Dual-targeting immune cells and methods of making, compositions, and uses thereof

Abstract

The application discloses a preparation method of double-targeted immune cells, which comprises the following steps: (1) transfecting immune cells with a nucleic acid vector by electroporation in an electrotransformation buffer, wherein the nucleic acid vector encodes at least one chimeric antigen receptor; (2) culturing the immune cells with non-natural sugars after recovering in an electrotransformation recovery solution for 2-5 hours, so that a binding site is generated on the surface of the immune cells; and (3) anchoring a nucleic acid aptamer on the surface of the immune cells expressing the chimeric antigen receptor by using the binding site, wherein the nucleic acid aptamer is combined with at least one target point on the surface of a target cell. The application can not only reduce the toxic effect of non-natural sugars on immune cells, but also simultaneously improve the expression level of CAR protein on the surface of the immune cells, the abundance of the binding site and the tumor killing function of CAR-T cells.

Description

Technical Field

[0001] This disclosure relates to a dual-targeting immune cell, its preparation method, composition, and application, belonging to the field of tumor cell immunotherapy. Background Technology

[0002] Immune checkpoint therapy strategies, represented by the PD-1 / PD-L1 axis, have achieved certain therapeutic results in clinical cancer treatment. However, significant heterogeneity exists among patients, and long-term efficacy is poor, suggesting that single immune checkpoint intervention strategies cannot fully overcome the complex immune escape networks of solid tumors. Recent research has found that long-term treatment targeting the PD-1 / PD-L1 axis may be a "depletionist" strategy. Although short-term PD-1 / PD-L1 blockade therapy has yielded clinical benefits, long-term treatment generally results in decreased efficacy and "immune exhaustion." This may be related to the abnormal precise regulation of PD-1 on T cell receptor signaling caused by long-term PD-1 / PD-L1 blockade therapy, which indirectly leads to the depletion of stem-like precursor T cells (TCF-1+PD-1+SLAMF6highCD8+ T cells). Therefore, how to rationally avoid the inhibitory effect of the PD-1 / PD-L1 axis on T cells, enhance the targeting affinity of blocking drugs for the natural PD-1 / PD-L1 axis, and avoid "immune depletion" are key issues for breaking through the bottleneck of immunotherapy for solid tumors in the future.

[0003] Besides immune checkpoints, cell therapy for solid tumors also focuses on addressing another key issue: improving the breadth of effector cell recognition and the persistence of tumor cell killing. The NKG2D / NKG2D-L axis, as a crucial component of tumor immune surveillance, exhibits enhanced stress response recognition characteristics in target design compared to traditional antigen-targeting approaches due to the upregulation of NKG2D-L expression under cellular stress and DNA damage. However, in solid tumors, NKG2D-L expression exhibits heterogeneity, and tumors can release sMICs via ligand cleavage to form an inhibitory protective layer around the tumor, thereby preventing immune cell recognition and inhibiting their killing activity. This is one of the main challenges facing engineered cells targeting the NKG2D / NKG2D-L axis in the treatment of solid tumors.

[0004] Although CAR-T cell therapy has achieved encouraging results in the treatment of hematologic malignancies, its application in solid tumors is still limited by factors such as tumor antigen heterogeneity, insufficient effector cell infiltration, and the immunosuppressive effects of the tumor microenvironment. Summary of the Invention

[0005] The technical problem to be solved by this invention is the low yield and decreased CAR-T immune function caused by the toxicity amplification of CAR-T cells prepared by electroporation and co-culture with non-natural sugars.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing dual-targeting immune cells includes the following steps:

[0008] (1) In an electroporation buffer, a nucleic acid vector is transfected into immune cells by electroporation, wherein the nucleic acid vector encodes at least one chimeric antigen receptor;

[0009] (2) After immune cells are recovered in electroporation recovery solution for 2-5 hours, they are co-cultured with non-natural sugars to generate binding sites on the surface of immune cells;

[0010] (3) Using the above-mentioned binding sites, the nucleic acid aptamer is anchored to the surface of an immune cell expressing the chimeric antigen receptor, wherein the nucleic acid aptamer binds to at least one target site on the surface of the target cell.

