Immune cells overexpressing alx3 and uses thereof

Abstract

The application belongs to the field of biological medicine and immunotherapy, and discloses immune cells overexpressing ALX3 and application thereof. The application discloses a novel anti-exhaustion use of ALX3 gene overexpression in CAR-T and TCR-T cells, and by endowing the engineered T cells with stronger stemness maintenance ability and lower exhaustion tendency, the ALX3 overexpression strategy is expected to significantly improve the efficacy of adoptive T cell immunotherapy in solid tumors and chronic inflammatory diseases. Compared with the existing anti-exhaustion strategies, the ALX3 overexpression shows a unique phenotype of "controlling initial activation and maintaining function for a long time", which can improve long-term efficacy and reduce the risk of CRS and other toxicities.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and immunotherapy technology, specifically relating to immune cells that overexpress ALX3 and their applications. Background Technology

[0002] Currently, engineered immunocellular therapies, represented by chimeric antigen receptor T cells (CAR-T), T cell receptor-engineered T cells (TCR-T), and CAR-NK cells, have demonstrated significant clinical efficacy in the treatment of malignant tumors (especially hematologic malignancies). However, traditional engineered immune cells still face many bottlenecks when dealing with solid tumors or complex in vivo immune microenvironments, such as insufficient infiltration capacity in the tumor microenvironment, easy immune exhaustion under continuous antigen stimulation, and poor survival and proliferation persistence in vivo. These limitations severely restrict further improvement in their clinical therapeutic effects.

[0003] Cell exhaustion is a state of immune cell dysfunction caused by chronic, continuous stimulation, characterized by severely impaired proliferation, gradual loss of effector function, increased co-expression of multiple inhibitory receptors (such as PD-1, Tim-3, and LAG3), and a shift in metabolic pattern from oxidative phosphorylation to glycolysis. In cell therapies such as CAR-T, CAR-NK, TCR-T, and TIL, cells reinfused into the body or directly modified cells in vivo (in vivo CAR / TCR-T) are subjected to continuous stimulation and are highly susceptible to entering a state of exhaustion characterized by poor cytotoxicity, low proliferation, and short survival time, resulting in poor therapeutic efficacy.

[0004] Enhancing the "stemness" of cells to improve their self-renewal capacity, multi-lineage differentiation potential, and functional durability is an effective strategy for improving the efficacy of cell therapy. [9]

[10] For example, stem-like T cell subsets can sustain immune responses through self-renewal and replenishment of terminally differentiated progeny. Current research attempts to improve cell function and reduce exhaustion through genetic engineering. For instance, knocking out genes such as SHP-1 / 2, PTPN2, and SH2D1A can reduce T cell exhaustion; or co-expressing transcription factors such as c-Jun in CAR-T cells can endow them with anti-exhaustion properties, thereby improving the efficacy of cell therapy.

[0005] The ALX3 (ALX homeobox 3) gene encodes a nucleoprotein with a homeobox DNA-binding domain, which functions as a transcriptional regulator involved in cell type differentiation and development. To date, there are no reports of ALX3 gene expression in immune cells improving the efficacy of cell therapy. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide immune cells overexpressing ALX3 and their applications.

[0007] The technical solution adopted in this invention is: A first aspect of the invention provides: an engineered immune cell that overexpresses ALX3 or a functionally active variant 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 ALX3 gene is selected from the nucleotide sequence shown in SEQ ID NO:1 or its conserved alternative sequences.

[0010] In some instances, the engineered immune cells are selected from T cells or NK cells.

[0011] 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.

[0012] In some instances, the nucleotide sequence of the chimeric antigen receptor comprises the sequence shown in SEQ ID NO:2; or the nucleotide sequence of the exogenous T cell receptor comprises the sequence shown in SEQ ID NO:4.

[0013] In some instances, the engineered immune cells are selected from CAR-T cells, TCR-T cells, tumor-infiltrating lymphocytes, CAR-NK cells, NK cells, γδ T cells, DC cells, or macrophages.

