A CD38 site-specific integration Her2-CAR-NK cell, its preparation method and application

Abstract

This invention relates to the field of cell therapy technology and discloses a Her2-CAR-NK cell with CD38 site-specific integration, its preparation method, and its applications. The preparation method includes: isolating and activating primary human NK cells; constructing a CRISPR / Cas9 gene editing system targeting the first exon of the CD38 gene; constructing an AAV6 donor vector carrying a homologous arm and a Her2-CAR expression cassette; introducing the Cas9 / gRNARNP complex into NK cells via electroporation, and using the AAV6 donor virus to site-specifically integrate the Her2-CAR expression cassette into the CD38 gene site through homologous recombination; and finally amplifying and culturing the CAR-NK cells to obtain them. This invention improves the safety and batch-to-batch consistency of the prepared CAR-NK cell product, enhances the persistence of cells in vivo, and improves the therapeutic potential for solid tumors.

Description

Technical Field

[0001] This invention relates to the field of cell therapy technology, specifically to a CD38 site-specific integrated Her2-CAR-NK cell, its preparation method, and its application. Background Technology

[0002] Cell therapy is a biological treatment method in the field of materials science and technology. It refers to the treatment method of repairing tissues and organs through autologous or somatic adult cells or stem cells. It is mainly divided into two types: stem cell therapy and immune cell therapy.

[0003] Currently, in the preparation of CAR-modified natural killer cell products targeting solid tumors, retroviral or lentiviral vectors are typically used for random integration of the CAR gene. This random integration method can lead to abnormal activation of proto-oncogenes, posing potential tumorigenic safety risks. Furthermore, it is impossible to control the integration site and copy number of the CAR gene, resulting in differences in CAR expression levels among different cells, affecting the uniformity of cell products and the stability of therapeutic efficacy. At the same time, since the CAR gene is driven by an exogenous strong promoter, its expression level is not coordinated with the physiological state, which can lead to premature depletion or functional abnormalities of CAR-NK cells, making it difficult for them to survive in vivo for a long time and exert a sustained anti-tumor effect.

[0004] Therefore, we propose a method for CD38 site-specific integration of Her2-CAR-NK cells, its preparation, and its application to address the aforementioned problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides Her2-CAR-NK cells with CD38 site-directed integration, their preparation method, and applications. This solves the problems mentioned in the background art regarding the random integration of CAR genes using retroviruses or lentiviral vectors, and the difficulty in long-term survival in vivo and the inability to exert sustained anti-tumor effects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a CD38 site-specific integration Her2-CAR-NK cell, its preparation method, and its application. The preparation method includes the following steps:

[0007] Step 1: Isolation, purification, activation and expansion of primary NK cells. Primary NK cells are isolated and purified from human peripheral blood mononuclear cells and then activated and expanded in culture.

[0008] Step 2: Construction of a CRISPR / Cas9 editing system targeting the CD38 gene, designing and synthesizing guide ribonucleic acid targeting the first exon of the human CD38 gene, and combining it with Cas9 protein to form a ribonucleoprotein complex;

[0009] Step 3: Construction of CD38 site homologous recombinant adeno-associated virus serum type 6 donor vector. Centered on the guide ribonuclease site in the first exon of the human CD38 gene, left and right homologous arms were designed, and Her2-CAR expression cassettes were inserted between the homologous arms to construct the donor vector and then perform virus packaging and purification.

[0010] Step 4: Electroporation of activated primary NK cells and transduction of adeno-associated virus serotype 6. The activated primary NK cells obtained in Step 1 were mixed with the ribonucleoprotein complex prepared in Step 2 and then electroporated. Immediately afterwards, the adeno-associated virus serotype 6 donor virus prepared in Step 3 was added for transduction.

[0011] Step 5: Expansion and culture of Her2-CAR-NK cells with CD38 site-specific integration. The cells treated in Step 4 are cultured and expanded to obtain Her2-CAR-NK cells with CD38 site-specific integration.

[0012] Preferably, in step one, the activation and expansion of primary NK cells includes the following steps: purifying the primary NK cells and resuspending them in complete culture medium at a density of 0.5 x 10⁶ to 1.5 x 10⁶ cells per milliliter; adding feeder cells at a ratio of 0.5:1 to 2:1 between primary NK cells and irradiated feeder cells; and culturing the cells in an incubator at 37 degrees Celsius and 5% carbon dioxide for 5 to 7 days. The complete culture medium contains basal culture medium, 5% to 20% fetal bovine serum, and 100 to 200 units of interleukin-2 per liter.

[0013] Preferably, in step two, the target sequence of the guide ribonucleic acid is 5'-CTGAACTCGCAGTTGGCCAT-3', and the three bases at the 5' end and the three bases at the 3' end of the guide ribonucleic acid are modified by 2'-O-methyl 3' thiophosphorylation. The ribonucleoprotein complex is prepared by mixing the modified guide ribonucleic acid with SpCas9 protein at a molar ratio of 1.5:1 to 2.5:1 and incubating at room temperature for 15 to 25 minutes.

[0014] Preferably, in step three, the chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure adapted to natural killer cells, comprising, from the N-terminus to the C-terminus, a CD8a signal peptide sequence, a single-chain variable region antibody sequence targeting human epidermal growth factor receptor 2, a CD8a hinge region sequence, a CD8a transmembrane region sequence, a CD28 co-stimulatory domain sequence, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signal transduction domain sequence. The 5' end of the Her2-CAR expression cassette is connected to a P2A self-cleaving peptide encoding sequence, and the entire Her2-CAR expression cassette is regulated by the endogenous promoter of the human CD38 gene.

