Modified feeder cell and use thereof

Abstract

Provided are an HLA-E modified feeder cell and a method for culturing natural killer cells (NK cells). By means of culturing NK cells using the HLA-E-modified feeder cell, NK cells having a low expression of inhibitory receptor NKG2A and a high expression of activating receptors, such as NKG2C and NKp44, can be obtained, thereby significantly enhancing the killing activity of NK cells against target cells, particularly those with a high HLA-E expression.

Description

Modified feeder cells and their applications Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to modified NK feeder cells and their applications. Background Technology

[0002] In recent years, with the rapid development of biomedical technology, cell therapy has become a promising treatment method. NK cell therapy, as an emerging immunotherapy, has broad application prospects. In cancer treatment, NK cells can directly recognize and kill tumor cells, showing potential therapeutic effects against various cancers, including breast cancer, lung cancer, gastric cancer, and colorectal cancer. Furthermore, NK cell therapy can also be applied to the treatment of autoimmune diseases. By enhancing the function of NK cells, the balance of the immune system can be effectively regulated, thereby achieving the goal of treating autoimmune diseases. Additionally, NK cell therapy can be applied to the treatment of infectious diseases. NK cells can kill various pathogens, such as bacteria and viruses, and therefore can be used to treat certain diseases, such as AIDS and hepatitis. Finally, NK cell therapy can also be applied to the treatment of stem cell transplantation. By improving the body's immune function, diseases such as aplastic anemia that occur after stem cell transplantation can be prevented and treated. In conclusion, with in-depth research into NK cell biology and continuous optimization of CAR structure design, NK cell therapy is expected to become one of the important options for future cancer treatment and expand its applications in autoimmunity, infectious diseases, and stem cell transplantation.

[0003] NK cell therapy offers several advantages over traditional T cell therapy. First, NK cells possess a broader spectrum of anti-tumor activity; they can rapidly recognize and attack tumor cells without antigen presentation. Second, NK cell therapy is safer, avoiding serious side effects such as cytokine release syndrome or graft-versus-host disease, making it more reliable. Finally, because NK cells are readily available and do not require individualized treatment for each patient, treatment time is saved, making it more suitable for off-the-shelf therapies. Although NK cell therapy is relatively new, its unique advantages have garnered significant attention in cancer treatment. With in-depth research into NK cell biology and continuous optimization of CAR structural design, NK cell therapy is expected to become a crucial option for future cancer treatment.

[0004] Inhibitory receptors play a balancing role in NK cells. They bind to MHC-I molecules on the cell surface and transmit inhibitory signals, keeping NK cells "silent" even when normal cells express sufficient amounts of MHC-I molecules, thus preventing them from being "falsely killed." However, on the surface of tumor cells or senescent cells, the expression of MHC-I molecules is usually reduced, causing the inhibitory receptors of NK cells to lose their function, thereby activating NK cells.

[0005] HLA-E and NKG2A play important roles in the activation and regulation of natural killer (NK) cells. HLA-E plays a crucial role in both NK cell activation and inhibition. HLA-E molecules can bind to the NKG2A receptor, inhibiting NK cell activity by transmitting inhibitory signals. However, tumor cells and senescent cells often upregulate HLA-E expression, thereby escaping NK cell immune surveillance. Summary of the Invention

[0006] First, this invention provides a feeder cell line modified to express HLA-E. NK cells obtained by expanding feeder cells expressing HLA-E show lower expression of the NKG2A inhibitory receptor and higher expression of active receptors such as NKG2C and NKp44.

[0007] In some embodiments, the feeder cells are modified to simultaneously express HLA-E, B2M, and a polypeptide fragment that can be presented by HLA-E to form a stable complex structure. β2-microglobulin (B2M) is the β-chain (light chain) portion of human lymphocyte antigen (HLA) on the cell surface. Simultaneous expression of B2M facilitates the formation of a stable HLA-E-B2M MHC-I complex structure, enabling better binding to NKG2A on NK cells. The polypeptide fragments can be selected from the table below:

[0008] Table 1. List of peptide ligands presented by HLA-E

[0009] In one specific embodiment, the feeder cells are modified to express HLA-G SP-B2M-HLAE, the amino acid sequence of which is shown in SEQ ID NO: 21.