[0014] 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 step of introducing the ALX3 gene into immune cells in vitro or in vivo and causing it to be overexpressed.

[0015] In some instances, the ALX3 gene is introduced into immune cells via a viral vector or a non-viral vector; the viral vector is a lentiviral vector or a retroviral vector; the non-viral vector is a transposon system or mRNA.

[0016] 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.

[0017] 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.

[0018] In some instances, the disease treated by the drug is selected from tumors, autoimmune diseases, or inflammatory diseases.

[0019] The beneficial effects of this invention are: This invention reveals a novel application of ALX3 gene overexpression in anti-exhaustion techniques in CAR-T and TCR-T cells. By endowing engineered T cells with stronger stemness maintenance capabilities and lower exhaustion tendency, the ALX3 overexpression strategy holds promise for significantly improving the efficacy of adoptive T-cell immunotherapy in solid tumors and chronic inflammatory diseases. Compared with existing anti-exhaustion strategies, ALX3 overexpression exhibits a unique phenotype of "restraining initial activation and maintaining function persistently," potentially reducing the risk of toxicities such as CRS while improving long-term efficacy. Attached Figure Description

[0020] Figure 1 Figure 1 shows the construction and testing of ALX3-overexpressing CAR-T cells. Figure 2 shows the comparison of ALX3 expression levels, Figure 3 shows the in vitro expansion curve of ALX3-overexpressing CAR-T cells, Figure 4 shows the results of multiple rounds of killing experiments, Figure 5 shows the expression detection of exhaustion markers (PD-1, Tim-3, LAG3) after the fourth round of killing, and Figure 6 shows the amount of IFNγ secreted in the supernatant of multiple rounds of killing experiments.

[0021] Figure 2 The figures show the anti-tumor effects of ALX3-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 positive ratio of PD-1 and TIM-3 in infiltrating T cells in tumor tissue. Figure C compares the number of infiltrating CD8+ T cells in tumor tissue. Figure D compares the number of CAR-T cells in peripheral blood.

[0022] Figure 3 Figure 1 shows the construction and testing of ALX3-overexpressing TCR-T cells. Figure 2 shows the comparison of ALX3 expression levels, Figure 3 shows the in vitro expansion curve of ALX3-overexpressing TCR-T cells, Figure 4 shows the results of multiple rounds of killing experiments, Figure 5 shows the expression detection of exhaustion markers (PD-1, Tim-3, LAG3) after the fourth round of killing, and Figure 6 shows the amount of IFNγ secreted in the supernatant of multiple rounds of killing experiments.

[0023] Figure 4The figures show the anti-tumor effects of ALX3-overexpressing TCR-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 compares the number of CD8+ T cells infiltrating the tumor tissue. Figure C shows the positive ratio of PD-1 and TIM-3 in infiltrating T cells in the tumor tissue. Figure D compares the number of TCR-T cells in peripheral blood.

[0024] Figure 5 Figure 1 shows the construction and in vitro testing of ALX3-overexpressing TIL cells. Figure A shows the comparison of ALX3 expression levels, and Figure B shows the results of the in vitro TIL killing experiment.

[0025] Figure 6 Figure A shows the in vitro expansion curves of ALX3-overexpressing T cells and NK cells. Figure B shows the in vitro expansion curves of ALX3-overexpressing T cells and ALX3-overexpressing NK cells. Detailed Implementation

[0026] A first aspect of the invention provides: an engineered immune cell that overexpresses ALX3 or a functionally active variant thereof.

[0027] In some instances, the engineered immune cells also contain chimeric antigen receptors or exogenous T-cell receptors capable of recognizing target antigens.

[0028] In some instances, the nucleotide sequence of the ALX3 gene is selected from the nucleotide sequence shown in SEQ ID NO:1 or its conserved alternative sequences.

[0029] In some instances, the engineered immune cells are selected from common immune cells such as T cells or NK cells.

[0030] 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.

[0031] In some instances, the nucleotide sequence of the chimeric antigen receptor comprises the sequence shown in SEQ ID NO:2; or the nucleotide sequence of the exogenous T cell receptor comprises the sequence shown in SEQ ID NO:4.

