CN103288968B - An anti-tumor targeting complex and a preparation method and applications thereof - Google Patents

Abstract

The present invention provides an anti-tumor targeting complex which is a chemical crosslinked complex CTX-Onc obtained from chlorotoxin and onconase through disulfide bond crosslinking. Compared with existing anti-tumor CTX and Onc mixtures, the anti-tumor targeting complex of the invention shows better anti-tumor effects both at a cellular level and an animal tumor model level, and has good clinical application and development prospects. The invention also provides a method for preparing the complex and applications of the complex in the preparation of antitumor drugs.

Description

A kind of antineoplastic target mixture and its preparation method and application

Technical field

The present invention relates to the field of biological pharmacy that biological chemistry and genetic engineering technique are relevant, specifically, the present invention relates to a kind of antineoplastic target mixture, and preparation method thereof with this application of target mixture in oncotherapy.

Background technology

According to Ministry of Health's statistic data, malignant tumour has become China's first fatal disease.The routine treatment of malignant tumour, comprises operation, radiotherapy, chemotherapy, all has some limitations.Such as traditional anti-tumor medicine has larger side effect usually, while killing cancer cells, also cause very large damage to normal cell.Targeted drug utilizes the targeted molecular special to tumour cell, by antitumor drug specificity guiding tumour cell, therefore has higher killing-efficiency to tumour cell, then lower to Normocellular toxic side effect, the emphasis of antitumor drug research after being.

Frog's egg rnase (English name onconase is called for short Onc) is a kind of rnase (RNase) separated from a kind of wood frog (Rana pipiens) ovocyte for 1988, only has 104 amino-acid residues.The most outstanding feature of Onc is that it has cytotoxicity, can kill cancer cells, and immunogenicity is low.At the beginning of 2007, U.S. FDA has ratified independent Onc as the clinical treatment of orphan's medicine for malignant pleural mesothelioma.

TM-601 (English name chlorotoxin is called for short CTX) is the polypeptide toxin separated from the golden scorpion venom in Africa for 1993, only has 36 amino-acid residues.CTX can be attached on tumour cell by the MMP2 of malignant glioma cells surface specific high expression level and Annexin-2.U.S. FDA has ratified the CTX of radioactivity iodine 131 mark as the clinical treatment of orphan's medicine for glioblastoma.Glioblastoma is the modal a kind of malignant tumour of central nervous system, is also one of the most refractory current malignant tumour.

In prior art, be used alone the effect that Onc or CTX carry out antineoplaston and there is very large limitation.Such as, although CTX has tumor-targeting, itself be not enough to kill tumour cell; Although Onc can kill tumour cell, also can kill normal cell, selective killing effect is not had to tumour cell.Even if Onc and CTX is used in combination, do not reach good antitumous effect yet.

Summary of the invention

The object of the invention is to: a kind of antineoplastic target mixture is provided, there is the antitumor curative effect higher than any one or its mixture in existing antineoplastic biologic preparation Onc, CTX.

Another object of the present invention is to provide a kind of preparation method preparing above-mentioned antineoplastic target mixture.

Another object of the present invention is to provide described antineoplastic target mixture and is preparing the application in antitumor drug.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

There is provided a kind of antineoplastic target mixture, it is the chemically crosslinked mixture that TM-601 (CTX) and frog's egg rnase (Onc) are obtained by disulfide bond crosslinking, and we are denoted by " CTX-Onc ".

Described CTX is existing material, and ripe strategy can be facilitated to prepare ripe CTX based on Recombinant protein expression and vitro enzyme, the nucleotide sequence of 6 × His-CTX of chemosynthesis is shown in Seq ID NO.1, and aminoacid sequence is shown in Seq ID NO.2.

Described Onc is existing material, and ripe strategy can be facilitated to prepare ripe Onc based on Recombinant protein expression and vitro enzyme, the nucleotide sequence of 6 × His-Onc of chemosynthesis is shown in Seq ID NO.3, and aminoacid sequence is shown in Seq ID NO.4.

