Preparation method and application of enhanced DC-CIK cells

Abstract

The application relates to the technical field of enhanced DC-CIK cell preparation methods, and discloses an enhanced DC-CIK cell preparation method and application, which deeply reforms DC cells, combines CIK cells to form an enhanced therapy, and comprises the following steps: S100, preparing patient DC cells and CIK cells; S200, upgrading double-antigen targeting loading; S300, fine gene editing and multi-target point cooperation; S400, adaptive optimization of stage-by-stage maturation induction and a co-culture system; and S500, completing preparation.

Description

Technical Field

[0001] This invention relates to the technical field of enhanced DC-CIK cell preparation methods, specifically, to an enhanced DC-CIK cell preparation method. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a highly prevalent malignant tumor worldwide, with HBV-related HCC accounting for more than 70% of liver cancer cases in my country. The recurrence rate of these patients after surgery is as high as 60%-70%, and the median survival of advanced patients after first-line treatment is less than 2 years, indicating a huge unmet clinical need.

[0003] Traditional DC-CIK therapy suffers from drawbacks such as low efficiency of DC cell antigen presentation and poor tolerance to immunosuppression in the tumor microenvironment. Existing DC vaccine projects have demonstrated that personalized antigen loading and gene modification can significantly improve efficacy (such as mRNA transfection DC vaccines and TACE combined with DC therapy), but still face challenges such as high heterogeneity of HBV-related HCC antigens and insufficient specific immune response. Summary of the Invention

[0004] One of the objectives of this invention is to propose a method for preparing enhanced DC-CIK cells, which deeply modifies DC cells and combines them with CIK cells to form an enhanced therapy.

[0005] The technical solution of the present invention is as follows: A method for preparing enhanced DC-CIK cells, comprising the following steps: S100: Preparation of patient DC cells and CIK cells; S200: Upgraded dual-antigen targeted loading: Optimizes antigen sequence and precisely controls loading timing to address the antigenic heterogeneity problem of HBV-related HCC; Step S200 includes: S210: Precisely screen and optimize antigen sequences; S220: Employs a phased dual-mode loading method to precisely control the timing and method of antigen loading; S300: Refined gene editing and multi-target synergy: The addition of CCL21 gene overexpression forms a three-target synergistic editing of "immune activation + chemotactic recruitment + microenvironment remodeling"; Step S300 includes: S310: Functional synergistic design for multi-target gene editing; S320: Optimized gene editing process; S400: Adaptability optimization of the phased maturation induction and co-culture system: increase the dynamic regulation of phased maturation induction and DC-CIK co-culture, and refine the quality control nodes; Step S400 includes: S410: Staged induction of DC cell maturation; S420: Dynamically regulate the combination and time of DC-CIK co-culture; S430: Customized serum-free culture medium: using serum-free culture medium adapted from peripheral blood of HCC patients, with 5% autologous plasma added to the basic serum-free culture medium; S500: Preparation complete.

[0006] Furthermore, step S210 includes: HBV-specific antigens: Based on the sequencing results of the patient's HBV virus strain, individualized HBV mutant antigen epitopes are screened, while conserved HBcAg18-27 epitopes are preserved. Universal antigen for liver cancer: The original single GPC3 polypeptide has been upgraded to a GPC3+AFP dual epitope tandem peptide, covering both GPC3-positive and AFP-positive liver cancer subgroups. The dual epitope tandem peptide can simultaneously activate CD4+ and CD8+ T cells.

[0007] Furthermore, step S220 includes: S221: Phase 1: On day 3 after monocytes are induced into immature DCs, personalized HBV mutant antigen and GPC3 fusion mRNA is introduced via mRNA electroporation. S222: Second stage: After gene editing is completed, AFP peptide pulse loading is performed.

[0008] Furthermore, the mRNA in step S221 uses a self-amplifying vector.

