Genetically engineered human trophoblast cells, methods of making and using the same

Abstract

This invention belongs to the field of cell therapy and immunotherapy, and provides a genetically engineered human trophoblast cell, its preparation method, and its application. The human trophoblast cell uses K562 cells as the starting cell line and, after genetic engineering, stably expresses membrane-bound interleukin-21, CD137 ligand, and Delta-like ligand 1. The constructed K562 three-factor trophoblast cell can significantly enhance the proliferation efficiency, activation state, and functional stability of NK cells and γδ T cells. Its synergistic mechanism includes enhancing the proliferation, cytotoxicity, and stemness maintenance of NK cells and γδ T cells through the STAT3, NF-κB, and Notch signaling pathways, respectively. The human trophoblast cell has advantages such as good expression stability, significant functional enhancement, and maintaining high activity after cryopreservation and thawing.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy and immunotherapy, specifically relating to a genetically engineered human trophoblast cell, its preparation method, and its application. Background Technology

[0002] NK cells and γδ T cells are widely used in the immunotherapy of solid tumors, hematological malignancies and infectious diseases due to their natural killing function and MHC independence, and are suitable for development as allogeneic "off-the-shelf" cell therapy products.

[0003] In existing technologies, K562 cells are widely used to prepare feeder cells to support the expansion of NK cells or T cells. Earlier literature, such as Campbell et al. (J Immunol Methods, 2007, 318(1-2):1-14), reported a K562 cell line expressing mbIL-21 (K562-mbIL21), which can effectively expand CD56⁺ NK cells. However, the expanded NK cells exhibit functional exhaustion after long-term culture and have limited support for γδ T cells (especially the Vδ1 subset). Subsequently, Romee et al. (Nat Commun. 2016; 7:12198) constructed K562-mbIL21 / 4-1BBL dual-factor trophoblast cells. Although this system enhanced the activation function of NK cells, it still had the following shortcomings: (1) It did not involve Notch signaling, making it difficult to maintain the stemness of γδ T cells; (2) The phenotype and function of the expanded cells still showed batch fluctuations and a tendency to exhaustion; (3) The source of trophoblast cells did not have a multi-factor synergistic stimulation design, making it difficult to support the expansion of two types of cell systems at the same time; (4) The trophoblast cell construction method was mainly based on viral vectors, which had GMP adaptation barriers and potential safety risks.

[0004] No K562-derived cell lines expressing DLL1 or other Notch signaling ligands are currently publicly available. DLL1, as a classic Notch pathway ligand, plays a crucial role in the development of T / NK precursors. Although studies (such as Maekawa et al., Blood Advances, 2020) have demonstrated that DLL1 can promote the differentiation of human iPSC-derived NK or T cells, it has not yet been applied in in vitro expansion systems, especially not in combination with mbIL-21 and CD137L to enhance expansion efficiency and slow functional exhaustion.

[0005] Therefore, current technology lacks a K562-derived feeder cell that can simultaneously activate NK cells and γδ T cells (especially the Vδ1 subset) through the IL-21, CD137L and Notch pathways to improve expansion efficiency, inhibit depletion, and meet the needs of clinical cell therapy preparation. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides genetically engineered human trophoblast cells, their preparation method, and applications. The cells stably co-express three key immunomodulatory molecules on their surface, including membrane-bound interleukin-21 (mbIL-21), CD137 ligand (CD137L), and Delta-like ligand 1 (DLL1). This cell line, named JY511, aims to overcome the problems of low efficiency in expanding NK cells and γδ T cells, poor functional maintenance, and cell phenotype depletion in existing technologies.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a genetically engineered human trophoblast cell, wherein the human trophoblast cell is a K562 cell as the starting cell and is genetically engineered to stably express membrane-bound interleukin-21 (mbIL-21), CD137 ligand (CD137L), and Delta-like ligand 1 (DLL1); the amino acid sequence of the membrane-bound interleukin-21 is shown in SEQ ID NO:1, the amino acid sequence of the CD137 ligand is shown in SEQ ID NO:2, and the amino acid sequence of the Delta-like ligand 1 is shown in SEQ ID NO:3.

[0009] This invention, by constructing a three-factor synergistic stimulation system, achieves efficient expansion, activation, and stemness maintenance of NK cells and γδ T cells (especially the Vδ1 subset), thereby providing a higher quality, more functional, and more industrially suitable supporting cell system for cell immunotherapy products, meeting the urgent needs of current allogeneic "off-the-shelf" cell therapy product development and clinical preparation.

[0010] Preferably, the membrane-bound interleukin-21 is formed by the fusion of the extracellular domain of IL-21, the CD8α hinge region, and the CD8α transmembrane domain.

[0011] Preferably, the nucleotide sequences encoding the membrane-bound interleukin-21, CD137 ligand, and Delta-like ligand 1 are introduced into K562 cells via an expression vector.

[0012] Preferably, the expression vector includes a viral vector or a non-viral vector.

