Application of adipose factor C1q / TNF (Tumor Necrosis Factor) associated protein 9 (CTTRP-9) in enhancing T cell immunotherapy effect

Abstract

The invention discloses an application of a fat factor C1q / TNF (Tumor Necrosis Factor) associated protein 9 (CTRP-9) in enhancing T cell immunotherapy, and relates to a new application of the fat factor C1q / TNF associated protein 9 (CTRP-9) in regulating T cell functions, and the CTRP-9 is an immune regulation molecule secreted by fat tissues. According to the present invention, the CTRP-9 is exogenously supplemented, such that the effect intensity of the T cells in the anti-tumor immunoreaction can be effectively enhanced, and the activation and the proliferation of the T cells can be promoted; meanwhile, the CTRP-9 can also be used as a CAR-T cell enhancer, so that the immunotherapy effect is improved. The method can be applied to an immunotherapy system based on T cells, and provides a new regulation way and target for improving the T cell immunotherapy effect.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy and relates to a method for enhancing the effect of T cell immunotherapy, specifically involving the application of adipokines C1q / TNF-related protein 9 (CTRP-9) in enhancing the effect of T cell immunotherapy. Background Technology

[0002] T cells are the core effector cells in the adaptive immune response, playing a crucial role in clearing tumor cells, virus-infected cells, and maintaining immune homeostasis. In recent years, T cell-based immunotherapies (including tumor-infiltrating T cell therapy, adoptive T cell transfer, CAR-T / TCR-T cell therapy, etc.) have made significant progress and have become an important direction in cancer treatment. However, although these methods have achieved long-term remission in some patients, the overall efficacy remains highly variable. The main limitation lies in the fact that T cells are prone to functional exhaustion under long-term antigen stimulation or in an immunosuppressive microenvironment, manifesting as decreased proliferation, reduced cytokine secretion, and decreased cytotoxicity, thereby weakening the sustainability and effectiveness of treatment. Therefore, improving the activation level and duration of effect of T cells has become a key direction for enhancing the efficacy of immunotherapy.

[0003] Against this backdrop, researchers have begun to focus on the impact of immunomodulatory molecules on T cell function. Various cytokines, chemokines, and immune co-stimulatory signals have been shown to regulate T cell activation thresholds, differentiation patterns, and effector functions. In particular, regulatory molecules derived from non-immune systems, such as hormones and neurotransmitters, have been increasingly found to influence the intensity of T cell immune responses through the immunoendocrine network. This discovery expands the research boundaries of immune regulation and provides new insights for improving T cell-mediated immunotherapy.

[0004] CTRP-9 (C1q / TNF-related protein 9) is a multifunctional adipokine discovered in recent years, belonging to the CTRP protein family. Previous research has mainly focused on its physiological functions in metabolic homeostasis, insulin sensitivity, and cardiovascular protection. However, systematic research on the role of CTRP-9 in the regulation of the immune system, especially T cell function, is lacking, and its application in enhancing the efficacy of cellular immunotherapy has not yet been reported. Existing strategies for enhancing T cell function mostly rely on exogenous cytokine stimulation, metabolic pathway regulation, or epigenetic modification. While these methods can improve T cell activation and effector function to some extent, they generally suffer from poor safety, limited regulatory targets, and short-term functional maintenance. Therefore, current approaches still struggle to achieve an ideal balance between enhancing T cell activity, maintaining immune function stability, and ensuring treatment safety. Currently, there is a lack of a naturally derived, safe molecule with multi-target comprehensive regulatory effects to effectively improve the overall efficacy of T cell immunotherapy. There is an urgent need to find new immunomodulatory factors or intervention pathways to overcome the limitations of existing technologies and significantly improve the efficacy and durability of T cell immunotherapy. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a method for enhancing the efficacy of T-cell immunotherapy using the adipokines CTRP-9. This invention proposes that exogenous supplementation with CTRP-9 can significantly promote T-cell activation, proliferation, and cytokine secretion, enhancing their effector function and immune response strength, thereby strengthening T-cell-mediated anti-tumor effects. Further research indicates that CTRP-9 can also enhance the efficacy of T-cell-based adoptive immunotherapy, including significantly increasing cytokine secretion, cytotoxicity, and target cell clearance efficiency in CAR-T cell systems, thus improving the overall efficacy of immunotherapy. Therefore, this invention proposes that CTRP-9, as a safe and effective immunomodulatory molecule, can both enhance the functional activity of natural T cells and act as a functional enhancer for CAR-T cells, providing a new molecular target and regulatory strategy for improving the sustainability and anti-tumor efficacy of T-cell immunotherapy.

[0006] This invention provides insights into the effects of novel immunomodulatory factors such as CTRP-9 on T-cell immune responses, potentially offering a new biological basis and regulatory mechanisms for improving the efficacy of T-cell therapy. This invention delves into the mechanism of action of CTRP-9 in T-cell function regulation, potentially providing new theoretical support and molecular targets for enhancing the efficacy of T-cell immunotherapy.

