WO2022012292A1 - Peripheral blood tcr marker for pancreatic cancer, and detection kit and use thereof - Google Patents

Abstract

Provided are a peripheral blood TCR marker for pancreatic cancer, and a detection kit and the use thereof. The marker comprises at least one protein, the sequences of which are shown as SEQ ID NO. 1-100. The method is performed on the basis of a high-throughput sequencing method, wherein only a small amount of peripheral blood needs to be collected to extract RNA; a sample is treated to establish an immune map library; high-throughput sequencing and TCR data analysis are carried out to determine a characteristic TCR sequence in peripheral blood of pancreatic cancer; and the test result of a sample to be tested is then compared with the characteristic TCR sequence so as to determine whether pancreatic cancer is present or not.

Description

A peripheral blood TCR marker of pancreatic cancer and its detection kit and application technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a peripheral blood TCR marker of pancreatic cancer and a detection kit and application thereof.

Background technique

Pancreatic cancer is a malignant tumor of the digestive tract with a high degree of malignancy, which is difficult to diagnose and treat. About 90% of them originate from ductal adenocarcinoma of the glandular epithelium. The incidence of pancreatic cancer varies by region and averages about 5.1 per 100,000 people. Males have a higher incidence than females, with a ratio of about 1.5-2:1. The overall incidence has shown an upward trend in recent years. The latest statistics from the National Cancer Center of China show that pancreatic cancer ranks the 8th in the incidence of malignant tumors in Chinese urban males, and the 5th in the mortality rate of malignant tumors in Beijing and Shanghai populations.

The cause of pancreatic cancer is not well understood. Its occurrence is related to smoking, alcohol consumption, high-fat and high-protein diet, excessive coffee consumption, environmental pollution and genetic factors; recent survey reports have found that the incidence of pancreatic cancer in people with diabetes is significantly higher than that in the general population; There is a certain relationship between patients with pancreatitis and the incidence of pancreatic cancer. It is found that the proportion of patients with chronic pancreatitis has a significantly higher incidence of pancreatic cancer. In addition, there are many factors that are related to the occurrence of this disease, such as occupation, environment, and geography. Common clinical manifestations of pancreatic cancer are as follows:

1. Abdominal pain: Pain is the main symptom of pancreatic cancer, regardless of whether the cancer is located in the head or tail of the pancreas. In addition to pain in the middle abdomen, left upper quadrant, and right upper quadrant, a few cases complained of pain in the left and right lower quadrants, around the umbilicus or in the entire abdomen, and even testicular pain, which is easily confused with other diseases. When the cancer involves the visceral capsule, peritoneum, or retroperitoneal tissue, there may be tenderness at the corresponding site.

2. Jaundice: Jaundice is an important symptom of pancreatic cancer, especially pancreatic head cancer. Jaundice is obstructive, accompanied by dark yellow urine and clay-like stool, which is caused by the invasion or compression of the lower end of the common bile duct.

3. Gastrointestinal symptoms: the most common is loss of appetite, followed by nausea, vomiting, diarrhea or constipation or even black stools. Diarrhea is often steatorrhea.

4. Weight loss and fatigue: Different from other cancers, pancreatic cancer often has weight loss and fatigue in the early stage.

5. Abdominal mass: The pancreas is deep and difficult to touch in the posterior abdomen. The abdominal mass is the result of the development of the cancer itself and is located at the location of the lesion. If the mass is touched, it is mostly in the advanced or advanced stage. Chronic pancreatitis can also feel the mass, and pancreatic cancer is not easy to distinguish.

6. Symptomatic diabetes: A small number of patients initially manifested as symptoms of diabetes, that is, before the main symptoms of pancreatic cancer such as abdominal pain and jaundice appeared, they first developed diabetes, and the accompanying emaciation and weight loss were mistaken for diabetes. performance without considering pancreatic cancer.

7. Thrombophlebitis: migratory thrombophlebitis or arterial thrombosis occurs in patients with advanced pancreatic cancer.

8. Mental symptoms: Some patients with pancreatic cancer may present with mental symptoms such as anxiety, irritability, depression, and personality changes.

9. Ascites: generally appears in the late stage of pancreatic cancer, mostly caused by peritoneal infiltration and spread of cancer.

10. Others: In addition, patients often complain of fever and obvious fatigue. There may be high fever or even chills and other symptoms similar to cholangitis, so it is easy to be confused with cholelithiasis and cholangitis.

