A kit for quality control and efficacy prediction of CAR-T cells based on TNFR2 gene M196R mutation and genetically engineered CAR-T cells

Abstract

This invention belongs to the field of biomedical technology and provides a CAR-T cell quality control and efficacy prediction kit based on the TNFR2 gene M196R mutation, as well as genetically engineered CAR-T cells. The kit contains a specific detection reagent targeting the rs1061622 locus of the TNFR2 gene, detecting the subject's genotype at this locus and assessing CAR-T treatment-related indicators by determining whether the subject carries the M196R germline mutation. The kit provided by this invention fills the gap in existing technologies regarding the focus on the host's germline genetic background. It is easy to operate, provides reliable results, and can assist in patient risk stratification and treatment strategy optimization before CAR-T treatment, providing key technical support for improving treatment safety and efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a quality control and efficacy prediction kit for CAR-T cells based on the M196R mutation of the TNFR2 gene, and genetically engineered CAR-T cells. Background Technology

[0002] Chimeric antigen receptor T cell (CAR-T) therapy, a revolutionary adoptive immunotherapy technique, has achieved breakthrough efficacy in the treatment of hematologic malignancies such as non-Hodgkin's lymphoma and acute lymphoblastic leukemia, providing a new treatment option for patients who have failed traditional treatments or have relapsed. Its core principle is to modify the patient's own T cells using genetic engineering technology to express chimeric antigen receptors (CARs) that specifically recognize tumor antigens, thereby precisely killing tumor cells and achieving disease remission.

[0003] However, CAR-T therapy still faces two major challenges in clinical application, which seriously restrict the stability of its therapeutic effect and its widespread application: On the one hand, after CAR-T cells come into contact with tumor antigens in vivo, they are prone to activation-induced cell death (AICD), resulting in a rapid decrease in the number of effective cells and insufficient proliferation persistence, which directly affects the durability of anti-tumor efficacy; on the other hand, the quality control of CAR-T cell products lacks efficient and precise molecular targets. Existing quality control systems mostly focus on conventional indicators such as cell purity and CAR expression rate, which are difficult to directly correlate with the core functions of cells (such as anti-apoptosis ability and tumor killing activity) through simple detection. This leads to large differences in the efficacy of different batches of cell products, and some products cannot achieve the expected therapeutic effect due to functional defects.

[0004] Tumor necrosis factor receptor 2 (TNFR2), an important member of the tumor necrosis factor receptor family, is mainly expressed on the surface of immune cells such as T cells and regulatory T cells, playing a crucial role in the regulation of T cell function. It can promote T cell proliferation and survival by activating signaling pathways such as NF-κB and MAPK, and can also induce autoimmune disease-associated clonic encephalopathy (AICD) under continuous stimulation, participating in the negative regulation of immune responses. A germline missense mutation exists at the rs1061622 site of the TNFR2 gene (TNFRSF1B), which leads to the substitution of arginine for methionine at position 196 of the encoded protein (M196R mutation). This mutation has been shown to be associated with the occurrence of autoimmune diseases, but no research reports have been published on its application in the regulation and quality control of CAR-T cell function.

[0005] In existing technologies, the preparation of CAR-T cells largely relies on traditional genetic engineering techniques, lacking targeted modification strategies to "enhance the cell's anti-apoptotic ability and improve its functional stability." At the same time, quality control kits are mostly designed around general cell indicators, and no molecular detection tools that can specifically correlate with the core functions of CAR-T cells have been developed, which cannot meet the clinical needs for precise quality control and screening of high-quality cell products.

[0006] Therefore, developing a genetically engineered CAR-T cell based on key functional targets and its dedicated quality control kit is of great significance for solving the problems of insufficient cell function and low quality control efficiency in existing CAR-T therapies, and is also a technical bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0007] A first aspect of the present invention provides a kit for quality control and / or efficacy prediction of CAR-T cell therapy, the kit comprising reagents for detecting the rs1061622 site polymorphism of the TNFRSF1B gene in subject samples.

[0008] Specifically, the kit also includes a positive control, a negative control, and a reaction buffer required for detection. The positive control is a TNFR2-containing buffer. M196R The mutated plasmid, wherein the negative control is a plasmid containing the wild-type TNFRSF1B gene.

