A method for activating gamma delta t cells using ctc or derivatives thereof and uses thereof

Abstract

This invention relates to γδT cell culture technology, specifically a method and application for activating γδT cells using circulating tumor cells (CTCs) or their derivatives. The method includes obtaining donor-derived γδT cells; obtaining CTCs or their derivatives from cancer patients; and co-culturing the γδT cells with the CTCs or their derivatives in an in vitro co-culture system, thereby activating the γδT cells under the stimulation of the CTCs or their derivatives to obtain CTC-γδT cells. This invention utilizes patient-derived circulating tumor cells as the activation stimulus for γδT cells, enabling the resulting γδT cells to recognize tumor-associated antigens and personalized neoantigens, overcoming tumor heterogeneity and antigen escape problems.

Description

Technical Field

[0001] This invention relates to γδT cell culture technology, specifically a method and application for activating γδT cells using CTC or its derivatives. Background Technology

[0002] Based on the type of surface cell antigen receptor, T cells can be divided into αβT cells and γδT cells. In cancer immunotherapy, αβT cells mainly participate in the acquired immune process. γδT cells act as a bridge between the innate and adaptive immune systems, forming the first line of defense against tumors. γδT cells are considered potent cells for effectively resisting infection and malignant tumors because they are strategically distributed in mucosal tissues, can specifically recognize target cells, and respond rapidly after activation. Currently, the main methods for in vitro expansion and activation of γδT cells include using synthetic phosphate antigens (such as phosphonate bromide) in combination with interleukin-2. However, this method has limitations: 1) the expanded γδT cells lack tumor specificity and may trigger non-specific inflammatory responses; 2) the homing and killing effects on certain solid tumors are limited.

[0003] Circulating tumor cells (CTCs) are tumor cells shed from primary or metastatic solid tumors and enter the peripheral blood. They carry a complete spectrum of tumor antigens, including mutated neoantigens and tumor-associated antigens. Therefore, CTCs are an ideal antigen source for the preparation of personalized tumor vaccines and cell therapies. However, there is currently no mature method for efficiently using CTCs to activate and train γδT cells to prepare immunocellular agents with potent and specific antitumor activity. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for activating γδT cells using CTCs or their derivatives. The CTC-γδT cells obtained according to this method can broadly recognize tumor heterogeneity and overcome antigen escape.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: a method for activating γδT cells using CTC or its derivatives, comprising the following steps: (1) Obtaining donor-derived γδT cells; (2) Obtaining CTCs or their derivatives from cancer patients; (3) The γδT cells and the CTC or its derivatives are placed in an in vitro co-culture system for co-culture, so that the γδT cells are activated under the stimulation of CTC or its derivatives to obtain CTC-γδT cells.

[0006] Preferably, the CTCs activate γδT cells through their intact cellular form, cell surface molecules, cell-released soluble factors, and / or cell-derived subcellular structures.

[0007] Preferably, the γδT cells are derived from peripheral blood mononuclear cells from healthy donors, and the γδT cells are expanded or activated in vitro.

[0008] Preferably, the expansion or activation of the γδT cells is achieved through induction with phosphoantigens, bisphosphonates, and / or cytokines.

[0009] Preferably, the CTCs or their derivatives are derived from peripheral blood, body fluids, or combinations thereof from cancer patients. The CTC derivatives include lysates of CTCs, cell debris, cell membrane components, exosomes, microvesicles, soluble proteins, metabolites, or any combination thereof. The CTC derivatives are obtained through physical disruption, chemical lysis, freeze-thaw treatment, ultrasonic treatment, and / or centrifugation.

[0010] Preferably, the CTCs are cultured, maintained, or functionalized in vitro before co-culture, and the number of γδT cells in the co-culture system is higher than the number of CTCs.

[0011] Preferably, the co-culture system adopts a direct contact co-culture mode, a semi-contact co-culture mode, or a non-contact co-culture mode.

[0012] Preferably, the co-culture system uses a basic culture medium suitable for immune cell culture, which is selected from RPMI-1640, AIM-V, X-VIVO series culture media or combinations thereof.

[0013] Preferably, the co-culture system also employs cytokines that promote γδT cell survival, activation, and / or memory phenotype formation, wherein the cytokines include cytokine analogs, fusion proteins, or functional fragments having the same or similar biological functions, and / or are selected from at least one of the following: Factors related to the survival or expansion of γδT cells: IL-2, IL-7; Factors associated with γδT cell activation and enhanced effector function: IL-12, IL-15, IL-18; Factors related to the maintenance of γδT cell memory phenotype: IL-15, IL-21.

