Chimeric antigen receptor nanocomposite and preparation method therefor and use thereof in preparation of Anti-aging drug

Abstract

The present invention belongs to the technical field of gene therapy. Provided are a chimeric antigen receptor nanocomposite and a preparation method therefor and the use thereof in the preparation of an anti-aging drug. The provided chimeric antigen receptor nanocomposite comprises a chimeric antigen receptor DNA plasmid specifically recognizing senescent cells, a polymer coupled with a signal peptide, a free polymer and an antibody polymer targeting T cells, wherein the chimeric antigen receptor DNA plasmid, the polymer coupled with a signal peptide, the free polymer and the antibody polymer targeting T cells are sequentially mixed and incubated to form the chimeric antigen receptor nanocomposite. The chimeric antigen receptor nanocomposite can rapidly generate senescent cell-marked chimeric antigen T cells in vivo. The engineered T cells have the ability to specifically eliminate senescent cells in vivo, and can rapidly and durably eliminate senescent cells in vivo, which is irreplaceable and has wide application prospects.

Description

Chimeric antigen receptor nanocomplex, preparation method thereof, and application thereof in preparing anti-aging drugs Technical Field

[0001] The present invention belongs to the technical field of gene therapy, and in particular relates to a chimeric antigen receptor nanocomplex, a preparation method thereof, and an application thereof in the preparation of anti-aging drugs. Background Art

[0002] As individuals age, metabolic disorders become increasingly common, leading to various age-related diseases, including metabolic syndrome and diabetes. The accumulation of senescent cells is considered a key factor in the aging process. These cells fail to function properly and secrete pro-inflammatory cytokines, further damaging tissue health, leading to decreased mobility, metabolic disorders, disease, and a shortened lifespan. Senolytic therapy, which improves tissue function and slows the aging process by removing senescent cells, is considered to have great potential.

[0003] Currently, most research on senolytic therapies focuses on developing small molecule drugs that target senescent cells. However, these drugs require repeated use over time and have drawbacks due to their inherent targeting imprecision and significant side effects. Existing senolytic therapies also include ex vivo CAR T-cell therapy, an emerging approach to clear senescent cells. Ex vivo CAR T-cell therapy uses genes encoding disease-specific chimeric antigen receptors (CARs) to program autologous T cells and expand them in vitro, enabling them to clear senescent cells upon reinfusion. Although trials of this CAR T senescent cell-clearing therapy have made progress, the in vitro methods for generating large numbers of senescent-specific T cells are complex and expensive. Due to the high cost of ex vivo CAR T-cell therapy, its widespread application in senescent cell-clearing therapy is limited. Given the billions of elderly people worldwide, the demand for senescent cell-clearing therapies is enormous, and a low-cost, simple-to-use, and effective senescent cell-clearing therapy is urgently needed.

[0004] Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a chimeric antigen receptor nanocomplex, a preparation method thereof, and an application in the preparation of anti-aging drugs; the chimeric antigen receptor nanocomplex provided by the present invention is low-cost, accurate, efficient, and has a long-lasting efficacy in the application of senescent cell clearance therapy.

[0006] The present invention provides a chimeric antigen receptor DNA plasmid that specifically recognizes senescent cells, comprising a cell membrane extracellular region sequence, a transmembrane region sequence, and a co-stimulatory molecule region sequence connected in sequence; the cell membrane extracellular region sequence comprises an scFv fragment encoding a senescent cell marker;

[0007] The cell membrane extracellular region sequence, transmembrane region sequence and co-stimulatory molecule region sequence are cloned into the multiple cloning site of the initial plasmid.

[0008] Preferably, the senescent cell marker is selected from one or more of uPAR, DPP4 (CD26) and HMGB1 senescent cell surface proteins.

[0009] Preferably, the initial plasmid is selected from a non-viral plasmid or a viral plasmid; the non-viral plasmid includes a transposon plasmid.

[0010] Preferably, when the senescent cell marker is uPAR, the nucleotide sequence of the chimeric antigen receptor DNA plasmid is as shown in SEQ ID NO.1;

[0011] When the senescent cell marker is DPP4 (CD26), the nucleotide sequence of the chimeric antigen receptor DNA plasmid is replaced by SEQ ID NO.3 for positions 87-407 of SEQ ID NO.1, and by SEQ ID NO.4 for positions 477-818 of SEQ ID NO.1;

[0012] When the senescent cell marker is HMGB1, the nucleotide sequence of the chimeric antigen receptor DNA plasmid replaces positions 87-407 of SEQ ID NO.1 with SEQ ID NO.5, and replaces positions 477-818 of SEQ ID NO.1 with SEQ ID NO.6.

[0013] The present invention also provides a chimeric antigen receptor nanocomplex, comprising the chimeric antigen receptor DNA plasmid that specifically recognizes senescent cells, a polymer coupled with a signal peptide, a free polymer, and an antibody polymer targeting T cells;

[0014] The mass ratio of the chimeric antigen receptor DNA plasmid, the signal peptide-coupled polymer, the free polymer, and the T cell-targeting antibody polymer is 1:(14-16):(14-16):(2-3);

[0015] The chimeric antigen receptor DNA plasmid, the polymer coupled with the signal peptide, the free polymer and the antibody polymer targeting T cells are mixed and incubated in sequence to form a chimeric antigen receptor nanocomplex.