[0010] In some embodiments, the feeder cells are modified to simultaneously express the HLA-E gene and the CD58 gene, further and stably reducing the expression of NKG2A in NK cells over a long period. In one specific embodiment, the CD58 amino acid sequence is shown in SEQ ID NO: 23.

[0011] In some embodiments, the feeder cells are further modified to express or contain co-stimulatory molecules to stimulate NK cell expansion. In some embodiments, the co-stimulatory molecules are selected from one or more of 4-1BBL, OX40L, GITRL, CD27, CD40L, and ICOSL. For example, but not limited to, ligands of co-stimulatory receptors such as 4-1BBL, ICOS-L, CD27, and CD40L. The presence of the co-stimulatory ligands can further stimulate NK cell expansion.

[0012] In one specific embodiment, the co-stimulatory molecule is 4-1BBL, and the amino acid sequence of 4-1BBL is shown in SEQ ID NO: 19.

[0013] In some embodiments, the feeder cells are further modified to express cytokines, which can further stimulate NK cell proliferation. In some embodiments, the cytokines are selected from one or more of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or FLT3L. In some embodiments, the cytokines are secreted or membrane-bound; preferably, the cytokines are membrane-bound; more preferably, the membrane-bound cytokines include a hinge region and a transmembrane region. In some embodiments, the hinge region is selected from CD8a, CD28, or IgG4. In some embodiments, the transmembrane region is selected from CD8a, CD28, or CD4.

[0014] In some embodiments, the cytokine is selected from membrane-bound IL21 (mbIL21) or membrane-bound IL15 (mbIL15).

[0015] In one specific embodiment, the mbIL2 comprises an IgG4 hinge region and a CD4 transmembrane region, and its amino acid sequence is shown in SEQ ID NO: 20.

[0016] In one specific embodiment, the feeder cells are modified to express HLA-E, 4-1BBL, and mIL21; in another specific embodiment, the feeder cells are modified to express HLA-E, CD58, 4-1BBL, and mIL21.

[0017] In some embodiments, the feeder cells are tumor cells or stem cells. In some embodiments, the tumor cells can be hematologic malignancies or solid tumor cells. In some embodiments, the solid tumor cells are prostate cancer cells PC3; in some embodiments, the hematologic malignancies can be K562, 221, NALM6, or Raji.

[0018] In some implementations, the stem cells may be MSCs or other stem cells derived from iPSCs.

[0019] In some implementations, the source of the MSCs can be arbitrary; preferably, the MSCs are derived from the umbilical cord, bone marrow, iPSCs, or fat.

[0020] In another aspect, the present invention provides a method for in vitro culture of NK cells, using the aforementioned feeder cells and NK cells for co-culture; in some embodiments, the NK cells are genetically engineered; in some embodiments, the NK cells are CAR-NK.

[0021] In some embodiments, the NK cells are isolated from a patient or healthy volunteer; in some embodiments, the NK cells are derived from stem cells through induced differentiation; in some embodiments, the stem cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, iPS cells, etc.; in some embodiments, the NK cells are genetically engineered, the modification being the introduction, knockout, or downsampling of a chimeric antigen receptor gene or other genes. In one specific embodiment, the NK cells are CAR-NK cells. Furthermore, the present invention provides the application of the cells as a kit for culturing NK cells.

[0022] In another aspect, the present invention provides the application of NK cells prepared by the aforementioned feeder cells or kit culture in the preparation of drugs for treating human diseases.

[0023] The applicant unexpectedly discovered that by culturing NK cells using modified 4-1BBL and HLA-E feeder cells, NK cells with lower expression of the inhibitory receptors NKG2A and TIGIT, and higher expression of the activating receptors NKG2C, NKp44, CD16, and FceRI, could be obtained. The obtained NK cells exhibited better target cell killing activity, especially in killing target cells with high HLA-E expression. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 is a schematic diagram of the cells in each feeder layer;

[0026] Figure 2 shows the flow cytometry results of the expression levels of HLA-E, CD58, etc. on the K562 cell membrane;

[0027] Figure 3 shows the flow cytometry results of the NKG2A expression ratio in NK cells obtained from cell expansion in each feeder layer.

