Immune cells overexpressing plagl1 or plagl2 and uses thereof

Abstract

The application belongs to the field of biological medicine and immunotherapy, and discloses immune cells overexpressing PLAGL1 or PLAGL2 and application thereof. The application first discloses a novel biological function of PLAGL1 and PLAGL2 gene overexpression in regulation of anti-exhaustion function of engineered immune cells, proves that overexpression of PLAGL1 and / or PLAGL2 in CAR-T or TCR-T cells can significantly improve long-term killing capacity after multiple antigen stimulations without enhancing single-round short-term effect function. This phenotype characteristic of "maintaining stemness and delaying exhaustion" is in sharp contrast with the traditional strategy of enhancing effect function to improve anti-tumor activity. In addition, the application also first extends the PLAGL1 / PLAGL2 overexpression strategy to NK cells and CAR-NK cells, and confirms that it has broad applicability beyond the T cell lineage.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and immunotherapy technology, specifically relating to an engineered immune cell that overexpresses PLAGL1 or PLAGL2 and its application. Background Technology

[0002] Adoptive cell immunotherapy (such as CAR-T and TCR-T) has made groundbreaking progress in the treatment of hematologic malignancies, but it faces a key obstacle of T cell exhaustion when dealing with solid tumors. In the complex microenvironment of solid tumors, driven by continuous stimulation from chronic antigens, tumor-specific T cells or NK cells inevitably enter a state of functional exhaustion. This exhaustion is directly manifested as loss of proliferative capacity, decline in effector function, and increased co-expression of inhibitory receptors (PD-1, Tim-3, LAG3). Once engineered immune cells are trapped in this irreversible differentiation process, they cannot survive in vivo for a long time and continuously eliminate tumor cells. Therefore, how to reverse or delay the exhaustion process of immune cells has become a core clinical challenge that urgently needs to be overcome in the field of next-generation cell immunotherapy.

[0003] Overcoming immune cell exhaustion is challenging due to the complexity of its underlying molecular mechanisms and the irreversibility of epigenetic processes. As exhaustion progresses, immune cells exhibit a high and sustained upregulation of various inhibitory receptors (such as PD-1, Tim-3, and LAG3) on their surface. The co-expression network of these receptors synergistically blocks intracellular activation signals. Therefore, current research and core strategies in this field focus on endowing engineered T cells with stronger stem cell maintenance capabilities.

[0004] Although existing technologies have developed interventions such as knocking out inhibitory receptors or immune checkpoint inhibitor therapy, these methods often focus on relieving immunosuppression in the short term and cannot truly solve the problem of persistence.

[0005] PLAGL1 and PLAGL2 are both C2H2 type zinc finger transcription factors belonging to the PLAG family. They are structurally highly homologous, recognize conserved DNA sequences, and participate in the regulation of multiple key signaling pathways, including the imprinted gene network, Wnt / β-catenin, and IGF2. Current technologies have clearly demonstrated that PLAGL1 and PLAGL2 are involved in physiological development and the pathological processes of various diseases and tumors. However, current research still faces significant technical limitations. On the one hand, the molecular mechanisms of functional switching between the two in different tissues and pathological states remain unclear; on the other hand, the complete upstream and downstream signaling regulatory networks have not been fully elucidated. To date, there are no research reports on the role of PLAGL1 or PLAGL2 in regulating T cell / NK cell stemness maintenance, inhibiting cell exhaustion, or enhancing the persistence of engineered immune cells. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide an immune cell that overexpresses PLAGL1 or PLAGL2 and its application.

[0007] The technical solution adopted in this invention is: A first aspect of the invention provides: an engineered immune cell that overexpresses PLAGL1 and / or PLAGL2 or functionally active variants thereof.

[0008] In some instances, the engineered immune cells also contain chimeric antigen receptors or exogenous T-cell receptors capable of recognizing target antigens.

[0009] In some instances, the nucleotide sequence of the PLAGL1 gene is selected from the nucleotide sequence shown in SEQ ID NO:1 or its conserved alternative sequences.

[0010] In some instances, the nucleotide sequence of the PLAGL2 gene is selected from the nucleotide sequence shown in SEQ ID NO:2 or its conserved alternative sequences.

[0011] In some instances, the engineered immune cells are selected from T cells or NK cells.

[0012] In some instances, the chimeric antigen receptor comprises: an antigen-binding domain, a hinge region, a transmembrane domain, at least one co-stimulatory signaling domain, and a CD3ζ signaling domain; wherein the co-stimulatory signaling domain is selected from CD28, 4-1BB, ICOS, OX40, CD27, or a combination thereof.

[0013] In some instances, the nucleotide sequence of the chimeric antigen receptor comprises a sequence as shown in SEQ ID NO:3 or SEQ ID NO:6.

[0014] In some instances, the nucleotide sequence of the exogenous T-cell receptor comprises the sequence shown in SEQ ID NO:5.

[0015] In some instances, the engineered immune cells are selected from CAR-T cells, TCR-T cells, tumor-infiltrating lymphocytes, CAR-NK cells, γδ T cells, or purified NK cells.

[0016] A second aspect of the present invention provides a method for constructing engineered immune cells as described in the first aspect of the present invention, comprising the steps of introducing the PLAGL1 gene and / or the PLAGL2 gene into immune cells in vitro or in vivo and overexpressing them.

[0017] In some instances, the PLAGL1 and / or PLAGL2 genes are introduced into immune cells via viral or non-viral vectors; the viral vector is a lentiviral vector or a retroviral vector; the non-viral vector is a transposon system or mRNA.

[0018] A third aspect of the invention provides: a pharmaceutical composition comprising the engineered immune cells described in the first aspect of the invention and a pharmaceutically acceptable carrier or excipient.

[0019] A fourth aspect of the present invention provides the use of the engineered immune cells described in the first aspect of the present invention or the pharmaceutical composition described in the third aspect of the present invention in the preparation of an immunotherapeutic drug.

