Synergistic engineering tumor infiltrating lymphocyte and preparation method thereof

Abstract

The invention provides a synergistic engineering tumor infiltration lymphocyte and a preparation method thereof, and belongs to the technical field of biological medicines. The engineering tumor infiltrating lymphocyte capable of simultaneously expressing the activated fusion protein and the retention fusion protein is constructed, so that the recognition and killing capabilities of the engineering tumor infiltrating lymphocyte on tumor cells are remarkably improved, and meanwhile, the self-killing of the cells in an activated state is effectively avoided, so that the continuity and the safety of treatment are enhanced. The cell preparation shows a good application prospect in solid tumor treatment, and a new effective strategy is provided for overcoming the limitation of the existing immune cell therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to enhanced engineered tumor-infiltrating lymphocytes and their preparation methods. Background Technology

[0002] Tumor immunotherapy is a crucial direction in current cancer treatment, with cell therapy attracting significant attention due to its high specificity and relatively controllable side effects. Tumor-infiltrating lymphocytes (TILs) are a heterogeneous population of T cells isolated from patient tumor tissue. They naturally possess the ability to recognize tumor-associated antigens, thus showing unique potential in the treatment of solid tumors. TIL therapy generally involves steps such as isolating TIL cells from tumor tissue, expanding them in vitro, and then reinfusing them into the patient, aiming to eliminate tumor cells by enhancing the body's own immune system.

[0003] Despite the positive results achieved by TIL therapy in some clinical studies, its practical application still faces numerous challenges. For example, TIL cells are prone to functional exhaustion during in vitro expansion, manifested as decreased proliferation, reduced cytokine secretion, and weakened killing function. Furthermore, immunosuppressive factors in the tumor microenvironment, such as downregulation of MHC class I molecule expression by tumor cells, can also weaken the recognition and killing efficiency of TIL cells. On the other hand, activated T cells may transiently express certain activating ligands, leading to mutual recognition and even killing among T cells—a phenomenon known as "cell-to-cell cannibalism"—which further affects the survival rate and therapeutic efficacy of TIL cells. Therefore, enhancing the sustained activity of TIL cells, improving their tumor-specific killing ability, and avoiding harmful intercellular interactions have become critical issues that urgently need to be addressed in this field. Summary of the Invention

[0004] The purpose of this invention is to provide enhanced engineered tumor-infiltrating lymphocytes and their preparation method, which solves the technical problems of cell depletion, insufficient killing ability, and reduced efficacy caused by cell cannibalism in existing tumor-infiltrating lymphocyte therapies.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an engineered tumor-infiltrating lymphocyte, which is constructed by infection with a lentiviral vector and stably expresses an activating fusion protein and a retention fusion protein. The activating fusion protein is a DAP10-CD3 fusion protein formed by the direct linkage of the intracellular domain of DAP10 and the intracellular domain of CD3ζ chain. The retention fusion protein is an NKG2D-KDEL fusion protein formed by the linkage of the extracellular region of NKG2D and the endoplasmic reticulum retention signal peptide KDEL.

[0006] Preferably, the activating fusion protein comprises a signal peptide sequence, a DAP10 extracellular domain sequence, a DAP10 transmembrane domain sequence, a DAP10 intracellular domain sequence, and a CD3ζ chain intracellular domain sequence connected in sequence.

[0007] Preferably, the amino acid sequence of the activated fusion protein is shown in SEQ ID NO:1.

[0008] Preferably, the retention fusion protein comprises the NKG2D extracellular region sequence and the endoplasmic reticulum retention signal peptide KDEL sequence.

[0009] Preferably, the amino acid sequence of the retained fusion protein is shown in SEQ ID NO:2.

[0010] Preferably, the lentiviral vector is a pLVX-IRES-ZsGreen1 vector containing the activating fusion protein encoding gene and / or the residing fusion protein encoding gene.

[0011] This invention also provides a method for preparing the above-mentioned engineered tumor-infiltrating lymphocytes, comprising the following steps: The nucleic acid sequences encoding the activating fusion protein and the retention fusion protein are synthesized, and the nucleic acid sequences are respectively constructed into lentiviral vectors to obtain recombinant lentiviral vectors; The recombinant lentiviral vector was packaged and concentrated to obtain lentivirus; The lentivirus was used to infect tumor-infiltrating lymphocytes isolated and cultured from tumor tissue to obtain engineered tumor-infiltrating lymphocytes. The tumor-infiltrating lymphocytes stably expressed an activating fusion protein and a retention fusion protein. The activating fusion protein was a DAP10-CD3 fusion protein formed by the direct linkage of the intracellular domain of DAP10 and the intracellular domain of CD3ζ chain. The retention fusion protein was an NKG2D-KDEL fusion protein formed by the linkage of the extracellular domain of NKG2D and the endoplasmic reticulum retention signal peptide KDEL.

