An adoptive immunotherapy cell preparation, and methods of making and using the same

Abstract

The application relates to the technical field of tumor immune cell therapy, in particular to an adoptive immunotherapy cell preparation as well as a preparation method and application thereof. The cell preparation is an effector cell population obtained through in-vitro co-culture, and the effector cell population comprises gamma-delta T cells and CD8 + T cells. The synergistic activation mechanism between the gamma-delta T cells and the CD8 + T cells can be utilized to realize directional remodeling of the functional state of the CD8 + T cells, and promote the functional remodeling phenotype transformation of the CD8 + T cells into a high-IFN-gamma secretion, high-T SCM ratio and high-tumor-killing-capability type. Therefore, the anti-tumor immune effect and solid tumor clearance capability of the adoptive immunotherapy cell preparation are significantly improved, and the adoptive immunotherapy cell preparation has significant clinical application value and popularization prospect.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, specifically to an adoptive immunotherapy cell preparation, its preparation method, and its application. Background Technology

[0002] Ovarian cancer is one of the most malignant and recurrent tumors of the female reproductive system, characterized by its insidious onset, rapid progression, and high recurrence rate. Epidemiological studies show that over 70% of ovarian cancer patients are already in an advanced stage at initial diagnosis. Currently, cytoreductive surgery combined with platinum-based chemotherapy remains the standard treatment, but the five-year survival rate is still low, and the recurrence rate is high. There is an urgent need to develop new treatment strategies, especially immune system-based therapies, to improve the clinical outcomes of ovarian cancer patients.

[0003] Adoptive cell therapy (ACT), as an important tumor immunotherapy method, involves expanding and reinfusing immune cells with anti-tumor activity in vitro, and has shown potential in the treatment of various tumors. Among these, CD8+... + T cells, as the main effector cells in the anti-tumor immune response, directly mediate tumor cell killing by recognizing tumor antigens presented by MHC class I molecules. However, in the tumor microenvironment, especially the immunosuppressive microenvironment of ovarian cancer, CD8... + T cells generally exhibit functional exhaustion, manifested by decreased secretion of effector factors, reduced memory phenotypes, and high expression of immune checkpoint molecules (such as PD-1), which significantly limits their anti-tumor effects.

[0004] γδT cells are a subset of T cells possessing characteristics of both innate and adaptive immunity. They can recognize and kill tumor cells in a non-MHC-restricted manner, exhibiting strong cytotoxic effects against both autologous and allogeneic tumor cells. Activated γδT cells can secrete cytokines such as IFN-γ and TNF-α to remodel the microenvironment and influence the activation status of other immune cells through antigen presentation and immunomodulation. However, γδT cells have low in vitro expansion efficiency, insufficient persistence in vivo, and their antitumor efficacy when used alone is still limited by the immunosuppressive microenvironment.

[0005] Currently, some studies have attempted to combine different types of immune cells in adoptive immunotherapy to enhance anti-tumor effects. However, existing strategies mostly focus on simple cell mixing and reinfusion or independent expansion followed by combined infusion, and their core remains at the level of "cell type superposition." These strategies lack systematic in vitro optimization design targeting the interactions between different immune cells, especially γδT cells and CD8 cells. +The synergistic activation mechanism between T cells has not been fully utilized; a stable and effective co-culture system has not been established; and it is difficult to improve anti-tumor activity while maintaining effective cell function and expansion efficiency; resulting in the inadequacy of the acquired adoptive immunotherapy cell preparations in terms of sustained anti-tumor ability.

[0006] Therefore, there is an urgent need to construct an in vitro method that can effectively activate CD8. + A combined cell preparation that enhances T cells, increases their memory phenotype ratio and effector function, and significantly improves their killing activity against solid tumors such as ovarian cancer. Summary of the Invention

[0007] The purpose of this invention is to provide an adoptive immunotherapy cell preparation that can utilize γδT cells and CD8+. + The synergistic activation mechanism among T cells significantly enhances CD8 + T cell activation level, promotion of CD8 + T cells differentiate into stem-like memory phenotypes, thereby significantly enhancing their ability to kill and induce apoptosis in tumor cells.

[0008] To achieve the above objectives, the present invention provides the following technical solution: an adoptive immunotherapy cell preparation, wherein the cell preparation is an effector cell population obtained through in vitro co-culture, and the effector cell population includes γδT cells and CD8 cells. + T cells.

[0009] Furthermore, the CD8 + T cells possess a functional remodeling phenotype induced by the γδT cells; the functional remodeling phenotype includes at least one of the following: (1) Increased CD40L expression levels; (2) T SCM The proportion of stem cell-like memory T cells increased; (3) Increased proportion of IFN-γ positive expression cells and / or increased secretion levels; (4) It has enhanced cytotoxicity and apoptosis-inducing ability on tumor cells.

[0010] Furthermore, the cell preparation induced apoptosis in ovarian cancer SK-OV-3 cells at a higher rate than CD8+ alone. + T cell count increased by at least 40%.

[0011] This application provides a method for preparing the above-mentioned adoptive immunotherapy cell preparation, comprising the following steps: S1. Peripheral blood mononuclear cells were isolated from the donor's peripheral blood; S2. A portion of the peripheral blood mononuclear cells were placed in a γδT cell expansion culture system for in vitro induction and expansion to obtain γδT cells; S3. After short-term stimulation with anti-CD3 / CD28 antibody, the remaining peripheral blood mononuclear cells are placed in a culture system containing IL-2 cytokine for in vitro expansion to obtain activated CD8 cells. + T cells; S4. The activated CD8 obtained in step S3 + T cells and the γδT cells obtained in step S2 are mixed in a preset ratio and co-cultured at 37°C and 5% CO2. After co-culture is completed, the cell population is collected to obtain the adoptive immunotherapy cell preparation.