[0020] In some instances, the disease treated by the drug is selected from tumors, autoimmune diseases, or inflammatory diseases.

[0021] In some instances, the tumor is selected from hematologic malignancies or solid tumors; the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, or multiple sclerosis; and the inflammatory disease is selected from inflammatory bowel disease, chronic inflammation, or graft-versus-host disease.

[0022] The beneficial effects of this invention are: This invention reveals for the first time the novel biological function of PLAGL1 and PLAGL2 gene overexpression in regulating the anti-exhaustion function of engineered immune cells, demonstrating that overexpression of PLAGL1 and / or PLAGL2 in CAR-T or TCR-T cells can significantly enhance long-term killing ability after multiple rounds of antigen stimulation without enhancing short-term effector function in a single round. This phenotypic characteristic of "maintaining stemness and delaying exhaustion" contrasts sharply with the traditional strategy of enhancing antitumor activity by enhancing effector function, constituting the unique technical contribution of this invention. Furthermore, this invention also extends the PLAGL1 / PLAGL2 overexpression strategy to NK cells and CAR-NK cells for the first time, demonstrating its broad applicability beyond the T cell lineage. Attached Figure Description

[0023] Figure 1The images show the construction and testing of PLAGL1 / PLAGL2 gene-overexpressing CAR-T cells. Figure A compares the expression levels of PLAGL1, Figure B compares the expression levels of PLAGL2, Figure C shows the in vitro expansion of PLAGL1 / PLAGL2-overexpressing CAR-T cells, Figure D shows the results of multiple rounds of killing experiments, Figure E shows the amount of IFNγ secreted in the supernatant of multiple rounds of killing experiments, Figure F shows the expression detection of exhaustion markers (PD-1, Tim-3, LAG3) after the fourth round of killing, and Figure G shows the TCF-1 expression level of PLAGL1 / PLAGL2-overexpressing CAR-T cells after the fourth round of killing.

[0024] Figure 2 The graphs show the anti-tumor effects of PLAGL1 / PLAGL2 overexpressing CAR-T cells in an in vivo tumor model. Figure A shows the tumor growth curve and mouse survival curve in the in vivo xenograft tumor model. Figure B shows the comparison of the number of CAR-T cells in peripheral blood. Figure C shows the comparison of the number of CAR-T cells in tumor tissue. Figure D shows the detection of the positive proportion of exhaustion markers (PD-1, LAG3 and TIM-3) in tumor tissue infiltrating T cells.

[0025] Figure 3 Figure 1 shows the construction and anti-tumor effect testing of PLAGL1 / PLAGL2 overexpressing TCR-T cells. Figure 2 shows the comparison of PLAGL1 expression level, Figure 3 shows the comparison of PLAGL2 expression level, Figure 4 shows the in vitro expansion of PLAGL1 / PLAGL2 overexpressing TCR-T cells, Figure 5 shows the tumor growth curve and mouse survival curve in the in vivo xenograft tumor model, Figure 6 shows the comparison of the number of TCR-T cells in peripheral blood, Figure 7 shows the comparison of the number of TCR-T cells in tumor tissue, and Figure 8 shows the detection of the positive proportion of exhaustion markers (PD-1, LAG3 and TIM-3) in tumor infiltrating T cells.

[0026] Figure 4 Figure 1 shows the construction and in vitro testing of PLAGL1 / PLAGL2 overexpressing TIL cells. Figure A compares the expression levels of PLAGL1 / PLAGL2, Figure B shows the in vitro expansion of PLAGL1 / PLAGL2 overexpressing TIL cells, and Figure C shows the results of the in vitro TIL killing experiment.

[0027] Figure 5 The graph shows the effect of PLAGL1 / PLAGL2 overexpression on the anti-exhaustion function of NK / CAR-NK cells. In the graph, A shows the comparison of in vitro expansion of PLAGL1 / PLAGL2 overexpressing NK cells, B shows the results of multiple rounds of killing experiments, and C shows the expression detection of exhaustion markers (PD-1, Tim-3, LAG3) after the fourth round of killing. Detailed Implementation

[0028] A first aspect of the invention provides: an engineered immune cell that overexpresses PLAGL1 and / or PLAGL2 or functionally active variants thereof.

[0029] In some instances, the engineered immune cells also contain chimeric antigen receptors or exogenous T-cell receptors capable of recognizing target antigens.

[0030] In some instances, the nucleotide sequence of the PLAGL1 gene is selected from the nucleotide sequence shown in SEQ ID NO:1 or its conserved alternative sequences.

[0031] In some instances, the nucleotide sequence of the PLAGL2 gene is selected from the nucleotide sequence shown in SEQ ID NO:2 or its conserved alternative sequences.

[0032] In some instances, the engineered immune cells are selected from common immune cells such as T cells or NK cells.

[0033] There are no particular requirements for the structure of the chimeric antigen receptor, but a mature CAR structure is preferred. In some examples, the chimeric antigen receptor comprises: an antigen-binding domain, a hinge region, a transmembrane domain, at least one co-stimulatory signaling domain, and a CD3ζ signaling domain; the co-stimulatory signaling domain is selected from CD28, 4-1BB, ICOS, OX40, CD27, or a combination thereof.

[0034] In some instances, the nucleotide sequence of the chimeric antigen receptor comprises a sequence as shown in SEQ ID NO:3 or SEQ ID NO:6.

[0035] In some instances, the nucleotide sequence of the exogenous T-cell receptor comprises the sequence shown in SEQ ID NO:5.

[0036] In some instances, the engineered immune cells include, but are not limited to, CAR-T cells, TCR-T cells, tumor-infiltrating lymphocytes, CAR-NK cells, γδ T cells, or purified NK cells.

[0037] A second aspect of the present invention provides a method for constructing engineered immune cells as described in the first aspect of the present invention, comprising the steps of introducing the PLAGL1 gene and / or the PLAGL2 gene into immune cells in vitro or in vivo and overexpressing them.

