Preparation method of immune cells

Abstract

The present invention provides a method for preparing immune cells, comprising a step of treating immune cells with a culture medium containing NAD + or a precursor thereof. The culture medium can also contain PQQ and DHQ. By adding the small molecules into the culture medium, immune cell proliferation can be improved, and immune cell senescence can be inhibited.

Description

[0001] The present invention claims priority to the Chinese application filed on October 18, 2023, with application number CN202311351509.6 and invention name “A method for preparing immune cells”. Technical Field

[0002] The present invention relates to the technical field of immune cell therapy, and more specifically to a method for preparing immune cells. Background Art

[0003] Tumor is one of the major diseases that seriously threaten human health and life. Every year, more than 14 million people worldwide suffer from cancer, and more than 8 million people die from cancer. The high incidence and mortality rates reflect, to a certain extent, the lack of effective clinical prevention and treatment methods, which poses a challenge to the prevention and treatment of tumors. With the continuous deepening of the understanding of tumors, tumor treatment technology has made great progress and significantly improved the overall tumor treatment effect, but there are more or less side effects and easy to develop drug resistance. At present, tumor biological immunotherapy has become a new treatment method.

[0004] Immune cell therapy includes CAR-T immunotherapy, NK immunotherapy, DC immunotherapy, TIL immunotherapy, etc. Immune cell therapy requires cell proliferation, which generally includes cell recovery, cell activation, gene editing, subculture, etc. However, the immune cell status of tumor patients is poor, and they are generally frozen, which further reduces cell activity and affects the expansion multiples of immune cells.

[0005] NAD+ (nicotinamide adenine dinucleotide, referred to as coenzyme I) is a coenzyme of many dehydrogenases in the body, connecting the tricarboxylic acid cycle and the respiratory chain. Its function is to transfer hydrogen removed during metabolism to flavin proteins. NAD+ can provide cell energy supply and promote cell regeneration. Figure 1 As shown. CN112472710A discloses that supplementation of NAD+ precursors can enhance the sensitivity of various tumors to immune checkpoint inhibitors.

[0006] The effect of NAD+ on immune cell culture, especially the effect on immune cell culture in tumor patients, has not been reported in the literature. Summary of the invention

[0007] The purpose of the present invention is to use a culture medium containing NAD+ or its precursor to treat immune cells, thereby promoting the proliferation of immune cells and improving cell activity.

[0008] The present invention provides a method for preparing immune cells, characterized in that it comprises the step of treating the immune cells with a culture medium containing NAD+ or its precursor.

[0009] In some embodiments, the immune cell is an immune effector cell (killer cell) or an antigen presenting cell, for example selected from T cells, tumor infiltrating lymphocytes (TIL) cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer cells (CIK) cells, DC cells (dendritic cells), macrophages and B cells.

[0010] In some embodiments, the immune cells are immune effector cells and antigen presenting cells.

[0011] In some embodiments, the immune effector cells further contain a coding sequence for a chimeric antigen receptor and / or are capable of expressing a chimeric antigen receptor.

[0012] In some embodiments, the antigen presenting cells are also loaded with tumor antigens or contain coding sequences of tumor antigens.

[0013] In some embodiments, the immune cell further comprises a coding sequence for an antibody and / or is capable of expressing an antibody. In some embodiments, the antibody is a single domain antibody. In some embodiments, the single domain antibody is a multispecific single domain antibody.

[0014] In some embodiments, the method comprises the steps of:

[0015] (1) obtaining immune cells from a subject;

[0016] (2) treating the immune cells with a first culture medium containing an activator to obtain activated immune cells,

[0017] (3) introducing a nucleic acid containing a chimeric antigen receptor coding sequence into the activated immune cell to obtain a genetically modified immune cell;

[0018] (4) culturing the genetically modified immune cells using a second culture medium;

[0019] Wherein, the first culture medium and / or the second culture medium is a culture medium containing NAD+ or its precursor.

[0020] In some embodiments, the method comprises the steps of:

[0021] (1) Loading tumor antigens onto antigen-presenting cells or introducing coding sequences of tumor antigens;

[0022] (2) obtaining immune effector cells from the subject;

[0023] (3) Co-culturing the immune effector cells with the antigen presenting cells in a culture medium containing NAD+ or its precursor for a period of time sufficient to activate the immune effector cells, thereby obtaining activated immune effector cells.

[0024] In some embodiments, the immune effector cells are selected from T cells or NK cells, preferably T cells.

[0025] In some embodiments, the antigen presenting cells are cultured using a third culture medium before and / or after loading the tumor antigen or introducing the coding sequence of the tumor antigen. The third culture medium can also be a culture medium containing NAD+ or its precursor.

[0026] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or alimentarily or pharmaceutically acceptable salts, derivatives or prodrugs thereof; preferably nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR).

[0027] In some embodiments, the concentration of NAD+ or its precursor in the culture medium is 1-1500 μM, for example, 10 μM, 20 μM, 30 μM, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM, 1200 μM, 1300 μM, 1400 μM or 1500 μM.

[0028] In some specific embodiments, the concentration of NAD+ or its precursor in the culture medium is preferably 50-1200 μM, for example, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM or 1200 μM.

[0029] In some embodiments, the culture medium further contains flavonoids and / or coenzymes;

[0030] In some embodiments, the flavonoid is selected from one or more of quercetin, dihydroquercetin (DHQ), fisetin, catechin, gallin, resveratrol, hesperetin, preferably quercetin and / or dihydroquercetin;

[0031] In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, and coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

[0032] In some embodiments, the concentration of flavonoids in the culture medium is 0-200 μM, such as 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM or 200 μM.

[0033] In some embodiments, the concentration of the coenzyme in the culture medium is 0-200 μM, such as 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM or 200 μM.

[0034] In some embodiments, the subject is a healthy person or a tumor patient, preferably a tumor patient.

[0035] In some embodiments, the method has any one or more of the following features:

[0036] A. In step (2), the activator is selected from one or more of the following: CD3 antibody, CD28 antibody, 4-1BB antibody, 4-1BBL antigen,

[0037] B. In step (2), the treatment time is 12-84 hours, preferably 24-72 hours,

[0038] C. In step (3), the introduction method is electroporation.

[0039] D. In step (3), the nucleic acid containing the polypeptide coding sequence is a nucleic acid construct, such as an expression vector. Preferably, the expression vector is a non-viral vector.

[0040] E. In step (4), the culture time is 3-10 days.