[0015] Preferably, in step three, the left homologous arm is the genome sequence of 600 to 1000 base pairs upstream of the guide ribonucleic acid cleavage site, the right homologous arm is the genome sequence of 600 to 1000 base pairs downstream of the guide ribonucleic acid cleavage site, and the titer of the adeno-associated virus serotype 6 donor virus is not less than 1 x 10^12 viral genome per milliliter.

[0016] Preferably, step four includes the following steps: after washing the activated primary NK cells obtained in step one with buffer, resuspending them and adjusting the cell density to 2 x 10^7 to 8 x 10^7 cells per milliliter, taking 50 to 150 microliters of cell suspension, adding the pre-prepared ribonucleoprotein complex, mixing, and transferring to an electroporation cuvette for electroporation, the electroporation program is selected from a preset program capable of transfecting primary lymphocytes. After electroporation, immediately add preheated complete culture medium to 37 degrees Celsius to the electroporation cuvette to resuspend the cells, and transfer them to a culture container. After standing for 20 to 40 minutes, add the adeno-associated virus type 6 donor virus prepared in step three according to the multiplicity of infection of the viral genome of 1 x 10^4 to 5 x 10^5 per cell.

[0017] Preferably, in step five, the culture and amplification conditions are as follows: the culture is carried out in an incubator at 37 degrees Celsius with a carbon dioxide volume fraction of 5%, half the volume of complete culture medium is replaced every 2 to 3 days, and interleukin-2 is supplemented to a final concentration of 300 to 600 IU per milliliter, and the culture is continued for 10 to 16 days.

[0018] Preferably, the Her2-CAR-NK cells with CD38 site-specific integration obtained by the preparation method have a surface chimeric antigen receptor positivity rate of more than 70% and a CD38 gene knockout efficiency of more than 95%.

[0019] Preferably, the first exon site of the human CD38 gene in the cell genome integrates a Her2-CAR expression cassette through homologous targeted repair, thereby knocking out the endogenous CD38 gene. The chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure containing a single-chain variable region antibody targeting human epidermal growth factor receptor 2, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain, and is expressed by an endogenous CD38 promoter. The chimeric antigen receptor is stably expressed on the cell surface, and the CD38 protein is not expressed.

[0020] Preferably, the Her2-CAR-NK cells specifically integrated at the CD38 site are used in the preparation of a drug for treating human epidermal growth factor receptor 2 positive gastric cancer.

[0021] Compared with existing technologies, this invention provides Her2-CAR-NK cells with CD38 site-specific integration, their preparation method, and applications, which have the following beneficial effects:

[0022] 1. In this invention, by employing a strategy combining the CRISPR / Cas9 editing system with adeno-associated virus serotype 6 donor vector, the Her2-CAR expression cassette is site-specifically integrated into the first exon of the human CD38 gene, achieving CAR gene insertion into the genome. This avoids the oncogene activation and tumorigenicity risks caused by random integration with traditional retroviral or lentiviral vectors. Furthermore, this site-specific integration strategy ensures a single, stable copy of the CAR gene, resulting in highly uniform expression of Her2-CAR driven by the endogenous CD38 promoter in the cell population, thus improving the safety and batch-to-batch consistency of the prepared CAR-NK cell products.

[0023] 2. In this invention, by designing the Her2-CAR expression cassette to replace a portion of the endogenous CD38 gene sequence during homologous recombination, the integration of CAR and the knockout of the CD38 gene are simultaneously achieved. The resulting Her2-CAR-NK cells with site-specific integration at the CD38 site do not express CD38 protein, thereby reducing the cannibalistic phenomenon that may occur in the cell product due to residual CD38-positive cells, ensuring the purity and yield of the cell product. At the same time, this cell product can be used in combination with commonly used CD38-targeting monoclonal antibody drugs in clinical practice, avoiding mutual antagonism, providing a safe basis for combination therapy, and also enhancing the persistence of cells in vivo.

[0024] 3. In this invention, the designed Her2-CAR molecule contains CD28 and 4-1BB dual co-stimulatory domains. This structure is optimized to adapt to the function of natural killer cells. When this CAR molecule is expressed in NK cells driven by an endogenous promoter, it can provide activation and sustained survival signals, enabling cells to acquire in vitro expansion capacity and in vivo persistence. Combined with the editing method of CRISPR / Cas9 ribonucleoprotein complex electroporation and adeno-associated virus serum type 6 transduction in primary NK cells, the problems of difficult transfection and editing of primary NK cells are solved. The prepared cells show killing activity against Her2-positive gastric cancer cells and solid tumor target cells, enhancing the therapeutic potential for solid tumors. Attached Figure Description

[0025] Figure 1 This diagram shows the identification results of constructing CD38 Her2CAR-NK at the Cas9RNP electroporation site combined with AAV6, comprising 10 sub-figures:

[0026] 1A: Schematic diagram of the structure of Her2-CAR and control CD19-CAR, showing the composition of each functional element of the third-generation CAR, from N-terminus to C-terminus: CD8a signal peptide, antigen-specific scFv, CD8a hinge region and transmembrane region, CD28 co-stimulatory domain, 4-1BB co-stimulatory domain, CD3ζ signal domain, P2A self-cleaving peptide, and GFP reporter gene;

[0027] 1B: Flowchart of CAR-NK cell construction and expansion, clarifying the time nodes and operation steps of the entire process, including primary NK cell isolation on Day 0, activation of feeder cells twice on Day 0 and Day 5, gene editing on Day 7, and cell identification on Day 14.