[0012] Preferably, the synthesis of the nucleic acid sequences encoding the activating fusion protein and the residing fusion protein specifically includes: deducing the encoding nucleic acid sequences in reverse from the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and performing mammalian cell codon preference optimization; Constructing a nucleic acid sequence into a lentiviral vector includes the following steps: double digestion of the pLVX-IRES-ZsGreen1 vector with restriction endonucleases EcoRI and BamHI to obtain a linearized vector; ligation of the nucleic acid sequence encoding the activating fusion protein or the residing fusion protein with the linearized vector using T4 DNA ligase; transformation of the ligation product into competent Escherichia coli DH5α and screening to obtain the correct recombinant lentiviral vector.

[0013] Preferably, packaging and concentrating lentivirus includes the following steps: co-transfecting the recombinant lentiviral vector plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G into HEK293T cells using liposome transfection at a mass ratio of 4:3:1.

[0014] The present invention also provides the application of the above-described engineered tumor-infiltrating lymphocytes in the preparation of drugs for treating solid tumors.

[0015] The beneficial effects of this invention are: This invention significantly enhances the tumor-infiltrating lymphocytes' ability to recognize and kill tumor cells by constructing engineered cells that simultaneously express both activating and retaining fusion proteins, while effectively preventing cannibalism among activated cells, thus improving the sustainability and safety of treatment. This cell preparation shows promising application prospects in the treatment of solid tumors, providing a new and effective strategy to overcome the limitations of existing immunocellular therapies. Attached Figure Description

[0016] Figure 1 A graph validating the efficiency of lentivirus infection in tumor-infiltrating lymphocytes; Figure 2 Immunoblotting image of fusion protein expression in engineered tumor-infiltrating lymphocytes; Figure 3 Flow cytometry analysis of the expression of activated fusion proteins on the surface of engineered tumor-infiltrating lymphocytes; Figure 4 Figure showing the comparison of the killing activity of engineered tumor-infiltrating lymphocytes against target tumor cells; Figure 5 A comparison of in vitro killing curves of engineered and wild-type tumor-infiltrating lymphocytes at different effector-to-target ratios; Figure 6 Immunofluorescence confocal analysis of subcellular localization of retained fusion proteins in engineered tumor-infiltrating lymphocytes. Detailed Implementation

[0017] This invention provides an engineered tumor-infiltrating lymphocyte population. In this invention, "tumor-infiltrating lymphocytes" (TILs) refer to a heterogeneous population of lymphocytes, primarily comprising T cells, isolated from surgically removed tumor tissue from cancer patients. These cells naturally infiltrate the tumor microenvironment, can recognize tumor-associated antigens, and are an important source of effector cells in adoptive cellular immunotherapy. Their preparation generally involves digesting tumor tissue into a single-cell suspension, followed by selective expansion in a culture medium containing cytokines such as interleukin-2 (IL-2).

[0018] The term "engineered" refers to the genetic modification of cells through genetic engineering techniques. Specifically, the engineered tumor-infiltrating lymphocytes are genetically modified through infection with lentiviral vectors. Lentiviral vectors are commonly used gene delivery tools that can efficiently integrate exogenous genes into the host cell's genome, thereby achieving long-term, stable expression of the target gene.

[0019] The engineered tumor-infiltrating lymphocytes stably express two exogenous fusion proteins: an activating fusion protein and a retention fusion protein. The "activating fusion protein" aims to enhance T cell activation signaling. In this invention, this protein is a DAP10-CD3 fusion protein formed by the direct linkage of the intracellular domain of DAP10 and the intracellular domain of the CD3ζ chain. DAP10 (DNAX activating protein 10) is an immune cell transmembrane adaptor protein whose intracellular domain contains the YxxM motif, which can recruit and activate downstream signaling molecules such as PI3K. CD3ζ is a key component of the T cell receptor (TCR) complex, and its intracellular domain contains the immune receptor tyrosine activation motif (ITAM), playing a central role in signal transduction after TCR receives antigen stimulation. Directly fusing the intracellular domains of DAP10 and CD3ζ is a strategy for constructing a co-stimulatory signaling chimera, aiming to integrate or enhance T cell activation signaling pathways. Stable expression refers to the integration of exogenous genes into the host cell's chromosomal genome, enabling stable replication and inheritance with host cell division, and continuous synthesis of the target protein within the cell.

