A method for in vitro expansion of tcr cells targeting viral antigens

Abstract

This invention discloses a method for in vitro expansion and culture of TCR cells targeting viral antigens, belonging to the field of in vitro immune cell culture technology. The specific steps of this method are as follows: first, peripheral blood mononuclear cells are isolated, and the initial activation of targeted T cells is completed and a culture system is established to start expansion; samples are taken at preset nodes, and the abundance of targeted and non-targeted TCR clones is calculated by single-cell TCR sequencing; culture parameters are dynamically adjusted according to abundance thresholds, and targeted enrichment is performed when non-targeted clones exceed the limit; the operation is repeated until the end of the expansion cycle and the cells are harvested. This invention integrates targeted clone abundance detection into the culture process by using fixed-node micro-sampling sequencing throughout the expansion process, combined with three-level graded threshold dynamic control of culture parameters, and dual-detection abundance correction. When non-targeted clones exceed the limit, they are enriched in real time, locking in the proliferative advantage of targeted clones, reducing cell viability loss during sorting, minimizing batch quality differences, and stably producing high-purity targeted TCR cells that meet clinical requirements.

Description

Technical Field

[0001] This invention relates to the field of in vitro culture technology of immune cells, specifically a method for in vitro expansion and culture of TCR cells targeting viral antigens. Background Technology

[0002] TCR cell therapy targeting viral antigens is an important intervention for chronic viral infections and virus-related malignancies. Its core principle is to obtain a sufficient quantity of functional TCR cells capable of specifically recognizing and eliminating virus-infected cells through in vitro expansion. Currently, the industry-standard culture process typically involves first screening target TCR clones through genetic testing, then using fixed antigen stimulation conditions and cytokine ratios to complete the entire expansion process. Genetic testing is only used for pre-culture clone screening and final quality control after culture; during the culture process, only routine cell counting and viability testing are performed, without any targeted process intervention.

[0003] Existing static, fixed-parameter culture methods cannot adapt to the changing states of T cells during expansion. Non-targeted T cell clones proliferate faster, easily crowding out the culture resources and proliferation space of targeted clones. After culture, the proportion of targeted TCR clones is generally low, mostly only reaching 30%-60%. Purity can only be improved through terminal magnetic bead sorting, which not only results in significant cell loss but also significantly reduces cell viability. Furthermore, there are large batch-to-batch variations in cell quality, making it difficult to consistently produce high-purity, high-activity targeted TCR cells, thus failing to reliably meet clinical needs. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for in vitro expansion and culture of TCR cells targeting viral antigens. This method constructs a closed-loop culture process for in vitro expansion of TCR cells targeting viral antigens. First, peripheral blood mononuclear cells are isolated to initially activate the targeted T cells, establishing a basic expansion culture system to initiate culture. Then, at preset key points in the expansion, aseptic micro-sampling is performed, and single-cell TCR sequencing identifies clonal sequences and calculates the abundance of targeted and non-targeted clones. Culture parameters are dynamically adjusted based on abundance grading thresholds. When non-targeted clones exceed the limit, targeted positive enrichment is immediately performed. This cycle is repeated until the preset expansion cycle is completed, at which point the cells are harvested. This method overcomes the limitations of traditional static culture, integrating abundance detection into the entire culture process. It accurately identifies the proliferative advantage of targeted clones, reduces cell viability loss during sorting, and can further improve control precision through dual detection correction, reducing batch-to-batch quality differences. It can stably produce high-purity targeted TCR cells that meet clinical quality control standards.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for in vitro expansion and culture of TCR cells targeting viral antigens, the specific steps of which are as follows:

[0006] S1, Sample processing and initial activation: Peripheral blood mononuclear cells were isolated and obtained. The target viral antigen epitope peptide was used to complete the initial activation of the targeted T cells. A basic expansion culture system was established, and the activated T cells were placed in the culture system to start the expansion culture.

[0007] S2, Micro-sampling at key nodes: At the preset key nodes of amplification culture, aseptic micro-sampling is performed on the culture system, and an equal volume of preheated fresh basal culture medium is added after sampling.