[0009] Furthermore, the pulse conditions in step S222 are: incubation at 37°C and 5% CO2 for 4 hours, with a peptide concentration of 50 μg / mL; The adjuvant polyI:C was added to enhance DC cell activation.

[0010] Furthermore, step S310 includes: S311: PD-L1 gene knockout: The target sequence is a conserved region of the PD-L1 gene exon3, using a non-viral CRISPR-Cas9 ribonucleoprotein delivery system; S312: Overexpression of IL-12 gene: Secretory IL-12 mutant was selected and transfected via lentiviral vector; S313: New overexpression of CCL21 gene: CCL21 can specifically chemotactically attract naive T cells and memory T cells in lymph nodes to the tumor site, constructing an immune synergistic network.

[0011] Furthermore, step S320 includes: S321: Delivery system selection: PD-L1 knockout uses the RNP system, and IL-12 and CCL21 overexpression uses a non-integrating lentiviral vector; S322: Post-editing screening: After gene editing, mature DC cells are sorted by flow cytometry with a purity of ≥90% to ensure the functional uniformity of each batch of DC cells.

[0012] Furthermore, step S410 includes: The maturation process of DC cells is divided into two stages: Phase 1: After gene editing is completed, TNF-α and CD40L are added for 24 hours of induction; Phase 2: PolyI:C+IFN-γ was added to continue induction for 24 hours to enhance the expression of co-stimulatory molecules and cytokine secretion in DC cells.

[0013] Furthermore, step S420 includes: Formula and time optimization: The original fixed 1:100 ratio was adjusted to a phased dynamic ratio. For the first 3 days of culture, DC:CIK = 1:50, and for the next 4 days, it was adjusted to 1:100. A low concentration of IL-2 was added to the co-culture environment. Co-culture quality control points: Quality control tests are conducted on days 2, 4, and 7 of co-culture, including cell viability, endotoxins, mycoplasma, bacteria and fungi, and gene editing efficiency. A complete quality control report is issued for each batch.

[0014] Another objective of this invention is to provide an application of the above-described enhanced DC-CIK cell preparation method in tumor treatment.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention involves precise dual-antigen loading, multi-target gene editing, and staged maturation induction to deeply modify DC cells, which, combined with CIK cells, forms an enhanced DC-CIK therapy for postoperative adjuvant therapy and late-stage palliative treatment of HBV-related HCC. It is both innovative and clinically feasible. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1

[0019] This embodiment provides a method for preparing enhanced DC-CIK cells, which includes the following steps: S100: Preparation of patient DC cells and CIK cells; S200: Upgraded dual antigen targeted loading: In response to the antigenic heterogeneity problem of HBV-related HCC, the antigen sequence is optimized and the loading timing is precisely controlled based on the original dual antigens. Step S200 includes: S210: Precisely screen and optimize antigen sequences, including: HBV-specific antigens: Instead of using the universal HBsAg / HBcAg fusion peptide, we screen individualized HBV mutation epitopes (such as high-frequency mutation sites like sP120T and cI97L) based on the patient's HBV virus strain sequencing results, while retaining the conserved HBcAg18-27 epitopes. This ensures recognition of the patient's own virus-integrated liver cancer cells and avoids antigen escape caused by viral mutations. The logic of AI-assisted personalized antigen design can be referenced. Universal antigen for liver cancer: The original single GPC3 peptide has been upgraded to a dual epitope tandem peptide of GPC3 (298-306) + AFP (158-166), covering the liver cancer subgroups of GPC3 positive (about 70% HCC) and AFP positive (about 60% HCC). The dual epitope tandem peptide can simultaneously activate CD4+ and CD8+ T cells. Preclinical experiments have shown that its antigen presentation efficiency is 1.8 times higher than that of a single epitope.

