WO2024066115A1 - 增强t淋巴细胞抗肿瘤功能的方法及用途 - Google Patents

增强t淋巴细胞抗肿瘤功能的方法及用途 Download PDF

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WO2024066115A1
WO2024066115A1 PCT/CN2022/143783 CN2022143783W WO2024066115A1 WO 2024066115 A1 WO2024066115 A1 WO 2024066115A1 CN 2022143783 W CN2022143783 W CN 2022143783W WO 2024066115 A1 WO2024066115 A1 WO 2024066115A1
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lymphocytes
ferroelectric
enhancing
lymphocyte
cells
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French (fr)
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张学慧
邓旭亮
吕丹
宋佳
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北京大学口腔医学院
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for enhancing the anti-tumor function of T lymphocytes by utilizing physical electrical stimulation, and the T lymphocytes obtained thereby and their application.
  • Tumor immunotherapy includes immune checkpoint inhibitors, adoptive cell therapy, and tumor vaccines.
  • Chimeric antigen receptor (CAR)-T cell therapy is the most popular adoptive cell therapy at present, showing great potential in the treatment of hematological tumors, especially in the treatment of relapsed or refractory acute and chronic leukemias. Breakthrough progress has been made.
  • CAR-T cell therapy The general process of CAR-T cell therapy is: first, T lymphocytes are isolated from tumor patients, CAR is artificially synthesized, and then the designed CAR is transferred into T lymphocytes using viral vectors, and in vitro screening and amplification are performed, and finally, it is returned to the patient's body, and the antibodies expressed on the surface of CAR-T cells specifically recognize tumor cells and activate downstream signaling pathways at the same time, thereby achieving CAR-T cell activation and proliferation and specific killing of tumor cells.
  • CAR-T cells do not require the participation of MHC molecules and can directly kill tumor cells.
  • CAR-T cell therapy has significant effects on the treatment of hematological tumors
  • CAR-T cells have problems such as short survival time, poor killing function, and insufficient infiltration in the tumor microenvironment after entering the recipient patient, which limits its efficacy. Therefore, maintaining the survival of CAR-T cells in vivo, optimizing the CAR-T structure, and improving the state of the tumor microenvironment are the key to improving the accuracy of T cell targeting tumors and enhancing the anti-tumor effect.
  • CAR-T modified with immunomodulatory molecules such as cytokines, cytokine receptors, chemokines, chemokine receptors, and chimeric activation receptors have gradually been developed, thereby enhancing the persistence and killing of T lymphocytes.
  • T lymphocytes by gene editing has the disadvantages of being time-consuming and inefficient, and the random insertion of gene fragments will also involve biosafety issues.
  • the strategy of improving the tumor microenvironment is affected by individual differences in tumor patients and is not of universal significance.
  • the present invention provides a method for enhancing the anti-tumor function of T lymphocytes.
  • the present invention includes the following contents.
  • a method for enhancing the function of T lymphocytes comprising the step of culturing T lymphocytes under physical electrical stimulation conditions.
  • the physical electrical stimulation is generated by a charged matrix, a conductive matrix, direct current, alternating current, pulsed electricity, magnetoelectricity or a ferroelectric active material.
  • the method of the present invention can also be referred to as a method for enhancing T lymphocyte function based on the electrical characteristics of the material.
  • the piezoelectric constant of the ferroelectric active material is 1-40pC/N.
  • the ferroelectrically active material includes an organic ferroelectric polymer, which includes at least one of polyesters, polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene, polyvinylidene fluoride-hexafluoropropylene, polyvinylidene fluoride-tetrafluoroethylene, polymethyl methacrylate and polydimethylsiloxane, or a combination thereof.
  • the ferroelectric active material comprises inorganic ferroelectric particles, which include at least one of barium titanate, barium strontium titanate, lithium niobate, potassium sodium niobate and hydroxyapatite, or a combination thereof.
  • the surface of the inorganic ferroelectric particles is coated with a dopamine layer.
  • the method for enhancing T lymphocyte function according to the present invention further comprises the step of activating and amplifying T lymphocytes.
  • enhancing T lymphocyte function comprises at least one selected from the following:
  • the second aspect of the present invention provides a T lymphocyte with enhanced function, which is prepared by the method described in the first aspect.
  • the T lymphocytes include effector T cells, cytotoxic T cells, initial T cells and modified T lymphocytes, wherein the modified T lymphocytes include CAR-T.
  • the third aspect of the present invention provides a pharmaceutical composition comprising the T lymphocytes with enhanced function as described in the second aspect.
  • the method of the present invention does not involve traditional gene editing methods that are complex and have a long screening process, and can quickly and effectively enhance the anti-tumor function of T lymphocytes. Therefore, the present invention can be used to promote CAR-T cell therapy.
  • FIG. 1 exemplarily shows the experimental process of enhancing the anti-tumor function of T lymphocytes.
  • FIG. 2 shows the piezoelectric constant d 33 of the ferroelectric nanocomposite film material.
  • FIG. 3 shows the results of the ferroelectric nanocomposite film materials and T lymphocytes in the tumor volume of Example 1 and the comparative example.
  • Figure 4 is the result of flow cytometry detection of the expression levels of surface markers of CD8 + T lymphocytes in OT-1 mice, where MFI represents mean fluorescence intensity and DCFDA represents a cellular reactive oxygen species probe.
  • FIG. 5 shows the test results of the in vitro killing ability of ferroelectric nanocomposite film materials with different charges on T cells.
  • One aspect of the present invention provides a method for enhancing the function of T lymphocytes, and in particular, relates to an in vitro method for enhancing the function of T lymphocytes, comprising the step of culturing T lymphocytes in vitro under physical electrical stimulation.
  • the physical electrical stimulation is provided by an external electric field.
  • the external electric field is configured to cause the surface in contact with the T lymphocytes to be negatively charged.
  • the physical electrical stimulation is performed by making the surface negatively charged by a ferroelectric active material.
  • the piezoelectric constant of the ferroelectric active material is 1-40 pC/N, preferably 5-30 pC/N, more preferably 6-35 pC/N, and further preferably 8-30 pC/N, such as 10 pC/N, 15 pC/N, 18 pC/N, 20 pC/N.