[0009] Specifically, the detection method includes at least one of qPCR, Sanger sequencing, high-throughput sequencing, allele-specific hybridization array, digital PCR, or mass spectrometry genotyping.

[0010] Specifically, the reagents include at least one of qPCR primer-probe combinations, sequencing primers, genotyping chips, or digital PCR detection reagents.

[0011] More specifically, the steps for using the kit include: (1) Detect the genotype of the TNFR2 gene at the rs1061622 locus in the subject samples; (2) Based on genotype, assess the risk of developing refractory cytokine release syndrome and / or the potential for long-term remission in the subject.

[0012] Preferably, the test results include: G / G, G / T, or T / T. Subjects with test results of G / T or T / T are considered to have a high risk of developing refractory cytokine release syndrome and / or a high potential for long-term remission.

[0013] A second aspect of the invention provides the use of any of the above-described kits in the preparation of products for quality control of genetically engineered CAR-T cells.

[0014] A third aspect of the invention provides a genetically engineered CAR-T cell whose genome contains a mutation at the TNFRSF1B locus that results in an M196R amino acid substitution.

[0015] Specifically, the mutation is introduced through gene editing technology, which includes, but is not limited to, any one or more of CRISPR / Cas9, TALEN, zinc finger nuclease, base editor, or leader editor.

[0016] A fourth aspect of the present invention provides the application of the above-described genetically engineered CAR-T cells in the preparation of tumor therapeutic drugs.

[0017] Specifically, the tumor is a hematologic malignancy.

[0018] More specifically, the hematologic malignancies include, but are not limited to, any one or more of leukemia, lymphoma, and multiple myeloma.

[0019] A fifth aspect of the present invention provides a pharmaceutical composition comprising any of the genetically engineered CAR-T cells described above.

[0020] Specifically, the pharmaceutical composition further includes pharmaceutical excipients.

[0021] More specifically, the pharmaceutical excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0022] Specifically, the administration methods of the pharmaceutical composition include oral, injection, implantation, external application, spray, inhalation, or combinations thereof.

[0023] A sixth aspect of the present invention provides a personalized method for CAR-T therapy, the method comprising the following steps: (1) Detect the genotype of the TNFR2 gene at the rs1061622 locus in the subject samples; (2) For subjects whose test results are G / T or T / T, implement specific clinical monitoring protocols and / or preventive treatment strategies.

[0024] Specifically, the particular clinical monitoring protocol and / or preventive treatment strategy includes the following steps: S1. Enhanced pretreatment: Employing a more aggressive lymphocyte-clearing chemotherapy regimen to reduce the initial tumor burden and immunosuppressive environment; S2. Enhanced monitoring: More intensive monitoring of cytokine levels and CAR-T cell expansion kinetics after CAR-T infusion.

[0025] A seventh aspect of the present invention provides a method for treating tumors, the method comprising administering to a subject a therapeutically effective amount of any of the above-described genetically engineered CAR-T cells or pharmaceutical compositions.

[0026] Specifically, the tumor is a hematologic malignancy.

[0027] More specifically, the hematologic malignancies include, but are not limited to, any one or more of leukemia, lymphoma, and multiple myeloma.

[0028] The term "subject" includes living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0029] The terms “treat” or “treatment” or “ameliorate” refer to the medical management of a subject’s disease, condition, or undesirable condition. Treatment or preventative benefits include improved clinical outcomes; reduction or alleviation of symptoms associated with the disease, condition, or undesirable condition; reduced symptom occurrence; improved quality of life; longer disease-free status; reduction in the severity of the disease, condition, or undesirable condition; stabilization of the disease state; delay in disease progression; remission; survival; prolonged survival; or any combination thereof.

[0030] The term "therapeutic effective dose" refers to a pharmaceutically considered effective dosage, that is, an amount of active drug sufficient to significantly improve the condition without causing serious side effects. Dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose; therefore, the dosage of this invention can vary widely.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The SNP detection technology involved in this invention is mature, low-cost, and easily integrated into existing clinical pathways. The classification and intervention recommendations based on the test results are specific and actionable, enabling rapid translation from research findings into precise clinical practice. Attached Figure Description

[0032] Figure 1 This is a plasmid map of the lentiviral vector CAR19-TNFR2.