[0020] Preferably, the activating fusion protein contains complete protein domains to enable its proper transport to the cell membrane and function. More preferably, it comprises a signal peptide sequence, a DAP10 extracellular domain sequence, a DAP10 transmembrane domain sequence, a DAP10 intracellular domain sequence, and a CD3ζ chain intracellular domain sequence connected in sequence. The signal peptide guides the newly synthesized protein into the endoplasmic reticulum; the transmembrane domain is responsible for anchoring the protein to the cell membrane.

[0021] Further preferably, the activating fusion protein has a specific amino acid sequence as shown in SEQ ID NO:1. This sequence is designed and synthesized using genetic engineering methods. In practice, the gene sequence encoding this protein can be reverse-translated according to SEQ ID NO:1 and optimized according to the codon usage preferences of mammalian cells (such as human cells) to improve its expression efficiency in TIL cells.

[0022] The aforementioned "retention-type fusion protein" aims to interfere with specific ligand-receptor interactions to reduce adverse intercellular effects. In this invention, the protein is an NKG2D-KDEL fusion protein formed by linking the extracellular region of NKG2D with the endoplasmic reticulum retention signal peptide KDEL. NKG2D (Natural Killer 2 Group D) is an activating receptor primarily expressed on the surface of NK cells and activated T cells (including some TILs), and its ligand (NKG2DL) is typically regulated on the surface of certain stressed or diseased cells (such as tumor cells). The KDEL sequence (amino acid sequence Lys-Asp-Glu-Leu) is a commonly used retention signal for endoplasmic reticulum-resident proteins, which can retain the fusion protein primarily in the endoplasmic reticulum, preventing its transport to the cell membrane surface.

[0023] Preferably, the retention-type fusion protein comprises an extracellular NKG2D region sequence and an endoplasmic reticulum retention signal peptide KDEL sequence. The extracellular NKG2D region is responsible for recognizing and binding its ligand, while the KDEL sequence determines the organelle localization of the fusion protein.

[0024] More preferably, the retained fusion protein has a specific amino acid sequence as shown in SEQ ID NO:2. Similarly, its encoding gene can be designed and synthesized based on this sequence, and codon optimization can be performed.

[0025] Preferably, the lentiviral vector used to infect the TIL cells is the pLVX-IRES-ZsGreen1 vector containing the activating fusion protein encoding gene and / or the residing fusion protein encoding gene. pLVX-IRES-ZsGreen1 is a commercially available lentiviral expression vector backbone containing a multiple cloning site for inserting the target gene, and an internal ribosome entry site (IRES) that allows co-expression of the target gene with the green fluorescent protein reporter gene (ZsGreen1), facilitating fluorescence monitoring of infection efficiency. This vector and its analogues are commercially available.

[0026] The present invention also provides a method for preparing the above-described engineered tumor-infiltrating lymphocytes.

[0027] The preparation method includes the following core steps: synthesizing the nucleic acid sequence encoding the fusion protein and constructing a recombinant lentiviral vector; packaging and concentrating the lentivirus; and infecting TIL cells with the lentivirus.

[0028] Preferably, in the step of synthesizing the nucleic acid sequence encoding the fusion protein, the corresponding DNA coding sequence is deduced in reverse based on the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2. To further improve the expression level in human TIL cells, the deduced DNA sequence can be optimized for codon bias according to a well-known mammalian cell codon frequency table. The optimized gene sequence can be chemically synthesized by a professional biotechnology company.

[0029] Preferably, the step of constructing the nucleic acid sequence into the lentiviral vector can be performed using standard molecular cloning techniques. Specifically, this includes: double digestion of the selected pLVX-IRES-ZsGreen1 vector with restriction endonucleases (such as EcoRI and BamHI) to generate compatible linearized ends. The digestion reaction time can vary from 1 to 6 hours, preferably 3 to 5 hours, more preferably about 4 hours; in this example, 4 hours is used. The synthesized target gene fragment is then ligated to the linearized vector using a DNA ligase (such as T4 DNA ligase). For example, the ligation system may use 20-100 ng of linearized vector, add the target fragment at a molar ratio of 1:1 to 10:1 (fragment:vector), use 1-5 units of ligase, maintain a ligation temperature of 4-25°C, and ligation time of 1-16 hours; preferably, about 50 ng of vector, about 100 ng of target fragment, and 1-2 units of ligase are used for overnight ligation at 16°C (about 12-16 hours). The ligation product was then transformed into competent Escherichia coli (such as DH5α strain), and the correct recombinant lentiviral vector plasmid was identified by antibiotic screening (such as ampicillin) and colony PCR, plasmid digestion or sequencing.

