CN113238058A - Method for evaluating CAR-T treatment initiation T cells - Google Patents
Method for evaluating CAR-T treatment initiation T cells Download PDFInfo
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Abstract
The invention relates to the technical field of immune cell therapy, in particular to a method for evaluating CAR-T treatment initiation T cells. The invention provides a method for evaluating CAR-T treatment initiation T cells, which comprises the steps of carrying out first detection on peripheral blood to obtain a first test result comprising a CD4/CD8 ratio and the percentage of Treg cells in CD4+ T cells, carrying out second detection on the peripheral blood to obtain a second test result comprising the contents of perforin and granzyme in CD3+ CD8+ T cells, the content of PD-1 on the surfaces of CD3+ T cells and the content of PD-L1 on the surfaces of CD3+ T cells, judging whether the first test result meets a first preset model and whether the second test result meets a second preset model in sequence, and judging whether the CAR-T treatment initiation T cells in the peripheral blood are qualified or not, thereby evaluating whether patients can successfully prepare the CAR-T cells and the curative effect of the CAR-T cells.
Description
Technical Field
The invention relates to the technical field of immune cell therapy, in particular to a method for evaluating CAR-T treatment initiation T cells.
Background
Adoptive Cell Therapy (ACT) for cancer refers to the isolation of natural "starting immune cells" from patients or healthy volunteers, which are activated and amplified in vitro for a short period of time, then transformed (or not) by special genetic engineering techniques, and then continuously amplified to the amount needed for treatment and then infused back into the patients, thus achieving the purpose of enhancing immunity and killing tumor cells. Adoptive cell therapy is a brand-new 'anti-tumor active drug therapy technology' created on the basis of the traditional anti-cancer therapy method, and creates a new starting point for overcoming cancers. According to the history development process of adoptive cell therapy, the products mainly comprise early broad-spectrum nonspecific anti-tumor cells such as LAK, CIK, DC and NK cells and new products of targeted killing tumor cells such as CAR-T and TCR-T which are rapidly developed in recent years.
CAR-T therapy (Chimeric Antigen Receptor T-Cell Immunotherapy) refers to Chimeric Antigen Receptor T-Cell Immunotherapy, which is a CAR-T targeted immune Cell (CAR-T Cell for short) formed by genetically modifying a patient's T Cell (an immune Cell of the human body), namely an "initiating T Cell", so that the CAR-T targeted immune Cell can kill cancer cells in the body. At present, targeting immune cell therapy products represented by CAR-T cells obtain favorable curative effects on clinical antitumor application. Three CAR-T cell products are approved to be on the market in European and American countries, and a plurality of products are in clinical tests in China in formal clinical use, and the results show that the curative effect and the safety are similar to those of developed countries in European and American countries and are about to formally enter the clinic. Meanwhile, a plurality of clinical researches are carried out in a normative and orderly manner, and positive influences are brought to the benefit of patients and the drug development.
Although clinical data show that CAR-T products currently on the market or in clinical trials achieve encouraging results and have promising application prospects, some difficulties have been encountered in clinical trials. One of the key challenges is that some patients cannot successfully prepare and obtain the lowest therapeutic requirement of CAR-T cells in vitro, and few other patients, even if they obtain sufficient numbers of CAR-T cells, show immune tolerance or no response to therapy after reinfusion into the patient. In conclusion, failure of CAR-T cell production or reduced antitumor function in these patients would not benefit from this novel therapeutic approach. In addition, the clinical popularization and application of the therapy is limited because the success of CAR-T cell preparation and therapeutic effect of patients cannot be determined at an early stage. Therefore, early assessment of whether a patient can successfully prepare CAR-T cells and benefit from such therapy is a challenge to be solved, and there is no unified consensus on the current research in this regard.
In response to the above problems, applicants analyzed the therapeutic procedure of adoptive immune cell therapy "CAR-T cells". First, peripheral blood of a patient is collected to prepare peripheral blood mononuclear cells (PBMCs, cells having a mononuclear in the peripheral blood, including lymphocytes and monocytes) as seed cells of "CAR-T cells", i.e., "starting immune cells". Then, the "starting T cells" in these "starting immune cells" were genetically modified to prepare "CAR-T cells". Finally, the CAR-T cells are expanded to a therapeutic amount and infused back into the patient to kill tumor cells. By root tracing, the applicant found that the number and quality of "starting T cells" are undoubtedly very important, and the number and quality of the starting T cells are decisive factors for the success and the efficacy of the CAR-T cell preparation.
