CN107188959B

CN107188959B - Antigenic peptide T790M-6 and application thereof in preparation of medicines for treating non-small cell lung cancer

Info

Publication number: CN107188959B
Application number: CN201710546364.3A
Authority: CN
Inventors: 冷晓燕; 张超; 段云飞; 常菁; 曹巍; 虞强
Original assignee: Jiangsu Maijian Biotechnology Development Co ltd
Current assignee: Jiangsu Maijian Biotechnology Development Co ltd
Priority date: 2017-07-06
Filing date: 2017-07-06
Publication date: 2020-09-29
Anticipated expiration: 2037-07-06
Also published as: CN107188959A

Abstract

The invention provides an antigenic peptide T790M-6 and application thereof in preparing a medicament for treating non-small cell lung cancer, wherein the amino acid sequence of the antigenic peptide T790M-6 is shown as SEQ ID NO. 1. The antigenic peptide has the effect of activating T cells to kill non-small cell lung cancer cells in a targeted manner, and can provide a novel and promising immunotherapy method for preventing and/or treating EGFR T790M mutant non-small cell lung cancer patients (EGFR-TKIs therapy).

Description

Antigenic peptide T790M-6 and application thereof in preparation of medicines for treating non-small cell lung cancer

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an antigenic peptide T790M-6 and application thereof in preparation of a medicine for treating non-small cell lung cancer.

Background

Lung cancer is the leading cause of cancer death worldwide. Non-small cell lung cancer accounts for 80% of lung cancer cases. Although the current treatment methods are greatly developed, the prediction of lung cancer patients is still poor. But recently significant advances have been made in the treatment of this disease. In particular, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have been developed as novel therapeutic modalities for certain non-small cell lung cancers in which the tyrosine kinase domain of the EGFR gene is mutated. Preclinical studies performed with EGFR-TKI drugs on EGFR mutant non-small cell lung cancer patients (e.g., del E746-A750 and L858R) showed a high clinical response rate, about 80%. However, most patients develop acquired resistance to EGFR-TKIs over time. Intensive studies on non-small cell lung cancer have identified the occurrence of the secondary mutation T790M in 50% of patients resistant to EGFR-TKIs. However, there is no effective treatment for non-small cell lung cancer patients with such mutations.

In recent years, there has been considerable progress in the field of tumor immunology due to the identification of a variety of tumor-associated antigens. In particular, the development and clinical trials of various tumor immunotherapies, including tumor vaccines that utilize tumor-associated proteins or peptides. Although immunotherapy showed feasibility and low toxicity in early clinical trials, the latter randomized trials, with few exceptions, failed to show beneficial therapeutic effects compared to existing treatment modalities. These unexpected results may be attributed, at least in part, to the type of vaccine antigen. Currently, most vaccine antigens are isolated from non-mutated self-antigens, which do not exhibit high immunogenicity due to the presence of central and/or peripheral immune tolerance mechanisms. In contrast, tumor-specific neoantigens, by virtue of containing mutated amino acid sequences, are immunogenic in that they are recognized by the host immune system as foreign. In particular, vaccine antigens from driver mutations may be an ideal target for immunotherapy, as they are less likely to be lost from tumor cells. Although some studies have reported that immunotherapy targeting mutant antigens is feasible, only a small fraction of mutant antigens have been identified as potential targets for immunotherapy to date.

In non-small cell lung cancer, several T cell epitopes derived from mutant antigens have been reported. However, in our study we predicted T cell epitopes from HLA a x 0201(a2) restricted antigens of EGFR T790M resistant mutants that are strongly immunogenic to human T cells, and the predicted T cell epitopes may provide a new and promising immunotherapeutic approach for the prevention and/or treatment of non-small cell lung cancer patients with EGFR T790M mutations (EGFR-TKIs therapy).

Disclosure of Invention

The technical problem to be solved is as follows: the invention aims to provide an antigenic peptide T790M-6 and application thereof in preparing a medicament for treating non-small cell lung cancer, wherein the antigenic peptide has the function of activating T cells to kill non-small cell lung cancer cells in a targeted manner.

The technical scheme is as follows: the amino acid sequence of the antigen peptide T790M-6 is shown in SEQ ID NO. 1.

Furthermore, the amino acid sequence of the antigenic peptide T790M-6 has an amino acid sequence obtained by substituting, deleting or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

Further, the amino acid sequence of the antigenic peptide T790M-6 is an amino acid sequence obtained by substituting, deleting or adding one or more amino acid residues in the sequence.

A medicine for treating non-small cell lung cancer contains the antigenic peptide T790M-6.

The antigenic peptide T790M-6 is applied to the preparation of the drugs for preventing and/or treating EGFR T790M mutant non-small cell lung cancer.

The application of the antigenic peptide T790M-6 in preparing a medicament for inducing immunity to non-small cell lung cancer with EGFR T790M mutation.

