WO2021087839A1 - Tumor-specific polypeptide sequence and application thereof - Google Patents

Abstract

Provided are a tumor-specific polypeptide sequence and an application thereof. The polypeptide comprises at least any polypeptide in a first peptide group, and can optionally comprise at least any polypeptide in a second peptide group. The first peptide group comprises polypeptides having the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5; the second peptide group comprises derived peptides having the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5. The derived peptides comprise front, middle and back peptide segments connected in sequence. The middle peptide segment has at least 80% homology with the sequences shown in SEQ ID NO:1 to SEQ ID NO:5, and the sum of the lengths of the front and back peptide segments is 14-16 amino acids. Also provided are an isolated nucleic acid, a construct, an expression vector, a pharmaceutical composition, an antigen presenting cell, an immune effector cell, a tumor vaccine, and use of the polypeptide in the preparation of a drug for preventing or treating a tumor and a method for treating a tumor patient.

Description

Tumor-specific peptide sequence and its application Technical field

The present invention relates to the field of biomedicine, in particular to a tumor-specific polypeptide sequence and its application, and specifically to a set of isolated polypeptides, isolated nucleic acids, constructs, expression vectors, host cells, pharmaceutical compositions, antigen-presenting cells, and immunity Use of effector cells, tumor vaccines, and polypeptides in preparing drugs for preventing or treating tumors and methods for treating patient tumors.

Background technique

Cancer, as a disease in which gene mutations in cells cause cell proliferation to go out of control, has become a major threat to human health and a major cause of human death. According to the "Analysis of the Epidemiology of Malignant Tumors in China in 2015" issued by the National Cancer Center, the incidence of malignant tumors in China in 2015 was approximately 3.929 million and the deaths were approximately 2.338 million. The burden of cancer continues to rise. In the past 10 years, the incidence of malignant tumors has maintained an annual increase of about 3.9%, and the mortality rate has maintained an annual increase of 2.5%. Among them, the main high-incidence malignant tumors are lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer and esophageal cancer in order. Therefore, finding effective and specific cancer treatment methods has great clinical value.

By modulating the body’s immune system, immunotherapy enhances the tumor microenvironment’s anti-tumor immunity, so as to achieve the purpose of controlling and killing tumor cells. It has the advantages of high efficiency, strong specificity and good tolerance, and has broad applications in tumor treatment. prospect. Immunotherapy mainly includes cytokine therapy, immune checkpoint monoclonal antibodies, adoptive cell reinfusion, and tumor immunotherapy vaccines. Among them, tumor immunotherapy vaccines mainly include tumor cell vaccines, dendritic cell vaccines, protein & peptide vaccines, nucleic acid vaccines, genetic engineering vaccines and anti-idiotypic antibody vaccines. The main mechanism of these vaccines killing tumors is by causing patients to target The tumor's immune response allows T cells to recognize tumor cells and then kill tumor cells.

Tumor antigens targeted by tumor immunotherapy vaccines include tumor-associated antigens and neoantigens. Tumor-associated antigens are derived from proteins that are highly expressed in tumor tissues but are low or not expressed in normal tissues; and tumor neoantigens are derived from variant proteins produced by mutations in the tumor genome. Because tumor neoantigens only exist in tumor cells and not in normal cells, neoantigens can bypass the central immune tolerance and cause a strong T cell immune response, which has the characteristics of good effect; at the same time, tumor-specific characteristics The tumor neoantigen has the advantages of good safety and low side effects. However, the tumor neoantigens targeted by suitable tumor immunotherapy vaccines need to be further improved.

Summary of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, an object of the present invention is to propose a tumor-specific polypeptide sequence and its application, specifically related to a set of isolated polypeptides, isolated nucleic acids, constructs, expression vectors, host cells, pharmaceutical compositions, antigen-presenting cells, Use of immune effector cells, tumor vaccines, and polypeptides in preparing drugs for preventing or treating tumors and methods for treating tumor patients.

When performing tumor immunotherapy on patients, several options are often used: 1. Treat the patient by using tumor-associated antigens that are highly expressed in the patient’s tumor. This type of treatment may be due to the presence of tumor-associated antigens in some normal tissues. Expression, low immunogenicity, which makes the effect poor. 2. Use tumor-associated antigens or tumor neoantigens that have been experimentally identified in some patients for treatment. However, tumor mutations are patient-specific, and most tumor mutations will not recur in multiple patients. Therefore, the tumor-specific antigens identified in some patients have not been verified by their frequency in a large number of tumor patients. The probability of re-use in new patients is low, so that the number of patients who can be treated with these tumor neoantigens is small. 3. Perform individualized tumor neoantigen screening for each patient. For example, the patient’s tumor-specific mutations and the variant peptides that these mutations may produce can be obtained by analyzing the sequencing data of its genome and transcriptome, and then pass The machine learning algorithm predicts which variant peptides may be presented as antigens by MHC molecules, and then uses these predicted tumor neoantigens for patient treatment. A sequencing-based personalized tumor neoantigen screening program, although the genome and transcriptome sequencing of a certain patient can be used to screen out the tumor neoantigens that can treat a certain patient through sequencing data analysis and algorithm prediction, but the whole process The cost is high, the time is long, and because the accuracy of the current antigen prediction algorithm is not high, the false positives of the selected antigens are high, and some of the predicted antigens cannot effectively cause the immune response in the patient's body, thus leading to the poor efficacy of the patient. 4. Combining the above schemes, that is, using the identified tumor-associated antigens and tumor neoantigen collections, combined with individualized tumor neoantigen screening programs. For example, using the known antigens in Scheme 1 or Scheme 2 to perform the first stage of treatment for the patient, while referring to Scheme 3 for individualized antigen screening of the patient, and then use the antigen obtained from the screening for the second stage of treatment of the patient. Although this program can make up for the long time for individualized tumor neoantigen screening, the treatment cost still cannot be reduced because it also involves an individualized tumor neoantigen screening program.