[0007] This invention proposes the application of adipokines CTRP-9 in enhancing the efficacy of T-cell immunotherapy. This is the first time this has been proposed, as there are no existing reports on CTRP-9's role in regulating T-cell immune function or enhancing the efficacy of T-cell therapy. Previous research on CTRP-9 has mainly focused on energy metabolism, cardiovascular protection, and insulin sensitivity; no studies have revealed its mechanism of action in T-cell immune regulation, nor has its potential application as an immune-enhancing factor in cell therapy systems been recognized. This invention is not a simple extension of existing immune-enhancing strategies, but rather, based on a systematic study of CTRP-9's characteristics, proposes that this molecule can significantly enhance T-cell proliferation and effector function by promoting phosphorylation of T-cell activation signaling pathways, while maintaining the persistence and functional stability of T-cell activation.

[0008] In proposing this invention, the inventors, based on a systematic exploration of the immunomodulatory effects of adipokines, proposed the theoretical concept that "metabolic secretory molecules can serve as exogenous immune regulatory signals to enhance T cell function." This theory breaks through the limitations of traditional immune enhancement strategies that rely solely on cytokines, metabolic inhibitors, or epigenetic modifications, providing innovative ideas for discovering new innate immune regulatory molecules. This invention is the first to apply this molecule to T cell and CAR-T cell immunotherapy systems, providing new molecular targets and intervention strategies for improving the efficacy of cell therapy.

[0009] In this invention, CTRP-9 refers to a secretory glycoprotein encoded by genes of the CTRP family, belonging to the C1q / TNF-related protein superfamily. It possesses a typical signal peptide region, a glycine-rich collagen-like domain, and a C-terminal globular C1q-like domain. This protein can be secreted from adipose tissue into the peripheral circulation and exhibits good solubility and thermostability. The CTRP-9 described in this invention can be derived from a natural or recombinant mouse form, the nucleotide sequence of which is shown in SEQ ID NO.3, and the amino acid sequence of which is shown in SEQ ID NO.4. Preferably, the recombinant mouse CTRP-9 protein (mCTRP9) used in the embodiments of this invention retains its biological activity after prokaryotic expression, purification, and gradient dialysis refolding.

[0010] This invention also proposes the application of CTRP-9 in the preparation of drugs that regulate T cell function, etc.

[0011] This invention also proposes the application of CTRP-9 in the preparation of drugs that enhance the efficacy of T-cell immunotherapy.

[0012] This invention also proposes the application of CTRP-9 in the preparation of drugs that enhance the effect intensity of T cells in anti-tumor immune responses.

[0013] This invention also proposes the application of CTRP-9 in the preparation of drugs that promote T cell activation and proliferation.

[0014] The present invention also proposes the application of CTRP-9 in the preparation of drugs that enhance the function of CAR-T cells.

[0015] This invention also proposes the application of CTRP-9 in the preparation of drugs that enhance the killing ability of CAR-T cells against tumor cells.

[0016] The present invention also proposes a regulator / promoter / activator, which includes CTRP-9 as described above.

[0017] The present invention also proposes CAR-T cell enhancers, including CTRP-9 as described above.

[0018] The present invention also proposes a reagent / kit containing one or more of the following: CTRP-9 as described above, regulators / promoters / activators as described above, and CAR-T cell enhancers as described above.

[0019] The present invention also proposes a drug / drug composition for enhancing the efficacy of T-cell immunotherapy, wherein the drug / drug composition comprises one or more of the following: CTRP-9 as described above, the regulator / promoter / activator as described above, the CAR-T cell enhancer as described above, and the reagent / kit as described above.

[0020] Furthermore, the drug / drug composition further includes a pharmaceutically acceptable carrier.

[0021] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0022] Specifically, the drug / drug composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0023] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0024] Specifically, the drug / drug composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0025] Specifically, the drug / drug composition can be formulated into injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field. In the above pharmaceutical formulation, the dosage form of the drug / drug composition is an injection.

[0026] Specifically, the drug / drug composition can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, via injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it can be introduced into the body after being mixed with or encapsulated by other substances. Preferably, it is administered by injection. The drug / drug composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0027] The dosage level of the drug / drug composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments thereof. The selected dosage level and regimen are rationally adjusted based on various factors including the activity and stability (i.e., half-life) of the cellular drug / drug composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0028] The therapeutically effective dose of the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0029] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0030] Specifically, the drug / drug composition may be used alone and / or in combination with one or more of other therapeutic agents, including immune checkpoint inhibitors, cytokine drugs, targeted therapies, antioxidants or metabolic modulators.

[0031] The present invention also proposes a method for preventing / inhibiting / alleviating / reducing / treating a disease, the method comprising administering to a subject in need an effective amount of one or more of the following: CTRP-9 as described above, a modulator / promoter / activator as described above, a CAR-T cell enhancer as described above, a reagent / kit as described above, a drug / pharmaceutical composition as described above. The method may also be in vitro or non-therapeutic.