[0038] In some instances, the ALX3 gene is introduced into immune cells via viral or non-viral vectors.

[0039] In some instances, the viral vector is a lentiviral vector or a retroviral vector.

[0040] In some instances, the non-viral vector is a transposon system or mRNA.

[0041] A third aspect of the invention provides: a pharmaceutical composition comprising the engineered immune cells described in the first aspect of the invention and a pharmaceutically acceptable carrier or excipient.

[0042] A fourth aspect of the present invention provides the use of the engineered immune cells described in the first aspect of the present invention or the pharmaceutical composition described in the third aspect of the present invention in the preparation of an immunotherapeutic drug.

[0043] In some instances, the disease treated by the drug is selected from tumors, autoimmune diseases, or inflammatory diseases.

[0044] In some instances, the tumor is selected from hematologic malignancies or solid tumors; the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, or multiple sclerosis; and the inflammatory disease is selected from inflammatory bowel disease, chronic inflammation, or graft-versus-host disease.

[0045] The following disclosure provides many different embodiments or examples for implementing various schemes of the present invention. The amino acid sequences corresponding to the nucleic acid sequences SEQ ID NO:1-6 are SEQ ID NO:7-12. Example 1: Construction of PLAGL1 / PLAGL2 gene overexpression CAR-T cells

[0046] 1.1 Construction of CAR and PLAGL1 / PLAGL2 expression vectors (1) Download the CDS sequences of PLAGL1 and PLAGL2 genes from NCBI, and then perform codon optimization on some gene sequences to enhance their expression. These optimization methods include, but are not limited to, human codon usage preference, appropriate GC content, stable mRNA secondary structure, etc., eliminating repetitive sequences and hidden splicing sites and unnecessary restriction enzyme sites, while preventing the depletion of the tRNA library in the cell to obtain the coding sequences (CDS) of human PLAGL1 and PLAGL2 genes. Finally, their nucleic acid sequences are shown as SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0047] (2) Construct a CAR (chimeric antigen receptor) sequence targeting MSLN (mesothelin), wherein the CAR comprises, from N-terminus to C-terminus, a signal peptide, a single-chain antibody (scFv) against tumor-associated antigen, a hinge region, a transmembrane region, a co-stimulatory signaling domain and a CD3ζ signaling domain, and its nucleic acid sequence is shown in SEQ ID NO:3.

[0048] (3) The CAR expression cassette and PLAGL1 / PLAGL2 expression cassette were cloned into a lentiviral expression vector (pCDH vector). The CAR and PLAGL1 / PLAGL2 were linked by a P2A self-cleaving peptide (SEQ ID NO:4) to construct vectors named pLV-CAR-P2A-PLAGL1 and pLV-CAR-P2A-PLAGL2, respectively.

[0049] The sequence SEQ ID NO:1 is as follows:

[0050] The sequence SEQ ID NO:2 is as follows:

[0051] The sequence SEQ ID NO:3 is as follows:

[0052] The sequence SEQ ID NO:4 is: ggatccGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT.

[0053] 1.2 Lentiviral Preparation HEK-293T cells were transfected with the lentiviral vector containing the PLAGL1 / PLAGL2 gene, the psPAX2 vector, and the pMD2.G vector plasmid obtained in Section 1.1. The transfection system was prepared as follows: (1) Centrifuge tube A: Opti-MEM 500 μL + main plasmid 10 μg + pMD2.G 5 μg + psPAX2 5 μg + p3000 40 μL; (2) Centrifuge tube B: Opti-MEM 500 μL + Lipo3000 40 μL. Slowly drop the mixture in centrifuge tube B into centrifuge tube A, gently mix with a pipette, and let stand at room temperature for 15-20 min.

[0054] The transfection system was added to HEK-293T cells. After 5 hours of transfection, the supernatant was discarded, and 10 mL of DMEM complete medium was added to each dish. The cells were then incubated at 37°C with 5% CO2 for 48 hours. The supernatant of the transfected 293T cells was collected and centrifuged at 400g for 5 minutes. The cells were then filtered through a 0.45 μm filter, and the resulting filtrate was the original solution of the recombinant lentivirus. The lentivirus was concentrated using an Ultra-15 centrifuge and filtration device, and centrifuged at 5000 rpm for 50 minutes. The concentrated lentivirus was aliquoted and stored at -80°C for later use.

[0055] 1.3 Lentiviral titer determination Cell counting was performed on the washed and digested HEK-293T cells. The lentiviral concentrate was diluted 10-fold serially (50×, 500×, 5000×, 50000×). 2.5 × 10⁻⁶ cells were collected. 5 Each HEK-293T cell was added to a corresponding centrifuge tube containing 500 μL of culture medium, virus, and cell mixture. After mixing, the mixture was transferred to a 24-well plate and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then replaced. After 48 hours, infected HEK-293T cells were collected, and the proportion of transgene-positive cells was detected by flow cytometry to calculate the viral titer.

[0056] Lentiviral titer calculation method: , where m is the proportion of cells that are positive for transgene expression.

[0057] 1.4 Isolation, activation, and transduction of T cells (1) Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy donors or autologous blood of patients and CD3+ T cells were sorted using CD3 microbeads.

[0058] (2) The sorted T cells were activated with Dynabeads CD3 / CD28 and cultured in T cell culture medium (X-VIVO 15 with 5% human AB serum) containing IL-2 (50 IU / mL).

[0059] (3) After activation for 24-48 hours, concentrated recombinant lentivirus was added at an MOI of 5-50 for transduction. After transduction, the cells were cultured for 6-8 days to obtain PLAGL1-overexpressing CAR-T cells (named CAR-T PLAGL1 cells) and PLAGL2-overexpressing CAR-T cells (named CAR-T PLAGL2 cells). At the same time, CAR-T cells transduced only with CAR but not PLAGL1 / PLAGL2 were prepared as controls (named CAR-T Control cells).

