阅读说明：本技术 肠癌细胞免疫禽类获得针对肠癌IgY抗体偶联广谱抗癌药物制备方法和防治肠癌应用 (Preparation method of coupled broad-spectrum anti-cancer drug for obtaining IgY antibody aiming at intestinal cancer from poultry immunized by intestinal cancer cells and application of coupled broad-) 是由 万积成 于 2019-08-28 设计创作，主要内容包括：本发明专利的目的在于,提供一种抗体药物偶联物新药,Igγ抗体偶联广谱抗癌药物的制备方法。以肠癌细胞做为免疫原,免疫禽类获得针对肠癌Igγ抗体,用多种化疗药物、多种不同的方法,修饰抗大肠癌的鸡Igγ抗体,提供一种可口服的双功能抗体的制备方法。口服抗体药物偶联物新药后,可与大肠癌结合且具有抗大肠癌的细胞毒性,降低化疗药物的耐药性和毒副作用。(The invention aims to provide a novel antibody drug conjugate, and a preparation method of an Ig gamma antibody conjugate broad-spectrum anti-cancer drug. Intestinal cancer cells are used as immunogen to immunize poultry to obtain an Ig gamma antibody aiming at intestinal cancer, and the chicken Ig gamma antibody resisting the intestinal cancer is modified by using a plurality of chemotherapeutic drugs and a plurality of different methods, thereby providing a preparation method of an orally-administrable bifunctional antibody. After the novel antibody drug conjugate is orally taken, the novel antibody drug conjugate can be combined with colorectal cancer and has cytotoxicity resistance to the colorectal cancer, and drug resistance and toxic and side effects of chemotherapeutic drugs are reduced.)

1. A method for preparing antibody conjugate medicine for treating carcinoma of large intestine is provided.

2. The antibody of claim 1, which is an egg yolk IgY antibody.

3. The method for preparing IgY antibody of claim 2

Comprises a preparation method of a carcinoma cell antigen of colorectal cancer,

including the establishment of cancer cell lines.

Including all digestive tract tumors;

including other sites of tumors.

4. The method for preparing IgY antibody of claim 2

Including suitable immunological adjuvants, is a heat labile toxin of Escherichia coli.

5. A conjugate of the antibody of claim 1, said compound being selected from the group consisting of:

comprising compounds defined as broad spectrum anticancer agents: such as 5-fluorouracil, capecitabine, oxaliplatin, irinotecan, daunorubicin and the like.

6. All methods of coupling carboxyl on the activated chemical substance with amino on the amino acid chain of the anti-colorectal cancer IgY antibody are utilized.

7. And (3) all methods for coupling the hydroxyl on the activated chemical substance with the amino on the amino acid chain of the anti-colorectal cancer antibody by adopting a sodium periodate oxidation method.

8. The active substance prepared by the method can inhibit the growth of colorectal cancer cells.

9. The above materials can be made into various dosage forms such as capsule, aqua, tablet, powder, etc.

The technical field is as follows: the invention relates to the field of therapeutic bifunctional antibodies, and relates to a chicken IgY antibody for resisting colorectal cancer and a modification method thereof.

Background art:

colorectal cancer (CRC) is one of the most common malignant tumors worldwide, the incidence rate accounts for the front of gastrointestinal tumors, and the incidence rate of colorectal cancer in China tends to increase year by year along with the improvement of economic development and the living standard of people and the change of life style and dietary structure. In 2008, there are about 120 ten thousand new cases of colorectal cancer, which account for about 10% of all new cancers, and the number of related deaths exceeds 60 ten thousand. Colorectal cancer is very common in economically developed countries and regions such as North America, Western Europe and Australia, the coarse incidence rate reaches 4/ten thousand, and accounts for more than two thirds of all new cases. In addition, the mortality rate of colorectal cancer is very high, mainly due to frequent postoperative metastasis and multiple drug resistance to chemotherapy drugs, so that the search for effective drugs for treating colorectal cancer is an important problem to be solved urgently in clinic.