[0030] Preferably, the step of packaging and concentrating lentivirus is typically performed in packaging cells (such as human embryonic kidney cells HEK293T) using a three-plasmid co-transfection system. Specifically, the recombinant lentiviral vector plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G are co-transfected into HEK293T cells using liposome transfection. The mixing mass ratio of each plasmid can be adjusted within a certain range; for example, the ratio of recombinant vector:psPAX2:pMD2.G can be (3-5):(2-4):(0.8-1.2), preferably 4:3:1. Transfection can be performed using commercially available liposome transfection reagents (such as Lipofectamine 2000, Lipofectamine 3000, etc.) according to their instructions. Approximately 48-72 hours after transfection, the cell culture supernatant containing lentiviral particles is collected. Virus concentration can be achieved using methods such as ultracentrifugation, ultrafiltration, or polyethylene glycol precipitation. When using ultracentrifugation, the centrifugal force can range from 50,000×g to 150,000×g, and the centrifugation time can range from 1.5 to 3 hours; the preferred conditions are 4°C and centrifugation at approximately 100,000×g for approximately 2 hours. The concentrated virus solution can be resuspended in a suitable buffer (such as PBS containing a small amount of serum), and its titer (expressed as transduction units per milliliter, TU / mL) can be determined using the limiting dilution method. After aliquoting, the solution is stored at -80°C.

[0031] Preferably, the step of infecting tumor-infiltrating lymphocytes isolated and cultured from tumor tissue with lentivirus involves two steps: preparation and infection of TILs. The isolation and culture of TILs can be performed using the following method: cutting the tumor tissue into pieces approximately 1-3 mm in size... 3 Small pieces of cells are digested at 37°C for 1-3 hours using a digestion solution containing collagenase (e.g., collagenase IV, concentration 1-3 mg / mL), hyaluronidase, and / or DNase (e.g., DNase I, concentration 0.05-0.2 mg / mL), preferably for about 2 hours using a digestion solution containing 2 mg / mL collagenase IV and 0.1 mg / mL DNase I. After digestion, the cells are filtered to obtain a single-cell suspension, washed, and cultured in complete medium (e.g., RPMI-1640, supplemented with 10% fetal bovine serum) containing a high concentration of IL-2 (e.g., 200-600 IU / mL, preferably about 300 IU / mL) to selectively expand TIL cells. The culture time is usually 7-14 days, preferably 7-10 days. For infection, TIL cells in the logarithmic growth phase are taken, and the cell density is adjusted, for example, to 1×10⁶ cells / mL. 5 Up to 1×10 6 Cells / mL, preferably about 5 × 10⁻⁶ 5Cells / mL. Add an appropriate amount of concentrated lentivirus; the multiple of infection (MOI) can be selected between 1 and 50, preferably 5-20, and more preferably about 10, depending on the viral titer and cell type. To enhance viral infection efficiency, a viral infection enhancer such as polybrene can be added simultaneously, with a final concentration of 5-10 μg / mL, preferably about 8 μg / mL. The infection process typically lasts 12-48 hours, preferably about 24 hours, after which fresh medium is replaced and cultured again. Infection efficiency can be assessed by observing the expression of the fluorescent reporter gene (such as ZsGreen1) carried by the vector. When the proportion of fluorescently positive cells exceeds a certain threshold (e.g., 50%, preferably above 70%), infection is considered successful, and engineered TIL cells are obtained.

[0032] The present invention also provides the use of the above-described engineered tumor-infiltrating lymphocytes in the preparation of a medicament for treating solid tumors.

