WO2024092629A1 - 烟酰胺单核苷酸增强car-t细胞寿命的用途 - Google Patents

烟酰胺单核苷酸增强car-t细胞寿命的用途 Download PDF

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WO2024092629A1
WO2024092629A1 PCT/CN2022/129589 CN2022129589W WO2024092629A1 WO 2024092629 A1 WO2024092629 A1 WO 2024092629A1 CN 2022129589 W CN2022129589 W CN 2022129589W WO 2024092629 A1 WO2024092629 A1 WO 2024092629A1
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钟晓松
白玥
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卡瑞济(北京)生命科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • the present invention relates to the field of CAR-T cell immunotherapy, in particular, the present invention relates to CAR-T cells with enhanced efficacy and lifespan, and the use of nicotinamide mononucleotide to enhance the efficacy and lifespan of CAR-T cells. In addition, the present invention also relates to the use of these cells and nicotinamide mononucleotide in treating tumors.
  • Chimeric antigen receptor T (CAR-T) cell therapy is considered an effective solution for the treatment of relapsed or refractory tumors, especially hematological malignancies.
  • CAR-T cell therapy there are multiple challenges in treating tumors with CAR-T cell therapy, such as poor T cell persistence, cell senescence, and T cell exhaustion, which make long-term tumor remission still uncommon and most patients prone to relapse.
  • Cell senescence is characterized by reduced telomerase length, ⁇ -galactosidase aggregation, and increased apoptosis. Improving the persistence of CAR-T cells is crucial to their in vivo efficacy, which is determined by the proliferation ability and cell death of CAR-T cells.
  • Telomeres are nucleoprotein complexes composed of thousands of bases of TTAGGG repeats, located at the ends of eukaryotic chromosomes and protecting chromosome stability. Telomere length has been shown to be an important feature of aging.
  • Cell senescence is a complex physiological and pathological process involving multiple genes under the regulation of the cell cycle. Senescent cells have significant characteristics. Their cells usually become larger in size. ⁇ -galactosidase (SA- ⁇ -Gal) is a typical aging biomarker, and its expression in senescent cells increases significantly. By detecting SA- ⁇ -gal, the aging of cells or tissues can be observed under an ordinary optical microscope. Cell apoptosis is one of the important indicators of cell senescence.
  • SA- ⁇ -Gal ⁇ -galactosidase
  • NMN (nicotinamide mononucleotide) is a precursor of NAD+ (nicotinamide adenine dinucleotide), a biologically active nucleotide formed by the reaction of a phosphate group with a nucleoside containing ribose and nicotinamide.
  • NAD+ nicotinamide adenine dinucleotide
  • NMN is known to boost cellular NAD+ production and counteract age-related pathologies associated with decreased tissue NAD+ levels, including cardiovascular disease, neurodegenerative diseases, metabolic diseases, and others. Aging is associated with cellular NAD+ depletion, and new evidence suggests that systemic NMN administration in mice effectively enhances NAD+ biosynthesis in various peripheral tissues.
  • NAD+ also has a key role in regulating the activity of NAD+-consuming enzymes, sirtuins, which are involved in the regulation of cellular aging processes.
  • sirtuins are involved in the regulation of cellular aging processes.
  • enhancing NAD+ biosynthesis by NMN treatment reversed age-related dysfunction in multiple organs, including the eye, skeletal muscle, and peripheral arteries.
  • Sirtuins are an evolutionarily conserved family of NAD+-dependent deacetylases and ADP-ribosyltransferases that play important roles in a variety of biological activities. However, Sirtuins can only function in the presence of NAD+, which is present in all living cells. There are seven members of the mammalian Sirtuins family (SIRT1-7), which mainly have deacetylase activity and can deacetylate a variety of proteins, thereby participating in many biological processes such as DNA damage repair, gene transcriptional regulation, apoptosis, metabolism and aging.
  • SIRT1-7 mammalian Sirtuins family
  • Sirt1 is the most intensively studied NAD+-dependent deacetylase, which plays a key role in a wide range of biological events, including metabolism, immune response and aging. Sirt1 can regulate a variety of cellular and organismal processes, including metabolism and aging. Early reports have shown that Sirt1 plays an important role in many normal and abnormal physiological processes, such as calorie restriction-related longevity, metabolism, DNA damage response (DDR), aging and tumorigenesis.
  • DDR DNA damage response
  • T cells are part of the adaptive immune system, characterized by the ability to initiate highly specific immune responses to certain antigens and lead to the formation of immune memory.
  • T cell subsets are important indicators of T cell differentiation, among which T memory stem cells (Tscm) represent a small group of Tcm cells with enhanced proliferation and differentiation characteristics, which have superior self-renewal ability, longevity and proliferation potential. It is believed that the maintenance of long-term immunity depends on Tscm, which is of great significance for cancer immunotherapy.
  • Central memory T cells (Tcm) can quickly trigger effector functions, kill infected cells and secrete inflammatory cytokines, and have better anti-tumor ability in vivo after adoptive transfer.
  • Tcm After secondary antigen exposure, since antigen-specific Tcm can differentiate into effector T cells, Tcm is considered to be the main mediator of maintaining and expanding T cell immunity. Therefore, increasing the ratio of Tscm and Tcm in CAR-T cells is an important aspect of enhancing antigen immune response.
  • NMN can promote the rejuvenation and proliferation of CD19 CAR-T cells by upregulating Sirt1, prolong the lifespan of CD19 CAR-T cells, and maintain the function of CD19 CAR-T cells, thereby advantageously improving the therapeutic efficacy of CAR-T cells in clinical practice.
  • the present invention provides a method for prolonging the lifespan of CAR-T cells and improving the proliferation ability of CAR-T cells, comprising treating the CAR-T cells with NMN.
  • the present invention provides a method for increasing the ratio of Tscm and Tcm in CAR-T cells, comprising treating the CAR-T cells with NMN.
  • the NMN treatment refers to incubating NMN with the CAR-T cells for at least 12, at least 24, at least 36, or at least 48 hours. In a specific embodiment, NMN is incubated with the CAR-T cells for at least 24 hours.
  • the CAR-T cells are treated with 10 ⁇ M-1 mM NMN.
  • the CAR-T cells are treated with 10 ⁇ M-100 ⁇ M NMN.
  • the CAR-T cells are derived from a subject who will receive therapy using genetically modified T cells, or are "derived” or "obtained” from a different subject.
  • the CAR-T cell is a CD19-CAR-T cell.
  • the present invention provides the use of NMN in the preparation of a drug for prolonging the lifespan of CAR-T cells, improving the proliferation ability of CAR-T cells, or increasing the ratio of Tscm and Tcm in CAR-T cells.
  • the present invention provides the use of NMN in the preparation of a medicament for enhancing the efficacy of CAR-T cell immunotherapy.
  • the CAR-T cells are treated with 10 ⁇ M-1 mM NMN. In a specific embodiment, the CAR-T cells are treated with 10 ⁇ M-100 ⁇ M NMN.
  • the NMN treatment refers to incubating NMN with the CAR-T cells for at least 12, at least 24, at least 36, or at least 48 hours. In a specific embodiment, NMN is incubated with the CAR-T cells for at least 24 hours.
  • the CAR-T cells are derived from a subject who will receive therapy using genetically modified T cells, or are "derived” or "obtained” from a different subject.
  • the CAR-T cell is a CD19-CAR-T cell.
  • the present invention provides a CAR-T cell population having prolonged cell lifespan and improved cell proliferation ability.
  • the present invention provides a CAR-T cell population with an increased proportion of Tscm and Tcm.
  • the Tscm and Tcm account for 3.37% and 0.9% of the CAR-T cell population, respectively.
  • the CAR-T cell is a CD19-CAR-T cell.
  • the CAR-T cells are derived from a subject who will receive therapy using genetically modified T cells, or are "derived” or "obtained” from a different subject.
  • the CAR-T cells are treated with the method described in the first aspect.
  • the present invention provides a method for treating cancer, comprising the step of administering the CAR-T cell population disclosed in the third aspect to a subject.
  • the method comprises administering to a subject a population of CAR-T cells comprising a high percentage of Tscm and Tcm.
  • the present invention provides a method for increasing the lifespan of CAR-T cells and improving their proliferation capacity in a cancer subject, comprising treating the CAR-T with NMN.
  • the CAR-T cell is a CD19-CAR-T cell.
  • the CAR-T cells are derived from a subject who will receive therapy using genetically modified T cells, or are "derived” or "obtained” from a different subject.
  • the cancer is a hematological malignancy.
  • the present invention provides a pharmaceutical composition comprising the CAR-T cell population described in the third aspect.
  • Figure 1 shows the effect of NMN on CAR-T cell proliferation and T cell effector function.
  • (a) Flow chart of the experimental protocol. The green arrow indicates that NMN was added on the same day to treat CD19-CAR-T cells. The blue arrow indicates that NALM-6 cells were added on the same day to co-culture with CD19-CAR-T cells. The dotted line indicates non-co-culture.