  • the ferroelectrically active material includes an organic ferroelectric polymer, which includes at least one of polyesters, polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene, polyvinylidene fluoride-hexafluoropropylene, polyvinylidene fluoride-tetrafluoroethylene, polymethyl methacrylate and polydimethylsiloxane, or a combination thereof.
  • Polyvinylidene fluoride is sometimes referred to herein as polyvinylidene fluoride (also referred to as PVDF); polymethyl methacrylate is sometimes referred to herein as PMMA; polydimethylsiloxane is sometimes referred to herein as PDMS.
  • polyesters include, but are not limited to, poly-L-lactide (also referred to as PLLA), polylactic acid-glycolic acid copolymer (also referred to as PLGA), and polycaprolactone (also referred to as PCL).
  • PLLA poly-L-lactide
  • PLA polylactic acid-glycolic acid copolymer
  • PCL polycaprolactone
  • the present invention has no particular limitation on the molecular weight of the ferroelectric polymer as long as the purpose of the present invention can be achieved.
  • the weight average molecular weights of the above-mentioned PVDF, polyvinylidene fluoride-trifluoroethylene, and polyvinylidene fluoride-hexafluoropropylene can be 150,000-300,000, 100,000-200,000, and 700,000-900,000, respectively.
  • the ferroelectric active material comprises inorganic ferroelectric particles, which include at least one of barium titanate, barium strontium titanate, lithium niobate, potassium sodium niobate and hydroxyapatite or a combination thereof.
  • the particle size of the inorganic ferroelectric particles is 0.1-500nm, and more preferably 10-50nm.
  • the ferroelectric active material of the present invention includes both ferroelectric polymer and ferroelectric particles, and the weight ratio of the ferroelectric polymer to the ferroelectric particles is 1:1-10, preferably 1:1-5, and further preferably 1:1-4.
  • the inorganic ferroelectric particles are further coated with a dopamine layer to obtain modified inorganic ferroelectric particles to provide excellent biocompatibility and ferroelectric properties.
  • the present invention coats the surface of the nano-ceramic particles with organic dopamine, so that a transition bonding layer is introduced between the ceramic particles and the ferroelectric polymer, thereby effectively improving the interface between the ceramic particles and the ferroelectric polymer, reducing defects caused by poor interface compatibility, achieving uniform dispersion of the ceramic particles in the ferroelectric polymer, and improving the ferroelectric properties of the material.
  • the thickness of the dopamine layer is not particularly limited, and can be 1-10 nm, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nm or any value therebetween.
  • the weight ratio of dopamine to ferroelectric particles is 1-10:1, preferably 2-8:1, and further preferably 3-7:1.
  • a special substrate for culturing T lymphocytes to enhance their anti-tumor function is prepared from the ferroelectrically active material of the present invention.
  • the special substrate comprises a first surface having a positive charge and a second surface having a negative charge.
  • the thickness of the substrate formed by the ferroelectrically active material is not particularly limited, preferably 10-80 ⁇ m, further preferably 15-50 ⁇ m, and further preferably 20-40 ⁇ m.
  • both the first surface and the second surface have good ferroelectric properties, it was found in the T cell in vitro killing ability experiment that the first surface with a positive charge could not enhance the anti-tumor function of T lymphocytes.
  • the second surface with a negative charge could not only enhance the anti-tumor function of T lymphocytes, inhibit tumor size, and increase the amount of biomarkers related to T lymphocyte survival, but also show a charge and dose-dependent trend.
  • the film preparation method is not particularly limited and can be prepared by methods known in the art.
  • the preparation method include but are not limited to spin coating, salivation, screen printing, dip coating, inkjet printing, and spray pyrolysis.
  • the ferroelectric active material is obtained by annealing and/or polarization treatment, and the order of annealing and polarization treatment is not particularly limited.
  • the film material is annealed at 50-150°C for 0.5h-3h, then cooled to room temperature, and the treated film material is subjected to corona polarization to obtain a ferroelectric active material, wherein the voltage of the corona polarization treatment is 10kV-50kV, the polarization temperature is 25°C-50°C, and the time is 10min-60min.
  • the temperature of the annealing treatment is 90-140°C
  • the time is 2h-3h
  • the voltage of the corona polarization treatment is 20kV-50kV
  • the temperature is 25-40°C
  • the time is 10min-40min.
  • the temperature of the annealing treatment can be 90°C, 100°C, 110°C, 120°C, 130°C, 140°C, or the range between any values therebetween
  • the time of the annealing treatment can be 0.5h, 1h, 1.5h, 2.0h, 2.5h, 3h, or the range between any values therebetween.
  • the voltage of the corona poling treatment can be 10KV, 20KV, 30KV, 40KV, 50KV or any range therebetween
  • the temperature of the corona poling treatment can be 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or any range therebetween
  • the time of the corona poling treatment can be 10min, 20min, 30min, 40min, 50min, 60min or any range therebetween.
  • the present invention subjects the film material to corona poling treatment, and then annealing treatment at 50-150°C for 0.5h-3h to obtain a ferroelectrically active material, wherein the voltage of the corona poling treatment is 10KV-50KV, the temperature is 25°C-50°C, and the time is 10min-60min; preferably, the voltage of the corona poling treatment is 20KV-50KV, the temperature is 25°C-40°C, and the time is 10min-40min; the temperature of the annealing treatment is 90-140°C, and the time is 0.5h-2h.
  • the voltage of the corona poling treatment can be 10KV, 20KV, 30KV, 40KV, 50KV or any range between them
  • the temperature of the corona poling treatment can be 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or any range between them
  • the time of the corona poling treatment can be 10min, 20min, 30min, 40min, 50min, 60min or any range between them.
  • the temperature of the annealing treatment can be 90°C, 100°C, 110°C, 120°C, 130°C, 140°C or any range between them
  • the time of the annealing treatment can be 0.5h, 1h, 1.5h, 2.0h, 2.5h, 3h or any range between them.
  • the above-mentioned corona polarization process can make the film material surface carry more polarization charges, and improve its ferroelectric properties.
  • the voltage of the corona polarization process is too low (for example, lower than 10KV)
  • the temperature is too low (for example, lower than 25°C) or the time is too short (for example, shorter than 10min)
  • the corona polarization process is incomplete, which directly affects the ferroelectric properties of the film material.
