TCR tumor associated antigen for pancreatic cancer and application thereof

Abstract

The invention relates to the technical field of tumor immunity, and discloses a TCR tumor-associated antigen for pancreatic cancer and application of the TCR tumor-associated antigen. The invention finds that DDX18 is highly expressed in pancreatic cancer, affects dryness, occurrence and development of pancreatic cancer, and regulates immune response. The antigen immunity of DDX18 is preliminarily verified on the basis of the mouse DDX18 gene, it is indicated that DDX18 can be used as a new tumor-associated antigen, and a new thought is provided for TCR-T treatment; according to the invention, the affinity of DDX18 and MHC is predicted by using a computer, and the actual affinity of the peptide fragment is detected and predicted by using ELISA. AAV-DDX18 adeno-associated virus is constructed by referring to ACTL treatment, killer T cells are obtained by adopting an in-vitro antigen presentation method, antigen-specific killer T cells are detected by adopting a tetramer technology, the antigen immunity of DDX18 is verified, and DDX18 can be used as a tumor-associated target antigen in TCR-T treatment.

Description

Technical Field

[0001] The present invention relates to the field of tumor immunology, and in particular to a TCR tumor-associated antigen for pancreatic cancer and its applications. Background Art

[0002] At present, tumor immunotherapy has developed rapidly and has become the fourth major treatment pillar after surgery, radiotherapy, and chemotherapy. T cell receptor-engineered T cell (TCR-T) therapy is one of the most effective ways of current tumor immunocyte therapy. In recent years, the research on TCR-T for the treatment of various solid tumors has increased significantly. In August 2024, the TCR-T therapy Afami-cel for synovial sarcoma developed by Adaptimmune Therapeutics was rapidly approved by the US Food and Drug Administration, which is the world's first approved TCR-T cell therapy. T cell (antigen) receptor (TCR) is a heterodimeric protein located on the surface of T cells and is a molecule that specifically recognizes antigens on the surface of T cells and mediates immune responses.

[0003] The basis for TCR-T cells to recognize tumor cells is to recognize pMHC generated after antigen processing. Therefore, the specificity of the antigen determines the accuracy of TCR-T cell killing. The recognition of tumor antigens is an important determinant of TCR-T cell therapy. An ideal TCR-T cell target antigen is a tumor-specific antigen (TSA). Such antigens only exist in tumor cells and are not expressed in normal cells and tissues. TSA is mainly a product of gene mutation. Theoretically, TSA is an ideal target for TCR-T cell therapy. The tumor specificity of TSA means that there is no immune self-tolerance, and the immune response against TSA will not damage normal tissues. However, there are some problems in the search for TCR studies targeting TSA. On the one hand, the immunogenicity of antigen peptides generated by gene mutation is relatively weak, and it is difficult to isolate high-affinity TCRs; on the other hand, TSA is often specific to individual tumors, or even specific to a certain time of an individual tumor. Targeting such antigens requires a very individualized TCR-T cell preparation process, which is currently difficult to apply in clinical treatment. The above reasons have greatly limited the application of the strategy of targeting TSA. Therefore, a class of protein molecules called tumor associated antigen (TAA) is the main antigen target in current TCR-T cell research.

[0004] TAAs mainly refer to antigen molecules present on normal cells or tumor cells, but they are not unique to tumor cells. Normal cells can synthesize them in small amounts, and they are usually highly expressed during tumor cell proliferation. These tumor-specific polypeptide-MHC (pMHC) complexes can be recognized by T cells and trigger an anti-tumor immune response in patients. So far, the peptide antigen targets for effective and safe immunotherapy using TCR-T in humans are still very limited. Most of the current targets are TAAs, which, although upregulated in tumor tissues, still maintain low levels of expression in normal tissues, which may lead to autoimmune toxicity. Therefore, neoantigens seem to be the safest targets for TCR-T cancer therapy. However, the main challenges in developing neoantigens for TCR-T clinical applications include: (1) Neoantigen-forming mutations are largely individualized and vary among cancer patients, making it difficult to develop immunotherapy products for widespread use; (2) The expression of neoantigens in tumor tissues is often heterogeneous. Therefore, there is an urgent need for more effective TCR-T antigens. Summary of the Invention

[0005] Selecting an ideal antigen is the key to improving anti-tumor efficiency and reducing related toxicity. Tumor specificity and immunogenicity are the primary considerations in antigen selection. Generally, human tumor antigens are mainly divided into two categories: tumor-associated antigens and tumor-specific antigens. Surface antigens are usually TAAs, and normal tissues can also express these antigens to affect functions, including cancer-testis antigens, overexpressed antigens, and differentiation antigens. Nearly 90% of solid tumor targeting depends on TSA, which can be divided into neoantigens and oncoviral antigens. Although TCR-T can target all tumor antigens, the diversity of tumor antigens often causes "on-target, off-tumor" toxicity. So far, the number of targets with sufficient safety and effectiveness that have been identified is still limited. Target antigens that are highly expressed in tumors but lowly expressed in normal tissues are usually selected to limit potential off-target effects. To make up for the deficiencies of the prior art, the present invention provides a TCR tumor-associated antigen for pancreatic cancer and its applications.