  • (b) NMN increases the expansion multiple of CAR-T cells.
  • (e) Flow cytometry results of NMN increasing CAR-T cell degranulation.
  • Figure 2 Effects of NMN on CD19 CAR-T cell phenotype and anti-aging.
  • NMN increased the NAD+/NADH ratio in CD19 CAR-T cells. NAD+/NADH levels were measured in the culture medium. Round0 and Round3 represent the number of co-cultures with NALM-6 cells.
  • NMN increased the percentages of Tcm and Tscm.
  • NMN increased the telomere activity of CD19 CAR-T cells.
  • NMN reduced the apoptosis of CD19 CAR-T cells (Scale bar: 100 ⁇ m).
  • NMN reduced the apoptosis of CD19 CAR-T cells. The results were analyzed by one-way ANOVA, and statistical significance was set as *p ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001.
  • FIG. 3 Gene ontology enrichment analysis of differentially expressed genes.
  • DEGs differentially expressed genes
  • FC>2 and adjusted p-value ⁇ 0.05 The horizontal axis represents the fold change, and the vertical axis represents the adjusted p-value.
  • the yellow and blue circles represent up-regulated and down-regulated genes, respectively, and the gene cluster diagram is shown on the right.
  • Protein-protein interaction diagram (c) Protein-protein interaction diagram.
  • FIG. 4 Q-PCR results of NMN regulating the expression of Sirt1 and aging-related genes.
  • NMN increased the expression of Sirt1, Sirt3, Sirt4, Sirt5, Sirt6 and Sirt7 in CD19 CAR-T cells.
  • NMN increased the expression level of Sirt1 in CD19-CAR-T cells.
  • NMN decreased the expression of TP53, NF- ⁇ B and Bax in CD19 CAR-T cells. The results were analyzed by one-way ANOVA, and the statistical significance was set as *p ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 5 shows the results of NMN enhancing the anti-tumor activity of CD19 CAR-T cells in a xenograft mouse model in vivo.
  • Figure 6 shows the action model of NMN in extending the lifespan of CD19 CAR-T cells.
  • Figure 7 shows the effect of different concentrations of NMN on CD19-CAR-T cell proliferation.
  • Figure 7a shows the effect of different concentrations of NMN on CD19-CAR-T cell proliferation observed by detecting relative OD450 values, *p ⁇ 0.01.
  • Figure 7b shows that NMN increased the lytic activity of CD19 CAR-T cells.
  • Figure 7c shows the ELISA test results for detecting INF- ⁇ secretion.
  • Figure 8 shows the denaturing polyacrylamide gel electrophoresis of NMN enhancing the telomerase activity of CD19 CAR-T cells, and the bands show the amplification products of TRAP PCR (i.e., telomerase products).
  • the bottom band (IC) represents the internal PCR control.
  • FIG9 shows the pathway of Sirt1 on the KEGG website.
  • Figure 10 NMN reduces the secretion of IL-6 and TNF- ⁇ by T cells.
  • Figures 10a and b show that NMN reduces the secretion of TNF- ⁇ and IL-6 by T cells, respectively.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” is used, unless otherwise indicated, the situation consisting of the stated elements, integers or steps is also covered.
  • an antibody variable region “comprising” a specific sequence it is also intended to cover the antibody variable region consisting of the specific sequence.
  • CAR-T cell refers to a T lymphocyte that is genetically engineered to express a CAR.
  • the definition of CAR-T cells covers all classes and subclasses of T lymphocytes, including CD4+, CD8+T cells, ⁇ T cells, as well as effector T cells, memory T cells, regulatory T cells, etc.
  • Genetically modified T lymphocytes can be "derived” or “obtained” from a subject who will receive therapy using genetically modified T cells, or they can be “derived” or “obtained” from a different subject.
  • treat refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.
  • the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.
  • prevention includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • the term "effective amount” refers to the amount or dosage of an antibody or conjugate or composition of the invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses.
  • the effective amount can be readily determined by the attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of mammal; body weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.
  • therapeutically effective amount refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time.
  • the therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary according to a variety of factors such as disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to stimulate the desired response in the individual.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%.
  • a measurable parameter e.g., tumor growth rate, tumor volume, etc.
  • the ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system that predicts efficacy in human tumors.
  • prophylactically effective amount refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • composition refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • Peripheral blood from healthy donors was diluted 1:1 with PBS solution, and the diluted peripheral blood was added to the lymphocyte separation agent (MP Biomedicals) and separated by Hypaque-Ficoll low-density centrifugation to prepare peripheral blood mononuclear cells (PBMC) and sort T cells.
  • T cells were transduced with retrovirus, activated with anti-CD3 and anti-CD28 beads according to the manufacturer's method, and transduced with Retronectin (Thermo) for 2 days by adding the virus to the carrier or container coated with Retronectin. Specifically: 0.5ml Retronectin (Thermo, 15ug/ml) was added to a 12-well plate and incubated at room temperature in the dark for 2h.
  • NALM-6 cell line was obtained from ATCC, is a B-cell leukemia cell line, and was cultured in RPMI-1640 medium (Lonza) supplemented with 10% fetal bovine serum (Biosera), 10,000 IU/mL penicillin/10,000 ⁇ g/mL streptomycin (EallBio Life Sciences) at 37°C in a humidified atmosphere incubator with 5% CO 2 .
  • NALM-6 cells were transduced to express firefly luciferase-GFP, and the obtained cells were named NALM-6-Luc cells.
  • NALM-6 cells expressing firefly luciferase-GFP were used as target cells.
  • Effector T cells (E) and target cells (T) were co-cultured in triplicate on 96-well plates, with a total volume of 200 ⁇ l of T cell culture medium per well, containing 1 ⁇ 10 4 target cells, and an E/T ratio of 10:1.
  • Target cells were plated separately at the same cell density to determine maximum luciferase expression.
  • 20 ⁇ l of luciferase substrate (Bright-Glo, Promega) was added to each well. Emitted light was detected on a Lunma IVIS Series III imaging system (Lunma) and quantified using Living Image software (Lunma).
  • NALM-6 cells expressing firefly luciferase-GFP were used as antigen presenting cells. 1 ⁇ 10 5 NALM-6 cells and 1 ⁇ 10 6 CAR-T cells were co-cultured in a 24-well plate containing XVIVO 15, human serum and 200 U IL-2/ml. Every 7 days, cells were counted with trypan blue and T cell culture medium was added to reach a concentration of 1 ⁇ 10 6 cells/ml.
  • Flow cytometry was performed and analyzed on a FACS Canto Plus instrument (BD Biosciences). Flow cytometry was used to detect the expression of CD45RO, CCR7, CD95, and CD27 molecules on the surface of CD19-CAR-T cells.
  • CD3-APC-CY7 (BD Biosciences, 560176); CD4-V450 (BD Biosciences 562424); CD8-FITC (BD Biosciences 555634); CCR7-PECY7 (BD Biosciences, 557648); 45RO-BV605 (BioLegend, 304238); PE-Cy5-labeled mouse anti-human CD95 (BD Biosciences); Alexa Fluo 700-labeled mouse anti-human CD27 (BD Biosciences).
  • the cells to be tested were divided into aliquots of 1 ⁇ 10 5 cells, washed, and incubated in PBS containing 2 ⁇ l of Annexin V-FITC (for early apoptosis detection) and 2 ul of 7-AAD/ml (for late apoptosis detection) in the dark for 15 minutes, and then the samples were analyzed by FAC-Scan flow cytometer.
  • NALM-6 cells and CAR-T cells treated with NMN were co-cultured at an E/T ratio of 10:1 for 24 hours, or NALM-6 cells and CAR-T cells not treated with NMN were co-cultured for 24 hours.
  • the culture medium from the co-culture was collected and centrifuged at 1500 rpm to collect the supernatant.
  • IFN- ⁇ , TNF- ⁇ , and IL-6 cytokines in the supernatant were measured using a commercial ELISA kit (R&D system) according to the manufacturer's instructions.
  • TRAP PCR was performed to amplify telomeres in a 50 ⁇ l volume containing the following components: 10X TRAP reaction buffer, 50X telomere dNTP mixture (dATP, dGTP, dTTP, dTCP), 1 ⁇ l TS Primer, 1 ⁇ l TRAP Primer Mix, 2 units of Taq polymerase, and ddH 2 O. 1 amole TSNT oligo was used as an internal quantification control.
  • PCR cycling conditions were as follows: 94°C 30s, followed by 32 cycles of 94°C 30s, 59°C 30s, and 72°C 60s.
  • TRAP PCR products were separated on a 12% PAGE gel in non-denaturing 1 ⁇ TBE buffer, and the amplified telomerase product was detected in a Gene Tools imaging system (SYNGEN).
  • the minimum band of the telomerase product was 50 bp, and the internal control (IC) band was 36 bp.