  • the voltage of the corona polarization process is too high (for example, lower than 50KV)
  • the temperature is too high (for example, higher than 50°C) or the time is too long (for example, longer than 60min)
  • the safety risk of actual operation is increased, but also the mechanical properties of the film material itself may be affected.
  • T lymphocytes include but are not limited to: at least one or a combination of effector T cells, cytotoxic T cells, naive T cells and modified T lymphocytes, wherein the modified T lymphocytes include CAR-T cells.
  • the naive T lymphocytes provided by the donor are further sorted to obtain CD8+T lymphocytes, which are then activated by OVA peptide (OVA peptide) and induced to expand by cytokines.
  • OVA peptide OVA peptide
  • naive T lymphocytes CD8+T lymphocytes, OVA peptide activation and cytokines are used for induced expansion, this does not mean a limitation on the specific type of T lymphocytes or the method of producing T lymphocytes. Those skilled in the art can select appropriate T lymphocytes, donors or activation methods as needed to obtain T lymphocytes that can inhibit tumors.
  • the functionally enhanced T lymphocytes include modified T lymphocytes.
  • modified T lymphocytes refers to the transfer of genetic material with specific antigen recognition domains and T cell activation signals into T cells through gene modification technology, so that T cells are directly activated by binding to specific antigens on the surface of tumor cells, directly killing tumor cells by releasing killer proteins, killer factors, etc., and also recruiting endogenous immune cells in the human body to kill tumor cells by releasing cytokines, thereby achieving the purpose of treating tumors, and further forming immune memory T cells, thereby obtaining a specific long-term anti-tumor mechanism.
  • the modified T lymphocytes are CAR-T cells.
  • the method for preparing enhanced T lymphocytes of the present invention comprises at least the following steps:
  • dopamine or its salt and inorganic ferroelectric particles are ultrasonically dispersed in water in proportion, stirred and placed in a vacuum dryer at 40-70°C to obtain modified ferroelectric particles.
  • a certain amount of organic ferroelectric polymer is mixed with an organic solvent and stirred, and the modified ferroelectric particles are ultrasonically dispersed in the organic solvent to obtain a dispersion.
  • the dispersion is added dropwise to an organic solution containing an organic ferroelectric polymer and stirred to obtain a uniform mixed solution.
  • a nanocomposite film of a specific thickness is obtained by a salivation method, and the organic solvent therein is dried and volatilized.
  • the concentration of the organic ferroelectric polymer in the organic solvent is 0.1-2 g/mL, preferably 0.1-1.5 g/mL.
  • the concentration of the modified ferroelectric particles in the organic solvent is 10-100 g/L, preferably 40-60 g/mL.
  • the organic solvent is not particularly limited, including but not limited to alcohol solution, N,N-dimethylformamide, dimethyl sulfoxide, etc.
  • the concentration of OVA peptide inducing T lymphocyte activation is 0.1-4 ⁇ g/ml, preferably 0.15-3 ⁇ g/ml.
  • cytokines are used for induction and amplification.
  • the cytokine is IL-2, and its concentration is 0.1-4 ng/ml, preferably 0.15-3 ng/ml.
  • the tumor size and tumor growth rate can be observed and measured, and the expression level of T lymphocyte surface markers or tumor cell survival rate can also be used to reflect the anti-tumor effect of T lymphocytes.
  • the marker determination method can adopt methods known in the art, including but not limited to flow cytometry, immunoblotting, etc.
  • the markers include CD25, a survival-related indicator, and DCFDA, an active oxygen metabolism-related indicator.
  • annealing treatment was selected to promote the crystallization of ⁇ phase in PVDF, so as to further increase the ⁇ phase content in the P(VDF-TrFE) ferroelectric film doped with BaTiO 3 nanoparticles.
  • the NC and LC group materials were annealed at a 55°C heating platform for 30 minutes, the MC group materials were annealed at a 90°C heating platform for 30 minutes, and the HC group materials were annealed at a 120°C heating platform for 30 minutes. After cooling to room temperature in the furnace, the film was gently separated from the quartz plate.
  • the prepared BaTiO 3 /P(VDF-TrFE) ferroelectric nanocomposite film material was placed on the metal bottom plate of a high voltage DC polarizer by corona poling method, with a loading voltage of 21 kV and a loading voltage of 21.5 kV for the HC group, and polarized at room temperature for 30 minutes.
  • OT-1 mice were killed by cervical vertebrae disconnection, fixed on a wax tray with a needle, and disinfected with 75% alcohol.
  • the abdominal skin of the mouse was grasped with ophthalmic forceps, and a small cut was made along the midline of the abdomen with ophthalmic scissors.
  • the peritoneum and skin were bluntly separated with ophthalmic scissors, and the skin was cut along the midline of the abdomen to expose the complete peritoneum.
  • the peritoneum was grasped with ophthalmic forceps, and the peritoneum was cut along the midline of the abdomen with ophthalmic scissors to expose the spleen tissue.
  • the spleen was bluntly separated from the surrounding tissues with ophthalmic forceps, and placed in a 10 cm culture dish containing 13 ml FACS buffer precooled at 4°C.
  • the 200-mesh screen cannot contact the bottom of the culture dish or be separated from the FACS buffer to avoid death of spleen cells.
  • Use a pipette to absorb the FACS buffer in the culture dish and repeatedly rinse the 200-mesh screen to collect residual cells.
  • the CD8 + T lymphocytes obtained in the previous step were plated and cultured on unpolarized (UP) and polarized (P) BaTiO 3 /P(VDF-TrFE) ferroelectric nanocomposite films, and the lymphocyte concentration was 2 ⁇ 10 6 /ml.
  • OT-1 mouse CD8 + T lymphocytes were activated by 2 ⁇ g/ml OVA peptide for 2 days, and then the dead cells produced during the activation process were removed by centrifugation without braking using mouse lymphocyte separation medium.
  • IL-2 2 ng/ml IL-2 was added to induce mouse CD8 + T lymphocyte expansion for 4 days. Cells were collected and counted every day to adjust the cell density to 2 ⁇ 10 6 /ml. Dead cells generated during the expansion process were then removed by centrifugation without braking using mouse lymphocyte separation fluid.
  • 3 ⁇ 10 6 LLC tumor cells overexpressing OVA were inoculated subcutaneously on the left side of NOD-SCID mice, and 5 ⁇ 10 5 OT-1 mouse CD8 + T lymphocytes activated and expanded in vitro were injected into the tail vein of tumor-bearing mice on the 7th day after tumor inoculation.
