DC (dendritic cell) for expressing multispecific T cell adapter as well as preparation method and application of DC

Abstract

The invention relates to a DC cell for expressing a multispecific T cell adapter, a preparation method and application thereof, and an immune preparation containing the DC cell. The preparation method comprises the following steps: preparing a nucleic acid construct for coding a multispecific T cell adapter, the multispecific T cell adapter comprising a first antigen binding domain for specifically binding with CD3 on the surface of a T cell to activate the T cell, and a second antigen binding domain for specifically binding with B7H3 on the surface of a tumor cell, and an immunomodulatory molecule interleukin-2, i.e., a third binding domain, capable of binding to an IL-2 receptor on the surface of an immune cell; and transferring the nucleic acid construct as an exogenous nucleic acid into the DC cell to obtain the DC cell for expressing the multispecific T cell adaptor. The DC cell expressing the multispecific T cell adapter can assist a human immune system in better and more targeted killing of tumor cells.

Description

Technical Field

[0001] This application relates to the field of immunology, and in particular to a DC cell expressing a multispecific T cell connector, its preparation method, and its application. Background Technology

[0002] Cancer is closely related to gene mutations, which are uncontrolled abnormal mutations in the body. Theoretically, the older an individual is and the longer the time elapsed, the higher the probability of developing cancer. With the improvement of living standards and the increase in life expectancy in my country, the incidence of cancer has also increased, leading to a growing demand for cancer treatment.

[0003] Therefore, how to assist the human immune system in killing tumor cells more effectively and in a more targeted manner has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] To assist the human immune system in killing tumor cells more effectively and in a more targeted manner, this application provides a DC cell expressing a multispecific T cell connective, its preparation method and application, and an immunomodulator.

[0005] This application provides a method for preparing DC cells expressing a multispecific T cell connective, including: Prepare a nucleic acid construct encoding a multispecific T cell adaptor, the multispecific T cell adaptor comprising a first antigen-binding domain for specifically binding to CD3 on the surface of T cells to activate T cells, and a second antigen-binding domain for specifically binding to B7H3 on the surface of tumor cells; The nucleic acid construct was transferred as an exogenous nucleic acid into DC cells to obtain DC cells expressing a multispecific T cell adaptor.

[0006] This application provides a dendritic cell (DC) expressing a multispecific T cell connector, which is prepared using the aforementioned method for preparing DCs expressing a multispecific T cell connector.

[0007] This application provides the use of the aforementioned DC cells expressing multispecific T cell connectives in the preparation of drugs or formulations for treating tumors. This application provides an immunomodulator comprising the aforementioned dendritic cells expressing a multispecific T-cell connective.

[0008] These dendritic cells (DCs) expressing multispecific T-cell connectives can assist the human immune system in killing tumor cells more effectively and in a more targeted manner. Attached Figure Description

[0009] Figure 1 Show the structural diagrams of TCE7, TCE9, and TCE9A; Figure 2 The flow cytometry results of Example 2 are shown in the figure. Figure 3 A statistical graph showing the results of in vitro expansion of T cells in PBMCs in Example 3; Figure 4 The image shows the results of CD8+ immunophenotyping analysis of T cells in PBMCs after in vitro expansion in Example 3. Figure 5 The statistical graph showing the results of the four experiments (A, B, C, and D) in Example 4 for killing tumor cells is displayed. Figure 6 The statistical graph showing the results of the four experiments (A, B, C, and D) in Example 5 for killing tumor cells is displayed. Figure 7 The statistical graph shows the results of the A and B experiments in Example 6, which showed the killing of tumor cells. Figure 8 The statistical graph shows the results of the A and B experiments in Example 7, which showed the killing of tumor cells. Detailed Implementation TCEs are a class of drugs that mediate cancer cell lysis by bringing non-specific polyclonal T cells close to target cancer cells, thereby stimulating T cell-dependent cytotoxicity (TDCC). Currently approved TCE targets include CD3×CD19, CD3×CD20, CD3×BCMA, CD3×GPRC5D, and CD3×DLL3. The first four are mainly for hematologic malignancies, while only the CD3×DLL3 TCE, approved in 2024, is a drug targeting solid tumors—small cell lung cancer. Due to the stromal barrier effect of solid tumors, higher doses of TCE drugs are required to ensure effective concentrations within the tumor tissue. However, higher peripheral exposure is more likely to produce "on target, off tumor" toxicity, making engineering and dosage design particularly challenging for solid tumor TCEs. Furthermore, when target-mediated T cell activation leads to cytokine overload, cytokine release syndrome (CRS) occurs. To date, almost all TCEs that have entered clinical trials have resulted in CRS, with symptoms ranging from mild flu-like symptoms to severe multi-organ failure. To mitigate these adverse reactions, researchers are actively exploring more specific targets, conditionally activated inactive TCE prodrugs, and improved delivery methods.