The research and evaluation technical guide principle of the cell therapy products and the quality control and guidance opinions of the CAR-T cell products are related to the consensus, and the quality control and guidance opinions are provided for the treatment of the CAR-T cell products from the blood collection of patients, the inspection of the blood collection, the enrichment of lymphocytes, the activation, transduction and expansion of T cells. Before CAR-T cell preparation, blood cell collection mainly emphasizes modes, components and limited applications, and mainly detects information such as cell survival rate, cell total amount, cell purity, B cell residual amount and the like. No study and guidance was given on the quantity and quality of the "starting T cells" collected, only the quality control of the CAR-T cell preparation.
Therefore, there is a need to provide a method for assessing CAR-T treatment initiating T cells.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, the present invention provides a method for evaluating CAR-T therapy-initiating T cells, comprising performing a first test on peripheral blood to obtain a first test result comprising a CD4/CD8 ratio and a percentage of Treg cells in CD4+ T cells, performing a second test on peripheral blood to obtain a second test result comprising a perforin and granzyme content in CD3+ CD8+ T cells, a CD3+ T cell surface PD-1 content, and a CD3+ T cell surface PD-L1 content, determining whether the first test result satisfies a first predetermined model and whether the second test result satisfies a second predetermined model, determining whether the CAR-T therapy-initiating T cells in peripheral blood are acceptable, thereby visually evaluating whether a patient can successfully prepare CAR-T cells and the efficacy of CAR-T cells, and evaluating whether a tumor patient can benefit from CAR-T cell therapy, the applicability of the therapy to patients is determined, the new immune cell therapy is promoted, more tumor patients can consider to select the therapy and benefit, part of patients are prompted to select the therapy with caution, and other treatment methods are actively sought, so that the time and economic cost of the patients are reduced, and the clinical significance and the value are important.
The purpose of the invention is realized by adopting the following technical scheme:
a method of assessing CAR-T treatment initiating T cells comprising the steps of:
s1: taking peripheral blood to perform first detection to obtain a first test result comprising a CD4/CD8 ratio and the percentage of Treg cells in CD4+ T cells;
s2: inputting the first test result into a first preset model, judging whether the first test result meets the first preset model, if not, judging that the CAR-T treatment starting T cells in the peripheral blood are unqualified, and if so, executing S3;
s3: performing second detection on the peripheral blood to obtain a second test result comprising the contents of perforin and granzyme in CD3+ CD8+ T cells, the content of PD-1 on the surfaces of CD3+ T cells and the content of PD-L1 on the surfaces of CD3+ T cells;
s4: inputting the second test result into a second preset model, judging whether the second test result meets the second preset model, and if not, judging that the CAR-T treatment starting T cells in the peripheral blood are unqualified;
wherein the first preset model is a correlation model of a first test result and the quality of the CAR-T treatment initiation T cells, and the second preset model is a correlation model of a second test result and the quality of the CAR-T treatment initiation T cells.
The "starting T cell" factor requires consideration of whether CAR-T cells can be successfully prepared and benefit the patient from the therapy. First, in terms of quantity, as an immune cell that can be engineered to effectively kill tumor cells, the "starting T cells" must reach a certain number to be considered for the preparation of CAR-T cells. In a second functional aspect, the "initiating T cells" with complete cellular immune function have the ability to kill tumor cells after modification. Perforin and granzyme are proteins that exert their associated functions in cell-mediated cytotoxicity. Perforin (also known as pore forming protein, PFP) and granzyme (granzyme) are exogenous serine proteases, which are stored in the cytosol of cytotoxic T cells and NK cells, trigger granule exocytosis by contact with target cells, and the released perforin forms pores on the surface of target cells by polymerization, and granzyme enters target cells through pores, triggering cell death and lysis through different pathways. PD-1 (programmed death receptor 1), also known as CD279, is an important immunosuppressive molecule that prevents the immune system from killing cancer cells by down-regulating the immune system's response to human cells, and by inhibiting T-cell inflammatory activity to regulate the immune system and promote self-tolerance. PD-L1 (programmed cell death-ligand 1), also known as CD274, binds to PD1 and transmits inhibitory signals that inhibit T cell activity and proliferation. In addition, Regulatory T cells (Tregs) are a subset of T cells that control autoimmune reactivity in vivo, and are also a type of immune negative immune cells. These inhibitory factors can inhibit differentiation, proliferation and killing functions of effector T cells, leading to late CAR-T cell failure or reduced antitumor function, thereby preventing such patients from benefiting from the novel treatment.
