Chimeric antigen receptor for enhancing tumor recognition specificity and application thereof

Abstract

The invention discloses a chimeric antigen receptor for enhancing tumor recognition specificity and application thereof, and belongs to the technical field of cell therapy. The invention provides a chimeric antigen receptor for enhancing tumor recognition specificity. The chimeric antigen receptor comprises a CD8 alpha signal peptide, an anti EGFR (epidermal growth factor receptor) scFv, a tag, a CD28Hinge, a CD28TM, a CD28 costimulatory domain, a P2A shearing peptide, a CD8 alpha signal peptide, a tag, an anti CD133scFv, a CD8 alpha Hinge, a CD8 alpha TM, CD3 zeta and ODD which are sequentially connected in series. The invention also provides a hypoxia response type double-target gene engineering immune cell. The hypoxia response double-antigen recognition immune cells prepared by the invention can be activated and play a role in killing tumor cells only under the conditions of hypoxia and simultaneous recognition of two antigens, damage to normal tissues caused by an off-target effect is avoided, and the hypoxia response double-antigen recognition immune cells have higher safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy, and specifically relates to a novel hypoxia-responsive dual-target CAR design, engineered NK cells based on the dual-target CAR, and their application in the treatment of 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 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, giving 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 and 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.

[0003] At present, many CAR-T products have been approved for marketing, which 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. Solid tumors account for 90% of all tumors, and there is a greater clinical demand for CAR-T products. Unlike hematological tumors, solid tumor cells lack tumor-specific antigens and do not have lineage antigens such as CD19 and CD22. They can only target tumor-associated antigens that are low in normal tissues and high in tumor cells. Tumor-associated antigens are usually used in solid tumors, that is, antigens that are highly expressed on tumor cells and also low 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 the treatment of a patient with colorectal cancer metastasis with third-generation CAR-T cells designed for human epidermal growth factor receptor II (Her2) reported by Morgan et al. 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, which also limits 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.

[0004] AND-gate dual-target CAR is a potential solution to the recognition specificity. By distributing the co-stimulatory domains required for activation of engineered immune cells downstream of the two antigen recognition domains, that is, constructing two first-generation CAR molecules, the purpose of activating immune cells by only recognizing two antigens at the same time can be achieved, which can improve the safety of engineered immune cells to a certain extent. However, since one of the first-generation CAR intracellular domains is CD3ζ, it provides the first activation signal to activate the immune cell toxicity response, which may lead to attacks on normal tissues that only express CD133, causing "on-target / off-tumor" risks. In view of this, the present invention discloses a method for preparing a hypoxia-responsive EGFR and CD133 dual-antigen recognition CAR-NK cell based on the hypoxic characteristics of the solid tumor microenvironment and its application, mainly involving the preparation of hypoxia-responsive EGFR and CD133 bispecific CAR, CAR-NK cells and their application in solid tumors.

[0005] The present invention provides a design scheme for hypoxia-responsive dual-target CAR that simultaneously targets EGFR and CD133, and selects safer NK cells that can be allogeneically reinfused as carriers to prepare hypoxia-responsive dual-target CAR-NK cells. The CD133 generation CAR molecule and the EGFR CAR form a dual-target CAR with complete structure and function, and add a hypoxia response element to the back of the CD133 generation CAR. Under normoxic conditions, the CD133 generation CAR is degraded, while in the hypoxic microenvironment of solid tumors, the CD133 generation CAR is normally expressed and exerts a killing function. The dual-target CAR-T or CAR-NK cells constructed based on this strategy can only activate the vector cells to the maximum extent when they recognize EGFR and CD133 at the same time under hypoxia, while the tissue cells in normoxic conditions or the tissue cells with low expression of a single target are not reactive, and have better safety than traditional single-target or ordinary dual-target CAR-T or CAR-NK cells. Summary of the invention

