CN107325172A - Antigenic Peptide T790M 2 and its application in the medicine for preparing treatment non-small cell lung cancer - Google Patents
Antigenic Peptide T790M 2 and its application in the medicine for preparing treatment non-small cell lung cancer Download PDFInfo
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- CN107325172A CN107325172A CN201710546363.9A CN201710546363A CN107325172A CN 107325172 A CN107325172 A CN 107325172A CN 201710546363 A CN201710546363 A CN 201710546363A CN 107325172 A CN107325172 A CN 107325172A
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- antigenic peptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
Abstract
Application the invention provides Antigenic Peptide T790M 2 and its in the medicine for preparing treatment non-small cell lung cancer, the amino acid sequence of the Antigenic Peptide T790M 2 is as shown in SEQ ID NO.1.The Antigenic Peptide has the effect that non-small cell lung cancer cell is killed in T cell targeting that activates, and can be the non-small cell lung cancer patient for preventing and/or treating EGFR T790M mutation（EGFR TKIs therapies）A kind of new, promising immunotherapy method is provided.
Description
Technical field
The invention belongs to biomedicine technical field, and in particular to Antigenic Peptide T790M-2 and its non-small thin preparing treatment
Application in the medicine of born of the same parents' lung cancer.
Background technology
Lung cancer is the main cause of global cancer mortality.And non-small cell lung cancer accounts for the 80% of cases of lung cancer.Although controlling now
Treatment means have to develop on a large scale very much, but the prediction of lung cancer patient still is apparent not enough.But nearest people are to this disease in treatment method
Achieve important progress.Particularly EGF-R ELISA-tyrosine kinase inhibitor (EGFR-TKIs), is replaced as Ji is non-
Buddhist nun, Erlotinib have been developed the new therapeutic modality being used as specific non-small cell lung cancer, these patients
The tyrosine kinase domain of EGFR gene is mutated.Using EGFR-TKI medicines to EGFR saltant type non-small cell lung carninomatosis
People is (such as
DelE746-A750 and L858R) implement preclinical research, show higher clinical response rate, about
80%.However, with the passage of time, most patient generates acquired drug resistance to EGFR-TKIs.To non-small cell lung
The further investigation of cancer is identified in the EGFR-TKIs resistance sufferers that secondary mutation T790M appears in 50%.But for there is this
For the non-small cell lung cancer sufferer for planting mutation, there is presently no effective therapeutic modality.
In recent years, due to the identification of kinds of tumors related antigen, tumor immunology field achieves noticeable progress.
The especially research and development and clinical test of kinds of tumors immunotherapy, using tumor correlated albumen or the tumor vaccine of peptide fragment.
Although immunotherapy shows feasibility and hypotoxicity, the random experiment in later stage, except minority in early clinic experiment
In addition, compared to existing therapeutic modality, fail to show beneficial therapeutic effect.These unexpected results may return
Because in being at least partly due to the type of vaccine antigen.At present, what most vaccine antigen was isolated from not undergoing mutation is autologous
Antigen, due to the presence of Central immunotolerance and/or peripheral immune tolerance mechanism, they do not show high immunogenicity.Phase
Than under, the neoantigen of tomour specific, due to the amino acid sequence containing mutation, with immunogenicity, because they can be by place
Main immune system is recognized as exotic.Particularly, the vaccine antigen for carrying out self-driven mutation is probably one of immunization therapy
Promising target, because they can seldom lose from tumour cell.Although there is some research reports to point out to target exempting from for mutant antigen
Epidemic disease treatment have feasibility, but up to now only have sub-fraction mutant antigen be identified be immunization therapy potential target spot.
In non-small cell lung cancer, some t cell epitopes from mutant antigen have been reported.But in our research
In, we predict the t cell epitope of HLA-A*0201 (A2) restricted antigen from EGFR T790M drug resistant mutants, it
Have very strong immunogenicity to the T cell of people, the t cell epitope predicted can be to prevent and/or treatment EGFR T790M dash forward
The non-small cell lung cancer patient (EGFR-TKIs therapies) of change provides a kind of new, promising immunotherapy method.
The content of the invention
The technical problem of solution：Non-small cell is treated it is an object of the invention to provide Antigenic Peptide T790M-2 and its preparing
Application in the medicine of lung cancer, the Antigenic Peptide has the effect of activation T cell targeting kill non-small cell lung cancer cell.
Technical scheme：Antigenic Peptide T790M-2, its amino acid sequence is as shown in SEQ ID NO.1.
Further, the amino acid sequence of the Antigenic Peptide T790M-2 has the amino acid sequence as shown in SEQ ID NO.1
Replace, lack or add the amino acid sequence that one or more amino acid residues are obtained in row.
