WO2023142244A1

WO2023142244A1 - Early screening device for acquired resistance to immunotherapy and use thereof

Info

Publication number: WO2023142244A1
Application number: PCT/CN2022/081515
Authority: WO
Inventors: 刘宝琳; 张园园; 张泽民
Original assignee: 北京大学
Priority date: 2022-01-26
Filing date: 2022-03-17
Publication date: 2023-08-03
Also published as: CN114107512A; CN114107512B

Abstract

The present application provides an early screening device for acquired resistance to immunotherapy and use thereof. The early screening device for acquired resistance to immunotherapy comprises a sequencing module, a screening module, a cluster analysis module, a monitoring module, and an analysis module. Through a comprehensive judgment based on the combination of the real-time monitoring of proportion change in cancer cell killer CD8 T cell clones in the peripheral blood with the proportion change of inexhaustible precursor T cells in the cancer cell killer CD8 T cell clones in biopsy samples, the present application can achieve very early screening of acquired resistance to immunotherapy.

Description

An early screening device for immunotherapy acquired drug resistance and its application

technical field

The application belongs to the technical field of immunotherapy, and in particular relates to an early screening device for immunotherapy acquired drug resistance and its application.

Background technique

Immune checkpoint blockade therapy represented by PD-1 antibody therapy has improved the treatment landscape of cancer. Some cancer patients respond to the drug after receiving immune checkpoint blockade therapy, and their condition gradually improves; however, 15% to 20% of the patients begin to get worse within a period of time after the treatment has an effect, and the tumor becomes larger and new tumors appear. Tumor metastasizes and develops acquired resistance to treatment.

At present, the hospital will evaluate the cancer patient three months after the first immunotherapy, and if there is no obvious disease progression or serious side effects, the immunotherapy will continue; if the cancer patient is found through imaging and other techniques After a period of improvement, if the tumor grows significantly, or new cancer metastasis appears, it is judged that the patient has acquired drug resistance, and new treatment strategies are adopted, such as immunotherapy and other treatment methods combined treatment.

Imaging-based methods for detecting acquired resistance to immunotherapy in cancer patients have certain drawbacks. On the one hand, this method has a hysteresis, and it is usually found that the patient’s condition is already serious when acquired drug resistance occurs, resulting in untimely treatment of the patient; Necessary medical expenses. Therefore, the untimely detection of acquired drug resistance by traditional technology will not only be detrimental to treatment, but also lead to economic losses for patients.

At present, there is no mature method for early screening of acquired resistance to immunotherapy. Therefore, how to provide a product and method for early screening of acquired drug resistance to immunotherapy has become an urgent problem to be solved.

Contents of the invention

The present application provides an early screening device for immunotherapy acquired drug resistance and its application. By monitoring the changes in the proportion of peripheral blood cancer cell killing CD8 T cell clones, combined with the changes in the proportion of non-exhausted precursor T cells in cancer cell killing CD8 T cell clones in biopsy samples, it was judged. The device provided by the application has accurate results and has practical application value.

In the first aspect, the present application provides an early screening device for immunotherapy acquired drug resistance, the early screening device for immunotherapy acquired drug resistance includes:

Sequencing module: take biopsy tissue for single-cell sequencing, classify cells according to T cell receptor sequence, and calculate the average value of each gene expression;

Screening module: According to the threshold of CD8A and CXCL13 expression, screen out CD8A and CXCL13 double-positive clones, which are cancer cell killing CD8 T cell clones;

Cluster analysis module: perform cluster analysis on the cancer cell killing CD8 T cell clones, screen out non-exhausted precursor T cells, and calculate the number of non-exhausted precursor T cells in the cancer cell killing CD8 The proportion of T cell clones, denoted as A1;

Monitoring module: Perform single-cell sequencing on peripheral blood, screen out CD8A and CXCL13 double-positive clones, that is, peripheral blood cancer cell killing CD8 T cell clones, calculate the proportion of peripheral blood cancer cell killing CD8 T cell clones, and perform monitor;

Analysis module: when the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is significantly decreased, the biopsy tissue is taken again for single-cell sequencing, and the ratio of non-exhausted precursor T cells in the killing CD8 T cell clones of the cancer cells is calculated. The ratio is recorded as A2, and the acquired drug resistance of the sample to immunotherapy is judged according to the difference between A1 and A2.

