Chimeric antigen receptor NK cell capable of simultaneously recognizing EGFR and CD133 double antigens as well as preparation method and application of chimeric antigen receptor NK cell
By designing dual antigen recognition CAR-NK cells to target EGFR and CD133, the lack of specific antigen and safety problems in solid tumor treatment has been solved, achieving more efficient and safer tumor immunotherapy effects.
Patent Information
- Application Number
- CN202411713865.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-28
AI Technical Summary
Existing CAR-T cell therapy faces great challenges in solid tumor treatment, including the lack of specific antigen targets, potential safety risks and side effects such as cytokine release syndrome (CRS), limiting its application in solid tumor treatment.
A dual-antigen recognition CAR-NK cell was designed, and two CAR molecules were connected through P2A self-shearing peptides, targeting EGFR and CD133 respectively. The receptor expression was controlled by the EF1α promoter, and safer NK cells were selected as vectors to prepare dual-target CAR-NK cells.
The dual-target CAR-NK cells were activated while simultaneously recognizing EGFR and CD133, avoiding attacks on normal tissue cells that low-express a single target, significantly improving safety and therapeutic efficacy.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation of EGFR and CD133 bispecific CAR and CAR-NK cells and their application in solid tumors. Background Art
[0002] Chimeric Antigen Receptor-T cell (CAR-T) therapy is a new method of tumor immunotherapy that has developed fastest in recent years. The core of CAR-T technology is to artificially express a chimeric receptor targeting a specific antigen on T cells through genetic engineering technology. The chimeric receptor is composed of a fusion of a specific antigen recognition domain on the cell surface (such as a single-chain fragment antibody scFv) and an intracellular T cell activation signal domain, which gives T cells the ability to specifically recognize and kill tumor cells. Clinical studies have confirmed that CAR-T cells can proliferate, survive for a long time and form immune memory in vivo, thereby obtaining a specific long-term anti-tumor mechanism. CAR-T therapy has a significant therapeutic effect in the treatment of acute leukemia and non-Hodgkin's lymphoma, and has shown good targeting, lethality and persistence in vitro and in clinical trials. Currently, a number of CAR-T products have been approved for marketing, which has completely changed the treatment pattern of hematological tumors. Despite this, CAR-T faces huge challenges in the treatment of solid tumors and has developed slowly. Unlike hematological tumors, solid tumors lack known tumor-specific antigens. At present, tumor-associated antigens are usually used in solid tumors, that is, antigens that are highly expressed on tumor cells and also lowly expressed in some normal tissue cells. CAR cells targeting a single antigen may attack normal tissues expressing the antigen at the same time after reinfusion, resulting in an "on-target / off-tumor" effect. As a living cell drug, the potential safety issues of CAR-T technology cannot be ignored. A typical case is that Morgan et al. reported that a patient with colorectal cancer metastasis was treated with third-generation CAR-T cells designed for human epidermal growth factor receptor II (Her2). The patient died 4 days after intravenous injection of CAR-T cells; an autopsy found that the death was caused by low-level expression of Her2 on the surface of lung epithelial cells, and the reinfused CAR-T cells attacked the patient's lungs, causing pulmonary toxicity. In addition, this potential safety risk also makes it impossible to improve the efficacy by increasing the dose, limiting the application of CAR cells in the treatment of solid tumors. Therefore, there is an urgent need to develop more efficient and safer CAR cell products or technologies in clinical practice.
