MLLT3 modified CAR-T structure and application thereof

Abstract

The invention belongs to the technical field of tumor cell immunotherapy, and particularly relates to an MLLT3 modified CAR-T structure and application thereof. The MLLT3 modified CAR-T provided by the invention has a structure as follows: CD8 [alpha]-TIGIT-CD8 [alpha] TM-41 BB-CD3 [zeta]-P2A-MLLT3, and a structural formula as shown in the specification, the amino acid sequence of the MLLT3 is as shown in SEQ ID NO. 1. The technical defect that in the prior art, when the CAR-T cells are used for preparing the medicine for treating the solid tumor, the T cells are rapidly depleted and die is overcome, the survival period of the CAR-T cells in the solid tumor tissue is effectively prolonged, and a technical foundation is laid for further improving the tumor killing capacity of the CAR-T cells; the method has good application potential and practical value.

Description

Technical Field

[0001] This invention belongs to the field of tumor cell immunotherapy technology, specifically involving MLLT3-modified CAR-T structures and their applications. Background Technology

[0002] In cancer prevention and treatment, CAR-T (Chimeric Antigen Receptor T-Cell) therapy, based on the immune system, has received considerable research and development in recent years due to its advantages such as fewer side effects and a wide range of indications. Clinically, CAR-T cells are used to target and eliminate CD19. + Tumor cells have shown good preventive and therapeutic effects in the treatment of acute lymphoblastic leukemia. However, the application of this technology in solid tumors is not yet satisfactory, and many problems need to be solved. One important problem is the rapid depletion and apoptosis of CAR-T cells in tumor tissue.

[0003] In solid tumors, despite adoptive T-cell therapy or PD-1 monoclonal antibody therapy, the cell body activity of CD8+ T cells declines, directly affecting cell proliferation and function. T cell depletion in the tumor microenvironment due to metabolic imbalance and prolonged antigen stimulation is considered one of the main reasons for the ineffectiveness of adoptive T-cell therapy. Tumor-infiltrating T memory cells are the main effector components of anti-tumor immunity in most tumor types, but they are often rapidly depleted due to the limitations of the tumor microenvironment. Similarly, CAR-T cells face similar challenges in the treatment of solid tumors due to the tumor microenvironment.

[0004] Australian patent AU2022487460A1 discloses a fusion gene containing a chimeric antigen receptor encoding gene and a chimeric switch receptor encoding gene, and its application. This fusion gene, through the chimeric switch receptor, converts the inhibitory signals of PD-L1 and TGFβ in tumors into CD27-mediated activation signals, blocking the inhibitory effects of the TGFβ and PD-1 pathways on CAR-T cells. Furthermore, the modified CAR-T cells have the ability to secrete cytokines IFN-γ and IFN-α, promoting the killing of tumor cells by other immune cells. This allows CAR-T cells to survive in a more favorable tumor microenvironment, thus enhancing their persistence in vivo. However, this patent does not enhance the survival ability of CAR-T cells themselves, and this activation depends on the levels of PD-L1 and TGF-β in the tumor microenvironment. Therefore, this method cannot directly solve the problem of CAR-T cell depletion in the tumor microenvironment, and excessive IFN-α can also cause harm and side effects.

[0005] Chinese patent CN120099104A discloses a method for preparing CAR-T cells overexpressing C1QBP and its applications. The CAR-T cells overexpressing C1QBP enhance the antitumor activity and long-term persistence of CAR-T cells by increasing mitochondrial oxidative phosphorylation, optimizing cellular energy metabolism, and promoting the formation of memory-like T cells. This method improves cell activity by enhancing the mitochondrial oxidative function of T cells, which are typically in a hypoxic state in the tumor microenvironment. However, this method has only been tested in hematologic malignancies, while the tumor microenvironment of solid tumors is complex, and its effectiveness is unknown.

[0006] In conclusion, existing technologies still require further development of CAR-T modification methods to enhance their survival ability in the tumor microenvironment and improve the depletion problem of CAR-T cells. Summary of the Invention

[0007] This invention aims to address the technical shortcomings of existing CAR-T cell therapies in solid tumor treatment, namely the rapid depletion and apoptosis of T cells. It provides an MLLT3-modified CAR-T structure and its applications. Based on the crucial regulatory role of MLLT3 in T cells, this invention uses MLLT3 as a "target" and, combined with the characteristics of CAR-T technology, designs targeted CARs to improve the survival rate of T cells in tumor tissues. This has significant technical implications for enhancing the application of CAR-T technology in solid tumors and further improving the therapeutic effects of tumors.