[0010] This application provides a design concept for a class of TCE drugs, combining them with dendritic cells (DCs). Using dendritic cells as production facilities and delivery carriers, these drugs migrate to the lungs, lymph nodes, or tumor tissues via DCs, releasing the antigens locally. This approach effectively lyses tumor cells without causing severe toxic side effects. Furthermore, the antigens released from local tumor lysis can be taken up and presented by nearby DCs, thereby inducing the generation of more antigen-specific T cells and enhancing the TCE effect, resulting in a synergistic anti-tumor effect.

[0011] This application provides a method for preparing dendritic cells (DCs) expressing a multispecific T cell engager, comprising the following steps: preparing a nucleic acid construct encoding a multispecific T cell engager (TCE), wherein the multispecific T cell engager includes a first antigen-binding domain for specifically binding to CD3 on the surface of T cells to activate T cells, and a second antigen-binding domain for specifically binding to B7H3 on the surface of tumor cells; transferring the nucleic acid construct as an exogenous nucleic acid into dendritic cells (DCs) to obtain DCs expressing the multispecific T cell engager.

[0012] In researching new TCE delivery methods, the industry has successively tried strategies such as in vivo delivery of TCE using mesenchymal stem cells (MSCs) and AAV delivery of TCE. However, MSCs themselves have immunomodulatory and inhibitory effects and are not suitable for cancer treatment; while AAV vectors are merely delivery tools, and in the past two years, pharmaceutical giants including Pfizer, Roche, Takeda, Vertex, and Biogen have abandoned AAV gene therapy due to its inherent immunogenicity and toxicity risks. Therefore, using AAV vectors to deliver TCE is not a good strategy either. Using dendritic cells (DCs) to generate and deliver transcranial encephalocytes (TCEs) is a relatively ideal strategy because DCs neither suppress the immune response of the innate immune system nor inhibit it. Instead, they enhance antigen presentation, promote the killing response of immune cells such as T cells and NK cells, and maintain the stability of the immune system. The dendritic cells that generate T-cell binders provided in this application, while fulfilling the original function of DCs in promoting immune responses, can also exert immunological effects by utilizing their multispecific T-cell binders generated in vivo and secreted extracellularly. Specifically, without antigen presentation, the T-cells (which can be bystander T cells or antigen-specific T cells) are activated and expanded by directly binding to the TCR-CD3 complex on the surface of T cells via the first antigen-binding domain (CD3 antibody terminus). The T cells are differentiated into cytotoxic T cells, and the specific binding of their first and second antigen-binding domains allows the T-cells to be targeted to tumor cells using TCE molecules, accelerating the process of T-cell targeted binding and killing of tumor cells. This is a relatively safe, effective, and novel method consistent with the principles of cancer immunotherapy.

[0013] Compared to other therapies, the T-cell conjugate generated within DC cells provided in this application has unique advantages. These advantages are mainly reflected in: (1) Safety. Compared with TCE protein drugs, the amount of TCE produced in DC cells is lower than that of TCE protein drugs used directly, so it is safer in the periphery.

[0014] (2) Target Specificity. On the one hand, this application selects B7H3, which is expressed in tumor cells but not expressed or expressed at very low levels in healthy tissues, as the key target for tumor therapy. Its high specificity and selectivity reduce its non-tumor-targeting toxicity. On the other hand, the specificity of TCE targets is largely determined by its delivery carrier, DC cells. The dendritic cells used in this application, after intravenous infusion, can be distributed not only to lymphatic organs but also to the lungs, liver, and kidneys, corresponding to the targeted distribution of tumors in which B7H3 is highly expressed in the above organs. In addition, the specificity of the target can also be limited by the targeting of local administration. For example, B7H3 is often highly expressed in gliomas, and DC-TCE can be precisely located by intrathecal or intracerebral administration. Therefore, in future applications, the administration method for different indications can be selected according to the targeting characteristics of DC cells, thereby minimizing the "on target, off tumor" effect.

[0015] (3) A virtuous cycle of DC cell delivery carrier-TCE-endogenous DC / T cells-improved immune microenvironment. After intravenous infusion, DC cells first enter the lungs and then distribute to organs such as lymph nodes and liver. If a tumor occurs in these organs, the TCE generated and secreted by the DC cell carrier will preferentially react, mobilizing various T cells in the organ to kill cancer cells before specific T cells take effect. Then, the lysed cancer cell protein fragments are re-taken up, processed and presented by endogenous DC cells and DC carrier cells in the organ, thereby promoting the generation of more specific T cells. The various cytokines released further chemotactically attract more immune cells into the tumor tissue, improving the tumor immunosuppressive microenvironment, which is more conducive to the anti-tumor effect of TCE and DC cell vaccines.

