CN1085910A - Anti-gastric cancer biological guiding medicine-immunoglobulin Y-phytotoxin A - Google Patents

Abstract

The present invention applies the knowledge of molecular genetics and oncogenetics and method synthesized anti-gastric cancer bio-directed drug-IgY-RicinA, and should Clinical research on gastric cancer patients, 28 cases have been treated, proving that the Bio-guided drugs have great potential for the treatment of tumors.

Description

The invention belongs to the field of molecular biology and biochemical medicine.

Gastric cancer is a malignant tumor with a high incidence. In order to diagnose and treat this disease, we have applied the knowledge and methods of molecular genetics and oncogenetics (Molecular genetics and Oncogenetics) to explore. IgY was reported by L.F.Mc Bee and O.J.Cotterill in 1979 (J.Food.Sci, 1979, 44:656), and J.C.Jensenius published an article in 1981 (J.lmmunol.Methods, 1981, 46:63), Japan Hajime Hatta et al. conducted research on IgY (Agric. Biol. Chem, 1990, 54: 2531-2535), but no one immunized with oncoalbumin as an antigen to obtain anti-cancer antibody IgY. Domestic Guiyang Medical College Wang He and others prepared anti-influenza antibody IgG in egg yolk (Shanghai Journal of Immunology, 1987, 7: 364-365).

Ricin and RicinA were reported in 1982 by R.J.Youle (J.Biol.Chem, 1982, 257:1598-1601), but we have not seen reports in China.

The toxic properties of immunotoxins were reported by Ellen. S. Vitetta and J.W, Uhr in 1985 (Cell, 1985, 41:653-654). Reported in Mehod Enzymology Vol, 112.

Our research uses two types of protein binding methods, that is, selecting an antibody that can recognize tumor cells but does not react with normal cells, and the other protein is a toxic protein that can kill cells. The combination of the two proteins forms a specific directed cancer drugs.

Antigen and antibody selection:

According to research on Oncogenes. It is believed that tumor cells are the result of two or more gene mutations, in which oncogenes produce a large number of oncoproteins, and antibodies obtained by immunizing with this oncoprotein as an antigen can specifically recognize cancer cells. To this end, we selected malignant tumor cells of the human stomach, and isolated and purified oncoproteins from poorly differentiated mucinous adenocarcinoma cells (Human stomach mucoid adenocarcinoma).

Purification of Oncoproteins:

The cultured human gastric cancer MGC-80-3 cells were suspended in buffer A (10mM Nacl, 1.5mM Mgcl ₂ , 10mM Tris-Hcl (pH8.0) 0.1mM PMSF, (phenylmethylsulfonyl fluoride) 0.1mM EDTA , sonicate the cells, add solid ammonium sulfate to make the broken cells 50% saturated, stir for 30 minutes, centrifuge at 2000rpm for 20 minutes, and dissolve the pellet in buffer B (20mM Tris-Hcl, pH8.0, 2%SDS, (Sodium dodecyl sulfate, 10 mM EDTA, 0.1% 2-mercaptoethanol) were extracted three times with water-saturated phenol, and the phenol phase was collected and dialyzed in buffer B (plus 0.25M sucrose to remove all phenol. The solution was passed through Sephorose - CL-4B antibody column affinity chromatography, (elution with Tris-Hel pH8.0, 1% NP40), collect the peak area. Gel electrophoresis analysis, the result is oncoprotein 110KD, used as an antigen for immunization.

Antibody acquisition:

The IgY antibody comes from the yolk of eggs laid by immunized hens, which is suitable for the analysis and research of conservative antigens, and the oncoprotein is a conserved protein in biological evolution. IgY is easy to obtain from egg yolk, and the yield is large. One egg can purify 50-100mg antibody IgY. The use of this antibody to treat tumors is our discovery. Because it can be obtained in large quantities, it provides the possibility for clinical treatment.

