CN118184759B - Preparation method and application of ATAP polypeptide and vaccine thereof - Google Patents
Preparation method and application of ATAP polypeptide and vaccine thereof Download PDFInfo
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- CN118184759B CN118184759B CN202410339834.9A CN202410339834A CN118184759B CN 118184759 B CN118184759 B CN 118184759B CN 202410339834 A CN202410339834 A CN 202410339834A CN 118184759 B CN118184759 B CN 118184759B
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- atap
- polypeptide
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention discloses a preparation method and application of an ATAP polypeptide and a vaccine thereof, wherein the ATAP polypeptide is protein of the following a) or b): a) As set forth in SEQ ID NO: 1.2, 3,4, wherein the amino acid sequence of SEQ ID NO: 1.2, 3,4, and b) a protein derived from a) wherein the amino acid sequence in a) is substituted, deleted or added with one or more amino acids and having a cancer preventing effect. The invention also discloses application of the ATAP polypeptide or pharmaceutically acceptable salt thereof in preparing medicines for preventing and treating cancers.
Description
Technical Field
The invention belongs to the technical field of biological medicines, in particular to a preparation method and application of an ATAP polypeptide and a vaccine thereof, and particularly relates to application of the ATAP polypeptide vaccine in preventing cancers, in particular to application of an ATAP-CD8T cell epitope polypeptide or pharmaceutically acceptable salt thereof in preparing a medicament for treating the cancers.
Background
ATAP (AMPHIPATHIC TAIL-Anchoring Peptide) is a novel parent tail anchor polypeptide derived from Bfl-1 (amino acids 147-175). Experiments show that ATAP can specifically target mitochondria to trigger apoptosis. ATAP has the unique advantage over other mitochondrially targeted peptide drugs of independently inducing potent apoptosis independent of Bcl-2 family proteins. In particular, ATAP may not permeate the mitochondrial membrane in the presence of Bax and Bak, which typically affect drug-carrying function and drug-carrying expression in cancer cells. In addition, ATAP-induced apoptosis is not affected by Bcl-2 or other anti-apoptotic family members, which are often over-expressed in cancer cells.
ATAP-M8 is a polypeptide containing 38 amino acid residues, which is optimized and engineered from the native ATAP sequence. To improve physicochemical properties and facilitate clinical use, amino acid substitutions and chemical modifications are made to the native ATAP sequence to increase its biological activity. ATAP-M8 is a fusion polypeptide obtained by fusing ATAP structure and iRGD sequence and modifying the structure. The iRGD sequence is capable of highly targeting integrin receptors that are highly expressed on the surface of many tumor cells (Integrin). ATAP-M8 can specifically recognize cell mitochondrial membranes, punch holes in the mitochondrial membranes through positioning of special amphipathic helical structures, physically destroy mitochondrial structures, and induce apoptosis. By linking ATAP to internalized RGD peptide (iggd), selective targeting of ATAP to tumor cells is achieved. ATAP can effectively identify cancer cells and normal cells, bring polypeptides into tumor cell areas in a directed manner, specifically kill tumor cells and reduce damage to normal cells.
The sequence of the obtained modified peptide ATAP-M8 is shown as follows:
Wherein: an intramolecular disulfide bond is formed between the two Cys residues at amino acid positions 30 and 38.
The molecular weight is as follows: 4375.20.
The structural formula is as follows:
Cancer vaccines are one of the important means of tumor immunotherapy, and are the most effective means for preventing and treating tumors currently. Searching for safe, effective and broad-spectrum cancer vaccines is a leading edge and hot spot problem of technological innovation in the medical community. Thus, applicants have attempted to investigate ATAP-M8 related cancer vaccine.
Disclosure of Invention
It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.
It is also an object of the invention to provide ATAP polypeptides.
It is still another object of the present invention to provide a method of preparing ATAP polypeptide vaccine.
It is another object of the present invention to provide the use of said ATAP polypeptide or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prophylaxis and treatment of cancer.
For this purpose, the technical scheme provided by the invention is as follows:
an ATAP polypeptide which provides a prophylactic and therapeutic effect on cancer, which ATAP polypeptide is a protein of a) or b) as follows:
a) As set forth in SEQ ID NO: 1.2, 34, and an amino acid sequence shown in FIG. 4 any one of the above-mentioned materials,
Wherein, SEQ ID NO: 1.2, 3,4, and between two Cys residues at amino acid positions 30 and 38, forms an intramolecular disulfide bond
B) A protein derived from a) wherein the amino acid sequence in a) is substituted, deleted or added with one or more amino acids and has a cancer-preventing effect.