[0041] On the other hand, the present application provides an immune cell culture medium containing NAD+ or its precursor.

[0042] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or alimentarily or pharmaceutically acceptable salts, derivatives or prodrugs thereof; preferably nicotinamide mononucleotide (NMN).

[0043] In some embodiments, the concentration of NAD+ or its precursor in the culture medium is 1-300 μM, such as 10 μM, 20 μM, 30 μM, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM or 300 μM.

[0044] In some embodiments, the culture medium further contains flavonoids and / or coenzymes.

[0045] In some embodiments, the flavonoid is selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallin, resveratrol, hesperetin, preferably quercetin and / or dihydroquercetin.

[0046] In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, and coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

[0047] In some embodiments, the concentration of flavonoids in the culture medium is 0-200 μM, such as 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM or 200 μM.

[0048] In some embodiments, the concentration of the coenzyme in the culture medium is 0-200 μM, such as 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM or 200 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of NAD+ metabolism in cells;

[0050] Figure 2 The results of NK cells cultured in different culture media killing skov-3 cells;

[0051] Figure 3 The results of TIL cells cultured in different culture media killing Hela cells.

[0052] Figure 4 Cell viability during the culture of CIK cells for NAD+ or its precursors;

[0053] Figure 5 Cell proliferation during the culture of CIK cells for NAD+ or its precursors;

[0054] Figure 6 Cell depletion of NAD+ or its precursors after culturing CIK cells for 14 days. DETAILED DESCRIPTION

[0055] definition

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0057] In the present invention, the term "expression cassette" refers to the complete elements required to express a gene, including a promoter and a gene coding sequence.

[0058] The term "coding sequence" is defined herein as the portion of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR, single-chain antibody, hinge region, and transmembrane region). The boundaries of the coding sequence are usually determined by the ribosome binding site (for prokaryotes) immediately upstream of the 5' end open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' end open reading frame of the mRNA. The coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0059] The term "co-stimulatory molecule" refers to a molecule present on the surface of antigen-presenting cells that can bind to the co-stimulatory molecule receptors on Th cells to produce a co-stimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens, but also the reception of co-stimulatory molecule signals. Co-stimulatory signals are transmitted to T cells mainly through the binding of co-stimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells to CD28 molecules on the surface of T cells. B cells can receive co-stimulatory signals through general pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells.

[0060] The term "specific binding" refers to the reaction between an antibody or antigen-binding fragment and the antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to the antigen with an affinity (KD) of less than about 10-5M, such as less than about 10-6M, 10-7M, 10-8M, 10-9M or 10-10M or less. "Specific recognition" has a similar meaning.

[0061] The term "chimeric antigen receptor" (CAR) is an artificially modified receptor that can anchor specific molecules (such as antibodies) that recognize cell surface antigens on immune cells, allowing immune cells to recognize cell surface antigens (such as tumor antigens or viral antigens) and kill tumor cells or virus-infected cells. CAR usually includes an optional signal peptide, an extracellular antigen binding domain (referred to as the extracellular recognition region, such as an antibody), a hinge region, a transmembrane region, and an intracellular signal region in sequence. Typically, a polypeptide that binds to a tumor cell membrane antigen can bind to a membrane antigen widely expressed by tumor cells with moderate affinity. The polypeptide that binds to a tumor cell membrane antigen can be a natural polypeptide or an artificially synthesized polypeptide.

[0062] The "heavy chain antibody" described herein is an antibody derived from a camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), and only contains 2 heavy chains consisting of a variable region (VHH) and other constant regions, and the variable region is connected to the constant region by a hinge region structure. Each heavy chain of the camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the cartilaginous fish heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragment of the heavy chain antibody includes VHH or a single-chain heavy chain antibody. By fusion with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0063] "Antibody fragments" include a portion of an intact antibody, preferably the antigen binding region and / or variable region of an intact antibody. Antibody fragments are preferably antigen binding fragments of antibodies. Examples of heavy chain antibody fragments include Fv fragments; diabodies; linear antibodies; single domain antibodies (VHH); single chain antibody molecules; scFv-Fc fragments; and any fragments that should be able to increase half-life by chemical modification or by incorporation into liposomes.

[0064] As used herein, the terms "nanoantibody", "single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH" are used interchangeably and refer to VHH that specifically recognizes and binds to an antigen. VHH is the variable region of a heavy chain antibody. Typically, VHH contains three CDRs and four FRs.

[0065] The term "NAD+ precursor" is any small molecule that results in an increase in NAD+, and the small molecule can exist in reduced or non-reduced form.

[0066] One of the purposes of the present invention is to use a culture medium containing NAD+ or its precursor to treat immune cells, thereby promoting the proliferation of immune cells and increasing cell activity.

[0067] The present invention provides a method for preparing immune cells, characterized in that it comprises the step of treating the immune cells with a culture medium containing NAD+ or its precursor.

[0068] NAD+ is a cofactor that plays a core role in metabolism, and NAD+ levels decrease with age. NAD+ can help repair DNA, regulate immune cell conduction, provide cell energy and reduce aging. During the in vitro preparation of immune cells, adding NAD+ treatment can provide sufficient energy for immune cell expansion, improve immune cell expansion and reduce cell aging indicators. NAD+ precursors are compounds that are converted into NAD+ through chemical conversion.

[0069] In some embodiments, the immune cell is an immune effector cell (killer cell) or an antigen presenting cell, for example selected from T cells, tumor infiltrating lymphocytes (TIL) cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer cells (CIK) cells, DC cells, macrophages and B cells.

[0070] In some embodiments, the immune cell is an immune effector cell (killer cell), and the killer cell is a T cell, a TIL cell, a NK cell, or a CIK cell.

[0071] In some embodiments, the killer cell also contains the coding sequence of a chimeric antigen receptor and / or can express a chimeric antigen receptor. The CAR contains an optional signal peptide sequence, an extracellular target recognition region (antigen binding domain), a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signal domain. The extracellular recognition region comprises an antibody targeting an antigen (eg, a tumor antigen), such as a full-length antibody, an antigen binding fragment, a single domain antibody (nanoantibody), a single-chain variable fragment (scFv).

