Application of IFN alpha / anti-PD-L1 bispecific fusion protein and TCR-T as fusion protein secretion carrier

Abstract

The invention provides an IFN alpha / anti-PD-L1 bispecific fusion protein and application of TCR-T as a fusion protein secretion carrier, and belongs to the technical field of biological medicine. The invention provides a bispecific fusion protein. The bispecific fusion protein comprises an anti-PD-L1 scFv, a flexible linker and a cytokine for activating T cells which are connected in sequence. The invention also provides a carrier capable of expressing the bispecific fusion protein, the bispecific fusion protein and a TCR structure are separated by utilizing IRES, and independent expression of the bispecific fusion protein and the TCR can be realized. The invention also constructs a novel antigen-specific TCR-T cell which is used as a secretion and delivery carrier and has a good anti-tumor function on various solid tumors. When the novel antigen specificity TCR-T is used for treating solid tumors, a synergistic enhancement effect can be generated, and finally, the solid tumors are safely and effectively treated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of IFNα / anti-PD-L1 bispecific fusion protein and TCR-T as a secretory fusion protein carrier. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human health and life. Currently, tumor immunotherapy has become the most promising treatment for malignant tumors after surgery, radiotherapy, chemotherapy, and targeted therapy. Among them, TCR-T involves the exogenous overexpression of TCR receptors on T cells, enabling them to specifically recognize tumor antigens presented by the major histocompatibility complex (MHC), and has the characteristics of good targeting, high sensitivity, and broad target range.

[0003] Tumor neoantigens serve as ideal targets for TCR-T therapy, exhibiting no off-target toxicity and a favorable safety profile. In August 2024, the TCR-T therapy Afami-cel became the world's first approved TCR-T therapy due to its significant objective response rate and duration of response in adult patients with advanced MAGE-A4-positive synovial sarcoma, fully demonstrating the unique advantages of TCR-T in the treatment of solid tumors.

[0004] However, the overall response rate of TCR-T therapy remains low, and its efficacy is unsatisfactory in various solid tumors such as pancreatic cancer, lung cancer, and colorectal cancer. Studies show that the main reasons are T cell dysfunction caused by the tumor immunosuppressive microenvironment and insufficient endogenous immune activation. There is an urgent need to enhance the anti-tumor function of TCR-T cells by increasing the secretion of pro-inflammatory factors, blocking suppressive immune checkpoints, and simultaneously activating endogenous immune cells, ultimately achieving effective treatment for various solid tumors. Summary of the Invention

[0005] This invention provides the application of IFNα / anti-PD-L1 bispecific fusion protein and TCR-T as secretory fusion protein carriers. The bispecific fusion protein can reduce the potential adverse reactions of IFNα and block IFNα-induced PD-L1 upregulation. The neoantigen-specific TCR-T expressing IFNα / anti-PD-L1 has good killing ability against solid tumors and good safety.

[0006] This invention provides a bispecific fusion protein comprising, in sequence, an anti-PD-L1 scFv-flexible linker-cytokine that activates T cells.

[0007] In a preferred embodiment of the present invention, the cytokines that activate T cells include interferon.

[0008] In a preferred embodiment of the present invention, the interferon includes IFNα.

[0009] The present invention also provides a vector comprising the above-mentioned bispecific fusion protein.

[0010] In a preferred embodiment of the present invention, the vector comprises a promoter-Igκ-the bispecific fusion protein-IRES-TCR structure connected in sequence.

[0011] The present invention also provides a TCR-T cell that secretes the above-mentioned bispecific fusion protein.

[0012] The present invention also provides a method for constructing the above-mentioned TCR-T cells, comprising constructing T cells using the above-mentioned vector to obtain the TCR-T cells that secrete the bispecific fusion protein.

[0013] The present invention also provides the use of the above-mentioned bispecific fusion protein, the above-mentioned vector, or the above-mentioned TCR-T cells in the preparation of drugs for treating tumors.

[0014] In a preferred embodiment of the present invention, the tumor includes a solid tumor.

[0015] The present invention also provides a drug for treating tumors, the active ingredient of which includes the above-mentioned TCR-T cells.

[0016] Beneficial Effects: This invention provides a bispecific fusion protein comprising, sequentially linked, an anti-PD-L1 scFv, a flexible linker, and a T-cell activating cytokine. The PD-L1 scFv recognizes human or mouse PD-L1 molecules, and the anti-PD-L1 scFv and the T-cell activating cytokine are connected by a flexible linker, maintaining their spatial connection while preserving their respective functions. This invention also provides a vector for expressing the bispecific fusion protein, the vector further comprising an Igκ and a TCR structure, wherein the Igκ sequence in the vector guides the extracellular secretion of the fusion protein; the bispecific fusion protein and the TCR structure are separated by an IRES, enabling independent expression of the bispecific fusion protein and the TCR.

[0017] This invention also constructs neoantigen-specific TCR-T cells formed by transfecting T cells with the aforementioned vector. Using these neoantigen-specific TCR-T cells as secretion and delivery vectors, they exhibit good anti-tumor function against various solid tumors. The neoantigen-specific TCR-T cells of this invention can efficiently secrete IFNα / anti-PD-L1, achieving safe and effective treatment of solid tumors. IFNα plays a key role in improving the tumor microenvironment and enhancing TCR-T efficacy. The IFNα / anti-PD-L1 combination utilizes the targeting ability of PD-L1 to enrich the fusion protein at the tumor site, reducing potential adverse reactions of IFNα, while simultaneously blocking the PD-L1 pathway and enhancing TCR-T efficacy. The neoantigen-specific TCR-T cells of this invention can act as a "living drug" for secreting and delivering the fusion protein. On the one hand, the dual targeting effect provided by neoantigen-specific TCR and PD-L1 ensures treatment safety; on the other hand, it solves the problem of short half-life of bispecific fusion proteins, requiring repeated infusions.

[0018] When using the neoantigen-specific TCR-T cells described in this invention to treat solid tumors, a synergistic enhancement effect can be produced. IFNα / anti-PD-L1 can promote the Th1 differentiation of TCR-T cells, activate the antigen recognition pathway, maintain their proliferation and survival ability, enhance TCR-T function, and at the same time upregulate the expression of innate immune-related molecules, promote NK cell activation and DC cell maturation, mobilize endogenous immune cells to play a synergistic role, and ultimately achieve safe and effective treatment of solid tumors. Attached Figure Description