[0013] Preferably, the non-viral vector includes a transposon subsystem or a site-specific integration system.

[0014] Preferably, the transposable subsystem includes the PiggyBac transposable subsystem.

[0015] Preferably, the membrane-bound interleukin-21 and CD137 ligands are linked by a self-cleaving peptide to form a co-expression unit, and the Delta-like ligand 1 is expressed by an independent promoter.

[0016] Preferably, the self-cleaving peptide includes any one of T2A, P2A, E2A, or F2A.

[0017] Preferably, the promoter driving the expression of the membrane-bound interleukin-21, CD137 ligand and / or Delta-like ligand 1 includes any one of EF1α, CMV, CAG, SFFV, PGK or UbC.

[0018] Preferably, the promoter of the Delta-like ligand 1 is the CAG promoter, and the promoters of the membrane-bound interleukin-21 and CD137 ligands are EF1α.

[0019] This invention addresses the problems of weak support capacity, low expansion efficiency, and easy depletion of NK or γδ T cells in existing technologies by providing a genetically engineered human trophoblast cell. Specifically, using the PiggyBac transposon system, the three genes mbIL-21, CD137L, and DLL1 are co-transfected into K-562 cells via a polycistronic expression vector. In vector construction, the EF1α promoter drives the expression of mbIL-21 and CD137L, and the CAG promoter drives the expression of CD137 ligand. After transfection, flow cytometry sorting yields a triple-positive expression monoclonal cell line, named JY511. This cell line maintains high expression stability after multiple passages, is GMP-compatible, and is suitable for industrial production.

[0020] In a specific embodiment of the present invention, the amplification effect of JY511 was comprehensively investigated. The results showed that JY511 can significantly enhance CD56 under antigen-free conditions. + The system exhibits a high NK cell ratio (>90%) and high expression of activating receptors such as NKG2D (>80%). It can also expand the Vδ1 cell population and enhance its cytotoxicity (IFN-γ and GzmB secretion). The expansion system demonstrates excellent cryopreservation and thawing capabilities, is suitable for multiple rounds of expansion, and shows promising application prospects in preclinical models.

[0021] In this invention, the genetically engineered human trophoblast cells maintain high expression levels of the three factors in at least 10 consecutive passages, with the proportion of triple-positive cells exceeding 85%.

[0022] In a second aspect, the present invention provides a method for constructing the genetically engineered human trophoblast cells described in the first aspect, the method comprising:

[0023] (1) Construct an expression vector containing nucleotide sequences encoding membrane-bound interleukin-21, CD137 ligand and Delta-like ligand 1;

[0024] (2) The expression vector is introduced into cells;

[0025] (3) Screen and isolate monoclonal cells that co-express membrane-bound interleukin-21, CD137 ligand and Delta-like ligand 1 to obtain the desired cells.

[0026] Preferably, the expression vector in step (1) includes the PiggyBac transposon vector.

[0027] Preferably, step (2) further includes co-transfection of a nucleotide sequence encoding the PiggyBac transposase.

[0028] Preferably, the amino acid sequence of the PiggyBac transposase is shown in SEQ ID NO:4.

[0029] Preferably, the nucleotide sequence encoding the PiggyBac transposase is shown in SEQ ID NO:5.

[0030] Preferably, the nucleotide sequences encoding membrane-bound interleukin-21, CD137 ligand and Delta-like ligand 1 are shown in SEQ ID NO:6-SEQ ID NO:8.

[0031] In this invention, the PiggyBac transposon system was used to stably co-express three immunomodulatory factors—mbIL-21, CD137L, and DLL1—in K562 cells to construct the JY511 cell line, achieving synergistic activation and expansion of NK cells and γδ T cells (especially the Vδ1 subset). Using the PiggyBac system for gene transfer avoids the potential biosafety risks associated with lentiviruses, while also possessing GMP-friendly characteristics such as low cost, high capacity, and low non-integrative toxicity. Genetically engineered human feeder cells enhance γδ T cell stemness maintenance through DLL1-Notch signaling, resulting in a higher proportion of central memory γδ T cells. Testing showed that the human feeder cells JY511 maintained high expression levels of the three factors (>85% triple-positive rate) even after more than 10 passages, supporting long-term cell preparation processes and facilitating industrial production. Human-derived feeder cells JY511 support the industrial expansion of iPSC-derived NK cells and γδ T cells, and can be used as universal support cells in CAR-NK, CAR-γδT, and immunoregenerative cell therapy systems. In summary, this invention develops an innovative feeder cell line with high expansion efficiency, functional durability, and production safety, significantly enhancing the clinical application potential of cell immunotherapy products.

[0032] Thirdly, the present invention provides the use of the genetically engineered human trophoblast cells described in the first aspect in the preparation of kits or compositions for in vitro expansion of NK cells or γδ T cells.