[0006] In view of the defects existing in the above-mentioned prior art, in order to solve the problems of "on-target / off-tumor" effect of CAR-T in the treatment of solid tumors, safety risks, inability to improve efficacy by increasing dosage, and the technical solution that requires simultaneous recognition of two antigens to activate immune cells, the following problems may exist: (1) the intracellular domain of the first generation CD133 CAR is CD3ζ, which provides the first activation signal leading to activation of immune cell toxicity reaction, posing a safety hazard; (2) under normoxic conditions, CD133 CAR will be degraded, resulting in the inability to assemble into a dual-target CAR. The purpose of the present invention is to design and provide a method for improving the specificity of NK or T cells to recognize tumor cells by targeting dual antigens and having a chimeric receptor with hypoxia response characteristics, thereby improving the accurate recognition of solid tumors.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a chimeric antigen receptor for enhancing tumor recognition specificity, wherein the chimeric antigen receptor comprises a CD8α signal peptide, a first antigen binding domain, a tag, a CD28 Hinge, a CD28 TM, a CD28 co-stimulatory domain, a P2A cleavage peptide, a CD8α signal peptide, a tag, a second antigen binding domain, CD8α Hinge, CD8α TM, CD3ζ, and ODD (Oxygen-dependent degradation domain in English).

[0009] The nucleic acid sequence of the ODD is shown in SEQ ID NO.15; ODD is a structural domain of the key hypoxia sensing protein HIF-1α, and the ODD structural domain can mediate the degradation of proteins connected thereto under normoxic conditions.

[0010] The first antigen binding domain is different from the second antigen binding domain;

[0011] Preferably, the first antigen binding domain and the second antigen binding domain are one of anti EGFR scFv or anti CD133 scFv.

[0012] The chimeric antigen receptor for enhancing tumor recognition specificity, the nucleic acid sequence of the CD8αSP is shown as SEQ ID NO.3;

[0013] The nucleic acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.4;

[0014] The nucleic acid sequence of the CD28 Hinge is shown in SEQ ID NO.6;

[0015] The nucleic acid sequence of the CD28 TM is shown in SEQ ID NO.7;

[0016] The nucleic acid sequence of the CD28 costimulatory domain is shown in SEQ ID NO.8;

[0017] The nucleic acid sequence of the P2A cleavage peptide is shown in SEQ ID NO.9;

[0018] The nucleic acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.11;

[0019] The nucleic acid sequence of the CD8αHinge is shown in SEQ ID NO.12;

[0020] The nucleic acid sequence of the CD8αTM is shown in SEQ ID NO.13;

[0021] The nucleic acid sequence of CD3ζ is shown in SEQ ID NO.14.

[0022] In a second aspect, the present invention provides a nucleic acid encoding the chimeric antigen receptor.

[0023] The nucleic acid has a nucleic acid sequence as shown in SEQ ID NO.2.

[0024] In a third aspect, the present invention provides a vector comprising the nucleic acid.

[0025] In a fourth aspect, the present invention provides the vector, which is a lentivirus, retrovirus, slow virus, adenovirus or adeno-associated virus containing the nucleic acid.

[0026] In a fifth aspect, the present invention provides a hypoxia-responsive dual-target genetically engineered immune cell, wherein the genetically engineered immune cell is transduced with the vector.

[0027] The hypoxia-responsive dual-target genetically engineered immune cell is a T cell, a NK cell or a NKT cell.

[0028] In a sixth aspect, the present invention provides the use of the hypoxia-responsive dual-target genetically engineered immune cells in the preparation of drugs for treating solid tumor diseases.

[0029] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the hypoxia-responsive dual-target genetically engineered immune cells as described in claim 7.