  • Genomic DNA was extracted from CD19 CAR-T cells treated with/without NMN using FastPure Blood DNA Isolation Mini Kit V2 (Vazyme, Nanjing Vazyme Biotech Co., Ltd, China) according to the manufacturer’s manual. DNA concentration was determined using NanoDrop 2000 spectrophotometer Thermo Fisher Scientific, Waltham, MA, USA.
  • telomere length was assessed using primers published in the literature Frequency of TERT Promoter Mutations in Real-World Analysis of 2,092 Thyroid Carcinoma Patients.
  • telomere length was measured by comparing the ratio of T repeat copy number to S copy number, expressed as the telomere length (T/S) ratio.
  • RNA was first extracted from 1 ⁇ 10 6 cells using TRIZOL reagent (Invitrogen), and the quantity and purity of RNA were measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). The extracted total RNA was then reverse transcribed to synthesize cDNA using a Prime Script RT Master Mix Kit (Thermo Fisher Scientific).
  • the primers for amplifying SIRT1 can be found in the literature The Role of Epigenetic Regulator SIRT1 in Balancing the Homeostasis and Preventing the Formation of Specific "Soil” of Metabolic Disorders and Related Cancers,
  • the PCR cycle conditions were 94°C for 2 min, 55°C for 1 min, and 72°C for 1 min, for a total of 35 cycles.
  • the samples were separated on 1% agarose gel and visualized with ethidium bromide.
  • the reverse transcription product was used as a template for Q-PCR amplification using SYBR Green PCR Master Mix (Thermo Fisher Scientific) with gene-specific primers. GAPDH was used as an internal control. The relative gene expression was calculated using the 2- ⁇ Ct method.
  • the primer information used in the Q-PCR analysis is as follows:
  • primers for amplifying sirt7 please refer to the literature Bone marrow mesenchymal stem cell-secreted exosomes carrying microRNA-125b protect against myocardial ischemia reperfusion injury via targeting SIRT7;
  • primers for amplifying TP53 please refer to the literature Empirical single-cell tracking and cell-fate simulation reveal dual roles of p53 in tumor suppression;
  • primers for amplifying NF-KB please refer to the literature PAUF Induces Migration of Human Pancreatic Cancer Cells Exclusively via the TLR4/MyD88/NF-kappaB Signaling Pathway;
  • primers for amplifying GAPDH please refer to the literature TKTL1 Knockdown Impairs Hypoxia-Induced Glucose-6-phosphate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase Overexpression.
  • NALM-6-Luc cells were prepared in 200 ⁇ l of PBS solution and inoculated into 6-12 week old NOD/SCID mice by tail vein injection the day before the experiment (-1 day), and then 2 ⁇ 10 7 CD19 CAR-T cells were injected for 3 consecutive days on days 0-2.
  • NALM-6 cells expressing firefly luciferase-GFP produced uniform tumor burden in mice, and NMN was injected intraperitoneally at 300 mg/kg/day. Mice were injected with luciferin via the tail vein every week for bioluminescence imaging and luminescence was quantified using IVIS Lunma Series III imaging. All operations were in accordance with the protocol approved by the Animal Care and Use Committee of Beijing Shijitan Hospital, Capital Medical University, and followed all relevant animal use guidelines and ethical regulations.
  • RNA Library Prep Kit #E7530L, NEB was used to generate sequencing libraries according to the manufacturer's method, and each sample containing 2 ⁇ g of total RNA was used as input material.
  • mRNA was purified from total RNA using magnetic beads attached with poly-T oligonucleotides. Fragmentation was performed using divalent cations at high temperature in NEB Next First Strand Synthesis Reaction Buffer (5X).
  • the first-strand cDNA was synthesized using random hexamer primers and RNase H.
  • the second-strand cDNA was then synthesized using buffer, dNTPs, DNA polymerase I, and RNase H.
  • the library fragments were purified using the QiaQuick PCR kit, eluted with EB buffer, and then terminal repair, poly A tail, and adapters were added. To complete the library, the product was recovered and PCR was performed. Index-encoded samples were clustered on the cBot Cluster Generation system using the TruSeq SR Cluster Kit v3-cBot-HS (Illumina Inc.) according to the manufacturer's method. Subsequently, the library was sequenced on the Illumina NovaSeq 6000System platform (Illumina Inc.). The raw data was first processed using a custom Perl script.
  • Clean data were read by removing reads containing poly-N and 5'-adapter contaminants, no 3'-adapter or inserted tags, or containing poly-A, -T, -G or -C, and low-quality reads from the raw data.
  • the online bioinformatics tool DAVID Bioinformatics Resources 6.8 was used to perform gene ontology (GO), KEGG pathway and subcellular localization analysis on the differentially expressed genes.
  • Example 2 NMN treatment can enhance CD19 CAR-T cell proliferation and maintain its effector function
  • CD19 CAR-T cells (Kochenderfer, JN, et al., Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood. 116(20): p. 4099-102) were cultured in X-VIVOTM15 medium at 37°C and counted. On day 0, 100 NMN was added to 1 ⁇ 10 4 CD19 CAR-T cells (E) and incubated at 37°C. NALM-6-Luc cells (T) were added at a 10:1 E/T ratio on days 7, 14, 21, and 28, respectively, and then the same amount of NMN was added weekly according to the time shown in Figure 1a.
  • the cytoplasm of cytotoxic T cells contains high concentrations of cytotoxic granules in the form of vesicles, and CD107a (lysosome-associated membrane protein 1) is the main component of vesicle membrane proteins.
  • CD107a lysosome-associated membrane protein 1
  • CD107a molecules are transported to the cell membrane surface, so CD107a molecules are a marker protein for T cell degranulation, which is directly related to cytotoxic activity and can reflect the level of T cell killing activity.
  • the present application detects CD107a by flow cytometry to calculate the degranulation of T cells, thereby identifying the killing activity of T cells (Fig. 1e).
  • the CD19 CAR-T cells treated with NMN proliferated significantly more than those not treated with NMN.
  • the number of CD19 CAR-T cells treated with NMN increased by 180 times compared with the starting number, while almost all CD19 CAR-T cells not treated with NMN died ( Figure 1b).
  • NMN can promote the proliferation of CD19 CAR-T cells.
  • the Cell Counting Kit-8 (CCK-8) kit to detect the effect of different concentrations of NMN on the proliferation of CD19-CAR-T cells by measuring the absorbance (OD450) at a wavelength of 450nm.
  • NMN-treated CD19 CAR-T cells reduced IL-6 expression but increased IFN- ⁇ expression relative to untreated cells ( Figure 1c and Figure 1d).
  • NMN treatment increased the anti-tumor activity in CD19 CAR-T cells, and NMN-treated and untreated CD19 CAR-T cells showed similar CD107a levels.
  • NMN-treated CD19 CAR-T cells showed higher CD107a levels, thereby having higher T cell killing activity.
  • NMN-treated CD19 CAR-T cells still had killing ability and high INF- ⁇ secretion levels even when untreated CD19 CAR-T cells died ( Figure 7b-7c).
  • the above results indicate that NMN can immediately enhance the anti-tumor activity of CD19 CAR-T cells and effectively delay the senescence of CD19 CAR-T cells.
  • Example 3 NMN treatment increases the percentage of Tscm and Tcm in CAR-T cells and delays cell senescence
  • telomere is essential in T cell division and function, as T cell differentiation, aging, and disease reduce telomerase expression.
  • TRAP PCR results of CD19 CAR-T cells treated with NMN after the third co-culture with 100 ⁇ M NMN, and the results showed that the telomerase activity of CD19 CAR-T cells treated with NMN was significantly higher than that of the control ( Figure 2c).
  • Annexin-V and 7-ADD are markers for evaluating cell apoptosis.
  • the apoptosis of CD19 CAR-T cells treated with NMN was detected by flow cytometry, and the results were subjected to one-way analysis of variance, with statistical significance set as *p ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001.
  • the results showed that NMN treatment resulted in lower apoptosis in CD19 CAR-T cells ( Figure 2e), which may partly explain the higher cell proliferation rate of CD19 CAR-T cells after NMN treatment.
  • NMN has the ability to resist CAR-T cell aging and can increase the lifespan of CD19 CAR-T cells. Similar effects were also observed in animal models (as shown below).
  • Example 4 changes the gene expression profile of CD19 CAR-T cells
  • this example used a high-throughput RNA sequence analysis method to examine the effect of NMN treatment on CAR-T cell gene expression.
  • Example 5 NMN regulates aging-related genes by upregulating Sirt1 expression
  • sirtuins may be necessary for providing protection to CD19 CAR-T cells after NMN administration.
  • Q-PCR we detected the expression differences of Sirt1, Sirt3, Sirt4, Sirt5, Sirt6 and Sirt7 at the mRNA level in CD19 CAR-T cells treated with 100 ⁇ M NMN.
  • TP53 NF- ⁇ B and tumor suppressor TP53.
  • Transcription factor TP53 acts as a hub to convert various stress signals into different cellular outcomes, such as DNA repair, cell cycle arrest, or cell death.
  • TNF- ⁇ and IL-6 are inflammatory factors secreted by cells.
