Method and kit for improving lentivirus transduction efficiency and transduction stability of TIL cells

Abstract

The invention relates to a method and a kit for improving lentivirus transduction efficiency and transduction stability of TIL cells. The invention relates to the technical field of biology. Specifically, the invention provides a method and a kit for promoting the lentivirus transduction efficiency and the transduction stability of TIL cells, BX-795 is added in the lentivirus infection process, the transduction efficiency can be improved to 60% or above, the transduction stability can be remarkably improved, and the viability of the TIL cells is not obviously influenced. The method for improving the lentivirus transduction efficiency and transduction stability of the TIL cells can be applied to obtaining genetically engineered TIL cells and promoting development and application of TIL cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and a kit for improving the lentiviral transduction efficiency and transduction stability of TIL cells. Background Art

[0002] Tumor infiltrating lymphocyte (TIL) therapy refers to the separation and extraction of immune cells from the patient's own tumor tissue, which are then cultured and amplified in vitro and then reinfused into the patient's body to attack and destroy tumor cells. TIL cells cultured and amplified in vitro have the advantages of stronger killing function, multiple targets, strong tumor tropism and infiltration ability, and few side effects, and thus show great application potential and value in tumor immunotherapy.

[0003] In clinical applications, the immunosuppressive tumor microenvironment often seriously weakens the full efficacy of the infused TIL cells in vivo. To solve this problem, gene modification or gene editing technology is often used to transform TIL cells to further improve their resistance to immunosuppression in the tumor microenvironment and enhance their efficacy against tumors. At present, in the research of immune cell therapy products represented by CAR-T cells, commonly used viral vectors include lentivirus and retroviral vectors, among which lentiviral vectors are widely used because of their better safety and technical maturity. CAR-T cells are often derived from peripheral blood T lymphocytes of patients or healthy donors, most of which are in an undifferentiated or immature state, and lentiviral vectors have a high and stable transduction efficiency for them.

[0004] However, TIL cells from tumor tissues, especially those from advanced patients, are often in a state of terminal differentiation and exhaustion, resulting in a generally low transduction efficiency of lentiviral vectors on TIL cells, which decreases with the proliferation of TIL cells, and there is a problem of unstable transduction efficiency. So far, there has been no case of efficient (e.g., transduction efficiency of 50% or more) and stable genetic modification and transformation of TIL cells using lentiviral vectors.

[0005] Therefore, there is an urgent need in the art to develop a method for efficient and stable genetic modification and transformation of TIL cells using lentiviral vectors. Summary of the invention

[0006] The purpose of the present invention is to provide a method for improving the lentiviral transduction efficiency and transduction stability of TIL cells.

[0007] In a first aspect of the present invention, a method for obtaining lentiviral transduced TIL cells is provided, comprising the steps of: adding a transduction-promoting agent to the transduction system of lentiviral transduced TIL cells, wherein the transduction-promoting agent comprises BX-795.

[0008] In another preferred embodiment, the transduction promoting agent further comprises: other protein kinase TBK1 / IKKε inhibitors except BX-795, such as Amlexanox, MRT67307, or a combination thereof.

[0009] In another preferred embodiment, the transduction promoting agent further includes: LentiBoost, Polybrene, Lentiviral Transduction Enhancer, or a combination thereof.

[0010] In another preferred embodiment, the method specifically comprises the steps of: A. Provide TIL cells and lentivirus to be transduced; B. inoculating the TIL cells to be transduced into a complete culture medium, and transducing the TIL cells with the lentivirus in the presence of a transduction-promoting agent; wherein the transduction-promoting agent includes BX-795; C. Collect transduced cells; D. Expand the cells from step C in complete expansion medium.

[0011] In another preferred embodiment, in step B, the final concentration of BX-795 in the reaction system is 1-10 μM, preferably 1.5-6 μM, such as 1.5-3 μM, or 3-6 μM.

[0012] In another preferred embodiment, in step B, the culture medium is a complete culture medium.

[0013] In another preferred embodiment, the components of the complete expansion medium include: cell basal medium, serum substitute, L-glutamine or its substitute, and IL-2.

[0014] In another preferred embodiment, the amount of IL-2 in the expansion complete medium is 100-600 IU / ml, preferably 300 IU / mL.

[0015] In another preferred embodiment, the cell basal culture medium is a culture medium suitable for mammalian cell culture.

[0016] In another preferred embodiment, the cell basal culture medium is selected from the group consisting of RPMI 1640 culture medium, AIM-V TM medium, SFM medium, X-VIVO medium, or a combination thereof.

[0017] In another preferred embodiment, the cell basal culture medium includes Advanced RPMI 1640 culture medium, CTS TMAIM-V TM Culture medium.

[0018] In another preferred embodiment, the cell basal culture medium is Advanced RPMI 1640 culture medium and CTS TMAIM-V TM culture medium 1:1 mixed with the resulting culture medium.

[0019] In another preferred embodiment, the L-glutamine substitute is Gluta Max TM .

[0020] In another preferred embodiment, the Gluta Max in the complete culture medium TM The content is 0.5-2%, preferably 1%.

[0021] In another preferred embodiment, the serum substitute is CTS TM Immune cell serum replacement.

[0022] In another preferred embodiment, the CTS in the complete culture medium TM The content of immune cell serum replacement is 1%-5%, preferably 2.5%.

[0023] In another preferred embodiment, in step A, the TIL cells to be transduced are derived from tumor tissue.

[0024] In another preferred embodiment, the tumor tissue is preferably tumor tissue from a patient with advanced tumor.

[0025] In another preferred embodiment, the patients with advanced tumors refer to patients in stage III or stage IV in TNM staging.

[0026] In another preferred embodiment, the TIL cells are TIL cells in a terminally differentiated and exhausted state.

[0027] In another preferred embodiment, the TIL cells to be transduced are TIL cells pretreated with BX-795, the concentration of the pretreated BX-795 is 1 μM, and the pretreatment time is 4 h to 16 h.

[0028] In another preferred embodiment, the TIL cells to be transduced are primary cultured TIL cells.

[0029] In another preferred embodiment, the primary culture is carried out in a primary culture medium, and the primary culture medium contains IL-2.