[0033] In this invention, the genetically engineered human trophoblast cells can be used to expand NK cells or γδ T cells under preclinical or GMP conditions, and are particularly suitable for the preparation of cell immunotherapy products, including but not limited to iPSC-NK, iPSC-γδT, PBMC-NK, CAR-NK, PBMC-γδT, CAR-γδT cell products, etc.

[0034] Fourthly, the present invention provides a method for in vitro expansion of NK cells or γδ T cells, the method comprising: co-culturing NK cells or γδ T cells with the genetically engineered human trophoblast cells described in the first aspect to obtain expanded NK cells or γδ T cells.

[0035] Preferably, the NK cells are derived from any one or a combination of at least two of human peripheral blood, umbilical cord blood, embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs); the γδ T cells are derived from any one or a combination of at least two of human peripheral blood, umbilical cord blood, embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs).

[0036] Preferably, the ratio of the NK cells or γδ T cells to the human trophoblast cells in co-culture is (0.2-5):1.

[0037] Preferably, the γδ T cells include γδ1 T cells or γδ2 T cells.

[0038] In this invention, the NK cells expanded using the cell line exhibit the following phenotypes: (1) high expression of NKG2D, NKp30, and CD16; (2) low expression of NKG2A; and (3) high IFN-γ secretion.

[0039] In this invention, the γδ T cells expanded using the cell line described above have the following characteristics: (1) a significantly increased proportion of Vδ1 or Vδ2 subsets; (2) enhanced expression of CD107a and IFN-γ.

[0040] In this invention, the mechanism by which the three factors synergistically promote NK cell proliferation and function is as follows:

[0041] (1) The mechanism of action of mIL-21 (membrane-type IL-21) factor is to continuously activate the IL-21R-JAK / STAT3 pathway. Its effects on NK cells include: enhancing proliferation capacity and inducing stem-like NK phenotype.

[0042] (2) The mechanism of action of CD137L (4-1BBL) factor is to activate the CD137 co-stimulatory pathway on the surface of NK cells. Its effects on NK cells include: increasing survival rate, enhancing IFN-γ secretion and killing activity.

[0043] (3) The mechanism of action of DLL1 (Notch ligand) factor is to activate Notch1 / Notch2-NICD nuclear translocation. Its effects on NK cells include: upregulation of activating factors (NKG2D, NKp30) and downregulation of negative regulatory factors such as NKG2A.

[0044] The three factors mentioned above activate the STAT3, NF-κB, and Notch pathways at the signaling level, respectively, synergistically enhancing NK cell activation and expansion. These three factors have synergistic advantages, including: high expansion efficiency and long duration; good phenotypic uniformity of expansion products; low expression of the inhibition / exhaustion marker (NKG2A); and sustained expansion capacity for NK and T cells.

[0045] In this invention, the mechanism by which the three factors synergistically promote the activation of γδ T cells is as follows:

[0046] (1) The mechanism of action of mIL-21 factor is to activate STAT3 / STAT5 and promote functional maturation. Its effects on γδ T cells (especially Vδ1) include: increasing the expression of Granzyme B and IFN-γ, and enhancing killing ability.

[0047] (2) The mechanism of action of CD137L factor is to bind to CD137 on the surface of γδ T cells. Its effects on γδ T cells (especially Vδ1) include: enhancing survival and effector function, and inhibiting early apoptosis.

[0048] (3) The mechanism of action of DLL1 factor is to activate Notch signaling and promote memory / stem maintenance. Its effects on γδ T cells (especially Vδ1) include: enhancing proliferative potential.

[0049] mbIL-21 (SEQ ID NO:1):

[0050] MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQ KHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0051] CD137L(SEQ ID NO:2):

[0052] MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE。

[0053] DLL1(SEQ ID NO:3):

[0054] MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWVAVCAGVILVLMLLLGCAAVVVCVRLRLQKHRPPADPCRGETETMNNLANCQREKDISVSIIGATQIKNTNKKADFHGDHSADKNGFKARYPAVDYNLVQDLKGDDTAVRDAHSKRDTKCQPQGSSGEEKGTPTTLRGGEASERKRPDSGCSTSKDTKYQSVYVISEEKDECVIATEV。

[0055] SEQ ID NO:4:

[0056] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF。

[0057] The nucleotide sequence of the PiggyBac transposase is shown in SEQ ID NO:5.