Thus, the method of the invention for assessing CAR-T treatment-initiating T cells provides a first test result comprising the ratio CD4/CD8 and the percentage of Treg cells in CD4+ T cells in peripheral blood, and a second test result comprising the contents of perforin and granzyme in CD3+ CD8+ T cells, the content of PD-1 on the surface of CD3+ T cells and the content of PD-L1 on the surface of CD3+ T cells in peripheral blood, wherein the test indices comprise the contents and ratios of various T lymphocytes, the contents of T lymphoblastin and granzyme, the expression levels of PD-1 and PD-L1, and the like.
Due to the effects of various factors such as tumor cell load, previous chemotherapy and the like, the quantity and quality of 'starting T cells' of patients have obvious difference, and the current detection and evaluation aiming at the 'starting T cells' still has no scientific and effective method. Thus, a method for assessing "starting T cells" in a patient was developed to assess early the success of the patient in producing CAR-T cells and the efficacy of CAR-T cells. The method for evaluating the CAR-T treatment initiation T cells can proactively evaluate whether tumor patients can benefit from CAR-T cell therapy, so that the applicability of the therapy to the patients is clear, the method is favorable for popularizing the emerging immune cell therapy, more tumor patients can consider selecting the therapy and benefit, part of the patients are prompted to select the therapy with caution, other treatment methods are actively sought, the time and economic cost of the patients are reduced, and the method has important clinical significance and value.
In the invention, the CAR-T treatment initiation T cells of patients who are intentionally subjected to CAR-T cell therapy can be evaluated only by collecting 3-5 mL of peripheral blood.
Preferably, S1 is preceded by:
taking a peripheral blood sample for detection, and collecting a first test result comprising a CD4/CD8 ratio and the percentage of Treg cells in CD4+ T cells and a second test result comprising the contents of perforin and granzyme in CD3+ CD8+ T cells, the content of PD-1 on the surfaces of CD3+ T cells and the content of PD-L1 on the surfaces of CD3+ T cells;
obtaining a trained first preset model by taking the first preset model as a first training model, taking the first test result as an input index of the first training model and taking the sample quality as an output result;
and taking a second preset model as a second training model, wherein the second test result is an input index of the second training model, and the sample quality is an output result, so as to obtain the trained second preset model.
Preferably, the CD4/CD8 ratio is calculated as the percentage of CD3+ CD4+ T cells to CD3+ CD8+ T cells.
Preferably, the first test result further comprises total T lymphocyte concentration;
the total T lymphocyte concentration is measured as CD3+ T cell concentration.
Preferably, in the first preset model, the ratio of CD4/CD8 is not less than 1.
Preferably, in the first pre-set model, the percentage of Treg cells in CD4+ T cells is no greater than 8.16%.
Preferably, in the first pre-set model, the concentration of CD3+ T cells is not less than 400/μ L.
Preferably, in the second pre-set model, the perforin content in CD3+ CD8+ T cells is not less than 25%, and the granzyme content in CD3+ CD8+ T cells is not less than 25%.
Preferably, in the second pre-set model, the content of PD-1 on the surface of CD3+ T cells is not more than 3.25%, and the content of PD-L1 on the surface of CD3+ T cells is not more than 0.73%.
The method for evaluating CAR-T treatment initiation T cells defines the reference ranges of all indexes in the first preset model and the second preset model, so that whether a patient can prepare CAR-T cells or not and the curative effect of the CAR-T cells are evaluated in a pre-visible manner.
Preferably, the first assay and the second assay are both flow cytometry assays.
Flow Cytometry (FCM) is a biological technique used to count or sort microscopic particles suspended in a fluid. The biological properties of single cells or organelles are rapidly determined by detecting the single cells marked with fluorescent signals flowing through an optical or electronic detector, and continuous multiple-parameter analysis is carried out, thereby realizing the technology of high-speed one-by-one quantitative analysis and sorting of cells. The method is characterized in that a plurality of important parameters such as cell DNA content, cell volume, protein content, enzyme activity, cell membrane receptor, surface antigen and the like are quantitatively determined by rapidly determining Coulter resistance, fluorescence, light scattering and light absorption. FCM plays a crucial role in diagnosis, prognosis and treatment of various hematological diseases by detecting and analyzing specific components, surface antigens or DNA in peripheral blood cells or bone marrow cells. The first detection and the second detection are flow cytometry detection, and the detection is rapid, accurate, scientific and efficient.