[0008] The present invention provides, in a first aspect, a CAR-T structure modified with MLLT3, wherein the MLLT3-modified CAR-T structure comprises, in sequence, a human CD8α signal peptide (CD8α), a human TIGIT extracellular region (TIGIT), a human CD8α transmembrane region (CD8αTM), a 41BB intracellular region (41BB), a human CD3ζ intracellular region (CD3ζ), a P2A linker sequence (P2A), and a coding sequence for MLLT3 (MLLT3), namely: CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3.

[0009] MLLT3, also known as AF9, is a key regulator of hematopoietic stem cells, highly enriched in human fetal, neonatal, and adult hematopoietic stem cells, but downregulated in culture. MLLT3 deficiency prevents the maintenance of transplantable hematopoietic stem cells in cultures, while stable MLLT3 expression in cultures can expand transplantable hematopoietic stem cells more than 12-fold (Calvanese V. MLLT3 governs human hematopoietic stem-cell self-renewal and engraftment. Nature. 2019 Dec.). However, whether MLLT3 plays an important role in T cells has not been studied. Therefore, this invention investigates the role of MLLT3 in T cell survival, aiming to develop a method to promote the survival of CAR-T cells in the tumor microenvironment and improve the problem of CAR-T cell depletion.

[0010] This invention reveals that MLLT3 expression in T cells promotes T cell survival and upregulates the expression of DOT1L and STAT5. Both DOT1L and STAT5 are genes that promote T cell survival. Studies by Bian Y et al. (CancerSLC43A2 alterers T cell methionine metabolism and histone methylation. Nature. 2020 Sep) showed that the loss of Dot1L in T cells leads to impaired immune responses and CD8+ T cell apoptosis. Studies by Adachi Y et al. (Cullin-5 deficiency promotes chimeric antigen receptor T cell effector functions potentially via the modulation of JAK / STAT signaling pathway. Nat Commun. 2024 Dec 10) showed that STAT5 plays an important role in regulating T cell survival and function. This further confirms the certainty of the results of this invention.

[0011] Furthermore, this invention has found that MLLT3 expression can promote the differentiation of CAR-T cells into more stem cell-like memory T cells, which is a key factor in the long-term survival of T cells in the tumor microenvironment. By promoting the differentiation of CAR-T cells into stem cell-like memory T cells and upregulating DOT1L and STAT5, this invention synergistically enhances the survival ability of CAR-T cells in the tumor microenvironment through multiple mechanisms, promotes the long-term maintenance of the anti-tumor activity of CAR-T cells, and enhances the therapeutic effect of CAR-T cells.

[0012] Preferably, the amino acid sequence of MLLT3 is shown in SEQ ID NO.1, which is as follows:

[0013] .

[0014] Preferably, the amino acid sequence of the transmembrane region of the human CD8α molecule is shown in SEQ ID NO.2, specifically as follows:

[0015] MALPVTALLLPLALLLHAARP.

[0016] Preferably, the amino acid sequence of the extracellular region of the human TIGIT is shown in SEQ ID NO.3, specifically as follows:

[0017] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPS FKDRVAPGPGGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP.

[0018] Preferably, the amino acid sequence of the human CD8α signal peptide is shown in SEQ ID NO.4, specifically as follows:

[0019] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0020] Preferably, the amino acid sequence of the intracellular region of the 41BB molecule is as shown in SEQ ID NO.5, specifically as follows:

[0021] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0022] Preferably, the amino acid sequence of the intracellular region of the human CD3ζ molecule is shown in SEQ ID NO.6, specifically as follows:

[0023] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0024] Preferably, the amino acid sequence of the P2A linker sequence is as shown in SEQ ID NO.7, specifically as follows:

[0025] GSGATNFSLLKQAGDVEENPGP.

[0026] Preferably, the amino acid sequence of the MLLT3-modified CAR-T structure is shown in SEQ ID NO.8, specifically as follows:

[0027] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPMALPVTALLLPLALLLHAARPKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMASSCAVQVKLELGHRAQVRKKPTVEGFTHDWMVFVRGPEHSNIQHFVEKVVFHLHESFPRPKRVCKDPPYKVEESGYAGFILPIEVYFKNKEEPRKVRFDYDLFLHLEGHPPVNHLRCEKLTFNNPTEDFRRKLLKAGGDPNRSIHTSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSTSFSKPHKLMKEHKEKPSKDSREHKSAFKEPSRDHNKSSKESSKKPKENKPLKEEKIVPKMAFKEPKPMSKEPKPDSNLLTITSGQDKKAPSKRPPISDSEELSAKKRKKSSSEALFKSFSSAPPLILTCSADKKQIKDKSHVKMGKVKIESETSEKKKSTLPPFDDIVDPNDSDVEENISSKSDSEQPSPASSSSSSSSSFTPSQTRQQGPLRSIMKDLHSDDNEEESDEVEDNDNDSEMERPVNRGGSRSRRVSLSDGSDSESSSASSPLHHEPPPPLLKTNNNQILEVKSPIKQSKSDKQIKNGECDKAYLDELVELHRRLMTLRERHILQQIVNLIEETGHFHITNTTFDFDLCSLDKTTVRKLQSYLETSGTS。