[0033] The term "solid tumor" refers to a malignant tumor that forms a solid mass, as opposed to non-solid tumors (such as leukemia), including but not limited to melanoma, lung cancer, liver cancer, breast cancer, colorectal cancer, head and neck cancer, and ovarian cancer. The term "medicine" refers to a pharmaceutical composition containing the engineered TIL cells as an active ingredient. This pharmaceutical composition is typically in the form of a cell suspension containing pharmaceutically acceptable carriers or excipients, such as cell cryopreservation solutions (containing DMSO and serum / albumin) or infusion solutions (such as physiological saline or buffer solutions containing human serum albumin). The preparation of the drug also includes the process of massively expanding the engineered TIL cells in vitro to reach the required therapeutic dose, and performing quality testing (such as sterility, mycoplasma, endotoxin testing, and potency testing) and formulation. Its purpose is to reinfuse the engineered TIL cells into the patient through adoptive cell immunotherapy to recognize and kill tumor cells.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example Preparation and Implementation Steps of Engineered TIL Cells (1) Synthesis of target nucleic acid sequence and vector construction Design and synthesis of target gene sequences: Based on the amino acid sequences of the activating fusion protein and the retention fusion protein, the corresponding coding nucleic acid sequences were deduced in reverse (codon preference was optimized to mammalian cell preferred codons). The above two nucleic acid sequences were synthesized by a gene synthesis company and named Sequence A (encoding the activating fusion protein) and Sequence B (encoding the retention fusion protein), respectively.

[0036] Sequence A (DAP10-CD3) amino acid sequence: MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRGMRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPORRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, as in SEQ Shown as ID NO.1; The sequence of the signal peptide is shown in positions 1-18, the sequence of the extracellular domain is shown in positions 19-48, the sequence of the transmembrane domain is shown in positions 49-69, the sequence of the DAP10 intracellular domain is shown in positions 70-92, and the sequence of CD3 is shown in positions 93-206.

[0037] The amino acid sequence of sequence B (NKG2D-KDEL) is shown below: MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMOKGDCALYASSFKGYIENCSTPNTYICMQRTVKDEL, as shown in SEQ ID NO.2; Among them, positions 22-156 are the extracellular region sequence of NKG2D; positions 157-160 are the KDEL sequence.

[0038] Lentiviral vector digestion and recovery: The lentiviral vector backbone pLVX-IRES-ZsGreen1 (Takara Bio Technology, 631253) was selected. The vector was double-digested using restriction endonucleases (such as EcoRI and BamHI, selected according to the multiple cloning site). The reaction system was as follows: 5 μL of 10× digestion buffer, 10 μL of lentiviral vector plasmid (1 μg / μL), 2 μL of EcoRI (10 U / μL), 2 μL of BamHI (10 U / μL), and enzyme-free pure water to a final volume of 50 μL. After incubation at 37℃ for 4 h, the digestion products were separated by 1% agarose gel electrophoresis. The linearized lentiviral vector fragment was recovered using a gel recovery kit, the concentration was determined, and the fragment was stored at -20℃.

[0039] Ligation of target sequences with vectors: Sequence A and sequence B were ligated with linearized lentiviral vectors, respectively. The ligation system was as follows: 2 μL of 10× ligation buffer, 2 μL of linearized lentiviral vector (50 ng / μL), 1 μL of sequence A / sequence B (100 ng / μL), 1 μL of T4 DNA ligase (5 U / μL), and enzyme-free pure water to a final volume of 20 μL; ligation was carried out overnight at 16°C to obtain recombinant lentiviral vectors pLVX-A (containing sequence A) and pLVX-B (containing sequence B).

[0040] Transformation and identification of recombinant vectors: The above ligation products were transformed into competent Escherichia coli DH5α cells. The specific procedures were as follows: 5 μL of ligation product was mixed with 50 μL of competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and then immediately incubated on ice for 2 min. 450 μL of LB liquid medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h with shaking. 100 μL of the culture was evenly spread on LB solid medium containing ampicillin (100 μg / mL) and incubated upside down at 37℃ for 12-16 h. Single colonies were picked and inoculated into LB liquid medium containing ampicillin (100 μg / mL) and cultured at 37℃ and 200 rpm for 12 h with shaking. Plasmids were extracted and verified by double enzyme digestion (the steps are the same as in "Lentinogenic vector digestion and recovery") and DNA sequencing to screen out recombinant lentiviral vectors with correct sequences.

[0041] (2) Packaging and concentration of lentiviruses HEK293T cell culture and plating: After resuscitation, HEK293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics at 37°C in a 5% CO2 incubator. When the cell confluence reached 70%-80%, the cells were passaged and the cell concentration was adjusted to 2×106 cells / mL. 10 mL of the cell suspension was seeded into a 10 cm cell culture dish and cultured for 24 h. When the cell confluence reached 80%-90%, the cells were used for transfection.

