Nouritrophoblast cell as well as preparation method and application thereof

Abstract

The invention provides a trophoblast cell as well as a preparation method and application thereof, and belongs to the technical field of biology. Effective molecules are expressed on the surfaces of the trophoblast cells, and the effector molecules comprise CD86 and 4-1BBL. By expressing a plurality of effector molecules on the surfaces of trophoblast cells, an effective costimulatory signal can be provided for amplification of tumor infiltrating lymphocytes (TILs), the activation and proliferation capacities of the TILs are remarkably enhanced, and particularly, the survival and function of effector T cells (such as CD8 + T cells) are promoted, so that the tumor targeting and killing efficiency of the amplified TILs are improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically, to a trophoblast cell, a preparation method thereof, and an application thereof. Background Art

[0002] Tumor-infiltrating lymphocytes (TILs) are infiltrating lymphocytes isolated from tumor tissues. They come from inside the tumor tissues, have stronger specific antigen targeting and recognition and killing capabilities for the tumor, and are not easily off-target. In recent years, TIL therapy has been used as an effective and safe method in early clinical experiments of solid tumors. According to the expression of CD8 and CD4 molecules on the surface of TILs, they are roughly classified, and their main subsets are CD8+TIL and CD4+TIL. CD8+T lymphocytes, also known as cytotoxic T lymphocytes (CTLs), as the main effector cells in the tumor-infiltrating T cell subset, have the ability to directly target and destroy tumor cells by binding to human leukocyte antigen (HLA) class I molecules, and are a stronger biomarker than CD3+.

[0003] The core of TIL therapy is to isolate tumor-specific T cells from the patient's tumor tissue, expand them in vitro, and then reinfuse them into the patient's body. However, since the TIL expansion process requires a certain trophic environment, its process stability and expansion efficiency are significantly limited. Traditional TIL expansion methods usually use peripheral blood mononuclear cells (PBMCs) from healthy donors as trophoblast cells to support TIL expansion by secreting cytokines and surface co-stimulatory molecules. However, donor-derived PBMCs have problems such as strong heterogeneity, large batch-to-batch differences, and unstable expansion efficiency, and it is difficult to meet the requirements of efficient and safe expansion. Summary of the Invention

[0004] This application aims to solve at least one of the existing problems. To this end, this application provides a trophoblast cell capable of efficiently and stably expanding tumor-infiltrating lymphocytes.

[0005] This application also provides a preparation method and an application of the foregoing trophoblast cell.

[0006] Specifically, this application provides the following technical solutions:

[0007] In the first aspect of the present application, a trophoblast cell is proposed. An effector molecule is expressed on the cell surface of the trophoblast cell, and the effector molecule includes CD86 and 4-1BBL. By expressing multiple effector molecules on the surface of trophoblast cells, it is possible to provide an effective co-stimulatory signal for the expansion of tumor-infiltrating lymphocytes (TILs), significantly enhancing the activation and proliferation ability of TILs. In particular, it has a promoting effect on the survival and function of effector T cells (such as CD8+ T cells), thereby improving the tumor targeting and killing efficiency of TILs after expansion.

[0008] In some examples of the present application, the foregoing trophoblast cell may further include at least one of the following additional technical features:

[0009] In some examples of the present application, the foregoing effector molecule includes at least one selected from the following: i) CD64; ii) mIL-21; iii) mIL-15 (WT-RA) or mIL-15 (Mu-RA); wherein, mIL-15 (WT-RA) is a chimeric protein in which IL-15 is linked to wild-type IL-15RA, and mIL-15 (Mu-RA) is a chimeric protein in which IL-15 is linked to mutant IL-15RA; mIL-21 is a chimeric protein in which IL-21 is linked to the transmembrane region of CD8a. By expressing multiple effector molecules on the surface of trophoblast cells, the proliferation ability of TILs is effectively enhanced.

[0010] The inventors found that the expression of specific effector factors on the surface of trophoblast cells can significantly improve the proliferation ability of tumor-infiltrating lymphocytes (TILs). Through research, the inventors determined several combinations of effector molecules that can effectively promote the proliferation of TILs and increase the proportion of CD8-positive cells in TILs.

[0011] In some examples of the present application, the effector molecules CD86, 4-1BBL, and CD64 are expressed on the cell surface of the trophoblast cell.