[0003] Cytokine release syndrome (CRS) is a systemic immune storm caused by the massive activation and dissolution of immune cells in the body during CAR-T injection and pretreatment. CRS can cause a significant increase in cytokine levels in the patient's blood, and may cause fever, headache, nausea, diarrhea, tachycardia, hypotension, hypoxia, epileptic seizures, mental confusion and other symptoms. In severe cases, it can cause multiple organ failure and death. CRS is currently the most common side effect of CAR-T cell therapy and a sign of CAR-T's effectiveness in the body. Therefore, compared with CAR-T, CAR-NK cell therapy has more advantages in tumor immunotherapy. It has been confirmed in clinical trials that CAR-NK does not cause CRS or neurotoxicity. Since CAR-NK cells do not need to match their own leukocyte antigens with tumors when they recognize tumor cells as effector cells, CAR-NK cells will not induce graft-versus-host reactions. In addition, CAR-NK cells have a variety of tumor killing mechanisms. In addition to CAR-mediated targeted killing, they can also kill tumor cells through NK cell receptor-dependent pathways and ADCC, thereby improving the effect of immunotherapy. In addition, the sources of CAR-NK cells are also wider, and they can be produced from peripheral blood (PB), umbilical cord blood (UCB), human embryonic stem cells (HESC), induced pluripotent stem cells (IPSC) and NK-92 cell lines. Therefore, CAR-NK cell immunotherapy has better safety than CAR-T cell immunotherapy and has broader application prospects in the treatment of solid tumors. Summary of the invention
[0004] In view of the above-mentioned prior art problems, the purpose of the present invention is to design and provide a dual-antigen recognition CAR-NK cell, preparation method and application thereof that simultaneously targets EGFR and CD133 for solid tumors. The present invention provides a design scheme of an "AND gate" dual-target CAR, in which the dual-antigen recognition chimeric receptor is composed of two CAR molecules that target EGFR and CD133 respectively, and the two CAR molecules are connected by a P2A self-cleavage peptide. The receptor expression is controlled by the EF1α promoter, and safer NK cells that can be allogeneically reinfused are selected as carriers to prepare dual-target CAR-NK cells. The dual-target CAR-NK cells constructed based on this strategy have better safety than traditional single-target CAR-T or CAR-NK cells.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] On the one hand, the present invention provides a chimeric antigen receptor that can simultaneously recognize EGFR and CD133 dual antigens, wherein the structure of the chimeric antigen receptor is CD8α signal peptide-anti EGFR scFv-tag-CD28 Hinge-CD28 TM-CD28co-stm-P2A cleavage peptide-CD8α signal peptide-tag-anti CD133 scFv-CD8αHinge-CD8αTM / T-CD3ζ.
[0007] The chimeric antigen receptor that simultaneously recognizes EGFR and CD133 dual antigens, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.19;
[0008] The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.20;
[0009] The amino acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.21;
[0010] The amino acid sequence of the CD28 Hinge is shown in SEQ ID NO.22;
[0011] The amino acid sequence of the CD28 TM is shown in SEQ ID NO.23;
[0012] The amino acid sequence of the CD28 co-stm is shown in SEQ ID NO.24;
[0013] The amino acid sequence of the P2A cleavage peptide is shown in SEQ ID NO.25;
[0014] The amino acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.26;
[0015] The amino acid sequence of the CD8αHinge is shown in SEQ ID NO.27;
[0016] The amino acid sequence of the CD8αTM is shown in SEQ ID NO.28;
[0017] The amino acid sequence of CD3ζ is shown in SEQ ID NO.29.
[0018] In a second aspect, the present invention provides a nucleic acid encoding the chimeric antigen receptor.
[0019] The nucleic acid, the sequence of the nucleic acid is shown in SEQ ID NO.3 or its degenerate sequence.
[0020] In a third aspect, the present invention provides a vector comprising the nucleic acid.
[0021] The vector is a lentivirus containing the nucleic acid.
[0022] In a fourth aspect, the present invention provides a CAR-NK cell that can simultaneously recognize EGFR and CD133 dual antigens, wherein the CAR-NK cell expresses the chimeric antigen receptor that can simultaneously recognize EGFR and CD133 dual antigens as described in claim 1 or 2.
[0023] The CAR-NK cell that can simultaneously recognize EGFR and CD133 dual antigens is obtained by transfecting HEK293T cells with the vector.
[0024] In a fifth aspect, the present invention provides the use of the CAR-NK cells that can simultaneously recognize dual antigens of EGFR and CD133 in the preparation of drugs for treating solid tumor diseases.
[0025] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the CAR-NK cells that can simultaneously recognize dual antigens of EGFR and CD133.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] The present invention provides a method for improving the specificity of NK or T cells to recognize tumor cells by targeting dual-antigen chimeric receptors. The dual-antigen recognition CAR-NK cells prepared based on the present invention can only be activated and play a role in killing tumor cells when they recognize both EGFR and CD133 antigens at the same time, and have no reactivity to normal tissue cells that low-express a single target, thus avoiding damage to normal tissues caused by off-target effects and having higher safety. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 It is a schematic diagram of the CAR structure;
[0029] Figure 2 This is a diagram of the CAR structure model;
[0030] Figure 3 Schematic diagram of antigen overexpression plasmid;
[0031] Figure 4 This is the result diagram of CAR expression on NK92MI cells;
[0032] Figure 5 The result diagram of antigen expression on target cells;
[0033] Figure 6This is a diagram showing the killing effect of CAR-NK92MI on target cells expressing different antigens. DETAILED DESCRIPTION
[0034] The present invention is described in detail below by specific examples, which are only used to illustrate the present invention and are not used to limit the scope of the present invention. The test methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used are all commercially available products.
