WO2022012293A1

WO2022012293A1 - Peripheral blood tcr marker for endometrial cancer, test kit using same, and application thereof

Info

Publication number: WO2022012293A1
Application number: PCT/CN2021/102042
Authority: WO
Inventors: 张志新; 杨鑫; 卓越; 钟雪梅; 杨诗涵; 高�浩
Original assignee: 成都益安博生物技术有限公司
Priority date: 2020-07-11
Filing date: 2021-06-24
Publication date: 2022-01-20
Also published as: CN111624341A

Abstract

A peripheral blood TCR marker for endometrial cancer, a test kit using same, and an application thereof. The marker comprises at least one of proteins shown with sequences SEQ ID NO. 1-100. A high-throughput sequencing method is employed such that only a small amount of peripheral blood is required to extract RNA. An immune profile library is created by processing the sample. Subsequently, after high-throughput sequencing and TCR data analysis, a characteristic TCR sequence in the peripheral blood of endometrial cancer is first determined, and then the test result of the sample to be tested is compared with the characteristic TCR sequence to determine whether a subject has endometrial cancer. The present invention can compare a huge amount of endometrial cancer-specific TCR sequences at the same time, and has higher specificity and accuracy and improves the diagnostic efficiency compared with separately testing of one or several markers.

Description

A kind of peripheral blood TCR marker of endometrial 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 endometrial cancer and a detection kit and application thereof.

Background technique

Endometrial cancer is a group of epithelial malignancies that occur in the endometrium, and is prevalent in perimenopausal and postmenopausal women. It is one of the most common female reproductive system tumors, with nearly 200,000 new cases every year, 75% to 95% recurrence within 2 to 3 years after surgery, and is the third most common gynecological malignancy leading to death. Because the etiology is not very clear. The key to improving prognosis lies in early detection, early diagnosis and early treatment. Currently, there is no screening method, and only clinical diagnosis, mainly imaging examination and endometrial biopsy, is performed under the guidance of a gynecologist when clinical symptoms such as vaginal bleeding, vaginal discharge, and lower abdominal pain are present.

At present, the diagnostic methods for ovarian cancer are mainly divided into imaging examination, endometrial biopsy and laboratory diagnosis.

1. Imaging examination methods

Gynecological B-ultrasound is a preliminary judgment, and transvaginal ultrasonography is the most commonly used non-invasive examination method. Endometrial biopsy is required when the endometrial thickness is greater than 5 mm.

Abdominal and pelvic magnetic resonance imaging (MRI) examination: Magnetic resonance imaging (MRI) is an imaging technique that uses the signal generated by the resonance of atomic nuclei in a strong magnetic field to reconstruct the image, and is a nuclear physical phenomenon. The size, location, depth of invasion, cervical and vaginal infiltration, infiltration of other pelvic and abdominal organs, and lymph node metastasis were determined. It is commonly used in imaging examinations and can be differentiated from submucosal uterine fibroids, endometrial polyps, uterine sarcomas, and cervical cancer. The disadvantage is that some patients are not suitable for magnetic resonance examination, which are contraindications and relative contraindications.

Preoperative imaging examination is very necessary. By understanding myometrial lesions and pelvic conditions, staging and treatment plan can be determined. Imaging examination contents: (1) Abdominal CT and pelvic MRI. (2) chest imaging examination (chest X-ray or CT). (3) Positron emission computed tomography (PET-CT) is selected if necessary, which can help to determine whether other parts have spread and clinical staging. (4) For patients with fertility preservation, chest and abdominal CT examinations to exclude suspicious lesions, pelvic MRI examinations to determine whether there is infiltration of the myometrium, and PET-CT examinations for distant metastatic lesions as a necessary option.

