System and method for determining amplification rate of CAR-T cells in body of lymphoma patient

Abstract

The embodiment of the invention discloses a system and a method for determining the amplification rate of CAR-T cells in a lymphoma patient, the system comprises the following formula: inputting an absolute count value of neutrophils to obtain the amplification rate of the CAR-T cells; the calculation formula is as follows: y = A1 * exp (-x / t1) + y0; wherein y < 0 > is equal to 0.06756 + / -0.06274, A < 1 > is equal to 0.77155 + / -0.14248, and t < 1 > is equal to 0.0826 + / -0.03373. According to the system, the amplification rate rho of the CAR-T cells in the lymphoma patient can be effectively predicted by using the absolute count value of the neutrophil, and the system is convenient, rapid and high in accuracy.

Description

Technical Field

[0001] Embodiments of the present invention relate to the field of biotechnology, and particularly to a system and method for measuring the amplification rate of CAR-T cells in lymphoma patients. Background Art

[0002] Lymphoma is a malignant tumor originating from the lymphohematopoietic system, mainly manifested as painless lymph node enlargement, hepatosplenomegaly, and all tissues and organs of the body can be involved, accompanied by systemic symptoms such as fever, night sweats, weight loss, and itching. Lymphoma has a high degree of heterogeneity, and the treatment effects vary greatly. There are significant differences in both the treatment intensity and prognosis for different pathological types and stages of lymphoma. The main treatment methods for lymphoma are as follows: (1) Radiotherapy: Some types of lymphoma can be treated with radiotherapy alone in the early stage. Radiotherapy can also be used for consolidation treatment after chemotherapy and adjuvant treatment during transplantation. (2) Chemotherapy with chemical drugs: Most lymphoma chemotherapies use combination chemotherapy, which can combine targeted therapeutic drugs and biological agents. In recent years, the chemotherapy regimens for lymphoma have been greatly improved, and the survival periods of many types of lymphoma have been significantly increased. (3) Bone marrow transplantation: For patients under 60 years old who can tolerate high-dose chemotherapy and are at high or intermediate risk, autologous hematopoietic stem cell transplantation can be considered. Allogeneic hematopoietic stem cell transplantation can also be considered for some young patients with recurrence or bone marrow invasion. (4) Surgical treatment: It is limited to biopsy or treatment of complications; for patients with hypersplenism and no contraindications, splenectomy can be performed if there are splenectomy indications to improve the blood picture and create favorable conditions for subsequent chemotherapy.

[0003] CAR-T cell immunotherapy refers to separating T cells from the patient's peripheral blood, genetically engineering them to carry a specific antigen recognition domain scfv and a co-stimulatory factor. After in vitro amplification, they are reinfused into the patient's body. These T cells can directly bind to the specific antigen on the surface of tumor cells and be activated to target and kill tumor cells, thereby achieving the purpose of treating tumors. CAR-T cell therapy has achieved remarkable results in the treatment of hematological malignancies in recent years and is expected to also make breakthroughs in solid tumors and autoimmune diseases.

[0004] Effective amplification of CAR-T cells in patients is the key to the success of CAR-T therapy. In addition, if CAR-T cells expand too fast, it is easy to cause side effects such as cytokine release syndrome (CRS) and immune cell therapy-induced neurotoxicity (ICANS). Effectively predicting the CAR-T amplification rate is crucial for predicting the efficacy of CAR-T and preventing side effects.

[0005] It is reported that monitoring the levels of peripheral blood IL-6 cytokine and ferritin can partly reflect the expansion of CAR-T. However, the disadvantage of this protocol is that IL-6 and ferritin are not specific and will also be upregulated in infected patients. In addition, the proliferation rate of CAR-T cannot be estimated by the levels of these two proteins.

[0006] Therefore, it is necessary to develop a system and method that can accurately and rapidly determine the expansion rate of CAR-T cells in lymphoma patients. Summary of the Invention

[0007] The object of the present invention is to provide a system and method for determining the expansion rate of CAR-T cells in lymphoma patients. Through this system, the absolute neutrophil count can be used to effectively predict the expansion rate ρ of CAR-T cells in lymphoma patients, which is convenient, rapid and has a high accuracy rate.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect of the embodiments of the present invention, a system for determining the expansion rate of CAR-T cells in lymphoma patients is provided. The system includes:

[0010] A processor and a memory, the memory is coupled to the processor, and the memory stores instructions. When the instructions are executed by the processor, the following steps are used:

[0011] Input the absolute neutrophil count into the following calculation formula to obtain the expansion rate of CAR-T cells; the calculation formula is as follows:

[0012] y = A1 × exp(-x / t1) + y0;

[0013] where y0 = 0.06756 ± 0.06274, A1 = 0.77155 ± 0.14248, t1 = 0.0826 ± 0.03373.