[0028] 1C: Flow cytometry analysis of CAR expression in NK cells transduced with lentiviral CD19-CAR and Her2-CAR, showing the CAR positivity rates of the three cell groups (untransduced, LV-CD19-CAR, and LV-Her2-CAR) as 0.14%, 54.6%, and 68.9%, respectively.

[0029] 1D: A statistical graph of the efficiency of CAR-NK cell expression transduced by lentivirus, quantitatively showing the statistical results of CAR positivity rate in three groups of cells;

[0030] 1E: A schematic diagram of the CRISPR / Cas9 combined with AAV6-mediated site-specific integration of Her2-CAR at the CD38 site, showing the molecular mechanism by which Cas9 / gRNA RNP targets the first exon of the CD38 gene to generate DSB, and the AAV6 donor vector integrates the Her2-CAR expression cassette into the CD38 site through homologous recombination.

[0031] 1F: Sanger sequencing peak diagram of CD38 gene knockout, comparing the sequences of CD38 wild type and knockout type, confirming that Cas9 / gRNA can achieve efficient gene cleavage and knockout at the expected site;

[0032] 1G: Flow cytometry analysis of CD38 expression on the surface of NK cells after knocking in Her2-CAR at the CD38 site, showing the CD38 expression levels of wild-type NK cells and CD38KI-Her2-CAR-NK cells. The positive rate of the former was 98.3%, while that of the latter decreased to 2.6%.

[0033] 1H: Expansion curves of NK cells in different groups, showing the fold increase in the number of unedited NK cells, LV-Her2-CAR-NK cells, and CD38KI-Her2-CAR-NK cells during the 14-day culture period;

[0034] 1I: Flow cytometry analysis of CAR expression after lentiviral transduction and site-specific integration at CD38 site, showing the CAR positivity rates of untransduced, LV-Her2-CAR, and CD38KI-Her2-CAR cells, which were 0.37%, 70.3%, and 81.0%, respectively.

[0035] 1J: Statistical graph of CAR expression efficiency by lentiviral transduction and CD38 site-specific integration, quantitatively showing the statistical results of CAR positivity rate in three groups of cells.

[0036] Figure 2 The results of immunophenotyping of CD38KI-Her2-CAR-NK cells in this invention include three sub-figures:

[0037] 2A: Flow cytometry and quantitative data of expression of CD38KI-Her2-CAR-NK and LV-Her2-CAR-NK cell surface activating receptors;

[0038] 2B: Flow cytometry and quantitative data of the expression of inhibitory receptors on the cell surface in two groups;

[0039] 2C: Statistical graph of receptor expression levels in the two groups of cells, with statistical differences marked.

[0040] Figure 3 The results of the in vitro anti-gastric cancer function verification of CD38KI-Her2-CAR-NK cells of this invention include 12 sub-figures, as follows:

[0041] 3A: Flow cytometry diagram of Her2 expression on the surface of target cells;

[0042] 3B-3D: Detection results of IL-2, TNF-α, and IFN-γ concentrations after co-culturing effector cells and target cells;

[0043] 3E-3F: Detection results of IL-1β and IL-6 concentrations after co-culturing effector cells and target cells;

[0044] 3G: Flow cytometry analysis of CD107a expression in effector cells and MKN-28 cells after 6 hours of co-culture;

[0045] 3H: Statistical graph of CD107a positivity rate after effector cells were co-cultured with three types of Her2-positive gastric cancer cells;

[0046] 3I-3L: Results of effector cell killing rate against K562, MKN-28, MKN-45 and NCI-N87 cells at different effector-to-target ratios.

[0047] Figure 4 The results of in vivo anti-gastric cancer activity verification of CD38KI-Her2-CAR-NK cells of the present invention include a flowchart of tumor-bearing mouse model construction and drug administration, tumor growth curves of mice in each group, tumor volume statistics, Kaplan-Meier curves of mouse survival, curves of mouse body weight change, HE / Tunel / Ki67 staining results of tumor tissue, and HE staining results of major organs of mice. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0049] Example 1: A CD38 site-specific integration Her2-CAR-NK cell, its preparation method, and its application. The preparation method includes the following steps:

[0050] Step 1: Isolation, purification, activation and expansion of primary NK cells. Primary NK cells are isolated and purified from human peripheral blood mononuclear cells and then activated and expanded in culture.

[0051] Step 2: Construction of a CRISPR / Cas9 editing system targeting the CD38 gene, designing and synthesizing guide ribonucleic acid targeting the first exon of the human CD38 gene, and combining it with Cas9 protein to form a ribonucleoprotein complex;

[0052] Step 3: Construction of CD38 site homologous recombinant adeno-associated virus serum type 6 donor vector. Centered on the guide ribonuclease site in the first exon of the human CD38 gene, left and right homologous arms were designed, and Her2-CAR expression cassettes were inserted between the homologous arms to construct the donor vector and then perform virus packaging and purification.

[0053] Step 4: Electroporation of activated primary NK cells and transduction of adeno-associated virus serotype 6. The activated primary NK cells obtained in Step 1 were mixed with the ribonucleoprotein complex prepared in Step 2 and then electroporated. Immediately afterwards, the adeno-associated virus serotype 6 donor virus prepared in Step 3 was added for transduction.