A medicament comprising one or more of said ATAP polypeptides or a pharmaceutically acceptable salt thereof.
Preferably, in the medicament, the medicament is a vaccine.
A DNA molecule encoding said ATAP polypeptide.
A method for preparing ATAP polypeptide vaccine, comprising the steps of:
preparing one or more ATAP polypeptides or pharmaceutically acceptable salts thereof according to claim 1 into a polypeptide mixed solution, and mixing the polypeptide mixed solution with an adjuvant with the same concentration according to the same volume to obtain the ATAP polypeptide vaccine.
Preferably, in the preparation method of the ATAP polypeptide vaccine, the adjuvant is: polyinosinic acid-polycytidylic acid or aluminum phosphate.
Preferably, in the method for preparing the ATAP polypeptide vaccine, the ATAP polypeptide is prepared into a 50mg/ml solution by using sterile water, and then the solution is prepared into a 15mg/ml solution by using PBS buffer to obtain the polypeptide mixed solution.
The application of the ATAP polypeptide or the pharmaceutically acceptable salt thereof in preparing medicaments for preventing and treating cancers.
Preferably, among the uses, the use includes use in the preparation of a vaccine for the prevention of cancer.
Preferably, in the use, the cancer is glioblastoma, esophageal squamous cell carcinoma, acute myelogenous leukemia, liver cancer, stomach cancer, kidney cancer, ovarian cancer, lung cancer, breast cancer and colon cancer.
The invention at least comprises the following beneficial effects:
In general, short peptide vaccines only induce specific Cytotoxic T Lymphocyte (CTL) cells, which cause insufficient immune response, and long peptides of corresponding design are needed to boost the immune response to achieve therapeutic effects. The ATAP polypeptide is 38 peptide, has better anticancer activity, enhances immune response by combining with CD8T cell epitope polypeptide, and can more rapidly and effectively induce a host to generate antitumor immunity after inoculation.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Previous studies by the inventors show that ATAP-M8 can effectively inhibit a variety of human cancer cells, including glioblastoma, esophageal squamous cell carcinoma, acute myelogenous leukemia, liver cancer, stomach cancer, kidney cancer, ovarian cancer, lung cancer (including non-small cell lung cancer), breast cancer, colon cancer and other 20 different source human cancer cells. Thus, APAP-M8 has a broad spectrum of anti-tumor effects.
The ATAP-M8 is used as a novel polypeptide antitumor drug, has a unique action mechanism, can specifically target and kill variant tumor cells, has no toxicity to normal tissue cells, and has higher safety than other types of small molecule cytotoxic drugs. And the metabolite is natural amino acid, which is a nutrient substance needed by human body. Animal experiments prove that the safety window of the ATAP-M8 is 98 times; the effective dose for human body is 7.5 mg, and the effective anti-tumor dose is 30 mg. The ATAP-M8 can be used as an anti-tumor drug at a therapeutic dose, and can obviously kill cancer cells and inhibit tumor growth; and under the dosage lower than the therapeutic dosage, the ATAP is administrated by intravenous drip, can specifically and accurately identify the variant tumor cells of human tissues in blood circulation, and has the immediate killing effect on the original trace tumor variant cells. Thus, ATAP-M8 can be used as a broad-spectrum antitumor vaccine for preventing the production of a variety of variant cancer cells.
The present invention provides an ATAP polypeptide which provides an effect of preventing and treating cancer, the ATAP polypeptide being a protein of a), b), or:
a) As set forth in SEQ ID NO: 1.2, 34, and an amino acid sequence shown in FIG. 4 any one of the above-mentioned materials,
B) A protein derived from a) wherein the amino acid sequence in a) is substituted, deleted or added with one or more amino acids and has a cancer-preventing effect.
The applicant predicts the epitope of CD 8+ T cells on the mutant polypeptide sequence of tumor-associated gene SWAP70, and picks out the mutant polypeptide sequence of the top 4, which are respectively:
KYQGLVVKQSV
DYVTGKMAV
YGLFSTFYM
GLFSTFYMR
The 4 mutant polypeptide sequences are respectively synthesized into polypeptide fragments, then the 4 polypeptide fragments are connected with ATAP, and then the polypeptide fragments are mixed with an adjuvant, and the amino acid sequence of the polypeptide fragments is shown as SEQ ID NO: 1.2, 3 and 4.