[0016] Preferably, the polymer is PBAE-447, and the sequence of the signal peptide in the polymer coupled with the signal peptide is shown as SEQ ID NO.2.

[0017] Preferably, the antibody polymer is polyglutamic acid coupled anti-CD3 antibody PGA-CD3.

[0018] The present invention also provides a method for preparing the chimeric antigen receptor nanocomplex, comprising the following steps:

[0019] The chimeric antigen receptor DNA plasmid was dissolved in sodium acetate buffer to obtain a plasmid solution;

[0020] mixing the polymer coupled with the signal peptide with the plasmid solution and incubating them for the first time to obtain a first complex;

[0021] mixing the free polymer with the first complex and incubating for a second time to obtain a second complex;

[0022] The antibody polymer is mixed with the second complex and incubated for a third time to obtain a chimeric antigen receptor nanocomplex.

[0023] Preferably, the concentration of the chimeric antigen receptor DNA plasmid in the plasmid solution is 0.05-0.15 mg / ml.

[0024] The present invention also provides the use of the chimeric antigen receptor nanocomplex in preparing a reagent for eliminating senescent cells.

[0025] The present invention also provides the use of the chimeric antigen receptor nanocomplex in preparing medicine for treating geriatric syndrome.

[0026] Preferably, the geriatric syndrome includes geriatric metabolic syndrome and liver cirrhosis.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a chimeric antigen receptor DNA plasmid that specifically recognizes senescent cells. The chimeric antigen receptor DNA plasmid, a polymer coupled with a signal peptide, a free polymer, and an antibody polymer targeting T cells are sequentially mixed and incubated to form a chimeric antigen receptor nanocomplex. The chimeric antigen receptor nanocomplex provided by the present invention can quickly generate chimeric antigen T cells marked by senescent cells in vivo. These engineered T cells have the ability to specifically eliminate senescent cells in the body, and can quickly and persistently eliminate senescent cells in the body. The chimeric antigen receptor nanocomplex provided by the present invention has a completely different technical route and technical effect from the current method for generating T cells with specific specificity for senescent cells. The chimeric antigen receptor nanocomplex provided by the present invention can be directly applied to individuals without the need to isolate T cells from autologous blood, and does not require complex and expensive laboratory procedures based on retroviral or lentiviral vectors for genetic modification and then re-infusion into the body.

[0029] Essentially, the present invention obtains chimeric antigen receptor nanocomplexes through clever design. After application to the body, the complex and expensive in vitro chimeric antigen (CAR) T cell preparation, expansion and reinfusion process can be completely transferred to the body, and the body itself becomes a CAR T cell preparation factory, thereby exponentially reducing costs and greatly shortening the time to prepare T cells. At the same time, it can also exponentially amplify production capacity, thus potentially benefiting everyone in need. Conservative estimates show that the present invention can reduce the current cost of CAR T from RMB 1 million to 2 million per case to RMB 10-20 per case. Considering that senolytic therapy is a need for almost all middle-aged or elderly people, numbering in the billions, and as the population continues to age, this number is still increasing rapidly, it is impossible to cover such a large population of people in need by producing CAR T cells in vitro, so the present invention has irreplaceable and broad application prospects.

[0030] According to the description in the examples, after the chimeric antigen receptor nanocomplex is applied to mice, it can reduce the aging biological markers (epigenetic age) of elderly mice, improve the exercise capacity of elderly mice, and reverse the increase in epigenetic age caused by a high-fat diet, improve glucose metabolism disorders and reverse liver fibrosis. Moreover, this therapeutic effect of reversing aging is better than the traditional in vitro cultured uPAR CAR T cell reversal effect targeting senescent cells.

[0031] Furthermore, the chimeric antigen receptor DNA plasmid of this invention utilizes a transposon / transposase system, which offers improved clinical safety compared to viral approaches like lentiviruses that potentially activate oncogenes. The transposon / transposase system has been used to safely introduce CAR transgenes into patients' T cells outside the body, and numerous Phase I clinical trials of transposon-based CAR T cells have demonstrated the safety of efficient transposon-based gene delivery. Compared to conventional lentiviral vectors, this transposase-mediated transgene integration is located in a safe locus, reducing the risk of potential mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a flowchart of the workflow for generating uPAR CAR-T cells in vivo.

[0033] Figure 2 shows that uPAR CAR-T cells produced in vivo can reverse the increase in aging markers (epigenetic age) and glucose tolerance test impairment caused by a high-fat diet, and are more effective than uPAR CAR-T produced in vitro.