[0072] The target antigen of the chimeric antigen receptor is not limited, including but not limited to: CD19, CD22, CD23, myeloproliferative leukemia protein (MPL), CD30, CD32, CD20, CD70, CD79b, CD99, CD123, CD138, CD179b, CD200R, CD276, CD324, Fc receptor-like 5 (FcRH5), CD171, CS-1 (signaling lymphocyte activation molecule family 7, SLAMF7), C-type lectin-like molecule-1 (CLL-1), CD33, cadherin 1, cadherin 6, cadherin 16, cadherin 17, cadherin 19, epidermal growth factor receptor variant III (EGFRviii), ganglioside GD2, ganglioside G D3, human leukocyte antigen A2 (HLA-A2), B cell maturation antigen (BCMA), Tn antigen, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), FMS-like tyrosine kinase 3 (FLT3), fibroblast activation protein (FAP), tumor-associated glycoprotein (TAG)-72, CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), B7-H3 (CD276), KIT, interleukin 13 receptor subunit alpha-2 (IL-13Ra2), interleukin 11 receptor subunit alpha (IL11Ra), mesothelin (MSLN), prostate stem cell antigen (PSCA), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis Y, CD24, platelet-derived growth factor receptor beta (PDGFR-β), protease serine 21 (PRSS21), sialoglycolipid stage-specific embryonic antigen 4 (SSEA-4), CD20, immunoglobulin Fc region, tissue factor, folate receptor alpha, epidermal growth factor receptor 2 (ERBB2), mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural small adhesion molecule (NCAM), protease, prostatic acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), Ephrin B2, insulin-like growth factor I receptor (IGF-1 receptor), carbonic anhydrase IX (CAIX), latent membrane protein 2 (LMP2), melanocyte protein gpl00, bcr-abl, tyrosinase, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), fucosylated monosialoganglioside (fucosylated GM1), sialyl Lewis a(sLea), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl GD2 ganglioside, folate receptor β, TEM1 / CD248, tumor endothelial marker-associated protein 7 (TEM7R), claudin6 (CLDN6), thyroid stimulating hormone receptor (TSHR), T cell receptor (TCR)-β1 constant chain, TCRβ2 constant chain, TCRγ-δ, G protein coupled receptor class C 5 group D member D (GPRC5D), CXORF61 protein, CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta specific 1 (PLAC1), carbohydrate antigen GloboH, breast differentiation antigen NY-BR-1, uroplakin-2 (UPK2), hepatitis A virus cell receptor 1 (H AVCR1), adrenergic receptor β3 (ADRB3), pan-nexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 family member K (LY6K), olfactory receptor family 51 subfamily E member 2 (OR51E2), T cell receptor gamma chain variable reading frame protein (TARP), Wilms tumor antigen 1 protein (WT1), tumor-testis antigen NY-ESO-1, tumor-testis antigen LAGE-1a, legumain, human papillomavirus (HPV) E6, HPV E7, human T lymphotrophic virus (HTLV1)-Tax, Kaposi's sarcoma-associated herpes virus glycoprotein (KSHV) K8.1 protein, Epstein-Barr virus (EBV)-encoded glycoprotein 350 (EBB gp350), HIV1 envelope glycoprotein gp120, multiplex automated genome engineering (MAGE)-A1, translocation-Ets-leukemia virus (ETV) protein 6-AML, sperm protein 17, X antigen family member (XAGE) 1, transmembrane tyrosine protein kinase receptor Tie 2. Melanoma tumor-testis antigen MAD-CT-1, melanoma tumor-testis antigen MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survival rate and telomerase, prostate cancer tumor antigen-1 (PCTA-1) / galectin 8, MelanA / MART1, Ras mutant, human telomerase reverse transcriptase (hTERT), delta-like 3 (DLL3), trophoblast cell surface antigen 2 (TROP2), protein tyrosine kinase 7 (PTK7), guanylate cyclase C (GCC), alpha-fetoprotein (AFP), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (TMPRSS2ETS fusion gene), N-acetylglucosyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytoma viral oncogene neuroblastoma derived homolog (MYCN), Ras homology family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), PAX5, preacrosome protein binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), RU2, intestinal carboxylesterase, heat shock protein 70-2 mutant (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), IgA receptor Fc fragment (FCAR), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2, EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), immunoglobulin lambda-like polypeptide 1 (IGLL1), FITC, luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), chorionic gonadotropin receptor (CGHR), CC chemokine receptor 4 (CCR4), ganglioside GD3, signaling lymphocyte activation molecule (SLAM) family member 6 (SLAMF6), SLAMF4, luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), chorionic gonadotropin receptor (CGHR), or any combination thereof.

[0073] In some embodiments, the extracellular recognition region is an anti-mesothelin single domain antibody, such as described in WO2022143550A1. In some embodiments, the extracellular recognition region is an anti-MUC1 single domain antibody. In some embodiments, the chimeric antigen receptor is as described in PCT / CN2022 / 143407. The above-mentioned documents The full text of the above-mentioned patent application is incorporated herein by reference to each embodiment thereof specifically and individually.

[0074] The optional signal peptide on CAR can be selected as needed. For example, CD8 signal peptide, CD28 signal peptide, CD4 signal peptide or light chain signal peptide, the sequence of which is within the knowledge of those skilled in the art. The CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used in CAR in the art.

[0075] The hinge region of CAR is selected from CD8α hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region or IgG4 Fc CH2CH3 hinge region, and its sequence is within the knowledge of those skilled in the art. The CD8 hinge region suitable for the present invention can be various human CD8 hinge region sequences commonly used in CAR in the art.

[0076] The transmembrane region of CAR is selected from one of CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region; preferably CD28 transmembrane region or CD8 transmembrane region, and its sequence is within the knowledge of those skilled in the art. The human CD28 transmembrane region or CD8 transmembrane region suitable for the present invention can be various human CD28 transmembrane region or CD8 transmembrane region sequences commonly used in CAR in the art.

[0077] Suitable intracellular co-stimulatory domains can be selected as needed, including intracellular domains with co-stimulatory signal molecules, such as CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS) and DNAX activation protein 10 intracellular domains, preferably CD28 co-stimulatory domains or 4-1BB co-stimulatory domains. Human CD28 co-stimulatory domains or 4-1BB co-stimulatory domains suitable for the present invention can be various human CD28 co-stimulatory domain sequences or 4-1BB co-stimulatory domain sequences commonly used in the art for CAR.

[0078] Similarly, the intracellular signaling domain of CAR can be selected as needed, including but not limited to CD3ζ intracellular signaling domain or FcεRIγ intracellular signaling domain. The CD3ζ intracellular signaling domain suitable for the present invention can be various CD3ζ intracellular signaling domains for CAR known in the art.