[0019] Figure 1 The diagram shows the construction pattern of the OT-1 TCR-T expressing IFNα / anti-PD-L1. A: Schematic diagram of the construction of the OT-1 TCR-T vector overexpressing IFNα / anti-PD-L1, IFNα, and anti-PD-L1 scFv; B: Schematic diagram of the construction of the OT-1 TCR and IFNα / anti-PD-L1 bispecific fusion protein vector; C: Schematic diagram of the IFNα / anti-PD-L1 bispecific fusion protein structure. Figure 2The figure illustrates the inhibitory effect of OT-1 TCR-T on the growth of large and small tumors. Figure A: Schematic diagram of the experimental protocol for OT-1 TCR-T treatment of solid tumors in C57BL / 6 mice; B: After inoculation with small tumors, the tumor volume changes of mice in different treatment groups were monitored regularly (n=3). PBS represented tail vein infusion of phosphate-buffered saline as a blank control, and NT represented tail vein infusion of T cells not transduced with OT-1 as a negative control; C: After inoculation with large tumors, the tumor volume changes of mice in each group were monitored regularly (n=3); D: At the treatment endpoint, tumors from each group of mice were collected, and the expression of 4-1BB and CD69 in tumor-infiltrating T cells was detected by flow cytometry; One-way ANOVA was used for BC difference analysis, and two-way ANOVA was used for EF difference analysis. Figure 3 To illustrate the infiltration effect of OT-1 TCR-T in large tumors, Figure A shows representative flow cytometry plots of the proportion of tumor-infiltrating T cells in different treatment groups in small and large tumors, detected by flow cytometry. Figure B shows the statistical results of three independent experiments using flow cytometry to detect the proportion of tumor-infiltrating T cells in mice from different treatment groups at the treatment endpoint. Figure 4 The figure shows the enrichment effect of OT-1 TCR-T on NK cell-activated M2 macrophages in large tumors. Figure A: Representative flow cytometry plots of CD69 expression levels in tumor-infiltrating NK cells from different treatment groups in small and large tumors; B: Representative flow cytometry plots of CD206 expression levels in tumor-infiltrating macrophages from different treatment groups in small and large tumors; C: Statistical results of three independent experiments using flow cytometry to detect CD69 expression levels in tumor-infiltrating NK cells from different treatment groups at the treatment endpoint; D: Statistical results of three independent experiments detecting CD206 expression levels in tumor-infiltrating macrophages from different treatment groups; E: Statistical analysis of the M2 / M1 genotype ratio of tumor-infiltrating macrophages in different treatment groups. Figure 5This diagram illustrates the effect of IFNα on the tumor microenvironment. Figure A shows patients divided into a poor prognosis group (Cluster 1) and a good prognosis group (Cluster 2) based on treatment efficacy, with GO enrichment analysis of differentially expressed genes between the two groups (n=22). Figure B shows the GO pathway enrichment between the two groups using GSEA. Figure C shows the KEGG pathway enrichment between the two groups using GSEA. Figure D shows the levels of Th1 and Th17 cytokines expressed in patients in the poor prognosis group (Cluster 1) and the good prognosis group (Cluster 2) (n=22). Figure E shows the correlation between IFNAR1 gene expression levels in tumor tissue and the prognosis of different tumor patients using Kaplan-Meier curve analysis. The sample size for pan-cancer is n=4420, for colon cancer n=1336, for gastric cancer n=881, and for lung cancer n=2167. Figure 6 The results of IFNα enhancing TCR-T function and activating endogenous immune cells are shown in the figure. AB: OT-1 TCR-T cells were stimulated with exogenous IL-12 or IFNα, and the expression of Ki67 (A) and T-bet (B) in TCR-T cells was detected after 12 h; C: Mouse NK cells were isolated and cultured, stimulated with exogenous IL-12 or IFNα, and the expression of CD69 in NK cells was detected after 12 h; D: Mouse macrophages were isolated and cultured with exogenous IL-12 or IFNα, and the expression of CD103 in macrophages was detected after 24 h; EF: Mouse DC cells were isolated and cultured with exogenous IL-12 or IFNα, and the expression of CD80 (E) and CD86 (F) in DC cells was detected after 24 h. Figure 7 The diagram shows the results of TCR-T cells secreting IFNα effectively controlling tumors and activating endogenous immunity. Figure A: Representative results of flow cytometry detection of perforin and granzyme B secretion by OT-1 TCR-T cells secreting IFNα and MC38 tumor cell line overexpressing OVA after co-culturing for 24 h; B: Using C57BL / 6 mice to establish a subcutaneous tumor model of MC38 overexpressing OVA, T cells with different treatments were infused via the tail vein, and changes in tumor volume were observed; C: At the treatment endpoint, tumors from different treatment groups were isolated, and flow cytometry was used to analyze the expression of CD69 in tumor-infiltrating NK cells and the M2 / M1 genotype ratio of macrophages. Figure 8The figure shows the results of TCR-T cells secreting IFNα causing systemic adverse reactions and upregulating PD-L1 in tumor tissue. Figure A: Tumors were removed from mice at the treatment endpoint, and tumor volume was measured in each treatment group (n=3-4). Figure B: Tumor tissues from mice in different treatment groups were isolated at the treatment endpoint, and the expression of CD80 and CD86 in tumor-infiltrating DC cells was detected by flow cytometry. Figure C: Changes in mouse body weight were monitored periodically. Figure D: Peripheral blood was collected from mice in different treatment groups at the treatment endpoint, and serum alanine aminotransferase (ALT) and serum creatinine (CREA) concentrations were detected using blood biochemistry analysis. Figure E: Immunohistochemical staining results of tumors in mice in different treatment groups. A suitable amount of excised tumor tissue was fixed and embedded in paraffin. The sections were stained with PD-L1 antibody. The scale bar in the figure is 100 μm. Figure F: The number of positive cells was counted using a 40x objective lens and five consecutive high-power fields (HPF) scans. Figure 9 The figures show the results of detecting the positivity rate of T cells transduced with different structures. A: Bright-field image of T cells transduced with different structures; B: Image of T cells transduced with different structures expressing green fluorescent protein under fluorescence excitation; C: Representative image of GFP expression in T cells transduced with different structures detected by flow cytometry; D: Statistical results of three independent experiments detecting GFP expression in T cells. All scale bars in the figures represent 100 μm. Figure 10 The figure shows the expression of the bispecific fusion protein in TCR-T cells. Figure A: The expression of the fusion protein was detected by IFNα flow cytometry antibody permeabilization staining; B: T cells with different structures were lysed and transduced, and the protein was extracted and detected by Western blot using V5-tagged antibody; C: The relative expression levels of different proteins were calculated; D: The trend of TCR-T secretion of different protein concentrations over time was detected using an IFNα ELISA kit. Figure 11 The diagram shows the results of the bispecific fusion protein recognizing and binding to PD-L1. In the diagram, A: MC38 tumor cells overexpressing PD-L1 were constructed by transducing the MC38 colorectal cancer tumor cell line using recombinant retroviruses, and PD-L1 expression was detected by flow cytometry; BC: The supernatant of TCR-T cell culture with different structures was concentrated and co-cultured with MC38-PD-L1 cells for 4 h, and the tumor cells were stained by flow cytometry using IFNα antibody or V5 tag antibody. Figure 12The results of TCR-T cells secreting IFNα / anti-PD-L1 effectively killing tumor cells are shown in Figure A: TCR-T cells with different structures were co-cultured with MC38-PD-L1 tumor cells at effector-to-target ratios of 1:3, 1:1, 3:1, and 9:1 for 12 h, 24 h, and 36 h, and the efficiency of TCR-T cells killing tumor cells was detected by fluorophore chemiluminescence assay; B: The trend of killing efficiency over time was statistically analyzed; C: TCR-T cells with different structures were co-cultured with MC38-PD-L1 tumor cells at an effector-to-target ratio of 9:1 for 24 h, and bright-field imaging was performed using an inverted microscope. The scale bar in the figure is 100 μm. The black arrows point to MC38-PD-L1 tumor cells, and the red arrows point to TCR-T cells. Figure 13 The figure shows the results of enhancing TCR-T activation function with IFNα / anti-PD-L1 bispecific fusion protein. In the figure, A: representative flow cytometry plots of TCR-T cells with different structures and MC38-PD-L1 tumor cells co-cultured at an effector-target ratio of 2:1 for 24 h, and the expression of 41BB, CD69, and OX40 in TCR-T cells detected by flow cytometry; BD: statistical results of detecting the expression levels of 41BB, CD69, and OX40 in TCR-T cells. Figure 14 The figure shows the results of reversing TCR-T depletion by the IFNα / anti-PD-L1 bispecific fusion protein. In the figure, A: representative results of the expression of PD-1, LAG-3 and TIM-3 in TCR-T cells after co-culturing with MC38-PD-L1 tumor cells by flow cytometry; BD: statistical results of the expression of depletion molecules in TCR-T cells. Figure 15 Figure A shows the results of maintaining the central memory phenotype of TCR-T cells with the IFNα / anti-PD-L1 bispecific fusion protein. In the figure, A: TCR-T cells with different structures were co-cultured with MC38-PD-L1 tumor cells at an effector-to-target ratio of 2:1 for 24 h. Flow cytometry was used to detect the expression levels of CD62L and CD44 in TCR-T cells and to count the proportion of each differentiation subtype. TN represents naive T cells, TCM represents central memory cells, TEff represents effector T cells, and TE represents exhausted T cells. BC: After co-culturing for 24 h, the proportion of each differentiation subtype of CD8 and CD4 positive TCR-T cells was counted. Figure 16 The figure shows the results of IFNα / anti-PD-L1 enhancing TCR-T proliferation and reducing apoptosis. Figure A: TCR-T cells with different structures were co-cultured with MC38-PD-L1 tumor cells at an effector-target ratio of 2:1 for 12 h, and the Ki67 expression level of TCR-T cells was detected by flow cytometry; Figure B: Statistical analysis of CD8 from three independent experiments. +Ki67 expression in TCR-T cells; C: Statistical analysis of CD4+ from three independent experiments. + TCR-T cell Ki67 expression; D: Annexin V / 7AAD apoptosis kit detection of early apoptotic TCR-T cells after co-culture (Annexin V) + 7AAD − ) and late apoptotic cells (Annexin V) + 7AAD + )Proportion; Figure 17 IFNα / anti-PD-L1 inhibits Treg cell proliferation and promotes CD4+. + The image shows the results of T-bet expression in T cells. In the image, AB represents CD25 expression in TCR-T cells co-cultured with MC38-PD-L1 tumor cells, detected by flow cytometry. + FOXP3 + Representative flow cytometry plots (A) and statistical results of three independent experiments on the proportion of Treg cells; Representative flow cytometry plots (C) and statistical results of three independent experiments on the proportion of T-bet positive cells to CD4 positive cells after co-culturing TCR-T and MC38-PD-L1 tumor cells. Figure 18 The results of TCR-T cells expressing IFNα / anti-PD-L1 secreting high levels of killing-related factors are shown in Figure AB: Representative flow cytometry (A) and statistical results of three independent experiments (B) were obtained by co-culturing TCR-T cells with different structures and MC38-PD-L1 tumor cells for 24 h, fixing and permeabilizing them, and detecting the secretion of granzyme B and perforin by intracellular staining using flow cytometry; C: The concentrations of IFNγ and IL-2 factors in the supernatant were detected by enzyme-linked immunosorbent assay after co-culturing TCR-T cells with different structures and MC38-PD-L1 tumor cells at effector-to-target ratios of 1:3, 1:1, 3:1, and 9:1 for 24 h. Figure 19 This diagram illustrates the results of IFNα / anti-PD-L1 effectively activating bystander T cells and maintaining their memory phenotype. Figure A: Schematic diagram of the Transwell assay, with the upper layer showing TCR-T cells transduced with different structures, and the lower layer showing a co-culture system of untransduced T cells and tumor cells, separated by a 40 μm membrane; Figure B: Representative flow cytometry plot showing the expression of 41BB and OX40 in the lower layer T cells after 24 h of co-culture in the Transwell assay; Figure C: Statistical results of detecting the expression levels of 41BB, CD69, and OX40 in the lower layer T cells; Figure D: Statistical analysis of the proportions of different differentiation subtypes of CD8 and CD4 positive T cells in the lower layer. Figure 20 The figure shows the results of IRF2-4 TCR-T cells expressing IFNα / anti-PD-L1 significantly enhancing tumor killing function. Figure A: IRF2-4 TCR-T cells with different transduced structures were co-cultured with MC38-PD-L1 tumor cells at effector-to-target ratios of 1:3, 1:1, 3:1, and 9:1 for 12 h, 24 h, and 36 h, and the tumor cell killing efficiency of IRF2-4 was detected by fluorophore chemiluminescence assay; Figure B: The trend of killing efficiency over time was statistically analyzed. Figure 21 The figure shows the results of TCR-T expressing IFNα / anti-PD-L1 significantly inhibiting tumor growth. Figure A: Schematic diagram of the experimental protocol for TCR-T treatment of mouse solid tumors; B: Tumor volume changes in each group of mice were monitored periodically, with Day 0 as the start of treatment (n=6); C: Tumors were removed from mice at the treatment endpoint, and the tumor volume of each treatment group was measured (n=3-4); D: Tumor mass was measured in each group at the treatment endpoint (n=3); E: Survival curves of the mouse solid tumor treatment model (n=5). Figure 22 Figure showing the results of TCR-T cells expressing IFNα / anti-PD-L1 effectively clearing solid tumors (n=4); Figure 23 The figure shows the results of TCR-T cells expressing IFNα / anti-PD-L1 exhibiting strong peripheral expansion capacity and good safety. Figure A: Peripheral blood samples were collected from mice at Day 7, Day 14, and Day 21, and the proportion of antigen-reactive TCR-T cells was analyzed by flow cytometry; B: Flow cytometry was used to detect the expression of 4-1BB in peripheral blood TCR-T cells; C: The concentration changes of aPDL1-IFNα or IFNα in mouse peripheral blood serum were monitored periodically; D: At the treatment endpoint, peripheral blood samples were collected from mice in different treatment groups, and serum biochemical analysis was used to detect the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (CREA), and creatine kinase (CK). Figure 24 The figure shows the results of IFNα / anti-PD-L1 enhancing T cell infiltration and blocking PD-L1 in tumor cells. In the figure, A: Tumor tissues from mice in each treatment group were fixed and embedded in paraffin at the treatment endpoint. H&E staining and immunohistochemical staining were performed using CD3, Ki67, and PD-L1 antibodies. The scale bar in the figure is 150 μm (20x magnification). BD: The number of positive cells was counted based on the CD3, Ki67, and PD-L1 staining results. Five consecutive high-power fields (HPF) scans were performed using a 40x objective lens. Figure 25The image shows the results of immunofluorescence imaging demonstrating the enhanced T cell infiltration and proliferation function of IFNα / anti-PD-L1. In the image, A: Tumor tissues from mice in each treatment group were fixed and frozen sections at the treatment endpoint, and immunofluorescence staining was performed using CD3 and Ki67 antibodies. The scale bar in the left image is 150 μm (20x magnification), and the scale bar in the right image is 50 μm (63x magnification). Red represents Ki67, and green represents CD3. B: The proportion of CD3-positive T cells was counted by scanning and counting four consecutive high-power fields (HPF) under a 20x objective lens. Figure 26 The results of IFNα / anti-PD-L1 enhancement of tumor-infiltrating T cell activation are shown in the figure. A: At the treatment endpoint, mouse tumor tissue was isolated, digested, and the activation of adoptive TCR-T and endogenous T cells was detected by flow cytometry. The figure shows a gate diagram. B: Flow cytometry detection of 4-1BB expression in CD8 and CD4 positive adoptive TCR-T cells in different treatment groups. C: Detection of CD69 expression in CD8 and CD4 positive adoptive TCR-T cells in different treatment groups. DF: Flow cytometry detection of 4-1BB, OX40, and CD69 expression in CD8 and CD4 positive endogenous T cells, respectively. Figure 27 The figure shows the results of IFNα / anti-PD-L1 reversal of tumor-infiltrating TCR-T cell exhaustion. Figure A: Representative results of flow cytometry detection of PD-1 and TIM-3 expression in adoptive TCR-T cells after tumor tissue digestion; B: Statistical results of three independent experiments detecting PD-1 and TIM-3 expression in TCR-T cells; C: Differentiation phenotype detection of tumor-infiltrating TCR-T cells in different treatment groups, with representative flow cytometry results shown; D: Flow cytometry detection of CD62L and CD44 expression levels in endogenous T cells and statistical analysis of the proportion of each differentiation subtype. N Represents naïve T cells, T CM Representing central memory cells, T Eff Representative effector T cells, T E Represents exhausted T cells; Figure 28 To illustrate the principal component analysis and cluster analysis results of TCR-T cells expressing IFNα / anti-PD-L1, TCR-T cells transduced with different structures were co-cultured with tumor cells overexpressing the neoantigen MC38 for 24 h, and then TCR-T cells underwent reference transcriptome sequencing (n=3). In the figure, A: Principal component analysis of gene expression in TCR-T cell populations of each treatment group; B: Unsupervised cluster analysis of immune-related gene sets in TCR-T cells of each group. Figure 29The gene expression profile of TCR-T cells expressing IFNα / anti-PD-L1 is similar to that of central memory T cells. Figure A: Upset plot showing the overlap of differentially expressed immune-related genes in each group of TCR-T cells; B: Expression heatmap of differentially expressed genes in each group of TCR-T cell populations in different functional gene sets; C: Relative abundance of eight T cell subsets analyzed using immuCellAI. Figure 30 The figure shows the results of TCR-T cells expressing IFNα / anti-PD-L1 upregulating Th1 differentiation and antigen recognition pathways. In the figure, A: GO analysis of differentially expressed genes in TCR-T cells that secrete bispecific fusion proteins and the corresponding control group (TCR-T cells that do not secrete bispecific fusion proteins); B: KEGG analysis of differentially expressed genes in TCR-T cells that secrete bispecific fusion proteins and the corresponding control group. Figure 31 The graph shows the results of upregulation of activation-related gene expression in TCR-T cells secreting IFNα / anti-PD-L1. Figure A: Heatmap of differentially expressed genes in TCR-T cells secreting bispecific fusion proteins and the corresponding control group; B: Volcano plot analysis of differentially expressed genes in TCR-T cells secreting bispecific fusion proteins and the corresponding control group; C: Expression of Th1 and antigen recognition pathway-related genes in TCR-T cells secreting bispecific fusion proteins and the corresponding control group. Sequencing samples n=3 in each group. Figure 32 This figure shows the expression results of endogenous immune-related genes activated by IFNα / anti-PD-L1. C57BL / 6 mice were subcutaneously inoculated unilaterally with MC38 tumor cell lines overexpressing neoantigens. Nine days later (Day 0), TCR-T cells with different structures were transduced via tail vein infusion. At the treatment endpoint, tumors from each group of mice were harvested for reference transcriptome sequencing (n=3). Figure A: Principal component analysis of gene expression in the endogenous immune cell population of each treatment group; B: Aggregate plot showing the overlap of differentially expressed genes in endogenous immune cells of each group; C: Expression heatmap of differentially expressed genes in endogenous immune cells of each group in different functional gene sets. Figure 33 The figure shows the results of IFNα / anti-PD-L1 activation of the innate immune-related pathway. Figure A: GO analysis of differentially expressed genes in endogenous immune cells of TCR-T cells secreting bispecific fusion protein and corresponding control group (TCR-T cells not secreting bispecific fusion protein); Figure B: KEGG analysis of differentially expressed genes in endogenous immune cells of TCR-T cells secreting bispecific fusion protein and corresponding control group. Figure 34 A diagram illustrating the pathways of IFNα / anti-PD-L1 upregulation of antigen peptide recognition, chemokine binding, and complement activation. Figure 35 The figure shows the results of IFNα / anti-PD-L1 promoting NK cell activation and DC cell maturation. Figure A: Statistical results of relative abundance of CD8 positive T cells, DC cells, NK cells, and M2 macrophage subsets analyzed using immuCellAI; Figure B: Violin plot showing the gene expression differences between the TCR-T treatment group and the corresponding control group expressing the bispecific fusion protein. Figure 36 This is a graph showing the results of IFNα / anti-PD-L1 inhibiting Treg cell proliferation and activating endogenous innate immunity. Figure A: Representative flow cytometry plots of T cells expressing CD25 and FOXP3 after digestion of tumor tissue from different treatment groups. + FOXP3 + A: Cell population consisted of Treg cells; B: Proportion of Treg cells among CD4-positive T cells; C: Detection of CD69 expression level in NK cells; D: Detection of the proportion of M2 macrophages among macrophages; E: Detection of CD103. + The proportion of cDC1 cells and their expression levels of CD80 and CD86; Figure 37 This figure shows the results of human TCR-T cells expressing IFNα / anti-PD-L1 secreting high levels of IFNγ and significantly killing tumor cells. Figure A: Flow cytometry analysis of the positive conversion rate of human TCR-T cells transduced with different structures; B: Statistical results of the positive conversion rate; C: Co-culturing TCR-T cells transduced with different structures with A375 and SW480 tumor cells at effector-to-target ratios of 1:3, 1:1, 3:1, and 9:1 for 24 h, and detecting the efficiency of TCR-T cell killing using fluorescein bioluminescence assay; D: ELISA analysis of IFNγ secretion in the supernatant after co-culture. Figure 38 The graph shows the results of enhanced activation function and maintenance of memory phenotype in human TCR-T cells expressing IFNα / anti-PD-L1. Figure A: Representative flow cytometry plots showing the upregulation of 4-1BB in TCR-T cells transduced with different structures after 24 h of co-culture with A375 tumor cells; Figure B: Representative flow cytometry plots showing the expression of CD62L and CD45RO in TCR-T cells after co-culture. N Represents naïve T cells, T CM Representing central memory cells, T Eff Representative effector T cells, T E C: Represents exhausted T cells; D: Statistical analysis of TCR-T expression of 4-1BB; E: Statistical analysis of the proportion of different TCR-T subtypes. Detailed Implementation

[0020] This invention provides a bispecific fusion protein comprising a cytokine sequentially linked to an anti-PD-L1 scFv-flexible linker-T cell activating agent.