2. Endometrial biopsy method

Endometrial biopsy is a method for the diagnosis of endometrial cancer, which is mainly divided into segmental curettage and hysteroscopic localization biopsy. Segmented curettage is to first scrape the tissue of the cervical canal, and then scrape the uterine body, and then send the scraped materials to the examination separately. The nature, location, and extent of involvement of the lesions were identified. Basically the same as general curettage, the difference is: before exploring the depth of the uterine cavity, first probe the depth of the cervical canal with a probe, generally 2.0-2.5cm deep, and then use a small curette to cure the cervical canal sequentially from the internal orifice of the cervix to the external orifice. One week, the scraped tissue is placed on gauze, and then the uterine cavity is scraped and the scraped material is divided into bottles and marked for inspection. The disadvantage is: when scratching the cervical canal, you must master the depth and strength, generally not more than 2-3cm deep, so as not to bring out the contents of the uterine cavity, and to avoid scraping and the external cervical tissue, resulting in false positives. During the operation, the uterus will be stretched, which may cause postoperative abdominal pain. Anti-inflammatory treatment is required to prevent infection. Generally speaking, it may have a certain impact only when it is operated during the operation, but it depends on the specific situation of the patient. In addition, the difficulty and duration of surgery may vary. For example, the patient's cervix is very narrow or the uterus is abnormally developed. In these cases, the operation will be more difficult. In the process of hysteroscopic biopsy, general anesthesia is implemented in the hospital, and then the perfusion system is activated to dilate the uterus, which can ensure the intrauterine pressure and also play a role in cooling and flushing. Hysteroscopy is further divided into examination hysteroscopy and surgical hysteroscopy. There are three different electrodes in the surgical hysteroscope, and their normal work is inseparable from the support of the energy system, that is, the current. In addition, for the operation to be accurate, a light source system and an imaging system are needed to help. During the operation, with clear lighting and monitoring by the imaging system, blurred vision can be avoided, and it can play a guiding role. The advantage is that it can directly observe the situation in the uterine cavity, and biopsy under direct vision, which is more accurate. The disadvantage is that the uterine distended fluid may cause some tumor cells to enter the abdominal cavity through the fallopian tube.

3. Laboratory diagnostic methods

Serum CA125: For patients with extrauterine lesions, CA125 is helpful for monitoring clinical treatment effects. It is worth noting that CA125 may be abnormally elevated in patients with peritoneal inflammation or radiation injury. In contrast, patients with solitary vaginal metastases do not have elevated CA125 and therefore cannot predict recurrence in the absence of other clinical findings.

There are no known sensitive tumor markers for diagnosis and follow-up of endometrial cancer.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a peripheral blood TCR marker of endometrial cancer and its detection kit and application in view of the deficiencies in the prior art, so as to accurately and quickly judge whether there is a higher uterine uterus in the sample to be tested. Patients at risk for endometrial cancer.

For achieving the above object, the technical scheme adopted in the present invention is:

A peripheral blood TCR marker for endometrial cancer, the TCR marker comprises at least one of the proteins whose sequences are shown in SEQ ID NO. 1-100.

Table 1 Marker sequences

Further, the protein sequence of the TCR marker is the sequence shown in SEQ ID NO. 1 to 100 after substitution, deletion and/or replacement of one or more bases, the protein with the same function can be expressed.

The application of the above TCR markers in the preparation of preparations for the treatment of endometrial cancer.

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

A kit for detecting endometrial cancer, comprising an antibody that can specifically bind to the above-mentioned peripheral blood TCR marker of endometrial cancer.

A preparation includes an antibody that can specifically bind to the above-mentioned peripheral blood TCR markers of endometrial cancer; the preparation can be used for diagnosis, prediction, detection or screening of endometrial cancer.

A protein chip for detecting endometrial cancer, the protein chip comprises a substrate and a specific antibody spotted on the substrate, and the specific antibody is an antibody that can specifically bind to the peripheral blood TCR marker of the endometrial cancer.

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 degraded 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.

Analysis of peripheral blood TCR sequences based on high-throughput sequencing to detect endometrial cancer, the specific steps are as follows:

(1) Collect the peripheral blood of the subjects and the control group, and obtain the antigenic determinant 3 (CDR3) amino acid sequences of the TCRs of the subjects and the control group by high-throughput sequencing to ensure the total number of CDR3 sequences of functional TCRs in each sample. Comprehensive not less than 30000;

(2) Randomly sample the CDR3 sequences of the TCR of each sample without replacement, so that the total number of CDR3 sequences in each sample is 30,000. For any particular CDR3 sequence X, the number of repetitions in this sample is counted as C _X ;

(3) TCR CDR3 data analysis: The sequence of endometrial cancer TCR marker CDR3 was determined by analysis:

a) Summarize and deduplicate all CDR3 sequences of the control group samples, and set them as a control sequence set;

b) Summarize and deduplicate all CDR3 sequences of the endometrial cancer sample, and then remove all sequences that duplicate the CDR3 sequences in the control sequence set, and set it as the endometrial cancer characteristic sequence set;

c) Collecting the characteristic sequences of endometrial cancer, CDR3 sequences that appear in two or more samples, press " _{the sum of the number of repeated occurrences of this sequence in all endometrial cancer samples C X} × the endometrium containing the sequence The number of cancer samples is sorted from high to low, and the top 100 are the sequences of the endometrial cancer TCR marker CDR3.