[0058] SEQ ID NO:5:

[0059]

[0060] Nucleotide sequence encoding mbIL-21 (SEQ ID NO:6):

[0061] atgagaagcagccccggcaacatggagagaatcgtgatctgcctgatggtgatcttcctgggcaccctggtgcacaagagcagcagccaaggccaagacagacacatgatcagaatgagacagctgatcgacatcgtggatcagctgaagaactacgtgaacgacctggtgcccgagttcctgcccgcccccgaggacgtggagaccaactgcgagtggagcgccttcagctgctttcagaaggctcagctgaagagcgccaacaccggcaacaacgagagaatcatcaacgtgagcatcaagaagctgaagagaaagccccctagcaccaacgccggcagaagacagaagcacagactgacctgccctagctgcgacagctacgagaagaagccccccaaggagttcctggagagattcaagagcctgctgcagaagatgatccatcagcacctgagcagcagaacacacgggtccgaagactccaccacaacacccgctcctagaccccccacccccgctcccaccatcgcctcccaacctctgagcctgcggcctgaggcctgtcggcccgctgccggcggggctgtgcacacaagaggcctggacttcgcctgcgacatctacatctgggcccccctggccggcacctgcggcgtgctgctcctgagcctggtgatcaccctgtactgc。

[0062] Nucleotide sequence encoding CD137L (SEQ ID NO:7):

[0063] atggaatacgcttccgatgcttccctcgaccccgaggctccttggcctcctgccccccgggctagagcctgcagagtcctcccctgggctctggtcgctggcctcctgctgctcctgctcctcgctgccgcctgcgctgtgttcctggcttgcccctgggccgtgtccggcgctagagcctcccccgggagcgctgcttccccccggctgcgggagggccctgagctgagccctgacgaccccgctggcctgctcgatctgagacaaggcatgttcgctcagctggtggctcagaacgtgctgctgatcgatggccccctgagctggtacagcgaccccgggctcgctggcgtgagcctgaccggcggcctgagctacaaggaggacaccaaggagctggtggtggccaaggccggcgtgtactacgtgttctttcagctggagctgagaagagtggtggccggcgagggcagcggctccgtgtccctggctctgcatctgcagcctctgagatccgccgccggggccgccgctctcgctctcacagtcgacctgcctcccgcctcctccgaggctagaaacagcgccttcggcttccaaggcagactgctgcacctgagcgccgggcagagactgggcgtgcacctgcacaccgaggctagagctagacacgcctggcagctgacccaaggcgccaccgtgctgggcctgttcagagtgacccccgagatccccgccggcctgcctagccctagaagcgag。

[0064] Nucleotide sequence encoding DLL1 (SEQ ID NO:8):

[0065]

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) The three-factor synergistic activation mechanism significantly enhances the cell proliferation capacity: JY511 cell line simultaneously expresses mbIL-21, CD137L and DLL1, which activate the STAT3, NF-κB and Notch pathways of NK cells and / or γδ T cells, respectively. This multi-signal synergistic stimulation can significantly enhance the cell proliferation capacity and functional maturity, avoiding uneven expansion or functional exhaustion caused by single-factor stimulation.

[0068] (2) Significantly enhances the amplification potential of Vδ1 subset in γδ T cells: Compared with the traditional IPP / Zoledronic acid system, JY511 particularly promotes the amplification of Vδ1 subset, and the amplification fold is significantly higher than that of the IPP / Zoledronic acid system.

[0069] (3) Stable expression, suitable for GMP standards and industrialization requirements: JY511 is constructed using a non-viral PiggyBac transposon system to avoid potential viral residues and insertion mutations. The cell bank has been validated through more than 10 consecutive generations, and the expression ratios of the three factors are stable, with good batch consistency and controllability, meeting the GMP standards for cell therapy raw materials.

[0070] (5) High versatility and can be widely applied to the development of allogeneic cell therapy products: This cell line is not only suitable for the expansion of NK cells and γδ T cells derived from peripheral blood and umbilical cord blood, but also suitable for the production platform of NK or γδ T cells derived from iPSC, providing a unified and efficient support system for "off-the-shelf" cell products.

[0071] (6) High expression and high function after cryopreservation and thawing: JY511 cells are cryopreserved at -130°C and the expression ratio of the three factors and the amplification support function remain stable after thawing, which is convenient for long-term storage and industrial use.

[0072] In summary, the JY511 three-factor trophoblast cells provided by this invention are superior to existing technologies in terms of expansion efficiency, functional maintenance, industrial compatibility, safety, and universal adaptability. They are a highly innovative, practical, and transformative key raw material for cell therapy. Attached Figure Description

[0073] Figure 1 The image shows the detection results of flow cytometry analysis of 4-1BBL.

[0074] Figure 2 The image shows the results of flow cytometry detection of IL-21.

[0075] Figure 3 The image shows the detection results of DLL-1 by flow cytometry.

[0076] Figure 4 The graph shows the expression results of the 4-1BBL transgene;

[0077] Figure 5 The graph shows the expression results of the IL-21 transgene.

[0078] Figure 6 The graph shows the expression results of the DLL-1 transgene;

[0079] Figure 7 The figure shows the results of a continuous kill test with an effective target ratio (E:T) of 1:1.

[0080] Figure 8 The figure shows the results of a continuous kill test with an effective target ratio (E:T) of 3:1.

[0081] Figure 9A Figure showing the in vitro killing effect of JY511 expanded NK cells on various hematological malignancies;

[0082] Figure 9B Figure showing the in vitro killing effect of JY511 expanded NK cells on various hematological malignancies;

[0083] Figure 10 The image shows the purity (CD56+ rate) of PBMC-NK cells expanded by JY511 for 7 days;

[0084] Figure 11 The image shows the purity (CD56+ rate) of PBMC-NK cells expanded from JY511 cells for 14 days.

