Method for inducing CIK cells and application thereof
By using lentiviral packaging to express the fusion protein of CD58 and IL-21 to induce CIK cells in PBMCs, the problems of low efficiency and safety hazards in traditional CIK cell preparation methods are solved, achieving efficient expansion and improved purity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- KAIRUI BIOTECH LTD
- Filing Date
- 2026-01-15
- Publication Date
- 2026-05-29
AI Technical Summary
Existing CIK cell preparation methods are cumbersome, time-consuming, have low expansion efficiency, and low cell viability and tumor killing rate. The use of animal-derived fetal bovine serum poses safety risks, and the cytokines in traditional methods have short half-lives, requiring multiple additions.
The fusion protein of CD58 and IL-21 was expressed by lentivirus packaging. CIK cells were induced in PBMCs by lentivirus particles. The membrane-expressed IL-21 protein was used to prolong the half-life, reduce the number of cytokine additions, and improve the amplification efficiency.
This study achieved efficient expansion of CIK cells, with a purity of 78% and an expansion fold of 160-800 times, reducing induction costs and providing a theoretical basis for CIK cell research.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for inducing CIK cells and its application. Background Technology
[0002] CIK cells (cytokine-induced killer cells) are a novel type of immune-active cell, as these cells simultaneously express CD3. + and CD56 + These two types of membrane protein molecules, hence they are also known as NK cell-like T lymphocytes, combining the potent antitumor activity of T lymphocytes with the non-MHC-restricted tumor-killing advantages of NK cells. Therefore, the application of CIK cells is considered the preferred option for next-generation adoptive cell immunotherapy for tumors.
[0003] However, the effector cells CD3 in CIK cells + and CD56 + CIK cells are rare in normal human peripheral blood PBMCs, accounting for only 1%-5%. Traditional CIK cell preparation methods are cumbersome, time-consuming, and involve the addition of multiple cytokines, such as IFN-γ, CD3 antibody, and interleukin-12. These cytokines or proteins need to be added multiple times during CIK cell induction and expansion because protein factors have short half-lives in solution and are easily degraded. CIK cell expansion is generally carried out after 3-4 weeks of culture, and the final expansion efficiency is usually around 100-fold on average. Existing induction techniques generally have disadvantages such as insignificant expansion effects, low cell viability in the later stages of growth, low killing rate against tumor cells, and certain safety risks associated with the use of animal-derived fetal bovine serum. Summary of the Invention
[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a fusion protein, wherein lentiviruses expressing the fusion protein can induce mononuclear cells to expand into CIK cells.
[0005] This invention also proposes a nucleic acid molecule.
[0006] This invention also proposes a biomaterial.
[0007] The present invention also proposes a reagent kit.
[0008] This invention also proposes a method for inducing CIK cells.
[0009] The present invention also proposes an application.
[0010] According to a first aspect of the invention, a fusion protein is provided, which comprises, from the N-terminus to the C-terminus, a CD58 protein, a linker, and an IL-21 protein.
[0011] In some embodiments of the present invention, the amino acid sequence of the CD58 protein includes: a) An amino acid sequence as shown in SEQ ID NO:4; or, b) An amino acid sequence that has more than 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:4 and has the function of the amino acid sequence defined in a).
[0012] In some embodiments of the present invention, the amino acid sequence of the IL-21 protein includes: c) The amino acid sequence as shown in SEQ ID NO:6; or, d) An amino acid sequence that has more than 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:6 and has the function of the amino acid sequence defined in c).
[0013] In some embodiments of the present invention, the amino acid sequence of the linker includes the sequence shown in SEQ ID NO:5.
[0014] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO:2.
[0015] The fusion protein is obtained by fusing CD58 protein with IL-21 (with its secretion signal peptide removed) via a linker. Since CD58 is a membrane protein, linking IL-21 (with its secretion signal peptide removed) to the cell membrane via a linker can fix the secreted cytokine IL-21 onto the cell membrane for continuous function.