[0028] In a second aspect, the present invention provides a nucleic acid encoding the MLLT3-modified CAR-T structure, as shown in SEQ ID NO.9, as follows:

[0029]

[0030] A third aspect of the present invention provides a lentiviral vector containing nucleic acid encoding the MLLT3-modified CAR-T structure.

[0031] Preferably, the starting vector of the lentiviral vector is pCDH-EF1-conGFP.

[0032] The present invention provides, in a fourth aspect, a method for preparing a lentiviral vector containing nucleic acid encoding the MLLT3-modified CAR-T structure, specifically comprising the following steps:

[0033] S1. Synthesize the nucleic acid fragment shown in SEQ ID NO.9;

[0034] S2. Using pCDH-EF1-conGFP plasmid as the starting vector, digest it with enzymes, and then ligate the nucleic acid fragment described in step S1 to the digested pCDH-EF1-conGFP.

[0035] S3. Transform the ligation product of step S2 into STABL3 competent cells, screen, expand culture, and then extract plasmids to obtain the lentiviral vector pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3 that can express the MLLT3-modified CAR-T structure.

[0036] Preferably, the ligation in step S2 is performed using In-Fusion HD Cloning Kits, Clontech, model 639649, whereby the nucleic acid fragment from step S1 is ligated with the enzyme-digested pCDH-EF1-conGFP vector.

[0037] The fifth aspect of the present invention provides a recombinant expression system for a lentiviral vector containing a nucleic acid encoding the MLLT3-modified CAR-T structure described above.

[0038] Preferably, the recombinant expression system uses T cells as the host cell.

[0039] The sixth aspect of the present invention provides the application of the above-described recombinant expression system in the preparation of antitumor drugs.

[0040] Preferably, the tumor is a solid tumor.

[0041] Preferably, the solid tumor is cervical cancer.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) This invention uses MLLT3 as a key regulatory factor to develop a CAR structure that resists T cell senescence and optimizes the survival persistence of T cells in the tumor microenvironment, thereby effectively addressing the technical bottleneck of rapid T cell depletion and apoptosis in the current CAR-T therapy in the field of solid tumor treatment.

[0044] (2) In vitro experiments have verified the killing effect of the MLLT3-modified CAR-T cells provided by this invention on cervical cancer cells, and its ability to cope with secondary antigen stimulation is good, thus addressing long-term antigen stimulation in the tumor microenvironment. In vivo animal experiments have also verified the ability of this invention to inhibit tumor growth. Therefore, the CAR-T structure provided by this invention effectively improves the survival period of CAR-T cells in cervical cancer tumor tissue, laying a good technical foundation for improving the application effect of CAR-T cells, and has good application potential and practical value.

[0045] (3) The present invention uses TIGIT’s natural extracellular domain to specifically recognize CD155, which does not require antibody screening and optimization like artificially constructed scFv, thus effectively shortening the research and development cycle. Attached Figure Description

[0046] Figure 1 (A) is a schematic diagram of the structure of the recombinant expression plasmid vector pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3 of the present invention. Figure 1 (B) is a flow cytometry result of CAR transfection efficiency detected using GFP;

[0047] Figure 2 Figure 1 shows the qPCR results of MLLT3 promoting the expression of DOT1L and STAT5 in T cells.

[0048] Figure 3 Flow cytometry results of MLLT3 promoting memory differentiation of T cells; specific cell types shown in the figure include: naive T cells ( T cells (TN), stem cell memory T cells (T cells) SCM Effector T cells (Recently activated effector memory or effector T cells, TEM), and exhausted T cells (TEX);

[0049] Figure 4To demonstrate that CAR-T cells targeting CD155 can effectively and specifically eliminate target cells. (A) Flow cytometry results showing the expression of CD155 on the surface of cervical cancer cell lines (HCA1, CAC1C) and human cervical immortalized epithelial cells (H8); (B) Graphs showing the specific killing effect of different groups of T cells on target cells using a lactate dehydrogenase assay kit; *P<0.05, ***P<0.0001;

[0050] Figure 5 (A) ELISA results of CAR-T cells targeting CD155 secreting the effector IL-2; (B) ELISA results of CAR-T cells targeting CD155 secreting the effector IFN-γ.

