Membrane binding type IL7 fusion protein, engineered immune cell expressing membrane binding type IL7 fusion protein and application

Abstract

The invention belongs to the field of biological medicine, and discloses a membrane binding type IL7 fusion protein, an engineered immune cell for expressing the membrane binding type IL7 fusion protein and application of the membrane binding type IL7 fusion protein. The fusion protein comprises an IL7 region and a transmembrane domain and can be expressed on a cell membrane, the tumor cell killing ability and the T cell survival ability of T cells expressing the fusion protein are both enhanced, and the aims of improving the tumor immune cell treatment effect and reducing the toxic and side effects are achieved. Particularly, the IL7 fusion protein anchors and expresses IL7 on the surface of a cell through transmembrane domains such as CD80 or PD-L1 and the like, so that (1) immune cells can be accurately regulated and controlled, the possibility that an excessive IL7 signal possibly causes autoimmune response or aggravates CRS is reduced, and the safety of the IL7 to immune cells such as T cells and the like is enhanced; (2) the half-life period of IL7 is prolonged, and the anti-tumor effect of adoptive immune cells is enhanced; and (3) the transmembrane fragment is linked with the IL7 through a hinge region of the flexible linker G4S, CD80 or PD-L1, so that the flexibility of the IL7 is enhanced, and the proliferation and killing functions of the IL7 on T cells are enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically gene engineering and tumor immunotherapy, particularly its application in adoptive T-cell therapy. Specifically, it relates to a method for fusion expression of the transmembrane domains of CD80 / PD-L1 / ICOSL / CD8a / CD8b / LFA1 / IL2Ra with IL7, thereby expressing IL7 on the cell membrane. T cells expressing this fusion protein exhibit enhanced tumor cell killing ability and T-cell survival. This invention achieves the goal of improving the efficacy of tumor immunotherapy while reducing toxic side effects. Background Technology

[0002] Tumor immunotherapy is one of the most groundbreaking technologies in cancer treatment in recent years. Its core principle is to genetically engineer or selectively activate the patient's own immune cells (such as T cells and NK cells) to specifically recognize and kill tumor cells. Currently, CAR-T cell therapy has achieved significant success in hematologic malignancies (such as leukemia, lymphoma, and multiple myeloma). However, immunotherapy still faces many challenges. In the treatment of solid tumors, the immunosuppressive nature of the tumor microenvironment (TME), antigenic heterogeneity, and physical barriers (such as dense stroma) severely limit its efficacy. Furthermore, T cell exhaustion (e.g., PD-1 / LAG-3 overexpression) and antigen escape (e.g., target downregulation) make it difficult to sustain efficacy.

[0003] The functional state of T cells is a key factor determining treatment efficacy. In recent years, regulating T cell activity using cytokines has become a research hotspot. Studies have shown that cytokines such as IL-7 play an important role in maintaining T cell stemness and anti-exhaustion properties, and can also enhance T cell proliferation and function, thus strengthening the immune response. IL-7 can significantly promote the survival and expansion of naïve T cells and memory T cell subsets, and inhibit apoptosis by upregulating BCL-2 expression. When T cells encounter antigens, IL-7 can enhance the T cell immune response to fight pathogens, including rescuing T cells from apoptosis, promoting the differentiation of naïve T cells into effector T cells, and increasing the production of memory T cells. IL-7 exerts its anti-tumor effect by regulating the release of cytokines such as IFN-γ, IL-1β, IL-1α, and TNF-α from immune cells. However, high levels of IL-7 in vivo, while maintaining T cell activity and inhibiting exhaustion, may also drive the survival of autoreactive T cells, leading to autoimmune diseases. To reduce cytokine-related toxicity and improve the survival and function of T cells in vivo, there is an urgent need in the field to develop engineered T cells with enhanced stemness, persistence, and antitumor activity, laying the foundation for the development of a new generation of T cell products with stronger antitumor activity and longer duration. Summary of the Invention

[0004] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a membrane-bound IL7 fusion protein and engineered immune cells expressing it, as well as its applications.

[0005] The technical solution adopted in this invention is: The first aspect of the present invention provides: An IL7 fusion protein comprising the following elements: Interleukin-7 element, composed of interleukin-7 or its functionally active fragment; The transmembrane domain is a transmembrane domain of CD80, PD-L1, ICOSL, CD8a, CD8b, LFA1, or IL2Ra protein.

[0006] In some instances, it also includes linkers for connecting different polypeptide fragments.

[0007] In some instances, the general structural formula of the IL7 fusion protein is X-IL7-H-TM, where each "-" independently represents a linker or no linker; X represents a signal peptide or no signal peptide; IL7 represents an interleukin-7 element; H represents the hinge region; and TM represents the transmembrane domain.

[0008] In some instances, it satisfies at least one of the following characteristics: The amino acid sequence of the interleukin-7 element is shown in SEQ ID NO.1; The amino acid sequence of the transmembrane domain of CD80 is shown in SEQ ID NO:4; The amino acid sequence of the PD-L1 transmembrane domain is shown in SEQ ID NO:5; The amino acid sequence of the transmembrane domain of the ICOSL is shown in SEQ ID NO:6; The amino acid sequence of the transmembrane domain of CD8a is shown in SEQ ID NO:7; The amino acid sequence of the transmembrane domain of CD8b is shown in SEQ ID NO:8; The amino acid sequence of the transmembrane domain of LEF1 is shown in SEQ ID NO:9; The amino acid sequence of the transmembrane domain of the IL2Ra is shown in SEQ ID NO:10.

[0009] In some instances, the linker is a flexible linker, preferably a GS linker. GS linkers include, but are not limited to, GSAS (SEQ ID NO.3), (GGCAGCGCCAGC)n, (GGCGGCGGCAGC)n, (GGCGGCGGCGGCAGC)n, (GGGS)n, (GGSG)n, (GGGGS)n, and (G)n, where 1 ≤ n ≤ 5, and n is an integer. In particular, the linker is G4S (linker), whose amino acid sequence is shown in SEQ ID:NO:2.

[0010] In some instances, it satisfies at least one of the following characteristics: X is a signal peptide, specifically the signal peptide of IL-7; The hinge region is the hinge region of CD80, PD-L1, ICOSL, CD8a, CD8b, LFA1, or IL2Ra protein, or an extended sequence thereof.