The clinical characteristics of pancreatic cancer are the short duration of the disease, the difficulty of early diagnosis and easy confusion with other diseases, and the rapid development and rapid deterioration of the disease in the middle and late stages. Therefore, pancreatic cancer is very difficult to treat and has a very poor prognosis. Because of the difficulty of early diagnosis, only 10% to 15% of patients have the chance of surgical resection. The average survival for pancreatic cancer is 2-3 months after diagnosis, with a one-year survival rate of 8% and a five-year survival rate of only 3%. Therefore, there is an urgent need to find effective diagnostic and therapeutic methods. At present, the diagnostic methods for pancreatic cancer are mainly divided into four categories: biochemical and molecular biological testing, genetic testing, imaging testing and pathological testing.

1. Biochemical and molecular biology testing:

1) Carbohydrate antigen CA19-9 is the most commonly used diagnostic marker for pancreatic cancer. It has the following clinical characteristics: taking serum CA19-9>37U/ml as a positive indicator, the sensitivity and specificity for the diagnosis of pancreatic cancer are 78.2% and 78.2%, respectively. 82.8%. About 10% of pancreatic cancer patients are Lewis antigen negative and CA19-9 is not elevated. At this time, it is necessary to combine other tumor markers such as CA125 and/or carcinoembryonic antigen (CarcinoEmbryonic Antigen, CEA) and other auxiliary diagnosis. For patients with elevated CA19-9, pancreatic cancer should be highly suspected after excluding factors such as biliary obstruction or biliary infection.

2) Monitoring of blood glucose changes: a) New-onset diabetes patients who are elderly, with low body mass index and no family history of diabetes should be alert to the occurrence of pancreatic cancer; b) Those who have suffered from diabetes for a long time in the past and have short-term blood sugar fluctuations that are difficult to control should also be alerted Occurrence of pancreatic cancer; c) Prospective study results show that for every 0.56 mmol/L increase in fasting blood glucose, the risk of pancreatic cancer increases by 14%; 2) Other biomarkers: peripheral blood microRNA, ctDNA, exosome Glypican- Level 1 also has potential clinical application prospects, but clinical application has yet to be confirmed by high-level evidence-based medical evidence.

2. Genetic testing: CDKN2A, BRCA1/2, PALB2 and other gene mutations have been confirmed to be closely related to the incidence of familial pancreatic cancer. But this kind of genetic testing can only assess cancer risk, not as a diagnostic standard.

3. Imaging examination

1) Enhanced three-dimensional dynamic CT thin-slice scanning: the most commonly used method for diagnosing pancreatic cancer at present, it can clearly display the size, location, density and blood supply of the tumor, and judge the tumor and blood vessels accordingly [Computed Tomography (Computed Tomography) is used when necessary] Angiography, CTA) examination], the adjacency relationship of adjacent organs, to guide the preoperative tumor resectability and the evaluation of the effect of neoadjuvant chemotherapy.

2) Magnetic resonance imaging (MRI): in addition to showing the anatomical features of pancreatic tumors, it can also clearly show the presence or absence of metastatic lesions in the lymph nodes adjacent to the pancreas and in the liver; and it is superior to CT in distinguishing from edematous or chronic mass pancreatitis Check. Magnetic resonance cholangiopancreatography (MRCP) combined with MRI thin-slice dynamic enhancement can help to identify cystic and solid pancreatic lesions (especially the differential diagnosis of cystadenoma and intraductal papillary mucinous tumor of the pancreas) , and to further clarify the expansion and invasion of the pancreatic duct and bile duct, the diagnostic value is higher.

3) Positron Emission Tomography (PET)-CT examination: It can display the metabolic activity and metabolic load of the tumor, and has obvious advantages in detecting extrapancreatic metastasis and evaluating the tumor load in the whole body.

4) Endoscopic UltraSonography (EUS): On the basis of endoscopic technology, ultrasound imaging is combined to improve the sensitivity and specificity of pancreatic cancer diagnosis; especially EUS-guided fine needle aspiration biopsy (EUS- FNA), has become the most accurate method for localization and qualitative diagnosis of pancreatic cancer. In addition, EUS is also helpful in judging tumor staging. The sensitivity and specificity for diagnosing T1-2 pancreatic cancer are 72% and 90%, respectively, and the sensitivity and specificity for diagnosing T3-4 pancreatic cancer are 90% and 72%, respectively.

4. Pathological examination: Histopathological and/or cytological examination is the "gold standard" for the diagnosis of pancreatic cancer. Except for the patients who plan to undergo surgical resection, the other patients should strive to make a clear pathological diagnosis before formulating a treatment plan. Current methods for obtaining histopathology or cytology specimens include:

1) EUS or CT-guided needle biopsy; 2) Ascites exfoliated cytology; 3) Exploratory biopsy under laparoscopic or open surgery.