[0012] In some examples of the present application, the trophoblast cell expresses the effector molecules CD86, 4-1BBL, CD64, and mIL-21.

[0013] In some examples of the present application, the trophoblast cell expresses the effector molecules CD86, 4-1BBL, CD64, and mIL-15 (WT-Ra).

[0014] In some examples of the present application, the trophoblast cell expresses the effector molecules CD86, 4-1BBL, CD64, and mIL-15 (Mu-Ra).

[0015] In some examples of the present application, the trophoblast cells express effector molecules CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (WT-Ra).

[0016] In some examples of the present application, the trophoblast cells express effector molecules CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (Mu-Ra).

[0017] In the above combination of effector molecules, the inventors verified through experiments that when three effector molecules, CD86, 4-1BBL, and CD64, are simultaneously expressed on the surface of trophoblast cells, the amplification of tumor-infiltrating lymphocytes (TILs) is dominant. Using such trophoblast cells for TILs amplification can not only stably and efficiently obtain a sufficient amount of tumor-infiltrating lymphocytes, but also increase the proportion of CD8-positive cells in tumor-infiltrating lymphocytes.

[0018] In some examples of the present application, the trophoblast cells are derived from K562 cells.

[0019] In some examples of the present application, the sequence of the effector molecule CD86 is as shown in SEQ ID NO:1.

[0020] In some examples of the present application, the sequence of the effector molecule 4-1BBL is as shown in SEQ ID NO:2.

[0021] In some examples of the present application, the sequence of the effector molecule CD64 is as shown in SEQ ID NO:3.

[0022] In some examples of the present application, the sequence of the effector molecule mIL-21 is as shown in SEQ ID NO:4.

[0023] In some examples of the present application, the sequence of the effector molecule mIL-15 (WT-RA) is as shown in SEQ ID NO:5.

[0024] In some examples of the present application, the sequence of the effector molecule mIL-15 (Mu-RA) is as shown in SEQ ID NO:6.

[0025] In the second aspect of the present application, the present application provides a method for preparing the trophoblast cells according to any example of the first aspect. According to the embodiments of the present application, the method includes: stably expressing effector molecules in a cell line, the effector molecules including: CD86 and 4-1BBL.

[0026] In some examples of the present application, the foregoing method may further include at least one of the following additional technical features:

[0027] In some examples of the present application, the aforementioned effector molecules include at least one selected from the following: i) CD64; ii) mIL-21; iii) mIL-15(WT-RA) or mIL-15(Mu-RA); wherein, mIL-15(WT-RA) is a chimeric protein in which IL-15 is linked to wild-type IL-15RA, and mIL-15(Mu-RA) is a chimeric protein in which IL-15 is linked to mutant IL-15RA; mIL-21 is a chimeric protein in which IL-21 is linked to the transmembrane region of CD8a.

[0028] The inventors verified the ability of the following combinations of effector molecules shown in 1)-7) to promote the proliferation of TILs respectively.

[0029] 1) CD86 and 4-1BBL;

[0030] 2) CD86, 4-1BBL and CD64;

[0031] 3) CD86, 4-1BBL, CD64 and mIL-21;

[0032] 4) CD86, 4-1BBL, CD64 and mIL-15(WT-Ra);

[0033] 5) CD86, 4-1BBL, CD64 and mIL-15(Mu-Ra);

[0034] 6) CD86, 4-1BBL, CD64, mIL-21 and mIL-15(WT-Ra);

[0035] 7) CD86, 4-1BBL, CD64, mIL-21 and mIL-15(Mu-Ra).

[0036] In the above combinations of effector molecules, the inventors found through experimental verification that when three effector molecules, CD86, 4-1BBL and CD64, are simultaneously expressed on the surface of trophoblast cells, the amplification of tumor-infiltrating lymphocytes (TILs) is dominant. Using this as a trophoblast cell for TILs amplification can not only stably and efficiently obtain a sufficient amount of tumor-infiltrating lymphocytes, but also increase the proportion of CD8-positive cells in tumor-infiltrating lymphocytes.

[0037] In some examples of the present application, the effector molecule CD86 sequence is as shown in SEQ ID NO:1.

[0038] In some examples of the present application, the effector molecule 4-1BBL sequence is as shown in SEQ ID NO:2.

[0039] In some examples of the present application, the effector molecule CD64 sequence is as shown in SEQ ID NO:3.