[0008] S3, Sequencing detection and abundance calculation: Single-cell TCR sequencing is performed on sampled cells to identify the CDR3 region sequence of TCR clones, and the TCR reference sequence library that matches the target viral antigen epitope is compared to screen TCR clones targeting viral antigens. The real-time abundance of the targeted TCR clones is calculated. The abundance of TCR clones of non-targeted viral antigens = 100% - real-time abundance of targeted TCR clones.

[0009] S4, Dynamic control of culture parameters: The real-time abundance of the targeted TCR clones is compared with the preset grading threshold, and the concentration of viral antigen epitope peptide stimulation and the ratio of cytokines in the culture system are dynamically adjusted according to the comparison results.

[0010] S5, Targeted Positive Clonal Enrichment: When the abundance of TCR clones of non-targeted viral antigens exceeds a preset threshold, targeted positive enrichment is performed on the cells in the culture system. After enrichment, the cells are returned to the culture system for further amplification.

[0011] S6, Closed-loop culture harvest: Repeat steps S1 to S6 to form a continuous closed-loop culture cycle until the preset expansion cycle is completed and the cells are harvested.

[0012] Furthermore, in step S1, the initial activation uses autologous dendritic cells loaded with the target viral antigen epitope peptide as antigen-presenting cells, with a dendritic cell to peripheral blood mononuclear cell ratio of 1:5-1:20, and an activation culture time of 24-72 h; the final concentration of the target viral antigen epitope peptide is 1-10 μg / mL.

[0013] Furthermore, in step S1, the basic amplification culture system uses a serum-free chemically defined culture medium as the base, and adds 1%-3% recombinant human serum albumin, 200-500 IU / mL IL-2, 5-20 ng / mL IL-7, and 5-20 ng / mL IL-15 to a final concentration. The pH of the system is controlled at 7.2-7.4, and the osmotic pressure is controlled at 280-320 mOsm / kg.

[0014] Furthermore, in step S2, the preset key nodes are four fixed nodes: day 0, day 3, day 7, and day 10 of the amplification culture; the sampling volume of each node is ≤100μL, the sampling volume accounts for ≤0.5% of the total culture system, and the fresh basal culture medium is preheated to 36.5℃-37.5℃.

[0015] Furthermore, after completing single-cell TCR sequencing at day 0 and obtaining the specific CDR3 region sequence of the targeted TCR clone, cell suspension samples are simultaneously collected at days 3, 7, and 10. The targeted TCR clone is absolutely quantified using droplet digital PCR. The quantification results are linearly fitted and corrected with the abundance results of single-cell TCR sequencing at the same node. The corrected abundance results are used as the final basis for adjusting the parameters in step S4.

[0016] Furthermore, the single-cell TCR sequencing in step S3 has a total cycle of ≤24h from sample preparation to abundance result output; the real-time abundance of the targeted TCR clone is calculated according to the following formula: Real-time abundance of targeted TCR clone = number of effective cells of targeted TCR clone / total number of effective cells sequenced × 100%.

[0017] Furthermore, the parameter adjustment operation in step S4 is completed within 48 hours after sampling at the same node; the preset thresholds mentioned in steps S4 and S5 are uniformly set to three-level hierarchical thresholds, and the corresponding adjustment strategy is as follows:

[0018] When the real-time abundance of the targeted TCR clone is ≥80%, the current concentration of viral antigen epitope peptide stimulation and the ratio of cytokines in the culture system are kept unchanged.

[0019] When the real-time abundance of the targeted TCR clone is 50%-80%, the concentration of viral antigen epitope peptide stimulation is increased to 110%-130% of the current concentration of the system before this sampling, and the concentration of IL-2 is increased to 1.2-1.8 times the current concentration of the system before this sampling, while the concentrations of IL-7 and IL-15 remain unchanged.

[0020] When the real-time abundance of the targeted TCR clone is <50%, keep the current culture system parameters unchanged, first perform the targeted positive enrichment operation in step S5. After the enrichment is completed, adjust the concentration of viral antigen epitope peptide stimulation to 130%-150% of the current concentration of the system before this sampling. At the same time, adjust the final concentration of IL-2 to 400-600 IU / mL, the final concentration of IL-7 to 15-25 ng / mL, and the final concentration of IL-15 to 15-25 ng / mL.

