A method for expanding bifunctional γδ T cells by time-phased administration of Cbl-b inhibitors, zoledronic acid, and IL-2, and its application.

Abstract

This application discloses a method and its application for expanding bifunctional γδ T cells by adding Cbl-b inhibitors, zoledronic acid, and IL-2 at specific times. The method includes: isolating human peripheral blood mononuclear cells; adding zoledronic acid and IL-2 on day 0 of expansion; supplementing with fresh culture medium containing zoledronic acid and IL-2 on day 4; supplementing with culture medium containing IL-2 to maintain cell density on day 6 and thereafter; adding Cbl-b inhibitors between day 6 and day 10 of expansion and maintaining this for at least one day. This method can significantly increase the in vitro expansion rate of γδ T cells, shifting them towards central memory T cells, enhancing the expression of the stem cell marker SLAMF6, upregulating TNF-α and IFN-γ secretion and CD86 and HLA-DR expression, enhancing the killing ability against tumor cells, and downregulating the LAG3 depletion marker under continuous antigen stimulation, maintaining long-term function. This application provides new technical support for γδ T cell adoptive immunotherapy.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a method and its application for time-segmented addition of Cbl-b inhibitors, zoledronic acid, and IL-2 to expand bifunctional γδ T cells. Background Technology

[0002] In recent years, the field of tumor immunotherapy has made groundbreaking progress, especially with the emergence and clinical application of therapeutic technologies such as immune checkpoint inhibitors (ICIs), cytokine therapy, tumor vaccines, and adoptive cell transfer therapy (ACT). ACT is a novel, personalized, immune-based approach to tumor treatment, primarily including tumor-infiltrating lymphocytes (TILs) therapy, engineered T-cell receptor (TCR) T-cell therapy (TCR-T cell) therapy, and chimeric antigen receptor T-cell (CAR-T) therapy. The principle of ACT is to isolate immune cells with anti-tumor potential from the patient's own body or a healthy donor, and then specifically modify them in vitro through activation, expansion, gene modification, or small molecule intervention to enhance their tumor recognition, proliferation, and killing abilities. These cells are then reinfused into the patient to eliminate tumor cells, reshape the body's anti-tumor immune microenvironment, and ultimately achieve the goal of tumor treatment.

[0003] Traditional ACT (antigen-activating enzyme) therapy primarily relies on αβ T cells, depending on MHC-restricted antigen recognition. However, the widespread immune escape mechanisms in tumors (such as low MHC expression or antigen mutations) pose a significant challenge to αβ T cells in treating recurrent tumors. Compared to αβ T cells, γδ T cells exhibit unique advantages in ACT due to their widespread tissue distribution, rapid antigen recognition capabilities, and efficient effector function. Most importantly, γδ T cells do not rely on MHC molecules for antigen recognition, allowing them to bypass tumor cell immune escape mechanisms. Currently, researchers are exploring various strategies to directly activate Vγ9Vδ2 T cells, including the use of aminobisphosphonates such as zoledronic acid (ZOL) and pamidronate, or synthetic pAg analogs.

[0004] However, existing methods for expanding γδ T cells still have significant limitations. In previous methods, γδ T cells often enter a plateau phase after reaching a certain expansion fold during in vitro expansion, resulting in low expansion rates. Furthermore, they are prone to functional suppression upon contact with tumor antigens, limiting their survival time in vivo and weakening their anti-tumor capabilities. These problems collectively restrict the further application and clinical translation of γδ T cells in cancer treatment.

[0005] Therefore, the urgent technical problem to be solved at this stage is to provide an in vitro expansion method that can effectively overcome the plateau limitation of γδ T cell expansion, increase the expansion fold, and enhance the durability of its anti-tumor function. Summary of the Invention

[0006] This application addresses the technical problems of low in vitro expansion efficiency, short in vivo survival time, and susceptibility to suppression of anti-tumor function in existing γδ T cell technologies. It provides a method and application for time-phased addition of a Cbl-b inhibitor, zoledronic acid, and IL-2 to expand bifunctional γδ T cells. This method employs a time-phased addition strategy, introducing a Cbl-b inhibitor at specific time points on top of a zoledronic acid and IL-2 expansion system, thereby achieving synergistic regulation of γδ T cells. This method can significantly increase the in vitro expansion rate of γδ T cells, optimize their memory differentiation phenotype (shifting towards TCM), enhance the expression of the stemness marker SLAMF6, and significantly improve their secretion of anti-tumor cytokines TNF-α and IFN-γ, as well as their killing effect on tumor cells. It also enhances their antigen-presenting potential and lays a solid foundation for the subsequent preparation of highly effective anti-tumor immune cell agents.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] In a first aspect, this application provides a method for time-phased addition of a Cbl-b inhibitor, zoledronic acid, and IL-2 to expand bifunctional γδ T cells, comprising the following steps:

[0009] (1) Collect human peripheral blood samples and isolate human peripheral blood mononuclear cells (PBMCs);

[0010] (2) PBMCs were inoculated into a culture medium containing fetal bovine serum, and zoledronic acid and IL-2 were added on day 0 of amplification for initial activation culture;

[0011] (3) On day 4, the cells were transferred to a new culture vessel, and fresh culture medium containing zoledronic acid and IL-2 was added. The cells were then cultured until day 6.

[0012] (4) On day 6 and in subsequent culture, fresh culture medium containing IL-2 was added regularly to maintain cell density;

[0013] (5) At any point between day 6 and day 10 of amplification, add the Cbl-b inhibitor to the culture medium and maintain its effective concentration in the culture medium for at least 1 day.

[0014] In some embodiments, the final concentration of zoledronic acid is 5 μM; the final concentration of IL-2 is 1000 IU / ml.

[0015] In some embodiments, the Cbl-b inhibitor is Cbl-b-IN-1 with a final concentration of 2 μM, and the structural formula of Cbl-b-IN-1 is as shown in Formula I. As shown.

[0016] In some preferred embodiments, in step (2), the inoculation density of the PBMCs is 2 × 10⁻⁶. 6 Cells / well; the culture medium contains 10% (v / v) fetal bovine serum.

[0017] In some implementations, in step (5), the Cbl-b inhibitor is added on day 6 of amplification and its effective concentration is maintained for continuous culture until day 14.

[0018] In some implementations, in step (5), the Cbl-b inhibitor is added on day 10 of amplification and its effective concentration is maintained for continuous culture until day 14.

[0019] In some implementations, in step (5), the Cbl-b inhibitor is added on day 10 of amplification and its effective concentration is maintained and cultured until day 11.

[0020] Secondly, this application provides bifunctional γδ T cells, which are obtained by the method described in the first aspect.

[0021] γδ T cells obtained by the method described in the first aspect have at least one of the following characteristics:

[0022] (a) The in vitro amplification fold was significantly increased;

[0023] (b) The memory differentiation phenotype shifts toward central memory T cells (TCM);

[0024] (c) The expression level of the stem marker SLAMF6 was significantly upregulated;

[0025] (d) The secretion capacity of anti-tumor cytokines TNF-α and IFN-γ was significantly enhanced;

[0026] (e) Significantly enhanced ability to induce late apoptosis in tumor cells;

[0027] (f) The expression levels of antigen-presenting molecules CD86 and HLA-DR were significantly upregulated;

[0028] (g) Under continuous tumor antigen stimulation, the expression of activation marker CD25 and proliferation marker Ki67 was upregulated, while the expression of depletion marker LAG3 was downregulated.