[0012] Further, in step S4, the γδT cells interact with the activated CD8+ cells. + T cells were mixed at a ratio of (0.8–2.2):1.

[0013] Furthermore, the co-culture period is 4 to 8 days; During the co-culture process, no CD3 / CD28 antibodies or other persistent T cell receptor stimulating factors are added to the culture medium.

[0014] Further, in step S4, the γδT cells induce the CD8+ cells through the CD40 / CD40L co-stimulatory signaling axis and the IFN-γ / IL-2 related cytokine network. + T cells turn to T cells SCM Phenotypic differentiation and enhancement of CD8 + The ability of T cells to secrete IFN-γ.

[0015] Further, in step S2, the γδT cell expansion culture system includes γδT cell expansion culture medium and zoledronic acid, and the γδT cell expansion culture medium includes IL-2 cytokine; The total duration of the in vitro induced amplification is 6-12 days. During the in vitro induced amplification process, when the total amplification time is 45%-50%, 200-300 μL of γδT cell amplification culture medium is added to the γδT cell amplification culture system, and thereafter, it is added every 2 days.

[0016] This application provides the use of the above-mentioned adoptive immunotherapy cell preparation in the preparation of a medicament for treating solid tumors, including ovarian cancer, platinum-resistant ovarian cancer, recurrent ovarian cancer, breast cancer, or lung cancer.

[0017] Furthermore, the cell preparation is used in combination with one or more of PD-1 / PD-L1 immune checkpoint inhibitors, CTLA-4 inhibitors, and PARP inhibitors.

[0018] The beneficial effects of this invention are as follows: The adoptive immunotherapy cell preparation provided in this application is an effector cell population obtained through in vitro co-culture. It utilizes γδT cells and CD8+ cells... + The synergistic activation mechanism between T cells enables the activation of CD8. + Directed remodeling of T cell functional state promotes CD8 + T cells secrete high levels of IFN-γ, and T cells secrete high levels of IFN-γ. SCM The phenotypic transformation results in a high proportion of tumor-killing cells and functional remodeling. This significantly enhances the anti-tumor immune effect and solid tumor clearance capacity of the adoptive immunotherapy cell preparation, giving it significant clinical application value and promising prospects for widespread application.

[0019] The method for preparing adoptive immunotherapy cell preparations provided in this application involves reacting γδT cells with activated CD8 cells. + Co-culturing T cells can significantly upregulate CD8. + The expression level of CD40L on the surface of T cells is increased, enhancing its co-stimulatory signaling and thereby increasing CD8 expression. + The overall immune activation state of T cells; it can also promote CD8. + T cells differentiate into a stem-like memory phenotype, thereby significantly increasing CD8 levels. + T cells T SCM The proportion of stem-like and memory T cells increases, thereby enhancing CD8 while maintaining basic effector function. + The long-term proliferative potential and sustained immune response of T cells provide the cellular basis for sustained anti-tumor effects in vivo. Furthermore, by combining γδ T cells with activated CD8... + T cell co-culture yielded adoptive immunotherapy cell preparations that significantly increased CD8 levels. + The expression levels of intracellular factors such as IFN-γ in T cells enable the final adoptive immunotherapy cell preparation to enhance function primarily by regulating the cytokine axis, rather than by achieving comprehensive and non-specific activation, thus achieving a targeted enhancement of the immune effect.

[0020] The method for preparing adoptive immunotherapy cell preparations provided in this application achieves the targeting of CD8 cells by constructing an in vitro co-culture process with a clearly defined source and controllable technology. + The targeted remodeling of T cell functional status significantly enhances their anti-tumor immune response. Furthermore, the preparation method has well-defined and reproducible steps, and the co-culture ratio and culture time can be precisely controlled, making it suitable for large-scale preparation under GMP conditions and possessing promising clinical translation prospects.

[0021] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0022] Figure 1 The CD8+ co-culture process shown in Example 1 of this invention + Changes in the number of T cells and γδT cells at different time points; Figure 2 The co-culture system and the single culture system of CD8 shown in Examples 1, 2 and Comparative Example 1 of this invention are examples of CD8 culture systems. + Flow cytometry results of T cell activation status and memory phenotype; Figure 3 The CD8+ cells in the adoptive immunotherapy cell preparations shown in Examples 1, 2, and Comparative Example 1 of this invention. + Expression of intracellular effector factors in T cells; Figure 4 The results show the in vitro killing effect of the adoptive immunotherapy cell preparations shown in Examples 1, 2 and Comparative Example 1 of this invention on ovarian cancer SK-OV-3 cells. Figure 5 The results show the detection effects of the adoptive immunotherapy cell preparations shown in Examples 1, 2 and Comparative Example 1 of this invention on the apoptosis-inducing effect of SK-OV-3 cells. Figure 6 The results show the detection levels of various cytokines under different culture systems in Examples 1, 2, 1, and 2 of this invention. Detailed Implementation

[0023] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] It should be noted that the experimental results of the specific embodiments of the present invention were all obtained using GraphPad Prism 10.0 statistical software. Quantitative data were expressed as mean ± standard deviation. The comparison between groups was performed using t-test or one-way ANOVA, and P < 0.05 was used to indicate that the difference was statistically significant.