Through a large amount of data analysis and experimental screening, the present invention has discovered the high-frequency mutation gene MUC3A (wild-type MUC3A gene encodes mucin 3A, which provides lubrication, cell signaling pathway and chemical barrier function), which is repeated in a variety of cancers. The high frequency mutation gene causes the amino acid at position 326 to be changed from serine (S) to threonine (T). The mutant polypeptide can be specifically and highly expressed in tumor tissues. The present invention verifies the high affinity of the mutant polypeptide with HLA-A11:01 typed molecules and the presentation in tumor cells through experiments. Further, the sequence of the mutant polypeptide was improved, and a large number of experiments were performed to screen out a mutant polypeptide that can be recognized by the same T cells as the original mutant polypeptide, but activates T cells and induces antigen-specific T cells to kill tumors.

Specifically, the present invention provides the following technical solutions:

In the first aspect of the present invention, the present invention proposes a set of isolated polypeptides. According to an embodiment of the present invention, the polypeptide includes at least any one polypeptide in the first peptide group, and may optionally include at least any one polypeptide in the second peptide group; the first peptide group includes a polypeptide having SEQ ID The polypeptides of the amino acid sequence shown in NO: 1 to SEQ ID NO: 5; the second peptide group includes derivative peptides of the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5, and the derivative peptides include sequentially connected peptides. The pro-peptide segment, the mid-peptide segment and the post-peptide segment, the mid-peptide segment has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5, the pro-peptide segment The sum of the length of the latter peptide segment is 14-16 amino acids.

The polypeptide sequence proposed by the present invention is a tumor-specific antigen and its variants generated by tumor gene mutations, and will not be expressed and presented in normal tissues, thus overcoming the problem of low safety in treatment with tumor-related antigens. At the same time, the proposed peptide sequence is derived from genes with high frequency mutations in a variety of cancers, and can be presented by HLA molecules frequently occurring in the population. Therefore, it can be repeated in the tumors of patients with multiple cancers and can cover currently known tumors. Patients whose antigen sequence cannot be covered.

According to an embodiment of the present invention, the isolated polypeptide described above may further include the following technical features:

In some embodiments of the present invention, the mid-peptide segment has at least 90% homology with the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5.

In some embodiments of the present invention, the mid-peptide segment is the same as the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5.

In some embodiments of the present invention, the derivative peptide has an amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 10.

In some embodiments of the present invention, the polypeptide is selected from at least one of the following:

(1) At least two polypeptides having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5;

(2) At least one polypeptide having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5, and at least one polypeptide having the amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 10.

In the second aspect of the present invention, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the above-mentioned polypeptide or its complementary sequence. As mentioned above, the above-mentioned polypeptides can be presented as antigens on the surface of tumor cells by HLA molecules that have affinity with them, and have the ability to activate T cells and direct these T cells to kill tumors, so they can encode the nucleic acid sequences of the above-mentioned polypeptides or these codes. The complementary sequence of the nucleic acid sequence of the above polypeptide can be used to prevent or treat tumors.

In the third aspect of the present invention, the present invention proposes a construct. According to an embodiment of the present invention, the construct comprises the nucleic acid according to the second aspect of the present invention and a control sequence, and the control sequence is operably linked to the nucleic acid. The constructs proposed in the embodiments of the present invention can efficiently express the above-mentioned polypeptides in suitable host cells under suitable conditions, and thus can be effectively used for the treatment or prevention of tumors. Wherein, the control sequence can instruct the nucleic acid to express the above-mentioned polypeptide in the host, and there can be one or more of these control sequences. These control sequences can be promoters, terminators, SD sequences, regulatory genes for regulating the expression of genes, etc., as required.

In the fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the construct according to the third aspect of the present invention. The expression vector provided by the present invention can efficiently express the above-mentioned polypeptide in a host under suitable conditions, and the expression vector can be effectively used for the treatment or prevention of tumors.

In the fifth aspect of the present invention, the present invention proposes a host cell. According to an embodiment of the present invention, the host cell carries the construct according to the third aspect of the present invention or the expression vector according to the fourth aspect of the present invention, and the host cell can be obtained by transfection or transformation of the aforementioned nucleic acid construct or expression vector of. The host cell can efficiently express the above-mentioned polypeptide under suitable conditions, and the host cell can be effectively used for the treatment or prevention of tumors.