[0029] Thirdly, this application provides the application of bifunctional γδ T cells in the preparation of drugs for treating tumors.

[0030] In some preferred embodiments, the tumor is cervical cancer.

[0031] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0032] 1. This application, by adding Cbl-b inhibitors at different times to the zoledronic acid and IL-2 amplification system, effectively overcomes the bottleneck of γδ T cell expansion reaching the plateau phase in existing technologies. Experimental results show that after treatment with Cbl-b inhibitors, Vδ2... + The total number of T cells and the expansion fold both increased significantly, with the expansion fold reaching nearly 3000 times. Short-term (24-hour) treatment also significantly enhanced cell proliferation.

[0033] 2. The method of this application can shift γδ T cells toward the central memory T cell (TCM) subset, while significantly increasing the expression level of the stemness marker SLAMF6, thereby enabling γδ T cells to maintain a durable immune response and long-term anti-tumor effect in vivo, and to have the potential to rapidly proliferate and differentiate into effector cells when they encounter antigens again.

[0034] 3. The method described in this application can significantly upregulate the secretion of anti-tumor cytokines TNF-α and IFN-γ in γδ T cells. After co-culturing with the cervical cancer cell line SiHa, the proportion of late-stage apoptosis in tumor cells significantly increased and the survival rate significantly decreased. Simultaneously, treatment with the Cbl-b inhibitor significantly enhanced the expression of antigen-presenting molecules CD86 and HLA-DR, enabling γδ T cells to possess both direct tumor-killing and antigen-presenting functions, which helps to form a synergistic anti-tumor immune response with αβ T cells.

[0035] 4. The expression of CD25, an activation marker of γδ T cells, and Ki67, a proliferation marker, prepared by the method of this application were significantly upregulated, and the expression of effector molecules Perforin, GZMB, TNF-α, and IFN-γ was increased. At the same time, the expression of the exhaustion marker LAG3 was significantly downregulated, indicating that the method of this application helps to maintain the long-term function and sustained anti-tumor ability of γδ T cells. Attached Figure Description

[0036] Figure 1The figure shows the results of how inhibiting Cbl-b can significantly improve the in vitro expansion efficiency of γδ T cells; in which, Figure 1 A is a schematic diagram of the experimental protocol. 1B shows the flow cytometry detection of CD3 amplification up to Day 14 after treating γδ T cells with DMSO or Cbl-b-IN-1 from Day 6 to Day 14. + Vδ2 + The proportion of T cells in the total number of cells (left), Vδ2 + The results show the number of T cells (middle) and the fold increase compared to Day 0 (right); 1C shows the flow cytometry analysis of CD3+ expansion to Day 14 after γδ T cells were treated with DMSO or Cbl-b-IN-1 from Day 10 to Day 14. + Vδ2 + The proportion of T cells in the total number of cells (left), Vδ2 + The results show the number of T cells (middle) and the fold increase compared to Day 0 (right); n=6, *, P<0.05; **, P<0.01.

[0037] Figure 2 The diagram shows the results of inhibiting Cbl-b to enhance the activation and proliferation of γδ T cells; in which, Figure 2 A is a schematic diagram of the experimental protocol: γδ T cells expanded in vitro to Day 10 were treated with DMSO or Cbl-b-IN-1 (2 μM) for 24 hours; 2B shows the flow cytometry detection of CD3. + Vδ2 + The expression results of CD25, a molecule related to T cell activation, are shown on the left (Vδ2). + CD25 + Cells account for Vδ2 + The percentage representation of cells is shown in the graph, with the right side being a statistical chart (n=9); 2C represents the CD3 count detected by flow cytometry. + Vδ2 + The expression results of Ki67, a T cell proliferation-related molecule, are shown on the left, while Vδ2 is on the right. + Ki67 + Vδ2 + The percentage representation of cells is shown in the graph, with the right side showing the statistical chart (n=6); 2D shows the CD3 count detected by flow cytometry. + Vδ2 + The proportion of T cells in the total number of cells (left), Vδ2 + The results show the number of T cells (middle) and the fold increase compared to Day 0 (right); n=9, *, P<0.05.

[0038] Figure 3 This is a diagram showing the effect of Cbl-b inhibition on γδ T cell memory differentiation; in which, Figure 3A is a representative graph showing the differentiation status of γδ T cells after treatment, as detected by flow cytometry; 3B shows the quantitative and statistical analysis of TN, TCM, TEM, and TEMRA at CD3 levels. + Vδ2 + Statistical chart of the proportion of T cells, n=9, *, P<0.05; **, P<0.01; ****, P<0.0001.

[0039] Figure 4 The figure shows the results of inhibiting Cbl-b to enhance the expression of γδ T cell stem-related molecules; in which, Figure 4 A. The left side shows a representative graph of the average fluorescence intensity (MFI) of SLAMF6, and the right side shows a statistical analysis graph, n=8; 4B shows LAG3. + Percentage and statistical chart of γδ T cells, n=9; 4C represents TIM3. + Percentage and statistical chart of γδ T cells, n=4; 4D represents PD-1. + Percentage and statistical graph of γδ T cells, n=4; **, P<0.001.

[0040] Figure 5 The figure shows the results of inhibiting Cbl-b upregulation of γδ T cell effector-related molecules; in which, Figure 5 A is Perforin + GZMB + Percentage of γδ T cells (left) and statistical graph (right), n=9; 5B represents TNF-α. + IFN-γ + Percentage of γδ T cells (left) and statistical graph (right), n=9; **, P<0.01.

[0041] Figure 6 The figure shows the results of inhibiting Cbl-b to enhance the anti-tumor ability of γδ T cells; in which Figure 6 A represents the co-culture of γδ T cells and SiHa cells at a 1:1 ratio for 12 h. Flow cytometry was used to detect SiHa cell apoptosis (left), and the proportion of late apoptosis was statistically analyzed (Annexin V). + 7-AAD + (Right) Result graph, n=8; 6B is the result graph of SiHa cell viability detected by CCK-8 assay after γδ T cells and SiHa cells were co-cultured at a 1:1 ratio for 12 h or 24 h, n=4; *, P<0.05; **, P<0.01.

[0042] Figure 7 The diagram shows the results of inhibiting Cbl-b to enhance the antigen-presenting potential of γδ T cells; in which, Figure 7 A shows the CD86 MFI representation chart on the left and the statistical analysis chart on the right; Figure 7 B shows the MFI representation plot of HLA-DR on the left and the statistical analysis plot on the right, n=9; *, P<0.05; **, P<0.01.

[0043] Figure 8 The figure shows the results of inhibiting Cbl-b to enhance the activation and proliferation levels of γδ T cells after continuous stimulation by tumor antigens; in which... Figure 8 A is a schematic diagram of the experimental protocol; 8B shows the flow cytometry detection of CD3. + Vδ2 + The expression results of CD25, a molecule related to T cell activation, are shown on the left. + Vδ2 + The percentage representation of T cells is shown in the graph, with statistical charts on the right; 8C represents CD3 detection by flow cytometry. + Vδ2 + The expression results of Ki67, a T cell proliferation-related molecule, are shown on the left. + Vδ2 + Percentage representation of T cells, with statistical chart on the right; n=9. ***, P<0.001.