[0021] The bispecific fusion protein IFNα / anti-PD-L1 described in this invention has Figure 1 The structure shown in SEQ ID No. A, the sequence of the anti-PD-L1 scFv is as shown in SEQ ID No. 1; its encoded amino acid sequence is as shown in SEQ ID No. 2, which can recognize human or mouse PD-L1 molecules. The scFv fragment is composed of a light chain variable region (VL) and a heavy chain variable region (VH), and VL and VH are connected by a (G4S)3 flexible linker to form a stable single-chain antibody structure. The nucleotide sequence of VL is 1-324 bp of the sequence shown in SEQ ID No. 1, and the amino acid sequence is 1-108 of the sequence shown in SEQ ID No. 2; the nucleotide sequence of VH is 370-723 bp of the sequence shown in SEQ ID No. 1, and the amino acid sequence is 124-241 of the sequence shown in SEQ ID No. 2.

[0022] The cytokine for activating T cells described in this invention can be interferon, such as IFNα in one embodiment, more specifically the murine α4 isoform, with its potential adverse reactions reduced by replacing the 20th amino acid from alanine (Ala) with leucine (Leu). After this substitution, the nucleotide sequence of the cytokine is shown in SEQ ID No. 3. This invention uses a (G4S)2 flexible linker to connect anti-PD-L1 scFv and IFNα, maintaining their spatial connection while ensuring their respective normal function.

[0023] The present invention also provides a vector comprising the above-mentioned bispecific fusion protein.

[0024] In one embodiment of this invention, the pMSGV1 recombinant retroviral vector was used, purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number P23481. This invention utilizes the pMSGV1 to construct a TCR-T structure expressing the aforementioned bispecific fusion protein, particularly expressing the IFNα / anti-PD-L1 bispecific fusion protein, as shown below. Figure 1 As shown in AC. In the vector described in this invention, LTR 3' is selected as the promoter, which can effectively initiate the expression of different gene structures and stably and efficiently transduce mouse T cells; and an Igκ sequence is inserted before the bispecific fusion protein, which can guide the extracellular secretion of the fusion protein. In one embodiment of this invention, the nucleotide sequence of the Igκ sequence is shown in SEQ ID No. 4.

[0025] The vector of this invention further includes an IRES sequence (internal ribosome entry site) to separate the bispecific fusion protein from the TCR structural sequence for independent expression. In one embodiment, the nucleotide sequence of the IRES sequence is shown in SEQ ID No. 5. The TCR structure of this invention is a structure that specifically recognizes the OVA antigen; for example, it may be the OT-1 TCR sequence in a mouse form and the KRAS sequence in a human form. G12V In this invention, the OT-1 TCR consists of a β chain and an α chain, both of which are composed of variable and constant regions connected together, and can specifically recognize OVA antigens. The nucleotide sequence of the β chain is shown in SEQ ID No. 6; the nucleotide sequence of the α chain is shown in SEQ ID No. 7.

[0026] In one embodiment of the present invention, the nucleotide sequence of the OT-1 TCR sequence is shown in SEQ ID No. 8; the KRAS G12V The nucleotide sequence is shown in SEQ ID No. 9.

[0027] In this invention, to facilitate subsequent detection of TCR-T cell transduction efficiency and verification of fusion protein expression, a green fluorescent protein (GFP) tag was added to the vector, and a V5 tag was added to the C-terminus of the bispecific fusion protein.

[0028] The present invention also provides a TCR-T cell that secretes the above-mentioned bispecific fusion protein.

[0029] This invention observed that the TCR-T cells, under bright-field conditions, were in good condition after transduction, exhibiting typical amplification and division morphology without obvious apoptosis, indicating that the viral transduction process had no significant impact on cell viability. Under fluorescence excitation conditions, TCR-T cells successfully transduced with the overexpression vector emitted green fluorescence, demonstrating that recombinant retroviruses can efficiently introduce vectors containing different target gene sequences into T cells.

[0030] The present invention also provides a method for constructing the above-mentioned TCR-T cells, comprising transforming T cells using the above-mentioned vector to obtain the TCR-T cells.

[0031] In this embodiment of the invention, TCR-T cells can be constructed by packaging recombinant retroviruses and introducing them into T cells.

[0032] The present invention also provides the use of the above-mentioned bispecific fusion protein, the above-mentioned vector, or the above-mentioned TCR-T cells in the preparation of drugs for treating tumors.

[0033] The TCR-T cells described in this invention can express and secrete a bispecific fusion protein, and the bispecific fusion protein can recognize IFNα receptor and PD-L1. The TCR-T cells described in this invention can recognize and target tumor cells, aggregate around the tumor cells, and exert a killing function, wherein the tumor cells are solid tumor cells.

[0034] This invention utilizes flow cytometry to detect the expression of activation markers in TCR-T cells co-cultured with tumor cells. Results show that the fusion protein significantly promotes 4-1BB expression and T cell activation. Furthermore, the bispecific fusion protein significantly upregulates TCR-T cell expression of OX40 and CD69, indicating that the bispecific fusion protein effectively enhances TCR-T cell activation, reduces cell exhaustion phenotype, maintains the central memory phenotype of TCR-T cells, significantly enhances TCR-T cell proliferation, reduces apoptosis, and significantly increases the proportion of Th1 cells. Moreover, the TCR-T cells described in this invention release higher levels of IFNγ and IL-2. IFNγ and IL-2 are important factors promoting T cell activation and killing function, activating bystander T cells and further enhancing the immune response.

[0035] In one embodiment of the present invention, a mouse model of subcutaneous colorectal cancer tumor was also constructed. After intervention with the TCR-T cells, tumor growth was significantly inhibited and the survival time of mice was significantly prolonged, demonstrating that the TCR-T cells secreting the bispecific fusion protein of the present invention have significant anti-tumor function and can effectively kill tumor cells.

[0036] The present invention also provides a drug for treating tumors, the active ingredient of which includes the above-mentioned TCR-T cells.

[0037] This invention demonstrates through in vivo experiments that TCR-T cells expressing IFNα / anti-PD-L1 exhibit good safety, effectively activate endogenous immunity, thus providing a guarantee for the formation of broader and more durable anti-tumor immunity, possess significant tumor-killing ability, and enhance TCR-T activation and factor secretion function. Therefore, the TCR-T cells described in this invention can effectively improve the tumor microenvironment, block PD-L1, and exert a synergistic effect by co-regulating endogenous immune cells while enhancing TCR-T cell function, ultimately achieving safe and effective clearance of solid tumors. Furthermore, IFNα / anti-PD-L1 and TCR-T can produce a synergistic effect. In TCR-T cells expressing IFNα / anti-PD-L1, pathways related to Th1 differentiation, antigen recognition, and presentation are significantly activated, and the key factor T-bet in the Th1 differentiation pathway is significantly upregulated, while key genes involved in T cell activation are also significantly upregulated. ZAP70 , IFNG , H2-AA and KLRD1Increased expression levels and in vitro experiments demonstrated that the IFNα / anti-PD-L1 bispecific fusion protein significantly enhanced the activation and killing functions of TCR-T cells. Furthermore, under the action of the bispecific fusion protein, TCR-T cells exhibited a gene expression profile similar to memory T cells, with upregulation of memory-related genes such as BCL2, CD28, MKI67, and CCL5. These genes play crucial roles in maintaining T cell survival and proliferation. Flow cytometry analysis also confirmed that TCR-T cells expressing IFNα / anti-PD-L1 had higher Ki67 expression levels and a more memory T cell phenotype. Simultaneously, the proportion of exhausted T cells in IFNα / anti-PD-L1-expressing TCR-T cells was reduced, indicating a higher proportion of T cell dysfunction. PDCD1 , TIGIT , HAVCR2 Genes such as TFN, CXCL2, CCL3, and EGR1 were downregulated. In addition, in vivo RNA-seq results showed that pathways related to NK cell killing, DC cell maturation, and complement activation in endogenous immune cells were significantly activated, and genes involved in innate immune activation, such as TFN, CXCL2, CCL3, and EGR1, were upregulated. Immune infiltration analysis showed that the proportion of DC cells and NK cells increased and the proportion of M2 macrophages decreased in tumor tissue.

[0038] To further illustrate the present invention, the following detailed description of the IFNα / anti-PD-L1 bispecific fusion protein and TCR-T provided by the present invention, with reference to the embodiments, is provided in detail, but should not be construed as limiting the scope of protection of the present invention.

[0039] The cell lines used in this invention include human embryonic kidney epithelial cells (HEK293T), human colon cancer cells (HCT116), human colon adenocarcinoma cells (SW480), human colon cancer cells (LoVo), mouse colon cancer cells (MC38), mouse colon cancer cells (CT26), human T-lymphocyte leukemia cells (Jurkat), and kidney cells transformed from African green monkey SV40 (COS-7), all of which are derived from ATCC.

[0040] The composition of each reagent in the embodiments of the present invention is as follows: LB medium (500 mL system): 5 g tryptone, 5 g sodium chloride and 2.5 g yeast extract; Agarose gel (100 mL system): 50×TAE 2.2 mL, agarose 1 g, Goldview 4 μL and H2O 107.8 mL; Adherent cell line culture medium (100 mL system): 89 mL DMEM medium, 1 mL Penicillin-Streptomycin Solution and 10 mL FBS; Suspension cell line culture medium (100 mL system): 89 mL RPMI-1640 medium, 1 mL Penicillin-Streptomycin Solution and 10 mL FBS; Cell cryopreservation solution (10 mL system): 9 mL FBS and 1 mL DMSO; 2×HBS solution (100 mL system): NaCl 280 mM, HEPES 50 mM, Glucose 12 mM, KCl 10 mM and Na2HPO4·2H2O 1.5 mM; Tumor tissue digestion solution (500 mL system): RPMI-1640 500 mL, DNase I 0.15 mg / mL, collagenase 3000 U / mL and hyaluronidase 1000 U / mL Mouse T cell expansion medium (500 mL system): RPMI-1640 440 mL, Penicillin-Streptomycin 5 mL, Primocin 500 μL, Glutamax 5 mL, FBS 50 mL, Human IL-2 500 U / mL; Mouse T cell activation medium (500 mL system): mouse T cell expansion medium (500 mL), Anti-mouse CD3 2.5 μg / mL and Anti-mouse CD28 1.25 μg / mL; Human T cell expansion culture medium (500 mL system): X-VIVO 440 mL, Penicillin-Streptomycin 5 mL, Primocin 500 μL, Glutamax 5 mL, FBS 50 mL and Human IL-2 500 U / mL; Human T cell activation medium (500 mL system): human T cell expansion medium (500 mL), Anti-human CD3 30 ng / mL and Anti-human CD28 500 ng / mL; T cell and DC cell co-culture medium: Human IL-2 2500 U / mL, Human IL-7 5 ng / mL, Human IL-15 5 ng / mL and Human IL-21 30 ng / mL; Mouse DC cell iDC induction medium (500 mL system): RPMI-1640 440 mL, Penicillin-Streptomycin 5 mL, Primocin 500 μL, Glutamax 5 mL, FBS 50 mL, Murine GM-CSF 20 ng / mL and Murine IL-4 10 ng / mL; Mouse DC cell mDC induction medium (500 mL system): mouse DC cell iDC induction medium (500 mL) and LPS 1 μg / mL; Human DC cell iDC induction medium (500 mL system): RPMI-1640 440 mL, Penicillin-Streptomycin 5 mL, Primocin 500 μL, Glutamax 5 mL, FBS 50 mL, Human GM-CSF 1000 U / mL and Human IL-4 500 U / mL; Human DC cell mDC induction medium (500 mL system): Human DC cell iDC induction medium (500 mL), Human TNF-α 10 ng / mL, Human IL-1β 10 ng / mL, Human IL-6 1000 U / mL and Human PGE2 1 μg / mL.

