T cell adapter constructed based on GAS6 as well as preparation method and application of T cell adapter

Abstract

The invention discloses a T cell adapter constructed based on GAS6 as well as a preparation method and application thereof, and relates to the technical field of cellular immunity, and the structure of the T cell adapter comprises an LG1 structural domain of GAS6, a CD3 binding domain, a linker, a marker protein and a fluorescent protein. Through a natural ligand-receptor targeting mechanism, multi-module collaborative design and structural optimization, the problems of high toxicity, single function, complex production process and the like of traditional CAR-T and scFv-BiTE are solved. Experimental data show that the T cell adapter has the advantages of high specificity, controllable immune activation, long-acting stability, easiness in large-scale production and the like, and provides a safer and more efficient immunotherapy strategy for AXL high-expression solid tumors (such as pancreatic cancer, breast cancer, liver cancer, colorectal cancer, lung cancer and the like) and hematoma (acute myelogenous leukemia, chronic lymphocytic leukemia and the like).

Description

Technical Field

[0001] The present invention relates to the technical field of cellular immunology, and particularly relates to a T cell engager constructed based on GAS6, its preparation method and application. Background Art

[0002] In recent years, chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable potential in cancer immunotherapy. Traditional CAR-T specifically recognizes tumor antigens through single-chain antibody fragments (scFv), but its high affinity may lead to "off-tumor targeting toxicity", that is, off-target effects on normal tissues expressing low levels of target antigens. For example, although CAR-T targeting CD19 is highly effective in hematological malignancies, it may cause side effects such as B cell depletion. To overcome this limitation, researchers have turned to using natural receptor / ligand interactions to construct CAR-T, as its moderate affinity can reduce damage to normal tissues. For example, based on the interaction between growth arrest-specific protein 6 (GAS6) and the receptor tyrosine kinase AXL, the previously developed GAS6-CAR-T showed specific killing of AXL-highly expressing tumor cells in a pancreatic cancer model, and no significant toxicity was observed (Fan J et al., J Hematol Oncol, 2023). However, CAR-T therapy still faces challenges such as high production costs, complex pharmacokinetics, and uncertain long-term safety.

[0003] As a novel bispecific / multispecific molecule, immune cell engager (ICE) can form a molecular bridge to activate immune killing by simultaneously binding immune cells (such as T cells, NK cells, and macrophages, etc.) and tumor cell surface antigens. Among them, T cell engager (TCE) has attracted much attention due to its advantages such as simple structure, convenient production, and controllable pharmacokinetics. However, existing TCEs mostly rely on antibody structures (such as scFv), and their high affinity may also lead to off-target effects, and the lack of cross-species conservation of antibodies may affect preclinical safety assessment. In addition, some TCEs have problems of insufficient affinity or poor stability when targeting specific solid tumor antigens.

[0004] The interaction between GAS6 and AXL provides a new idea for the development of novel TCEs. Research has shown that the LG1 domain (261-476AA) of GAS6 is the key region mediating its binding to AXL (Sasaki T et al., J Biol Chem, 2002). By using this domain to replace traditional antibody fragments, the natural conservation of receptor / ligand interaction can be exploited to enhance targeting specificity and reduce the risk of cross-species toxicity. However, the potential of the GAS6 domain in TCE design has not been fully explored in the prior art, especially in optimizing molecular linkage, enhancing stability, and improving tumor selectivity, where there are still technological gaps. Summary of the Invention

[0005] The present invention discloses a T cell engager constructed based on GAS6, which aims to achieve highly specific and low-toxicity tumor-targeted therapy by integrating the AXL-binding domain of GAS6 with the T cell activation domain, while overcoming the limitations of traditional CAR-T and existing TCEs.