[0011] There are no specific requirements for the CAR or TCR signal peptide; it can be any known signal peptide, such as the Mesothelin-specific CAR signal peptide (amino acid sequence as shown in SEQ ID NO:15) or the NY-ESO1-specific TCR signal peptide (amino acid sequence as shown in SEQ ID NO:16).

[0012] In some instances, the hinge region and the transmembrane domain originate from the same protein.

[0013] In some instances, it satisfies at least one of the following characteristics: The transmembrane domain is the CD80 hinge region outside the transmembrane domain, and the amino acid sequence of the CD80 hinge region is shown in SEQ ID NO:11; The transmembrane domain is an extension of the hinge region outside the CD80 transmembrane domain, and the amino acid sequence of the extended CD80 hinge region is shown in SEQ ID NO:12; The transmembrane domain is the hinge region outside the PD-L1 transmembrane domain, and the amino acid sequence of the PD-L1 hinge domain is shown in SEQ ID NO:13; The transmembrane domain is an extension of the hinge region outside the PD-L1 transmembrane domain, and the amino acid sequence of the extended PD-L1 hinge region is shown in SEQ ID NO:14.

[0014] These features can be combined arbitrarily as long as they do not conflict with each other.

[0015] A second aspect of the present invention provides: The nucleotide sequence encoding the IL7 fusion protein described in the first aspect of the present invention.

[0016] A third aspect of the present invention provides: A protein expression vector or expression system, comprising the nucleotide sequence described in the second aspect of this invention.

[0017] A fourth aspect of the present invention provides: An engineered immune cell comprising the IL7 fusion protein described in the first aspect of the present invention, or the nucleotide sequence described in the second aspect of the present invention.

[0018] In some instances, the immune cells are T cells or NK cells.

[0019] A fifth aspect of the present invention provides: Applications of the IL7 fusion protein described in the first aspect of this invention or the nucleotide sequence described in the second aspect of this invention, the applications including: To prepare drugs for the prevention, relief and / or treatment of tumors; To prepare drugs for alleviating and / or treating autoimmune diseases; Expanding immune cells; Prepare vaccine antigen enhancers.

[0020] Specifically, this includes: 1. Enhancing tumor immunotherapy, for example, by endowing TCRT and CAR-T cells with the ability to express membrane-bound IL7, enabling them to provide sustained survival signals to themselves and neighboring T cells, thereby significantly improving their persistence, expansion capacity, and anti-tumor effect in the tumor microenvironment; simultaneously, it can also be used to construct more powerful "artificial antigen-presenting cells" for efficient expansion of immune cells such as T cells and dendritic cells in vitro. 2. Developing novel vaccines, by co-expressing membrane-bound IL7 with vaccine antigens, a powerful "immune niche" can be created at the injection site, strongly activating and expanding antigen-specific T cells, thereby inducing a stronger and more durable cellular immune response. 3. Promoting immune reconstitution, in hematopoietic stem cell transplantation or the treatment of immunodeficiency diseases, engineered cells expressing membrane-bound IL7 can provide a supportive microenvironment for the development and maturation of newborn T cells, accelerating the recovery of the immune system. Despite challenges such as safety control and delivery technology, the local application of IL7 in membrane-bound form is undoubtedly a highly promising technological direction for breaking through the current bottlenecks of immunotherapy and improving efficacy.

[0021] The beneficial effects of this invention are: The membrane-bound IL7 fusion proteins of some examples of the present invention retain the biological activity of IL7, while having better stability and stronger ability to promote T cell proliferation and killing.

[0022] Some examples of the membrane-bound IL7 fusion protein of the present invention anchor IL7 on the cell surface through the transmembrane domain of CD80 or PD-L1, and can be used to conduct experiments for the following purposes: (1) enhance the safety of IL7 for immune cells such as T cells, and precisely regulate immune cells by anchoring to the cell membrane, reducing the possibility of excessive IL7 signaling triggering autoimmune reactions or aggravating cytokine release syndrome (CRS); (2) increase the half-life of IL7 by anchoring to the cell membrane, and enhance the anti-tumor effect of adoptive immune cells; (3) link the transmembrane fragment to IL7 through the flexible linker G4S and the hinge region of CD80 or PD-L1, and enhance the flexibility of IL7 while anchoring IL7 to the cell membrane, thereby enhancing the proliferation and killing function of IL7 on T cells. Attached Figure Description

[0023] Figure 1 The results are flow cytometry findings of co-expression of IL7 and CAR after lentiviral transfection, which are supported by different transmembrane domains supporting IL7 cell membrane expression.

[0024] Figure 2 This is a schematic diagram of the structure of IL7 fusion proteins expressed by different Mesothelin CAR-T membranes.

[0025] Figure 3 and Figure 4 This is the result of flow cytometry analysis of CAR-T cells prepared by transfecting T cells with lentivirus.

[0026] Figure 5 This is a comparison of the expression intensity of IL7 on the membrane surface of different CAR-T cells.

[0027] Figure 6 The difference lies in the fold increase of CAR-T cells transfected with different lentiviruses.

[0028] Figure 7 This is a comparison of the in vitro killing efficiency of different CAR-T cells.

[0029] Figure 8 This is a comparison of the in vitro killing effect of different CAR-T cells on IFNγ secretion.

[0030] Figure 9 This is a result of membrane-expressed IL7 enhancing CAR-T cell infiltration into tumors and its therapeutic effect within the tumor.

[0031] Figure 10 This is a schematic diagram of the structure of IL7 fusion protein expressed on the cell membranes of different TCR-T cells.

[0032] Figure 11 and Figure 12 This is the result of flow cytometry analysis of TCR-T cells prepared by transfecting T cells with lentivirus.

[0033] Figure 13 This is a comparison of the expression intensity of IL7 in the cell membrane of T cells after lentiviral transfection.

[0034] Figure 14 The difference lies in the amplification of different TCRT cells.

[0035] Figure 15 This is a comparison of the in vitro killing functions of different TCR-T cells.

[0036] Figure 16 It is the amount of IFN-γ secreted in the supernatant of different TCR-T in vitro killing agents. Figure 17 This is a result of membrane-expressed IL7 enhancing TCR-T cell infiltration into tumors and its therapeutic effect on tumors.