[0042] Plasmid transfection: The recombinant lentiviral vector plasmid (pLVX-A or pLVX-B), envelope plasmid (BioFone, 12259), and packaging plasmid psPAX2 (BioFone, 12260) were mixed at a mass ratio of 4:1:3 using liposome transfection. The specific procedure was as follows: A sterile EP tube was filled with 500 μL of serum-free DMEM medium, followed by the addition of the recombinant lentiviral vector plasmid (4 μg), pMD2.G plasmid (1 μg), and psPAX2 plasmid (3 μg). Gently mix the contents of the two tubes. In a separate sterile EP tube, add 500 μL of serum-free DMEM medium and 20 μL of liposome transfection reagent (such as Lipofectamine 3000), mix gently, and let stand at room temperature for 5 min. Mix the liquids from the two EP tubes and let stand at room temperature for 20 min to form a transfection complex. Slowly add the transfection complex to a HEK293T cell culture dish, gently shake the dish to distribute the liquid evenly, and continue to incubate in a 37°C, 5% CO2 incubator.

[0043] Lentiviral Collection and Concentration: HEK293T cell culture supernatant was collected at 48h and 72h post-transfection and centrifuged at 3000rpm for 10min at 4℃ to remove cell debris. The supernatants collected from both trials were combined, and the lentivirus was concentrated using ultracentrifugation at 100000×g for 2h at 4℃. The supernatant was discarded, and the virus pellet was resuspended in PBS buffer containing 2% FBS. The viral titer was determined using limiting dilution, calculated based on the proportion of GFP-positive cells, ensuring a viral titer ≥1×10⁻⁶. 8 (TU / mL), the concentrated lentivirus was aliquoted and stored at -80℃.

[0044] (3) Lentiviral infection of TIL cells TIL cell isolation and culture: TIL cells were isolated from surgically removed tumor tissue from cancer patients. The specific procedures were as follows: The tumor tissue was rinsed three times with PBS buffer to remove blood stains and necrotic tissue, and then cut into 1-2 mm pieces. 3 Small pieces of cells were added to RPMI-1640 medium containing collagenase IV (2 mg / mL) and DNase I (0.1 mg / mL), and digested at 37°C with shaking for 2 h. The cells were then filtered through a 70 μm cell sieve to collect the single-cell suspension, centrifuged at 1500 rpm for 5 min at 4°C, and the supernatant was discarded. The cells were resuspended in RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin antibiotics, and 300 IU / mL IL-2, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells were cultured at a density of 1 cell / mL in a 37°C, 5% CO2 incubator. The culture medium was changed every 2-3 days, and purified TIL cells were obtained after 7-10 days of culture.

[0045] Lentiviral infection procedure: Adjust the concentration of TIL cells cultured to logarithmic growth phase to 5 × 10⁻⁶. 5 Cells / mL were seeded into 24-well cell culture plates, with 500 μL of cell suspension added to each well; concentrated lentivirus (e.g., 5 μL of 1×10⁻⁶ titer per well) was added according to the viral titer at a ratio of MOI = 10. 8 The virus (TU / mL) was added, along with polybrene at a final concentration of 8 μg / mL to improve infection efficiency. After gentle mixing, the cells were incubated at 37°C in a 5% CO2 incubator for 24 h. The medium was then replaced with RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin antibiotics, and 300 IU / mL IL-2, and cultured for another 48 h. The proportion of GFP-positive cells was observed using a fluorescence microscope (≥70% was considered a successful infection), and engineered TIL cells were obtained (named TIL-A and TIL-B, corresponding to lentiviral infection products containing sequences A and B, respectively).

[0046] Implementation steps for cell viability and killing ability testing (1) Detection of engineered TIL cell viability based on MTT assay Cell seeding: The engineered TIL cells (TIL-A, TIL-B) and uninfected blank TIL cells (control group) prepared above were adjusted to a concentration of 2 × 10⁻⁶. 4 Cells / mL were seeded into 96-well cell culture plates, with 100 μL of cell suspension added to each well. Three replicates were set up for each group. At the same time, blank control wells containing only culture medium (100 μL of culture medium) were also set up.

[0047] Culture and MTT addition: After incubating the 96-well plate in a 37℃, 5% CO2 incubator for 24h, 48h, and 72h, add 20μL MTT solution (5mg / mL, prepared with PBS and filtered for sterilization) to each well and continue incubation for 4h.

