A synergist for enhancing car-t cell expansion and maintenance of memory phenotype and application thereof

Abstract

The application provides an enhancer for enhancing CAR-T cell expansion and memory phenotype maintenance and an application thereof, relates to the technical field of biological medicine, and adds a small molecule compound IWR-1-endo to the activation of T cells, virus transduction and / or a culture system in a subsequent expansion culture stage, so that the in-vitro expansion efficiency of CAR-T cells can be significantly improved, a larger number of effector cells can be obtained in the same culture time, T cells are effectively inhibited to differentiate into terminal effector cells, the development of the T cells to CAR-Tscm cells and CAR-Tcm subgroups with long-term activity and memory potential is guided and maintained, the product quality is improved, the specific killing capacity on target tumor cells is higher in-vitro, tumors are more effectively removed and long-term control is achieved in an animal model, stronger in-vivo persistence and anti-tumor efficacy are shown. A new strategy for efficiently expanding CAR-T cells in-vitro is provided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a synergist that enhances CAR-T cell expansion and memory phenotype maintenance, and its application. Background Technology

[0002] CAR-T cell therapy has achieved success in the treatment of hematologic malignancies. One of the key steps in this therapy is the large-scale expansion of genetically modified T cells in vitro to obtain a sufficient number of therapeutic cells. Currently, conventional CAR-T cell preparation procedures heavily rely on cytokines (such as interleukin-2 and IL-2) to stimulate T cell activation and proliferation.

[0003] However, existing technologies have significant limitations: First, while long-term or high-dose use of IL-2 can promote proliferation, it easily induces terminal differentiation of T cells, leading to T stem cell memory cells (Tscm) with long-term memory function and stronger reproliferative potential (CD62L). + CD45RA+ T cells), and central memory T cells (Tcm), (CD62L) + CD45RA - The proportion of CAR-T cells decreases, while the proportion of short-acting effector T cells increases. Secondly, this directly affects the persistence of CAR-T cells in the patient after infusion, potentially leading to disease relapse. Furthermore, the expansion efficiency sometimes still fails to meet clinical needs. Therefore, developing a novel strategy that can efficiently expand CAR-T cells in vitro while specifically promoting and maintaining their memory phenotype is crucial for improving the long-term efficacy of CAR-T therapy.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of IWR-1-endo in the preparation of CAR-T cells, in order to solve the technical problem that there is a lack of an synergist in the prior art that can efficiently prepare CAR-T cells and promote and maintain their memory phenotype.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the application of IWR-1-endo in the preparation of CAR-T cells.

[0007] Furthermore, the preparation of CAR-T cells includes at least one of the following: an in vitro activation stage of T cells, a viral transduction stage, or an expansion culture stage.

[0008] Furthermore, the working concentration of the IWR-1-endo is 0.5~10μM, preferably 0.5~5μM.

[0009] Secondly, the present invention provides a method for preparing CAR-T cells, comprising adding IWR-1-endo to a culture system in at least one of the A1 to A3 steps; A1. In vitro activation of T cells; A2. Introduction of exogenous genes into the T cell genome; A3. Expanding transduced CAR-T cells.

[0010] Furthermore, the final concentration of IWR-1-endo in the culture system is 0.5~10 μM, preferably 0.5~5 μM.

[0011] Furthermore, the exogenous gene contains a chimeric antigen receptor that targets CD19; Preferably, the chimeric antigen receptor comprises a 4-1BB co-stimulatory domain and / or CD19-GD2-28ζ.

[0012] Furthermore, the T cells include CD4. + T cells and / or CD8 + T cells.

[0013] Thirdly, the present invention provides a CAR-T cell prepared using the above-described preparation method.

[0014] Fourthly, the present invention provides the application of CAR-T cells prepared by the above-described method in the preparation of drugs for treating tumors.