In this application, the early screening device for immunotherapy acquired drug resistance monitors the change in the proportion of peripheral blood cancer cell killing CD8 T cell clones, and combines the non-exhausted precursor T cells in the biopsy sample in the cancer cell killing CD8 T cells. The change in the proportion of T cell clones, and then judge whether the sample has acquired drug resistance to immunotherapy, the result is accurate, and provides a theoretical basis for related immunotherapy; the collection of peripheral blood is more convenient, so it can achieve real-time monitoring. It is convenient to adjust the treatment plan in time, choose a more precise and targeted method, and at the same time avoid the damage caused by repeated biopsy and reduce the pain of the patient.

Preferably, the single-cell sequencing includes 10×Genomics 5' single-cell transcriptome library sequencing.

In this application, cells with the same T cell receptor (TCR) sequence come from the same cell clone, and the cells can be classified according to the T cell receptor sequence to confirm the clone to which each cell belongs.

In this application, the thresholds of the expression levels of CD8A and CXCL13 can be confirmed specifically for each sample. The method is: check the gene expression distributions of CD8A and CXCL13 in units of clones, and determine the inflection points of the two distributions as CD8A and CXCL13 respectively. expression threshold.

In this application, 0.9 can also be directly used as the threshold value of CD8A expression level, and 0.15 can be used as the threshold value of CXCL13 expression level. CD8 T cell clones can be screened in this way with an accuracy rate of 96.9%.

Preferably, said cluster analysis comprises unsupervised cluster analysis.

In the present application, the process of the unsupervised cluster analysis includes: standardization of gene expression, selection of differentially expressed genes, principal component analysis, batch effect removal and cluster analysis.

In this application, the cancer cell killing CD8 T cell clones can be divided into four different subgroups after unsupervised cluster analysis, namely, proliferative T cells (highly expressing genes such as MKI67 and STMN1), exhausted T cells (high expression of genes such as HAVCR2, ENTPD1, LAYN, PDCD1, TIGIT, LAG3 and CTLA4) and non-exhausted precursor (precursor) T cells (low expression of signal genes of exhausted T cells), in which non-exhausted precursor T cells further Divided into GZMK positive precursor T cells and IL7R positive precursor T cells (high expression of IL7R, TCF7, LEF1, CCR7 and SELL genes).

Preferably, the peripheral blood cancer cell killing CD8 T cell clone has the same T cell receptor sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue.

In this application, in the peripheral blood, the T cell clone with the same TCR sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue is the peripheral blood cancer cell killing CD8 T cell clone.

In the present application, the ratio of killing CD8 T cell clones of peripheral blood cancer cells refers to the ratio of killing CD8 T cell clones of peripheral blood cancer cells among all T cells in peripheral blood.

Preferably, the criteria for a significant decrease in the ratio of the peripheral blood cancer cell killing CD8 T cell clones are:

The proportion of the killing CD8 T cell clone of the peripheral blood cancer cells is less than 50% compared with the proportion during the effective period of treatment, and no significant decline occurs;

The ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is greater than or equal to 50% compared with the ratio during the effective treatment period, which is a significant decline.

In the present application, the ratio during the effective period of treatment is the ratio of peripheral blood cancer cell killing CD8 T cell clones when peripheral blood is sampled for the first time.