[0051] Figure 6 The figure shows the results of detecting the ability of CAR-T cells to respond to secondary antigen stimulation using a lactate dehydrogenase cytotoxicity assay kit.

[0052] Figure 7 (A) Schematic diagram of mouse experiment; (B) Statistical chart of mouse tumor volume measurement; (C) Survival curve of mouse. Detailed Implementation

[0053] The present invention will be further described below with reference to embodiments. These embodiments are provided to facilitate understanding of the invention and are not intended to limit the invention. Those skilled in the art can make various modifications based on the basic idea of ​​the invention, but all modifications are within the scope of the invention as long as they do not depart from its fundamental spirit.

[0054] The biological materials and components used in the specific implementation are as follows:

[0055] Non-obese diabetic / severe combined immunodeficiency (NOD / SCID) mice, female mice aged 6-8 weeks, were purchased from Beijing Vital River Co., Ltd. (Beijing, China) and housed in a sterile environment;

[0056] Human cervical cancer-related cell lines: HCA1, CAC1C, purchased from ATCC;

[0057] Human cervical immortalized cell line: H8, purchased from ATCC;

[0058] During the experiment, all cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM), which contained 10% fetal bovine serum (FBS, HyClone, Chicago, IL, USA) and 100 U / mL penicillin and 100 μg / mL streptomycin (Invitrogen, Carlsbad, CA, USA).

[0059] Fluorescent cell lines were obtained as follows: Before the experiment, tumor cells were transfected with viral supernatant containing luciferase-GFP, and the GFP channels were sorted using a FACS Aria™ cell sorter (BD Biosciences, San Jose, CA, USA) to finally obtain cell lines that stably express luciferase-green fluorescent protein (GFP).

[0060] Unless otherwise specified, the enzymes, primers, reagents and reaction conditions involved in this invention can be reasonably selected based on the experience of those skilled in the art. The reagents and consumables are common commercial products, and the detection methods and instruments involved are well known and skillfully mastered by those skilled in the art.

[0061] Example 1: Construction of MLLT3-modified CAR-T structure

[0062] This invention utilizes MLLT3 to modify CAR-T cells. First, a specific CAR structure is designed, and then the T cells are labeled. The specific process is as follows:

[0063] 1. CAR structure design

[0064] The MLLT3-modified CAR-T structure provided by this invention is an amino acid sequence consisting of several protein fragments linked together, named CD8α-TIGIT-41BB-CD3ζ-MLLT3. The specific structure is as follows: the human CD8α molecule signal peptide, the human TIGIT extracellular region, the human CD8α molecule transmembrane region, the 41BB molecule intracellular region, the human CD3ζ molecule intracellular region, the P2A linker sequence, and the coding sequence of MLLT3 are linked sequentially; that is: CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3. Wherein: the amino acid sequence of MLLT3 is shown in SEQ ID NO.1; the amino acid sequence of the human CD8α molecule signal peptide is shown in SEQ ID NO.2; the amino acid sequence of the extracellular region of the human TIGIT is shown in SEQ ID NO.3; the amino acid sequence of the transmembrane region of the human CD8α molecule is shown in SEQ ID NO.4; the amino acid sequence of the intracellular region of the 41BB molecule is shown in SEQ ID NO.5; the amino acid sequence of the intracellular region of the human CD3ζ molecule is shown in SEQ ID NO.6; and the amino acid sequence of the P2A linker sequence is shown in SEQ ID NO.7.

[0065] The final designed amino acid sequence of the MLLT3-modified CAR-T structure is shown in SEQ ID NO. 8, and the nucleotide sequence encoding the amino acid sequence of the MLLT3-modified CAR-T structure is shown in SEQ ID NO. 9. MLLT3 is located at the 3' end. Based on the above CAR structure design, the nucleic acid fragment shown in SEQ ID NO. 9 was synthesized by Sangon Biotech Co., Ltd.

[0066] 2. Construction of recombinant lentiviral expression plasmid vector

[0067] Based on the characteristics of CAR-T technology, this invention uses pCDH-EF1-conGFP plasmid as an expression vector to further construct a recombinant lentiviral expression plasmid vector, which facilitates subsequent use for infecting T cells to prepare CAR-T cells. The specific lentiviral expression vector construction process is described below:

[0068] (1) The coding sequence DNA of the MLLT3 modified CAR-T structure was synthesized by Sangon Biotech Co., Ltd., as shown in SEQ ID NO.9 above.

[0069] (2) Using pCDH-EF1-conGFP plasmid (purchased from Novopro, model V007087) as the expression vector, the restriction endonuclease EcoRI (purchased from New England Biolabs, model R0101V) was digested at 37℃. Subsequently, referring to the instructions, the coding sequence DNA in step S1 was integrated into the pCDH-EF1-conGFP plasmid using In-Fusion HD Cloning Kits (purchased from Clontech, model 639649).