[0037] Figure 18 This is a schematic diagram of the structure of IL7 fusion protein expressed in the cell membranes of different TILs.

[0038] Figure 19 and Figure 20 These are the results of flow cytometry analysis of lentivirus-infected TIL cells.

[0039] Figure 21 This is a comparison of the expression intensity of IL7 in membranes of different TILs.

[0040] Figure 22 This is a comparison of the tumor organoid killing function of TIL cells expressing membrane-bound IL7. Detailed Implementation

[0041] The technical solution of the present invention will be further illustrated below with examples and experimental results. The names and sequence numbers of each sequence in the examples of the present invention are shown in Table 1.

[0042] Table 1. Sequence Names and Numbers

[0043] Example 1: Comparison of lentiviral titers supporting IL7 cell membrane expression by different gene transmembrane domains 1. Plasmid construction To compare the differences in viral packaging titers caused by different gene transmembrane domains (CD80 / PD-L1 / ICOSL / CD8a / CD8b / LFA1 / IL2Ra) supporting IL7 cell membrane expression, a Mesothelin CAR-T cell line supporting IL7 cell membrane expression with CD80 / PD-L1 / ICOSL / CD8a / CD8b / LFA1 / IL2Ra was designed and constructed using existing Mesothelin-specific CAR-T cells (CAR signal peptide amino acid sequence shown in SEQ ID NO:15). The construction steps are as follows: (1) Download the CDS sequences of relevant genes from NCBI, and then optimize the codons of some gene sequences to enhance their expression. These optimization methods include, but are not limited to: human codon usage preference, appropriate GC content, stable mRNA secondary structure, etc., eliminating repetitive sequences and hidden splice sites and unnecessary restriction enzyme sites, while preventing the depletion of tRNA library in cells. (2) After optimization, the sequence was directly cloned into the lentiviral shuttle vector pLV-EF1a-cMYC-IRES-EGFP by GenScript. The EGFP sequence in the vector was replaced with the target sequence. The newly constructed vector was named pLV-EF1a-CAR-T-XTM-IL7. XTM represents the transmembrane segment of different genes, such as CD80TM representing the transmembrane domain and hinge region of the CD80 gene.

[0044] 2. Lentiviral preparation HEK-293T cells were transfected with the pLV-EF1a-CAR-T-X-IL7, psPAX2, and pMD2.G vector plasmids containing the IL7 gene obtained in step 1. The transfection system was prepared as follows: (1) Centrifuge tube A: Opti-MEM 500 ul + main plasmid 10 μg + pMD2.G 5 μg + psPAX2 5 μg + p3000 40 ul; (2) Centrifuge tube B: Opti-MEM 500 ul + Lipo3000 40 ul. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, and after being gently mixed with a pipette, it was allowed to stand at room temperature for 15-20 min. The transfection system was added to HEK-293T cells. After 5 hours of transfection, the supernatant was discarded, and 10 ml of DMEM complete medium was added to each dish. The cells were then incubated at 37°C with 5% CO2 for 48 hours. The supernatant of the transfected 293T cells was collected and centrifuged at 400g for 5 minutes. The cells were then filtered through a 0.45 μm filter, and the resulting filtrate was the original solution of the recombinant lentivirus. The lentivirus was concentrated using an Ultra-15 centrifuge and filtration device, and centrifuged at 5000 rpm for 50 minutes. The concentrated lentivirus was aliquoted and stored at -80°C for later use.

[0045] 3. Lentiviral titer determination Cell counting was performed on the washed and digested HEK-293T cells. The lentiviral concentrate was serially diluted 10-fold (50×, 500×, 5000×, 50000×). 2.5 × 10⁻⁶ cells were collected. 5 HEK-293T cells were added to corresponding centrifuge tubes, each containing 500 μl of culture medium, virus, and cell mixture. After mixing, the mixture was transferred to 24-well plates and incubated at 37°C with 5% CO2 for 24 h. The culture medium was then replaced. After 48 h, infected HEK-293T cells were collected, and the proportion of transgene-positive cells was detected using flow cytometry to calculate the viral titer. Lentiviral titer was calculated as: viral titer = (m × 2.5 × 10 × dilution factor) / transfection volume, where m is the proportion of transgene-positive cells. The lentiviral titers of CD80 / PD-L1 / ICOSL / CD8a / CD8b / LFA1 / IL2Ra supporting IL7 cell membrane expression are shown in Table 2. The lentiviral packaging titers of CD80 transmembrane domain and PD-L1 transmembrane domain supporting IL7 were higher than those of other groups.

[0046] Table 2. Titers of lentiviruses supporting IL7 cell membrane expression by different transmembrane domains

[0047] 4. Comparison of the transmembrane domains of various genes supporting the expression intensity of IL7 on the cell membrane. T cells were activated with CD3 / CD28 magnetic nanobeads for 24 hours, and CAR-T cells were prepared by lentiviral transfection of T cells. Flow cytometry analysis of IL7 and CAR co-expression results is as follows: Figure 1 As shown, the CD80 transmembrane domain and the PD-L1 transmembrane domain support the highest IL7 positivity rate in IL7 cell membrane expression, indicating that the CD80 transmembrane domain and the PD-L1 transmembrane domain have a better ability to support IL7 cell membrane expression than other gene transmembrane domains.

[0048] Example 2: Comparison of antitumor activity of IL7-expressing CAR-T cells 1. Plasmid construction To further optimize IL7 expression in CAR-T cell membranes, and to investigate the antitumor activity of IL7 in CAR-T cells and the differences in IL7 function between the CD80 transmembrane domain and the PD-L1 transmembrane domain, seven Mesothelin-specific CAR-T cells were designed and constructed using existing Mesothelin-specific CAR-T cells. The amino acid sequences of these Mesothelin CAR-T cells are shown in SEQ ID NO: 10, and each structure was named CAR-T 001, 002, 003, 004, 005, 006, and 007, respectively. Figure 2 As shown; constructs 002, 003, and 004 use the CD80 transmembrane domain (SEQ ID NO:4), and constructs 005, 006, and 007 use the PD-L1 transmembrane domain (SEQ ID NO:5). IL7 expression is achieved by using extracellular hinge regions of different lengths from CD80 or PD-L1 and G4S linkers, allowing IL7 to be expressed on the cell membrane surface via the transmembrane domain. Simultaneously, IL7 is linked using hinge regions of different lengths and linkers such as G4S. CAR-T 001, 002, 003, 004, 005, 006, and 007 are constructed using the EF1a promoter. The plasmid construction methods for the seven constructs are as follows: (1) Download the CDS sequences of relevant genes from NCBI, and then optimize the codons of some gene sequences to enhance their expression. These optimization methods include, but are not limited to: human codon usage preference, appropriate GC content, stable mRNA secondary structure, etc., eliminating repetitive sequences and hidden splice sites and unnecessary restriction enzyme sites, while preventing the depletion of tRNA library in cells. (2) The optimized sequence was directly cloned into the lentiviral shuttle vector pLV-EF1a-cMYC-IRES-EGFP by GenScript, and the EGFP sequence in the vector was replaced with the target sequence. The newly constructed vector was named pLV-EF1a-CAR-T-IL7.