[0033] Figure 2The results of the quantitative apoptosis experiment are as follows: A represents the apoptotic response of Jurkat cells to TNF-α under normal culture conditions; B represents the apoptotic response of Jurkat cells to TNF-α under CD3 stimulation conditions; and C represents the apoptotic response of Jurkat cells to TNF-α under tumor antigen stimulation conditions.

[0034] Figure 3 The effect of TNFα stimulation at different time points after CD3 stimulation on the number of Jurkat cells.

[0035] Figure 4 CAR19-TNFR2 WT -Jurkat or CAR19-TNFR2 M196R -Jurkat cells and Nalm6 cells were co-cultured at a 1:1 E:T ratio for 24 h, and simultaneously stimulated with different concentrations of TNFα (0, 1 ng / ml). The expression of Ki67 was different.

[0036] Figure 5 To detect differential expression of phosphorylated p65 using Western blotting.

[0037] Figure 6 To detect the surface dynamics of TNFR2 expression in naïve and CD3 / CD28 activated T cells by flow cytometry.

[0038] Figure 7 TNFR2 WT -OE-CAR-T and TNFR2 M196R -OE-CAR-T cells were co-cultured with Nalm6 cells at an E:T ratio of 1:20, and the difference in apoptosis was detected by flow cytometry.

[0039] Figure 8 TNFR2 WT -OE-CAR-T and TNFR2 M196R -OE-CAR-T was co-cultured with Nalm6 at a ratio of 1:20 for 24 hours, and TNFα and IFNγ were detected by ELISA.

[0040] Figure 9 TNFR2 for different TNFR2 genotypes M196R -OE and TNFR2 WT Cytotoxicity and viability of OE CAR-T cells after co-culturing with Nalm6 for 24 hours.

[0041] Figure 10 Sequencing profiles of the TNFR2 genotypes (WT, MR, and RR).

[0042] Figure 11 To use conventional, TNFR2WT -OE or TNFR2 M196R After multiple rounds of killing experiments were conducted on OE CAR-T cells and Nalm6 cells at a ratio of 1:5, the killing ability of CAR-T cells on Nalm6 cells and the number of remaining CAR-T cells were detected by flow cytometry.

[0043] Figure 12 To determine the effect of co-culturing TNFR2 with Nalm6 cells at a 1:1 E:T ratio. WT -OE-CAR-T and TNFR2 M196R Ki67 expression in -OE-CAR-T cells.

[0044] Figure 13 Raji cells and targeted knock-in of TNFR2 WT or TNFR2 M196R Cytotoxicity and CAR-T cell count analysis after 24 hours of co-culturing CAR-T cells at a 1:2 E:T ratio.

[0045] Figure 14 For targeted knock-in of TNFR2 WT or TNFR2 M196R CAR-T cells were subjected to multiple rounds of killing experiments with Raji cells at a 1:1 E:T ratio, and the number of remaining Raji cells and CAR-T cells was detected by flow cytometry. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] In a specific embodiment of the present invention, the abbreviation "OE" represents overexpression; TNFR2 WT -OE-CAR-T and TNFR2 M196R -OE-CAR-T represents overexpression of TNFR2 in cells. WT or TNFR2 M196R abbreviation TNFR2 WT CAR-T or TNFR2 M196R CAR-T cells represent site-specific knock-in of TNFR2 WT or TNFR2 M196R .

[0048] Example 1: Construction of CAR-TNFR2 co-expression vector Objective: To obtain the lentiviral vector CAR19-TNFR2, which can simultaneously express CD19-CAR and full-length TNFR2 in T cells, for subsequent CAR-T construction and functional validation.

[0049] Vector framework and element selection: see plasmid map. Figure 1 The third-generation self-inactivated (SIN) lentiviral vector backbone was selected, containing 5′LTR, 3′LTR, self-inactivated U3 region, packaging signal ψ, and WPRE enhancement elements. The promoter is preferably the human EF1α promoter to ensure stable expression in T cells. The CAR structure sequentially includes: scFv derived from the anti-CD19 monoclonal antibody; CD8α hinge region; CD8α transmembrane region; 4-1BB co-stimulatory intracellular domain; and CD3ζ signaling domain. The TNFR2 structure is TNFR2. WT Or CAR19-TNFR2 M196R .