[0011] The nucleic acid vector encodes at least one chimeric antigen receptor, including one or more of a promoter, a polyadenylation signal, an enhancer, and a reporter gene for detecting gene expression.

[0012] The immune cells include one or more of T cells, B cells, NK cells, monocytes, macrophages, and dendritic cells;

[0013] The non-natural sugars refer to monosaccharide analogs with bioorthogonal reactive groups, which can be taken up by cells and integrated into the cell membrane surface via endogenous sugar metabolism pathways, generating binding sites on the surface of immune cells.

[0014] The binding site is an azide group.

[0015] The nucleic acid vector includes mRNA or circRNA.

[0016] The electroporation buffer comprises the following components: 8.2-8.7 mM disodium hydrogen phosphate, 8.3-9.1 mM sodium dihydrogen phosphate, 3.2-3.8 mM potassium chloride, 10.0-11.0 mM magnesium chloride, 7.0-7.3 mM sodium succinate, 14.0-14.5 mM mannitol, 19.5-20.3 mM d-glucose, 0.4-0.5 mM sodium pyruvate, 0.2-0.3 mM inositol, and 4.0-4.3 mM GlutaMAX.

[0017] The electroporation recovery solution comprises KBM-581 containing the following components: 9-15 v / v% human AB serum and 100-500 IU / mL IL-2.

[0018] Furthermore, a dual-targeting immune cell prepared based on the aforementioned preparation method is provided.

[0019] Furthermore, a composition is provided comprising the dual-targeting immune cells.

[0020] Furthermore, the composition is provided for use in the preparation of tumor cell immunotherapy drugs.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention involves culturing immune cells in an electroporation recovery solution for 2-5 hours after electroporation, followed by co-culturing with non-natural sugars. This not only reduces the toxic effects of non-natural sugars on immune cells but also simultaneously increases the expression level of CAR proteins on the surface of immune cells, the abundance of binding sites, and the tumor-killing function of CAR-T cells. Attached Figure Description

[0023] Figure 1 The difference in anti-apoptotic ability of cells under empty rotation conditions in Example 4 and opti-MEM;

[0024] Figure 2 AO / PI staining was used to verify cell viability;

[0025] Figure 3 To verify the effects of different electroporation buffers on T cell apoptosis by electroporation with different nucleic acid vectors using flow cytometry;

[0026] Figure 4 To verify the differences in circRNA expression among different groups using flow cytometry;

[0027] Figure 5 To verify the differences in the proportion of azide-positive cells among different groups using flow cytometry;

[0028] Figure 6 To verify the differences in MFI groups of the azide group using flow cytometry;

[0029] Figure 7 To verify the differences in cell killing ability among the groups using RTCA. Detailed Implementation

[0030] Unless otherwise stated, the terms used in this disclosure have the meanings commonly understood by those skilled in the art in fields such as organic chemistry, medicine, pharmacy, molecular biology, and microbiology. Below are definitions of some terms used in this disclosure, and these definitions supersede the common understanding found in this disclosure.

[0031] In this invention, a chimeric antigen receptor (also referred to herein as CAR) is a protein containing a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain contains an antigen recognition site that specifically binds to the target, such as an scFv fragment containing a monoclonal antibody against the target; or, when the target is a receptor, the extracellular domain contains a ligand that binds to that receptor.

[0032] The targets are selected from antigens or receptors that are significantly expressed in tumor cells compared to adjacent normal tissue or other normal human tissues. When the target is an antigen, the target includes, but is not limited to, EpCAM, EGFR, MSLN, CD155, MUC1, CLDN18.2, HER2, B7-H3, etc.; or antigens present in the extracellular matrix, such as CEA present in tumor necrosis regions; or mutant proteins (neoantigens) identified through tumor genomic analysis and / or differential expression studies. When the target is a receptor, the receptor ligand can be used as an antigen recognition site to replace scFv, such as using the extracellular domains of NKG2D-L, PD-L1, CD27, NKp30, etc.