[0079] The above-mentioned parts forming the chimeric antigen receptor of the present invention, such as CD8 signal peptide, antigen binding domain, CD8 hinge region, CD28 transmembrane region, CD28 co-stimulatory domain, CD3ζ intracellular signal domain, etc., can be directly connected to each other, or can be connected through a linker sequence. The linker sequence can be a linker sequence suitable for antibodies known in the art, such as a linker sequence containing G and S. Generally, the linker contains one or more motifs repeated before and after. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motif is adjacent in the linker sequence, and no amino acid residues are inserted between the repeats. The linker sequence can be composed of 1, 2, 3, 4 or 5 repeated motifs. The length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine residues in the linker sequence is not particularly limited, and is generally 2 to 20, such as 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. In certain embodiments, the linker sequence is (GGGGS)n, where n is an integer of 1 to 5.

[0080] In an exemplary embodiment, CAR contains, from N-terminus to C-terminus, a CD8 signal peptide, an anti-MSLN single domain antibody or an anti-MUC1 single domain antibody, a CD8 hinge region, a CD28 transmembrane region or a CD8 transmembrane region, a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0081] In some embodiments, the immune cell is an antigen presenting cell.

[0082] In some embodiments, the immune cells are immune effector cells and antigen presenting cells. Antigen presenting cells take up and process antigens and present the antigens to killer cells, thereby activating the killer cells. Co-culturing killer cells and antigen presenting cells can be used to activate killer cells.

[0083] In some embodiments, the antigen presenting cells are also loaded with tumor antigens or contain coding sequences of tumor antigens.

[0084] In some embodiments, the immune cell further comprises a coding sequence for an antibody and / or is capable of expressing an antibody. In some embodiments, the antibody is a single domain antibody. In some embodiments, the single domain antibody is a multispecific single domain antibody.

[0085] The tumor antigen is selected from tumor-associated antigens and tumor neoantigens; preferably, the antigen-presenting cell expresses the tumor antigen; more preferably, the tumor antigen is selected from one or more of the following: hTERT, p53, Her2, Survivin, CEA, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-C1, MAGE-C2, MUC1, Wilms tumor 1 (WT1), Her2-neu, P53, NY-ESO-1, hTERT, Mammaglobin-A, Folate Receptorα (FR-α), HPV16 / 18-E6, HPV16 / 18-E7, alpha-fetoprotein (AFP), Glypican3 (GPC3), prostate specific antigen (PSA), prostate acid phosphatase (PAP), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate 6 transmembrane epithelial antigen 1 (STEAP1), B cell maturation antigen (BCMA), CMV pp65, gp100, PRAME; further preferably, the tumor antigens include any two or three of CEA, survivin and p53.

[0086] In some embodiments, the coding sequence of the tumor antigen is RNA, preferably mRNA.

[0087] In some embodiments, the multispecific single domain antibody contains multiple functional regions that respectively target multiple targets, and the multiple functional regions are respectively single domain antibodies.

[0088] In some embodiments, the multiple targets are selected from: immune checkpoint proteins, immune cell-associated antigens, tumor-associated antigens, immune co-stimulatory molecules or their receptors. Immune checkpoint proteins include but are not limited to: PD-1, CTLA4, PDL1, PDL2, PDL3, TIM3, LAG3, CD47, BTLA, TIGIT, CD160, LAIR1, B7-H1, B7-1, VSIR, CD244. Immune cell-associated antigens include but are not limited to: CD28, CD137, CD134, CD40, CD40L, ICOS, HVEM, CD2, CD27, CD30, GITR, LIGHT, DR3, SLAM, CD226, CD80, CD86. Tumor-associated antigens include but are not limited to: EIIIB fibronectin, Siglecl5, VEGF (R), HER2, PSMA, AXL, MUC1, MUC16.

[0089] In one or more embodiments, at least one of the multiple functional regions is an immune checkpoint inhibitory single domain antibody.

[0090] In one or more embodiments, at least one of the plurality of functional domains is an activating antibody to an immune co-stimulatory molecule or its receptor.

[0091] In one or more embodiments, the multiple functional regions are different immune checkpoint inhibitory single domain antibodies.

[0092] In one or more embodiments, the multispecific single-domain antibody contains two or three functional regions that target two or three targets, respectively.

[0093] In one or more preferred embodiments, the multispecific single-domain antibody contains: a first functional region targeting PD-1, and a second functional region targeting CTLA4; the first functional region is an anti-PD-1 single-domain antibody, and the second functional region is an anti-CTLA-4 single-domain antibody. Exemplary PD-1 single-domain antibodies are described in WO2022143552A1, and exemplary CTLA-4 single-domain antibodies are described in WO2023051618A1. The full text of the above-mentioned patent application is incorporated herein by reference to each embodiment thereof specifically and individually.

[0094] In some embodiments, the coding sequence of the multispecific single-domain antibody is DNA or RNA, preferably mRNA or saRNA (self-replicating RNA).

[0095] In one or more embodiments, the multispecific Nanobody further contains an Fc region; preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4; the IgG1, IgG2, IgG3 or IgG4 is of human origin.

[0096] Since the Fc region can cause significant ADCC and CDC effects, it may cause immune cell damage and have negative pharmacological effects. Therefore, Fc is usually modified (also referred to as variant Fc herein), and the affinity constant of multispecific nano antibodies to FcγRIIIa and / or C1q is reduced compared to before mutation through site mutation, thereby improving the efficacy of antibody drugs. The currently disclosed mutation sites, according to the EU numbering system, include: Fc region mutation sites in IgG1 include L234A, L235A, L235E, L235G, G236A, G237A, N297A, G318A, L320A, L322A; Fc region mutation sites in IgG3 include V234A, G237A, P238S, H28A, V309L , A330S, P331S; Fc region mutation sites in IgG3 include Leu281, Leu282, Gly283, Gly284, Asn344, Pro378; Fc region mutation sites in IgG4 include S228P, E233P, F234V, L235A, F243L, D254A, R292P, Y300L, L309V, R409K. The variant Fc herein includes but is not limited to the Fc of each IgG having the above mutation sites.

[0097] In one or more embodiments, the Fc region is an Fc region of IgG1, and according to the EU numbering system, the Fc region has one or more of the following mutations: L234A, L235A, G237A. In one or more embodiments, the Fc region is an Fc region of IgG4, and according to the EU numbering system, the Fc region has one or more of the following mutations: S228P, E233P, F234V, L235A, D254A, L309V, R409K.