[0016] In one possible implementation, the multispecific T-cell adaptor is a bispecific TCE, comprising a first antigen-binding domain for specifically binding to CD3 on the surface of T cells to activate T cells, and a second antigen-binding domain for specifically binding to B7H3 on the surface of tumor cells. Further, the DNA sequence of the aforementioned bispecific T-cell adaptor is shown in SEQ ID No: 3, the amino acid sequence is shown in SEQ ID No: 4, and it is named TCE9. The second antigen-binding domain of TCE9 is B7H3.

[0017] It should be noted that B7H3 (also known as CD276) is a type I transmembrane glycoprotein, a member of the B7 superfamily, and a newly discovered immune checkpoint in recent years. It is not expressed in monocytes, granulocytes, or normal human tissues, but is abnormally highly expressed in various tumor tissues, including non-small cell lung cancer, pancreatic cancer, primary liver cancer, kidney cancer, glioma, colorectal cancer, breast cancer, prostate cancer, pharyngitis, melanoma, sarcoma, and cervical cancer. It is also expressed on the surface of stromal cells, fibroblasts, and epithelial cells in the tumor microenvironment (TME). Overexpression of B7H3 in tumor tissues is often associated with poor prognosis and shorter overall survival and progression-free survival. The significant difference in B7H3 protein expression levels between normal and tumor tissues suggests that targeting B7H3 with drugs could lead to tumor-specific toxicity, minimizing damage to healthy cells. This makes B7H3 a promising target for cancer treatment. Therefore, the B7H3 antibody with the second antigen-binding domain of the multispecific TCE involved in this application specifically binds to the B7H3 antigen on the surface of tumor cells, which is highly targeted and can target T cells to the vicinity of the target cells, shorten the distance, skip a series of immune cascade reactions such as antigen presentation, and is fast and efficient. It can promote the immune killing of tumor cells by T cells, and has little impact on healthy cells in normal tissues and few side effects.

[0018] In another possible implementation, the multispecific T-cell connector is a trispecific T-cell connector. Preferably, this application provides a trispecific T-cell connector with the DNA sequence shown in SEQ ID No: 5 and the amino acid sequence shown in SEQ ID No: 6, named TCE9A. The second antigen-binding domain of TCE9A is a B7H3 antibody, and the third binding domain is the immunomodulatory molecule interleukin-2 (IL-2).

[0019] To illustrate the function and specificity of TCE9 and TCE9A, this application provides a comparative example: a CD3×CD19 TCE structure. It has a first antigen-binding domain that specifically binds to CD3 on the T cell surface to activate T cells, and a second antigen-binding domain for specific binding to CD19 on the tumor cell surface. Preferably, this application provides a bispecific T cell binder, the DNA sequence of which is shown in SEQ ID No: 1, the amino acid sequence of which is shown in SEQ ID No: 2, and named TCE7. Furthermore, the structures of TCE7, TCE9, and TCE9A are as follows... Figure 1 As shown.

[0020] In one possible implementation, the nucleic acid construct is transduced into dendritic cells (DCs) using a viral vector to obtain dendritic cells that generate T-cell connectives. Preferably, after preparing the nucleic acid construct, it is transferred into the DCs using a lentiviral packaging system to obtain dendritic cells that generate T-cell connectives, wherein the nucleic acid construct is a transfer plasmid in the lentiviral packaging system.

[0021] In one possible implementation, the nucleic acid construct and lentiviral packaging plasmids (in a broad sense, lentiviral packaging plasmids actually include packaging plasmids and envelope plasmids in a narrow sense) are co-transfected into host cells, the transfected host cells are cultured, the virus synthesized by the host cells is collected, and the collected virus is used to transduce the nucleic acid construct into DC cells to obtain DC cells expressing multispecific T cell connectives.

[0022] In one possible implementation, after the nucleic acid construct is transferred as an exogenous nucleic acid into DC cells, the DC cells expressing the multispecific T cell adaptor are cultured in RPMI 1640 medium.

[0023] This application provides the use of the aforementioned dendritic cells (DCs) expressing multispecific T-cell connectives in the preparation of drugs or formulations for treating tumors. Preferably, the prepared DCs (after undergoing harmless treatment, such as changing the medium to remove culture medium components unsuitable for injection into the human body) are used as a blood injection agent. This allows them to activate T cells to kill tumor cells.

[0024] In one possible implementation, the aforementioned DC cells expressing multispecific T cell connectives are used in the preparation of drugs or formulations for the treatment of any one of human melanoma, human cervical cancer, human osteosarcoma, and human sarcoma.

[0025] This application provides an immunomodulator, characterized in that it comprises the aforementioned DC cells expressing a multispecific T cell connector.