(SEQ ID NOs: 1, 2, 3, 4 in this order from top to bottom)
The present invention also provides a medicament comprising one or more of said ATAP polypeptides or a pharmaceutically acceptable salt thereof.
In some embodiments of the invention, the agent is a vaccine and the ATAP polypeptide vaccine comprises one or more of the ATAP polypeptides or a pharmaceutically acceptable salt thereof.
The invention also provides a DNA molecule which encodes the ATAP polypeptide.
The invention also provides a preparation method of the ATAP polypeptide vaccine, which comprises the following steps:
preparing one or more ATAP polypeptides or pharmaceutically acceptable salts thereof according to claim 1 into a polypeptide mixed solution, and mixing the polypeptide mixed solution with an adjuvant with the same concentration according to the same volume to obtain the ATAP polypeptide vaccine.
In the above embodiment, preferably, the adjuvant is: polyinosinic acid-polycytidylic acid or aluminum phosphate.
In the above-described embodiment, preferably, the ATAP polypeptide is prepared as a 50mg/ml solution using sterile water, and then prepared to a concentration of 15mg/ml using PBS buffer, to obtain the polypeptide mixture.
The invention also provides application of the ATAP polypeptide or pharmaceutically acceptable salt thereof in preparing medicaments for preventing and treating cancers.
In the above-described scheme, preferably, the use includes use in the preparation of a vaccine for preventing cancer.
In the above-described scheme, preferably, the cancer is glioblastoma, esophageal squamous cell carcinoma, acute myelogenous leukemia, liver cancer, stomach cancer, kidney cancer, ovarian cancer, lung cancer, breast cancer and colon cancer.
The ATAP-M8 can be used as a cancer vaccine due to a special accurate targeting mutated tumor cell mechanism and safe, efficient and broad-spectrum antitumor effects, and can effectively kill the original tumor mutated cells in vivo by intravenous drip administration for 1-2 times per year under the dosage condition of safety to human bodies, thereby achieving the effect of preventing cancers.
The present invention relates to SEQ ID NO:1-4 or a pharmaceutically acceptable salt thereof, wherein the cancer is selected from glioblastoma, esophageal squamous cell carcinoma, acute myelogenous leukemia, liver cancer, gastric cancer, renal cancer, ovarian cancer, lung cancer (including non-small cell lung cancer), breast cancer, colon cancer and the like, and the integrin receptor is highly expressed.
The invention also relates to a polypeptide comprising SEQ ID NO:1-4 or a pharmaceutically acceptable salt thereof, wherein the cancer is selected from the group consisting of liver cancer, stomach cancer, kidney cancer, ovarian cancer, lung cancer (including non-small cell lung cancer), breast cancer and colon cancer, in the preparation of a vaccine medicament for preventing the cancer.
In the invention, the polypeptide shown in SEQ ID NO.1-4 or pharmaceutically acceptable salt thereof can be used as a vaccine active ingredient for preventing cancers, and can also be combined with additional pharmaceutically active compounds for preventing and treating cancers.
The SEQ ID NO:1-4 can be prepared using Fmoc solid phase synthesis.
In the present invention, the unit "M" represents mol/L, and "μM" represents μmol/L.
In the present invention, "pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the subject compound and exhibit minimal undesirable toxicological effects. These pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compound or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively.
The dosage and method of use of the polypeptides of the invention will depend upon a number of factors including the age, weight, sex, natural health, nutritional status, the activity of the compound, the time of administration, the metabolic rate, the severity of the condition and the subjective judgment of the treating physician. The preferred dosage is between 0.001-100mg/kg body weight/day. The amount is administered as a single dose per day or as several sub-doses per day, e.g. 2,3, 4,5 or 6 doses per day. Alternatively, the administration may be intermittent, for example, once every other day, once a week or once a month. The therapeutically effective amount of a salt or solvate, etc. may be determined as a proportion of the therapeutically effective amount of the polypeptide itself.
In some embodiments, the pharmaceutical composition may optionally further comprise one or more additional pharmaceutically active compounds.