[0041] The experimental methods used in the embodiments of this invention are as follows: 1. Establishment of a co-culture system of mouse T cells and DC cells The C57BL / 6 mice used in this invention were provided by the Department of Animal Science, Peking University School of Medicine. This study has been reviewed and approved by the Experimental Animal Research Ethics Committee of Peking University School of Medicine, ethics approval number: DLASBE0272.

[0042] 1.1 Acquisition and culture of T cells derived from mouse spleen, dissociation of mouse tumor tissue samples and culture of TILs, and acquisition and culture of DC cells derived from mouse bone marrow (refer to the article Che X, Zheng S, Sun Y, Wang X, Zhang P, Cao J, Bai Y. Multi-engineered T cell vaccine boosting TCR-T cell therapy enhances anti-tumor function and eradicates heterogeneous solid tumors. MolTher. 2025 Sep 3;33(9):4529-4551. doi: 10.1016 / j.ymthe.2025.05.036.) 1.2 Co-culture of mouse T cells and DC cells 1) Take mature cultured DC cells and administer at a ratio of 2×10⁻⁶. 5 The cells were seeded at a density of 1 / mL into 24-well plates and cultured in DC cell culture medium for 12 h.

[0043] 2) After the DC cells have completely adhered to the wall, remove mouse T cells isolated from the spleen or tumor tissue, centrifuge at 350 g for 5 minutes at room temperature, discard the supernatant, and add 1×10⁻⁶ cells. 6 Resuspend the cells in T-cell culture medium at a density of / mL.

[0044] 3) Gently aspirate the DC cell culture medium, add the T cells with the adjusted density to the DC cell culture wells, and incubate at 37°C.

[0045] 4) Replace the culture medium with fresh medium every 2-3 days based on the color change.

[0046] 5) Observe the cell status and expansion every day, and use flow cytometry to detect cell phenotype or conduct functional experiments as needed.

[0047] 2. Establishment of a co-culture system for human T cells and DC cells The peripheral blood from healthy individuals used in this invention was obtained from Shanghai Yayu Biotechnology Co., Ltd., and has been reviewed by the ethics committee. The ethics approval number is: Yuanlunkuai

[2022] 09.

[0048] 2.1 Isolation of peripheral blood mononuclear cells (PBMCs), acquisition and culture of PBMC-derived T cells, and acquisition and culture of PBMC-derived dendritic cells (see article: Che X, Zheng S, Sun Y, Wang X, Zhang P, Cao J, Bai Y. Multi-engineered T cell vaccine boosting TCR-T cell therapy enhances anti-tumor function and eradicates heterogeneous solid tumors. Mol Ther. 2025 Sep 3;33(9):4529-4551. doi: 10.1016 / j.ymthe.2025.05.036.) 2.2 Sorting of naive T cells and memory T cells 1) Resuscitate CD3+ T cells derived from PBMCs and allow them to rest for 6 hours.

[0049] 2) T cells were stained using flow cytometry antibodies. The antibodies used for staining were CD8, CD62L, and CD45RO, with an antibody staining concentration of 10 μL / mL. The cells were washed with PBS after 30 minutes.

[0050] 3) Centrifuge at 350 g for 5 minutes at room temperature.

[0051] 4) According to each 1×10 7 Resuspend cells in PBS at a ratio of 300 μL per cell, add 100 μL of 7AAD antibody, pass through a 40 μm cell sieve, transfer to flow cytometry tubes, and perform flow cytometry sorting of naive T cells and memory T cells.

[0052] 5) Use 5 mL flow cytometry tubes for cell sorting and reception. 1 mL of human serum containing streptomycin and penicillin should be added to the flow cytometry tubes in advance to maintain T cell activity and prevent cell contamination.

[0053] 6) After sorting, transfer the cells to a 15 mL centrifuge tube, centrifuge at 300 g for 10 minutes, discard the supernatant, and wash twice with PBS.

[0054] 7) Resuspend the cells in T cell expansion medium, and add IL-7 and IL-15 to the initial T cells for culture. Incubate at 37°C for 12 h.

[0055] 2.3 Co-culture of human T cells and DC cells 1) Resuscitate mature mDC cells and resuspend them in a culture medium co-incubated with T cells and DC cells, at a concentration of 1×10⁻⁶. 6Cells were seeded at a density of 1 / mL into 96-well U-shaped plates and incubated at 37°C for 4 h.

[0056] 2) After the mDC cells have completely adhered to the wall, according to the experimental design, the initial T cells or memory T cells obtained by sorting are aspirated into a 15 mL centrifuge tube, centrifuged at 350 g for 5 minutes, the supernatant is discarded, and the cells are resuspended in the culture medium for co-incubation of T cells and DC cells.

[0057] 3) Add culture medium to adjust the density ratio of T cells to DC cells to 4:1, and gently pipette to mix.

[0058] 4) According to the experimental design, add an equal volume of T cells to the mDC cell culture wells and add 10 μL / mL of antigen peptide. Gently shake left and right to distribute the cells evenly.

[0059] 5) Place the 96-well cell culture plate in a 37°C co-culture environment.

[0060] 6) Observe the T cell status and expansion every day, and add or replace fresh culture medium according to the color of the culture medium. When the T cells have filled the entire well, transfer the co-culture system to a 48-well cell culture plate to continue co-culture.

[0061] 7) Every 5 days, add mDC cells to the co-culture system at a ratio of 4:1 for T cells and DC cells, and add 10 μL / mL of antigen peptide.

[0062] 3. TCR single-cell sequencing 3.1 Tumor antigen-reactive T cell sorting (Reference: Che X, Zheng S, Sun Y, Wang X, Zhang P, Cao J, Bai Y. Multi-engineered T cell vaccine boosting TCR-T celltherapy enhances anti-tumor function and eradicates heterogeneous solidtumors. Mol Ther. 2025 Sep 3;33(9):4529-4551. doi: 10.1016 / j.ymthe.2025.05.036.) 4. Packaging, concentration, and titer detection of recombinant retroviral vectors 5.1 Plasmid extraction and purification (using the PureYield PlasmidMaxiprep System kit manufactured by Promega, catalog number A2393).

[0063] 5.2 Culture of HEK293T 1) The frozen HEK293T cells were rapidly thawed at 37°C, then transferred to centrifuge tubes with a pipette, 5 mL of DMEM complete medium preheated to 37°C was added, and then centrifuged (300 g for 4 minutes) and the supernatant was discarded.

[0064] 2) Add 10 mL of DMEM complete medium to resuspend the cells, gently mix with a pipette, and transfer the cell suspension to a 100 cm³ container. 2 Place the cells in a culture dish. Incubate at 37°C and ensure daily passage (18-24 hours) to avoid excessive cell density.

[0065] 3) Continue cell culture and passage, closely observing cell growth. When the cell number reaches the experimental requirements, perform plating and then conduct plasmid transduction experiments.

[0066] 5.3 Cell passage 1) Wash the cell culture wells once with PBS buffer, discard the washing solution, and avoid blowing the cells during the washing process.

[0067] 2) Add trypsin digestion solution to the cell culture wells and incubate at 37°C for 5 minutes. Observe the cell digestion regularly. Stop digestion when cells detach when the culture dish is gently shaken.

[0068] 3) Add serum-containing culture medium to stop digestion, gently pipette the cells off, and collect the cell suspension.

[0069] 4) Centrifuge at 300 g for 5 minutes at room temperature, discard the supernatant, add complete culture medium to resuspend the cells, gently pipette to mix, and then transfer to a culture dish for adherent culture at 37°C.

[0070] 5.4 Recombinant Retroviral Packaging 1) Cell preparation: Take out HEK293T cells in the logarithmic growth phase. At this time, the cells should have clear boundaries, not aggregate, and form a monolayer. The cells should look plump, with few granules and 3-4 protrusions. The protrusions should not be too long. Packaging can be carried out when the confluence reaches 60-80%.

[0071] 2) Plasmid preparation: Three types are used: the main plasmid (to be packaged), and the helper plasmids psPAX2 (X2) and PMD2.G (2G). After large-scale extraction, the plasmids are precipitated with ethanol and dissolved in sterile, endotoxin-free deionized water. The plasmid concentration is then measured for later use. For each 10 cm cell culture dish, the dosage of each plasmid is 16 μg of the main plasmid, 12 μg of X2, and 4 μg of 2G.

[0072] 3) Prepare reagents: 2.5 mmol / L CaCl2, 2× HBS (pH 7.05), and ultrapure water. All reagents must be equilibrated to room temperature in advance.

[0073] 4) Replace HEK293T cells with fresh culture medium (DMEM containing 10% FBS) before coating with the virus.

[0074] 5) Mix the main plasmid, X2 and 2G in a ratio of 16:12:4, add 10% volume of 2.5 mmol / L CaCl2 solution, and bring the volume to 500 μL with ultrapure water.

[0075] 6) Transform the plasmid mixture into HEK293T cells using the calcium phosphate method. Take a 5 mL flow cytometry tube, add 500 μL of 2× HBS, and add an equal volume of the mixture dropwise to the 2× HBS at a rate of one drop per second, shaking immediately after each drop. After mixing, the solution will appear milky white. Vortex the solution 8 times to thoroughly mix.

[0076] 7) Add 1 mL of the mixture along the side wall of the culture dish to HEK293T medium, shake gently to mix, and place in a 37℃ incubator for further incubation.

[0077] 8) Change the medium after 8-12 hours and add fresh culture medium.

[0078] 5.5 Detection of recombinant retrovirus titers 1) After changing the medium for 12 h, collect the viral supernatant, aspirate the supernatant into a 15 mL centrifuge tube, filter it using a 0.22 μm cell filter, freeze it at −80℃, and add 5 mL of fresh culture medium to the cell culture dish to continue culturing.

[0079] 2) Collect the viral supernatant again after 12 h, aspirate it into a 15 mL centrifuge tube, filter and freeze. Each time the supernatant is collected, set aside about 100 μL for titer measurement.

[0080] 3) Digest and count HEK293T cells with 0.25% trypsin, and seed cells into 12-well plates at a ratio of 2×10⁵ / well.

[0081] 4) Incubate the cells at 37°C in a 5% CO2 incubator until the confluence reaches 50%.

[0082] 5) Take the virus supernatant and add 0.5 μL, 1 μL, 5 μL, 10 μL and 20 μL to 1.5 mL centrifuge tubes respectively. Add polybrene to a final concentration of 8 μg / mL and fresh culture medium to a final volume of 1 mL.

[0083] 6) Discard the HEK293T cell supernatant, add the above-mentioned virus mixtures of different concentrations to the twelve-well plates, and incubate them at 37°C in a 5% CO2 incubator for 48 h.

[0084] 7) HEK293T cells were digested, and the positive rate of cell transduction was detected by flow cytometry.

[0085] 8) Viral titer (TU / mL) = (cell positivity rate × total number of cells / volume of added viral supernatant) × 1000.

[0086] 9) To avoid errors, select wells with a cell positivity rate of 10-20% for titer calculation, and determine the virus titer based on the average value of each group.

[0087] 5.6 Recombinant Retrovirus Concentration 1) Filter the supernatant of the recombinant lentivirus using a 0.22 µm filter, and transfer the filtered virus solution into centrifuge tubes with a volume of approximately 35 mL per tube.

[0088] 2) Insert the pipette into the bottom of the recombinant lentivirus solution and slowly add 2 mL of 20% glucose solution to form a buffer layer.

[0089] 3) Assemble the centrifuge tube tubing onto the sleeve and centrifuge at 24000 g for 1 h at 4°C.

[0090] 4) After centrifugation, discard the supernatant, add 2 mL of fresh culture medium, and resuspend the virus particles.

[0091] 4) Clean centrifuge tubes and related equipment promptly, and use 75% ethanol for cleaning.

[0092] 5) Dispense the concentrated virus solution into 1.5 mL centrifuge tubes and store at −80℃ for later use.

[0093] 6. Recombinant retrovirus transduction of T cells 6.1 T cell activation 1) The sorted CD8+ T cells were processed at a rate of 1×10⁻⁶. 6 Cells were resuspended in T-cell activation medium at a concentration of 1 cell / mL and transferred to a six-well plate, with 2 mL of cell suspension added to each well.

[0094] 2) Place the six-well plate in a CO2 incubator for incubation.

[0095] 3) During culture, the growth status of T cells and changes in the color of the culture medium should be closely observed. If rehydration is required, IL-2, anti-CD3 antibody, and anti-CD28 antibody should be added to fresh culture medium in proportion to the total volume after rehydration. Recombinant retrovirus transduction should be performed within a 40-48 hour time window after cell activation.

[0096] 6.2 Recombinant retrovirus transduction of mouse T cells 1) Count the activated T cells and resuspend them to 4 × 10⁶ cells / day using preheated T cell activation medium. 5 The concentration was set at 4 μL / mL, and then Polybrene was added to the cell suspension to improve the transduction efficiency of recombinant reverse transcription.

[0097] 2) Add the above T cell suspension to a six-well plate, 0.5 mL per well.

[0098] 3) Add 50 μL of the prepared concentrated recombinant retrovirus supernatant to each well of T cell suspension.

[0099] 4) Use sealing film to wrap around the edge of the six-well plate to prevent cell contamination.