The present invention also provides the method for CTX and the Onc chemically crosslinked mixture (CTX-Onc) described in preparation, and its step is as follows:

1) restructuring preparation ripe CTX and amino specific modifier SPDP (50mmol/l sodium phosphate in phosphoric acid buffer, 150mmol/l NaCl, pH8.0) by the mol ratio of 1:1 in room temperature reaction 1h, be then adjusted to pH3.0 with TFA, purify with HPLC, collect modify CTX and through Mass Spectrometric Identification;

2) restructuring preparation ripe Onc and amino specific modifier 2-Iminothiolane (50mmol/l sodium phosphate in phosphoric acid buffer, 150mmol/l NaCl, 2.4mmol/l mercaptoethanol, pH8.0) by the mol ratio of 1:20 at room temperature reaction 1h, then be adjusted to pH3.0 with acetic acid, remove excessive modifier with Sephadex G-25 molecular sieve;

3) CTX step 1) modified and step 2) Onc that modifies is by mol ratio (50mmol/l sodium phosphate in phosphoric acid buffer of 1:1,150mmol/l NaCl, pH8.0) in room temperature reaction 0.5h in, then pH3.0 is adjusted to acetic acid, salt is removed with Sephadex G-25 molecular sieve, with 10% acetic acid wash-out, obtain described CTX and Onc chemically crosslinked mixture, namely described antineoplastic target mixture.

The present invention also provides described antineoplastic target mixture preparing the application in antitumor drug.

In the present invention, we utilize CTX can the characteristic of specific binding tumour cell, by its with the Onc with tumor cytotoxicity effect by together with reversible disulfide bond crosslinking, thus will the Onc target tumor tissue of killing functions of immunocytes be had.Reversible disulfide bond be cross-linked be conducive to CTX delivery Onc in the release of tumor locus.As compared to the mixture of existing antitumorigenic substance CTX with Onc, this novel targeted mixture all demonstrates better antitumous effect on a cellular level with on animal tumor model, has good clinical application DEVELOPMENT PROSPECT.

Accompanying drawing explanation

Fig. 1 is the electrophoresis result figure by SDS-PAGE electroresis appraisal embodiment 3 made disulfide bond crosslinking CTX-Onc target mixture.

Fig. 2 is that the made crosslinked target mixture CTX-Onc of embodiment 3 and control group (mixture of CTX and Onc) are to the lethal effect comparison diagram of 4 kinds of tumour cells.

Fig. 3 is the made crosslinked target mixture CTX-Onc of embodiment 3 and control group (mixture of CTX and the Onc) effect contrast figure on model of nude mice bearing tumor.Inoculated tumour cell is glioma cell of U251, and injected dose is 2.5mg/kg.

Fig. 4 is the made crosslinked target mixture CTX-Onc of embodiment 3 and control group (mixture of CTX and the Onc) effect contrast figure on model of nude mice bearing tumor.Inoculated tumour cell is glioma cell SHG-44, and injected dose is 2.5mg/kg.

Embodiment

Embodiment 1: the ripe CTX of Recombinant protein expression

The abduction delivering of GST-6 × His-CTX and separation and purification: the N-that encodes holds the gene chemical method of the CTX precursor carrying 6 × His label and enterokinase cleavage site point to synthesize, use intestinal bacteria preference codon (Seq ID NO.1).This chemosynthesis gene clone in coli expression carrier pGEX-4T-1, and confirms through DNA sequencing.This expression vector pGEX-4T-1/6 × His-CTX is transformed in e. coli bl21 (DE3), by IPTG abduction delivering (16 ° of C overnight incubation) in TB substratum.Collected by centrifugation thalline, and be resuspended in (50mmol/l phosphoric acid salt, 0.5mol/l NaCl, pH7.5) in lysis buffer, utilize the broken thalline of pressure application, add DNA enzymatic I degradation of dna.Add solid sodium sulfite subsequently to final concentration 0.2mol/L, adding solid sodium tetrathionate to final concentration is 0.15mol/L, slowly shakes 2-3h by reversibly modified for the sulfydryl sulfonic group in CTX in room temperature.Centrifugal and filter after, Sulfonated GST-6 × His-CTX metal chelate chromatography (Ni ²⁺-post) purifying, with 100mmol/L imidazoles wash-out, elution fraction SDS-PAGE detects.