In the sixth aspect of the present invention, the present invention proposes the use of polypeptides in preparing drugs for preventing or treating tumors or preparing kits for diagnosing tumors. If the tumor expresses the above-mentioned mutant gene MUC3A, the high-frequency mutant gene causes the amino acid at position 326 to be changed from serine (S) to threonine (T), and expresses the HLA-A11:01 type that has affinity with the polypeptide. HLA molecules, the above-mentioned polypeptides have the ability of HLA molecules typed by HLA-A11:01 with affinity to be presented on the surface of tumor cells as antigens, activating T cells and directing these T cells to kill tumors. Therefore, the proposed polypeptide can be used to prevent and control tumors. At the same time, as mentioned above, since the polypeptide proposed by the present invention is specifically expressed in tumor cells, it is used for the treatment or prevention of tumors and has good safety. It can also be used in the preparation of kits for diagnosing tumors.

In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the aforementioned polypeptide and pharmaceutically usable excipients. The pharmaceutical composition containing the aforementioned polypeptides and adjuvants can significantly stimulate the proliferation of tumor-specific T cells and the secretion of cytokines of these T cells, thereby killing tumor cells expressing corresponding mutant genes, and can be used for the treatment or prevention of tumors. Of course, the pharmaceutical composition provided can also include some pharmaceutically usable adjuvants. These adjuvants act as non-specific immune enhancers. When injected together with the aforementioned polypeptides or injected into the body in advance, they can enhance the body’s ability to respond to polypeptide antigens. Immune response or change the type of immune response. Useful adjuvants include but are not limited to PD-1 inhibitors.

In the eighth aspect of the present invention, the present invention provides an antigen presenting cell. According to an embodiment of the present invention, the antigen-presenting cell can present the aforementioned polypeptide. Antigen-presenting cells can be obtained by loading the polypeptide, transfecting or transforming the aforementioned nucleic acid construct or expression vector, or phagocytosing the aforementioned host cell. According to an embodiment of the present invention, the antigen-presenting cells presenting the aforementioned polypeptides significantly stimulate the proliferation of tumor-specific T cells and the secretion of cytokines of these T cells, thereby killing tumor cells expressing corresponding mutant genes, and can be used for the treatment or prevention of tumors. . These available antigen-presenting cells can be dendritic cells, macrophages, B cells and the like.

In the ninth aspect of the present invention, the present invention proposes an immune effector cell. According to an embodiment of the present invention, the immune effector cell can recognize the aforementioned polypeptide or the antigen-presenting cell described in the eighth aspect of the present invention. The immune effector cells can be induced by the aforementioned polypeptides or the aforementioned antigen-presenting cells. The immune effector cells can specifically kill tumor cells expressing the corresponding mutant genes, and are used for the treatment or prevention of tumors. These available immune effector cells can be T cells, effector T cells, NK cells and the like.

In the tenth aspect of the present invention, the present invention proposes a tumor vaccine. According to an embodiment of the present invention, the tumor vaccine comprises the aforementioned nucleic acid, or the aforementioned nucleic acid construct, or the aforementioned expression vector, or the aforementioned host cell, or the aforementioned antigen presentation Cells, or immune effector cells as described above.

In the eleventh aspect of the present invention, the present invention provides a method for treating patients with tumors, the method comprising administering to the patients an effective amount of a pharmaceutical composition or an effective amount of a tumor vaccine, the pharmaceutical composition being the seventh aspect of the present invention In the pharmaceutical composition, the tumor vaccine is the tumor vaccine according to the tenth aspect of the present invention. Among them, the "effective amount" of the pharmaceutical composition means that as long as it can achieve the purpose of inhibiting tumor growth or interfering with tumor proliferation. "Effective amount" of tumor vaccine refers to the introduction of these tumor vaccines into the patient's body, which can overcome the immunosuppressive state caused by the tumor and activate the patient's own immune system, so as to achieve the purpose of controlling or eliminating the tumor.

Description of the drawings

Fig. 1 is a diagram of the results of the mass spectrometry identification of mutant polypeptides provided by an embodiment of the present invention.

Figure 2 is a flow cytometric verification result diagram of the affinity of a polypeptide to T2 according to an embodiment of the present invention.

Fig. 3 is a diagram of the detection results of polypeptides and in vitro immunogenic ELISPOTs according to an embodiment of the present invention.

Figure 4 is a diagram showing the results of the polypeptide vaccine provided according to an embodiment of the present invention in controlling tumor growth in mice.

Figure 5 is a diagram showing the results of the polypeptide DC vaccine provided according to an embodiment of the present invention in controlling tumor growth in mice.

Figure 6 is a diagram showing the results of the DC-CTL vaccine provided in accordance with an embodiment of the present invention in controlling tumor growth in mice.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that these embodiments are exemplary and are intended to explain the present invention, but should not be understood as limiting the present invention.

At the same time, in order to facilitate the understanding of those skilled in the art, some terms of the present invention are explained and described. It should be noted that these explanations and descriptions are only used to help the understanding of the technical solutions of the present invention, and should not be regarded as Limitations on the scope of protection of the present invention.