According to the invention, the pharmaceutical composition comprises the polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may be administered by a route such as oral or parenteral. The pharmaceutical compositions of the present invention may be formulated into various dosage forms, including but not limited to tablets, capsules, solutions, suspensions, granules or injections, etc., by conventional methods in the art, for example, by oral or parenteral routes.
The pharmaceutical compositions of the present invention may be presented in unit dosage forms containing a predetermined amount of active ingredient per unit dose. Such units may contain 0.001 to 1000mg, for example ,0.05mg、0.1mg、0.5mg、1mg、2mg、2.5mg、5mg、7.5mg、10mg、12.5mg、15mg、17.5mg、20mg、22.5mg、25mg、27.5mg、30mg、35mg、37.5mg、40mg、42.5mg、45mg、47.5mg、50mg、60mg、70mg、80mg、100mg、150mg、200mg、250mg、300mg、500mg、750mg or 1g of a compound of the invention, depending on the disease to be treated, the route of administration and the age, weight and symptoms of the subject, or the pharmaceutical composition may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose. In another embodiment, the unit dose compositions are those containing daily doses or sub-doses as described herein or an appropriate fraction thereof of the active ingredient. In addition, such pharmaceutical compositions may be prepared by any method known to those skilled in the art.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1 in vitro pharmacodynamic evaluation
Evaluating the test drug SEQ ID NO:1-4 on human tumor cells and normal tissue cells of different tissue sources. The tumor cell lines and human normal hepatocytes used in this example were from the university of Zhejiang college of medicine cell bank.
Cell culture and seeding: the experimental tumor cell line was cultured in RPMI1640 medium (HyClone, SH30809.01B,500 mL) containing 10% serum, 100IU/mL penicillin and 100IU/mL streptomycin, and incubated in a 5% CO 2 incubator at 37 ℃. A white 96-well plate was seeded with a certain amount of tumor cells according to the cell growth rate.
Administration: the test drug group and the positive control group were experimentally set. Each test drug group was set with 8 concentration gradients, half-dilution, three duplicate wells per concentration. Highest initial concentration of test drug: 100. Mu.M. Experiments were repeated three times.
CellTiter assay 50. Mu.L of CellTiter-Glo reagent (from Promega) was added to each well 48h after the test drug and positive control cells. Incubated at room temperature for 10min under light-protected conditions, and chemiluminescent signals were detected by an enzyme-labeled instrument (SpectraMax M5 model, available from Molecular Devices). The results were recorded, cell growth curves were plotted with drug concentration on the abscissa and absorbance on the ordinate, and IC 50 values were calculated.
And (3) result processing:
According to the OD value measured by the enzyme label instrument, the inhibition rate is calculated according to the following formula:
Inhibition (%) = (1-OD Administration of drugs /OD control ) x 100%,
If the inhibition rate is less than or equal to 0%, the inhibition rate is recorded as 0%.
IC 50 was calculated and the experiment was repeated three times, the mean and standard deviation were calculated and the data were expressed as: the mean.+ -. Standard deviation experimental results are shown in Table 1.
TABLE 1 inhibition of tumor cell proliferation by ATAP-M8
Note that: NA: > 100nM.
Test article SEQ ID NO:1-4 shows broad-spectrum antitumor activity: has obvious proliferation inhibition effect on 20 human tumor cells from different tissues, and the half inhibition concentration (IC 50) is between 0.83 mu M and 9.92 mu M.
Example 2SEQ ID NO:1-4 Effect on mitochondrial injury and apoptosis of gastric cancer HGC27 cells
Flow cytometry detection results showed that SEQ ID NO:1-4 shows positive correlation with the expression level of Integrin alpha V beta 3 on the surface of tumor cells, namely, the higher the expression level of INTERGRIN alpha V beta 3 of the tumor cells is, the higher the sensitivity of ATAP-M8 is, and the lower the IC 50 is.
SEQ ID NO:1-4 (5, 10 and 20 mu M) can induce the reduction of the mitochondrial membrane potential of Integrin αvβ3 high-expression gastric cancer cell HGC27 cells in a concentration-dependent manner after 4h, the ratio of JC-1 green fluorescent cells is 11.39+/-6.70%, 33.32+/-7.55%, 34.70 +/-5.49% (negative Control 11.94+/-6.77%) and the apoptosis rate of the positive Control Taxol is 5.93+/-1.93%. Under the same conditions, the effect of obviously reducing Integrin alpha V beta 3 low-expression intestinal cancer HT29 cells is not shown, the JC-1 green fluorescent cell ratio is 5.34+/-3.22%, 5.74+/-1.08%, 11.60+/-6.50% (negative Control 3.50+/-1.80%) and the positive Control Taxol apoptosis rate is 7.10+/-5.31%.