[0098] In some embodiments, the method comprises the steps of:

[0099] (1) obtaining immune cells from a subject;

[0100] (2) treating the immune cells with a first culture medium containing an activator to obtain activated immune cells,

[0101] (3) introducing a nucleic acid containing a chimeric antigen receptor coding sequence into the activated immune cell to obtain a genetically modified immune cell;

[0102] (4) culturing the genetically modified immune cells using a second culture medium;

[0103] Wherein, the first culture medium and / or the second culture medium is a culture medium containing NAD+ or its precursor.

[0104] In some embodiments, the method comprises the steps of:

[0105] (1) Loading tumor antigens onto antigen-presenting cells or introducing coding sequences of tumor antigens;

[0106] (2) obtaining immune effector cells from the subject;

[0107] (3) Co-culturing the immune effector cells with the antigen presenting cells in a culture medium containing NAD+ or its precursor for a period of time sufficient to activate the immune effector cells, thereby obtaining activated immune effector cells.

[0108] In some embodiments, the immune effector cells are selected from T cells or NK cells, preferably T cells.

[0109] In some embodiments, the antigen presenting cell is a DC cell, and the immune effector cell is selected from a T cell or a NK cell, preferably a T cell.

[0110] In some embodiments, the antigen presenting cells are cultured using a third culture medium before and / or after loading the tumor antigen or introducing the coding sequence of the tumor antigen. The third culture medium can also be a culture medium containing NAD+ or its precursor.

[0111] Herein, "activator" is used to activate immune cells before the polypeptide coding sequence is introduced into immune cells, which helps to alleviate cell damage in cell preparation and improve cell viability and survival ratio. In theory, any reagent known in the art that can be used for immune cell activation can be used. In an exemplary embodiment where the immune cell is a T cell (particularly CD3+T cell), the activator includes one or more selected from the following: CD3 antibody, CD28 antibody, 4-1BB antibody, 4-1BBL antigen.

[0112] The activation process includes incubating immune cells under conditions where the activator and the cells are in contact. In the incubation mixture, the final concentration of the activator, the concentration of the immune cells, and the ratio of the two are not limited. For example, the activator can be coated on a solid phase carrier, and the final concentration of the coating solution can be 1-20 μg / ml, such as 1, 5, 10, 15 or 20 μg / ml. The concentration ratio of the activator to the immune cells can be 1-20 μg / ml: 2.45-2.8×10 8 The activation temperature is any temperature suitable for the growth of immune cells, preferably 27-45° C., more preferably 37° C. Usually, the activation is performed in an environment containing CO 2 , such as 5% CO 2 .

[0113] The culture medium required for activation incubation can be any commercial or homemade culture medium suitable for the growth of immune cells (e.g., T cells). In one or more embodiments, AIM-V culture medium containing or not containing 5% serum or its substitute is used for activation incubation. Preferably, the culture medium also contains cytokines, such as IL-2, IL-15, IL-21, IL-7, IL-6, LSD1 inhibitor, MALT1 inhibitor. When the immune cells are T cells, the cytokines are IL-7 and / or IL-15, preferably, the final concentration of IL-7 is 1-50ng / mL, and the final concentration of IL-15 is 1-50ng / mL. When the immune cells are NK cells, the cytokines are IL-2 and / or IL-15.

[0114] The activator may be present in the incubation mixture in the form of a solute, or it may be fixed on a solid support. Solid supports that can be used to fix activators (e.g., antibodies) are well known in the art, such as magnetic beads or container walls. In some embodiments, the activator is a CD3 antibody and a CD28 antibody fixed on magnetic beads; preferably, the activator is MiltenyiMACS GMP TransAct CD3 / 28 magnetic beads and / or CTSDynabeads CD3 / 28. In some embodiments, the activator is a CD3 antibody, a CD3 antibody and a CD28 antibody, a CD3 antibody and a 4-1BB antibody, or a CD3 antibody and a 4-1BBL antigen fixed on the container wall; preferably, the container is a T75 bottle.

[0115] In some embodiments, the sorted T cells are activated by container-coated CD3 antibody and then electroporated 24-72 hours later, preferably 48 hours later; the sorted T cells are activated by container-coated CD3 antibody and CD28 antibody and then electroporated 24-72 hours later, preferably 48 hours later; the sorted T cells are activated by container-coated CD3 antibody and 4-1BBL antigen and then electroporated 24-72 hours later, preferably 48 hours later; the sorted T cells are activated by container-coated CD3 antibody and 4-1BB antibody and then electroporated 24-72 hours later; the sorted T cells are activated by Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads coated with CD3 antibody and CD28 antibody and then electroporated 24-72 hours later, preferably 72 hours later; the sorted T cells are activated by DynabeadsCD3 / 28 magnetic beads coated with CD3 antibody and CD28 antibody and then electroporated 24-72 hours later, preferably 48-72 hours later. In one or more embodiments, the final concentration of Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads is 4 mL / 1×10 8 T cells, 6mL / 1×10 8 T cells, 8mL / 1×108 In one or more embodiments, the ratio of CTSDynabeads CD3 / 28 magnetic beads to T cells is 1:1.

[0116] Before activation, immune cells are sorted, such as T cells, NK cells. Methods commonly used to sort immune cells, especially T cells, are known in the art, such as antibody sorting or flow cytometry. In one or more embodiments, the T cells are CD3+ T cells.

[0117] Herein, nucleic acid conversion is preferably electroporation. Typically, the electroporation mixture is electroporated in an electroporation device. Exemplarily, the electroporation mixture comprises: nucleic acid, immune cells activated by the method described herein, and electroporation solution. In a specific embodiment, the electroporation device is a Lonza Nucleofactor 4D or Maxcyte electroporator (the electroporation solution of the Lonza device is V4XP-3024; the electroporation solution of the Maxcyte device is Gibco Opti-MEM TM I Reduced Serum Medium), and the electrotransfer program is FI-115 or Resting T / Expand T4. Those skilled in the art can choose different electrotransfer instruments and select electrotransfer programs and parameters according to the nucleic acids and cells to be electrotransferred.

[0118] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof; preferably nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR). As a direct precursor of NAD+, NMN can be easily directly converted into NAD+ by NMNAT enzyme and easily absorbed by cells.

[0119] In some embodiments, the concentration of NAD+ or its precursor in the culture medium is 1-1500 μM, for example, 10 μM, 20 μM, 30 μM, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM, 1200 μM, 1300 μM, 1400 μM or 1500 μM.