Although colorectal cancer still bears a heavy burden on the health and society of people, great progress is made in the treatment field in the last two decades, the 5-year survival rate is improved from 50% to 63% in the past, and the life quality of patients is also remarkably improved. The above results are largely due to the breakthrough progress made in the drug therapy of colorectal cancer and the rational and standardized medication. Fluorouracil has been widely used in clinical practice as a basic chemotherapeutic drug for the treatment of colorectal cancer since the 50 s of the 20 th century. In the middle and later 90 s, new high-efficiency chemotherapeutic drugs such as oxaliplatin, irinotecan, capecitabine, and molecular targeted drugs cetuximab and bevacizumab are developed and marketed successively, so that colorectal cancer makes great progress in the aspect of drug treatment.

In recent years, various antibody drugs for treating cancer targeting antigen proteins on cancer cells have been developed worldwide, and have attracted attention as cancer-specific therapeutic agents with a certain drug efficacy. An antibody targeting a cancer antigen specifically expressed on the surface of a cancer cell is expected to be a drug for drug therapy with fewer side effects. Antibody drugs have the advantages of high specificity, high stability and low toxicity, and become the mainstream of modern biological drug development, and almost every antibody is born with the hope of being a 'heavy drug'. In anti-tumor therapy, antibody drugs have also become standard therapies, but their efficacy alone is often unsatisfactory. In recent years, the breakthrough progress of the new antibody drug conjugate, namely the antibody and small molecule drug integrated acetoin, is obtained, and the new generation of heavy drugs in antibody tumor therapy is very likely to grow.

Antibody-drug conjugates (ADCs) are drugs that couple small molecule cytotoxic drugs to antibodies, and consist of a monoclonal antibody, a chemical drug, and a linker, 3 parts together. ADCs deliver small-molecule drugs to tumor sites in a targeted manner by using monoclonal antibodies, enter lysosomes through endocytosis effect, and release cytotoxic small-molecule drugs, so that the effect of specifically killing tumor cells without damaging normal tissue cells is achieved, and the high specificity of the antibodies and the high toxicity of cytotoxic drugs to tumors are achieved.

Chicken yolk IgY antibody study: after the laying hens are immunized with the antigen, the hens can produce corresponding antibodies and transfer and accumulate the antibodies in egg yolks, and the main component of the antibodies extracted from the egg yolks is IgY. IgY is convenient and economical in source and can be produced in large scale. At present, antibodies against viral infectious diseases of various chickens, such as anti-infectious bursal disease egg yolk antibodies, anti-newcastle disease antibodies and the like, are widely applied and have good control effect. In the fields of medicine and food science, a specific pathogen is utilized to immunize chickens to generate specific antibodies, and the antibodies are used as oral preparations and food additives and have good effects on preventing and treating infantile rotavirus diarrhea, colibacillosis diarrhea, decayed teeth and the like. Therefore, how to develop and utilize IgY will be more and more appreciated.

The study of physicochemical properties and biological characteristics of the egg yolk IgY: (1) the effect of temperature on the activity of IgY has been shown by experiments that the titer of IgY extracted from newcastle disease virus resistant egg yolk is maintained substantially unchanged after 1h treatment at 65 ℃. This suggests that treatment with a certain sterilization process such as pasteurization (60 c, 30 minutes) can both kill certain pathogens and maintain antibody activity, which provides guidance for the extraction and further processing of egg yolk IgY. (2) In an acidic environment, tests show that the IgY still maintains higher activity in an environment with the pH of 3-11, which indicates that the IgY has strong acid-base resistance. (3) In an in vitro simulation test, after being treated by pepsin and trypsin, the IgY still keeps higher activity, which indicates that the IgY has certain digestion-resistant protease enzymolysis effect; can keep activity in digestive tract after taking orally, effectively kill pathogens existing in digestive tract of animals and human. (4) Is suitable for large-scale production. The Hansen et al research shows that the titer level of the high immunity yolk can be maintained for 2 months in the laying hens.

This provides a better theoretical basis for the treatment of diseases by oral administration of IgY, and makes it possible to prepare the medicament from the yolk IgY. After oral administration, IgY adheres to the cell walls of the gastrointestinal tract, forming a complex. The traditional Chinese medicine composition has the advantages of strong pertinence, almost no side effect, wide application range, effective therapeutic effect on gastrointestinal tracts, no drug resistance trouble and capability of providing a new solution for colorectal cancer treatment.