[0014] In the second aspect of the embodiments of the present invention, a computer-readable storage medium is provided. A computer program is stored on the computer-readable storage medium, and when the computer program is executed by a processor, the above steps are used.

[0015] In the third aspect of the embodiments of the present invention, an application of the system for determining the expansion rate of CAR-T cells in lymphoma patients or the computer-readable storage medium, the prediction system, in the preparation of products for determining the expansion rate of CAR-T cells in lymphoma patients or products for predicting the efficacy of CAR-T and preventing side effects is provided.

[0016] In the fourth aspect of the embodiments of the present invention, a product for measuring the expansion rate of CAR-T cells in lymphoma patients is provided, including:

[0017] A product for measuring the absolute neutrophil count;

[0018] And the system for measuring the expansion rate of CAR-T cells or the computer-readable storage medium as described above.

[0019] The product for measuring the absolute neutrophil count includes a hematology analyzer, or other kits or products capable of measuring the absolute neutrophil count.

[0020] In the fifth aspect of the embodiments of the present invention, a method for measuring the expansion rate of CAR-T cells in lymphoma patients is provided. The method includes: inputting the absolute neutrophil count into the system for measuring the expansion rate of CAR-T cells in lymphoma patients or the computer-readable storage medium to obtain the expansion rate of CAR-T cells.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] The system and method for measuring the expansion rate of CAR-T cells in lymphoma patients provided by the embodiments of the present invention can effectively predict the expansion rate ρ of CAR-T cells in lymphoma patients with the absolute neutrophil count, which is convenient, fast and has a high accuracy rate. At the same time, the neutrophil count is directly obtained from the patient's blood routine data, which has the characteristics of non-invasive, low cost, timely results, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 For the data results of Example 1, where Figure 1 A - B is a graph showing the change results of the absolute neutrophil count in peripheral blood of 45 patients from day 0 to day 14 after CAR-T infusion; Figure 1 C - E is a graph showing the pharmacokinetic results of peripheral blood CAR-T cells after the patients are grouped according to the degree of lack of absolute neutrophil count;

[0025] Figure 2 For the correlation analysis and calculation formula results graph of peripheral blood neutrophils and CAR-T expansion rate (ρ) in lymphoma patients;

[0026] Figure 3 ROC curve of the verification result of the prediction accuracy of the absolute neutrophil count for the CAR-T cell expansion rate (ρ). Detailed implementation mode

[0027] The embodiments of the present invention will be specifically described below in combination with the detailed implementation mode and embodiments, and the advantages and various effects of the embodiments of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation modes and embodiments are used to illustrate the embodiments of the present invention, rather than limiting the embodiments of the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as the general understanding of those skilled in the art to which the embodiments of the present invention belong. In case of contradiction, this specification shall prevail.

[0029] The general idea of the technical solution of the present invention is as follows:

[0030] The inventors of the present application found from the clinical data of dozens of patients receiving CAR-T treatment that after the CAR-T infusion, among the patients with severe neutropenia, the CAR-T expansion was better. We fitted a curve with the neutrophil count and the CAR-T cell expansion rate ρ, and the calculation formula of the curve is as follows:

[0031] y = A1×exp(-x / t1)+y0;

[0032] where y0 = 0.06756±0.06274, A1 = 0.77155±0.14248, t1 = 0.0826±0.03373; P = 5.43522 E -14, R-squared value (Predicted R-squared) = 0.25327;

[0033] The inventors of the present application found that through the above curve, neutrophils can effectively predict the CAR-T cell expansion rate ρ. Effectively predicting the CAR-T expansion rate is crucial for predicting the CAR-T efficacy and preventing side effects.

[0034] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the embodiments of the present invention can be obtained through market purchase or can be prepared by existing methods.

[0035] The following will detail a system and method for measuring the CAR-T cell expansion rate of the present application in combination with experimental data.