The yolks of eggs laid after immunization were harvested and diluted (1:1 v/v) with buffer C (0.1M NaCl, 0.01% NaN ₃ , 0.01M sodium phosphate). Add PEG (polyethylene glycol Mr.6000) after dilution, add 7 grams of PEG to 100ml egg yolk dilution, stir to dissolve the PEG, centrifuge at 12000rpm for 10 minutes, take the supernatant, filter it with four layers of gauze, add PEG in In the filtrate, make it 12% concentration, dissolve and then centrifuge as above, take the precipitate, dissolve it in buffer C, the volume is the same as the original volume, repeat this operation three times, finally, dissolve the precipitate in normal saline, if a longer time For storage, add 25% glycerol and 0.02% NaN ₃ to normal saline and store at -4°C.

Ricin is a kind of poison in plant tissue, it can inhibit the synthesis of protein, it is a heterogeneous double body combined with disulfide bonds, composed of two peptide bonds of A and B, and the B chain is a plant lectin The nature of the substance is similar to that of galactose, which promotes the binding of poisons to the cell membrane. The A chain is an enzymatic peptide chain that binds to ribosomes, inhibits the synthesis of cellular proteins, and kills cells. We chose ricin as a poison, replaced its B chain with antibody IgY, and retained the killing effect of the A chain.

Take Ricinus Commnnis Costor peeled off the seed coat, soak 350 grams of seeds in 1000ml of 5% acetic acid, stir overnight at room temperature, beat the soaked material, add 1000ml of 5% acetic acid after homogenization, and the liquid is at 3000rpm Centrifuge for 20 minutes, pass the orange liquid through four layers of gauze and then filter it with Xinhua No. 1 filter paper, add solid ammonium sulfate to the filtrate to 80% saturation, stir for 40 minutes and centrifuge at 3000rpm for 30 minutes, dissolve the precipitate in 150ml double distilled water, and Dialyzed in double distilled water for 20 hours to obtain 350ml dark yellow solution.

The solution was chromatographed on DEAE-Sephadex A50, and the ricin was not combined with the chromatographic agent. The column liquid was collected, and ammonium sulfate was added to reach 80% saturation, stirred at 4°C, centrifuged as above, the precipitate was collected, dissolved in double distilled water, and Dialyzed in double distilled water for 48 hours, then chromatographed on Sephadex G100, eluted with buffer D (0.01M sodium phosphate, 0.1M galactose) and collected step by step, collected from the poisonous peak area of ricinum, and saturated with ammonium sulfate at 50% Precipitate ricin, dissolve the precipitate in double distilled water, dialyze in double distilled water, and then freeze-dry to obtain purified Ricin (ricin).

Dissolve Ricin in buffer E (0.1M Tris-Hcl pH 8.5, 0.5M lactose, 5% ethyl-mercaptoethanol), stir at room temperature (20-25°C) for 12 hours, and let stand for 10 hours. After DEAE-cellulose DE52 chromatography, the peak area was dialyzed overnight at 4°C in buffer F (0.01M lactose, 0.1% ethylene-mercaptoethanol, 5mM sodium phosphate (pH6.5). The product was RicinA. Then CM-sephaoles CM50 chromatography, 0-0.15M Nacl buffer F gradient elution, peak area collection, to obtain pure RicinA.

IgY-RicinA conjugation

Antibody IgY is coupled with ricin to form an artificial hybrid protein. Linking two protein peptides from different sources requires a bifunctional reagent that prevents intramolecular crosslinking. Especially toxic proteins are easy to form cohesion, we choose SPDP [N-Succinimidyl-3 (2-pyridyldithio) propionate) as the linker, it provides pyridyl disulfide bond group and thiol gene for the protein, so that the two proteins can be combined to form Disulfide bonded conjugates.

This conjugate replaces the B bond of ricin with an antibody, which increases the specific recognition of the conjugate, and the conjugated toxin may form a sulfolipid substance to reduce toxicity. If it is combined with the full poison of ricinum (A.B chain), it is highly toxic, so we only use the A chain. According to our research, IgY has a good affinity with RicinA, so it can make RicinA play a good role. Strong ability to kill tumor cells.