[0032] Specifically, the subject or individual undergoing therapeutic or preventative treatment is preferably a mammal, including but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, and pigs), pets (such as dogs and cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, and hamsters), or captured wild animals (such as foxes and deer). Preferably, the subject is a primate. Most preferably, the subject is a human.

[0033] Specifically, the subject may be a patient with the disease or an individual who wishes to prevent the disease. One or more of the following, as described above: CTRP-9, as described above: regulators / promoters / activators, as described above: CAR-T cell enhancers, as described above: reagents / kits, as described above: drugs / drug compositions, etc., may be administered to the subject before, during, or after receiving disease treatment.

[0034] In one specific embodiment, the method for preventing / inhibiting / alleviating / reducing / treating the disease includes methods for enhancing the efficacy of T-cell immunotherapy. Specifically, the method for enhancing the efficacy of T-cell immunotherapy includes administering an effective amount of adipokines CTRP-9 to the subject's in vivo or in vitro T-cell system to promote T-cell activation, proliferation, and cytokine secretion, thereby increasing the intensity of its anti-tumor immune response. This method, based on the multi-target regulatory effect of CTRP-9 on T-cell signaling pathways, can simultaneously enhance the effector function and functional persistence of T cells, overcoming the problems of poor safety, short activation time, and functional exhaustion existing in current enhancement strategies.

[0035] In this invention, the disease includes a variety of diseases caused by low or imbalanced T-cell immune function, including but not limited to various malignant tumors, autoimmune diseases, chronic viral infections, and immunodeficiency-related diseases. In a preferred embodiment, the disease is CD19-expressing B-cell lymphoma.

[0036] The present invention also proposes the application of CTRP-9 as described above, or the regulator / promoter / activator as described above, or the CAR-T cell enhancer as described above, or the reagent / kit as described above, or the drug / drug composition as described above, or the method as described above, in the preparation of drugs that regulate T cell function, enhance the effect of T cell immunotherapy, enhance the effect strength of T cells in anti-tumor immune response, promote T cell activation and proliferation, enhance the function of CAR-T cells, and enhance the killing ability of CAR-T cells against tumor cells.

[0037] Compared with existing technologies, the beneficial effects of this invention include: proposing a novel function of CTRP-9 in immunotherapy, providing new targets and methods of action, and proposing that CTRP-9 can enhance T cell function in a multi-level and all-round way; it also proposes that CTRP-9 can be used as a universal enhancer to significantly improve the anti-tumor function of CAR-T cells. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 Figure showing the construction results of mouse CTRP-9 recombinant protein;

[0040] Figure 2 The graph shows the results of Western blot analysis of molecular phosphorylation levels in mouse spleen cells.

[0041] Figure 3 For the detection of CD4 by flow cytometry + and CD8 + Graph showing the expression levels of specific molecules in T cells;

[0042] Figure 4 For the detection of CD4 by flow cytometry + and CD8 + Graph showing the results of T cell proliferation;

[0043] Figure 5 For the detection of CD4 by flow cytometry + and CD8 + Figure showing the secretion levels of IL-2 and IFN-γ in T cells;

[0044] Figure 6 A schematic diagram illustrating the construction of the mouse CD19 CAR molecule;

[0045] Figure 7 Design diagram for in vitro co-culture experiment of anti-CD19 CAR-T cells and A20 cells;

[0046] Figure 8 This diagram illustrates the flow cytometry analysis strategy for the co-culture of the two cell types mentioned above in vitro.

[0047] Figure 9 Figure showing the results of flow cytometry analysis of apoptosis levels in A20 cells;

[0048] Figure 10 Figure showing the results of flow cytometry detection of cleaved caspase-3 levels in A20 cells;

[0049] Figure 11 For flow cytometry detection of co-cultured CD4 + or CD8 + Graph showing IFN-γ secretion levels in CAR-T cells;

[0050] Figure 12 For flow cytometry detection of co-cultured CD4 + or CD8 + Graph showing Granzyme B secretion levels in CAR-T cells;

[0051] Figure 13 For flow cytometry detection of co-cultured CD4 + or CD8 + The proportion of CD107a cells in CAR-T cells. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0053] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0054] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0055] This invention provides a method for enhancing the efficacy of T-cell immunotherapy, involving a novel application of adipokines C1q / TNF-related protein 9 (CTRP-9) in regulating T-cell function. CTRP-9 is an immunomodulatory molecule secreted by adipose tissue. This invention proposes that exogenous supplementation with CTRP-9 can effectively enhance the effector strength of T cells in anti-tumor immune responses and promote T-cell activation and proliferation. Simultaneously, CTRP-9 can also act as a CAR-T cell enhancer, thereby improving the efficacy of immunotherapy. This method can be applied to T-cell-based immunotherapy systems, providing a new regulatory pathway and target for improving the therapeutic effect of T-cell immunotherapy.

[0056] In this embodiment of the invention, the sequence of the mouse mCTRP9 gene fragment is shown in SEQ ID NO.3.