[0028] Figure 4 shows the flow cytometry results of the NKG2C expression ratio in NK cells obtained from cell expansion in each feeder layer.

[0029] Figure 5. Effects of different modified feeder layers on NK cell expansion and phenotype.

[0030] Figure 6. Effects of feeder cells modified with 4-1BBL+IL21 and 4-1BBL+IL21+CD58+HLA-E on various NK cell phenotypes.

[0031] Figure 7. Killing function of NK cells cultured in different feeder layers against K562 and K562-HLA-E.

[0032] Figure 8. NK cell phenotypes after multiple rounds of stimulation of feeder cells.

[0033] Figure 9. Effects of feeder layer cells on the expansion of NK cells initiated from different components.

[0034] Figure 10 Comparison of the cytotoxic ability of NK or CAR-NK cells cultured in different feeder cell layers against target cells.

[0035] Figure 11. NK cell amplification and phenotype in feeder cell culture based on NALM6.

[0036] Figure 12. In vitro function of NK cells cultured from feeder cells constructed based on NALM6.

[0037] Figure 13 ADCC function of NK cells cultured from feeder cells constructed based on NALM6.

[0038] Figure 14 Comparison of proliferation curves of feeder cells constructed based on MSCs and NK cells expanded by K562-41BBL.

[0039] Figure 15. Detection of surface markers such as NKG2A expressed in NK cells expanded from feeder cells constructed based on MSCs. Detailed Implementation

[0040] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0041] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0042] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0043] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0044] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0045] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity greater than or equal to 2.

[0046] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0047] The term "modified" refers to cells that have undergone genetic engineering or other types of modification, including but not limited to: the introduction of one or more genes, gene knockout, gene knockdown, etc.

[0048] The HLA-E, CD58, and B2M described in this invention can be natural full-length sequences, truncated fragments that retain their original functions, or full-length mutants or truncated mutants that retain their original functions.

[0049] The co-stimulatory molecules for stimulating NK cell expansion in this invention include, but are not limited to: 4-1BBL, OX40L, GITRL, CD27, CD40L, ICOSL, etc., which can be natural full-length sequences, truncated fragments that basically retain the original function, or full-length mutants or truncated mutants that basically retain the original function.

[0050] The cytokines described in this invention can be secreted or membrane-bound. The cytokines can be natural full-length sequences, truncated fragments that retain their original functions, or full-length mutants or truncated mutants that retain their original functions.

[0051] In some embodiments, the membrane-bound cytokine comprises a hinge region and a transmembrane region; the hinge region and transmembrane region can be any suitable hinge region and transmembrane region; preferably, the hinge region is selected from CD8a, CD28, or IgG4; preferably, the transmembrane region is selected from CD8a, CD28, or CD4. The hinge region and transmembrane region can be a natural full-length sequence, a truncated fragment that substantially retains its original function, or a full-length mutant or truncated mutant that substantially retains its original function.

[0052] The feeder cells of this invention can be tumor cells or stem cells; the tumor cells can be solid tumor cells or hematologic malignancy cells; in some embodiments, the feeder cells are prostate cancer cells PC3, a solid tumor; in some embodiments, the feeder cells are hematologic malignancy cells K562, 221, or Raji. In some embodiments, the feeder cells are MSCs, and the source of the MSCs can be arbitrary; preferably, the MSCs are derived from umbilical cord, bone marrow, iPSCs, or adipose tissue.

[0053] The NK cells described in this invention can be isolated from patients or healthy volunteers, or derived from stem cells through induced differentiation. In some embodiments, the stem cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, iPS cells, etc. In some embodiments, the NK cells are genetically engineered, and the modification may be the introduction, knockout, or knockdown of a chimeric antigen receptor gene or other genes. In one specific embodiment, the NK is a CAR-NK. As used herein, a "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a polypeptide different from the derived extracellular domain, and at least one intracellular domain. A "chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor" or a "chimeric immune receptor (CIR)". An "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals in the cell to induce activation or inhibition of biological processes.