SEQ ID NO:1-4 (5, 10 and 20 mu M) induces the apoptosis of Integrin αVβ3 high-expression gastric cancer cells HGC27 in a concentration-dependent manner, the apoptosis rate of the drug action for 24 hours is 16.06+/-4.37%, 29.50+/-11.61%, 41.85 +/-25.42% (negative Control 11.34+/-1.82%) and the apoptosis rate of the positive Control Taxol is 33.32+/-18.09%. SEQ ID NO: the apoptosis rate of 1-4 for 48h is 11.86+ -6.65%, 30.70 + -5.95%, 53.98+ -17.93% (negative Control 8.69+ -4.45%), and the apoptosis rate of positive Control Taxol is 60.20+ -9.53%. The ATAP-M8 (5, 10 and 20 mu M) does not induce apoptosis of Integrin αVβ3 low-expression intestinal cancer cell HT-29 cells, the apoptosis rate of 24h is 7.11+/-3.19%, 6.79+/-2.53%, 10.03+/-2.99% (Control 7.95+/-0.73%) and the apoptosis rate of the positive Control Taxol is 25.69+/-16.47%. SEQ ID NO: the apoptosis rate of 1-4 for 48h is 6.79+ -2.37%, 7.60+ -2.33%, 9.51+ -3.99% (negative Control 5.04+ -2.06%), and the apoptosis rate of positive Control Taxol is 33.19+ -17.10%.
EXAMPLE 3 in vivo evaluation of anti-tumor pharmacodynamics
Different doses of SEQ ID NO:1 on the experimental treatment effect of human tumor cell nude mice subcutaneous transplantation tumor.
Establishing a nude mouse transplantation tumor model: the in vivo antitumor activity was evaluated using different human cancer cells (cell bank of university of Zhejiang college of pharmacy). Injecting 1×10 7 human tumor cells into armpit of nude mice (purchased from Beijing Vermilion laboratory animal technologies Co., ltd.) and after the tumor character is stabilized (generally 2-3 passages), dissecting the tumor mass of the seed mice, placing into a glass dish containing normal saline, peeling off the surface blood vessel, cutting to remove necrotic region, cutting the tumor mass into 1-2mm 3, and accessing the left armpit of the nude mice with a trocar.
Grouping and administration of animals: after the tumors grow to an average volume of 100-300mm 3, 5-6 tumors per group.
Data measurement and processing: tumor volume was weighed and measured 2 times per week and on the next day of last dose, the nude mice were sacrificed and tumor mass weighed after weighing and measuring tumor volume, and Relative Tumor Volume (RTV), relative tumor proliferation rate (T/C) and percent tumor Inhibition (IR) were calculated for statistical detection. Tumor tissue was photographed and recorded.
Data determination and statistics:
Tumor volumes were weighed and measured 2 times per week for 21 days of the dosing cycle, weight was measured on day 22 and tumor mass weights were sacrificed after tumor volume measurement for bare mice, and Relative Tumor Volumes (RTV), relative tumor proliferation rates (T/C) and percent tumor Inhibition (IR) were calculated for statistical detection. The calculation formula is as follows:
(1) Tumor volume: TV (tumor volume) = 1/2 x a x b 2, where a, b represent the length and width (cm) of the tumour respectively;
(2) Tumor volume ratio: RTV (relative tumor volume) = V t/V0, where V 0 is the tumor volume measured at the time of group administration (i.e., d 0), vt is the tumor volume at each measurement;
(3) Relative tumor proliferation rate: T/C (%) = T RTV/CRTV x 100%, where T RTV is RTV in the treatment group and C RTV is RTV in the solvent control group;
(4) Percent tumor inhibition: IR (%) = (1-TW t/TWc) ×100%, where TW t is the tumor weight of the treatment group and TW c is the tumor weight of the solvent control group.