[0057] In this embodiment of the invention, the experimental materials used were: Buffer 1: 50 mM PBS, 29.3 g / L NaCl, 480 g / L urea, NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH=7.4), 1‰ protease inhibitor, 1‰ PMSF, 1% phosphorylase inhibitor, phospho-S6 (Ser240 / 244, Catalog #5364), phospho-ERK1 / 2 (Thr202 / Tyr204, Catalog #4370), phospho-AKT (Thr308, Catalog #13038), phospho-JNK (Thr183 / Thr183 / Thr221, Catalog #AF1762), β-actin (Catalog #4970), phospho-p65 (Ser536, Catalog #AF1762), β-actin (Catalog #4970), and phospho-p65 (Ser536, Catalog #AF1762). #AF5881), Annexin V antibody (Catalog #640920, BioLegend), Annexin V Binding buffer (0.01 M HEPES / NaOH, 0.14 M NaCl, 2.5 mM CaCl2), 7-AAD (Catalog #A1310, Invitrogen), CFSE (Catalog #C34554, Invitrogen), APC-conjugated anti-mouse CD69 (Catalog #104513, BioLegend), PerCP / Cyanine 5.5-conjugated anti-mouse CD25 (Catalog #101911, BioLegend), FITC-conjugated anti-mouse CD4 (Catalog #100509, BioLegend), PE-conjugated anti-mouse CD8 (Catalog #162303, BioLegend), BV421-conjugated anti-mouseCD107a (Catalog #121617, BioLegend), BD cytofix / cytoperm buffer (BDBiosciences), 1× BD perm / wash buffer (BD Biosciences), cleaved caspase 3 (Catalog #9664, Cell Signaling Technology), cleaved caspase 8 (Catalog #9496, Cell Signaling Technology), APC-conjugated anti-mouse IL-2 (Catalog #503809,BioLegend)、Brilliant Violet 421-conjugated anti-mouse IFN-γ (Catalog #505829, BioLegend), PE anti-human / mouse Granzyme B Recombinant (Catalog #372207, BioLegend). .

[0058] Example 1:

[0059] This embodiment provides a method for preparing recombinant mouse adipokines CTRP-9 protein. The mouse strain used is BALB / c.

[0060] Furthermore, the preparation method of the above-mentioned recombinant protein includes the following steps:

[0061] Cloning of the mouse adipokines CTRP-9 gene:

[0062] Total RNA was extracted from mouse leukocytes, and samples meeting quality standards were reverse transcribed into cDNA. Specific primer pairs were designed: forward primer F1 (SEQ ID NO.1: CCCAAGCTTGCCAGGACACCTGCCGGCAAGG) and reverse primer R1 (SEQ ID NO.2: CCGCTCGAGTCAAGAGCTGCTGAACAGCA). Using cDNA as a template, a 30-50 μL PCR reaction system was established, with F1 and R1 each accounting for 4-5%, 2×PrimerSTAR DNA Polymerase 45-50%, cDNA template 4-5%, and the remainder supplemented with dH2O. Amplification conditions were: 95-98℃ for 10-12 s, 55-58℃ for 10-15 s, 70-75℃ for 8-10 s, for 32-40 cycles; followed by the addition of 1 μL rTaq and extension at 72℃ for 20 min. The amplified product was recovered by gel electrophoresis to obtain the mouse CTRP-9 gene fragment (SEQ ID NO.3), which has been isolated and purified from natural genomic DNA and related proteins.

[0063] Construction of CTRP-9 gene expression vector:

[0064] 4.5 μL of the recovered product was mixed with the vector (0.5 μL PMD19T vector, 5 μL Solution Ⅰ) and ligated at 16℃ for 10–15 h to obtain the first ligation system (ligation product). 10 μL of the ligation product was transferred into 50 μL of DH5α competent cells, placed on ice for 30 min, heat-shocked at 42℃ for 45 s, and immediately incubated on ice for 2 min. 200–400 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 37℃ with shaking for 1 h. Then, 50–200 μL of the medium was plated on LB agar plates containing ampicillin and cultured overnight at 37℃. Positive clones were picked and sequenced, and the PMD19T plasmid containing the CTRP-9 gene (CTRP-9-PMD19T) was extracted.

[0065] Add 1 μg each of CTRP-9-PMD19T and pET-28a plasmids to a double digestion system (1 μL BamHI, 1 μL HindIII, 4 μL Cutsmart buffer, to a final volume of 40 μL), and digest at 37°C for 4 h to obtain two digestion products. Mix 4.5 μL of each product with a DNA ligation system (1 μL T4 DNA Ligase, 1 μL Buffer), and ligate at 15-18°C for 10-12 h to obtain the second ligation system. Transform the second ligation product into 50-100 μL of Transetta competent cells, incubate on ice for 30 min, heat shock at 42°C for 40-45 s, incubate on ice for 2 min, then add 300-500 μL of LB broth medium and incubate with shaking for 1 h. Spread 50-200 μL of the bacterial culture onto LB agar plates containing kanamycin and incubate overnight at 37°C. The next day, positive clones were selected, cultured, and plasmids were extracted to obtain the pET28a-CTRP-9 expression vector.