[0035] Embodiment 1:
[0036] 1. Plasmid structure design and construction
[0037] according to Figure 1 The structure shown in the figure inserts the corresponding nucleic acid sequence into the lentiviral packaging backbone plasmid pWPXLd to construct a CAR expression plasmid named EGFR-28z (nucleic acid sequence as shown in SEQ ID NO.1), CD133-28z (nucleic acid sequence as shown in SEQ ID NO.2), and antiEGFR-CD133-dualCAR (abbreviated as dualCAR, nucleic acid sequence as shown in SEQ ID NO.3). Among them, the nucleic acid sequence of CD8αSP (or CD8α signal peptide) is shown in SEQ ID NO.4; the nucleic acid sequence of anti-EGFR scFv is shown in SEQ ID NO.5; the nucleic acid sequence of Myc tag is shown in SEQ ID NO.6; the nucleic acid sequence of CD28 Hinge (or CD28 hinge region) is shown in SEQ ID NO.7; the nucleic acid sequence of CD28 TM (or CD28 transmembrane domain) is shown in SEQ ID NO.8; the nucleic acid sequence of CD28 co-stm (or CD28 co-stimulatory domain) is shown in SEQ ID NO.9; the nucleic acid sequence of P2A cleavage peptide is shown in SEQ ID NO.10; the nucleic acid sequence of Flag tag is shown in SEQ ID NO.11; the nucleic acid sequence of antiCD133 scFv is shown in SEQ ID NO.12; the nucleic acid sequence of CD8αHinge (or CD8α hinge region) is shown in SEQ ID NO.13; the nucleic acid sequence of CD8αTM (or CD8α transmembrane domain) is shown in SEQ ID The nucleic acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO.14; the nucleic acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO.15. Figure 2Shown are structural schemata of EGFR-28z, CD133-28z and dualCAR, showing the structural schemata of the CAR molecules shown in the present invention expressed on NK cells. The antigen recognition domains anti-EGFR scFv and anti-CD133 scFv are both expressed on the surface of NK cells, while the CD28 co-stimulatory domain and CD3ζ signaling domain are expressed in the NK cell membrane. The extracellular antigen recognition domain is connected to the intracellular signaling domain and co-stimulatory domain through the two transmembrane domains CD28 TM and CD8 TM.
[0038] according to Figure 3 The structure shown inserts the CDS sequences of human EGFR (nucleic acid sequence as shown in SEQ ID NO.16) and CD133 (nucleic acid sequence as shown in SEQ ID NO.17) into the lentiviral packaging backbone plasmid pLV3 to construct antigen expression plasmids named pLV3-EGFR and pLV3-CD133.
[0039] The gRNA coding sequence targeting human EGFR (nucleic acid sequence as shown in SEQ ID NO.18) was inserted into the CRISPR-Cas9 plasmid to construct a gene knockout plasmid named EGFR-KO.
[0040] 2. Lentivirus packaging and concentration
[0041] 1) HEK293T cells were seeded in 10 cm 2 Incubate the cells in a CO2 incubator overnight in a cell culture dish to allow the cell confluence to reach 70%-90% on the next day;
[0042] 2) Liposome transfection: Add the target plasmid (10 μg), lentiviral packaging plasmid psPAX2 (7.5 μg), and envelope plasmid pMD2.G (2.5 μg) to 1 mL of opti-MEM medium at a ratio of 4:3:1, then add 60 μL of PEI transfection reagent, mix well, let stand at room temperature for 15 min, slowly add to HEK293T cells, gently shake the culture dish to mix, place in a 37°C incubator, and replace with fresh complete medium after 6 hours of culture;
[0043] 3) After 48 hours, the culture medium in the culture dish was collected and filtered through a 0.45 μm filter to obtain the lentiviral supernatant;
[0044] 4) Add 2 mL of 5×PEG8000 to every 8 mL of viral supernatant and shake at 4°C overnight;
[0045] 5) Centrifuge at 4000 g for 30 min at 4°C, remove the supernatant completely, and resuspend the virus precipitate in 100 μL PBS to obtain the concentrated lentivirus;
[0046] 3. Preparation of CAR-NK cells
[0047] 2 × 10 5 For NK92MI cells, add concentrated EGFR-28z, CD133-28z and dualCAR lentivirus directly into the wells, add polybrene with a final concentration of 8 μg / mL to enhance infection, replace with fresh culture medium the next day, and detect the CAR positivity rate after another two days.