[0060] 1.5 Detection of PLAGL1 / PLAGL2 overexpression Total RNA was extracted from CAR-T cells and CAR-T Control cells, reverse transcribed into cDNA, and then detected by qRT-PCR using PLAGL1 and PLAGL2 specific primers, with GAPDH as an internal control. The results showed that the expression levels of the corresponding mRNAs in CAR-T PLAGL1 and CAR-T PLAGL2 cells were significantly upregulated compared to CAR-T Control cells, with the experimental groups showing an 8-12 fold increase compared to the control group. Figure 1 A\B). Example 2: In vitro expansion capacity of PLAGL1 / PLAGL2 overexpressing CAR-T cells

[0061] The CAR-T cells constructed in Example 1 were expanded and cultured in vitro in complete medium containing IL-2 (100 IU / mL). The cell count was counted and passaged every 2-3 days, and the cells were observed continuously for 14 days.

[0062] Results: Compared with control CAR-T cells, PLAGL1-overexpressing and PLAGL2-overexpressing CAR-T cells showed significantly enhanced proliferative capacity throughout the culture period. Figure 1 C). PLAGL1-CAR-T cells expanded 3.4 times more than the control group on day 14 (P < 0.001); PLAGL2-CAR-T cells expanded 3.2 times more than the control group (P < 0.001). Example 3: Comparison of effector functions of PLAGL1 / PLAGL2 overexpressing CAR-T cells

[0063] To verify whether PLAGL1 / PLAGL2 overexpression affects the direct effector function of CAR-T cells, CAR-T cells were co-cultured with target antigen-positive tumor cells at an effector cell:target cell ratio of 1:1. Fresh target cells were replaced every 72 hours, and the number of effector cells was counted. Four rounds of killing experiments were performed to assess the cumulative killing capacity and effector cell survival rate after multiple rounds of repeated antigen stimulation. The supernatant was collected to detect the secretion level of IFN-γ effector cytokines.

[0064] Results: In the first round of killing, there was no significant difference in the killing rate among the groups (approximately 97%). However, with each round, the killing ability of the control CAR-T cells decreased significantly—from 97.8% in the first round to only 22.9% in the fourth round. In contrast, PLAGL1-CAR-T and PLAGL2-CAR-T cells maintained sustained killing function, maintaining killing rates of approximately 77.6% and 65.8%, respectively, in the fourth round. Figure 1 D).

[0065] The supernatants from the co-cultures of the CAR-T control group were collected after multiple rounds of in vitro cell killing, and the secretion level of IFN-γ was detected using an ELISA kit. The results showed that during the multiple rounds of cell killing, the IFN-γ secretion level in the co-culture supernatant of the CAR-T control group gradually decreased, while the IFN-γ secretion level in the CAR-T PLAGL1 / PLAGL2 group remained relatively stable after each round of cell killing. Figure 1 E). There was no significant difference in IFN-γ between the two groups during the first round of killing, but after multiple rounds of killing, the PLAGL1 / PLAGL2 group showed an overwhelming advantage. This difference pattern fully reflects the unique mechanism by which PLAGL1 / PLAGL2 exerts its effects by maintaining dryness and reducing exhaustion rather than enhancing the initial effect function. Example 4: Expression of exhaustion markers in PLAGL1 / PLAGL2 overexpressing CAR-T cells

[0066] After four rounds of killing experiments, T cells from each experimental group and control group were collected, and the expression of exhaustion-related markers and stemness-related molecules was detected by flow cytometry and RT-qPCR.

[0067] The results showed that the positive rates of exhaustion markers, PLAGL1 overexpression, PLAGL2 overexpression, PD-1, Tim-3, and LAG3 on the surface of CAR-T cells were significantly lower than those in the control group. Figure 1 F).

[0068] Stem cell-related molecules: The mRNA expression level of T cell stem cell-related transcription factor TCF1 in CAR-T cells of all overexpression groups was significantly increased. Figure 1 G). TCF1 is a core transcription factor that maintains the stem-like exhaustion state of T cells. Its high expression is a key molecular marker for T cells to maintain their self-renewal capacity and long-term functional durability. For the first time, it has been confirmed that PLAGL1 and PLAGL2 are involved in the maintenance of T cell stemness and anti-exhaustion effects. Example 5: Anti-tumor effect of PLAGL1 / PLAGL2 overexpressing CAR-T cells in an in vivo tumor model

[0069] A human tumor xenograft model was established subcutaneously in NSG mice. The tumor volume reached approximately 100 mm. 3 Mice were randomly divided into five groups (n=5 / group): (1) PBS control group; (2) control CAR-T cell group; (3) PLAGL1-CAR-T cell group; (4) PLAGL2-CAR-T cell group. Each group received a tail vein injection of the corresponding CAR-T cell (1×10⁻⁶ cells). 6 (cells / mouse), observe tumor growth curves and mouse survival time.

[0070] The following is an analysis of T cell infiltration and exhaustion status in tumor tissue: (1) After the mice were euthanized, tumor tissue was taken and a single-cell suspension was prepared.

[0071] (2) Flow cytometry staining was performed using antibodies against human CD45, CD3, CD8, PD-1, TIM-3, etc., to detect the number and exhaustion status of tumor-infiltrating T cells.

[0072] result: Tumor growth inhibition: Tumors in the PBS group mice continued to grow rapidly; compared with the CAR-T control group, the PLAGL1-CAR-T and PLAGL2-CAR-T groups maintained significant tumor inhibition effects; the PLAGL1-CAR-T group showed the best effect. Figure 2 A).

[0073] CAR-T cells proliferate rapidly upon encountering target cells, resulting in a significant increase in their proportion and number in peripheral blood. The proportion and number of CAR-T cells in mouse peripheral blood directly reflect the viability and proliferative potential of CAR-T cells, and can be used to assess the in vivo expansion and proliferation capacity of CAR-T cells. The number of CAR-T cells in the peripheral blood of the PLAGL1-overexpressing CAR-T cell group was significantly higher than that of the control group ( Figure 2 B) indicates that PLAGL1-overexpressing CART cells have better viability and proliferative potential than the control group.