[0100] 5) Place the six-well plate in a centrifuge and centrifuge at 350 g for 1 h at room temperature, using a slow acceleration and slow deceleration mode.

[0101] 6) After centrifugation, remove the six-well plate, remove the sealing film, and gently place it into the CO2 incubator. After standing for 12 hours, replace the culture medium.

[0102] 7) The transduction efficiency of the recombinant retrovirus was detected 48-72 h after transduction.

[0103] 8) If necessary, successfully transduced CD8+ T cells can be sorted using a flow cytometer.

[0104] 7. TCR-T cell function test 7.1 Detection of TCR-T killing function and factor secretion 1) Digest tumor cell lines overexpressing GFP-luciferase, centrifuge at 350 g for 5 minutes at room temperature, discard the supernatant, resuspend in fresh culture medium and count.

[0105] 2) Adjust the tumor cell density to 1×10⁻⁶ 5 Cells / mL, the cell suspension was transferred to 96-well white plates at a ratio of 100 μL per well.

[0106] 3) Place the white plate in a 5% CO2, 37℃ incubator and incubate for 7 h. Once the cells have adhered, they can be used for subsequent killing detection.

[0107] 4) Take TCR-T cells, centrifuge at 300 g for 5 minutes at room temperature, resuspend the cells in T cell culture medium and count them.

[0108] 5) Adjust the initial T cell density to 2×10⁻⁶.6 cells / mL, 4×10 5 cells / mL and 2×10 5 per mL.

[0109] 6) Set up different gradients according to the three cell concentrations mentioned above, with each concentration containing three parallel wells. Use a pipette to transfer 200 μL of T cells to a 96-well white plate, and adjust the kill effect-target ratio to 10:1, 2:1, and 1:1 respectively.

[0110] 7) Place the white plate in a 5% CO2, 37℃ incubator and continue culturing for 12-36 h.

[0111] 8) Remove the white plate and centrifuge at 400 g for 2 minutes at room temperature.

[0112] 9) Pipette 150 μL of supernatant into another clean 96-well plate. It can be used immediately for ELISA testing, or stored at −80°C for subsequent testing. Follow the instructions for ELISA testing.

[0113] 10) Add luciferase luminescent substrate to the 96-well white plate. First, dilute the luciferase to a concentration of 20 mg / mL using PBS at a ratio of 1:50. Then, quickly add 50 μL of the diluent to each well using a multi-channel pipette. Note that the entire operation must be performed under light-protected conditions.

[0114] 11) The white board with the substrate was tested for luminescence using a microplate luminescence detector. The killing efficiency was calculated using the following formula: Killing efficiency (%) = (Luminescence intensity of control well - Luminescence intensity of test well) / Luminescence intensity of control well × 100%.

[0115] 7.2 ELISPOT 1) The microplate was surface activated using basal culture medium (serum-free medium or RPMI-1640). 200 μL of culture medium was added to each well and equilibrated at room temperature for 5 minutes. The liquid in the well was then poured out.

[0116] 2) Add samples and stimulants according to the experimental design, and transfer the culture plate to a 37°C constant temperature incubator for 16 h.

[0117] 3) After incubation, add 200 μL of 4℃ deionized water to lyse the cells.

[0118] 4) Wash each well 5 times with buffer (200 μL / well, 30-60 s each time), then add biotin-labeled antibody working solution (100 μL / well) to each well and incubate at 37°C for 60 minutes.

[0119] 5) After 5 rounds of buffer washing, add enzyme-labeled streptavidin working solution (100 μL / well) to each well and incubate for 60 minutes.

[0120] 6) Add 100 μL of freshly prepared colorimetric solution to each well under dark conditions, and adjust the reaction time according to the ambient temperature. If the room temperature is above 20℃, let it stand for 15-45 minutes; otherwise, transfer it to a 37℃ incubator and check the color development every 5 minutes.

[0121] 7) After pouring out the liquid from the well, rinse the front and back of the culture plate and the base with deionized water. Place the detection plate in a well-ventilated and dark environment and allow it to dry naturally before taking spot readings.

[0122] 7.3 Immunofluorescence staining 1) The sections were placed in environmentally friendly dewaxing solution I, environmentally friendly dewaxing solution II, and environmentally friendly dewaxing solution III for 10 minutes each, and then in anhydrous ethanol I, anhydrous ethanol II, and anhydrous ethanol III for 5 minutes each to dewax the paraffin sections, and then washed with distilled water.

[0123] 2) Place the slide in EDTA (pH 9.0) and microwave on medium heat for 8 minutes. After turning off the microwave, microwave on medium-low heat for 7 minutes to perform antigen retrieval. During the retrieval process, avoid excessive evaporation of the buffer solution. After retrieval, allow the slide to cool naturally. Place the slide in PBS and wash three times using a shaker.

[0124] 3) After slightly drying the sections, use a histochemical pen to draw circles around the tissue, then add BSA to block for 30 minutes.

[0125] 4) Add primary antibody mixing reagent: Mix primary antibodies from different species, add the prepared primary antibody dropwise, place the slide in a humidified chamber, and incubate overnight at 4°C.

[0126] 5) Add secondary antibody reagent: Place the slide in PBS, wash it with a shaker, add the corresponding secondary antibody, and incubate at room temperature in the dark for 50 minutes.

[0127] 6) Counterstaining cell nuclei with DAPI: After washing, add DAPI staining solution and incubate at room temperature in the dark for 10 minutes.

[0128] 7) After washing, add self-fluorescent quencher solution B for 5 minutes, then rinse with running water for 10 minutes.

[0129] 8) Use anti-fluorescence quenching mounting medium for mounting.

[0130] 9) Image acquisition: DAPI excitation wavelength 330-380 nm, emission wavelength 420 nm; 488 excitation wavelength 465-495 nm, emission wavelength 515-555 nm; CY3 excitation wavelength 510-560 nm, emission wavelength 590 nm; CY5 excitation wavelength 608-648 nm, emission wavelength 672-712 nm.

[0131] Unless otherwise specified, statistical methods used for the data in this embodiment of the invention include Student's t-test, One-way ANOVA, or Two-way ANOVA for difference analysis. All statistical analyses were performed using GraphPad Prism 10.1 software. The p-values ​​represent the following: ns: not significant. p ≤ 0.05, p ≤ 0.01, p ≤ 0.001.

[0132] Example 1 Testing the in vivo antitumor efficacy of TCR-T 1.1 Detection of the killing ability and cellular function of TCR-T cells under different tumor volume conditions First, an in vivo functional validation platform was established using OT-1 TCR-T targeting the OVA model antigen. For example... Figure 2 As shown in Figure A, 5 × 10⁶ mice were subcutaneously injected on one side of each side of 6-8 week old C57BL / 6 mice. 5 A mouse subcutaneous colorectal cancer solid tumor model was constructed using MC38 tumor cells overexpressing OVA antigen. The tumors were allowed to grow to a size of 30-50 mm. 3 (Small tumor) or 75-100mm 3 (For large tumors) use 1×10 7 OT-1 TCR-T, NT (T cells not transduced with OT-1), or an equal volume of PBS were administered via tail vein in two separate infusions to observe the tumor-suppressing effect of TCR-T infusion under different initial tumor volumes.

[0133] like Figure 2 As shown in the middle BC diagram, OT-1 TCR-T cells effectively inhibited tumor growth in small tumors, but failed to inhibit tumor progression in large tumors. This was achieved by digesting tumor tissue and detecting the activation phenotype of tumor-infiltrating T cells using flow cytometry. Figure 2As shown in the data from the DF study, tumor-infiltrating T cells treated with OT-1 TCR-T expressed higher levels of 4-1BB and CD69 activation markers in the small tumor group, while in the large tumor group, there was no significant difference in the expression levels of activation markers between tumor-infiltrating T cells treated with OT-1 TCR-T and those treated with control T cells (NT). This indicates that even with overexpression of the target antigen, TCR-T is still unable to effectively kill larger solid tumors.

[0134] The study examined T cell infiltration capacity after TCR-T infusion under different initial tumor volumes. The proportion of CD3-positive T cells in tumor tissue was measured at the treatment endpoint, as shown in Figure 3A-B. In the small tumor group, the proportions of tumor-infiltrating T cells after PBS, NT, and OT1-TCR-T treatment were 29.8%, 35.5%, and 45.6%, respectively; in the large tumor group, these proportions were 27.4%, 26.7%, and 36.9%, respectively. Comparing the large and small tumor groups, the proportion of tumor-infiltrating T cells after OT-1 TCR-T treatment was lower in large tumors than in small tumors. These results indicate that OT-1 TCR-T has a poor infiltration effect on large tumors. Although the OT-1 TCR-T treatment group showed a higher proportion of infiltration compared to the PBS and NT treatment groups, OT-1 TCR-T infusion still failed to effectively inhibit the progression of large tumors, suggesting that the tumor microenvironment in large tumors may affect the therapeutic efficacy of TCR-T.

[0135] 1.2 Analysis of the differences in innate immune cell phenotypes under different tumor volumes Flow cytometry was used to detect the innate immune cell phenotypes of tumor infiltration in small and large tumors after OT-1 TCR-T treatment.

[0136] like Figure 4 As shown in Figure A, the positive rate of CD69 expression by NK cells was 71.9% in the small tumor group, while it was only 55.6% in the large tumor group. Figure 4 As shown in Figure B, macrophages in small tumors expressed lower levels of CD206. CD206 is a typing marker for M2 macrophages, which have anti-inflammatory effects and can promote tumor growth. This suggests that insufficient NK cell activation and the enrichment of M2 macrophages in the tumor microenvironment may be the reason why OT-1 TCR-T cannot inhibit the progression of large tumors. Further comparison of NK cell activation levels and macrophage typing percentages among different treatment groups, such as... Figure 4 As shown in Figure C, tumor-infiltrating NK cells in the small tumor group expressed higher levels of CD69 after OT-1 TCR-T treatment compared to the control group. Figure 4 As shown in the DE assay, although there was no significant difference in the proportion of macrophage differentiation phenotypes among the treatment groups in small tumors, the expression level of CD206 in macrophages in each group was lower than that in the large tumor group. These results indicate insufficient NK cell activation and enrichment of M2-type macrophages in large tumors.

[0137] Therefore, TCR-T dysfunction and insufficient endogenous immune activation are the main reasons why TCR-T cannot inhibit the progression of large tumors.

[0138] Example 2 Evaluate the efficacy and safety of TCR-T therapy that secretes IFNα. 2.1 Screening for key factors that improve the tumor microenvironment Transcriptome sequencing data of tumor samples from multiple colorectal cancer patients who received TCR-T clinical treatment were obtained from the GEO database. Based on the median survival, the samples were divided into a poor prognosis group and a good prognosis group. For example... Figure 5 As shown in Figure AB, in samples with better prognosis, pathways related to T cell activation, innate immune activation, and antigen uptake were significantly upregulated, while pathways negatively regulating T cell proliferation were downregulated. Enrichment analysis of the KEGG pathway was performed, as shown... Figure 5 In the sample with a better prognosis shown in Figure C, the Th1 and Th17 cell differentiation pathways were activated. Th1 and Th17 cells expressed and produced Th1 and Th17 cytokines, respectively, and studies have shown that these two types of factors play an important role in anti-tumor immunity.

[0139] Further analysis was conducted on the expression of Th1 and Th17 cytokine-related genes in the two groups of samples, such as... Figure 5 As shown in Figure D, samples with better prognosis expressed higher levels of IL12A, IL21R, CSF2, TNFRSF1A, and IFNAR1, with IFNAR1 showing the most significant difference between the two groups (p ≤ 0.001). The study indicates that IFNα and IL-12 can enhance the anti-tumor function of TCR-T cells while activating endogenous immune cells. Specifically, IFNα promotes T cell activation, enhances the ability of dendritic cells to take up and present antigenic peptides, activates NK cells, and induces polarization of M1 tumor-associated macrophages. IL-12 promotes T cell differentiation into Th1 cells, stimulates the activation of CD8+ T cells and NK cells, and enhances both innate and adoptive immune responses. Figure 5 As shown in Figure E, Kaplan-Meier survival curve analysis revealed the correlation between IFNAR1 expression and prognostic outcomes in cancer patients. The results showed that IFNAR1 was positively correlated with good prognosis in patients with pan-cancer, colorectal cancer, gastric cancer, and lung cancer. These findings suggest that IFNα may play a crucial role in improving the tumor microenvironment and enhancing the efficacy of TCR-T therapy.

[0140] Comparison of two cytokines, IL-12 and IFNα, screened using transcriptome sequencing data. First, TCR-T cells were stimulated exogenously with either IL-12 or IFNα, such as... Figure 6 As shown in Figure AB, compared to IL-12, IFNα significantly upregulated Ki67 and T-bet expression in TCR-T cells, indicating that IFNα can enhance the proliferative function of TCR-T cells and promote their differentiation into Th1 cells. Further examination of the phenotype of NK cells after exogenous IL-12 or IFNα stimulation was conducted, such as... Figure 6 As shown in Figure C, IFNα significantly upregulated CD69 expression in NK cells, with a positive rate of 20.05%. Furthermore, as... Figure 6 As shown in the middle DF, IFNα promotes M1 macrophage polarization and upregulates CD80 and CD86 expression in dendritic cells. These results suggest that combined TCR-T therapy with IFNα may significantly improve the tumor immunosuppressive microenvironment, enhance the anti-tumor function of endogenous T cells, and mobilize NK cells and M1 macrophages to synergistically eliminate solid tumors.