[0033] In a preferred embodiment, the target is selected from NKG2D-L specifically expressed on the surface of solid tumors, and the extracellular domain used is selected from human-derived NKG2D 82-216aa. In a more preferred embodiment, the amino acid sequence of the extracellular domain is LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO:1), or has this amino acid sequence, or is an amino acid sequence with at least 90% sequence identity and retaining the same function as this amino acid sequence.

[0034] The extracellular domain typically contains a leader sequence (signal peptide) that promotes the translocation of the CAR protein to the cell membrane surface. In a preferred embodiment, CD8α is used as the leader sequence of the CAR protein. In a more preferred embodiment, the amino acid sequence of the leader sequence is MALPVTALLLPLALLLHAARP (SEQ ID NO:2), or has this amino acid sequence, or has at least 90% sequence identity with this amino acid sequence and retains the same function.

[0035] The extracellular domain and the transmembrane domain generally contain a specially designed hinge sequence. This hinge sequence not only connects the extracellular domain and the transmembrane domain, but also, through the design of amino acids at specific sites in the sequence, enables the CAR protein to form homodimers, control the distance between the CAR protein and the cell membrane, or promote the binding of the CAR protein to the target antigen. The hinge sequence can be selected from the CD8α hinge sequence, the CD28 hinge sequence, or the human IgG antibody Fc fragment (hinge sequence). In a preferred embodiment, the hinge sequence is selected from the CD8α hinge sequence. In a more preferred embodiment, the amino acid sequence of the hinge sequence is TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:3), or has this amino acid sequence, or is an amino acid sequence with at least 90% sequence identity and retaining the same function as this amino acid sequence.

[0036] The transmembrane domain is located between the extracellular domain and the intracellular co-stimulatory signaling domain. The transmembrane domain can be, but is not limited to, transmembrane domains of CD28, CD8, CD4, etc., or an artificially constructed polypeptide structure. In a preferred embodiment, the transmembrane domain of CD28 is used as the transmembrane domain of the CAR protein. In a more preferred embodiment, the amino acid sequence of the transmembrane domain is FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:4), or has this amino acid sequence, or is an amino acid sequence with at least 90% sequence identity and retaining the same function as this amino acid sequence.

[0037] Intracellular signaling domains are used to transmit essential signals for the effector functions of immune cells. For second-generation CAR-T cells, intracellular signaling domains generally include a first domain for transmitting signals mediated by the TCR complex, such as CD3ζ or FcεRIγ; and a second domain for transmitting co-stimulatory signals, such as 4-1BB (CD137), OX-40, CD28, etc. In this invention, the first and second domains are allowed to be freely combined from any existing examples, and their connection order, connection method (direct connection or adapter connection), and number of connections are not limited. In a preferred embodiment, the amino acid sequence of the first domain is RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:5), or has this amino acid sequence, or has an amino acid sequence with at least 90% sequence identity and retains the same function as this amino acid sequence; in another preferred embodiment, the amino acid sequence of the second domain is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:6), or has this amino acid sequence, or has an amino acid sequence with at least 90% sequence identity and retains the same function as this amino acid sequence; in a more preferred embodiment, the second domain in the intracellular signaling domain is connected to the hinge sequence and the first domain, respectively.

[0038] In this invention, immune cells expressing the chimeric antigen receptor are prepared by transfecting the gene encoding the chimeric antigen receptor into immune cells using a nucleic acid vector. The nucleic acid vector refers to a nucleic acid molecule capable of stably transfecting and expressing the chimeric antigen receptor into immune cells via electroporation. In this invention, the nucleic acid vector allows RNA of any origin, such as mRNA or circRNA. The nucleic acid vector can be prepared using conventional methods or commercially available kits. The nucleic acid vector generally includes the target gene sequence encoding the chimeric antigen receptor and related components, such as a promoter, a polyadenylate addition signal, an enhancer, and a reporter gene (e.g., eGFP) for detecting gene expression.