[0040] In some examples of the present application, the effector molecule mIL-21 sequence is as shown in SEQ ID NO:4.

[0041] In some examples of the present application, the effector molecule mIL-15(WT-RA) sequence is as shown in SEQ ID NO:5.

[0042] In some examples of the present application, the effector molecule mIL-15(Mu-RA) sequence is as shown in SEQ ID NO:6.

[0043] In some examples of the present application, the cell line is derived from K562 cells.

[0044] In some examples of the present application, the stable expression of effector molecules in animal cell lines includes: S1. Constructing a lentiviral vector; S2. Co-transfecting the lentiviral recombinant vector and the helper plasmid in step S1 into 293T cells to package the corresponding lentivirus; S3. Infecting K562 cells with the lentivirus in step S2, and centrifuging and collecting the cells on the 1st - 3rd day after infection for phenotypic analysis; S4. Taking the infected K562 cells after centrifugation in step S3, performing flow cytometry detection and sorting, sorting out K562 cells with high expression of effector molecules, and sorting them into 96-well plates for monoclonal screening; S5. After monoclonal selection, monoclonal cells with an effector molecule expression level > 95% are selected for amplification culture to obtain K562 stable transfection strain cells of the effector molecule, and inactivating them to obtain feeder cells that can be used to amplify tumor-infiltrating lymphocytes.

[0045] In some examples of the present application, step S1 includes: connecting CD86 and 4-1BBL through a cleavage peptide to obtain a chimeric sequence; connecting IL-15 and IL-15RA to obtain a chimeric gene mIL-15; connecting IL-21 and the CD8a transmembrane region to obtain a chimeric gene mIL-21; constructing the chimeric gene co-expressing CD86 and 4-1BBL into the same lentiviral vector, and constructing the corresponding lentiviral vectors for the remaining effector molecules respectively. In step S1, the cleavage peptide is selected from P2A or T2A. The P2A or T2A sequence is a conventional sequence used in biological experiments in the art and will not be elaborated here.

[0046] In some examples of the present application, in step S1, the foreign gene promoter is selected from CMV, and its sequence is as shown in SEQ ID NO:7.

[0047] In some examples of the present application, the preparation method of the lentivirus in step S2 is to resuscitate and passage 293T cells to make the cell growth confluence reach 80%-90%. Mix the two lentiviral vectors prepared in step S1 with the lentiviral packaging auxiliary plasmid and PEI to form a PEI-plasmid mixture. Transfect the above PEI-plasmid mixture into 293T cells, and collect the virus supernatants at 36-48 h and 60-84 h respectively.

[0048] In some examples of the present application, the inactivation treatment is irradiation.

[0049] In some examples of the present application, the irradiation intensity is 140 Gy - 160 Gy, optionally 100 Gy, 110 Gy, 120 Gy, 130 Gy, 140 Gy, 150 Gy or 160 Gy. In some preferred examples of the present application, the aforementioned irradiation intensity is 100 Gy, 120 Gy or 150 Gy. In some more preferred examples of the present application, the aforementioned irradiation intensity is 150 Gy.

[0050] The above preparation method of trophoblast cells has the advantages of being simple, easy to operate, strong operability, stable production conditions, etc., and the preparation cost is relatively low, and batch production can be achieved.

[0051] In the third aspect of the present application, the present application proposes a method for amplifying tumor-infiltrating lymphocytes. According to the embodiments of the present application, the method includes: amplifying trophoblast cells and tumor-infiltrating lymphocytes according to a predetermined quantitative ratio; wherein, the trophoblast cells are as shown in any example of the first aspect or are prepared by the method of any example of the second aspect. This method can be used to stably and efficiently amplify tumor-infiltrating lymphocytes.

[0052] In some examples of the present application, the aforementioned method further includes at least one of the following additional technical features:

[0053] In some examples of the present application, the combination of effector molecules expressed by the trophoblast cells is selected from any one of 1)-7):

[0054] 1) CD86 and 4-1BBL;

[0055] 2) CD86, 4-1BBL and CD64;

[0056] 3) CD86, 4-1BBL, CD64 and mIL-21;

[0057] 4) CD86, 4-1BBL, CD64 and mIL-15(WT-Ra);

[0058] 5) CD86, 4-1BBL, CD64 and mIL-15(Mu-Ra);

[0059] 6) CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (WT-Ra);

[0060] 7) CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (Mu-Ra).

