Preparation method of universal CAR-T cell for targeting CD19 mediated by umbilical cord blood

Abstract

The invention relates to the technical field of cellular immunotherapy, in particular to a preparation method of universal CAR-T (Chimeric Antigen Receptor-T) cells for targeting CD19 mediated by umbilical cord blood, and provides a standardized and large-scale preparation scheme aiming at the problems of long preparation period, high cost, large immune risk, limited applicable population and the like of the existing autologous CAR-T cells. The method comprises the steps of umbilical cord blood collection and storage, T cell separation and purification, CD19-targeted CAR gene construction and lentivirus transduction, TCR and HLA-I type molecule knockout through gene editing, CAR-T cell amplification and quality control and the like. The advantages of low immunogenicity and high multiplication capacity of the umbilical cord blood T cells are utilized, an optimized gene modification and culture system is combined, batch production of the CAR-T cells is achieved, the preparation period is shortened, the cost is reduced, adverse reactions such as GVHD and CRS are reduced, the applicable crowd for treatment is expanded, and an efficient and safe new strategy is provided for treatment of malignant B cell diseases.

Description

Technical Field

[0001] This invention relates to the field of cell immunotherapy technology, specifically a method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood. Background Technology

[0002] Malignant B-cell diseases are a group of hematologic malignancies that seriously threaten human health, mainly including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma. With changing environmental factors and an aging population, their incidence is increasing year by year, placing a heavy medical burden on patients' families and society. Traditional treatments such as chemotherapy, radiotherapy, and hematopoietic stem cell transplantation are widely used in clinical practice, but they have significant limitations. Chemotherapy works by non-specifically killing tumor cells, but it also damages normal cells, leading to serious adverse reactions such as hair loss, decreased immunity, and gastrointestinal reactions, greatly affecting patients' quality of life. Radiotherapy, while precisely targeting the tumor site, causes significant radiation damage to surrounding normal tissues, and its efficacy against metastases is limited. Hematopoietic stem cell transplantation, while potentially curative, faces risks such as low matching success rates, post-transplant infection, and rejection, making it difficult for most patients to benefit. For patients with relapsed or refractory malignant B-cell diseases, traditional treatments are even less effective, with extremely poor prognoses and significantly shortened survival time. Therefore, there is an urgent clinical need for more efficient and safer treatment methods.

[0003] The development of cell immunotherapy has brought revolutionary breakthroughs to the treatment of malignant B-cell diseases, with CAR-T cell therapy being particularly prominent. CAR-T cell therapy uses genetic engineering technology to modify T cells to express chimeric antigen receptors (CARs), thereby specifically recognizing and killing tumor cells. However, currently used CAR-T products are all autologous, and there are many problems that urgently need to be solved: First, the preparation cycle is long, requiring the collection of T cells from the patient for individualized modification, which takes about a month. Relapsed / refractory patients face a very high risk of disease progression during the waiting period. Second, the cost is high; the "personalized customization" model cannot achieve large-scale production, resulting in treatment costs reaching millions of yuan, placing a heavy financial burden on patients. Third, cell quality depends on the patient's own condition; some advanced-stage patients lose the opportunity for treatment due to insufficient T cell numbers or impaired function caused by radiotherapy and chemotherapy. Fourth, there are significant safety risks, including adverse reactions such as immune rejection, graft-versus-host disease (GVHD), cytokine release syndrome (CRS), and neurotoxicity, which can be life-threatening in severe cases.

[0004] Umbilical cord blood, as a biological resource rich in hematopoietic stem cells and immune cells, is increasingly attracting attention for its potential in CAR-T therapy. Compared with peripheral blood-derived T cells, umbilical cord blood T cells have unique advantages: stronger proliferative capacity, allowing for large-scale in vitro expansion; lower immunogenicity, reducing post-transplant immune rejection; and higher plasticity, making them easier to genetically modify. Domestic and international research on umbilical cord-derived CAR-T cells has been conducted, enhancing cell activity through optimized gene transduction technology and cell culture systems, and preliminary clinical trials have confirmed their therapeutic potential. However, existing research still has shortcomings: a lack of long-term safety and efficacy data; significant differences in preparation processes and treatment regimens among different studies, making it difficult to establish standardized procedures; and inadequate measures for the prevention and control of adverse reactions. Therefore, developing a standardized, large-scale, safe, and effective universal method for preparing CD19-targeting CAR-T cells mediated by umbilical cord blood is of great significance for promoting the development of immunotherapy for malignant B-cell diseases. Summary of the Invention

[0005] This invention aims to address the problems of long preparation cycles, high costs, unstable cell quality, high immune risks, and limited applicable populations associated with existing autologous CAR-T cell preparation methods. It provides a method for preparing universal umbilical cord blood-mediated CD19-targeting CAR-T cells that can be mass-produced, has high safety, and reliable efficacy, in order to meet the clinical treatment needs of patients with malignant B-cell diseases.