[0044] Figure 9 The figure shows the results of inhibiting Cbl-b upregulation of the expression of effector molecules in γδ T cells after continuous stimulation by tumor antigens; among them, Figure 9 A is Perforin + GZMB + Percentage of γδ T cells (left) and statistical chart (right); 9B represents TNF-α. + IFN-γ + Percentage of γδ T cells (left) and statistical graph (right); n=9. *, P<0.05; **, P<0.01.

[0045] Figure 10 The figure shows the effect of inhibiting Cbl-b on the expression of depleted molecules in γδ T cells after continuous stimulation by tumor antigens; among them, Figure 10 A is LAG3 + Percentage of γδ T cells (left) and statistical graph (right), n=9; 10B represents TIM3. + Percentage of γδ T cells (left) and statistical graph (right), n=6; 10C represents PD-1. + Percentage of γδ T cells (left) and statistical graph (right), n=5; **, P<0.01.

[0046] Figure 11 The figure shows the results of downregulating LAG3 in mediating the inhibition of Cbl-b and enhancing γδ T cell function; in which, Figure 11 A represents the detection of CD3 by flow cytometry.+ Vδ2 + Perforin in T cells + GZMB + The percentage of γδ T cells; 11B is for CD3 detection by flow cytometry. + Vδ2 + TNF-α in T cells + IFN-γ + The percentage of γδ T cells; 11C is used to detect SiHa cell apoptosis by flow cytometry (left), and late apoptotic cells (Annexin V) are counted. + 7-AAD + The results of the proportion (right) are shown in the graph; n=4, *, P<0.05; **, P<0.01.

[0047] Figure 12 The figure shows the results of inhibiting Cbl-b downregulation of LAG3 expression in γδ T cells stimulated with anti-CD3 / CD28; in which, Figure 12 A represents LAG3 for each group. + Percentage representation of γδ T cells; 12B represents LAG3 in each group. + Statistical graph of the percentage of γδ T cells; n=5. *, P<0.05.

[0048] Figure 13 The diagram shows the results of inhibiting Cbl-b by downregulating LAG3 expression in γδ T cells through transcriptional repression; in which, Figure 13 A represents the flow cytometry detection of CD3 after the addition of CHX. + Vδ2 + LAG3 in T cells + The graph shows the change in the percentage of γδ T cells over time, n=4; 13B shows the flow cytometry results of CD3 after the addition of Act D. + Vδ2 + LAG3 in T cells + The results of the change in the percentage of γδ T cells over time, n=4; 13C is the result of RT-qPCR detection of LAG3 mRNA level, n=4; **, P<0.01.

[0049] Figure 14 The transcriptome analysis results show that inhibiting Cbl-b upregulates γδ T cell function and stemness-related gene expression; among them, Figure 14A is a volcano plot, showing the differentially expressed genes between the Cbl-b-IN-1 treatment group and the DMSO group after screening for P<0.05 and |log2(Fold Change)|>1. Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes with no difference. 14B is a gene enrichment analysis plot, showing the differences in some T cell activation, effector, stemness, and exhaustion-related genes. Red dots represent upregulated genes in the Cbl-b-IN-1 treatment group compared to the DMSO group, and blue dots represent downregulated genes.

[0050] Figure 15 The figure shows the results of GO and KEGG pathway enrichment analysis in γδ T cells treated with Cbl-b-IN-1 and DMSO; among them, Figure 15 A is the GO analysis result of the biological process (BP); 15B is the KEGG analysis result; n=3.

[0051] Figure 16 The figure shows the results of the PI3K-AKT signaling pathway mediating the inhibition of Cbl-b-induced downregulation of LAG3 expression; among them, Figure 16 A represents the detection of CD3 by flow cytometry. + Vδ2 + LAG3 in T cells + Percentage representation of γδ T cells; 16B represents LAG3 in each group. + Statistical graph of the percentage of γδ T cells; n=4. *, P<0.05; **, P<0.01; ***, P<0.001.

[0052] Figure 17 This is a Western blot image showing the results of Cbl-b regulating LAG3 expression via the PI3K-AKT-GSK-3β-T-bet axis; where, Figure 17 Figure A shows the results of Western blot analysis of changes in the protein levels of PI3K, AKT, p-AKT, GSK-3β, p-GSK-3β (S9), T-bet, and LAG3, and the calculation of their relative expression levels; Figure 17B shows the results of Western blot analysis of the changes in the protein levels of the above proteins after the addition of LY294002.

[0053] Figure 18 This diagram illustrates the signaling pathway by which inhibiting Cbl-b enhances the anti-tumor function of γδ T cells by downregulating LAG3 expression. Cbl-b-IN-1, by inhibiting Cbl-b, activates the PI3K-AKT-GSK-3β-T-bet signaling pathway, downregulates LAG3 expression, and enhances activation, stemness maintenance, and effector function, thereby strengthening the anti-tumor function of γδ T cells. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0055] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0056] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0057] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following explanations of terminology are provided to better understand this application.

[0059] 1. Cbl-b: refers to an E3 ubiquitin ligase that negatively regulates T cell activation. Cbl-b inhibits the transduction of T cell receptors (TCRs) and co-stimulatory signaling pathways by ubiquitinizing and degrading key signaling molecules, thereby limiting T cell activation, proliferation, and effector functions.

[0060] 2. Cbl-b inhibitor: refers to a compound or biomolecule that can inhibit Cbl-b enzyme activity or downregulate its expression level. An exemplary Cbl-b inhibitor in this application is Cbl-b-IN-1 (CAS: 2368841-84-1, molecular formula: C...). 29 H 34 N6O2 (molecular weight: 498.62), structural formula is: It can be obtained through commercial channels; this application was made from MedChemExpress, catalog number HY-136339. By inhibiting Cbl-b, its negative regulation of T cell signaling can be relieved, enhancing the activation, expansion, and anti-tumor function of γδ T cells.

[0061] 3. γδ T cells: These are a subset of T lymphocytes that express T cell receptors (TCRs) composed of γ and δ chains, distinct from traditional αβ T cells. γδ T cells do not rely on MHC molecules to recognize antigens and are characterized by rapid response, widespread tissue distribution, and dual functions of innate and adaptive immunity.

[0062] 4. Time-Stage Addition: In the amplification method of this application, zoledronic acid, IL-2, and Cbl-b inhibitor are not added to the culture medium at the same time point, but are added stepwise according to a specific time sequence. Specifically, in this application, zoledronic acid and IL-2 are added on day 0 for initial activation, and then the Cbl-b inhibitor is added at any time point between day 6 and day 10. This time-stage addition strategy is a key process feature for achieving synergistic effects.

[0063] 5. Dual function: This refers to γδ T cells that, after being expanded using the method described in this application, possess two anti-tumor functions: one is a direct killing function, which directly kills tumor cells by secreting perforin, granzyme B (GZMB), and cytokines TNF-α and IFN-γ; the other is an antigen-presenting function, which activates αβ T cells by expressing antigen-presenting molecules such as CD86 and HLA-DR, thereby forming a synergistic anti-tumor immune response.

[0064] 6. SLAMF6: This refers to a co-stimulatory molecule expressed on the surface of immune cells, which is associated with stemness maintenance and long-term survival in T cells. T cells that highly express SLAMF6 have stronger proliferative capacity and more durable anti-tumor effects.