Herein, the terms "first peptide group" or "second peptide group" respectively refer to polypeptides containing different amino acid sequences.

The term "derived peptide" is used to indicate a polypeptide sequence derived from a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5. These derived sequences are from N-terminus to C-terminus, including the connected propeptide segments in turn , The mid-peptide segment and the post-peptide segment, wherein the mid-peptide segment has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5, preferably at least 90% homology, The sum of the length of the front and back peptides is 14-16 amino acids. There are no special restrictions on the specific types of amino acids in the pre- and post-peptide segments. In at least some embodiments, these derived peptides may be long peptide sequences with a total length of 23mer-25mer obtained by extending the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5 to both sides. In some preferred embodiments, these derived peptides may be polypeptides having the amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 10.

In at least some embodiments of the present invention, the isolated polypeptide provided by the present invention is selected from at least one of the following groups: 1) A polypeptide having the amino acid sequence shown in SEQ ID NO: 1-SEQ ID NO: 5; (2) ) At least any polypeptide having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5, and at least any polypeptide having the amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 10.

The polypeptides of the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 10 are shown in Table 1 below.

Table 1 Polypeptides and their variant sequences

SEQ ID	Peptide sequence	Mutation information	SEQ ID	Derived peptide sequence
NO:1	TILPTTITK	MUC3A_S326T	NO:6	PLSTLVTILPTTITKSTPTSETT
NO:2	TSLPTTITK	MUC3A_S326T	NO:7	PLSTLVTSLPTTITKSTPTSETT
NO:3	TTLPTTITR	MUC3A_S326T	NO:8	PLSTLVTTLPTTITRSTPTSETT
NO:4	TTLPTTITK	MUC3A_S326T	NO:9	PLSTLVTTLPTTITKSTPTSETT
NO:5	TVLPTTITK	MUC3A_S326T	NO:10	PLSTLVTVLPTTITKSTPTSETT

These peptide sequences are derived from tumor-specific antigens produced by tumor gene mutations, and will not be expressed and presented in normal tissues. Therefore, they have higher specificity and cause immune responses with higher specificity. They are safe for treatment. The side effect is small, and its structure is simple, and it is easy to artificially synthesize. At the same time, these peptide sequences have the characteristics of affinity with HLA molecules, the ability to stimulate T cell expansion and secretion of cytokines, and the ability to induce antigen-specific T cells to kill target cells, without changing the relationship between them and T cells. Specificity and better tumor control effect.

The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, it shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased on the market.

Example one

Based on a large number of tumor mutation data of public databases such as TCGA/ICGC, count the high-frequency mutations among them, predict, screen, and experimentally verify the high-frequency typing of the Chinese population, and obtain a polypeptide sequence, such as SEQ ID NO :1 ~ SEQ ID NO: 5, and also use tumor mutation data to obtain derivative peptides. The derivative peptide sequence is a polypeptide sequence formed by the polypeptide sequence extending to a length of 23 amino acids on both sides. The derivative peptide sequence is as SEQ ID NO: 6 ~ SEQ ID NO: 10.

In the following examples, various polypeptide sequences and derived peptide sequences are studied. For the convenience of expression, the sequence shown in SEQ ID NO: 3 can be called a mutant polypeptide, and the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5 can be called a variant. Peptides.

Example 2 Mass spectrometry experiments verify that polypeptides are presented by HLA molecules on the surface of tumor cells

In the present invention, the polypeptide sequence and derived peptide coding gene obtained in Example 1 are transfected into tumor cells by lentivirus, and then the polypeptide-MHC complex on the cell surface is enriched by the combined method of co-immunoprecipitation and mass spectrometry. Whether the MHC molecules on the cell surface presented the mutant polypeptide was identified. The specific method is as follows:

1. Separation and purification of MHC-I restricted T cell epitope peptides:

Use pan-MHC-I A/B/C antibody (clone number: w6/32) to bind protein A molecule sepharose CL-4B beads on the surface for 1 hour at 4°C, use NanoDrop to detect the residual antibody content in the supernatant, Antibody binding rate >90% is considered qualified, prepare pan-MHC-I A/B/C combined sepharose, 4℃ for use. Add 40ml RIPA lysis buffer to the cell sample, incubate at 4°C for 1 hour, centrifuge at 12000 rpm for 30 minutes, add sepharose CL-4B beads to the supernatant for pre-hybridization, and incubate at 4°C for 1 hour; centrifuge to remove the beads, and add pan-MHC-A to the supernatant. B/C combined with sepharose CL-4B beads, incubated overnight (16-18 hours) at 4°C. Wash the beads with 4°C pre-cooled PBS, repeat three times; wash the beads with ultrapure water; remove the washing solution by centrifugation, and use 0.1N acetic acid to elute the antibody-MHC-I protein complex and antibody-MHC-I protein on the surface of the beads The complex is dissociated under acidic conditions, and further, the protein and peptides in the eluted product are separated and purified using a 3kDa ultrafiltration tube or a c18 solid phase extraction column (25mg, waters), and the purified product is concentrated using a refrigerated vacuum centrifuge. The concentrated product is stored at -20°C to the mass spectrometer.