[0036] Example 1. Exploration of the relationship between absolute neutrophil count and CAR-T cell expansion rate

[0037] 1. Obtain the copy number of CAR-T cells and the absolute neutrophil count in peripheral blood:

[0038] Collect blood samples from 17 patients with multiple myeloma and 28 patients with lymphoma. A total of 45 peripheral blood samples from the day of CAR-T cell infusion (day 0) to day 14 were collected for lentivirus copy number detection. Based on the obtained copy number data, the CAR-T cell expansion rate (ρ) was calculated using the published rate calculation model (see the literature Frontiers in pharmacology vol.13 803693.2 Feb. 2022). At the same time, a fully automated hematology analyzer was used for blood cell detection, and the absolute neutrophil count was recorded. The results of the absolute neutrophil count are as Figure 1 shown.

[0039] The change results of the absolute neutrophil count in peripheral blood of 45 patients from day 0 to day 14 after CAR-T cell infusion are shown in Figure 1 A - B. It can be seen that after CAR-T cell infusion, patients will experience varying degrees of neutropenia, and it mostly occurs within 14 days after infusion.

[0040] The results of the pharmacokinetics of CAR-T cells in peripheral blood after grouping patients according to the degree of absolute neutrophil count deficiency are shown in Figure 1 C - E. It can be seen that if patients show more severe neutropenia after CAR-T cell infusion, this group of patients often has better CAR-T cell expansion.

[0041] 2. Correlation analysis between severe neutropenia and CAR-T cell expansion:

[0042] Using the results of the absolute neutrophil count data in peripheral blood, patients were divided into a neutropenia group and a non-neutropenia group, and pharmacokinetic analysis of CAR-T cells was performed on the two groups of patients to further reflect the CAR-T cell expansion situation.

[0043] From Figure 2 it can be seen that through differential analysis, patients in the neutropenia group have higher CAR-T cell expansion. At the same time, through correlation analysis, there is a clear negative correlation between the absolute neutrophil count and the CAR-T expansion rate (ρ).

[0044] 3. Model for measuring the CAR-T cell expansion rate

[0045] We fitted the neutrophil count and the CAR-T cell expansion rate ρ into a curve, and the calculation formula of the curve is as follows:

[0046] y = A1 × exp(-x / t1) + y0;

[0047] Wherein, y0 = 0.06756 ± 0.06274, A1 = 0.77155 ± 0.14248, t1 = 0.0826 ± 0.03373. In this curve, P = 5.43522 E -14, R-squared value (Predicted R-squared) = 0.25327, indicating that the curve has a good fitting degree.

[0048] Example 2. System for measuring the expansion rate of CAR-T cells

[0049] An embodiment of the present invention provides a system for measuring the expansion rate of CAR-T cells, and the system includes:

[0050] A processor and a memory, the memory is coupled to the processor, and the memory stores instructions that use the following steps when executed by the processor:

[0051] Input the absolute neutrophil count value into the model described in Example 1 to obtain the expansion rate of CAR-T cells.

[0052] Example 3. Computer-readable storage medium

[0053] An embodiment of the present invention provides a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, it implements the steps of the method in Example 1 and / or the method in Example 2.

[0054] Of course, for a storage medium provided by an embodiment of the present invention that contains computer-executable instructions, the computer-executable instructions are not limited to the method operations described above, and can also execute related operations in the methods provided by any embodiment of the present invention.

[0055] Through the above description of the embodiments, those skilled in the art can clearly understand that the present invention can be implemented by means of software and necessary general-purpose hardware. Of course, it can also be implemented by hardware, but in many cases the former is a better implementation method. Based on such an understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as a floppy disk, read-only memory (ROM), random access memory (RAM), flash memory (FLASH), hard disk, or optical disc of a computer, etc., including several instructions for causing an electronic device (which can be a personal computer, server, or network device, etc.) to execute the methods described in various embodiments of the present invention.

[0056] It should be noted that in the above embodiments, the various units and modules included are only divided according to functional logic, but are not limited to the above division, as long as the corresponding functions can be achieved; in addition, the specific names of the functional units are only for the convenience of mutual distinction and do not limit the protection scope of the present invention.

[0057] Experimental Example 1. Verification of the prediction ability of the model by the ROC curve

[0058] The diagnostic value of the system was evaluated using the Receiver-operator characteristic (ROC) curve. All statistical analyses were performed using GraphPad Prism 9.0 and IBM SPSS Statistics 26.0. A P < 0.05 was considered statistically significant.