(4) Use the endometrial cancer characteristic index to determine the risk of endometrial cancer in unknown subjects:

a) According to the immune profiles of healthy controls, non-tumor patients, non-endometrial cancer tumor patients, endometrial cancer patients, and subjects with unknown health status, the endometrial cancer characteristic indices were analyzed.

The endometrial cancer characteristic index is defined as: in a certain sample, the sum _{of the repeated occurrence times C X of all CDR3 sequences belonging to the endometrial cancer characteristic sequence set in the sample.}

b) When a subject of unknown health status has a characteristic index of endometrial cancer that is higher than or close to the mean of the "other tumors" group + 2 x SD, the person is at higher risk of developing endometrial cancer; such as in the uterus Endometrial cancer characteristics index close to the average of healthy people or non-neoplastic disease group, indicating a low risk of endometrial cancer.

To sum up, due to the adoption of the above-mentioned technical solutions, 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-endometrial cancer control samples and the TCR high-throughput sequencing data of 25 endometrial cancer patients. Sexual TCR sequence comparison can clearly determine whether there is a higher risk of endometrial cancer in the sample to be tested;

2. Analysis of TCR changes through high-throughput sequencing can detect very early endometrial cancer. Using endometrial cancer-specific TCR CDR3 sequences to analyze the response of T cells in the human immune system to endometrial cancer is a kind of new detection methods;

3. In the present invention, due to the use of high-throughput sequencing technology and the simultaneous comparison of a huge number of specific TCR sequences, it 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 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 also lower than that of a large number of cytology detection, therefore, the present invention greatly reduces the detection cost;

5. The present invention only needs to take a small amount of peripheral blood, and the sampling is simple and safe;

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

Description of drawings

Figure 1 shows the CDR3 sequence of the control group and the characteristic sequence of endometrial cancer in the present invention. Abscissa represents a particular combination of amino acid sequences are added to the control CDR3 sequence set or endometrial cancer wherein the sequence of the sequence set, the ordinate represents the logarithm of the number of times C _X duplication sequence in a sample; Endometrial The immune profiles of cancer patients have multiple types of endometrial cancer signature sequences with high repetitions, healthy people rarely have endometrial cancer signature sequences, and unknown subjects have more obvious endometrial cancer signatures, indicating that the patient has uterine cancer. The risk of endometrial cancer is higher.

Fig. 2 is in the present invention, for the endometrial cancer characteristic sequence set, calculate the endometrial cancer characteristic index of healthy people, non-tumor patients, non-endometrial cancer tumor patients and endometrial cancer patients, healthy people, non-cancer patients The endometrial cancer characteristic indices of tumor patients and non-endometrial cancer tumor patients were significantly different from those of endometrial cancer patients, which proved the specificity of the endometrial cancer signature sequence set. Based on this, it can be determined whether the unknown subject suffers from endometrial cancer.

detailed description

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The components of the embodiments of the invention generally described and illustrated in the drawings herein may be arranged and designed in a variety of different configurations. Thus, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present invention. The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1: Obtaining a sequence set of endometrial cancer TCR marker CDR3 by immunographic analysis

1. Sampling and immunographic analysis

Collected 1301 control groups (including healthy and non-tumor disease patients, 1300 were used for model establishment, 1 healthy person was used for validation), 21 endometrial cancer patients (20 were used for model establishment, 1 was used for verification) and peripheral blood (10 mL per person) of a subject with unknown health status, the TCR antigenic determinant 3 (CDR3) amino acid sequences of the subjects and the control group were obtained by high-throughput sequencing to ensure that each sample was The total number of CDR3 sequences of functional TCRs should not be less than 30,000;

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 endometrial cancer TCR marker CDR3 by analyzing TCR CDR3 data:

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

b) All CDR3 sequences of the 25 endometrial cancer samples used to establish the model were aggregated and deduplicated, and then all sequences containing sequence duplication with the control sequence set were removed, and set as the endometrial 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 certain amino acid combination is added to the control sequence set or the endometrial cancer characteristic sequence set, and the ordinate represents the sequence repeated in a certain sample. The logarithmic value of the degree C _X.