[0030] In another preferred embodiment, the primary culture medium further comprises the following components: TIL cell basal medium, buffer, serum or serum substitute, L-glutamine or its substitute, antibiotics, and optional TIL cell activation reagent.

[0031] In another preferred embodiment, the concentration of IL-2 is 100-6000 IU / mL.

[0032] In another preferred example, the TIL cell basal culture medium includes: RPMI 1640 culture medium, SFM culture medium, and X-VIVO culture medium.

[0033] In another preferred embodiment, the TIL cell activation reagent includes: anti-CD3 monoclonal antibody, anti-4-1BB humanized monoclonal antibody, or a combination thereof.

[0034] In another preferred embodiment, the final concentration of the anti-CD3 monoclonal antibody is 10-100 ng / mL, and the final concentration of the anti-4-1BB humanized monoclonal antibody is 1-50 μg / mL.

[0035] In another preferred embodiment, the primary culture medium comprises: RPMI 1640 medium, Hepes buffer with a final concentration of 2.5% V / V, serum replacement with a final concentration of 1% to 10% V / V, GlutaMax with a final concentration of 1% V / V and 0.1% V / V gentamicin, and 6000 IU / mL of IL-2.

[0036] In another preferred embodiment, the primary culture medium further comprises: 500-3000 IU / mL of IL-2, 10-100 ng / mL of anti-CD3 monoclonal antibody, and 1-50 μg / mL of anti-4-1BB humanized monoclonal antibody.

[0037] In another preferred embodiment, the lentivirus is VSV-G lentivirus.

[0038] In another preferred embodiment, the lentivirus is a recombinant lentivirus carrying an exogenous target gene.

[0039] In another preferred embodiment, the exogenous target gene includes: a gene encoding an element that enhances TIL cell function, and the element of TIL cell function includes: modified or unmodified exogenous cytokines, modified or unmodified exogenous membrane proteins, modified or unmodified exogenous secretory proteins, modified or unmodified exogenous antibodies.

[0040] In another preferred embodiment, the multiplicity of infection MOI of the lentivirus is 0.1-100, preferably 1-50, and more preferably 5-20.

[0041] In another preferred embodiment, in step B, the infection time is 12 to 48 hours, such as 24 to 48 hours, preferably 24 hours ± 2 hours.

[0042] In another preferred embodiment, in step C, the method for collecting cells is a method known in the art, such as centrifugation, discarding the supernatant, and collecting the cell precipitate.

[0043] In another preferred embodiment, in step D, the expansion culture is carried out in a complete expansion culture medium.

[0044] In another preferred embodiment, the complete culture medium in step D is the same as the complete culture medium in the reaction system.

[0045] In another preferred embodiment, the expansion culture is carried out in the presence of feeder cells.

[0046] In another preferred embodiment, the amplification culture is carried out in the presence of BX-795, the concentration of BX-795 is 0.1 μM, and the treatment time of BX-795 is 48 to 72 hours.

[0047] In another preferred embodiment, the feeder cells are K562 cells, preferably irradiated feeder cells.

[0048] In another preferred embodiment, the feeder cells are engineered K562 cells expressing membrane-bound IL-21 and 4-1BBL.

[0049] In another preferred embodiment, the ratio of the TIL cells to the feeder cells is 1:2.5 to 1:50.

[0050] In another preferred embodiment, the conditions for the expansion culture are 37°C ± 3°C, 5% CO 2 .

[0051] In another preferred embodiment, the expansion culture time is 5 to 12 days, preferably 7 to 10 days.

[0052] In another preferred embodiment, during the amplification culture, the medium is changed every 2-3.5 days.

[0053] In another preferred embodiment, the transduction efficiency of the cells collected in step C is E0, E0≥50%, preferably E0≥55%, and more preferably E0≥60%.

[0054] In another preferred embodiment, the transduction efficiency of the cells after expansion and culture in step D is E1, E0≥50%, preferably E0≥55%, and more preferably E0≥60%.

[0055] In another preferred embodiment, the transduction efficiency does not decrease after expansion culture, or the decrease range (E0-E1) / E0 is ≤20%, preferably ≤15%, and more preferably ≤10%.

[0056] In another preferred embodiment, the viability of the cells after expansion and culture in step D is ≥60%, preferably ≥70%, and more preferably ≥80%.

[0057] In a second aspect of the present invention, a use of BX-795 is provided for preparing a reagent, which is added to a reaction system of lentivirus-infected TIL cells to improve the lentiviral transduction efficiency and transduction stability of TIL.

[0058] In another preferred embodiment, in the reaction system, the concentration of BX-795 is 1-10 μM, preferably 1.5-6 μM, and more preferably 3-6 μM.

[0059] In another preferred embodiment, the reagent is added before, during, and / or after the reaction of lentivirus infection of TIL cells, preferably before and / or during the reaction.

[0060] In another preferred embodiment, the reagent further includes: other protein kinase TBK1 / IKKε inhibitors except BX-795, such as Amlexanox, MRT67307; and / or LentiBoost.

[0061] In another preferred embodiment, the transduction efficiency is: the ratio of TIL cells that successfully express the exogenous target gene to the total TIL cells.

[0062] In another preferred embodiment, the high transduction stability means that the transduction efficiency of the transduced TIL cells after expansion culture is almost not decreased or the decrease is ≤20%, preferably ≤15%, and more preferably ≤10% compared with the transduction efficiency before expansion culture.

[0063] In a third aspect of the present invention, a kit for lentiviral transduction of TIL cells is provided, the kit comprising: (a) a transduction-promoting reagent, wherein the transduction-promoting reagent comprises BX-795; (b) a complete expansion medium; and (c) instructions.

[0064] In another preferred embodiment, the instructions describe the method for using the kit: A. Provide TIL cells and lentivirus to be transduced; B. inoculating the TIL cells to be transduced into a complete culture medium, and transducing the TIL cells with the lentivirus in the presence of a transduction-promoting agent, wherein the transduction-promoting agent includes BX-795, and the final concentration of BX-795 in the transduction system is 1.5-6 μM; C. Collect transduced cells; D. Expand the cells from step C in complete expansion medium.