Compared with the prior art, the invention has the beneficial effects that:
according to the method for evaluating CAR-T treatment initiation T cells, the first test result and the second test result comprise 'initiation T cell' indexes which influence the successful preparation and curative effect of CAR-T cells, and the method has definite meanings, judges whether the first test result meets the first preset model and whether the second test result meets the second preset model in sequence, and judges whether the CAR-T treatment initiation T cells are qualified, so that whether patients can successfully prepare the CAR-T cells and the curative effect of the CAR-T cells are preliminarily evaluated.
The method for evaluating the CAR-T treatment initiation T cells has important clinical practical value, CAR-T cell therapy is a new anti-tumor therapy, brings new hopes for tumor patients, can determine the applicability of the therapy to the patients by rapidly evaluating the initiation T cells of the patients, can consider to select the therapy and benefit more tumor patients for popularizing the new immune cell therapy, and has important clinical significance.
The method for evaluating the CAR-T treatment initiation T cells can quickly evaluate the initiation T cells, can preliminarily evaluate whether tumor patients can benefit from CAR-T cell therapy, evaluates patients with poor curative effect of the initiation T cells, and requires doctors and patients to carefully select the therapy and actively consider other treatment strategies, so that the time and economic cost of the patients are reduced, and the method has important clinical significance and value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a graph showing the results of flow cytometry for detection of CD3+ T cells in example 2;
FIG. 2 is a graph showing the results of flow cytometry detection of the ratio of CD4/CD8 in example 2;
FIG. 3 is a graph of the results of flow cytometry for the determination of the percentage of Treg cells in CD4+ T cells in example 2;
FIG. 4 is a graph showing the results of flow cytometry for detecting CD3+ T cells in example 3;
FIG. 5 is a graph showing the results of flow cytometry detection of the ratio of CD4/CD8 in example 3;
FIG. 6 is a graph of the results of flow cytometry for the detection of the percentage of Treg cells in CD4+ T cells in example 3;
FIG. 7 is a graph showing the results of flow cytometry for the determination of perforin content in CD3+ CD8+ T cells in example 3;
FIG. 8 is a graph showing the results of flow cytometry for detecting the granzyme content in CD3+ CD8+ T cells in example 3;
FIG. 9 is a graph showing the results of measuring the surface PD-1 content of CD3+ T cells by flow cytometry in example 3;
FIG. 10 is a graph showing the results of measuring the PDL-1 content on the surface of CD3+ T cells by flow cytometry in example 3.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment. The following are specific examples of the present invention, and raw materials, equipments and the like used in the following examples can be obtained by purchasing them unless otherwise specified.
Example 1
The embodiment provides a method for constructing a first preset model and a second preset model.
After signing an informed consent, carrying out detection before conventional treatment and evaluation of the state of a patient who is clinically and intentionally subjected to CAR-T cell immune cell treatment, wherein in the conventional blood detection, the number of white blood cells of the patient is 4-10 multiplied by 109Within a normal medical reference value range of individuals/L. When CAR-T cell preparation is carried out and patient cells are collected, 3-5 mL of peripheral blood is collected through a heparin tube vein. And 3-5 ml of peripheral blood of normal volunteers is collected by using a heparin tube vein. Detecting the origin of peripheral blood of a patient and a normal person by adopting a flow cytometer within 2-6 hoursStarting T cell "number and quality, detection metrics include: peripheral blood total T lymphocytes (CD3+ T cells), helper T lymphocytes (CD3+ CD4+ T cells), cytotoxic T lymphocytes (CD3+ CD8+ T cells), regulatory T cells (Treg cells), CD4/CD8 ratio, PD-1 and PD-L1 content on different T cells, and granzyme and perforin content.
1. Flow cytometry detection of T cell subsets
Taking 100-200 mu L of whole blood, adding antibodies CD4-FITC, CD8-PE, CD3-PC5 and CD45-PC7 into 5-15 mu L of whole blood, reacting at room temperature for 15min under the condition of keeping out of the sun, adding 900 mu L of hemolytic agent, standing at room temperature for 10min under the condition of keeping out of the sun, detecting by using a flow cytometer, analyzing the percentage of CD3+ T cells in all nucleated cells, and calculating the ratio of CD4/CD8 according to the percentage of CD3+ CD4+ T cells to CD3+ CD8+ T cells. The total nucleated cell number is counted conventionally, and the absolute number of CD3+ T cells is calculated according to the formula: CD3+ T cells absolute value-total nucleated cell number × lymphocyte proportion (total nucleated cell proportion) × CD3+ T cells proportion (lymphocyte proportion).