[0070] (3) The ligation product from step S2 was transformed into STABL3 competent cells (purchased from Weidibio, catalog number Stbl3). Before transformation, the competent cells were removed from -80℃ and placed on ice. The entire ligation system was added to the competent cells, and the cells were kept on ice for 2 hours. After standing, the competent cells were quickly transferred to a 42℃ water bath and heat-shocked for 45 seconds. Then, they were quickly returned to ice and kept on ice for 2 minutes. 500 μL of LB culture medium was added, and the cells were shaken at 37℃ for 1 hour. An appropriate volume of cells was plated onto an LB gel culture plate containing antibiotics. The plate was inverted and cultured at 37℃ for 12 hours for screening. After screening and expansion culture, the plasmid was further extracted to prepare pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3.

[0071] The schematic diagram of the constructed lentiviral expression plasmid pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3 is shown below. Figure 1 As shown in (A), the relevant operations during the construction process can be referred to the conventional operations of existing technologies, and will not be described in detail here.

[0072] As a control, the present invention prepared a control recombinant plasmid pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ using the above method, which was used for subsequent experimental control.

[0073] Example 2: Preparation of CAR-T cells

[0074] (I) Lentiviral Packaging

[0075] 293T cells were used as the initial target cells for transfection, as detailed below:

[0076] First, 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences, model SCSP-502) were seeded in a six-well plate and incubated for 24 h (37℃, DMEM complete medium (purchased from Thermo Fisher Scientific, model 12491015)).

[0077] Subsequently, the culture medium was replaced with OPTI-MEM medium (purchased from Thermo Fisher Scientific, model 31985070), and 1.5 g of the lentiviral expression plasmid constructed in Example 1, 1.5 g of the packaging plasmid psPAX2, and 1 g of Pmd2.G were added, along with 8 μL of liposome transfection reagent. The mixture was thoroughly mixed to transfect the target cells 293T.

[0078] Replace with normal DMEM medium 12 hours after transfection;

[0079] Finally, after culturing for another 48 hours, the viral supernatant (i.e., the packaged virus-like particles) was collected, centrifuged at 1500 rpm for 10 minutes, and the supernatant was stored at -80°C for later use in T cell infection.

[0080] The control group virus was prepared using the above method, and the lentiviral plasmid was pCDH-EF1-CD8α-TIGIT-41BB-CD3ζ.

[0081] The experimental group virus was prepared using the above method, and the lentiviral plasmid was pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3.

[0082] (II) Preparation of purified CD3 +T cells

[0083] First, mononuclear cells were isolated from human peripheral blood using density gradient centrifugation.

[0084] Then, T-cell separation magnetic beads were used for separation and purification to obtain purified CD3. + T cells;

[0085] Next, the purified CD3 + T cells were cultured in 24-well cell culture plates using RPMI-1640 containing IL-2 (100 IU / mL, PeproTech, Suzhou, Jiangsu, China) and L-glutamine (2 mM, Gibco / Life Technologies / Thermo Fisher Scientific, Waltham, MA, USA).

[0086] Finally, before use, CD3 + T cells were activated with CD3 / CD28 antibody (1 μl / 10) 7 Cells were stimulated and activated for 2 days.

[0087] For specific operational methods, please refer to existing technologies (Zhang Kai. (2024). IL-24 improves efficacy of CAR-T cell therapy by targeting stemness of tumor cells. British Journal of Cancer.), which will not be elaborated further.

[0088] (III) T-cell infection

[0089] Using a 24-well cell culture plate, add 10 T cells activated for 2 days in step S2 to each well. 6 / well), and add 1 mL of the viral supernatant collected in step (I) and polybrene (8 μg / mL) to each well, and incubate overnight for infection;

[0090] After 24 hours, the culture was replaced with normal RPMI-1640 containing IL-2 (100 IU / mL) and L-glutamine (2 mM).

[0091] (iv) T cell expansion

[0092] To facilitate subsequent experimental applications, further analysis was conducted on the CD3 cells incubated after infection in step (iii). +T cells were centrifuged and washed three times, then added to RPMI 1640 medium containing 1000 U IL-2 and 5% fetal bovine serum (the medium was changed every 2-3 days) to further expand the T cells. The prepared cells were denoted as:

[0093] TIGIT.CAR-T (containing the control lentiviral vector plasmid pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ) and TIGIT.CAR.MLLT3-T (containing the lentiviral vector plasmid pCDH-EF1-CD8α-TIGIT-CD8αTM-41BB-CD3ζ-P2A-MLLT3 of the present invention).