[0085] Figure 12 Image showing the residual PBMC-NK cells from JY511 expanded for 14 days as feeder cells;

[0086] Figure 13 Figure showing the killing effect of JY511 expanded PBMC-NK cells on tumor target cells after 14 days.

[0087] Figure 14 Image showing the results of autonomous interferon secretion from PBMC-NK cells amplified by JY511 for 14 days;

[0088] Figure 15 Figure showing the interferon-specific secretion results of PBMC-NK cells expanded from JY511 for 14 days after activation by tumor target cells;

[0089] Figure 16The results of JY511 PBMC-NK cell phenotype (CD337+, CD158b, CD16+, CD107a expression) after 14 days of expansion.

[0090] Figure 17 Flow cytometry plot of PBMC-γδT cells amplified by JY511 cells for 14 days;

[0091] Figure 18 The results show the ability of co-expression of the three genes of JY511 to promote the killing of tumor cells by iPSC-derived NK cells;

[0092] Figure 19 Figure showing the fold expansion of γδT cells differentiated from iPSCs after 7 days of expansion using JY511 feeder cells.

[0093] Figure 20 The figure shows the proportion of CD3+γδTCR+ cells in γδT cells differentiated from iPSCs after 7 days of expansion using JY511 trophoblast cells. Detailed Implementation

[0094] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0095] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0096] Example 1

[0097] Construction and screening of JY511 cell line

[0098] (1) Origin and identification of starting cells.

[0099] JY511 trophoblast cells are derived from human chronic myeloid leukemia K-562 cells (ATCC® CCL-243™, batch number 70056075). These cells have been species-identified as human and have been identified through STR typing. They are free from bacterial and mycoplasma contamination, and tests for human viruses such as HIV, HBV, EBV, and CMV are all negative.

[0100] (2) PiggyBac transposon system was constructed to express the strain.

[0101] Using the PiggyBac transposon system, three genes encoding mbIL-21, CD137L, and DLL1 were stably integrated into the K-562 genome.

[0102] Transposon plasmid PJY0105-1: The transposon plasmid contains the following core elements connected in sequence: a 5' terminal inverted repeat sequence (5' ITR), an insulator, a human Delta-like ligand 1 (DLL1) coding sequence driven by the CAG promoter, a human membrane-bound interleukin-21 (mbIL-21) coding sequence driven by the EF-1α promoter, a T2A self-cleaving peptide sequence, a human 4-1BB ligand (CD137L) coding sequence, and a 3' terminal inverted repeat sequence (3' ITR). The mbIL-21 is formed by the fusion of the extracellular domain of human IL-21 with the hinge region and transmembrane region of the human CD8a molecule.

[0103] Transposase plasmid pJY0119: Expresses PiggyBac transposase to mediate gene integration. The above plasmid was constructed using molecular cloning technology, and its correctness was verified by sequencing. After extraction and purification, the purity, sterility, and endotoxin levels of the plasmid met the requirements for cell transfection.

[0104] (3) Cell transfection and transgene integration.

[0105] Electroporation was performed using Lonza's 4D Nucleofector™ MAX X-unit. mbIL-21 was obtained after FACS sorting. + / CD137L + / DLL1 + Triple-positive cell population.

[0106] K562 cells were electroporated using the Lonza 4D-Nucleofector system (protocol: SE buffer + FF-120 program). 1–2 μg of expression vector and 0.5 μg of transposase plasmid were co-transfected. Single clones were sorted by flow cytometry, and clones with high transgene expression were selected to establish single-clonal cell lines.

[0107] (4) Single clone screening and identification.

[0108] The sorted single clones were expanded cultured for cryopreservation of seed cell lines and for the screening and identification of clones. The process of single clone expansion culture and screening and identification is shown in Table 1 below.

[0109] Table 1

[0110]

[0111]

[0112] 3.1 Transgenic expression intensity in 32 monoclonal cells.

[0113] Based on the growth status of 39 monoclonal cell lines, 32 monoclonal cell lines in good condition were selected, sampled, and the expression of 4-1BBL (i.e., CD137L), IL-21, and DLL-1 was detected by flow cytometry. The results are as follows: Figures 1-3 As shown.

[0114] Figure 1 The results are from flow cytometry analysis of 4-1BBL. Figure 2 The results are from flow cytometry analysis of IL-21. Figure 3 This is the result of flow cytometry detection of DLL-1.

[0115] The mean fluorescence intensity (MFI) of the 4-1BBL (i.e., CD137L), IL-21, and DLL-1 transgenes are shown in Table 2 below.