[0021] Furthermore, in step S5, the targeted positive enrichment operation is completed using a positive sorting method that combines biotin-labeled viral antigen epitope peptide-MHC tetramer with streptavidin immunomagnetic beads. After sorting, the proportion of targeted TCR positive cells is ≥90%, and the cell viability is ≥90%.

[0022] Furthermore, the preset amplification cycle in step S6 is 12-16 days, and the qualified criteria for harvesting cells are: total cell amplification ≥ 1000-fold, target TCR clone abundance ≥ 90%, cell viability ≥ 95%, and negative results for sterility, endotoxin, and mycoplasma.

[0023] Compared with existing technologies, this method for in vitro expansion and culture of TCR cells targeting viral antigens has the following advantages:

[0024] I. This invention integrates targeted clonal abundance detection directly into the culture process by amplifying micro-sampling at four fixed nodes throughout the entire process and tracking single-cell TCR sequencing in real time, rather than just performing screening or quality control before and after culture. It can read the changes in the proportion of TCR clones targeting viral antigens in real time throughout the entire process. Once non-target clones proliferate excessively, the antigen stimulation intensity and cytokine ratio can be adjusted in time to lock in the proliferation advantage of the targeted clones in advance. This can not only stably increase the final proportion of targeted clones, but also reduce the cell viability loss caused by sorting.

[0025] Second, this invention provides a clear quantitative basis for adjusting culture parameters at each step by using a precise adjustment rule of three-level grading thresholds, combined with bidirectional correction of abundance results from single-cell sequencing and digital PCR. It eliminates the need to apply a fixed set of culture parameters throughout the process, allowing for matching of corresponding culture conditions based on the actual cell expansion status, reducing ineffective culture operations, and avoiding the abundance calculation deviation caused by a single sequencing method. This makes the culture process controllable for each batch, effectively reducing cell quality differences between batches and stably producing cells that meet clinical requirements.

[0026] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0028] Figure 1 This is a schematic diagram of the overall process of the in vitro expansion and culture method of TCR cells targeting viral antigens according to the present invention.

[0029] Figure 2 This is a schematic diagram of the process for micro-sampling and dual-detection correction, which are key nodes of the present invention.

[0030] Figure 3 This is a schematic diagram of the dynamic control process of the threshold for targeting TCR clonal abundance classification in this invention. Detailed Implementation

[0031] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0032] Example 1:

[0033] This embodiment describes the in vitro expansion and culture of TCR cells targeting the EBV LMP2A antigen. A closed-loop culture method was employed, using micro-sampling and single-cell TCR sequencing at four fixed points throughout the expansion process to track changes in the abundance of targeted TCR clones in real time. Culture parameters were dynamically adjusted using a three-level grading threshold, and targeted enrichment was performed immediately when non-targeted clones exceeded the limit. This embodiment verifies the effectiveness of this method in increasing the terminal proportion of targeted TCR clones, and also verifies whether the cell expansion fold, viability, and various quality control indicators meet clinical requirements.

[0034] like Figure 1 As shown in the figure, an embodiment of the present invention provides a method for in vitro expansion and culture of TCR cells targeting viral antigens. The method includes steps S1 to S6, and the specific implementation process is as follows:

[0035] S1, Sample processing and initial activation.

[0036] 50 mL of autologous peripheral blood was collected from EBV-positive patients. Peripheral blood mononuclear cells were separated using density gradient centrifugation with Ficoll-Paque PLUS separation buffer. The centrifugation parameters were 400 g, 20 degrees Celsius, and 25 minutes. After centrifugation, peripheral blood mononuclear cells in the white membrane layer were aspirated and washed twice with sterile phosphate buffer. The centrifugation parameters were 300 g and 5 minutes each time. The cell viability was 96% as determined by trypan blue staining, which met the requirements for subsequent procedures.

[0037] Autologous dendritic cells loaded with EBV LMP2A antigenic epitope peptide were used as antigen-presenting cells. The ratio of dendritic cells to peripheral blood mononuclear cells was 1:10. The final concentration of EBV LMP2A antigenic epitope peptide was 5 μg / mL. The cells were cultured in a saturated humidity incubator at 37°C and 5% carbon dioxide for 48 hours to complete the initial activation of targeted T cells.