[0020] S220: Employs a phased dual-mode loading method to precisely control the timing and method of antigen loading, including: S221: Phase 1 (Immature DC phase): On day 3 after monocytes are induced into immature DCs, personalized HBV mutant antigen and GPC3 fusion mRNA is introduced via mRNA electrotransfection. Taking advantage of the high uptake efficiency of immature DCs, stable endogenous expression of the antigen is achieved. The mRNA used in step S221 is a self-amplifying vector (saRNA), which can continuously replicate and express the antigen in DC cells, extending the expression duration from 72 hours for conventional mRNA to 14 days.

[0021] S222: Second stage (semi-mature DC phase): On day 5 after gene editing, AFP peptide pulse loading is performed.

[0022] The pulse conditions in step S222 are precisely controlled as follows: incubation at 37°C and 5% CO2 for 4 hours, with a peptide concentration of 50 μg / mL. The adjuvant polyI:C was added to enhance DC cell activation. This loading stage can supplement exogenous antigen stimulation and further improve DC cell maturity. Preclinical data showed that this loading time sequence can increase the expression rate of CD83 (maturity marker) in DC cells from 75% to 92%.

[0023] S300: Refined gene editing and multi-target synergy: Based on the original PD-L1 knockout + IL-12 overexpression, CCL21 gene overexpression is added to form a three-target synergistic editing of "immune activation + chemotactic recruitment + microenvironment remodeling". At the same time, the gene editing process is optimized to improve editing efficiency and safety. Step S300 includes: S310: Functional synergistic design for multi-target gene editing, including: S311: PD-L1 gene knockout: The target sequence is the conserved region of exon 3 of the PD-L1 gene (sgRNA sequence: 5'-GGAATTCGACGATGCCACCT-3'). A non-viral CRISPR-Cas9 ribonucleoprotein (RNP) delivery system is used, achieving an editing efficiency of over 85% and an off-target rate of <0.1% (verified by whole-genome sequencing). Knockout can block the binding of dendritic cells (DCs) to PD-1+ T cells in the tumor microenvironment, preventing DC functional exhaustion and increasing CTL induction activity by 40%. S312: Overexpression of IL-12 gene: Secretory IL-12 (sIL-12) mutant (retaining p35 / p40 heterodimer activity and reducing systemic toxicity) was selected and transfected through lentiviral vector (modified with replication defects, with no risk of integration). The transfection efficiency reached 78%. sIL-12 can be locally secreted and activate the NKG2D receptor of CIK cells, which increases the killing activity of CIK cells against liver cancer cells by 52% and promotes Th1 immune response. S313: New overexpression of CCL21 gene: CCL21 can specifically chemotactically attract naive T cells and memory T cells in lymph nodes to the tumor site, constructing an immune synergistic network of "DC cells-T cells-CIK cells". Preclinical experiments show that DC cells overexpressing CCL21 can increase the infiltration density of CD8+ T cells in the tumor site by 3.2 times, solving the pain point of "low cell homing efficiency" in traditional DC-CIK therapy.

[0024] S320: Optimizes gene editing processes, including: S321: Delivery system selection: PD-L1 knockout uses the RNP system (no risk of integration), and IL-12 and CCL21 overexpression uses a non-integrating lentiviral vector (Sin-LV) to avoid genomic instability caused by random integration; S322: Post-editing screening: After gene editing, mature DC cells of “PD-L1⁻CD83⁺IL-12⁺CCL21⁺” are sorted by flow cytometry with a purity of ≥90% to ensure the functional uniformity of each batch of DC cells and reduce individual differences in clinical treatment.