[0116] Table 2

[0117]

[0118] Based on the expression intensity of the transgene, 12 monoclonal strains, namely 1-B9, 2-B6, 2-B7, 2-E11, 2-G8, 3-C8, 3-C9, 3-G10, 4-F11, 5-B9, 6-C5, and 6-G4, were screened for amplification culture and cryopreservation.

[0119] 3.2 Expression stability of 12 monoclonal transgenic strains.

[0120] Two weeks after passage of 12 monoclonal strains, transgene expression was re-tested to assess its stability. The results are as follows: Figures 4-6 As shown.

[0121] Figure 4 The results show the expression of the 4-1BBL transgene. Figure 5 The results show the expression of the IL-21 transgene. Figure 6 The results show the expression of the DLL-1 transgene.

[0122] The mean fluorescence intensity (MFI) of the 4-1BBL (i.e., CD137L), IL-21, and DLL-1 transgenes are shown in Table 3 below.

[0123] Table 3

[0124]

[0125] The results show that the expression of 4-1BBL (i.e. CD137L) in the 4-F11 monoclonal strain was significantly reduced. Therefore, this monoclonal strain was discarded, and the remaining 11 monoclonal strains were retained for subsequent experiments.

[0126] 3.3 Detection of 11 monoclonal iNK cell lines for expansion.

[0127] To evaluate the role of different monoclonal cells as feeder cells in NK cell expansion, 11 monoclonal cell lines were irradiated with 100 Gy of X-rays. The irradiated monoclonal cells were then used to promote iNK cell expansion. On days 0 and 8, different monoclonal cell lines were co-incubated with AI-iNK cells (AI-iNK cells are NK cells induced by iPSCs that integrate the IL15RF gene via HDR) at a 2:1 ratio. AI-iNK cell counts were performed on days 8 and 14 to calculate the fold increase. On day 14, IL15RF expression and cytotoxic function on expanded AI-iNK cells were assessed. Table 4 below shows the fold increase and IL15RF expression data of AI-iNK cells on days 8 and 14. The data show that all 11 monoclonal cells effectively expanded iNK cells (>50-fold) in the first round of stimulation. After the second round of stimulation, the different clones showed differences in NK cell expansion.

[0128] Table 4

[0129]

[0130] The amplified AI-iNK groups were subjected to continuous killing experiments: 2×10⁻⁶ ions were added to each well of a 96-well flat plate. 4 K562-Luc-GFP live cells were added; iNK live cells were added according to E:T=1:1 and E:T=3:1 respectively, mixed well, and incubated in an Incucyte S3 instrument, with continuous photography taken every 3 hours. Approximately every 24 hours, the 96-well plate was removed, 80 μL of supernatant was aspirated from each well, and 100 μL of fresh K562-Luc-GFP cell suspension (containing 2 × 10⁶ cells / well) was added. 4 (1 live cell), after mixing, was placed in the Incucyte S3 instrument for imaging. The fluorescence intensity of each well was statistically analyzed using the built-in software of the Incucyte S3 instrument to compare the killing ability of iNK cells expanded by different monoclonal strains against K562-Luc-GFP.

[0131] The results of the E:T=1:1 continuous lethality test are as follows: Figure 7 As shown. The results of the E:T=3:1 continuous lethality test are as follows. Figure 8 As shown.

[0132] Based on in vitro functional tests, and combined with the results of AI-iNK amplification and expression detection, monoclonal 2-B7 was selected from 11 monoclonal strains.

[0133] (4) After expanding and culturing 2-B7 cells, they were irradiated with X-rays at a dose of 100 Gy for 30 min. The irradiated cells were then cryopreserved and became JY511 feeder cells.

[0134] Example 2

[0135] JY511 amplification of NK cells derived from PBMCs.

[0136] PBMCs were isolated from peripheral blood and counted. Based on the counting results, the PBMCs were resuspended in NK medium at a density of 1×10⁻⁶. 6 The JY511 feeder cells prepared in Example 1 were revived and added to PBMC cells for co-culture at a ratio of 1:1. After 7 days of expansion, a second batch of JY511 feeder cells was added, maintaining a 1:1 ratio with PBMCs. The total expansion and culture time was 14 days. After the expansion culture was completed, NK cells expanded from JY511 feeder cells were collected. These are NK cells derived from PBMCs prepared by JY511 expansion. Meanwhile, the control group consisted of NK cells derived from PBMCs expanded from Zhongying feeder cells, prepared under the same conditions as NK cells prepared by JY511.

[0137] Cell killing assay: Multiple hematologic malignancies were resuscitated and cultured as target cells for the killing assay, including K562, HL60, THP1, MoLT-4, Nalm6, Kasumi-1, KG-1α, Jurkat, and Molm-13 (a total of nine target cell types). Effector-to-target ratios were 2.5:1, 5:1, and 10:1. NK cells expanded from JY511 cells were used as effector cells, and co-cultured with the above nine target cell types at different effector-to-target ratios. Experimental results are as follows: Figures 9A-9B As shown, NK cells significantly killed the aforementioned nine types of tumor cells (target cells) at different effector-to-target ratios. Generally, the killing rate of NK cells increased with increasing effector-to-target ratio. This experiment demonstrates that JY511-amplified NK cells have a significant in vitro killing effect on hematological malignancies.