[0065] 7. LAG3: This refers to an immune checkpoint molecule whose expression is upregulated when T cells are continuously stimulated by antigens, transmitting inhibitory signals and leading to T cell exhaustion. In this application, by inhibiting Cbl-b, LAG3 expression can be downregulated through the PI3K-AKT-GSK-3β-T-bet axis, thereby delaying γδ T cell exhaustion.

[0066] The following are specific examples:

[0067] Example 1: Isolation of human peripheral blood mononuclear cells and initial expansion of γδ T cells

[0068] Human peripheral blood lymphocyte separation medium (Lanjieke, catalog number: LTS1077) was used to separate PBMCs: An equal volume of blood was mixed with PBS and slowly added along the tube wall to a centrifuge tube containing 5 ml of separation medium; centrifuged at 900×g, 24 °C, with an ascending speed of 1 and a descending speed of 0 for 30 min, and the white membrane layer (mononuclear cells) was collected and transferred to 10 ml of PBS; centrifuged at 500×g, 24 °C for 5 min, and the supernatant was discarded; the cells were resuspended in 10 ml of RPMI 1640 medium containing 10% FBS, and centrifuged under the same conditions for 5 min; the supernatant was discarded, 2 ml of erythrocyte lysis buffer was added, and the cells were treated at 4 °C for 10 min, then 10 ml of RPMI 1640 medium containing 10% FBS was added to stop the treatment, and the cells were centrifuged at 500×g, 24 °C for 5 min to obtain PBMCs;

[0069] PBMCs were 2×10 per well. 6 Cells were seeded at a density of 1,000 cells per well in 24-well plates and cultured in X-VIVO medium (Lonza, catalog number: 04-418Q) containing 10% (v / v) fetal bovine serum (FBS, Gibco, catalog number: A2663916CP).

[0070] On day 0 of amplification, 5 μM zoledronic acid (ZOL, Sigma, SML0223) and 1000 IU / ml recombinant human IL-2 (Beijing Sihuan Biopharmaceutical Co., Ltd.) were added to the culture medium and cultured until day 4. On day 4, the cells were transferred to 6-well plates, and 1 ml of fresh culture medium containing 5 μM ZOL and 1000 IU / ml recombinant human IL-2 was added. The cells were cultured until day 6.

[0071] On Day 6 and thereafter, cells were counted daily, and fresh X-VIVO medium containing 1000 IU / ml IL-2 was added to maintain a cell density of 0.8–1.2 × 10⁻⁶ cells / day. 6 Within the range of / ml.

[0072] Example 2: Effect of Cbl-b inhibitor addition at different time points on γδ T cell expansion efficiency

[0073] Based on the culture protocol in Example 1 above, the Cbl-b inhibitor Cbl-b-IN-1, with a final concentration of 2 μM, was added at different time points:

[0074] Option 1: When culturing to Day 6, add Cbl-b-IN-1 to maintain a final concentration of 2 μM, and continue culturing to Day 14.

[0075] Option 2: When culturing to Day 10, add Cbl-b-IN-1 to maintain a final concentration of 2 μM, and continue culturing to Day 14.

[0076] Option 3: When culturing to Day 10, add Cbl-b-IN-1 to maintain a final concentration of 2 μM, and continue culturing to Day 11.

[0077] Figure 1 A is a schematic diagram of the experimental design, such as... Figure 1 As shown in A, with 2×10 6 Starting with 1000 IU / ml recombinant human IL-2 and 5 μM ZOL on Day 0, cells were cultured until Day 6. During subsequent culture, cells were counted daily, and fresh medium containing 1000 IU / ml IL-2 was added to maintain a cell density of 0.8–1.2 × 10⁶ cells / ml. 6 Within the range of / ml, cultured until Day 14; in γδ T cells expanded to Day 6 or Day 10, DMSO (control group) or Cbl-b-IN-1 (2 μM, experimental group) were added and treated until Day 14.

[0078] CD3 was detected using flow cytometry. + Vδ2 + The percentage of T cells and the total number of cells were compared with the number of cells on Day 0, and CD3 were calculated. + Vδ2 + The final fold expansion of T cells.

[0079] Flow cytometry results showed that, in the Day 6 to Day 14 treatment groups, the total number of cells and the fold increase in the Cbl-b-IN-1 group were significantly increased, suggesting that inhibiting Cbl-b can promote cell proliferation. Figure 1 B). In the Day 10 to Day 14 treatment groups, the Vδ2 of the Cbl-b-IN-1 group... + The total number of T cells also increased significantly, and the expansion rate was also significantly improved. Figure 1 C). In the Day 10 to Day 11 treatment groups: even after only 24 hours of treatment, Cbl-b-IN-1 treatment significantly enhanced cell proliferation ( Figure 2 D).

[0080] The above results show that even though γδ T cells at different proliferation stages exhibit varying sensitivity to Cbl-b inhibition, the addition of Cbl-b inhibitors at different starting time points significantly enhances Vδ2. + The expansion capacity of γδ T cells was observed. Specifically, the expansion effect of adding the drug on Day 10 to Day 14 (scheme 2) was slightly better than that of adding the drug on Day 6 to Day 14 (scheme 1). These results collectively demonstrate that inhibiting Cbl-b significantly increases Vδ2. +The efficiency of in vitro expansion of T cells lays the foundation for further enhancing their function for anti-tumor research.

[0081] Example 3: Effect of inhibiting Cbl-b on γδ T cell immunophenotype

[0082] 3.1 Inhibition of Cbl-b enhances the activation and proliferation of γδ T cells

[0083] Figure 2 Figure A shows a schematic diagram of the experimental protocol. On Day 10, γδ T cells were treated with either DMSO or Cbl-b-IN-1 (2 μM) for 24 hours. After treatment, the expression of CD25 (activation marker) and Ki67 (proliferation marker) was detected by flow cytometry.

[0084] The flow cytometry results are as follows:

[0085] (1) CD25 expression in the Cbl-b-IN-1 treatment group was significantly higher than that in the DMSO group. Figure 2 B);

[0086] (2) Although there was no statistically significant difference in Ki67 expression (P=0.0725), it also showed an increasing trend in the Cbl-b-IN-1 treatment group. Figure 2 C).

[0087] (3) Cbl-b-IN-1 treatment can significantly increase Vδ2 + The total number and expansion fold of T cells can be increased, with the expansion fold reaching nearly 3000-fold. Consistent with the results of long-term Cbl-b-IN-1 treatment, short-term (24-hour) Cbl-b-IN-1 treatment can still enhance cell proliferation. Figure 2 D), and can enhance the activation of γδ T cells.

[0088] 3.2 Inhibition of Cbl-b affects the memory differentiation of γδ T cells

[0089] After Day 10 γδ T cells were treated with DMSO or Cbl-b-IN-1 for 24 hours, their differentiation phenotypes were detected by flow cytometry, including: naive T cells (TN), central memory T cells (TCM), effector memory T cells (TEM), and terminally differentiated effector memory T cells (TEMRA).

[0090] Figure 3 The figure shows the effect of Cbl-b inhibition on γδ T cell memory differentiation. Compared with the DMSO group, the proportion of TCM in the Cbl-b-IN-1 treatment group was significantly increased, while the proportions of TEMRA and TEM decreased, and the proportion of TN changed little. Figure 3 A, 3B).

[0091] These results indicate that inhibiting Cbl-b can cause γδ T cells to shift towards the central memory T cell (TCM) subset, which is beneficial for maintaining a long-lasting immune response in vivo.