[0026] To verify the enhancing effect of dendritic cells that generate T-cell connectives on the immune response to kill tumor cells, this application conducted the following experiments on dendritic cells that generate T-cell connectives: The experimental materials and reagents involved include: 48-well plates, 96-well white plates, 1640 medium, X-VIVO medium, fetal bovine serum, interleukin-2, serum substitutes, trypsin, 1×PBS, CCRL lysis buffer, LAR substrate, B7H3 antibody, CD4 antibody, and CD8 antibody.

[0027] Cell lines: DC (human dendritic cells); DC-TCE7 (engineered human dendritic cells expressing TCE7, the DNA sequence encoding TCE7 is shown in SEQ ID NO: 1, the amino acid sequence of TCE7 is shown in SEQ ID NO: 2, TCE7 is a bispecific T cell connector, the first antigen-binding domain specifically binds to CD3, and the second antigen-binding domain is a CD19 antibody that can specifically bind to the CD19 antigen of tumor cells) DC-TCE9 (engineered human dendritic cells expressing TCE9, the DNA sequence encoding TCE9 is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4, and the second antigen-binding domain of TCE9 is a B7H3 antibody) DC-TCE9A (engineered human dendritic cells expressing TCE9A, the DNA sequence encoding TCE9A is shown in SEQ ID NO: 5, the amino acid sequence is shown in SEQ ID NO: 6, the second antigen-binding domain of TCE9A is B7H3 antibody, and the third binding domain is interleukin-2) PBMCs (human peripheral blood mononuclear cells) A375L (engineered human melanoma cell line expressing luciferase) HelaL (engineered human cervical cancer cell line expressing luciferase) U2OSL (engineered human osteosarcoma cell line expressing luciferase) HT1080L (engineered human sarcoma cell line expressing luciferase) Data Analysis: All experimental data are expressed as mean ± standard deviation (SD). Significance analysis was performed using t-tests. P < 0.05 was considered statistically significant, and P < 0.01 indicated highly significant differences.

[0028] The present invention will be further illustrated below with reference to embodiments.

[0029] Example 1: Preparation of dendritic cells that generate T cell connectives Referring to the sequence listing, the DNA sequence of TCE7 is shown in SEQ ID NO: 1, and the amino acid sequence of TCE7 is shown in SEQ ID NO: 2. The DNA sequence of TCE9 is shown in SEQ ID NO: 3, and the amino acid sequence of TCE9 is shown in SEQ ID NO: 4. The DNA sequence of TCE9A is shown in SEQ ID NO: 5, and the amino acid sequence of TCE9A is shown in SEQ ID NO: 6. Example 1 set up three experimental groups to prepare DC-TCE7, DC-TCE9, and DC-TCE9A, respectively. Except for the gene sequences, all experimental procedures were kept consistent across the three experimental groups.

[0030] (1) Obtain the gene sequences of TCE7, TCE9 and TCE9A, and send them to a third-party company for gene synthesis. After enzyme digestion and ligation, the plasmids obtained by enzyme digestion and ligation are transformed into E. coli. After the sequencing is correct, the bacteria are shaken and the plasmids are extracted (the plasmids extracted and collected here are the aforementioned nucleic acid constructs, plasmids with TCE7, TCE9 or TCE9A sequences) for later use.

[0031] (2) Resuscitate 293T cells, culture and passage them, and administer them at a dose of 5×10⁻⁶ cells per cell line the day before proceeding to step (3). 6 Cells were seeded at a density of 10 cells / dish in culture dishes and incubated overnight at 37°C.

[0032] (3) Transfection: Mix the lentiviral packaging plasmid and the target plasmid (7 μg each) at a 1:1 ratio, add 500 μL of serum-free RPMI 1640 medium, and incubate at room temperature for 15 min at a target plasmid: PEI (mass:volume) ratio of 1:3. Replace the 293T cells with fresh RPMI 1640 complete medium in advance, and gently add the transfection mixture dropwise into the culture dish, gently mix, and incubate at 37°C.

[0033] (4) Collect the virus at 24 h, 48 h, and 72 h. Specifically: After culturing for 24 h, collect the supernatant into a 50 mL centrifuge tube and store it at 4 ℃ as the collected virus solution. Then, add 12 mL of 1640 medium containing 8% FBS to the remaining 293T cells. After culturing for 48 h, collect the supernatant into a 50 mL centrifuge tube and store it at 4 ℃ as the collected virus solution. Then, add 12 mL of 1640 medium containing 8% FBS to the 293T cells. After culturing for 72 h, collect the supernatant and mix it with the virus solutions from 24 h and 48 h. Centrifuge at 3500 rpm for 10 min and collect the centrifuged supernatant as the virus collected in step (4).

[0034] (5) Add virus concentrate to the collected virus, mix well, and let stand overnight at 4°C to allow the virus to precipitate. The next day, centrifuge and discard the supernatant. Add fresh culture medium to the centrifuged precipitate at 1 / 10 of the volume of the solution before centrifugation (the amount of culture medium added), resuspend the virus, aliquot and store at -80°C for later use.