[0141] 2.2 Detecting the efficacy of TCR-T therapy by detecting IFNα secretion Studies have shown that direct infusion of IFNα can induce an inflammatory response in normal tissues. This invention uses a recombinant retrovirus to transduce the IFNα structure into OT-1 TCR-T cells, constructing IFNα-secreting OT-1 TCR-T cells (IFNα-OT-1). Untransduced OT-1 T cells (NT) and OT-1 TCR-T cells that do not express IFNα (OT-1) were used as controls to observe the synergistic effect of IFNα and TCR-T cells.

[0142] First, OT-1 TCR-T cells secreting IFNα, along with two control groups, were co-cultured in vitro with MC38 tumor cells overexpressing OVA at a 2:1 effector-target ratio for 24 h. The T cells were then fixed, permeabilized, and stained intracellularly to detect the expression of the cytotoxic factors perforin and granzyme B. For example... Figure 7 As shown in Figure A, compared to the control group, TCR-T cells secreting IFNα significantly upregulated the secretion of perforin and granzyme B after tumor stimulation, demonstrating that IFNα can enhance the killing function of TCR-T cells.

[0143] Simultaneously, this invention utilizes the MC38 colorectal cancer mouse subcutaneous tumor model to perform tail vein infusion of TCR-T cells secreting IFNα, such as... Figure 7 As shown in Figure B, compared to the control group, TCR-T cells secreting IFNα effectively inhibited tumor growth. To further elucidate the role of IFNα in endogenous immune cells, such as... Figure 7As shown in Figure C, flow cytometry analysis of NK cell and macrophage phenotypes in tumor tissue revealed that, compared to the OT-1 TCR-T treatment group, infusion of IFNα-secreting TCR-T cells significantly upregulated CD69 expression in tumor-infiltrating NK cells, while simultaneously increasing the proportion of M1 macrophages associated with pro-inflammatory responses. This indicates that IFNα secreted by TCR-T cells can effectively activate endogenous immune cells and enhance the efficacy of TCR-T therapy.

[0144] 2.3 Assess the safety of TCR-T therapy that secretes IFNα. like Figure 8 As shown in Figure AB, TCR-T cells secreting IFNα can effectively control tumor progression. Furthermore, it was found that in the TCR-T group overexpressing IFNα, the tumor tissue dendritic cells expressed higher levels of CD80 and CD86, indicating that IFNα enhances the antigen-presenting function of dendritic cells. However, as... Figure 8 As shown in Figure C, we monitored the body weight of mice that received tail vein infusions of different cells and observed that the body weight of the TCR-T group mice that secreted IFNα decreased significantly during the treatment process.

[0145] At the treatment endpoint, multiple biochemical indicators in the peripheral blood of mice were measured, such as... Figure 8 As shown in Figure D, compared to the control group, mice in the TCR-T group overexpressing IFNα had higher concentrations of alanine aminotransferase (ALT) and serum creatinine in their serum. ALT is an indicator of liver function, and serum creatinine is an indicator of kidney function; these elevated levels suggest that IFNα may cause damage to the liver and kidneys during its breakdown and excretion in vivo. Furthermore, immunohistochemical staining of mouse tumors with anti-PD-L1 yielded the following results: Figure 8 As shown in the middle EF, tumor cells in the TCR-T group overexpressing IFNα upregulated PD-L1 expression compared to the control group, and PD-L1 is considered a key molecule causing T cell exhaustion. Therefore, although TCR-T overexpression of IFNα can enhance the killing function of TCR-T and activate endogenous immune cells, in vivo treatment can lead to systemic adverse reactions and cause tumor cells to upregulate PD-L1.

[0146] Example 3 Constructing a novel antigen-specific TCR-T expressing IFNα / anti-PD-L1 3.1 Design of TCR-T vectors overexpressing IFNα / anti-PD-L1 This invention uses the pMSGV1 recombinant retroviral vector to construct a TCR-T structure expressing an IFNα / anti-PD-L1 bispecific fusion protein (vector construction as follows). Figure 1(As shown in AC). The promoter used for construction was LTR 3'. An Igκ sequence was added before the fusion protein sequence. The fusion protein sequence includes anti-PD-L1 scFv and IFNα. The IFNα is a mouse α4 isoform, and its 20th amino acid was replaced with leucine (Leu) instead of alanine (Ala). The anti-PD-L1 scFv and IFNα are connected by a (G4S)2 flexible linker to maintain their spatial connection while ensuring their respective functions.

[0147] To achieve independent expression of the bispecific fusion protein and the OT-1 TCR, an IRES sequence (internal ribosome entry site) was introduced into the vector to separate the fusion protein sequence from the OT-1 TCR sequence. To facilitate subsequent detection of TCR-T cell transduction efficiency and verification of fusion protein expression, a green fluorescent protein (GFP) tag was added to the vector, and a V5 tag was added to the C-terminus of the bispecific fusion protein.

[0148] As controls, TCR-T vectors expressing the fusion protein (OT-1), IFNα (IFNα-OT-1), and anti-PD-L1 (aPDL1-OT-1) were designed. These vectors were then packaged with recombinant retroviruses and transduced into T cells to construct OT-1 TCR-T cells expressing different protein structures.

[0149] After transducing T cells using recombinant retroviruses, the TCR-T cell status was observed using an inverted fluorescence microscope. Figure 9 As shown in Figures A and B, under bright-field conditions, the transduced TCR-T cells were clearly in good condition, exhibiting typical amplification and division morphology, with no obvious apoptosis, indicating that the viral transduction process had no significant impact on cell viability. Under fluorescence excitation conditions, the successfully transduced TCR-T cells overexpressing the vector emitted green fluorescence, demonstrating that recombinant retroviruses can efficiently introduce vectors containing different target gene sequences into T cells.

[0150] Flow cytometry was used to detect the transduction positivity rates of different structures. Representative flow cytometry plots and statistical analysis results are presented in [the table / document / etc.]. Figure 9 In C and D, the test results showed that although the vectors containing different target gene sequences differed in base length, this did not significantly affect the positive rate of transduction. This indicates that the LTR 3' promoter can effectively initiate the expression of different gene structures, and also proves that recombinant retroviruses can stably and efficiently transduce mouse T cells.

[0151] 3.2 Testing the recognition function of bispecific fusion proteins for their corresponding ligands To examine the binding and recognition capabilities of the bispecific fusion protein to its corresponding ligand, this invention first investigated the expression and secretion of the IFNα / anti-PD-L1 bispecific fusion protein by TCR-T cells. TCR-T cells expressing different structures were fixed and stained using IFNα antibody, and the results are as follows: Figure 10 As shown in Figure A, successful expression of the bispecific fusion protein in TCR-T cells can be detected. Furthermore, Western blot analysis was used to detect the expression of the fusion protein in TCR-T cells, as shown... Figure 10 As shown in Figure B, the TCR-T transduced with IFNα / anti-PD-L1 was able to express the protein of the expected molecular weight and exhibited a single band. The relative expression levels of different protein structures were statistically analyzed by comparing band brightness. Figure 10 As shown in Figure C, the relative expression level of the IFNα / anti-PD-L1 bispecific fusion protein was slightly lower than that of anti-PD-L1, but higher than that of IFNα. Enzyme-linked immunosorbent assay (EILSA) was used to detect the increasing protein concentration over time in the supernatant of TCR-T cells expressing the bispecific fusion protein, as shown in Figure C. Figure 10 As shown in Figure D, the transduced TCR-T cells can successfully express and secrete a bispecific fusion protein, and the fusion protein can recognize the IFNα receptor.

[0152] To detect the recognition ability of the bispecific fusion protein for PD-L1, MC38 tumor cells were transduced using a recombinant retrovirus to overexpress PD-L1. Purified MC38 tumor cell line overexpressing PD-L1 (MC38-PD-L1) was obtained by flow cytometry sorting. The flow cytometry results of PD-L1 expression in tumor cells are shown below. Figure 11 As shown in Figure A. MC38-PD-L1 cells were cultured for 4 h in a concentrated supernatant containing the bispecific fusion protein to allow the fusion protein to fully contact and bind to the cells. Flow cytometry staining of the tumor cells was then performed using IFNα antibody or V5-tagged antibody to detect the binding ability of the anti-PD-L1 on the bispecific fusion protein to the corresponding target. Figure 11 As shown in Figure BC, the positive rate of binding of the bispecific fusion protein to MC38-PD-L1 cells detected by IFNα antibody was 17.3%, and the positive rate detected by V5 tag antibody was 24.7%. Although the positive rates detected by IFNα antibody or V5 tag antibody were lower than the positive rates of actual PD-L1 expression in MC38-PD-L1 cells, the bispecific fusion protein showed specific binding to MC38-PD-L1 cells compared to the IFNα control group without anti-PD-L1.

[0153] Example 4 Detection of in vitro antitumor function of TCR-T cells expressing IFNα / anti-PD-L1 4.1 Evaluation of TCR-T tumor-killing function expressing IFNα / anti-PD-L1 First, TCR-T cells transduced with different structures were co-cultured with MC38-PD-L1 cells, such as... Figure 12 As shown in Figure AB, the killing efficiency of TCR-T cells expressing IFNα / anti-PD-L1 was significantly higher than that of the control group at 12 h, 24 h, and 36 h, and the killing efficiency gradually increased with the increase of the effector-to-target ratio. Figure 12 As shown in Figure C, at an effector-to-target ratio of 9:1, TCR-T cells expressing IFNα / anti-PD-L1 were observed to better recognize and target tumor cells, accumulating around the tumor cells and exerting their killing function. This indicates that IFNα / anti-PD-L1 can enhance the targeting ability of TCR-T cells to tumor cells and simultaneously strengthen their tumor-killing function.

[0154] 4.2 Analysis of TCR-T activation function expressed with IFNα / anti-PD-L1 First, flow cytometry was used to detect the expression of activation markers after co-culturing TCR-T cells with tumor cells transduced with different structures, such as... Figure 13 As shown in Figure AB, the proportions of NT, OT-1, IFNα-OT-1, aPDL1-OT-1, and aPDL1-IFNα-OT-1 expressing 4-1BB were 0.7%, 3.4%, 24.4%, 25.1%, and 38.9%, respectively. 4-1BB is considered an important indicator of T cell activation levels. In addition, such as... Figure 13 As shown in the CD, IFNα / anti-PD-L1 can significantly upregulate the expression levels of OX40 and CD69 in TCR-T cells, indicating that the bispecific fusion protein can effectively enhance the activation function of TCR-T cells.

[0155] 4.3 Detection of TCR-T depletion phenotype expressing IFNα / anti-PD-L1 Detecting the levels of TCR-T expression depletion markers after co-culture, such as Figure 14 As shown in Figure AB, TCR-T cells secreting the fusion protein expressed lower levels of PD-1 compared to TCR-T cells secreting only IFNα, indicating that anti-PD-L1 is beneficial in reversing TCR-T cell exhaustion. Furthermore, the detection results for LAG-3 and TIM-3 (e.g., ...) Figure 14 The results showed that TCR-T cells secreting the fusion protein expressed lower levels of LAG-3 and TIM-3. These results indicate that the IFNα / anti-PD-L1 bispecific fusion protein can effectively activate TCR-T cells while reducing the exhaustion phenotype of cells.

[0156] 4.4 Analysis of TCR-T differentiation phenotypes expressing IFNα / anti-PD-L1 Flow cytometry was used to detect the expression of CD62L and CD44 in co-cultured TCR-T cells to analyze the differentiation phenotype changes of TCR-T cells during tumor killing. Figure 15 As shown in the AC diagram, the proportions of memory cells in NT, OT-1, IFNα-OT-1, aPDL1-OT-1, and aPDL1-IFNα-OT-1 were 62.9%, 59.6%, 51.6%, 83.7%, and 82.1%, respectively. By detecting the proportions of different differentiation subtypes in CD8-positive or CD4-positive T cells in each group, the results showed that the proportion of central memory cells in the TCR-T group secreting the bispecific fusion protein was significantly higher than that in the control group, while the proportion of exhausted T cells was lower than that in the control group. These results indicate that the IFNα / anti-PD-L1 bispecific fusion protein plays a role in maintaining the central memory phenotype of TCR-T cells.

[0157] 4.5 Evaluation of TCR-T proliferation function expressed with IFNα / anti-PD-L1 The co-cultured TCR-T cells were perforated and stained to assess their proliferation and apoptosis levels. Figure 16 As shown in Figure A, the proportions of Ki67 expression by NT, OT-1, IFNα-OT-1, aPDL1-OT-1, and aPDL1-IFNα-OT-1 were 2.38%, 4.82%, 5.32%, 8.42%, and 15.3%, respectively. Statistical results are as follows: Figure 16 As shown in BC, CD8 cells secreting IFNα / anti-PD-L1 + TCR-T and CD4 + TCR-T cells showed significantly upregulated Ki67 expression. Furthermore, as... Figure 16 As shown in Figure D, the Annexin V / 7AAD kit was used to detect TCR-T cell apoptosis. The results showed that aPDL1-IFNα-OT-1 had a lower proportion of early and late apoptotic cells compared to IFNα-OT-1. These results indicate that IFNα / anti-PD-L1 significantly enhances the proliferation function of TCR-T cells and reduces their apoptosis level.