The invention content is as follows:

the invention aims to provide a novel antibody drug conjugate drug, and a preparation method of an IgY antibody conjugate broad-spectrum anti-cancer drug. Intestinal cancer cells are used as immunogen to immunize poultry to obtain an intestinal cancer IgY antibody, and the chicken IgY antibody for resisting the large intestinal cancer is modified by a plurality of chemotherapeutic drugs and a plurality of different methods, thereby providing a preparation method of an orally-administrable bifunctional antibody. After the novel antibody drug conjugate is orally taken, the novel antibody drug conjugate can be combined with colorectal cancer and has cytotoxicity resistance to the colorectal cancer, and drug resistance and toxic and side effects of chemotherapeutic drugs are reduced.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

1. intestinal cancer cells are used as immunogen to immunize poultry to obtain IgY antibody aiming at intestinal cancer.

2. Activating carboxyl on the chemotherapeutic drug, and coupling with amino on the amino acid chain of the anti-colorectal cancer IgY antibody.

3. Activating the hydroxyl on the chemotherapeutic drug, and coupling with the carboxyl on the amino acid chain of the anti-colorectal cancer IgY antibody.

The implementation mode is as follows:

patent embodiment 1:

preparation of anti-colorectal cancer IgY antibody

Culturing in vitro large intestine cancer cells according to the formula 10⁸cells/immunized chicken, immunizing part of wing under skin, mixing with adjuvant for the first immunization, enhancing immunity every 15 days for 4 times, collecting ovum, and detecting the binding activity of IgY antibody and colon cancer cellAnd (4) sex.

The IgY antibody is purified by a water dilution method: diluting luteum body with 10 times of distilled water, adjusting pH of distilled water to 5.0 with 0.1% hydrochloric acid, stirring for 15min, standing at 4 deg.C overnight, naturally precipitating lipoprotein, removing lipid by ultracentrifugation at 10000r/min at low temperature for 25min, and collecting supernatant to obtain IgY antibody for resisting carcinoma of large intestine.

Patent embodiment 2:

anti-colorectal cancer IgY antibody coupled with 5-fluorouracil (5-FU)

1) Weighing 20mg of iodoacetyl succinimide ester (NHSIA) and 40mg of 5-FU to react for 18 hours in the presence of potassium carbonate in DMF, adding water, extracting with ethyl acetate, evaporating an organic layer to dryness to obtain a 5-FU derivative, weighing 50mg of IgY, dissolving in 1mL of 0.01moL/L PBS (pH7.4), adding 20mg of SPDP, stirring at room temperature for reacting for 5 hours to obtain an IgY-PDP solution, adding 40mg of Dithiothreitol (DTT) to reduce overnight to obtain HS-IgY, dialyzing to obtain a purified HS-IgY solution, reacting with the 5-FU derivative overnight, filling into a dialysis bag, dialyzing with 0.01moL/L PBS solution at 4 ℃ for 3 days, and changing the solution 3 times every day to obtain the 5-FU-IgY.

Patent embodiment 3:

anti-colorectal cancer IgY antibody coupled capecitabine

40mg of capecitabine is dissolved in 1mL of 0.01mol/L PBS (pH7.4), 17mg of NaIO4 is added at room temperature, and the mixture is stirred at room temperature in the dark for reaction for 1 hour, and the excess NaIO4 is eliminated by using glycerol with the final concentration of 0.05 mol/L. Adding 1ml of the mixture into 0.15mol/L (pH9.5) K₂CO₃IgY in buffer was stirred at 37 ℃ for 1 hour to form Schiff base. NaBH4 was added in a molar excess of NaIO4 corresponding to capecitabine oxide, and the mixture was incubated in an incubator at 37 ℃ for 2 hours to stabilize Schiff's base. Centrifuging, dialyzing to obtain a filtrate, loading into dialysis bag, and standing at 4 deg.CDialyzing with 0.01moL/L PBS solution for 3 days, and changing the solution 3 times per day to obtain capecitabine-IgY conjugate