(1) Dissolve SPDP in absolute ethanol at a concentration of 20mM.

(2) Dilute IgY to 10mg/ml with PBS (0.15M Nacl, 0.01M sodium phosphate).

(3) 750ml IgY plus 1ml SPDP (7.5g IgY: 6.25mg SPDP) was stirred at room temperature (20-25°C) for 30 minutes.

(4) Adjust RicinA to 10 mg per ml, and add 2-mercaptoethanol to 0.1%. Stir at room temperature for 30 minutes.

(5) Slowly add the treated RicinA to the treated IgY (according to IgY:RicinA = 2:1) in an ice bath, stir the mixture gently, and let it stand in the ice bath for 5 minutes, then place it at 20°C-25°C Leave overnight (20 hours).

(6) IgY-RicinA was separated by Sephacryl S-200 chromatography, the peak area was collected and concentrated. Become a finished anti-gastric cancer antibody toxin-oriented drug.

The IgY antibody has a selective recognition effect on cells, which provides an important guiding role for the antibody to treat tumors. IgY fluorescence immunoassay in cultured cells can only react with gastric cancer cells. No cross-immunity with other cells. Immunofluorescence assays from patient tumor tissues proved that IgY can recognize gastric cancer cells and do not react with normal tissue cells. IgY antibody was labeled with radioactive isotope ¹⁶⁹ Yb and tracked in animals. The results showed that IgY was mainly concentrated in tumor tissues, and the content in other organs was very small. At the same time, we compared the recognition effect of anti-gastric cancer monoclonal antibody and IgY antibody. The positive rate of IgY to gastric cancer tissues was 87.1% by ABC staining method. The positive rate of anti-gastric cancer monoclonal antibody MG9 to gastric cancer tissues was 80.6%. There was no significant difference between the two, indicating that IgY has a specific recognition effect on monoclonal antibody release.

The IgY-RicinA conjugate has a strong inhibitory effect on the protein synthesis of gastric cancer cells. When 10 micrograms of IgY-RicinA are contained in each milliliter of culture, the protein cannot be synthesized at all, and the cells die. With 0.1 micrograms of IgY-RicinA per milliliter of culture fluid in cell culture, the survival rate of gastric cancer cells is only 0.1%, while the survival rate of other cells is above 75%. The ultrastructural observation of the reaction of IgY-RicinA to gastric cancer cells showed that the membrane structure of gastric cancer cells melted and destroyed, the nuclear membrane disappeared, and the nucleus was destroyed, causing the cells to collapse.

IgY-RicinA has a good therapeutic effect on experimental tumors in mice. The tumor cure rate is above 83.3%.

Clinical research on IgY-RicinA treatment of gastric cancer and other tumor patients. A total of 28 cases were treated from February 1989 to March 1992. These patients were diagnosed as gastric cancer in 25 cases, esophageal cancer in 1 case, laryngeal cancer in 1 case, and liver cancer in 1 case through pathological examination. Among these patients, the 2-year survival period accounted for 52.3%. Among the dead cases, 6 survived for half a year, 3 survived for half a year to one year, and 1 survived for nearly 2 years. It proves that the bio-guided drug has great potential for treating tumors.

Claims (5)

1, a kind of synthetic synthesis step that it is characterized in that of anti-cancer of the stomach bio-guide medicine comprises the purifying of cancer protein, and antibody obtains, the purifying of RicinA, the coupling of IgY-RicinA.