In this application, the proportion of killing CD8 T cell clones of peripheral blood cancer cells is used as the monitoring index. If the proportion of killing CD8 T cell clones of peripheral blood cancer cells decreases significantly, it is considered that acquired drug resistance may have occurred, and a new biopsy is required. Samples were sequenced and analyzed for further verification and confirmation; if the proportion of cytotoxic CD8 T cell clones in peripheral blood cancer cells did not decrease significantly, it was considered that acquired drug resistance did not appear, and biopsy sampling was not required. Because the sampling of peripheral blood is more convenient, it is possible to collect and analyze the peripheral blood at multiple time points to achieve the effect of real-time monitoring. On the one hand, it is beneficial to adjust the treatment plan in time according to the inspection results, and on the other hand, it can also avoid repeated biopsies. The damage caused reduces the pain of the patient.

Preferably, the criteria for the judgment are:

The difference between A1 and A2 is greater than or equal to 0.39, and the sample has acquired drug resistance to immunotherapy;

The difference between A1 and A2 is less than 0.39, and the sample has no acquired drug resistance to immunotherapy.

As a preferred technical solution, the early screening device for immunotherapy acquired drug resistance described in this application includes:

Sequencing module: Take the biopsy tissue during the effective period of treatment and perform 10×Genomics 5' single-cell transcriptional library sequencing to obtain single-cell gene expression data and T-cell receptor sequencing data, and classify the cells according to the T-cell receptor sequence, and Calculate the average value of each gene expression;

Clustering analysis module: perform unsupervised clustering analysis on the cancer cell killing CD8 T cell clones, screen out non-exhausted precursor T cells, and calculate the rate of killing of the cancer cells by the non-exhausted precursor T cells Proportion of positive CD8 T cell clones, denoted as A1;

Monitoring module: Perform 10×Genomics 5'single-cell transcriptional library sequencing on peripheral blood to screen out clones that are double positive for CD8A and CXCL13 and have the same T cell receptor sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue , which is the killing CD8 T cell clone of peripheral blood cancer cells, the ratio of the killing CD8 T cell clones of peripheral blood cancer cells is calculated and monitored;

Analysis module: when the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is greater than or equal to 50% compared with the ratio during the effective treatment period, the biopsy tissue is taken again for 10×Genomics 5'single-cell transcriptional library sequencing, and the calculation The proportion of non-exhausted precursor T cells in the cancer cell-killing CD8 T cell clone is recorded as A2, and the acquired drug resistance of the sample to immunotherapy is judged according to the difference between A1 and A2. The standard is:

In this application, the early screening device for immunotherapy acquired drug resistance can be used for breast cancer, esophageal cancer, liver cancer, lung cancer, melanoma, colorectal cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, kidney cancer and Screening of various samples, including endometrial cancer, has strong applicability and broad application prospects.

In a second aspect, the present application provides an early screening method for immunotherapy acquired drug resistance for the purpose of non-disease diagnosis and/or treatment, and the early screening method includes:

Take the biopsy tissue for single-cell sequencing, classify the cells according to the T cell receptor sequence, and calculate the average value of each gene expression;

According to the threshold of the expression of CD8A and CXCL13, clones that are double positive for CD8A and CXCL13 are screened out, which are cancer cell killing CD8 T cell clones;

Carrying out cluster analysis on the cancer cell killing CD8 T cell clones, screening out non-exhausted precursor T cells, and calculating the ratio of the non-exhausted precursor T cells in the cancer cell killing CD8 T cell clones Ratio, denoted as A1;

Perform single-cell sequencing on peripheral blood, screen out CD8A and CXCL13 double-positive clones, which are peripheral blood cancer cell killing CD8 T cell clones, calculate the ratio of peripheral blood cancer cell killing CD8 T cell clones, and monitor;

When the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells decreased significantly, the biopsy tissue was taken again for single-cell sequencing, and the ratio of non-exhausted precursor T cells in the killing CD8 T cell clones of the cancer cells was calculated, and recorded as It is A2. According to the difference between A1 and A2, the acquired drug resistance of the sample to immunotherapy is judged.