[0048] Crystallization dissolution and absorbance detection: Carefully aspirate the supernatant from each well, add 150 μL of dimethyl sulfoxide (DMSO) to each well, and place on a shaker to shake at low speed for 10 min to completely dissolve the purple formazan crystals; use an ELISA reader to measure the absorbance value (OD490) of each well at a wavelength of 490 nm.

[0049] Cell viability calculation: Cell viability (%) = (OD490 value of experimental group - OD490 value of blank control well) / (OD490 value of control group - OD490 value of blank control well) × 100%. The viability of engineered TIL cells was evaluated by the calculation results.

[0050] (2) Detection of the killing ability of engineered TIL cells (co-culture method) Tumor cell preparation: Select tumor cell lines corresponding to the tumor tissue from which TIL cells are derived (e.g., HepG2 cells for liver cancer), culture them in DMEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics until the logarithmic growth phase, digest with 0.25% trypsin, centrifuge at 1500 rpm for 5 min at 4°C, and discard the supernatant; resuspend the cells in RPMI-1640 medium containing 10% FBS and adjust the concentration to 1×10⁻⁶ cells / mL. 4 Cells / mL were used as target cells.

[0051] Construction of the co-culture system: Target cells were seeded into 96-well cell culture plates, with 100 μL of cell suspension added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. The concentrations of engineered TIL cells (TIL-A, TIL-B) and blank TIL cells (control group) were adjusted to 1 × 10⁻⁶ cells / well. 5 Effector cells / mL (effector cell ratio = 10:1), add 100 μL of effector cell suspension to each well, and set up 3 replicates per group; at the same time, set up target cell control wells containing only target cells (100 μL target cell suspension + 100 μL culture medium) and effector cell control wells containing only effector cells (100 μL effector cell suspension + 100 μL culture medium).

[0052] Co-culture and survival detection: The 96-well plate was placed in a 37℃, 5% CO2 incubator and cultured for 24 h. The supernatant of each well was aspirated, and 100 μL of medium containing 10% CCK-8 reagent was added to each well (or the MTT method was used, with the same steps as "Cultivation and MTT Addition" and "Crystallization Dissolution and Absorbance Detection"). The plate was cultured for another 2 h. The absorbance value (OD450) of each well was measured at 450 nm using a microplate reader.

[0053] Tumor cell survival rate calculation: Tumor cell survival rate (%) = (OD450 value of experimental group - OD450 value of effector cell control well) / (OD450 value of target cell control well - OD450 value of blank control well) × 100%. The survival rate is used to evaluate the killing ability of engineered TIL cells against tumor cells (the lower the survival rate, the stronger the killing ability).

[0054] The experimental results are as follows: Figure 1 shows the statistical results of GFP positivity rate of TIL cells infected with lentiviral vector in different samples. Among them, TIL-A and TIL-B represent two batches of independently prepared engineered TIL cells, with GFP positivity rates of approximately 76.5% and 74.8%, respectively, both significantly higher than those of the uninfected control group (GFP positivity rates of approximately 3.9% and 2.8%).

[0055] like Figure 2As shown, the activating DAP10-CD3 fusion protein was significantly expressed in TIL-A cells, while its expression was low in TIL-B cells; the retained NKG2D-KDEL fusion protein was detectable in both TIL-A and TIL-B cells.

[0056] Figure 3 shows the expression analysis of the DAP10–CD3 activating fusion protein on the cell membrane surface of engineered TIL cells. Dual-parameter flow cytometry analysis using anti-DAP10 and anti-CD3 assays demonstrated that the engineered TIL cells constructed in this invention can stably express the DAP10–CD3 activating fusion protein on the cell membrane. The proportion of double-positive cells in TIL-A reached 50.2%, significantly higher than the 28.7% in TIL-B.

[0057] Figure 4 The results show the comparison of the target cell killing activities of engineered TIL-A cells, TIL-B cells, and control cells.

[0058] Figure 5 The cell killing curves are for engineered TIL vs WT TIL. Under different effector-to-target ratios (E:T = 1:1, 5:1, 10:1), engineered TIL-A showed significantly higher tumor cell killing ability than WT TIL and TIL-B.

[0059] Figure 6 shows the immunofluorescence detection of the retention-type fusion protein NKG2D-KDEL of the present invention in engineered TIL. The results show that the fluorescence signal of NKG2D-KDEL significantly co-localizes with the endoplasmic reticulum marker Calnexin, proving that the fusion protein is effectively retained in the endoplasmic reticulum.