The enzyme of GST-6 × His-CTX cuts the oxidative folding with CTX: the elution peak of above-mentioned collection with remove portion imidazoles by ultrafiltration and concentration, is then added appropriate enteropeptidase and cuts through night to remove GST and 6 × His label in 25 ° of C enzymes.Sulfonated CTX and the GST cut down are divided out by molecular sieve (Sephadex G-50) by digestion products.Adding DTT to final concentration in the CTX component of collecting is 10mmol/l, room temperature reaction 1-2h, to remove the sulfonic group modified, then 20 is diluted to (0.5mol/l arginine, 1.0mmol/l Sleep-promoting factor B in the folding buffered liquid of precooling, pH8.5), folding 1-2h in ice bath.Then folded product trifluoroacetic acid (TFA) adjusts pH to 3.0, purifies with C18 reversed-phase column HPLC, collects elution peak, lyophilize, and uses mass spectroscopy molecular weight.

Embodiment 2: the ripe Onc of Recombinant protein expression

6 × His-Onc abduction delivering and separation and purification: N-holds the Onc gene carrying 6 × His label to be synthesized by chemical method, selects intestinal bacteria preference codon (Seq ID NO.3).This gene to be connected in expression vector pET and to be confirmed by DNA sequencing.Then this expression vector pET/6 × His-Onc is transformed in e. coli bl21 (DE3), and in TB substratum, uses IPTG in 37 ° of C abduction deliverings.Thalline is broken by supersonic method, by collected by centrifugation inclusion body.Inclusion body 6mol/l guanidine hydrochloride dissolution, and to add final concentration be that the S-WAT of 0.2mol/l and the sodium tetrathionate of 0.15mol/l are with the sulfydryl in reversibly modified Onc.After room temperature slowly shakes 2-3h, Sulfonated 6 × His-Onc metal chelate chromatography purifying, the wash-out of imidazoles.

6 × His-Onc enzyme cuts the maturation with Onc: dialysed to water by 6 × His-Onc elutriant of above-mentioned collection, to remove Guanidinium hydrochloride wherein and imidazoles.The 50mmol/l Tris-Cl(pH8.5 of 6 × His-Onc precipitation containing 2.4mol/l Guanidinium hydrochloride separated out after dialysis) dissolve, 1:2000 adds Areanomas aminopeptidase and pawpaw glutamine cyclase in molar ratio, spends the night in 37 ° of C water-baths.Then add the DTT that final concentration is 50mol/l, room temperature places 30min, and 100 times are diluted in the folding reaction solution (50mM Tris-Cl, 1.0mmol/l GSSG, 1.0mmol/l EDTA, pH8.5) of precooling subsequently.Folding 2-3h in ice bath, folding mixture TFA is adjusted to pH3.0, reverse HPLC-purified with C18.

The chemically crosslinked of embodiment 3:CTX and Onc

Recombinate embodiment 1 the ripe CTX and amino specific modifier SPDP (50mmol/l sodium phosphate in phosphoric acid buffer that prepare, 150mmol/l NaCl, pH8.0) by the mol ratio of 1:1 in room temperature reaction 1h, then pH3.0 is adjusted to TFA, purify with HPLC, collect modify CTX and through Mass Spectrometric Identification.Recombinate embodiment 2 the ripe Onc and amino specific modifier 2-Iminothiolane (50mmol/l sodium phosphate in phosphoric acid buffer that prepare, 150mmol/l NaCl, 2.4mmol/l mercaptoethanol, pH8.0) by the mol ratio of 1:20 at room temperature reaction 1h, then be adjusted to pH3.0 with acetic acid, remove excessive modifier with Sephadex G-25 molecular sieve.The CTX of above-mentioned modification and the Onc of modification presses the mol ratio of 1:1 (50mmol/l sodium phosphate in phosphoric acid buffer, 150mmol/l NaCl, pH8.0) in room temperature reaction 0.5h, be then adjusted to pH3.0 with acetic acid, salt is removed, with 10% acetic acid wash-out with Sephadex G-25 molecular sieve.Eluted product lyophilize, and identify with tricine SDS-PAGE, result is as shown in Figure 1.