  • the length ⁇ width 2 of the subcutaneous tumor of the mouse was taken as the tumor volume, and the tumor size was continuously observed and measured.
  • the expression level of surface markers of CD8 + T lymphocytes in OT-1 mice was detected by flow cytometry.
  • CD8 + T lymphocytes from OT-1 mice were obtained and cultured on ferroelectric nanocomposite membranes with different charges.
  • the lymphocyte concentration was 2 ⁇ 10 6 /ml.
  • 2 ⁇ g/ml OVA peptide was used to induce CD8 + T lymphocyte activation for 2 days.
  • the activated CD8 + T lymphocytes were added to tumor cells at a ratio of 1:1, 0.5:1, and 0.25:1 (T cells: tumor cells), and tumor cell survival was detected after 24 hours.
  • the BaTiO 3 /P(VDF-TrFE) ferroelectric nanocomposite film was dried on a heating platform at a constant temperature of 55° C. without polarization.
  • the other steps were the same as those in Example 1.
  • the piezoelectric constant d 33 of the ferroelectric nanocomposite film material is shown in Figure 2, where NC represents no charge, LC+ represents low charge, positive charge (annealing temperature 55 degrees, polarization voltage 21kV), LC- represents low charge, negative charge (annealing temperature 55 degrees, polarization voltage 21kV), MC- represents medium charge, negative charge (annealing temperature 90 degrees, polarization voltage 21kV), HC- represents high charge, negative charge (annealing temperature 120 degrees, polarization voltage 21.5kV).
  • the piezoelectric constant d 33 of the ferroelectric nanocomposite film material prepared in the comparative example was -4.678 ⁇ 0.2525 (LC+), 7.322 ⁇ 0.1637 (LC-), 10.27 ⁇ 0.3297 (MC-), 17.27 ⁇ 0.2864 (HC-).
  • low power refers to 4-8pC/N
  • medium power refers to 9-12pC/N
  • high power refers to 15-20pC/N.
  • T lymphocytes cultured on the surface of polarized ferroelectric nanocomposite membrane materials were infused back into mice, they had stronger anti-tumor ability, as shown by smaller tumor volume and slower tumor growth rate ( Figure 3).
  • T lymphocytes cultured on the surface of unpolarized ferroelectric nanocomposite membrane materials had weaker anti-tumor ability after being infused back into mice, as shown by larger tumor volume and faster tumor growth rate ( Figure 3).
  • Flow cytometry was used to detect the expression levels of surface markers of CD8 + T lymphocytes in OT-1 mice.
  • OT-1 mouse CD8 + T lymphocytes were obtained and cultured on ferroelectric nanocomposite membranes with different charges to induce CD8 + T lymphocyte activation.
  • the activated CD8 + T lymphocytes were added to tumor cells according to different T cell: tumor cell ratios.