[0006] The technical solution adopted by the present invention is as follows:

[0007] One of the objectives of the present invention is to provide a T cell engager constructed based on GAS6, and the T cell engager comprises a structure shown in formula (I) or formula (II):

[0008] S-D1-L1-D2-T1-T2-CopGFP (I);

[0009] S-D2-L1-D1-T1-T2-CopGFP (II);

[0010] Wherein,

[0011] Each "-" is independently a linker peptide or a peptide bond;

[0012] S is a signal peptide sequence, selected from the IgK signal peptide or the GAS6 signal peptide, the amino acid sequence of the IgK signal peptide is as shown in SEQ ID NO: 1, and the amino acid sequence of the GAS6 signal peptide is as shown in SEQ ID NO: 2;

[0013] D1 is a fragment of the extracellular binding domain of GAS6 that specifically binds to AXL, and its amino acid sequence is as shown in SEQ ID NO: 3;

[0014] L1 is a (GGGGS)n linker, and its amino acid sequence is as shown in SEQ ID NO: 4;

[0015] D2 is a structure that specifically binds to T cells in the CD3 binding domain, and its amino acid sequence is as shown in SEQ ID NO: 5;

[0016] T1 is a labeled protein selected from the FC: CH2 and CH3 domains of IgG1, and its amino acid sequence is as shown in SEQ ID NO: 6;

[0017] T2 is a labeled protein selected from His tag or FLAG tag, and its amino acid sequence is as shown in SEQ ID NO: 7;

[0018] CopGFP is a green fluorescent protein, and its amino acid sequence is as shown in SEQ ID NO: 8.

[0019] SEQ ID NO: 1 METDTLLLWVLLLWVPGSTGD;

[0020] SEQ ID NO: 2 MAPSLSPGPAALRRAPQLLLLLLAAECALA;

[0021] SEQ ID NO: 3 HCDGRGGLKLSQDMDTCEDIIPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTER;

[0022] SEQ ID NO: 4 GGGGSGGGGSGGGGS;

[0023] SEQ ID NO: 5 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL;

[0024] SEQ ID NO: 6 DKTHTCPPCPAPELLGGPSVfLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSITCLVKGFYPSDLAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK:

[0025] SEQ ID NO: 7 HHHHHH;

[0026] SEQ ID No: 8 PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTR[EKYEDGGVLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA。

[0027] A second object of the present invention is to provide a method for preparing the T cell engager constructed based on GAS6, comprising the following steps:

[0028] (1) Obtain the base sequences of fragments S, D1, D2, L1, T1, T2, and CopGFP through artificial synthesis or PCR amplification, as shown in SEQ ID NO: 9 to SEQ ID NO: 16 respectively, and then splice fragments S, D1, D2, L1, T1, T2, and CopGFP into a complete coding sequence in the order of formula (I) or (II) by overlap extension PCR;

[0029] (2) Clone the spliced complete coding sequence into the lentiviral expression vector pCDH-CMV-MCS-EF1α-Puro to obtain a recombinant lentiviral vector;

[0030] (3) Transfect the recombinant lentiviral vector into HEK-293F cells cultured in suspension, culture using SMM 293-TII medium, and purify to obtain a T cell adapter after the culture is completed.

[0031] SEQ ID NO: 9 ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC;

[0032] SEQ ID NO: 10 ATGGCCCCATCCCTGTCACCTGGTCCAGCTGCTCTGCGGAGAGCCCCACAGCTGCTCCTCTTGCTCCTTGCCGCCGAGTGCGCTCTGGCT;

[0033] SEQ ID NO: 11 CACTGTGACGGGCGTGGGGGCCTCAAGCTGTCCCAGGACATGGACACCTGTGAGGACATCTTGCCGTGCGTGCCCTTCAGCGTGGCCAAGAGTGTGAAGTCCTTGTACCTGGGCCGGATGTTCAGTGGGACCCCCGTGATCCGACTGCGCTTCAAGAGGCTGCAGCCCACCAGGCTGGTAGCTGAGTTTGACTTCCGGACCTTTGACCCCGAGGGCATCCTCCTCTTTGCCGGAGGCCACCAGGACAGCACCTGGATCGTGCTGGCCCTGAGAGCCGGCCGGCTGGAGCTGCAGCTGCGCTACAACGGTGTCGGCCGTGTCACCAGCAGCGGCCCGGTCATCAACCATGGCATGTGGCAGACAATCTCTGTTGAGGAGCTGGCGCGGAATCTGGTCATCAAGGTCAACAGGGATGCTGTCATGAAAATCGCGGTGGCCGGGGACTTGTTCCAACCGGAGCGAGGACTGTATCATCTGAACCTGACCGTGGGAGGTATTCCCTTCCATGAGAAGGACCTCGTGCAGCCTATAAACCCTCGTCTGGATGGCTGCATGAGGAGCTGGAACTGGCTGAACGGAGAAGACACCACCATCCAGGAAACGGTGAAAGTGAACACGAGGATGCAGTGCTTCTCGGTGACGGAGAGA;