[0066] Preparation of His-tagged recombinant proteins:

[0067] Positive clones were inoculated into 300 mL of LB liquid medium containing kanamycin and cultured at 37 °C and 200 rpm until OD600 = 0.6. IPTG (final concentration 0.5 mmol / L) was added for induction for 4 h, and the bacterial pellet was collected. The pellet was resuspended in 30 mL of Buffer 1 (50 mM PBS, 29.3 g / L NaCl, 480 g / L urea, pH 7.4), sonicated on ice, and centrifuged at 10,000 rpm and 4 °C for 10 min. The supernatant was collected.

[0068] Protein purification: The nickel column was first rinsed with water to remove ethanol, then activated with 0.2 M nickel sulfate and washed with water until the baseline was stable. After equilibration with Buffer 1, the column was loaded with the sample. Unbound protein was washed away with Buffer 1, followed by fractionation elution with 50 mM imidazole buffer (to remove contaminating proteins) and 400 mM imidazole buffer (to elute the target protein). The fraction containing the target protein mCTRP9 was collected. Finally, the column was washed sequentially with 1 M NaOH, pure water, and 20% ethanol and stored.

[0069] Detection: Take an appropriate amount of the target protein and add it to 5×SDS loading buffer. Heat in a boiling water bath for 5 min to denature the target protein. Detect the purity of the target protein by 12% SDS-PAGE.

[0070] The amino acid sequence of the recombinant protein mCTRP9 is shown in SEQ ID NO.4, and its sequence is consistent with the known mouse CTRP-9 native protein sequence.

[0071] Refolding: The purified target protein (mCTRP9) sample from the "Preparation of His-tagged Recombinant Protein" step was placed in a dialysis bag and dialyzed sequentially with dialysis buffers at pH 9 and urea concentrations of 6 M, 4 M, 2 M, 1 M, and 0 M at 4°C, each step lasting 12 hours. Subsequently, the dialysis bag was transferred to pre-cooled Tris-HCl buffer (pH 8.5) and dialyzed for another 12 hours. After dialysis, the sample was centrifuged (10,000 rpm, 4°C, 10 min), and the supernatant was collected and concentrated to the target volume using a 10 kDa ultrafiltration tube (4,000 rpm, 4°C). Finally, the target protein concentration was determined using the BCA method, and the sample was mixed with 5×SDS loading buffer, boiled for denaturation, and the target protein purity was analyzed by 12% SDS-PAGE. The results are shown below. Figure 1 As shown.

[0072] Example 2:

[0073] This embodiment provides an application of CTRP-9 to enhance T-cell immunity, including the following steps:

[0074] To detect T cell activation, mouse spleen leukocytes were isolated (1 × 10⁶ cells per well). 6Cells were seeded in 24-well plates and stimulated for 6 h with 10 μg / mL mCTRP9 (mCTRP9 here is the purified and refolded high-purity, biologically active mouse CTRP-9 recombinant protein finally obtained in the "His-tag recombinant protein preparation" and "renaturation" steps of Example 1 of this invention). Cells were then resuspended in 40 μL NP-40 lysis buffer (1‰ protease inhibitor, 1‰ PMSF, 1% phosphorylase inhibitor) and lysed on ice for 30 min. After centrifugation at 12000 rpm and 4℃ for 10 min, the supernatant was collected, and 10 μL of 5×SDS loading buffer was added. The mixture was then boiled in a water bath for 5 min to obtain protein samples for each group. The samples were subjected to SDS-PAGE electrophoresis, transfer, blocking, and PBST washing three times for 10 min each time. The target antibody was added and incubated overnight at 4℃, followed by PBST washing three times for 10 min each time. Alexa Fluor 800-conjugated goat protein was added at a 1:20000 concentration. Anti-rabbit IgG H&L (Abcam) was incubated at room temperature for 1 h, followed by washing three times with PBST for 10 min each time. The NC membrane was scanned using an Odyssey CLX imaging system. For flow cytometry experiments, three groups were compared: ① Ctrl group (negative control group): spleen cells without any stimulation; ② αCD3 / 28 group (activation control group): stimulated only with CD3 / CD28 monoclonal antibody; ③ αCD3 / 28 + mCTRP-9 group (treatment group of this invention): stimulated with CD3 / CD28 monoclonal antibody plus 10 μg / mL mCTRP-9. After 12 h of stimulation, approximately 5 × 10⁶ cells were collected. 6 Cells were placed in 96-well V-plates and centrifuged at 2500 rpm for 3 min, discarding the supernatant. Mouse spleen cells were then incubated on ice for 30 min with 1:400 diluted FITC-labeled anti-mouse CD4, PE-labeled anti-mouse CD8, APC-labeled anti-mouse CD69, or PerCP / Cyanine 5.5-labeled anti-mouse CD25. After washing twice with FACS buffer (PBS containing 2% FBS), cells were resuspended in FACS buffer. All samples were collected using a BD LSR Fortessa flow cytometer and analyzed using Flowjo software.