  • the 24-hour tumor cell survival results showed that when CD8 + T lymphocytes were spread on the negatively charged side of the ferroelectric nanocomposite membrane, they significantly inhibited tumor cell survival.
  • this inhibitory effect showed a charge and dose-dependent trend.
  • the ratio of T cells to tumor cells was close to 1:1, the inhibitory effect on tumor cell survival became more obvious.
  • the increase of charge or the increase of piezoelectric constant the inhibitory effect on tumor cell survival became more obvious.

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Abstract

公开增强T淋巴细胞抗肿瘤功能的方法及用途。该方法包括使T淋巴细胞在物理电刺激条件下进行培养的步骤，其中，T淋巴细胞包括初始T细胞或修饰的T淋巴细胞。方法不涉及复杂、筛选流程长的传统基因编辑手段，能够快速有效增强T淋巴细胞抗肿瘤功能，可用于促进CAR-T细胞疗法。

Description

增强T淋巴细胞抗肿瘤功能的方法及用途 技术领域
本发明涉及生物技术领域，具体地涉及利用物理电刺激来增强T淋巴细胞抗肿瘤功能的方法及由此得到的T淋巴细胞及应用。
背景技术
随着传染病死亡率的大幅下降，癌症已成为威胁人类健康的第二大死因。并且，癌症的发病率呈逐年上升和年轻化趋势。肿瘤的常规治疗方法包括手术、放疗、化疗、介入等。虽然这些方法对于减轻肿瘤负荷见效快，但是存在治疗不彻底，对机体毒副作用较大等弊端。随着生物技术研究的进展，肿瘤免疫疗法在肿瘤治疗中取得重大突破。肿瘤免疫治疗包含免疫检查点抑制剂、过继性细胞疗法以及肿瘤疫苗等。嵌合抗原受体(chimeric antigen receptor，CAR)-T细胞疗法是目前最受关注的过继性细胞治疗，在治疗血液系统肿瘤方面表现出巨大潜力，尤其在治疗复发或难治性急、慢性白血病上已取得突破性进展。
CAR-T细胞疗法的一般流程是：首先从肿瘤患者体内分离获得T淋巴细胞，人工合成CAR，之后利用病毒载体将设计好的CAR转入T淋巴细胞中，并进行体外筛选与扩增，最后回输到患者体内，通过CAR-T细胞表面表达的抗体特异性识别肿瘤细胞，并同时激活下游信号通路，从而实现CAR-T细胞活化增殖以及对肿瘤细胞特异性杀伤的作用。相较于传统T细胞过继疗法，CAR-T细胞不需要MHC分子参与，能够直接对肿瘤细胞发挥杀伤作用。
尽管CAR-T细胞疗法对于血液系统肿瘤治疗效果显著，但是CAR-T细胞进入受体患者后存在存活时间短、杀伤功能差、肿瘤微环境中浸润不足等问题，使其疗效受限。因此，维持CAR-T细胞的体内存活、优化CAR-T结构、改善肿瘤微环境状态是提高T细胞靶向肿瘤的精确性、增强抗肿瘤效果的关键。随着研究的深入，修饰细胞因子、细胞因子受体、趋化因子、趋化 因子受体和嵌合激活受体等免疫调节分子的CAR-T逐渐开发出来，从而增强T淋巴细胞的持久性和杀伤性。通过基因编辑的方式来改造T淋巴细胞存在耗时长、效率低等缺点，基因片段随机性插入还会涉及生物安全性问题。改善肿瘤微环境的策略受到肿瘤患者个体差异的影响，不具有普遍意义。
背景技术中的信息仅仅在于说明本发明的总体背景，不应视为承认或以任何形式暗示这些信息构成本领域一般技术人员所公知的现有技术。
发明内容
为解决现有技术中的技术问题，尤其是现有利用基因编辑手段增强CAR-T细胞疗法耗时长、效率低、筛选流程较长的技术问题，本发明提供一种增强T淋巴细胞抗肿瘤功能的方法。具体地，本发明包括以下内容。
本发明的第一方面，提供一种增强T淋巴细胞功能的方法，其包括使T淋巴细胞在物理电刺激条件下进行培养的步骤。
在某些实施方案中，根据本发明所述的增强T淋巴细胞功能的方法，其中，所述物理电刺激通过带电基质、导电基质、直流电、交流电、脉冲电、磁电或铁电活性材料产生。此时，本发明的方法也可称作基于材料电学特征增强T淋巴细胞功能的方法。优选地，所述铁电活性材料的压电常数为1-40pC/N。
在某些实施方案中，根据本发明所述的增强T淋巴细胞功能的方法，其中，所述铁电活性材料包括有机铁电聚合物，其包括聚酯类、聚偏氟乙烯、聚偏氟-三氟乙烯、聚偏氟乙烯-六氟丙烯、聚偏氟乙烯-四氟乙烯、聚甲基丙烯酸甲酯和聚二甲基硅氧烷中的至少一种或其组合。
在某些实施方案中，根据本发明所述的增强T淋巴细胞功能的方法，其中，所述铁电活性材料包括无机铁电颗粒，其包括钛酸钡、钛酸锶钡、铌酸锂、铌酸钾钠和羟基磷灰石中的至少一种或其组合。
在某些实施方案中，根据本发明所述的增强T淋巴细胞功能的方法，其中，所述无机铁电颗粒表面包覆多巴胺层。
在某些实施方案中，根据本发明所述的增强T淋巴细胞功能的方法，其中，进一步包括对T淋巴细胞进行活化和扩增的步骤。
在某些实施方案中，根据本发明所述的增强T淋巴细胞功能的方法，其中，增强T淋巴细胞功能包括选自下述至少之一：
a.存活时间更长；
b.增殖性增强；
c.抗肿瘤活性提高；
d.CD25和/或DCFDA表达升高。
本发明的第二方面，提供一种功能增强的T淋巴细胞，其通过第一方面所述的方法制备得到。
在某些实施方案中，根据本发明所述的功能增强的T淋巴细胞，其中，所述T淋巴细胞包括效应T细胞、细胞毒性T细胞、初始T细胞及修饰的T淋巴细胞，其中所述修饰的T淋巴细胞包括CAR-T。
本发明的第三方面，提供一种药物组合物，其包括第二方面所述的功能增强的T淋巴细胞。
本发明的方法不涉及复杂、筛选流程长的传统基因编辑手段，能够快速有效增强T淋巴细胞抗肿瘤功能。因此，本发明可用于促进CAR-T细胞疗法。