  • the secretion of TNF- ⁇ and IL-6 is the main manifestation of inflammatory response.
  • CD19 CAR-T cells treated with NMN were co-cultured with NALM-6 cells at 37°C for 24 hours, and the TNF- ⁇ level or IL-6 level in the culture supernatant was detected by ELISA.
  • Sirt1 inhibitor EX527 10 ⁇ M
  • NMN reduced the level of TNF- ⁇ (Figure 10a) or IL-6 (Figure 10b) secreted by CAR-T cells.
  • IL-6 Figure 10b
  • NF- ⁇ B is a family of inducible transcription factors that play a central role in the emergence of the inflammation- and senescence-associated secretory phenotype (SASP).
  • NMN delays the senescence of CD19-CAR-T cells by increasing the expression level of Sirt1 and the subsequently regulated related genes. Therefore, NMN, which can effectively intervene in the senescence of CAR-T cells, has important clinical value in CAR-T cell immunotherapy for the treatment of cancer.
  • Example 6 In vivo NMN treatment enhances the antitumor activity of CD19 CAR-T cells in a B-ALL xenograft mouse model
  • NALM-6-Luc cells were subcutaneously injected into NOD-SCID mice one day before the experiment (-1 day) to produce a xenograft mouse model, and the mice were randomly divided into 3 groups: PBMC negative control group (NT), CD19 CAR-T cell treatment group (CD19 CAR-T), CD19 CAR-T cell treatment group treated with NMN (CD19 CAR-T+NMN), 5 mice in each group.
  • PBMC negative control group NT
  • CD19 CAR-T cell treatment group CD19 CAR-T
  • CD19 CAR-T+NMN CD19 CAR-T+NMN
  • CD19 CAR-T cells treated with NMN showed a more persistent tumor inhibitory effect in mice.
  • the tumor burden of mice treated with CD19 CAR-T cells treated with NMN was significantly lower than that of mice treated with CD19 CAR-T cells not treated with NMN. It can be seen that NMN can increase the lifespan of CD19 CAR-T cells and thereby regulate the treatment of B lymphocytic leukemia, and therefore can play an important clinical value in CAR-T cell immunotherapy for the treatment of cancer.

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Abstract

本发明涉及具有增强的功效和寿命的CAR-T细胞，烟酰胺单核苷酸增强CAR-T细胞功效和寿命的用途。此外，本发明还涉及这些细胞以及烟酰胺单核苷酸在治疗肿瘤中的用途。

Description

烟酰胺单核苷酸增强CAR-T细胞寿命的用途 技术领域
本发明涉及CAR-T细胞免疫疗法领域，具体地，本发明涉及具有增强的功效和寿命的CAR-T细胞，烟酰胺单核苷酸增强CAR-T细胞功效和寿命的用途。此外，本发明还涉及这些细胞以及烟酰胺单核苷酸在治疗肿瘤中的用途。
背景技术
嵌合抗原受体T(CAR-T)细胞疗法被认为是治疗复发或难治性肿瘤，特别是血液系统恶性肿瘤的有效解决方案。然而，用CAR-T细胞疗法治疗肿瘤存在多重挑战，例如T细胞持久性差、细胞衰老、T细胞耗竭等，使得肿瘤的长期缓解仍不常见，并且多数患者容易复发。细胞衰老的特征是端粒酶长度减少、β-半乳糖苷酶聚集和细胞凋亡增加。提高CAR-T细胞的持久性对其体内功效至关重要，这是由CAR-T细胞的增殖能力和细胞死亡共同决定的。
端粒是由数千个碱基的TTAGGG重复组成的核蛋白复合物，位于真核生物染色体的末端，可保护染色体的稳定性。端粒长度已被证明是衰老的一个重要特征。
细胞衰老是细胞周期调控下多基因参与的复杂的生理病理过程，衰老细胞有着显著的特性，其细胞通常体积变大，β-半乳糖苷酶(SA-β-Gal)是典型的衰老生物标志物，其在衰老细胞中的表达明显增加。通过检测SA-β-gal可以在普通的光学显微镜下观测细胞或组织的衰老情况。细胞凋亡是细胞衰老的重要指标之一。
NMN(烟酰胺单核苷酸)是NAD+(烟酰胺腺嘌呤二核苷酸)的前体，是一种具有生物活性的核苷酸，由磷酸基团与含有核糖和烟酰胺的核苷反应形成。已知NMN可促进细胞NAD+的产生并抵消与组织NAD+水平下降相关的与年龄相关的病理，包括心血管疾病、神经退行性疾病、代谢疾病等。衰老与细胞NAD+耗竭有关，新证据表明，在小鼠中全身性NMN给药有效地增强了各种外周组织中NAD+的生物合成。NAD+在调节NAD+消耗酶-sirtuins的活性方面也具有关键作用，这些酶与细胞衰老过程的调节有关。在老年小鼠中，通过NMN治疗增强NAD+生物合成可逆转多个器官中与年龄相关的功能障碍，包括眼睛、骨骼肌 和外周动脉。
Sirtuins是一个进化上保守的NAD+依赖性去乙酰酶和ADP-核糖基转移酶家族，在多种生物活动中发挥重要作用。但是，Sirtuins仅在存在于所有活细胞中的NAD+存在下才能发挥功能。哺乳动物Sirtuins家族成员共有7个(SIRT1-7)，其主要具有去乙酰化酶的活性，可以使多种蛋白发生去乙酰化，进而参与DNA的损伤修复、基因的转录调控、细胞凋亡、代谢及衰老等诸多生物进程。其中，Sirt1是研究最深入的NAD+依赖性去乙酰酶，在广泛的生物事件中发挥关键作用，包括新陈代谢、免疫反应和衰老。Sirt1可调节多种细胞和有机体过程，包括新陈代谢和衰老。早期报告表明，Sirt1在许多正常和异常生理过程中发挥重要作用，例如热量限制相关的长寿、新陈代谢、DNA损伤反应(DDR)、衰老和肿瘤发生。
T细胞是适应性免疫系统的一部分，其特点是能够发起对某些抗原高度特异性的免疫反应并导致免疫记忆的形成。T细胞亚群是T细胞分化的重要指标，其中T记忆干细胞(Tscm)代表一小群具有增强的增殖和分化特征的Tcm细胞，其具有优越的自我更新能力、长寿和增殖潜力，认为长期免疫的维持依赖于Tscm，这对癌症免疫治疗具有重要意义。中央记忆型T细胞(Tcm)可以迅速触发效应功能，杀死受感染的细胞并分泌炎性细胞因子，在过继转移后在体内具有更好地抗肿瘤能力。在二次抗原暴露后，由于抗原特异性Tcm能够分化成效应T细胞，因此认为Tcm是维持和扩大T细胞免疫的主要介质。因此，增加Tscm和Tcm在CAR-T细胞中的比例是增强抗原免疫反应的一个重要方面。
在目前的CAR-T细胞疗法中，仍然迫切需要提高CAR-T细胞的治疗效果，尤其是迫切需要提高对血液系统恶性肿瘤的治疗效果。本发明满足了这些需求。
在本发明中，申请人发现NMN可以通过上调Sirt1来促进CD19 CAR-T细胞复壮和增殖，延长了CD19 CAR-T细胞的寿命，同时维持了CD19 CAR-T细胞的功能，从而在有利地提高了CAR-T细胞在临床上的治疗功效。
发明内容
第一方面，本发明提供了一种延长CAR-T细胞寿命、改善CAR-T细胞增殖能力的方法，包括用NMN处理所述CAR-T细胞。
在一个实施方案中，本发明提供了一种增加Tscm和Tcm在CAR-T细胞中 的比例的方法，包括用NMN处理所述CAR-T细胞。
在一个实施方案中，所述NMN处理是指将NMN与所述CAR-T细胞共孵育至少12、至少24、至少36、至少48小时。在一个具体的实施方案中，将NMN与所述CAR-T细胞共孵育至少24小时。
在一个具体实施方案中，使用10μM-1mM的NMN处理所述CAR-T细胞。