[0054] In some embodiments, the chimeric antigen receptor includes a hinge region, a transmembrane region, and an intracellular signal transduction region.

[0055] As used herein, the “region” or “domain” contained in the chimeric antigen receptor refers to a region of a polypeptide that can fold into a specific structure independently of other regions. These “regions” or “domains” can be murine or other animal-derived sequences, preferably human sequences. Furthermore, unless otherwise specified or emphasized, “region” or “domain” should be understood as a well-known sequence, which can be a full-length or partially active segment.

[0056] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0057] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0058] Example 1: Construction of plasmid vector

[0059] The CD137L (4-1BBL) and Blastidin (BSD) sequence fragments were artificially synthesized and sequentially linked. The fragments were cloned into the pIMHEF vector to obtain a plasmid expressing 4-1BBL.

[0060] IL21 and puromycin were synthesized in sequence and cloned into the pIMHEF vector to obtain a plasmid expressing IL21.

[0061] The HLA-G signal peptide (9AA), B2M, HLAE and Blastidin (BSD) were synthesized in sequence and cloned into the pIMHEF vector to obtain a plasmid expressing HLAE.

[0062] CD58, HLA-G signal peptide (9AA), B2M, HLAE and Blastidin (BSD) were synthesized in sequence and cloned into the pIMHEF vector to obtain plasmids expressing CD58 and HLAE.

[0063] CD137L(4-1BBL) amino acid sequence (SEQ ID NO: 19):

[0064] mbIL21 amino acid sequence (IL21+IgG4+CD4TM, SEQ ID NO: 20):

[0065] HLA-G SP-B2M-HLAE amino acid sequence (SEQ ID NO: 21):

[0066] CD58+HLA-G SP-B2M-HLAE amino acid sequence (SEQ ID NO: 22):

[0067] CD58 amino acid sequence (SEQ ID NO: 23):

[0068] Example 2: Lentiviral Packaging

[0069] 1. Take 293F cells in good growth condition and transfect the target plasmid and helper plasmid with PEI;

[0070] 2. 40-48 h after transfection, collect the supernatant, centrifuge to concentrate, resuspend the virus pellet in AIM-V / X-vivo medium, aliquot, and store at -80℃;

[0071] 3. Use the collected concentrated virus to infect Jurkat cells and detect the viral titer.

[0072] Example 3: Construction of K562 feeder cells

[0073] K562 cells were infected with the lentiviral vector expressing 4-1BBL packaged in Example 2, and BSD selection was performed to obtain K562-4-1BBL cells with high expression of 4-1BBL.

[0074] The K562-4-1BBL cells constructed above were infected with a virus expressing mbIL21. After selection with puromycin, K562-4-1BBL+mbIL21 cells were obtained. Mitomycin or irradiation was added to inactivate the cells and feeder cells for NK cell activation were obtained.

[0075] The constructed K562-4-1BBL+mbIL21 cells were infected with viruses expressing HLAE or CD58+HLAE. After BSD selection and flow cytometry sorting, K562-4-1BBL+mbIL21+HLAE and K562-4-1BBL+mbIL21+CD58+HLAE cells were obtained. These cells were then inactivated by mitomycin C or irradiation to obtain feeder cells for NK cell activation. A schematic diagram of the constructed K562 feeder cells is shown in Figure 1, and the flow cytometry analysis is shown in Figure 2.

[0076] Example 4: Phenotypic detection of NK cell expansion from K562 feeder cells

[0077] ① PBMCs were sorted to remove CD3, and the cell density was adjusted to 1.5E6 / ml using NK medium. Inactivated K562-4-1BBL-mbIL21, K562-4-1BBL+mbIL21+HLAE, and K562-4-1BBL+mbIL21+CD58+HLAE were added in a certain proportion, mixed in a culture dish, and then cultured in a 37℃, 5% CO2 incubator.

[0078] ② Day 3: Replace half the medium or adjust the medium replacement ratio according to the color of the medium. Gently aspirate the supernatant and slowly add fresh medium. Do not blow away the cells at the bottom.

[0079] ③Count NK cells on days 5-7, add fluid to bring the NK cell concentration to about 1.0×10E6 Cell / ml, and add plasma at 5%;

[0080] ④ Observe or count cells daily from day 8 to 10. Add plasma at 1% to make the cell concentration slightly lower than 1.0×10E6 Cell / ml for amplification.