[0034] DNA-nanoparticle treatment involves the in vivo generation of uPAR CAR-T cells using the chimeric antigen receptor nanocomplexes provided by the present invention. (A): In vivo generation of uPAR CAR T cells reverses the increase in epigenetic age induced by a high-fat diet, demonstrating superior efficacy compared to in vitro CAR T cell treatment. (B): In vivo generation of uPAR CAR T cells corrects metabolic abnormalities induced by a high-fat diet, demonstrating superior efficacy compared to in vitro CAR T cell treatment. *P<0.05, **P<0.01, ***P<0.001.

[0035] Figure 3 shows that uPAR CAR-T cells produced in vivo can reverse aging markers (epigenetic age) and improve the exercise capacity of elderly mice, and are more effective than uPAR CAR-T produced in vitro.

[0036] DNA-nanoparticle treatment involves the in vivo generation of uPAR CAR-T cells using the chimeric antigen receptor nanocomplexes provided by the present invention. (A): In vivo generation of uPAR CAR T cells reversed the epigenetic age of elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment. (B): In vivo generation of uPAR CAR T cells improved treadmill endurance in elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment. (C): In vivo generation of uPAR CAR T cells increased the maximum speed of elderly mice on a treadmill, demonstrating superior efficacy compared to in vitro CAR T cell treatment. *P < 0.05, **P < 0.01, ***P < 0.001.

[0037] Figure 4 shows that uPAR CAR-T cells produced in vivo can reverse CCL4-induced liver cirrhosis in mice and are more effective than uPAR CAR-T produced in vitro.

[0038] DNA-nanoparticle treatment involves the in vivo generation of uPAR CAR-T cells using the chimeric antigen receptor nanocomplexes provided by the present invention. (A): In vivo generation of uPAR CAR T cells reduced Sirius Red-positive areas, demonstrating a reversal of liver fibrosis and superior efficacy compared to in vitro CAR T cell treatment. (B): In vivo generation of uPAR CAR T cells reduced the proportion of SA-β-gal-positive cells, demonstrating a superior efficacy compared to in vitro CAR T cell treatment. *P < 0.05, **P < 0.01, ***P < 0.001.

[0039] Figure 5 shows that in vivo generation of DPP4 CAR-T cells can reverse aging markers (epigenetic age) and improve exercise capacity in elderly mice, with superior efficacy compared to in vitro generation of DPP4 CAR-T cells. DNA-nanoparticle treatment involves the use of the chimeric antigen receptor nanocomplex provided by the present invention to generate DPP4 CAR-T cells in vivo. (A) In vivo generation of uPAR CAR T cells reverses epigenetic age in elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment. (B) In vivo generation of DPP4 CAR T cells improves treadmill endurance in elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment. (C) In vivo generation of DPP4 CAR T cells increases the maximum speed of a treadmill in elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment. *P<0.05, **P<0.01, ***P<0.001.

[0040] Figure 6 shows that HMGB1 CAR-T cells produced in vivo can reverse aging markers (epigenetic age) and improve the exercise capacity of elderly mice, and are more effective than HMGB1 CAR-T produced in vitro.

[0041] DNA-nanoparticle treatment involves the in vivo generation of HMGB1 CAR-T cells using the chimeric antigen receptor nanocomplexes provided by the present invention. (A): In vivo generation of uPAR CAR T cells reverses epigenetic age in elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment; (B): In vivo generation of HMGB1 CAR T cells improves motorized treadmill endurance in elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment; (C): In vivo generation of DPP4 CAR T cells increases the maximum speed of a motorized treadmill in elderly mice, demonstrating superior efficacy compared to in vitro CAR T cell treatment. *P<0.05, **P<0.01, ***P<0.001.

[0042] FIG7 is a transposon plasmid map for generating uPAR CAR T cells in vivo according to the present invention;

[0043] FIG8 is a transposon plasmid map of the present invention for in vivo production of DPP4 CAR T cells;

[0044] FIG9 is a transposon plasmid map for generating HMGB1CAR T cells in vivo according to the present invention. DETAILED DESCRIPTION

[0045] The present invention provides a chimeric antigen receptor DNA plasmid that specifically recognizes senescent cells, comprising an extracellular membrane region sequence, a transmembrane region sequence, and a costimulatory molecule region sequence connected in sequence; the extracellular membrane region sequence comprises an scFv fragment encoding a senescent cell marker; the extracellular membrane region sequence, the transmembrane region sequence, and the costimulatory molecule region sequence are cloned into the multiple cloning site of the initial plasmid.

[0046] In the present invention, the senescent cell marker is selected from one or more of uPAR, DPP4 (CD26) and HMGB1 senescent cell surface proteins, more preferably uPAR. In the present invention, the costimulatory molecule region sequence preferably includes CD28 and CD3ζ.

[0047] In the present invention, the initial plasmid is selected from a non-viral plasmid or a viral plasmid; the non-viral plasmid preferably includes a transposon plasmid. The non-viral vector may also be selected from liposomes, polymer nanoparticles, gold nanoparticles, and cell-penetrating peptides; the viral vector may be a non-replicating viral vector that has been genetically engineered to target T cells in vivo, such as adenovirus, adeno-associated virus, lentivirus, herpes virus, and Sendai virus.