[0015] Furthermore, the tumor includes CD19. + Tumor; Preferably, the CD19 + Tumors include CD19 + B-cell malignant tumors; Preferably, the drug comprises CAR-T cells prepared by the above-described method; Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0016] This invention relates to the application of IWR-1-endo in the preparation of CAR-T cells. The invention reveals that adding the small molecule compound IWR-1-endo to the culture system during the activation, viral transduction, and / or subsequent expansion stages of T cells in the in vitro preparation of CAR-T cells significantly improves the in vitro expansion efficiency of CAR-T cells, resulting in a greater number of effector cells within the same culture time. It effectively inhibits the differentiation of T cells into terminal effector cells, guiding and maintaining their development into CAR-Tscm cells and CAR-Tcm subsets with greater long-term viability and memory potential, thereby improving product quality. CAR-T cells prepared by treatment with the small molecule compound IWR-1-endo exhibit stronger specific killing ability against target tumor cells in vitro, and can more effectively eliminate tumors and achieve long-term control in animal models, demonstrating stronger in vivo persistence and anti-tumor efficacy. The small molecule compound IWR-1-endo can serve as a synergist to enhance CAR-T cell expansion and maintain memory phenotype, providing a new strategy for efficient in vitro expansion of CAR-T cells. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the experimental process timeline for CAR-T cell preparation and evaluation provided in an embodiment of the present invention; Figure 2 The phenotypic detection results of CAR-T cells provided in the embodiments of the present invention are as follows: Q1 is terminal effector T cells, Q2 is stem cell memory T cells, Q3 is central memory T cells, and Q4 is effector memory cells. Figure 3 Statistical results of CAR-Tscm cell expansion ratio and cell number expansion fold provided in the embodiments of the present invention; Figure 4 Statistical results of CAR-Tcm cell expansion ratio and cell number expansion fold provided in the embodiments of the present invention; Figure 5 The in vitro tumor suppression effect of CAR-T cells under different effector-target ratios provided in the embodiments of the present invention; Figure 6 The results of CAR-T cell anti-tumor detection in animals provided in the embodiments of the present invention. Detailed Implementation

[0019] In this document, "prepared from" and "comprising" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0020] In this document, the conjunction "composed of" excludes any unspecified elements, steps, or components. If used in a claim, this phrase will make the claim closed, meaning it does not contain any materials other than those described, except for conventional impurities associated with them.

[0021] In this document, when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1~5” is disclosed, the described range should be interpreted as including the ranges “1~4”, “1~3”, “1~2”, “1~2 and 4~5”, “1~3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0022] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0023] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbering I...V; A1, A2...A7; first, second...fifth, etc.

[0024] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0025] In this article, “each…independently selected”, “…independently selected respectively”, and “…independently selected” are interchangeable and should all be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention provides, in one aspect, the application of IWR-1-endo in the preparation of CAR-T cells.

[0028] This invention discovers that adding the small molecule compound IWR-1-endo to the culture system during the T cell activation, viral transduction, and / or subsequent expansion stages of CAR-T cell preparation significantly enhances the in vitro expansion efficiency of CAR-T cells, resulting in a greater number of effector cells within the same culture time. It effectively inhibits T cell differentiation into terminal effector cells, guiding and maintaining their development into CAR-Tscm cells and CAR-Tcm subsets with greater long-term viability and memory potential, thereby improving product quality. CAR-T cells prepared using the small molecule compound IWR-1-endo exhibit stronger specific killing ability against target tumor cells in vitro, and can more effectively eliminate tumors and achieve long-term control in animal models, demonstrating stronger in vivo persistence and anti-tumor efficacy. The small molecule compound IWR-1-endo can serve as a synergist to enhance CAR-T cell expansion and maintain memory phenotype, providing a new strategy for efficient in vitro expansion of CAR-T cells.

[0029] In some specific embodiments, the preparation of CAR-T cells includes at least one of the following: an in vitro activation phase of T cells, a viral transduction phase, or an expansion culture phase.

[0030] In some specific embodiments, the working concentration of the IWR-1-endo is 0.5~10 μM.

[0031] The working concentration of IWR-1-endo can be, but is not limited to, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 5 μM, 5 μM, or 10 μM, or can be 0.5~10 μM, preferably 0.5~5 μM.

[0032] According to another aspect of the present invention, a method for preparing CAR-T cells is also provided, comprising adding IWR-1-endo to a culture system in at least one of the A1 to A3 steps; A1. In vitro activation of T cells; A2. Introduction of exogenous genes into the T cell genome; A3. Expanding transduced CAR-T cells.

[0033] This preparation method only requires the addition of the small molecule compound IWR-1-endo to the existing standard culture system. It is simple to operate, requires no complex process changes, and is easy to scale up and produce under conditions that comply with Good Manufacturing Practices (GMP). It has high feasibility for clinical translation.

[0034] In some specific embodiments, the final concentration of IWR-1-endo in the culture system is 0.5~10 μM, preferably 0.5~5 μM.

[0035] In some specific embodiments, the exogenous gene comprises a chimeric antigen receptor targeting CD19; in some specific embodiments, the chimeric antigen receptor comprises a 4-1BB co-stimulatory domain and / or CD19-GD2-28ζ.

[0036] In some specific embodiments, the T cells include CD4. + T cells and / or CD8 + T cells.

[0037] According to another aspect of the present invention, a CAR-T cell prepared by the above-described preparation method is also provided.

[0038] According to another aspect of the present invention, the application of CAR-T cells prepared by the above-described method in the preparation of drugs for treating tumors is also provided.