[0120] In some specific embodiments, the concentration of NAD+ or its precursor in the culture medium is preferably 50-1200 μM, for example, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM or 1200 μM.

[0121] In some embodiments, the culture medium further contains flavonoids and / or coenzymes.

[0122] Flavonoids have anti-free radical and antioxidant effects, and can delay cell apoptosis. In some embodiments, the flavonoids are selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallin, resveratrol, and hesperidin, preferably quercetin and / or dihydroquercetin. Among them, quercetin is a plant flavonol from the flavonoid group of polyphenols, which is present in many fruits, vegetables, leaves, seeds and grains. It is a naturally occurring polar auxin transport inhibitor. Dihydroquercetin is a dihydroflavonol compound belonging to the vitamin P family. It is a widely used bioactive agent with multiple biological activities in the human body, including antioxidant, free radical scavenging and other effects.

[0123] Coenzyme is a general term for a large class of organic cofactors, which are essential factors for enzyme-catalyzed redox reactions, group transfer and isomerization reactions. In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10. Among them, pyrroloquinoline quinone (PQQ) is a redox enzyme cofactor with a chemical formula of C 14 H6N2O8 participates in a variety of redox reactions and, as a coenzyme, mediates a series of cellular and molecular biochemical reactions, thereby affecting the physiological and metabolic processes of the human body.

[0124] In some embodiments, the concentration of flavonoids in the culture medium is 0-200 μM, such as 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM or 200 μM.

[0125] In some embodiments, the concentration of the coenzyme in the culture medium is 0-200 μM, such as 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM or 200 μM.

[0126] In some embodiments, the subject is a healthy person or a tumor patient, preferably a tumor patient. The obtained immune cells are freshly collected or frozen and resuscitated cells.

[0127] In some embodiments, in step (2), the activator is selected from one or more of the following: CD3 antibody, CD28 antibody, 4-1BB antibody, 4-1BBL antigen.

[0128] In some embodiments, in step (2), the treatment time is 12-84 hours, preferably 24-72 hours.

[0129] In some embodiments, in step (3), the introduction method is electroporation.

[0130] In some embodiments, in step (3), the nucleic acid containing the polypeptide coding sequence is a nucleic acid construct, such as an expression vector. Preferably, the expression vector is a non-viral vector.

[0131] In some embodiments, in step (4), the culturing time is 3-10 days.

[0132] Adding NAD+ or its precursors, flavonoids and / or coenzymes to the immune cell culture medium can increase the intracellular NAD+ content, increase the mitochondrial content, provide sufficient energy to enhance cell function, improve cell metabolism and activity, thereby promoting cell proliferation, inhibiting cell aging, increasing cell telomeres and longevity proteins, nucleic acid transduction positivity, increasing cytokine secretion and target cell killing ability, etc.

[0133] The present invention also provides a pharmaceutical composition, comprising the immune cells prepared by the preparation method of the present invention, and pharmaceutically acceptable excipients.

[0134] In some embodiments, the pharmaceutical composition is a CAR-T preparation.

[0135] In some embodiments, the pharmaceutical composition is a NK cell preparation.

[0136] In some embodiments, the pharmaceutical composition is a TIL cell preparation.

[0137] In some embodiments, the pharmaceutical composition is a CIK cell preparation.

[0138] In some embodiments, the pharmaceutical composition is a vaccine composition, preferably a DC vaccine.

[0139] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.

[0140] Experimental methods:

[0141] Example 1, PBMC resuscitation and NAD+ detection

[0142] PBMCs of two donors (tumor patients) were resuscitated, and PBMCs were resuscitated using AIM-V medium (containing 2% FBS), inoculated into culture bottles at 5E6 / mL, and placed in an incubator (37°C; 5% CO2). NAD+ detection was performed on the day of resuscitation using an NAD+ / NADH detection kit (WST-8 method, Bio-Tech). The NAD+ detection results are shown in Table 1. Based on the test results, S01 with a lower NAD+ level was selected from patient S01 (0.13uM) and patient S02 (0.19uM) for the preparation of immune cells such as T cells, DC cells, and NK cells.

[0143] Table 1: NAD+ detection results of S01 and S02 PBMC

[0144]

[0145] Example 2: CAR-T cell preparation method:

[0146] Use culture medium and 5ug / mL anti-CD3 and anti-CD28 to coat 6-well plates or culture flasks at 4°C overnight. Inoculate the revived PBMCs into the coated 6-well plates or culture flasks and stimulate for 48 hours for activation. Collect the cells and electroporate them using the Lonza 4D electroporator. The electroporation process is optimized according to the electroporator manual. Resuspend 5×10 6 to 7×10 6For PBMC cells, 6ug pC23S-1444 was added to the cell suspension, and 20ug PB mRNA (the amino acid sequence of the PB enzyme is as described in CN105154473A) and 200IU Rnaseinhibitor were added to the electroporation system, and electroporation was performed using the EO-115 program. After electroporation, the cells were allowed to stand at room temperature for 10 minutes. After that, the PBMCs were transferred to a 6-well plate containing the culture medium of the corresponding group (need to be preheated to 37°C in advance), placed in an incubator (37°C; 5% CO2) and cultured for 24h-48h, and then the culture medium was supplemented to 4mL. The first subculture was performed on the third day after electroporation, the second subculture was performed on the sixth day, and the CAR-T cells were prepared on the tenth day. The PBMCs during the subculture process were cultured in the culture medium of the corresponding group shown in Table 2, and the subculture cell density was 5×10 5 / ml. During and after the CAR-T cell preparation process, flow cytometry was used to detect the cell surface CAR positivity rate and surface markers.

[0147] Among them, in order to verify the effects of NMN and the combination of NMN with PQQ and DHQ on CAR-T cell culture, during the CAR-T culture process, the culture medium was grouped as follows: CAR-T culture medium, CAR-T culture medium + NMN, CAR-T culture medium + NMN + PQQ, CAR-T culture medium + NMN + DHQ and CAR-T culture medium + NMN + PQQ + DHQ; in addition, CAR-T culture medium + NMN + PQQ + DHQ was divided into different concentration groups.

[0148] The above-mentioned pC23S-1444 plasmid is a plasmid containing the coding sequence of 1444CAR (CD8 signal peptide, 1444 anti-mesothelin nanobody, CD8 hinge region, CD8 transmembrane region, 4-1BB costimulatory domain, CD3ζ intracellular signal domain from N-terminus to C-terminus). The nucleotide sequence of the pC23S-1444 plasmid is shown in SEQ ID NO:1.