[0049] 2. Lentiviral preparation HEK-293T cells were transfected with pLV-EF1a-CAR-T-IL7, psPAX2 vector and pMD2.G vector plasmid containing the IL7 gene obtained in step 1. The transfection system was prepared as follows: (1) Centrifuge tube A: Opti-MEM 500 ul + main plasmid 10 μg + pMD2.G 5 μg + psPAX2 5 μg + p3000 40 ul; (2) Centrifuge tube B: Opti-MEM 500 ul + Lipo3000 40 ul. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, and after being gently mixed with a pipette, it was allowed to stand at room temperature for 15-20 min. The transfection system was added to HEK-293T cells. After 5 hours of transfection, the supernatant was discarded, and 10 ml of DMEM complete medium was added to each dish. The cells were then incubated at 37°C with 5% CO2 for 48 hours. The supernatant of the transfected 293T cells was collected and centrifuged at 400g for 5 minutes. The cells were then filtered through a 0.45 μm filter, and the resulting filtrate was the original solution of the recombinant lentivirus. The lentivirus was concentrated using an Ultra-15 centrifuge and filtration device, and centrifuged at 5000 rpm for 50 minutes. The concentrated lentivirus was aliquoted and stored at -80°C for later use.

[0050] 4. Lentiviral titer determination Cell counting was performed on the washed and digested HEK-293T cells. The lentiviral concentrate was serially diluted 10-fold (50×, 500×, 5000×, 50000×). 2.5 × 10⁻⁶ cells were collected. 5 HEK-293T cells were added to corresponding centrifuge tubes, each containing 500 μl of culture medium, virus, and cell mixture. After thorough mixing, the mixture was transferred to 24-well plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then replaced. After 48 hours, infected HEK-293T cells were collected, and the proportion of transgene-positive cells was detected using flow cytometry to calculate the viral titer. Lentiviral titer was calculated as follows: Viral titer = (m × 2.5 × 10 × dilution factor) / transfection volume, where m is the proportion of transgene-positive cells.

[0051] (1) Expression intensity of IL7 in CAR-T cell membrane and its effect on T cell proliferation CAR-T cells were prepared by activating T cells with CD3 / CD28 magnetic nanobeads for 24 hours, followed by lentivirus transfection. Flow cytometry results are as follows. Figure 3 and Figure 4 As shown, IL7 can be expressed on the cell membrane surface, and longer hinges and linkers show higher MFI of IL7 expression, such as... Figure 5 As shown; T cell counts were performed on days 6, 9, and 12 after lentiviral transfection to compare the fold expansion of different CAR-T cells. It was found that the fold expansion of CAR-T 004 and CAR-T 007 was significantly higher than that of other groups, such as... Figure 6 As shown.

[0052] (2) Comparison of in vitro killing function of IL7-expressing CAR-T cells on cell membrane CAR-T cells were co-cultured with A549-mesothelin-treated tumor cells. Tumor-killing function was compared using the LDH release assay, and the secretion of IL-7 and IFN-γ by T cells was detected using ELISA. The experimental method is as follows: 1×10⁻⁶ cells were used... 5A549-mesothelin cells were seeded in 24-well plates, and 1 ml of CAR-T cell suspension (i.e., 1.5 × 10⁻⁶ cells) was added to each well. 6 CAR-T cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The cell culture supernatant was then collected. Tumor-killing function was compared using the LDH release assay. The results were as follows: Figure 7 As shown, membrane-expressed IL7 enhances the in vitro killing function of CAR-T cells, and IL7-linked CAR-T cells exhibit even stronger killing ability when connected with longer hinges and linkers.

[0053] (3) ELISA detection of IFN-γ secretion in in vitro cytotoxic supernatant Centrifuge the collected cell culture supernatant at 400g for 5 min, and transfer the supernatant to a new centrifuge tube. Determine the required number of strips based on the number of experimental wells (blank and standard). Add 100 μl of diluted standard and sample, and 100 μl of 1× dilution buffer R as a control well. Add 100 μl of biotinylated human IFN-γ antibody (Dayou, 1110002) working solution. Cover with sealing film and incubate at room temperature (18-25℃) for 1 h. Wash the plate: remove the liquid from the wells, add 300 μl of 1× washing buffer working solution, let stand for 1 min, and then discard the liquid from the wells. Repeat 3 times, patting dry on filter paper each time. Add 100 μl of streptavidin-HRP working solution. Cover with sealing film and incubate at room temperature (18-25℃) in the dark for 30 min. Terminate the reaction: quickly add 100 μl of stop solution to terminate the reaction. The results are as follows. Figure 8 The membrane expresses IL7, which enhances the secretion function of CAR-T IFNγ, and the secretion function of CAR-T 004 and CAR-T 007 is even stronger.