[0016] According to a second aspect of the invention, a nucleic acid molecule is provided that encodes a fusion protein as described in the first aspect of the invention.
[0017] According to a third aspect of the present invention, a biomaterial is provided, said biomaterial comprising at least one of (1) to (11): (1) An expression cassette comprising a nucleic acid molecule as described in the second aspect of the present invention; (2) A carrier comprising a nucleic acid molecule as described in the second aspect of the present invention; (3) A carrier comprising the expression box described in (1); (4) Transgenic cells comprising nucleic acid molecules as described in the second aspect of the present invention; (5) Transgenic cells containing the expression cassette described in (1); (6) Transgenic cells containing the vector described in (2); (7) Transgenic cells containing the vector described in (3); (8) Lentiviral particles comprising nucleic acid molecules as described in the second aspect of the present invention; (9) Lentiviral particles containing the expression cassette described in (1); (10) Lentiviral particles containing the vector described in (2); (11) Lentiviral particles containing the vector described in (3).
[0018] In some embodiments of the present invention, the transgenic cells do not contain reproductive material.
[0019] In some embodiments of the present invention, the transgenic cells are selected from eukaryotic cells or prokaryotic cells.
[0020] In some embodiments of the present invention, the eukaryotic cells are selected from mouse cells or human cells.
[0021] According to a fourth aspect of the present invention, a kit is provided comprising at least one of the fusion protein as described in the first aspect of the present invention, the fusion protein encoded by the nucleic acid molecule as described in the second aspect of the present invention, and the biomaterial as described in the third aspect of the present invention.
[0022] According to a fifth aspect of the present invention, a method for inducing CIK cells is provided, the preparation method comprising the following steps: Lentiviral particles containing the fusion protein described in the first aspect of the present invention, IL-2, and a cell suspension of mononuclear cells were mixed and cultured. On days 6 to 8, lentiviral particles containing the fusion protein described in the first aspect of the present invention were added again, and the cells were cultured for another 4 to 6 days to induce CIK cells.
[0023] In some embodiments of the present invention, the titer of the lentiviral particles in the culture system is 5 × 10⁻⁶. 5 ~6×10 6 TU / mL.
[0024] In some embodiments of the present invention, the concentration of IL-2 in the culture system is 800~1200 IU.
[0025] In some embodiments of the present invention, the culture medium used for the cell suspension includes GT-T551-H3 lymphocyte serum-free culture medium.
[0026] In some embodiments of the present invention, the fusion protein is expressed on the membrane surface of the lentiviral particle.
[0027] In some embodiments of the present invention, the cell concentration of the cell suspension at the time of inoculation is 0.8 × 10⁻⁶. 6 ~1.2×10 6 per mL.
[0028] The present invention has at least the following beneficial effects: The CIK cell induction method proposed in this invention innovatively packages and constructs lentiviruses that can express human CD58 and human IL-21 on the membrane surface. The half-lives of the expressed factors are longer. Lentiviral virus is added once on day 1 and once on day 7 of PBMC culture for induction stimulation, and CIK cells (CD3+) can be effectively induced and expanded on day 12. + and CD56 + The purity can reach 78%, and the amplification fold can reach 160-800 times. Compared with existing methods, it greatly improves the induction efficiency and reduces the induction cost, providing a theoretical basis and technical support for in vivo and in vitro research of CIK cells. Attached Figure Description
[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the structure of the PCDH-CD58-IL21 carrier in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the structure of the PCDH-CD58-IL21 carrier in Comparative Example 1 of the present invention; Figure 3 This is a microscopic image of the induction result on the seventh day in the experimental example of this invention; the magnification is 40x. Figure 4 This is a microscopic image of the induction result on the twelfth day in the experimental example of this invention; the magnification is 40x. Figure 5 This is a statistical curve of the total number of live cells in the experimental examples of this invention; Figure 6 This is a graph showing the detection results of flow cytometry in the experimental examples of this invention. Detailed Implementation
[0030] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.
[0031] The source information of the reagents and materials used in the following examples is shown in Table 1.