However, the above existing detection methods have their limitations. For example, biochemical and molecular biological testing, due to the small number of markers, it is difficult to ensure specificity and sensitivity at the same time; imaging testing is expensive; pathological testing requires puncture or ascites extraction, and the patient suffers greatly. Moreover, all existing detection methods are difficult to distinguish early pancreatic cancer from other types of diseases, and patients often miss the opportunity of early diagnosis. Therefore, a simple and easy diagnostic method for pancreatic cancer with higher sensitivity and specificity is urgently needed.

SUMMARY OF THE INVENTION

In view of the above deficiencies in the prior art, the present invention provides a peripheral blood TCR marker for pancreatic cancer and a detection kit and application thereof, which can accurately and quickly determine whether there is a patient with a higher risk of pancreatic cancer in the sample to be tested.

For realizing the above-mentioned purpose, the technical scheme that the present invention solves its technical problem adopts is:

A peripheral blood TCR marker of pancreatic cancer, the marker comprises at least one of the proteins whose sequence is shown in SEQ ID NO.1～100, and the specific sequence is shown in Table 1.

Table 1 Marker sequences

serial number protein sequence SEQ ID NO.1 Ala Ser Ser Ala Pro Arg Thr Gly Asp Pro Gly Glu Leu Phe SEQ ID NO.2 Ala Ser Ser Gln Asp Val Leu Tyr SEQ ID NO.3 Ala Ser Ser Gln Asp Gly Thr Ala Asp Leu Tyr Gln Glu Thr Gln Tyr SEQ ID NO.4 Ala Ser Ser Ser Asp Arg Arg Arg Asp Tyr Glu Gln Tyr SEQ ID NO.5 Ala Ser Ser Leu Glu Tyr Ser Trp Ser Ser Tyr Asn Glu Gln Phe SEQ ID NO.6 Ala Ser Ser Leu Phe Gly Gly Met Asn Thr Gln Tyr SEQ ID NO.7 Ala Ile Arg Ser Gly Gln Asp Asn Gly Tyr Thr SEQ ID NO.8 Ala Ser Ser Leu Val Gly Thr Gly Glu Gly Asp Glu Gln Phe SEQ ID NO.9 Leu Val Val Cys Pro Pro Ser Trp Arg Pro Ser Thr SEQ ID NO.10 Ala Ser Arg Asp Trp Gly Gly Thr Ser Asp Asn Glu Gln Phe SEQ ID NO.11 Ala Ser Ser Pro Asn Asp Pro Ser Arg Thr Lys Asp Trp Gly Glu Gln Tyr SEQ ID NO.12 Ala Ser Ser Leu Pro Gly Leu Tyr Tyr Asn Glu Gln Phe SEQ ID NO.13 Ala Ser Ser Arg Asp Arg Gly Lys Gln Glu Thr Gln Tyr SEQ ID NO.14 Ala Ser Ser Leu Asp Lys Leu Ala Gly Pro Thr Tyr Tyr Glu Gln Tyr SEQ ID NO.15 Ala Ser Ser Pro Asn Thr Gly Gly Ile Asn Gln Pro Gln His SEQ ID NO.16 Ala Ser Ser Pro Arg Gln Gly Ile Lys Thr Gln Tyr SEQ ID NO.17 Ala Ser Ser Pro Leu Ile Gly Thr Asp Ile Thr Asp Asp Gly Tyr Thr SEQ ID NO.18 Ala Ser Val Thr Gly Ala Val Tyr Glu Gln Tyr SEQ ID NO.19 Ala Ser Ser Pro Asp Arg Gly Lys Thr Glu Thr Gln Tyr SEQ ID NO.20 Ala Ser Ser Ala Ser Gly Pro Ala Gly Asn Thr Ile Tyr SEQ ID NO.21 Ala Ser Ser Pro Lys Arg Gln Gly Arg Asn Gln Pro Gln His SEQ ID NO.22 Ala Ser Gly Leu Val Leu Gly Gln Ala Tyr Gly Tyr Thr