[0065] In another preferred embodiment, the complete expansion medium comprises the following components: cell basal medium, serum substitute, L-glutamine or its substitute, and IL-2.

[0066] In another preferred embodiment, the amount of IL-2 in the expansion complete medium is 100-600 IU / ml, preferably 300 IU / mL.

[0067] In another preferred embodiment, the cell basal culture medium is Advanced RPMI 1640 culture medium and CTS TMAIM-V TM culture medium 1:1 mixed with the resulting culture medium.

[0068] In another preferred embodiment, the L-glutamine substitute is Gluta Max TM , the content is 0.5-2%, preferably 1%.

[0069] In another preferred embodiment, the serum substitute is CTS TM Immune cell serum replacement, the content is 1%-5%, preferably 2.5%.

[0070] In another preferred embodiment, the transduction promoting agent further comprises: other protein kinase TBK1 / IKKε inhibitors except BX-795, such as Amlexanox, MRT67307, or a combination thereof.

[0071] In another preferred embodiment, the transduction promoting agent further includes: LentiBoost, Polybrene, Lentiviral Transduction Enhancer, or a combination thereof.

[0072] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0073] The beneficial effects of the present invention include: 1. The present invention provides a method for significantly improving the transduction efficiency of lentivirus on TIL cells, which can increase the transduction efficiency from about 20% to about 60%, an increase of up to 3 times.

[0074] 2. The method of adding BX-795 of the present invention can improve the stability of lentivirus transduction of TIL cells. Even after a 9-day expansion stage, the transduction efficiency of TIL cells can still be maintained at a high level, with no significant decrease compared to before expansion.

[0075] 3. After expansion, the TIL cells obtained by the method of adding BX-795 of the present invention recovered to a high level of cell viability, which was comparable to that of the control group without adding BX-795. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The results show the transduction efficiency of TIL cells from donor 1 after they were infected with lentivirus in the presence or absence of BX-795 before and after expansion.

[0077] Figure 2 The results show the cell viability of TIL cells from donor 1 before and after expansion after they were infected with lentivirus in the presence or absence of BX-795.

[0078] Figure 3 The results show the transduction efficiency of TIL cells from donor 2 after they were infected with lentivirus in the presence or absence of BX-795 before and after expansion.

[0079] Figure 4 The results show the cell viability test results before and after expansion of TIL cells from donor 2 after they were infected with lentivirus in the presence or absence of BX-795.

[0080] Figure 5 The results show the total number of transduction-positive cells detected after TIL cells from donor 1 were infected with lentivirus in the presence or absence of BX-795 and then expanded.

[0081] Figure 6 The results show the total number of transduction-positive cells detected after TIL cells from donor 2 were infected with lentivirus in the presence or absence of BX-795 and then expanded.

[0082] Figure 7 The figure shows the flow cytometric profiles of transduction efficiency of TIL cells from donor 1 before and after expansion after they were infected with lentivirus in the presence or absence of BX-795.

[0083] Figure 8 The figure shows the flow cytometric profiles of transduction efficiency of TIL cells from donor 2 before and after expansion after they were infected with lentivirus in the presence or absence of BX-795.

[0084] Fig. 9 The results show the transduction efficiency of TIL cells from donor 3 after they were infected with lentivirus under different concentrations of BX-795 before and after expansion. DETAILED DESCRIPTION

[0085] After extensive and in-depth research, the inventors have provided a method for promoting the lentiviral transduction efficiency and transduction stability of TIL cells, including adding BX-795 during lentiviral infection, which can increase the transduction efficiency to more than 60%, and the transduction efficiency is increased by more than 3 times compared with the control group without adding BX-795. In addition to greatly improving the transduction efficiency, the method of the present invention also significantly improves the stability of transduction, that is, the transduced TIL cells will not cause a significant decrease in transduction efficiency after the expansion culture stage.

[0086] The inventors also unexpectedly found that after the TIL cells obtained by the method of adding BX-795 of the present invention were expanded and cultured, the cell viability was improved and reached a level equivalent to that of the control group without adding BX-795. The method of the present invention efficiently promotes the lentiviral transduction of cells without affecting the cell viability and improves the transduction stability, and is particularly suitable for transduction of TIL cells in a terminally differentiated or exhausted state (e.g., TIL cells from patients with advanced cancer).

[0087] The present invention has been completed on this basis.

[0088] the term Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0089] As used herein, the term "tumor infiltrating lymphocytes" or "TIL cells" is a type of immune cell present in the tumor microenvironment. TIL cells have significant advantages in the field of cancer treatment. First, they can be directly extracted from the patient's body and re-injected into the patient's body after in vitro amplification and activation, thereby achieving personalized treatment. Secondly, since TIL cells are derived from the patient himself, there is no risk of allogeneic rejection and the safety is higher. In addition, TIL cell therapy has shown significant therapeutic effects on certain refractory tumors (such as melanoma) in clinical trials.

[0090] As used herein, the term "primary culture" is also referred to as "preliminary culture" or "pre-REP", and refers to the first stage of culturing the initial TIL cells in the tumor tissue to obtain activated TIL cells with expansion potential. In the present invention, the culture medium used in the initial culture stage is referred to as "primary culture medium" or "primary culture complete medium", and the TIL cells obtained after the completion of the initial culture are referred to as "TIL primary culture cells".

[0091] In one embodiment, the components of the primary culture complete medium used in the primary culture stage are: RPMI 1640 medium, Hepes with a final concentration of 2.5% V / V, serum replacement with a final concentration of 5% V / V, GlutaMax with a final concentration of 1% V / V and 0.1% V / V gentamicin (concentration of 10 μg / ml), anti-CD3 monoclonal antibody with a final concentration of 30 ng / mL, anti-4-1BB humanized monoclonal antibody with a final concentration of 5 μg / mL, and human interleukin-2 injection (IL-2) with a final concentration of 1000 IU / mL.