[0054] (5) In vivo comparison of the anti-tumor function of CAR-T cells expressing IL7 in cell membranes. Six- to eight-week-old female severely immunodeficient mice (B-NOG) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in specific pathogen-free (SPF) cages and provided with sterilized feed and drinking water. When constructing a subcutaneous tumor model, equal amounts of Ovar3 cells (1×10⁻⁶) were used. 7 / mouse) were subcutaneously injected into the right groin of B-NDG mice (suspended in 100 μl PBS). Fourteen days after tumor implantation, all mice were randomly divided into 5 groups (n=6 per group), and each group received a single treatment of the following: 1×10 7 Mock CAR-T cells and MSLNCAR-T cells from different groups (001, 002, 003, 004, 005, 006, 007) (1×10⁻⁶) 7(One) treatment. Results as follows: Figure 9 As shown in Figure C, IL7 expression on the membrane surface can enhance the in vivo therapeutic effect of CAR-T cells, especially the CAR-T 004 and CAR-T 007 structures, which showed the strongest in vivo therapeutic effect. Furthermore, membrane expression of IL7 enhanced the infiltration of CAR-T cells into tumors, as shown in Figure C. Figure 9 A and Figure 9 As shown in B.

[0055] Example 3: Comparison of antitumor activity of IL7-expressing TCR-T cells 1. Plasmid construction To verify whether IL7 expression on the TCR-T cell membrane promotes its anti-tumor activity and the differences in the function of IL7 between the CD80 transmembrane domain and the PD-L1 transmembrane domain, seven TCRTs were designed and constructed using existing NY-ESO-1 specific TCR-T cells. The amino acid sequence of the NY-ESO-1 TCRT is shown in SEQ ID NO:16, and each structure was named TCRT008, 009, 010, 011, 012, 013, and 014, respectively. The structures are shown in the attached figure. Figure 10 As shown; constructs 009, 010, and 011 use the CD80 transmembrane domain (SEQ ID NO:4), and constructs 012, 013, and 014 use the PD-L1 transmembrane domain (SEQ ID NO:5). IL7 expression is achieved by using extracellular hinge regions of different lengths from CD80 or PD-L1 and G4S linkers, allowing IL7 to be expressed on the cell membrane surface via the transmembrane domain. Simultaneously, IL7 is linked using hinge regions of different lengths and linkers such as G4S. The EF1a promoter is used to construct TCRT008, 009, 010, 011, 012, 013, and 014, respectively. The plasmid construction methods for the seven constructs are as follows: (1) Download the CDS sequences of relevant genes from NCBI, and then optimize the codons of some gene sequences to enhance their expression. These optimization methods include, but are not limited to: human codon usage preference, appropriate GC content, stable mRNA secondary structure, etc., eliminating repetitive sequences and hidden splice sites and unnecessary restriction enzyme sites, while preventing the depletion of tRNA library in cells. (2) The optimized sequence was directly cloned into the lentiviral shuttle vector pLV-EF1a-cMYC-IRES-EGFP by GenScript, and the EGFP sequence in the vector was replaced with the target sequence. The newly constructed vector was named pLV-EF1a-TCRT-IL7.

[0056] 2. Lentiviral preparation HEK-293T cells were transfected with pLV-EF1a-TCRT-IL7, psPAX2 vector and pMD2.G vector plasmid containing the IL7 gene obtained in step 1. The transfection system was prepared as follows: (1) Centrifuge tube A: Opti-MEM 500 ul + main plasmid 10 μg + pMD2.G 5 μg + psPAX2 5 μg + p3000 40 ul; (2) Centrifuge tube B: Opti-MEM 500 ul + Lipo3000 40 ul. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, and after being gently mixed with a pipette, it was allowed to stand at room temperature for 15-20 min. The transfection system was added to HEK-293T cells. After 5 hours of transfection, the supernatant was discarded, and 10 ml of DMEM complete medium was added to each dish. The cells were then incubated at 37°C with 5% CO2 for 48 hours. The supernatant of the transfected 293T cells was collected and centrifuged at 400g for 5 minutes. The cells were then filtered through a 0.45 μm filter, and the resulting filtrate was the original solution of the recombinant lentivirus. The lentivirus was concentrated using an Ultra-15 centrifuge and filtration device, and centrifuged at 5000 rpm for 50 minutes. The concentrated lentivirus was aliquoted and stored at -80°C for later use.

[0057] 5. Lentiviral titer determination Cell counting was performed on the washed and digested HEK-293T cells. The lentiviral concentrate was serially diluted 10-fold (50×, 500×, 5000×, 50000×). 2.5 × 10⁻⁶ cells were collected. 5 HEK-293T cells were added to corresponding centrifuge tubes, each containing 500 μl of culture medium, virus, and cell mixture. After thorough mixing, the mixture was transferred to 24-well plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then replaced. After 48 hours, infected HEK-293T cells were collected, and the proportion of transgene-positive cells was detected using flow cytometry to calculate the viral titer. Lentiviral titer was calculated as follows: Viral titer = (m × 2.5 × 10 × dilution factor) / transfection volume, where m is the proportion of transgene-positive cells.

[0058] (1) IL7 expression intensity in TCRT cell membrane and its effect on T cell proliferation Following the method disclosed in Example 1, lentivirus packaging and viral titer testing were performed, and TCR-T cells were prepared by transfecting T cells. Flow cytometry was used to detect the positive rate of transduced T cells and IL7 cell membrane expression. TCR-T cells were co-cultured with A375 tumor cells, and LDH and ELISA methods were used to detect the TCRT killing function and TCRT IFN-γ secretion capacity. The experimental methods are as follows: (1) IL7 expression intensity in TCRT cell membrane and its effect on T cell proliferation T cells were activated with CD3 / CD28 magnetic nanobeads for 24 hours, and TCR-T cells were prepared by transfecting T cells with lentivirus. Flow cytometry results are as follows. Figure 11 and Figure 12 As shown, IL7 can be expressed on the cell membrane surface, and longer hinges and linkers show higher MFI of IL7 expression, such as... Figure 13 As shown; T cell counts were performed on days 6, 9, and 12 after lentiviral transfection to compare the fold increase of different TCRT groups. It was found that the fold increase of TCRT 011 and TCRT 014 was significantly higher than that of other groups. Figure 14 As shown.

[0059] (2) Comparison of in vitro killing functions of IL7-TCRT expressed in cell membranes Take 1×10 5 A375 cells were seeded in 24-well plates, and 1 ml of CAR-T cell suspension (i.e., 1.5 × 10⁻⁶ cells) was added to each well. 6 TCR-T cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The cell culture supernatant was collected, and the cells were washed once with PBS. The in vitro killing function of TCRT was detected using the LDH method. The results are as follows: Figure 15 As shown: Membrane expression of IL7 enhances the ability of TCRT to kill tumor cells in vitro.