[0092] 3.3 Inhibition of Cbl-b enhances the expression of γδ T cell stemness-related molecules

[0093] This experiment aimed to evaluate whether inhibiting Cbl-b would regulate γδ T cell stemness and exhaustion. The specific steps were as follows:

[0094] On Day 10 Vδ2 + After 24 hours of treatment with DMSO or Cbl-b-IN-1 in γδ T cells, the expression of stemness marker SLAMF6 and exhaustion markers LAG3, TIM3, and PD-1 was detected by flow cytometry.

[0095] Figure 4 The figure shows the results of inhibiting Cbl-b to enhance the expression of γδ T cell stemness-related molecules. The following results can be drawn from the figure:

[0096] (1) Cbl-b-IN-1 treatment significantly increased the expression level of SLAMF6 in γδ T cells ( Figure 4 A);

[0097] (2) The expression of exhaustion-related molecules LAG3, TIM3, and PD-1 did not differ significantly between the DMSO and Cbl-b-IN-1 treatment groups. Figure 4 (B, 4C, 4D).

[0098] These results indicate that inhibiting Cbl-b can enhance the stemness of γδ T cells, giving them the potential for long-term survival and differentiation into effector cells, thus providing a potential advantage for maintaining long-term anti-tumor effects in vivo after adoptive infusion.

[0099] 3.4 Inhibition of Cbl-b upregulation of γδ T cell effector-related molecules

[0100] This experiment aimed to evaluate the effect of Cbl-b inhibition on the effector function of γδ T cells. The specific steps were as follows: On Day 10, γδ2... + After 24 hours of treatment with DMSO or Cbl-b-IN-1 in γδ T cells, the expression of cytotoxic molecules Perforin and GZMB, as well as cytokines TNF-α and IFN-γ, was detected by flow cytometry.

[0101] Figure 5 The figure shows the results of inhibiting Cbl-b upregulation of γδ T cell effector-related molecules. From the figure, we can see that:

[0102] (1) Cbl-b-IN-1 processing group Perforin + GZMB + The proportion of γδ T cells was not significantly different from that of the control group. Figure 5 A);

[0103] (2) TNF-α in the Cbl-b-IN-1 treatment group + IFN-γ + The proportion of γδ T cells increased significantly ( Figure 5 B).

[0104] The above results indicate that Cbl-b-IN-1 treatment of γδ T cells can significantly enhance the expression of cytotoxic molecules such as TNF-α and IFN-γ while maintaining high levels of cytotoxicity-related molecules, thereby improving overall effector function.

[0105] 3.5 Inhibition of Cbl-b enhances the killing ability of γδ T cells against tumor cells

[0106] This experiment aims to further verify whether the inhibition of Cbl-b-induced downregulation of γδ T cell exhaustion-related molecules and upregulation of effector-related molecules can be translated into an actual killing effect on tumor cells. The specific method is as follows:

[0107] On Day 10 Vδ2 + After γδ T cells were treated with DMSO or Cbl-b-IN-1 for 24 hours, they were co-cultured with the cervical cancer cell line SiHa (ATCC, HTB-35), and the following assays were performed:

[0108] (1) Apoptosis detection: Effector cells (γδ T cells) and target cells (SiHa cells) were co-cultured at a 1:1 ratio for 12 hours, and tumor cell apoptosis was detected according to the instructions using the Annexin V / 7-AAD apoptosis detection kit (Biolegend, 640914).

[0109] (2) Survival rate detection: Effector cells and target cells were co-cultured at a 1:1 ratio for 12 hours and 24 hours, and the survival rate of tumor cells was detected by CCK-8 kit (Nanjing Novizan Biotechnology Co., Ltd., A311-01) according to the instructions.

[0110] Figure 6 The figure shows the results of inhibiting Cbl-b to enhance the anti-tumor ability of γδ T cells. From the figure, we can see that:

[0111] (1) Flow cytometry results showed that the proportion of late apoptosis in SiHa cells in the Cbl-b-IN-1 treatment group (Annexin V) + 7-AAD+ ) significantly increased ( Figure 6 A).

[0112] (2) The results of CCK-8 assays further confirmed that Cbl-b-IN-1 treatment significantly reduced the survival rate of SiHa cells in both 12-hour and 24-hour co-culture systems, and the killing effect was more significant at 24 hours. Figure 6 B).

[0113] The above results indicate that inhibiting Cbl-b can significantly enhance the killing ability of γδ T cells against tumor cells.

[0114] 3.6 Inhibition of Cbl-b enhances the antigen presentation potential of γδ T cells

[0115] This experiment aimed to investigate the effect of inhibiting Cbl-b on the antigen-presenting potential of γδ T cells. The specific steps were as follows:

[0116] On Day 10 Vδ2 + After 24 hours of treatment with DMSO or Cbl-b-IN-1 in γδ T cells, the expression levels of classical antigen-presenting molecules CD86 and HLA-DR were detected by flow cytometry.

[0117] Figure 7 The result of inhibiting Cbl-b to enhance the antigen presentation potential of γδ T cells is shown in the figure. From the figure, we can see that:

[0118] Cbl-b-IN-1 treatment significantly enhances CD86 ( Figure 7 A) and HLA-DR ( Figure 7 The expression of B).

[0119] The above results indicate that Cbl-b inhibition can effectively enhance Vδ2. + The antigen-presenting potential of γδ T cells gives them the dual function of directly killing tumor cells and activating αβ T cells.

[0120] The results of this embodiment demonstrate that short-term Cbl-b-IN-1 treatment can significantly enhance Day 10 Vδ2. +The Cbl-b-IN-1 treatment group enhanced the activation level and in vitro expansion capacity of γδ T cells, while also reshaping their differentiation profile, shifting them towards the TCM (transmissible cell mutational pathway), and significantly increasing the T cell stemness index SLAMF6. Furthermore, the Cbl-b-IN-1 treatment group also increased the secretion of cytokines TNF-α and IFN-γ, their direct tumor-killing ability, and their antigen-presenting potential. These results suggest that short-term Cbl-b-IN-1 treatment can maintain the high functional state of γδ T cells, improve cell stemness, prolong cell survival time, and enable them to form a durable and efficient anti-tumor immune response. These results indicate that Cbl-b inhibition not only enhances the in vitro function of γδ T cells but also optimizes their potential in vivo anti-tumor capacity, providing a strong foundation for the clinical application of γδ T cells.

[0121] Example 4: Inhibiting Cbl-b to enhance the function of γδ T cells after continuous stimulation by tumor antigens

[0122] The results of the foregoing embodiments systematically reveal the regulatory effect of Cbl-b inhibition on γδ T cell activation, proliferation, differentiation, stemness, exhaustion, and effector function. To fully reflect the functional state of γδ T cells under long-term exposure to antigen stimulation in the tumor microenvironment, this embodiment simulates continuous tumor-associated antigen stimulation by establishing a long-term co-culture system of Cbl-b-IN-1 treated γδ T cells and SiHa cells to observe the effects of Cbl-b inhibition on the activation state, anti-tumor ability, and exhaustion molecules of γδ T cells after subsequent continuous stimulation by tumor antigens.

[0123] Figure 8 A is a schematic diagram of the experimental scheme, as shown in the figure. Day 10 Vδ2 + γδ T cells were treated with DMSO or Cbl-b-IN-1 for 24 hours and then co-cultured with the cervical cancer cell line SiHa at a 1:1 ratio for 4 days.