A kind of medicine for being used to treat non-small cell lung cancer, the medicine contains above-mentioned Antigenic Peptide T790M-2.
Medicines of the above-mentioned Antigenic Peptide T790M-2 in the non-small cell lung cancer for preparing prevention and/or treatment EGFR T790M mutation
Application in thing.
Above-mentioned Antigenic Peptide T790M-2 is being prepared for inducing to exempting from the EGFR T790M non-small cell lung cancers being mutated
Application in the medicine of epidemic disease.
Applications of the above-mentioned Antigenic Peptide T790M-2 in the medicine for induced tumor reaction-ive T cell is prepared, the tumour
Reaction-ive T cell is the T cell for the non-small cell lung cancer cell growth that can have EGFR T790M to be mutated with targeted inhibition.
Beneficial effect：There is the Antigenic Peptide T790M-2 of the present invention activation T cell targeting to kill non-small cell lung cancer cell
Effect, provides a kind of new to prevent and/or treating the non-small cell lung cancer patient (EGFR-TKIs therapies) of EGFR T790M mutation
, promising immunotherapy method.
Brief description of the drawings
Fig. 1 is that the CTL cells of maturation DC inductions in embodiment 2 are united to H1975-A2 and H1975 cytotoxic activity column
Meter figure；
Fig. 2 is the nude mice body weight column statistical chart of CTL killing experiments in embodiment 2；
Fig. 3 is the nude mice pathologic section-H＆E coloration results of CTL killing experiments in embodiment 2.
Embodiment
Invention is described in further detail below by embodiment.But it will be understood to those of skill in the art that under
Row embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
Embodiment 1
Antigenic Peptide T790M-2 is predicted and synthesis
Recent motif method analysis display, SYFPEITHI hyper-base sequence methods are effective HLA-A2 binding peptides forecasting systems.Cause
This is used for the HLA-A2 binding peptides for predicting the 9-mer from EGFR-T790M.Using HLA-s of the SYFPEITHI to EGF-T790M
A*02:01 restricted CTL epitope carries out remotely predicting, obtains the nonapeptide (L I M Q L M P F G) of score value higher (13 points).
Above-mentioned Antigenic Peptide is prepared using the method for chemical synthesis, i.e., highly developed solid-phase peptide well known in the art is closed
Into method, both Fmoc methods can also be used using Boc methods.Specific practice is exactly that protected amino acid is even one by one
It is linked to inertia solid phase carrier up, is then cleaved from carrier peptide chain using strong acid, while removes side chain protected.Prepare
Obtain the peptide that amino acid sequence is L I M Q L M P F G.
Embodiment 2
1. PMBC separating experiment
HLA-A*02 is taken under aseptic condition:01 positive normal health donor limosis vein blood 20mL, uses separation of lymphocytes
Liquid separates monocyte, cell suspension is prepared, using whole mass concentration as 2 × 109/ L is placed in blake bottle, is placed in 37 DEG C, 5%CO2
Incubator is incubated after 2h, washes away non-adherent cell；Collect adherent cell (monocyte) standby.
2. maturation DC is induced and antigen load
Above-mentioned monocyte presses whole mass concentration and adds human granulocyte-macrophage colony stimulating factor (rhGM-CsF)
1000mg/L, recombinant human interleukin--4 (rhIL-4) 500mg/L, add the cell factor of 1/2 concentration first, culture is extremely every other day
(A groups) Antigenic Peptide is added within 5th day, (B groups) only adds culture medium as a control group, in 37 DEG C, 5%CO2Under the conditions of cultivate 2 days, more
Nutrient solution is changed, TNF (TNF)-α (10mg/L) is added, continues to cultivate 48h, cell is collected and supernatant is standby.
3.DC surface markers are detected
Collect the DC cells of different antigen inductions and 10min is centrifuged with 1500r/min, extract cell precipitation and slow with phosphate
Fliud flushing (PBS) is washed 3 times, and 30min is incubated at room temperature, reuse PBS wash after 1 time add FITC- mouse anti-human CD80, CD83, CD86,
CDla, HLA DR sites (HLA-DR), using the Ig of isotype fluorescent staining as control, use flow cytometer
The expression of cell surface molecule is analyzed, one is shown in Table.