(5) Tumor (volume) inhibition The Tumor Growth Inhibition value (TGI). Is used for evaluating the inhibition of the test drugs on tumor growth in vivo (i.e. animal experiments). Tgi= (1-treated tumor weight/control tumor weight) ×100%
Efficacy evaluation criteria: T/C (%) >40%, IR (%). T/C (%) is less than or equal to 40%, IR (%) is more than or equal to 60%, and p <0.05 is effective after statistical treatment.
Experimental results: see Table 2
Table 2 test therapeutic Effect of ATAP polypeptide on human different tumor nude mice transplantable tumors
SEQ ID NO:1-4 selectively induces the reduction of the mitochondrial membrane potential of Integrin alpha V beta 3 high-expression tumor cells, damages mitochondria and induces apoptosis of the tumor cells so as to play an anti-tumor role. In vitro experiments show SEQ ID NO:1-4 showed broad spectrum antitumor activity with half inhibition concentrations (IC 50) of less than 10. Mu.M. In vivo experiments show that SEQ ID NO:1-4 can inhibit a plurality of human tumor nude mice transplanted tumors in a dose-dependent manner, has obvious curative effects on stomach cancer, prostatic cancer, osteosarcoma and breast cancer, and has a minimum effective dose of 5.0mg/kg for sensitive tumor strains.
Example 4 cynomolgus monkey intravenous injection of SEQ ID NO: toxicity test for 1 week
Purpose of test
Cynomolgus monkey is given intravenous administration of SEQ ID NO: and (3) after 14 weeks, stopping taking the medicine and recovering for 4 weeks, observing the property, degree, dose effect, aging relation and reversibility of the possibly caused toxic reaction of the test sample, judging a toxic target organ or target tissue, and simultaneously researching the toxic kinetics characteristics of the target organ or target tissue to know the relation between exposure dose and toxicology result in toxicity research.
Experimental animal
Species: cynomolgus monkey;
grade: a normal stage;
number and sex of animals purchased and used: 40, male and female halves;
Age: 3 to 5 years old;
weight of: the weight of the animals is 2.40-4.10 kg when the animals enter the adaptation period, the average weight of the animals is 3.01kg when the animals are grouped in a test mode, and the individual weight values of the rest animals except a few animals are within the average weight of +/-20%;
The source is as follows: guangxi Guidong primate development experiments Co., ltd (production license number: SCXK cassia 2016-0001). Quality certification issuing and printing units: guangxi Zhuang autonomous area science and technology hall. Quality certification number: 45001200000334;
Grouping animals
Group design: control group, SEQ ID NO:1 low, medium, high dose group;
Number of animals: 10 pieces/group, total 40 pieces;
sex ratio: the male and female halves;
the grouping method comprises the following steps: random grouping according to weight division gender of the monkeys by adopting PRISTIMA7.2.0 edition data system;
dose design
SEQ ID NO:1 the effective dose in the mouse tumor-bearing model is 5 mg/kg/day, which is converted into the cynomolgus monkey equivalent dose of 1.25 mg/kg/day.
The test design of the ATAP-M8 low, medium and high dose groups is 3, 10 and 30mg/kg respectively. A control group was also set up and given by intravenous injection an equal volume of 0.9% sodium chloride injection.
The specific dose designs are shown in table 3.
Table 3 dose design table
Administration of drugs
Route of administration: intravenous injection;
the route of administration was chosen for: the clinical application path is consistent with that of the clinical application;
frequency and period of administration: the administration is carried out for 4 weeks for 1 time a day, and the recovery period of stopping administration is 4 weeks;
drug administration site: bilateral upper limb vein and lower limb vein administration (left and right alternation);
dosing volume: 15mL/kg;
drug administration rate: set to 2 mL/min (allow ± 0.3 mL/min deviation, administer with microinjection pump);
Administration syringe: syringe gauge 50mL, syringe lot number 180106;
Notice that: except the control group, each group of new syringes for administration can be rinsed with a certain amount of the test article administration preparation with corresponding concentration before use;
The dose of each animal was adjusted according to the weight of the last measurement.
The day of first dosing was defined as day 1 of the trial.