The application of the antigenic peptide T790M-6 in preparing a medicament for inducing tumor-reactive T cells, wherein the tumor-reactive T cells are T cells capable of targeting and inhibiting the growth of non-small cell lung cancer cells with EGFR T790M mutation.

Has the advantages that: the antigenic peptide T790M-6 has the function of activating T cells to kill non-small cell lung cancer cells in a targeted manner, and provides a novel and promising immunotherapy method for preventing and/or treating EGFR T790M mutant non-small cell lung cancer patients (EGFR-TKIs therapy).

Drawings

FIG. 1 is a histogram of the cytotoxic activity of mature DC-induced CTL cells against H1975-A2 and H1975 in example 2;

FIG. 2 is a histogram of the body weight of nude mice in CTL killing experiment of example 2;

FIG. 3 shows the pathological section-H & E staining results of the lung tissue of nude mice in CTL killing experiment in example 2.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments. However, those skilled in the art will appreciate that the following examples are illustrative only and should not be construed to limit the scope of the present invention.

Example 1

Antigenic peptide T790M-6 prediction and synthesis

Recent motif analysis showed that the SYFPEITHI hyper-motif method is a potent HLA-a2 binding peptide prediction system. And thus used to predict HLA-A2 binding peptides from the 9-mer of EGFR-T790M. The HLA-A02: 01 restricted CTL epitope of EGF-T790M was remotely predicted by SYFPEITHI to obtain decapeptide (L T S T V Q L I MQ) with higher score (10).

The above-mentioned antigenic peptides are prepared by chemical synthesis, i.e., by well-established solid phase peptide synthesis methods known in the art, either Boc or Fmoc. The specific method is to couple the protected amino acids to an inert solid phase carrier one by one, then crack the peptide chain from the carrier by using strong acid, and simultaneously remove the side chain protection. The peptide having amino acid sequence L T S T V Q L I M Q was prepared.

Example 2

1. Peripheral blood mononuclear cell separation experiment

Taking 20mL of fasting venous blood of HLA-A02: 01 positive normal healthy donor under aseptic condition, separating mononuclear cells with lymphocyte separation solution, preparing cell suspension to final mass concentration of 2 × 10⁹The culture flask is filled with/L of 5% CO at 37 deg.C₂After the incubator is incubated for 2 hours, non-adherent cells are washed away; adherent cells (monocytes) were collected for use.

2. Mature DC induction and antigen Loading

Adding human granulocyte-macrophage colony stimulating factor (rhGM-CsF) 1000mg/L and recombinant human interleukin-4 (rhIL-4)500mg/L into the above monocyte at final mass concentration, and adding every other dayThe first 1/2 concentration of cytokine, cultured until 5 days, added (group A) antigen peptide, and (group B) culture medium alone as control group, at 37 deg.C and 5% CO₂Culturing for 2 days under the condition, changing culture solution, adding Tumor Necrosis Factor (TNF) - α (10mg/L), culturing for 48h, and collecting cells and supernatant.

DC surface marker detection

DC cells induced by different antigens were collected and centrifuged at 1500r/min for 10min, cell pellets were extracted and washed 3 times with Phosphate Buffered Saline (PBS), incubated at room temperature for 30min, washed 1 time with PBS and then added with FITC-mouse anti-human CD80, CD83, CD86, CDla, human leukocyte antigen DR site (HLA-DR), isotype fluorescent stained Ig was used as a control, and cell surface molecule expression was analyzed by flow cytometry, see Table one.

Table-antigen peptide load analysis% of DC cell surface molecular marker, n-3,

)

group of	CD1a	CD 80	CD 83	HLA-DR	CD86
						A	38.1±4.9	59.4±3.2	50.9±5.0	72.9±2.0	72.0±2.8
B	25.4±3.7	51.3±4.1	41.1±1.9	62.8±1.9	71.8±3.7

4. Mature DC-induced CTL cells

Peripheral blood from the same donor, adding 4mL of autologous anticoagulation blood into a centrifuge tube of T lymphocyte separation solution, carrying out layered centrifugation, sucking mononuclear cells, and adding 2 × 10⁸And co-culturing the L monocytes and the antigen-loaded DCs for 5d, and removing adherent cells for later use.

Determination of killing Activity of CTL by MTT color development

(1) Mature DC-induced CTL cells as effector cells, human non-small cell lung carcinoma cells H1975 (HLA-A2)^-T790M⁺)、H1975-A2(H1975transfected with HlA-A2，HLA-A2⁺T790M⁺) As target cells. Adjusting the effective target ratio of the effector cells to the target cells to be 3:1 and 10: l.

(2) 96-well culture plates (pre-UV irradiation for 20min) were used. Adding effector cells and target fine liquid into each hole of the experimental group, respectively adding 100 mu L of effector cells and target fine liquid, uniformly mixing, and adding three multiple holes in total; setting effector cell and target cell controls, setting three multiple wells, taking 100 μ L each, adding RPMI-1640 culture solution 100 μ L (containing 10% fetal calf serum) into all control wells, at 37 deg.C, 5% CO₂Co-cultivating for 24h under the condition.