2. Cytometric detection of Treg cells
And (3) taking 100-200 mu L of whole blood, adding 5-15 mu L of each of antibodies CD25-FITC, CD127-PE and CD4-PC5, reacting for 15min at room temperature in a dark condition, adding 900 mu L of hemolytic agent, standing for 10min at room temperature in a dark condition, and detecting by using a flow cytometer. The percentage of Treg cells (CD4+ CD25+ CD127low) on CD4+ T cells was analyzed.
3. Detection of perforin and granzyme content in CD3+ CD8+ T cells
Taking 100-200 mu L of whole blood, adding 5-15 mu L of each of antibodies CD3-FITC, CD8-ECD, CD56-APC, CD4-APC A700 and CD45-PB, reacting at room temperature for 15min under a dark condition, adding 1100 mu L of reagent in a Beckmann membrane-breaking perforating kit, reacting at a dark condition for 15min at room temperature, adding 4mL of 0.9% physiological saline, centrifuging at 1500 rpm for 5min, then discarding supernatant, adding 2100 mu L of reagent, reacting at room temperature for 5min under a dark condition, adding 5-15 mu L of each of granzyme-Percyc5.5 and perforin-PE, reacting at room temperature for 15min under a dark condition, adding 4mL of 0.9% physiological saline, centrifuging at 1500 rpm for 5min, then discarding supernatant, detecting by a flow cytometer, and analyzing the contents of perforin CD3+ CD8+ T cells by analyzing the contents of perforin the CD3+ CD8+ T cells.
4. Detecting the content of PD-1 and PD-L1 on the surface of CD3+ T cells
Taking 100-200 mu L of whole blood, adding 10uL of each of antibodies CD3-FITC, CD4-PE, CD8-ECD, CD279-APC, CD274-PC7 and CD45-PB, reacting at room temperature for 15min under the condition of avoiding light, adding 900 mu L of hemolytic agent, standing at room temperature for 10min under the condition of avoiding light, centrifuging for 5min under the condition of 1500 r/min, then removing supernatant, carrying out flow cytometry detection, and analyzing the content of PD-1 and PDL-1 on the surfaces of CD3+ T cells.
According to whether the CAR-T cells are successfully prepared in the later stage and the curative effect of the CAR-T cells, the CAR-T cells are divided into a CAR-T cell preparation failure group, a CAR-T treatment poor group and a CAR-T treatment effective group, and the expression levels and the differences of all indexes among the three groups are compared. And (3) establishing the expression level and the reference range of the indexes of the normal person by detecting the indexes of the normal person.
First, CD3+ T cells require a certain number to genetically modify and subsequently expand "starting T cells". According to the blood routine and flow cytometry detection, the number of CD3+ T cells of a patient is calculated to reach more than 400 cells/microliter, and then the CAR-T cells can be prepared.
After statistical analysis of the CAR-T cell preparation failure group, the CAR-T treatment poor group and the CAR-T treatment effective group, the finding that the reduction of the ratio of CD4/CD8 to the increase of the proportion of Treg cells is a negative factor of the preparation failure. The content of PD-1 and PD-L1 and the content of granzyme and perforin on the T cells are important factors influencing the therapeutic effect of the CAR-T cells, wherein the increased expression of PD-1 and PD-L1 and the reduced content of granzyme and perforin are important reasons for the poor therapeutic effect of the CAR-T cells. Therefore, the detection values of the CAR-T cell treated patients and normal people are analyzed, and a first preset model and a second preset model which comprise the standard reference values of the indexes are established and used for evaluating whether the patients can successfully prepare the CAR-T cells and the curative effect of the CAR-T cells.
The CD4/CD8 ratio of less than 1 and the proportion of Treg cells exceeding 8.16% (in CD4+ T cells) are disadvantageous factors in the failure of CAR-T cell production, and it is important to note that the presence of either element can cause failure of CAR-T cell production. The content of perforin and granzyme in CD3+ CD8+ T cells is less than 25%, the proportion of PD-1 on CD3+ T cells is more than 3.25%, and the proportion of PD-L1 on CD-L1 is more than 0.73%, which are adverse factors of the reduction of the anti-tumor function of CAR-T cells and the non-response of treatment, and are shown in Table 1.