SEQ ID NO.23 Ala Ser Arg Ser Arg Gly Thr Asn Tyr Asn Glu Gln Phe SEQ ID NO.24 Ala Ser Ser Arg Asn Pro Gly Pro Tyr Glu Gln Tyr SEQ ID NO.25 Ala Ser ArgLeu Asp Arg Gly Lys Gly Thr Glu Ala Phe SEQ ID NO.26 Ala Ser Ser Leu Val Pro Thr Ala Glu Gln Tyr SEQ ID NO.27 Ala Ser Ser Phe Ser Gly Gly Ser Gln Ser Thr Asp Thr Gln Tyr SEQ ID NO.28 Ala Ser Lys Val Glu Gly Ile Thr Glu Ala Phe SEQ ID NO.29 Gln Gln Phe Ile Gln Gly Cys Gly Asp Pro Val SEQ ID NO.30 Ser Thr Asp Glu Arg Gly Phe SEQ ID NO.31 Ala Ser Ser Ala Pro Gly Thr Gly Trp His Glu Gln Phe SEQ ID NO.32 Ala Ser Ser Leu Leu Gln Thr Gly Gly Leu Asp Glu Gln Tyr SEQ ID NO.33 Ala Ser Ser Leu Val Gln Gly Thr Leu Ser Tyr Glu Gln Tyr SEQ ID NO.34 Ala Ser Ser Pro Gln Pro Ala Val Lys Thr Gln Tyr SEQ ID NO.35 Ala Ser Ser Pro Asn Asp Arg Val His Thr Ile Tyr SEQ ID NO.36 Ala Ser Ser Leu Val Gly Leu Ala Gly Gly Ala Gln Tyr SEQ ID NO.37 Ser Ala Arg Val Arg Glu Ala Asn Glu Gln Phe SEQ ID NO.38 Ala Ser Ser Leu Leu Gly Phe Ser His Tyr Gly Tyr Thr SEQ ID NO.39 Ala Ser Ser Leu Leu Thr Gly Gly Ala Gly Ile Tyr Asn Glu Gln Phe SEQ ID NO.40 Ala Ser Ser Ser Gln Ala Gly Val Asp Lys Thr Gln Tyr SEQ ID NO.41 Ala Ser Thr Thr Asp Gly Arg Glu Thr Gln Tyr SEQ ID NO.42 Ala Ser Lys Ser Trp Thr Ser Gln Glu Thr Gln Tyr SEQ ID NO.43 Ala Ser Ser Leu Asn Ser Gly Gln Gly Ala Glu Thr Gln Tyr SEQ ID NO.44 Ala Ser Ser His Trp Ala Gly Lys Ser Gly Asn Thr Ile Tyr SEQ ID NO.45 Ala Ser Ser Leu Glu Gly Gln Arg Glu Pro Gln His SEQ ID NO.46 Ala Ser Ser Pro Ser Thr Thr Gly Asp Thr Glu Ala Phe SEQ ID NO.47 Gln Gln Phe Arg Pro Lys Thr Arg Leu Arg Ala Val SEQ ID NO.48 Ala Ser Ser Leu Tyr Pro Gly Gln Gly Lys Ala Phe SEQ ID NO.49 Ala Ser Ser Leu Val Gln Gly Ile Glu Glu Gln Tyr SEQ ID NO.50 Ala Ser Ser Ser Gly Gln Asp Tyr Gly Gln Tyr SEQ ID NO.51 Ala Ser Ser Glu Ser Arg Thr Gly Leu Asn Thr Ile Tyr SEQ ID NO.52 Ala Ser Ser Leu Leu Gly Leu Glu Arg Asp Thr Gln Tyr SEQ ID NO.53 Ala Ser Ser Leu Leu Arg Pro Gly Ser Ser Glu Gln Tyr SEQ ID NO.54 Ala Ser Ser Leu Ser Phe Gly Ala Thr Gly Asn Gly Gln Glu Thr Gln Tyr SEQ ID NO.55 Ala Ser Ser Pro Leu Leu Arg Asp Tyr Asn Glu Gln Phe SEQ ID NO.56 Ala Gln Gly Gly Arg Gly Ser Tyr Gly Tyr Thr SEQ ID NO.57 Ala Ser Thr Gln Asp Trp Gly Thr Gly Glu Leu Phe SEQ ID NO.58 Ser Ala Leu Arg Arg Gly Gln Arg Asn Ser Pro Leu His SEQ ID NO.59 Ala Ser Ser Leu Glu Ser Ala Thr Gln Ala Phe SEQ ID NO.60 Ala Ser Ser Lys Leu Thr Pro Thr Glu Pro Gln His SEQ ID NO.61 Ala Ser Ser Val Ser Pro Asp Pro Ser Gly Asn Thr Ile Tyr