[0006] The present invention is achieved by the following technical solutions: In a first aspect, the present invention provides a TCR tumor-associated antigen for pancreatic cancer, and this antigen is the DDX18 protein. The peptide segments with strong binding ability of the DDX18 protein are any one of KVPPFVDL, TSFASLSNL, VKYHYELL, RAYDSHSL, and QIFNVNNL.

[0007] In a second aspect, the present invention provides the use of the TCR tumor-associated antigen DDX18 protein in constructing an AAV-DDX18 recombinant adeno-associated virus. After constructing the AAV-DDX18 recombinant adeno-associated virus, mouse dendritic cells are transfected and used to stimulate T lymphocytes in vitro to generate cytotoxic T lymphocytes that can effectively kill tumor cells.

[0008] In a third aspect, the present invention provides the use of the TCR tumor-associated antigen DDX18 protein in preparing immunological reagents. Five peptide segments, namely KVPPFVDL, TSFASLSNL, VKYHYELL, RAYDSHSL, and QIFNVNNL, are used to synthesize their respective tetramers. The tetramers are labeled with PE fluorescence, and the tetramer detection technique is used to detect antigen-specific killer T cells against DDX18 by flow cytometry.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention discovers that DDX18 is highly expressed in pancreatic cancer, affects the stemness, occurrence, and development of pancreatic cancer, and regulates the immune response. Based on the murine DDX18 gene, the present invention initially verifies the antigen immunity of DDX18, indicating that it can be used as a new tumor-associated antigen, providing a new idea for TCR-T therapy; 2. The present invention uses a computer to predict the affinity between DDX18 and MHC, and uses ELISA to detect the actual affinity of the predicted peptide segments. Referring to ACTL therapy, an AAV-DDX18 adeno-associated virus is constructed, and cytotoxic T cells are obtained by an in vitro antigen presentation method. The tetramer technique is used to detect antigen-specific killer T cells, verifying the antigen immunity of DDX18, which may be used as a tumor-associated target antigen in TCR-T therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The present invention will be further described below with reference to the accompanying drawings.

[0011] Figure 1 To analyze the expression of DDX18 in normal and cancerous pancreatic tissues through the TCGA and GTEx databases; Figure 2 To analyze the level of DDX18 during the survival period of PDAC patients. Figure 2 In [A], the overall survival rate of a tissue microarray using 90 pairs of paraffin-embedded PDAC and matched normal tissues is shown. The ( Figure 2 In [B]) disease-free survival and ( Figure 2 In [C]) overall survival of 45 pairs of PDAC patient samples in the database; Figure 3 Are organoids from two KPC mice, where Figure 3 In [A] and [B], [T] shows pictures of tumor tissues; Figure 3The pictures of N in C and D are adjacent tissues; Figure 4 For detecting the protein level of DDX18 in organoids by Western blot; Figure 5 For calculating and predicting the binding affinity between MHC of KPC mice and DDX18 polypeptide; Figure 6 For the computer-predicted affinity results; Figure 7 For the ELISA detection results; among which Figure 7 A and B in it are the detection result graph and the detection result bar graph respectively; Figure 8 For detecting the transfection of DC cells with AV-DDX18 recombinant adeno-associated virus by GFP fluorescence, among which Figure 8 A in it is the control group, Figure 8 B in it is the experimental group; Figure 9 For detecting the protein level of DDX18 in transfected DC cells by Western blot; Figure 10 For the control group using the tetramer detection technique to flow detect antigen-specific cytotoxic T cells against DDX18; among which Figure 10 A and B in it are the lymphocyte population and the CD3 T cell population respectively, Figure 10 C and D in it are to stain with tetramer (pe fluorescence), circle out specific CTL cells, and verify DDX18 photoreceptor-specific TCR T cells; Figure 11 For the experimental group using the tetramer detection technique to flow detect antigen-specific cytotoxic T cells against DDX18; among which Figure 11 A and B in it are the lymphocyte population and the CD3 T cell population respectively, Figure 11 C and D in it are to stain with tetramer (pe fluorescence), circle out specific CTL cells, and verify DDX18 photoreceptor-specific TCR T cells. Specific implementation manner

[0012] The present invention will be further described below in conjunction with the accompanying drawings.

[0013] Example 1 In this example, it is found that DDX18 is highly expressed in pancreatic cancer, lowly expressed in normal tissues, and affects the prognosis of patients.

[0014] For the database result analysis, see Figure 1 、 Figure 2 。

[0015] Example 2 The laboratory KPC mouse model is a transgenic mouse model used to study pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDAC). KPC represents two key gene mutations: Kras and P53. KPC mice carry a pancreas-specifically activated mutant Kras gene (usually Kras^G12D) and an inactivated P53 gene (usually P53^R172H or P53^R270H). These gene mutations are common in many pancreatic cancer patients, so KPC mice have become an important model for studying the mechanisms of pancreatic cancer occurrence and development.