Preferably, said cluster analysis comprises unsupervised cluster analysis.

Preferably, the criteria for the judgment are:

As a preferred technical solution, the early screening method for immunotherapy-acquired drug resistance for the purpose of non-disease diagnosis and/or treatment described in this application includes the following steps, and its flow chart is shown in Figure 1:

Biopsy tissues during the effective period of treatment were taken for 10×Genomics 5' single-cell transcriptional library sequencing to obtain single-cell gene expression data and T-cell receptor sequencing data. Cells were classified according to the T-cell receptor sequence, and each The average value of gene expression;

Carrying out unsupervised clustering analysis on the cancer cell killing CD8 T cell clones, screening out non-exhausted precursor T cells, and calculating the number of non-exhausted precursor T cells in the cancer cell killing CD8 T cell clones The ratio in is recorded as A1;

Perform 10×Genomics 5'single-cell transcriptional library sequencing on peripheral blood, and screen out clones that are double positive for CD8A and CXCL13 and have the same T cell receptor sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue, namely Killing CD8 T cell clones of peripheral blood cancer cells, calculating and monitoring the ratio of killing CD8 T cell clones of peripheral blood cancer cells;

When the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is greater than or equal to 50% compared with the ratio during the effective period of treatment, the biopsy tissue is taken again for 10×Genomics 5'single-cell transcriptional library sequencing, and the non-depletion rate is calculated. The proportion of precursor T cells in the cancer cell killing CD8 T cell clone is recorded as A2, and the acquired drug resistance of the sample to immunotherapy is judged according to the difference between A1 and A2, and the criteria for the judgment are:

In the third aspect, the present application provides the early screening device for immunotherapy acquired drug resistance described in the first aspect and/or the immunotherapy acquired resistance device for the purpose of non-disease diagnosis and/or treatment described in the second aspect. The application of the early screening method of drugs in the preparation of immunotherapy acquired drug resistance screening products.

Compared with the prior art, the present application has the following beneficial effects:

This application monitors the proportion of killing CD8 T cell clones of peripheral blood cancer cells, combined with the change of the proportion of non-exhausted precursor T cells in the killing CD8 T cell clones of cancer cells in biopsy samples, to determine the acquired drug resistance of samples It provides a reference for relevant treatment and medication; the early screening device for immunotherapy acquired drug resistance is simple to operate, easy to use, and can avoid the damage and pain caused by repeated biopsy to patients; All types of samples can be effectively detected, and the applicability is strong; the samples can be screened for very early acquired drug resistance, and before the patient's condition worsens, that is, before the tumor relapses, enlarges and metastasizes, it can be judged in time whether the patient will respond to immunotherapy Acquired drug resistance is generated to facilitate the follow-up treatment of patients; it has good sensitivity, specificity and accuracy, and can predict the acquired drug resistance of immunotherapy in samples, with an accuracy rate of over 93.3%, which is convenient for clinical detection Results Adjust the treatment plan as soon as possible, and the application prospect is broad.

Description of drawings

Figure 1 is a schematic flow chart of the early screening method for immunotherapy acquired drug resistance for the purpose of non-disease diagnosis and/or treatment in this application;

Figure 2 is a picture of the analysis results of single-cell sequencing data of tumor samples that did not receive immunotherapy, did not respond after immunotherapy, and responded after immunotherapy in Example 1 of the present application;

Figure 3 is a picture of the classification results of different types of T cell clones in Example 2 of the present application;

Figure 4 is a picture of the cluster analysis and gene expression results of Exhaustion ^hi CXCL13 ⁺ CD8A ⁺ T cells and Exhaustion ^low CXCL13 ⁺ CD8A ⁺ T cells in Example 3 of the present application;

Fig. 5 is a result picture of the correlation between the ratio of cancer cell killing CD8 T cells having the same TCR sequence in tumor and peripheral blood in Example 4 of the present application;

Figure 6 is a picture of the statistical results of the proportion of peripheral blood cancer cell killer CD8 T cells in response to treatment and acquired drug resistance or non-response samples in Example 4 of the present application;

7 is a picture of the statistical results of the ratio of non-exhausted precursor T cells in cancer cell killing CD8 T cells in response to treatment and acquired drug resistance or non-response samples in Example 4 of the present application;

Fig. 8 is a picture of the statistical results of the prediction accuracy of immunotherapy acquired drug resistance in Example 6 of the present application.