[0035] (6) Add 1640 culture medium (0.5 mL per well) to the wells of the six-well plate; take DC cells and add them at a concentration of 1×10⁻⁶. 6 DC cells (referred to as the original DC cells of Example 1 in the following examples) were seeded into 6-well plates at a density of 0.5 mL / well. Virus solutions of TCE7, TCE9, and TCE9A were added to each well, and polybrene was added to a final concentration of 10 μg / mL. After mixing, the plates were incubated at 37 °C for 6 h, and then fresh 1640 complete culture medium was added.

[0036] (7) Collect the cultured cells. The first group is a mixture of DC-TCE7 and a small amount of original DC cells. In the following examples, the cells collected in this group will be referred to as DC-TCE7 cells prepared in Example 1. The second group is a mixture of DC-TCE9 and a small amount of original DC cells. In the following examples, the cells collected in this group will be referred to as DC-TCE9 cells prepared in Example 1. The third group is a mixture of DC-TCE9A and a small amount of original DC cells. In the following examples, the cells collected in this group will be referred to as DC-TCE9A cells prepared in Example 1.

[0037] Example 2: Preparation of various engineered tumor cells expressing luciferase: (1) Take A375, HeLa, U2OS, and HT1080 cells, and mix them at a ratio of 5 × 10⁻⁶. 5 One cell per well was seeded into a 6-well plate and cultured for 3 hours until the cells adhered.

[0038] (2) Prepare and collect the virus using a lentiviral system (the transfer plasmid can express luciferase). Add polybrene to the virus solution to a final concentration of 10 μg / mL.

[0039] (3) Add the mixture obtained in step (2) into the well at a density of 0.5 mL / well. After culturing for 6 h, add fresh 1640 complete medium to obtain A375L, HelaL, U2OSL and HT1080L cells expressing luciferase.

[0040] In this embodiment, the tumor cells obtained endogenously express B7H3 molecules. The tumor cells prepared in Example 2 were subjected to flow cytometry antibody staining using an antibody targeting B7H3 for detection. The detection results are as follows: Figure 1 As shown, tumor cells were found to positively express the B7H3 molecule, which can be used for subsequent experiments.

[0041] Example 3: In vitro expansion of T cells in PBMCs induced by DC-TCE9 and DC-TCE9A cells: This embodiment sets up five experimental groups: blank control group, original DC cell group, DC-TCE7 group, DC-TCE9 group, and DC-TCE9A group. These five experimental groups are identical except for the following step (1): whether DC cells are added and the specific cells added. The specific operating steps are as follows: (1) The blank control group did not add DC cells, while the other four groups used the original DC cells from Example 1, the DC-TCE7 cells, DC-TCE9 cells, and DC-TCE9A cells prepared in Example 1, respectively. Subsequently, they were processed at a rate of 1×10⁻⁶. 6 At a density of cells / mL, the collected cells were seeded in X-VIVO medium containing 8% serum substitute and cultured for about 20 h. (2) Centrifuge the cells from step (1) at 1200 rpm for 3 min and collect the supernatant for later use.

[0042] (3) Recover PBMC, according to 1×10 6 500 μL of the supernatant was seeded into 5 wells of a 12-well plate; the supernatant from step (2) was diluted 4-fold and added to PBMCs, and fresh culture medium was added to 1 mL. The supernatant from step (2) was added every two days at a 4-fold dilution, and interleukin-2 was added daily (the final concentration of interleukin-2 after addition was 500 U / mL). Cell counts were performed every two days during the culture process, and the count results are as follows: Figure 3 As shown.

[0043] (4) On day 18 of culture, a portion of the cells were taken for CD4+CD8+ immunophenotypic analysis. The results are as follows: Figure 4As shown.

[0044] Depend on Figure 3 It can be seen that from the 4th day of culture, the number of cells in the DC-TCE7 group, DC-TCE9 and DC-TCE9A group began to increase. From the 6th day, the cells entered the logarithmic growth phase and the cells were in good condition. The total number of cells increased by about 45 times on the 18th day, while the control group cells did not increase at all.

[0045] like Figure 4 As shown, the CD8+ T cell content in the blank control group was only 28.7%, while the CD8+ T cell content in the DC-TCE7 and DC-TCE9 groups was 77.8% and 73%, respectively. The CD8+ T cell content in the DC-TCE9A group was 92.5%. This indicates that TCE7, TCE9, and TCE9A all have the effect of activating a large number of T cells and inducing T cells to differentiate into CD8+ T cells, with the induced cells being predominantly CD8+ T cells. Interleukin-2 can further increase the CD8+ T cell content because IL-2 regulates the immune response in vivo through multiple mechanisms, including activating and proliferating immune cells, promoting cytokine production, and directly participating in anti-tumor effects.