[0124] 4.1 Detection of activation and proliferation levels

[0125] After 4 days of co-culture, flow cytometry was used to detect the cell activation marker CD25 and the proliferation marker Ki67.

[0126] The results are as follows Figure 8 As shown in the figure. It can be seen from the figure that:

[0127] (1) Under co-culture conditions, the CD25 expression of γδ T cells in the Cbl-b-IN-1 treatment group was significantly higher than that in the DMSO control group ( Figure 8 B); This suggests that its activation state continues to increase after continuous exposure to tumor antigens.

[0128] (2) Ki67 did not show significant differences under previous short-term treatments, but after co-culturing with SiHa cells for 4 days, Ki67 in γδ T cells of the Cbl-b-IN-1 treatment group was significantly increased. Figure 8 C); therefore, it is believed that continuous stimulation by tumor antigens inhibits Cbl-b, maintains cell activation, and promotes the long-term proliferative potential of γδ T cells.

[0129] The above results indicate that γδ T cells that have been temporarily suppressed by Cbl-b beforehand can maintain a high activation phenotype and enhance cell proliferation after being continuously stimulated by tumor antigens in vitro. This suggests that the approach may improve the survival time and tumor-killing ability of γδ T cells after reinfusion into the body in the tumor microenvironment.

[0130] 5.2 Detection of effect-related molecular expression

[0131] Since Cbl-b inhibition was observed to maintain the highly activated state of γδ T cells during continuous co-culture, suggesting that they may maintain their anti-tumor potential in vivo, this experiment further evaluated the effector function of γδ T cells under continuous antigen stimulation. Flow cytometry was used to detect the expression of key cytotoxic molecules Perforin and GZMB, and anti-tumor cytokines TNF-α and IFN-γ.

[0132] The results are as follows Figure 9 As shown in the figure:

[0133] (1) Perforin in γδ T cells of the Cbl-b-IN-1 treatment group + GZMB + The proportion of γδ T cells was significantly higher than that in the DMSO control group ( Figure 9 (A) This change was not observed with previous short-term antigen stimulation.

[0134] (2) TNF-α + and IFN-γ + The proportion of cells also increased significantly. Figure 9 Therefore, it is believed that inhibiting Cbl-b not only maintains the secretion of anti-tumor cytokines, but also enhances the expression of cytotoxic molecules.

[0135] These results indicate that, under conditions of continuous antigen stimulation in vivo, Cbl-b inhibition can significantly enhance the antitumor effect of γδT cells.

[0136] 5.3 Detection of Depleted Molecular Expression

[0137] This experiment further evaluated the role of Cbl-b inhibition in maintaining the functional stability of γδ T cells and whether it affects the expression of depleted molecules under prolonged antigen stimulation. Flow cytometry was used to detect Day 10 Vδ2.+ Expression of PD-1, TIM3 and LAG3, T cell exhaustion markers, in γδ T cells after treatment with DMSO or Cbl-b-IN-1 for 24 hours and co-culture with SiHa for 4 days.

[0138] The results are as follows Figure 10 As shown in the figure:

[0139] (1) The expression of LAG3 in γδ T cells of the Cbl-b-IN-1 treatment group was significantly lower than that in the DMSO control group. Figure 10 A) suggests that Cbl-b inhibitors can reduce the expression of LAG3 in γδ T cells under continuous antigen stimulation.

[0140] (2) TIM3 ( Figure 10 B) and PD-1 ( Figure 10 C) The expression was low in both groups, and the difference was not significant.

[0141] These results indicate that inhibiting Cbl-b not only maintains the activation and cytotoxic function of γδ T cells in a simulated in vivo environment of continuous antigen stimulation, but also downregulates the expression of the exhaustion marker LAG3, contributing to the maintenance of long-term cell function and sustained anti-tumor ability. This finding has significant implications for the clinical translation of γδ T cell adoptive infusion therapy. Infused γδ T cells often face challenges in the tumor microenvironment, including limited proliferation, short lifespan, decreased cytotoxic activity, and increased exhaustion. Treating γδ T cells with Cbl-b inhibitors before adoptive infusion shows promise as an effective strategy to alleviate these challenges, providing new insights for optimizing γδ T cell adoptive immunotherapy and improving clinical efficacy in solid tumors.

[0142] Example 5: Downregulation of LAG3 participates in mediating the inhibition of Cbl-b-enhanced γδ T cell function.

[0143] This embodiment aims to verify whether the ability to inhibit Cbl-b's enhancement of γδT function is related to its reduction of the depletion marker LAG3. The specific method is as follows:

[0144] On Day 10 Vδ2 + γδ T cells were treated with DMSO (control group) or Cbl-b-IN-1 for 24 hours, with or without the LAG3 blocking antibody Relatlimab (MedChemExpress, HY-P99156), and then co-cultured with SiHa for 4 days. The expression of cytotoxic and function-related molecules Perforin, GZMB, TNF-α, and IFN-γ was detected. Furthermore, after 4 days of co-culture, γδ T cells were co-cultured with SiHa again for 12 hours, and tumor cell apoptosis was detected.

[0145] The results are as follows Figure 11 As shown in the figure:

[0146] Compared to the DMSO group, the perforin levels in the Cbl-b-IN-1, DMSO+Relatlimab, and Cbl-b-IN-1+Relatlimab groups were significantly lower. + GZMB + γδ T cell ratio and TNF-α + IFN-γ + The proportion of γδ T cells was significantly increased ( Figure 11 (A, B) indicates that inhibiting Cbl-b or directly blocking LAG3 can enhance the cytotoxic molecular levels of γδ T and the expression of anti-tumor cytokines. Corresponding tumor cell apoptosis experiments also showed that, compared with the DMSO control group, the other three treatments significantly increased the late apoptosis rate of SiHa (…). Figure 11 C), indicating that the above-mentioned functional enhancements can be translated into actual lethality.

[0147] The above indicators did not show significant differences among the Cbl-b-IN-1, DMSO+Relatlimab, and Cbl-b-IN-1+Relatlimab groups, suggesting that there may be redundancy in the mechanisms of inhibiting Cbl-b and blocking LAG3-mediated enhancement of γδ T cell anti-tumor function. Combined with the aforementioned results of Cbl-b upregulation of LAG3 expression, this suggests that inhibiting Cbl-b depends at least partially on downregulating LAG3 to enhance the anti-tumor function of γδ T cells. In clinical applications, compared to the requirement for continuous use of LAG3 antibodies after γδ T cell reinfusion, the strategy of transiently inhibiting Cbl-b during in vitro expansion is simpler and therefore has greater translational potential.

[0148] Example 6: Inhibition of Cbl-b downregulation of anti-CD3 / CD28-stimulated LAG3 expression in γδ T cells

[0149] Since the above embodiments suggest that inhibiting Cbl-b can enhance the anti-tumor ability of γδ T cells by downregulating the expression of the exhaustion marker LAG3, this embodiment uses short-term anti-CD3 / CD28 treatment to replace continuous stimulation by tumor cells to simplify the model and explore the molecular mechanism of Cbl-b inhibition downregulating LAG3 in γδ T cells. Anti-CD3 provides the first signal to initiate T cell activation, while anti-CD28 transmits co-stimulatory signals. This stimulation regimen, combined with IL-2 treatment, provides the three essential elements for complete T cell activation, which is stronger than the IL-2 regimen used previously. Therefore, it can better simulate continuous antigen stimulation and eliminates the confounding factors caused by tumor cell death in continuous tumor cell stimulation.