[0060] (3) ELISA was used to detect the secretion of IFN-γ in the in vitro cytotoxic supernatant. Centrifuge the collected cell culture supernatant at 400g for 5 min, and transfer the supernatant to a new centrifuge tube. Determine the required number of strips based on the number of experimental wells (blank and standard). Add 100 μl of diluted standard and sample, and 100 μl of 1× dilution buffer R as a control well. Add 100 μl of biotinylated human IFN-γ antibody (Dayou, 1110002) working solution. Cover with a sealing film and incubate at room temperature (18-25℃) for 1 h. Wash the plate: remove the liquid from the wells, add 300 μl of 1× washing buffer working solution, let stand for 1 min, and then discard the liquid from the wells. Repeat 3 times, patting dry on filter paper each time. Add 100 μl of streptavidin-HRP working solution. Cover with a sealing film and incubate at room temperature (18-25℃) in the dark for 30 min. Terminate the reaction: quickly add 100 μl of stop solution to terminate the reaction. The results are as follows. Figure 16 As shown, membrane expression of IL7 enhances the IFNγ secretion function of TCRT.

[0061] (4) In vivo comparison of the anti-tumor function of IL7 TCR-T cells expressed in cell membranes. Six- to eight-week-old female severely immunodeficient mice (B-NOG) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in specific pathogen-free (SPF) cages and provided with sterilized feed and drinking water. When constructing a subcutaneous tumor model, equal amounts of Ovar3 cells (1×10⁻⁶) were used. 7 Each mouse was subcutaneously inoculated into the right groin of a B-NDG mouse (suspended in 100 μl PBS). Fourteen days after tumor implantation, all mice were randomly divided into 5 groups (n=6 per group), and each group received a single dose of the following treatment: 1 × 10⁻⁶ saturated PBS. 7 Mock T cells and NY-ESO-1 specific TCR-T cells from different groups (TCRT 008, 009, 010, 011, 012, 013, 014) (1×10⁻⁶) 7 (One) treatment, results as follows Figure 17 C: Membrane surface expression of IL7 can enhance the in vivo therapeutic effect of TCRT, especially the O14 structure (PD-L1 transmembrane domain structure) with the strongest in vivo therapeutic effect. Membrane expression of IL7 enhances the infiltration of TCRT cells into the tumor, as shown in the results. Figure 17 A and Figure 17 B.

[0062] Example 4: Infection of TIL cells with lentivirus containing the target gene 1. Plasmid construction Six structures were designed and constructed, named constructs 015, 016, 017, 018, 019, and 020, and their whole genomes were synthesized. Figure 18 As shown. The amino acid sequence of IL7 in each construct is as described in SEQ ID NO:1; constructs 015, 016, and 017 use a CD80 transmembrane structure, with different hinge regions and G4S linkers to connect with IL7, and the EF1a promoter is used; constructs 018, 019, and 020 use the PD-L1 transmembrane domain and the extracellular hinge region of PD-L1. The plasmid construction methods for each of the six constructs are as follows: (1) Download the CDS sequences of relevant genes from NCBI, and then optimize the codons of some gene sequences to enhance their expression. These optimization methods include, but are not limited to: human codon usage preference, appropriate GC content, stable mRNA secondary structure, etc., eliminating repetitive sequences and hidden splice sites and unnecessary restriction enzyme sites, while preventing the depletion of tRNA library in cells. (2) After optimization, the sequence was directly cloned into the lentiviral shuttle vector pLV-EF1a-cMYC-IRES-EGFP by GenScript. The EGFP sequence in the vector was replaced with the target sequence. The newly constructed vector was named pLV-EF1a-IL7.

[0063] 2. Lentiviral preparation HEK-293T cells were transfected with the pLV-EF1a-IL7, psPAX2, and pMD2.G vectors containing the IL7 gene obtained in step 1. The transfection system was prepared as follows: (1) Centrifuge tube A: Opti-MEM 500 ul + main plasmid 10 μg + pMD2.G 5 μg + psPAX2 5 μg + p3000 40 ul; (2) Centrifuge tube B: Opti-MEM 500 ul + Lipo3000 40 ul. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, and after being gently mixed with a pipette, it was allowed to stand at room temperature for 15-20 min. The transfection system was added to HEK-293T cells. After 5 hours of transfection, the supernatant was discarded, and 10 ml of DMEM complete medium was added to each dish. The cells were then incubated at 37°C with 5% CO2 for 48 hours. The supernatant of the transfected 293T cells was collected and centrifuged at 400g for 5 minutes. The cells were then filtered through a 0.45 μm filter, and the resulting filtrate was the original solution of the recombinant lentivirus. The lentivirus was concentrated using an Ultra-15 centrifuge and filtration device, and centrifuged at 5000 rpm for 50 minutes. The concentrated lentivirus was aliquoted and stored at -80°C for later use.

[0064] 3. Lentiviral titer determination Cell counting was performed on the washed and digested HEK-293T cells. The lentiviral concentrate was serially diluted 10-fold (50×, 500×, 5000×, 50000×). 2.5 × 10⁻⁶ cells were collected. 5 HEK-293T cells were added to corresponding centrifuge tubes, each containing 500 μl of culture medium, virus, and cell mixture. After thorough mixing, the mixture was transferred to 24-well plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then replaced. After 48 hours, infected HEK-293T cells were collected, and the proportion of transgene-positive cells was detected using flow cytometry to calculate the viral titer. Lentiviral titer was calculated as follows: Viral titer = (m × 2.5 × 10 × dilution factor) / transfection volume, where m is the proportion of transgene-positive cells.

[0065] 4. Preparation of lung cancer TIL cells Lung cancer tumor fragments were placed in 100mm culture dishes and washed with PBS. Necrotic areas and connective tissue were removed from the tumor fragments using sterile ophthalmic scissors or a scalpel, and the fragments were then cut into 1-3mm pieces. The tumor fragments were cultured in RPMI 1640 medium containing 10% FBS serum, 1% glutamine, 1% penicillin-dextrose antibody, and 6000 IU / mL IL2. Microscopic observation revealed no obvious adherent cells; the medium was changed every other day. If the lymphocyte count did not gradually increase or the cancer cell count did not decrease, the medium was changed again. If the lymphocyte density significantly increased and cell clusters appeared, the wells were expanded, and the cells were incubated at 37°C with 5% CO2 for further culture. Culture was continued for no more than 10 days, followed by TIL cell collection. The collected cell suspension was filtered through a 100μm filter to remove tumor fragments; the cells were centrifuged at 1000 rpm for 5 min, resuspended in an appropriate amount of complete T-cell culture medium, and set aside for later use.