[0048] In the present invention, the plasmid map of the chimeric antigen receptor DNA plasmid is preferably as shown in FIG1 , and the nucleotide sequence of the chimeric antigen receptor DNA plasmid is as shown in SEQ ID NO. 1, specifically as follows:

[0049] The present invention also provides a chimeric antigen receptor nanocomplex, comprising the chimeric antigen receptor DNA plasmid that specifically recognizes senescent cells, a polymer coupled with a signal peptide, a free polymer, and an antibody polymer targeting T cells; the mass ratio of the chimeric antigen receptor DNA plasmid, the polymer coupled with a signal peptide, the free polymer, and the antibody polymer targeting T cells is 1:(14-16):(14-16):(2-3); the chimeric antigen receptor DNA plasmid, the polymer coupled with a signal peptide, the free polymer, and the antibody polymer targeting T cells are mixed and incubated in sequence to form a chimeric antigen receptor nanocomplex.

[0050] In the present invention, the polymer is preferably PBAE-447, and the sequence of the signal peptide in the polymer coupled with the signal peptide is shown in SEQ ID NO. 2, including the microtubule-associated sequence MTAS and the nuclear microtubule signal sequence NLS; specifically as follows:

[0051] The present invention preferably adds a cysteine ​​to the N segment of the signal peptide sequence for connection with PBAE-447.

[0052] In the present invention, the antibody polymer is polyglutamic acid coupled anti-CD3 antibody PGA-CD3.

[0053] In the present invention, the mass ratio of the chimeric antigen receptor DNA plasmid, the signal peptide-coupled polymer, the free polymer and the T cell-targeting antibody polymer is preferably 1:(14.5-15.5):(14.5-15.5):(2.2-2.8), more preferably 1:15:15:2.5.

[0054] The present invention also provides a method for preparing the chimeric antigen receptor nanocomplex, comprising the following steps:

[0055] The chimeric antigen receptor DNA plasmid was dissolved in sodium acetate buffer to obtain a plasmid solution;

[0056] mixing the polymer coupled with the signal peptide with the plasmid solution and incubating them for the first time to obtain a first complex;

[0057] mixing the free polymer with the first complex and incubating for a second time to obtain a second complex;

[0058] The antibody polymer is mixed with the second complex and incubated for a third time to obtain a chimeric antigen receptor nanocomplex.

[0059] In the present invention, the chimeric antigen receptor DNA plasmid is dissolved in a sodium acetate buffer to obtain a plasmid solution; the concentration of the chimeric antigen receptor DNA plasmid in the plasmid solution is preferably 0.05-0.15 mg / ml, more preferably 0.05-0.12 mg / ml, and most preferably 0.1 mg / ml; the concentration of the sodium acetate buffer is preferably 20-30 mM, more preferably 22-28 mM, and most preferably 25 mM; the pH of the sodium acetate buffer is preferably 5.1-5.3, more preferably 5.2.

[0060] After obtaining the plasmid solution, the present invention mixes the polymer coupled with the signal peptide with the plasmid solution and performs a first incubation to obtain a first complex; the mixing is followed by slight oscillation for 8 to 12 seconds; the first incubation time is preferably 1 to 3 minutes, preferably 1.5 to 2.5 minutes.

[0061] After obtaining the first complex, the present invention mixes the free polymer with the first complex and performs a second incubation to obtain a second complex; the mixing is followed by gentle shaking for 8 to 12 seconds; the second incubation time is preferably 4 to 6 minutes, preferably 4.5 to 5.5 minutes.

[0062] After obtaining the second complex, the present invention mixes the antibody polymer with the second complex and performs a third incubation to obtain a chimeric antigen receptor nanocomplex. The mixing is followed by gentle shaking for 8 to 12 seconds; the second incubation time is preferably 4 to 6 minutes, preferably 4.5 to 5.5 minutes.

[0063] After obtaining the chimeric antigen receptor nanocomplex of the present invention, it is preferably lyophilized. Before the lyophilization, a lyoprotectant is preferably added, preferably sucrose. The final concentration of sucrose in the lyophilized material is preferably 25-35 mg / ml, preferably 28-32 mg / ml, and more preferably 30 mg / ml. When used, the lyophilized particles of the chimeric antigen receptor nanocomplex obtained by the present invention are preferably resuspended in water at 1 / 3 of the pre-lyophilization volume.

[0064] The present invention also provides the use of the chimeric antigen receptor nanocomplex in preparing a reagent for eliminating senescent cells.

[0065] The present invention also provides the use of the chimeric antigen receptor nanocomplex in the preparation of a medicament for treating geriatric syndromes. In the present invention, the geriatric syndromes preferably include geriatric metabolic syndrome and liver cirrhosis. Upon application to an organism, the chimeric antigen receptor nanocomplex of the present invention acquires the ability to specifically eliminate senescent cells, thereby reducing biomarkers of aging (epigenetic age) in the subject, improving exercise capacity, ameliorating metabolic disorders induced by a high-fat diet, such as glucose tolerance, and reversing liver fibrosis.