[0054] Step 5: Expansion and culture of Her2-CAR-NK cells with CD38 site-specific integration. The cells treated in Step 4 are cultured and expanded to obtain Her2-CAR-NK cells with CD38 site-specific integration.

[0055] In step one, the activation and expansion of primary NK cells includes the following steps: the purified primary NK cells are resuspended in complete culture medium at a density of 0.5 x 10^6 cells per milliliter, and feeder cells are added at a ratio of 0.5:1 between primary NK cells and irradiated feeder cells. The cells are then cultured for 5 days in an incubator at 37 degrees Celsius and 5% CO2. The complete culture medium contains basal culture medium, 5% fetal bovine serum, and 100 units of interleukin-2 per liter.

[0056] In step two, the target sequence of the guide ribonucleic acid is 5'-CTGAACTCGCAGTTGGCCAT-3'. The three bases at the 5' end and the three bases at the 3' end of the guide ribonucleic acid are modified by 2'-O-methyl 3' thiophosphorylation. The ribonucleoprotein complex is prepared by mixing the modified guide ribonucleic acid with SpCas9 protein at a molar ratio of 1.5:1 and incubating at room temperature for 15 minutes.

[0057] In step three, the chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure adapted to natural killer cells. From the N-terminus to the C-terminus, it includes the CD8a signal peptide sequence, the single-chain variable region antibody sequence targeting human epidermal growth factor receptor 2, the CD8a hinge region sequence, the CD8a transmembrane region sequence, the CD28 co-stimulatory domain sequence, the 4-1BB co-stimulatory domain sequence, and the CD3ζ signal transduction domain sequence. The 5' end of the Her2-CAR expression cassette is connected to the P2A self-cleaving peptide encoding sequence, and the entire Her2-CAR expression cassette is under the endogenous promoter regulation of the human CD38 gene.

[0058] In step three, the left homologous arm is the genome sequence of 600 base pairs upstream of the guide ribonuclease cleavage site, and the right homologous arm is the genome sequence of 600 base pairs downstream of the guide ribonuclease cleavage site. The titer of adeno-associated virus type 6 donor virus is not less than 1 x 10^12 viral genome per milliliter.

[0059] Step four includes the following steps: After washing the activated primary NK cells obtained in step one with buffer, resuspend them and adjust the cell density to 2 x 10^7 cells per milliliter. Take 50 μL of cell suspension, add the pre-prepared ribonucleoprotein complex, mix, and transfer to an electroporation cuvette for electroporation. The electroporation program is selected from a preset program that can transfect primary lymphocytes. After electroporation, immediately add preheated complete culture medium to 37 degrees Celsius to the electroporation cuvette to resuspend the cells, and transfer them to a culture container. After standing for 20 minutes, add the adeno-associated virus type 6 donor virus prepared in step three at a viral genome multiplicity of infection of 1 x 10^4 per cell.

[0060] In step five, the culture and amplification conditions are as follows: the culture is carried out in an incubator at 37 degrees Celsius with a carbon dioxide volume fraction of 5%. Half of the complete culture medium is replaced every two days, and interleukin-2 is added until the final concentration is 300 IU per milliliter. The culture is continued for 10 days.

[0061] Her2-CAR-NK cells with CD38 site-specific integration obtained by the preparation method have a surface chimeric antigen receptor positivity rate of more than 70% and a CD38 gene knockout efficiency of more than 95%.

[0062] The first exon site of the human CD38 gene in the cell genome integrates the Her2-CAR expression cassette through homologous targeted repair, resulting in the knockout of the endogenous CD38 gene. The chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure containing a single-chain variable region antibody targeting human epidermal growth factor receptor 2, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. Its expression is driven by the endogenous CD38 promoter. The chimeric antigen receptor is stably expressed on the cell surface, and the CD38 protein is not expressed.

[0063] Application of Her2-CAR-NK cells that integrate at the CD38 site in the preparation of drugs for the treatment of human epidermal growth factor receptor 2 positive gastric cancer.

[0064] Example 2: A CD38 site-specific integration Her2-CAR-NK cell, its preparation method, and its application. The preparation method includes the following steps:

[0065] Step 1: Isolation, purification, activation and expansion of primary NK cells. Primary NK cells are isolated and purified from human peripheral blood mononuclear cells and then activated and expanded in culture.

[0066] Step 2: Construction of a CRISPR / Cas9 editing system targeting the CD38 gene, designing and synthesizing guide ribonucleic acid targeting the first exon of the human CD38 gene, and combining it with Cas9 protein to form a ribonucleoprotein complex;

[0067] Step 3: Construction of CD38 site homologous recombinant adeno-associated virus serum type 6 donor vector. Centered on the guide ribonuclease site in the first exon of the human CD38 gene, left and right homologous arms were designed, and Her2-CAR expression cassettes were inserted between the homologous arms to construct the donor vector and then perform virus packaging and purification.

[0068] Step 4: Electroporation of activated primary NK cells and transduction of adeno-associated virus serotype 6. The activated primary NK cells obtained in Step 1 were mixed with the ribonucleoprotein complex prepared in Step 2 and then electroporated. Immediately afterwards, the adeno-associated virus serotype 6 donor virus prepared in Step 3 was added for transduction.