[0075] The results are as follows Figure 2-3 As shown, mCTRP9 treatment of mouse spleen cells significantly increased the phosphorylation levels of JNK, AKT, ERK1 / 2, NF-κB, and S6 (the phosphorylation of the aforementioned factors corresponds to p-JNK, p-AKT, p-ERK1 / 2, p-NF-κB, and p-S6, respectively). Figure 2This indicates enhanced T cell activation. This was further confirmed by a T cell activation assay, in which mCTRP9 administration increased CD25 and CD69 expression induced by the CD3 / CD28 monoclonal antibody in T cells, particularly on CD4. + T cells ( Figure 3 ).

[0076] To detect T cell proliferation, mouse spleen leukocytes were labeled with 10 μM CFSE for 9 minutes at room temperature. After washing twice with DMEM, the labeled cells were cultured in DMEM with or without 10 μg / mL mCTRP9, and T cell proliferation was induced with 2 μg / mL anti-mouse CD3 and 2 μg / mL anti-mouse CD28. After culturing at 28°C for 48 hours, the cells were stained with 1:400 diluted FITC-labeled anti-mouse CD4 monoclonal antibody and 1:400 diluted PE-labeled anti-mouse CD8 monoclonal antibody. 1:400 diluted 7-AAD was added before flow cytometry to distinguish between live and dead cells.

[0077] The results are as follows Figure 4 As shown, mCTRP9 confers CD4 upon CD3 / CD28 mAb stimulation. + and CD8 + T cells have a stronger proliferative capacity.

[0078] When detecting cytokine levels, take approximately 5 × 10⁻⁶ samples. 6 Mouse spleen leukocytes were placed in 96-well V-plates and centrifuged at 2500 rpm for 3 min, discarding the supernatant. A 1:400 dilution of anti-mouse CD4 antibody, anti-mouse CD8 antibody, and PE-Cy7-labeled anti-mouse CD19 antibody were added, and the cells were incubated on ice in the dark for 30 min. After centrifugation at 2500 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 200 μL FACSBuffer (L-15, 2% FBS), centrifuged again, and the resulting cell pellet was washed twice. For intracellular staining, cells were fixed on ice for 30 min with BD fixation / permeabilization buffer and washed twice with 1× BD permeabilization / washing buffer. Then, cells were stained on ice for 30 min with cAPC-labeled anti-mouse IL-2 and Brilliant Violet 421-labeled anti-mouse IFN-γ. After washing twice with 1× BD permeabilization / washing buffer, the cells were resuspended in 1× BD permeabilization / washing buffer. All samples were collected using a BD LSR Fortessa flow cytometer and analyzed using Flowjo software.

[0079] The results are as follows Figure 5As shown, mCTRP9 significantly enhances CD4 under P+I stimulation ("P+I" is a commonly used and highly effective T cell stimulation protocol in immunological experiments, consisting of a combination of two reagents: P: Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), mimicking an important intracellular signal in the T cell activation process; I: Ionomycin, another key signal required for T cell activation). + IL-2 is produced in T cells, but it is not effective against CD8. + The production of IL-2 in T cells was neither affected nor inhibited, and the same trend was observed for the production of IFN-γ.

[0080] Example 3:

[0081] This embodiment provides a method for preparing anti-CD19 CAR-T cells. Further, the above-mentioned method for preparing CAR-T cells includes the following steps:

[0082] (1) Construction of a CAR molecule targeting mouse CD19

[0083] A post-transcriptional regulatory element (WPRE) of marmot hepatitis virus was inserted downstream of the transgene expression cassette in the MSCV retroviral backbone (Clonetech). The anti-mouse CD19 CAR structure was synthesized by GenScript and consisted of: a single-chain variable region fragment derived from the monoclonal antibody 1D3, a mouse 4-1BB intracellular domain sequence fragment (TNFRSF9, Uniprot: Q60765), and a mouse CD3ζ intracellular region (CD247, Uniprot: P24161). The anti-mouse CD19 CAR was separated from green fluorescent protein (GFP) (Uniprot: Q9BYP9) via a P2A self-cleaving polypeptide linker (sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO.5)) and co-cloned into the MSCV plasmid to obtain the mCD19-GFP-CAR plasmid. The vector carrying GFP was used as a reporter gene control. A schematic diagram of the specific construction is shown below. Figure 6 As shown.

[0084] (2) Preparation of anti-CD19 CAR-T cells

[0085] 10 μg of mCD19-GFP-CAR plasmid targeting mouse CD19 or GFP-CAR control plasmid (the preparation method for the control plasmid is similar to that described above. Using the MSCV retroviral plasmid backbone as a vector, green fluorescent protein was amplified by PCR and ligated to the MSCV empty vector plasmid via a P2A self-cleaving peptide to construct the GFP-CAR control plasmid), and co-transfected BOSC23 packaging cells with 5 μg of PCL-ECO helper plasmid. Cell supernatant rich in retroviral particles was collected 72 hours after transfection. 2 × 10⁻⁶ cells were then used. 6 Mouse spleen cells were resuspended in RPMI 1640 medium and seeded in 24-well plates. Activation culture was initiated with 2 μg / ml anti-CD3 antibody, 2 μg / ml anti-CD28 antibody, and 100 U / ml IL-2. After 24 hours, the activated cells were transferred to 6-well plates, and 1 ml of viral supernatant containing 8 μg / ml polybrene was added. The plates were centrifuged at 2000 rpm for 60 minutes and then incubated at 37°C for 8 hours. The medium was then replaced with fresh medium containing 100 U / ml IL-2. Cells were collected 72 hours after viral infection, and GFP was detected by flow cytometry. + The proportion of CAR-T cells. The specific procedure is as follows: Figure 7 As shown.