TABLE 1 CAR-T cell failure to prepare or treatment efficacy deficiency factors and reference values
In conclusion, in the first preset model, the concentration of the CD3+ T cells is not less than 400/muL, the ratio of the CD4/CD8 is not less than 1, and the percentage of Treg cells in the CD4+ T cells is not more than 8.16%; in the second preset model, the perforin content of CD3+ CD8+ T cells is not less than 25%, the granzyme content of CD3+ CD8+ T cells is not less than 25%, the PD-1 content on the surfaces of CD3+ T cells is not more than 3.25%, and the PD-L1 content on the surfaces of CD3+ T cells is not more than 0.73%. Predicting that the patient is likely to have failed CAR-T production or will not respond to treatment if the patient has one or more adverse factors, recommending no CAR-T treatment, and seeking other treatment; conversely, that the patient is not suffering from the above-mentioned disadvantages, can benefit from CAR-T cell therapy.
Example 2
1. Collecting peripheral blood of clinical patient
Tumor patients who were clinically willing to receive CAR-T cell immune cell therapy signed an informed consent and after routine pre-treatment testing and patient status assessment, 3mL of peripheral blood was collected intravenously using heparin tubes.
2. Flow cytometry detection of relevant indexes
After receiving the blood samples, total T lymphocytes (CD3+ T cells), helper T lymphocytes (CD3+ CD4+ T cells), cytotoxic T lymphocytes (CD3+ CD8+ T cells), regulatory T cells (Treg cells), CD4/CD8 ratio, PD-1 and PD-L1 content on CD3+ T cells, and granzyme and perforin content were measured, respectively, as follows:
2.1 flow cytometry detection of T cell subsets
Taking 100 mu L of whole blood, adding 10 mu L of each antibody CD4-FITC, CD8-PE, CD3-PC5 and CD45-PC7, reacting at room temperature for 15min under the condition of keeping out of the sun, adding 900 mu L of hemolytic agent, standing at room temperature for 10min under the condition of keeping out of the sun, detecting by a flow cytometer, analyzing the percentage of CD3+ T cells in all nucleated cells, and calculating the ratio of CD4/CD8 according to the percentage of CD3+ CD4+ T cells to CD3+ CD8+ T cells. The total nucleated cell number is counted conventionally, and the absolute number of CD3+ T cells is calculated according to the formula: CD3+ T cells absolute value-total nucleated cell number × lymphocyte proportion (total nucleated cell proportion) × CD3+ T cells proportion (lymphocyte proportion).
2.2 flow cytometry detection of Treg cells
And (3) adding 10 mul of each of antibodies CD25-FITC, CD127-PE and CD4-PC5 monoclonal antibody into 100 mul of whole blood, reacting for 15min at room temperature under the condition of keeping out of the light, adding 900 mul of hemolytic agent, standing for 10min at room temperature under the condition of keeping out of the light, and detecting by a flow cytometer. The percentage of Treg cells to CD4+ T cells was analyzed.
2.3 detection of perforin and granzyme levels in CD3+ CD8+ T cells
Taking 100 mu L of whole blood, adding 10 mu L of each of antibodies CD3-FITC, CD8-ECD, CD56-APC, CD4-APC A700 and CD45-PB monoclonal antibodies, reacting at room temperature for 15min under a light-proof condition, adding 1100 mu L of reagent in a Beckmann membrane-rupturing perforating kit, reacting at room temperature for 15min under a light-proof condition, adding 4mL of 0.9% physiological saline, centrifuging at 1500 rpm for 5min, then discarding supernatant, adding 2100 mu L of reagent, reacting at room temperature under a light-proof condition for 5min, adding 5 mu L of each of granzyme-Percyc5.5 and perforin-PE, reacting at room temperature under a light-proof condition for 15min, adding 4mL of 0.9% physiological saline, centrifuging at 1500 rpm for 5min, then discarding supernatant, detecting by a flow cytometer, and analyzing the contents of CD3+ CD8+ T cell pigment and granzyme.
2.4 detection of the content of PD-1 and PD-L1 on the surface of CD3+ T cells
Taking 100 mu L of whole blood, adding 10 mu L of each of antibodies CD3-FITC, CD4-PE, CD8-ECD, CD279-APC, CD274-PC7 and CD45-PB, reacting at room temperature for 15min under the condition of avoiding light, adding 900 mu L of hemolytic agent, standing at room temperature for 10min under the condition of avoiding light, centrifuging for 5min under the condition of 1500 r/min, removing supernatant, detecting by a flow cytometer, and analyzing the content of PD-1 and PDL-1 on the surfaces of CD3+ T cells.