[0066] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] uPAR CAR design: Design of plasmid DNA sequence for CAR that recognizes senescent T cells as chimeric antigens

[0069] Design principles of the second generation CAR

[0070] Extracellular region sequence (uPAR scFC region):

[0071] VL DNA sequence SEQ.ID NO.1: positions 87-407

[0072] VH DNA sequence SEQ.ID NO.1: positions 477-818

[0073] VL-VH linker sequence SEQ.ID NO.1: positions 408-467

[0074] Transmembrane region sequence:

[0075] CH2CH3 DNA sequence SEQ.ID NO.1: 852-1547

[0076] Costimulatory molecule domain sequence

[0077] CD28 DNA sequence SEQ.IDNO.1: positions 1560-1763

[0078] CD3ζ DNA sequence SEQ.ID NO.1: positions 1764-2102

[0079] Plasmid synthesis: The CAR plasmid DNA sequence was inserted into the empty transposon plasmid sequence to form a complete transposon plasmid DNA sequence. This was then submitted to Vector Builder (https: / / en.vectorbuilder.com) for synthesis of the corresponding plasmid. The complete plasmid map is shown in Figure 2, and the complete sequence is shown in SEQ.ID NO. 1. The highly active transposon enzyme plasmid was purchased from System Bioscience (Cat. No. PB210PA-1).

[0080] Preparation of DNA-nanoparticles targeting T cells (DNA nanoparticles refer to chimeric antigen receptor nanocomplexes):

[0081] The signal peptide (microtubule-associated sequence MTAS and nuclear localization signal sequence NLS, SEQ ID No. 2) GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR was custom synthesized by AnaSpec, and a cysteine ​​was added to the N-terminus of the peptide for attachment to the PBAE-447 polymer.

[0082] Nanoparticle preparation:

[0083] Preparation of PBAE Polymer: Prepared using the method described in reference (PMID: 25643235): 1,4-Butanediol diacrylate and 4-amino-1-butanol were mixed in a molar ratio of 1.1:1. This mixture was heated with stirring at 90°C for 24 hours to produce acrylate-terminated poly(4-amino-1-butanol-1,4-butanediol diacrylate). 2.3 g of the polymer was dissolved in 2 ml of tetrahydrofuran (THF). To form the piperazine-terminated 447 polymer, 786 mg of 1-(3-aminopropyl)-4-methylpiperazine was dissolved in 13 ml of THF and added to the polymer / THF solution. The resulting solution was stirred at room temperature for 2 hours. The piperazine-terminated polymer was precipitated with 5 volumes of diethyl ether. The diethyl ether was decanted, and the collected polymer was washed with 2 volumes of fresh diethyl ether. The polymer residue was dried in vacuo for 2 days. Pure polymer was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mg / ml and stored at -20°C.

[0084] Conjugation of PBAE polymers and signal peptides: A solution of 12 mg of 4-(maleimido)phenylisocyanate (PMPI) (20 mg / ml) in dimethyl sulfoxide (DMSO) was added to a solution of 447 polymer (100 mg / ml) in 86 mg of DMSO. Mixing was allowed to proceed for 3 h at room temperature. The 447-maleimide derivative was added to a solution of 100 mg of NLS-MTAS peptide in 5.3 ml of DMSO containing tris(2-hydroxyethyl)phosphinothioate hydrochloride (TCEP·HCl; 3 mg / ml). Mixing was allowed to proceed for 3 h at room temperature, followed by filtration through a 7k Zeba spin column filled with DMSO. The DMSO was evaporated in vacuo overnight. The 447-peptide conjugate was dissolved in DMSO to 100 mg / ml and stored at -20°C.

[0085] Polyglutamic acid (PGA)-CD3 antibody conjugation: Polyglutamic acid (PGA) was dissolved in water at a concentration of 20 mg / ml and sonicated in a waterbath for 10 minutes. An equal volume of ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (4 mg / ml, 16 equivalents) was added and mixed at room temperature for 5 minutes. The resulting activated PGA was added to an antibody solution (InVivoMAb anti-mouse CD3ε F(ab')2 fragment, from Bioxcell.com; catalog number BE0001-1FAB) in phosphate-buffered saline (PBS) at a 4:1 molar ratio and mixed at room temperature for 6 hours. Excess reagents were removed by dialysis (20,000 MWCO Slide-A-Lyzer Dialysis Cassette). The dialyzate was dialyzed against PBS for 24 hours and then filtered through a 40k Zeba spin column. Antibody concentrations were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

[0086] DNA-nanoparticle preparation: All components were diluted in sodium acetate buffer (25 mM, pH 5.2) at the following concentrations: plasmid DNA, 0.1 mg / ml; PBAE 447 and 447-NLS-MTAS, 3 mg / ml; PGA-antibody, 0.45 mg / ml antibody.