The Antigenic Peptide of table one loads analysis %, the n=3 of DC cell surface molecule marks,)
| Group | CD1a | CD 80 | CD 83 | HLA-DR | CD86 |
| A | 37.5±2.3 | 59.0±4.3 | 51.3±2.9 | 73.8±2.2 | 72.6±4.2 |
| B | 25.2±2.1 | 50.7±2.3 | 38.5±4.4 | 64.2±3.9 | 70.9±4.1 |
4. the CTL cells of maturation DC inductions
Peripheral blood comes from same blood donor.The autologous anticoagulations of 4mL are added to the centrifuge tube of T lymphocyte separation mediums, layering
Centrifugation, draws monocyte.By 2 × 108The DC of/L monocyte and Antigen co-cultures 5d, then to remove attached cell standby
With.
5.MTT determination of color CTL killing activity
(1) the CTL cells for inducing ripe DC are used as effector cell, Non-small cell lung carcinoma cell H1975 (HLA-A2-
T790M+), H1975-A2 (H1975transfected with HlA-A2, HLA-A2+T790M+) it is used as target cell.Adjustment effect
Cell is answered, the effect target ratio of target cell is 3:1、10:l.
(2) using 96 well culture plates (advance ultraviolet irradiation 20min).Experimental group adds effector cell per hole and the thin liquid of target is each
100 μ L are mixed, and three multiple holes are added altogether；Laying effect cell and target cell control, are all provided with three wells, respectively take 100 μ L, all simultaneously
It is each in control wells to add the μ L of RPMI-1640 (containing 10% hyclone) nutrient solution 100,37 DEG C, 5%CO2Under the conditions of educate 24h altogether.
(3) after 24h, the μ L of supernatant 100 are abandoned in suction, and adding MTT liquid storages, (5mg/mL is dissolved in 0.02M, pH7.4,0.05% Portugal
In the PBS of grape sugar) 20 μ L, 37 DEG C, 5%CO2Under the conditions of, then 2~4h is educated altogether.
(4) carefully inhale and abandon the addition μ L of dimethyl sulfoxide (DMSO) 150 after supernatant, place 20min.
(5) OD490nm values are determined in enzyme mapping.
Killer cell activity is represented with killing cytotoxic activity percentage, is calculated as follows：
As a result as shown in Table 2：
The CTL cells of the maturation of table two DC inductions to H1975-A2 and H1975 cytotoxic activity (%, n=3,)
Result above shows that the CTL cells of maturation DC inductions are higher than H1975 to H1975-A2 cytotoxic activity, shows this
The CTL cells of Maturation induction are recognized in mode restricted a kind of HLA-A2 with the non-small of EGFR T790M mutation in invention
Cell lung cancer cell.The gap of each group more can intuitively be compared by Fig. 1 cytotoxic activity columns statistical chart.
6.CTL killing experiments：
(1) foundation of non-small cell lung cancer nude mice model：Cellar culture mouse source non-small cell lung cancer cell (H1975-A2),
In exponential phase 1 × 10 is made using physiological saline7/ mL cell suspension, and cell tail vein was transplanted in 5~8 weeks
Female BAl BIc/c nude mices, interval is transplanted once for 3 days again, continuous 2-3 weeks, and modeling is successful on the whole after the 1-2 months.The side of blood is taken with eye socket
Formula obtains nude mice venous blood, monocyte is separated, according to " PMBC separating experiment " and " detection of DC surface markers "
Method induction CTLs.
(2) experiment packet：It is randomly divided into 5 groups (body weight state is homogeneous), every group 5：(A groups) blank control group：Do not appoint
The control group of where reason；(B groups) model control group：Transplant mouse source non-small cell lung cancer cell (H1975-A2), later stage injection life
Reason salt solution does negative control；(C groups) CTL groups, transplanting mouse source non-small cell lung cancer cell (H1975-A2), later stage injection load is anti-
The CTLs of the DC inductions of former peptide, daily can defeated 1 × 106Individual cell, continuous processing one week；(D groups) pure medicine group：Transplant mouse source non-
Small cell lung cancer cell (H1975-A2), later stage injection Gefitinib medicine, injects a 50mg/kg, continuous processing one daily
Week；(E groups) medicine+CTL groups, transplanting mouse source non-small cell lung cancer cell (H1975-A2), later stage per injection Gefitinib
CTLs1 × 10 of the DC inductions of 50mg/kg and Loading peptides6, continuous processing one week.
(3) Testing index：After medicine and CTL are handled 2 weeks, observation nude mice changes of weight and nude mice lung pathologies change feelings
Condition.As knowable to the data of table three, the nude mouse of normal nude mice control group, model control group and treated with gefitinib group is done with blank group
Weight substantially mitigates；And CTL treatment groups and CTL+ treated with gefitinib groups nude mouse weight relative drop are less.By Fig. 2 body weight columns
Statistical chart more can intuitively compare the gap of each group.As shown in Figure 3, the disease of the nude mice of model control group and treated with gefitinib group
Manage result and understand that various pulmonary lesions are more obvious, there is obvious focus, and invade the substantial amounts of tumour cell of profit in the tissue；CTL
Treatment group and CTL+ treated with gefitinib group nude mice focuses are considerably lighter, and the tumor cell number in tissue is substantially reduced.