[0030] The dual antigen recognition chimeric receptor of the present invention is composed of two first-generation CAR molecules targeting EGFR and CD133 respectively, wherein EGFR CAR is composed of anti-EGFR scFv, CD28 hinge / transmembrane region and CD28 co-stimulatory domain, and CD133CAR is composed of anti-CD133 scFv, CD8α hinge / transmembrane region, CD3ζ co-stimulatory domain and hypoxia response element ODD. The two CAR molecules are connected by P2A self-cleavage peptide, and receptor expression is controlled by EF1α promoter. Under normoxic conditions, CD133CAR will be degraded, resulting in the inability to assemble into dual-target CAR; while under hypoxia, the stability of CD133 CAR protein is improved, and it can be assembled with EGFRCAR into a fully functional dual-target CAR, which can effectively activate engineered immune cells when recognizing EGFR and CD133 at the same time. Since the inside of solid tumors is in a hypoxic state, while normal tissues are in a normoxic state, the hypoxia-responsive dual-antigen recognition CAR-NK cells prepared based on the present invention can only be activated and play a role in killing tumor cells under hypoxia and simultaneous recognition of both EGFR and CD133 antigens, avoiding damage to normal tissues caused by off-target effects, and having higher safety.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The hypoxia-responsive dual-target CAR provided by the present invention can only activate immune cells to the greatest extent when hypoxia occurs and the target cells express two antigens at the same time, recognize a single target or do not activate or activate at a low level under normoxic conditions, and can improve the ability of CAR immune cells to distinguish between tumor cells and normal tissue cells. The dual-target CAR-NK cells or CAR-T cells constructed based on the structure of the present invention have better safety than traditional CAR immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the structure diagram of CAR plasmid;

[0034] Figure 2 This is a diagram of the CAR structure model;

[0035] Figure 3 Schematic diagram of antigen overexpression plasmid;

[0036] Figure 4 The figure shows the expression results of dualCAR and dualCAR-ODD on NK92MI cells;

[0037] Figure 5 The figure shows the expression results of dualCAR-ODD in Jurkat cells;

[0038] Figure 6The figure shows the expression results of dualCAR and dualCAR-ODD on primary human T cells;

[0039] Figure 7 This is the result diagram of target cell antigen expression;

[0040] Figure 8 The CoCl2-induced CAR-NK92MI hypoxia model was constructed, where A shows the effect of different concentrations of CoCl2 on NK92MI cell viability, B shows the changes in CD133 CAR expression levels induced by different concentrations of CoCl2, C shows the changes in CD133 CAR expression levels induced by different CoCl2 treatment times, D shows the expression of EGFR CAR and CD133CAR of dualCAR and dualCAR-ODD under normoxic conditions, and the CD133 CAR expression of dualCAR-ODD induced by the selected 150μM CoCl2 treatment for 24h;

[0041] Fig. 9 The CoCl2-induced CAR-Jurkat hypoxia model was constructed, where A shows the effect of different concentrations of CoCl2 on Jurkat cell viability, B shows the changes in CD133 CAR expression levels induced by different concentrations of CoCl2, and C shows the expression of marker CD69 activated after dualCAR-ODD was co-cultured with target cells under 100 μM CoCl2 treatment;

[0042] Fig.10 Figure 1 is a diagram showing the killing effect of CAR-NK92MI on target cells under normoxic and hypoxic conditions, wherein A is the killing result of CAR-NK92MI on target cells under normoxic conditions, and B is the killing result of CAR-NK92MI on target cells under hypoxic conditions;

[0043] Fig.11 This is a diagram showing the killing effect of CAR-T on target cells under normoxia;

[0044] Fig.12 Figure 2 is a diagram showing the cytokine secretion effect of CAR-NK92MI co-cultured with target cells under normoxic and hypoxic conditions, where A is the cytokine secretion effect of CAR-NK92MI co-cultured with MDA-MB-231 under normoxic and hypoxic conditions EGFR+CD133+ Cytokine secretion results after co-culture, B is CAR-NK92MI and Huh7 under normoxic and hypoxic conditions EGFR+CD133dim Results of cytokine secretion after co-culture;

[0045] Fig.13 Figure 2 is the cytokine secretion effect diagram of CAR-T co-cultured with target cells under normoxia, where A is the cytokine secretion effect diagram of CAR-T co-cultured with MDA-MB-231 under normoxia and hypoxia EGFR+CD133+Cytokine secretion results after co-culture, B is CAR-T and MDA-MB-468 under normoxic and hypoxic conditions EGFR+CD133dim Results of cytokine secretion after co-culture;