[0032] Table 1. Information on the source of reagents and materials
[0033] Unless otherwise specified, the instruments and reagents used in the following examples are all commercially available instruments and reagents obtained through conventional purchasing channels.
[0034] Example 1 This embodiment provides a method for inducing CIK cells, the specific steps of which are as follows: 1. Lentiviral packaging: 1) Adjust the density of HEK293T suspension acclimatized cells to 4 × 10⁶ cells using KOP293 medium. 6 Cells / mL, cell viability ≥95%, prepare 100 mL of cell suspension in an Erlenmeyer flask for transfection.
[0035] 2) Prepare solutions A and B according to the formula shown in Table 2, mix them thoroughly, and let them stand for 5 min. Add solution B to solution A, shake to mix, and let stand at room temperature for 15 min to obtain the mixed transfection solution. The structural diagram of the PCDH-CD58-IL21 vector is shown below. Figure 1 As shown, the target fragment is a fusion gene obtained by fusing the human CD58 gene with the IL-21 gene (with its secretory signal peptide removed) via a linker. The nucleic acid sequence and fusion protein sequence information of this fusion gene are as follows: Amino acid sequence of CD58-linker-IL-21 fusion protein: mvagsdagralgvlsvvcllhcfgfiscfsqqiygvvygnvtfhvpsnvplkevlwkkqkdkvaelensefrafssfknrvy ldtvsgsltiynltssdedeyemespnitdtmkfflyvleslpsptltcaltngsievqcmipehynshrglimyswdcpmeqckrnstsiyfkmendlpqkiqctlsnp lfnttssiilttcipssghsrhryalipiplavittcivlymngilkcdrkpdrtnsnGSSGGSGGGGSGGGGSGGGGSSGQGQDRHMIRMRQLIDIVDQLKNYVNDLVP EFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQID NO:2); The amino acid sequence of the CD58 membrane localization signal peptide is: MVAGSDAGRALGVLSVVCLLHCFGFISC (SEQ ID NO:3). The amino acid sequence of CD58 with the membrane localization signal peptide is: mvagsdagralgvlsvvcllhcfgfiscfsqqiygvvygnvtfhvpsnvplkevlwkkqkdkvaelensefrafssfknrvyldtvsgsltiynltssdedeyemespnitdtmkfflyvleslpsptltcaltngsievqcmipehynshrglimyswdcpmeqckrnstsiyfkmendlpqkiqctlsnplfnttssiilttcipssghsrhryalipiplavittcivlymngilkcdrkpdrtnsn (SEQ ID NO:4); The amino acid sequence of the linker is: GSSGGSGGGGSGGGGGSGGGGSSG (SEQ ID NO:5); The amino acid sequence of IL-21 with the secretion signal peptide removed: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO:6).
[0036] Table 2 Transfection system formulation
[0037] 3) Using a pipette, take about 11 mL of the mixed transfection solution obtained in step 2) and add it dropwise onto the surface of the cell suspension prepared in step 1). Shake well while adding the solution, and then place it in a cell culture shaker at 37°C and 5% CO2 for constant temperature shaking at 120 rpm. Start timing from the completion of transfection and culture for 72 h. After 72 h, collect the culture system and centrifuge at 4°C and 5000 rpm for 30 min to remove cells and cell debris. Harvest the virus supernatant and use a 0.45 μm filter membrane for vacuum filtration to further remove cell debris. At this point, the CD58-M21 virus stock solution harvest is complete.
[0038] 2. Virus concentration: 1) Using a 100 kDa ultrafiltration tube (Jetbio, catalog number FTT500500), 50 mL of viral stock solution was ultrafiltered and concentrated to a volume of less than 1 mL. Each time, 15 mL of viral stock solution was added to the ultrafiltration tube, and centrifuged at 4℃, 4000 g for 15 min at an ascent speed of 9 and a descent speed of 9. This centrifugation was repeated approximately 3 times until all viral stock solution was filtered. The final centrifugation for 30 min yielded concentrated virus. The physical titer of the concentrated virus was detected using a lentivirus P24 antigen detection ELISA kit, and the titer was 1.18 × 10⁻⁶. 7 -1.18×10 8 Between TU / mL.