[0094] In addition, as a blank control, referring to the above operation, the present invention packages the pCDH-EF1 empty plasmid containing GFP into a lentivirus and then directly infects T cells. The prepared cells are denoted as: GFP-T (equivalent to the blank control T cell group that only expresses GFP).

[0095] Example 3: Cell and Animal Experiments

[0096] Based on Example 2, the lentiviral expression plasmid constructed in this invention was transfected into CAR-T cells, and preliminary cell experiments were further conducted. The specific experimental process is as follows:

[0097] (1) Virus packaging detection

[0098] The expression of CAR on the surface of T cells was detected by flow cytometry after 5 days of expansion in step (iv) of Example 2 above.

[0099] Experimental results are as follows Figure 1 As shown in (B), it can be seen that the CAR expression positivity rate reached 60% to 70%. This result indicates that the CAR expression plasmid constructed in this invention was successfully packaged into lentiviral particles and infected T cells, and was well expressed, which can be used for subsequent experiments.

[0100] (2) Detection of DOT1L and STAT5 expression in different T cells

[0101] Collect T cells amplified for 5 days as described in step (iv) of Example 2 above. Total RNA was extracted using an RNA extraction kit (Beyotime, catalog number R0018S), and cDNA was obtained using a reverse transcription kit (TAKARA, catalog number RR047Q). The above procedures were performed according to the instructions of each kit. The obtained cDNA was then further analyzed by RT-PCR.

[0102] The amplification primers for DOT1L are: forward primer: TCAGATGACCGACGACGAC, reverse primer: CACCTCAGGACCAAAGGCAA. The kit used is SYBR. TM Green Universal Master Mix (purchased from Thermo Fisher Scientific, item number 4309155);

[0103] RT-PCR detection of STAT5 was performed using Human STAT5AqPCR Primer Pair (purchased from Beyotime, catalog number QH05769S). The above procedures were performed in accordance with the instructions of each kit.

[0104] Experimental results are as follows Figure 2 As shown, compared to the TIGIT.CAR-T group cells, the expression of DOT1L and STAT5 in the TIGIT.CAR.MLLT3-T group cells changed significantly in the presence of MLLT3. The expression of DOT1L and STAT5 in the TIGIT.CAR.MLLT3-T group cells was significantly higher than that in the TIGIT.CAR-T group cells. In other words, MLLT3 promotes the expression of DOT1L and STAT5 in T cells, and the increased expression of DOT1L and STAT5 can enable T cells to survive better in the tumor microenvironment, thereby allowing CAR-T cells to better exert their anti-tumor function and improve the therapeutic effect.

[0105] (3) Detection of differentiation status of different T cells

[0106] In the process of evaluating CAR expression in T cells, the differentiation of T cells was further detected and analyzed.

[0107] Collect T cells that have been expanded for 5 days in step (iv) of Example 2 above, resuspend the cell pellet in PBS, and then add 1 μL CD3 antibody, 1 μL CD45RA antibody, and 1 μL CD62L antibody. Incubate at 4°C in the dark for 15 minutes. Analyze the data using a C6 flow cytometer (Becton Dickinson). FlowJo software (FlowJo, LLC, Ashland, Covington, KY, USA) was used for analysis.

[0108] Experimental results are as follows Figure 3 As shown, CD45RA is highly expressed in naive T cells and terminally differentiated effector T cells, but lowly expressed in effector memory T cells. CD62L is highly expressed in naive T cells and central memory T cells, but lowly expressed in effector memory T cells (Tem) and terminal effector T cells. Therefore... Figure 3In this context, Q1 represents: Central Memory T Cells (Tcm). Q2 represents: Naïve T Cells (Tn). Q3 represents: Terminally Differentiated Effector Memory T Cells (Tem). Q4 represents: Effector Memory T Cells (Temra). Figure 3 As can be seen, compared with the control group TIGIT.CAR-T cells, the proportion of different cell types in the TIGIT.CAR.MLLT3-T group cells changed significantly. The proliferating cells Tcm and Tn increased, while the proliferating cells Temra, which were in a state of near depletion, decreased. In other words, this invention promotes T cell differentiation into Tcm and Tm by adding MLLT3 to the existing CAR-T structure, enabling T cells to survive better in the tumor microenvironment. This allows CAR-T cells to better exert their anti-tumor function, thereby improving the therapeutic effect.