[0069] Step 5: Expansion and culture of Her2-CAR-NK cells with CD38 site-specific integration. The cells treated in Step 4 are cultured and expanded to obtain Her2-CAR-NK cells with CD38 site-specific integration.

[0070] In step one, the activation and expansion of primary NK cells includes the following steps: the purified primary NK cells are resuspended in complete culture medium at a density of 1.0 x 10^6 cells per milliliter, and feeder cells are added at a ratio of 5:4 between primary NK cells and irradiated feeder cells. The cells are then cultured for 6 days in an incubator at 37 degrees Celsius and 5% carbon dioxide. The complete culture medium contains basal culture medium, 12.5% ​​fetal bovine serum, and 150 units of interleukin-2 per liter.

[0071] In step two, the target sequence of the guide ribonucleic acid is 5'-CTGAACTCGCAGTTGGCCAT-3'. The three bases at the 5' end and the three bases at the 3' end of the guide ribonucleic acid are modified by 2'-O-methyl 3' thiophosphorylation. The ribonucleoprotein complex is prepared by mixing the modified guide ribonucleic acid with SpCas9 protein at a molar ratio of 2.0:1 and incubating at room temperature for 20 minutes.

[0072] In step three, the chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure adapted to natural killer cells. From the N-terminus to the C-terminus, it includes the CD8a signal peptide sequence, the single-chain variable region antibody sequence targeting human epidermal growth factor receptor 2, the CD8a hinge region sequence, the CD8a transmembrane region sequence, the CD28 co-stimulatory domain sequence, the 4-1BB co-stimulatory domain sequence, and the CD3ζ signal transduction domain sequence. The 5' end of the Her2-CAR expression cassette is connected to the P2A self-cleaving peptide encoding sequence, and the entire Her2-CAR expression cassette is under the endogenous promoter regulation of the human CD38 gene.

[0073] In step three, the left homologous arm is the genome sequence of 800 base pairs upstream of the guide ribonuclease cleavage site, and the right homologous arm is the genome sequence of 800 base pairs downstream of the guide ribonuclease cleavage site. The titer of adeno-associated virus type 6 donor virus is not less than 1 x 10^12 viral genome per milliliter.

[0074] Step four includes the following steps: After washing the activated primary NK cells obtained in step one with buffer, resuspend them and adjust the cell density to 5 x 10^7 cells per milliliter. Take 100 μL of cell suspension, add the pre-prepared ribonucleoprotein complex, mix, and transfer to an electroporation cuvette for electroporation. The electroporation program is selected from a preset program that can transfect primary lymphocytes. After electroporation, immediately add preheated complete culture medium to 37 degrees Celsius to the electroporation cuvette to resuspend the cells, and transfer them to a culture container. After standing for 30 minutes, add the adeno-associated virus type 6 donor virus prepared in step three according to the multiplicity of infection of the viral genome per cell of 2.45 x 10^5.

[0075] In step five, the culture and amplification conditions are as follows: the culture is carried out in an incubator at 37 degrees Celsius with a carbon dioxide volume fraction of 5%. Half of the complete culture medium is replaced every 2.5 days, and interleukin-2 is supplemented to a final concentration of 450 IU per milliliter. The culture is continued for 13 days.

[0076] Her2-CAR-NK cells with CD38 site-specific integration obtained by the preparation method have a surface chimeric antigen receptor positivity rate of more than 70% and a CD38 gene knockout efficiency of more than 95%.

[0077] The first exon site of the human CD38 gene in the cell genome integrates the Her2-CAR expression cassette through homologous targeted repair, resulting in the knockout of the endogenous CD38 gene. The chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure containing a single-chain variable region antibody targeting human epidermal growth factor receptor 2, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. Its expression is driven by the endogenous CD38 promoter. The chimeric antigen receptor is stably expressed on the cell surface, and the CD38 protein is not expressed.

[0078] Application of Her2-CAR-NK cells that integrate at the CD38 site in the preparation of drugs for the treatment of human epidermal growth factor receptor 2 positive gastric cancer.

[0079] Example 3: A CD38 site-specific integration Her2-CAR-NK cell, its preparation method, and its application. The preparation method includes the following steps:

[0080] Step 1: Isolation, purification, activation and expansion of primary NK cells. Primary NK cells are isolated and purified from human peripheral blood mononuclear cells and then activated and expanded in culture.

[0081] Step 2: Construction of a CRISPR / Cas9 editing system targeting the CD38 gene, designing and synthesizing guide ribonucleic acid targeting the first exon of the human CD38 gene, and combining it with Cas9 protein to form a ribonucleoprotein complex;

[0082] Step 3: Construction of CD38 site homologous recombinant adeno-associated virus serum type 6 donor vector. Centered on the guide ribonuclease site in the first exon of the human CD38 gene, left and right homologous arms were designed, and Her2-CAR expression cassettes were inserted between the homologous arms to construct the donor vector and then perform virus packaging and purification.

[0083] Step 4: Electroporation of activated primary NK cells and transduction of adeno-associated virus serotype 6. The activated primary NK cells obtained in Step 1 were mixed with the ribonucleoprotein complex prepared in Step 2 and then electroporated. Immediately afterwards, the adeno-associated virus serotype 6 donor virus prepared in Step 3 was added for transduction.

[0084] Step 5: Expansion and culture of Her2-CAR-NK cells with CD38 site-specific integration. The cells treated in Step 4 are cultured and expanded to obtain Her2-CAR-NK cells with CD38 site-specific integration.