[0087] The specific steps are as follows: 447-NLS-MTAS is added to DNA at a PBAE:DNA mass ratio of 15:1. The mixture is gently shaken for 10 seconds and then incubated at room temperature for 2 minutes. Unbound 447 is then added to the complex at a PBAE:DNA mass ratio of 15:1. The mixture is gently shaken for 10 seconds and then incubated at room temperature for 5 minutes. PGA-antibody is then added at an Ab:DNA mass ratio of 2.5:1. The mixture is gently shaken for 10 seconds and then incubated at room temperature for 5 minutes. Sucrose is added as a cryoprotectant at a final concentration of 30 mg / ml. The mixture is gently shaken and then frozen in liquid nitrogen and freeze-dried using a FreeZone 2.5-liter freeze-drying system (Labconco). The freeze-dried particles are resuspended in water at 1 / 3 of the pre-freeze-drying volume.

[0088] DNA-nanoparticle identification: The number, average hydrodynamic radius, and concentration of nanoparticles were determined using a NanoSight NS300 instrument (Malvern Instruments). Lyophilized particles were resuspended in water at the same concentration used for transfection. After gentle shaking, the particles were incubated on ice for 10 minutes to fully hydrate. The suspension was centrifuged at 2,400g for 3 minutes, and the supernatant was diluted 5-fold for nanoparticle tracking analysis. The zeta potential of the particles was determined using a ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments Corporation). Freshly prepared nanoparticles were centrifuged at 1,000g for 1 minute, and the supernatant was diluted 14-fold in PBS for measurement.

[0089] Experimental Example 1 uPAR CAR T cell elimination therapy

[0090] In vitro uPAR CAR T cell preparation for the control treatment group: Transposon CAR-T cell preparation, expansion, and treatment were performed as described in reference (PMID: 35732992). The corresponding number of mice were euthanized, spleens were collected, and after tissue dissection and erythrocyte lysis, primary mouse T cells were purified using the Mouse Pan T Cell Isolation Kit (Miltenyi Biotec). Purified T cells were cultured in RPMI-1640 medium supplemented with 10% FBS (HyClone), 10 mM HEPES (Invitrogen), 2 mM l-glutamine (Invitrogen), MEM non-essential amino acids 1× (Invitrogen), 55 μM β-mercaptoethanol, 1 mM sodium pyruvate (Invitrogen), 100 IU ml-1 recombinant human IL-2 (Proleukin; Novartis), and mouse anti-CD3 / 28 Dynabeads (Gibco) at a bead (Dynabeads): cell ratio of 1:2. T cells were electroporated with the same transposon plasmid as described above 24 h after initial T cell activation (Lonza). 3-4 days later, uPAR magnetic beads (Miltenyi Biotec) were used to enrich and amplify the cells to obtain sufficient numbers of uPAR CAR T cells for use.

[0091] In an elderly mouse model (12-month-old mice, purchased from Jaxson Lab), the efficacy of intravenous infusion of DNA-nanoparticles (DNA nanoparticles prepared in Example 1) to eliminate senescent cells was compared with that of in vitro CAR T therapy.

[0092] DNA-nanoparticle intravenous treatment: 3 × 10 11 A suspension of DNA-nanoparticles (or control particles, i.e., the transposon DNA plasmid encoding CAR was replaced by the reporter gene GFP) was slowly injected into the tail vein of 18-20 month old C57BL / 6 mice, once a day for 5 consecutive days.

[0093] Efficacy of conventional in vitro expanded uPAR CAR T cell therapy: 5 million uPAR CART cells were transduced with exosome uPAR CAR transposon vectors and injected intravenously into 18-20 month old C57BL / 6 mice as a single dose. Cyclophosphamide (200 mg kg -1 ).

[0094] Senescence marker detection: After 6 weeks of DNA-nanoparticle treatment and in vitro uPAR CAR T cell treatment, mouse livers were collected and analyzed by the kit. Liver genomic DNA was extracted and purified using a genomic DNA extraction and purification kit (New England Labs, NEB #T3050) and instructions. Genomic methylation profiles were scanned using an Illumina RRBS array. Aging biomarkers (epigenetic age) were calculated according to reference PMID 24138928 and the corresponding biological age clock (https: / / dnamage.genetics.ucla.edu / home). The results showed that after six weeks of in vivo uPAR CAR T cell treatment, the epigenetic age of mice significantly decreased, surpassing the effect of in vitro uPAR CAR T cells.

[0095] Exercise capacity was assessed 6 weeks after DNA-nanoparticle treatment and in vitro uPAR CAR T cell treatment: Exercise capacity was assessed using a motorized treadmill (model 1050EXER 3 / 6; Columbus Instruments). Three days before the test, mice were gradually acclimated to the treadmill (mice walked on the treadmill at a speed of 10 m / min for 10 to 15 minutes per day). After acclimation, all mice underwent exercise capacity testing on consecutive days. At the beginning of the test, mice walked at a speed of 10 m / min, increasing the speed by 2 m / min every 2 minutes until endurance was exhausted (despite repeated encouragement, mice could no longer reach the maximum speed of the treadmill). The primary endpoints included the time to endurance exhaustion and the maximum speed. The experimental results suggest that after 6 weeks of in vivo uPAR CAR T cell treatment, the motorized treadmill tolerance time and maximum speed of mice were significantly improved, which was superior to the effect of in vitro uPAR CAR T cells.