[0061] The aforementioned combination of effector molecules can effectively improve the proliferation ability of tumor-infiltrating lymphocytes.

[0062] In some preferred examples of the present application, the aforementioned combination of effector molecules is selected from 2) CD86, 4-1BBL, and CD64. Compared with other combinations of effector molecules, when using the combination of effector molecules 2) for the expansion of tumor-infiltrating lymphocytes, the expansion effect is better, the positive proportion of CD8 cells is higher, and the tumor cell killing effect is better.

[0063] In some examples of the present application, the aforementioned predetermined quantity ratio is selected from 1:(10 - 200), optionally 1:10, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, or 1:200. In some preferred examples of the present application, the aforementioned predetermined quantity ratio is 1:50.

[0064] In some examples of the present application, the aforementioned amplification treatment includes: separating tumor-infiltrating lymphocytes; mixing and culturing trophoblast cells with tumor-infiltrating lymphocytes according to the aforementioned predetermined quantity ratio.

[0065] In some examples of the present application, the aforementioned mixing culture time is selected from 10d - 14d. By mixing and culturing for 7d using the aforementioned culture method, the number of tumor-infiltrating lymphocytes is increased by at least 50 times; by mixing and culturing for 12d - 14d, the number of tumor-infiltrating lymphocytes is increased by at least 500 - 1500 times. Compared with the existing amplification methods, the amplification method of the present application can significantly increase the number of tumor-infiltrating lymphocytes.

[0066] In the fourth aspect of the present application, the present application proposes a kit for amplifying tumor-infiltrating lymphocytes. According to the embodiments of the present application, the kit includes: trophoblast cells of any example in the first aspect. This kit has portability and can be used for efficient temperature amplification of tumor-infiltrating lymphocytes, which is convenient for clinical application.

[0067] In the fifth aspect of the present application, a pharmaceutical composition is proposed. According to an embodiment of the present application, the pharmaceutical composition includes: trophoblast cells of any example in the first aspect. The pharmaceutical composition containing trophoblast cells can be used to increase the number of TILs in a patient's body and help improve the immune system's response ability to tumors.

[0068] In some examples of the present application, the aforementioned pharmaceutical composition may further include at least one of the following additional technical features:

[0069] In some examples of the present application, the aforementioned pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0070] In some examples of the present application, the excipient includes: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0071] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space, as long as they can act together to achieve the purpose of the present application. For example, the components contained in the composition can be administered to the subject as a whole, or separately. When the components contained in the composition are administered to the subject separately, the individual components can be administered to the subject simultaneously or sequentially.

[0072] According to an embodiment of the present application, the pharmaceutical composition of the present application contains a safe and effective amount of the active ingredient of the present application and a pharmaceutically acceptable excipient.

[0073] The effective amount of the active ingredient described in the present application may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment situation, several separate doses can be administered daily, or the dose can be proportionally reduced.

[0074] In the sixth aspect of the present application, the use of trophoblast cells of any example in the first aspect in the preparation of a drug is proposed, and the aforementioned drug is used for the prevention and / or treatment of tumors. The drug prepared based on trophoblast cells can be effectively used for the prevention and / or treatment of tumors.

[0075] In some examples of the present application, the aforementioned use may further include at least one of the following additional technical features:

[0076] In some examples of the present application, the tumor includes: at least one of melanoma, lung cancer, gastric cancer, pancreatic cancer or ovarian cancer.

[0077] Additional aspects and advantages of the present application will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0079] Figure 1 is a schematic diagram of the serum-free domestication of K562 as trophoblast cells provided by an embodiment of the present application;

[0080] Figure 2 is a schematic structural diagram of K562 trophoblast cells expressing CD86, CD64, 4-1BBL, mIL-15, and mIL-21 provided by an embodiment of the present application;

[0081] Figure 3 is a schematic flow cytometry detection diagram of trophoblast cells expressing CD86, 4-1BBL, CD64, mIL21, and mIL15 provided by an embodiment of the present application;

[0082] Figure 4 is a schematic diagram of the survival rate curve of trophoblast cells after irradiation provided by an embodiment of the present application;

[0083] Figure 5 is a schematic diagram of the amplification comparison curve of different ratios of TILs cells:K562 trophoblast cells provided by an embodiment of the present application;

[0084] Figure 6 is a schematic diagram of the amplification curve of the second cell population of tumor infiltrating lymphocytes (TILs) by trophoblast cells provided by an embodiment of the present application;

[0085] Figure 7 is a schematic diagram of the total cell number of the CD8+ phenotype of TILs amplified by trophoblast cells and control cells provided by an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0086] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts fall within the scope of protection of the present application.