[0038] A basic expansion culture system was established using X-VIVO 15 medium with serum-free chemical components as the base, supplemented with 2% recombinant human serum albumin, 300 IU / mL IL-2, 10 ng / mL IL-7, and 10 ng / mL IL-15. The pH of the system was controlled between 7.2 and 7.4, and the osmotic pressure was controlled between 280 and 320 mOsm / kg. The activated T cells were adjusted to an initial seeding density of 1×10⁶ cells / mL and placed in an incubator to start the expansion culture.

[0039] S2, micro-sampling at key nodes.

[0040] Four fixed detection points were set at day 0, day 3, day 7, and day 10 of the amplification culture. At each point, aseptic operation was performed in a Class A biosafety cabinet. 100 μL of cell suspension was taken from the culture system as the detection sample. The sampling volume accounted for 0.4% of the total culture system. Immediately after sampling, an equal volume of fresh basal culture medium preheated to 37 degrees Celsius was added to the culture system to avoid large fluctuations in the volume, nutrient composition, and temperature of the culture system.

[0041] S3, sequencing detection and abundance calculation.

[0042] Single-cell TCR sequencing was performed on sampled cells. Cells were washed twice with sterile phosphate-buffered saline (PBS) to adjust the cell concentration to 1 × 10⁶ cells / mL. A single-cell immunoassay kit was used for single-cell capture, mRNA reverse transcription, specific amplification of the TCR α and β chains CDR3 region, and library construction. After library quality control, high-throughput sequencing was performed with a sequencing depth of at least 5000 reads / cell. Following sequencing, professional bioinformatics analysis software was used to identify TCR clone types, assemble CDR3 region sequences, and annotate the V(D)J gene rearrangement. The obtained TCR clone CDR3 region sequences were compared with a pre-constructed TCR reference sequence library matching the EBV LMP2A antigen epitope using the IEDB immunoepitaxy database to screen for TCR clones targeting the EBV antigen. The above micro-sampling and single-cell TCR sequencing process is as follows: Figure 2 As shown.

[0043] The real-time abundance of targeted TCR clones and the TCR clone abundance of non-targeted viral antigens were calculated separately according to preset fixed formulas. The formula for calculating the real-time abundance of targeted TCR clones is: Real-time abundance of targeted TCR clones = Effective number of targeted TCR clones / Total effective number of sequencing cells × 100%; The formula for calculating the TCR clone abundance of non-targeted viral antigens is: TCR clone abundance of non-targeted viral antigens = 100% - Real-time abundance of targeted TCR clones. The entire process of single-cell TCR sequencing from sample preparation to abundance result output is controlled within 22 hours, which meets the preset requirements.

[0044] S4, dynamic adjustment of culture parameters.

[0045] The measured real-time abundance of targeted TCR clones was compared with the preset three-level classification threshold, such as... Figure 3 As shown. Based on the comparison results, the concentration of viral epitope peptide stimulation and the cytokine ratio in the culture system were dynamically adjusted. All parameter adjustments were completed within 48 hours of sampling at the same node. At day 0, the initial abundance of the targeted TCR clone was 12%, and culture was started according to the basic culture system parameters. At day 3, the real-time abundance of the targeted TCR clone was 62%, falling within the 50% to 80% range. The concentration of EB virus epitope peptide stimulation was increased to 120% of the current concentration before this sampling, and the IL-2 concentration was increased to 1.5 times the current concentration before this sampling, while the IL-7 and IL-15 concentrations remained unchanged. At day 7, the real-time abundance of the targeted TCR clone was 86%, greater than or equal to 80%, and the current viral epitope peptide stimulation concentration and cytokine ratio in the culture system were maintained unchanged. At day 10, the real-time abundance of the targeted TCR clone was 91%, greater than or equal to 80%, and the current culture system parameters were maintained unchanged.

[0046] S5, targeting positive clone enrichment.