附图说明
图1示例性地示出了增强T淋巴细胞抗肿瘤功能的实验过程。
图2为铁电纳米复合膜材料压电常数d 33。
图3为实施例1和比较例的铁电纳米复合膜材料与T淋巴细胞在肿瘤体积中的结果。
图4为流式细胞术检测OT-1小鼠CD8 +T淋巴细胞表面标志的表达水平结果。其中MFI表示平均荧光强度，DCFDA表示细胞活性氧探针。
图5为不同带电量的铁电纳米复合膜材料对T细胞体外杀伤能力检测结果。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为具体公开了该范围的上限和下限以及它们之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。除非另有说明，否则％为基于重量的百分数。
本发明的一个方面，提供一种增强T淋巴细胞功能的方法，尤其是涉及增强T淋巴细胞功能的体外方法，包括使T淋巴细胞在物理电刺激下进行体外培养的步骤。
在某些实施方案中，物理电刺激通过外源电场提供。外源电场设置为使得与T淋巴细胞接触的表面呈负电状态。
在某些实施方案中，通过铁电活性材料使表面带负电，从而进行所述物理电刺激。所述铁电活性材料的压电常数为1-40pC/N，优选5-30pC/N，还优选为6-35pC/N，进一步优选为8-30pC/N，如10pC/N、15pC/N、18pC/N、20pC/N。
在某些实施方案中，所述铁电活性材料包括有机铁电聚合物，其包括聚酯类、聚偏氟乙烯、聚偏氟-三氟乙烯、聚偏氟乙烯-六氟丙烯、聚偏氟乙烯-四氟乙烯、聚甲基丙烯酸甲酯和聚二甲基硅氧烷中的至少一种或其组合。聚偏氟乙烯本文有时也称为聚偏二氟乙烯(也简称PVDF)；聚甲基丙烯酸甲酯本文有时也简称为PMMA；聚二甲基硅氧烷本文有时也简称为PDMS。本发明中，聚酯类的实例包括但不限于聚L-丙交酯(也简称PLLA)、聚乳酸-乙醇酸共聚物(也简称PLGA)、聚己内酯(也简称PCL)。
本发明对铁电聚合物的分子量没有特别限制，只要能够实现本发明的目的即可，例如上述PVDF、聚偏氟乙烯-三氟乙烯、聚偏氟乙烯-六氟丙烯的重均分子量依次可以为15万-30万、10万-20万、70万-90万。
本发明中，铁电活性材料包括无机铁电颗粒，其包括钛酸钡、钛酸锶钡、铌酸锂、铌酸钾钠和羟基磷灰石中的至少一种或其组合。优选地，所述无机铁电颗粒的粒径为0.1-500nm，还优选为10-50nm。
示例性实施方案中，本发明的铁电活性材料包括铁电聚合物与铁电颗粒两者，且铁电聚合物与铁电颗粒的重量比为1:1-10，优选为1:1-5，还优选为1:1-4。
本发明中，无机铁电颗粒进一步通过包覆多巴胺层得到改性的无机铁电颗粒，以提供优异的生物相容性以及铁电性能。本发明通过在纳米陶瓷颗粒表面包覆有机物多巴胺，使得陶瓷颗粒与铁电聚合物之间引入过渡粘结层，有效改善了陶瓷颗粒与铁电聚合物之间的界面，减少了由界面相容性差引发的缺陷，实现了陶瓷颗粒在铁电聚合物中的均匀分散，提高了材料的铁电性能。
本发明中，多巴胺层的厚度不特别限定，可以是1-10nm，例如1、2、3、4、5、6、7、8、9、10nm或其间任意数值的厚度。
本发明中，多巴胺与铁电颗粒的重量比为1-10:1，优选为2-8:1，还优选为3-7:1。
在某些实施方案中，由本发明的铁电活性材料制备得到用于培养T淋巴细胞，从而增强其抗肿瘤功能的专用基材。优选地，所述专用基材包括具有正电的第一面和具有负电的第二面。由铁电活性材料形成的基材厚度不特别限定，优选为10-80μm，还优选为15-50μm，进一步优选为20-40μm。虽然上述第一面和第二面均具有良好的铁电性能，但是在T细胞体外杀伤能力实验中发现，具有正电的第一面并不能够增强T淋巴细胞的抗肿瘤功能，相反，具有负电的第二面不仅能够增强T淋巴细胞的抗肿瘤功能，抑制肿瘤大小，提高T淋巴细胞与存活相关的生物标志物的量，并且呈现带电量以及剂量依赖性趋势。
在本发明中，膜制备方法不特别限定，可以采用本领域已知的方法进行制备，制备方法的实例包括但不限于如旋涂法、流涎法、丝网印刷法、浸涂法、喷墨打印法及喷雾热解法等。
本发明中，铁电活性材料通过退火和/或极化处理得到，退火和极化处理的顺序不特别限定。
在某些实施方案中，将膜材料在50-150℃的下退火处理0.5h-3h，然后降至室温，对处理后的膜材料进行电晕极化处理得到铁电活性材料，电晕极化处理的电压为10kV-50kV、极化温度为25℃-50℃、时间为10min-60min。优选地，退火处理的温度为90-140℃、时间为2h-3h，电晕极化处理的电压为20kV-50kV、温度为25-40℃、时间为10min-40min。例如，退火处理的温度可以为90℃、100℃、110℃、120℃、130℃、140℃或为其间任意数值之间的范围，退火处理的时间可以为0.5h、1h、1.5h、2.0h、2.5h、3h或为其间任意数值之间的范围。例如，电晕极化处理的电压可以为10KV、20KV、30KV、40KV、50KV或为其间任意数值之间的范围，电晕极化处理的温度可以为25℃、30℃、35℃、40℃、45℃、50℃或为其间任意数值之间的范围，电晕极化处理的时间可以为10min、20min、30min、40min、50min、60min或为其间任意数值之间的范围。
在某些实施方案中，本发明将膜材料进行电晕极化处理，然后在50-150℃下退火处理0.5h-3h得到铁电活性材料，电晕极化处理的电压为10KV-50KV、温度为25℃-50℃、时间为10min-60min；优选地，电晕极化处理的电压为20KV-50KV、温度为25℃-40℃、时间为10min-40min；退火处理的温度为90-140℃、时间为0.5h-2h。例如，电晕极化处理的电压可以为10KV、20KV、30KV、40KV、50KV或为其间任意数值之间的范围，电晕极化处理的温度可以为25℃、30℃、35℃、40℃、45℃、50℃或为其间任意数值之间的范围，电晕极化处理的时间可以为10min、20min、30min、40min、50min、60min或为其间任意数值之间的范围。例如，退火处理的温度可以为90℃、100℃、110℃、120℃、130℃、140℃或为其间任意数值之间的范围，退火处理的时间可以为0.5h、1h、1.5h、2.0h、2.5h、3h或为其间任意数值之间的范围。