[0014] The present invention also provides a method for obtaining CTC-γδT cells by activating γδT cells using CTCs or their derivatives, and its use in the preparation of immune cell preparations for the treatment of solid tumors, or in the preparation of drugs for personalized anti-tumor immunotherapy.

[0015] Compared with the prior art, the present invention has the following technical effects: 1) This invention utilizes circulating tumor cells derived from patients as the source of activation stimulation for γδT cells, enabling the resulting γδT cells to recognize tumor-associated antigens and personalized neoantigens, thus overcoming the problems of tumor heterogeneity and antigen escape.

[0016] 2) Compared with γδT cells induced solely by phosphate antigens or bisphosphonates, γδT cells activated by CTCs showed significantly improved in vitro cytotoxic activity, functional persistence, and proportion of memory phenotypes.

[0017] 3) The method of the present invention can use γδT cells from allogeneic healthy donors, which is less likely to induce graft-versus-host disease and is suitable for large-scale preparation and development of ready-to-use immune cell preparations. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the method of the present invention.

[0019] Figure 2 This is a schematic diagram showing the comparison before and after γδT cell activation in this invention.

[0020] Figure 3 This is a comparison of the killing activity of comparative γδT cells and CTC-γδT cells against DU145 and LNCaP cells.

[0021] Figure 4 This is a comparative study of IFN-γ expression (CD3 gating) in γδT cells and CTC-γδT cells.

[0022] Figure 5 To compare the expression of IFN-γ (Vγ9 gating) in γδT cells and CTC-γδT cells.

[0023] Figure 6 IFN-γ expression (CD3 gating) in different replicate experiments.

[0024] Figure 7 IFN-γ expression (Vγ9 gating) in different replicate experiments. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0026] To address the problems of existing in vitro γδT cell expansion methods that generally rely on phosphate antigens or bisphosphonates, resulting in insufficient tumor specificity and limited killing effect on solid tumors, this invention provides a method for in vitro activation of γδT cells using CTCs derived from cancer patients, thereby obtaining γδT cells with both broad-spectrum anti-tumor activity and personalized tumor recognition capabilities.

[0027] like Figure 1 and Figure 2 As shown, the present invention provides a method for activating γδT cells using CTCs, comprising the following steps: 1) Obtain donor-derived γδT cells, wherein the γδT cells are derived from peripheral blood mononuclear cells of healthy donors, and perform in vitro expansion or activation treatment on the γδT cells, which is induced by phosphoantigen, bisphosphonate compounds and / or cytokines.

[0028] 2) Obtaining tumor cells (CTCs) from cancer patients. CTCs are derived from peripheral blood, body fluids, or a combination thereof from cancer patients. Before co-culturing, CTCs undergo in vitro culture, maintenance, or functional treatment to enhance their activation capacity for γδT cells; and the number of γδT cells is higher than the number of CTCs in the co-culture system. The ratio of γδT cells to CTCs in the co-culture system can be adjusted according to the experimental purpose, preferably generally 10:1. In some embodiments, the ratio can be dynamically adjusted during co-culture to simulate the changes in the ratio of immune cells to tumor cells during an in vivo immune response.

[0029] 3) The γδT cells and CTCs are co-cultured in an in vitro co-culture system, allowing the γδT cells to be activated under the stimulation of circulating tumor cells, thereby obtaining CTC-activated γδT cells, i.e., CTC-γδT cells. During this process, CTCs can activate γδT cells through their intact cell form, cell surface molecules, soluble factors released by the cells, and / or cell-derived subcellular structures. Specifically, the activation effect of CTCs on γδT cells is not limited to co-culture with intact living cells; activation can also be achieved through CTC cell surface molecules, soluble factors released by the cells, and subcellular structures derived from circulating tumor cells, including but not limited to cell debris, lysates, exosomes, or microvesicles. All of the above forms can activate or train γδT cells in an in vitro co-culture system.

[0030] The co-culture system of this invention employs a direct contact co-culture mode, a semi-contact co-culture mode, or a non-contact co-culture mode. In the direct contact co-culture mode, γδT cells and CTCs are in direct contact within the same culture space; in the semi-contact co-culture mode, γδT cells and CTCs are spatially separated by a porous membrane structure; in the non-contact co-culture mode, γδT cells are activated by stimulation through the conditioned medium of CTCs or CTC derivatives.