[0158] 4.6 Detection of CD4+ TCR-T cell phenotype expressing IFNα / anti-PD-L1 The proportions of Treg cells and Th1 cells in CD4+ TCR-T cells were detected. Figure 17 As shown in Figure AB, CD25 in the groups NT, OT-1, IFNα-OT-1, aPDL1-OT-1, and aPDL1-IFNα-OT-1 + FOXP3 +The proportions of TCR-T cells were 0.6%, 9.7%, 13.8%, 2.1%, and 2.8%, respectively. During co-culture, TCR-T cells expressing IFNα significantly upregulated the proportion of Treg cells. However, because the anti-PD-L1 component of the bispecific fusion protein can reverse T cell exhaustion, the proportion of Treg cells in TCR-T cells expressing IFNα / anti-PD-L1 was significantly lower. Given that IFNα can upregulate the Th1 pathway in T cells, and T-bet is a key transcription factor in the Th1 pathway, CD4+ was measured... + Analysis of TCR-T expression and T-bet status, and the proportion of Th1 cells. For example... Figure 17 As shown in CD, T-bet in the groups NT, OT-1, IFNα-OT-1, aPDL1-OT-1 and aPDL1-IFNα-OT-1 + The proportions of TCR-T cells were 52.9%, 59.7%, 79.0%, 59.9%, and 81.0%, respectively, indicating that the proportion of Th1 cells in TCR-T cells expressing IFNα was significantly increased.

[0159] 4.7 Assess the secretory function of TCR-T factors expressing IFNα / anti-PD-L1. Flow cytometry was used to detect the expression of cytotoxic factors in TCR-T cells with different structures after co-culturing with tumor cells, such as... Figure 18 As shown in Figure AB, after co-culturing with MC38-PD-L1 tumor cells, the expression levels of granzyme B in OT-1 and aPDL1-IFNα-OT-1 were 32.3% and 44.0%, respectively, and the expression levels of perforin were 10.7% and 27.4%, respectively. Compared with other control groups, aPDL1-IFNα-OT-1 significantly upregulated the expression of granzyme B and perforin. Furthermore, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokines, such as... Figure 18 As shown in Figure C, TCR-T cells expressing aPDL1-IFNα were observed to release higher levels of IFNγ and IL-2. IFNγ and IL-2 are important factors that promote T cell activation and killing function, and can activate bystander T cells, further enhancing the immune response.

[0160] 4.8 Study on the activation effect of IFNα / anti-PD-L1 on bystander T cells To investigate the effects of IFNα / anti-PD-L1 on bystander T cells, such as... Figure 19As shown in Figure A, a Transwell system was used to separate TCR-T cells secreting fusion proteins from bystander T cells. The upper layer of the Transwell chamber contained TCR-T cells secreting different structures, while the lower layer contained a co-culture system of untransduced T cells and tumor cells. A thin membrane between the two layers prevented transmembrane cell movement but allowed protein structures to pass through. This system was used to mimic the effect of IFNα / anti-PD-L1 on bystander T cells. Flow cytometry results showed that, compared to TCR-T cells (OT-1) without fusion protein expression, TCR-T cells expressing IFNα / anti-PD-L1 induced higher levels of 4-1BB, OX40, and CD69 activation markers (such as...) in the lower layer of T cells. Figure 19 (As shown in BC). Furthermore, as... Figure 19 As shown in Figure D, IFNα / anti-PD-L1 can maintain the central memory and effector phenotypes of bystander T cells and reduce T cell exhaustion. In contrast, although IFNα can also activate bystander T cells, it tends to exhaust their phenotype, further demonstrating the important role of anti-PD-L1 in reversing T cell exhaustion and maintaining early T cell phenotypes.

[0161] 4.9 Detection of tumor-killing function of neoantigen-specific TCR-T cells expressing IFNα / anti-PD-L1 The IFNα / anti-PD-L1 structure was transduced into IRF2-4 cells to construct a neoantigen-specific TCR-T cell expressing a bispecific fusion protein (aPDL1-IFNα-IRF2-4). As a control, IRF2-4 cells secreting only IFNα (IFNα-IRF2-4) and those secreting anti-PD-L1 (aPDL1-IRF2-4) were also constructed. These TCR-T cells transduced with different structures were co-cultured with MC38-PD-L1 tumor cells, as shown in the figure. Figure 20 As shown in Figure AB, the tumor cell killing efficiency of aPDL1-IFNα-IRF2-4 was significantly higher than that of the control group at 12 h, 24 h, and 36 h, and the killing efficiency gradually increased with the increase of the effector-to-target ratio. At an effector-to-target ratio of 9:1, aPDL1-IFNα-IRF2-4 had a significantly enhanced tumor killing function compared with other control groups.

[0162] Example 5 Evaluation of in vivo efficacy of TCR-T therapy expressing IFNα / anti-PD-L1 5.1 Monitoring the in vivo tumor-suppressing effect of TCR-T cells expressing IFNα / anti-PD-L1 A subcutaneous colorectal cancer tumor model overexpressing OVA and PD-L1 was established using C57BL / 6 mice, such as... Figure 21As shown in Figure A, 1×10⁻⁶ mice were subcutaneously injected on one side. 6 The MC38OVA-PD-L1 tumor cell line was used until the tumor volume reached 75-100 mm. 3 On Day 0, mice were infused with tail vein T cells transduced with different structures. As a control, mice were treated with different methods, including tail vein infusion of non-transduced T cells (NT), non-synthesis-secreting TCR-T cells (OT-1), IFNα-secreting TCR-T cells (IFNα-OT-1), and anti-PD-L1-secreting TCR-T cells (aPDL1-IFNα-OT-1). Mice were monitored regularly for 27 days post-infusion, and various indicators were measured. Results showed that TCR-T cells expressing the IFNα / anti-PD-L1 bispecific fusion protein significantly inhibited tumor growth (e.g., TCR-T cells expressing the IFNα / anti-PD-L1 bispecific fusion protein). Figure 21 (As shown in BC). Figure 21 As shown in Figure D, at the treatment endpoint, mice in the TCR-T group that received infusion of the IFNα / anti-PD-L1 bispecific fusion protein had lower tumor weight, and the infusion of the TCR-T group that received the bispecific fusion protein significantly prolonged the survival of mice (e.g., Figure 21 (E).

[0163] By performing in vivo fluorescence imaging on mice, such as Figure 22 As shown, tail vein infusion of TCR-T cells expressing IFNα / anti-PD-L1 exhibited a significant tumor-suppressive effect. On day 14, compared to the control group, mice infused with IFNα / anti-PD-L1 TCR-T cells showed significantly smaller tumor volume. These results indicate that IFNα / anti-PD-L1-secreting TCR-T cells possess significant anti-tumor function and can effectively kill tumor cells.

[0164] 5.2 Analysis of peripheral blood T cell phenotype and fusion protein secretion pMHC loaded with OVA antigen peptides was prepared using tetramer technology, and antigen-specific T cell detection was performed on peripheral blood, such as... Figure 23 As shown in Figure AB, mice in the TCR-T treatment group secreting the fusion protein exhibited higher frequency antigen-reactive TCR-T cells in their peripheral blood, and these cells expressed higher levels of 4-1BB. This indicates that the bispecific fusion protein can promote the amplification and persistence of tumor antigen-reactive TCR-T cells in vivo, maintaining long-term efficacy. Furthermore, the protein concentration in peripheral blood... Figure 23 As shown in Figure C, the concentration of the bispecific fusion protein was lower than that of IFNα, indicating that the design of the IFNα / anti-PD-L1 bispecific fusion protein is beneficial in reducing the concentration in the peripheral circulation, suggesting that infusion of TCR-T expressing the fusion protein may have better safety. To further clarify the response of mice to TCR-T infusion under different treatments, peripheral blood biochemical indicators of mice in different treatment groups were detected. Figure 23 As shown in Figure D, compared to the control group, mice in the TCR-T group overexpressing IFNα had higher concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (creatine kinase), and serum creatinine in their serum. Creatine kinase is an indicator of myocardial function, and serum creatinine is an indicator of renal function. The elevation of these indicators suggests that IFNα may cause damage to normal organs during its circulation, breakdown, and excretion in vivo. The bispecific fusion protein, due to its better targeting, does not exhibit systemic adverse reactions.

[0165] 5.3 Monitoring T cell infiltration levels and PD-L1 expression in tumor cells. Immunohistochemical staining was used to stain tumor samples from mice in different treatment groups for CD3, Ki67, and PD-L1, such as... Figure 24 As shown in Figure AB, the number of CD3-positive cells in the TCR-T group secreting the IFNα / anti-PD-L1 bispecific fusion protein was significantly increased, and obvious CD3-positive T cell infiltration was observed in the tumor tissue. Ki67 staining results are as follows... Figure 24 As shown in Figure C, compared with the TCR-T group that secretes only IFNα or anti-PD-L1, the tumor cells in the TCR-T group that secrete the bispecific fusion protein express lower levels of Ki67, indicating that their proliferation ability is significantly inhibited.

[0166] Furthermore, the level of PD-L1 expression in TCR-T cells secreting the bispecific fusion protein was significantly lower than that in TCR-T cells secreting only IFNα. Figure 24 The presence of IFNα / anti-PD-L1 may be due to IFNα / anti-PD-L1 mediating the specific killing of PD-L1-positive tumor cells by TCR-T cells. These results indicate that TCR-T cells expressing the IFNα / anti-PD-L1 bispecific fusion protein can enhance T cell infiltration and expansion, inhibit tumor cell proliferation, and relieve the inhibitory effect of PD-L1 on T cells.

[0167] 5.4 Immunofluorescence detection of tumor-infiltrating T cell proliferation CD3 and Ki67 levels in mice from different treatment groups were detected using immunofluorescence staining techniques. Figure 25 As shown in Figure AB, except for the TCR-T group expressing IFNα / anti-PD-L1, tumor cells in other control groups exhibited a significant proliferative phenotype. Furthermore, the TCR-T group expressing IFNα / anti-PD-L1 showed significant CD3 cell infiltration, and CD3-positive T cells expressed high levels of Ki67, indicating that IFNα / anti-PD-L1 enhanced T cell infiltration and proliferation.

[0168] 5.5 Detection of activation function of tumor-infiltrating T cells To further clarify the functional phenotype of infiltrating T cells in tumor tissues of mice in different treatment groups, tumor tissues were collected from mice in each group at the treatment endpoint, digested using a prepared digestive solution, and analyzed using methods such as... Figure 26 The gating method shown in Figure A was used to perform flow cytometry analysis on the phenotype of tumor-infiltrating T cells in each group. The activation status of adoptive TCR-T cells in tumor tissue infiltration is shown below. Figure 26 As shown in the middle BC diagram, the TCR-T treatment group secreting the bispecific fusion protein exhibited higher levels of 4-1BB and CD69 in both CD8-positive and CD4-positive TCR-T cells. In particular, compared to the TCR-T treatment group secreting only IFNα, the TCR-T activation level in the fusion protein-secreting group was significantly increased, indicating that the bispecific fusion protein possesses better functional activity. The phenotype of endogenous T cells infiltrating tumor tissue is as follows: Figure 26 As shown in the data from the DF study, compared to the TCR-T group that did not express the fusion protein, the TCR-T treatment group secreting the bispecific fusion protein exhibited higher levels of 4-1BB and CD69 expression in both CD8-positive and CD4-positive endogenous T cells. However, for OX40, there was no difference in expression levels among the groups in CD8-positive endogenous T cells. In CD4-positive T cells, OX40 expression showed a significant difference, indicating that the bispecific fusion protein can effectively activate CD4-positive T cells.

[0169] 5.6 Detecting the exhaustion level of tumor-infiltrating T cells The study aimed to detect whether TCR-T cells infiltrating tumor tissue showed an increase in exhaustion markers after activation. Flow cytometry was used to analyze the expression of exhaustion markers in adoptive TCR-T cells, such as... Figure 27 As shown in Figures A and B, the expression levels of PD-1 and TIM-3 in TCR-T cells of the TCR-T treatment group secreting the fusion protein were essentially the same as those in the control T cell treatment group. Notably, the expression levels of PD-1 and TIM-3 in TCR-T cells of the TCR-T treatment group expressing only IFNα were significantly increased, indicating that the bispecific fusion protein can also reverse the TCR-T exhaustion state in vivo. This suggests that anti-PD-L1 may also maintain the central memory phenotype of TCR-T cells; therefore, flow cytometry was used to detect the proportions of tumor-infiltrating TCR-T naive T cells, central memory T cells, effector T cells, and exhausted T cells. Figure 27In the tumor tissue shown in the CD, effector T cells constituted the majority of TCR-T cells. However, in TCR-T cells expressing only IFNα, the proportion of exhausted T cells exceeded that of effector T cells, reaching 51.2%. Although the proportion of effector T cells in the TCR-T treatment group secreting the fusion protein was significantly higher than that in the TCR-T treatment group not expressing the fusion protein, the proportion of exhausted T cells was only 21.2%, far lower than that in the control group. This indicates that the bispecific fusion protein can maintain the functional state of TCR-T cells in vivo, thereby achieving a better tumor-suppressive effect.