[0074] The results showed that the number of CD8+ T cells in the tumor tissue of the PLAGL1 / PLAGL2 overexpressing T cell group was significantly higher than that in the control group. Figure 2 C), and the proportion of PD-1 and TIM-3 positive cells in infiltrating T cells was significantly reduced (C). Figure 2 (D) indicates that PLAGL1 / PLAGL2 overexpression promotes T cell infiltration and functional maintenance in tumor tissues. Example 6: Validation of the anti-tumor effect in PLAGL1 / PLAGL2 overexpressing TCR-T cells

[0075] To further verify the effect of PLAGL1 / PLAGL2 overexpression on T cell function, this embodiment applies the PLAGL1 / PLAGL2 overexpression strategy to TCR-T cells. TCR-T cells targeting the tumor antigen NY-ESO-1 (TCR-T) were constructed, and simultaneously overexpressed the PLAGL1 / PLAGL2 genes (PLAGL1-TCR-T / PLAGL2-TCR-T). Control TCR-T cells were also included.

[0076] 6.1 Construction of TCR and PLAGL1 / PLAGL2 expression vectors (1) Download the CDS sequence of PLAGL1 / PLAGL2 gene from NCBI, and then perform codon optimization on some gene sequences to enhance their expression. These optimization methods include, but are not limited to: human codon usage preference, appropriate GC content, stable mRNA secondary structure, elimination of repetitive sequences and hidden splice sites and unnecessary restriction enzyme sites, while preventing the depletion of tRNA library in cells. The coding sequence (CDS) of human PLAGL1 / PLAGL2 gene is obtained, and its nucleic acid sequence is shown in SEQ ID NO:1 / 2.

[0077] (2) Construct a TCR (exogenous T cell receptor) sequence targeting NY-ESO-1, the nucleic acid sequence of which is shown in SEQ ID NO:5. The TCR expression cassette and PLAGL1 / PLAGL2 expression cassette are cloned into a lentiviral expression vector (pCDH vector). The TCR and PLAGL1 / PLAGL2 are linked by a P2A self-cleaving peptide (SEQ ID NO:3). The constructed vector is named pLV-TCR-P2A-PLAGL1 / pLV-TCR-P2A-PLAGL2.

[0078] The sequence SEQ ID NO:5 is:

[0079] 6.2 Lentiviral Preparation HEK-293T cells were transfected with the pLV-TCR-P2A-PLAGL1 / pLV-TCR-P2A-PLAGL2, psPAX2, and pMD2.G vectors containing the PLAGL1 / PLAGL2 genes obtained in Section 6.1. The transfection system was prepared as follows: (1) Centrifuge tube A: Opti-MEM 500 μL + main plasmid 10 μg + pMD2.G 5 μg + psPAX 25 μg + p3000 40 μL; (2) Centrifuge tube B: 500 μL OPti-MEM + 40 μL Lipo3000. Slowly drop the mixture in centrifuge tube B into centrifuge tube A, gently mix with a pipette, and let stand at room temperature for 15-20 min; The transfection system was added to HEK-293T cells. After 5 h of transfection, the supernatant was discarded, and 10 ml of DMEM complete medium was added to each dish. The cells were then incubated at 37°C with 5% CO2 for 48 h. The supernatant of the transfected 293T cells was collected and centrifuged at 400 g for 5 min. The cells were then filtered through a 0.45 μm filter, and the filtrate obtained was the original solution of the recombinant lentivirus. The lentivirus was concentrated using an Ultra-15 centrifuge and filtration device, and centrifuged at 5000 rpm for 50 min. The concentrated lentivirus was aliquoted and stored at -80°C for later use.

[0080] 6.3 Lentiviral titer determination Cell counting was performed on the washed and digested HEK-293T cells. The lentiviral concentrate was diluted 10-fold serially (50×, 500×, 5000×, 50000×). 2.5 × 10⁻⁶ cells were collected. 5 Each HEK-293T cell was added to a corresponding centrifuge tube containing 500 μL of culture medium, virus, and cell mixture. After mixing, the mixture was transferred to a 24-well plate and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then replaced. After 48 hours, infected HEK-293T cells were collected, and the proportion of transgene-positive cells was detected by flow cytometry to calculate the viral titer.

[0081] Lentiviral titer calculation method: , where m is the proportion of cells that are positive for transgene expression.

[0082] 6.4 Isolation, activation, and transduction of T cells (1) Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy donors or autologous blood of patients and CD3+ T cells were sorted using CD3 microbeads.

[0083] (2) The sorted T cells were activated with Dynabeads CD3 / CD28 and cultured in T cell culture medium (X-VIVO 15 with 5% human AB serum) containing IL-2 (50 IU / mL).

[0084] (3) After activation for 24-48 hours, concentrated recombinant lentivirus was added at an MOI of 5-50 for transduction. After transduction, the cells were cultured for 6-8 days to obtain PLAGL1 / PLAGL2 overexpressing TCR-T cells, which were named TCR-T-PLAGL1 / PLAGL2 cells. At the same time, TCR-T cells transduced only TCR without PLAGL1 / PLAGL2 were prepared as a control (named TCR-T Control cells).

[0085] 6.5 Detection of PLAGL1 / PLAGL2 overexpression Total RNA was extracted from PLAGL1-TCR-T / PLAGL2-TCR-T cells and TCR-T Control cells, reverse transcribed into cDNA, and then detected by qRT-PCR using PLAGL1 / PLAGL2-specific primers, with GAPDH as an internal control. The results showed that the expression level of PLAGL1 / PLAGL2 mRNA in PLAGL1-TCR-T / PLAGL2-TCR-T cells was significantly upregulated compared to that in TCR-T Control cells. Figure 3 A\3B).