[0032] 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, NK cells, γδ T cells, DC cells, or macrophages.

[0033] 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 step of introducing the ALX3 gene into immune cells in vitro or in vivo and causing it to be overexpressed.

[0034] In some instances, the ALX3 gene is introduced into immune cells via viral or non-viral vectors.

[0035] In some instances, the viral vector is a lentiviral vector or a retroviral vector.

[0036] In some instances, the non-viral vector is a transposon system or mRNA.

[0037] 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.

[0038] 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.

[0039] In some instances, the disease treated by the drug is selected from tumors, autoimmune diseases, or inflammatory diseases.

[0040] 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:1-4 are SEQ ID NO:5-8. Example 1: Construction and functional detection of CAR-T cells overexpressing ALX3

[0041] 1.1 Construction of CAR and ALX3 expression vectors (1) Download the CDS sequence of the ALX3 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 the tRNA library in the cell to obtain the coding sequence (CDS) of the human ALX3 gene, whose nucleic acid sequence is shown in SEQ ID NO:1.

[0042] (2) Construct a CAR sequence targeting MSLN, wherein the CAR comprises, from N-terminus to C-terminus, a signal peptide, a single-chain antibody against tumor-associated antigen (scFv), 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:2.

[0043] (3) The CAR expression cassette and the ALX3 expression cassette were cloned into a lentiviral expression vector (pCDH vector). The CAR and ALX3 were linked by a P2A self-cleaving peptide (SEQ ID NO:3). The constructed vector was named pLV-CAR-P2A-ALX3.

[0044] The sequence SEQ ID NO:1 is as follows:

[0045] The sequence SEQ ID NO:2 is as follows:

[0046] The sequence SEQ ID NO:3 is as follows: ggatccGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT.

[0047] 1.2 Lentiviral Preparation HEK-293T cells were transfected with pLV-CAR-P2A-ALX3, psPAX2, and pMD2.G vectors containing the ALX3 gene 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. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, and after being gently mixed with a pipette, it was allowed to stand at room temperature for 15-20 min. 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.

[0048] 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 HEK-293T cells were added to corresponding centrifuge tubes, each containing 500 μl of culture medium, virus, and cell mixture. After thorough mixing, the mixture was transferred to 24-well plates 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 using flow cytometry to calculate the viral titer. Lentiviral titer calculation method: , where m is the proportion of cells that are positive for transgene expression.

[0049] 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.

[0050] (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).

[0051] (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 CAR-T cells overexpressing ALX3, which were named CAR-T ALX3 cells. At the same time, CAR-T cells transduced only CAR and not ALX3 were prepared as a control (named CAR-T Control cells).

[0052] 1.5 Detection of ALX3 overexpression Total RNA was extracted from CAR-T ALX3 cells and CAR-T Control cells, reverse transcribed into cDNA, and then detected by qRT-PCR using ALX3-specific primers, with GAPDH or β-actin as internal controls. The results showed that the expression level of ALX3 mRNA in CAR-T ALX3 cells was significantly upregulated compared to CAR-T Control cells. Figure 1 A).

[0053] 1.6 In vitro proliferation capacity detection (1) CAR-T Control cells and CAR-T ALX3 cells were taken separately. During the culture process, T cells were counted, and the differences in the fold expansion of different CAR-T cells were compared. It was found that the fold expansion of CAR-T ALX3 cells was significantly higher than that of the CAR-T Control group, such as... Figure 1 Results B showed that the proliferation rate of CAR-T ALX3 cells was significantly higher than that of CAR-T Control cells ( ). Figure 1 B).