[0092] In a preferred embodiment, the components of the primary culture complete medium used in the primary culture stage are: RPMI 1640 medium, containing Hepes with a final concentration of 2.5% V / V, serum replacement with a final concentration of 5% V / V, GlutaMax with a final concentration of 1% V / V and gentamicin with a concentration of 0.1% V / V (concentration of 10 μg / ml), and human interleukin-2 injection (IL-2) with a final concentration of 6000 IU / mL.

[0093] As used herein, the terms "expansion culture", "expansion culture", "rapid expansion culture" and "REP" are used interchangeably and refer to the expansion culture of primary cultured cells to quickly obtain a large number of TIL cells.

[0094] In the present invention, the method of the expansion stage is not limited, and conventional expansion methods known in the art can be selected to rapidly expand the cells that have undergone initial culture.

[0095] A preferred expansion method is: expansion in a complete expansion medium, wherein the complete expansion medium contains the following components: IL-2, cell basal medium, serum replacement, L-glutamine or its replacement. The amount of IL-2 is 100-600 IU / ml, preferably, the amount of IL-2 is 200-400 IU / ml, and more preferably 300 IU / ml.

[0096] In a preferred embodiment, the cell basal medium is a medium suitable for mammalian cell culture, such as RPMI 1640 medium, AIM-V TM In a preferred embodiment, the cell basal culture medium of the present invention is Advanced RPMI 1640 culture medium and CTS TM AIM-V TM culture medium 1:1 mixed with the resulting culture medium.

[0097] In a preferred embodiment, the contents of other components in the expansion complete medium are as follows: 0.5-2% L-glutamine substitute, preferably the L-glutamine substitute is Gluta Max™; ​​1%-5% serum substitute, such as CTS™ immune cell serum substitute.

[0098] In the present invention, feeder cells may or may not be added during the expansion stage. Feeder cells include feeder cells commonly used in the art, especially irradiated feeder cells. Preferred feeder cells are PBMC or K562 cells, and more preferred feeder cells are engineered feeder cells, such as feeder cells expressing mbIL21 and 4-1BBL. In a preferred embodiment of the present invention, the feeder cells used are irradiated K562 cells expressing mbIL21 and 4-1BBL. When feeder cells are added, the ratio of feeder cells to TIL cells is (2.5-100):1.

[0099] The term "transduction" refers to the transfer of nucleic acid into a host cell, such as the transfer of an exogenous target gene into a host cell. As used herein, the term includes all techniques for introducing nucleic acid into a cell, including but not limited to transformation with a plasmid vector, infection with a viral vector or viral particles, and introduction of naked DNA by electroporation, nuclear transfection, lipofection, or a particle gun.

[0100] The term "pseudotype (vector)" refers to a vector particle carrying an envelope glycoprotein from other enveloped viruses. In a non-limiting embodiment, the lentiviral vector used to transduce TIL cells is a VSV-G pseudotype lentiviral vector.

[0101] The term "intrinsic antiviral activity" refers to the activity of the innate immune system of the host cell to inhibit viral replication and / or viral gene expression in the host cell. It is well known in the art that dsRNA or dsDNA checkpoints in the cytoplasm (e.g., retinoic acid-inducible gene I (RIG I), cyclic GMP AMP synthase) can recognize viral nucleic acids and trigger the host cell to enter an antiviral state by inducing a type I interferon response. Therefore, "agents capable of inhibiting intrinsic antiviral activity" refer to inhibitors that can inhibit the development of an antiviral state in the host. In a non-limiting embodiment, the agent is an inhibitor for IκB kinase (IKKε) or TANK binding kinase 1 (TBK1), such as BX795.

[0102] The term "BX-795" is a commonly used bioactive compound with the chemical name ethyl 2-amino-5-(1-ethyl-2-hydroxypropyl)-3,4-dioxazol-1-yl acetate. It is often used as an effective and relatively specific inhibitor of TBK1 and IKKε, acting downstream of RIG-I, MDA-5 and TLR3, and can effectively inhibit the signaling pathway of innate immunity against viral infection.

[0103] The term "transduction efficiency" is also called "transduction positive rate", which refers to the ratio of TIL cells that successfully express the exogenous target gene to the total TIL cells. The transduction efficiency can be detected by conventional methods in the art such as flow cytometry.

[0104] The term "transduction stability" refers to the change in transduction efficiency of infected TIL cells before and after expansion. For transduced TIL, the transduction efficiency usually decreases after expansion. When the decrease in transduction efficiency after expansion is very small (e.g., the decrease is ≤20%), the transduction stability can be considered high.

[0105] Methods for improving the efficiency of lentiviral transduction of TIL cells In the present invention, a method for improving the transduction efficiency of lentivirus on TIL cells is provided, comprising adding BX-795 to the reaction system of lentivirus-infected TIL cells.

[0106] The method of the present invention specifically includes three stages: pre-infection, infection and post-infection. Pre-infection: preparing the lentivirus and TIL cells to be transduced; during infection: performing a reaction of lentivirus infection of TIL cells in a culture medium; post-infection: harvesting the infected TIL cells and amplifying and culturing the TIL cells.

[0107] Before infection: First, it is necessary to prepare a lentiviral vector and perform preliminary culture of TIL cells. In the present invention, the lentiviral vector is a VSV-G type lentiviral vector, wherein the lentivirus carries an exogenous target gene. The preparation of the lentivirus can be carried out by any method known in the art, such as a three-plasmid packaging method, but is not limited thereto.

[0108] In the present invention, the source of TIL cells is not limited, and they are generally TIL cells isolated from tumor tissues in tumor patients. Preferably, TIL cells will be initially cultured to obtain a sufficient number of isolated TIL cells for subsequent transduction. The initial culture method of the present invention is not limited, for example, the initial culture can be performed in an initial culture medium known in the art. In a preferred embodiment, the components of the initial culture medium used in the present invention include: TIL cell basal culture medium, buffer, serum or serum substitute, L-glutamine or its substitute, antibiotics, IL-2, and optional TIL cell activation reagents.

[0109] In the present invention, the ratio of the total number of lentiviruses to the total number of TIL cells, i.e., the range of the multiplicity of infection MOI is not limited, and those skilled in the art can routinely adjust it as needed. Preferably, the MOI is 0.1-100, such as 1-50, 5-20.