[0081] ⑤ The proportions of NKG2A and NKG2C on the surface of NK cells were detected, as shown in Figures 3 and 4. It can be seen that the proportion of NKG2A expressed by NK cells expanded from K562-4-1BBL+mbIL21+CD58+HLAE as feeder cells was significantly lower than that of NK cells expanded from K562-4-1BBL-mbIL21 or K562-4-1BBL+mbIL21+HLAE feeder cells (Figures 3A and 3B), and the proportion of NKG2C was higher than that of NK cells expanded from K562-4-1BBL-mbIL21 or K562-4-1BBL+mbIL21+HLAE feeder cells (Figure 4).

[0082] Example 5: Effects of CD58 and CD58+HLAE on NK cell expansion and phenotype

[0083] Given the different effects of K562-4-1BBL+mbIL21+HLAE and K562-4-1BBL+mbIL21+CD58+HLAE on NK cells in Example 4, we further investigated the role of CD58 in NK cell amplification and phenotype. Based on K562, we constructed K562-4-1BBL+mbIL21+CD58, K562-CD58, and K562-CD58+HLAE, and compared the differences in NK cell amplification and phenotype among these feeder cells. The experimental methods were the same as in Example 4. As shown in Figure 5B, K562-CD58 or K562-CD58+HLAE alone cannot effectively activate and expand NK cells. When 4-1BBL+mbIL21 is constructed simultaneously, K562-4-1BBL+mbIL21+CD58 and K562-4-1BBL+mbIL21+CD58+HLAE can effectively activate NK cells and promote their expansion. However, when CD58 is used alone in combination with 4-1BBL+mbIL21, the expression of NKG2A, NKG2C, NKp46, and CD62L on NK cells is not significantly different from that of traditional feeder cells. Only when CD58 and HLAE are used in combination with 4-1BBL+mbIL21 do NK cells show significant advantages in terms of NKG2A phenotype and other aspects, and NK cells can be effectively expanded (as shown in Figure 5).

[0084] Example 6: Further study on the effects of CD58 and CD58+HLAE on NK cell phenotype

[0085] Given the differences in the effects of feeder cells with different molecular constructs on NK cells observed in Examples 4 and 5, we further investigated their effects on other phenotypes of NK cells. The experimental methods were the same as in Example 4, and the results are shown in Figure 6. Except for the differences in NKG2A and NKG2C observed in K562-4-1BBL+mbIL21 and K562-4-1BBL+mbIL21+CD58+HLAE as in the previous examples, the NK cells expanded by K562-4-1BBL+mbIL21+CD58+HLAE showed higher expression of NKp44 and CD16 activating receptors compared to the traditional molecular combination 4-1BBL+mbIL21, and lower expression of FceRI and TIGIT inhibitory receptors and molecules. There was no significant difference in NKp46 expression levels.

[0086] Example 7: Killing function of NK cells with different phenotypes against K562 and K562-HLAE

[0087] Because tumor cells and senescent cells often upregulate HLA-E expression, thereby escaping NK cell immune surveillance, we constructed the K562-HLAE cell line, which highly expresses HLAE, in vitro and prepared NK cells from two different donor sources. We then examined the differences in NK cell expression levels at different NKG2A levels when killing K562-HLAE cells. The experimental results are shown in Figure 7. When killing K562 cells, NK cells cultured using K562-4-1BBL+mbIL21+CD58+HLAE as feeder cells showed a certain advantage over conventional K562-4-1BBL+mbIL21. When killing the K562-HLAE cell line, which highly expresses HLAE, the NK cells expanded from K562-4-1BBL+mbIL21+CD58+HLAE showed a significant increase compared to conventional feeder cells. NK cells prepared from PBMC cells from different donor sources showed consistent performance.