[0085] In step one, the activation and expansion of primary NK cells includes the following steps: the purified primary NK cells are resuspended in complete culture medium at a density of 1.5 x 10^6 cells, and feeder cells are added at a ratio of 2:1 between primary NK cells and irradiated feeder cells. The cells are then cultured for 7 days in an incubator at 37 degrees Celsius and 5% carbon dioxide. The complete culture medium contains basal culture medium, 20% fetal bovine serum, and 200 units of interleukin-2 per liter.

[0086] In step two, the target sequence of the guide ribonucleic acid is 5'-CTGAACTCGCAGTTGGCCAT-3'. The three bases at the 5' end and the three bases at the 3' end of the guide ribonucleic acid are modified by 2'-O-methyl 3' thiophosphorylation. The ribonucleoprotein complex is prepared by mixing the modified guide ribonucleic acid with SpCas9 protein at a molar ratio of 2.5:1 and incubating at room temperature for 25 minutes.

[0087] In step three, the chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure adapted to natural killer cells. From the N-terminus to the C-terminus, it includes the CD8a signal peptide sequence, the single-chain variable region antibody sequence targeting human epidermal growth factor receptor 2, the CD8a hinge region sequence, the CD8a transmembrane region sequence, the CD28 co-stimulatory domain sequence, the 4-1BB co-stimulatory domain sequence, and the CD3ζ signal transduction domain sequence. The 5' end of the Her2-CAR expression cassette is connected to the P2A self-cleaving peptide encoding sequence, and the entire Her2-CAR expression cassette is under the endogenous promoter regulation of the human CD38 gene.

[0088] In step three, the left homologous arm is the genome sequence of 1000 base pairs upstream of the guide ribonuclease cleavage site, and the right homologous arm is the genome sequence of 1000 base pairs downstream of the guide ribonuclease cleavage site. The titer of adeno-associated virus type 6 donor virus is not less than 1 x 10^12 viral genome per milliliter.

[0089] Step four includes the following steps: After washing the activated primary NK cells obtained in step one with buffer, resuspend them and adjust the cell density to 8 x 10^7 cells. Take 150 μL of cell suspension, add the pre-prepared ribonucleoprotein complex, mix, and transfer to an electroporation cuvette for electroporation. The electroporation program is selected from a preset program that can transfect primary lymphocytes. After electroporation, immediately add preheated complete culture medium to 37 degrees Celsius to the electroporation cuvette to resuspend the cells, and transfer them to a culture container. After standing for 40 minutes, add the adeno-associated virus type 6 donor virus prepared in step three at a viral genome multiplicity of infection of 5 x 10^5 per cell.

[0090] In step five, the culture and amplification conditions are as follows: the culture is carried out in an incubator at 37 degrees Celsius with a carbon dioxide volume fraction of 5%. Half of the complete culture medium is replaced every 3 days, and interleukin-2 is added until the final concentration is 600 IU per milliliter. The culture is continued for 16 days.

[0091] Her2-CAR-NK cells with CD38 site-specific integration obtained by the preparation method have a surface chimeric antigen receptor positivity rate of more than 70% and a CD38 gene knockout efficiency of more than 95%.

[0092] The first exon site of the human CD38 gene in the cell genome integrates the Her2-CAR expression cassette through homologous targeted repair, resulting in the knockout of the endogenous CD38 gene. The chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure containing a single-chain variable region antibody targeting human epidermal growth factor receptor 2, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. Its expression is driven by the endogenous CD38 promoter. The chimeric antigen receptor is stably expressed on the cell surface, and the CD38 protein is not expressed.

[0093] Application of Her2-CAR-NK cells that integrate at the CD38 site in the preparation of drugs for the treatment of human epidermal growth factor receptor 2 positive gastric cancer.

[0094] Comparative Example 1: The difference between this comparative example and Example 1 is that in preparing Her2-CAR-NK cells, this comparative example does not use CRISPR / Cas9-mediated CD38 site-specific integration technology, but uses a third-generation lentiviral vector to randomly integrate the same Her2-CAR expression cassette into the genome of primary NK cells, and does not perform CD38 gene knockout.

[0095] Comparative Example 2 differs from Example 2 in that: in the gene editing process, this comparative example does not use adeno-associated virus serotype 6 as a homologous recombination donor vector, but instead co-delivers the double-stranded DNA donor template and the CRISPR / Cas9 ribonucleoprotein complex into primary NK cells via electroporation.

[0096] Comparative Example 3 differs from Example 3 in that the chimeric antigen receptor molecule used in constructing the Her2-CAR expression cassette in this comparative example does not contain the CD28 and 4-1BB dual co-stimulatory domains, but retains only the CD3ζ signal transduction domain as the first-generation CAR structure.

[0097] Comparative Example 4 differs from Example 1 in that, in preparing CAR-NK cells, although CRISPR / Cas9 was used to integrate the Her2-CAR expression cassette at the CD38 site, the guide RNA used targeted the non-coding region of the CD38 gene, so that after the Her2-CAR expression cassette was integrated, the endogenous CD38 gene was not knocked out, and the cells still expressed the CD38 protein.

[0098] Performance tests were conducted on Her2-CAR-NK cells with CD38 site-specific integration, their preparation methods, and applications as described in Examples 1-3 and Comparative Examples 1-4. The test items and methods are as follows:

[0099] Gene editing efficiency and CAR expression detection: Flow cytometry was used to detect the expression loss rate of CD38 protein in the cell population to assess gene knockout efficiency, and the expression positivity rate of Her2-CAR was detected. The correctness of CAR gene integration at the CD38 site was analyzed by PCR and sequencing.