[0025] It should be noted that the main reagents used in the specific embodiments of this invention, including CD3-PE-CY5.5, CD8-PE, CD62L-APC-CY7, CD95-PE-CY7, CD8-FITC, CCR7-FITC, CD45RO-APC, CD40L-PE, OX40-PE, PD-1-PE-CY7, Tim3-APC, TCF-1-AF647, IFN-γ-PE, granzyme B-PE antibody, and granzyme K-PE antibody, are all commercially available. The Annexin V / 7-AAD apoptosis detection kit, CBA detection kit, human peripheral blood lymphocyte separation medium, and recombinant human IL-2 for injection are also commercially available. The enhanced RPMI-1640 culture medium is also commercially available. The 1640 culture medium, Australian fetal bovine serum, L-glutamine, non-essential amino acid solutions, zoledronic acid, and β-mercaptoethanol are all commercially available products. Furthermore, reagents, tools, and instruments not specifically mentioned are standard products in this field and will not be described further.

[0026] A preferred embodiment of this application provides an adoptive immunotherapy cell preparation. This cell preparation is an effector cell population obtained through in vitro co-culture. The effector cell population comprises γδT cells and CD8+ cells. + T cells. Through in vitro co-culture, γδ T cells and CD8+ were utilized. + Synergistic activation mechanisms among T cells, directed remodeling of CD8 + This adoptive immunotherapy cell preparation enables the functional synergy and mutual activation between two types of immune cells without genetic modification or complex engineering, thereby enhancing overall anti-tumor immune activity and overcoming the limitations of single-cell therapy. In this embodiment or other embodiments, the effector cell population consists of expanded γδT cells and activated CD8+ cells. + T cells were obtained by mixing them at a ratio of (0.8–2.2):1 and then co-culturing them in vitro. This was achieved by limiting the interaction between γδ T cells and activated CD8+. + The optimal mixing ratio of T cells ensures the best combination of the two cell types in the co-culture system, thus balancing cell proliferation and synergistic effects. This results in a cell formulation that combines the MHC-unrestricted killing advantage of γδ T cells with that of CD8+ cells. + T cell-specific anti-tumor activity.

[0027] In one embodiment, CD8 + T cells exhibit a functional remodeling phenotype induced by γδ T cells. This functional remodeling phenotype includes at least one of the following: (1) Increased CD40L expression levels; (2) T SCM The proportion of stem cell-like memory T cells increased; (3) Increased proportion of IFN-γ positive expression cells and / or increased secretion levels; (4) It has enhanced cytotoxicity and apoptosis-inducing ability on tumor cells.

[0028] These functional remodeling phenotypes are generated by γδT cells through cell contact-dependent mechanisms and / or soluble factor-mediated mechanisms, and are used to achieve CD8 activation. + The directed transformation of T cells from a "functional exhaustion state" to a "stem-like memory enhancement state" enhances their sustained proliferation capacity, immune response capacity, and anti-tumor effect, thereby significantly improving the problem of insufficient T cell function during adoptive immunotherapy.

[0029] In one embodiment, the cell preparation induced apoptosis in ovarian cancer SK-OV-3 cells at a higher rate than CD8+ alone. + T cell count increased by ≥40%. This indicates that γδ T cells and CD8+ cells are involved. + During co-culture in an in vitro system, T cells, under the immune regulation of γδ T cells, can activate CD8+. + The anti-tumor effector function of T cells was significantly enhanced, thereby significantly improving their ability to induce apoptosis in tumor cells. This effectively enhanced the overall cytotoxic activity and anti-tumor immune effect of the obtained effector cell population, making it superior to CD8+ alone under in vitro conditions. + The anti-tumor effects of T cells have great application potential.

[0030] One embodiment provides a method for preparing the above-mentioned adoptive immunotherapy cell preparation, the preparation method comprising the following steps: S1. Peripheral blood mononuclear cells (PBMCs) are isolated from the peripheral blood of the donor. S2. A portion of the peripheral blood mononuclear cells were placed in a γδT cell expansion culture system for in vitro induction and expansion to obtain γδT cells; S3. After short-term stimulation with anti-CD3 / CD28 antibody, the remaining peripheral blood mononuclear cells are placed in a culture system containing IL-2 cytokine for in vitro expansion to obtain activated CD8 cells. + T cells; S4. The activated CD8 obtained in step S3 + T cells and the γδT cells obtained in step S2 are mixed in a preset ratio and co-cultured at 37°C and 5% CO2. After co-culture is completed, the cell population is collected to obtain the adoptive immunotherapy cell preparation.

[0031] This preparation method was used to construct γδT cells and CD8+ cells. + An in vitro co-culture system for T cells, thereby achieving the control of CD8 + The targeted regulation and remodeling of T cell functional status transforms them from a relatively low-responsive or exhausted state into a highly efficient anti-tumor effector cell population with enhanced IFN-γ secretion and stem cell memory phenotype characteristics, thereby significantly improving their killing ability and apoptosis-inducing ability against solid tumor cells. This preparation method establishes an integrated process for the isolation, targeted expansion, and co-culture of peripheral blood mononuclear cells, forming a standardized and reproducible cell preparation system. This ensures that the adoptive immunotherapy cell preparation has controllable sources, clear operational steps, and good stability. It not only enables the efficient preparation of functionally enhanced immune cells but also possesses good feasibility for industrial production and potential for clinical translational applications.