[0054] 1.7 In vitro cytotoxic activity assay To compare the differences in tumor cell killing function between CAR-T Control cells and CAR-T ALX3 cells, CAR-T cells were co-cultured with A549-mesothelin tumor cells for multiple rounds of tumor cell killing, and the differences in tumor cell killing function among the CAR-T groups were compared. The experimental method is as follows: 2 × 10⁶ cells were used... 4A549-mesothelin cells were seeded in 48-well plates, and 100 μL of CAR-T cell suspension was added to each well at an effector-to-target ratio of 1:2. After incubation at 37°C and 5% CO2 for 48 hours, images were taken under a microscope to compare the differences in tumor cell killing effects among the groups. The killed T cells were then removed for further rounds of killing, and the differences in multi-target cell killing function of CAR-T cells among the groups were compared after multiple rounds of killing. Results are as follows: Figure 1 As shown in Figure C, there was no significant difference in the kill rate between the two groups in the first round of CAR-T cell stimulation (approximately 95%). However, with each round, the killing ability of the control CAR-T cells decreased significantly—from 95% in the first round to only 28% in the fourth round, indicating that the control CAR-T cells rapidly entered a state of exhaustion under repeated antigen stimulation. In contrast, ALX3-CAR-T cells maintained sustained killing function, maintaining a kill rate of approximately 90% in the fourth round, which was three times that of the control group. Flow cytometry analysis showed that the co-expression ratios of exhaustion markers PD-1, Tim-3, and LAG3 in ALX3-CAR-T cells were significantly lower than those in the control group (reduced by approximately 52%, 47%, and 45%, respectively, P < 0.01). These results indicate that ALX3 overexpression can effectively inhibit the functional exhaustion of CAR-T cells after multiple rounds of antigen stimulation, enabling engineered T cells to maintain stronger stemness characteristics, higher survival rates, and more durable anti-tumor functions. Figure 1 D).

[0055] 1.8 Cytokine secretion detection 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 secretion of IFN-γ in the co-culture supernatant of the CAR-T control group gradually decreased, while the secretion of IFN-γ in the CAR-T ALX3 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 ALX3 group showed an overwhelming advantage. This pattern of difference fully reflects the unique mechanism by which ALX3 works by maintaining dryness and reducing exhaustion rather than enhancing the initial effect function.

[0056] 1.9 In vivo tumor treatment capability detection (1) The tumor cell line A549-mesothelin was subcutaneously injected into NSG immunodeficient mice to establish a tumor-bearing model.

[0057] (2) When the tumor volume reaches approximately 50-100 mm 3 At that time, the tumor-bearing mice were randomly divided into the following three groups: Group 1: PBS control group (PBS injected via tail vein) Group 2: Control T cell group (CAR-T control cells injected via tail vein) Group 3: ALX3 overexpressing T cell group (CAR-T ALX3 cells injected via tail vein) (3) Measure tumor volume and mouse weight every 3-4 days and plot tumor growth curve.

[0058] (4) The results showed that, compared with the CAR-T control group, the tumor volume of mice in the ALX3 overexpressing T cell group was significantly reduced, and tumor growth was effectively inhibited. Figure 2 A) 1.10 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.

[0059] (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.

[0060] (3) The results showed that the number of CD8+ T cells in the tumor tissue of the ALX3 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 (B) indicates that ALX3 overexpression promotes T cell infiltration and functional maintenance in tumor tissues.

[0061] 1.11 Comparison of CART numbers in peripheral blood of mice 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 ALX3-overexpressing CAR-T cell group was significantly higher than that of the control group (…). Figure 2 (D) indicates that the vitality and proliferative potential of ALX3-overexpressing CART cells are superior to those of the control group. Example 2: Validation of the anti-exhaustion effect of ALX3-overexpressing TCR-T cells

[0062] In addition to CAR-T cells, this embodiment applies an ALX3 overexpression strategy to TCR-T cells. TCR-T cells targeting the tumor antigen NY-ESO-1 (TCR-T) were constructed, and simultaneously overexpressed the ALX3 gene (ALX3-TCR-T). Control TCR-T cells were also included.

[0063] 2.1 Construction of TCR and ALX3 expression vectors (1) Download the CDS sequence of the ALX3 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 the tRNA library in the cell to obtain the coding sequence (CDS) of the human ALX3 gene, whose nucleic acid sequence is shown in SEQ ID NO:1.