[0039] In some specific embodiments, the tumor includes CD19. + Tumor; in some specific embodiments, the CD19 + Tumors include CD19 + B-cell malignant tumors.

[0040] In some specific embodiments, the drug comprises CAR-T cells prepared by the above-described preparation method; in some specific embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0041] The pharmaceutically acceptable carriers include, but are not limited to, saline, buffer solutions, glucose, and combinations thereof.

[0042] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] All data are presented as mean ± standard error. Statistical significance was assessed using an unpaired t-test, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0044] Example 1 The specific process for preparing CAR-T in this embodiment is as follows: Figure 1 As shown.

[0045] 1. Materials: Peripheral blood from healthy donors, CD3 / CD28 activating magnetic beads, CD19-41BBz CAR lentivirus, IWR-1-endo (purchased from MedChemExpress (MCE), batch number HY-12238), recombinant human IL-2, RPMI 1640 medium, and fetal bovine serum.

[0046] 2. Grouping Experimental group: 1 μM IWR-1-endo was added to the standard culture system (RPMI 1640 + 10% FBS + 200 U / mL IL-2).

[0047] Control group: using only the standard culture system.

[0048] 3. T cell isolation (1) Mononuclear cell acquisition: Peripheral blood from the donor is taken and diluted with an equal volume of PBS solution in an anticoagulated blood collection tube and mixed well; mononuclear cells are separated using Ficoll lymphocyte separation solution at a speed of 400g for 20-25 minutes (25 minutes in this example), with the centrifuge acceleration being 2 up and 0 down, and the white membrane layer is collected as mononuclear cells; the collected blood sample can also be directly treated with red blood cell lysis buffer to remove red blood cells and obtain red blood cell-free mononuclear cells.

[0049] (2) T cell purification: CD3 T cell negative selection was performed using the EasySep™ Human T Cell Negative Selection Kit (STEM CELL, Canada) according to the kit instructions. Specifically, the appropriate amount of negative selection antibody mixture was added according to the cell quantity, and incubated for 5 minutes; rapid spheres were vortexed for 30 seconds to homogenize the cells, and the appropriate amount of rapid spheres were added according to the instructions to bring the total volume to 2.5 ml. The mixture was then mixed and allowed to stand for 3 minutes. The test tube containing the cells was placed in a magnetic rack and allowed to stand for 3 minutes. The cells that were not bound by the magnetic beads were poured out, and the cells were centrifuged at 300g for 5 minutes. The collected cells were the T cells.

[0050] 4. CAR-T cell preparation (1) T cell activation: Anti-CD3 / CD28 magnetic beads activate T cells at a ratio of 3:1, meaning 3 beads correspond to one T cell. Before use, the protective agent inside the anti-CD3 / CD28 magnetic beads needs to be removed by washing them three times with PBS. The T cells mixed with anti-CD3 / CD28 were seeded in 24-well plates at a cell density of 1~1.5×10⁻⁶ cells / wells. 6 Cells / well (Cell density in this example is 1×10⁻⁶)6 (cells / pores).

[0051] (2) Viral transfection to prepare CAR-T cells: The centrifuge was preheated to 32℃~37℃ (in this example, it was preheated to 32℃), and the high-speed preheating was 3500rpm for about 0.5 hours; after T cells were activated for 24 hours, the T cells were centrifuged to remove the supernatant, and 500ul of viral culture medium and polybrene (10 mg / ml, 1:1000) were added; the centrifuge was set at 2000rpm, the rate of increase was 2, the rate of decrease was 0, and the temperature was 32℃~37℃ (in this example, it was 32℃) for 1 hour; then it was placed in a 37℃ carbon dioxide incubator for 6 hours, and 500ul of complete culture medium was gently added; after 24 hours of culture, the culture medium containing the virus was removed by centrifugation, and the control group was replaced with standard culture medium (RPMI 1640 + 10% FBS + 200 U / mL IL-2), and the experimental group was replaced with standard culture system + 1 μM IWR-1-endo.

[0052] (3) CAR-T cell sorting and culture: Three days after viral transfection, i.e., the fourth day of T cell culture, the viral infection efficiency was detected by flow cytometry. CAR-T cells were sorted by flow cytometry and cultured for the later stages. The medium was changed every 2-3 days thereafter (in this example, the medium change interval was 2 days) to maintain the concentrations of IL-2 and IWR-1-endo. IWR-1-endo was added to the experimental group to a final concentration of 1 μM.

[0053] 5. Phenotypic and functional analysis of CAR-T cells Samples were taken and counted on days 4, 7, 10, 14 and 20 of culture, and CAR-T cell phenotype and function were analyzed.