2,, it is characterized in that the people's Gastric Cancer MGC-80-3 cell that will cultivate is suspended from (10mM NaCl, 1.5mM MgCl in the buffer A according to the said method of claim 1 ₂10mM Tris-HCl(pH8.0) 0.1mM PMSF(phenylmethylsulfonyl fluoride), 0.1mMEDTA) ultrasonication, add solid ammonium sulfate and become 50% saturation ratio, stirred 30 minutes centrifugal 20 minutes at 2000rpm, precipitation is dissolved in (20mM Tris-HClpH8.0 in the buffer B, 2%SDS(sulfuric acid dodecyl sodium) 10mMEDTA, the 0.1%2-mercaptoethanol), with water-saturated phenol extracting three times, collection phenol (adds 0.25-M sucrose) in buffer B dialysis, solution process Sephorase-cL-4B chromatography, (elutriant Tris-HClpH8.0,1%NP40), collect the peak district, gel electrophoresis analysis cancer protein 110KD is used for immunity.

3,, it is characterized in that yellow damping fluid C(0.1MNaCl, the 0.01%NaN of using of immune egg according to the said method of claim 1 ₃, the 0.01M sodium phosphate) and dilution, add the PEG(polyoxyethylene glycol after the dilution, Mr6000), the 100ml yolk diluent adds 7gPEG, centrifugal 10 minutes of 12000rpm, the clear liquid filtration adds PEG and becomes 12% concentration, goes up the centrifuging and taking precipitation behind molten Jie for another example, is dissolved among the damping fluid C.Triplicate, precipitation is dissolved in the physiological saline at last, as the long period preservation, adds 25% glycerine in physiological saline, 0.02%NaN ₃-4 ℃ of preservations.

4, according to the said method of claim 1, it is characterized in that getting castor seeds 350 grams of peeling kind of skin off is soaked among the 5% acetate 1000ml, stir under the room temperature and spend the night, add 1000ml5% acetate after the making beating on 3000rpm centrifugal 20 minutes, filter, filtrate adds solid ammonium sulfate and becomes 80% saturation ratio to stir 40 minutes, centrifugal 30 minutes of 3000rpm, precipitation is dissolved in the 150ml distilled water, dialysed 20 hours, solution is through DEAE-Sephadex A50 chromatography, collect chromatographic solution, add ammonium sulfate and become 80% saturation ratio, 4 ℃ of stirrings, centrifugally precipitation is dissolved in distilled water and dialysed 48 hours, again through Sephadex G100 chromatography, with damping fluid D(0.01M sodium phosphate, 0.1M semi-lactosi) wash-out, substep is collected, with ammonium sulfate 50% saturation ratio precipitation ricin (Ricin), be dissolved in distilled water dialysis, frost drying, the Ricin of purifying, get Ricin and be dissolved in damping fluid E(0.1MTris-HCl pH8.5,0.5M lactose, the 5%2-mercaptoethanol), room temperature (20-25 ℃) stirred 12 hours, left standstill 10 hours, through DEAE-Cellulose DE52 chromatography, the peak district is (0.01M lactose, 0.1%2-mercaptoethanol in damping fluid F, 4 ℃ of dialysis of 5mM sodium phosphate (pH6.5 ℃), 0-0.15M NaCl, damping fluid F gradient elution, the peak district collect pure RicinA.

5, it is characterized in that according to the said method of claim 1 IgY-Ricin A coupling selects SPDP(N-Succinimidyl-3-(2 pyridyldithio for use) propionate) be coupler, by 20mM concentration SPDP is dissolved in dehydrated alcohol, use PBS(0.15M NaCl, 0.01M sodium phosphate) IgY is diluted to 10mg/ml, 750ml IgY adds 1ml SPDP stirring at room 30 minutes, the every 10mg/ml of Ricin A is added 2 mercapto ethanol become 0.1%, stirring at room 30 minutes, in ice bath, the Ricin A that handles slowly is added to (IgY: Ricin A=2: 1) among the IgY of processing, mild stirring, leave standstill 5 minutes 20-25 ℃ of placement 20 hours, with Sephaery S-200 chromatographic separation IgY-Ricin A, collect in the peak district, concentrates, and gets the anti-cancer of the stomach targeted drug of finished product.