在一个具体实施方案中，使用10μM-100μM的NMN处理所述CAR-T细胞。
在一个具体实施方案中，所述CAR-T细胞来源于将接受使用基因修饰的T细胞治疗的受试者，或者来源于不同的受试者“衍生”或“获得”。
在一个具体实施方案中，所述CAR-T细胞是CD19-CAR-T细胞。
第二方面，本发明提供了NMN在制备用于延长CAR-T细胞寿命、改善CAR-T细胞增殖能力、或提高Tscm和Tcm在CAR-T细胞中的比例的药物中的用途。
在一个实施方案中，本发明提供了NMN在制备用于增强CAR-T细胞免疫疗法疗效的药物中的用途。
在一个具体实施方案中，使用10μM-1mM的NMN处理所述CAR-T细胞。在一个具体实施方案中，使用10μM-100μM的NMN处理所述CAR-T细胞。
在一个实施方案中，所述NMN处理是指将NMN与所述CAR-T细胞共孵育至少12、至少24、至少36、至少48小时。在一个具体的实施方案中，将NMN与所述CAR-T细胞共孵育至少24小时。
在一个具体实施方案中，所述CAR-T细胞来源于将接受使用基因修饰的T细胞治疗的受试者，或者来源于不同的受试者“衍生”或“获得”。
在一个具体实施方案中，所述CAR-T细胞是CD19-CAR-T细胞。
第三方面，本发明提供了一种CAR-T细胞群，其具有延长的细胞寿命和改善的细胞增殖能力。
在一个实施方案中，本发明提供了一种Tscm和Tcm占比提高的CAR-T细胞群。在一个具体实施方案中，所述Tscm和Tcm占CAR-T细胞群的3.37％和0.9，
在一个具体实施方案中，所述CAR-T细胞是CD19-CAR-T细胞。
在一个具体实施方案中，所述CAR-T细胞来源于将接受使用基因修饰的T细胞治疗的受试者，或者来源于不同的受试者“衍生”或“获得”。
在一个具体实施方案中，所述CAR-T细胞经过第一方面所述方法的处理。
第四方面，本发明提供了一种治疗癌症的方法，包括将第三方面公开的CAR-T细胞群施用给受试者的步骤。
在一个具体实施方案中，所述方法包括向受试者施用包含高Tscm和Tcm百分比的CAR-T细胞群。
在一个实施方案中，本发明提供了一种增加癌症受试者中CAR-T细胞寿命并改善其增殖能力的方法，包括用NMN处理所述CAR-T。
在一个具体实施方案中，所述CAR-T细胞是CD19-CAR-T细胞。
在一个具体实施方案中，所述CAR-T细胞来源于将接受使用基因修饰的T细胞治疗的受试者，或者来源于不同的受试者“衍生”或“获得”。
在一个具体实施方案中，所述癌症是血液系统恶性肿瘤。
第五方面，本发明提供了一种药物组合物，其包含第三方面所述的CAR-T细胞群。
附图说明
图1显示了NMN对CAR-T细胞增殖和T细胞效应功能的影响。(a)实验方案流程图。绿色箭头表示当天加入NMN以处理CD19-CAR-T细胞。蓝色箭头表示当天加入NALM-6细胞与CD19-CAR-T细胞共培养。虚线表示非共培养。(b)NMN增加CAR-T细胞的扩增倍数。(c.d)IFN-γ水平和IL-6水平的ELISA检测结果。(e)NMN增加CAR-T细胞脱颗粒的流式细胞术检测结果。(f)NMN增加CAR-T细胞对靶细胞的裂解活性。结果通过单因素方差分析进行分析，统计显著性设定为*p<0.05,**P<0.01,***p<0.001。
图2 NMN对CD19 CAR-T细胞表型和抗衰老的影响。(a)NMN提高了CD19 CAR-T细胞中的NAD+/NADH比率。NAD+/NADH水平在培养基中测量。Round0和Round3代表与NALM-6细胞共培养的次数。(b)NMN提高了Tcm和Tscm百分比。(c)NMN提高了CD19 CAR-T细胞端粒活性。(d)NMN降低了 CD19 CAR-T细胞的凋亡(比例尺：100μm)。(e)NMN降低了CD19 CAR-T细胞的凋亡。结果通过单因素方差分析进行分析，统计显著性设定为*p<0.05,**P<0.01,***p<0.001。
图3.差异表达基因的基因本体富集分析。(a)差异表达基因(DEG)的火山图(FC>2和调整后的p值<0.05)。横轴表示倍数变化，纵轴表示调整后的p值。黄色和蓝色圆圈分别表示上调和下调基因，基因簇图如右图。(b)对差异表达的基因进行GO分析的结果。(c)蛋白质-蛋白质相互作用图。
图4.NMN调节Sirt1和衰老相关基因的表达的Q-PCR结果。(a)NMN提高了CD19 CAR-T细胞中Sirt1、Sirt3、Sirt4、Sirt5、Sirt6和Sirt7的表达。(b-c)NMN增加了CD19-CAR-T细胞中Sirt1的表达水平。(d.e.f)NMN降低了CD19 CAR-T细胞中TP53、NF-κB和Bax的表达。结果通过单因素方差分析进行分析，统计显著性设定为*p<0.05,**P<0.01,***p<0.001。
图5显示NMN体内增强异种移植小鼠模型中CD19 CAR-T细胞的抗肿瘤活性的结果。(a)NMN处理小鼠的操作流程图。(b)小鼠负荷肿瘤经量化的生物发光信号结果。(c.d)小鼠负荷肿瘤以荧光值表示的统计结果。
图6显示NMN延长CD19 CAR-T细胞的寿命的作用模型。
图7显示了不同浓度的NMN对CD19-CAR-T细胞增殖的作用。图7a显示了通过检测相对OD450值观测的不同浓度NMN对CD19-CAR-T细胞增殖的影响，*p<0.01。图7b显示NMN提高了CD19 CAR-T细胞的裂解活性。图7c显示检测INF-γ分泌的ELISA检测结果。
图8显示了NMN增强CD19 CAR-T细胞的端粒酶活性的变性聚丙烯酰胺凝胶电泳图，条带显示TRAP PCR的扩增产物(即端粒酶产物)。底部条带(IC)代表内部PCR对照。
图9显示KEGG网站上Sirt1的通路。
图10 NMN减少了T细胞分泌的IL-6、TNF-α。图10a和b分别显示NMN减少了T细胞分泌的TNF-α和IL-6。
具体实施方式
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说 明特定实施方案之用，而不意欲为限制性的。
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“CAR-T细胞”是指经基因工程化以表达CAR的T淋巴细胞。CAR-T细胞的定义涵盖T淋巴细胞的所有类别和亚类，包括CD4+、CD8+T细胞、γδT细胞以及效应T细胞、记忆T细胞、调节性T细胞等。经基因修饰的T淋巴细胞可以从将接受使用基因修饰的T细胞治疗的受试者“衍生”或“获得”，或者它们可以从不同受试者“衍生”或“获得”。
术语“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体用来延缓疾病发展或用来减慢疾病的进展。
术语“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“有效量”指本发明的抗体或缀合物或组合物的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动 物的物种；体重、年龄和一般健康状况；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
术语“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其缀合物或组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率、肿瘤体积等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。
术语“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
实施例
以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。
下面实施例中述及的实验方法，如无特殊说明，均为本领域采用默认参数、步骤等的常规方法；所用的实验材料，如无特殊说明，均为市售产品。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照相应产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道 购买得到的常规产品。
实施例1材料制备、基本实验方法及鉴定
1.细胞培养和NMN处理
用PBS溶液1:1稀释来自健康供体的人外周血，将稀释的外周血加在淋巴细胞分离剂(MP Biomedicals)上通过Hypaque-Ficoll低密度离心分离，制备外周血单个核细胞(PBMC)并分选T细胞。采用逆转录病毒对T细胞进行转导，根据制造商的方法，用抗CD3和抗CD28珠激活T细胞，并通过将病毒加入到用Retronectin(Thermo)包被载体或容器，用Retronectin转导T细胞2天。具体为：0.5ml Retronectin(Thermo,15ug/ml)加入12孔板，室温避光孵育2h。弃掉上清，加入0.5％human AB血清(Biosera)(PBS配制)，温育30min,弃掉上清。加入0.5ml T细胞(1.6×106/ml)与0.5ml病毒液体，用封口膜封闭孔板，700g，离心1h后，放入37℃培养箱培养，获得抗原特异性的基因修饰T细胞。转导后6天，将T细胞在含有5％Gem Cell TM人AB血清(Gemini Bio)和IL-2(SL PHARM)的X-VIVOTM15无血清培养基(Lonza)中培养。用100μM NMN(St.Louis,MO,USA N3501)处理T细胞进行每周刺激。
本研究经北京世纪坛医院机构审查委员会批准，并获得所有参与者的知情同意。
2.细胞系