[0046] Example 4: Results of co-culturing DC-TCE7, DC-TCE9, and DC-TCE9A cells with CD3 monoclonal antibody-activated T cells to kill tumor cells.

[0047] This embodiment sets up four experiments: A, B, C, and D. The experimental content of each group for each experiment is shown in Tables 1, 2, 3, and 4.

[0048] Table 1: Item A Table 2: Item B Table 3: Item C Table 4: Item D TCE-derived cells were co-cultured with effector cells and tumor cells, and the survival rate of tumor cells was detected. The specific procedure is as follows: (1) Digest tumor cells, count them, and measure them at a rate of 2.5~3×10⁻⁶. 4 One cell / well was seeded in a 48-well plate with three parallel wells per group and cultured overnight to allow the tumor cells to adhere to the plate. (2) Take effector cells. Effector cells are mixed cells that have been expanded and cultured after activating PBMCs with CD3 monoclonal antibody. The test results show that the T cell content is about 95%. Count the cells and add the corresponding TCE source cells and CD3 monoclonal antibody activated T cells into the target cells at the same time according to the effector-target ratio in the table and the ratio of TCE source (cells): effector cells = 1:1. Mix and culture at 37℃ for 20 h. (3) Remove the suspended cells, wash the plate gently with PBS 2-3 times, and aspirate the liquid from the wells; (4) Add 75 μL of cell lysis buffer to each well and lyse on ice for 10 min to allow live cells to release luciferase protein; (5) Aspirate the lysis product into a 1.5 mL EP tube and centrifuge at 12000 g for 30 s; (6) Take 20 μL of the centrifuged supernatant into each of the 96 wells, and add 20 μL of the substrate for the luminescent enzyme reaction to each well: (7) The tumor cells were detected using an ELISA reader. The tumor cell survival rate was calculated based on the fluorescence intensity, and the killing rate of the effector cells against the tumor cells was obtained. The statistical results of the killing rates of each item are as follows: Figure 5 As shown.

[0049] according to Figure 5 We know that in the group involving DC-TCE7 cells, effector cells showed weak killing effect on tumor cells. However, effector cells induced by DC-TCE9 cells with high TCE9 expression exhibited the strongest tumor-killing effect. Specifically, at an effector-to-target ratio of 0.5:1, the killing rates against U2OSL and HeLaL were 80% and 60%, respectively (see...). Figure 5 A and D) also have a killing effect of about 50% on A375L cells. Figure 5 C), has a killing effect of about 15% on HT1080L cells ( Figure 5 (B) The difference was statistically significant (mean ± SD), P < 0.05. At an effector-to-target ratio of 1:1, the killing rates of these four tumor cell lines were 95%, 75%, 62%, and 25%, respectively.

[0050] At an effector-to-target ratio of 0.5:1, DC-TCE9A achieved cell killing rates of 35%-60% against these four tumor cell lines, which were significantly different from the control group (mean ± SD), P < 0.05. At an effector-to-target ratio of 1:1, the cell killing rates against U2OSL, HeLaL, and A375L were 80%, 52%, and 35%, respectively. Figure 5 (A, C, D). This indicates that TCE9 and TCE9A activate a large number of T cells in PBMCs through CD3 ScFv, and at the same time kill tumor cells by targeting the B7H3 antigen on the surface of tumor cells.

[0051] Example 5: Results of co-culturing supernatants secreted by DC-TCE7, DC-TCE9, and DC-TCE9A with CD3 monoclonal antibody-activated T cells to kill tumor cells. In this embodiment, in order to verify that TCE9 and TCE9A are secreted cytokines, five experiments (A, B, C, and D) were designed. The experimental contents of each group are shown in Tables 5, 6, 7, and 8.

[0052] Table 5: Item A Table 6: Item B Table 7: Item C Table 8: Item D According to Tables 5-8, the supernatant secreted by TCE-derived cells, effector cells, and target cells were co-cultured, and the survival of tumor cells was detected. The specific procedure is as follows: (1) Digest tumor cells, count them, and measure them at a rate of 2.5~3×10⁻⁶. 4 10 cells / well were seeded in a 48-well plate with three parallel wells per group and cultured overnight to allow tumor cells to adhere to the plate. (2) Take 4 groups of DC cells, count them, and perform a 1×10⁻⁶ test. 6 Inoculate at a density of 1640 cells / mL in 1640 medium containing 8% FBS and incubate at 37°C for 20 h. (3) Take the supernatant from step (2) into a 1.5 mL EP tube, centrifuge at 1200 rpm for 3 min, and co-culture at effector cell:tumor cell ratios of 0.5:1 and 1:1, respectively. At the same time, add the supernatant obtained by centrifugation to the tube after a 4-fold dilution and mix culture. The culture system is 300 μL of 1640 medium containing 8% FBS and mixed culture at 37 ℃ for 20 h. (4) Remove the suspended solution, wash the plate gently with PBS 2-3 times, and aspirate the liquid from the wells; (5) Add 75 μL of cell lysis buffer to each well and lyse on ice for 10 min to allow live tumor cells to release fluorescent protein; (6) Aspirate the lysis product into a 1.5 mL EP tube and centrifuge at 12000 g for 30 s; (7) Take 20 μL of centrifuged supernatant into each of the 96 wells, and add 20 μL of the substrate for the luminescent enzyme reaction to each well; (8) Detection was performed using an ELISA reader. The killing rate of effector cells against tumor cells was calculated based on the fluorescence intensity. The statistical results are as follows: Figure 6 As shown.