[0086] Example 4:

[0087] This embodiment provides an application of CTRP-9 in promoting anti-CD19 CAR-T cell tumor killing, and the specific operation steps are as follows: Figure 7 As shown, the flow cytometry staining strategy is as follows: Figure 8 As shown, it includes the following steps:

[0088] The anti-CD19 CAR-T cells prepared in Example 3 of this invention were mixed with A20 B-cell lymphoma cells at a 1:1 ratio (5 × 10 cells per well). 5 A20 cells were seeded in 24-well plates and co-cultured for the specified time. For the stimulation group, 10 μg / mL mCTRP9 was added for co-incubation. Flow cytometry analysis showed that CD19... + Apoptosis and cleaved caspase-3 staining were performed on A20 cells (i.e., A20 B-cell lymphoma cells expressing CD19 positive in a co-culture system).

[0089] CD19 + The detection of apoptosis levels in A20 cells after co-culturing with CAR-T cells included the following steps:

[0090] When detecting apoptosis levels, approximately 5 × 10⁻⁶ samples were taken. 6Co-cultured cells were placed in 96-well V-plates and centrifuged at 2500 rpm for 3 min, discarding the supernatant. After staining with PE-Cy7-labeled anti-mouse CD19 antibody as described above, APC-Annexin V diluted 1:400 in 1× Binding Buffer was added, and the cells were incubated at room temperature for 15 min. After incubation, 100 μL of 1× Binding Buffer was added. Subsequently, 7-AAD was added at a 1:400 ratio, and cell apoptosis was immediately detected. For intracellular staining, cells were fixed on ice for 30 min with BD fixation / permeabilization buffer and washed twice with 1× BD permeabilization / washing buffer. Cells were then stained on ice with cleaved caspase-3 for 30 min; after washing twice with 1× BD permeabilization / washing buffer, the cells were resuspended in 1× BD permeabilization / washing buffer. All samples were collected using a BD LSRFortessa flow cytometer and analyzed using Flowjo software.

[0091] The results are as follows Figure 9-10 As shown, the control group's retrovirus-transduced T cells (using the MSCV empty retrovirus plasmid backbone as a vector, co-transfecting BOSC23 packaging cells with PCL-ECO helper plasmid to generate retrovirus particles, and then using this virus to transduce mouse T cells, followed by expansion and purification to obtain control retrovirus-transduced T cells) had a weak apoptosis-inducing effect on A20B lymphoma cells; while anti-CD19 CAR-T cells could effectively kill target cells, and mCTRP9 treatment further enhanced CAR-T cell-mediated target cell death ( Figure 9 This phenomenon was also confirmed in A20 B-cell lymphoma cells through increased caspase-3 cleavage levels. Figure 10 ).

[0092] Example 5:

[0093] This embodiment provides an application of CTRP-9 to enhance the anti-CD19 CAR-T cell anti-tumor function, with specific flow cytometry staining strategies as follows: Figure 8 As shown, it includes the following steps:

[0094] The anti-CD19 CAR-T cells prepared in Example 3 of this invention were mixed with A20 B-cell lymphoma cells at a 1:1 ratio (5 × 10 cells per well). 5 A20 B-cell lymphoma cells were seeded into 24-well plates and co-cultured for 12 h. For the stimulation group, 10 μg / mL mCTRP9 was added for co-incubation (37 °C, 12 h). Flow cytometry analysis showed that the cells stimulated to produce GFP... + CD4 + or GFP+ CD8 + CAR-T cells were stained for IL-2, IFN-γ, TNF-α, Granzyme B, and CD107a. The cytotoxicity of CAR-T cells was assessed using the LDH release assay: the Cytotoxicity Detection Kit Plus was used according to the manufacturer's instructions.