[0110] Infection: The reaction of lentivirus infection of TIL cells is usually carried out in a TIL cell culture medium, to which BX-795 is added. In the present invention, the TIL cell culture medium is a culture medium that is conducive to the growth and expansion of TIL cells, and its components are not particularly limited. In a preferred embodiment, the reaction of lentivirus infection of TIL cells is carried out in a TIL cell expansion culture medium.

[0111] In a preferred embodiment, the concentration of BX-795 is 1 to 5 μM, preferably 2 to 4 μM, and more preferably 2.5 to 3.5 μM. The infection time is 12 to 48 hours, preferably 24 hours ± 2 hours. After the infection, the TIL cells are collected by centrifugation, and the transduction efficiency and cell viability, i.e., the transduction efficiency and cell viability before expansion, are detected.

[0112] Compared with the control group without adding BX-795 and with the same other conditions, the transduction efficiency of the method of the present invention is more than 3 times that of the control group, but the cell viability is reduced.

[0113] After infection: The TIL cells collected after the infection are expanded and cultured to obtain a large number of TIL cells. The method of expansion and culture of the present invention is not limited, and can be any method known in the art for TIL cell expansion and culture. The expansion and culture time is usually 7 to 12 days. The expansion and culture can be carried out under the condition of feeder cells or without feeder cells, preferably under the condition of feeder cells.

[0114] After the expansion culture is completed, the transduction efficiency and cell viability of the TIL cells are tested again, that is, the transduction efficiency and cell viability after the expansion culture. Compared with the transduction efficiency before the expansion culture, the transduction efficiency after the expansion culture hardly decreases or decreases very little, and the decrease is ≤20%, such as ≤15%, or ≤10%.

[0115] The present invention unexpectedly found that compared with a control group without BX-795 and with the same other conditions, the cell viability of the present invention after adding BX-795 and expanding the culture was equivalent to that of the control group, that is, the effect of BX-795 on cell viability was restored after expansion.

[0116] application The present invention also provides a use of BX-795 in preparing a reagent for adding to a transduction system for lentiviral transduction of TIL cells to improve the lentiviral transduction efficiency and transduction stability of TIL.

[0117] In the present invention, a culture medium for TIL cells infected with a lentivirus is further provided, wherein the culture medium is supplemented with a transduction-promoting agent, including BX-795, and optionally other agents that can promote transduction, such as other protein kinase TBK1 / IKKε inhibitors other than BX-795, such as Amlexanox, MRT67307; and / or LentiBoost.

[0118] In the present invention, BX-795 can be used to promote the infection of lentivirus to TIL cells of various sources. In the present invention, the tumor type of TIL cell source is not limited, for example, solid tumors, hematological tumors, preferably solid tumors. In a preferred embodiment, the tumor includes but is not limited to: lung cancer, melanoma, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer.

[0119] More preferably, BX-795 is particularly suitable for transduction of TIL cells derived from advanced tumor tissues, wherein the advanced stage refers to patients with clinical stage III or IV. TIL cells derived from patients with advanced tumors are usually in a terminally differentiated or exhausted state, and are difficult to transduce and have low efficiency. However, the transduction method of the present invention can significantly improve the lentiviral transduction efficiency of TIL cells derived from patients with advanced tumors.

[0120] The method of improving the transduction efficiency of lentivirus on TIL cells or the culture medium for lentivirus-infected TIL cells of the present invention can be applied to transforming TIL cells, obtaining genetically engineered TIL cells and applying them to TIL cell therapy.

[0121] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0122] Example 1: Initial culture of TIL cells Source of TIL cells: The TIL cells used in the examples are TIL cells derived from tumor tissues of tumor patients, wherein donor 1 is a patient with squamous cell carcinoma of the lung (stage III), donor 2 is a patient with melanoma (stage IV), and donor 3 is a patient with mucosal melanoma (stage III).

[0123] Initial culture complete medium: RPMI 1640 medium, supplemented with Hepes at a final concentration of 2.5% V / V, serum replacement at a final concentration of 5% V / V, GlutaMax at a final concentration of 1% V / V and gentamicin (concentration of 10 μg / ml) at 0.1% V / V, anti-CD3 monoclonal antibody at a final concentration of 30 ng / mL, anti-4-1BB humanized monoclonal antibody at a final concentration of 5 μg / mL, and human interleukin-2 injection (IL-2) at a final concentration of 1000 IU / mL.

[0124] Initial culture steps: transfer the tumor tissue to a 50 mL centrifuge tube, add tissue flushing solution to wash the tissue and soak for 1 to 5 minutes; transfer to a 10 cm 2 In the culture dish, remove the blood stains and unusable tissue on the surface, and use tissue flushing solution to rinse the tumor tissue; transfer the tissue to a 10 cm 2 In the culture dish, add tissue flushing solution to wash and soak for 1 to 5 minutes, 1 to 5 times in total; after washing, cut all sample tissues into pieces of about 1 to 3 mm 3 Take 0.02-0.1 g of tumor tissue and transfer it to a breathable culture bag or culture bottle, add 100 mL of primary culture complete medium 1; put the breathable culture bag or culture bottle into a carbon dioxide incubator (37°C, 5% CO 2 ) for 7-14 days.

[0125] Example 2: Preparation of lentiviral vector An endotoxin-free plasmid extraction kit (purchased from Qiagen) was used to prepare the expression plasmid containing the target gene required for transfection (the pLV-HRE-mIL-15 expression plasmid of the mIL-15 gene used in this example was from patent application CN117820493A).

[0126] HEK-293T cells were transfected with expression plasmids pLV-HRE-mIL-15, psPAX2 and pMD2.G. The transfection system was configured as follows: (1) Centrifuge tube A: Opti-MEM 500 μL, pLV-HRE-mIL-15 plasmid 10 μg + pMD2.G plasmid 5 μg + psPAX2 plasmid 5 μg and p3000 40 μL; (2) Centrifuge tube B: Opti-MEM 500 μL and Lipo3000 40 μL. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, gently mixed with a pipette, and allowed to stand at room temperature for 15-20 min.