[0066] 5. Lentiviral infection of TIL cells Based on the lentivirus titer, TIL cells were infected with MOI=10; lentivirus (MOI=10) and TIL cells (5×10⁻⁶) were then inoculated. 5 Add 200 μl of culture medium, virus, and cell mixture to each centrifuge tube, mix well, and transfer to a 48-well plate. Incubate at 37°C with 5% CO2. After 24 h of infection, collect the cells, centrifuge at 1000 rpm, and discard the virus solution. Resuspend the cells in an appropriate amount of REP complete culture medium, transfer to a 24-well plate, and incubate at 37°C with 5% CO2.

[0067] 6. Flow cytometry Take the cell suspension from step 5 for cell counting, and take 5 × 10⁶ cells. 5 Centrifuge at 400g for 5 min, resuspend cells in 100 μL of PBS containing 2% FBS, then add 2.5 μL of Fc receptor blocker and incubate at room temperature for 10 min. Divide the cells into two groups: a full-negative group and a test group, with each group containing 50 μL of cells (2.5 × 10⁻⁶ cells / mL). 5 Cells were collected; 2.5 μL of IL7-FITC antibody was added to the test group and incubated at 4°C for 30 min; after incubation, 1 ml of PBS containing 2% FBS was added and centrifuged at 400g for 5 min; cells were resuspended in 300 μL of PBS containing 2% FBS in the test group and detected by flow cytometry.

[0068] 7. Flow Cytometry Results TIL cell suspensions infected with lentiviruses containing the target gene were stained by flow cytometry and analyzed using a BD flow cytometer. Results are as follows: Figure 19 and Figure 20As shown, plasmid construction and lentivirus infection were successful, yielding TIL-015, TIL-016, TIL-017, TIL-018, TIL-019, and TIL-020, respectively. Figure 21 Compared with uninfected TILs, IL7 expression could be detected by staining the cell membrane surface in both cases.

[0069] 8. Detection of tumor organoid killing function of membrane-bound IL7-expressing TIL cells Tumor cells extracted from lung cancer tissue were cultured in 3D using Matrigel (e.g., Matrigel). Organ-specific culture media supplemented with growth factors (e.g., EGF, Wnt3a, Noggin, etc.) were used. The tumor characteristics of the organoids were confirmed by HE staining and immunofluorescence (e.g., CK, EpCAM labeling). TIL cells obtained in step 7 were co-cultured with organoids and divided into eight groups: organoid control group, TIL control group, TIL-015, TIL-016, TIL-017, TIL-018, TIL-019, and TIL-020. After 24 hours, LDH release in the co-culture supernatant was compared to assess the killing effect. Results are as follows: Figure 22 As shown, membrane expression can enhance the in vitro killing function of TILs.

[0070] discuss Interleukin-7 (IL-7) plays a crucial role in anti-tumor immunotherapy, particularly in adoptive T-cell therapies such as CAR-T and TCRT, where its mechanism of action and clinical translational value are receiving increasing attention. As a core regulator of T-cell homeostasis and function, IL-7 activates key signaling pathways such as JAK-STAT5, PI3K-Akt, and MAPK by binding to the IL-7Rα (CD127) and γc chain (CD132) receptor complex, thereby exerting a wide range of influences on multiple levels of T-cell development, survival, metabolic adaptation, and functional maintenance. In the peripheral immune system, IL-7 maintains the survival of naïve T cells and memory T cells by upregulating the expression of the anti-apoptotic protein BCL-2, which is essential for long-term immune surveillance. Furthermore, IL-7 can significantly enhance the formation of central memory T cells (TCM) and stem cell-like memory T cells (TSCM). These cell subsets possess stronger self-renewal capabilities and long-lasting anti-tumor activity, making them key determinants of the durable efficacy of adoptive T-cell therapy.

[0071] Within the tumor microenvironment (TME), IL-7 plays a particularly prominent role. Since solid tumors are typically highly immunosuppressive, T cells are prone to functional exhaustion (e.g., high expression of PD-1 and TIM-3) and metabolic limitations (e.g., inhibited glycolysis). IL-7 can enhance glucose uptake by upregulating GLUT1 and promote mitochondrial metabolism, providing continuous energy support for T cells and thus partially reversing TME-induced T cell dysfunction. Furthermore, IL-7 can promote T cell infiltration into tumor tissues by enhancing the expression of chemokine receptors such as CXCR3 and inhibit the immunosuppressive effects of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), thereby reshaping the tumor immune microenvironment and transforming it from a "cold tumor" to a "hot tumor." These properties make IL-7 an important strategy for optimizing CAR-T and TIL therapies, especially in the treatment of solid tumors, where its potential value is even more significant.

[0072] In adoptive T-cell therapy, the application of IL-7 is mainly reflected in the following aspects: First, in the in vitro culture stage, IL-7 can significantly improve the expansion efficiency and survival rate of T cells, while maintaining their stemness and undifferentiated state, thereby enhancing the persistence after reinfusion. For example, recent studies have shown that compared with T cells expanded by traditional IL-2, CAR-T cells cultured with IL-7 combined with IL-15 have higher metabolic flexibility and anti-apoptotic ability, longer survival time in vivo, and more significant anti-tumor effects. Second, in the in vivo treatment stage, IL-7 can further support the survival and function of reinfused T cells through systemic or local delivery (such as intratumoral injection). However, the clinical application of IL-7 still faces several challenges. On the one hand, excessive IL-7 signaling may trigger autoimmune responses or exacerbate cytokine release syndrome (CRS), especially in patients with high tumor burden. On the other hand, the short half-life of IL-7 (only 4-6 hours for natural IL-7) limits its duration of action, thus requiring the development of long-acting formulations (such as Fc fusion proteins or membrane-bound IL-7) to optimize the dosing regimen. Furthermore, the effects of IL7 on different T cell subsets are heterogeneous, and further research is needed to explore how to precisely regulate its effects to maximize therapeutic efficacy.