[0047] In this embodiment, the real-time abundance of targeted TCR clones did not fall below 50% throughout the entire cycle, and the targeted positive enrichment operation was not triggered. If the real-time abundance of targeted TCR clones falls below 50%, the current culture system parameters are maintained unchanged. Targeted positive enrichment is then performed using a positive sorting method combining biotin-labeled EB virus antigen epitope peptide-MHC tetramer with streptavidin immunomagnetic beads. After sorting, the proportion of targeted TCR positive cells is greater than or equal to 90%, and the cell viability is greater than or equal to 90%. After enrichment, the culture system parameters are adjusted, and the cells are returned to the culture system for further expansion.

[0048] S6, harvested from closed-loop cyclic culture.

[0049] Repeat steps S2 to S5 to form a continuous closed-loop culture cycle. The preset amplification period is 14 days, and cells are harvested after 14 days of culture. The test results of the harvested cells are as follows: the total cell amplification is 1380-fold, the abundance of targeted TCR clones is 92.3%, the cell viability is 94.8%, and the results of sterility testing, endotoxin testing, and mycoplasma testing are all negative, meeting the preset qualification standards.

[0050] This embodiment successfully completed the in vitro expansion and culture of EB virus antigen-targeted TCR cells. Through a closed-loop culture process, the final abundance of targeted TCR clones was increased from an initial 12% to 92.3%, achieving a total cell expansion fold of 1380-fold. Simultaneously, no unnecessary sorting was performed throughout the process, maintaining a high cell viability. This embodiment verifies that this method can effectively lock in the proliferative advantage of targeted TCR clones, consistently increasing the proportion of targeted clones in the final product, solving the problem of low purity of targeted clones in traditional static culture methods, and producing cells that consistently meet clinical requirements.

[0051] Example 2:

[0052] This embodiment focuses on the in vitro expansion and culture of TCR cells targeted by human cytomegalovirus pp65 antigen. Based on a closed-loop culture method, droplet digital PCR was added for absolute quantification of the targeted TCR clones. The quantification results were linearly fitted and corrected with the abundance results from single-cell TCR sequencing, and the corrected abundance results were used as the final basis for adjusting culture parameters. This embodiment verifies the effect of the dual detection method on improving the accuracy of abundance calculation, and the optimization effect of precise control on the amplification effect and batch stability of targeted TCR clones.

[0053] The specific implementation process is as follows:

[0054] S1, Sample processing and initial activation.

[0055] 40 mL of peripheral blood from a human cytomegalovirus-positive donor was collected. Peripheral blood mononuclear cells were isolated using density gradient centrifugation with Ficoll-Paque PLUS separation buffer. The centrifugation parameters were set to 400 g, 20 degrees Celsius, and 25 minutes. After centrifugation, peripheral blood mononuclear cells in the white membrane layer were aspirated and washed twice with sterile phosphate buffer. The centrifugation parameters were 300 g and 5 minutes each time. The cell viability was 95.5% as determined by trypan blue staining, which met the requirements for subsequent operations.

[0056] Autologous dendritic cells loaded with human cytomegalovirus pp65 antigenic epitope peptide were used as antigen-presenting cells. The ratio of dendritic cells to peripheral blood mononuclear cells was 1:10. The final concentration of human cytomegalovirus pp65 antigenic epitope peptide was 5 μg / mL. The cells were cultured in a saturated humidity incubator at 37 degrees Celsius and 5% carbon dioxide for 48 hours to complete the initial activation of targeted T cells.

[0057] A basic expansion culture system was established using X-VIVO 15 medium with serum-free chemical components as the base, supplemented with 2% recombinant human serum albumin, 300 IU / mL IL-2, 10 ng / mL IL-7, and 10 ng / mL IL-15. The pH of the system was controlled between 7.2 and 7.4, and the osmotic pressure was controlled between 280 and 320 mOsm / kg. The activated T cells were adjusted to an initial seeding density of 1×10⁶ cells / mL and placed in an incubator to start the expansion culture.

[0058] S2, micro-sampling at key nodes.