c) According to the same mapping method, the immune profiles of 1 healthy person, 1 patient with endometrial cancer and 1 subject with unknown health status are mapped with reference to the set of control sequences and the set of endometrial cancer characteristic sequences, See Figures 1B-D. It can be seen from the figure that the immune maps of patients with endometrial cancer contain a large number of endometrial cancer characteristic sequences with a high number of repetitions; the immune maps of healthy people contain only a very small number of endometrial cancer characteristic sequences; However, subjects of unknown health status had higher endometrial cancer signature sequences than healthy subjects, indicating that this person had a higher risk of developing endometrial cancer.

d) Set the characteristic sequences of endometrial cancer, and put all CDR3 sequences that appear in two or more samples of endometrial cancer involved in modeling, and click “repeat in a single sample of this sequence in all samples of endometrial cancer involved in modeling.” The total number of occurrences C _X × the number of endometrial cancer samples that contain the sequence involved in modeling" is sorted from high to low, and the top 100 are the sequence of the endometrial cancer TCR marker CDR3, the specific sequence is as shown in SEQ ID NO. 1 to 100.

Example 2: Validation of the specificity of the endometrial cancer TCR marker CDR3 sequence set

1. Sampling and immunographic analysis

The peripheral blood (10 mL per person) of 319 non-endometrial cancer tumor patients and 6 subjects with unknown health status was collected, and the TCR epitope 3 (CDR3) of the subjects and the control group was obtained by high-throughput sequencing. ) amino acid sequence, to ensure that the total number of CDR3 sequences of functional TCRs in each sample is not less than 30,000; the CDR3 sequences of TCRs in each sample are randomly sampled without replacement, so that the total number of CDR3 sequences in each sample is 30,000 .

1. 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, 25 endometrial cancer patients from Example 1, and 319 non-endometrial cancer tumor patients and 6 unknown health conditions newly acquired in Example 2 Immune profiles of subjects analyzed for indices characteristic of endometrial cancer.

The analysis results are shown in Table 2 below and accompanying drawing 2. The endometrial cancer group was significantly different from healthy individuals (p=6.6E-71), non-neoplastic disease (p=7.7E-37), and other tumors (p=1.8E-172), demonstrating that the endometrium Specificity of cancer signature sequence sets.

Table 2. Endometrial cancer characteristic index of different sample groups

4. The endometrial cancer characteristic index of each group was analyzed (Table 3). Among subjects with unknown health status (test samples), the endometrial cancer characteristic index of the top 1 person was higher than the average value of the "other tumors" group+ 2×SD (232+2×664=1560), this person has a higher risk of developing endometrial cancer; the endometrial cancer characteristic index of the last 4 people is close to the average value of healthy people or non-tumor disease group, suffering from endometrial cancer Low risk. Compared with the results of clinical physical examination, the first one was a patient with endometrial cancer, and the last four were healthy. This example demonstrates the feasibility of using the endometrial cancer signature sequence set and the endometrial cancer signature index to predict the risk of endometrial cancer in a subject.

Table 3. Analysis of Endometrial Cancer Characteristic Index

To sum up, the CDR3 sequence of the endometrial cancer TCR marker of the present invention does have significant endometrial cancer specificity, and can not only be used for endometrial cancer to predict the risk of endometrial cancer in subjects, but also in the future. It can also be used for biological immunotherapy of endometrial cancer.

Claims

The peripheral blood TCR marker of endometrial cancer is characterized in that, the TCR marker comprises at least one of the proteins shown in the sequence of SEQ ID NO. 1-100.
The TCR marker of endometrial cancer peripheral blood according to claim 1, wherein the protein sequence of the TCR marker is the sequence shown in SEQ ID NO. 1-100, which is substituted, deleted and/or replaced After one or more bases, the protein with the same function can be expressed.
Application of the peripheral blood TCR marker of endometrial cancer according to claim 1 in the preparation of a preparation for the treatment of endometrial cancer.
The application according to claim 3, wherein the preparation comprises a T cell receptor containing the TCR marker, or a plasmid, viral vector or nucleic acid fragment capable of expressing the T cell receptor producing the TCR marker.
A kit for detecting endometrial cancer, characterized by comprising an antibody that can specifically bind to the peripheral blood TCR marker of endometrial cancer according to claim 1.
A preparation, characterized by comprising an antibody capable of specifically binding to the peripheral blood TCR marker of the endometrial cancer of claim 1.
A protein chip for detecting endometrial cancer, characterized in that the protein chip comprises a substrate and a specific antibody spotted on the substrate, and the specific antibody is the same as the endometrial cancer of claim 1. Antibodies that specifically bind to peripheral blood TCR markers.