CAR (chimeric antigen receptor) modified immune cell as well as preparation method and application thereof

Abstract

The invention discloses a CAR expression vector, the CAR expression vector comprises a vector and a CAR accessed to the vector, the CAR comprises an extracellular antigen binding region, a hinge region and an intracellular signal transduction region, the extracellular antigen binding region is a gene sequence of an anti-HER2 single-domain antibody for binding HER2 protein, and the nucleotide sequence of the gene sequence is as shown in SEQ ID NO.2; the hinge region is CD8alpha; and the intracellular signal transduction region is CD28-ICOS-CD3 [zeta]. The invention further discloses a CAR-KN cell, and the CAR-KN cell contains the CAR expression vector. The CAR-NK cell prepared by the preparation method disclosed by the invention shows a remarkable tumor killing capability, and particularly has a killing effect on HER2 positive tumor cells. The CAR-NK cell prepared by the invention has potential to become an effective new strategy for cancer immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a CAR-modified immune cell, a preparation method thereof, and an application thereof. Background Art

[0002] With the development of immunotherapy technology, chimeric antigen receptor T cell (CAR-T) therapy has achieved remarkable results, especially in the treatment of hematological malignancies, showing a strong anti-tumor effect. However, CAR-T therapy still faces many challenges in the treatment of solid tumors, including tumor microenvironment suppression, immune escape mechanisms, and poor persistence of CAR-T cells. To address these issues, researchers have begun to apply CAR technology to other immune cells, such as natural killer (NK) cells, dendritic cells (DCs), etc., in order to overcome the limitations of CAR-T in solid tumors.

[0003] As an immune cell with natural immune activity, NK cells can recognize and kill tumor cells without antigen presentation and are not affected by the problem of T cell immune tolerance. CAR-NK cells are obtained by transducing the CAR gene into NK cells, endowing them with the ability to specifically recognize tumor antigens, which can not only retain the natural immune function of NK cells but also enhance their tumor targeting. Therefore, CAR-modified NK cells have great potential in tumor treatment, especially in the treatment of solid tumors, and may break through the bottleneck of CAR-T therapy.

[0004] The purpose of the present invention is to provide an improved CAR-modified immune cell and a preparation method thereof, which can improve the treatment effect in tumor immunotherapy, especially in solid tumors, showing stronger targeting and sustained immune response. Summary of the Invention

[0005] The purpose of the present invention is to provide an improved CAR-modified immune cell and a preparation method thereof.

[0006] Therefore, on the one hand, the present invention discloses an anti-HER2 single-domain antibody, and the VNAR sequence of the anti-HER2 single-domain antibody is as shown in SEQ ID NO.1.

[0007] On the one hand, the present invention also discloses a CAR expression vector, which includes a vector and a CAR inserted into the vector. The CAR includes an extracellular antigen-binding region, a hinge region, and an intracellular signal transduction region. The extracellular antigen-binding region is the gene sequence of an anti-HER2 single-domain antibody for binding to the HER2 protein, and its nucleotide sequence is as shown in SEQ ID NO.2; the hinge region is CD8α; the intracellular signal transduction region is CD28-ICOS-CD3ζ.

[0008] In one aspect, the present invention also discloses a CAR-KN cell, and the CAR-KN cell contains the CAR expression vector described above.

[0009] In one aspect, the present invention also discloses the use of the anti-HER2 single-domain antibody described above in the preparation of a CAR expression vector.

[0010] In one aspect, the present invention also discloses the use of the CAR expression vector described above in the preparation of CAR-KN cells.

[0011] The CAR-NK cells prepared by the present invention exhibit significant tumor-killing ability, especially in the killing effect on HER2-positive tumor cells. The killing rate of CAR-NK cells in vitro is 72%, while the killing rate of unmodified NK cells is only 30%. The treatment results in a mouse model also show that the CAR-NK cell treatment group significantly inhibits tumor growth, and the tumor volume is much lower than that of the control group and the blank group, indicating that CAR-NK cells have the potential to become an effective cancer immunotherapy strategy. Description of the Drawings

[0012] Figure 1 SDS-PAGE detection results of the anti-HER2 single-domain antibody, where M is the Marker and 1 is the anti-HER2 single-domain antibody. Detailed Embodiments

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0014] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0015] Example 1: Preparation of CAR-NK Cells

[0016] I. Cell Source and Isolation

[0017] 1. Blood collection: 10 mL of peripheral blood was collected from healthy volunteers.

