Application of itraconazole in preparation of product for amplifying induced T cells into central memory T cells in vitro

Abstract

The invention belongs to the technical field of new application of medicines, and particularly relates to application of itraconazole in preparation of a product for amplifying an induced T cell into a central memory T cell in vitro. It is found through experiments that itraconazole is additionally added into a CAR-T cell culture medium, CAR-T cells can be rapidly induced, differentiated and amplified into central memory CAR-T cells in vitro, and the proportion of the central memory CAR-T cells in the CAR-T cells is obviously increased. Itraconazole is a clinical medicine, pharmacokinetic and toxicological studies are clear, the safety is high, good technical support is provided for tumor cell treatment, and the itraconazole has important significance.

Description

Technical Field

[0001] This invention belongs to the field of new drug use technology, specifically relating to the application of itraconazole in the preparation of products that induce T cells to be central memory T cells through in vitro expansion. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy holds immense potential in tumor immunotherapy. This therapy involves genetically engineering a patient's own T cells to express receptors that specifically recognize tumor antigens, thereby achieving precise targeting and elimination of cancer cells. However, CAR-T therapy still faces challenges in terms of durability and efficacy stability.

[0003] One of the key factors limiting the long-term efficacy of CAR-T therapy lies in the in vivo state of the reinfused cells. Studies show that CAR-T cells expanded and activated in vitro are prone to terminal differentiation after reinfusion and rapidly enter a state of functional exhaustion. CAR-T cells in a terminally differentiated and exhausted state not only have insufficient tumor infiltration capacity but also poor in vivo persistence, making it difficult to form long-term immune memory, ultimately leading to tumor immune escape and disease progression. Central memory T cells (TCMs) have superior in vivo persistence, self-renewal capacity, and anti-tumor activity compared to terminally differentiated effector cells. If CAR-T cells can be directed to the TCM phenotype during the in vitro preparation stage, it is hoped that the in vivo persistence of reinfused cells can be extended, thereby improving their long-term efficacy.

[0004] Currently, methods for in vitro induction of TCM or stem cell-like memory T cell (TSCM) differentiation have many limitations: some small molecule inhibitors are expensive and highly toxic; cytokine combination protocols are complex and prone to non-specific activation; and novel inducers require lengthy preclinical safety validation. Therefore, finding a safe, efficient, economical, and easily clinically translatable in vitro TCM inducer has become a key need to improve the efficacy of CAR-T therapy. Summary of the Invention

[0005] To address the shortcomings of the prior art, the purpose of this invention is to provide the application of itraconazole in the preparation of products that induce T cells to be central memory T cells through in vitro expansion.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the invention provides the use of itraconazole in the preparation of products that induce T cells to be central memory T cells through in vitro expansion.

[0007] A second aspect of the present invention provides a method for in vitro expansion and induction of T cells into central memory T cells, comprising the following steps: T cells are suspended in a T cell culture medium containing rIL-2 and itraconazole for induction culture to obtain central memory T cells.

[0008] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention has shown through experiments that adding itraconazole to the CAR-T cell culture medium can rapidly induce CAR-T cells to differentiate into central memory CAR-T cells in vitro and expand them. Furthermore, the proportion of central memory CAR-T cells in CAR-T cells is significantly increased, and the central memory CAR-T cells obtained through induced differentiation retain the characteristics and functions of central memory CAR-T cells.

[0009] Itraconazole, as a mature drug, has the advantages of low cost and good safety. The central memory CAR-T cells induced by itraconazole can be used for adoptive transfer without special treatment, providing a strong practical basis for tumor cell therapy and having important development value and promotion significance. Attached Figure Description

[0010] Figure 1 CD45RO / CCR7 expression was detected after T cells were treated with itraconazole; where A is a flow cytometry typical plot of CD45RO / CCR7 expression in T cells, and B is a statistical plot of the percentage of CD45RO / CCR7 expression in T cells.

[0011] Figure 2 This is a schematic diagram of the CD19 CAR molecular structure.

[0012] Figure 3 To analyze the expression of FMC63 in CAR molecules using flow cytometry.

[0013] Figure 4 A schematic diagram illustrating the formation of central memory in CD19 CAR-T cells induced by itraconazole.

[0014] Figure 5 Cell viability of CD19 CAR-T cells after itraconazole treatment.

[0015] Figure 6 CD45RO / CCR7 expression was detected after CD19 CAR-T cells were treated with itraconazole; where A is a flow cytometry typical plot of CD45RO / CCR7 expression in CD19 CAR-T cells, and B is a statistical graph of the percentage of CD45RO / CCR7 expression in CD19 CAR-T cells.