[0060] As demonstrated by the above embodiments, this invention provides engineered tumor-infiltrating lymphocytes capable of simultaneously and stably expressing the activating fusion protein DAP10-CD3 and the retaining fusion protein NKG2D-KDEL. These cells exhibited good transduction efficiency and protein expression in in vitro experiments, and further demonstrated significantly enhanced tumor cell killing ability while effectively avoiding intercellular killing. This indicates that the engineered TIL cells constructed according to this invention have a clear and positive effect on enhancing anti-tumor immune function.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An engineered tumor-infiltrating lymphocyte, characterized in that, The tumor-infiltrating lymphocytes were constructed by infection with a lentiviral vector and stably expressed an activating fusion protein and a retention fusion protein. The activating fusion protein was the DAP10-CD3 fusion protein formed by the direct connection of the intracellular domain of DAP10 and the intracellular domain of CD3ζ chain. The retention fusion protein was the NKG2D-KDEL fusion protein formed by the connection of the extracellular domain of NKG2D and the endoplasmic reticulum retention signal peptide KDEL.

2. The engineered tumor-infiltrating lymphocytes according to claim 1, characterized in that, The activating fusion protein comprises a signal peptide sequence, a DAP10 extracellular domain sequence, a DAP10 transmembrane domain sequence, a DAP10 intracellular domain sequence, and a CD3ζ chain intracellular domain sequence connected in sequence.

3. The engineered tumor-infiltrating lymphocytes according to claim 2, characterized in that, The amino acid sequence of the activated fusion protein is shown in SEQ ID NO:

1.

4. The engineered tumor-infiltrating lymphocytes according to claim 1, characterized in that, The retention-type fusion protein contains the NKG2D extracellular region sequence and the endoplasmic reticulum retention signal peptide KDEL sequence.

5. The engineered tumor-infiltrating lymphocytes according to claim 4, characterized in that, The amino acid sequence of the retained fusion protein is shown in SEQ ID NO:

2.

6. The engineered tumor-infiltrating lymphocytes according to claim 1, characterized in that, The lentiviral vector is the pLVX-IRES-ZsGreen1 vector containing the activating fusion protein encoding gene and / or the residing fusion protein encoding gene.

7. The method for preparing engineered tumor-infiltrating lymphocytes according to any one of claims 1 to 6, characterized in that, Includes the following steps: The nucleic acid sequences encoding the activating fusion protein and the retention fusion protein are synthesized, and the nucleic acid sequences are respectively constructed into lentiviral vectors to obtain recombinant lentiviral vectors; The recombinant lentiviral vector was packaged and concentrated to obtain lentivirus; The lentivirus was used to infect tumor-infiltrating lymphocytes isolated and cultured from tumor tissue to obtain engineered tumor-infiltrating lymphocytes. The tumor-infiltrating lymphocytes stably expressed an activating fusion protein and a retention fusion protein. The activating fusion protein was a DAP10-CD3 fusion protein formed by the direct linkage of the intracellular domain of DAP10 and the intracellular domain of CD3ζ chain. The retention fusion protein was an NKG2D-KDEL fusion protein formed by the linkage of the extracellular domain of NKG2D and the endoplasmic reticulum retention signal peptide KDEL.

8. The preparation method according to claim 7, characterized in that, The synthesis of the nucleic acid sequences encoding the activating fusion protein and the residing fusion protein specifically includes: deducing their encoding nucleic acid sequences in reverse from the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and performing mammalian cell codon preference optimization; Constructing a nucleic acid sequence into a lentiviral vector includes the following steps: double digestion of the pLVX-IRES-ZsGreen1 vector with restriction endonucleases EcoRI and BamHI to obtain a linearized vector; ligation of the nucleic acid sequence encoding the activating fusion protein or the residing fusion protein with the linearized vector using T4 DNA ligase; transformation of the ligation product into competent Escherichia coli DH5α and screening to obtain the correct recombinant lentiviral vector.

9. The preparation method according to claim 7, characterized in that, The packaging and concentration of lentivirus includes the following steps: the recombinant lentiviral vector plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G are co-transfected into HEK293T cells at a mass ratio of 4:3:1 using liposome transfection.

10. The use of engineered tumor-infiltrating lymphocytes according to any one of claims 1 to 6 in the preparation of drugs for treating solid tumors.

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