[0100] In vitro cell proliferation and persistence test: Cells were continuously cultured in complete medium containing interleukin-2 under antigen-free conditions, and cell growth curves were plotted periodically to evaluate the in vitro expansion capacity and long-term survival capacity of cells.

[0101] In vitro specific killing function test: Using the standard chromium-51 release assay or real-time cell analysis technology, Her2 positive gastric cancer cell lines were used as target cells and Her2 negative cell lines were used as controls. The cells were co-cultured at different effector-target ratios to calculate the specific killing activity of CAR-NK cells on target cells.

[0102] Anti-cannibalism ability and compatibility with targeted drugs: Edited NK cells were co-cultured with unedited NK cells, or cells were treated with anti-CD38 antibodies. The apoptosis rate was detected by flow cytometry to evaluate the effect of CD38 knockout on preventing cell-cell cannibalism and its compatibility with CD38 targeted drugs.

[0103] The test data of Her2-CAR-NK cells with CD38 site-directed integration, their preparation methods, and applications from Examples 1-3 and Comparative Examples 1-4 are recorded in the table below:

[0104] Test Project CD38 gene knockout efficiency Her2-CAR expression positivity rate CAR expression homogeneity In vitro amplification capacity Killing activity against Her2+ gastric cancer cells Anti-fratricide capability Genomic integration safety Example 1 Extremely high high Excellent powerful Excellent Extremely strong Excellent (fixed point) Example 2 Extremely high high Excellent powerful Excellent Extremely strong Excellent (fixed point) Example 3 Extremely high high Excellent powerful Excellent Extremely strong Excellent (fixed point) Comparative Example 1 none medium Difference medium medium none Poor (random) Comparative Example 2 Extremely high Low medium weak weak Extremely strong Excellent (fixed point) Comparative Example 3 Extremely high high Excellent medium medium Extremely strong Excellent (fixed point) Comparative Example 4 none high Excellent powerful Excellent none Excellent (fixed point)

[0105] Comparison and analysis of the data in the table show that the Her2-CAR-NK cells and their preparation methods and applications using the CD38 site-specific integration method in Examples 1-3 are superior to those using the same method in Comparative Examples 1-4. This indicates that by employing a strategy combining the CRISPR / Cas9 editing system with adeno-associated virus serotype 6 donor vector, the Her2-CAR expression cassette is site-specifically integrated into the first exon of the human CD38 gene, achieving CAR gene insertion into the genome. This avoids the oncogene activation and tumorigenic risk caused by random integration with traditional retroviral or lentiviral vectors. Furthermore, this site-specific integration strategy ensures a single, stable copy of the CAR gene, resulting in highly uniform expression of Her2-CAR driven by the endogenous CD38 promoter in the cell population. This improves the safety and batch-to-batch consistency of the prepared CAR-NK cell products. By designing the Her2-CAR expression cassette to replace a portion of the endogenous CD38 gene sequence during homologous recombination, simultaneous CAR integration and... Knockout of the CD38 gene results in Her2-CAR-NK cells that integrate at the CD38 site and do not express CD38 protein. This reduces cannibalism caused by residual CD38-positive cells in the cell product, ensuring purity and yield. Furthermore, this cell product can be used in combination with commonly used CD38-targeting monoclonal antibodies, avoiding antagonism and providing a safe basis for combination therapy. It also enhances the persistence of the cells in vivo. The designed Her2-CAR molecule contains dual co-stimulation of CD28 and 4-1BB. The CAR molecule, optimized for natural killer cell function, provides activation and sustained survival signals when expressed in NK cells by an endogenous promoter, enabling cells to achieve in vitro expansion and in vivo persistence. Combined with the editing method of CRISPR / Cas9 ribonucleoprotein complex electroporation and adeno-associated virus serum type 6 transduction in primary NK cells, the problems of difficult transfection and editing of primary NK cells have been solved. The prepared cells exhibited killing activity against Her2-positive gastric cancer cells and solid tumor target cells, enhancing the therapeutic potential for solid tumors.

[0106] By comparing and analyzing the relevant data in the table, it can be seen that the Her2-CAR-NK cells with CD38 site-specific integration, the preparation method and application of the present invention have superior comprehensive performance.

[0107] In the description of this specification, references to terms such as "an embodiment," "example," and "specific example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0108] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing Her2-CAR-NK cells with CD38 site-specific integration, characterized in that, Includes the following steps: Step 1: Isolation, purification, activation and expansion of primary NK cells. Primary NK cells are isolated and purified from human peripheral blood mononuclear cells and then activated and expanded in culture. Step 2: Construction of a CRISPR / Cas9 editing system targeting the CD38 gene, designing and synthesizing guide ribonucleic acid targeting the first exon of the human CD38 gene, and combining it with Cas9 protein to form a ribonucleoprotein complex; Step 3: Construction of CD38 site homologous recombinant adeno-associated virus serum type 6 donor vector. Centered on the guide ribonuclease site in the first exon of the human CD38 gene, left and right homologous arms were designed, and Her2-CAR expression cassettes were inserted between the homologous arms to construct the donor vector and then perform virus packaging and purification. Step 4: Electroporation of activated primary NK cells and transduction of adeno-associated virus serotype 6. The activated primary NK cells obtained in Step 1 were mixed with the ribonucleoprotein complex prepared in Step 2 and then electroporated. Immediately afterwards, the adeno-associated virus serotype 6 donor virus prepared in Step 3 was added for transduction. Step 5: Expansion and culture of Her2-CAR-NK cells with CD38 site-specific integration. The cells treated in Step 4 are cultured and expanded to obtain Her2-CAR-NK cells with CD38 site-specific integration.