2.5 inputting the first test result and the second test result into the first preset model and the second preset model in sequence
Referring to fig. 1 to 3, fig. 1 is a graph showing the results of flow cytometry for detecting CD3+ T cells in example 2, fig. 2 is a graph showing the results of flow cytometry for detecting the ratio of CD4/CD8 in example 2, and fig. 3 is a graph showing the results of flow cytometry for detecting the percentage of Treg cells in CD4+ T cells in example 2. The result shows that the ratio of CD4/CD8 is 0.55 and less than 1, the percentage of Treg cells in CD4+ T cells is 12.59 percent and more than 8.16 percent, the Treg cells are input into a first preset model, the first test result is judged not to meet the first preset model, the CAR-T treatment starting T cells are judged to be unqualified, and the subsequent CAR-T cells cannot be successfully prepared.
Example 3
1. Collecting peripheral blood of clinical patient
Tumor patients who were clinically willing to receive CAR-T cell immune cell therapy signed an informed consent and after routine pre-treatment testing and patient status assessment 4mL of peripheral blood was collected intravenously using heparin tubes.
2. Flow cytometry detection of relevant indexes
After receiving the blood samples, total T lymphocytes (CD3+ T cells), helper T lymphocytes (CD3+ CD4+ T cells), cytotoxic T lymphocytes (CD3+ CD8+ T cells), regulatory T cells (Treg cells), CD4/CD8 ratio, PD-1 and PD-L1 content on CD3+ T cells, and granzyme and perforin content were measured, respectively, as follows:
2.1 flow cytometry detection of T cell subsets
Taking 100 mu L of whole blood, adding antibodies CD4-FITC, CD8-PE, CD3-PC5 and CD45-PC7 into 5 mu L of whole blood, reacting at room temperature for 15min under the condition of keeping out of the sun, adding 900 mu L of hemolytic agent, standing at room temperature for 10min under the condition of keeping out of the sun, detecting by a flow cytometer, analyzing the percentage of CD3+ T cells in all nucleated cells, and calculating the ratio of CD4/CD8 according to the percentage of CD3+ CD4+ T cells to CD3+ CD8+ T cells. The total nucleated cell number is counted conventionally, and the absolute number of CD3+ T cells is calculated according to the formula: CD3+ T cells absolute value-total nucleated cell number × lymphocyte proportion (total nucleated cell proportion) × CD3+ T cells proportion (lymphocyte proportion).
2.2 flow cytometry detection of Treg cells
And (3) adding 5 mul of each of antibodies CD25-FITC, CD127-PE and CD4-PC5 monoclonal antibody into 100 mul of whole blood, reacting for 15min at room temperature under the condition of keeping out of the light, adding 900 mul of hemolytic agent, standing for 10min at room temperature under the condition of keeping out of the light, and detecting by a flow cytometer. The percentage of Treg cells to CD4+ T cells was analyzed.
2.3 detection of perforin and granzyme levels in CD3+ CD8+ T cells
Taking 100 mu L of whole blood, adding antibodies CD3-FITC, CD8-ECD, CD56-APC, CD4-APC A700 and CD45-PB monoclonal antibodies each of which is 5 mu L, reacting at room temperature for 15min under a light-proof condition, adding 1100 mu L of reagent in a Beckmann membrane-rupturing perforating agent box, reacting at room temperature for 15min under a light-proof condition, adding 3mL of 0.9% physiological saline, centrifuging at 1500 rpm for 5min, then discarding the supernatant, adding 2100 mu L of reagent, reacting at room temperature under a light-proof condition for 5min, adding 5 mu L of granzyme-Percyc5.5 and perforin-PE, reacting at room temperature under a light-proof condition for 15min, adding 3mL of 0.9% physiological saline, centrifuging at 1500 rpm for 5min, then discarding the supernatant, detecting by a flow cytometer, and analyzing the contents of CD3+ CD8+ T cell pigment and granzyme.
2.4 detection of the content of PD-1 and PD-L1 on the surface of CD3+ T cells
And (2) adding antibodies CD3-FITC, CD4-PE, CD8-ECD, CD279-APC, CD274-PC7 and CD45-PB into 100 mu L of whole blood, reacting at room temperature for 15min under a light-shielding condition, adding 900 mu L of hemolytic agent, standing at room temperature for 10min under a light-shielding condition, centrifuging for 5min under a 1500 rpm condition, removing supernatant, detecting by a flow cytometer, and analyzing the content of PD-1 and PDL-1 on the surfaces of CD3+ T cells.
2.5 inputting the first test result and the second test result into the first preset model and the second preset model in sequence
Referring to fig. 4 to 6, fig. 4 is a graph showing the results of flow cytometry for detecting CD3+ T cells in example 3, fig. 5 is a graph showing the results of flow cytometry for detecting the ratio of CD4/CD8 in example 3, and fig. 6 is a graph showing the results of flow cytometry for detecting the percentage of Treg cells in CD4+ T cells in example 3. The result shows that the ratio of CD4/CD8 is 1.52 and is larger than 1, the percentage of Treg cells in CD4+ T cells is 4.12 percent and is smaller than 8.16 percent, the Treg cells are input into the first preset model, and the first test result is judged to meet the first preset model.