[0087] In this text, unless otherwise specified, the terms "first", "second", etc. in the description, claims, and the above-mentioned drawings of this application are used to distinguish similar objects, and do not necessarily need to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances, so that the embodiments of this application described here can be implemented in an order other than those illustrated or described here. In this application, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or server that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices. In the description of this application, unless otherwise stated, "a plurality" means two or more than two.

[0088] The sequences in this application are shown in Table 1.

[0089] Table 1

[0090]

[0091]

[0092]

[0093] The solution of this application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate this application and should not be regarded as limiting the scope of this application. For those not specified in the embodiments in terms of specific technology or conditions, they shall be carried out according to the technology or conditions described in the literature in this field or according to the product manual. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0094] Example 1: Preparation of trophoblast cells

[0095] In this example, the preparation of trophoblast cells is exemplarily demonstrated through the K562 cell line. The specific steps are as follows:

[0096] (1) Construction of the vector plasmid

[0097] The gene amino acid sequence information used in this example is shown in Table 1.

[0098] The chimeric sequence was obtained by linking CD86 and 4-1BBL through the P2A cleavage peptide; the recombinant sequence mIL-15 was obtained by linking IL-15 to the transmembrane region of IL-15RA through the (G4S)5 linker; the chimeric gene mIL-21 was obtained by linking IL-21 to the transmembrane region of CD8a; the chimeric gene expressing CD86 and 4-1BBL was constructed into the same lentiviral vector; CD64 was constructed into a lentiviral vector; the recombinant sequence mIL-15 was constructed into a lentiviral vector; meanwhile, the recombinant sequence mIL-21 was constructed into a lentiviral vector.

[0099] (2) Preparation of recombinant lentivirus

[0100] Resuscitate 293T cells and passage them to make the cell growth confluence reach 80%-90%. Mix the corresponding lentiviral vectors prepared above with lentiviral packaging helper plasmids and PEI to make a PEI-plasmid mixture. Transfect the 293T cells with the above PEI-plasmid mixture, and collect the virus supernatants at 36-48 h and 60-84 h respectively.

[0101] (3) Serum-free acclimation of K562 and infection of K562 cells with recombinant lentivirus, and sorting to obtain target cells for monoclonal

[0102] After resuscitation, K562 cells were cultured in IMDM complete medium with 10% SR (serum replacement), passaged every 2-3 days, changed to IMDM complete medium with 7.5% SR on the 9th day, changed to IMDM complete medium with 5% SR after 10 days of culture, and changed to IMDM complete medium with 2% SR on the 31st day and cultured continuously. During this period, the viability was detected during each passage. The viability tended to be stable under the culture conditions of IMDM complete medium with 2% SR, and there was no obvious difference from the culture conditions of IMDM complete medium with 10% FBS. The change in viability during the whole process is as Figure 1 shown.

[0103] Before transduction with recombinant lentivirus, preheat the centrifuge to 32 °C, collect K562 cells into a 15 mL centrifuge tube, centrifuge at 500 g for 3 min, discard the supernatant, add an appropriate amount of IMDM complete medium to resuspend and count, adjust the cell density to 1×10 6 cells / mL, inoculate the above cells into 2 wells of a 6-well plate, 5 mL / well (5×10 6cells / well). Add 50 μL of the virus stock solution of the corresponding effector molecule, and leave another well untransduced as a control group. Mix the virus stock solution and cell culture medium, then place it in an incubator and continue culturing until 48 h later. Observe the cell status under a microscope, centrifuge at 500 g for 3 min to collect the cells into a centrifuge tube, discard the supernatant, add the flow cytometry antibody of the corresponding transduction molecule for detection and flow sorting. Seed the sorted positive cells directly into a 96-well plate as single cells, and continue culturing in the incubator using complete medium. After about 7 - 14 days, perform flow cytometry on the cells with successful monoclonal growth in the 96-well plate, and select the cells with a transduction molecule positive rate > 95% as the stable transfected strain of this effector molecule, and expand the culture and cryopreserve in batches.