[0059] The ROC curve graph of the verification result of the prediction accuracy of the absolute neutrophil count for the CAR-T cell expansion rate (ρ) is as Figure 3 shown. The ROC curve analysis results show that the absolute neutrophil count can be used to predict the CAR-T cell expansion rate (AUC = 0.63, P < 0.0001), and the area under the ROC curve shows good prediction ability.

[0060] Application Example 1. Method for measuring the CAR-T cell expansion rate

[0061] 1. Collect the peripheral blood of B-cell hematological tumor patients who clinically receive the reinfusion of CAR-T cell therapy and detect the absolute neutrophil count. Then input the absolute neutrophil count value into the prediction formula established by the present invention to obtain the CAR-T cell expansion rate. The calculation formula is as follows:

[0062] y = A1 × exp(-x / t1) + y0;

[0063] In some of these embodiments, peripheral blood is collected from clinical patients, and the absolute neutrophil count value obtained by detection with a hematology analyzer is 0.5*10 9 / L. Substitute it into the above curve, and the CAR-T cell expansion rates obtained by calculation are 0.0693732716 respectively.

[0064] 2. At the same time, detect the lentivirus copy number of the peripheral blood sample, and calculate the CAR-T cell expansion rate (ρ) = 0.061891988 based on the published rate calculation model according to the obtained copy number data.

[0065] It can be seen from this that the system and method of the present invention can successfully and quickly detect the CAR-T amplification rate with high accuracy.

[0066] After calculating the CAR-T amplification rate of the patient, the doctor evaluates the patient's curative effect according to the amplification rate value and gives clinical intervention treatment as soon as necessary.

[0067] It is reported that monitoring the levels of peripheral blood IL-6 cytokine and ferritin can partly reflect the amplification of CAR-T. However, the disadvantage of this scheme is that IL-6 and ferritin are not specific and will also be up-regulated in infected patients. In addition, the CAR-T proliferation rate cannot be estimated through the levels of these two proteins.

[0068] In summary, the present invention can not only accurately predict the CAR-T amplification rate, but also minimize the influence of external factors to the greatest extent. And we have verified this model, and the area under the ROC curve shows good predictive ability.

[0069] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0070] Although the preferred embodiments of the embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0071] Obviously, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if these modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalent technologies, the embodiments of the present invention are also intended to include these changes and modifications.

Claims

1. A system for measuring the amplification rate of CAR-T cells in lymphoma patients, characterized in that, The system includes: a processor and a memory, the memory being coupled to the processor, the memory storing instructions which, when executed by the processor, use the following steps: input the absolute neutrophil count value into the following calculation formula to obtain the CAR-T cell amplification rate; the calculation formula is as follows: y = A1 × exp(-x / t1) + y0; wherein, y0 = 0.06756 ± 0.06274, A1 = 0.77155 ± 0.14248, t1 = 0.0826 ± 0.03373.

2. The system for measuring the amplification rate of CAR-T cells in lymphoma patients according to claim 1, wherein The method for measuring the absolute neutrophil count value includes: performing peripheral blood collection on a clinical patient, and detecting to obtain the absolute neutrophil count through a blood cell analyzer.

3. A computer-readable storage medium, characterized in that, A computer program is stored on the computer-readable storage medium, and when the computer program is executed by a processor, the steps described in claim 1 are used.

4. Use of the system for measuring the CAR-T cell amplification rate in a lymphoma patient according to any one of claims 1-2 or the prediction system of the computer-readable storage medium according to claim 3 in the preparation of a product for measuring the CAR-T cell amplification rate or a product for predicting the CAR-T efficacy and preventing side effects.

5. A product for measuring the amplification rate of CAR-T cells in lymphoma patients, characterized in that, including: a product for measuring the absolute neutrophil count value; and the system for measuring the CAR-T cell amplification rate in a lymphoma patient according to any one of claims 1-2 or the computer-readable storage medium according to claim 3.

6. A method for measuring the amplification rate of CAR-T cells in lymphoma patients, characterized in that, The method includes: inputting the absolute neutrophil count value into the system for measuring the CAR-T cell amplification rate in a lymphoma patient according to any one of claims 1-2 or the computer-readable storage medium according to claim 3 to obtain the CAR-T cell amplification rate.

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