上述电晕极化处理，能够使得膜材料表面带有较多的极化电荷，提升其铁电性能。当电晕极化处理的电压过低(例如低于10KV)、温度过低(例如低于25℃)或时间过短(例如短于10min)时，电晕极化处理不完全，直接影响膜材料的铁电性能改善。当电晕极化处理的电压过高(例如低于50KV)、温度过高(例如高于50℃)或时间过长(例如长于60min)时，不仅增加实际操作的安全风险，而且可能影响膜材料本身的力学性能。
本发明中，T淋巴细胞包括但不限于：效应T细胞、细胞毒性T细胞、初始T细胞及修饰的T淋巴细胞中的至少一种或其组合，其中所述修饰的T淋巴细胞包括CAR-T细胞。本发明中，通过供体提供的初始T淋巴细胞，并进一步进行分选得到CD8+T淋巴细胞，然后通过OVA肽(OVA peptide)活化以及细胞因子进行诱导扩增。可以理解的是，虽然在某些实施方案中，使用了初始T淋巴细胞、CD8+T淋巴细胞、OVA肽活化以及细胞因子进行诱导扩增，但这不意味着是对T淋巴细胞具体类型或T淋巴细胞产生方法的限制，本领域技术人员可以根据需要选择合适的T淋巴细胞、供体或活化方法，从而得到能够抑制肿瘤的T淋巴细胞。
本发明中，功能增强的T淋巴细胞包括修饰的T淋巴细胞，术语“修饰的T淋巴细胞”是指通过基因修饰技术，将带有特异性抗原识别结构域、T细胞激活信号的遗传物质转入T细胞，使T细胞直接与肿瘤细胞表面的特异性抗原相结合而被激活，通过释放杀伤蛋白、杀伤因子等直接杀伤肿瘤细胞，同时还通过释放细胞因子募集人体内源性免疫细胞杀伤肿瘤细胞，从而达到治疗肿瘤的目的，进一步可形成免疫记忆T细胞，从而获得特异性的抗肿瘤长效机制。优选地，修饰的T淋巴细胞为CAR-T细胞。
在示例性实施方案中，本发明的增强T淋巴细胞的制备方法至少包括以下步骤：
1)制备铁电纳米复合膜材料；
2)获取供体初始T淋巴细胞；
3)将T淋巴细胞于铁电纳米复合膜材料表面培养；
4)T淋巴细胞活化以及扩增。
首先，将多巴胺或其盐与无机铁电颗粒按比例在水中超声分散，搅拌后置于40-70℃真空干燥，得到改性的铁电颗粒。然后，取一定量的有机铁电聚合物与有机溶剂混合并搅拌，将改性的铁电颗粒在有机溶剂中超声分散得到分散液。将分散液滴加到含有有机铁电聚合物的有机溶液中搅拌得到均匀的混合液。采用流涎法，得到特定厚度的纳米复合膜，烘干挥发其中的有机溶剂。
在本发明的制备方法中，有机铁电聚合物在有机溶剂中的浓度为0.1-2g/mL，优选为0.1-1.5g/mL。改性的铁电颗粒在有机溶剂中的浓度为10-100g/L，优选为40-60g/mL。有机溶剂不特别限定，包括但不限于醇溶液、N,N-二甲基甲酰胺、二甲基亚砜等。
在本发明的制备方法中，诱导T淋巴细胞活化的OVA peptide的浓度为0.1-4μg/ml，优选0.15-3μg/ml。
在本发明的制备方法中，采用细胞因子进行诱导扩增，优选地，细胞因 子为IL-2，其浓度为0.1-4ng/ml，优选0.15-3ng/ml。
对于T淋巴细胞抗肿瘤功能检测，可以对肿瘤大小和肿瘤生长速度进行观察和测量，也可以通过T淋巴细胞表面标志的表达水平或肿瘤细胞存活率来反映T淋巴细胞抗肿瘤效果，标志物测定方法可以采用本领域已知的方法，包括但不限于例如流式细胞术、免疫印迹等。在某些实施方案中，标志物包括存活相关的指标CD25、活性氧代谢相关指标DCFDA。
本领域技术人员应理解，只要能够实现本发明的目的，在上述步骤(1)-(6)前后，或这些任意步骤之间还可包含其他步骤或操作，例如进一步优化和/或改善本发明所述的方法。
实施例
(1)制备铁电纳米复合膜材料
称取0.3g盐酸多巴胺粉末，加入180ml去离子水，搅拌均匀后加入6g钛酸钡纳米粒，超声30min使钛酸钡纳米颗粒均匀分散，搅拌10-12小时。静置溶液至出现明显分层，吸弃上清，用无水乙醇和去离子水反复清洗至上清澄澈无明显浑浊。将经过盐酸多巴胺改性的钛酸钡纳米颗粒置于55℃恒温真空干燥箱中完全干燥。
称取5g P(VDF-TrFE)粉末，加入35ml N,N-二甲基甲酰胺(DMF)中，搅拌3小时。同时称取盐酸多巴胺改性的钛酸钡纳米颗粒0.89g加入15ml DMF中，搅拌30分钟，超声30分钟，重复3个循环，使材料充分混合均匀。将超声分散后的盐酸多巴胺改性的钛酸钡纳米颗粒混悬液滴加到P(VDF-TrFE)溶液中，搅拌12小时，得到均匀、稳定的BaTiO 3/P(VDF-TrFE)混合液。使用流涎法，在石英玻璃上铺成厚度为30μm的BaTiO 3/P(VDF-TrFE)铁电纳米复合膜，并于55℃恒温的加热平台上烘干，至溶剂完全挥发。
本实验中选择退火处理促进PVDF内结晶形成β相，以进一步提高掺杂BaTiO 3纳米颗粒的P(VDF-TrFE)铁电薄膜中β相含量。NC、LC组材料于55℃加热平台退火30分钟，MC组材料于90℃加热平台退火30分钟，HC组材 料于120℃加热平台退火30分钟。随炉冷却至室温后，将薄膜与石英板轻轻分离。
采用电晕极化法将制备的BaTiO 3/P(VDF-TrFE)铁电纳米复合膜材料置于高压直流极化仪的金属底板上，加载电压为21kV，HC组加载电压为21.5kV，室温极化30分钟。
(2)获取供体初始T淋巴细胞
如图1所示，用离断颈椎的方法处死OT-1小鼠，用针头将小鼠固定于蜡盘上，并使用75％的酒精对小鼠进行消毒。用眼科镊夹起小鼠腹部皮肤，并用眼科剪沿腹中线剪一小口。用眼科剪钝性分离腹膜与皮肤，将皮肤沿腹中线剪开，暴露完整腹膜。用眼科镊夹起腹膜，并用眼科剪沿腹中线剪开腹膜，暴露脾脏组织，用眼科镊将脾脏与周围组织钝性分离，并放置于装有提前4℃预冷的13ml FACS缓冲液的10cm培养皿中。
将200目筛网放置于10cm培养皿中，浸没入FACS缓冲液。用眼科镊将脾脏组织转移至200目筛网上，并用眼科剪将其剪碎。用研磨棒在200目筛网上对剪碎的脾脏组织向同一方向进行研磨，直至不可见完整组织块为止。此过程中需注意200目筛网不可与培养皿底接触，也不可脱离FACS缓冲液，以免造成脾脏细胞死亡。使用移液器吸取培养皿中的FACS缓冲液，反复冲洗200目筛网，以收集残留的细胞。用移液器将培养皿中的FACS缓冲液全部转移入15ml离心管中，使用水平转子离心机，1600rpm 4℃离心5min，弃上清。使用1ml ACK裂解液室温裂解5分钟。加入10ml FACS缓冲液终止裂解。离心后用1ml FACS缓冲液重悬沉淀细胞，磁珠分选CD8 +T淋巴细胞，用于后续实验。