[0086] 6.6 In vitro proliferation capacity detection (1) TCR-T Control cells and PLAGL1-TCR-T / PLAGL2-TCR-T cells were taken and co-cultured with tumor cells A549-mesothelin at a ratio of 1:1.

[0087] (2) On day 3 of co-culture, the cultured T cells were counted, and the differences in the fold expansion of different TCR-T cells were compared. It was found that the fold expansion of PLAGL1-TCR-T / PLAGL2-TCR-T was significantly higher than that of the TCR-T Control group, such as Figure 3 The results showed that the proliferation rate of PLAGL1-TCR-T / PLAGL2-TCR-T cells was significantly higher than that of TCR-T control cells. 6.7 Establish a human tumor xenograft model subcutaneously in NSG mice.

[0088] When the tumor volume reaches approximately 100 mm 3Mice were randomly divided into five groups (n=5 / group): (1) PBS control group; (2) control TCR-T cell group; (3) PLAGL1-TCR-T cell group; (4) PLAGL2-TCR-T cell group. Each group received a tail vein injection of the corresponding TCR-T cells (1×10⁻⁶). 6 (cells / mouse), observe tumor growth curves and mouse survival time.

[0089] Results: Tumors in the PBS group mice continued to grow rapidly; compared with the TCR-T control group, the PLAGL1-TCR-T and PLAGL2-TCR-T groups maintained significant tumor inhibition effects; the PLAGL1-TCR-T group showed the best effect. Figure 3 D).

[0090] 6.8 Comparison of TCR-T numbers in peripheral blood of mice TCR-T cells proliferate rapidly upon encountering target cells, resulting in a significant increase in their proportion and number in peripheral blood. The proportion and number of TCR-T cells in mouse peripheral blood directly reflect the viability and proliferative potential of TCR-T cells, and can be used to assess the in vivo expansion and proliferation capacity of TCR-T cells. The number of TCR-T cells in the peripheral blood of the PLAGL1-overexpressing TCR-T cell group was significantly higher than that of the control group ( Figure 3 E) indicates that PLAGL1-overexpressing TCR-T cells have better viability and proliferative potential than the control group.

[0091] 6.9 Detection of T cell infiltration and exhaustion status in tumor tissue (1) After the mice were euthanized, tumor tissue was taken and a single-cell suspension was prepared.

[0092] (2) Flow cytometry staining was performed using antibodies against human CD45, CD3, CD8, PD-1, TIM-3, etc., to detect the number and exhaustion status of tumor-infiltrating T cells.

[0093] (3) The results showed that the number of CD8+ T cells in the tumor tissue of the PLAGL1 / PLAGL2 overexpressing T cell group was significantly higher than that in the control group. Figure 3 F), and the proportion of PD-1 and TIM-3 positive cells in infiltrating T cells was significantly reduced (F). Figure 3 (G), indicating that PLAGL1 / PLAGL2 overexpression promotes T cell infiltration and functional maintenance in tumor tissue. Example 7: Construction of TIL cells overexpressing PLAGL1 / PLAGL2

[0094] 3.1 Isolation and Culture of TIL Cells (1) Take tumor tissue blocks from the tumor tissue surgically removed from patients with solid tumors, cut them into pieces, and prepare single-cell suspensions by digestion with collagenase / DNase.

[0095] (2) Inoculate the tumor cell suspension into TIL medium containing a high dose of IL-2 (e.g., 6000 IU / mL) and culture it in a 37°C, 5% CO2 incubator. Replace half of the medium and supplement with IL-2 every 2-3 days.

[0096] (3) After culturing for approximately 14-28 days, TIL cells will expand to a sufficient number (e.g., 1×10⁻⁶). 8 (The above) was further amplified using a rapid amplification protocol.

[0097] 3.2 PLAGL1 / PLAGL2 overexpression transduction and functional detection in TIL cells (1) Construct a lentiviral expression vector to express the PLAGL1 / PLAGL2 gene, driven by the EF1α promoter, and named pLV-EF1α-PLAGL1 / pLV-EF1α-PLAGL2.

[0098] (2) Packaging lentivirus (method as in Example 1 1.2).

[0099] (3) The expanded TIL cells were activated with anti-CD3 / CD28 antibody. After activation for 24-48 hours, recombinant lentivirus was added at an MOI of 5-50 for transduction. After transduction, the cells were cultured for another 7-10 days to obtain PLAGL1 / PLAGL2 overexpressing TIL cells, which were named TIL-PLAGL1 / TIL-PLAGL2 cells. At the same time, TIL cells transduced with empty vector were prepared as a control (named TIL-Control cells).

[0100] 3.3 Detection of PLAGL1 / PLAGL2 overexpression The expression levels of TIL-PLAGL1 / TIL-PLAGL2 in TIL-PLAGL1 / TIL-PLAGL2 cells were detected by qRT-PCR (method as in Example 1.1.5). The results showed that the expression levels of PLAGL1 / PLAGL2 in TIL-PLAGL1 / TIL-PLAGL2 cells were significantly higher than those in TIL-Control cells (…). Figure 4 A).

[0101] 3.4 TIL in vitro amplification detection Cells from each group were expanded in vitro in a complete culture medium containing IL-2 (100 IU / mL). Cell counts were performed every 2-3 days, and observations were conducted continuously for 21 days.

[0102] The results are as follows Figure 4B: T cell expansion: PLAGL1 / PLAGL2 overexpressing T cells expanded 1.5 times more than the control group on day 14 (P < 0.01); and 2.4 times more than the control group on day 21 (P < 0.001). Figure 4 C).

[0103] 3.5 In vitro cytotoxic activity assay Take organoids embedded in matrix gel, add effector cells to each well (calculate the number of cells according to the preset effector-target ratio), and mix gently.