[0095] The detection of IL-2, IFN-γ, TNF-α, Granzyme B, CD107a levels in CAR-T cells and cleaved caspase-3 levels in A20 cells includes the following steps:

[0096] Take approximately 5 × 10 6 Cells were placed in 96-well V-plates and centrifuged at 2500 rpm for 3 min, discarding the supernatant. A 1:400 dilution of anti-mouse CD4 antibody, anti-mouse CD8 antibody, and PE-Cy7-labeled anti-mouse CD19 antibody were added, and the cells were incubated on ice in the dark for 30 min. After centrifugation at 2500 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 200 μL FACS Buffer (L-15, 2% FBS), centrifuged again, and the resulting cell pellet was washed twice. For CD107a staining, BV421-labeled anti-mouse CD107a was added simultaneously during the first staining step. For intracellular staining, cells were fixed on ice for 30 min with BD fixation / permeabilization buffer and washed twice with 1× BD permeabilization / washing buffer. Cells were then stained on ice for 30 minutes with cleaved caspase 3, APC-labeled anti-mouse IL-2, PE-labeled anti-mouse TNF-α, Brilliant Violet 421-labeled anti-mouse IFN-γ, or PE-labeled anti-human / mouse antibodies. After washing twice with 1× BD permeabilization / washing buffer, the cells were resuspended in 1× BD permeabilization / washing buffer. All samples were collected using a BD LSRFortessa flow cytometer and analyzed using Flowjo software.

[0097] The results are as follows Figure 11-13 As shown, mCTRP9 treatment significantly upregulated CD4 in anti-CD19 CAR-T cells. + and CD8 + Subgroups of IFN-γ and Granzyme B ( Figure 11-12 The expression level of CTRP9 was increased, indicating that CTRP9 can enhance CAR-T cell function. Cytotoxicity experiments further validated this: anti-CD19 CAR-T cells treated with mCTRP9 showed significantly increased CD107a degranulation levels and stronger cell lysis activity, indicating enhanced cytotoxicity. Figure 13).

[0098] In summary, this invention reveals for the first time the role of adipokines CTRP-9 in enhancing T-cell immunotherapy. Traditionally, research on CTRP-9 has focused on the metabolic and cardiovascular fields. This invention makes a breakthrough by discovering that exogenous CTRP-9 can significantly promote T-cell activation, proliferation, and cytokine secretion, and effectively enhance the cytotoxic function and anti-tumor effects of CAR-T cells. This provides a novel molecular target and combination strategy for overcoming T-cell dysfunction and improving the durability of immunotherapy. This discovery has significant clinical application potential and market value, laying the theoretical and technological foundation for the development of next-generation cell-mediated immune enhancement agents.

[0099] Sequence information in this invention:

[0100] SEQ ID NO. 3:

[0101]

[0102] SEQ ID NO. 4:

[0103] MRIWWLLLVMGACTRSVFSQDTCRQGHSGIPGNPGHNGLPGRDGRDGAKGDKGDAGEPGHPGGPGKDGIRGEKGEPGADGRVEAKGIKGDPGSRGSPGKHGPKGSIGPTGEQGLPGETGPQGQKGDKGEVGPTGPEGLMGSTGPLGPKGLPGPMGPIGKPGPRGEA GPMGPQGEPGVRGMRGWKGDRGEKGKVGEAPLVPKSAFTVGLTVISKFPPPDAPIKFDKILYNELNHYNVATGKFTCHVAGVYYFTYHITVFSRNVQVSLVKNGVKVLHTKDSYMSSEDQASGGIVQELKLGDEVWMQVTGGERFNGLFADEDDDTTFTGFLLFSSS

[0104] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0105] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0106] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of CTRP-9 in preparation of a drug for enhancing T cell immunotherapy effect, regulating T cell function, enhancing the effect strength of T cells in anti-tumor immune response, promoting T cell activation and proliferation.

2. Application of CTRP-9 in preparation of a drug for enhancing the function of CAR-T cells and enhancing the killing ability of CAR-T cells on tumor cells.

3. Use according to claim 1 or 2, characterized in that, The nucleotide sequence of the CTRP-9 is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO.

4.

4. A modulator / accelerator / activator, characterized in that, The modulator / promoter / activator comprises CTRP-9.

5. A CAR-T cell enhancer, characterized in that, The CAR-T cell enhancer comprises CTRP-9.

6. A reagent / kit characterized in that, The reagent / kit comprises one or more of CTRP-9, the modulator / promoter / activator of claim 4, the CAR-T cell enhancer of claim 5.

7. A medicament / pharmaceutical composition for enhancing the effect of T cell immunotherapy, characterized by, The drug / drug composition comprises one or more of CTRP-9, the modulator / promoter / activator of claim 4, the CAR-T cell enhancer of claim 5, or the reagent / kit of claim 6.

8. The modulator / accelerator / activator of claim 4, the CAR-T cell enhancer of claim 5, the reagent / kit of claim 6, or the drug / drug composition of claim 7, wherein, The nucleotide sequence of the CTRP-9 is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO.

4.

9. Application of the modulator / promoter / activator of claim 4, the CAR-T cell enhancer of claim 5, the reagent / kit of claim 6, or the drug / drug composition of claim 7 in preparation of a drug for enhancing T cell immunotherapy effect, regulating T cell function, enhancing the effect strength of T cells in anti-tumor immune response, promoting T cell activation and proliferation.

10. Application of the modulator / promoter / activator of claim 4, the CAR-T cell enhancer of claim 5, the reagent / kit of claim 6, or the drug / drug composition of claim 7 in preparation of a drug for enhancing the function of CAR-T cells and enhancing the killing ability of CAR-T cells on tumor cells.

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