Conclusion:
under the test conditions, cynomolgus monkeys were continuously injected intravenously for 4 weeks with 3, 10, 30mg/kg doses of SEQ ID NO:1, stopping the medicine and recovering for 4 weeks. The 30mg/kg group of monkeys mainly had reduced adrenal tract vacuoles, spleen and lymph node foam-like macrophage infiltration, high serum ALT, AST, urea, crea liters, slightly reduced peripheral red blood cells, WBC and LYM, and reduced part of monkey PLT and bone marrow producing plate cells. Stopping the medicine for 4 weeks, and recovering the adrenal lesions of 30mg/kg group monkey. In addition, no significant abnormal changes were seen in the body temperature, blood pressure, II-lead electrocardiogram, circulating immune complex, immunoglobulin, lymphocyte subpopulation, cytokine, ophthalmic examination, etc. of each group.
In the dosage range of 3-30 mg/kg, SEQ ID NO:1. there was no significant sex difference in exposure, and the exposure increase was lower than the dose increase ratio, with no accumulation for 4 weeks of continuous administration.
Cynomolgus monkey is given intravenous administration of SEQ ID NO:1.4 weeks, the highest non-severe toxic dose (HNSTD) was 30mg/kg (after last dose, ATAP-M8 average AUC females were 133h. μg/mL, male monkeys were 147h. μg/mL), and the non-toxic response dose (NOAEL) was 10mg/kg (after last dose, ATAP-M8 average AUC females were 262h. μg/mL, male monkeys were 274h. μg/mL).
Example 5SEQ ID NO:1. genotoxicity
50, 150, 500, 1500, 5000 Μg/dish of SEQ ID NO:1. none of the tested strains had caused genetic mutations. I.e., the Ames test results were negative.
About 4 hours of exposure with or without the S9 metabolic activation system, 80, 200, 500 μg/mL of ATAP-M8 did not cause a significant increase in chromosomal structural aberration rate of CHL cells; about 24 hours of exposure to the S9-free metabolic activation system, 32, 80, 200. Mu.g/mL of ATAP-M8 also did not cause a significant increase in chromosomal structural aberration rate of CHL cells. The result of the ATAP-M8 in vitro CHL cell chromosome aberration test is negative.
NIH mice were given 6.25, 12.5, 25mg/kg of ATAP-M8 by intravenous injection 1 time a day for 3 consecutive days, and the results of the bone marrow micronucleus test of the ATAP-M8 mice were negative without causing damage to the chromosomal integrity of bone marrow cells or causing abnormalities in chromosomal segregation.
EXAMPLE 6 preparation of ATAP polypeptide vaccine
A method for preparing ATAP polypeptide vaccine, comprising the steps of:
Respectively taking the ATAP polypeptides described in SEQ ID NO. 1-4, respectively preparing four ATAP polypeptides into 50mg/ml solutions by using sterile water, preparing into 15mg/ml solution by using PBS buffer agent to obtain four polypeptide mixed solutions, and respectively mixing the four polypeptide mixed solutions with the adjuvant with the same concentration according to the equal volume to obtain the four ATAP polypeptide vaccines. The adjuvant is as follows: polyinosinic acid-polycytidylic acid or aluminum phosphate.
Example 7.SEQ ID NO:1-4 polypeptide vaccine elicits a specific Cytotoxic T (CTL) response.
1.1 In vitro induction of PBMC antigen specific Effect CTL responses
Peripheral Blood Mononuclear Cells (PBMCs) were prepared: peripheral blood of healthy people is taken and separated by a layering liquid gradient centrifugation method, and polysucrose-diatrizoic glucamine is adopted, wherein the cell number is 5X 10 5 ml/L. With SEQ ID NO:1-4 polypeptide stimulated PBMC 3 times, the final mass concentration of polypeptide was 5nmol/ml, 1 time every 5 days, and IL-2 30U/ml was added the next day. On day 4 after the end, the supernatants were collected for IFN-gamma detection.
Specific CTL cytotoxic effects were detected using a standard 51 Cr release assay: t2 cells or Caski cells were used as target cells, respectively, and PBMCs were collected as effector cells after 6 days. The polypeptide sample was added so that the final mass concentration was 5nmol/ml, caski cells were washed and the cell number was 5X 10 5 ml/L for use as target cells.
According to the detection result, the method shows that:
(1) Epitope-loaded peptide T 2 cells are target cells, and SEQ ID NO:1-4 polypeptide all induced stronger CTL activity (P <0.01 compared to control group), wherein the amino acid sequence of SEQ ID NO:4 (P <0.05 compared to SEQ ID NO. 1-3).
(2) Control experiment T 2 cells which are not loaded with epitope peptide are target cells, and SEQ ID NO: the CTL induced by the 1-4 polypeptide has a specific lysis rate of less than 10%.