[0034] SEQ ID NO: 12 GGTGGCGGCGGCTCCGGGGGGGGCGGTTCTGGCGGCGGTGGCTCC;

[0035] SEQ ID NO: 13 GAAGTTCAGTTGGTGGAGAGCGGGGGCGGACTTGTGCAGCCCGGCGGCAGCCTGAAGCTGTCTTGCGCTGCTTCCGGATTCACGTTCAATAAGTACGCCATGAACTGGGTGCGACAGGCGCCTGGGAAAGGACTGGAATGGGTAGCGAGGATTAGATCTAAGTATAATAACTATGCAACCTACTACGCGGATTCCGTGAAAGACCGGTTTACCATTTCCAGAGACGACAGCAAGAATACTGCATATCTTCAGATGAATAATCTGAAGACCGAGGACACTGCTGTGTACTATTGCGTTAGACATGGCAATTTTGGCAATTCTTACATTTCCTACTGGGCCTACTGGGGCCAGGGCACCCTTGTCACCGTCTCTTCTGGCGGGGGCGGTTCCGGCGGGGGCGGCTCAGGGGGAGGAGGGTCCCAGACTGTGGTGACTCAGGAACCAAGCTTGACCGTGTCACCAGGGGGAACTGTCACCTTGACCTGCGGGTCTAGTACAGGTGCAGTCACAAGCGGAAACTACCCCAACTGGGTTCAGCAAAAACCTGGACAGGCCCCTCGCGGGCTGATTGGAGGCACTAAGTTTCTCGCACCAGGCACCCCGGCTAGATTCAGTGGTAGTCTGCTTGGGGGAAAGGCCGCCTTGACCCTCTCCGGTGTCCAGCCCGAGGATGAGGCTGAGTACTACTGTGTGCTCTGGTACTCTAATAGATGGGTGTTCGGCGGGGGCACAAAGCTCACCGTGCTG;

[0036]

[0037] SEQ ID NO: 15 CACCACCATCACCATCAC;

[0038] SEQ ID NO: 16 CCCGCCATGGAGATCGAGTGCCGCATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCCAAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCGCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGGTGGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGCAGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCACCTTCAGCCTGCGCGACGGCGGCTACTACAGCTTCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCGCGTGGAGGAGCTGCACAGCAACACCGAGCTGGGCATCGTGGAGTACCAGCACGCCTTCAAGACCCCCATCGCCTTCGCC。

[0039] The third object of the present invention is to disclose the application of the above-mentioned T cell engager constructed based on GAS6 in the preparation of anti-tumor drugs.

[0040] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0041] (1) The T cell engager prepared by the present invention has high targeting specificity and significantly reduces off-target toxicity. Based on the natural ligand-receptor interaction between the LG1 domain (261-476AA) of GAS6 and the AXL receptor, it has moderate affinity compared with the traditional scFv antibody structure, can accurately recognize AXL highly expressed on the surface of tumor cells, and avoid non-specific killing of normal tissues. In addition, the GAS6 / AXL interaction is highly conserved in primates, and the preclinical safety evaluation is more predictive, which can reduce the off-target risk during treatment.

[0042] (2) The T cell engager prepared by the present invention forms a T cell-tumor cell immune synapse through the bispecific binding of GAS6 (D1) and CD3 (D2), and activates the specific killing of T cells.