[0054] 1) Flow cytometry was used to detect phenotypic markers (CD62L, CD45RA). CAR-T cell phenotypic results are as follows: Figure 2 As shown, the proportions of Q1 and Q4 in the experimental group were consistently significantly lower than those in the control group, while the proportions of Q2 and Q3 were consistently significantly higher than those in the control group. This indicates that the addition of IWR-1-endo to the experimental group can maintain optimal stemness of cells, improve their self-renewal capacity, make them sensitive to cytokines, enable them to survive for a long time, and provide a lasting anti-tumor effect.

[0055] 2) Amplification efficiency, such as Figure 3 As shown, by day 7 of culture, the proportion of Tscm cells in both the control and experimental groups showed an increasing trend. From day 10 to day 20, the proportion of Tscm cells in the control group decreased significantly, while the proportion of Tscm cells in the experimental group decreased slightly, and the expansion fold showed an increasing trend. By day 20, the proportion of Tscm cells in the experimental group was more than twice that of the control group; indicating that IWR-1-endo significantly promoted cell proliferation.

[0056] 3) Memory phenotype analysis, such as Figure 4 As shown, on day 7 of culture, there was no significant difference in the proportion of Tcm cells between the experimental and control groups. From day 10 to day 20, the proportion of Tcm cells in the control group decreased rapidly, but the proportion in the experimental group stopped decreasing from day 14 and remained at approximately 20%, significantly higher than that in the control group. The fold increase in the number of Tcm cells in the experimental group was generally better than that in the control group. Figure 3 Long-term in vitro culture data further showed that the high proportion of Tcm in the experimental group could be maintained for a longer period of time.

[0057] Example 2 In vitro killing experiment With CD19 + Raji lymphoma cells were used as target cells (E), and CAR-T cells (T) were co-cultured with target cells (E) at different effector-to-target ratios. The results of D-fluorescein potassium salt (Nanjing Novizan Biotechnology Co., Ltd., catalog number: DD1210, working concentration for cell treatment 1.5 mg / mL) detection are as follows... Figure 5 As shown, with the same number of effector cells, the killing efficiency of CAR-T cells in the experimental group was significantly higher than that in the control group.

[0058] Example 3: In vivo antitumor experiment A Raji lymphoma model was established in NOD-SCID mice, and equal numbers of CAR-T cells were infused into either the experimental or control groups. Tumor status was periodically monitored using potassium D-luciferin (Nanjing Novizan Biotechnology Co., Ltd., catalog number: DD1210, working concentration for mouse treatment: 15 mg / mL, 300 μL / mouse). Results are as follows: Figure 6 As shown, tumor growth in the experimental group mice was completely inhibited, survival was significantly prolonged, and high levels of CAR-T cells could still be detected in peripheral blood at longer time points after infusion, demonstrating superior in vivo persistence.

[0059] In summary, integrating the small molecule IWR-1-endo into the CAR-T cell preparation process has successfully established a synergistic preparation method that can simultaneously increase cell yield, improve cell quality (enrichment of memory phenotype), and enhance the function of the final product. This method is simple, effective, and has significant clinical application value.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of IWR-1-endo in the preparation of CAR-T cells.

2. The application according to claim 1, characterized in that, The preparation of CAR-T cells includes at least one of the following: in vitro activation of T cells, viral transduction, or amplification culture.

3. The application according to claim 1 or 2, characterized in that, The working concentration of the IWR-1-endo is 0.5~10 μM.

4. A method for preparing CAR-T cells, characterized in that, This includes adding IWR-1-endo to the culture system in at least one step of A1 to A3; A1. In vitro activation of T cells; A2. Introduction of exogenous genes into the T cell genome; A3. Expanding transduced CAR-T cells.

5. The preparation method according to claim 4, characterized in that, The final concentration of IWR-1-endo in the culture system is 0.5~10 μM.

6. The preparation method according to claim 4, characterized in that, The exogenous gene contains a chimeric antigen receptor that targets CD19; The chimeric antigen receptor contains a 4-1BB co-stimulatory domain and / or CD19-GD2-28ζ.

7. The preparation method according to any one of claims 4 to 6, characterized in that, The T cells include CD4 + T cells and / or CD8 + T cells.

8. A CAR-T cell, characterized in that, It is prepared by the preparation method according to any one of claims 4 to 7.

9. The use of CAR-T cells prepared by the method according to any one of claims 4 to 7 in the preparation of drugs for treating tumors.

10. The application according to claim 9, characterized in that, The tumor includes CD19 + Tumor; The CD19 + Tumors include CD19 + B-cell malignant tumors; The drug comprises CAR-T cells prepared by the preparation method according to any one of claims 4 to 6; The drug also includes a pharmaceutically acceptable carrier.

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