[0039] In this invention, immune cells, after undergoing electroporation, can accommodate the nucleic acid vector and achieve stable expression of the chimeric antigen receptor. The immune cells can be selected from T cells (CD4+). + CD8 - T, CD4 - CD8 +The immune cells include T cells (T, αβ-T, γδ-T, or NKT), B cells, NK cells, monocytes, macrophages, dendritic cells (DCs), or combinations thereof. The immune cells are derived from human cells, such as iPS cells or PBMCs. The immune cells can be autologous or allogeneic cells. In a preferred embodiment, the immune cells are derived from a population of T cells isolated and activated from autologous PBMCs.

[0040] In this invention, "transfection" refers to the process by which the nucleic acid vector enters the cytoplasm of immune cells through micropores formed by electroporation on the surface of the immune cell membrane.

[0041] In this invention, "electroporation" refers to the technique of creating transient, reversible micropores on the cell membrane surface using an external electric field to deliver exogenous substances (such as the nucleic acid carrier) into cells. The electroporation parameters may vary slightly depending on the instrument model, but it can stably deliver the nucleic acid carrier into immune cells without causing widespread cell death (e.g., a positive rate of more than 10% for dead cells in AO / PI staining) or apoptosis (e.g., Annexin V). + / 7-AAD + Cells with a cell count of 20% or higher are all suitable for this invention. In a preferred embodiment, the electroporation is performed using an X-Porator H1 electroporator (Suzhou Yida Biotechnology Co., Ltd.), with the following electroporation parameters: voltage 200 V, number of cycles 4, pulse 1000 μs, and pulse interval 635 ms.

[0042] In this invention, non-natural sugars refer to monosaccharide analogs with bioorthogonal reactive groups, which can be taken up by cells and integrated onto the cell membrane surface via endogenous sugar metabolism pathways, providing binding sites for subsequent click chemistry reactions. The non-natural sugars also include salts, solvates, hydrates, and precursors of non-natural sugars with protecting groups (such as acetyl or trimethylsilyl groups), provided that the precursors can be converted into the non-natural sugars that achieve the purposes of this invention, either intracellularly or extracellularly. The non-natural sugars are selected from Ac4ManNAz, Ac4GlcNAz, or Ac4GalNAz. In a preferred embodiment, the non-natural sugar is Ac4ManNAz.

[0043] This invention provides a method for preparing dual-targeted immune cells, comprising transfecting immune cells with a nucleic acid vector via electroporation in an electroporation buffer, wherein the immune cells are recovered in an electroporation recovery solution for 2-5 hours and then co-cultured with non-natural sugars; wherein the nucleic acid vector encodes at least one chimeric antigen receptor; and wherein the non-natural sugars generate binding sites on the surface of the immune cells through a sugar metabolism process.

[0044] The electroporation buffer serves as the environmental medium for co-incubating immune cells and nucleic acid vectors, and also as the buffer medium for electroporation in an electroporation instrument (such as an electroporation cup matched to the instrument). The osmotic pressure and ionic composition of the electroporation buffer determine the delivery efficiency of the nucleic acid vector, and maintain the osmotic pressure balance between the inside and outside of the immune cells with micropores on their cell membranes after electroporation, preventing rapid loss of cell contents and promoting rapid recovery of the micropores. After electroporation, the immune cells are cultured in the electroporation recovery solution for 2-5 hours, which stabilizes the cell state, promotes full closure of the micropores, reduces the toxicity of subsequent non-natural sugar treatments to the immune cells, and facilitates the effective expression and integration of the chimeric antigen receptor and its binding sites on the cell membrane. In a preferred embodiment, the chimeric antigen receptor targets NKG2D-L on tumor cells.