SEQ ID NO.62 Ala Ser Thr Phe Ser Gly Val Glu Gln Phe SEQ ID NO.63 Ala Ser Ser Ser Gly Gln Asp Tyr Glu Arg Tyr SEQ ID NO.64 Ala Ser Ser Asp Arg Ser Gly Tyr Pro Tyr Glu Gln Tyr SEQ ID NO.65 Ala Ser Ser Asn Arg Asp Arg Asp Ser Ser Glu Gln Tyr SEQ ID NO.66 Ala Ser Ser Leu Glu Gly Thr Ser Gly Arg Asn Phe Asn Thr Gln Tyr SEQ ID NO.67 Ala Ser Ser Leu Ala Leu Thr Gly Ser His Pro Glu Ser SEQ ID NO.68 Ala Ser Ser Tyr Ser Val Thr Gly Ile Leu Thr Gly Glu Leu Phe SEQ ID NO.69 Ala Ser Ser Ser Gly Gln Asp Tyr Glu Gln Cys SEQ ID NO.70 Ala Ser Ser Pro Ala Gly Ala Ser Gly Thr Glu Gln Phe SEQ ID NO.71 Ala Ser Ser Pro Gly Pro Lys Ser Arg Tyr Glu Gln Tyr SEQ ID NO.72 Ser Ala Arg Asp Gln Ala Thr Leu Val Asp Glu Gln Tyr SEQ ID NO.73 Ala Ser Arg Arg Asp Gly Glu Gly Tyr Thr SEQ ID NO.74 Ala Ser Ser Leu Gly Thr Met Tyr Thr Glu Ala Phe SEQ ID NO.75 Ala Ser Ile Arg Ser Gly Val Arg Tyr Asn Glu Gln Phe SEQ ID NO.76 Ala Ser Ser Arg Pro Arg Ala Ser Gly Ser Tyr Glu Gln Tyr SEQ ID NO.77 Ala Ser Ser Asp Ala Pro Gly Gly Ala Leu Trp Glu Gln Tyr SEQ ID NO.78 Ala Ser Ser Ser Gln Arg Glu Lys Glu Thr Gln Tyr SEQ ID NO.79 Ala Ser Ser Leu Val Pro Arg Thr Gly Leu Ser Ser Tyr Glu Gln Tyr SEQ ID NO.80 Ala Ser Ser Val Glu Ala Asp Ser Trp Asn Ser Pro Leu His SEQ ID NO.81 Ala Ser Ser Thr Pro Thr Ser Asp Leu Pro Tyr Glu Gln Tyr SEQ ID NO.82 Ala Ser Ser Glu Gln Gly Ala Ile Ser Tyr Gly Tyr Thr SEQ ID NO.83 Ala Ser Ser Leu Ala Gly Gln Gly Glu Ser Tyr Asn Tyr Gly Tyr Thr SEQ ID NO.84 Ala Ser Ser Ser Lys Gly Pro Thr Gly Thr Asn Glu Gln Phe SEQ ID NO.85 Ala Thr Gly Ala Gly Thr Gly Ala Ser Pro Leu His SEQ ID NO.86 Ala Ser Gly Phe Gly Thr Glu Phe Tyr Glu Gln Tyr SEQ ID NO.87 Ala Ser Ser Asn Asn Arg Gly Arg Asn Glu Lys Leu Phe SEQ ID NO.88 Ala Ser Ser Tyr Asp Arg Ile Leu Thr Asp Thr Gln Tyr SEQ ID NO.89 Ala Ser Ser Leu Ala Ala Ala Ala Gly Tyr Gly Tyr Thr SEQ ID NO.90 Pro Ala Arg Gln Gly Glu Glu Arg Pro Ser Thr SEQ ID NO.91 Ser Val Gly Ala Ala Gln Gly Asp Thr Glu Ala Phe SEQ ID NO.92 Ala Ser Arg Gln Gly Gln Gly Thr Ser Pro Leu His SEQ ID NO.93 Ala Ser Ser Leu Asn Pro Gly Arg Pro Thr Tyr Glu Gln Tyr SEQ ID NO.94 Ala Ser Ser Arg Arg Gln Ala Leu Ser Thr Asp Thr Gln Tyr SEQ ID NO.95 Ala Ser Ser Leu Asp Arg Gly Gln Ile Gly Thr Glu Ala Phe SEQ ID NO.96 Ala Ser Ser Thr His Pro Arg Ser Gln Tyr SEQ ID NO.97 Ala Ser Ser Tyr Gly Gln Gly Lys Met Asn Thr Glu Ala Phe SEQ ID NO.98 Ala Ser Ser Leu Asp Leu Gly Leu Val Asn Thr Gly Glu Leu Phe SEQ ID NO.99 Ala Ser Ser Phe Gly Pro Val Gln Ala Asn Tyr Gly Tyr Thr SEQ ID NO.100 Ala Ser Ser Ser Leu Phe Pro Gly Gln Gly Arg Gly Glu Gln Phe

Further, the protein sequence of the marker is the sequence shown in SEQ ID NO. 1-100, after one or more bases are substituted, deleted and/or replaced, the protein with the same function can be expressed.