[0032] In one embodiment, in step S4, the γδT cells and activated CD8 + T cells are mixed at a ratio of (0.8–2.2):1. In this embodiment or other embodiments, the co-culture time in step S4 is 4–8 days. Furthermore, during co-culture, no CD3 / CD28 antibodies or other persistent T cell receptor (TCR) stimulating factors are added to the culture medium. By limiting the co-culture process to avoid the addition of persistent TCR stimulating factors, the excessive differentiation or exhaustion of T cells caused by strong continuous TCR stimulation can be effectively avoided, thus maintaining CD8+. + By leveraging the stem-like memory characteristics and long-term proliferative potential of T cells, the durable anti-tumor ability of the final cell preparation can be enhanced.

[0033] In one embodiment, in step S4, γδT cells induce CD8+ activation through the CD40 / CD40L co-stimulatory signaling axis and the IFN-γ / IL-2 related cytokine network. + T cells turn to T cells SCM Phenotypic differentiation and enhanced CD8 + This reveals the IFN-γ secretion capacity of T cells. + The immune regulatory mechanism of T cells, thereby achieving the control of CD8 + Targeted reprogramming of T cell functional state, thereby significantly enhancing its IFN-γ secretion capacity and T cell function. SCM Sample phenotype formation ability.

[0034] In one embodiment, in step S2, the γδT cell expansion culture system includes γδT cell expansion medium and zoledronic acid. The γδT cell expansion medium includes IL-2 cytokine. In this embodiment or other embodiments, the total expansion time for in vitro induction is preferably 6-12 days. Furthermore, during in vitro induction expansion, at 45%-50% of the total expansion time, 200-300 μL of fresh γδT cell expansion medium is added to the γδT cell expansion culture system, and thereafter, this is done every 2 days. By setting a specific replenishment time window during γδT cell expansion, i.e., at 45%-50% of the total culture time, the nutrient consumption and metabolic balance in the cell culture system can be optimized, thereby maintaining high proliferative activity and survival rate of γδT cells. This culture strategy also helps improve the expansion efficiency and functional consistency of γδT cells, resulting in higher phenotypic stability between different batches of cells, thereby enhancing the subsequent γδT cell and CD8... + The overall stability and reproducibility of the T cell co-culture system.

[0035] In one embodiment, in step S3, the concentration of IL-2 cytokine in the culture system is any value between 100 U / mL and 200 U / mL. The in vitro expansion time is 1–3 days. This is achieved by analyzing CD8... + In T cell culture systems, the concentration of IL-2 is limited, and combined with short-term stimulation by CD3 / CD28 antibodies, this can ensure the preservation of CD8 levels. + While T cells receive effective initial activation signals, they avoid terminal differentiation or functional exhaustion caused by excessive or continuous stimulation. This results in the acquisition of CD8 cells with high proliferative potential and phenotypic plasticity. + The T cell population provides the basic cellular conditions for functional remodeling during subsequent co-culture with γδT cells, and helps improve the functional consistency and therapeutic stability of the final adoptive immunotherapy cell preparation.

[0036] One embodiment provides the application of the aforementioned adoptive immunotherapy cell preparation in the preparation of a drug for treating solid tumors. Solid tumors include, but are not limited to, ovarian cancer, platinum-resistant ovarian cancer, recurrent ovarian cancer, breast cancer, or lung cancer. By applying this adoptive immunotherapy cell preparation to the treatment of multiple types of solid tumors, its applicability can be expanded, no longer limited to a single tumor type, thereby improving its clinical applicability and indication coverage. Simultaneously, because this cell preparation has enhanced CD8... + The T-cell's ability to remodel its function and its strong tumor cell killing activity enable it to exert certain immune effects under different tumor microenvironment conditions, thereby enhancing its application potential and translational value as a broad-spectrum anti-tumor immunotherapy.

[0037] In one embodiment, the cell preparation is used in combination with one or more of PD-1 / PD-L1 immune checkpoint inhibitors, CTLA-4 inhibitors, and PARP inhibitors. By combining this cell preparation with the aforementioned immune checkpoint inhibitors or DNA damage repair inhibitors, a synergistic enhancement of anti-tumor effects can be achieved at different mechanism levels. Specifically, PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors can relieve the immunosuppressive state of T cells in the tumor microenvironment, enhancing T cell activation and sustained response; PARP inhibitors, by interfering with the DNA damage repair process of tumor cells, increase the immunogenicity of tumor cells, thereby promoting the recognition and clearance of tumor cells by the immune system. This combined treatment strategy can produce synergistic effects at multiple levels, including enhanced cellular immunity, relief of tumor immunosuppression, and increased tumor cell vulnerability, thereby further enhancing the overall anti-tumor therapeutic effect and improving the treatment response rate and potential clinical efficacy.

[0038] Example 1

[0039] S1. Obtain peripheral blood from healthy donors and separate the obtained peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation.

[0040] S2. The partially isolated peripheral blood mononuclear cells were resuspended in γδT cell expansion medium to a concentration of 1.5 × 10⁻⁶. 6 Cells / mL were seeded into 24-well culture plates. Zoledronic acid was then added to each well as a stimulating factor to construct a γδT cell expansion culture system, and in vitro induction and expansion were performed. During in vitro induction and expansion, 250 μL of fresh γδT cell expansion medium was added to each well on day 4 of culture, and thereafter, fresh medium was added every 2 days until day 9 of culture, yielding expanded γδT cells. Each 50 mL γδT cell expansion medium consisted of: 45 mL Advanced 1640 basal medium, 5 mL fetal bovine serum, 500 μL of penicillin-antibody solution, 500 μL of L-glutamine solution, 500 μL of MEM non-essential amino acid solution, 7500 U IL-2 cytokine, and 0.38 μL β-mercaptoethanol.