[0016] Figure 7The expression of TIM-3 in CD19 CAR-T cells was detected by flow cytometry. Among them, A is a typical flow cytometry plot of TIM-3 expression in CD19 CAR-T cells, B is a statistical plot of the percentage of TIM-3 expression in CD19 CAR-T cells, and C is a statistical plot of the average fluorescence intensity of TIM-3 expression in CD19 CAR-T cells.

[0017] Figure 8 The expression of CD69 in CD19 CAR-T cells and SU-DHL-4 cells was detected by flow cytometry after 6 hours of co-incubation. In the figure, A is a typical flow cytometry plot of CD69 expression in CD19 CAR-T cells, and B is a statistical graph of the percentage of CD69 expression in CD19 CAR-T cells.

[0018] Figure 9 Principal component analysis (PCA) plot of bulk RNA-seq of itraconazole-treated CD19 CAR-T cells.

[0019] Figure 10 A heatmap of genes related to memory. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0022] Terminology Definition The term "CAR-T cells" refers to chimeric antigen receptor T cells, a personalized immunotherapy that uses genetically engineered T cells to attack cancer cells.

[0023] The term "central memory" refers to a subset of memory T cells generated after naïve T cells are activated by antigens. These cells are biomarked as being double-positive for CCR7 and CD45RO and have the ability to hom to lymph nodes.

[0024] The term "rIL-2" refers to recombinant human interleukin-2 for injection, a cytokine produced through genetic engineering. Its core function is to activate and promote the proliferation and differentiation of immune cells (especially T cells), and it is a commonly used cytokine supplement in clinical immunotherapy.

[0025] The term "FMC63" refers to a mouse-derived monoclonal antibody that specifically targets the CD19 antigen on the surface of B cells. In CAR-T therapy, its single-chain antibody variable region fragment is a key component in constructing a chimeric antigen receptor that targets CD19.

[0026] The term "TIM-3" refers to T cell immunoglobulin and mucin domain protein-3, an immune checkpoint molecule expressed on the surface of activated T cells, natural killer cells, and other immune cells. It negatively regulates the immune response by transmitting inhibitory signals and is closely related to T cell exhaustion.

[0027] The term "CD69" refers to an early marker of T cell activation, a transmembrane protein belonging to the C-type lectin superfamily. It is rapidly expressed after T cells are activated by antigens and participates in regulating lymphocyte retention, proliferation, and effector functions.

[0028] The term "bulk RNA-seq" refers to bulk RNA sequencing technology. This is a method for extracting and sequencing whole RNA from populations containing a large number of heterogeneous cells to obtain the average gene expression profile of a sample under specific conditions.

[0029] In a typical embodiment of the present invention, the use of itraconazole in the preparation of a product for in vitro expansion of induced T cells into central memory T cells is provided.

[0030] In some embodiments, the T cells are CD3. + T cell subsets.

[0031] In some embodiments, the CD3 + The T cell subset consists of CAR-T cells modified with CAR.

[0032] In some embodiments, the CAR-T cells are human CAR-T cells that target CD19.

[0033] In some embodiments, itraconazole is the sole active ingredient or one of the active ingredients in the product.

[0034] In some embodiments, the product may be a central memory CAR-T cell culture medium or an in vitro cell expansion inducer.

[0035] In some embodiments, the final in vitro induction concentration of itraconazole in the central memory CAR-T cell in vitro expansion inducer is 2~10 μM, specifically 2.5 μM, 5 μM or 10 μM.

[0036] In another typical embodiment of the present invention, a method for in vitro expansion and induction of T cells into central memory T cells is provided, comprising the following steps: T cells are suspended in a T cell culture medium containing rIL-2 and itraconazole for induction culture to obtain central memory T cells; wherein the T cells are CD3+. + T cell subsets.

[0037] In some implementations, during the induction culture process, rIL-2 and itraconazole are added to the T cell culture medium every 2 to 3 days, and the culture time is 3 to 4 days.

[0038] In some implementations, during the induction culture, rIL-2 and itraconazole are added to the T cell culture medium every 2 days for a total culture time of 3 days.

[0039] In some implementations, the concentration of rIL-2 used in the T cell culture medium is 20 ng / mL.

[0040] In some implementations, the concentration of itraconazole used in the T cell culture medium is 2 to 10 μM, specifically 2.5 μM, 5 μM, or 10 μM.