[0059] Four fixed detection points were set at days 0, 3, 7, and 10 of the amplification culture. At each point, aseptic procedures were performed in a Class A biosafety cabinet. 100 μL of cell suspension was taken from the culture system as the detection sample for single-cell TCR sequencing, representing 0.4% of the total culture volume. Immediately after sampling, an equal volume of fresh basal culture medium preheated to 37°C was added to the culture system. After single-cell TCR sequencing was completed at day 0, and the specific CDR3 region sequence of the targeted TCR clone was obtained, 30 μL of cell suspension samples were simultaneously taken at days 3, 7, and 10 for droplet digital PCR detection.

[0060] S3, sequencing detection and abundance calculation.

[0061] Single-cell TCR sequencing was performed on the sampled cells. The sampled cells were washed twice with sterile phosphate buffer and the cell concentration was adjusted to 1×106 cells / mL. Single-cell immunoassay kits were used to complete single-cell capture, mRNA reverse transcription, specific amplification of the TCR α and β chain CDR3 regions, and library construction. After the library passed quality control, high-throughput sequencing was performed with a sequencing depth of not less than 5000 reads / cell.

[0062] After sequencing, professional bioinformatics analysis software was used to identify TCR clone types, assemble CDR3 region sequences, and annotate V(D)J gene rearrangements. The obtained TCR clone CDR3 region sequences were compared with a TCR reference sequence library that matched human cytomegalovirus pp65 antigen epitopes constructed in advance using the IEDB immune epitope database to screen TCR clones targeting human cytomegalovirus antigens.

[0063] The real-time abundance of targeted TCR clones and the TCR clone abundance of non-targeted viral antigens were calculated separately according to preset fixed formulas. The formula for calculating the real-time abundance of targeted TCR clones is: Real-time abundance of targeted TCR clones = Effective number of targeted TCR clones / Total effective number of sequencing cells × 100%; The formula for calculating the TCR clone abundance of non-targeted viral antigens is: TCR clone abundance of non-targeted viral antigens = 100% - Real-time abundance of targeted TCR clones. The entire process of single-cell TCR sequencing from sample preparation to abundance result output is controlled within 23 hours, which meets the preset requirements.

[0064] For samples collected simultaneously on days 3, 7, and 10, absolute quantification of targeted TCR clones was performed using droplet digital PCR. Specific upstream and downstream primers and a TaqMan fluorescent probe were designed based on the CDR3 region sequence of the targeted TCR clone obtained on day 0. The probe was labeled with a FAM fluorescent group at its 5' end and a BHQ1 quencher group at its 3' end. Total RNA was extracted from the synchronously sampled cells and reverse transcribed to synthesize cDNA as a reaction template. A 20 μL reaction system was prepared containing 10 μL of 2× droplet digital PCR premix, 1 μL each of 10 μmol / L upstream and downstream primers, 0.5 μL of 10 μmol / L probe, 2 μL of cDNA template, and enzyme-free water to a final volume of 20 μL. The reaction system was added to the droplet generation card to complete the droplet preparation. After being transferred to a 96-well PCR plate and sealed, the droplets were amplified. The amplification program was as follows: 95°C pre-denaturation for 10 minutes, 94°C denaturation for 30 seconds, 60°C annealing extension for 60 seconds, for a total of 40 cycles, followed by enzyme inactivation at 98°C for 10 minutes and incubation at 4°C.

[0065] After amplification, the fluorescence signal was read using a droplet reader to analyze the absolute copy number of the targeted TCR clone and simultaneously detect the copy number of the internal reference gene GAPDH. The relative abundance of the targeted TCR clone by droplet digital PCR was calculated, and the quantitative results were linearly fitted and corrected with the abundance results of single-cell TCR sequencing at the same node. The corrected abundance results were used as the final basis for adjusting the culture parameters.

[0066] S4, dynamic adjustment of culture parameters.