[0006] The technical solution adopted by this invention to solve its technical problem is: a method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood, comprising the following steps: Step 1, Collection, transportation and storage of umbilical cord blood: Umbilical cord blood is collected after the newborn is delivered and the umbilical cord is cut, but before the placental circulation has terminated. After health screening and aseptic treatment, it is stored at low temperatures for a specified period of time. Step 2, T cell isolation and purification: A cell layer containing T cells was obtained from umbilical cord blood using density gradient centrifugation, and then purified to obtain high-purity T cells using specific magnetic bead sorting technology; Step 3, CAR gene construction and transduction: Construct a CAR gene containing an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain targeting CD19, load it into a lentiviral vector, and transduce it into the T cells purified in Step 2. Step 4, gene editing: Use gene editing technology to knock out the T cell receptor (TCR) and human leukocyte antigen-I molecules (HLA-I molecules) in the transduced T cells of Step 3. Step 5, CAR-T cell expansion: The T cells edited in step 4 are expanded and cultured in a medium containing immune cytokines; Step 6, Quality Control: The expanded CAR-T cells are tested for purity, phenotype, cytotoxic activity and safety, and universal CAR-T cells that meet the standards are screened.

[0007] Specifically, the health screening in step one includes infectious disease screening, blood routine and coagulation function tests; the specified time is within 6-8 hours after collection; and the deep cryopreservation is in a liquid nitrogen environment.

[0008] Specifically, in step two, density gradient centrifugation achieves cell stratification using lymphocyte separation fluid, and the specific magnetic bead sorting technology targets T cell surface characteristic antigens, resulting in T cell purity ≥90% after purification.

[0009] Specifically, in step three, the antigen-binding domain of the CAR gene is the variable region (scFv) of the anti-CD19 single-chain antibody; the intracellular signal transduction domain includes a T cell activation signaling domain and a co-stimulatory signaling domain, wherein the T cell activation signaling domain is CD3ζ and the co-stimulatory signaling domain is CD28 or 4-1BB.

[0010] Specifically, in step three, the lentiviral vector is the pLVX-EF1α-CAR19 vector, and the transduction process achieves efficient transduction by optimizing the viral titer and incubation conditions.

[0011] Specifically, the gene editing technology in step four is CRISPR / Cas9 technology.

[0012] Specifically, in step five, the culture medium is a serum-free culture medium or a human serum culture medium containing at least one of interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin-15 (IL-15).

[0013] Specifically, in step six, purity detection is performed by flow cytometry, with CAR-T cells accounting for ≥90%; phenotypic detection includes CD4⁺ / CD8⁺ T cell ratio detection, where the ratio is 1:1-2:1.

[0014] A lentiviral expression vector, specifically the pLVX-EF1α-CAR19 vector carrying the CAR gene.

[0015] The beneficial effects of this invention are: The preparation cycle is significantly shortened: By using pre-stored umbilical cord blood as the cell source, the patient's own T cell collection is eliminated. Combined with a large-scale production process, the preparation time is greatly shortened, meeting the emergency treatment needs of relapsed / refractory patients.

[0016] Reduced treatment costs: Multiple CAR-T products can be prepared from a single unit of umbilical cord blood, enabling large-scale production, reducing unit product costs, and improving patient accessibility.

[0017] Expanded applicable population: It does not rely on the quality of the patient's own T cells and is suitable for patients with insufficient white blood cell count, failure of autologous T cell collection, or impaired function, thus overcoming the limitations of traditional autologous CAR-T.

[0018] Improved safety: Utilizing the natural advantage of low immunogenicity of umbilical cord blood T cells, combined with gene editing to knock out TCR and HLA-I molecules, the risks of GVHD, immune rejection, etc. are significantly reduced; clinical trials have confirmed that the incidence of CRS and neurotoxicity is low and the safety is controllable.