[0088] Example 8: NK cell phenotype after multiple rounds of stimulation of feeder cells

[0089] Traditional NK cell culture often employs two or more rounds of feeder cell stimulation to increase NK cell proliferation. Therefore, we investigated the differences in NK cell proliferation capacity and phenotype between two-stage stimulation and single-stage activation. The experimental results are shown in Figure 8. In the K562-4-1BBL+mbIL21+CD58+HLAE group, single-stage activation resulted in lower NKG2A and higher NKG2C expression compared to traditional feeder cells. Consistent with previous experiments, when K562-4-1BBL+mbIL21+CD58+HLAE feeder cells were added again on day 7, the NK cells activated twice by K562-4-1BBL+mbIL21+CD58+HLAE showed stronger proliferation capacity compared to single-stage activation. The expression ratio of the inhibitory receptor NKG2A was further reduced, while the expression of the activating receptor NKG2C was increased. The proportion of cells reaching the target level further increased on day 21. There was no significant difference in CD16 levels among the groups, indicating that the feeder layer cells of K562-4-1BBL+mbIL21+CD58+HLAE could effectively stimulate NK cell proliferation and maintain certain NKG2A and NKG2C dominant phenotypes, regardless of whether they were activated multiple times or once. Two activations further improved the NK cell proliferation capacity while maintaining or further enhancing the NKG2A and NKG2C phenotypes, demonstrating the applicability of the feeder layer K562-4-1BBL+mbIL21+CD58+HLAE in traditional NK cell culture processes.

[0090] Example 9: Effect of feeder cells on NK cells after expansion of different component-based starting cells

[0091] In NK cell culture, CD3-removed cells are often used as starting cells or directly sorted and purified NK cells are used as seed cells. To optimize the NK cell culture process and expand the application range of feeder cells, and to compare the differences in NK cell expansion and phenotype between cells from different starting components, we used CD3-removed NK cells from PBMCs and purified NK cells as seed cells to activate and culture NK cells. The experimental results are shown in Figure 9. Both CD3-removed and purified NK cells from PBMCs as seed cells effectively expanded NK cells and achieved high cell viability. Compared to CD3-removed seed cells, purified NK cells had a higher expansion capacity, and the differences between different feeder cell types were small. Regarding phenotype, the NK cells expanded from seed cells of different sources were consistent with the aforementioned results. NK cells expanded from K562-4-1BBL+mbIL21+CD58+HLAE maintained a lower NKG2A ratio and a higher NKG2C ratio, while there was no significant difference in NKG2D among the groups. The K562-4-1BBL+mbIL21+CD58+HLAE feeder cells can be used in different starting seed cells and can effectively expand NK cells, making them convenient and widely applicable.

[0092] Example 10: Specific killing function of CAR-NK cells with different phenotypes against target cells

[0093] ① CD19 CAR lentivirus was packaged in 293T cells, concentrated by centrifugation at 10000g for 4 hours, and the titer was detected.

[0094] ②Retronectin was coated on 24-well plates overnight. On Day 7, CD19 CAR lentivirus was used to infect NK cells cultured in different feeder layers with an MOI of 4. After mixing, the cells were incubated for more than 20 minutes to allow them to adhere to the plate. The plates were then centrifuged at 1000g for 10 minutes and cultured in a 37°C, 5% CO2 incubator.

[0095] ③ Change the medium after 24 hours, expand the cells according to their state and density, and detect the expression of CD19 CAR after 48 hours.

[0096] ④ Count NK cells on days 5-7, add fluid to bring the NK cell concentration to about 1.0×10E6 Cell / ml, and add plasma at 5%;

[0097] ⑤ Observe or count cells daily from day 8 to 10. Add plasma at 1% to make the cell concentration slightly lower than 1.0×10E6 Cell / ml for amplification.

[0098] ⑥ Detection of NK and CD19 CAR-NK cytotoxic target cell Raji-luc function: First, Raji-luc target cells were collected, washed, and resuspended in culture medium. 5E4 cells / 100 μL / well were added to round-bottomed plates, with two replicates per group. A control target cell group was also prepared without effector cells. Effector cells were collected and added to the corresponding wells at E:T ratios of 0.1:1, 0.3:1, and 1:1. After mixing the cells, they were centrifuged at 200g for 2 minutes and incubated overnight at 37℃ for approximately 18-24 hours.