[0096] In a high-fat diet mouse model, intravenous infusion of DNA-nanoparticle complexes to clear senescent cell therapy improved aging markers (epigenetic age) and glucose tolerance test and was compared head-to-head with in vitro CAR T treatment: male C57BL / 6J wild-type (WT) mice, three to four per cage, with free access to food and water. Approximately 4 months before the start of the experiment, the mice were switched to a diet (LabDiet 5053, purchased from Labdiet). At the beginning of the experiment, the mice were fed a high-fat diet (the mice were switched to a high-fat AIN-93G diet, which provided 60% of their calories from fat by adding hydrogenated coconut oil, to establish a high-fat diet mouse model); after 5 months of high-fat feeding, DNA-nanoparticle treatment and in vitro uPAR CAR T cell treatment were performed, respectively. The experiments started when the tested mice were 4 months of calendar age. After 5 months, the livers of one group of mice were collected for aging biomarker (epigenetic age) detection (see the above steps), and one group of mice was fasted for 8-12 hours and then injected intraperitoneally with glucose (Sigma-Aldrich; 2 g per kg of body weight was used for aging experiments and 1 g per kg of body weight was used for high-fat diet experiments). Blood samples were collected at 0, 15, 30, 60 and 120 minutes after injection. Insulin concentrations were measured in serum collected at 0 and 15 minutes using the UltraSensitive Mouse Insulin ELISA kit (Crystal Chem, 90080). The results suggest that after six weeks of in vivo uPAR CAR T cell treatment, glucose tolerance on a high-fat diet in mice was significantly improved, surpassing that achieved with in vitro uPAR CAR T cell treatment.

[0097] In a mouse model of liver fibrosis, intravenous administration of DNA-nanoparticle complexes to clear senescence cells reversed liver fibrosis and compared the efficacy with in vitro CAR T therapy: 6-8 week old C57BL / 6 mice were intraperitoneally injected with 1 ml kg twice a week. -1Carbon tetrachloride (CCl4) was injected 12 times to induce liver fibrosis. DNA-nanoparticle treatment and in vitro uPAR in vitro CAR T cell treatment were performed as above, with CCl4 administered continuously at the same dose and interval. 48-72 hours after the last CCl4 injection, the mouse livers were harvested. One group of mice had their livers harvested for aging biomarker (epigenetic age) detection (see above), and one group of mice underwent liver SA-β-gal staining: SA-β-gal staining was performed as previously described (PMID: 30573629). Fresh frozen tissue sections were fixed with phosphate-buffered saline (PBS) containing 0.5% glutaraldehyde for 15 minutes at pH 5.5, washed with PBS containing 1 mM MgCl2, and stained for 5-8 hours in PBS containing 1 mM MgCl2, 1 mg ml-1X-β-gal, 5 mM potassium ferrocyanide, and 5 mM potassium ferrocyanide. Tissue sections were stained with eosin. Five high-power fields of view for each well or section were counted and averaged to quantify the percentage of SA-β-gal-positive cells. A set of mouse liver tissue specimens were fixed overnight in 10% formalin, then embedded in paraffin and cut into 5-μm-thick sections. The sections were stained with hematoxylin-eosin (H&E) and Sirius Red for the detection of fibrosis. For quantitative analysis of fibrosis, at least three complete sections from each mouse were scanned, and the images were then quantitatively analyzed using NIH ImageJ software. The amount of fibrotic tissue was calculated relative to the total analyzed liver area. The experimental results suggest that after 6 weeks of in vivo uPAR CAR T cell treatment, liver fibrosis in mice was significantly improved, which was better than that of in vitro uPAR CAR T cells.

[0098] Experimental Example 2: DPP4 CAR T cell elimination therapy for senescent cells

[0099] The first step is to design and prepare a chimeric antigen for DPP4 CAR T to recognize senescent cells, except for the DNA sequence encoding DPP4 VL.

[0100] SEQ ID No.3:

[0101] DNA sequence encoding DPP4 VH:

[0102] SEQ ID No.4:

[0103] ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGGTGTCCACTCCGCAAGCACCAAAGGCCCATCGGTATTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA) except replacing the VL and VH sequences corresponding to uPAR, the rest of the method was the same as that of Experimental Example 1.

[0104] In the second step, DPP4 CAR DNA-nanoparticles (with the VL and VH of uPAR replaced by DPP4) were administered intravenously to eliminate senescence cells, and compared head-to-head with in vitro CAR T cell therapy to reduce epigenetic age and enhance exercise capacity. The results (Figure 5) showed that after six weeks of in vivo DPP4 CAR T cell treatment, the mice's epigenetic age was significantly reduced, and their motorized treadmill endurance time and maximum speed were significantly improved, surpassing the in vitro DPP4 CAR T cell effect.