[0050] (2) Construct a restructuring vehicle through a commercial company.

[0051] Example 2 Preparation of CAR-TNFR2-T (1) Isolation and activation of peripheral blood T cells ① Collect peripheral blood from healthy donors or patients (containing anticoagulants, such as vacuum blood collection tubes containing EDTA or heparin).

[0052] ② Dilute with PBS at a 1:1 ratio and slowly spread the whole blood along the tube wall onto the upper layer of the Ficoll-Paque density gradient solution.

[0053] ③ Centrifuge at 800 g for 40 min at room temperature, and transfer the PBMC layer to a new 50 mL centrifuge tube.

[0054] ④ Wash PBMCs twice with PBS, 300 g each time, for 5-10 min.

[0055] ⑤ Count PBMCs and use 0.5-1×10 6 Cells / mL were seeded in 24-well or 6-well plates using X-VIVO15 or AIM-V medium (containing 5%–10% autologous inactivated serum or human AB serum).

[0056] ⑥ Add anti-CD3 / CD28 magnetic beads (or antibody-coated plates) and activate T cells at a ratio of 1:1 or 1:2 (beads:cells), and culture for 48 h; then add IL-2.

[0057] (2) Lentiviral packaging and titer determination ① Seed HEK293T cells in 10 cm dishes one day in advance to ensure confluence of 70%-80% by the time of transfection the next day.

[0058] ② Using standard liposome transfection reagents, CAR19-TNFR2 was transfected. WT Or CAR19-TNFR2 M196R Lentiviral plasmids and packaging plasmids (such as psPAX2, pMD2.G) are mixed and transfected at a mass ratio of 4:3:1.

[0059] ③ Replace with 12 mL of fresh complete culture medium DMEM 6 h after transfection.

[0060] ④ Add 6 mL of DMEM solution after 48 h, collect the supernatant after 72 h, and filter it through a 0.45 μm filter membrane; if a high titer is required, it can be concentrated by ultracentrifugation or PEG precipitation.

[0061] ⑤ The viral titer can be roughly estimated by infecting indicator cells and detecting reporter gene expression or by qPCR.

[0062] 3. T-cell lentiviral transduction ① T cells activated for 24-48 hours were resuspended in a culture medium containing IL-2.

[0063] ② Add the viral supernatant according to the predetermined MOI. Polycationic polymers (such as polyaluminum) can be added to improve infection efficiency.

[0064] ③ Retronectin-coated plates can be used: dilute Retronectin and coat the bottom of a 6-well or 24-well plate; add viral supernatant, centrifuge at 2000 g for 2 h to allow the virus to adsorb onto the bottom of the plate; then add activated T cells, centrifuge at 1000 g for 10 min, and incubate at 37℃.

[0065] ④ Replace with fresh culture medium 24 hours after transduction.

[0066] ⑤ Three days later, the expression of CAR and TNFR2 was detected by flow cytometry.

[0067] 4. Construction of AAV vector Objective: To obtain a method to package the CDS sequence of a mutated gene into AAV viral particles and express the mutated gene using the promoter of endogenous TNFR2. This will enable subsequent targeted knock-in using the CRISPR / Cas9 ribonucleoprotein complex in conjunction with AAV6 to deliver homologous recombination repair templates. AAV recombinant vectors and viral packaging were constructed using commercial methods.

[0068] 5. Targeted knock-in of TNFR2 in T cells WT / TNFR2 M196R ① After peripheral blood T cells are isolated and activated for 24-48 hours, sgRNA and cas9 are mixed at a 1:1 molar ratio and incubated for 5-10 minutes to form the RNP complex.

[0069] ② The RNP complex is introduced into T cells using electroporation or reagents.

[0070] ③ Add AAV adeno-associated virus (MOI: 2E4-5E4) to (2) and incubate at 37°C for 45-60 min.

[0071] ④ Add T cell culture medium containing 5% FBS and 100-300 U IL-2 to the incubated cell mixture to make the cell density 2-3E6 / mL.

[0072] ⑤ After culturing for 4-5 days, the cell editing efficiency was tested. The CAR positivity rate represents the knock-in efficiency.