2. Mass spectrometry identification of MHC-I restricted T cell epitope peptides:

The concentrated MHC-I restricted epitope peptide solution was analyzed by Q Exactive mass spectrometer (Thermo Fisher Scientific) connected to nanoflow HPLC (Thermo Fisher Scientific) online, and the ReproSil-Pur C18-AQ 1.9um filler was manually filled with a length of 15 cm, A separation column with an inner diameter of 75um was used for separation, and a linear gradient of 2-30% buffer B (80% ACN/0.5% acetic acid) was used to elute the peptides. The flow rate was set to 250 nl/min, and the elution time was 90 min. The secondary mass spectrometer uses HCD for fragmentation, and data acquisition selects the "Top 20" method on which the data depends. The acquisition resolution of MS spectrum is 70,000, 200m/z, and the target value is 3E6 ion; the top 10 ions in ion intensity are usually separated and accumulated with a maximum injection time of 120ms until the value of the automatic gain controller is displayed as 1E5. The peptide matching option is set to "disable", and the MS/MS resolution is set to 17,500 (200m/z).

3. Mass spectrometry data analysis of MHC-I restricted T cell epitope peptides:

Data analysis uses MaxQuant (version 1.3.10.15) to compare mass spectra with human whole protein library (Uniprot, 86,749 proteins), tumor-associated antigens, tumor-specific mutant peptides, and one that contains 247 common pollutants (keratin, cattle A list of maps generated from the data set of serum proteins and proteases. Variable modification detection settings: N-terminal acetylation and methionine oxidation. The second peptide identification setting: enable; specific enzyme digestion setting: unspecific; peptide identification FDR (false discovery rate) is set to 1%, protein identification FDR is not set; the sequence matching length limit is set to 8-15aa, and the maximum peptide quality is set to 1500Da, the maximum charge state is set to 3. The initial allowable mass deviation of the lead ion is set to 6 ppm, and the maximum fragment mass deviation is set to 20 ppm. The "match between runs" setting is enabled. The output of the identification result is saved in the "peptide.txt" file, the peptides matching the anti-library and the contaminated library are removed, and the rest are the identification results of the MHC-I restricted epitope.

The experimental results showed that surface mutant polypeptides, modified polypeptides and various derived peptide sequences can be expressed and presented on HLA molecules on the cell surface. Taking the mutant polypeptide SEQ ID NO: 3 as an example, the mass spectrum of the polypeptide is shown in Figure 1. The results show that the above-mentioned polypeptide can be expressed and presented on the cell surface HLA molecules.

Example 3 Verification of polypeptide T2 affinity

T2 cells are essential antigen polypeptide transporter-deficient cell strains in the endogenous antigen presentation pathway, and can be used to study the process of antigen presentation and the strength of the mutual recognition of MHC molecules.

In order to test the affinity of exogenous peptides with T2 cells, a peptide that has been confirmed to have a strong affinity with T2 cells was used as a positive control, and T2 cells without added peptides were used as a background control, and the exogenous peptides were combined with the surface MHC of T2 cells. The binding of class I molecules can increase the expression of MHC class I molecules on the surface. The more stable the combination of the two, the more MHC class I molecules can be detected. Finally, the average fluorescence intensity is used as the detection index, and the fluorescence coefficient (FI) As a measure. Based on this, the affinity between the polypeptide and T2 cells is judged. The higher the FI value, the stronger the affinity between the polypeptide and T2 cells, which is conducive to the subsequent identification of specific CD8 ⁺ T cells.

In the experiment, the synthesized polypeptide was added to 2*10 ⁵ T2 cells, and human β2 microglobulin (final concentration, 3ug/ml) was added, and cultured in a 24-well plate, in an incubator (37℃, 5%). CO ₂ ), incubate overnight. T2 cells without added peptides were used as background control, and CMV peptide (its sequence is NLVPMVATV, which is a viral peptide, is a known peptide that has been confirmed to have a strong affinity with T2 cells) as a positive control, experiment setting 2 Replicate holes and take the average value.

Take 200g of the cultured cells and centrifuge for 5 minutes to collect the cells. After the cells were washed twice with PBS, the cells were directly incubated with FITC-labeled anti-HLA typing (HLA-A*11:01) monoclonal antibody, and maintained at 4°C for 30 minutes. Then use a flow cytometer (BD FACSJazz ^TM ) and its software to detect and analyze the average fluorescence intensity, as shown in Figure 2. The T2 affinity results are shown in Table 2 below.

Table 2 T2 affinity results

Peptide sequence	FI	in conclusion
Positive control	1.63	To
TTLPTTITR	4.09	High affinity

It can be seen from Table 2 that compared with the positive control, the provided polypeptide TTLPTTITR shows a very high affinity.