Detailed ways

In order to further illustrate the technical means and effects adopted by the present application, the present application will be further described below in conjunction with the embodiments and accompanying drawings. It can be understood that the specific implementation manners described here are only used to explain the present application, but not to limit the present application.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through regular channels.

Example 1

In this example, the data of tumor samples corresponding to 33 cancer patients (head and neck cancer, lung cancer, and breast cancer) who did not receive immunotherapy, did not respond after immunotherapy, and responded after immunotherapy were collected, and the single-cell sequencing data of T cells were analyzed. . Among them, immunotherapy is PD-1 antibody therapy or PD-L1 antibody therapy. The antigen specificity of each CD8 T cell clone analyzed has been verified by in vitro experiments or relative computational analysis based on the results of in vitro experiments.

The result is shown in Figure 2. It can be seen that all CD8 T cell clones are divided into cancer cell killing CD8 T cells that can recognize tumor antigens, and non-cancer cell killing CD8 T cells that recognize specific influenza virus antigens. The results showed that cancer cell-killing CD8 T cell clones from tumors that did not receive immunotherapy and did not respond after treatment highly expressed depletion signaling genes (HAVCR2, ENTPD1, LAYN, PDCD1, TIGIT, LAG3 and CTLA4) and CXCL13, marked by Exhaustion ^hi CXCL13 ⁺ CD8A ⁺ T cells. Cancer cell-killing CD8 T cells from tumors that respond after treatment have low expression of exhaustion signaling genes and high expression of CXCL13, which are labeled as Exhaustion ^low CXCL13 ⁺ CD8A ⁺ T cells.

Example 2

The expression of CXCL13 and CD8A of all CD4 T cell clones and CD8 T cell clones of the sample in Example 1 was analyzed. The results are shown in Figure 3, the expression levels of CD8A and CXCL13 can effectively identify cancer cell killing CD8 T cell clones. In this embodiment, 0.9 is directly used as the threshold value of CD8A expression level, and 0.15 is used as the threshold value of CXCL13 expression level. If the expression level of CD8A is greater than or equal to 0.9 and the expression level of CXCL13 is greater than or equal to 0.15, it is considered to be a cancer cell killing CD8 T cell clone. Using this method to screen T cell clones, the accuracy rate can reach 96.9%.

Example 3

Unsupervised clustering analysis was performed on all Exhaustion ^hi CXCL13 ⁺ CD8A ⁺ T cells and Exhaustion ^low CXCL13 ⁺ CD8A ⁺ T cells in Example 1, and it was found that these cancer cell killing CD8 T cells can be divided into 4 subgroups (as shown in 4), including proliferative T cells (high expression of MKI67 and STMN1 and other genes), exhausted T cells (high expression of HAVCR2, ENTPD1, LAYN, PDCD1, TIGIT, LAG3 and CTLA4 and other genes) and non-exhausted precursor T cells , non-exhausted precursor T cells can be further divided into GZMK-positive precursor T cells and IL7R-positive precursor T cells (high expression of IL7R, TCF7, LEF1, CCR7 and SELL genes).

Example 4

In this example, the single-cell sequencing data of 9 tumor samples from 33 patients and the corresponding 9 peripheral blood TCR sequencing data were analyzed, and the cancer cell killing CD8 of non-exhausted precursor T cells in the tumor were calculated respectively. The proportion of T cells, and the overall proportion of the clones with the same TCR sequence in the peripheral blood corresponding to this group of cells, it was found that the proportion of cancer cell killing CD8 T cells in the peripheral blood was significantly correlated with the proportion in the tumor (As shown in FIG. 5 ), it shows that the change of the killing CD8 T cells of the cancer cells in the tumor can be judged by detecting the changes of the killing CD8 T cells of the peripheral blood cancer cells.