(3) Caski cells are target cells, SEQ ID NO:1-4 can kill Caski cells (P <0.01, compared with control group) by CTL induced in vitro.
As shown in table 4:
1.2 Induction of antigen-specific Effect CTL responses in vivo
Female transgenic mice of 10 weeks of age were selected, randomly divided into 7 groups of 10 animals each, and the tail root was injected with SEQ ID NO:1-4 polypeptide, at a dose of 10nmol each, the control group was then injected with the same concentration of ATAP and Phosphate Buffered Saline (PBS) fully emulsified with IFA and boosted 1 week later.
Spleen cells were prepared from spleens of mice 10 days after the last immunization, the number of cells was 5X 10 5ml-1, the final mass concentration of polypeptide was 2nmol/ml, IL-2 30U/ml was added the next day, incubation was performed at 37℃with 5% CO 2 for 4 days, and IFN-gamma was detected.
51 Cr release assay detects specific CTL cytotoxic effects: cells were collected after 10 days and used as effector cells. The JurkatA2.1/K b cell line was used as target cells, the concentration of the polypeptide sample was 10: 10nmoL/ml, the incubation was 100 minutes at 37℃with 5% CO 2, and the cell number was 5X 10 5ml-1.
The experimental detection results show that:
(1) The JurkatA2.1/K b loaded with epitope peptide is the target cell SEQ ID NO:4 induces stronger CTL activity, at E/t=100:1, target cell specific lysis rate is 78.0% (P <0.01 compared to control and SEQ ID NOs: 1-3); CTL activity was also induced by No.1-3, with a target cell specific lysis rate of 58-62% (P <0.01 compared to control) at E/t=100:1.
(2) The JurkatA2.1/K b without epitope peptide is target cell, and the specific lysis rate of CTL induced by the SEQ ID NO 1-4 polypeptide is less than 10%.
As shown in table 5:
example 8 detection of extracellular IFN-gamma of SEQ ID NO.1-4 polypeptide vaccine
2.1. ELISA Spot (ELl-Spot) detection
ELI-Spot experiments can detect the number of IFN-gamma secreting cells at the single cell level. The activated T lymphocytes are secreted with high concentration of IFN-Y cytokines, so that ELISA can be performed to count the number of cytokine-secreting cells. Cell numbers secreting IFN-gamma following stimulation of PBMC with the polypeptide were tested by ELI-Spot experiments.
The ELl-Spot detection antigen specificity CTL of IFN-gamma adds lymphocytes into ELl-Spot detection plate with the dosage of 3 multiplied by 10 5 cells/hole, adds a stimulator to make the final concentration 10 mug/mL, incubate for 30h, and the subsequent operation is carried out according to the specification of ELl-Spot; the immune spot analyzer scans the plate, counts the spots and performs statistical analysis.
The detection results show that the SEQ ID NO. 1-4 polypeptides can effectively stimulate Peripheral Blood Mononuclear Cells (PBMC) to generate IFN-gamma secretion cells (P <0.01, compared with a control group), wherein the effect of the SEQ ID NO.4 is strongest, and the values of the blank group PBS and the original drug ATAP are very small.
TABLE 6 ELI-Spot method for analysis of IFN-Y amount secreted by cells
* P <0.01, # P <0.01, compared to control, compared to SEQ ID NO 1-3.
2.2. Detection of extracellular IFN-gamma secretion values
The concentration of IFN-gamma outside the cell can be used to reflect the proliferation of IFN-gamma secreting cells. The IFN-gamma secretion values for each group can be determined based on a standard curve. As can be seen from ELISA measurements, the polypeptides of SEQ ID NO. 1-4 all induced secretion of IFN-gamma by PBMC (P <0.01 compared with the control group), wherein the effect of SEQ ID NO. 3 was strongest, and the values of PBS and ATAP were small in the blank group.
TABLE 7 detection of extracellular IFN-Y secretion by PBMC
* P <0.01, # P <0.01, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO. 4.
Data analysis experimental data are expressed as mean ± standard deviation (mean ± SD) and plotted using Graphpad software, statistical analysis was performed on experimental data using one-factor variance, and comparison of group-to-group differences was performed with t-test, with P <0.05 being considered as a significant difference between the two groups.
The number of modules and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (10)
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