[0016] In this invention, fresh tissues were extracted from the KPC pancreatic cancer mouse model, and organoids of tumors and their adjacent tissues were successfully constructed. Western Blot experiments verified that the protein level of DDX18 was higher in organoids derived from tumors. This invention verified the high expression of DDX18 in the organoid model of KPC mice (a spontaneous mouse pancreatic cancer model), as shown in Figure 3 、 Figure 4 。

[0017] Example 3 Prediction of antigen affinity: For neoantigens processed and presented by MHC molecules, many online prediction tools have been created, including NetChop, NetCTL, and NetCTLpan. In this example, the binding affinity between MHC of KPC mice and DDX18 polypeptide was calculated and predicted. Six peptide segments with strong binding affinity to mouse MHC H2-Kb were selected, as shown in Figure 5 ,The computer prediction affinity results are shown in Figure 6 。

[0018] Computer prediction: DDX18 peptide segments with strong binding affinity to mouse H2-Kb.

[0019] Detection of antigen affinity: In this example, ELISA was used to detect the predicted peptide segments with strong binding affinity to verify their actual affinity. The results showed that 5 of these peptide segments had strong binding affinity to mouse H2-Kb, which was basically the same as the positive reference. As shown in Figure 7 They are KVPPFVDL, TSFASLSNL, VKYHYELL, RAYDSHSL, and QIFNVNNL respectively.

[0020] Example 4 Antigen immunogenicity detection: In this example, in vitro antigen immunogenicity verification was carried out with reference to the ACTL therapy. The ACTL tumor cell-targeted therapy technology is to reconstruct the non-pathogenic wild-type adeno-associated virus (AAV) into a recombinant adeno-associated virus carrying a specific tumor-associated antigen determinant gene through genetic recombination technology, infect the patient's peripheral blood mononuclear cells, and through cytokine induction, the mononuclear cells are transformed into dendritic cells (DC cells) with powerful antigen presentation functions. The dendritic cells (DC cells) obtained by this technology stimulate the patient's T lymphocytes in vitro to produce cytotoxic T lymphocytes (CTLs) that can effectively kill tumor cells. The CTLs produced have tumor antigen specificity, that is, targeting.

[0021] According to the above process, in this example, the AAV-DDX18 recombinant adeno-associated virus was constructed: the foreign gene DDX18 was cloned into the viral vector (the vector carries the GFP tag). The DDX18 recombinant expression plasmid was co-transfected into AAV-293 cells together with pHelper (carrying genes derived from adenovirus) and pAAV-RC (carrying AAV replication and capsid genes). After 2 to 3 days of transfection, the recombinant AAV was assembled in the packaging cells. The AAV virus particles were collected from the infected AAV-293 cells, concentrated and purified to obtain the AAV-DDX18 recombinant adeno-associated virus, which was used to transfect mouse dendritic cells (DC cells) (induced differentiation in vitro). The transfection effect was obvious. Compared with the control group, there was obvious GFP fluorescence in the experimental group, and the Western Blot experiment verified the high expression of the DDX18 protein in the experimental group. In this example, mouse DC cells with high expression of the DDX18 gene were constructed.

[0022] The transfection of AAV-DDX18 recombinant adeno-associated virus into DC cells is shown in Figure 8 , and the Westernblot detection of the DDX18 protein level after transfection of DC cells is shown in Figure 9 .

[0023] In vitro antigen presentation: Using the above DC cells with high expression of DDX18, mouse T lymphocytes were stimulated in vitro to produce cytotoxic T lymphocytes (CTLs) that can effectively kill tumor cells.

[0024] Example 5 MHC Tetramer is a type of immunological reagent, which consists of four monomeric molecules formed by the binding of the Major Histocompatibility Complex (MHC) to antigenic peptides and is labeled with fluorescence. It is a rapid and simple method for qualitative and quantitative analysis of antigen-specific T cells. For the above-mentioned 5 peptide segments (KVPPFVDL, TSFASLSNL, VKYHYELL, RAYDSHSL, QIFNVNNL) with strong binding affinity to the mouse MHC locus (H2-Kb locus), their respective tetramers (the tetramers are labeled with PE fluorescence) were synthesized. The tetramer detection technique was used to detect antigen-specific cytotoxic T cells against DDX18 by flow cytometry. Compared with the control group, the experimental group significantly produced cytotoxic T lymphocytes (CTLs) that killed tumor cells targeting the DDX18 target, as shown in Figure 10 - 11 .

Claims

1. A TCR tumor-associated antigen for pancreatic cancer, characterized in that: The antigen is DDX18 protein, and the strong binding peptide segment of DDX18 protein is any one of KVPPFVDL, TSFASLSNL, VKYHYELL, RAYDSHSL, and QIFNVNNL.

2. Use of the TCR tumor-associated antigen DDX18 protein as described in claim 1 in constructing AAV-DDX18 recombinant adeno-associated virus.

3. Use of the TCR tumor-associated antigen DDX18 protein as claimed in claim 1 in the preparation of immunological reagents, characterized in that: Five peptides, KVPPFVDL, TSFASLSNL, VKYHYELL, RAYDSHSL, and QIFNVNNL, were used to synthesize their respective tetramers, which were labeled with PE fluorescence. The tetramer detection technology was used to flow cytometry detect antigen-specific killing T cells against DDX18.

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