The proportion of cytotoxic CD8 T cells in the peripheral blood of cancer patients in the response phase of cancer patients during treatment was further compared with the overall proportion of clones with the same TCR sequence in the peripheral blood of this group of cells in the phase of acquired drug resistance or non-response in patients, It was found that when patients developed acquired drug resistance, the proportion of cancer-killing CD8 T cells in peripheral blood was significantly reduced (as shown in Figure 6).

And calculated the ratio of the four subsets of cancer cell killing CD8 T cells in the tumor, and found that non-exhausted precursor T cells (including GZMK positive precursor T cells and IL7R positive precursor T cells) The proportion of T cell clones was higher in tumors that responded to treatment and lower in tumors that developed acquired resistance after treatment and in tumors that did not respond to treatment (as shown in Figure 7), indicating that The proportion of non-exhausted precursor T cells in cancer cell killing CD8 T cells in tumors combined with the change in the proportion of peripheral blood cancer cell killing CD8 T cells can be a good early estimate of whether patients have acquired resistance to immunotherapy. screening.

Example 5

This embodiment provides an early screening device for acquired drug resistance in immunotherapy, which includes:

Clustering analysis module: perform unsupervised clustering analysis on the cancer cell killing CD8 T cell clones, screen out non-exhausted precursor T cells, and calculate the rate of killing of the cancer cells by the non-exhausted precursor T cells The proportion of positive CD8 T cell clones, denoted as A1;

Example 6

This example uses the early screening device for immunotherapy acquired drug resistance in Example 5, and 15 samples that respond to immunotherapy without drug resistance and 15 samples that respond to immunotherapy have drug resistance or treatment non-response The samples were analyzed and the accuracy of the results was evaluated. The results are shown in FIG. 8 . The early screening device for immunotherapy acquired drug resistance was used to predict the status of immunotherapy acquired drug resistance of samples, and the accuracy rate reached 93.3%.

In summary, this application monitors the changes in the proportion of peripheral blood cancer cell killing CD8 T cell clones, combined with the change in the proportion of non-exhausted precursor T cells in cancer cell killing CD8 T cell clones in biopsy samples, can It is very early screening for acquired drug resistance to immunotherapy, which is convenient for adjusting the treatment plan in time. It is easy to operate, accurate in results, and can relieve the suffering of patients, which has extremely high application value.

The applicant declares that the present application illustrates the detailed method of the present application through the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the application must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of each raw material of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

Claims

An early screening device for immunotherapy acquired drug resistance, which includes:

Sequencing module: take biopsy tissue for single-cell sequencing, classify cells according to T cell receptor sequence, and calculate the average value of each gene expression;

Screening module: According to the threshold of CD8A and CXCL13 expression, screen out CD8A and CXCL13 double-positive clones, which are cancer cell killing CD8 T cell clones;

Cluster analysis module: perform cluster analysis on the cancer cell killing CD8 T cell clones, screen out non-exhausted precursor T cells, and calculate the number of non-exhausted precursor T cells in the cancer cell killing CD8 The proportion of T cell clones, denoted as A1;

Monitoring module: Perform single-cell sequencing on peripheral blood, screen out CD8A and CXCL13 double-positive clones, that is, peripheral blood cancer cell killing CD8 T cell clones, calculate the proportion of peripheral blood cancer cell killing CD8 T cell clones, and perform monitor;