[0127] Add transfection system to HEK-293T cells. After 4 h of transfection, discard the supernatant and add 10 mL of DMEM complete medium to each dish. Place in 37°C, 5% CO 2 Continue to culture in the incubator; after 48 hours, collect the culture supernatant of the transfected HEK-293T cells; centrifuge at 400g for 5 min; filter with a 0.45 μm filter, and the filtrate obtained is the original solution of the recombinant lentivirus; use an Utra 15 centrifugal filter device to concentrate the lentivirus, and centrifuge at 5000 rpm for 50 min; divide the concentrated lentivirus into aliquots and store in a refrigerator at 80°C for later use.

[0128] Example 3: Lentivirus titer determination Count the cells of HEK-293T cells after washing and digestion. Dilute the concentrated lentiviral stock solution in 10-fold gradient dilution (10×, 100×, 1000×, 10000×). Take 2.5×10 5 Each HEK-293T cell was added to the corresponding centrifuge tube, and each centrifuge tube contained 500 μL of culture medium-virus-cell mixture. After mixing, it was transferred to a 24-well plate and placed in a 37°C, 5% CO 2 After 24 h of culture in the incubator, the culture medium was replaced. After 48 h, the infected HEK-293T cells were collected and the proportion of cells with positive transgene expression was detected by flow cytometry to calculate the virus titer. Lentivirus titer calculation method: virus titer = (m × 2.5 × 10 5 × dilution factor) / transfected volume, where m is the proportion of cells positive for transgene expression.

[0129] Example 4: Lentivirus infection of TIL primary culture cells The TIL primary culture cells obtained in Example 1 and the lentivirus prepared in Example 2 were used for infection in this example. The specific steps are as follows:

[0130] Complete culture medium: Prepare a 1:1 mixed culture medium of Advanced RPMI 1640 and CTS AIM-VMedium containing 1% Gluta Max, 2.5% CTS Immune Cell SerumReplacement, 300 IU / ml recombinant human interleukin-2 for injection as the complete culture medium.

[0131] Lentivirus (MOI = 10), TIL primary culture cells (1 × 10 6 ) were inoculated into 24-well cell culture plates, and 2 wells were inoculated. BX795 was added to only one well (final concentration was 3.0 μM), and BX795 was not added to the other well (i.e., the final concentration of BX795 was 0 μM), and the volume was supplemented to 200 μL with expansion complete medium and placed at 37°C, 5% CO 2 Culture in an incubator; 24 h after infection, collect the cells, centrifuge at 1000 rpm, discard the supernatant, and retain the cell pellet; add an appropriate amount of expansion complete medium to resuspend, which is the cell before expansion.

[0132] Example 5: Expansion of TIL cells after lentiviral infection In this example, the TIL cells infected with the lentivirus obtained in Example 4 were expanded and cultured using a complete expansion culture medium in the presence of feeder cells. The complete expansion culture medium is described in Example 4.

[0133] The feeder cells used in the present invention are K562 cells expressing mbIL21 and 4-1BBL, which are irradiated at a dose of 100 Gy and cryopreserved after irradiation, and are represented as K562-mIL21-4-1BBL cells.

[0134] The K562-mIL21-4-1BBL cells were revived with the complete culture medium in Example 4; the TIL cells obtained before culture in Example 4 were co-cultured with the K562-mIL21-4-1BBL cells at a ratio of 1:25 using a G-REX 24-well plate; and the cells were placed at 37°C and 5% CO 2 Culture in an incubator; change half of the medium every 2-3 days for 9 days. Harvest the expanded cells.

[0135] Example 6: Flow cytometry detection of transduction efficiency before and after TIL cell expansion Experimental Materials: Cells before expansion: Take a certain volume of the cell suspension obtained in Example 4 and inoculate it into a cell culture plate, and place it at 37°C and 5% CO. 2 The cells were cultured in the incubator for 48 h, and the obtained cells were used for subsequent detection of transduction efficiency and cell viability.

[0136] Cells after expansion: The cells obtained in Example 5 were taken to perform cell counting, transduction efficiency and cell viability tests.

[0137] Experimental steps: The steps for detecting transduction efficiency are as follows: 1×10 6 The cells were centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL of 1× PBS solution containing 2% FBS, followed by addition of 5 μL of Fc blocking antibody for blocking and incubation at room temperature for 10 min; the cells were divided into two groups, control tube and experimental tube, with a cell volume of 50 μL in each group, i.e. 5×10 5 cells; 17.5 μL of antibody mixture (including BV510 anti-human CD3 ((Biolegend, 317332), PB anti-human CD4 (Biolegend, 317429), APC / Cy7 anti-human CD8 (Biolegend, 344714), AF700 anti-human CD45 (Biolegend, 368514), PE / Cy7 anti-human CD56 (Biolegend, 362510), eF660 EGFR (eBioscience, 50-9509-42), 2.5 μL of each of the above antibodies, mixed evenly) was added to the control tube and the experimental tube, and incubated at room temperature for 8 min; after the incubation; 50 μL of 7-AAD mixture prepared with 1× PBS solution containing 2% FBS was added to the cells in the experimental tube only, and incubated at room temperature for 7 min; 1 ml of 1× PBS solution containing 2% FBS was added to the control tube and the experimental tube, and centrifuged at 400 g for 5 min. min; discard the supernatant, add 200 μL of 1× PBS solution containing 2% FBS to resuspend the cells, and test on the machine. The transduction efficiency obtained by testing the cells before expansion is the initial transduction efficiency of TIL cells; the transduction efficiency obtained by testing the cells after expansion is the transduction efficiency of TIL cells after expansion. The flow cytometric profiles of the transduction efficiency of each group of cells are shown in Figure 7 and Figure 8 .

[0138] The steps for detecting cell viability and cell number are as follows: take samples from each experimental well in the G-REX 24-well plate, pipette 20 μL into a 1.5mL centrifuge tube, add 20 μL AO / PI dye solution, dilute and stain at a 1:1 ratio, mix well, pipette 20 μL of the mixture into the well of the Countstar counting plate, let it stand for 2 minutes, and then insert it into the injection slot of the Countstar Rigel fully automatic cell fluorescence analyzer. Select the corresponding counting mode according to the cell type, and enter the information of each well on the measurement information page. After confirming the information, click the start button, Countstar Rigel automatically measures, and the measurement is completed. The measurement results are displayed on the screen, including information such as cell viability and cell density.