[0031] Preferably, the co-culture system uses a basal culture medium suitable for immune cell culture, selected from RPMI-1640, AIM-V, X-VIVO series culture media, or combinations thereof. In some embodiments, the culture medium further contains serum substitutes, human albumin, or serum-free additives to improve system stability and suitability for clinical-grade preparation. The co-culture system also employs cytokines that promote γδT cell survival, activation, and / or memory phenotype formation. These cytokines include cytokine analogs, fusion proteins, or functional fragments with the same or similar biological functions, and / or are selected from at least one of the following: Factors related to the survival or expansion of γδT cells: IL-2, IL-7; Factors associated with γδT cell activation and enhanced effector function: IL-12, IL-15, IL-18; Factors related to the maintenance of γδT cell memory phenotype: IL-15, IL-21.

[0032] Besides using CTCs to activate γδT cells, CTC derivatives can also be used. CTC derivatives retain the antigenic information and molecular characteristics of tumor cells, enabling them to stimulate immune cells without the participation of intact living cells. Based on this understanding, this invention also proposes a method for activating γδT cells using CTC derivatives to obtain CTC-γδT cells, thereby reducing biosafety risks, improving operational stability, and expanding the application of CTCs in immune cell preparation. In practice, the specific method for activating γδT cells using CTC derivatives is basically the same as the method described above, except that the CTC derivatives include CTC lysates, cell fragments, cell membrane components, exosomes, microvesicles, soluble proteins, metabolites, or any combination thereof, and the CTC derivatives are obtained through physical disruption, chemical lysis, freeze-thaw treatment, ultrasonic treatment, and / or centrifugation.

[0033] CTC-γδT cells obtained by activating γδT cells using CTCs or their derivatives can be used in the preparation of immunocellular agents for solid tumor treatment, and also in the preparation of personalized anti-tumor immunotherapy drugs. Furthermore, quality control testing of CTC-γδT cells can be performed using the following methods: 1. Phenotypic detection: Activation markers: CD69, CD25; Memory-related biomarkers: CD45RO, CCR7; Kill-related receptors: NKG2D, DNAM-1.

[0034] 2️ Functional testing: The ability to secrete effector factors such as IFN-γ and TNF-α; In vitro killing activity against tumor cells; Cell proliferation capacity or ability to survive.

[0035] 3️ Safety testing: Detection of residual circulating tumor cells or tumor-related components.

[0036] Microscopic examination, flow cytometry, and gene testing can be used to detect the presence of no tumor gene traces.

[0037] Example: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood. γδT cells were induced to expand and activate in a culture medium containing phosphate antigens or bisphosphonates and cytokines to obtain a γδT cell population. Simultaneously, circulating tumor cells (CTCs) were isolated from peripheral blood samples of cancer patients and subjected to short-term in vitro culture or maintenance treatment to preserve their activity and tumor-related characteristics. The γδT cells and CTCs were co-cultured in an RPMI-1640, IL-2, IL-15, and IL-18 co-culture system at a ratio where the number of γδT cells was higher than the number of circulating tumor cells. After co-culture, CTC-γδT cells were obtained.

[0038] Comparative example: Using existing techniques, γδT cells were isolated from healthy donor PBMCs and induced in vitro using only phosphoantigen and IL-2 without co-culturing with CTCs to obtain conventionally induced γδT cells.

[0039] The CTC-γδT cells obtained in the examples were compared with the conventional γδT cells obtained in the comparative examples for in vitro functional testing (n=3). The results are as follows: (1) Cytotoxicity detection, such as Figure 3 , Under the condition of an effectiveness-to-target ratio of 10:1: CTC-γδT cells achieved a killing rate of 85-90% against DU145 and LNCaP cells. The comparative γδT cell killed only about 75% of DU145 and LNCaP cells.

[0040] The results showed that the killing activity of CTC-γδT cells against tumor cells was significantly higher than that of the control group (P < 0.01).

[0041] Cytokine secretion assay, such as Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown: In the CD3⁺T cell population, the proportion of IFN-γ positive cells in the CTC-γδT cell group (48.2%) was significantly higher than that in the control group γδT cell group (22.6%). Furthermore, repeated experiments showed that the proportion of IFN-γ positive cells in the CTC-γδT cell group (47.7%) was significantly higher than that in the control group γδT cell group (23.8%).