[0109] (4) Detection of apoptosis in different tumor cells

[0110] Using tumor cells highly expressing CD155 as the experimental subject and CAR-T cells as the experimental "drug," the technical efficacy of the constructed CAR was detected and evaluated through tumor cell apoptosis. A brief description of the specific experimental procedure is as follows:

[0111] Human cervical cancer cell lines HCA1 and CAC1C, which highly express CD155, and the CD155-negative human cervical immortalized cell line H8 were used as target cells, respectively. Figure 4 A) Using T cells with different treatments as effector cells (GFP-T, TIGIT.CAR-T, TIGIT.CAR.MLLT3-T), T cells and target cells were co-incubated in 96-well plates for 6 hours at an effector-target ratio of 5:1, with three replicates for each group.

[0112] After incubation, apoptosis of tumor cells was detected using a lactate dehydrogenase (LDH) cytotoxicity assay kit (purchased from Beyotime Biotechnology, product number: C0016). The cell culture plates were centrifuged at 400g for 5 min using a multi-well plate centrifuge. Remove as much supernatant as possible, add 150 μL of the LDH release reagent provided in the kit (diluted 10-fold with PBS to 1 volume of LDH release reagent and mix well), gently shake the culture plate to mix, and then continue incubation in a cell culture incubator for 1 hour. Then centrifuge the cell culture plate at 400g for 5 min. Take 120 μL of the supernatant from each well and add it to the corresponding well of a new 96-well plate, and then proceed with sample measurement. Add 60 μL of LDH detection working solution to each well. Mix well and incubate at room temperature (approximately 25°C) in the dark for 30 min (it can be wrapped in aluminum foil and placed on a horizontal shaker or a side-swinging shaker with gentle shaking). Then measure the absorbance at 490 nm. Use any wavelength of 600 nm or greater as a reference wavelength for dual-wavelength measurement.

[0113] Cytotoxicity or mortality rate (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100.

[0114] Experimental results are as follows Figure 4 As shown in Figure B, it can be seen that compared with the blank control group GFP-T group, the TIGIT.CAR-T group can specifically kill CD155-positive HCA1 and CAC1C tumor cells. At the same ratio, TIGIT.CAR.MLLT3-T cells can kill CD155-positive tumor cells more effectively. This result indicates that the presence of MLLT3 can enhance the killing effect of CAR-T cells on CD155 antigen-positive tumor cells.

[0115] Furthermore, the culture supernatant was collected after 6 hours of co-incubation, and the secretion of effector factors in the culture supernatant was detected. ELISA results showed that CAR-T cells produced a large amount of effector factor IL-2 after co-incubation with tumor cells expressing CD155. Figure 5 A) and IFN-γ Figure 5 B), while co-incubation with CD155-negative cells did not have this effect; co-incubation of control GFP-T cells with tumor cells did not produce a large number of effector factors. Compared with TIGIT.CAR-T cells, TIGIT.CAR.MLLT3-T cells were able to secrete more effector factors. This result further illustrates that in the presence of MLLT3, by increasing the secretion of effector factors IL-2 and IFN-γ, the killing effect of CAR-T cells on CD155 antigen-positive tumor cells can be enhanced. (5) Detection of the response of different CAR-T cells to secondary antigen stimulation.

[0116] In the evaluation of CAR-T cell killing of tumor cells, the present invention further detected and analyzed the response of T cells to secondary antigen stimulation.

[0117] Two groups of CAR-T cells (TIGIT.CAR-T and TIGIT.CAR.MLLT3-T) were co-incubated with target cells (HCA1 and CAC1C) (effect-to-target ratio 5:1). After 8 hours, the CAR-T cells were collected and co-incubated with new target cells again. After 8 hours, tumor cell apoptosis was detected using a lactate dehydrogenase cytotoxicity assay kit (purchased from Beyotime Biotechnology, product number: C0016). Absorbance was measured at 490 nm. Dual-wavelength measurements were performed using either 600 nm or a wavelength greater than 600 nm as a reference wavelength.

[0118] Cytotoxicity or mortality rate (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100.

[0119] Experimental results are as follows Figure 6 As shown in the figure, it can be seen that compared to the TIGIT.CAR-T group, the TIGIT.CAR.MLLT3-T group was able to effectively kill more tumor cells during the second incubation with tumor cells after two long-term antigen stimulations, indicating that TIGIT.CAR.MLLT3-T cells can better cope with secondary antigen stimulation. This result preliminarily suggests that MLLT3 expression can improve CAR-T cell exhaustion caused by long-term antigen stimulation in the tumor microenvironment.

[0120] Long-term antigen stimulation in the tumor microenvironment is a significant cause of T cell exhaustion and apoptosis. After prolonged antigen stimulation, CAR-T cells exhibit functional exhaustion, elevated expression of inhibitory markers, decreased proliferation capacity, and metabolic abnormalities. This state not only weakens the immune system's anti-cancer ability but is also a major obstacle to CAR-T therapy in treating solid tumors.