Further, the marker is the peripheral blood TCR CDR3 sequence.

The application of the above markers in the preparation of preparations for treating pancreatic cancer.

Further, the preparation includes a T cell receptor containing the marker, or a plasmid, viral vector or nucleic acid fragment capable of expressing the T cell receptor producing the marker.

A kit for detecting pancreatic cancer, comprising antibodies that can specifically bind to the above markers.

A preparation comprising an antibody that can specifically bind to the above-mentioned marker; the preparation can be used for diagnosing, predicting, detecting or screening pancreatic cancer.

A protein chip for detecting pancreatic cancer, the protein chip comprises a substrate and a specific antibody spotted on the substrate, the specific antibody being an antibody that can specifically bind to the above marker.

The principle of the present invention is as follows: B lymphocytes and T lymphocytes in the human body are two important types of cells in the acquired immune system. B cells recognize antigens through the B cell receptor (BCR) on the cell surface. Later, when B cells differentiate into plasma cells, BCR is expressed as an antibody and secreted outside the cell. T cells recognize antigens through T cell receptors (TCRs) on the cell surface. The diversity of BCR and TCR is the basis for the establishment of the adaptive immune system. The theoretical value of BCR diversity is 10 ¹⁸ and the theoretical value of TCR diversity is 10 ¹⁴ . Among BCR and TCR sequences, antigenic determinant 3 (CDR3) is the most important part in determining its antigenic specificity, so the sequence of CDR3 is considered to represent the properties of BCR and TCR sequences.

In various diseases, the diversity or expression levels of BCR and TCR will change with different antigenic stimulation. Therefore, the occurrence and development of diseases can be tracked by using BCR or TCR high-throughput sequencing results. In human cells, after degradation of senescent proteins, their fragments are transported to the cell surface and presented to T cells in the immune system through histocompatibility antigen II (MCHII). Antigen fragments presented by normal cells do not cause an immune response due to immune tolerance. Once normal cells become cancerous, the abnormal protein expressed by the mutated gene and its fragments are presented on the cell surface, which will cause the human immune system to produce a targeted immune response. Therefore, analyzing the changes of BCR or TCR can detect the occurrence and development of tumors.

The beneficial effects of the present invention are:

1. In the present invention, an artificial intelligence analysis model was first established by using 1300 non-pancreatic cancer control samples and the TCR high-throughput sequencing data of 6 pancreatic cancer patients. Clearly determine whether there is a higher risk of pancreatic cancer in the sample to be tested.

2. Analysis of TCR changes through high-throughput sequencing can detect very early pancreatic cancer. Using the unique TCR CDR3 sequence of pancreatic cancer to analyze the response of T cells in the human immune system to pancreatic cancer is a new detection method.

3. By using high-throughput sequencing technology, the present invention can compare a huge number of specific TCR sequences at the same time, and has higher specificity and accuracy than detecting one or several markers alone.

4. The cost of the high-throughput sequencing instrument used in the present invention is lower than that of large-scale imaging equipment, and it can be outsourced to a third party. In addition, the labor cost of sampling and processing is lower than the simultaneous detection of multiple markers, and it is also lower than that of a large number of cytology Therefore, the present invention greatly reduces the cost of detection; moreover, the present invention only needs to take a small amount of peripheral blood, and the sampling is simple and safe.

5. The TCR CDR3 sequence described in the present invention can be used for immunotherapy of pancreatic cancer.

Description of drawings

Figure 1 shows the CDR3 sequence of the control group and the characteristic sequence of pancreatic cancer in the present invention. The abscissa represents the sequence in which the CDR3 sequence of a specific amino acid combination was added to the control sequence set or the pancreatic cancer characteristic sequence set, and the ordinate represents the _{logarithm of the number of repetitions of the sequence in a sample, C X} ; the immune response of pancreatic cancer patients The atlas has multiple types of pancreatic cancer characteristic sequences with a high number of repetitions. There are very few pancreatic cancer characteristic sequences in healthy people, while the pancreatic cancer characteristics of unknown subjects are more obvious, indicating a higher risk of pancreatic cancer.

Fig. 2 is the pancreatic cancer characteristic index of healthy people, non-tumor patients, non-pancreatic cancer tumor patients and pancreatic cancer patients according to the pancreatic cancer feature sequence set in the present invention, healthy people, non-tumor patients, non-pancreatic cancer tumor patients The characteristic indices of pancreatic cancer were significantly different from those of pancreatic cancer patients, demonstrating the specificity of the pancreatic cancer characteristic sequence set. Based on this, it can be determined whether the unknown subject suffers from pancreatic cancer.

detailed description

The specific embodiments of the present invention are described below to facilitate those skilled in the art to understand the present invention, but it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes Such changes are obvious within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the inventive concept are within the scope of protection.