[0073] Example 1: TNFR2 M196R Functional mechanism verification of germline mutation 1. Validation of anti-apoptotic function: (1) In Jurkat cells (which do not express TNFR1 / 2 receptors), CAR19 and CAR19-TNFR2 were constructed by lentiviral infection as described in Example 2. WT CAR19-TNFR2 M196R The Jurkat cell lines were named CAR19-Jurkat and CAR19-TNFR2, respectively. WT -Jurkat and CAR19-TNFR2 M196R -Jurkat.

[0074] The three types of Jurkat cells constructed above were stimulated with 0, 1, and 10 ng / ml TNFα in a 37°C incubator for 24 h, respectively. The differences in cell apoptosis were detected by flow cytometry. The specific steps are as follows: Jurkat cells and Nalm6 tumor cells were co-cultured for 24 hours at a specified effective-target ratio. Before co-culture, tumor cells were labeled with the live cell dye Fixed Viability Dye eFluor™ 450 to distinguish them from Jurkat cells.

[0075] Cells were collected, washed, and then co-stained with Annexin V and 7-AAD.

[0076] (3) After 15 min, the cell apoptosis was detected by flow cytometry.

[0077] The results are as follows Figures 2-4 As shown, TNFR2 is expressed M196R The apoptosis rate of the cells was significantly lower than that of the wild-type control group, and a higher number of viable cells were maintained, demonstrating that this mutation conferred the ability of T cells to resist TNFα / TNFR2 axis-mediated apoptosis.

[0078] 2. Verification of enhanced affinity: (1) Ligand immobilization: The analyte molecules TNFR2 (M196R) and TNFR2 (WT) were immobilized on the surface of a metal thin film to form a sensing chip. (2) Analyte injection: The sample containing the analyte TNFα protein was injected into the flow cell to contact the molecules immobilized on the chip surface. The intermolecular interactions were monitored in real time using the SPR instrument Biacore, and the changes in reflected light intensity during binding and dissociation were recorded. (3) Chip regeneration: The chip surface was cleaned with regeneration buffer to thoroughly wash away the analytes that were still bound to the ligands after spontaneous dissociation, so that the target surface was restored to its initial state. This process was repeated until all the corresponding concentrations of the compounds were run. (4) Data analysis: By analyzing the sensing signal of the change in reflected light intensity over time, parameters such as binding constant (Ka), dissociation constant (Kd), and affinity (KD) were obtained. The experimental results are shown in Table 1.

[0079] Surface plasmon resonance (SPR) technology confirmed that the M196R mutation increased the affinity of TNFR2 for TNFα by about 2 times, which structurally explains the reason for the signal enhancement.

[0080] Table 1

[0081] Example 2 TNFR2 M196R Enhance CAR-T anti-tumor activity This embodiment utilizes lentiviral vectors or CRISPR / Cas9-mediated site-directed knock-in technology to overexpress TNFR2 in primary human CAR-T cells targeting CD19. WT or TNFR2 M196R The mutations were named TNFR2. WT -OE-CAR-T and TNFR2 M196R -OE-CAR-T was used for subsequent experiments. TNFR2 was validated through long-term, multi-round tumor-killing experiments. M196R Enhance the anti-tumor activity of CAR-T cells.

[0082] 1. CAR-T anti-tumor efficacy testing Objective: To compare the effects of TNFR2 carriers WT or TNFR2 M196R Differences in the antitumor efficacy of CAR-T cells from different receptors.

[0083] (1) Label tumor cells Nalm6 and CAR-T cells with cell dyes respectively.

[0084] (2) Set different effective target ratios.

[0085] (3) Cells were collected 24 hours later, and CAR-T survival and tumor lysis rate were statistically analyzed by flow cytometry.

[0086] 2. Detection of TNFα and IFNγ cytokine secretion (1) After co-culturing CAR-T and Nalm6 at a ratio of 1:5 for 24 h, the culture supernatant was collected by centrifugation at 300g for 10 minutes. 300 μl of 1× washing buffer was added to each well of the microplate to be used, and the plate was allowed to stand for 30 seconds. After discarding the washing buffer, the microplate was patted dry on absorbent paper. The microplate should be used immediately after washing; do not allow it to dry.