[0045] The preparation method further includes anchoring the nucleic acid aptamer to the surface of immune cells expressing the chimeric antigen receptor using the binding site, wherein the nucleic acid aptamer binds to at least one target site on the target cell surface. In a preferred embodiment, the binding target site is an azide group, and the nucleic acid aptamer is anchored to the surface of immune cells expressing the chimeric antigen receptor via the azide group. More specifically, one end of the nucleic acid aptamer is connected to a DBCO group, i.e., the cell surface anchoring of the nucleic acid aptamer is achieved through a click chemical reaction between the DBCO group and the azide group. The nucleic acid aptamer is a nucleic acid sequence with specific binding ability to target sites on the target cell surface. That is, in this invention, any of the existing examples of nucleic acid aptamers targeting cancer cell-specific targets are applicable. In an exemplary embodiment, the nucleic acid aptamer targets the PD-L1 epitope of tumor cells. In a preferred embodiment, the nucleotide sequence of the nucleic acid aptamer is ATACCAGCTTATTCAATTGTAGAGTATAAAAAGAGTGATGATCTTTTGTAGGTTTTTTAGATAGTAAGTGCAATCT (SEQ ID NO:7), or has this nucleotide sequence, or has a nucleotide sequence with at least 90% sequence identity and retains the same function as this nucleotide sequence.

[0046] In a preferred embodiment, the nucleic acid vector is selected from mRNA or circRNA. In a more preferred embodiment, the nucleic acid vector is circRNA. In this invention, any circRNA containing the gene encoding the chimeric antigen receptor and capable of entering immune cells through micropores formed by electroporation and being normally expressed therein is applicable to this invention.

[0047] In one embodiment, the electroporation buffer comprises the following components: 8.2-8.7 mM disodium hydrogen phosphate, 8.3-9.1 mM sodium dihydrogen phosphate, 3.2-3.8 mM potassium chloride, 10.0-11.0 mM magnesium chloride, 7.0-7.3 mM sodium succinate, 14.0-14.5 mM mannitol, 19.5-20.3 mM d-glucose, 0.4-0.5 mM sodium pyruvate, 0.2-0.3 mM inositol, and 4.0-4.3 mM GlutaMAX. The pH of the electroporation buffer is controlled at 7.20 ± 0.05, and it is filtered through a 0.22 μm filter membrane. In a preferred embodiment, the electroporation buffer comprises the following components: 8.5-8.7 mM disodium hydrogen phosphate, 8.5-9.0 mM sodium dihydrogen phosphate, 3.2-3.6 mM potassium chloride, 10.5-11.0 mM magnesium chloride, 7.2-7.3 mM sodium succinate, 14.0-14.6 mM mannitol, 19.8-20.3 mM d-glucose, 0.4-0.5 mM sodium pyruvate, 0.2-0.3 mM inositol, and 4.0-4.1 mM GlutaMAX. In a preferred embodiment, the electroporation buffer comprises the following components: 8.7 mM disodium hydrogen phosphate, 8.7 mM sodium dihydrogen phosphate, 3.6 mM potassium chloride, 10.9 mM magnesium chloride, 7.2 mM sodium succinate, 14.5 mM mannitol, 20.0 mM d-glucose, 0.5 mM sodium pyruvate, 0.3 mM inositol, and 4.0 mM GlutaMAX.

[0048] In one embodiment, the electroporation recovery solution comprises KBM-581 containing 9-15 v / v% human AB serum and 100-500 IU / mL IL-2. This solution provides basic nutrition to immune cells with micropores on their membrane surface after electroporation, accelerating cell membrane recovery, promoting the expression of the chimeric antigen receptor, and inhibiting the depletion process of immune cells. In a preferred embodiment, the electroporation recovery solution comprises KBM-581 containing 9-12 v / v% human AB serum and 100-300 IU / mL IL-2. In a most preferred embodiment, the electroporation recovery solution comprises KBM-581 containing 10 v / v% human AB serum and 200 U / mL IL-2.

[0049] The present invention further provides a dual-targeting immune cell prepared by the preparation method described above.

[0050] In one embodiment, the dual-targeting immune cells simultaneously target PD-L1 and NKG2D-L. Specifically, the chimeric antigen receptor targets NKG2D-L, and the aptamer targets PD-L1.