[0099] ⑦ After co-incubation, centrifuge to remove 160 μL of supernatant, leaving 40 μL of solution in each well (containing cells). Add 40 μL of Steady-Glo mixture to each well (mixed...). Buffer and Substrate). Gently shake. Incubate in the dark at room temperature for 10 minutes. Transfer the solution to an opaque white plate, avoiding air bubbles.

[0100] ⑧ The chemiluminescence was detected using an ELISA reader, and the killing efficiency percentage was calculated as (target cells - experimental wells) / target cells * 100%. The results are shown in Figure 10. CD19 CAR-NK cells prepared from NK cells expanded using K562-4-1BBL+mbIL21+CD58+HLAE as feeder cells showed a significant advantage in killing target cells compared to K562-4-1BBL+mbIL21 as feeder cells, exhibiting stronger killing ability. This indicates that K562-4-1BBL+mbIL21+CD58+HLAE as feeder cells also has the potential for application in CAR-NK.

[0101] Example 11: NK cell amplification and phenotype of feeder cell culture based on NALM6

[0102] Building upon the previous comparison of feeder cells constructed based on K562, to further determine its broad applicability, we constructed NALM6-41BBL and NALM6-41BBL+HLAE on NALM6 cells and compared their effects on NK cell expansion and phenotype. The specific culture method is described in Example 4. The results are shown in Figure 11A. NK cells cultured with NALM6-41BBL+HLAE showed significantly better total cell expansion and NK cell expansion rate than those cultured with NALM6-41BBL. Regarding NK cell phenotype, NK cells cultured with NALM6-41BBL+HLAE showed a higher NK cell proportion at day 13 compared to the NALM6-41BBL group, and significantly lower expression of the inhibitory receptor NKG2A compared to the NALM6-41BBL group. Expression of activating receptors and memory phenotype receptors NKG2C, CD16, and CD57 was higher in the NALM6-41BBL group, while NKG2D and CD226 (DNAM-1) showed no significant differences (Figure 11B).

[0103] Example 12: In vitro functionalization of NK cells cultured from feeder cells constructed based on NALM6

[0104] Since tumor cells and senescent cells often upregulate HLA-E expression, thereby escaping NK cell immune surveillance, we examined the cytotoxic function of NK cells cultured from NALM6 feeder cells against K562 cells and the K562-HLAE cell line, which highly expresses HLAE. The results are shown in Figure 12. When killing K562 cells, NK cells cultured from NALM6-41BBL+HLAE showed a certain advantage over the NALM6-41BBL group. When killing the K562-HLAE cell line, the NK cells expanded from NALM6-41BBL+HLAE showed a more significant cytotoxic advantage, indicating that the NALM6-41BBL+HLAE feeder cells also have potential applications in anti-aging and other fields.

[0105] Example 13: ADCC function of NK cells cultured from feeder cells constructed based on NALM6

[0106] To investigate the ADCC function of NK cells cultured from NALM6 feeder cells, we used NK cells cultured in Example 11, combined with a certain concentration of the CD20 monoclonal antibody Obinutuzumab, and co-incubated with CD20-positive target cells Raji at a certain effector-to-target ratio for approximately 4 hours. The proportions of Annexin V and PI on the surface of Raji cells were then measured, and the ADCC killing function of NK cells against target cells was assessed. The results are shown in Figure 13. NK cells cultured from NALM6-41BBL+FLT3L as feeder cells exhibited stronger ADCC function in killing Raji cells than the NALM6-41BBL group, indicating that NK cells cultured from NALM6-41BBL+FLT3L as feeder cells also have higher application potential in target-specific ADCC function.

[0107] Example 14 Effects of different feeder cell structures constructed based on MSC-41BBL on NK cell expansion and phenotype

[0108] ① After sorting PBMCs to remove CD3, inactivated MSC-41BBL, MSC-41BBL+FLT3L, MSC-41BBL+CD112, MSC-41BBL+mTNF-α and MSC-41BBL+HLAE were added respectively. After mixing, they were placed in an incubator at 37℃ and 5% CO2.

[0109] ② Add culture medium (0.8-1.0×1E6 Cell / ml) according to the color of the cell suspension or the number of cells.