[0043] (3) The present invention incorporates the CopGFP fluorescent protein to achieve real-time tracking of the expression of the T cell engager and optimize the treatment dose and timing.

[0044] (4) The present invention introduces the IgG1 Fc domain (T1) to extend the protein half-life and effectively reduce the dosing frequency.

[0045] (5) The present invention uses the (GGGGS)3 flexible linker peptide (SEQ ID NO: 4) to balance the steric hindrance between the GAS6 and CD3 binding domains. After optimizing the linker length, the T cell activation efficiency is improved.

[0046] (6) The present invention helps to develop a more economical, safe and effective anti-tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagrams of the structures of CD19-CAR, GAS6-CAR, Mock / CD3 BiTE, GAS6 / CD3 IgK_SP BiTE, and GAS6 / CD3G6_SP BiTE cells.

[0048] Figure 2 Bar graphs showing the killing effects of GAS6-CAR, GAS6 / CD3 IgK_SP BiTE, and GAS6 / CD3 G6_SP BiTE cells on MIA PaCa2 with high AXL expression.

[0049] Figure 3 Bar graphs showing the killing effects of GAS6-CAR, GAS6 / CD3 IgK_SP BiTE, and GAS6 / CD3 G6_SP BiTE cells on PANC1 with high AXL expression.

[0050] Figure 4Results of the binding ability test of Mock / CD3 BiTE and GAS6 / CD3 G6_SP BiTE to normal T cells after co-incubation.

[0051] Figure 5 Results of the binding ability test of Mock / CD3 BiTE and GAS6 / CD3 G6_SP BiTE to tumor cells MIA PaCa2, PANC1, and ASPC1 after co-incubation.

[0052] Figure 6 Results of the killing ability test of Mock / CD3 BiTE and GAS6 / CD3 G6_SP BiTE against MIA PaCa2 with high AXL expression.

[0053] Figure 7 Results of the killing ability test of Mock / CD3 BiTE and GAS6 / CD3 G6_SP BiTE against PANC1 with high AXL expression.

[0054] Figure 8 Results of the killing ability test of Mock / CD3 BiTE and GAS6 / CD3 G6_SP BiTE against ASPC1 with low AXL expression. Detailed implementation methods

[0055] The present invention will be specifically described below in combination with specific implementation methods and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific implementation methods and examples are used to illustrate the present invention, rather than limiting the present invention.

[0056] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0057] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0058] The synthesis and extraction of all plasmids in the following examples were completed by Tsingke Biological Company.

[0059] The present invention constructs CAR-T and TCE based on the extracellular domain of GAS6, Figure 1 which are schematic diagrams of the sequences of CD19-CAR, GAS6-CAR, Mock / CD3BiTE, GAS6 / CD3 IgK_SP BiTE, and GAS6 / CD3 G6_SP BiTE.

[0060] The vector of CAR-T is PGC-EF1α-P2A-EGFP, where EF1α is a promoter element, 261-476AA is the extracellular domain of GAS6, the hinge region and transmembrane region are both derived from human CD8 molecule, 4-1BB is from human CD137, CD3ζ is from human CD3, and mKate2 is a fluorescent label for detecting CAR expression.

[0061] The vector of TCE is pCDH-CMV-MCS-EF1α-Puro, where CMV and EF1α are promoter elements, and the signal peptides are respectively from IgK or GAS6; the extracellular domain of GAS6 is 261-476AA; CD3-ScFV is the human CD3 binding domain that specifically binds to T cells, and the two are connected by a (GGGGS)3 linker; CH2-CH3 is the FC fragment of IgG1 for extending the half-life of the protein; His tag is used for protein purification; CopGFP is used for monitoring the infection efficiency.