[0046] Fig.14 Figure 2 is the result of CD107a expression of CAR-NK92MI co-cultured with target cells under normoxic and hypoxic conditions, where A is the result of CAR-NK92MI co-cultured with MDA-MB-231 under normoxic conditions. EGFR+CD133+ 、MDA-MB-468 EGFR+CD133dim and Huh7 EGFR+CD133dim The expression results of CD107a in co-culture. B is the expression of CD107a in co-cultured CAR-NK92MI and MDA-MB-231 under hypoxic conditions. EGFR+CD133+ 、MDA-MB-468 EGFR+CD133dim and Huh7 EGFR+CD133dim CD107a expression results of co-culture;

[0047] Fig.15 Figure 2 is the result of CD69 expression of CAR-NK92MI co-cultured with target cells under normoxic and hypoxic conditions, where A is the result of CAR-NK92MI co-cultured with MDA-MB-231 under normoxic conditions. EGFR+CD133+ 、MDA-MB-468 EGFR+CD133dim and Huh7 EGFR+CD133dim The expression results of CD69 in co-culture. B is the expression of CD69 in co-cultured CAR-NK92MI and MDA-MB-231 under hypoxic conditions. EGFR +CD133+ 、MDA-MB-468 EGFR+CD133dim and Huh7 EGFR+CD133dim CD69 expression results of co-culture;

[0048] Fig.16 This is a diagram showing the effect of dualCAR-ODD-Jurkat co-cultured with target cells under normoxic and hypoxic conditions;

[0049] Fig.17 The figure is a diagram of the in vivo anti-tumor effect of CAR-NK92MI on MDA-MB-231 orthotopic tumor, wherein A is a schematic diagram of the experimental scheme, B is the weight of the mouse, C is the tumor volume, D is a tumor picture, E is the tumor weight, F is the proportion of humanCD56+ cells in the lung tissue of the mouse, G is the proportion of Myc+ cells of human CD56+ cells in the lung tissue of the mouse, and H is the proportion of Flag+ cells of human CD56+ cells in the lung tissue of the mouse;

[0050] Fig.18The in vivo anti-tumor effect of CAR-NK92MI on Huh7 subcutaneous tumors, wherein A is a schematic diagram of the experimental scheme, B is the weight of mice, C is the tumor volume, D is a tumor picture, and E is the tumor weight;

[0051] Fig.19 The figure is a diagram of the in vivo anti-tumor effect of CAR-NK92MI on Nalm6 hematological tumor, wherein A is the expression of EGFR and CD133 in the constructed Nalm6 cells, B is the killing of Nalm6 cells overexpressing EGFR and CD133 by CAR-NK92MI in vitro, C is a schematic diagram of the in vivo experimental scheme, D is the in vivo imaging result of tumor-bearing mice, E is the weight of mice, F is the survival curve of mice, and G is the statistics of in vivo imaging data of tumor-bearing mice. DETAILED DESCRIPTION

[0052] The present invention is described in detail below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples, unless otherwise specified, are conventional methods, and the experimental materials used are all commercially available products.

[0053] Embodiment 1:

[0054] 1. Plasmid structure design and construction

[0055] according to Figure 1The 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 antiEGFR-CD133-dualCAR (abbreviated as dualCAR, the nucleic acid sequence is shown in SEQ ID NO.1) and antiEGFR-CD133-dualCAR-ODD (abbreviated as dualCAR-ODD, the nucleic acid sequence is shown in SEQ ID NO.2). Among them, the nucleic acid sequence of CD8αSP (or CD8α signal peptide) is shown in SEQ ID NO.3; the nucleic acid sequence of anti-EGFR scFv is shown in SEQ ID NO.4; the nucleic acid sequence of Myc tag is shown in SEQ ID NO.5; wherein CD28H / T is split into CD28Hinge and CD28 TM, and the nucleic acid sequence of CD28 Hinge (or CD28 hinge region) is shown in SEQ ID NO.6; the nucleic acid sequence of CD28TM (or CD28 transmembrane domain) is shown in SEQ ID NO.7; the nucleic acid sequence of CD28 co-stm (or CD28 co-stimulatory domain) is shown in SEQ ID NO.8; the nucleic acid sequence of P2A cleavage peptide is shown in SEQ ID NO.9; the nucleic acid sequence of Flag tag is shown in SEQ ID NO.10; the nucleic acid sequence of antiCD133 scFv is shown in SEQ ID NO.11; wherein CD8αH / T is split into CD8αHinge and CD8αTM, and the nucleic acid sequence of CD8αHinge is shown in SEQ ID The nucleic acid sequence of CD8αTM (or CD8α transmembrane domain) is shown in SEQ ID NO.12; the nucleic acid sequence of CD8αTM (or CD8α transmembrane domain) is shown in SEQ ID NO.13; the nucleic acid sequence of CD3ζ signal domain is shown in SEQ ID NO.14; the nucleic acid sequence of ODD is shown in SEQ ID NO.15. Figure 2 Shown are structural schematic diagrams of dualCAR and dualCAR-ODD, showing the structural schematic diagram of the CAR molecule shown in the present invention expressed on the cell membrane, the antigen recognition domains anti-EGFR scFv and anti-CD133 scFv are both located outside the cell, the CD28 costimulatory domain and the CD3ζ signaling domain are located inside the cell, and the extracellular antigen recognition domain is connected to the intracellular signaling domain and costimulatory domain through the two transmembrane domains CD28 TM and CD8 TM, and the hypoxia response element ODD is connected behind CD3ζ of dualCAR-ODD to mediate the degradation of this generation of CAR under normoxia.

[0056] according to Figure 3The 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.

[0057] 2. Lentivirus packaging and concentration

[0058] 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;

[0059] 2) Liposome transfection: core plasmid (10 μg), lentiviral packaging plasmid psPAX2 (7.5 μg), and envelope plasmid pMD2.G (2.5 μg) were added to 1 mL of opti-MEM medium at a ratio of 4:3:1, and then 60 μL of PEI transfection reagent was added. After mixing, the mixture was allowed to stand at room temperature for 15 min, and then slowly added to HEK293T cells. The culture dish was gently shaken to mix, and then placed in a 37°C incubator. After 6 hours of culture, fresh complete medium was replaced.

[0060] 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;

[0061] 4) Add 2 mL of 5×PEG8000 to every 8 mL of viral supernatant and shake at 4°C overnight;

[0062] 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.

[0063] 3. Preparation of CAR-NK and CAR-T cells

[0064] For the preparation of CAR-NK cells, 2 × 10 5 For NK92MI cells, add the concentrated dualCAR and dualCAR-ODD lentiviruses directly into the wells, add polybrene at a final concentration of 8 μg / mL to enhance infection, replace the culture medium with fresh one the next day, and detect the CAR positivity rate after another two days.

[0065] For the preparation of CAR-Jurkat cells, 4 × 10 5Jurkat cells were cultured by adding 500 μL of unconcentrated dualCAR and dualCAR-ODD lentiviral supernatant to each well, and polybrene at a final concentration of 10 μg / mL was added to enhance infection. Fresh culture medium was replaced the next day, and the CAR positivity rate was tested after another two days.

[0066] For the preparation of primary CAR-T cells, PBMC cells were routinely revived, stained with trypan blue and counted for later use. Vortex and mix CD3CD28 activated magnetic beads (Cat. No.: GMP-TL601). According to the number of live PBMC cells counted, 3 times the number of PBMC cells were washed with 1 mL PBS (containing 1% FBS) and added to PBMC. Incubate slowly at room temperature on a shaker for 30-60 minutes. Then add culture medium to a cell density of 0.5-1×10 6 / mL, add rhIL-2 (Cat. No.: GMP-TL777) to a final concentration of 100 IU / mL, and place in a cell culture incubator for 1 day. After 1 day, 6-well plates can be plated and infected with lentivirus, 2×10 6 cells / well, add concentrated dualCAR and dualCAR-ODD lentivirus, add 8μg / mL polybrene to enhance infection, replace with fresh culture medium 12h after infection, and detect the CAR positivity rate after another two days.