[0149] Example 3: DC+T cell co-culture preparation method:

[0150] Human monocyte isolation kit (MojoSort TMHuman CD14+Monocytes Isolation Kit, Biolegend, USA) was used to enrich monocytes by magnetic bead negative selection. The purity of the isolated monocytes was measured, and the surface markers were CD14+CD116-. Cytokines IL-4 and GM-CSF were added to the monocyte suspension to induce monocyte differentiation. The cells were cultured in a 37°C, 5% CO2 incubator for 5 days using culture medium, and maturation factors (IFN-γ, PolyI:C, R848, PGE2) were added for cell maturation. The cells were collected on the 6th day to obtain mature DC cells.

[0151] Electroporation was used to electroporate the anti-PD-1 / CTLA-4 bispecific nanoantibody mRNA and three antigen mRNAs (CEA antigen mRNA, Survivin antigen mRNA, and P53 antigen mRNA) into mature DCs. The electroporation concentrations of the three antigen mRNAs were 5ug / 2.5×10 6 The electroporation concentrations of cells and nanoantibody mRNA were 60ug / 2.5×10 6 cell.

[0152] Human T Cell Isolation Kit (Stemcell) was used to enrich T cells by magnetic bead negative selection, and CD3 antibody was used to detect the purity of T cells after sorting. Preheat the co-culture medium and resuspend the T cell pellet. After 24 hours of electroporation, three antigen-loaded mDCs were collected, centrifuged, resuspended in co-culture medium, and co-cultured with T cells at a ratio of 1:10, respectively, and cultured in a 37°C, 5% CO2 incubator for 3 days, and the supernatant and cells were collected according to experimental requirements.

[0153] Among them, in order to verify the effects of NMN and the combination of NMN with PQQ and DHQ on DC cell culture, in addition to the addition of cytokines during DC induction and cell maturation culture, the culture medium was divided into the following groups: DC culture medium, DC culture medium + NMN, DC culture medium + NMN + PQQ, DC culture medium + NMN + DHQ, and DC culture medium + NMN + PQQ + DHQ. In addition, DC culture medium + NMN + PQQ + DHQ was divided into different concentration groups.

[0154] In order to verify the effects of NMN and the combination of NMN with PQQ and DHQ on the co-culture of DC cells and T cells, the culture medium was grouped as follows: co-culture medium, co-culture medium + NMN, co-culture medium + NMN + PQQ, co-culture medium + NMN + DHQ, and co-culture medium + NMN + PQQ + DHQ; in addition, the co-culture medium + NMN + PQQ + DHQ was divided into different concentration groups.

[0155] Example 4: NK cell preparation method:

[0156] Peripheral blood mononuclear cells were isolated by Ficoll separation and used NK cell expansion kit (ExCell Bio) is used to expand NK cells in vitro. Pre-coat the culture plate with factor I, add 10% hAB and factor II to the NK basal medium to prepare the induction medium, use the NK induction medium to resuspend an appropriate amount of mononuclear cells, and culture at 37°C 5% CO2 for five days. Avoid disturbing the cells during the process. On the third day, observe the cell state and add an appropriate amount of fresh medium. Add 5% hAB and factor III to the NK basal medium to prepare the expansion medium. Use NK expansion medium for subsequent culture and passage, and it can be cultured for 2-3 weeks. The purity of NK cells is detected at the end of the culture, and the surface marker is CD3-CD56+.

[0157] Among them, in order to verify the effects of NMN and the combination of NMN with PQQ and DHQ on NK cell culture, in addition to the addition of cytokines during NK induction and cell expansion culture, the culture medium was grouped as follows: NK culture medium, NK culture medium + NMN, NK culture medium + NMN + PQQ, NK culture medium + NMN + DHQ and NK culture medium + NMN + PQQ + DHQ; In addition, NK culture medium + NMN + PQQ + DHQ was divided into different concentration groups, as shown in Table 2. The Excell culture medium in group 1# refers to the culture medium according to NK cell expansion kit with NK induction and cell expansion medium.

[0158] Table 2: NK culture medium grouping

[0159] Group Medium composition #1 Excell #2 Excell+10μM NMN #3 Excell+30μM NMN #4 Excell+60μM NMN #5 Excell+10μM NMN+0.1nM PQQ+5μg / ml DHQ #6 Excell+30μM NMN+1nM PQQ+10μg / ml DHQ #7 Excell+60μM NMN+10nM PQQ+20μg / ml DHQ

[0160] The NK cells on the 21st day of culture were co-cultured with skov-3 cells for 4 h. The killing of tumor cells was detected using the xCELLigence RTCA device (Roche Applied Science, Canada). The detection process was carried out according to the device operating instructions. The results are shown in Figure 2 shown. Figure 2 The results showed that the killing of group 5# tumor cells by 10μM NMN+0.1nM PQQ+5μg / ml DHQ was better than that of other groups, and the other combinations of adding NMN alone or adding high concentrations were not much different from the NK cell culture without addition. Therefore, the combination of lower concentrations of NMN, PQQ and DHQ is suitable for culturing NK cells.

[0161] Example 5: TIL cell preparation method:

[0162] Tumor tissue samples were obtained through surgery. After removing blood vessels, capsule and necrotic tissue using ophthalmic scissors and ophthalmic forceps, the tumor tissue samples were cut into 2-3 mm 3 Small pieces of TIL cells were separated and expanded using TIL cell culture medium. According to the number and growth status of TIL cells, cytokines were added or the medium was changed every two days. The culture period was about 3 weeks. The TIL cell subpopulations (purity), activation and exhaustion were detected at the end of the culture period. The markers were CD3+CD4+, CD3+CD8+, CD3+CD4+CD69+, CD3+CD8+CD69+, CD3+CD4+PD1+CTLA4+ and CD3+CD8+PD1+CTLA4+.

[0163] Among them, in order to verify the effects of NMN and the combination of NMN with PQQ and DHQ on TIL cell culture, during the TIL culture process, the culture medium was grouped as follows: TIL culture medium, TIL culture medium + NMN, TIL culture medium + NMN + PQQ, TIL culture medium + NMN + DHQ and TIL culture medium + NMN + PQQ + DHQ; In addition, TIL culture medium + NMN + PQQ + DHQ was divided into different concentration groups. The grouping is shown in Table 3, and the M17 culture medium is the culture medium B in Example 1 in CN202110054608.2.