[0062] And the TCE and CAR-T in the following examples are constructed according to the following steps:

[0063] 1. Construction of TCE

[0064] (1) Based on the nucleotide sequences of the LG1 (261-476) domain of GAS6 shown in SEQ ID NO: 9 to SEQ ID NO: 16, the human IgK or GAS6 signal peptide, (G4S)3 linker, human CD3-ScFV, the CH2 and CH3 of the FC domain of human IgG1, 6His and other gene sequence information, the corresponding nucleotide sequences are obtained by artificial synthesis method or PCR method, and then the above sequences are spliced into a complete coding sequence in the structural order shown in Figure 1 and the obtained complete coding sequence is cloned into the lentiviral vector pCDH-CMV-MCS-EF1α-Puro to obtain a recombinant lentiviral vector.

[0065] (2) Protein expression (culturing and transfection of 293F cells)

[0066] (a) Cell culture

[0067] Culture medium: SMM 293-TII Expression Medium (Serum free, complete medium).

[0068] Culture conditions: 37°C, 8% CO2, shaker speed 120 - 140 rpm (suspension culture).

[0069] The cell density is maintained at 0.5 - 3×10 6 cells / mL, and the survival rate > 90%.

[0070] Amplification: Inoculate into a large-volume (such as 1 L) shake flask or bioreactor.

[0071] (b) Transfection

[0072] Transfection procedure:

[0073] When the cell density reaches 1 - 1.5×10 6 cells / mL, perform transfection.

[0074] Mix PEI (polyethyleneimine) with the recombinant lentiviral vector constructed above (the ratio is usually 1:2 - 1:3, w / w), and incubate at room temperature for 15 - 20 minutes.

[0075] Add the mixture dropwise into the cell culture.

[0076] 4 - 6 hours after transfection, add a supplement (1% FBS) to enhance expression.

[0077] (c) Expression monitoring

[0078] Time: Usually 4 - 7 days, regularly take samples to detect protein expression (SDS-PAGE, ELISA).

[0079] Harvest: Centrifuge (3500 rpm, 20 minutes) to remove cell debris and collect the supernatant.

[0080] (3) Protein purification

[0081] (a) Pretreatment of the supernatant

[0082] Filtration: Filter with a 0.45 μm or 0.22 μm filter membrane to remove residual particles.

[0083] Buffer adjustment: Add a protease inhibitor (such as PMSF) and adjust the pH to 7.0 - 7.5.

[0084] (b) Affinity chromatography

[0085] Column: HisTrap HP column

[0086] Buffer preparation:

[0087] Binding buffer: 20 - 50 mM sodium phosphate or Tris-HCl, 300 mM NaCl, 10 - 20 mM imidazole, pH 7.4.

[0088] Elution buffer: 20 - 50 mM sodium phosphate or Tris-HCl, 300 mM NaCl, 250 - 500 mM imidazole, pH 7.4.

[0089] The steps are as follows:

[0090] Column equilibration: Equilibrate with 5 - 10 column volumes (CV) of binding buffer.

[0091] Sample loading: Load the filtered supernatant onto the column at a flow rate of 1 - 2 mL / min.

[0092] Washing: Remove impurity proteins with a buffer containing 20 - 50 mM imidazole (5 - 10 CV).

[0093] Elution: Perform gradient elution (250 - 500 mM imidazole), collect the protein peak to obtain GAS6-TCE.

[0094] 2. CAR-T construction

[0095] (1) Lentivirus preparation

[0096] (a) Resuscitate HEK-293T cells into a 15-cm culture dish. When the cell density reaches 90%, digest with trypsin and passage at a ratio of 1:2.

[0097] (b) When the cell density reaches about 80%, perform transfection. Add the following components to 2 mL of serum-free DMEM medium per 15-cm dish: core plasmid (CD19-CAR, GAS6-CAR, Mock / CD3 BiTE, GAS6 / CD3 IgK_SP BiTE, GAS6 / CD3 G6_SP BiTE, etc.) (20 μg): pCMV-dR8.91 (10 μg): pMD2.G (4 μg), and simultaneously add 68 μL of lipo8000. Mix well and add to the culture dish.

[0098] (c) Supplement each 15-cm dish with 13 mL of DMEM medium containing 5% FBS, and gently shake well.