[0067] The real-time abundance of the corrected targeted TCR clones was compared with the preset three-level grading threshold. Based on the comparison results, the concentration of viral antigen epitope peptide stimulation and the cytokine ratio in the culture system were dynamically adjusted. All parameter adjustments were completed within 48 hours after sampling at the same node. At day 0, the initial abundance of the targeted TCR clones was 10.5%, and culture was started according to the basic culture system parameters. At day 3, the real-time abundance of the corrected targeted TCR clones was 42%, which was less than 50%. The current culture system parameters were maintained unchanged, and targeted positive enrichment was performed. After enrichment, the concentration of human cytomegalovirus antigen epitope peptide stimulation was adjusted to 140% of the current concentration of the system before this sampling. At the same time, the final concentrations of IL-2, IL-7, and IL-15 were adjusted to 500 IU / mL, 20 ng / mL, and 20 ng / mL, respectively. On day 7, the real-time abundance of targeted TCR clones after node correction was 72%, falling within the 50% to 80% range. The concentration of human cytomegalovirus epitope peptide stimulation was increased to 120% of the current concentration in the system before this sampling, while the IL-2 concentration was increased to 1.5 times the current concentration in the system before this sampling. The concentrations of IL-7 and IL-15 remained unchanged. On day 10, the real-time abundance of targeted TCR clones after node correction was 93%, greater than or equal to 80%. The current concentration of viral epitope peptide stimulation and the cytokine ratio in the culture system were maintained unchanged.

[0068] S5, targeting positive clone enrichment.

[0069] On day 3, the real-time abundance of the target TCR clones was less than 50%, triggering a targeted positive enrichment operation. Enrichment was achieved using a positive sorting method combining biotin-labeled human cytomegalovirus epitope peptide-MHC tetramer with streptavidin immunomagnetic beads. Labeling and binding were completed by incubation at 4°C in the dark. After washing, the cell suspension was added to a magnetic sorting column for positive sorting. Target TCR-positive cells bound to the magnetic beads were collected, washed, and the magnetic beads were removed. After sorting, the percentage of target TCR-positive cells was 92.6%, and the cell viability was 91.2%, meeting the preset requirements. After enrichment, the cells were returned to the culture system for further expansion.

[0070] S6, harvested from closed-loop cyclic culture.

[0071] Repeat steps S2 to S5 to form a continuous closed-loop culture cycle. The preset amplification period is 14 days, and cells are harvested after 14 days of culture. The test results of the harvested cells are as follows: the total cell amplification is 1260-fold, the abundance of targeted TCR clones is 95.7%, the cell viability is 94.2%, and the results of sterility testing, endotoxin testing, and mycoplasma testing are all negative, meeting the preset qualification standards.

[0072] This embodiment successfully completed the in vitro expansion and culture of human cytomegalovirus antigen-targeted TCR cells. Dual detection and correction using droplet digital PCR and single-cell TCR sequencing further improved the accuracy of abundance calculation and parameter control, ultimately achieving a final TCR clone abundance of 95.7% and a total cell expansion fold of 1260-fold. This embodiment verifies that this method can effectively address situations with low initial target clone abundance through precise process control, while further reducing batch-to-batch cell quality variations, stably producing high-purity, highly active targeted TCR cells, and meeting the full-process requirements of clinical-grade cell preparation.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for in vitro expansion and culture of TCR cells targeting viral antigens, characterized in that, The specific steps of this method are as follows: S1, Sample processing and initial activation: Peripheral blood mononuclear cells were isolated and obtained. The target viral antigen epitope peptide was used to complete the initial activation of the targeted T cells. A basic expansion culture system was established, and the activated T cells were placed in the culture system to start the expansion culture. S2, Micro-sampling at key nodes: At the preset key nodes of amplification culture, aseptic micro-sampling is performed on the culture system, and an equal volume of preheated fresh basal culture medium is added after sampling. S3, Sequencing detection and abundance calculation: Single-cell TCR sequencing is performed on sampled cells to identify the CDR3 region sequence of TCR clones, and the TCR reference sequence library that matches the target viral antigen epitope is compared to screen TCR clones targeting viral antigens. The real-time abundance of the targeted TCR clones is calculated. The abundance of TCR clones of non-targeted viral antigens = 100% - real-time abundance of targeted TCR clones. S4, Dynamic control of culture parameters: The real-time abundance of the targeted TCR clones is compared with the preset grading threshold, and the concentration of viral antigen epitope peptide stimulation and the ratio of cytokines in the culture system are dynamically adjusted according to the comparison results. S5, Targeted Positive Clonal Enrichment: When the abundance of TCR clones of non-targeted viral antigens exceeds a preset threshold, targeted positive enrichment is performed on the cells in the culture system. After enrichment, the cells are returned to the culture system for further amplification. S6, Closed-loop culture harvest: Repeat steps S1 to S6 to form a continuous closed-loop culture cycle until the preset expansion cycle is completed and the cells are harvested.

2. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 1, characterized in that, In step S1, the initial activation uses autologous dendritic cells loaded with the target viral antigen epitope peptide as antigen-presenting cells, with a dendritic cell to peripheral blood mononuclear cell ratio of 1:5-1:20, and an activation culture time of 24-72 h; the final concentration of the target viral antigen epitope peptide is 1-10 μg / mL.

3. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 1, characterized in that, In step S1, the basic amplification culture system uses a serum-free chemically defined culture medium as the base, and adds 1%-3% recombinant human serum albumin, 200-500 IU / mL IL-2, 5-20 ng / mL IL-7, and 5-20 ng / mL IL-15 to a final concentration. The pH of the system is controlled at 7.2-7.4, and the osmotic pressure is controlled at 280-320 mOsm / kg.

4. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 1, characterized in that, In step S2, the preset key nodes are four fixed nodes: day 0, day 3, day 7, and day 10 of the amplification culture; the sampling volume of each node is ≤100μL, and the sampling volume accounts for ≤0.5% of the total culture system. The fresh basal culture medium is preheated to 36.5℃-37.5℃.

5. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 4, characterized in that, After completing single-cell TCR sequencing at day 0 and obtaining the specific CDR3 region sequence of the targeted TCR clone, cell suspension samples were simultaneously collected at days 3, 7, and 10. The targeted TCR clone was absolutely quantified using droplet digital PCR. The quantification results were linearly fitted and corrected with the abundance results of single-cell TCR sequencing at the same node. The corrected abundance results were used as the final basis for adjusting the parameters in step S4.

6. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 1, characterized in that, The single-cell TCR sequencing in step S3, from sample preparation to abundance result output, has a total cycle time of ≤24h; the real-time abundance of the targeted TCR clone is calculated according to the following formula: .

7. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 1, characterized in that, The parameter adjustment operation in step S4 shall be completed within 48 hours after sampling at the same node; the preset thresholds mentioned in steps S4 and S5 shall be uniformly set to three-level hierarchical thresholds, and the corresponding adjustment strategy shall be as follows: When the real-time abundance of the targeted TCR clone is ≥80%, the current concentration of viral antigen epitope peptide stimulation and the ratio of cytokines in the culture system are kept unchanged. When the real-time abundance of the targeted TCR clone is 50%-80%, the concentration of viral antigen epitope peptide stimulation is increased to 110%-130% of the current concentration of the system before this sampling, and the concentration of IL-2 is increased to 1.2-1.8 times the current concentration of the system before this sampling, while the concentrations of IL-7 and IL-15 remain unchanged. When the real-time abundance of the targeted TCR clone is <50%, keep the current culture system parameters unchanged, first perform the targeted positive enrichment operation in step S5. After the enrichment is completed, adjust the concentration of viral antigen epitope peptide stimulation to 130%-150% of the current concentration of the system before this sampling. At the same time, adjust the final concentration of IL-2 to 400-600 IU / mL, the final concentration of IL-7 to 15-25 ng / mL, and the final concentration of IL-15 to 15-25 ng / mL.

8. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 1, characterized in that, In step S5, the targeted positive enrichment operation is completed by a positive sorting method using biotin-labeled viral antigen epitope peptide-MHC tetramer combined with streptavidin immunomagnetic beads. After sorting, the proportion of targeted TCR positive cells is ≥90%, and the cell viability is ≥90%.

9. The method for in vitro expansion and culture of TCR cells targeting viral antigens according to claim 1, characterized in that, The preset amplification period in step S6 is 12-16 days. The qualified criteria for harvesting cells are: total cell amplification ≥ 1000-fold, target TCR clone abundance ≥ 90%, cell viability ≥ 95%, and negative results for sterility, endotoxin, and mycoplasma.

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