[0048] 4. Target cell construction
[0049] The expression of EGFR and CD133 in wild-type MCF-7 and MDA-MB-231 is as follows: EGFRdimCD133- and MDA-MB-231 EGFR+CD133- , you need to build MCF-7 EGFR+CD133- 、MCF-7 EGFR+CD133+ , and MDA-MB-231 EGFR-CD133+ Target cells. Add 2×10 5 After MCF-7 or MDA-MB-231 cells adhere overnight, add 1 mL of unconcentrated pLV3-EGFR, pLV3-CD133 or EGFR-KO lentiviral supernatant according to the antigen phenotype to be constructed, add polybrene with a final concentration of 10 μg / mL to enhance infection, replace fresh culture medium the next day, and detect the expression of EGFR and CD133 after another two days.
[0050] 5. Detection of CAR, EGFR and CD133 expression by flow cytometry
[0051] 1) Collect infected CAR-NK92MI, MCF-7 and MDA-MB-231 cells in 1.5 mL centrifuge tubes, and collect wild-type cells as negative controls, centrifuge at 4°C, 200 g for 3 min, and remove the culture medium;
[0052] 2) Resuspend the cell pellet in 1 mL of pre-cooled PBS, centrifuge at 200 g for 3 min at 4°C, and remove the supernatant;
[0053] 3) For CAR detection, add 100 μL of diluted anti-Myc or anti-Flag antibody working solution to each sample; for target antigen detection, add 100 μL of diluted anti-EGFR or anti-CD133 antibody working solution to each sample, protect from light, and incubate at 4°C for 20 min;
[0054] 4) Add 1 mL of pre-cooled PBS, centrifuge at 4°C, 200 g for 3 min, remove the supernatant, and repeat once;
[0055] 5) Add 200 μL PBS and resuspend, then perform flow cytometry analysis to analyze the expression of CAR and target antigen.
[0056] 6. Flow cytometry sorting of CAR-NK or target cells
[0057] 1) Collect approximately 5 × 10 6 The infected CAR-NK92MI, MCF-7 and MDA-MB-231 cells were centrifuged at 200 g for 3 min at 4°C, and the culture medium was removed.
[0058] 2) Resuspend the cell pellet in 5 mL of pre-cooled PBS, centrifuge at 200 g for 3 min, and remove the supernatant;
[0059] 3) For CAR-NK92MI sorting, add 500 μL of diluted anti-Myc or anti-Flag antibody working solution to each sample; for target cell sorting, add 500 μL of diluted anti-EGFR or anti-CD133 antibody working solution to each sample and incubate at 4°C for 20 min in a dark environment;
[0060] 4) Add 5 mL of pre-cooled PBS to resuspend, centrifuge at 200 g for 3 min, remove the supernatant, and repeat once;
[0061] 5) Resuspend in 500 μL complete medium and transfer to a 15 mL centrifuge tube and place on ice away from light;
[0062] 6) In the flow cytometer, determine the gate of positive cells based on the negative control and sort out the target cells;
[0063] 7) The sorted cells were centrifuged at 300g for 5 min, resuspended in complete culture medium containing 3% cyan / chain dual antibody, and cultured in a CO2 incubator to obtain completely positive CAR-NK92MI and target cells expressing different antigens. Figure 4 As shown in Figure 2, it can be seen that the CAR-NK92MI cells after sorting are all CAR-positive cells. The expression of EGFR and CD133 on the target cells after sorting is shown in Figure 2 Figure 5 As shown, it can be seen that high-purity target cells expressing different antigens were obtained after sorting.