Example 1 Obtaining the Pancreatic Cancer TCR Marker CDR3 Sequence Set by Immunographic Analysis

1. Sampling and immunographic analysis (refer to patent ZL201910300069.9 for the method)

Collected 1301 controls (including healthy and non-tumor disease patients, 1300 were used for model establishment, 1 healthy person was used for validation), 7 pancreatic cancer patients (6 people were used for model establishment, 1 was used for validation) And the peripheral blood (10mL per person) of a subject with unknown health status, the TCR antigenic determinant 3 (CDR3) amino acid sequence of the subject and the control group was obtained by high-throughput sequencing to ensure the functionality of each sample The total number of CDR3 sequences of TCR is not less than 30000;

2. Perform random non-replacement sampling on the CDR3 sequences of the TCR of each sample, so that the total number of CDR3 sequences in each sample is 30,000. For any specific CDR3 sequence X, the number of repetitions in the single-sample sequencing result is counted as C _X ;

3. Determine the sequence of pancreatic cancer TCR marker CDR3 by analyzing TCR CDR3 data:

a) All CDR3 sequences of the 1300 control group samples used to build the model were aggregated and deduplicated, and set as a control sequence set;

b) Summarize and deduplicate all CDR3 sequences of the 6 pancreatic cancer samples used to establish the model, and then remove all sequences containing sequence duplications with the control sequence set, and set it as a pancreatic cancer characteristic sequence set. The drawing is shown in Figure 1A, in which the abscissa represents the sequence in which the CDR3 sequence of a specific amino acid combination is added to the control sequence set or the pancreatic cancer characteristic sequence set, and the ordinate represents the number of repetitions C of the sequence in a certain sample. The logarithmic value of _X.

c) According to the same mapping method, the immune profiles of 1 healthy person, 1 pancreatic cancer patient and 1 subject with unknown health status are mapped with reference to the control sequence set and the pancreatic cancer characteristic sequence set, see Figure 1B -D. It can be seen from the figure that the immune maps of patients with pancreatic cancer contain many types of pancreatic cancer characteristic sequences with a high number of repetitions; the immune maps of healthy people contain only a very small number of pancreatic cancer characteristic sequences; while subjects with unknown health status , have higher pancreatic cancer characteristic sequences than healthy people, indicating that this person has a higher risk of developing pancreatic cancer.

d) Set the characteristic sequences of pancreatic cancer, and put all CDR3 sequences that appear in two or more pancreatic cancer samples participating in the modeling, and press the “sum of the _{number of repeated occurrences of this sequence in a single sample of all pancreatic cancer samples participating in the modeling C X”} ×Number of pancreatic cancer samples that contain this sequence and participate in modeling" in order from high to low, and the top 100 are the pancreatic cancer TCR marker CDR3 sequences. The specific sequences are shown in SEQ ID NO. 1-100.

Example 2 Verification of the specificity of the pancreatic cancer TCR marker CDR3 sequence set

1. Sampling and immunographic analysis (refer to patent ZL201910300069.9 for the method)

The peripheral blood (10 mL per person) of 349 non-pancreatic cancer tumor patients and 2 subjects with unknown health status was collected, and the antigenic determinant 3 (CDR3) amino acids of TCR of the subjects and the control group were obtained by high-throughput sequencing Sequence, to ensure that the total number of CDR3 sequences of functional TCRs of each sample is not less than 30,000; the CDR3 sequences of TCR of each sample are randomly sampled without replacement, so that the total number of CDR3 sequences of each sample is 30,000.

2. 100 healthy people and 45 non-tumor disease patients were randomly selected from the control group in Example 1.

3. According to 100 healthy people, 45 non-tumor disease patients, 6 pancreatic cancer patients from Example 1, and 349 non-pancreatic cancer tumor patients and 2 subjects with unknown health conditions newly obtained in Example 2. Immune profiling to analyze the characteristic index of pancreatic cancer.

Among them, the characteristic index of pancreatic cancer is defined as: in a certain sample, the sum _{of the repeated occurrence times C X of all CDR3 sequences belonging to the characteristic sequence set of pancreatic cancer in the sample.} The analysis results are shown in Table 2 below and accompanying drawing 2. The pancreatic cancer group was significantly different from healthy people (p=1.9E-78), non-tumor diseases (p=6.6E-43), and other tumors (p=9.9E-162), which proved that the pancreatic cancer feature sequence set specificity.