[0138] To verify the purity of NK cells expanded from JY511 trophoblast cells, NK cells expanded for 7 and 14 days were collected, analyzed by flow cytometry, and compared with the control group NK cells (expanded from Zhongying trophoblast cells). Results are as follows: Figure 10 and Figure 11 As shown, after 7 days of expansion, JY511 expanded NK cells (CD3+) - CD56 + The proportion of NK cells was 95.07%, while the control NK cell proportion was 83.15%. After 14 days of amplification, the proportion of NK cells amplified by JY511 was 94.96%, while the control NK cell proportion was 86.89%. Simultaneously, flow cytometry was used to detect the residual trophoblast cells after 14 days of amplification. Figure 12 As shown, the trophoblast cell residue in JY511-expanded NK cells was also lower than that in the control group NK cells. This experiment demonstrates that JY511-expanded NK cells possess high purity and low residue.

[0139] To further verify the function of NK cells expanded by JY511 trophoblast cells, NK cells expanded for 14 days were used in a killing experiment. K562 cells were used as the target cells, and effector-to-target ratios were 2:1, 5:1, and 10:1. The killing ability of JY511 expanded NK cells was compared with that of control NK cells. The results are as follows: Figure 13 As shown, in three different effector-to-target ratios, the killing ability of JY511-amplified NK cells was significantly higher than that of the control group. IFN-γ is one of the key cytokines for evaluating NK effector function. Studies have shown that Notch signaling can enhance IL-2 and IL-15 responses, promote JAK / STAT5 and STAT1 / 4 signaling activity, and enhance cytotoxicity and IFN-γ secretion. Cell supernatant collected during the killing process was used for IFN-γ detection, and the results are as follows: Figure 14 and Figure 15 As shown, the IFN-γ secreted by JY511 expanded NK cells was significantly higher than that of the control group, and even in the presence of tumor cells (K562), the IFN-γ secreted by JY511 expanded NK cells was still significantly higher than that of the control group.

[0140] To further explore the function of JY511-expanded NK cells, flow cytometry was used to detect the phenotype of NK-related active cells. The results are as follows: Figure 16 As shown, the viable cell phenotypes of NK cells expanded by JY511 were higher than those of the control group, especially CD337. + The proportion was 94.48%, while the control group was 88.97%; CD16 + The proportion was 90.80%, while the control group was 82.39%; CD107a + The proportion was 90.88%, while the control group was 73.09%. Only CD158b was slightly lower than the control group. + The proportion was 37.5%, while the control group was 44.35%.

[0141] Example 3

[0142] JY511 expands γδT cells derived from PBMCs.

[0143] PBMCs isolated from peripheral blood of donor #185 were counted and their viability was determined. The cell viability was 91.23%, and the proportion of CD3-positive and δ1-positive cells was 14.3%. The cells were then cultured at a concentration of 1.5 × 10⁻⁶. 5Live cells were seeded and expanded with anti-γδTCR antibody. Cells were harvested 7 days after expansion, and cell viability was assessed. Cells were divided into two groups: Group A received the antibody, and Group B received the antibody and JY511 feeder cells at a 1:1 ratio. Cells were cultured separately. Cell harvesting, cell viability, cell number, and the ratio of CD3 cells to δ1T cells were assessed on day 14. The results are shown in Table 5. In Group A, the proportion of δ1T cells was 55.13%, the total cell expansion was 0.4-fold, and the δ1T cell expansion was 1.4-fold. In Group B, the proportion of δ1T cells was 98.58%, the total cell expansion was 91.3-fold, and the δ1T cell expansion was 629.6-fold. Under the same culture conditions, JY511 significantly increased the proportion of δ1T cells and enabled large-scale in vitro expansion of δ1T cells. γδT cells expanded for 14 days were characterized using CD45RA and CD27. Figure 17 As shown, the data indicates that the number of central memory cells in the γδT cells expanded by the B group was significantly higher than that in the A group.

[0144] Table 5

[0145]

[0146] Example 4

[0147] JY511 amplification of iPSC-derived NK cells.

[0148] Human induced pluripotent stem cells (iPSCs) were differentiated into NK cells. After differentiation, NK cells were collected and expanded. NK cells were added to 24-well G-Rex culture tanks, 1E6 cells per well, and feeder cells were added for co-culture at a ratio of 1:2. The feeder cells were JY511, and the control feeder cells were K562-mIL21-CD137L. Seven days after expansion, feeder cells were added again at the 1:2 ratio, and expansion continued for 14 days. After expansion, NK cells were collected for cytotoxicity testing. NK cells were co-cultured with tumor cells (Raji and Nalm6) for 4 hours at an effector-to-target ratio of 1:1, and then the cytotoxicity was detected by flow cytometry. The results are as follows: Figure 18 As shown, NK cells expanded from JY511 cells exhibited significantly enhanced killing ability compared to NK cells expanded from control trophoblast cells.