(3)将T淋巴细胞于铁电纳米复合膜材料表面培养
将上一步获取的CD8 +T淋巴细胞铺于未经极化(UP)和极化(P)的BaTiO 3/P(VDF-TrFE)铁电纳米复合膜上培养，淋巴细胞浓度为2×10 6/ml。
(4)T淋巴细胞活化以及扩增
利用2μg/ml OVA peptide诱导OT-1小鼠CD8 +T淋巴细胞活化2天，之后通过小鼠淋巴细胞分离液，无制动离心，去除活化过程中产生的死细胞。
加入2ng/ml IL-2诱导鼠CD8 +T淋巴细胞扩增4天，每天收集细胞，对细胞进行计数，调整细胞密度为2×10 6/ml，之后通过小鼠淋巴细胞分离液，无制动离心，去除扩增过程中产生的死细胞。
(5)将T淋巴细胞回输给荷瘤受体
给NOD-SCID小鼠左侧皮下接种3×10 6个过表达OVA的LLC肿瘤细胞，在接种肿瘤第7天给荷瘤小鼠尾静脉注射5×10 5个经体外活化扩增的OT-1小鼠CD8 +T淋巴细胞。
(6)T淋巴细胞抗肿瘤功能检测
将小鼠皮下肿瘤的长×宽 2作为肿瘤的体积，持续对肿瘤大小进行观察和测量。利用流式细胞术检测OT-1小鼠CD8 +T淋巴细胞表面标志的表达水平。
(7)铁电纳米复合膜材料对T细胞体外杀伤能力检测
获取OT-1小鼠CD8 +T淋巴，细胞铺于不同带电量铁电纳米复合膜上培养，淋巴细胞浓度为2×10 6/ml，利用2μg/ml OVA peptide诱导CD8 +T淋巴细胞活化2天。将活化后的CD8 +T淋巴细胞按照1:1、0.5:1、0.25:1(T细胞：肿瘤细胞)的比例加入肿瘤细胞中，24小时后检测肿瘤细胞存活。
比较例
BaTiO 3/P(VDF-TrFE)铁电纳米复合膜，于55℃恒温的加热平台上烘干后不极化，其他步骤与实施例1相同。
测试例
铁电纳米复合膜材料压电常数d 33结果如图2所示，其中NC代表不带电，LC+代表低电量，正电(退火温度55温，极化电压21kV)，LC-代表低电量，负电(退火温度55温，极化电压21kV)，MC-代表中电量，负电(退火温度90温，极化电压21kV)，HC-代表高电量，负电(退火温度120度，极化电压21.5kV)。此外，经测定，比较例中制备的铁电纳米复合膜材料压电常 数d 33分别为-4.678±0.2525(LC+)，7.322±0.1637(LC-)，10.27±0.3297(MC-)，17.27±0.2864(HC-)。其中，低电量是指4-8pC/N，中电量指9-12pC/N，高电量15-20pC/N。
在极化的铁电纳米复合膜材料表面培养过的T淋巴细胞给小鼠回输后，抗肿瘤能力更强，表现为肿瘤体积更小，肿瘤生长速度更慢(图3)。相较于极化的铁电纳米复合膜材料，在未极化的铁电纳米复合膜材料表面培养过的T淋巴细胞给小鼠回输后，抗肿瘤能力更弱，表现为肿瘤体积更大，肿瘤生长速度更快(图3)。利用流式细胞术检测OT-1小鼠CD8 +T淋巴细胞表面标志的表达水平，结果显示在极化的铁电纳米复合膜材料表面培养的CD8 +T淋巴与存活相关的指标CD25、活性氧代谢相关指标DCFDA有所升高。相较于极化的铁电纳米复合膜材料，未极化的铁电纳米复合膜材料表面培养的CD8+T淋巴与存活相关的指标CD25、活性氧代谢相关指标DCFDA更低(图4)。
如图5所示，在铁电纳米复合膜材料对T细胞体外杀伤能力检测实验中，获取OT-1小鼠CD8 +T淋巴细胞铺于不同带电量铁电纳米复合膜上培养，诱导CD8 +T淋巴细胞活化。将活化后的CD8 +T淋巴细胞按照不同T细胞：肿瘤细胞比例加入肿瘤细胞中，24小时肿瘤细胞存活结果可以发现，CD8 +T淋巴细胞铺于铁电纳米复合膜携带负电一侧时，具有显著抑制肿瘤细胞存活，另外也观察到这种抑制效果呈现带电量以及剂量依赖性趋势，在一方面，当T细胞：肿瘤细胞比例接近1:1时，肿瘤细胞存活的抑制效果愈加明显。在另一方面，随着带电量增加或压电常数的增加，肿瘤细胞存活的抑制效果愈加明显。
尽管本发明已经参考示例性实施方案进行了描述，但应理解本发明不限于公开的示例性实施方案。在不背离本发明的范围或精神的情况下，可对本发明说明书的示例性实施方案做多种调整或变化。权利要求的范围应基于最宽的解释以涵盖所有修改和等同结构与功能。

Claims (10)

  1. 一种增强T淋巴细胞功能的方法，其特征在于，包括使T淋巴细胞在物理电刺激条件下进行培养的步骤。
  2. 根据权利要求1所述的增强T淋巴细胞功能的方法，其特征在于，物理电刺激通过带电基质、导电基质、直流电、交流电、脉冲电、磁电或铁电活性材料产生，且所述铁电活性材料的压电常数为1-40pC/N。
  3. 根据权利要求2所述的增强T淋巴细胞功能的方法，其特征在于，所述铁电活性材料包括有机铁电聚合物，其包括聚酯类、聚偏氟乙烯、聚偏氟-三氟乙烯、聚偏氟乙烯-六氟丙烯、聚偏氟乙烯-四氟乙烯、聚甲基丙烯酸甲酯和聚二甲基硅氧烷中的至少一种或其组合。
  4. 根据权利要求2或3所述的增强T淋巴细胞功能的方法，其特征在于，所述铁电活性材料包括无机铁电颗粒，其包括钛酸钡、钛酸锶钡、铌酸锂、铌酸钾钠和羟基磷灰石中的至少一种或其组合。
  5. 根据权利要求4所述的增强T淋巴细胞功能的方法，其特征在于，所述无机铁电颗粒表面包覆多巴胺层。
  6. 根据权利要求1所述的增强T淋巴细胞功能的方法，其特征在于，增强T淋巴细胞功能包括选自下述至少之一：
    a.存活时间更长；
    b.增殖性增强；
    c.抗肿瘤活性提高；
    d.CD25和/或DCFDA表达升高。
  7. 铁电活性材料在制备专用于增强T淋巴细胞功能的培养基材中的用途。
  8. 一种功能增强的T淋巴细胞，其特征在于，其通过根据权利要求1-7任一项所述的方法制备得到。
  9. 根据权利要求8所述的功能增强的T淋巴细胞，其中，所述T淋巴细胞包括效应T细胞、细胞毒性T细胞、初始T细胞及修饰的T淋巴细胞， 其中所述修饰的T淋巴细胞包括CAR-T细胞。
  10. 药物组合物，其特征在于，包括权利要求8或9所述的功能增强的T淋巴细胞。
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