[0019] Reliable therapeutic effect: Umbilical cord blood T cells have strong proliferative capacity and high activity. In vitro experiments show that they have a significant killing effect on CD19⁺ tumor cells. In clinical trials, the objective response rate is high and some patients can achieve long-term remission. Attached Figure Description

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Figure 1 This is a schematic diagram of the CAR19 backbone vector structure, showing the composition of the antigen-binding domain, transmembrane domain, and intracellular signal transduction domain of the CAR gene; Figure 2 The image shows the flow cytometry results of T cells infected with CAR19, illustrating the transduction efficiency of the CAR gene in T cells. Figure 3 The graph shows the killing effect of CAR19 cells on primary ALL cells, demonstrating the lysis efficiency of CAR-T cells on tumor cells at different effector-to-target ratios. Figure 4 The bar chart shows the release levels of cytokines after CAR19 stimulation, including cytokine-related and CRS-related cytokines. Detailed Implementation

[0022] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0023] like Figures 1-4 As shown in this embodiment, a method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood includes the following steps: Step 1: Collection, Transportation, and Storage of Umbilical Cord Blood: Collection should be performed after the newborn is delivered and the umbilical cord is cut, while the placenta is still circulating blood, as the cell activity in the cord blood is optimal at this time. Before collection, a comprehensive health screening of the mother is necessary, including screening for infectious diseases (such as hepatitis B, hepatitis C, syphilis, and HIV), complete blood count, and coagulation function tests to ensure the safety of the cord blood. The collection process strictly adheres to aseptic techniques: First, disinfect the area 10-15 cm from the umbilical cord stump towards the placenta with iodine. Then, puncture a engorged umbilical vein 3-5 cm from the stump. After successful puncture, connect the blood collection bag containing anticoagulant to the puncture needle. Allow the cord blood to flow naturally into the collection bag using gravity, gently shaking the bag during this process to ensure thorough mixing of the anticoagulant and blood and prevent coagulation. After collection, label the blood collection bag and record key information such as the mother's name, newborn information, and collection time. The collected cord blood needs to be delivered to the storage facility within 6-8 hours and preserved in a liquid nitrogen cryogenic environment (-196℃). Temperature-controlled transport equipment is used during transportation to monitor the temperature in real time and ensure cell viability.

[0024] Step 2, T cell isolation and purification: T cells were isolated and purified using density gradient centrifugation combined with magnetic bead sorting. First, frozen umbilical cord blood was thawed. After thorough mixing with an anticoagulant, the cord blood was slowly layered on top of the lymphocyte separation medium, avoiding mixing of the two liquids. The centrifuge tubes were placed in a centrifuge and centrifuged under suitable conditions. After centrifugation, the liquids separated into layers according to density, with the middle white membrane layer rich in T cells. The white membrane layer cells were collected, washed with buffer, and then magnetic beads that bind to specific antigens on the T cell surface were added. The mixture was incubated at a suitable temperature to allow the magnetic beads to fully bind to the T cells. The cell suspension was then passed through a magnetic field. T cells bound to the magnetic beads were adsorbed into the magnetic field, while unbound impurity cells flowed out, thus obtaining high-purity T cells.

[0025] Step 3: Construction and Transduction of the CAR Gene: The structural design of the CAR gene includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. The antigen-binding domain uses the variable region (scFv) of a single-chain antibody targeting CD19, which can specifically recognize the CD19 antigen on the surface of tumor cells; the transmembrane domain is composed of hydrophobic amino acids, ensuring stable expression and localization of the CAR on the T cell membrane; the intracellular signal transduction domain includes a T cell activation signaling domain (such as CD3ζ) and a co-stimulatory signaling domain (such as CD28 or 4-1BB), where CD3ζ can initiate the killing signal of T cells, and the co-stimulatory signaling domain can enhance the proliferation capacity and survival time of T cells. The constructed CAR gene is inserted into a lentiviral vector (such as pLVX-EF1α-CAR19), and lentiviral particles are obtained through viral packaging. The lentiviral particles are co-incubated with the T cells purified in Step 2. By optimizing conditions such as viral titer, incubation temperature, and time, the transduction efficiency of the CAR gene is improved, enabling T cells to stably express the CAR.