[0104] The stable transfected strains obtained after flow sorting of monoclonal cells can obtain trophoblast cells with high expression of CD86, 4-1BBL, CD64, mIL-21, and mIL-15. The schematic diagram is as Figure 2 shown, and the constructed different effector factor combinations are:

[0105] 1) CD86 and 4-1BBL;

[0106] 2) CD86, 4-1BBL, and CD64;

[0107] 3) CD86, 4-1BBL, CD64, and mIL-21;

[0108] 4) CD86, 4-1BBL, CD64, and mIL-15 (WT-Ra);

[0109] 5) CD86, 4-1BBL, CD64, and mIL-15 (Mu-Ra);

[0110] 6) CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (WT-Ra);

[0111] 7) CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (Mu-Ra).

[0112] The above combinations were all successfully constructed under the same preparation method. Flow cytometry was used to detect the expression of CD86, 4-1BBL, CD64, mIL21, and mIL15 in trophoblast cells, and the results are as Figure 3 shown. All five effector proteins can be highly expressed on the cell membrane surface.

[0113] (4) Irradiation inactivation of sorted trophoblast cells

[0114] After centrifuging and collecting the trophoblast cells after monoclonal screening and culturing in large quantities, resuspend them with IMDM complete medium to adjust to 5×10 7The density of cells / mL, a total of 10 mL was placed in a T25 culture flask for irradiation treatment. Irradiation intensity: 100 Gy, 120 Gy, and 150 Gy, and the irradiation time was about 12 - 25 minutes. After irradiation, 5×10 6 cells were placed in the corresponding medium for culture to detect the change in their viability as Figure 4 shown. At the irradiation intensity of 150 Gy, the viability of K562 cells was the lowest, and the remaining cells were irradiated, centrifuged, and cryopreserved.

[0115] Example 2: Expansion of tumor - infiltrating lymphocytes with different ratios of trophoblast cells

[0116] (1) Preparation of the first cell population of tumor - infiltrating lymphocytes: The obtained patient tissue was cut into tumor tissue pieces of 1 - 3 mm 3 in size, one piece per well was placed in a 24 - well plate and 2 mL of medium was added for culture. After 12 - 14 days of culture, the first cell population was harvested and cryopreserved for rapid expansion in the second stage.

[0117] (2) REP stage: According to different ratios of TILs cells:K562 trophoblast cells of 1:10, 1:50, 1:100, 1:150, the corresponding number of K562 trophoblast cells was added. The trophoblast cells were

[0118] K562 - CD86 / 4 - 1BBL. The ratio of TILs cells:PBMC trophoblast cells was 1:200. The two types of cells were mixed and cultured for 12 - 14 days, and then harvested and counted as Figure 5 shown. When the ratio of TILs cells:K562 trophoblast cells was 1:50, it was more beneficial for the expansion of TILs cells.

[0119] (3) The number of the above - mentioned TIL cells was 2000, and the number of the K562 - CD86 / 4 - 1BBL trophoblast cells was 2×10 4 ~2×10 5 cells.

[0120] Example 3: Influence of different effector factor combinations on the expansion of tumor - infiltrating lymphocytes

[0121] (1) Preparation of the first cell population of tumor - infiltrating lymphocytes: The obtained patient tissue was cut into tumor tissue pieces of 1 - 3 mm 3 in size, one piece per well was placed in a 24 - well plate and 2 mL of medium was added for culture. After 12 - 14 days of culture, it was harvested and cryopreserved for rapid expansion in the second stage.

[0122] (2) REP stage: Add K562 feeder cells with different effector molecule combinations at a ratio of TIL cells:K562 feeder cells of 1:50, which are: A: K562-WT; B: CD86 and 4-1BBL; C: CD86, 4-1BBL and CD64; D: CD86, 4-1BBL, CD64 and mIL-21; E: CD86, 4-1BBL, CD64 and mIL-15(WT-Ra); F: CD86, 4-1BBL, CD64 and mIL-15(Mu-Ra); G: CD86, 4-1BBL, CD64, mIL-21 and mIL-15(WT-Ra); H: CD86, 4-1BBL, CD64, mIL-21 and mIL-15(Mu-Ra); I: PBMC. At the same time, use PBMC feeder layer as the control group. After culturing for 12 - 14 days, perform counting harvest and flow cytometry detection, and the results are as Figure 6 and Figure 7 shown. The proportion of CD8-positive cells in each combination is higher than that of the control group. Among them, the combination C has the highest proportion of CD8-positive cells.