Example 4 Polypeptide stimulates and expands CD8+ T cells in vitro

Take the PBMC cells of the volunteers with positive polypeptide corresponding subtypes, 2×10 ⁷ PBMC cells, and separate the monocytes by the adherence method (sticking for 3 hours), and the CD8 magnetic beads method to separate CD8+ T cells. Use GM-CSF (1000U/ml) and IL-4 (1000U/ml) to induce adherent monocytes to immature DC, and then use IFN-gamma (100U/ml), LPS (10ng/ml), and various peptides The induced mature DC cells are polypeptide-specific mature DC cells. The obtained polypeptide-specific mature DC cells ^{were co-cultured with CD8 +} T cells of volunteers, and IL-21 was added. After 3 days, IL-2 and IL-7 were supplemented. After that, IL-2 and IL-7 were supplemented once on the 5th and 7th day, and the co-cultured cells were taken for counting on the 10th day, and the follow-up ELISPOTs and LDH detection were performed.

Example 5 ELISPOTs method verifies that polypeptide activates CD8+ T cell immune response

The ELISPOT method, known as Enzyme-linked immunospot assay, can detect the cytokines secreted by a single cell. In the experiment, the culture plate is coated with specific monoclonal antibodies, and then the cells to be tested and antigen stimuli are added for culture. Under the stimulation of the stimuli, T cells secrete corresponding cytokines, and the secreted cytokines are coated Captured by the antibody on the culture plate. After washing away the cells, the captured cytokines can be combined with the fluorescently labeled secondary antibody to form spots. That is, the coated antibody can be used to capture the cytokines secreted by the cells in culture and present them in the form of enzyme-linked spots to detect and verify the strength of the immune response of the polypeptide to activate CD8+ T cells.

Refer to the description in the ELISPOTs kit instructions, combine the cultured cells in Experimental Example 4 with the experimental polypeptide (ie TTLPTTITR) and irrelevant polypeptides (referring to polypeptides that do not stimulate T cells to secrete IFN-gamma interferon, the specific sequence is LSYRNKPSI (The irrelevant polypeptides used in the following examples are also of this sequence) T2 cells were added to the ELISPOTs plate for culture, and the ELISPOTs detection was performed 20 hours later (see the kit instructions). The results of ELISPOTs are shown in Figure 3, and the results are summarized in Table 3 below:

Table 3 Peptides stimulate specific CD8+ T cells to secrete IFN-gamma interferon

Among them, the second and third columns in Table 3 respectively represent the number of spots detected using experimental peptides as stimuli or irrelevant polypeptides as stimuli, and the multiples in the fourth column represent the use of experimental peptides as stimuli and irrelevant peptides. As the ratio of the number of spots produced by the stimulus. Generally speaking, when the ratio exceeds a certain multiple (>=2), it is regarded as immunogenic, and the higher the ratio, the stronger the immunogenicity of the polypeptide.

Example 6 LDH release experiment proves the specific killing activity of ^{CD8 + T cell polypeptide}

LDH (lactate dehydrogenase) is an enzyme that exists in the cytoplasm. When the cell membrane is damaged, LDH is released into the culture medium. Since the released LDH is stable, the amount of LDH in the detection medium can be used as an indicator to determine the number of dead cells and damaged cells.

The cells cultured in Experimental Example 4 are co-cultured with T2 cells that have been loaded with experimental polypeptides or unrelated polypeptides or not loaded with polypeptides. In the experiment, the maximum release hole, volume correction hole, medium control hole, spontaneous release hole, and different targets are set in the experiment. The ratio (the ratio of the number of T cells to T2 cells) and other controls, each group set 3 replicate wells, 4h later, take out 50μl of the co-cultured cell supernatant and add it to 50ul LDH substrate mixture to make the cell supernatant catalyze LDH substrate reaction, finally read the 490nm wavelength and 680nm reference wavelength, and calculate the killing activity of target cells to kill T2 based on the control well. The results are shown in Table 4 below. The larger the value shown in Table 4, the stronger the killing effect.

The results show that CD8 ⁺ T cells stimulated by these polypeptides have polypeptide-specific killing activity.

Table 4 T cells specifically recognize and kill target cells that present experimental polypeptides

The results show that CD8 ⁺ T cells stimulated by TTLPTTITR polypeptide have polypeptide-specific killing activity.

Example 7 Establishment of mouse subcutaneous transplantation tumor model

In this example, a mouse subcutaneous xenograft tumor model was constructed. This model is used to verify the tumor control effect of the polypeptide drug combination, antigen presenting cell, and vaccine proposed in the present invention.

1. The coding gene of each polypeptide is introduced by means of lentiviral transfection, and a recombinant lentivirus expressing the aforementioned mutant polypeptide or its modification is constructed and packaged.

2. Establishment of human lung cancer cell line expressing polypeptide

The human lung cancer cell line HCC827 was purchased from ATCC (number: CRL-2868), and its HLA subtype is HLA-A*1101 positive. The cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and streptomycin. Cultivate in a 37°C, 5% CO _{2 incubator.} The packaged lentivirus was transfected into the HCC827 cell line, and Puromycin antibiotic (puromycin) was used to continuously screen the surviving HCC827 cell line, and finally the HCC827 cell line expressing the polypeptide was established.