[0064] Example 2: Luciferase chemiluminescence method to determine the tumor killing effect of CAR-NK cells
[0065] 1) The tumor target cells with good growth status were digested and resuspended and counted at 1×10 per well. 4 The cells were seeded in a black, light-proof 96-well ELISA plate and cultured overnight;
[0066] 2) Remove the target cell culture medium, add 100 μL of different CAR-NK92MI cells according to different effector-target ratios, and culture them in a CO2 incubator for 4-6 hours. Set up 3 replicates for each effector-target ratio in each group, and set up a target cell culture group (negative control group) and a culture medium group (blank group);
[0067] 3) Prepare D-luciferin potassium salt working solution: Dilute 30 mg / mL D-luciferin potassium salt stock solution to 0.15 mg / mL with PBS and keep away from light for subsequent operations;
[0068] 4) Remove the cell culture medium, add 100 μL of D-luciferin potassium salt working solution to each well using an 8-channel pipette, and incubate at 37°C for 5 min;
[0069] 5) The fluorescence intensity was collected in a microplate reader, the blank group signal was subtracted from the signal in all wells, and then the killing rate of CAR-NK on target cells was calculated according to the following formula: (1-co-culture group reading / negative control group reading)×100%.
[0070] like Figure 6 The results show the killing effect of dualCAR-NK92MI cells on different target cells under different effector-target ratios. EGFRdimCD133- and MCF-7 EGFR+CD133- ), EGFR-28zCAR-NK92MI can mediate significant killing (about 40%-60%), while dualCAR-NK92MI has no killing function; for CD133 single positive MDA-MB-231 (MDA-MB-231 EGFR-CD133+ ), CD133-28z CAR-NK92MI mediated significant killing (about 90%), while dualCAR-NK92MI significantly reduced killing (about 50%); for EGFR and CD133 double-positive MCF-7 cells (MCF-7 EGFR+CD133+ ), EGFR-28z, CD133-28z, and dualCAR-NK92MI can all mediate significant killing (about 60%-80%).
[0071] This result verifies that dualCAR can only play a complete killing function in the case of double positive of EGFR and CD133. For single positive target cells, dualCAR cannot mediate killing or can only mediate weak killing, which verifies the improvement of antiEGFR-CD133-dualCAR disclosed in the present invention in terms of specificity and safety.
[0072] Conclusion: The "AND gate" dual-target CAR involved in the present invention can only activate immune cells to the greatest extent when the target cells express two antigens at the same time, and can improve the ability of CAR immune cells to distinguish tumor cells from normal tissue cells. Therefore, the dual-target CAR-NK cells or CAR-T cells constructed based on this structure have better safety than traditional CAR immune cells.
Claims
1. A chimeric antigen receptor capable of simultaneously recognizing EGFR and CD133 dual antigens, characterized in that: The structure of the chimeric antigen receptor is CD8α signal peptide-anti EGFR scFv-tag-CD28 Hinge-CD28 TM-CD28 co-stm-P2A cleavage peptide-CD8α signal peptide-tag-anti CD133 scFv-CD8αHinge-CD8αTM / T-CD3ζ.
2. A chimeric antigen receptor that simultaneously recognizes EGFR and CD133 dual antigens as claimed in claim 1, characterized in that: The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.19; The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.20; The amino acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.21; The amino acid sequence of the CD28 Hinge is shown in SEQ ID NO.22; The amino acid sequence of the CD28 TM is shown in SEQ ID NO.23; The amino acid sequence of the CD28 co-stm is shown in SEQ ID NO.24; The amino acid sequence of the P2A cleavage peptide is shown in SEQ ID NO.25; The amino acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.26; The amino acid sequence of the CD8αHinge is shown in SEQ ID NO.27; The amino acid sequence of the CD8αTM is shown in SEQ ID NO.28; The amino acid sequence of CD3ζ is shown in SEQ ID NO.
29.
3. A nucleic acid encoding the chimeric antigen receptor according to claim 1 or 2.
4. The nucleic acid according to claim 3, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO. 3 or its degenerate sequence.
5. A carrier, characterized in that The vector comprises the nucleic acid according to claim 3 or 4.
6. The vector according to claim 5, characterized in that The vector is a lentivirus containing the nucleic acid.
7. A CAR-NK cell capable of simultaneously recognizing dual antigens of EGFR and CD133, characterized in that: The CAR-NK cells express the chimeric antigen receptor that can simultaneously recognize EGFR and CD133 dual antigens as described in claim 1 or 2.
8. The CAR-NK cell capable of simultaneously recognizing dual antigens of EGFR and CD133 according to claim 7, characterized in that: The CAR-NK cells are obtained by transfecting HEK293T cells with the vector as described in claim 3.
9. Use of a CAR-NK cell capable of simultaneously recognizing dual antigens of EGFR and CD133 as claimed in claim 7 in the preparation of a drug for treating solid tumor diseases.
10. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the CAR-NK cells that can simultaneously recognize EGFR and CD133 dual antigens as described in claim 7.