[0026] Step four, gene editing: Gene editing technologies (such as CRISPR / Cas9) are used to edit the transduced T cells, knocking out the T cell receptor (TCR) and human leukocyte antigen-I molecules (HLA-I molecules) on the T cell surface. TCR knockout reduces T cell recognition of host tissues, lowering the risk of GVHD; HLA-I molecule knockout reduces the host's immune rejection of CAR-T cells, increasing cell survival time. Flow cytometry is used to detect gene editing efficiency, ensuring a significant reduction in the expression levels of TCR and HLA-I molecules.

[0027] Step 5, CAR-T cell expansion: Gene-edited T cells are seeded into a culture medium for expansion. The culture medium used is a basal medium (such as AIMV medium), supplemented with serum substitutes and cytokines (such as IL-2, IL-7, and IL-15). IL-2 promotes T cell proliferation, while IL-7 and IL-15 enhance T cell survival and activity. During culture, a suitable temperature and gas environment are controlled, and cell growth is observed regularly. Supplementation or passage is performed as needed to achieve large-scale expansion of CAR-T cells.

[0028] Step Six, Quality Control and Activity Assay: A comprehensive quality assessment of the expanded CAR-T cells is performed, including: cell purity testing (using flow cytometry to determine the proportion of CAR-T cells in the total cell count to ensure purity meets specified standards); phenotypic analysis (analyzing the ratio of CD4⁺ / CD8⁺ T cells and CAR expression levels to ensure appropriate T cell subset proportions and stable CAR expression); cytotoxic activity assay (evaluating the lytic ability of CAR-T cells against CD19⁺ tumor cells through in vitro cytotoxicity experiments); and safety testing (including pathogen detection (e.g., bacteria, viruses), gene mutation detection, and cytokine release assays to ensure cells are free from pathogen contamination, potential gene mutation risks, and that cytokine release levels related to adverse reactions such as CRS are within safe limits). CAR-T cells meeting the quality standards are then selected for subsequent treatment.

[0029] Specifically, the preparation of umbilical cord blood-mediated CD19-targeting universal CAR-T cells includes the following steps: Umbilical cord blood collection and storage: Healthy mothers, after passing infectious disease screening (negative for hepatitis B, hepatitis C, syphilis, and HIV), and with normal blood routine and coagulation function tests, will have their umbilical cord blood collected within 30 minutes of the newborn's umbilical cord being cut. During collection, disinfect the area from the umbilical cord stump to the placenta using povidone-iodine. Puncture the umbilical vein 4 cm from the stump, and collect the blood into a blood bag containing anticoagulant. Gently agitate the bag to mix the blood and anticoagulant evenly. A total of 75-85 ml of umbilical cord blood will be collected. After collection, label the mother's name, hospital number, newborn's sex, and collection time. The blood should be delivered to the laboratory within 4 hours. After passing quality testing, it will be stored in liquid nitrogen at -196℃.

[0030] T cell isolation and purification: After reviving umbilical cord blood stored in liquid nitrogen, the blood was mixed with an anticoagulant in a specific ratio and slowly layered onto the upper layer of lymphocyte separation medium. After centrifugation, the cells in the middle white membrane layer were collected. The white membrane layer cells were washed twice with buffer, and T cell-specific magnetic beads were added and incubated at room temperature for 20 minutes to allow the magnetic beads to fully bind to the T cells. The cell suspension was then passed through a magnetic field sorting column, and the T cells adsorbed in the magnetic field were collected; these were the purified T cells. Flow cytometry analysis showed that the T cell purity was 92%-94%.

[0031] Construction and transduction of the CAR gene: A CAR gene containing CD19 targeting scFv, a transmembrane domain, a CD3ζ activation signaling domain, and a CD28 co-stimulatory signaling domain was constructed and inserted into the pLVX-EF1α vector to construct the pLVX-EF1α-CAR19 lentiviral vector (vector structure shown in the attached figure). Figure 1 (As shown). Lentiviral particles were obtained through viral packaging and co-incubated with T cells purified in step 2. Different multiples of infection (MOI) were set, with MOI=5 showing the best transduction efficiency. Flow cytometry analysis showed that CAR expression on the surface of T cells was 42%-45% (results are attached). Figure 2 (As shown).

[0032] Gene editing: CRISPR / Cas9 technology was used to edit transduced T cells, targeting and knocking out TCR and HLA-I molecules. Flow cytometry analysis after editing showed that the proportion of TCR⁺ cells decreased from over 90% before editing to below 5%, and the proportion of HLA-I molecule⁺ cells decreased from over 95% to below 8%.