[0025] S400: Adaptability optimization of the phased maturation induction and co-culture system: Based on the original culture process, dynamic control of phased maturation induction and DC-CIK co-culture is added, while quality control nodes are refined to ensure compliance of clinical application and product stability. Step S400 includes: S410: Staged induction of DC cell maturation, including: The maturation process of dendritic cells (DCs) is divided into two stages to avoid premature maturation leading to a decrease in antigen uptake capacity. Phase 1 (semi-maturation induction, day 5): After gene editing, TNF-α (50 ng / mL) and CD40L (100 ng / mL) were added to induce DC cells for 24 hours to achieve initial maturation and retain some antigen uptake capacity. Phase 2 (complete maturation induction, day 6): PolyI:C (20 μg / mL) + IFN-γ (10 ng / mL) were added to continue induction for 24 hours to enhance the expression of co-stimulatory molecules and cytokine secretion in DC cells. The positive rates of CD80 / CD86 / HLA-DR in the final mature DC cells were ≥90%, and the IL-12 secretion reached 850 pg / mL (2.1 times higher than the traditional induction method).

[0026] S420: Dynamically controlling the combination and time of DC-CIK co-culture, including: Optimization of formulation and time: The original fixed 1:100 formulation was adjusted to a phased dynamic formulation. For the first 3 days of culture, the DC:CIK ratio was 1:50 (high DC ratio to enhance antigen presentation), and for the next 4 days it was adjusted to 1:100 (to ensure CIK cell expansion). A low concentration of IL-2 (100 IU / mL) was added to the co-culture environment, which promoted CIK cell proliferation while avoiding the toxic side effects caused by high doses of IL-2. Ultimately, the CIK cell expansion reached more than 35 times (compared to 20 times in the traditional protocol), and the proportion of CD3+CD56+ double-positive cells was ≥45%. Co-culture quality control points: Quality control tests are performed on days 2, 4, and 7 of co-culture, including cell viability (≥95%), endotoxin (<0.5 EU / mL), mycoplasma (negative), bacteria and fungi (negative), and gene editing efficiency (≥80%). A complete quality control report is issued for each batch, which complies with GMP production standards.

[0027] S430: Customized serum-free culture medium: Using peripheral blood from HCC patients as the adaptable serum-free culture medium, 5% autologous plasma (after irradiation inactivation treatment) is added to the basic serum-free culture medium, which avoids the risk of immune rejection from heterologous serum and improves the survival and function of DC cells in the patient's individualized environment. S500: Preparation complete.

[0028] The table below shows the data on DC maturation and antigen presentation function, which verifies whether DC vaccines or DC-based immunotherapies have been successfully prepared and have the expected functions.

[0029]

[0030] The table below shows gene editing efficiency and functional validation data, used to assess and confirm whether the gene editing operation was successful and accurately achieved the expected goals, and to examine whether the edited cell function has undergone corresponding changes.

[0031]

[0032] The table below shows the results of an in vitro tumor killing experiment, used to verify whether DC cells can effectively kill tumor cells.

[0033]

[0034] The table below shows the efficacy data of the animal model, used to verify the ability to recognize and kill tumor cells in an in vitro environment.

[0035]

[0036] The table below shows key data for safety evaluation, used to verify the safety, reliability, and quality control of cell-based therapeutic products, drugs, or therapies during application.

[0037]

[0038] Example 2

[0039] Based on Example 1, this example applies the above-described enhanced DC-CIK cell preparation method to tumor treatment, thereby enhancing the therapeutic effect.

[0040] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing enhanced DC-CIK cells, characterized in that, The following steps are used: S100: Preparation of patient DC cells and CIK cells; S200: Upgraded dual-antigen targeted loading: Optimizes antigen sequence and precisely controls loading timing to address the antigenic heterogeneity problem of HBV-related HCC; Step S200 includes: S210: Precisely screen and optimize antigen sequences; S220: Employs a phased dual-mode loading method to precisely control the timing and method of antigen loading; S300: Refined gene editing and multi-target synergy: The addition of CCL21 gene overexpression forms a three-target synergistic editing of "immune activation + chemotactic recruitment + microenvironment remodeling"; Step S300 includes: S310: Functional synergistic design for multi-target gene editing; S320: Optimized gene editing process; S400: Adaptability optimization of the phased maturation induction and co-culture system: increase the dynamic regulation of phased maturation induction and DC-CIK co-culture, and refine the quality control nodes; Step S400 includes: S410: Staged induction of DC cell maturation; S420: Dynamically regulate the combination and time of DC-CIK co-culture; S430: Customized serum-free culture medium: using serum-free culture medium adapted from peripheral blood of HCC patients, with 5% autologous plasma added to the basic serum-free culture medium; S500: Preparation complete.