Analysis module: when the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is significantly decreased, the biopsy tissue is taken again for single-cell sequencing, and the proportion of non-exhausted precursor T cells in the killing CD8 T cell clones of the cancer cells is calculated. The ratio is recorded as A2, and the acquired drug resistance of the sample to immunotherapy is judged according to the difference between A1 and A2.
The device for early screening of immunotherapy-acquired drug resistance according to claim 1, wherein the single-cell sequencing includes 10×Genomics 5' single-cell transcriptome library sequencing;

The cluster analysis includes unsupervised cluster analysis;

The peripheral blood cancer cell killing CD8 T cell clone has the same T cell receptor sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue.
The device for early screening of immunotherapy-acquired drug resistance according to claim 1, wherein the criteria for a significant decrease in the ratio of peripheral blood cancer cell killer CD8 T cell clones are:

The proportion of the killing CD8 T cell clone of the peripheral blood cancer cells is less than 50% compared with the proportion during the effective period of treatment, and no significant decline occurs;

The ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is greater than or equal to 50% compared with the ratio during the effective treatment period, which is a significant decline.
The early screening device for immunotherapy acquired drug resistance according to claim 1, wherein the criteria for judgment are:

The difference between A1 and A2 is greater than or equal to 0.39, and the sample has acquired drug resistance to immunotherapy;

The difference between A1 and A2 is less than 0.39, and the sample has no acquired drug resistance to immunotherapy.
The early screening device for immunotherapy acquired drug resistance according to claim 1, wherein the early screening device for immunotherapy acquired drug resistance comprises:

Sequencing module: Take the biopsy tissue during the effective period of treatment and perform 10×Genomics 5' single-cell transcriptional library sequencing to obtain single-cell gene expression data and T-cell receptor sequencing data, and classify the cells according to the T-cell receptor sequence, and Calculate the average value of each gene expression;

Screening module: According to the threshold of CD8A and CXCL13 expression, screen out CD8A and CXCL13 double-positive clones, which are cancer cell killing CD8 T cell clones;

Clustering analysis module: perform unsupervised clustering analysis on the cancer cell killing CD8 T cell clones, screen out non-exhausted precursor T cells, and calculate the rate of killing of the cancer cells by the non-exhausted precursor T cells The proportion of positive CD8 T cell clones, denoted as A1;

Monitoring module: Perform 10×Genomics 5'single-cell transcriptional library sequencing on peripheral blood to screen out clones that are double positive for CD8A and CXCL13 and have the same T cell receptor sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue , which is the killing CD8 T cell clone of peripheral blood cancer cells, the ratio of the killing CD8 T cell clones of peripheral blood cancer cells is calculated and monitored;

Analysis module: when the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is greater than or equal to 50% compared with the ratio during the effective treatment period, the biopsy tissue is taken again for 10×Genomics 5'single-cell transcriptional library sequencing, and the calculation The proportion of non-exhausted precursor T cells in the cancer cell-killing CD8 T cell clone is recorded as A2, and the acquired drug resistance of the sample to immunotherapy is judged according to the difference between A1 and A2. The standard is:

The difference between A1 and A2 is greater than or equal to 0.39, and the sample has acquired drug resistance to immunotherapy;

The difference between A1 and A2 is less than 0.39, and the sample has no acquired drug resistance to immunotherapy.
A method for early screening of immunotherapy acquired drug resistance for the purpose of non-disease diagnosis and/or treatment, comprising:

Take the biopsy tissue for single-cell sequencing, classify the cells according to the T cell receptor sequence, and calculate the average value of each gene expression;

According to the threshold of the expression of CD8A and CXCL13, clones that are double positive for CD8A and CXCL13 are screened out, which are cancer cell killing CD8 T cell clones;

Carrying out cluster analysis on the cancer cell killing CD8 T cell clones, screening out non-exhausted precursor T cells, and calculating the ratio of the non-exhausted precursor T cells in the cancer cell killing CD8 T cell clones Ratio, denoted as A1;

Perform single-cell sequencing on peripheral blood, screen out CD8A and CXCL13 double-positive clones, which are peripheral blood cancer cell killing CD8 T cell clones, calculate the ratio of peripheral blood cancer cell killing CD8 T cell clones, and monitor;