[0039] 2) Resuspend the above concentrated virus using DPBS, adjust the concentrated virus volume to 1 ml, and then sterilize and dispense it using a 0.22 μm filter membrane to make 50 μL / tube virus working solution, and store at -80℃ for later use.
[0040] 3. Cell induction: Resuscitate PBMCs and count them, then resuspend them in GT-T551-H3 serum-free lymphocyte medium to a concentration of 1×10⁻⁶. 6Cells were seeded at a density of 1 / mL into 24-well plates, with 1 mL of cell suspension in each well. Then, 100 μL of recombinant IL-2 (the final concentration of IL-2 in the culture system was 1000 IU) and 50 μL of the viral working solution obtained in step 2 were added to each well. The mixture was then incubated at 37°C, 5% CO2, and 70% humidity for 7 days. Another 50 μL of the viral working solution obtained in step 2 was added to each well, and the cells were incubated for 12 days to induce CIK cells.
[0041] Comparative Example 1 This comparative example provides a method for inducing CIK cells. The only difference between this method and Example 1 is that the PCDH-CD58-IL21 vector in solution A of the transfection system in step 1 is replaced with an equal amount of the PCDH-4BL-M21-M15 vector. All other steps are the same as in Example 1. The target fragment of the PCDH-4BL-M21-M15 vector is a fusion gene constructed from the human 4-1BB-L gene via a linker with IL-21 and IL-15 (with the secretory signal peptides removed). A schematic diagram of the structure of this vector is shown below. Figure 2 As shown.
[0042] The amino acid sequence of the fusion protein 4BL-M21-M15 expressed by the PCDH-4BL-M21-M15 vector is: MVAGSDAGRALGVLSVVCLLHCFGFISCEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGSSGGSGGGGSGGGGSGGGGSSGQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGSSGGSGGGGSGGGGSGGGGSSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSHHHHHH (seq ID no:8); Amino acid sequence of 4BL: EYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE(seq ID no:9); Amino acid sequence of M15: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVSGGSGGGGSGGGSGGGGSLQNWVN VISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (seq ID no:10); Amino acid sequence of M21: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (seq ID no: 11).
[0043] Comparative Example 2 This comparative example provides a method for inducing CIK cells. The only difference between this method and Example 1 is that the second viral inoculation on day 7 in step 3 is omitted. All other steps are consistent with Example 1.
[0044] Test case This experiment examined the cells induced by the induction methods provided in Example 1, Comparative Example 1, and Comparative Example 2. The specific experimental methods and results are as follows: 1. Observe cell morphology under a microscope: In Example 1 and Comparative Examples 1 and 2, cell activation and expansion were observed under a microscope on day 7 (before secondary virus inoculation) and day 12. The results are as follows: Figure 3 and Figure 4 As shown.
[0045] Depend on Figure 3 and Figure 4 It can be seen that, compared with Comparative Example 1, the cells in Example 1 were well activated and expanded, with many clusters of activated cell groups.
[0046] 2. Total number of viable cells: Viable cell counts were performed on days 3, 5, 7, 10, and 12 during the induction process of Examples 1, 1, and 2, using a Beckman Coulter cell technique (trypan blue staining). The results are shown below. Figure 5 As shown in the figure. In the figure, Example 1-1 and Example 1-2 represent two repeated experiments, and Comparative Example 1-1 and Comparative Example 1-2 represent two repeated experiments.
[0047] Depend on Figure 5 As can be seen, during the entire cell culture period in Example 1, the cells were in an amplified state and grew well, with the total amplification reaching 2.4 × 10⁻⁶ on day 12. 7 Each cell.