[0064] (2) Construct a TCR sequence targeting NY-ESO1, the nucleic acid sequence of which is shown in SEQ ID NO:4. The TCR expression cassette and ALX3 expression cassette are cloned into a lentiviral expression vector (pCDH vector). TCR and ALX3 are linked by a P2A self-cleaving peptide (SEQ ID NO:3). The constructed vector is named pLV-TCR-P2A-ALX3.

[0065] The sequence SEQ ID NO:4 is as follows:

[0066] 2.2 Lentiviral Preparation HEK-293T cells were transfected with pLV-TCR-P2A-ALX3, psPAX2, and pMD2.G vectors containing the ALX3 gene obtained in Section 2.1. 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. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, and after being gently mixed with a pipette, it was allowed to stand at room temperature for 15-20 min. 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.

[0067] 2.3 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 HEK-293T cells were added to corresponding centrifuge tubes, each containing 500 μl of culture medium, virus, and cell mixture. After thorough mixing, the mixture was transferred to 24-well plates 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 using flow cytometry to calculate the viral titer. The lentivirus titer was calculated as follows: Virus titer = (m × 2.5 × 10 × dilution factor) / transfection volume, where m is the proportion of transgene-positive cells.

[0068] 2.4 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.

[0069] (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).

[0070] (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 ALX3-overexpressing TCR-T cells, which were named TCR-T-ALX3 cells. At the same time, TCR-T cells that were transduced only for CAR and not for ALX3 were prepared as a control (named TCR-T Control cells).

[0071] 2.5 Detection of ALX3 overexpression Total RNA was extracted from TCR-T-ALX3 cells and TCR-T Control cells, reverse transcribed into cDNA, and then detected by qRT-PCR using ALX3-specific primers, with GAPDH or β-actin as internal controls. The results showed that the expression level of ALX3 mRNA in TCR-T-ALX3 cells was significantly upregulated compared to TCR-T Control cells. Figure 3 A).

[0072] 2.6 In vitro proliferation capacity detection (1) TCR-T Control cells and TCR-T-ALX3 cells were taken and co-cultured with tumor cells A549-mesothelin at a ratio of 1:1.

[0073] (2) T cell counts were performed on days 3, 5, and 7 of co-culture, and the fold increase of different TCR-T cells was compared. It was found that the fold increase of TCR-T-ALX3 cells was significantly higher than that of the TCR-T Control group. Figure 3 Results B showed that the proliferation rate of TCR-T-ALX3 cells was significantly higher than that of TCR-T Control cells. 2.7 In vitro cytotoxic activity assay To compare the tumor cell killing function of TCR-T Control cells and TCR-T ALX3 cells, TCR-T cells were co-cultured with A375 tumor cells for multiple rounds of tumor cell killing, and the differences in tumor cell killing function among the TCR-T groups were compared. The experimental method is as follows: 2 × 10⁶ cells were used... 4A375 cells were seeded in 48-well plates, and 100 μL of TCR-T cell suspension was added to each well at an effector-to-target ratio of 1:2. After incubation at 37°C and 5% CO2 for 48 hours, images were taken under a microscope to compare the differences in tumor cell killing effects among the groups. Killed T cells were then removed for further rounds of killing, and the differences in multi-target cell killing function of TCR-T cells among the groups were compared after multiple rounds of killing. Results are as follows: Figure 2 As shown in C, during multiple rounds of killing, the killing ability of T cells in the TCR-T Control group gradually decreased, while the killing efficiency of T cells in the TCR-T ALX3 group remained at a high level after 4 rounds of killing. Figure 3 C). In the fourth round, it maintained a kill rate of approximately 90%, three times that of the control group. Flow cytometry analysis showed that the co-expression ratios of exhaustion markers PD-1, Tim-3, and LAG3 in ALX3-TCR-T cells were significantly lower than those in the control group. Figure 3 D). The above results indicate that ALX3 overexpression can effectively inhibit the functional exhaustion of TCR-T cells after multiple rounds of antigen stimulation, enabling engineered T cells to maintain stronger stemness characteristics, higher survival rate, and more durable anti-tumor function.