[0051] The present invention further provides a composition comprising the aforementioned dual-targeting immune cells. The composition also includes, but is not limited to, nutrient media for maintaining the basic survival of the dual-targeting immune cells, antibiotics to prevent contamination, and other nutrient components (such as vitamins, cytokines, growth factors, etc.).

[0052] This invention provides the use of the composition in the preparation of tumor cell immunotherapy drugs. The tumor cell immunotherapy drugs are used to treat cancer, particularly cancers that highly express the dual-target immune cells. The cancers can be hematologic malignancies or solid tumors, including but not limited to B-cell lymphoma, myeloproliferative neoplasms, acute myeloid leukemia, small cell lung cancer, non-small cell lung cancer, melanoma, renal cell carcinoma, gastric cancer, pancreatic cancer, prostate cancer, and colorectal cancer. In a preferred embodiment, the cancer is a solid tumor. In a more preferred embodiment, the cancer is gastric cancer, more specifically gastric adenocarcinoma. The tumor cell immunotherapy drugs are administered to a subject at a therapeutically effective dose. The therapeutically effective dose is determined based on the subject's sex, age, weight, and symptoms. The subject is preferably a cancer patient. The administration methods of the tumor cell immunotherapy drugs include, but are not limited to, intravenous injection, intra-arterial injection, portal vein injection, subcutaneous injection, and intramuscular injection. In this invention, the administration methods can be systemic or local administration of the tumor cell immunotherapy drugs. The number of administrations of the tumor cell immunotherapy drug is determined based on factors such as the administration method and the subject, such as single administration, multiple administrations, or continuous administration, or simultaneous or sequential administration when the tumor cell immunotherapy drug is used in combination with any of the existing immunotherapy drugs.

[0053] Examples 1-7

[0054] Table 1 Electroporation buffer

[0055]

[0056] Table 2 Electroporation Recovery Fluid

[0057]

[0058] Example 8

[0059] 2×10 7One T cell was mixed with 52 μL of mRNA (working solution concentration of 1.0 μg / μL, containing CAR protein expression units SEQ ID NO:1-6) in 100 μL of electroporation buffer (Example 4), transferred to an electroporation cuvette, and electroporated using an electroporator (voltage 200 V, number of cycles 4, pulse 1000 μs, pulse interval 635 ms). The electroporated T cells were then transferred to electroporation recovery medium (Example 7), cultured at 37°C with 5% CO2 for 4 h, and then Ac4ManNAz was added and the concentration adjusted to 50 μM. The cells were then cultured at 37°C with 5% CO2 for another 24 h. After the culture was complete, the culture suspension was centrifuged, resuspended in PBS, and then aptamers (SEQ ID NO:7) were added to adjust the aptamer concentration to 500 μM. The cells were incubated at 37°C for 30 min to obtain Apt-CAR-T cells.

[0060] Example 9

[0061] 2×10 7 One T cell was mixed with 40 μL of circRNA (working solution concentration 1.3 μg / μL, CAR protein expression unit consistent with Example 8) in 100 μL of electroporation buffer (Example 4), transferred to an electroporation cuvette, and electroporated using an electroporator (voltage 200 V, number of cycles 4, pulse 1000 μs, pulse interval 635 ms). The electroporated T cells were then transferred to electroporation recovery medium (Example 7), cultured at 37°C for 2 h with 5% CO2, and Ac4ManNAz was added to adjust the concentration to 50 μM. The cells were then cultured at 37°C for another 24 h with 5% CO2. After the culture was complete, the culture suspension was centrifuged, resuspended in PBS, and an aptamer (SEQ ID NO: 7) was added to adjust the aptamer concentration to 500 μM. The cells were then incubated at 37°C for 30 min to obtain Apt-CAR-T cells.