[0150] In this embodiment, γδ T cells were cultured for 24 h without IL-2 to maintain low LAG3 expression, which served as the unstimulated baseline. The other two groups were subsequently treated with anti-CD3 / CD28+DMSO or anti-CD3 / CD28+Cbl-b-IN-1 for 24 h in the presence of IL-2, and changes in LAG3 expression were then detected.

[0151] The results are as follows Figure 12 As shown in the figure:

[0152] LAG3 expression was lowest in the baseline group. After anti-CD3 / CD28 stimulation, the expression level in the DMSO group was upregulated, while the LAG3 expression in the Cbl-b inhibition group was significantly reduced to the baseline level before stimulation. Figure 12 A, B).

[0153] These results indicate that TCR-mediated activation signaling leads to the upregulation of LAG3 in γδ T cells, and Cbl-b inhibition can reduce the expression of this exhaustion marker and delay functional inhibition, further supporting the hypothesis that Cbl-b inhibition maintains long-term activation and function of γδ T cells by regulating LAG3. Furthermore, since this result is consistent with the results obtained in the previous γδ T cell-SiHa cell co-culture system, this experimental system was used in subsequent examples to further explore the molecular mechanism of Cbl-b downregulation of LAG3 expression.

[0154] Example 7: Inhibition of Cbl-b downregulates LAG3 membrane expression by inhibiting transcription.

[0155] LAG3 expression is regulated at multiple levels, including transcription, translation, and post-translational modification. To further clarify the specific mechanism by which Cbl-b downregulates LAG3 expression in γδ T cells, this study stimulated Day 10 γδ T cells with anti-CD3 / CD28 and added either DMSO or Cbl-b-IN-1. Cycloheximide (CHX, MedChemExpress, HY-12320) was added during culture to inhibit protein synthesis. LAG3 expression levels were detected after 4, 8, and 12 hours of treatment.

[0156] Actinomycin D (Act D) is a classic transcription inhibitor that inhibits the synthesis of new mRNA by binding to the DNA strand and blocking RNA polymerase activity. To further clarify whether the regulation of LAG3 in γδ T cells by Cbl-b inhibition depends on the transcriptional level, this study administered Act D (Shanghai Aladdin, A432787) to Day 10 γδ T cells simultaneously with anti-CD3 / CD28 stimulation to inhibit transcription. LAG3 expression levels were then measured after treatment with DMSO or Cbl-b-IN-1 for 4, 8, and 12 hours, respectively.

[0157] The results are as follows Figure 13 As shown in the figure:

[0158] In the presence of CHX, there was no significant difference in LAG3 expression at 4, 8, and 12 hours in either the DMSO group or the Cbl-b-IN-1 group. Figure 13 A) indicates that the protein degradation rates of LAG3 in the two groups were essentially equivalent. The results suggest that the mechanism by which Cbl-b inhibits the downregulation of LAG3 expression in γδ T cells depends on the synthesis of new proteins: it may reduce LAG3 membrane surface expression by downregulating the transcriptional or translational levels of LAG3, rather than by accelerating the degradation of already synthesized proteins.

[0159] In the presence of Act D, LAG3 expression showed no significant difference at 4, 8, and 12 hours in both the DMSO and Cbl-b-IN-1 groups, and the rate of decrease in LAG3 expression was comparable over time. Figure 13B) indicates that transcriptional inhibition eliminated the effect of Cbl-b-IN-1 in downregulating LAG3 expression in γδ T cells, suggesting that the mechanism by which Cbl-b-IN-1 downregulates LAG3 expression in γδ T cells occurs at the transcriptional rather than translational level. This hypothesis was confirmed by RT-qPCR results: under anti-CD3 / CD28 stimulation, the mRNA content of LAG3 in Vδ2+ T cells treated with DMSO or Cbl-b-IN-1 for 24 h was significantly lower in the Cbl-b-IN-1 treatment group than in the DMSO group. Figure 13 C).

[0160] The above results collectively indicate that the effect of inhibiting Cbl-b in downregulating LAG3 expression in γδ T cells is mainly achieved by reducing LAG3 transcriptional levels, rather than translational or post-translational regulation.

[0161] Example 8: Effect of inhibiting Cbl-b on the γδ T cell transcriptome

[0162] To systematically evaluate the effect of Cbl-b inhibition on the global transcriptional state of γδ T cells and further explore the possible regulatory pathways for downregulating LAG3 expression in γδ T cells by Cbl-b inhibition, RNA was extracted from Day 10 γδ T cells after treatment with DMSO or Cbl-b-IN-1 for 24 hours under anti-CD3 / CD28 stimulation and then subjected to transcriptome sequencing.

[0163] Figure 14 The figure shows the transcriptome analysis results of Cbl-b inhibition upregulating γδ T cell function and stemness-related gene expression. Figure 14 A is a volcano plot, showing the differentially expressed genes between the Cbl-b-IN-1 treatment group and the DMSO group after screening for P < 0.05 and |log2(Fold Change)| > 1. Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes with no difference. Figure 14 B is a gene enrichment analysis plot, showing differences in genes related to T cell activation, effector function, stemness, and exhaustion. Red indicates genes upregulated in the Cbl-b-IN-1 treatment group compared to the DMSO group, and blue indicates downregulated genes. The volcano plot and gene enrichment analysis show (…). Figure 14 Compared to the DMSO control group (A and B), the Cbl-b-IN-1 treatment group showed significantly upregulated expression of multiple activation and effector-related genes (such as IL2, TNF, IFNG, and GZMB), indicating enhanced γδ T cell activation and cytotoxicity. Simultaneously, inhibition of Cbl-b also significantly upregulated stemness-related genes BACH2, ID3, KLF2, CXCR5, and XCL1. In summary, transcriptome analysis indicates that inhibition of Cbl-b can promote the maintenance of central memory or stemness phenotype in γδ T cells.

[0164] Meanwhile, previous studies have shown that in CD8 + There is a positive feedback regulation between NR4A3 and TOX in T cells, and they jointly characterize the exhaustion state of T cells. In this example, NR4A3 expression was significantly downregulated in the Cbl-b-IN-1 treatment group, which further supports the idea that inhibiting Cbl-b can reduce the exhaustion of γδ T cells.

[0165] Example 9: GO and KEGG pathway enrichment analysis reveals the signaling pathways upregulated by γδ T cells after Cbl-b inhibition.

[0166] To further explore the potential molecular mechanisms by which Cbl-b inhibits and regulates γδ T cell function and stemness, this study performed GO enrichment analysis on differentially expressed genes obtained from transcriptome sequencing. The results showed that genes upregulated in the Cbl-b-IN-1 treatment group were significantly enriched in multiple pathways related to immune activation, particularly T cell activation pathways, suggesting that γδ T cell activation is enhanced at the transcriptional level. Furthermore, lymphocyte differentiation was also enriched. Figure 15 A) suggests that Cbl-b inhibition may promote the maintenance of γδ T cell activation, function, and stemness, while delaying exhaustion.