[0149] Example 5

[0150] JY511 expands γδT cells derived from iPSCs.

[0151] After the differentiation of human induced pluripotent stem cells (iPSCs) into γδT cells, γδT cell expansion experiments were performed. Different expansion protocols were used, with cells divided into four groups:

[0152] (1) KBM 581 lymphocyte serum-free medium was used as the amplification medium and Zoledronic acid was added. Human γδT cells can be activated by phosphorylated antigens and aminobisphosphonates (such as zoledronic acid), which can cause γδT proliferation.

[0153] (2) T cell culture medium was used as the amplification medium, and Zoledronic acid and IL-2 were added.

[0154] (3) T cell culture medium was used as the amplification medium, and CD3 / 28 magnetic beads were added as activation magnetic beads.

[0155] (4) KBM 581 lymphocyte serum-free medium was used as the amplification medium, and JY511 trophoblast cells were added at a ratio of 1:1.

[0156] Four expansion protocols were used, with each protocol starting with γδT cells differentiated from iPSCs from the same batch. The initial cell quantity was either 8E5 or 6.5E5. Seven days after expansion, cells were collected for viable cell counting and flow cytometry analysis to determine the positive rates of CD3 and γδTCR. The results are as follows: Figure 19 and Figure 20 As shown, after 7 days of amplification, the fourth group, namely the group with added JY511 trophoblast cells, had an amplification fold of 32-fold, which was significantly higher than the other three groups (0.8-1.4-fold). The proportion of CD3 and γδTCR double positive cells was 46.5%, which was also significantly higher than the other three groups (11-12%).

[0157] In summary, the JY511 three-factor K562 cell line of this invention exhibits excellent performance in terms of expansion efficiency, functional activation, low exhaustion characteristics, and GMP compatibility, and can be widely used in the in vitro expansion systems of cell therapy products such as CAR-NK, iPSC-NK, and VδT cells.

[0158] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A genetically engineered human trophoblast cell, characterized in that, The human-derived trophoblast cells, starting with K562 cells, were genetically engineered to stably express membrane-bound interleukin-21, CD137 ligand, and Delta-like ligand 1. The amino acid sequence of the membrane-bound interleukin-21 is shown in SEQ ID NO:1, the amino acid sequence of the CD137 ligand is shown in SEQ ID NO:2, and the amino acid sequence of the Delta-like ligand 1 is shown in SEQ ID NO:

3.

2. The genetically engineered human trophoblast cells according to claim 1, characterized in that, The membrane-bound interleukin-21 is formed by the fusion of the extracellular domain of IL-21, the CD8α hinge region, and the CD8α transmembrane domain.

3. The genetically engineered human trophoblast cells according to claim 1, characterized in that, Nucleotide sequences encoding the membrane-bound interleukin-21, CD137 ligand, and Delta-like ligand 1 were introduced into K562 cells via an expression vector, which may be a viral vector or a non-viral vector.

4. The genetically engineered human trophoblast cells according to claim 3, characterized in that, The non-viral vectors include transposon subsystems or site-specific integration systems.

5. A method for constructing genetically engineered human trophoblast cells according to any one of claims 1-4, characterized in that, The method includes: (1) Construct an expression vector containing nucleotide sequences encoding membrane-bound interleukin-21, CD137 ligand and Delta-like ligand 1; (2) The expression vector is introduced into cells; (3) Screen and isolate monoclonal cells that co-express membrane-bound interleukin-21, CD137 ligand and Delta-like ligand 1 to obtain the desired cells.

6. The method according to claim 5, characterized in that, The expression vector mentioned in step (1) includes the PiggyBac transposon vector; the nucleotide sequences encoding membrane-bound interleukin-21, CD137 ligand and Delta-like ligand 1 are shown in SEQ ID NO:6-SEQ ID NO:

8.

7. Use of genetically engineered human trophoblast cells according to any one of claims 1-4 in the preparation of kits or compositions for in vitro expansion of NK cells or γδ T cells.

8. A method for in vitro expansion of NK cells or γδ T cells, characterized in that, The method includes: co-culturing NK cells or γδ T cells with genetically engineered human trophoblast cells according to any one of claims 1-4 to obtain expanded NK cells or γδ T cells.

9. The method according to claim 8, characterized in that, The NK cells are derived from any one or a combination of at least two of human peripheral blood, umbilical cord blood, embryonic stem cells, or human induced pluripotent stem cells; the γδ T cells are derived from any one or a combination of at least two of human peripheral blood, umbilical cord blood, embryonic stem cells, or human induced pluripotent stem cells.

10. The method according to claim 8, characterized in that, The ratio of NK cells or γδ T cells to human trophoblast cells in co-culture is (0.2-5):1; the γδ T cells include γδ1 T cells or γδ2 T cells.

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