NALM-6细胞株获自ATCC，是一种B-细胞性白血病细胞株，在补充有10％胎牛血清(Biosera)、10,000IU/mL青霉素/10,000μg/mL链霉素(EallBio Life Sciences)的RPMI-1640培养基(Lonza)中于37℃、5％CO 2、湿润气氛的培养箱中培养。转导NALM-6细胞以表达萤火虫荧光素酶-GFP，获得的细胞命名为NALM-6-Luc细胞。
3.细胞毒性测定
以表达萤火虫荧光素酶-GFP的NALM-6细胞作为靶细胞。在96孔板上进行效应细胞T细胞(E)和靶细胞(T)共培养，一式三份，其中每孔T细胞培养基总体积为200μl，包含1×10 4个靶细胞，E/T比为10:1。以相同细胞密度的靶细胞 单独铺板以确定最大荧光素酶表达。24小时后，向每孔加入20μl荧光素酶底物(Bright-Glo，Promega)。在Lunma IVIS系列III成像系统(Lunma)上检测发射光，并使用Living Image软件(Lunma)进行量化。
4.抗原提供和增殖测定
表达萤火虫萤光素酶-GFP的NALM-6细胞用作抗原呈递细胞。将1×10 5NALM-6细胞和1×10 6CAR-T细胞在包含XVIVO 15、人血清和200U IL-2/ml的24孔板中共培养。每7天，用台盼蓝对细胞进行计数，并添加T细胞培养基以达到1×10 6个细胞/ml的浓度。
5.流式细胞术
在FACS Canto Plus仪器(BD Biosciences)上进行流式细胞术并进行分析。采用流式细胞仪检测CD19-CAR-T细胞表面CD45RO、CCR7、CD95和CD27分子的表达。以下抗体用于流式细胞术检测相应抗原表达：CD3-APC-CY7(BD Biosciences，560176)；CD4-V450(BD生物科学562424)；CD8-FITC(BD生物科学555634)；CCR7-PECY7(BD Biosciences,557648)；45RO-BV605(生物传奇，304238)；PE-Cy5标记的小鼠抗人CD95(BD Biosciences)；Alexa Fluo 700标记的小鼠抗人CD27(BD Biosciences)。当采用流式细胞术检测细胞凋亡时，将待测细胞配置成1×10 5个细胞的等分试样，洗涤，在含有2μl膜联蛋白V-FITC(检测早期凋亡)和2ul 7-AAD/ml(检测晚期细胞凋亡)的PBS中于黑暗中孵育15分钟，然后通过FAC-Scan流式细胞仪分析样品。
6.酶联免疫吸附检测(ELISA检测)
分别以10:1的E/T比共培养NALM-6细胞和用NMN处理的CAR-T细胞24小时，或共培养NALM-6细胞和未用NMN处理的CAR-T细胞24小时。分别收集来自共培养的培养基并以1500rpm离心以收集上清液。根据制造商的说明，使用商业ELISA试剂盒(R&D system)测量上清液中的IFN-γ、TNF-α和IL-6细胞因子。
7.NAD/NADH比率测定
用冷PBS溶液收获和清洗1×10 6个待测细胞。通过低速离心在管中沉淀细 胞并丢弃上清液。用匀浆器研磨15-30次匀浆细胞，加入100ul NAD提取缓冲液用于NAD测定，或加入100ul NADH提取缓冲液用于NADH测定。离心细胞并将提取的NAD/NADH上清液转移到新试管中，将提取物在60℃加热5min，然后加入20ul Assay Buffer和100ul反向提取缓冲液(opposite extraction buffer)以中和提取物。保持在冰上进行NAD/NADH测定。用酶板分析仪测定吸光度值。
8.端粒酶活性测量
分别收集1×10 6个用NMN处理的CD19 CAR-T细胞和收集1×10 6个未用NMN处理的CD19 CAR-T细胞，并在补充有0.1mM PMSF的200μl CHAPS裂解缓冲液中裂解收集的细胞。在包含以下成分的50μl体积中进行TRAP PCR以扩增端粒：10X TRAP反应缓冲液、50X端粒dNTP混合物(dATP、dGTP、dTTP、dTCP)、1μl TS Primer、1μl TRAP Primer Mix、2单位Taq聚合酶和ddH 2O。1amole TSNT oligo作为内部量化对照。PCR循环条件如下：94℃30s，然后是94℃ 30s、59℃ 30s、72℃ 60s的32个循环。TRAP PCR产物在非变性1×TBE缓冲的12％PAGE凝胶上分离，并在Gene Tools成像系统(SYNGEN)中检测到扩增的端粒酶产物。端粒酶产物的最小条带为50bp，内控(IC)条带为36bp。
9.DNA分离和端粒长度测量
根据制造商的手册，使用FastPure Blood DNA Isolation Mini Kit V2(Vazyme，南京Vazyme Biotech Co.，Ltd，China)从用/不用NMN处理的CD19 CAR-T细胞中提取基因组DNA。使用NanoDrop 2000分光光度计Thermo Fisher Scientific，Waltham，MA，USA测定DNA浓度。
实时定量PCR用于评估端粒长度。简而言之，采用文献Frequency of TERT Promoter Mutations in Real-World Analysis of 2,092Thyroid Carcinoma Patients中公开的引物扩增CAR-T细胞的端粒(T)和(S)。
通过比较T重复拷贝数和S拷贝数的比率来测量相对端粒长度，表示为端粒长度(T/S)比率。
10.SA-β-gal染色
使用SA-β-gal染色试剂盒(Cell Signaling Technology)研究了用/不用NMN处理的CD19CAR-T细胞的SA-β-gal染色。根据制造商的说明，将NMN处理或未处理的CD19CAR-T细胞在1.5ml试管中固定和洗涤，并在37℃下与染色溶液一起孵育过夜。使用相差显微镜(Olympus)对每个孔的四个随机视野进行成像。SA-β-Gal染色呈深蓝色，计算SA-β-gal阳性细胞的平均百分比。
11.逆转录-PCR和定量实时聚合酶链式反应(Q-PCR)
按照制造商的说明，首先用TRIZOL试剂(Invitrogen)从1×10 6细胞中提取总RNA，使用Nanodrop One分光光度计(Thermo Fisher Scientific)测量RNA的数量和纯度。然后使用Prime Script RT Master Mix Kit(Thermo Fisher Scientific)对提取的总RNA进行逆转录合成cDNA。
其中扩增SIRT1的引物请参考文献The Role of Epigenetic Regulator SIRT1 in Balancing the Homeostasis and Preventing the Formation of Specific"Soil"of Metabolic Disorders and Related Cancers，
PCR循环条件为94℃2min、55℃1min、72℃1min，共35个循环。样品在1％琼脂糖凝胶上分离，并用溴化乙锭观察。
将逆转录产物用作模板，使用带有基因特异性引物的SYBR Green PCR Master Mix(Thermo Fisher Scientific)进行Q-PCR扩增。GAPDH用作内部对照。使用2-ΔCt方法计算相对基因表达。Q-PCR分析中使用的引物信息如下：
扩增sirt3的引物请参考文献Rescue of Mitochondrial SIRT3 Ameliorates Ischemia-like Injury in Human Endothelial Cells；
扩增sirt4的引物请参考文献Deacetylation of MTHFD2 by SIRT4 senses stress signal to inhibit cancer cell growth by remodeling folate metabolism；
扩增sirt5的引物请参考文献Sirtuin5 protects colorectal cancer from DNA damage by keeping nucleotide availability；
扩增sirt6的引物请参考文献Sirt6 deficiency contributes to mitochondrial fission and oxidative damage in podocytes via ROCK1-Drp1 signalling pathway；
扩增sirt7的引物请参考文献Bone marrow mesenchymal stem cell-secreted exosomes carrying microRNA-125b protect against myocardial ischemia reperfusion  injury via targeting SIRT7；
扩增TP53的引物请参考文献Empirical single-cell tracking and cell-fate simulation reveal dual roles of p53 in tumor suppression；
扩增NF-KB的引物请参考文献PAUF Induces Migration of Human Pancreatic Cancer Cells Exclusively via the TLR4/MyD88/NF-kappaB Signaling Pathway；
扩增BAX的引物请参考文献The role of FASL,BCL-2 and BAX polymorphisms in brazilian patients with prostate cancer and benign prostatic hyperplasia；