[0041] S3. Remaining peripheral blood mononuclear cells were briefly stimulated with a culture medium containing anti-CD3 / CD28 antibodies for 24 hours. Subsequently, the peripheral blood mononuclear cells stimulated with anti-CD3 / CD28 antibodies were placed in a culture system containing IL-2 cytokines for in vitro expansion. After culturing for the second day, activated CD8 cells were obtained. + T cells. The concentration of IL-2 cytokine in this culture system was 150 U / mL.

[0042] S4. The activated CD8 obtained in step S3 + T cells were mixed with the γδT cells obtained in step S2 at a 1:1 ratio. They were then co-cultured in an incubator at 37°C and 5% CO2. After 6 days of co-culture, the cell population was collected to obtain the adoptive immunotherapy cell preparation.

[0043] During the co-culture process, the cell growth status was dynamically observed; and on day 6 of co-culture, a portion of the co-culture system was taken for further culture and expansion, with simultaneous observation of changes in cell growth status. The results are as follows: Figure 1 As shown.

[0044] Depend on Figure 1 It can be seen that, under the same culture conditions, both cell types showed different degrees of proliferation trends with prolonged culture time, among which CD8... + The number of T cells was significantly higher than that of γδ T cells at all time points. From day 3 to day 6, both cell types were in a relatively slow proliferation phase, with a relatively gradual increase in cell number; after day 9, CD8... + T cells entered a rapid proliferation phase, with a significant increase in cell number, reaching a level significantly higher than that of γδ T cells by day 12. In contrast, although γδ T cells also gradually increased with prolonged culture time, the overall expansion rate was lower, and the expansion was limited. These results indicate that under the same or similar culture conditions, CD8+... + T cells have a stronger ability to proliferate in vitro, and their cell number increases at a significantly higher rate than that of γδT cells, providing experimental evidence for achieving functional complementarity and synergistic regulation between the two cell types through a co-culture system.

[0045] Example 2

[0046] The difference between this embodiment and Embodiment 1 is that in step S4, the activated CD8 obtained in step S3 is... + T cells were mixed with the γδT cells obtained in step S2 at a ratio of 2:1 and co-cultured. Ultimately, an adoptive immunotherapy cell preparation was obtained.

[0047] Comparative Example 1 Peripheral blood was obtained from healthy donors and separated into peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation. The separated PBMCs were then subjected to short-term spikes in a culture medium containing anti-CD3 / CD28 antibodies. Subsequently, the PBMCs short-term spiked with anti-CD3 / CD28 antibodies were placed in a culture system containing IL-2 cytokines for in vitro expansion. After culturing for two days, activated CD8+ cells were obtained. + T cells. The obtained activated CD8 +T cells were placed in an incubator and cultured at 37°C and 5% CO2 for 6 days. The cell population was then collected to obtain a cell preparation containing a single cell component.

[0048] Comparative Example 2 Peripheral blood was obtained from healthy donors and separated into peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation. The separated PBMCs were resuspended in γδT cell expansion medium to a concentration of 1.5 × 10⁻⁶. 6 Cells / mL were seeded into 24-well culture plates. Zoledronic acid was then added to each well as a stimulating factor to construct a γδT cell expansion culture system, and in vitro induction and expansion were performed. During in vitro expansion, 250 μL of fresh γδT cell expansion medium was added to each well on day 4, and thereafter, fresh medium was added every 2 days until day 9, yielding expanded γδT cells. The obtained γδT cells were placed in an incubator and co-cultured at 37°C and 5% CO2. After 6 days of co-culture, the cell population was collected to obtain a cell preparation containing a single cell component.

[0049] Flow cytometry was used to detect the effect of the co-culture systems of Examples 1 and 2 on CD8. + The influence of T cell activation status and memory phenotype, and on CD8 single cell activation status. + The T-cell culture system served as a control. Specifically, on day 6 of co-culture, cell suspensions from each group were collected, centrifuged to remove the supernatant, and then resuspended in PBS (containing 1% fetal bovine serum). Corresponding fluorescently labeled antibodies were added, and the cells were incubated at room temperature in the dark for 30 min. After washing, the cells were resuspended and analyzed by flow cytometry. This assay included detection of CD8+. + The expression level of CD40L on the surface of T cells was detected, and memory T cell subsets were analyzed; the results are as follows: Figure 2 As shown. Among them, CD8 can be determined based on the expression of CCR7 and CD45RO. + T cells are classified into: naive / stem-like memory T cells (T cells) SCM CCR7 + CD45RO - ), central memory T cells (T CM CCR7 + CD45RO + ) and effector memory T cells (T EM CCR7 - CD45RO + Subgroups such as )

[0050] Depend on Figure 2 From A and B, we can see that CD8 cultured alone in Comparative Example 1... +The expression level of CD40L on the surface of T cells was low; CD8 in Examples 1 and 2 was low. + After co-culturing T cells with γδ T cells, the proportion of CD40L positivity was significantly increased in both cases (all P < 0.0001), indicating that co-culturing CD80L with γδ T cells significantly increased the proportion of CD40L positivity. + When T cells are co-cultured with γδT cells, regardless of whether the mixing ratio is 1:1 or 2:1, CD8 + The expression levels of CD40L on the surface of T cells were significantly increased, and there was no statistically significant difference between the two ratios (P > 0.05). This indicates that the co-culture system within this ratio range can effectively enhance CD8 expression. + The activation state of T cells. (From...) Figure 2 As shown in C and D, compared with CD8 cultured alone in Comparative Example 1... + Compared to T cells, CD8 in Examples 1 and 2 + In the co-culture system of T cells and γδT cells, T SCM The proportion of subpopulations was significantly increased (P < 0.05), indicating that the cells were in a more stem and long-term proliferative potential functional state. Compared with the single-culture system in Example 1 and the co-culture systems in Examples 1 and 2, T... CM The variation in subgroup proportions is relatively limited, and T EM No significant differences were observed among the subgroups. This result indicates that by combining γδT cells with CD8... + T cell co-culture can significantly enhance CD8 + T cell activation levels, such as upregulated CD40L expression, promote their differentiation into stem-like memory phenotypes, such as T cells. SCM In this way, while maintaining basic effector functions, its long-term immune response potential is enhanced.

