}}=({\mathrm{\ϵ}}_{\mathrm{Fab}}^{232.5}-{\mathrm{R\ϵ}}_{\mathrm{Fab}}^{280})/({\mathrm{R\ϵ}}_{\mathrm{ADM}}^{280}-{\mathrm{\ϵ}}_{\mathrm{ADM}}^{232.5})$ R＝A _232.5/A ₂₈₀

In the above-mentioned formula, A represents absorbance, A=ε bc, and wherein ε is specific absorbance, and the L/ of unit (gcm), b are liquid layer thickness (being generally the thickness of cuvette), and the cm of unit, c are solution concentration, the g/L of unit.

1.2.1.6 non-reduced SDS-PAGE detects

Adopt non-reduced SDS-PAGE to detect the size of conjugate.Resolving gel concentration is 7.5%, and concentrated gum concentration is 5%, and electrophoretic voltage is 100V, and the time is 80min.

1.2.2 the body outer cell proliferation inhibition test of conjugate

Select mtt assay to detect.The take the logarithm BEL-7402 cell of trophophase, counting and to adjust cell concentration be 3000/100 μ L, every hole adds 100 μ L in 96 porocyte culture plates, at 37 ℃, 5%CO ₂Incubator in cultivate 24h.Adding is with ADM and each 10 μ L/ hole of conjugate Fab-ADM of serum-free DMEM doubling dilution, and each concentration is established 3 parallel holes, establishes simultaneously the blank hole, and negative control hole adds serum-free DMEM culture fluid 10 μ L.Continue to cultivate 72h, every hole adds 10 μ L MTT solution, in cell culture incubator, hatch 4h after, every hole adds 100 μ L Formanzan lysates, continues to hatch about 4h, in 570nm place mensuration absorbance and calculate tumor control rate and the IC50 value.Suppression ratio (%)=(1 one experimental group OD values/matched group OD value) * 100%.And calculate using dosage in the body of conjugate according to the IC50 value: actual dosage in the coupling object=free ADM dosage * (the external IC50/ of conjugate dissociate the external IC50 of ADM).The computational methods of IC50 are improvement Kou Shifa:

lgIC50＝Xm-I(P-(3-Pm-Pn)/4)

Xm:1g maximal dose wherein

I:1g (maximal dose/face mutually dosage)

P: positive reaction rate sum

Pm: maximum positive reaction rate

Pn: minimum positive reaction rate

1.2.3 the in-vivo tumour inhibition test of conjugate

Kunming mice, male, body weight 18-22g, random packet, 10 every group.Murine hepatocarcinoma cell strain H22 goes down to posterity once through mouse peritoneal, collects ascites cells, with normal saline by dilution in 1: 3 after, be inoculated in the right axil of mice subcutaneous, 0.2ml/ only (approximately 2 * 10 ⁶Individual cell/only).Treat that gross tumor volume grows into when approximately 4 * 6mm is big or small the beginning medication.Administering mode is tail vein injection, and matched group gives normal saline 0.2ml/ only, and ADM dosage is 0.4mg/kg, and consumption such as is expressed as at cytotoxicity dosage: the 0.4mg/kg in the coupling object, and Fab dosage also is 0.4mg/kg.Be administered once weekly continuous 3 all administrations.Experimental session is measured weekly major diameter a and the minor axis b of 2 tumors, with formula V=0.52ab ²Calculate tumor volume (V), draw tumor growth curve, and calculate tumour inhibiting rate.Observe the animal survival situation, calculate median survival time with Kap lan-Meier method.

The gross tumor volume of each treated animal of statistical procedures all uses mean ± SD to represent, analyzes between group and adopts the t check.

2 results

2.1 the molecular proportion of Fab and ADM is calculated in the conjugate

The production standard curve is obtained the ε value, R=0.722/0.298 ≈ 2.42, cADM/cFab ≈ 2, molecular proportion: Fab: ADM=1: 2.The molecular proportion of Fab and ADM is 1: 2 in the conjugate, illustrates that a Fab molecule combines two ADM, according to the characteristic of MBS, also should contain two MBS molecules in the conjugate, and according to this hypothesis, the molecular weight that can estimate conjugate is M _Fab+ 2M _ADM+ 2M _MBS≈ 51788Da.

2.2 non-reduced SDS-PAGE detects

The molecular weight of conjugate roughly conforms to estimation.

2.3 the tumor cell in vitro inhibited proliferation of conjugate Fab-ADM

Measure Fab-ADM conjugate and ADM to the cytotoxicity of the BEL-7402 hepatoma carcinoma cell of In vitro culture with mtt assay.The results are shown in Figure 2.Calculate ADM and Fab-ADM conjugate according to formula the IC50 that the propagation of hepatoma cell line BEL-7402 suppresses is respectively 3.15ug/ml and 124ug/ml.If the cytotoxicity dosage such as be calculated as, the IC50 value of Fab-ADM is 2.78, and the IC50 value difference of this numerical value and free ADM is different little, and this may not be that the target cell of Endoglin is relevant with BEL-7402.