扩增GAPDH的引物请参考文献TKTL1Knockdown Impairs Hypoxia-Induced Glucose-6-phosphate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase Overexpression。
12.动物实验
将2×10 6个NALM-6-Luc细胞配置在200μl的PBS溶液中，并在实验前一天(-1天)通过尾静脉注射向6-12周龄的NOD/SCID小鼠接种，然后在第0-2天连续3天注射2×10 7CD19 CAR-T细胞。表达萤火虫萤光素酶-GFP的NALM-6细胞在小鼠中产生均匀的肿瘤负荷，NMN以300mg/kg/天进行腹膜内注射。每周通过尾静脉向小鼠注射荧光素来进行生物发光成像并使用IVIS Lunma Series III成像量化发光，所有操作都根据首都医科大学北京世纪坛医院动物护理和使用委员会批准的方案，遵循所有相关的动物使用指南和伦理法规。
13.RNA序列分析
使用
Figure PCTCN2022129589-appb-000001
RNA Library Prep Kit(#E7530L,NEB)按照制造商提供的方法，每个含有2μg总RNA的样品作为输入材料生成测序文库。简而言之，使用附有poly-T寡核苷酸的磁珠从总RNA中纯化mRNA。在NEB Next First Strand Synthesis Reaction Buffer(5X)中，在高温下使用二价阳离子进行片段化。使用随机六聚体引物和RNase H合成第一链cDNA。随后使用缓冲液、dNTP、DNA聚合酶I和RNase H合成第二链cDNA。文库片段用QiaQuick PCR试剂盒纯化，用EB缓冲液洗脱，然后实施了终端修复、添加Poly A尾和适配子(adapters)。为了完成文库，回收产品并进行了PCR。根据制造商的方法，使用 TruSeq SR Cluster试剂盒v3-cBot-HS(Illumina Inc.)在cBot Cluster Generation系统上对索引编码样本进行聚类。随后，在Illumina NovaSeq 6000System平台(Illumina Inc.)对该文库进行测序。原始数据首先使用自定义Perl脚本进行处理。通过从原始数据中去除含有poly-N和5'-接头污染物、没有3'-接头或插入标签、或包含poly-A、-T、-G或-C的读数，以及低质量读数来读取清洁数据(清洁读数)。
使用在线生物信息学工具：DAVID Bioinformatics Resources 6.8，对差异表达的基因进行基因本体论(GO)、KEGG通路和亚细胞定位分析。
14.统计分析
两组之间差异的统计显著性通过2尾配对或非配对t检验进行评估。多于两组的比较通过单因素方差分析和多重比较测试进行。数据显示为平均值±标准差(SD)。差异标记为NS，P>0.05；*P<0.05；**P<0.01，和***P<0.001。从研究中获得的所有数据均使用SPSS 22.0(IBM,USA)进行分析。
实施例2.NMN治疗可增强CD19 CAR-T细胞增殖并维持其效应器功能
为了研究NMN对CAR-T细胞的功能，我们研究了NMN对CD19 CAR-T细胞的增殖和靶细胞杀伤能力的影响。
在37℃、X-VIVOTM15培养基中培养CD19 CAR-T细胞(Kochenderfer,J.N.,et al.,Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19.Blood.116(20):p.4099-102)并计数。第0天，向1×10 4的CD19 CAR-T细胞(E)中加入100NMN，37℃孵育，按照图1a所示时间，在第7天、14天、21天和28天分别按照10:1的E/T比例加入NALM-6-Luc细胞(T)共培养，之后按照图1a所示时间依次每周加入等量的NMN。
CD19-CAR-T细胞与NALM-6-Luc细胞共培养28天。在与NALM-6-Luc靶细胞以10:1的E:T比率共培养后，每周用100NMN处理细胞。采用台盼蓝排斥法确定存活的CAR-T细胞数，并计算CAR-T细胞的扩增倍数(图1b)。在24小时后收集上清液，通过ELISA方法(E:T＝0.5:1)评估细胞表达的IFN-γ水平和IL-6水平。在CD19 CAR-T细胞与NALM-6-Luc细胞系(E:T比率＝0.5:1)共 培养24小时后，测量存活的NALM-6细胞所产生的荧光素酶信号。结果通过单因素方差分析进行分析，统计显著性设定为*p<0.05,**P<0.01,***p<0.001(图1f)。
细胞毒T细胞细胞质内含有高浓度以囊泡形式存在的细胞毒性颗粒，CD107a(溶酶体相关膜蛋白l)是囊泡膜蛋白的主要成分。在杀伤靶细胞时，T细胞的毒性颗粒将到达细胞膜并与细胞膜融合，引起颗粒内容物释放导致靶细胞死亡，此时CD107a分子被转运到细胞膜表面，因此CD107a分子是T细胞脱颗粒的一种标志蛋白，与细胞毒活性直接相关，可反映T细胞杀伤活性水平。本申请通过流式细胞术检测CD107a从而计算T细胞的脱颗粒，由此鉴定T细胞的杀伤活性(图1e)。
结果如图1所示，经NMN处理的CD19 CAR-T细胞比未经NMN处理的CD19 CAR-T细胞增殖明显更多。与NALM-6细胞第三次共培养后，NMN处理的CD19 CAR-T细胞数量比起始数量增加了180倍，而未经NMN处理的CD19 CAR-T细胞几乎全部死亡(图1b)。可见，NMN可以促进CD19 CAR-T细胞增殖。接着，我们采用Cell Counting Kit-8(CCK-8)试剂盒通过测量450nm波长处的吸光度(OD450)检测了不同浓度的NMN对CD19-CAR-T细胞增殖的作用。CCK8检测结果表明，不同浓度的NMN均增加了CD19-CAR-T细胞的增殖，其中100μM的NMN增殖作用最大，在处理96小时时，NMN处理后CD19 CAR-T细胞的OD450值是未处理组的两倍(图7a)，*p<0.01。与NALM-6第三次共培养后，NMN处理的CD19 CAR-T细胞仍然表现出高特异性裂解(图7b)。
100μM NMN处理之后，我们观察到经NMN处理的CD19 CAR-T细胞相对于未处理的细胞减少了IL-6的表达，却增加了IFN-γ的表达(图1c和图1d)。如图1e和图1f所示，NMN处理增加了CD19 CAR-T细胞中的抗肿瘤活性，NMN处理和未处理的CD19 CAR-T细胞表现出相似的CD107a水平，甚至在与NALM-6细胞第二次和第三次共培养后，经NMN处理的CD19 CAR-T细胞表现出更高的CD107a水平，由此具有更高的T细胞杀伤活性。令人鼓舞的是，在与NALM-6细胞第三次共培养后，即便未经处理的CD19 CAR-T细胞死亡时，经NMN处理的CD19 CAR-T细胞仍然具有杀伤能力和高INF-γ分泌水平(图7b-7c)。上述结果表明，NMN可以立即增强CD19 CAR-T细胞的抗肿瘤活性并 有效延缓CD19 CAR-T细胞的衰老。
实施例3.NMN治疗增加了CAR-T细胞中Tscm、Tcm的百分比并延缓细胞衰老
在本实施例中，为了研究NMN对CAR-T细胞寿命和治疗活性的影响，我们检测了NMN处理的CD19 CAR-T细胞中与细胞寿命相关的指标：NAD/NADH比值、端粒酶活性、SA-β-gal水平和凋亡标记物；以及与T细胞治疗活性和记忆能力相关的指标：Tscm和Tcm在总T细胞中的占比。
随着年龄的增长，小鼠和人类的全身NAD+可用性降低。作为NAD+的前体，NMN过度会导致细胞内NAD+水平的升高。在本研究中，添加的NMN确实增加了CD19 CAR-T细胞中NAD+的表达。具体地，我们检测了经NMN处理的CD19 CAR-T细胞中的NAD/NADH比值，结果发现相对应对照，NMN增加了T细胞中的NAD/NADH比值(图2a)。
端粒酶在T细胞分裂和功能中至关重要，因为T细胞分化、衰老和疾病降低端粒酶表达。在本研究中，我们观察到NMN处理的CD19 CAR-T细胞的端粒长度更长，且端粒酶活性增加。如图8所示，我们检测了在与100μM NMN第三次共培养后，经NMN处理的CD19 CAR-T细胞的TRAP PCR结果，结果显示经NMN处理的CD19 CAR-T细胞的端粒酶活性明显高于对照(图2c)。
经NMN处理的CD19 CAR-T细胞中Tscm亚群和Tcm亚群的百分比高于未经NMN处理的CD19 CAR-T细胞，在与NALM-6第三次共培养后，NMN处理的CD19 CAR-T细胞具有更高百分比的Tcm亚群和Tscm亚群(图2b)。这表明NMN可以抑制CAR-T细胞的分化，从而可以长期维持CAR-T细胞的免疫能力。
在与NALM-6第三次共培养后，检测经NMN处理的CD19 CAR-T细胞的β-半乳糖苷酶水平，发现相对于对照，视野中的深蓝色较少，表明较少的β-半乳糖苷酶积累(图2d)，可见NMN减少了CD19 CAR-T细胞的凋亡。
Annexin-V和7-ADD是评估细胞凋亡的标记物，本实施例通过流式细胞术检测了经NMN处理的CD19 CAR-T细胞的凋亡情况，并对结果进行单因素方差分析，统计显著性设定为*p<0.05,**P<0.01,***p<0.001。结果显示NMN处理使得CD19 CAR-T细胞具有较低的细胞凋亡(图2e)，这可能部分解释了NMN处理使得CD19 CAR-T细胞具有较高的细胞增殖率。这些结果表明NMN具有抗 CAR-T细胞衰老能力、可以提高CD19 CAR-T细胞的寿命。在动物模型中，也观察到了类似的效果(如下所示)。
实施例4.NMN处理改变了CD19 CAR-T细胞的基因表达谱