[0018] 2. Isolation of peripheral blood mononuclear cells (PBMC): Add peripheral blood into a centrifuge tube containing 0.2% heparin. Use density gradient centrifugation (Ficoll-Paque, GE Healthcare) to isolate PBMC. The centrifugation conditions are: 400 g, 20 °C, 30 minutes, ensuring clear separation of plasma, monocyte layer, and red blood cell layer.

[0019] 3. NK cell sorting: Use an NK cell sorting kit (Human NK Cell Isolation Kit, Miltenyi Biotec) to sort NK cells by magnetic bead method.

[0020] 4. Quality control standard: Examine the expression of NK cell surface markers CD56 and CD3. Flow cytometry analysis results show that the proportion of CD56+CD3− positive cells > 90%. This indicates that the isolated NK cells are in good condition.

[0021] II. Construction of CAR expression vector

[0022] Use a commercial CAR design platform (such as the CAR construction template provided by Addgene or prepare according to Example 1 of Chinese Invention Patent 201610600015.0) to construct a CAR targeting HER2, and synthesize and clone the CAR gene into a lentiviral vector under laboratory conditions, which is the CAR expression vector. The CAR targeting HER2 mainly includes the following components:

[0023] (1) Single-domain antibody: Select the anti-HER2 single-domain antibody prepared in the present invention (such as prepared in Example 4) as the source of the targeting antibody. According to the known antibody sequence (shown in SEQ ID NO.1), use bioinformatics tools (Benchling or SnapGene) to design the single-domain antibody gene to ensure its high-efficiency expression when cloned into the lentiviral vector. The designed anti-HER2 single-domain antibody gene sequence is shown in SEQ ID NO.2.

[0024] (2) Linker region: Use CD8α. The human CD8α leader chain can be extracted from human CD8α cells or obtained from corresponding suppliers. Primers for the human CD8α leader chain can be provided by corresponding suppliers;

[0025] (3) Signal transduction domain: Use CD28-ICOS-CD3ζ. The sequence and primer sequence of CD28-ICOS-CD3ζ can be found in GenBank of NCBI, or prepared by corresponding suppliers, or designed by oneself.

[0026] III. CAR gene transduction

[0027] 1. Virus packaging: Co-transfect HEK293T cells with the CAR lentiviral vector and the packaging plasmid (pVSV-G). Collect the virus supernatant after 48 hours, concentrate the virus by ultracentrifugation, and store it at -80°C.

[0028] 2. NK cell transduction: Transfer the CAR gene into NK cells using the virus transduction method with optimized transduction conditions (MOI = 5). During transduction, add Polybrene (8 μg / mL) to improve the virus infection efficiency. The electroporation method (Nucleofector, Lonza) can be used to improve the transduction efficiency.

[0029] 3. CAR expression detection: Detect the surface expression of CAR in the transduced cells by flow cytometry, and label them with anti-HER2 single-domain antibody or CAR-specific antibody. Detect the expression level of CAR. The results show that the transduction efficiency can reach more than 80%.

[0030] IV. Amplification and culture

[0031] 1. Culture conditions: Use a specific medium for NK cells (NK Cell Expansion Medium, StemCell Technologies), and add IL-2 (50 IU / mL) and IL-15 (20 ng / mL) to support the proliferation and function of NK cells. Replace the medium every 2 - 3 days to maintain the cell concentration at 1×10 6 cells / mL.

[0032] 2. Cell monitoring: Observe the morphological changes of the cells using an inverted microscope every 2 - 3 days to ensure good cell viability. At the same time, regularly monitor the surface markers of the cells by flow cytometry to confirm the purity of CAR-NK cells and the surface CAR expression rate.

[0033] 3. The detection results show that: The viability of the cultured cells remains above 90%, and the purity of CAR-NK cells reaches above 85%.

[0034] 4. Further expand the above-cultured cells for cryopreservation or use in subsequent experiments.

[0035] In addition, prepare according to Example 1 of Chinese Invention Patent 201610600015.0, replace the anti-HER2 single-domain antibody of the present invention with a commonly used anti-HER2 monoclonal antibody, and conduct subsequent comparative experiments.