[0139] The number of cells harvested after expansion culture was counted, and the total number of transduction-positive cells after expansion culture was calculated by the following formula: total number of transduction-positive cells after expansion culture = number of cells harvested after expansion culture × transduction efficiency after expansion culture.

[0140] The cell transduction efficiency, cell viability and total number of transduction-positive cells after expansion were tested for the BX795 = 3.0 μM group and the BX795 = 0 μM group before and after expansion, respectively. The results are shown in Tables 1 and Figures 1 to 6 shown.

[0141] Table 1

[0142] (1) Effect on transduction efficiency For donor 1, if Figure 1 As shown, when BX-795 (0 μM) was not added, the initial transduction efficiency of lentivirus on TIL cells was 24.87%; when BX-795 = 3.0 μM concentration was added, the initial transduction efficiency could reach 75.34%, which was 3 times higher than that of the group without BX-795 addition.

[0143] For donor 2, if Figure 3 As shown, when BX-795 (0 μM) was not added, the initial transduction efficiency of lentivirus on TIL cells was 20.44%; when BX-795 = 3.0 μM concentration was added, the initial transduction efficiency could reach 66.08%, which was 3.2 times higher than that of the group without BX-795 addition.

[0144] The above results show that the addition of BX-795 can significantly improve the transduction efficiency of lentivirus-infected TIL cells before expansion (i.e., the initial transduction efficiency), with the increase reaching more than 3 times.

[0145] (2) Effect on transduction stability For donor 1, see Table 1 and Figure 1 As shown, when BX-795 (0 μM) was not added, the transduction efficiency of lentivirus-infected TIL cells was 14.82% after 9 days of expansion, a decrease of 40.4% compared with before expansion (24.87%). The transduction efficiency decreased significantly after expansion.

[0146] When BX-795 was added at a concentration of 3.0 μM, the transduction efficiency was 68.66% after 9 days of culture, a decrease of 8.9% compared to before culture (75.34%). Compared to no addition of BX-795, the transduction efficiency did not decrease significantly after culture when BX-795 was added at a concentration of 3.0 μM.

[0147] For donor 2, see Table 1 and Figure 3 As shown, when BX-795 (0 μM) was not added, the transduction efficiency of lentivirus-infected TIL cells was 9.82% after 9 days of expansion, a decrease of 52% compared with before expansion (20.44%). The transduction efficiency decreased significantly after expansion.

[0148] When BX-795 was added at a concentration of 3.0 μM, the transduction efficiency was 61.76% after 9 days of culture, a decrease of 6.5% compared to before culture (66.08%). Compared to no addition of BX-795, the transduction efficiency did not decrease significantly after culture when BX-795 was added at a concentration of 3.0 μM.

[0149] The above results indicate that the addition of BX-795 can significantly improve the transduction stability of lentivirus-infected TIL cells, that is, the transduction efficiency did not decrease significantly after 9 days of expansion.

[0150] (3) Effect on cell viability The results are as follows Figure 2 and Figure 4 As shown. For TIL cells after lentiviral transfection and before expansion, the cell viability of the group with BX-795 added was lower than that of the control group without BX-795 added, that is, after adding BX-795, the cell viability dropped from 81.42% to 72.20% (donor 1) and from 60.82% to 43.29% (donor 2), indicating that the addition of BX-795 would lead to a decrease in TIL cell viability to a certain extent.

[0151] However, after 9 days of expansion using the method of the present invention, the cell viability of the group with BX-795 added was comparable to that of the control group without BX-795 added (donor 1: 87.50% vs 84.12%, donor 2: 76.53% vs 77.61%). This indicates that after the infected TIL cells were expanded, the adverse effect of BX-795 on cell viability was restored.

[0152] The above results show that although the addition of BX-795 will reduce the viability of TIL cells just after transduction, the cell viability will recover to a very high level after 9 days of expansion, indicating that the scheme of the present invention of adding BX-795 for transduction and expansion can ensure the high viability of TIL cells.

[0153] (4) Effect on the total number of transduction-positive cells For donor 1, see Table 1 and Figure 5 As shown, in the group with BX-795 added, the total number of positive cells harvested after transduction and expansion increased by about 2.6 times compared with the group without BX-795 added.

[0154] For donor 2, see Table 1 and Figure 6As shown, in the group with BX-795 added, the total number of positive cells harvested after transduction and expansion increased by about 4.9 times compared with the group without BX-795 added.

[0155] The above results show that, when other conditions are the same, adding BX-795 during transduction will significantly increase the total number of harvested positive cells, with an increase of about 2 to 5 times.

[0156] Example 7: Transduction-promoting effect of different concentrations of BX-795 The TIL primary culture cells from donor 3 obtained in Example 1 and the lentivirus prepared in Example 2 were used for lentivirus infection in this example. The specific steps are as follows:

[0157] Complete culture medium: Prepare a 1:1 mixed culture medium of Advanced RPMI 1640 and CTS AIM-VMedium containing 1% Gluta Max, 2.5% CTS Immune Cell SerumReplacement, 300 IU / ml recombinant human interleukin-2 for injection as the complete culture medium.

[0158] Lentivirus (MOI = 10), TIL primary culture cells (1 × 10 6 ) were inoculated into 24-well cell culture plates, and 4 wells were inoculated. No BX-795 was added to one well (i.e., the final concentration of BX-795 was 0 μM), and different concentrations of BX-795 were added to the other three wells (the final concentrations of BX-795 in each well were 1.5 μM, 3.0 μM, and 6.0 μM, respectively), and the volume was supplemented to 200 μL with expansion complete medium, and placed at 37°C, 5% CO 2 Culture in an incubator; 24 h after infection, collect the cells, centrifuge at 1000 rpm, discard the supernatant, and retain the cell pellet; add an appropriate amount of expansion complete medium to resuspend, which is the cell before expansion.