[0033] CAR-T cell expansion: Edited T cells were seeded in AIMV medium containing serum substitute and IL-2 and cultured in a 37°C, 5% CO2 incubator. Medium and cytokines were added as needed during the culture process. After 14 days of culture, the cell count increased 50-60 times.

[0034] Quality control and activity assays: Flow cytometry analysis showed that the CAR-T cell purity was 92%-95%, and the CD4⁺ / CD8⁺ T cell ratio was 1.2:1-1.8:1. In in vitro killing experiments, when the effector-target ratio was 5:1, 10:1, and 20:1, the lysis efficiency of CAR-T cells against CD19⁺ tumor cells was 28%-32%, 60%-65%, and 70%-73%, respectively (killing effect as shown in the attached figure). Figure 3 (As shown in the attached image); Cytokine detection showed that the release levels of killing-related cytokines such as IL-2 and IL-17A were significantly increased, while the release levels of CRS-related cytokines such as IL-6 were low (detection results are attached). Figure 4 As shown in the figure); pathogen detection and gene sequencing showed no abnormalities, meeting the quality standards.

[0035] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood, characterized in that, Includes the following steps: Step 1, Collection, transportation and storage of umbilical cord blood: Umbilical cord blood is collected after the newborn is delivered and the umbilical cord is cut, but before the placental circulation has terminated. After health screening and aseptic treatment, it is stored at low temperatures for a specified period of time. Step 2, T cell isolation and purification: A cell layer containing T cells was obtained from umbilical cord blood using density gradient centrifugation, and then purified to obtain high-purity T cells using specific magnetic bead sorting technology; Step 3, CAR gene construction and transduction: Construct a CAR gene containing an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain targeting CD19, load it into a lentiviral vector, and transduce it into the T cells purified in Step 2. Step 4, gene editing: using gene editing technology to knock out the T cell receptor and human leukocyte antigen-I molecules in the transduced T cells of Step 3; Step 5, CAR-T cell expansion: The T cells edited in step 4 are expanded and cultured in a medium containing immune cytokines; Step 6, Quality Control: The expanded CAR-T cells are tested for purity, phenotype, cytotoxic activity and safety, and universal CAR-T cells that meet the standards are screened.

2. The method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood according to claim 1, characterized in that: The health screening in step one includes infectious disease screening, routine blood tests, and coagulation function tests; the specified time is within 6-8 hours after collection; and the deep cryopreservation is performed in a liquid nitrogen environment.

3. The method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood according to claim 1, characterized in that: In step two, density gradient centrifugation achieves cell stratification using lymphocyte separation fluid, and the specific magnetic bead sorting technology targets T cell surface characteristic antigens, resulting in T cell purity ≥90% after purification.

4. The method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood according to claim 1, characterized in that: In step three, the antigen-binding domain of the CAR gene is the variable region of the anti-CD19 single-chain antibody; the intracellular signal transduction domain includes a T cell activation signaling domain and a co-stimulatory signaling domain, wherein the T cell activation signaling domain is CD3ζ and the co-stimulatory signaling domain is CD28 or 4-1BB.

5. The method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood according to claim 1, characterized in that: In step three, the lentiviral vector is the pLVX-EF1α-CAR19 vector, and the transduction process achieves efficient transduction by optimizing the viral titer and incubation conditions.

6. The method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood according to claim 1, characterized in that: The gene editing technology used in step four is CRISPR / Cas9 technology.

7. The method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood according to claim 1, characterized in that: In step five, the culture medium is a serum-free culture medium or a human serum culture medium containing at least one of interleukin-2, interleukin-7 and interleukin-15.

8. The method for preparing universal CD19-targeting CAR-T cells mediated by umbilical cord blood according to claim 1, characterized in that: In step six, purity detection is performed by flow cytometry, with CAR-T cells accounting for ≥90%; phenotypic detection includes CD4⁺ / CD8⁺ T cell ratio detection, where the ratio is 1:1-2:

1.

9. A lentiviral expression vector, characterized in that, This refers to the pLVX-EF1α-CAR19 vector carrying the CAR gene.

10. A universal CAR-T cell targeting CD19 prepared using the method for preparing universal CAR-T cells mediated by umbilical cord blood according to any one of claims 1-8.

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