[0051] Flow cytometry was used to analyze the CD8+ levels in the adoptive immunotherapy cell preparations obtained in Examples 1-2 and the single-cell component cell preparation obtained in Comparative Example 1. +The expression levels of intracellular effector molecules and in vitro killing function of T cells were detected. Specifically, the adoptive immunotherapy cell preparations prepared in Examples 1-2 and the single-cell component cell preparation prepared in Comparative Example 1 were stimulated with PMA (Phorbol 12-myristate 13-acetate) and iomycin, respectively, to induce intracellular cytokine expression. After 1 h of stimulation, GolgiStop was added to inhibit protein secretion, and incubation continued for 3-4 h. Subsequently, cells from each group were collected, and the collected cells were sequentially stained with surface antibodies, fixed, and permeabilized. Finally, intracellular anti-IFN-γ, granzyme B, and granzyme K antibodies were added for staining. After washing, flow cytometry signals were detected to analyze the intracellular IFN-γ expression and the expression of cytotoxic molecules granzyme B and granzyme K. The detection results are as follows: Figure 3 As shown.

[0052] Depend on Figure 3 From A and B, we know that in CD8 + Regarding the expression level of IFN-γ in T cells, in Comparative Example 1, CD8 cells cultured alone... + T cells showed a low proportion of IFN-γ positivity. In Examples 1 and 2, CD8... + After co-culturing T cells with γδT cells, CD8 + The proportion of T cells positive for IFN-γ was significantly increased (P < 0.05). Example 1: CD8+ after co-culture + The proportion of T cells positive for IFN-γ and CD8+ after co-culture in Example 2 + There was no statistically significant difference in the proportion of IFN-γ-positive T cells compared to the previous system (P > 0.05). This indicates that the co-culture system can enhance CD8+. + The ability of T cells to produce IFN-γ was observed, but within this range, the increase in IFN-γ production showed a plateauing trend under different formulation conditions. Figure 3 As can be seen from A, in Examples 1 and 2, CD8+ γδT cells were co-cultured. + T cells and CD8 cells cultured alone in Comparative Example 1 + In T cells, the overall expression ratio of granzyme B to granzyme K did not change significantly, and no statistically significant difference was observed (P > 0.05). This result indicates that γδ T cells and CD8... + T cell co-culture system mainly enhances CD8 + The co-culture system effectively inhibits the IFN-γ-related effector function of T cells, while having a relatively limited impact on the expression levels of granzyme B and granzyme K. This suggests that the co-culture system has a positive effect on CD8+. + The regulation of T cell effector function is selective.

[0053] The in vitro killing effect of the adoptive immunotherapy cell preparations prepared in Examples 1 and 2, and the single-cell component cell preparation prepared in Comparative Example 1, on ovarian cancer SK-OV-3 cells was detected using the CCK-8 assay. Specifically, the adoptive immunotherapy cell preparations prepared in Examples 1 and 2, and the single-cell component cell preparation prepared in Comparative Example 1, were used as effector cells, and SK-OV-3 cells were used as target cells. SK-OV-3 cells in logarithmic growth phase were cultured at 5 × 10⁻⁶ cells per cell line. 3 Cells were seeded per well in 96-well plates and cultured at 37°C until stable adhesion. Effector cells from each group were then added at a 10:1 effector-to-target ratio and co-cultured for 24 h. After culture, CCK-8 reagent was added to each well, and incubation continued for 2 h. The absorbance (OD) was then measured at 450 nm. The cell killing rate was calculated using the following formula: Kill rate (%) = 100 - 100 × [(OD) （T细胞+肿瘤细胞） -OD T细胞单独 ) / OD 肿瘤细胞单独 The test results are as follows: Figure 4 As shown.

[0054] Depend on Figure 4 It can be seen that, compared with the single CD8 in Comparative Example 1 + Compared to the effector cell population of the T component, the γδT cells and CD8 cells in Examples 1 and 2 + The effector cell population obtained from T cell co-culture significantly enhanced the killing ability of SK-OV-3 cells. This further demonstrates that co-culture with γδT cells can significantly improve the overall anti-tumor effect. Specifically, in Example 1, γδT cells and CD8+... + The effector cell population obtained by co-culturing T cells in a 1:1 ratio showed a significantly higher killing rate against SK-OV-3 cells than that of CD8 monoclonal antibodies in Comparative Example 1. + The killing rate of T component effector cell population against SK-OV-3 cells was (P < 0.05). Although, in Example 2, γδT cells and CD8... + The effector cell population obtained by co-culturing T cells at a 1:2 ratio also showed a significantly higher killing rate against SK-OV-3 cells than that of CD8 monoclonal antibodies in Comparative Example 1. + The effector cell population of the T component showed a higher killing rate against SK-OV-3 cells, but the overall increase was lower than that of γδT cells and CD8 cells in Example 1. + The effector cell population obtained by co-culturing T cells in a 1:1 ratio. This result indicates that γδT cells and CD8+... + Effector cell populations obtained by co-culturing T cells at different ratios all exhibited strong cytotoxic effects against ovarian cancer SK-OV-3 cells under in vitro conditions. Among these, γδ T cells and CD8+ cells showed particularly strong cytotoxic effects. +The effector cell population obtained by co-culturing T cells in a 1:1 ratio showed the best killing effect, indicating that the co-culture system at this ratio has a better synergistic effect in enhancing anti-tumor effects.