[0087] (2) Add standard: Add 100 μl of 2-fold serially diluted standard to the standard well. Add 100 μl of standard dilution or culture supernatant to the blank well.

[0088] (3) Add sample: Add 100 μl of cell culture supernatant to each well.

[0089] (4) Add test sample: Add 50 μl of “detection antibody working solution” to each test well.

[0090] (5) The addition of standards, samples and detection antibodies should be completed within 15 minutes, continuously and without interruption.

[0091] (6) Incubation: Use a sealing plate to seal the plate. Shake at 100-300 rpm and incubate at room temperature for 2 hours.

[0092] (7) Washing: Discard the liquid, add 300 μl of washing solution to each well and wash the plate 6 times.

[0093] (8) Add enzyme: Add 100 μl of "streptavidin working solution" to each well.

[0094] (9) Incubation: Seal the plate with a new sealing film. Shake at 100-300 rpm and incubate at room temperature for 45 minutes.

[0095] (10) Repeat step (7).

[0096] (11) Add substrate for color development: Add 100 μl of color development substrate to each well, and incubate at room temperature for 5-30 minutes in the dark.

[0097] (12) Add stop solution: Add 100 μl of stop solution to each well. The color changes from blue to yellow.

[0098] (13) Detection reading: Within 30 minutes, use an ELISA reader to perform dual-wavelength detection, measuring the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm or 630 nm. The calibrated OD value is the measured value at 450 nm minus the measured value at 570 nm or 630 nm.

[0099] 3. Multiple rounds of lethality experiments (1) Label tumor cells with cell dyes (Nalm6, Raji) (2) CAR-T: Nalm6 was co-cultured at a ratio of 1:5, and the remaining Nalm6 and CAR-T were detected by flow cytometry every 24 hours. After the tumor cells were eliminated, Nalm6 was added again at a ratio of 1:5, and the remaining Nalm6 and CAR-T were detected by flow cytometry.

[0100] CAR-T and Raji cells were co-cultured at a ratio of 1:1, and the remaining Raji and CAR-T cells were detected by flow cytometry every 48 hours. Then, the same number of Raji cells were added, and the process was repeated three times, with the remaining Nalm6 and CAR-T cells detected by flow cytometry.

[0101] Ki67 detection Collect the cell pellet by centrifugation, add 1 ml of Foxp3 fixation and permeabilization working solution to the tube, mix well, and incubate at room temperature in the dark for 30-60 min.

[0102] Add 2 ml of 1× membrane rupture buffer to the tube and centrifuge at 400-600 g for 5 min at room temperature. Discard the supernatant.

[0103] Resuspend the precipitate in the remaining volume of 1× membrane-breaking solution, typically 100 μl.

[0104] Add 5 μl of Ki67 flow cytometry antibody to the tube and incubate at room temperature in the dark for at least 30 minutes.

[0105] Add 2 ml of 1× membrane rupture buffer to the tube and centrifuge at 400-600 g for 5 min at room temperature. Discard the supernatant.

[0106] Repeat step (5).

[0107] Resuspend the stained cells in an appropriate volume of PBS.

[0108] Samples were analyzed using flow cytometry.

[0109] Western blot analysis of differential expression of phosphorylated p65 Collect the cell pellet and add protease inhibitors and phosphatase inhibitors to pre-chilled RIPA lysis buffer. Lyse on ice for 30 min, vortexing several times during the process, then centrifuge at 12000-14000g for 15 min at 4°C and collect the supernatant (i.e., protein sample).

[0110] (2) Protein quantification, denaturation and loading Quantitative analysis: The BCA method is recommended for protein quantification.

[0111] Sample preparation: Mix the protein sample with an appropriate amount of 5× loading buffer. The buffer should contain β-mercaptoethanol (to reduce disulfide bonds) or dithiothreitol.

[0112] Denaturation: Place the mixed sample in a 95-100℃ metal bath and heat for 5-10 minutes to fully denature the protein.

[0113] Loading amount: Depending on the abundance of the target protein, 20-40 μg total protein / well is usually a safe starting point. Pre-stained protein markers must be set.