(3) After 24h, 100. mu.L of the supernatant was aspirated off, and 20. mu.L of MTT stock solution (5mg/mL in 0.02M, pH7.4, 0.05% glucose in PBS) was added at 37 ℃ with 5% CO₂And (5) co-cultivating for 2-4 h under the condition.

(4) Carefully remove the supernatant by aspiration, add 150. mu.L of dimethyl sulfoxide, and stand for 20 min.

(5) OD490nm values were determined on an enzyme label.

The killer cell activity is expressed as a percentage of killer cytotoxic activity and is calculated according to the following formula:

the results are shown in table two:

table two cytotoxic activities of mature DC-induced CTL cells against H1975-a2 and H1975 (%, n-3,

)

the above results show that the cytotoxic activity of mature DC-induced CTL cells against H1975-A2 is higher than that of H1975, indicating that the mature induced CTL cells of the present invention recognize non-small cell lung cancer cells carrying EGFR T790M mutation in an HLA-A2-restricted manner. The differences between groups can be more intuitively compared from the histogram of cytotoxic activity of FIG. 1.

CTL killing assay:

(1) establishment of non-small cell lung cancer nude mouse model by culturing murine non-small cell lung cancer cells (H1975-A2) and preparing into 1 × 10 with physiological saline at logarithmic growth phase⁷And transplanting the tail veins of the cells into female BALB/c nude mice for 5-8 weeks, wherein the transplantation is performed once every 3 days, the continuous period is 2-3 weeks, and the molding is basically successful after 1-2 months. Obtaining nude mouse venous blood by a way of orbital bleeding, separating mononuclear cells, and inducing CTLs according to methods of 'peripheral blood mononuclear cell separation experiment' and 'DC surface marker detection'.

(2) Grouping experiments: randomized into 5 groups (uniform body weight), 5 per group: (group a) blank control group: control group without any treatment; (group B) model control group: transplanting murine non-small cell lung cancer cells (H1975-A2), and injecting physiological saline at the later stage to serve as a negative control; (group C) CTL group transplanted with murine non-small cell lung carcinomaCells (H1975-A2), late-injected DC-induced CTLs loaded with antigenic peptides, delivered 1 × 10 daily⁶One cell for continuous treatment for one week, (D) pure drug group, transplantation of murine non-small cell lung cancer cells (H1975-A2), late injection of gefitinib drug once daily for one week, drug + CTL group, transplantation of murine non-small cell lung cancer cells (H1975-A2), late injection of gefitinib drug 50mg/kg and antigen peptide-loaded DC-induced CTLs1 × 10⁶Continuously treating for one week.

(3) Detection indexes are as follows: after 2 weeks of drug and CTL treatment, the weight change and lung pathological changes of the nude mice were observed. As can be seen from the data in Table three, the weight of the nude mice in the model control group and the gefitinib-treated group is obviously reduced by taking the blank group as the normal nude mouse control group; and the weight of the nude mice in the CTL treatment group and the CTL + gefitinib treatment group is relatively reduced. The weight histogram of fig. 2 makes it more intuitive to compare the differences between groups. As shown in fig. 3, the pathological results of the nude mice of the model control group and the gefitinib-treated group revealed that lung tissue lesion was more pronounced, there was a pronounced lesion, and a large number of tumor cells were infiltrated in the tissue; the CTL treatment group and the CTL + gefitinib treatment group of nude mice have obviously lighter lesions, and the number of tumor cells in tissues is obviously reduced.

Weight comparison of the three models after two weeks of nudity

SEQUENCE LISTING

<110> Jiangsu maijian biotechnological development corporation

<120> antigenic peptide T790M-6 and application thereof in preparation of drugs for treating non-small cell lung cancer

<130>20170630

<160>1

<170>PatentIn version 3.3

<210>1

<211>10

<212>PRT

<213> Artificial sequence

<400>1

Leu Thr Ser Thr Val Gln Leu Ile Met Gln

1 5 10

Claims

1. Antigenic peptide T790M-6, characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

2. A medicament for the treatment of non-small cell lung cancer, characterized by: the pharmaceutical composition comprising the antigenic peptide T790M-6 of claim 1.

3. The use of the antigenic peptide T790M-6 of claim 1 in the manufacture of a medicament for the treatment of EGFR T790M mutant non-small cell lung cancer.

4. Use of the antigenic peptide T790M-6 of claim 1 in the manufacture of a medicament for inducing immunity to non-small cell lung cancer having the EGFR T790M mutation.

5. Use of the antigenic peptide T790M-6 according to claim 1, for the preparation of a medicament for the induction of tumor-reactive T cells, characterized in that: the tumor-reactive T cells are T cells that can be targeted to inhibit the growth of non-small cell lung cancer cells having the EGFR T790M mutation.