[0053] according to Figure 6 We know that the group involving DC-TCE7 cells showed weak killing effect on tumor cells. However, the supernatant secreted by DC-TCE9 cells induced effector cells to kill A375L and HeLaL cells more effectively than DC-TCE9A cells. At an effector-to-target ratio of 1:1, the supernatant secreted by DC-TCE9 cells induced effector cells to kill 75% and 85% of A375L and HeLaL cells, respectively, while the latter's killing rate was 48% and 75%, respectively. These differences were statistically significant (mean ± SD), P < 0.05. Figure 6 C, D). The killing effects of the two methods on HT1080L and U2OSL cells were not significantly different. Effector-target ratios of 0.5:1 and 1:1 showed approximately 80% and 92% killing rates for U2OSL cells, respectively, and approximately 50% and 75% killing rates for HT1080L cells, respectively. These differences were statistically significant (mean ± SD), P < 0.05. Figure 6 A, B).

[0054] Example 6: Results of co-culturing DC-TCE7, DC-TCE9, and DC-TCE9A cells with PBMCs to kill tumor cells.

[0055] Examples 4 and 5 verified that TCE9 and TCE9A can induce CD3 monoclonal antibody-activated T cells to kill tumor cells. In this example, to verify that this type of TCE can also activate T cells in PBMCs to exert a tumor-killing effect, the following experiment was conducted: This embodiment includes two tests, A and B, and the experiments for each group are shown in Table 9.

[0056] (1) Digest and count tumor cells, according to 2.5-3×10 4 10 cells / well were seeded in a 48-well plate with three parallel wells per group and allowed to adhere overnight. (2) Take 4 groups of DC cells, count them, and add TCE-derived cells and PBMC cells into the target cells at the same time according to the effector-target ratio and the ratio of TCE-derived cells to effector cells = 1:1, and mix and culture at 37℃ for 20 h. (3) Remove the suspended cells, wash the plate gently with PBS 2-3 times, and aspirate the liquid from the wells; (4) Add 75 μL of cell lysis buffer to each well and lyse on ice for 10 min to allow live cells to release luciferase protein; (5) Aspirate the lysis product into a 1.5 mL EP tube and centrifuge at 12000 g for 30 s; (6) Take 20 μL of lysis supernatant into each of the 96 wells, and add 20 μL of luciferase luminescent substrate (LAR substrate) to each well: (7) The enzyme-linked immunosorbent assay (ELISA) was used for detection. The killing rate of effector cells against tumor cells was calculated based on the fluorescence intensity. The statistical results are as follows: Figure 7 As shown.

[0057] according to Figure 7 We know that DC-TCE7 cells induce weak killing of tumor cells by effector cells. Compared to DC-TCE9A cells, DC-TCE9 cells induce stronger tumor cell killing ability. Specifically, at an effector-to-target ratio of 0.5:1, DC-TCE9 cells induce approximately 80% and 40% killing rates against U2OSL and A375L cells, respectively; at an effector-to-target ratio of 1:1, they induce 95% and 72% killing rates against both, respectively, which are statistically significant (mean ± SD), P < 0.05. Figure 7 (A, B) This indicates that DC-TCE9 and DC-TCE9A can activate both CD3 monoclonal antibody-derived T cells and T cells in PBMCs to kill tumor cells, and there is no significant difference between the two.

[0058] Example 7: Results of DC-TCE7, DC-TCE9 and DC-TCE9A cells inducing antigen-specific T cells to kill tumor cells.

[0059] This embodiment includes two tests, A and B, and the experiments for each group are shown in Table 10.