[0099] (d) Collect the cell culture supernatant 48 h after transfection, replace with 15 mL of fresh medium; and collect the culture supernatant at 72 h.

[0100] (e) Take the supernatant of the (4) culture medium, centrifuge at 3500 rpm at 4 °C for 15 min, and filter through a 0.45 μm filter membrane; for each ultracentrifuge tube, add 30 mL of supernatant + 5 mL of 20% sucrose (spread at the bottom of the tube), centrifuge at 25000 rpm at 4 °C for 2.5 h; discard the supernatant, invert and dry the precipitate for 5 min, add 50 μL of lentivirus lysate, and dissolve the virus overnight at 4 °C;

[0101] (f) Obtain lentivirus particles, aliquot the virus solution, and store at -80 °C for later use.

[0102] (2) Preparation of human CAR-T cells

[0103] (a) T cell isolation: Use RosetteSep TM gradient centrifugation tube and Human T Cell Enrichment Cocktail kit to isolate T cells from the peripheral blood of healthy volunteers. The specific steps are as follows:

[0104] (I) Add 20 mL of peripheral blood + 800 μL of RosetteSep TM Cocktail, mix well and let stand for 10 min, then dilute 1-fold with PBS (containing 2% FBS);

[0105] (II) Take 15 mL of Ficol Lymphoprep in a RosetteSep TM centrifuge tube, and add (I) to (II), and perform gradient centrifugation at 1200 g for 10 min;

[0106] (III) Take the supernatant and transfer it to a 50 mL centrifuge tube, add an equal volume of PBS (containing 2% FBS), and perform gradient centrifugation at 300 g for 10 min;

[0107] (IV) Discard the supernatant, add cryopreservation solution (70% Advanced RPMI 1640 medium + 20% FBS + 10% DMSO) for cryopreservation, and detect CD3 expression by flow cytometry.

[0108] (b) Preparation of CAR-T cells:

[0109] (I) Activate T cells in the culture medium (5 mL of FBS + 10 μL of IL-2 + 500 μL of Gluta-MAX + 500 μL of P / S + 44 mL of Advanced RPMI 1640) + Human T-Activator CD3 / CD28 magnetic beads (25 μL / 1×10 6 T cells) for 72 h;

[0110] (II) Centrifuge at 500 g for 5 min and count. Infect CAR lentivirus particles using a 96-well plate: 1×105 T cells + 1 μL LentiBoost (1 μg / mL) + CAR lentiviral particles (MOI = 100). After 24 h of infection, centrifuge at 500 g for 5 min, discard the supernatant and transfer to a 24-well plate. On the 3rd day after CAR-T cell infection, monitor the infection efficiency of CAR-T by flow cytometry, and the obtained CAR-T cells are used for subsequent experimental studies.

[0111] Example 1

[0112] To evaluate the killing ability of GAS6-TCE on tumor cells. In this example, first, the CD19-CAR, GAS6-CAR, Mock / CD3 BiTE, GAS6 / CD3 IgK_SP BiTE, GAS6 / CD3 G6_SP BiTE lentiviral particles as shown in Figure 1 were used to infect T cells. After 72 h, co-culture with luciferase-labeled MIA PaCa2 and PANC1 cell lines at an effector-to-target ratio of 2:1, and detect the luciferase activity after 24 h. As shown in Figures 2 - 3 the results indicate that GAS6-CAR-T, GAS6 / CD3 IgK_SP BiTE, and GAS6 / CD3 G6_SP BiTE cells can all effectively kill MIA PaCa2 and PANC1, and the killing effect of GAS6 / CD3 G6_SP BiTE is better than that of GAS6 / CD3 IgK_SP BiTE. Therefore, the AXL-targeted GAS6 bispecific T cell engager is successfully constructed and effective, and the subsequent invention uses GAS6 / CD3 G6_SP BiTE as the core (abbreviated as GAS6 / CD3).