[0105] Experimental Example 3: HMGB1 CAR T cell elimination therapy for senescent cells

[0106] The first step is to design and prepare a chimeric antigen for HMGB1 CAR T cells that recognizes senescent cells, except for the DNA sequence encoding HMGB1 VL:

[0107] SEQ ID No.5:

[0108] DNA sequence encoding HMGB1 VH, SEQ ID No. 6:

[0109] The remaining methods were the same as those in Experimental Example 1 except that the VL and VH sequences corresponding to uPAR were replaced.

[0110] In the second step, HMGB1CAR DNA-nanoparticles (with HMGB1 VL and VH replacing uPAR VL and VH) were administered intravenously in an aged mouse model to eliminate senescence cells and compare their efficacy with in vitro CAR T cell therapy in reducing epigenetic age and enhancing exercise capacity. The results (Figure 6) showed that after six weeks of in vivo HMGB1 CAR T cell treatment, the mice's epigenetic age was significantly reduced, and their motorized treadmill endurance time and maximum speed were significantly improved, surpassing the in vitro HMGB1 CAR T cell effects.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor DNA plasmid that specifically recognizes senescent cells, characterized in that: It includes an extracellular membrane region sequence, a transmembrane region sequence, and a co-stimulatory molecule region sequence connected in sequence; the extracellular membrane region sequence includes an scFv fragment encoding a senescent cell marker; The cell membrane extracellular region sequence, transmembrane region sequence and co-stimulatory molecule region sequence are cloned into the multiple cloning site of the initial plasmid.

2. The chimeric antigen receptor DNA plasmid according to claim 1, characterized in that The senescent cell marker is selected from one or more of uPAR, DPP4 (CD26), and HMGB1 senescent cell surface specific proteins.

3. The chimeric antigen receptor DNA plasmid according to claim 1 or 2, characterized in that The initial plasmid is selected from a non-viral plasmid or a viral plasmid; the non-viral plasmid includes a transposon plasmid.

4. The chimeric antigen receptor DNA plasmid according to claim 3, characterized in that When the senescent cell marker is uPAR, the nucleotide sequence of the chimeric antigen receptor DNA plasmid is as shown in SEQ ID NO.1; When the senescent cell marker is DPP4 (CD26), the nucleotide sequence of the chimeric antigen receptor DNA plasmid is replaced by SEQ ID NO.3 for positions 87-407 of SEQ ID NO.1, and by SEQ ID NO.4 for positions 477-818 of SEQ ID NO.1; When the senescent cell marker is HMGB1, the nucleotide sequence of the chimeric antigen receptor DNA plasmid replaces positions 87-407 of SEQ ID NO.1 with SEQ ID NO.5, and replaces positions 477-818 of SEQ ID NO.1 with SEQ ID NO.

6.

5. A chimeric antigen receptor nanocomplex, characterized in that The invention comprises the chimeric antigen receptor DNA plasmid for specifically recognizing senescent cells according to any one of claims 1 to 4, a polymer coupled with a signal peptide, a free polymer, and an antibody polymer targeting T cells; The mass ratio of the chimeric antigen receptor DNA plasmid, the signal peptide-coupled polymer, the free polymer, and the T cell-targeting antibody polymer is 1:(14-16):(14-16):(2-3); The chimeric antigen receptor DNA plasmid, the polymer coupled with the signal peptide, the free polymer and the antibody polymer targeting T cells are mixed and incubated in sequence to form a chimeric antigen receptor nanocomplex.

6. The chimeric antigen receptor nanocomplex according to claim 5, characterized in that The polymer is PBAE-447, and the sequence of the signal peptide in the polymer coupled with the signal peptide is shown in SEQ ID NO.

2.

7. The chimeric antigen receptor nanocomplex according to claim 5, characterized in that The antibody polymer is polyglutamic acid coupled to the anti-CD3 antibody PGA-CD3.

8. The method for preparing a chimeric antigen receptor nanocomplex according to any one of claims 5 to 7, characterized in that: The following steps are involved: The chimeric antigen receptor DNA plasmid was dissolved in sodium acetate buffer to obtain a plasmid solution; mixing the polymer coupled with the signal peptide with the plasmid solution and incubating them for the first time to obtain a first complex; mixing the free polymer with the first complex and incubating for a second time to obtain a second complex; The antibody polymer is mixed with the second complex and incubated for a third time to obtain a chimeric antigen receptor nanocomplex.

9. The preparation method according to claim 8, characterized in that The concentration of the chimeric antigen receptor DNA plasmid in the plasmid solution is 0.05-0.15 mg / ml.

10. Use of the chimeric antigen receptor nanocomplex according to any one of claims 5 to 7 or the chimeric antigen receptor nanocomplex prepared by the preparation method according to claim 8 or 9 in preparing an agent for eliminating senescent cells.

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