2. The method for preparing enhanced DC-CIK cells according to claim 1, characterized in that, Step S210 includes: HBV-specific antigens: Based on the sequencing results of the patient's HBV virus strain, individualized HBV mutant antigen epitopes are screened, while conserved HBcAg18-27 epitopes are preserved. Universal antigen for liver cancer: The original single GPC3 polypeptide has been upgraded to a GPC3+AFP dual epitope tandem peptide, covering both GPC3-positive and AFP-positive liver cancer subgroups. The dual epitope tandem peptide can simultaneously activate CD4+ and CD8+ T cells.

3. The method for preparing enhanced DC-CIK cells according to claim 2, characterized in that, Step S220 includes: S221: Phase 1: On day 3 after monocytes are induced into immature DCs, personalized HBV mutant antigen and GPC3 fusion mRNA is introduced via mRNA electroporation. S222: Second stage: After gene editing is completed, AFP peptide pulse loading is performed.

4. The method for preparing enhanced DC-CIK cells according to claim 3, characterized in that, The mRNA in step S221 uses a self-amplifying vector.

5. The method for preparing enhanced DC-CIK cells according to claim 4, characterized in that, The pulse conditions in step S222 are: incubation at 37°C and 5% CO2 for 4 hours, with a peptide concentration of 50 μg / mL; The adjuvant polyI:C was added to enhance DC cell activation.

6. The method for preparing enhanced DC-CIK cells according to claim 5, characterized in that, Step S310 includes: S311: PD-L1 gene knockout: The target sequence is a conserved region of the PD-L1 gene exon3, using a non-viral CRISPR-Cas9 ribonucleoprotein delivery system; S312: Overexpression of IL-12 gene: Secretory IL-12 mutant was selected and transfected via lentiviral vector; S313: New overexpression of CCL21 gene: CCL21 can specifically chemotactically attract naive T cells and memory T cells in lymph nodes to the tumor site, constructing an immune synergistic network.

7. The method for preparing enhanced DC-CIK cells according to claim 6, characterized in that, Step S320 includes: S321: Delivery system selection: PD-L1 knockout uses the RNP system, and IL-12 and CCL21 overexpression uses a non-integrating lentiviral vector; S322: Post-editing screening: After gene editing, mature DC cells are sorted by flow cytometry with a purity of ≥90% to ensure the functional uniformity of each batch of DC cells.

8. The method for preparing enhanced DC-CIK cells according to claim 7, characterized in that, Step S410 includes: The maturation process of DC cells is divided into two stages: Phase 1: After gene editing is completed, TNF-α and CD40L are added for 24 hours of induction; Phase 2: PolyI:C+IFN-γ was added to continue induction for 24 hours to enhance the expression of co-stimulatory molecules and cytokine secretion in DC cells.

9. The method for preparing enhanced DC-CIK cells according to claim 8, characterized in that, Step S420 includes: Formula and time optimization: The original fixed 1:100 ratio was adjusted to a phased dynamic ratio. For the first 3 days of culture, DC:CIK = 1:50, and for the next 4 days, it was adjusted to 1:

100. A low concentration of IL-2 was added to the co-culture environment. Co-culture quality control points: Quality control tests are conducted on days 2, 4, and 7 of co-culture, including cell viability, endotoxins, mycoplasma, bacteria and fungi, and gene editing efficiency. A complete quality control report is issued for each batch.

10. The application of the enhanced DC-CIK cell preparation method as described in any one of claims 1-9 in tumor treatment.

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