The model of table three after naked two weeks weight ratio compared with
SEQUENCE LISTING
<110>Jiangsu Mai Jian biotechnologies Development stock Co., Ltd
<120>Antigenic Peptide T790M-2 and its application in the medicine for preparing treatment non-small cell lung cancer
<130> 20170629
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213>Artificial sequence
<400> 1
Leu Ile Met Gln Leu Met Pro Phe Gly
1 5
Claims (6)
1. Antigenic Peptide T790M-2, it is characterised in that：Its amino acid sequence is as shown in SEQ ID NO.1.
2. Antigenic Peptide T790M-2, it is characterised in that：Its amino acid sequence is in the amino acid sequence as shown in SEQ ID NO.1
Replace, lack or add the amino acid sequence that one or more amino acid residues are obtained.
3. a kind of medicine for being used to treat non-small cell lung cancer, it is characterised in that：The medicine contains described in claim 1 or 2
Antigenic Peptide T790M-2.
4. the Antigenic Peptide T790M-2 described in claim 1 or 2 is preparing prevention and/or is treating the non-small of EGFR T790M mutation
Application in the medicine of cell lung cancer.
5. Antigenic Peptide T790M-2 described in claim 1 or 2 prepare be used to inducing to EGFR T790M be mutated it is non-
Application in the immune medicine of ED-SCLC.
6. the Antigenic Peptide T790M-2 described in claim 1 or 2 is in the medicine for induced tumor reaction-ive T cell is prepared
Using, it is characterised in that：The tumor-reactive T cells are can have EGFR T790M to be mutated with targeted inhibition non-small thin
The T cell of born of the same parents' lung cancer cell growth.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109887553A (en) * | 2019-01-29 | 2019-06-14 | 杭州纽安津生物科技有限公司 | For the polypeptide vaccine and its design method in tumor-targeting drug drug resistance site |
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| CN102153644A (en) * | 2010-12-31 | 2011-08-17 | 广东药学院 | Antigenic peptide for dimerization of epidermal growth factor receptor |
| EP2883884A1 (en) * | 2012-08-10 | 2015-06-17 | Kanagawa Prefectural Hospital Organization | Antigen peptide originated from t790m point-mutated sequence of epidermal growth factor receptor |
| CN109310739A (en) * | 2016-03-31 | 2019-02-05 | 内恩疗法公司 | Neoantigen and its application method |
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2017
- 2017-07-06 CN CN201710546363.9A patent/CN107325172A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102153644A (en) * | 2010-12-31 | 2011-08-17 | 广东药学院 | Antigenic peptide for dimerization of epidermal growth factor receptor |
| EP2883884A1 (en) * | 2012-08-10 | 2015-06-17 | Kanagawa Prefectural Hospital Organization | Antigen peptide originated from t790m point-mutated sequence of epidermal growth factor receptor |
| CN109310739A (en) * | 2016-03-31 | 2019-02-05 | 内恩疗法公司 | Neoantigen and its application method |
Non-Patent Citations (2)
| Title |
|---|
| KAZUYA OFUJI ET AL: "A peptide antigen derived from EGFR T790M is immunogenic in non‑small cell lung cancer", 《INTERNATIONAL JOURNAL OF ONCOLOGY》 * |
| TEPPEI YAMADA ET AL: "EGFR T790M Mutation as a Possible Target for Immunotherapy; Identification of HLA-A*0201-Restricted T Cell Epitopes Derived from the EGFR T790M Mutation", 《PLOS ONE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109887553A (en) * | 2019-01-29 | 2019-06-14 | 杭州纽安津生物科技有限公司 | For the polypeptide vaccine and its design method in tumor-targeting drug drug resistance site |
| CN112687325A (en) * | 2019-01-29 | 2021-04-20 | 杭州纽安津生物科技有限公司 | Polypeptide vaccine composition for targeted drugs Aleptinib, Ceritinib and crizotinib and design method thereof |
| CN112687325B (en) * | 2019-01-29 | 2024-01-26 | 杭州纽安津生物科技有限公司 | Polypeptide vaccine composition aiming at targeted drugs of alatinib, ceritinib and crizotinib and design method thereof |
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Application publication date: 20171107 |