[0073] This invention anchors IL7 to the cell membrane through a hinge region and a G4S linker. Compared with designs that rigidly connect IL7 to the hinge region without using any flexible linkers, the introduction of the G4S linker brings about a fundamental performance improvement, and IL7 has a stronger ability to promote T cell proliferation and killing.

[0074] Analysis suggests that without G4S, the rigid connection between the IL7 molecule and the hinge region creates significant steric hindrance, severely restricting the IL7's freedom of movement and making it difficult for it to effectively find and dock with IL7 receptors on the cell membrane surface, thus significantly weakening signal transduction efficiency. In contrast, the G4S linker acts as a crucial "molecular joint," its superior flexibility allowing IL7 to swing and rotate freely, ensuring its efficient binding to receptors—something rigid connections cannot achieve. Regarding protein structural integrity, the rigid connection without G4S easily leads to interference between the two functional domains, potentially forcing IL7 to adopt an unnatural, incorrect conformation, significantly reducing or even completely eliminating its biological activity. The G4S linker, as a neutral, biocompatible spacer, physically separates IL7 from the hinge region, providing IL7 with an independent and undisturbed folding environment, preserving its native structure and full biological activity to the greatest extent possible. Furthermore, from a safety perspective, a structurally damaged, low-activity fusion protein may be more easily recognized as an abnormal protein by the immune system, triggering an unnecessary immune response. The G4S linker is composed of natural amino acids, and its low immunogenicity further reduces this risk, ensuring the persistence of therapeutic T cells in vivo. In in vivo mouse experiments, groups with longer hinge regions and G4S showed higher proportions of CAR-T and TCRT in peripheral blood, further validating this hypothesis.

[0075] This invention anchors IL7 on the cell surface via the CD80 or PD-L1 transmembrane domain, enabling the following experimental objectives: (1) enhancing the safety of IL7 for immune cells such as T cells by precisely regulating immune cells through anchoring to the cell membrane, reducing the potential for excessive IL7 signaling to trigger autoimmune reactions or exacerbate cytokine release syndrome (CRS); (2) increasing the half-life of IL7 by anchoring to the cell membrane, thereby enhancing the anti-tumor effect of adoptive immune cells; (3) linking transmembrane fragments to IL7 via the flexible linker G4S and the hinge region of CD80 or PD-L1, thereby enhancing the flexibility of IL7 while anchoring it to the cell membrane, and enhancing the proliferation and killing function of IL7 against T cells.

[0076] In conclusion, cell membrane-anchored IL7 expression offers a new breakthrough in overcoming the limitations of current adoptive immunotherapy methods such as CAR-T and TCRT in cancer treatment, and is expected to bring long-term survival benefits to more patients with refractory cancers.

[0077] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. An IL7 fusion protein, characterized in that, The IL7 fusion protein comprises the following elements: an interleukin 7 element, which is composed of interleukin 7 or a functionally active fragment thereof; a transmembrane domain, which is a transmembrane domain of CD80, PD-L1, ICOSL, CD8a, CD8b, LFA1 or IL2Ra protein.

2. The IL7 fusion protein of claim 1, wherein, It also comprises a linker for connecting different polypeptide fragments.

3. The IL7 fusion protein of claim 1, wherein, The structural general formula of the IL7 fusion protein is X-IL7-H-TM, wherein each of "_" is independently null or a linker; X is null or a signal peptide; IL7 is an interleukin 7 element; H is a hinge region; and TM is a transmembrane domain.

4. The IL7 fusion protein of claim 1, wherein, It satisfies at least one of the following characteristics: The amino acid sequence of the interleukin 7 element is shown in SEQ ID NO. 1; The amino acid sequence of the CD80 transmembrane domain is shown in SEQ ID NO: 4; The amino acid sequence of the PD-L1 transmembrane domain is shown in SEQ ID NO: 5; The amino acid sequence of the ICOSL transmembrane domain is shown in SEQ ID NO: 6; The amino acid sequence of the CD8a transmembrane domain is shown in SEQ ID NO: 7; The amino acid sequence of the CD8b transmembrane domain is shown in SEQ ID NO: 8; The amino acid sequence of the LEF1 transmembrane domain is shown in SEQ ID NO: 9; The amino acid sequence of the IL2Ra transmembrane domain is shown in SEQ ID NO:

10.

5. The IL7 fusion protein of claim 2 or 3, wherein, The linker is a flexible linker, preferably a GS linker.

6. The IL7 fusion protein of claim 3, wherein, It satisfies at least one of the following characteristics: The X is a signal peptide, which is a signal peptide of IL7; The hinge region is a hinge region of CD80, PD-L1, ICOSL, CD8a, CD8b, LFA1 or IL2Ra protein or an extended sequence thereof.

7. The IL7 fusion protein of claim 6, wherein, It satisfies at least one of the following characteristics: The transmembrane domain is a CD80 transmembrane domain outside the hinge region, and the amino acid sequence of the CD80 hinge domain is shown in SEQ ID NO: 11; The transmembrane domain is an extension of the CD80 transmembrane domain outside the hinge region, and the amino acid sequence of the extended CD80 hinge region is shown in SEQ ID NO: 12; The transmembrane domain is a PD-L1 transmembrane domain outside the hinge region, and the amino acid sequence of the PD-L1 hinge domain is shown in SEQ ID NO: 13; The transmembrane domain is an extension of the PD-L1 transmembrane domain outside the hinge region, and the amino acid sequence of the extended PD-L1 hinge region is shown in SEQ ID NO:

14.

8. A nucleotide sequence encoding the IL7 fusion protein of any one of claims 1-7.

9. A protein expression vector or expression system, characterized by, A nucleotide sequence according to claim 8.

10. An engineered immune cell, characterized in that, The immune cells comprise the IL7 fusion protein of any one of claims 1-7 or the nucleotide sequence of claim 8; preferably, the immune cells are T cells or NK cells.

11. Use of the IL7 fusion protein of any one of claims 1-7 or the nucleotide sequence of claim 8, which use comprises: Preparation of a medicament for preventing, alleviating and / or treating a tumor; Preparation of a medicament for alleviating and / or treating an autoimmune disease; Expansion of immune cells; Preparation of a vaccine antigen booster.

Images