[0104] Set the following molecular groups: Target organ control group: organoids Effector cell control group: TIL Control group: Organoid + TIL-control group Experimental group 1: Organoid + TIL-PLAGL1 group; Experimental group 2: Organoids + TIL-PLAGL2 group; The co-culture plates were incubated at 37°C in a 5% CO2 incubator. According to the experimental design, the co-culture supernatant was collected after 24 hours. Following the LDH detection kit instructions, the absorbance was read at 490 nm using a microplate reader, and the killing rate was calculated using the following formula:

[0105] The results showed that the organoid killing rate of cells overexpressing TIL-PLAGL1 / TIL-PLAGL2 was significantly higher than that of TIL Control cells. Figure 4 C) The amplification fold and killing function of PLAGL1-TIL and PLAGL2-TIL were significantly better than those of the control group, indicating that PLAGL1 / PLAGL2 overexpression is also suitable for TIL therapy. Example 8: Effects of PLAGL1 / PLAGL2 overexpression on the anti-exhaustion function of NK cells and CAR-NK cells

[0106] CD56⁺NK cells were isolated from PBMCs of healthy individuals and constructed into the following four groups: (1) CAR-NK cells targeting CD19 (CAR-NK) (the nucleic acid sequence of which is shown in SEQ ID NO:6); (2) PLAGL1 / PLAGL2 overexpressing CAR-NK cells (PLAGL1-CAR-NK / PLAGL2-CAR-NK), and the expansion rate of each group was compared.

[0107] The sequence SEQ ID NO:6 is:

[0108] Results: Overexpression of both PLAGL1 and PLAGL2 effectively promoted the in vitro expansion of CAR-NK cells. Figure 5 A), and this promoting effect was already apparent early and lasted until day 21; among them, PLAGL1 had a slightly better growth-promoting effect than PLAGL2. CAR-NK cells overexpressing PLAGL2 were significantly better than the control group, but slightly lower than the PLAGL1 group. The values ​​on days 7, 14, and 21 were 81.0±4.8, 216.0±19.8, and 324.0±24.7, respectively. On day 21, it was about 2.7 times that of the control group, and about 10% to 15% lower than the PLAGL1 group.

[0109] To verify the difference in CAR-NK killing function after PLAGL1 / PLAGL2 overexpression, NK cells from each group were co-cultured with Raji cells (target antigen-positive tumor cells) for multiple rounds of killing experiments (effect-to-target ratio 1:1, target cells replaced every 72 hours, for a total of 4 rounds). Figure 5 B). Based on data from four rounds of continuous killing experiments, the killing activity of all three groups of CAR-NK cells decreased with increasing rounds, but the magnitude of the decrease and the maintenance level differed significantly. The control group (Ctrl-CAR-NK) showed a killing rate of 71.8%, 55.4%, 33.9%, and 20.9% in rounds 1 to 4, respectively, exhibiting a sharp decline, reaching approximately 29% of the initial level by round 4. In contrast, the killing rates of PLAGL1-overexpressing CAR-NK cells were 71.3%, 63.3%, 62.9%, and 58.5% in rounds 1 to 4, respectively, maintaining high and stable activity throughout, decreasing by only about 12.8 percentage points from round 1 to 4, and almost remaining the same in round 3 compared to round 2 (62.9% vs 63.3%), demonstrating extremely strong and sustained killing ability. CAR-NK cells overexpressing PLAGL2 showed 72.0%, 63.3%, 57.4%, and 52.4% cytokines in rounds 1 to 4, respectively. While still superior to the control group, the reduction was greater than that in the PLAGL1 group (approximately 19.6 percentage points), especially in rounds 3 and 4 where the percentages were significantly lower than in the PLAGL1 group (57.4% vs 62.9%, 52.4% vs 58.5%). These results indicate that PLAGL1 overexpression not only effectively promotes the proliferation of CAR-NK cells (as mentioned above) but also significantly enhances their durable cytotoxic function. While PLAGL2 also showed improvement, the effect was slightly less pronounced than that of PLAGL1.

[0110] Comparison of exhaustion status after multiple rounds of NK cell killing of target cells: The mean PD-1⁺ positivity rate of the control group (Ctrl-CAR-NK) was 76.5% (range 73.6%–80.2%), and the mean Tim-3⁺ positivity rate was 71.5% (68.7%–73.0%), indicating that unmodified CAR-NK cells exhibited a significant exhaustion phenotype after long-term culture or continuous stimulation. In contrast, the PD-1⁺ and Tim-3⁺ positivity rates of CAR-NK cells overexpressing PLAGL1 decreased to 38.6% and 38.1%, respectively, a decrease of nearly half; the PD-1⁺ and Tim-3⁺ positivity rates of CAR-NK cells overexpressing PLAGL2 were 42.9% and 43.3%, respectively, also significantly lower than those of the control group (Ctrl-CAR-NK). Figure 5 C). The positive rates of the PLAGL1 group on both markers were slightly lower than those of the PLAGL2 group (PD-1: 38.6% vs 42.9%; Tim-3: 38.1% vs 43.3%), suggesting that PLAGL1 has a better effect in inhibiting the expression of exhaustion markers. Overall, overexpression of both PLAGL1 and PLAGL2 can effectively downregulate the levels of PD-1 and Tim-3 in CAR-NK cells, thereby delaying or alleviating cell exhaustion. Among them, the effect of PLAGL1 is more prominent, which is consistent with its better proliferative capacity and sustained killing function mentioned above, further supporting that PLAGL1 modification can significantly enhance the anti-exhaustion ability and effector function maintenance of CAR-NK cells.

[0111] The above results indicate that PLAGL1 / PLAGL2 overexpression not only enhances the anti-exhaustion ability of T cell-based products, but is also applicable to NK cells and CAR-NK cells, significantly improving the durable killing function of NK cells after repeated antigen exposure.

[0112] References [1] Baessler A, Vignali DAA. T Cell Exhaustion. Annu Rev Immunol,2024, 42: 179-206.

[0113] [2] Dong C, et al. Regulators of CD8⁺T cell exhaustion. Nat RevImmunol, 2025.