[0036] Example 2: Application of CAR-NK cells in cells

[0037] I. Tumor cell killing experiment

[0038] 1. Tumor cell preparation: Use HER2-positive A431 cells as target cells, and label A431 cells with CFSE (Carboxyfluorescein diacetate, succinimidyl ester) (at a concentration of 2 μM and incubated for 30 minutes).

[0039] 2. Co-culture: Co-incubate CAR-NK cells (anti-HER2 single-domain antibody or anti-HER2 monoclonal antibody) with CFSE-labeled A431 cells at a ratio of 1:1 for 48 hours. Use flow cytometry to detect the apoptosis rate of CFSE-positive cells in A431 cells.

[0040] 3. Experimental results: In the co-culture experiment of CAR-NK cells with A431 cells, significant killing effects were demonstrated, and CAR-NK cells prepared with anti-HER2 single-domain antibodies had a higher killing rate. The killing rates of unmodified NK cells and the blank group were significantly lower, verifying the effect of CAR. This shows that the CAR-NK cells prepared with anti-HER2 single-domain antibodies in the present invention have better application effects than CAR-NK cells prepared with existing anti-HER2 monoclonal antibodies. The specific data are shown in Table 1.

[0041] Table 1 Results of tumor cell killing experiment

[0042]

[0043] II. Detection of cytokine release

[0044] 1. Collection of culture supernatant: Collect the culture supernatants of the above two types of CAR-NK cells and unmodified NK cells, and detect the concentrations of cytokines such as IFN-γ and TNF-α respectively.

[0045] 2. ELISA detection: Use a commercial ELISA kit to detect the concentrations of IFN-γ and TNF-α.

[0046] 3. Experimental results: The concentrations of IFN-γ and TNF-α released by CAR-NK cells were significantly higher than those of unmodified NK cells and the blank group, further demonstrating the functional activation effect of CAR-NK cells, and the values of CAR-NK cells prepared with anti-HER2 single-domain antibodies were higher. The above results indicate that the CAR-NK cells prepared with anti-HER2 single-domain antibodies in the present invention have better application effects than CAR-NK cells prepared with existing anti-HER2 monoclonal antibodies. The specific results are shown in Table 2.

[0047] Table 2 Results of cytokine release detection

[0048]

[0049] Example 3: Therapeutic Effect of CAR-NK Cells on Mouse Tumor Model

[0050] 1. Animal Model: Twenty 6-8-week-old BALB / c nude mice were subcutaneously inoculated with HER2-positive A431 tumor cells (5×10 6 cells / mouse).

[0051] 2. Grouping:

[0052] (1) Treatment Group 1 (5 mice): Injected with CAR-NK cells (prepared with anti-HER2 single-domain antibody) (1×10 6 cells / mouse), once a week for 3 consecutive weeks.

[0053] (2) Treatment Group 2 (5 mice): Injected with CAR-NK cells (prepared with anti-HER2 monoclonal antibody) (1×10 6 cells / mouse), once a week for 3 consecutive weeks.

[0054] (3) Control Group (5 mice): Injected with unmodified NK cells (1×10 6 cells / mouse), once a week for 3 consecutive weeks.

[0055] (4) Blank Group (5 mice): Injected with normal saline.

[0056] 3. Procedures:

[0057] (1) Tumor Growth Monitoring: The tumor volume was measured every 3 days (V = (a×b 2 ) / 2, where a is the long diameter of the tumor and b is the short diameter). The body weight and general health status were recorded weekly.

[0058] (2) Endpoint Analysis: At the end of the experiment (the 4th week), the mice were sacrificed, and the tumor tissues were taken for immunohistochemical staining analysis to evaluate the apoptosis of tumor cells.

[0059] 4. Experimental Results: CAR-NK cells significantly inhibited tumor growth. The tumor volume and weight were significantly lower than those of unmodified NK cells and the blank group. At the same time, the tumor apoptosis rate was higher, and the CAR-NK cells prepared with anti-HER2 single-domain antibody had a better effect. The above results indicate that the CAR-NK cells prepared with anti-HER2 single-domain antibody of the present invention have better application effects than the existing CAR-NK cells prepared with anti-HER2 monoclonal antibody. Specifically, as shown in Table 3.