[0042] In the Vγ9⁺T cell population, the proportion of IFN-γ positive cells in the CTC-γδT cell group (49.3%) was significantly higher than that in the control group γδT cell group (23.1%). Furthermore, repeated experiments showed that the proportion of IFN-γ positive cells in the CTC-γδT cell group (62.7%) was significantly higher than that in the control group γδT cell group (24.2%).

[0043] (3) Memory phenotype analysis (flow cytometry) Proportion of central memory γδT cells (CD45RO⁺ CCR7⁺): CTC-γδT: (42.6 ± 3.5)% Comparative example: (21.4 ± 2.8)% Significantly improved (P < 0.01).

[0044] (4) Activating biomarker expression CD69⁺ γδT cell ratio: CTC-γδT: (58.2 ± 5.1)% Comparative example: (29.7 ± 4.3)% In summary, γδT cells activated by circulating tumor cells are significantly superior to conventionally induced γδT cells in terms of cytotoxic activity, cytokine secretion capacity, and the proportion of memory phenotypes, indicating that the method of the present invention can effectively improve the anti-tumor function of γδT cells.

Claims

1. A method for activating γδT cells using CTCs or their derivatives, characterized in that... Includes the following steps: (1) Obtaining donor-derived γδT cells; (2) Obtaining CTCs or their derivatives from cancer patients; (3) The γδT cells and the CTC or its derivatives are co-cultured in an in vitro co-culture system to activate the γδT cells under the stimulation of CTC or its derivatives, thereby obtaining CTC-γδT cells.

2. The method for activating γδT cells using CTC or its derivatives according to claim 1, characterized in that: The CTCs activate γδT cells through their intact cellular form, cell surface molecules, cell-released soluble factors, and / or cell-derived subcellular structures.

3. The method for activating γδT cells using CTC or its derivatives according to claim 1, characterized in that: The γδT cells are derived from peripheral blood mononuclear cells from healthy donors, and the γδT cells are expanded or activated in vitro.

4. The method for activating γδT cells using CTC or its derivatives according to claim 3, characterized in that: The amplification or activation of γδT cells was achieved through induction with phosphoantigens, bisphosphonates, and / or cytokines.

5. The method for activating γδT cells using CTC or its derivatives according to claim 1, characterized in that: The CTCs or their derivatives are derived from peripheral blood, body fluids, or combinations thereof from cancer patients. The CTC derivatives include lysates of CTCs, cell debris, cell membrane components, exosomes, microvesicles, soluble proteins, metabolites, or any combination thereof. The CTC derivatives are obtained through physical disruption, chemical lysis, freeze-thaw treatment, ultrasonic treatment, and / or centrifugation.

6. The method for activating γδT cells using CTC or its derivatives according to claim 1, characterized in that: The CTCs were cultured, maintained, or functionalized in vitro before co-culture, and the number of γδT cells in the co-culture system was higher than the number of CTCs.

7. The method for activating γδT cells using CTCs or their derivatives according to any one of claims 1 to 6, characterized in that: The co-culture system adopts a direct contact co-culture mode, a semi-contact co-culture mode, or a non-contact co-culture mode.

8. The method for activating γδT cells using CTC or its derivatives according to claim 1, characterized in that: The co-culture system uses a basic culture medium suitable for immune cell culture, which is selected from RPMI-1640, AIM-V, X-VIVO series culture media or combinations thereof.

9. The method for activating γδT cells using CTC or its derivatives according to claim 8, characterized in that: The co-culture system also employs cytokines that promote γδT cell survival, activation, and / or memory phenotype formation. These cytokines include cytokine analogs, fusion proteins, or functional fragments with the same or similar biological functions, and / or are selected from at least one of the following: Factors related to the survival or expansion of γδT cells: IL-2, IL-7; Factors associated with γδT cell activation and enhanced effector function: IL-12, IL-15, IL-18; Factors related to the maintenance of γδT cell memory phenotype: IL-15, IL-21.

10. The use of CTC-γδT cells obtained by the method of activating γδT cells using CTCs or their derivatives as described in any one of claims 1 to 9 in the preparation of an immune cell preparation for the treatment of solid tumors, or in the preparation of a drug for personalized antitumor immunotherapy.

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