[0167] To investigate the molecular mechanism by which Cbl-b inhibits and maintains γδ T cell activation and stemness, this study focused on enriching pathways related to T cell function and exhaustion regulation from differentially expressed genes identified by RNA-seq. KEGG enrichment analysis showed that the differentially expressed genes in the Cbl-b-IN-1 treatment group were significantly enriched in multiple classical T cell signaling pathways, including the NF-κB signaling pathway, JAK-STAT signaling pathway, and PI3K-AKT signaling pathway. In addition, they were also enriched in other immune-related pathways, such as those related to stemness or proliferation. These pathways are all closely related to γδ T cell activation, enhanced function, and delayed exhaustion. Figure 15 B).

[0168] Example 10: PI3K-AKT signaling pathway mediates inhibition of Cbl-b-induced downregulation of LAG3 expression.

[0169] To verify the role of the above pathways in this process, this embodiment selected corresponding specific inhibitors for intervention experiments, including JSH-23, which inhibits NF-κB nuclear translocation and transcriptional activity and blocks the NF-κB signaling pathway; Stattic, which inhibits STAT3 phosphorylation and dimerization, thereby blocking the JAK-STAT signaling pathway; LY294002, which can specifically inhibit PI3K kinase activity, thereby blocking the downstream signal of PI3K-AKT; and Rapamycin, which inhibits the downstream mTORC1 complex of PI3K-AKT.

[0170] In this embodiment, anti-CD3 / CD28 stimulation, DMSO or Cbl-b-IN-1, and LAG3 expression were detected after 24 hours of treatment with the above inhibitors in Day 10 γδ T cells to evaluate the role of different signaling pathways in Cbl-b regulation of γδ T cell exhaustion.

[0171] Experimental results are as follows Figure 16 As shown in the figure:

[0172] After treatment with JSH-23 or Static, the LAG3 expression level in the Cbl-b-IN-1 treatment group remained significantly lower than that in the DMSO group, indicating that the NF-κB and JAK-STAT signaling pathways are not involved in the regulation of LAG3 by Cbl-b. In contrast, the difference in LAG3 expression between the DMSO group and the Cbl-b-IN-1 group disappeared only when the PI3K-AKT inhibitor LY294002 was added, suggesting that the PI3K-AKT signaling pathway is a key downstream pathway for Cbl-b inhibition and regulation of LAG3 expression. Interestingly, although mTOR is downstream of PI3K-AKT, no significant difference was observed between the Rapamycin treatment group and the control group, indicating that mTOR is not the main pathway mediating LAG3 regulation (16A, B), and Cbl-b may exert its regulatory role through other downstream branches of the PI3K-AKT signaling pathway. In summary, Cbl-b inhibition of LAG3 expression in γδ T cells mainly relies on the PI3K-AKT signaling pathway, but may not exert its effect through the downstream mTOR pathway of PI3K-AKT.

[0173] Example 11: Cbl-b modulates LAG3 via the PI3K-AKT-GSK-3β-T-bet axis

[0174] The results of the above examples indicate that the PI3K-AKT signaling pathway may be a key pathway for Cbl-b to regulate LAG3 expression on γδ T cells. However, its classic downstream pathway, mTOR, is not involved. Therefore, this example explores other downstream pathways. Previous studies have shown that GSK3 is the first reported AKT substrate, and AKT can repressively phosphorylate the S9 site of GSK-3β, altering its spatial conformation and thus hindering the binding of GSK-3β to its substrate. In mouse CD8... +In T cells, GSK-3β inactivation promotes Tbx21 transcription and increases T-bet expression, thereby inhibiting the expression of the T cell exhaustion marker PD-1. Furthermore, another study demonstrated that GSK-3β inhibition upregulates T-bet expression, and T-bet binds to the LAG3 gene promoter, inhibiting its transcription while simultaneously promoting the expression of effector molecules GZMB and IFN-γ. Based on this, this embodiment hypothesizes that Cbl-b inhibition may regulate LAG3 expression in γδ T cells through the PI3K-AKT-GSK-3β-T-bet signaling axis.

[0175] In this embodiment, Day 10 γδ T cells were stimulated with anti-CD3 / CD28 and treated with either DMSO or Cbl-b-IN-1 for 24 hours. Proteins were then extracted and Western blot analysis was performed to detect key molecules in the PI3K-AKT-GSK-3β-T-bet signaling axis, including PI3K, AKT, p-AKT, GSK-3β, p-GSK-3β (S9), T-bet, and LAG3. The results showed that compared with the DMSO control, the levels of p-AKT and p-GSK-3β (S9) were significantly upregulated in the Cbl-b-IN-1 treatment group, indicating that GSK-3β was inactivated by AKT phosphorylation. Simultaneously, T-bet expression increased, while LAG3 expression decreased. Figure 17 A). When the PI3K-AKT inhibitor LY294002 was added, the changes in the above molecules completely disappeared. Figure 17 (B) This further supports the key role of this signal axis in Cbl-b regulation of LAG3 expression.

[0176] The above experimental results indicate that Cbl-b inhibition activates the PI3K-AKT signaling pathway, leading to the inhibitory phosphorylation and inactivation of GSK-3β, thereby relieving the inhibition of T-bet and promoting T-bet upregulation while inhibiting LAG3 expression. This signaling axis explains the molecular mechanism underlying the previously observed downregulation of LAG3, enhanced activation, maintenance of stemness, and improved effector function in γδ T cells. Figure 18 This provides a possible molecular basis for Cbl-b regulating the long-term anti-tumor ability of γδ T cells.

[0177] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for time-phased addition of Cbl-b inhibitor, zoledronic acid, and IL-2 to expand bifunctional γδ T cells, characterized in that, Includes the following steps: (1) Collect human peripheral blood samples and isolate human peripheral blood mononuclear cells; (2) Human peripheral blood mononuclear cells were seeded in a culture medium containing fetal bovine serum, and zoledronic acid and IL-2 were added on day 0 of amplification for initial activation culture; (3) On day 4, the cells were transferred to a new culture vessel, and fresh culture medium containing zoledronic acid and IL-2 was added. The cells were then cultured until day 6. (4) On day 6 and thereafter, fresh culture medium containing IL-2 was added regularly to maintain cell density; (5) At any point between day 6 and day 10 of amplification, add the Cbl-b inhibitor to the culture medium and maintain its effective concentration in the culture medium for at least 1 day.

2. The method according to claim 1, characterized in that, The final concentration of zoledronic acid is 5 μM; the final concentration of IL-2 is 1000 IU / ml.

3. The method according to claim 1, characterized in that, The Cbl-b inhibitor is Cbl-b-IN-1, with a final concentration of 2 μM. The structural formula of Cbl-b-IN-1 is shown in Formula I. As shown.

4. The method according to claim 1, characterized in that, In step (2), the inoculation density of the PBMCs is 2 × 10⁻⁶. 6 Each well contains 10% fetal bovine serum (FBS) by volume.

5. The method according to claim 1, characterized in that, In step (5), the Cbl-b inhibitor is added on day 6 of amplification and its effective concentration is maintained and cultured until day 14.

6. The method according to claim 1, characterized in that, In step (5), the Cbl-b inhibitor is added on the 10th day of amplification and its effective concentration is maintained and cultured until the 14th day.

7. The method according to claim 1, characterized in that, In step (5), the Cbl-b inhibitor is added on the 10th day of amplification and its effective concentration is maintained and cultured until the 11th day.

8. Bifunctional γδ T cells, characterized in that, It is obtained by amplification using any one of the methods described in claims 1-7.

9. The use of the bifunctional γδ T cells according to claim 8 in the preparation of a medicament for treating tumors.

10. The application according to claim 9, wherein the tumor is cervical cancer.

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