[0123] (3) The number of the above TIL cells is 1×10 5 ~1×10 6 cells, and the number of K562 feeder cells is 5×10 6 ~5×10 7 cells.

[0124] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0125] Although the embodiments of this application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting this application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this application without departing from the principle and purpose of this application.

Claims

1. A trophoblast cell, characterized in that: The cell surfaces of the trophoblast cells express effector molecules, which include CD86 and 4-1BBL.

2. The trophoblast cell according to claim 1, characterized in that The effector molecule comprises at least one selected from the following: i) CD64; ii) mIL-21; iii)mIL-15(WT-RA) or mIL-15(Mu-RA); Among them, mIL-15(WT-RA) is a chimeric protein of IL-15 connected to wild-type IL-15RA, mIL-15(Mu-RA) is a chimeric protein of IL-15 connected to mutant IL-15RA; mIL-21 is a chimeric protein of IL-21 connected to the transmembrane region of CD8a.

3. The trophoblast cell according to claim 2, characterized in that The cell surfaces of the trophoblast cells express effector molecules CD86, 4-1BBL and CD64; Optionally, the trophoblast cells express effector molecules CD86, 4-1BBL, CD64 and mIL-21; Optionally, the trophoblast cells express effector molecules CD86, 4-1BBL, CD64 and mIL-15 (WT-Ra); Optionally, the trophoblast cells express effector molecules CD86, 4-1BBL, CD64 and mIL-15 (Mu-Ra); Optionally, the trophoblast cells express effector molecules CD86, 4-1BBL, CD64, mIL-21 and mIL-15 (WT-Ra); Optionally, the trophoblast cells express effector molecules CD86, 4-1BBL, CD64, mIL-21 and mIL-15 (Mu-Ra).

4. The trophoblast cell according to any one of claims 1 to 3, characterized in that The trophoblast cells are derived from K562 cells; Optionally, the effector molecule CD86 sequence is as shown in SEQ ID NO: 1; Optionally, the effector molecule 4-1BBL sequence is as shown in SEQ ID NO: 2; Optionally, the effector molecule CD64 sequence is as shown in SEQ ID NO: 3; Optionally, the effector molecule mIL-21 sequence is as shown in SEQ ID NO: 4; Optionally, the effector molecule mIL-15 (WT-RA) sequence is as shown in SEQ ID NO: 5; Optionally, the effector molecule mIL-15 (Mu-RA) sequence is as shown in SEQ ID NO:

6.

5. A method for preparing the trophoblast cells according to any one of claims 1 to 4, characterized in that: include: The cell lines were made to stably express effector molecules including CD86 and 4-1BBL.

6. The method according to claim 5, characterized in that The effector molecule comprises at least one selected from the following: i) CD64; ii) mIL-21; iii)mIL-15(WT-RA) or mIL-15(Mu-RA); Among them, mIL-15(WT-RA) is a chimeric protein of IL-15 connected to wild-type IL-15RA, mIL-15(Mu-RA) is a chimeric protein of IL-15 connected to mutant IL-15RA; mIL-21 is a chimeric protein of IL-21 connected to the transmembrane region of CD8a.

7. The method according to claim 6, characterized in that The effector molecule combination is selected from any one of 1)-7): 1) CD86 and 4-1BBL; 2) CD86, 4-1BBL and CD64; 3) CD86, 4-1BBL, CD64 and mIL-21; 4) CD86, 4-1BBL, CD64, and mIL-15 (WT-Ra); 5) CD86, 4-1BBL, CD64 and mIL-15 (Mu-Ra); 6) CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (WT-Ra); 7) CD86, 4-1BBL, CD64, mIL-21 and mIL-15 (Mu-Ra); Optionally, the effector molecule CD86 sequence is as shown in SEQ ID NO: 1; Optionally, the effector molecule 4-1BBL sequence is as shown in SEQ ID NO: 2; Optionally, the effector molecule CD64 sequence is as shown in SEQ ID NO: 3; Optionally, the effector molecule mIL-21 sequence is as shown in SEQ ID NO: 4; Optionally, the effector molecule mIL-15 (WT-RA) sequence is as shown in SEQ ID NO: 5; Optionally, the effector molecule mIL-15 (Mu-RA) sequence is as shown in SEQ ID NO:

6.