[0074] 2.8 Cytokine secretion detection The supernatants from the co-cultures after multiple rounds of in vitro cell killing were collected, and the secretion level of IFN-γ was detected using an ELISA kit. The results showed that during multiple rounds of cell killing, the IFN-γ secretion level in the co-culture supernatant of the TCR-T Control group gradually decreased, while the IFN-γ secretion level in the TCR-T ALX3 group remained relatively stable after each round of cell killing. Figure 3 E).

[0075] 2.9 In vivo tumor treatment capability detection (1) The tumor cell line A375 was subcutaneously injected into NSG immunodeficient mice to establish a tumor-bearing model.

[0076] (2) When the tumor volume reaches approximately 50-100 mm 3 At that time, the tumor-bearing mice were randomly divided into the following three groups (n=5-10 in each group): Group 1: PBS control group (PBS injected via tail vein) Group 2: Control T cell group (TCR-T Control cells injected via tail vein) Group 3: ALX3 overexpressing T cell group (tail vein injection of TCR-T-ALX3 T cells) (3) Measure tumor volume and mouse weight every 3-4 days and plot tumor growth curve.

[0077] (4) The results showed that, compared with the control group, the tumor volume of mice in the ALX3 overexpressing T cell group was significantly reduced, and tumor growth was effectively inhibited. Figure 4 A) 2.10 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.

[0078] (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.

[0079] (3) The results showed that the number of CD8+ T cells in the tumor tissue of the ALX3 overexpressing T cell group was significantly higher than that in the control group. Figure 4 B), the proportion of PD-1 and TIM-3 positive CD8+ T cells in tumor tissue was significantly reduced in the ALX3-overexpressing T cell group. Figure 4 (C) indicates that ALX3 overexpression promotes T cell infiltration and functional maintenance in tumor tissues.

[0080] 2.11 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 TCRT 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 TCRTs. The number of TCRT cells in the peripheral blood of the ALX3-overexpressing TCRT cell group was significantly higher than that of the control group ( Figure 4 (D) indicates that the viability and proliferative potential of ALX3-overexpressing TCRT cells are superior to those of the control group. Example 3: Construction of TIL cells overexpressing ALX3

[0081] 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.

[0082] (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.

[0083] (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.

[0084] 3.2 ALX3 overexpression transduction and functional detection in TIL cells (1) A lentiviral expression vector was constructed to express the ALX3 gene, driven by the EF1α promoter, and named pLV-EF1α-ALX3.

[0085] (2) Packaging lentivirus (method as in Example 1.2).

[0086] (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 7-10 days to obtain ALX3-overexpressing TIL cells, which were named TIL-ALX3 cells. At the same time, TIL cells transduced with empty vector were prepared as a control (named TIL-Control cells).

[0087] 3.3 Detection of ALX3 overexpression The expression level of ALX3 in TIL-ALX3 cells was detected by qRT-PCR (method as in Example 1.5). The results showed that the expression level of ALX3 mRNA in TIL-ALX3 cells was significantly higher than that in TIL-Control cells. Figure 5 A).

[0088] 3.4 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.

[0089] Set the following molecular groups: Target organ control group: organoids Effector cell control group: TIL Control group: Organoid + TIL-control group Experimental group: organoid + TIL-ALX3 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:

[0090] The results showed that the TIL ALX3 overexpression group had a significantly higher organoid killing rate than the TIL control group. Figure 5 B) Example 4: The general enhancement of the in vitro expansion capacity of T cells and NK cells by ALX3 overexpression.

[0091] To verify whether ALX3 overexpression has a broad-spectrum amplification and enhancement effect, CD3⁺ T cells and CD56⁺ NK cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. The isolated T cells and NK cells were transduced with lentiviral vectors to obtain the ALX3 gene (ALX3-T cells and ALX3-NK cells), while a control group with an empty vector (Ctrl-T and Ctrl-NK) was also included. On day 3 after transduction, the ALX3 overexpression efficiency was verified by RT-qPCR (the experimental groups showed a 6-10 fold increase compared to the control groups). Subsequently, the cells from each group were cultured in vitro in complete medium containing IL-2 (100 IU / mL), and the cell count was performed every 2-3 days for 21 consecutive days.