为了探究NMN延缓CAR-T细胞衰老的作用机制，本实施例采用高通量RNA序列分析方法检查了NMN处理对CAR-T细胞基因表达的影响。
与对照细胞相比，我们在NMN处理的CD19 CAR-T细胞中发现了7889个上调基因和5976个下调基因(图3a)。使用在线生物信息学工具(DAVID Bioinformatics Resources 6.8)对前400个差异(200个上调，200个下调)表达的基因进行基因本体分析(GO分析)，其中BP：生物过程；CC：细胞成分；MF：分子功能；使用Fisher精确检验进行基因富集分析。我们发现，在NMN处理的细胞中，调节NAD依赖性蛋白去乙酰酶活性、细胞增殖的正调节、细胞衰老的负调节、适应性免疫反应的正调节和响应DNA损伤的内在凋亡信号通路的基因上调(图3b)。经过蛋白质-蛋白质相互作用(PPI)分析，我们发现了两个基因簇，一个主要是关于NAD+转化的，另一个主要是关于作为NAD+和功能调节基因(例如PPI网络中的TP53，Bax和NF-κB)之间焦点的Sirtuins家族(图3c)。该结论与KEGG网站上关于Sirt1作为焦点基因有一条关于细胞寿命的通路(图9)相一致。
实施例5.NMN通过上调Sirt1表达调控衰老相关基因
为了进一步阐明NMN在CD19 CAR-T细胞中的抗衰老作用机制，我们通过Q-PCR方法检测了sirtuins及相关分子的表达，发现NMN处理的D19 CAR-T中sirtuins表达上调。因此，sirtuins可能是NMN给药后提供对CD19 CAR-T细胞的保护所必需的。通过Q-PCR方法，我们检测了经100μM的NMN处理的CD19 CAR-T细胞中Sirt1、Sirt3、Sirt4、Sirt5、Sirt6和Sirt7在mRNA水平上的表达差异，结果表明NMN处理的CD19 CAR-T细胞具有更高的Sirtuins基因表达(图4a)，琼脂糖凝胶电泳结果显示NMN增加了CD19-CAR-T细胞中Sirt1的表达水平(图4b)。
许多主要的转录因子和辅因子因依赖于Sirt1的去乙酰化而失活，例如：NF-κB和肿瘤抑制因子TP53。转录因子TP53作为中枢发挥作用，将各种应激信号 转化为不同的细胞结果，例如DNA修复、细胞周期停滞、或细胞死亡。我们发现当采用NMN处理CD19-CAR-T细胞后，一些Sirt1相关基因(如TP53、NF-κB和Bax)也发生了显著变化，在本研究中，通过Q-PCR方法检测相关基因的mRNA水平，结果通过单因素方差分析进行分析，统计显著性设定为*p<0.05,**P<0.01,***p<0.001。我们发现NMN对Sirt1的上调导致TP53mRNA下调、NF-κB mRNA下调和Bax mRNA下调。进一步地，采用Sirt1抑制剂EX527(10μM)进一步检测了NMN的作用。加入Sirt1抑制剂EX527(10μM)之后抑制了NMN诱导的TP53mRNA下调、NF-κB mRNA下调和Bax mRNA下调，表明NMN通过上调Sirt1并导致TP53降低来影响CD19 CAR-T细胞的增殖。(图4c-4f)，这些基因在KEGG网站上证明与细胞寿命有关。
我们还检测了NMN对细胞分泌的炎症因子(TNF-α和IL-6)的影响，TNF-α和IL-6的分泌是炎症反应的主要表现。按照E/T为10:1的比例，37℃共培养经NMN处理的CD19 CAR-T细胞与NALM-6细胞24小时，通过ELISA方法检测培养上清液中的TNF-α水平或IL-6水平。之后添加Sirt1抑制剂(EX527 10μM)再培养24小时，再次检测培养上清液中的TNF-α水平或IL-6水平。结果表明NMN降低了CAR-T细胞分泌的TNF-α水平(图10a)或IL-6水平(图10b)，添加Sirt1抑制剂(EX527 10μM)后，TNF-α水平或IL-6水平显著升高，表明加入的EX527抑制了NMN诱导的IL-6、TNF-α下调。NF-κB是一个诱导型转录因子家族，在炎症和衰老相关分泌表型(SASP)的出现中起核心作用。NF-κB的下调导致TNF-α和IL-6的下降，降低了炎症反应，这可能表明NMN减少了在免疫治疗中的炎症反应。基于本文建立的结果，我们提出了一个NMN-Sirt1轴模型(图6)。
总体而言，这些结果表明，在CD19-CAR-T细胞中，NMN通过增加Sirt1的表达水平和随后受调控的相关基因来延缓CD19-CAR-T细胞的衰老。因此，可以有效干预CAR-T细胞衰老的NMN在治疗癌症的CAR-T细胞免疫治疗中有重要的临床价值。
实施例6.体内NMN治疗增强B-ALL异种移植小鼠模型中CD19 CAR-T细胞的抗肿瘤活性
在本实施例中，我们检测了NMN对异种移植小鼠模型中CD19 CAR-T细胞 的抗肿瘤活性的影响。将NALM-6-Luc细胞在实验前一天(-1天)皮下注射到NOD-SCID小鼠中以产生异种移植小鼠模型，并将小鼠随机分为3组：PBMC阴性对照组(NT)，CD19 CAR-T细胞处理组(CD19 CAR-T)，经NMN处理的CD19 CAR-T细胞处理组(CD19 CAR-T+NMN)，每组5只小鼠。在第0-2天，每天通过尾静脉分别向各组注射100μl的包含1×10 7个的PBMC细胞、CD19 CAR-T细胞和经NMN处理的CD19 CAR-T细胞。从第2天开始每天腹膜内注射NMN(300mg NMN/kg体重)。每周用IVIS系统监测肿瘤发展，操作流程图参见图5a。随着时间的推移，每只小鼠的生物发光信号显示身体内的肿瘤负荷。图5b显示生物发光经IVIS Lunma Series III成像的结果，图5c-d显示量化辐射后的结果，其中图5c表示每只小鼠的检测结果，图5d显示各组经拟合标准化后的结果。如图5b-d所示，经NMN处理的CD19 CAR-T细胞在小鼠体内表现出更持久的肿瘤抑制作用。用经NMN处理的CD19 CAR-T细胞治疗的小鼠的肿瘤负荷明显低于未经NMN处理的CD19 CAR-T细胞治疗的小鼠的肿瘤负荷。可见，NMN可提高CD19 CAR-T细胞的寿命并由此调节B淋巴细胞白血病的治疗，因此可以在治疗癌症的CAR-T细胞免疫治疗中发挥重要的临床价值。

Claims (18)

  1. 一种延长CAR-T细胞寿命、改善CAR-T细胞增殖能力的方法，包括用10μM-1mM的NMN室温孵育所述CAR-T细胞至少12小时。
  2. 一种增加CAR-T细胞中Tscm和Tcm的比例的方法，包括用10μM-1mM的NMN室温孵育所述CAR-T细胞至少12小时。
  3. 权利要求1或2所述的方法，其中用10μM-100μM的NMN与所述CAR-T细胞孵育。
  4. 权利要求1或2所述的方法，其中所述NMN与所述CAR-T细胞孵育至少24小时。
  5. 权利要求1-4中任一项所述的方法，其中所述CAR-T细胞来源于将接受使用基因修饰的T细胞治疗的受试者，或者来源于不同的受试者“衍生”或“获得”。
  6. 权利要求1-4中任一项所述的方法，其中所述CAR-T细胞是CD19-CAR-T细胞。
  7. NMN在制备用于延长CAR-T细胞寿命、改善CAR-T细胞增殖能力的药物中的用途。
  8. NMN在制备用于提高CAR-T细胞中Tscm和Tcm比例的药物中的用途。
  9. NMN在制备用于增强CAR-T细胞免疫疗法疗效的药物中的用途。
  10. 权利要求7-9中任一项所述的用途，其中所述CAR-T细胞来源于将接受使用基因修饰的T细胞治疗的受试者，或者来源于不同的受试者“衍生”或“获得”。
  11. 权利要求7-9中任一项所述的用途，其中所述CAR-T细胞是CD19-CAR-T细胞。
  12. 一种CAR-T细胞群，其由权利要求1-6中任一项所述的方法获得，且具有延长的细胞寿命和改善的细胞增殖能力。
  13. 一种Tscm和Tcm占比提高的CAR-T细胞群，其由权利要求1-6中任一项所述的方法获得，其中所述Tscm和Tcm占CAR-T细胞群的3.37％和0.9％，
  14. 权利要求12或13所述的CAR-T细胞群，其中所述CAR-T细胞来源于将接受使用基因修饰的T细胞治疗的受试者，或者来源于不同的受试者“衍生”或 “获得”。
  15. 权利要求12-14中任一项所述的CAR-T细胞群，其是CD19-CAR-T细胞群。
  16. 一种治疗癌症的方法，包括将权利要求12-15中任一项所述的CAR-T细胞群施用给受试者。
  17. 权利要求16所述的方法，其中所述癌症是血液系统恶性肿瘤。
  18. 一种药物组合物，其包含权利要求12-15中任一项所述的CAR-T细胞群。
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WO2020243911A1 (zh) * 2019-06-04 2020-12-10 上海科技大学 Nad+和/或nad+抑制剂和/或nad+激动剂的用途及其联合制剂
US20220062314A1 (en) * 2018-12-17 2022-03-03 MitoPower, LLC Nicotinyl riboside compounds and their uses

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US20220062314A1 (en) * 2018-12-17 2022-03-03 MitoPower, LLC Nicotinyl riboside compounds and their uses
WO2020243911A1 (zh) * 2019-06-04 2020-12-10 上海科技大学 Nad+和/或nad+抑制剂和/或nad+激动剂的用途及其联合制剂

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