8. The method according to any one of claims 5 to 7, characterized in that: The cell line is derived from K562 cells; Optionally, the step of causing the cell line to stably express the effector molecule comprises: S1. Construction of lentiviral vector; S2. Co-transfecting the lentiviral recombinant vector and the auxiliary plasmid in step S1 into 293T cells to package and obtain the corresponding lentivirus; S3. Infect K562 cells with the lentivirus in step S2, and collect the cells by centrifugation on day 1-3 after infection for phenotypic analysis; S4. Take the infected K562 cells after centrifugation in step S3, perform flow cytometry detection and sorting, sort out the K562 cells with high expression of effector molecules, and sort them into 96-well plates for monoclonal screening; S5. After monocloning, monoclonal cells with an expression level of effector molecules greater than 95% are selected for amplification and culture to obtain K562 stably transfected cells of the effector molecule, which are inactivated to obtain trophoblast cells that can be used to amplify tumor infiltrating lymphocytes; Optionally, step S1 includes: connecting CD86 and 4-1BBL by cleavage peptide to obtain a chimeric sequence; connecting IL-15 and IL-15RA to obtain a chimeric gene mIL-15; connecting IL-21 and the CD8a transmembrane region to obtain a chimeric gene mIL-21; constructing the chimeric gene co-expressing CD86 and 4-1BBL into the same lentiviral vector, and constructing corresponding lentiviral vectors for the remaining effector molecules; Optionally, in step S1, the cleavage peptide is selected from P2A or T2A; Optionally, in step S1, the exogenous gene promoter is selected from CMV; Optionally, the method for preparing the lentivirus in step S2 is to resuscitate 293T cells and passage them to make the cell growth confluence reach 80%-90%, mix the two lentiviral vectors prepared in step S1 with the lentiviral packaging helper plasmid and PEI to prepare a PEI-plasmid mixture, transfect 293T cells with the PEI-plasmid mixture, and collect the virus supernatant at 36-48h and 60-84h respectively; Optionally, the inactivation treatment is irradiation; Optionally, the irradiation intensity is 140 Gy-160 Gy, preferably 100 Gy, 120 Gy or 150 Gy, more preferably 150 Gy.

9. A method for amplifying tumor infiltrating lymphocytes, characterized in that: include: amplifying the trophoblast cells and the tumor infiltrating lymphocytes according to a predetermined quantity ratio; Wherein, the trophoblast cells are prepared as described in any one of claims 1-4 or by the method described in any one of claims 5-8.

10. The method according to claim 9, characterized in that The effector molecule combination expressed by the trophoblast cells is selected from any one of 1)-7): 1) CD86 and 4-1BBL; 2) CD86, 4-1BBL and CD64; 3) CD86, 4-1BBL, CD64 and mIL-21; 4) CD86, 4-1BBL, CD64, and mIL-15 (WT-Ra); 5) CD86, 4-1BBL, CD64 and mIL-15 (Mu-Ra); 6) CD86, 4-1BBL, CD64, mIL-21, and mIL-15 (WT-Ra); 7) CD86, 4-1BBL, CD64, mIL-21 and mIL-15 (Mu-Ra); Preferably, the effector molecule combination is selected from 2) CD86, 4-1BBL and CD64; Optionally, the predetermined quantity ratio is selected from 1:(10-200), preferably 1:

50.

11. The method according to claim 9, characterized in that The amplification process comprises: Isolation of tumor-infiltrating lymphocytes; Mixing and culturing the trophoblast cells and the tumor infiltrating lymphocytes according to the predetermined quantitative ratio; Optionally, the mixed culture time is selected from 10d-14d.

12. The method according to claim 11, characterized in that After 7 days of mixed culture, the number of tumor infiltrating lymphocytes increased by at least 50 times; after 12 days to 14 days of mixed culture, the number of tumor infiltrating lymphocytes increased by at least 500-1500 times.

13. A kit for amplifying tumor infiltrating lymphocytes, characterized in that: include: The trophoblast cell according to any one of claims 1 to 4.

14. A pharmaceutical composition, characterized in that include: The trophoblast cell according to any one of claims 1 to 4; Optionally, a pharmaceutically acceptable excipient is further included.

15. Use of the trophoblast cells according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating tumors; Optionally, the tumor comprises: At least one of melanoma, lung cancer, gastric cancer, pancreatic cancer or ovarian cancer.

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