[0062] Example 10

[0063] 2×10 7One T cell was mixed with 40 μL of circRNA (working solution concentration 1.3 μg / μL, CAR protein expression unit consistent with Example 8) in 100 μL of electroporation buffer (Example 4), transferred to an electroporation cuvette, and electroporated using an electroporator (voltage 200 V, number of cycles 4, pulse 1000 μs, pulse interval 635 ms). The electroporated T cells were then transferred to electroporation recovery medium (Example 7), cultured at 5% CO2 and 37°C for 4 h, and then Ac4ManNAz was added and the concentration adjusted to 50 μM. The cells were then cultured at 5% CO2 and 37°C for another 24 h. After the culture was complete, the culture suspension was centrifuged, resuspended in PBS, and then an aptamer (SEQ ID NO: 7) was added, adjusting the aptamer concentration to 500 μM. The cells were incubated at 37°C for 30 min to obtain Apt-CAR-T cells.

[0064] Comparative Example 1

[0065] 1×10 7 One T cell was resuspended in 100 μL of electroporation buffer (Example 4), transferred to an electroporation cuvette, and electroporated using an electroporator (voltage 200 V, number of cycles 4, pulse 1000 μs, pulse interval 635 ms). The electroporated T cells were then transferred to KBM-581 medium and cultured at 37°C with 5% CO2 for 4 h.

[0066] Comparative Example 2

[0067] 1×10 7 One T cell was resuspended in 100 μL of electroporation buffer (opti-MEM), transferred to an electroporation cuvette, and electroporated using an electroporator (voltage 200 V, number of cycles 4, pulse 1000 μs, pulse interval 635 ms). The electroporated T cells were then transferred to KBM-581 medium and cultured at 37°C with 5% CO2 for 4 h.

[0068] Comparative Example 3

[0069] 2×10 7T cells were cultured in KBM-581 medium, with Ac4ManNAz added to adjust the concentration to 50 μM, and cultured for 24 h at 37°C with 5% CO2. After culture, cells were harvested and mixed with 40 μL of circRNA (working solution concentration of 1.3 μg / μL, CAR protein expression unit consistent with Example 10) in 100 μL of electroporation buffer (Example 4). The mixture was then transferred to an electroporation cuvette and electroporated using an electroporator (voltage 200 V, 4 cycles, pulse 1000 μs, pulse interval 635 ms). The electroporated T cells were then transferred to electroporation recovery medium (Example 7) and cultured for 24 h at 37°C with 5% CO2. After culture, the culture suspension was centrifuged, resuspended in PBS, and aptamer (SEQ ID NO: 7) was added to adjust the aptamer concentration to 500 μM. The cells were incubated at 37°C for 30 min to obtain Apt-CAR-T cells.

[0070] Detection Example 1

[0071] Empty T cells were prepared according to the methods of Comparative Examples 1 and 2, and apoptosis was detected by flow cytometry (Annexin V). + / 7-AAD + The result is as follows: Figure 1 As shown, compared with conventional electroporation buffer (Opti-MEM), cells treated with the electroporation buffer described in this invention (Example 4) have significantly improved anti-apoptotic ability.

[0072] Detection Example 2

[0073] CAR-T cells (i.e., T cells after electroporation with nucleic acid vectors) were prepared according to the methods in Examples 8 and 10, respectively. The cells were cultured in different electroporation recovery media for 24 hours. Cell viability was detected by AO / PI staining, and apoptosis was detected by flow cytometry (Annexin V). + / 7-AAD + The result is as follows: Figure 2 and Figure 3 As shown, compared with KBM-581 basal medium, the addition of 200 IU / mL IL-2 and / or 10% (v / v) human AB serum significantly improved cell viability and inhibited apoptosis.

[0074] In the circRNA group, there was no significant difference in cell viability between the two groups that received human AB serum alone and those that received IL-2 and human AB serum in combination; however, the combined group showed a more significant inhibitory effect on apoptosis of circRNA-electroplated CAR-T cells.

[0075] In the mRNA group, there was no statistically significant difference in cell viability and apoptosis levels between the group that received human AB serum alone and the group that received IL-2 and human AB serum together.