[0164] Table 3: TIL culture medium grouping

[0165] Group Medium composition #1 M17 medium #2 M17 medium + NMN (200uM) #3 M17 medium + NMN (200uM) + PQQ (1nM) #4 M17 medium + NMN (200uM) + DHQ (5nM) #5 M17 medium + NMN (200uM) + PQQ (1nM) + DHQ (5nM) #6 M17 medium + NMN (50uM) + PQQ (0.1nM) + DHQ (1nM)

[0166] TIL cells on the 12th day of culture were co-cultured with Hela cells at an effector-target ratio of 10:1 and 5:1 for 4 h. The killing of tumor cells was detected using the xCELLigence RTCA device (Roche Applied Science, Canada). The detection process was carried out according to the device operating instructions. The results are shown in Figure 3 shown. Figure 3 It shows that the combination of NMN with PQQ and / or DHQ can promote TIL cells to kill Hela cells, which is more obvious under the high-efficiency target ratio of 10:1; among all groups, Group 5 had the highest killing rate, so the combination of high concentrations of NMN, PQQ and DHQ has the best effect on TIL cell culture.

[0167] Example 6: CIK cell culture

[0168] 6.1 Thaw frozen PBMC cells and plate PBMC at 1-2×10 6The cells were suspended in the culture medium at a concentration of 1,000 U / mL of recombinant human IFN-γ and cultured in a 37°C, 5% CO2 incubator; 24 hours later, 50 ng / mL of CD3 monoclonal antibody and 300 U / mL of recombinant human IL-2 were added to stimulate the growth of CIK cells; the culture medium was subsequently added for subculture, and the viability and cell mass were detected by AO / PI (Nasilon Bio Instrument Co., Ltd.) double fluorescence staining, and LAG3+ depletion indicators were detected by flow cytometry. Different concentrations of anti-aging substances (NMN and NR) were added to the culture medium used in this process, and the group settings are shown in Table 4.

[0169] Table 4: CIK cell culture group settings

[0170]

[0171] 6.3 Experimental results Figure 4-6 As shown, Figure 4 The cell viability during the culture process was significantly higher in the NR and NMN groups than in the control group (CIK-0, i.e., dosage 0), which proves that the method of the present invention can significantly improve the cell viability.

[0172] Figure 5 The cell proliferation during the culture process, the NR and NMN groups were significantly higher than the control group (CIK-0, i.e., dosage 0), proving that the method of the present invention can significantly increase the cell proliferation rate.

[0173] Figure 6 It is the cell exhaustion status after 14 days of culture. The exhaustion index LAG3 of the NR and NMN groups were significantly lower than that of the control group (CIK-0, i.e., dosage 0), which proves that the method of the present invention can significantly reduce cell exhaustion.

[0174] The above experiments show that the method provided by the invention can improve CIK cell viability, increase cell proliferation rate, and reduce cell exhaustion.

Claims

1. A method for preparing immune cells, characterized in that: include: A step of treating immune cells with a culture medium containing NAD+ or its precursor, wherein the immune cells are immune effector cells and / or antigen presenting cells, wherein the immune effector cells are selected from tumor infiltrating lymphocytes (TIL) cells, natural killer (NK) cells, natural killer T (NKT) cells or cytokine-induced killer cells (CIK) cells, and the antigen presenting cells are selected from DC cells, macrophages or B cells.

2. The method according to claim 1, characterized in that The immune effector cells also contain a coding sequence for a chimeric antigen receptor and / or are capable of expressing a chimeric antigen receptor.

3. The method according to claim 2, characterized in that The method comprises the following steps: (1) obtaining immune cells from a subject; (2) treating the immune cells with a first culture medium containing an activator to obtain activated immune cells, (3) introducing a nucleic acid containing a chimeric antigen receptor coding sequence into the activated immune cell to obtain a genetically modified immune cell; (4) culturing the genetically modified immune cells using a second culture medium; Wherein, the first culture medium and / or the second culture medium is a culture medium containing NAD+ or its precursor.

4. The method according to claim 1 or 2, characterized in that: The antigen presenting cells are also loaded with tumor antigens or contain coding sequences of tumor antigens.

5. The method according to claim 4, characterized in that The method comprises the following steps: (1) Loading tumor antigens onto antigen-presenting cells or introducing coding sequences of tumor antigens; (2) obtaining immune effector cells from the subject; (3) Co-culturing the immune effector cells with the antigen presenting cells in a culture medium containing NAD+ or its precursor for a period of time sufficient to activate the immune effector cells, thereby obtaining activated immune effector cells.

6. The method according to claim 5, characterized in that The antigen-presenting cells are cultured using a third culture medium before and / or after being loaded with a tumor antigen or introducing a coding sequence of a tumor antigen, and the third culture medium is a culture medium containing NAD+ or a precursor thereof.

7. The method according to any one of claims 1 to 6, characterized in that The immune cell also contains an antibody coding sequence and / or is capable of expressing an antibody; preferably, the antibody is a single domain antibody, and more preferably, the single domain antibody is a multispecific single domain antibody.

8. The method according to any one of claims 1 to 7, characterized in that The NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof; preferably nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR).

9. The method according to any one of claims 1 to 8, characterized in that The concentration of NAD+ or its precursor in the culture medium is 1-1500 μM, preferably 50-1200 μM.

10. The method according to any one of claims 1 to 8, characterized in that The culture medium further contains flavonoids and / or coenzymes; Preferably, the flavonoid is selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallin, resveratrol, hesperetin, preferably quercetin and / or dihydroquercetin; Preferably, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, and coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

11. The method according to claim 10, characterized in that The concentration of flavonoids in the culture medium is 0-200 μM.

12. The method according to any one of claims 10 to 11, characterized in that The concentration of the coenzyme in the culture medium is 0-200 μM.

13. The method according to any one of claims 3 to 12, characterized in that The subject is a healthy person or a tumor patient, preferably a tumor patient.

14. The method according to any one of claims 3 to 13, characterized in that: Possess any one or more of the following AE characteristics: A. In step (2), the activator is selected from one or more of the following: CD3 antibody, CD28 antibody, 4-1BB antibody, 4-1BBL antigen, B. In step (2), the treatment time is 12-84 hours, preferably 24-72 hours, C. In step (3), the introduction method is electroporation. D. In step (3), the nucleic acid containing the polypeptide coding sequence is a nucleic acid construct, such as an expression vector. Preferably, the expression vector is a non-viral vector. E. In step (4), the culture time is 3-10 days.

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