[0055] The apoptosis-inducing effect of the adoptive immunotherapy cell preparations prepared in Examples 1 and 2, and the single-cell component cell preparation prepared in Comparative Example 1, on ovarian cancer SK-OV-3 cells was detected using an annexin V / 7-AAD or AV / 7-AAD double staining method. Specifically, the adoptive immunotherapy cell preparations prepared in Examples 1 and 2, and the single-cell component cell preparation prepared in Comparative Example 1, were used as effector cells, and logarithmically growing SK-OV-3 cells were used as target cells. Effector cells and SK-OV-3 cells were co-cultured for 15 h at an effector-to-target ratio of 10:1. After culture, adherent tumor cells were collected and washed with PBS. Subsequently, they were resuspended in binding buffer, and annexin V and 7-AAD dye were added. The cells were incubated at room temperature in the dark for 15 min. Flow cytometry was performed within 1 h after incubation. Apoptosis rates were statistically analyzed by clustering annexin V and 7-AAD staining results, including early apoptosis (annexin V). + / 7-AAD - ) and late apoptosis (annexin V) + / 7-AAD + The test results are as follows: Figure 5 As shown.

[0056] Depend on Figure 5 From A and B, we can see that the single CD8 prepared in Comparative Example 1 + Among the effector cell population of T cells, SK-OV-3 cells had a low total apoptosis rate of approximately 10.51%; the proportions of early apoptosis and late apoptosis were relatively similar.

[0057] Compared with the single CD8 prepared in Comparative Example 1 + Compared to the effector cell population of T cell components, γδ T cells and CD8 in Examples 1 and 2 + The effector cell population obtained by co-culturing T cells significantly enhanced the apoptosis-inducing effect on SK-OV-3 cells (P < 0.05). In Example 1, γδ T cells and CD8... + In the effector cell population obtained by co-culturing T cells in a 1:1 ratio, the total apoptosis rate of SK-OV-3 cells increased to approximately 17.54%, an increase of approximately 66%; while in Example 2, γδT cells and CD8... +In the effector cell population obtained by co-culturing T cells at a 1:2 ratio, the total apoptosis rate of SK-OV-3 cells also showed an increased apoptosis rate, but the overall level was slightly lower than that in the effector cell population of Example 1. This indicates that γδT cells and CD8+... + The T cell co-culture system significantly enhanced the apoptosis-inducing ability of effector cell populations on SK-OV-3 ovarian cancer cells, among which γδ T cells and CD8+ T cells significantly increased the apoptosis-inducing ability of effector cell populations. + The effector cell population obtained by co-culturing T cells in a 1:1 ratio showed superior pro-apoptotic effects, which is more conducive to the formation of a highly efficient anti-tumor immune effector cell population. Furthermore, in terms of the distribution of early and late apoptosis, γδ T cells and CD8+ cells showed... + The proportion of annexin V-positive cells in the effector cell population obtained after T cells were mixed and co-cultured in a certain proportion increased overall, indicating that the initiation and progression of apoptosis were enhanced.

[0058] The secretion levels of various cytokines in the adoptive immunotherapy cell preparations prepared in Examples 1 and 2, as well as the single-cell component cell preparations prepared in Comparative Examples 1 and 2, were detected using the CBA (Cytometric Bead Array) method to evaluate the interaction between γδ T cells and CD8+. + Immunomodulatory effects of T cell co-culture system. Specifically, in Examples 1, 2, Comparative Example 1, and Comparative Example 2, the supernatant of the co-culture system or culture system was collected after 6 days of co-culture. Simultaneously, γδT cells were cultured separately, and the obtained supernatant was centrifuged to remove cell debris, obtaining supernatant samples. The supernatant samples were mixed with capture microspheres and PE-labeled detection antibodies and incubated under light-protected conditions to allow cytokines to specifically bind to the capture microspheres. After incubation, the samples were washed, and event signals of the capture microspheres were collected using flow cytometry. The concentrations of each cytokine were calculated based on a standard curve. The cytokines included IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17. The detection results are as follows: Figure 6 As shown.

[0059] Depend on Figure 6 It can be seen that the secretion levels of various cytokines differed in the adoptive immunotherapy cell preparations obtained in Examples 1 and 2, as well as in the single-cell component cell preparations obtained in Comparative Examples 1 and 2. Among them, the changes in IFN-γ cytokines were the most significant. Compared with the single CD8+ cytokine in Comparative Example 1... + Compared to the T cell culture system, the γδT cells in Examples 1 and 2 are similar to CD8 cells. + In the T cell co-culture system, the overall IFN-γ secretion level was significantly increased (P < 0.05). In Example 1, γδ T cells and CD8...+ In the T cell co-culture system with a 1:1 ratio, the overall IFN-γ secretion level was slightly higher than that of γδT cells and CD8 in Example 2. + The IFN-γ secretion levels in a co-culture system where T cells were mixed at a 1:2 ratio showed limited difference between the two. This indicates that co-culture systems within this ratio range can all improve IFN-γ secretion levels to some extent.