3. Human immune reconstitution in NOD SCID mice

Collect 600 to 900 ml of anticoagulated peripheral blood from healthy volunteers. Ficoll separates peripheral blood mononuclear cells (peripheral blood mononuclear, PBMC), collects the cells for later use. Take 300 NOD SCID mice that exclude immune leakage and inject PBMC 2*10 ⁷ /0.5ml into the intraperitoneal cavity of NOD SCID mice for human immune reconstitution. Four weeks later, mice were selected to be inoculated with human lung cancer cell line models.

4. Construction of human lung cancer tumor model

The established human lung cancer cell line was cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and streptomycin. Cultivate in a 37°C, 5% CO _{2 incubator.} The tumor cells were collected, centrifuged at 3000 rpm, and the tumor cells were washed 3 times with sterile saline. Make proper dilution, take 40 microliters of cell suspension, add 10 microliters of 0.4% trypanol blue to stain and count under microscopy to make ^{a tumor cell suspension with a concentration of 1*10 8} cells/ml, and select the NOD after immune reconstitution. SCID mice, each mouse is inoculated subcutaneously with 100 ml of tumor cell suspension. After the inoculation is completed, observe daily for infection at the inoculation site, and whether the tumor has spontaneously subsided after growth. After 7 days, the mouse subcutaneous tumor can feel the tumor about the size of a grain of rice. The subcutaneous tumor model NOD/SCID mice with immune reconstitution for 4 weeks were treated with DC vaccine respectively, and the tumor volume was recorded every 3-4 days.

Example 8 Preparation and treatment plan of polypeptide vaccine

The HCC827 subcutaneous tumor model NOD/SCID mice with immune reconstitution for 4 weeks were randomly divided into 4 groups: adjuvant + wild-type peptide group (the wild-type peptide is TTLPTTISR), adjuvant + blank peptide group (that is, only containing adjuvant) , Adjuvant + mutant polypeptide group (the mutant polypeptide is TTLPTTITR), adjuvant + modified polypeptide group (which can be divided into four groups according to the different modified polypeptides used, and the modified polypeptides used are TILPTTITK, TSLPTTITK) , TTLPTTITK, TVLPTTITK), 6 in each group. The adjuvant used is Freund's adjuvant.

The first immunization dose of each group of peptides is 100ml/head. After the above-mentioned polypeptide was resuspended in PBS, mixed with 150ml/head Freund's complete adjuvant, adjusted to 300ml/head with PBS, and injected into the back subcutaneously at two points. Two weeks later, the same dose was used for booster immunization (complete Freund's adjuvant for the first time, and incomplete Freund's adjuvant for the first time) for a total of 4 immunizations. After the injection, the vital signs of the mice were observed, and the vertical and horizontal size of the tumor was measured with a vernier caliper every 3-4 days. Tumor volume was calculated as: tumor volume = 1/2 * L * W ^2. At the same time, the changes in the weight of the mice were recorded. The results are shown in Figure 4.

The results show that compared to the adjuvant+wild-type polypeptide-loaded polypeptide vaccine group and the adjuvant+blank polypeptide group, the adjuvant+mutant polypeptide or adjuvant+modified polypeptide-loaded polypeptide vaccine group can significantly slow down the growth of mouse tumors.

Example 9 Preparation and treatment plan of DC polypeptide vaccine

Collect 100 to 150ml of anticoagulated peripheral blood from healthy volunteers. Ficoll separates peripheral blood mononuclear cells (PBMC), collects PBMC cells, ^{resuspends them in RPMI 1640 medium at 2-3*10 6} /ml, and incubates at 37°C for 2 hours. Adherent cells are DCs. Aspirate Non-adherent cells are peripheral blood lymphocytes (PBL) for use. Adopt GM-CSF (1000U/ml), IL-4 (1000U/ml) to induce adherent monocytes to immature DC, then add IFN-gamma (100U/ml), CD40L (10ng/ml), and finally respectively The wild-type peptide combination and the mutant peptide combination (concentration of 10 μg/ml) were added to induce the adherent cells to become mature DC cells, and the mature DC were harvested and washed 3 times with physiological saline. The DC loaded with the polypeptide was adjusted to (4.0±0.5)*10 ⁷ /ml with physiological saline for subsequent experiments. The mice were randomly divided into 4 groups: DC-loaded wild-type polypeptide group (the wild-type polypeptide is TTLPTTISR), DC-loaded mutant polypeptide group (the mutant polypeptide is TTLPTTITR), DC-loaded modified polypeptide group (wherein according to the used The denatured peptides obtained are different and can be divided into four groups. The denatured peptides used are TILPTTITK, TSLPTTITK, TTLPTTITK, TVLPTTITK), blank peptide group (that is, no peptide group loaded), each with 6 mice. Preparation of DC-loaded wild-type polypeptide, DC-loaded mutant polypeptide, DC-loaded modified polypeptide and DC-loaded blank polypeptide polypeptide cell suspension. The mice were injected intracutaneously into the inner thighs near the groin, 0.1ml on each side, once a week. The dose is (4.0±0.5)*10 ⁶ cells/time, with a total of 2 injections. After the injection, the vital signs of the mice were observed, and the vertical and horizontal size of the tumor was measured with a vernier caliper every 3-4 days. Tumor volume was calculated as: tumor volume = 1/2 * L * W ^2. At the same time, the changes in the weight of the mice were recorded. The results are shown in Figure 5.