In the zoopery, free ADM adopts the dosage of 0.4mg/kg, and the actual using dosage of extrapolating conjugate Fab-ADM according to the IC50 value is 15.75mg/kg, but is expressed as the dosage such as cytotoxicity such as grade of 0.4mg/kg.

2.4 the in-vivo tumour inhibition test of conjugate

Behind the mouse hypodermic inoculation hepatocarcinoma H22, treat that gross tumor volume grows into when approximately 4 * 6mm is big or small, beginning medication treatment.Tumor growth curve as shown in Figure 3.Compare with free ADM, wait the conjugate Fab-ADM of cytotoxicity dosage can significantly suppress the growth of H22.Calculate the tumour inhibiting rate of medicine according to gross tumor volume: medication the 14th day, the suppression ratio of ADM (0.4mg/kg) is 25%, the suppression ratio of conjugate (0.4mg/kg waits cytotoxicity dosage) reaches 91.94%; And the 21st day the time, the tumor-bearing mice of ADM treatment is all dead, and the suppression ratio of conjugate still is 87.00%, and inhibitory action significantly strengthens (P＜0.05) than ADM.

Observe survival condition and the life span of animal, the result shows (Fig. 4): the median survival time of conjugate Fab-ADM treatment group is about 32 days, and the ADM treatment group be about 14 days.This explanation is compared with the ADM that dissociates, and conjugate Fab-ADM not only has stronger tumor suppression effect, but the life span of significant prolongation tumor animal (P＜0.01) also.

3 conclusions

The Endoglin antibody passage Fab is through the big or small approximately 51788Da of the coupled product Fab-ADM of MBS and amycin, and in the experiment, Fab-ADM can significantly suppress the growth of H22 in the body.Calculate the tumour inhibiting rate of medicine according to gross tumor volume: medication the 14th day, the suppression ratio of ADM (0.4mg/kg) is 25%, the suppression ratio of conjugate (0.4mg/kg waits cytotoxicity dosage) reaches 91.94%; And the 21st day the time, the tumor-bearing mice of ADM treatment is all dead, and the suppression ratio of conjugate still is 87.00%, and inhibitory action significantly strengthens (P＜0.05) than ADM.The median survival time of conjugate Fab-ADM treatment group is about 32 days, and the ADM treatment group be about 14 days.This explanation is compared with the ADM that dissociates, and conjugate Fab-ADM not only has stronger tumor suppression effect, but the life span of significant prolongation tumor animal (P＜0.01) also.

Above disclosed only is preferred embodiment of the present invention, certainly can not limit with this interest field of the present invention, and the equivalent variations of therefore doing according to claim of the present invention still belongs to the scope that the present invention is contained.

Claims (2)

1. the conjugate of miniaturization Endoglin antibody and amycin, it is characterized in that: conjugate Fab-ADM is as cross-linking agent by N-hydroxy-succinamide base-meta-(N-dimaleoyl imino) benzoate, N-hydroxy-succinamide base-meta-(N-dimaleoyl imino) benzoate at first with one contain-molecule of NH2 is that amycin reaction and stable bond are active intermediate product, and then contain with one-molecule of S-is combined, this molecule is through pepsin digestion, the antibody passage Fab of dithiothreitol, DTT reduction, the end-product that reaction obtains is conjugate; The molecular proportion of Fab and amycin is 1: 2.

2. the preparation method of the conjugate of miniaturization Endoglin antibody as claimed in claim 1 and amycin: may further comprise the steps:

(1), select N-hydroxy-succinamide base-meta-(N-dimaleoyl imino) benzoate for subsequent use as cross-linking agent;

(2), the MBS of amycin modifies:

The adjustment doxorubicin concentration is 10mg/ml, N-hydroxy-succinamide base-meta-(N-dimaleoyl imino) benzoate concentration is 2mg/ml, adjusted reaction system as 10: 1 take N-hydroxy-succinamide base-meta-(N-dimaleoyl imino) benzoate and amycin mol ratio, behind room temperature reaction 30min, mix with the monoclonal antibody of sulfhydrylation immediately;

(3), the sulfhydrylation of Endoglin antibody passage Fab

Through the monoclonal antibody molecule that saturated ammonium sulphate and propylene polydextran gel S-200HR purification obtain, the molecular proportion when reacting with pepsin is about 20: 1, and 37 ℃, to react 3-6 hour, pH value of reaction system is 4.5; After reaction finishes, add the Tris of 2M, the pH8.0 cessation reaction; Adjustment is 10mg/ml through F (ab ') 2 concentration of propylene polydextran gel S-200HR gel column purification gained, add the dithiothreitol, DTT reduction, its final concentration is 50mM, room temperature reaction 30min, after the desalination immediately with step (2) in the MBS amycin coupling of modifying;

(4), the preparation of conjugate

The amycin that MBS modifies and the mol ratio of sulfhydrylation monoclonal antibody are approximately 15: 1, room temperature reaction 18h; Conjugate is crossed propylene polydextran gel S-200HR gel column purification and ultrafiltration and concentration, measures protein concentration in absorbance 280nm place.

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