Patent embodiment 4:

anti-colorectal cancer IgY antibody coupled with oxaliplatin

Weighing 1 g of glucan T40(MW4 ten thousand) and dissolving in 200mL of distilled water, adding 0.44 g of sodium periodate to react at room temperature in a dark place overnight to obtain aldehyde dextran (PAD), dialyzing the distilled water sufficiently to remove periodate, and freeze-drying to obtain white flocculent powder and storing at 4 ℃. 20mg PAD is weighed and dissolved in 1mL PBS solution with pH7.2, 20mg oxaliplatin is added to react for 20 hours at room temperature in the dark, 20mg IgY is added to react for 20 hours at 4 ℃ with stirring, and the reaction mixture is reduced for 2 hours at 37 ℃ by sodium borohydride (1/3 with the amount of sodium periodate). Dialyzing after centrifugation to obtain oxaliplatin-DEX-IgY conjugate

Patent embodiment 5:

anti-colorectal cancer IgY antibody-conjugated irinotecan

1) Weighing 40mg g of irinotecan, placing the irinotecan into a reaction bottle, adding 2mL of tetrahydrofuran to dissolve the irinotecan, weighing 20mg of succinic anhydride, placing the succinic anhydride into the reaction bottle, adding 10 microliters of concentrated sulfuric acid into the reaction bottle, and heating and stirring the mixture at 50 ℃ for reaction for 5 hours; and (3) carrying out reduced pressure rotary evaporation, concentrating to obtain an off-white solid, soaking and washing with petroleum ether, and drying to obtain a carboxylated irinotecan product.

2) NHS 20mg, EDC 30mg, irinotecan carboxyl modification 40mg, and 500. mu.l DMF were weighed out accurately, and the solution was called solution A. Solution A was incubated for 24 hours at room temperature with stirring in the dark.

3) IgY is weighed out exactly 50mg and 3mL of 0.01moL/L (pH 7.4) Phosphate Buffered Saline (PBS) is added, this solution being designated as solution B. And (3) dropwise adding the incubated solution A into the human solution B, stirring while adding, stirring at room temperature for 3 hours, after the reaction is finished, filling into a dialysis bag, dialyzing for 3 days at 4 ℃ by using 0.01moL/L PBS solution, and changing the solution 3 times every day to obtain the irinotecan-IgY conjugate.

Patent embodiment 6:

anti-colorectal cancer IgY antibody coupled daunorubicin

Weighing 10mg of daunorubicin, dissolving in 1mL of 0.01moL/L (pH 7.4) Phosphate Buffer Solution (PBS), adding 5mg of sodium periodate at room temperature, reacting for 1 hour in a dark place, removing excess sodium periodate with glycerol, adding 10mL of IGY (100mg) dissolved in 0.15 moL/L-1 pH9.5 potassium carbonate solution, reacting for 1 hour at 37 ℃, adding 5mg of sodium borohydride, reducing at 37 ℃, continuing to react for 2 hours, centrifuging to remove insoluble substances, filling the supernatant into a dialysis bag, dialyzing for 3 days at 4 ℃ with 0.01moL/L PBS solution, and changing the solution 3 times every day to obtain the daunorubicin-IgY conjugate.

Patent embodiment 7:

anti-colorectal cancer IgY antibody coupled daunorubicin

Weighing 1 g of glucan T40(MW4 ten thousand) and dissolving in 200mL of distilled water, adding 0.44 g of sodium periodate to react at room temperature in a dark place overnight to obtain aldehyde dextran (PAD), dialyzing the distilled water sufficiently to remove periodate, and freeze-drying to obtain white flocculent powder and storing at 4 ℃. Weighing 12mg PAD, dissolving in 1mL PBS (pH7.2), adding 1mg daunorubicin, reacting for 20 hours at room temperature in the dark, adding 20mg IgY, reacting for 20 hours at 4 ℃ with stirring, and reducing the reaction mixture with sodium borohydride (1/3 with the amount of sodium periodate) at 37 ℃ for 2 hours. After centrifugation, the daunorubicin-DEX-IgY conjugate is obtained by dialysis.