[0170] Example 6 Analysis of the mechanism by which TCR-T cells expressing IFNα / anti-PD-L1 enhance the therapeutic effect against solid tumors 6.1 Detection of gene expression profiles in TCR-T cells expressing IFNα / anti-PD-L1 TCR-T cells secreting different structures were co-cultured with MC38 tumor cells, and the TCR-T cells were isolated for RNA sequencing. For example... Figure 28 As shown in Figure A, principal component analysis of the TCR-T gene expression profiles of each group revealed that the samples were divided into five clusters. The correlation between samples within each group was good, while differences existed between samples between groups. Unsupervised clustering analysis was then performed on the TCR-T expression data of each group based on the immunological characteristic gene set in the MsigDB database, as shown below. Figure 28 As shown in Figure B, the expression profiles of immune-related genes of TCR-T expressing the IFNα / anti-PD-L1 bispecific fusion protein are similar to those of TCR-T expressing only IFNα or anti-PD-L1, but are significantly different from those of TCR-T that do not secrete fusion proteins.

[0171] Analyze the differential expression of TCR-T immune-related genes in each group, such as Figure 29 As shown in Figure A, TCR-T cells secreting the bispecific fusion protein exhibited significant differences in immune function compared to other control groups. To explore the differences in immune function among TCR-T cells in each group, a graph was drawn based on T cell functional characteristics as shown in Figure A. Figure 29The gene expression heatmaps of TCR-T cells in each group, shown in Figure B, reveal that TCR-T cells secreting bispecific fusion proteins express higher levels of memory-related genes, such as BCL2, CD28, MKI67, and CCL5, compared to other groups. These genes play crucial roles in maintaining T cell survival and proliferation. TCR-T cells secreting bispecific fusion proteins also express higher levels of effector genes, such as SMAD7, STAT1, IFNAR1, GZMB, and KLRD1, while expressing lower levels of exhaustion genes, such as PDCD1, TIGIT, and HAVCR2, compared to TCR-T cells secreting only IFNα. This indicates that the bispecific fusion protein enhances TCR-T activation while mitigating exhaustion. The relative abundance of different T cell subsets in each TCR-T group was analyzed using ImmuCellAI. Figure 29 As shown in Figure C, it was also found that compared to TCR-T cells that secrete only IFNα, TCR-T cells that secrete the bispecific fusion protein had a higher proportion of CD8-positive naive T cells and CD4-positive memory T cells, and a lower proportion of CD8-positive exhausted T cells. These results indicate that TCR-T cells secreting the bispecific fusion protein exhibit a gene expression profile similar to that of memory T cells and effector T cells.

[0172] 6.2 Analysis of the upregulation of the key TCR-T pathway in IFNα / anti-PD-L1 expression GO enrichment analysis was performed on differentially expressed genes of TCR-T cells that secrete bispecific fusion proteins and those that do not. Figure 30 As shown in Figure A, pathways related to T cell activation, such as adaptive immunity, antigen recognition and presentation, and granzyme-mediated killing, differed between the two groups. KEGG enrichment analysis was performed, as shown in Figure A. Figure 30 As shown in Figure B, the differentially expressed genes are mainly enriched in complement activation, Th1 and Th17 cell differentiation, antigen recognition and presentation, and IL-17 signaling-related pathways.

[0173] 6.3 Analysis of the expression of key TCR-T molecules expressing IFNα / anti-PD-L1 Heatmap analysis of these differentially expressed genes, such as Figure 31 As shown in Figure A, compared with the control group, TCR-T cells secreting the bispecific fusion protein expressed higher levels of activation-related genes such as ZAP70, TLR9, GZMB, and CXCL11. Figure 31 As shown in Figure B, volcano plot analysis of the two groups of differentially expressed genes revealed that 51 genes were upregulated in TCR-T, which secretes bispecific fusion proteins, while 35 genes were downregulated. Figure 31As shown in Figure C, key genes in the Th1 differentiation pathway, ZAP70 and IFNG, as well as key genes in the antigen recognition and presentation pathway, H2-AA and KLRD1, are all upregulated in TCR-T cells that secrete bispecific fusion proteins. These results indicate that TCR-T cells expressing the IFNα / anti-PD-L1 bispecific fusion protein enhance their anti-tumor function by upregulating T cell Th1 differentiation and antigen recognition-related pathways.

[0174] 6.4 Detection of endogenous immune cell transcriptome expression At the treatment endpoint, mouse tumor tissue was collected for reference transcriptome sequencing. For example... Figure 32 As shown in Figure A, principal component analysis results indicate that the tumor tissue samples within each treatment group showed good correlation, while differences existed between the groups. Figure 32 As shown in the BC diagram, endogenous cells treated with IFNα / anti-PD-L1 TCR-T therapy exhibited significant differences in immune function compared to other control groups. Genes related to NK cell activation, including NKG7, CD69, CXCL2, and ID2; DC cell maturation, including CD40 and CD83; and M1 macrophage activation, including EGR1, CSF1, CD14, and MCL1, were all upregulated after IFNα / anti-PD-L1 TCR-T therapy.

[0175] 6.5 Detecting the upregulation of key pathways in endogenous immune cells GO enrichment analysis was performed on differentially expressed genes in endogenous immune cells between the TCR-T treatment group expressing the IFNα / anti-PD-L1 bispecific fusion protein and the TCR-T treatment group that did not secrete the fusion protein. Figure 33 As shown in Figure A, differences were found between the two groups in pathways related to endogenous innate immunity, such as B cell activation, antigen binding, and complement activation. KEGG enrichment analysis was performed on the differentially expressed genes in the two groups, as shown in Figure A. Figure 33 As shown in Figure B, the differentially expressed genes are mainly enriched in the TNF signaling pathway, the cytokine and its receptor binding pathway, and the IL-17 signaling pathway.

[0176] The GSEA method was used to analyze the enrichment of these differentially expressed genes in the Reactome gene set, such as... Figure 34 As shown, differentially expressed genes were found to be enriched mainly in antigen peptide recognition, chemokine receptor, lymphocyte-non-lymphocyte interaction, and complement activation-related pathways, and these pathways were all upregulated in the TCR-T group that secreted bispecific fusion proteins.

[0177] 6.6 Analysis of the proportion of endogenous immune cell infiltration and the expression of key molecules. ImmuCellAI was used to analyze the relative abundance of different immune cell subsets in each TCR-T treatment group, such as... Figure 35 As shown in Figure A, the proportions of CD8-positive T cells, DC cells, and NK cells were upregulated, while the proportion of M2 macrophages was downregulated, suggesting that TCR-T cells secreting bispecific fusion proteins are conducive to the enrichment of pro-inflammatory cells. Genes showing significant differences between the TCR-T treatment group expressing IFNα / anti-PD-L1 bispecific fusion protein and the TCR-T treatment group not secreting the fusion protein were analyzed, such as... Figure 35 As shown in Figure B, compared with the control group, the TCR-T treatment group secreting the bispecific fusion protein expressed higher levels of pro-inflammatory and chemokine genes such as TNF, CXCL2, CCL3, and CCL6, as well as genes involved in innate immune function such as RGS1, EGR1, FOS, and JUN. These results suggest that IFNα / anti-PD-L1 improves the tumor microenvironment by promoting NK cell activation and DC cell maturation, and inducing M1 macrophage differentiation, thereby enhancing the efficacy of TCR-T therapy.

[0178] 6.7 Detection of tumor tissue infiltrating Treg cells and innate immune cell phenotypes Given the low proportion of Treg cells in TCR-T cells expressing IFNα / anti-PD-L1 observed in in vitro experiments, the proportion of Treg cells among tumor-infiltrating T cells in each group was examined. Figure 36 As shown in Figures A and B, the proportions of Treg cells in each group were 1.3%, 2.73%, 17.4%, 17.6%, and 3.26%, respectively. The proportion of Treg cells in the tumor tissue of the TCR-T treatment group secreting the fusion protein was even lower, indicating that the IFNα / anti-PD-L1 bispecific fusion protein can effectively inhibit Treg cell proliferation. Furthermore, flow cytometry was used to detect the proportions and activation levels of different innate immune cell subsets in the tumor tissue of the TCR-T treatment group expressing the fusion protein, such as... Figure 36 As shown in the CE, the TCR-T therapy group secreting the bispecific fusion protein exhibited higher levels of CD69 expression in infiltrating NK cells and a lower proportion of M2 macrophages in tumor tissue. Simultaneously, the proportion of type I classical dendritic cells (cDC1), which mediate tumor immunity, was significantly increased in the TCR-T therapy group secreting the fusion protein, and cDC1 cells expressed higher levels of CD80 and CD86. These results indicate that TCR-T cells secreting the IFNα / anti-PD-L1 bispecific fusion protein can effectively activate endogenous immunity. Notably, cDC1 cells, as antigen-presenting cells that effectively activate T cells, suggest that cDC1 cells may play a role in activating endogenous T cells in tumor tissue, thereby synergistically enhancing the anti-tumor effect.

[0179] Example 7 Constructing a novel antigen-specific TCR-T expressing human IFNα / anti-PD-L1 7.1 Detection of the secretion and cytotoxic function of human TCR-T factor expressing IFNα / anti-PD-L1. To evaluate the clinical translational application prospects of IFNα / anti-PD-L1, targeted therapies targeting NY-ESO-1 (NYE TCR) and KRAS were used, respectively. G12V The TCRs of (RAS TCRs) were constructed to create novel antigen-specific TCRs for human expression of IFNα / anti-PD-L1 (aPD-L1-IFNα-NYE and aPD-L1-IFNα-RAS). The NYE TCR and RAS TCR are known to specifically recognize the NY-ESO-1 antigen peptide and KRAS with high affinity. G12V Antigenic peptides.

[0180] This invention utilizes the natural expression of NY-ESO-1 and HLA-A The A375 human melanoma cell line at 02:01 was used as the target cell for NYETCR-T. In addition, the SW480 human colorectal cancer cell line, which naturally expresses the KRASG12V mutation, was overexpressed with HLA-A using recombinant lentivirus. 11:01, thus enabling it to be recognized by RAS TCR. In summary, this invention utilizes co-culture of aPD-L1-IFNα-NYE cells and A375 cells, leveraging aPD-L1-IFNα-RAS and overexpressing HLA-A... SW480 cells were co-cultured at a ratio of 11:01 to detect the TCR-T killing function of cells expressing aPD-L1-IFNα.

[0181] The positive rate of IFNα / anti-PD-L1 transduced T cells is as follows: Figure 37 As shown in Figures A and B, the positive rates of RAS, IFNα-RAS, aPD-L1-RAS, and aPD-L1-IFNα-RAS transduction were 45.9%, 35.4%, 45.7%, and 36.0%, respectively. There were no significant differences in the results of the three independent experiments. Therefore, recombinant lentivirus can successfully transduce IFNα / anti-PD-L1 into human T cells. The efficiency of TCR-T cell killing of tumor cells after co-culture with different structures is shown in Figures B. Figure 37 As shown in Figure C, both NYETCR-T and RAS TCR-T expressing IFNα / anti-PD-L1 significantly killed tumor cells, with killing efficiencies exceeding 90%. Furthermore, the IFNγ secretion by TCR-T during co-culture was as follows... Figure 37 As shown in Figure D, T cells transduced with aPD-L1-IFNα-RAS secrete high levels of IFNγ.

[0182] 7.2 Detection of TCR-T activation function and differentiation phenotype in human patients expressing IFNα / anti-PD-L1 The activation function and differentiation phenotype of TCR-T cells expressing aPD-L1-IFNα were examined, and the results are as follows: Figure 38 As shown in Figure A, the proportions of T cells expressing 4-1BB in untransduced T cells (NT), T cells transducing RAS TCR (RAS), RAS cells secreting IFNα (IFNα-RAS), RAS cells secreting aPDL1 (aPDL1-RAS), and RAS cells secreting IFNα / anti-aPDL1 (aPDL1-IFNα-RAS) were 3.45%, 8.80%, 12.36%, 8.49%, and 21.70%, respectively. TCR-T cells expressing IFNα / anti-aPDL1 showed significant activation (e.g., ...). Figure 38 In addition, the proportion of TCR-T depleted cells secreting IFNα increases (e.g., ...). Figure 38 In the case of T cells (B), the percentage reached 9.43%, while IFNα / anti-aPDL1 reversed T cell exhaustion, with the proportion of exhausted cells at only 2.12%. Figure 38 As shown in Figure D, the proportion of TCR-T memory cells expressing IFNα / anti-aPDL1 was significantly increased. In summary, a neoantigen-specific TCR-T cell line overexpressing IFNα / anti-aPDL1 was constructed using human T cells, demonstrating its significant anti-tumor function.

[0183] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bispecific fusion protein, characterized in that, This includes sequentially linked anti-PD-L1 scFv-flexible linker-T cell-activating cytokines.

2. The bispecific fusion protein according to claim 1, characterized in that, The cytokines that activate T cells include interferon.

3. The bispecific fusion protein according to claim 2, characterized in that, The interferon includes IFNα.

4. A vector comprising the bispecific fusion protein according to any one of claims 1 to 3.

5. The carrier according to claim 4, characterized in that, The vector includes a promoter-Igκ-the bispecific fusion protein-IRES-TCR structure connected in sequence.

6. A TCR-T cell that secretes the bispecific fusion protein of any one of claims 1 to 3.

7. The method for constructing TCR-T cells according to claim 6, characterized in that, This includes constructing TCR-T cells using the vector described in claim 4 or 5 to obtain the TCR-T cells that secrete the bispecific fusion protein.

8. The use of the bispecific fusion protein according to any one of claims 1 to 3, the vector according to claim 4 or 5, or the TCR-T cell according to claim 6 in the preparation of a medicament for treating tumors.

9. The application according to claim 8, characterized in that, The tumors include solid tumors.

10. A drug for treating tumors, characterized in that, The active ingredient includes the TCR-T cells as described in claim 6.

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