Referring to fig. 7 to 10, fig. 7 is a graph showing the results of flow cytometry for detecting the content of perforin in CD3+ CD8+ T cells in example 3, fig. 8 is a graph showing the results of flow cytometry for detecting the content of granzyme in CD3+ CD8+ T cells in example 3, fig. 9 is a graph showing the results of flow cytometry for detecting the content of PD-1 on the surface of CD3+ T cells in example 3, and fig. 10 is a graph showing the results of flow cytometry for detecting the content of CD3+ PDL-1 on the surface of T cells in example 3. The results show that the CD3+ CD8+ T cell perforin content is 2.43%, less than 25%; the content of CD3+ CD8+ T cell granzyme is 5.95% and is less than 25%; the content of PD-1 on the surface of CD3+ T cells is 53.01 percent and is more than 3.25 percent; the content of CD3+ T cell PD-L1 is 5.98 percent and is more than 0.73 percent, the CD3+ T cell PD-L1 is input into a second preset model, the second test result is judged not to meet the second preset model, the CAR-T treatment starting T cell is judged to be unqualified, and the subsequent CAR-T cell treatment effect is not good.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method of assessing CAR-T treatment initiating T cells comprising the steps of:
s1: taking peripheral blood to perform first detection to obtain a first test result comprising a CD4/CD8 ratio and the percentage of Treg cells in CD4+ T cells;
s2: inputting the first test result into a first preset model, judging whether the first test result meets the first preset model, if not, judging that the CAR-T treatment starting T cells in the peripheral blood are unqualified, and if so, executing S3;
s3: performing second detection on the peripheral blood to obtain a second test result comprising the contents of perforin and granzyme in CD3+ CD8+ T cells, the content of PD-1 on the surfaces of CD3+ T cells and the content of PD-L1 on the surfaces of CD3+ T cells;
s4: inputting the second test result into a second preset model, judging whether the second test result meets the second preset model, and if not, judging that the CAR-T treatment starting T cells in the peripheral blood are unqualified;
wherein the first preset model is a correlation model of a first test result and the quality of the CAR-T treatment initiation T cells, and the second preset model is a correlation model of a second test result and the quality of the CAR-T treatment initiation T cells.
2. The method of claim 1, wherein S1 is preceded by the step of:
taking a peripheral blood sample for detection, and collecting a first test result comprising a CD4/CD8 ratio and the percentage of Treg cells in CD4+ T cells and a second test result comprising the contents of perforin and granzyme in CD3+ CD8+ T cells, the content of PD-1 on the surfaces of CD3+ T cells and the content of PD-L1 on the surfaces of CD3+ T cells;
obtaining a trained first preset model by taking the first preset model as a first training model, taking the first test result as an input index of the first training model and taking the sample quality as an output result;
and taking a second preset model as a second training model, wherein the second test result is an input index of the second training model, and the sample quality is an output result, so as to obtain the trained second preset model.
3. The method of claim 1, wherein said CD4/CD8 ratio is calculated as the percentage of CD3+ CD4+ T cells to CD3+ CD8+ T cells.
4. The method of claim 1, wherein said first test result further comprises total T lymphocyte concentration;
the total T lymphocyte concentration is measured as CD3+ T cell concentration.
5. The method of claim 1, wherein in said first predetermined model the CD4/CD8 ratio is not less than 1.
6. The method of claim 1, wherein the percentage of Treg cells in CD4+ T cells in said first pre-established model is no greater than 8.16%.
7. The method of claim 4, wherein said CD3+ T cell concentration in said first pre-set model is not less than 400/μ L.
8. The method of claim 1, wherein the perforin content of CD3+ CD8+ T cells and granzyme content of CD3+ CD8+ T cells is not less than 25% in said second pre-set model.
9. The method of claim 1, wherein the second pre-set model comprises no more than 3.25% PD-1 on the surface of CD3+ T cells and no more than 0.73% PD-L1 on the surface of CD3+ T cells.
10. The method of claim 1, wherein said first assay and said second assay are both flow cytometry assays.
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| US20200061113A1 (en) * | 2018-04-12 | 2020-02-27 | Novartis Ag | Chimeric antigen receptor therapy characterization assays and uses thereof |
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