[0041] In some embodiments, the CD3 + The T cell subset consists of CAR-T cells modified with CAR.

[0042] In some embodiments, the preparation of the CAR-T cells includes: suspending T cells in a T cell culture medium containing rIL-2, adding Anti-CD3 antibody and Anti-CD28 antibody to the T cell culture medium for culturing, and modifying the T cells with CAR to obtain CAR-T cells.

[0043] In some embodiments, the CAR-T cells are human CAR-T cells that target CD19.

[0044] In some embodiments, the sequence of the CAR is as shown in SEQ ID NO:1.

[0045] In some embodiments, the concentration of rIL-2 in the T cell culture medium is 20 ng / mL, the concentration of Anti-CD3 is 10 μg / mL, and the concentration of Anti-CD28 is 10 μg / mL.

[0046] In another typical embodiment of the present invention, central memory T cells obtained according to the above method are also provided.

[0047] In another typical embodiment of the present invention, the use of the above-mentioned central memory T cells in the preparation of a medicament for treating cancer is provided.

[0048] In some implementations, the cancer is a solid tumor, such as ovarian tumor, melanoma, lung tumor, liver cancer, gastrointestinal tumor, or breast tumor.

[0049] In some implementations, the tumor is a liquid tumor, such as leukemia or lymphoma.

[0050] In some implementations, the lymphoma is diffuse large B-cell lymphoma.

[0051] The present invention will be further described in detail below with reference to the accompanying drawings and specific experiments. Unless otherwise specified, the present invention and the reagents, instruments, equipment and methods used are all conventional commercially available reagents, instruments, equipment and methods in this technical field.

[0052] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0053] Example 1: Itraconazole induces central memory formation in T cells (1) CD3 was obtained by sorting. + T cells, at a rate of 0.3 × 10 6 The cells were seeded into 96-well plates, and 300 µL of RPMI-1640 complete medium (containing 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES, 1% sodium pyruvate and 1% penicillin-streptomycin solution) was added to each well.

[0054] (2) Add 10 μg / mL anti-CD3 antibody (Biolegend, CAS#300438), 10 μg / mL anti-CD28 antibody (Biolegend, CAS#377804) and 20 ng / mL rIL-2 (Beijing Biolegend, CAS#IL2-H5215) directly to the culture medium, and supplement with 20 ng / mL rIL-2 every two days.

[0055] (3) CD3 +On day 5 of T cell stimulation and expansion, itraconazole (Shanghai Taoshu Biotechnology, CAS#T1011) was added to the culture medium. Itraconazole treatment groups of 2.5 μM, 5 μM, and 10 μM were set up, and DMSO was added as a control group. The induction culture continued. During the induction culture, rIL-2 was supplemented every two days to maintain the concentration at 20 ng / mL, and itraconazole was supplemented to maintain the concentration at the initial treatment concentration.

[0056] (4) After culturing for 72 hours, CD3 was detected by flow cytometry. + Expression levels of CD45RO and CCR7 on T cells.

[0057] The results are as follows Figure 1 As shown in A and B, itraconazole significantly increased the number of central memory T cells (CD45RO). + CCR7 + ) proportion.

[0058] Example 2: Construction of CD19 CAR-T cells (1) CD3 was obtained by sorting. + T cells, at a rate of 0.3 × 10 6 The cells were seeded into 96-well plates, and 300 µL of RPMI-1640 complete medium (containing 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES, 1% sodium pyruvate and 1% penicillin-streptomycin solution) was added to each well.

[0059] (2) Add 10 μg / mL anti-CD3 antibody (Biolegend, CAS#300438), 10 μg / mL anti-CD28 antibody (Biolegend, CAS#377804) and 20 ng / mL rIL-2 (Beijing Biolegend, CAS#IL2-H5215) directly to the culture medium, and supplement with 20 ng / mL rIL-2 every two days.

[0060] (3) On the third day of T cell stimulation, CAR virus solution (moi=5) was added to the cells, and after 6 hours of continuous infection, 1 times the culture medium was added.

[0061] Among them, the CAR virus mentioned above is a lentivirus that targets CD19. The schematic diagram of the CD19 CAR molecular structure is shown below. Figure 2 As shown in the figure, its CAR structure comprises an anti-CD19 single-chain antibody (FMC63), a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. The sequence of the CD19 CAR structure is shown in SEQ ID NO:1, and the sequence of the constructed lentiviral vector is shown in SEQ ID NO:2.