[0060] Table 10 Following the steps outlined in Table 10, pp65 antigen-specific CTLs, tumor cells, and TCE-derived cells from each group were mixed, and the survival rate of the tumor cells was assessed. The specific procedure is as follows: (1) Digest tumor cells, count them, and measure them at a rate of 2.5~3×10⁻⁶. 4 10 cells / well were seeded in a 48-well plate with three parallel wells per group and cultured overnight to allow tumor cells to adhere to the plate. (2) Take 4 groups of DC cells, count them, and add TCE-derived cells and effector cells into the tumor cells at the same time according to the effector-target ratio and the ratio of TCE-derived cells to effector cells = 1:1, according to Table 10. Mix and culture at 37 ℃ for 20 h. (3) Remove the suspended cells, wash the plate gently with PBS 2-3 times, and aspirate the liquid from the wells; (4) Add 75 μL of cell lysis buffer to each well and lyse on ice for 10 min to allow live tumor cells to release fluorescent enzyme protein; (5) Aspirate the lysis product into a 1.5 mL EP tube and centrifuge at 12000 g for 30 s; (6) Take 20 μL of the centrifuged supernatant into each of the 96 wells, and add 20 μL of the luminescent enzyme substrate to each well: (7) The enzyme-linked immunosorbent assay (ELISA) was used for detection. The killing rate of effector cells against tumor cells was calculated based on the fluorescence intensity. The statistical results are as follows: Figure 8 As shown, DC-TCE7 cells induced weak killing of tumor cells by effector cells. However, at an effector-to-target ratio of 0.5:1, DC-TCE9 cells induced approximately 75% and 38% killing rates against U2OSL and A375L cells, respectively. At an effector-to-target ratio of 1:1, the killing rates against both were 97% and 65%, respectively, which were statistically significant (mean ± SD), P < 0.05. Figure 8 A, B). Compared to DC-TCE9 cells, DC-TCE9A-induced effector cells showed weaker tumor-killing activity. Specifically, at an effector-to-target ratio of 1:1, the killing rates against U2OSL and A375L cells were 92% and 45%, respectively, which was statistically significant (mean ± SD), P < 0.05. Figure 8 (A, B). This indicates that DC-TCE9 cells and DC-TCE9A cells can kill tumor cells by simultaneously activating CD3 monoclonal antibody-activated T cells, T cells in PBMCs, and antigen-specific T cells through the secretion of TCE9 and TCE9A, and there is no significant difference among the three.

[0061] sequence list SEQ ID NO: 1 SEQ ID NO: 2 MALPVTALLLPLALLLHAARPDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGG GGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVVSSTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDASAVYCARYYDDH YCLDYWGQGTTLTVSSVEGGSGGSSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK SEQ ID NO: 3 SEQ ID NO: 4 MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNYPFTFGQGTKLEIKGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYSSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVVSSTTTPAPRPPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDASAVYCARYYDDHY CLDYWGQGTTLTVSSVEGGSGGSSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK SEQ ID NO: 5 SEQ IDNO:6 MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSIISTLT

Claims

1. A method for preparing DC cells expressing a multispecific T cell connective, characterized in that, include: Prepare a nucleic acid construct encoding a multispecific T cell adaptor, the multispecific T cell adaptor comprising a first antigen-binding domain for specifically binding to CD3 on the surface of T cells to activate T cells, and a second antigen-binding domain for specifically binding to B7H3 on the surface of tumor cells; The nucleic acid construct was transferred as an exogenous nucleic acid into DC cells to obtain DC cells expressing a multispecific T cell adaptor.

2. The method for preparing DC cells expressing multispecific T cell connectives according to claim 1, characterized in that, The amino acid sequence of the multispecific T cell connector is shown in SEQ ID No:

4.

3. The method for preparing DC cells expressing multispecific T cell connectives according to claim 1, characterized in that, The multispecific T cell connector is a trispecific T cell connector, comprising a first antigen-binding domain for specifically binding to CD3 on the surface of T cells to activate T cells, a second antigen-binding domain for specifically binding to B7H3 on the surface of tumor cells, and a third binding domain, wherein the third binding domain is interleukin-2.

4. The method for preparing DC cells expressing a multispecific T cell connector according to claim 3, characterized in that, The amino acid sequence of the multispecific T cell connector is shown in SEQ ID No:

6.

5. The method for preparing DC cells expressing multispecific T cell connectives according to claim 1, characterized in that, After the nucleic acid construct was prepared, it was transduced into dendritic cells using a viral vector to obtain dendritic cells that generate T cell connectives.

6. The method for preparing DC cells expressing a multispecific T cell connector according to claim 5, characterized in that, After the nucleic acid construct is prepared, it is transferred to the DC cells using a lentiviral packaging system to obtain dendritic cells that generate T cell connectors, wherein the nucleic acid construct is a transfer plasmid in the lentiviral packaging system.

7. A DC cell expressing a multispecific T cell adaptor, characterized in that, The DC cells expressing a multispecific T cell connective were prepared using the method described in any one of claims 1 to 6.

8. The application of a DC cell expressing a multispecific T cell connective, characterized in that, The use of the DC cells expressing multispecific T cell connectives as described in claim 7 in the preparation of drugs or formulations for treating tumors.

9. An immunomodulatory agent, characterized in that, Includes DC cells expressing a multispecific T cell adaptor as described in claim 7.

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