[0114] (3) SDS-PAGE electrophoresis Gel concentration: p65 has a molecular weight of approximately 65 kDa, and a 10% separating gel can achieve good separation results.

[0115] Electrophoresis conditions: Constant voltage electrophoresis, typically 80V for the stacking gel and adjusted to 120V after entering the separating gel. Stop electrophoresis when the indicator (bromophenol blue) reaches the bottom of the gel.

[0116] (4) Transfer (wet transfer method recommended) Membrane selection: PVDF membranes are recommended for phosphorylated protein detection due to their strong protein binding capacity and high mechanical strength. They need to be activated by soaking in methanol for 1-2 minutes before use.

[0117] Transfer conditions: Use pre-cooled transfer buffer and place in an ice bath. For p65, a constant voltage of 100V for 90 minutes is recommended. Conditions need to be optimized according to the specific equipment in your laboratory.

[0118] (5) Closed Blocking was performed using 5% BSA (bovine serum albumin) TBST solution on a shaker at room temperature for 1 hour.

[0119] (6) Incubation of primary and secondary antibodies Phosphorylated p65 antibody: Recognizes the phosphorylated form of the p65 protein at a specific serine site (Ser536).

[0120] Total p65 antibody: Recognizes all forms of p65 protein and serves as an internal control.

[0121] Incubation conditions: Primary antibody incubation: Dilute the primary antibody with TBST containing 5% BSA. Incubate slowly overnight on a shaker at 4°C (for best results). After incubation, wash the membrane three times with TBST at room temperature for 10 minutes each time.

[0122] Secondary antibody incubation: Dilute the HRP-labeled secondary antibody of the corresponding species with TBST and incubate on a shaker at room temperature for 1 hour. Wash the membrane three times with TBST, 10 minutes each time.

[0123] Internal reference selection: In addition to total p65, GAPDH is also a commonly used internal reference.

[0124] (7) Chemiluminescence, development and data analysis Development: The ECL working solution is evenly dropped onto the membrane, and after reacting for 1-2 minutes, the signal is acquired using a chemiluminescence imaging system.

[0125] The experimental results are shown in Figure 5-14 : (1) The number of CAR-T cells decreased more slowly under repeated stimulation by tumor antigens (Nalm6, Raji), showing stronger proliferation persistence (CAR-T:Nalm6=1:5, CAR-T:Raji=1:1).

[0126] (2) Secreting higher levels of cytokines (such as TNFα, IFN-γ) in co-culture systems.

[0127] (3) It has a stronger or longer killing effect on tumor cell lines (such as Nalm6, Raji).

[0128] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for quality control and / or efficacy prediction of CAR-T cell therapy, characterized in that, The kit contains reagents for detecting the rs1061622 site polymorphism of the TNFRSF1B gene in subject samples.

2. The reagent kit according to claim 1, characterized in that, The detection methods include at least one of qPCR, Sanger sequencing, high-throughput sequencing, allele-specific hybridization arrays, digital PCR, or mass spectrometry genotyping.

3. The reagent kit according to claim 2, characterized in that, The reagents include at least one of qPCR primer-probe combinations, sequencing primers, genotyping chips, or digital PCR detection reagents.

4. The reagent kit according to any one of claims 1-3, characterized in that, The steps for using the kit include: (1) Detect the genotype of the TNFR2 gene at the rs1061622 locus in the subject samples; (2) Based on genotype, assess the risk of developing refractory cytokine release syndrome and / or the potential for long-term remission in the subject.

5. The use of the kit according to any one of claims 1-4 in the preparation of products for quality control of genetically engineered CAR-T cells.

6. A genetically engineered CAR-T cell, characterized in that, The TNFRSF1B locus in the CAR-T cell genome contains a mutation that causes an amino acid substitution at M196R.

7. The genetically engineered CAR-T cells according to claim 6, characterized in that, The mutation is introduced through gene editing technology, including CRISPR / Cas9, TALEN, zinc finger nucleases, base editors, or leader editors.

8. The use of the genetically engineered CAR-T cells according to any one of claims 6-7 in the preparation of tumor therapeutic drugs.

9. The application according to claim 8, characterized in that, The tumor is a hematologic malignancy.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the genetically engineered CAR-T cells as described in any one of claims 6-7.

Images