When the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells decreased significantly, the biopsy tissue was taken again for single-cell sequencing, and the ratio of non-exhausted precursor T cells in the killing CD8 T cell clones of the cancer cells was calculated, and recorded as It is A2. According to the difference between A1 and A2, the acquired drug resistance of the sample to immunotherapy is judged.
The method for early screening of acquired drug resistance to immunotherapy for the purpose of non-disease diagnosis and/or treatment according to claim 6, wherein the single-cell sequencing includes 10×Genomics 5' single-cell transcriptome library sequencing;

The cluster analysis includes unsupervised cluster analysis;

The peripheral blood cancer cell killing CD8 T cell clone has the same T cell receptor sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue.
The method for early screening of immunotherapy acquired drug resistance for the purpose of non-disease diagnosis and/or treatment according to claim 6, wherein the criterion for a significant decrease in the ratio of the peripheral blood cancer cell killing CD8 T cell clones is :

The proportion of the killing CD8 T cell clone of the peripheral blood cancer cells is less than 50% compared with the proportion during the effective period of treatment, and no significant decline occurs;

The proportion of the killing CD8 T cell clone of the peripheral blood cancer cells is greater than or equal to 50% compared with the proportion during the effective period of treatment, and a significant decrease occurs;

The criteria for the judgment are:

The difference between A1 and A2 is greater than or equal to 0.39, and the sample has acquired drug resistance to immunotherapy;

The difference between A1 and A2 is less than 0.39, and the sample has no acquired drug resistance to immunotherapy.
The early screening method for immunotherapy acquired drug resistance for the purpose of non-disease diagnosis and/or treatment according to claim 6, wherein the early screening method comprises:

Biopsy tissues during the effective period of treatment were taken for 10×Genomics 5' single-cell transcriptional library sequencing to obtain single-cell gene expression data and T-cell receptor sequencing data. Cells were classified according to the T-cell receptor sequence, and each The average value of gene expression;

According to the threshold of the expression of CD8A and CXCL13, clones that are double positive for CD8A and CXCL13 are screened out, which are cancer cell killing CD8 T cell clones;

Carrying out unsupervised clustering analysis on the cancer cell killing CD8 T cell clones, screening out non-exhausted precursor T cells, and calculating the number of non-exhausted precursor T cells in the cancer cell killing CD8 T cell clones The ratio in is recorded as A1;

Perform 10×Genomics 5'single-cell transcriptional library sequencing on peripheral blood, and screen out clones that are double positive for CD8A and CXCL13 and have the same T cell receptor sequence as the cancer cell killing CD8 T cell clone in the biopsy tissue, namely Killing CD8 T cell clones of peripheral blood cancer cells, calculating and monitoring the ratio of killing CD8 T cell clones of peripheral blood cancer cells;

When the ratio of the killing CD8 T cell clones of the peripheral blood cancer cells is greater than or equal to 50% compared with the ratio during the effective period of treatment, the biopsy tissue is taken again for 10×Genomics 5'single-cell transcriptional library sequencing, and the non-depletion rate is calculated. The proportion of precursor T cells in the cancer cell killing CD8 T cell clone is recorded as A2, and the acquired drug resistance of the sample to immunotherapy is judged according to the difference between A1 and A2, and the criteria for the judgment are:

The difference between A1 and A2 is greater than or equal to 0.39, and the sample has acquired drug resistance to immunotherapy;

The difference between A1 and A2 is less than 0.39, and the sample has no acquired drug resistance to immunotherapy.
The early screening device for immunotherapy-acquired drug resistance according to any one of claims 1-5 and/or the acquisition of immunotherapy for the purpose of non-disease diagnosis and/or treatment according to any one of claims 6-9 The application of the early screening method of sexual drug resistance in the preparation of immunotherapy acquired drug resistance screening products.