[0114] [3] Zhang YX, Pan YZ. Research progress in T-cell exhaustion. ChineseJournal of Pathophysiology, 2024, 40(8): 1548-1553.

[0115] [4] Ngiow SF, et al. PD-1 and LAG-3 synergistically foster T cellexhaustion. Signal Transduct Target Ther, 2024, 9: 291.

[0116] [5] Wei J, et al. Mechanisms of CAR-T cell exhaustion and itsapplication for next-generation CAR-T cells to overcome solid tumors. DrugDiscov Ther, 2025, 4(2): 93-106.

[0117] [6] Wen SQ, et al. TCF-1 maintains CD8⁺ T cell stemness in tumormicroenvironment. J Leukoc Biol, 2021, 110(3): 485-491.

[0118] [7] Ni L, et al. Potential mechanisms of cancer stem-like progenitorT-cell bio-behaviours. Clin Transl Med, 2024, 14: e1817.

[0119] [8] Lynn RC, et al. c-Jun overexpression in CAR T cells inducesexhaustion resistance. Nature, 2019, 576(7786): 293-300.

[0120] [9] Snyder RA, et al. Modulating AP-1 enables CAR T cells toestablish an intratumoral stemlike reservoir and overcomes resistance to PD-1blockade. Sci Immunol, 2026, 11(105): eadw7685.

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[10] Spengler D, et al. A novel zinc finger protein, ZAC, is a potenttranscriptional activator and induces apoptosis and cell cycle arrest. J BiolChem, 1997, 272(36): 22578-22585.

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[11] Varrault A, et al. ZAC1 is a transcriptional coactivator for p53and induces apoptosis. Proc Natl Acad Sci USA, 1998, 95(15): 8835-8840.

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[12] Valleley EM, et al. PLAGL1 encodes a zinc finger DNA-bindingtranscription factor that can regulate apoptosis and cell cycle arrestconcurrently. Hum Mol Genet, 2007, 16(3): 453-460.

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[13] The Zinc Finger Transcription Factor PLAGL2 Enhances Stem CellFate and Activates Expression of ASCL2 in Intestinal Epithelial Cells. 2018.

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[14] PLAGL2 promotes the proliferation and migration of diffuse largeB-cell lymphoma cells via Wnt / β-catenin pathway. Ann Clin Lab Sci, 2022.

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[15] Ferreira LM, et al. A tissue-specific promoter derived from aSINE retrotransposon drives biallelic expression of PLAGL1 in humanlymphocytes. PLoS One, 2017, 12(9): e0185678.

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[16] Kamiya M, et al. The cell cycle control gene ZAC / PLAGL1 isimprinted--a strong candidate gene for transient neonatal diabetes. Hum MolGenet, 2000, 9(3): 453-460.

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[17] PLAGL2 is a characteristic marker of mucinous colorectal cancerassociated with MUC2 expression. Cell Biochem Funct.

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[18] National Center for Biotechnology Information. PLAGL2 PLAG1 likezinc finger 2 [Homo sapiens (human)]. Bethesda (MD): NCBI.

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[19] Carpenter RO, et al. Recursive antigen exposure in vitro revealsextended effector function, viability, and proliferation capacity in BCMA CART cells. Blood, 2018, 132(Suppl 1): 1976.

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[20] Fischer J, et al. Serial killing assay using longitudinalimpedance-based tumor cell viability measurement. J Vis Exp, 2025, (226):e69623.

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[21] Fraietta JA, et al. In vitro re-challenge of CAR T cells. Methods Cell Biol, 2022, 167: 231-250.

[0133] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. An engineered immune cell, characterized in that, The engineered immune cells overexpress PLAGL1 and / or PLAGL2 or their functionally active variants.

2. The engineered immune cells according to claim 1, characterized in that, The engineered immune cells also contain chimeric antigen receptors or exogenous T cell receptors capable of recognizing target antigens; and / or The nucleotide sequence of the PLAGL1 gene is selected from the nucleotide sequence shown in SEQ ID NO:1 or its conserved alternative sequences; and / or The nucleotide sequence of the PLAGL2 gene is selected from the nucleotide sequence shown in SEQ ID NO:2 or its conserved alternative sequences; and / or The engineered immune cells are selected from T cells or NK cells.

3. The engineered immune cells according to claim 2, characterized in that, The chimeric antigen receptor comprises: an antigen-binding domain, a hinge region, a transmembrane domain, at least one co-stimulatory signaling domain, and a CD3ζ signaling domain; the co-stimulatory signaling domain is selected from CD28, 4-1BB, ICOS, OX40, CD27, or a combination thereof.

4. The engineered immune cells according to claim 3, characterized in that, The nucleotide sequence of the chimeric antigen receptor comprises the sequence shown in SEQ ID NO:3 or SEQ ID NO:6; or the nucleotide sequence of the exogenous T cell receptor comprises the sequence shown in SEQ ID NO:

5.

5. The engineered immune cells according to any one of claims 1-4, characterized in that, The engineered immune cells are selected from CAR-T cells, TCR-T cells, tumor-infiltrating lymphocytes, CAR-NK cells, γδ T cells, or purified NK cells.

6. The method for constructing engineered immune cells according to any one of claims 1 to 5, characterized in that, include: The steps of introducing the PLAGL1 gene and / or PLAGL2 gene into immune cells in vitro or in vivo and overexpressing them.

7. The construction method according to claim 6, characterized in that, The PLAGL1 and / or PLAGL2 genes are introduced into immune cells via viral or non-viral vectors; the viral vector is a lentiviral vector or a retroviral vector; the non-viral vector is a transposon system or mRNA.

8. A pharmaceutical composition, characterized in that, It comprises the engineered immune cells as described in any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient.

9. The use of the engineered immune cells according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 8 in the preparation of an immunotherapeutic drug.

10. The application according to claim 9, characterized in that, The diseases treated by the drug are selected from tumors, autoimmune diseases, or inflammatory diseases.

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