The results show that compared with the DC vaccine group loaded with wild-type polypeptides and the DC vaccine group loaded with blank polypeptides, the DC vaccine group loaded with mutant polypeptides or deformed polypeptides can significantly slow down the growth of mouse tumors.

Example 10 Preparation and treatment plan of polypeptide-specific DC-CTL vaccine

Example 9 The collected PBLs were sorted by magnetic beads to obtain CD8 ⁺ T cells and DCs loaded with blank polypeptides, DCs loaded with wild-type polypeptides, DCs loaded with mutant polypeptides, and DCs loaded with modified polypeptides were co-incubated and sensitized, and the ratio of cells was DC :CD8 ⁺ T cells=1:4. Add 500IU/ml IL-2 and 50ng/ml IL-7 to the _{culture medium and incubate together in a 37V 5% CO 2} incubator. After one week of culture, perform cell count; in the second week, use DC loaded with blank polypeptide and loaded with wild-type Polypeptide DCs, DCs loaded with mutant polypeptides, and DCs loaded with modified polypeptides are subjected to the second round of stimulation. Three rounds of co-stimulation, appropriate medium was added during the culture period. The number of lymphocytes was counted on the 0, 7, 14 and 21 days of culture, and the cell proliferation index (PI) was calculated. Wherein, PI=number of cells after expansion/number of cells inoculated. After 21 days of culture, cytotoxic T lymphocytes (CTL) were harvested. The cells were resuspended with saline, resuspended volume of 0.2ml, via the tail vein transfusion, each tumor model mouse cells reinfusion of about l * l0 ⁸ cells. After the injection, the vital signs of the mice were observed, and the vertical and horizontal size of the tumor was measured with a vernier caliper every 3-4 days. Tumor volume was calculated as: tumor volume = 1/2 * L * W ^2. At the same time, the changes in the weight of the mice were recorded. The results are shown in Figure 6.

The results show that, compared with the blank polypeptide control group and the wild-type polypeptide group, the DC-CTL vaccine activated by the mutant polypeptide or the deformed polypeptide can significantly slow down the growth of mouse tumors.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structure, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above-mentioned terms are not necessarily directed to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned embodiments within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims (15)

1. A set of isolated polypeptides, characterized in that the polypeptides include at least any one polypeptide in the first peptide group, and can optionally include at least any one polypeptide in the second peptide group;

   The first peptide group includes polypeptides having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5;

   The second peptide group includes derivative peptides of the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5. The derivative peptide includes a pre-peptide segment, a mid-peptide segment, and a post-peptide segment that are sequentially connected, and the mid-peptide The segment has at least 80% homology with the amino acid sequence shown in SEQ ID NO:1～SEQ ID NO:5, and the sum of the length of the pro-peptide segment and the back-peptide segment is 14-16 amino acids .
2. The polypeptide of claim 1, wherein the mid-peptide segment has at least 90% homology with the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5.
3. The polypeptide of claim 1, wherein the mid-peptide segment is the same as the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5.
4. The polypeptide of claim 1, wherein the derivative peptide has an amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 10.
5. The polypeptide of claim 1, wherein the polypeptide is selected from at least one of the following:

   (1) At least two polypeptides having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5;

   (2) At least one polypeptide having the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5, and at least one polypeptide having the amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 10.
6. An isolated nucleic acid, characterized in that the nucleic acid encodes the polypeptide of any one of claims 1 to 5 or its complementary sequence.
7. A construct, characterized in that the construct comprises the nucleic acid according to claim 6 and a control sequence, and the control sequence is operably linked to the nucleic acid.
8. An expression vector, characterized in that the expression vector comprises the construct of claim 7.
9. A host cell, characterized in that the host cell carries the construct according to claim 7 or the expression vector according to claim 8.
10. Use of the polypeptide according to any one of claims 1 to 5 in the preparation of drugs for preventing or treating tumors or in the preparation of kits for diagnosing tumors.
11. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the polypeptide according to any one of claims 1 to 5 and pharmaceutically usable excipients.
12. An antigen-presenting cell, characterized in that the antigen-presenting cell presents the polypeptide according to any one of claims 1 to 5.
13. An immune effector cell, characterized in that the immune effector cell can recognize the polypeptide of any one of claims 1 to 5 or recognize the antigen-presenting cell of claim 12.
14. A tumor vaccine, characterized in that the tumor vaccine comprises the polypeptide according to any one of claims 1 to 5, or the nucleic acid according to claim 6, or the construct according to claim 7, or The expression vector according to claim 8, or the host cell according to claim 9, or the antigen presenting cell according to claim 12, or the immune effector cell according to claim 13.
15. A method for treating a tumor patient, characterized in that it comprises administering an effective amount of a pharmaceutical composition or an effective amount of a tumor vaccine to the patient, the pharmaceutical composition being the pharmaceutical composition according to claim 11, and the vaccine is The tumor vaccine of claim 14.

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