2. The method for preparing Her2-CAR-NK cells with CD38 site-specific integration according to claim 1, characterized in that, In step one, the activation and expansion of primary NK cells includes the following steps: the purified primary NK cells are resuspended in complete culture medium at a density of 0.5 x 10⁶ to 1.5 x 10⁶ cells per milliliter, and feeder cells are added at a ratio of 0.5:1 to 2:1 between primary NK cells and irradiated feeder cells. The cells are then cultured in an incubator at 37 degrees Celsius with a carbon dioxide volume fraction of 5% for 5 to 7 days. The complete culture medium contains basal culture medium, fetal bovine serum at a volume fraction of 5% to 20%, and interleukin-2 at a mass-volume concentration of 100 to 200 units per liter.

3. The method for preparing Her2-CAR-NK cells with CD38 site-specific integration according to claim 1, characterized in that, In step two, the target sequence of the guide ribonucleic acid is 5'-CTGAACTCGCAGTTGGCCAT-3', and the three bases at the 5' end and the three bases at the 3' end of the guide ribonucleic acid are modified by 2'-O-methyl 3' thiophosphorylation. The ribonucleoprotein complex is prepared by mixing the modified guide ribonucleic acid with SpCas9 protein at a molar ratio of 1.5:1 to 2.5:1 and incubating at room temperature for 15 to 25 minutes.

4. The method for preparing Her2-CAR-NK cells with CD38 site-specific integration according to claim 1, characterized in that, In step three, the chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure adapted to natural killer cells. From the N-terminus to the C-terminus, it includes a CD8a signal peptide sequence, a single-chain variable region antibody sequence targeting human epidermal growth factor receptor 2, a CD8a hinge region sequence, a CD8a transmembrane region sequence, a CD28 co-stimulatory domain sequence, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signal transduction domain sequence. The 5' end of the Her2-CAR expression cassette is connected to a P2A self-cleaving peptide encoding sequence, and the entire Her2-CAR expression cassette is under the endogenous promoter regulation of the human CD38 gene.

5. The method for preparing Her2-CAR-NK cells with CD38 site-specific integration according to claim 1, characterized in that, In step three, the left homologous arm is the genome sequence of 600 to 1000 base pairs upstream of the guide ribonucleic acid cleavage site, the right homologous arm is the genome sequence of 600 to 1000 base pairs downstream of the guide ribonucleic acid cleavage site, and the titer of the adeno-associated virus serotype 6 donor virus is not less than 1 x 10^12 viral genome per milliliter.

6. The method for preparing Her2-CAR-NK cells with CD38 site-specific integration according to claim 1, characterized in that, Step four includes the following steps: After washing the activated primary NK cells obtained in step one with buffer, resuspend them and adjust the cell density to 2 x 10⁷ to 8 x 10⁷ cells per milliliter. Take 50 to 150 μL of cell suspension, add the pre-prepared ribonucleoprotein complex, mix, and transfer to an electroporation cuvette for electroporation. The electroporation program is selected from a preset program that can transfect primary lymphocytes. After electroporation, immediately add preheated complete culture medium to 37 degrees Celsius to resuspend the cells in the electroporation cuvette and transfer them to a culture container. After standing for 20 to 40 minutes, add the adeno-associated virus type 6 donor virus prepared in step three according to the multiplicity of infection of the viral genome of 1 x 10⁴ to 5 x 10⁵ cells per cell.

7. The method for preparing Her2-CAR-NK cells with CD38 site-specific integration according to claim 1, characterized in that, In step five, the culture and amplification conditions are as follows: the culture is carried out in an incubator at 37 degrees Celsius with a carbon dioxide volume fraction of 5%, half of the complete culture medium is replaced every 2 to 3 days, and interleukin-2 is supplemented to a final concentration of 300 to 600 IU per milliliter, and the culture is continued for 10 to 16 days.

8. The method for preparing Her2-CAR-NK cells with CD38 site-specific integration according to claim 1, characterized in that, Her2-CAR-NK cells with CD38 site-specific integration obtained by the preparation method have a surface chimeric antigen receptor positivity rate of more than 70% and a CD38 gene knockout efficiency of more than 95%.

9. A CD38 site-specific integration Her2-CAR-NK cell, prepared by any one of the methods for preparing a CD38 site-specific integration Her2-CAR-NK cell according to claims 1 to 8, characterized in that, The first exon site of the human CD38 gene in the genome of the cell integrates the Her2-CAR expression cassette through homologous targeted repair, thereby knocking out the endogenous CD38 gene. The chimeric antigen receptor encoded by the Her2-CAR expression cassette is a third-generation structure containing a single-chain variable region antibody targeting human epidermal growth factor receptor 2, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain, and is expressed by the endogenous CD38 promoter. The chimeric antigen receptor is stably expressed on the cell surface, and the CD38 protein is not expressed.

10. The use of the CD38 site-specific integrated Her2-CAR-NK cells according to claim 9 in the preparation of a drug for treating human epidermal growth factor receptor 2 positive gastric cancer.

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