[0159] The expansion operation of TIL cells after lentivirus infection is referred to Example 5. The determination of transduction efficiency before and after expansion of TIL cells infected with lentivirus is referred to Example 6.

[0160] The cell transduction efficiency before and after expansion of the groups with different concentrations of BX-795 was tested, and the results are shown in Tables 2 and Figure 7 .

[0161] Table 2

[0162] like Figure 7As shown, when the concentration of BX-795=0 μM, the initial transduction efficiency of lentivirus on TIL cells was 44.70%, and the transduction efficiency was 20.13% after 9 days of expansion, a decrease of 55% compared with before expansion. The transduction efficiency decreased significantly after expansion.

[0163] When BX-795=1.5 μM concentration was added, the initial transduction efficiency could reach 62.11%, which was 39% higher than that of the group without BX-795 addition; the transduction efficiency was 47.15% after 9 days of culture expansion, which was 24% lower than that before culture expansion. Compared with the group without BX-795 addition, the decrease in transduction efficiency after culture expansion was significantly reduced when BX-795=1.5 μM concentration was added.

[0164] When BX-795=3.0 μM concentration was added, the initial transduction efficiency could reach 64.55%, which was 44% higher than that of the group without BX-795 addition; the transduction efficiency was 67.97% after 9 days of expansion, which was 5% higher than that before expansion. Compared with the group without BX-795 addition, there was no significant difference in transduction efficiency after expansion when BX-795=3.0 μM concentration was added.

[0165] When BX-795=6.0 μM concentration was added, the initial transduction efficiency could reach 66.13%, which was 48% higher than that of the group without BX-795 addition; the transduction efficiency was 66.25% after 9 days of expansion, which was the same as that before expansion. Compared with the group without BX-795 addition, the transduction efficiency remained basically unchanged after expansion when BX-795=6.0 μM concentration was added.

[0166] The results of donor 3 showed that within the concentration range of BX-795≤6.0 μM, the addition of BX-795 could significantly improve the initial transduction efficiency of lentivirus-infected TIL primary culture cells, with an increase of about 40% or more. At the same time, there was no significant difference in the increase in initial transduction efficiency among BX-795=1.5, 3.0, and 6.0 μM concentrations. In addition, the addition of BX-795 could significantly improve the stability of the transduction efficiency of lentivirus-infected TIL primary culture cells after expansion, especially at a concentration of BX-795=3.0 μM, when the transduction efficiency was maintained best.

[0167] discuss Lentiviral transduction technology has important applications in the field of cell therapy. However, the efficiency of lentiviral transduction of cells is often affected by the cell type, resulting in very low transduction efficiency. For example, TIL cells from tumor tissues, especially TIL cells from tumor tissues of advanced patients, are often in a state of terminal differentiation and exhaustion, resulting in a generally low transduction efficiency of lentiviral vectors to TIL cells, generally around 20%, and with large individual differences. In addition, the transduction efficiency will continue to decrease with the proliferation of TIL cells, and there is a problem of unstable transduction efficiency.

[0168] Adding a transduction-promoting agent during lentiviral transduction can improve the transduction efficiency to a certain extent. However, for special cell populations such as TIL cells, there are few reports in the prior art on which agent can effectively promote transduction. In addition, the addition of transduction-promoting chemical reagents will lead to a decrease in cell viability and affect the quality of cell products. Therefore, there is an urgent need in the art to screen suitable transduction-promoting agents and transduce TIL cells under appropriate methods, while improving the transduction efficiency of lentivirus to TIL cells, and ensuring the high viability of TIL cells and the stability of transduction.

[0169] In the present invention, a method for promoting lentivirus transduction of TIL cells is provided, which can increase the transduction efficiency by more than 3 times, and the transfected cells can maintain a cell viability comparable to that of the control group after expansion. The results of the present invention comprehensively show that the method provided by the present invention can not only improve the transfection efficiency of TIL cells, but also provide transfection stability and ensure cell viability, and is particularly suitable for producing high-quality TIL cell products in TIL cell therapy.

[0170] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for improving the lentiviral transduction efficiency and transduction stability of TIL cells, characterized in that: Includes steps: A. Provide TIL cells and lentivirus to be transduced; B. inoculating the TIL cells to be transduced into a complete culture medium, and transducing the TIL cells with the lentivirus in the presence of a transduction-promoting agent; wherein the transduction-promoting agent includes BX-795, and the final concentration of BX-795 in the transduction system is 1.5-6 μM; C. Collect transduced cells; D. Expand the cells from step C in complete expansion medium.

2. The method according to claim 1, characterized in that The transduction time for step B is 24-48 hours.

3. The method according to claim 1, characterized in that The final concentration of BX-795 in the transduction system of step B is 3-6 μM.

4. The method according to claim 1, characterized in that The TIL cells to be transduced are derived from tumor tissue.

5. The method according to claim 1, characterized in that The components of the complete expansion culture medium include: cell basal culture medium, serum substitute, L-glutamine or its substitute, and IL-2.

6. The method according to claim 5, characterized in that The amount of IL-2 in the complete culture medium is 100-600 IU / ml.

7. The method according to claim 1, characterized in that The amplification culture time is 7 to 10 days.

8. The method according to claim 1, characterized in that The method has one or more characteristics selected from the group consisting of: (1) The transduction efficiency of the cells collected in step C is E0, E0 ≥ 50%; (2) The transduction efficiency of the cells expanded and cultured in step D is E1, E1 ≥ 50%; (3) After the expansion culture in step D, the transduction efficiency of the cells does not decrease, or the decrease is (E0-E1) / E0≤20%; (4) The cell viability after expansion culture in step D is ≥ 60%.

9. Use of BX-795 in the preparation of a reagent for adding to a transduction system for lentiviral transduction of TIL cells to improve the lentiviral transduction efficiency and transduction stability of TIL.

10. A kit for improving the lentiviral transduction efficiency and transduction stability of TIL cells, characterized in that: The kit comprises: (a) a transduction-promoting reagent, wherein the transduction-promoting reagent comprises BX-795; (b) a complete culture medium; and (c) an instruction manual, wherein the instruction manual describes the method for using the kit.

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