[0113] Example 2

[0114] Infect the constructed GAS6-TCE into 293F cells, collect the culture supernatant, and perform protein purification. Figure 4 It is indicated that Mock / CD3 BiTE and GAS6 / CD3 G6_SP BiTE have a high binding ability with T cells. Figure 5 It shows that Mock / CD3 BiTE and GAS6 / CD3 G6_SP BiTE have a high binding ability with tumor cells MIA PaCa2 and PANC1 with high AXL expression, and a low binding ability with ASPC1 with low AXL expression.

[0115] Example 3

[0116] To evaluate the killing ability of GAS6-TCE protein on tumor cells. In this example, the purified Mock / CD3BiTE (Mock / CD3), GAS6 / CD3 G6_SP BiTE (GAS6 / CD3) proteins were added to the co-culture system of tumor cells (MIA PaCa2, PANC1, ASPC1) and T cells. The T cells and target cells were co-cultured at an effector-to-target ratio of 2:1, and Mock / CD3 BiTE (Mock / CD3), GAS6 / CD3 G6_SPBiTE (GAS6 / CD3) proteins at different concentration gradients (0, 10 -3 、10 -2 、10 -1 、10 0 、10 1 、10 2 、10 3 ng / mL) were added. After 24 h, the killing effect on target cells was detected using the Luciferase Assay System. The results are as Figures 6 - 8 shown. GAS6 / CD3 G6_SP BiTE cells effectively killed MIA PaCa2 and PANC1 with high AXL expression in a dose-dependent manner. However, there was no obvious killing effect on ASPC1 with low AXL expression.

[0117] Finally, it should also be noted that the term "comprising", "including" or any other variant is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0118] The above-described embodiments only represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several variations and improvements can be made, and these all belong to the protection scope of the present application.

Claims

1. A T cell engager constructed based on GAS6, characterized in that, The T cell adapter comprises a structure shown in formula (I) or formula (II): S-D1-L1-D2-T1-T2-CopGFP (I); S-D2-L1-D1-T1-T2-CopGFP (II); wherein, each "-" is independently a linker peptide or a peptide bond; S is a signal peptide sequence selected from an IgK signal peptide or a GAS6 signal peptide, the amino acid sequence of the IgK signal peptide is as shown in SEQ ID NO: 1, and the amino acid sequence of the GAS6 signal peptide is as shown in SEQ ID NO: 2; D1 is a fragment of the extracellular binding domain of GAS6 that specifically binds to AXL, and its amino acid sequence is as shown in SEQ ID NO: 3; L1 is a (GGGGS)n linker, and its amino acid sequence is as shown in SEQ ID NO: 4; D2 is a structure of the CD3 binding domain that specifically binds to T cells, and its amino acid sequence is as shown in SEQ ID NO: 5: T1 is a marker protein selected from the FC: CH2 and CH3 domains of IgG1, and its amino acid sequence is as shown in SEQ ID NO: 6; T2 is a marker protein selected from a His tag or a FLAG tag, and its amino acid sequence is as shown in SEQ ID NO: 7; CopGFP is a green fluorescent protein, and its amino acid sequence is as shown in SEQ ID NO:

8.

2. The preparation method of the T cell engager constructed based on GAS6 according to claim 1, wherein, Comprising the following steps: (1) Obtain the base sequences of fragments S, D1, D2, L1, T1, T2, and CopGFP by artificial synthesis or PCR amplification, which are respectively as shown in SEQ ID NO: 9 to SEQ ID NO: 16, and then splice the fragments S, D1, D2, L1, T1, T2, and CopGFP into a complete coding sequence in the order of formula (I) or (II) by overlap extension PCR; (2) Clone the spliced complete coding sequence into the lentiviral expression vector pCDH-CMV-MCS-EF1α-Puro to obtain a recombinant lentiviral vector; (3) Transfect the recombinant lentiviral vector into HEK-293F cells cultured in suspension, culture using SMM 293-TII medium, and purify to obtain the T cell adapter after the culture is completed.

3. Use of the T cell adapter constructed based on GAS6 as described in claim 1 in the preparation of an anti-tumor drug.

Images