Embodiment 4: the antitumous effect detecting CTX-Onc target mixture on a cellular level

Cultured tumor cells is inoculated in 96 well culture plates (every hole about 2 × 10 ³individual cell), add crosslinked target mixture (CTX-Onc) and mixing contrast (CTX+Onc, namely CTX and Onc simply mixes) process 36h in substratum respectively, then detect cell viability by MTT method.

This experiment has carried out simultaneous test for U251, U87, SGH-44 and A549 tetra-kinds of tumour cells respectively, and experimental result as shown in Figure 2.Result display CTX target Onc(CTX-Onc) fragmentation effect of above-mentioned 4 kinds of tumour cells is significantly higher than to the mixture (CTX+Onc) of CTX and Onc, show that CTX serves the effect of Onc targets neoplastic cells.

Embodiment 5: the antitumous effect of CTX-Onc target mixture on mouse model

By culturing cell suspension (the SHG-44 cell count 5 × 10 containing 50% matrigel ⁷; U251 cell count 2.5 × 10 ⁸) be subcutaneously injected into 4 ~ 5 week age nude mice on the right side of abdomen back.Gross tumor volume to be seeded is about 100 ~ 200mm ³afterwards (after injection about 4 ~ 5 weeks), respectively with target CTX-Onc mixture, mixture (CTX+Onc) and physiological saline tail vein injection prepared by embodiment 3.Per-Hop behavior twice, successive administration 2 ~ 3 weeks, periodic measurement mouse tumor volume and Mouse Weight, by sacrifice after experiment terminates, knurl body is weighed and is fixed, cuts into slices.Experimental result as shown in Figure 3 and Figure 4.

Sequence table

The equal Bioisystech Co., Ltd in <110> Shanghai

<120> antineoplastic target mixture and its preparation method and application

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<210>1

<211>150

<212>DNA

<213> artificial sequence

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<223> synthesizes the intestinal bacteria preference codon of CTX

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Cys Tyr Gly Pro Gln Cys Leu Cys Arg

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<210>2

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<213> TM-601 (CTX)

<400>2

Met His His His His His His Met Asp Asp Asp Asp Lys Met Cys Met Pro Cys Phe Thr Thr Asp His

1 5 10 15 20

Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln Cys

25 30 35 40 45

Leu Cys Arg

<210>3

<211>339

<212>DNA

<213> artificial sequence

<220>

<223> synthesizes the intestinal bacteria preference codon of Onc

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atg cat cac cat cac cat cat atg cag gat tgg ctg acc ttt caa aaa aaa cat att acg 60

Met His His His His His His Met Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr

1 5 10 15 20

aac act cgt gat gtg gac tgt gat aac atc atg agc act aac ctg ttt cac tgc aaa gat 120

Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp

25 30 35 40

aaa aat acc ttc atc tat tct cgc ccg gaa ccg gtt aaa gcg att tgc aaa ggc att atc 180

Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile

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gcc tcc aaa aat gtc ctg acc acg agc gag ttt tac ctg agc gac tgt aac gta acc tcg 240

Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser

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cgc ccg tgc aaa tat aaa ctg aaa aag agc acc aat aaa ttt tgt gtt acg tgc gaa aac 300

Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn

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cag gca ccg gtc cac ttc gtg ggc gtt ggc tct tgt taa 339

Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys

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<210>4

<211>111

<212>PRT

<213> frog's egg rnase (Onc)

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Met His His His His His His Met Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr

1 5 10 15 20

Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp

25 30 35 40

Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile

45 50 55 60

Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser

65 70 75 80

Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn

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Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys

105 110

Claims (3)

1. an antineoplastic target mixture, it is the chemically crosslinked mixture CTX-Onc that TM-601 CTX and frog's egg rnase Onc is obtained by disulfide bond crosslinking; Described CTX is the ripe CTX facilitating ripe strategy to prepare based on Recombinant protein expression and vitro enzyme, and 6 × His-CTX of chemosynthesis that aminoacid sequence with Seq IDNO.2 identical identical with Seq ID NO.1 by nucleotide sequence remove N ?hold hexahistidine tag to obtain; Described Onc is the ripe Onc facilitating ripe strategy to prepare based on Recombinant protein expression and vitro enzyme, and 6 × His-Onc of chemosynthesis that aminoacid sequence with Seq ID NO.4 identical identical with Seq ID NO.3 by nucleotide sequence remove N ?hold hexahistidine tag to obtain;

Described antineoplastic target mixture is prepared by following methods:

1) the ripe CTX prepared and amino specific modifier SPDP that recombinates presses the mol ratio of 1:1 in room temperature reaction 1h in phosphoric acid buffer, described phosphoric acid buffer contains 50mmol/l sodium phosphate and 150mmol/l NaCl, and pH=8.0, then pH3.0 is adjusted to TFA, purify with HPLC, collect modify CTX and through Mass Spectrometric Identification;

2) the ripe Onc prepared and amino specific modifier 2-Iminothiolane that recombinates presses the mol ratio of 1:20 at room temperature reaction 1h in phosphoric acid buffer, described phosphoric acid buffer contains 50mmol/l sodium phosphate, 150mmol/l NaCl and 2.4mmol/l mercaptoethanol, and pH=8.0, then be adjusted to pH3.0 with acetic acid, remove excessive modifier with Sephadex G-25 molecular sieve;

3) by step 1) CTX that modifies and step 2) Onc that modifies by the mol ratio of 1:1 in phosphoric acid buffer in room temperature reaction 0.5h, described phosphoric acid buffer contains 50mmol/l sodium phosphate and 150mmol/l NaCl, and pH=8.0, then pH3.0 is adjusted to acetic acid, salt is removed with Sephadex G-25 molecular sieve, with 10% acetic acid wash-out, obtain described CTX and Onc chemically crosslinked mixture, namely described antineoplastic target mixture.

2. the preparation method of antineoplastic target mixture according to claim 1, its step is as follows:

1) the ripe CTX prepared and amino specific modifier SPDP that recombinates presses the mol ratio of 1:1 in room temperature reaction 1h in phosphoric acid buffer, described phosphoric acid buffer contains 50mmol/l sodium phosphate and 150mmol/l NaCl, and pH=8.0, then pH3.0 is adjusted to TFA, purify with HPLC, collect modify CTX and through Mass Spectrometric Identification;

2) the ripe Onc prepared and amino specific modifier 2-Iminothiolane that recombinates presses the mol ratio of 1:20 at room temperature reaction 1h in phosphoric acid buffer, described phosphoric acid buffer contains 50mmol/l sodium phosphate, 150mmol/l NaCl and 2.4mmol/l mercaptoethanol, and pH=8.0, then be adjusted to pH3.0 with acetic acid, remove excessive modifier with Sephadex G-25 molecular sieve;

3) by step 1) CTX that modifies and step 2) Onc that modifies by the mol ratio of 1:1 in phosphoric acid buffer in room temperature reaction 0.5h, described phosphoric acid buffer contains 50mmol/l sodium phosphate and 150mmol/l NaCl, and pH=8.0, then pH3.0 is adjusted to acetic acid, salt is removed with Sephadex G-25 molecular sieve, with 10% acetic acid wash-out, obtain described CTX and Onc chemically crosslinked mixture, namely described antineoplastic target mixture.

3. antineoplastic target mixture according to claim 1 is preparing the application in antitumor drug, it is characterized in that: described antitumor drug is the medicine of anti-glioma or anti-lung gland cancer.