[0060] Table 3 Detection Results of Therapeutic Effects on Mouse Tumor Model

[0061]

[0062] Example 4: Preparation of Anti-HER2 Single-Domain Antibody

[0063] 1. Vaccine Preparation and Immunization

[0064] (1) Mix the recombinant HER2 protein (MCE, Catalog No.: HY-P78680) with complete Freund's adjuvant (FCA) to prepare a vaccine, with each milliliter of the vaccine containing 0.5 mg of HER2 protein.

[0065] (2) Subcutaneously inject the above-prepared vaccine into sharks at a dose of 0.5 mL per fish. Two weeks after the first immunization, immunize the sharks a second time with the vaccine emulsified with recombinant HER2 protein and FIA adjuvant at the same dose and method. Two weeks after the second immunization, perform a third immunization at the same dose and method (the vaccine is the same as that used for the second immunization).

[0066] (3) Seven days after the third immunization, collect the peripheral blood of the sharks and isolate lymphocytes. Then use TRIzol reagent to extract the total RNA of B cells and reverse transcribe it into cDNA according to the kit instructions.

[0067] (4) Amplify the shark single-domain antibody (VNAR) fragment by PCR, with the amplification conditions as follows: pre-denature at 94 °C for 5 minutes, followed by 35 cycles (94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 1 minute), and finally perform an extension at 72 °C for 5 minutes.

[0068] 2. Cloning and Sequencing

[0069] (1) Clone the amplified VNAR fragment into the pGEM-T vector and transform it into DH5α Escherichia coli competent cells.

[0070] (2) After screening for positive clones, extract the plasmid and perform sequencing. Finally, the VNAR sequence of the anti-HER2 single-domain antibody of the present invention was obtained, labeled as SEQ ID NO.1.

[0071] 3. Expression and Purification

[0072] (1) Clone the codon-optimized VNAR sequence of the anti-HER2 single-domain antibody into the pET28a vector and add a 6His tag at its C-terminus for subsequent protein purification.

[0073] (2) Transform the vector into BL21(DE3) Escherichia coli competent cells. When the OD600 reaches 0.6 during cultivation at 37 °C, add 1 mM IPTG for induction and continue to cultivate for 4 to 6 hours, then collect the cells.

[0074] (3) Soluble proteins were extracted using ultrasonic lysis and purified by nickel column affinity chromatography. The purified antibody was dialyzed to remove salts, sterilized through a 220 nm filter membrane, and then stored at -80 °C.

[0075] 4. Purity analysis and ELISA detection

[0076] (1) SDS-PAGE was used to analyze the purity of the purified anti-HER2 single-domain antibody, and the results showed that the purity of the antibody reached over 90%. As Figure 1 shown.

[0077] (2) The anti-HER2 single-domain antibody and the commercial monoclonal antibody (MCE, HY-P9907A) were respectively diluted to 1 μg / mL, and ELISA detection was performed on the recombinant HER2 protein. The results showed that at a 10,000-fold dilution, the OD450nm value of the anti-HER2 single-domain antibody was 1.98, while that of the commercial monoclonal antibody was 1.56, indicating that the anti-HER2 single-domain antibody prepared by the present invention showed better performance in terms of sensitivity and specificity.

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An anti-HER2 single domain antibody, characterized in that The anti-HER2 single-domain antibody VNAR sequence is shown in SEQ ID NO.

1.

2. A CAR expression vector, comprising a vector and a CAR connected to the vector, wherein the CAR comprises an extracellular antigen binding region, a hinge region and an intracellular signal transduction region, characterized in that: The extracellular antigen binding region is the gene sequence of the anti-HER2 single-domain antibody according to claim 1 for binding to HER2 protein, and its nucleotide sequence is shown in SEQ ID NO.2; the hinge region is CD8α; and the intracellular signal transduction region is CD28-ICOS-CD3ζ.

3. A CAR-KN cell, characterized in that: The CAR-KN cells contain the CAR expression vector of claim 2.

4. Use of the anti-HER2 single domain antibody as described in claim 1 in preparing a CAR expression vector.

5. Use of the CAR expression vector as described in claim 2 in preparing CAR-KN cells.

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