Table 2 Pancreatic cancer characteristic indices in different sample groups

healthy person non-neoplastic disease other tumors Pancreatic cancer Test samples risk 18 60 644 14715 2222 higher 10 52 298 12976 2508 higher 15 33 278 11511     17 twenty one 110 10136     112 20 76 9235     3 18 53 7299     9 17 47       17 15 44       10 13 44       4 13 43       twenty three 12 43       5 12 43      

17 12 40       6 11 40       13 11 39       9 10 37       10 10 35       15 10 35       7 10 32       6 9 32       6 9 32       16 9 31       17 8 31       5 7 30       twenty four 7 30       11 7 29       14 7 28       6 6 28       3 6 28       4 6 27       13 5 27       20 4 27       2 4 26       12 3 25       12 3 25       10 3 25       1 2 25       2 2 twenty four       17 1 twenty four       7 1 twenty three       7 0 twenty three       3 0 twenty three      

36 0 twenty three       14 0 twenty two       9 0 twenty two       4   twenty two       6   twenty two       7   twenty two       14   twenty two       4   twenty two       13   twenty one       10   twenty one       5   twenty one       6   20       4   20       28   20       17   20       4   20       14   20       14   20       11   19       6   19       5   19       5   19       7   19       3   19       6   19       25   18       7   18       6   18       5   18       7   18      

15   18       11   18       10   17       11   17       17   17       15   17       3   17       9   17       13   17       17   17       4   17       7   17       12   16       11   16       17   16       6   16       12   16       15   16       16   16       16   16       17   16       7   16       14   16       16   16       13   16       9   16       12   15       twenty four   15           15           15      

    15           15           15           15           15           15           15           15           15           15           14           14           14           14           14           14           14           14           14           14           14           14           14           14           14           14           14           14           14           13      

    13           13           13           13           13           13           13           13           13           13           13           13           13           12           12           12           12           12           12           12           12           12           12           12           12           12           12           12           12           12      

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    6           6           6           6           6           6           6           6           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5           5      

    5           5           5           5           4           4           4           4           4           4           4           4           4           4           3           3           3           3           3           3           3           3           3           3           3           3           3           3           3           3      

    3           3           3           3           3           3           3           3           2           2           2           2           2           2           2           2           2           2           2           2           2           2           1           1           1           1           1      

4. Analyze the pancreatic cancer characteristic index of each group (Table 3). Among the subjects with unknown health status (test samples), the pancreatic cancer characteristic index of 2 people is higher than the average value of the "other tumors" group + 2 × SD ( 16.1+2×41.1=98.2), these 2 people have a higher risk of developing pancreatic cancer. After comparing with the clinical physical examination results, these two people were indeed pancreatic cancer patients. This example demonstrates the feasibility of predicting the risk of pancreatic cancer in a subject using the pancreatic cancer signature sequence set and the pancreatic cancer signature index.

Table 3 Analysis of the characteristic index of pancreatic cancer

  healthy person non-neoplastic disease other tumors Pancreatic cancer Test samples Mean 11.89 10.42 16.06 10978.67 2365.00 SD 11.90 11.97 41.08 2665.46 202.23 mean+2SD 35.69 34.36 98.23    

In summary, the pancreatic cancer TCR marker CDR3 sequence of the present invention does have significant pancreatic cancer specificity, and can not only be used for pancreatic cancer to predict the risk of pancreatic cancer in subjects, but can also be used for biological immunity of pancreatic cancer in the future. treat.

Claims (7)

A peripheral blood TCR marker for pancreatic cancer, characterized in that the marker comprises at least one of the proteins whose sequences are shown in SEQ ID NO. 1-100. The peripheral blood TCR marker for pancreatic cancer according to claim 1, wherein the protein sequence of the marker is the sequence shown in SEQ ID NO. After a few bases, a protein with the same function can be expressed. The application of the marker of claim 1 in the preparation of a preparation for treating pancreatic cancer. The use according to claim 3, wherein the preparation comprises a T cell receptor containing the marker, or a plasmid, viral vector or nucleic acid fragment capable of expressing the T cell receptor producing the marker. A kit for detection of pancreatic cancer, characterized by comprising an antibody that can specifically bind to the marker of claim 1. A preparation, characterized in that it comprises an antibody that can specifically bind to the marker of claim 1. A protein chip for detecting pancreatic cancer, characterized in that the protein chip comprises a substrate and a specific antibody spotted on the substrate, the specific antibody being capable of specifically binding to the marker of claim 1 antibody.

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