[0062] (4) T cells were centrifuged and resuspended in fresh RPMI-1640 complete medium at a density of 1×10⁻⁶ cells 24 hours after infection. 6 / mL; (5) Three days after infection, the infection efficiency of CAR molecules was detected by flow cytometry.

[0063] like Figure 3 As shown, the constructed CD19 CAR-T cells have an efficiency greater than 80%.

[0064] Example 3: Itraconazole-induced central memory formation in CD19 CAR-T cells A schematic diagram illustrating itraconazole-induced central memory formation in CD19 CAR-T cells is shown below. Figure 4 As shown.

[0065] (1) The CD19 CAR-T cells prepared in Example 2 were mixed at a ratio of 0.3 × 10⁻⁶. 6 The cells were seeded into 96-well plates, and 300 µL of RPMI-1640 complete medium containing rIL-2 (20 ng / mL) was added to each well.

[0066] (2) Add itraconazole (Shanghai Taoshu Biotechnology, CAS# T1011) to the culture medium and set up itraconazole treatment groups of 2.5 μM, 5 μM and 10 μM respectively. Add DMSO as the control group and continue to induce culture. During the induction culture, add rIL-2 every two days to maintain the concentration at 20 ng / mL and add itraconazole to maintain the concentration at the initial treatment concentration.

[0067] (3) After culturing for 72 hours, the phenotype and function of CAR-T cells were detected by flow cytometry, principal component analysis (PCA) was performed using bulk RNA-seq technology, and the expression of memory-related genes was analyzed.

[0068] (4) Further, CD19 CAR-T cells (5×10⁻⁶) cultured for 72 hours were... 5 / mL) and SU-DHL-4 cells (5×10 5 The cells were co-cultured ( / mL) for 6 hours, and the expression of the cell activation marker CD69 was detected by flow cytometry.

[0069] like Figure 5 As shown, itraconazole at concentrations of 2.5 μM to 10 μM had no effect on the cell viability of CD19 CAR-T cells.

[0070] like Figure 6 As shown in A and B, itraconazole significantly increased the number of central memory CAR-T cells (CD45RO) in CD19 CAR-T cells. + CCR7+ ) proportion.

[0071] like Figure 7 As shown in A, B, and C, itraconazole reduced TIM-3 expression.

[0072] like Figure 8 As shown in A and B, itraconazole increased the expression of CD69 in CD19 CAR-T cells.

[0073] like Figure 9 As shown, itraconazole-treated CD19 CAR-T cells showed significant PCA clustering.

[0074] like Figure 10 As shown, itraconazole increased the expression of memory-related genes in CD19 CAR-T cells.

[0075] The above results indicate that the addition of itraconazole to the CAR-T cell culture medium can rapidly induce CAR-T cells to differentiate and expand into central memory CAR-T cells, and the proportion of central memory CAR-T cells in CAR-T cells is significantly increased. The induced and differentiated central memory CAR-T cells maintain the characteristics and functions of central memory CAR-T cells.

[0076] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of itraconazole in the preparation of products that induce T cells to become central memory T cells through in vitro expansion, characterized in that, The T cells are CD3. + T cell subsets.

2. A method for in vitro expansion and induction of T cells into central memory T cells, characterized in that, Includes the following steps: T cells were suspended in a T cell culture medium containing rIL-2 and itraconazole for induction culture to obtain central memory T cells; the T cells were CD3+. + T cell subsets.

3. The method as described in claim 2, characterized in that, The concentration of rIL-2 was 20 ng / mL.

4. The method as described in claim 2, characterized in that, The concentration of itraconazole is 2-10 μM.

5. The method as described in claim 2, characterized in that, During the induction culture process, rIL-2 and itraconazole were added to the T cell culture medium every 2-3 days.

6. The method as described in claim 2, characterized in that, The CD3 + The T cell subset consists of CAR-T cells modified with CAR.

7. The method as described in claim 6, characterized in that, The preparation of the CAR-T cells includes: suspending T cells in a T cell culture medium containing rIL-2, adding Anti-CD3 antibody and Anti-CD28 antibody to the T cell culture medium for culture, and modifying the T cells with CAR to obtain CAR-T cells.

8. The method as described in claim 7, characterized in that, The CAR-T cells are human CAR-T cells targeting CD19; the sequence of the CAR is shown in SEQ ID NO:

1.

9. The method as described in claim 7, characterized in that, The concentration of rIL-2 was 20 ng / mL.

10. The method as described in claim 7, characterized in that, The concentration of the Anti-CD3 antibody is 10 μg / mL, and the concentration of the Anti-CD28 antibody is 10 μg / mL.

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