[0121] This invention utilizes MLLT3 expression to promote the differentiation of CAR-T cells into more stem cell-like memory T cells, and regulates the increase of DOT1L and STAT5 expression. These are key factors for T cells to survive for a long time in the tumor microenvironment, thereby maintaining the anti-tumor activity of CAR-T cells in multiple ways, achieving better therapeutic effects, and ultimately exerting better anti-tumor activity.

[0122] (6) Detection of tumor control in mice

[0123] Based on the above experiments, further animal experiments were conducted on mice. The specific process is briefly described below:

[0124] Mouse experimental model diagram as follows Figure 7 As shown in Figure A, this invention utilizes NOD / SCID immunodeficient mice, purchased from Vital Rivers, which were subcutaneously inoculated with 1×10⁻⁶ mice resuspended in PBS. 5 HCA1 cells (100 μL) were used to construct a mouse subcutaneous xenograft tumor model. The appearance of tumors in the mice indicated successful model establishment. Seven days later, 5 × 10⁵ HCA1 cells were injected via the tail vein. 6 Mice were treated with 100 μL of different groups of T cells (GFP-T, TIGIT.CAR-T, TIGIT.CAR.MLLT3-T) (n=5 per group). Tumor size was measured on the day of tail vein injection of T cells and calculated using the formula V = 0.52 × L × W. 2 Where L is the major axis and W is the minor axis. The tumor volume in mice was measured every other day. Figure 7 B) When the tumor volume reaches 1500 mm 3 Mice were considered dead at a certain time, and their survival time was recorded and statistically analyzed. Figure 7 C).

[0125] Experimental results are as follows Figure 7 As shown in the figure. The statistical results and analysis show that:

[0126] CAR-T cells co-expressing MLLT3 were better able to respond to tumors in mice, and the survival time of the mice was prolonged. These results preliminarily confirm that MLLT3 expression can promote CAR-T cells to better respond to tumor growth in mice.

[0127] In summary, the experimental results show that this invention, through further modification of the existing CD155-targeting CAR structure, utilizes MLLT3 to further promote T cell subtype differentiation, thereby improving T cell exhaustion and thus further enhancing the prevention and treatment of related tumors.

[0128] In summary, MLLT3 plays a role similar to hematopoietic stem cells in T cells. MLLT3 expression in T cells enhances the maintenance of T cell stemness and is a key factor in this maintenance, supporting the sustained anti-tumor activity of T cells. Designing CARs based on MLLT3 and thereby improving the durability of CAR-T cell survival in tumor tissues is of great technical significance for enhancing the application of CAR-T technology in solid tumors and further improving the therapeutic effect of tumors.

[0129] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A MLLT3-modified CAR-T structure, characterized in that, The MLLT3-modified CAR-T structure is specifically composed of: a human CD8α molecule signal peptide, a human TIGIT extracellular region, a human CD8α molecule transmembrane region, a 41BB molecule intracellular region, a human CD3ζ molecule intracellular region, a P2A linker sequence, and an MLLT3 sequence connected sequentially.

2. The MLLT3-modified CAR-T structure according to claim 1, characterized in that, The amino acid sequence of the MLLT3 is shown in SEQ ID NO.1; The amino acid sequence of the transmembrane region of the human CD8α molecule is shown in SEQ ID NO.2; The amino acid sequence of the extracellular region of the human TIGIT is shown in SEQ ID NO.3; The amino acid sequence of the human CD8α molecular signal peptide is shown in SEQ ID NO.4; The amino acid sequence of the intracellular region of the 41BB molecule is shown in SEQ ID NO.5; The amino acid sequence of the intracellular region of the human CD3ζ molecule is shown in SEQ ID NO. 6; The amino acid sequence of the P2A linker sequence is shown in SEQ ID NO.

7.

3. The MLLT3-modified CAR-T structure according to claim 1, characterized in that, The amino acid sequence of the MLLT3-modified CAR-T structure is shown in SEQ ID NO.

8.

4. A nucleic acid encoding the MLLT3-modified CAR-T structure as described in claim 3, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO.

9.

5. A lentiviral vector, characterized in that, It contains the nucleic acid as described in claim 4.

6. The lentiviral vector according to claim 5, characterized in that, The starting vector for the lentiviral vector is pCDH-EF1-conGFP.

7. A recombinant expression system, characterized in that, It includes the lentiviral vector as described in any one of claims 5-6.

8. The use of the recombinant expression system according to claim 7 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumor is a solid tumor.

10. The application according to claim 9, characterized in that, The solid tumor is one of cervical cancer, ovarian cancer, or breast cancer.

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