[0110] ①Count NK cells on day 5-day 7, add fluid to make the NK cell concentration 1.0×10E6 Cell / ml, and add plasma at 5%;

[0111] ② Observe or count daily from day 8 to 10. Add plasma at 1% to make the cell concentration slightly lower than 1.0×10E6 Cell / ml. Expand to day 14 and count the NK amplification curve as shown in Figure 14. MSCs in each group, as feeder cells, all have a certain amplification effect on NK. The lowest amplification on day 14 was 424-fold (MSC-41BBL+FLT3L), with the highest amplification of 3477-fold in the MSC-41BBL+HLAE group.

[0112] ③ On day 8 and day 12, the surface markers of NK cells, such as NKG2A and NKG2C, were detected, as shown in Figure 15. It can be seen that on day 8, the proportion of NK cells expanded from MSCs as feeder cells in each group expressing NKG2A was lower than that of MSC-41BBL, with the lowest being 34.66% (MSC-41BBL+HLAE). The highest NKG2A expression proportion was in the MSC-41BBL group, at 68.6% (Figure 15A). The NKG2C expression proportion in the MSC-41BBL+HLAE group was significantly higher than that in other groups at different time points, with expression proportions of 34.76% (day 8) and 75.47% (day 12), respectively (Figure 15B). There was no significant difference in the expression proportions of NKG2D and CD56 among the groups (Figure 15C, 15D).

Claims

1. A feeder cell, wherein the feeder cell is modified to express HLA-E.

2. The feeder cells according to claim 1, further modified to express B2M and HLA-E presented polypeptide fragments.

3. The feeder cell according to claim 2, wherein the HLA-E presented polypeptide fragment is selected from: HLA-I class molecular signal peptide, HSP60 signal peptide, HCMV UL40 signal peptide, HIV Gag signal peptide, TCR Vβ1, TCR Vβ2, influenza A virus M protein, EBV BZLF-1 protein, and HCV core protein.

4. The feeder cell according to claim 3, wherein the HLA-I class molecular signal peptide is an HLA-G signal peptide, the amino acid sequence of which is shown in SEQ ID NO:

2.

5. The feeder cells according to claim 4, wherein the feeder cells are modified to express HLA-G SP-B2M-HLA-E; preferably, the amino acid sequence of the HLA-G SP-B2M-HLA-E is as shown in SEQ ID NO:

21.

6. The feeder cells according to any one of claims 1-5, further modified to express co-stimulatory molecules.

7. The feeder cell according to claim 7, wherein the co-stimulatory molecule is selected from at least one of 4-1BBL, CD8, OX40L, GITRL, CD27, CD40L, and ICOSL.

8. The feeder cells according to any one of claims 1-7, wherein the feeder cells are modified to express cytokines.

9. The feeder cells according to claim 8, wherein the cytokine is selected from one or more of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 or FLT3L.

10. The feeder cells according to claim 8 or 9, wherein the cytokine is secreted or membrane-bound; preferably, the cytokine is membrane-bound; more preferably, the cytokine is membrane-bound IL21 (mbIL21) or membrane-bound IL15 (mbIL15).

11. The feeder cells according to any one of claims 1-10, wherein the feeder cells are modified to express HLA-E and 4-1BBL; or modified to express HLA-E, 4-1BBL and mIL21; or modified to express HLA-E, 4-1BBL, mIL21 and CD58.

12. The feeder layer cells according to any one of claims 1 to 11, characterized in that The feeder cells are selected from tumor cells or stem cells.

13. The feeder layer cells of claim 12, wherein The tumor cells are hematologic tumor cells or solid tumor cells.

14. The feeder layer cells of claim 13, wherein The hematologic tumor cells were selected from K562, 221, NALM6, or Raji.

15. The feeder layer cells according to claim 14, characterized in that... The stem cells mentioned are MSCs.

16. A method for in vitro culture of NK cells, characterized in that... The feeder cells and NK cells described in any one of claims 1-15 are co-cultured; preferably, the NK cells are genetically engineered; more preferably, the NK cells are CAR-NK.

17. A reagent kit, characterized in that... It contains the feeder layer cells as described in any one of claims 1-15 and an instruction manual.

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