[0060] Besides IFN-γ, the secretion levels of other cytokines such as IL-2, IL-4, IL-6, IL-10, TNF-α, and IL-17 showed relatively small overall changes. Among them, some inflammatory factors, such as TNF-α and IL-17, only showed a slight increase; anti-inflammatory factors (such as IL-10) did not show significant inhibition or abnormal decrease in the co-culture system. This result indicates that γδT cells and CD8+... + The T cell co-culture system primarily promoted the enhanced secretion of IFN-γ, a key Th1 effector cytokine, without causing overall disturbances to the multifactorial spectrum. This indicates that the immunomodulatory effect of this co-culture system has a clear directionality and selectivity. It mainly manifests as the targeted enhancement of the anti-tumor-related effect axis centered on IFN-γ while maintaining the relative stability of the overall cytokine network. This distinguishes it from non-specific immune activation or inflammatory amplification effects, further demonstrating its specific regulatory characteristics and technological advantages in immune function reprogramming.

[0061] The adoptive immunotherapy cell preparation provided in this application, through the construction of γδT cells and CD8... + An in vitro co-culture system for T cells to achieve CD8 + Directional remodeling of T cell functional state, transforming it from a basal activated state to one with enhanced IFN-γ secretion capacity; SCM The high-efficiency effect state is characterized by an increased proportion of stem cell-like memory phenotypes and a significantly enhanced tumor cell killing ability.

[0062] Compared to methods that rely solely on the expansion of a single T cell or simple cell-mixed reinfusion, this application achieves synergistic enhancement at the functional level through intercellular interactions, rather than simply adding up the numbers, thereby significantly improving its immune clearance efficiency against solid tumor cells.

[0063] In addition, this γδT cell interacts with CD8 + The T-cell co-culture system achieves functional enhancement while maintaining the relative stability of the cytokine spectrum and the reproducibility of the preparation process. It has good process controllability and potential for large-scale preparation, and shows high feasibility and promotion value in clinical translational applications.

[0064] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An adoptive immunotherapy cell preparation, characterized in that, The cell preparation is an effector cell population obtained through in vitro co-culture, and the effector cell population includes γδT cells and CD8 cells. + T cells.

2. The adoptive immunotherapy cell preparation as described in claim 1, characterized in that, The CD8 + T cells possess a functional remodeling phenotype induced by the γδT cells; the functional remodeling phenotype includes at least one of the following: (1) Increased CD40L expression levels; (2) T SCM The proportion of stem cell-like memory T cells increased; (3) Increased proportion of IFN-γ positive expression cells and / or increased secretion levels; (4) It has enhanced cytotoxicity and apoptosis-inducing ability on tumor cells.

3. The adoptive immunotherapy cell preparation as described in claim 2, characterized in that, The cell preparation induced apoptosis in SK-OV-3 ovarian cancer cells at a higher rate than CD8+ alone. + T cell count increased by at least 40%.

4. The method for preparing the adoptive immunotherapy cell preparation according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Peripheral blood mononuclear cells were isolated from the donor's peripheral blood; S2. A portion of the peripheral blood mononuclear cells were placed in a γδT cell expansion culture system for in vitro induction and expansion to obtain γδT cells; S3. After short-term stimulation with anti-CD3 / CD28 antibody, the remaining peripheral blood mononuclear cells are placed in a culture system containing IL-2 cytokine for in vitro expansion to obtain activated CD8 cells. + T cells; S4. The activated CD8 obtained in step S3 + T cells and the γδT cells obtained in step S2 are mixed in a preset ratio and co-cultured at 37°C and 5% CO2. After co-culture is completed, the cell population is collected to obtain the adoptive immunotherapy cell preparation.

5. The preparation method according to claim 4, characterized in that, In step S4, the γδT cells interact with the activated CD8+ cells. + T cells were mixed at a ratio of (0.8–2.2):

1.

6. The preparation method according to claim 4, characterized in that, In step S4, the co-culture time is 4 to 8 days; During the co-culture process, no CD3 / CD28 antibodies or other persistent T cell receptor stimulating factors are added to the culture medium.

7. The preparation method according to claim 4, characterized in that, In step S4, the γδT cells induce CD8+ through the CD40 / CD40L co-stimulatory signaling axis and the IFN-γ / IL-2 related cytokine network. + T cells turn to T cells SCM Phenotypic differentiation and enhancement of CD8 + The ability of T cells to secrete IFN-γ.

8. The preparation method according to claim 4, characterized in that, In step S2, the γδT cell expansion culture system includes γδT cell expansion culture medium and zoledronic acid, and the γδT cell expansion culture medium includes IL-2 cytokine; The total duration of the in vitro induced amplification is 6-12 days. During the in vitro induced amplification process, when the total amplification time is 45%-50%, 200-300 μL of γδT cell amplification culture medium is added to the γδT cell amplification culture system, and thereafter, it is added every 2 days.

9. The use of the adoptive immunotherapy cell preparation according to any one of claims 1-3 in the preparation of a medicament for treating solid tumors, characterized in that, The solid tumors include ovarian cancer, platinum-resistant ovarian cancer, recurrent ovarian cancer, breast cancer, or lung cancer.

10. The application as described in claim 9, characterized in that, The cell preparation is intended for use in combination with one or more of PD-1 / PD-L1 immune checkpoint inhibitors, CTLA-4 inhibitors, and PARP inhibitors.

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