[0048] 3. Flow cytometry detection: 1) After the induction process in Example 1 and Comparative Example 2, 5 × 10⁻⁶ samples were taken from each sample. 5 Add 1 mL of PBS buffer to each cell, mix well, centrifuge at 1500 rpm for 5 minutes, wash once, and remove the supernatant; 2) Add CD3 and CD56 fluorescently labeled flow cytometry antibodies according to the recommended dosage in the instructions, mix well, and incubate at 4°C in the dark for 30 minutes.
[0049] 3) Add 1 mL of PBS buffer and mix well. Centrifuge at 1500 rpm for 5 minutes and remove the supernatant. Repeat the operation 3 times.
[0050] 4) Resuspend the cells in 200 μL of PBS buffer, and perform flow cytometry analysis. The results are as follows: Figure 6 As shown in the figure. Among them, the isotype control is the result of staining with isotype control antibodies (purchased from eLabscience, catalog numbers E-AB-F09802C and E-AB-F09792D). Since isotype control antibodies generally do not bind to cells, they can be used as a negative control reference.
[0051] Depend on Figure 6 It can be seen that, compared with Comparative Example 2, which was treated with the virus only once, Example 1 was able to amplify more CD3+ on day 12 of induction.+ CD56 + The CIK cells had a purity of 78.21%.
[0052] The induction effect of the induction method provided in Comparative Example 1 is not as good as that in Example 1. This may be because the vector provided in Comparative Example 1 has certain design flaws. The 4BL-M21-M15 vector is designed for tandem expression. On the one hand, the sequence is too long, leading to a decrease in viral packaging titer and affecting the induction effect; on the other hand, tandem expression uses two linkers, which may affect protein folding and obscure functional active regions, resulting in reduced activation. The PCDH-CD58-IL21 vector provided in this invention does not have these problems.
[0053] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.
Claims
1. A fusion protein, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, CD58 protein, a linker, and IL-21 protein.
2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the CD58 protein includes: a) An amino acid sequence as shown in SEQ ID NO:4; or, b) An amino acid sequence that has more than 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:4 and has the function of the amino acid sequence defined in a).
3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the IL-21 protein includes: c) The amino acid sequence as shown in SEQ ID NO:6; or, d) An amino acid sequence that has more than 80% sequence similarity to the amino acid sequence shown in SEQ ID NO:6 and has the function of the amino acid sequence defined in c).
4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein as described in any one of claims 1 to 3.
5. A biomaterial, characterized in that, The biomaterial includes at least one of (1) to (11): (1) An expression cassette comprising the nucleic acid molecule as described in claim 4; (2) A carrier comprising the nucleic acid molecule as described in claim 4; (3) A carrier comprising the expression box described in (1); (4) Transgenic cells comprising the nucleic acid molecules as described in claim 4; (5) Transgenic cells containing the expression cassette described in (1); (6) Transgenic cells containing the vector described in (2); (7) Transgenic cells containing the vector described in (3); (8) Lentiviral particles comprising the nucleic acid molecules as described in claim 4; (9) Lentiviral particles containing the expression cassette described in (1); (10) Lentiviral particles containing the vector described in (2); (11) Lentiviral particles containing the vector described in (3).
6. A reagent kit, characterized in that, The kit comprises at least one of the fusion protein as described in any one of claims 1 to 3, the fusion protein encoded by the nucleic acid molecule as described in claim 4, and the biomaterial as described in claim 5.
7. A method for inducing CIK cells, characterized in that, The preparation method includes the following steps: Lentiviral particles containing the fusion protein of any one of claims 1 to 3, IL-2, and mononuclear cell suspensions are mixed and cultured. On days 6 to 8, lentiviral particles containing the fusion protein of any one of claims 1 to 3 are added again, and CIK cells are induced by continuing culture for 4 to 6 days.
8. The induction method according to claim 7, characterized in that, The titer of the lentivirus particles in the culture system was 5 × 10⁻⁶. 5 ~6×10 6 TU / mL.
9. The induction method according to claim 7, characterized in that, The concentration of IL-2 in the culture system is 800~1200 IU.
10. The use of any one of the fusion protein according to any one of claims 1 to 3, the fusion protein encoded by the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, and the kit according to claim 6 in induced CIK cells.