[0092] The results are as follows Figure 4 T cell expansion: ALX3-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 6 A).

[0093] NK cell expansion: ALX3-overexpressing NK cells expanded 1.5 times more than the control group on day 14 (P < 0.01); and 3.1 times more than the control group on day 21 (P < 0.001). Figure 6 B) The above results demonstrate that ALX3 overexpression not only enhances the expansion of CAR-T / TCR-T cells (see Examples 1 / 2), but also has a significant proliferative effect on unengineered natural T cells and NK cells. This finding implies that ALX3 overexpression is universally applicable to all cell therapy products based on T cells or NK cells (including CAR-T, TCR-T, TIL, CAR-NK, etc.), providing fundamental support for their widespread application in various adoptive cell therapies.

[0094] References: [1] Zhang YX, Pan YZ. Research progress in T-cell exhaustion. ChineseJournal of Pathophysiology, 2024, 40(8): 1548–1553.

[0095] [2] Baessler A, Vignali DAA. T Cell Exhaustion. Annu Rev Immunol, 2024, 42: 179–206.

[0096] [3] Dong C, et al. Regulators of CD8⁺ T cell exhaustion. Nat RevImmunol, 2025. (Online publication) [4] Ngiow SF, et al. PD-1 and LAG-3 synergistically foster T cellexhaustion. Signal Transduct Target Ther, 2024, 9: 291.

[0097] [5] Li J, et al. Exhausted T cells and epigenetic status. Cancer BiolMed, 2020, 17(4): 923–936.

[0098] [6] 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.

[0099] [7] Liu B, et al. Overcoming T cell exhaustion and senescence in CART cell therapy for solid tumors. Clin Transl Oncol, 2026. (Onlinepublication) [8] He HX, Fan H, Yang J. Mechanism and strategy of CAR-T celltherapy for T cell exhaustion in tumor. J Huazhong Univ Sci Tech (Med Sci),2024, 53(5): 705–710.

[0100] [9] Wen SQ, et al. TCF-1 maintains CD8⁺ T cell stemness in tumormicroenvironment. J Leukoc Biol, 2021, 110(3): 485–491.

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

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[11] 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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[12] Fischer J, et al. Serial killing assay using longitudinalimpedance-based tumor cell viability measurement—A useful method to assess Tcell performance. J Vis Exp, 2025, (226): e69623.

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

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

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

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[16] National Center for Biotechnology Information. ALX3 ALX homeobox3 [Homo sapiens (human)]. Bethesda (MD): NCBI.

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[17] National Center for Biotechnology Information. Alx3 ALX homeobox3 [Rattus norvegicus (Norway rat)]. Bethesda (MD): NCBI.

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[18] Illumina BaseSpace. QuickView for ALX3 (gene) (human). San Diego (CA): Illumina.

[0110] 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 ALX3 or its functionally active variants.

2. The engineered immune cells according to claim 1, characterized in that, The engineered immune cells further comprise a chimeric antigen receptor or exogenous T cell receptor capable of recognizing the target antigen; and / or the nucleotide sequence of the ALX3 gene is selected from the nucleotide sequence shown in SEQ ID NO:1 or its conserved alternative sequence; 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 2 or 3, characterized in that, The nucleotide sequence of the chimeric antigen receptor comprises the sequence shown in SEQ ID NO:2; or the nucleotide sequence of the exogenous T cell receptor comprises the sequence shown in SEQ ID NO:

4.

5. The engineered immune cells according to claim 1, characterized in that, The engineered immune cells are selected from CAR-T cells, TCR-T cells, tumor-infiltrating lymphocytes, CAR-NK cells, NK cells, γδ T cells, DC cells, or macrophages.

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 ALX3 gene into immune cells in vitro or in vivo and overexpressing it.

7. The construction method according to claim 6, characterized in that, The ALX3 gene is introduced into immune cells via a viral vector or a non-viral vector; 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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