[0076] Detection Example 3

[0077] N3-CAR-T cells (without aptamer ligation) and CAR-T cells (T cells after circRNA electroporation) were prepared according to the methods of Example 10 and Comparative Example 3, respectively. CircRNA expression (using GFP as the PCR signal) was detected by flow cytometry. + The expression of the azide group (-N3) and its expression status are shown in the figure. Figure 4 As shown, compared with Comparative Example 3 (pre-Man CAR-T), the CAR-T cells obtained in Example 10 (post-Man CAR-T) had higher circRNA expression levels; at the same time, the addition of Ac4ManNAz could increase circRNA expression.

[0078] Regarding the azide group, Example 10 employed a strategy of adding Ac4ManNAz after electroporation. Although there was no significant difference in the azide-positive cell rate compared to Comparative Example 3, the abundance of the azide group (expressed as mean fluorescence intensity MFI) was significantly increased (e.g. Figure 5 , Figure 6 The results showed that the CAR-T cells prepared in Example 10 had more azide sites on each cell, thus enabling them to bind more aptamers.

[0079] Furthermore, Apt-CAR-T cells were prepared according to the methods of Example 10 and Comparative Example 3, respectively, using MKN-74 cells (PD-L1). + / NKG2D-L + Using [cell type] as the target cells, and under an effector-to-target ratio of 1:1, RTCA (Real-Time Cellular Analysis) was used to detect cell-killing activity. The results are as follows: Figure 7 As shown: Compared with Comparative Example 3, the killing ability of Apt-CAR-T cells prepared in Example 10 was significantly improved.

[0080] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing dual-targeted immune cells, characterized in that, Includes the following steps: (1) In an electroporation buffer, a nucleic acid vector is transfected into immune cells by electroporation, wherein the nucleic acid vector encodes at least one chimeric antigen receptor; (2) After immune cells are recovered in electroporation recovery solution for 2-5 hours, they are co-cultured with non-natural sugars to generate binding sites on the surface of immune cells; (3) Using the above-mentioned binding sites, the nucleic acid aptamer is anchored to the surface of an immune cell expressing the chimeric antigen receptor, wherein the nucleic acid aptamer binds to at least one target site on the surface of the target cell.

2. The preparation method according to claim 1, characterized in that, The nucleic acid vector encodes at least one chimeric antigen receptor, including one or more of a promoter, a polyadenylation signal, an enhancer, and a reporter gene for detecting gene expression. The immune cells include one or more of T cells, B cells, NK cells, monocytes, macrophages, and dendritic cells; The non-natural sugars refer to monosaccharide analogs with bioorthogonal reactive groups, which can be taken up by cells and integrated into the cell membrane surface via endogenous sugar metabolism pathways, generating binding sites on the surface of immune cells.

3. The preparation method according to claim 1, characterized in that, The binding site is an azide group.

4. The preparation method according to claim 1, characterized in that, The nucleic acid vector includes mRNA or circRNA.

5. The preparation method according to claim 1, characterized in that, The electroporation buffer comprises the following components: 8.2-8.7 mM disodium hydrogen phosphate, 8.3-9.1 mM sodium dihydrogen phosphate, 3.2-3.8 mM potassium chloride, 10.0-11.0 mM magnesium chloride, 7.0-7.3 mM sodium succinate, 14.0-14.5 mM mannitol, 19.5-20.3 mM d-glucose, 0.4-0.5 mM sodium pyruvate, 0.2-0.3 mM inositol, and 4.0-4.3 mM GlutaMAX.

6. The preparation method according to claim 1, characterized in that, The electroporation recovery solution comprises KBM-581 containing the following components: 9-15 v / v% human AB serum and 100-500 IU / mL IL-2.

7. Dual-targeting immune cells prepared by the preparation method according to any one of claims 1 to 6.

8. The dual-targeting immune cells according to claim 7, characterized in that, The dual-targeting immune cells simultaneously target PD-L1 and NKG2D-L.

9. A composition, characterized in that, Includes the dual-targeting immune cells described in claim 7.

10. The use of the composition according to claim 9 in the preparation of tumor cell immunotherapy drugs.

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