[0067] 4. Target cell construction

[0068] The EGFR and CD133 expression of wild-type Huh7, MCF-7 and MDA-MB-231 are: Huh7EGFR+CD133dim, MCF-7EGFRdimCD133- and MDA-MB-231EGFR+CD133-. It is necessary to construct MCF-7EGFR+CD133+ and MDA-MB-231EGFR+CD133+ target cells.

[0069] Add 2×105 MCF-7 or MDA-MB-231 cells to each well of a 6-well plate. After overnight attachment, add 1 mL of unconcentrated pLV3-EGFR or pLV3-CD133 lentiviral supernatant according to the antigen phenotype to be constructed. Add polybrene at a final concentration of 10 μg / mL to enhance infection. Replace with fresh culture medium the next day, and detect the expression of EGFR and CD133 two days later.

[0070] 5. Detection of CAR, EGFR and CD133 expression by flow cytometry

[0071] 1) Collect infected cells in a 1.5 mL centrifuge tube and collect wild-type cells as a negative control. Centrifuge at 4°C, 200 g for 3 min and remove the culture medium.

[0072] 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;

[0073] 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;

[0074] 4) Add 1 mL of pre-cooled PBS, centrifuge at 4°C, 200 g for 3 min, remove the supernatant, and repeat once;

[0075] 5) After adding 200 μL PBS and resuspending, flow cytometry was performed to analyze the expression of CAR and target antigen.

[0076] 6. Flow cytometry sorting of CAR-NK92MI, CAR-Jurkat or target cells

[0077] 1) Collect approximately 5 × 10 6 The infected CAR-NK92MI, CAR-Jurkat, MCF-7 and MDA-MB-231 cells were centrifuged at 200 g for 3 min at 4°C, and the culture medium was removed.

[0078] 2) Resuspend the cell pellet in 5 mL of pre-cooled PBS, centrifuge at 200 g for 3 min, and remove the supernatant;

[0079] 3) For the sorting of CAR-NK92MI and CAR-Jurkat, add 500 μL of diluted anti-Myc or anti-Flag antibody working solution to each sample; for the sorting of target cells, 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 the dark;

[0080] 4) Add 5 mL of pre-cooled PBS to resuspend, centrifuge at 200 g for 3 min, remove the supernatant, and repeat once;

[0081] 5) Resuspend in 500 μL complete medium and transfer to a 15 mL centrifuge tube and place on ice away from light;

[0082] 6) In the flow cytometer, determine the gate of positive cells based on the negative control and sort out the target cells;

[0083] 7) The sorted cells were centrifuged at 300g for 5 minutes, resuspended in complete culture medium containing 3% cyan / chain dual antibody, and cultured in a CO2 incubator to obtain completely positive CAR-NK92MI, CAR-Jurkat and target cells expressing different antigens. Figure 4 As shown, the expression of CAR on the sorted CAR-Jurkat cells is as follows Figure 5 As shown in Figure 2, it can be seen that the sorted CAR-NK92MI and CAR-Jurkat cells are all CAR-positive cells, while the primary CAR-T cells are not sorted, and the CAR positive rate is as follows: Figure 6 The expression of EGFR and CD133 on the sorted target cells is shown in Figure 7 As shown, it can be seen that high-purity target cells expressing different antigens were obtained after sorting.

[0084] 7. Construction of in vitro hypoxia model

[0085] In vitro chemical hypoxia was induced by CoCl2 treatment. For CAR-NK92MI cells, cell death was detected by flow cytometry with 7-AAD after treatment with different concentrations of CoCl2 for 24 h. It can be seen that CoCl2 concentrations not exceeding 400 μM were non-toxic to CAR-NK92MI cells (e.g. Figure 8 A), then different CoCl2 concentration gradients and time gradients were set at non-toxic concentrations to detect the expression of the CD133-CD3ζ structure connected to the ODD. It was found that with the increase of CoCl2 concentration and the extension of treatment time, the expression of the CD133-CD3ζ structure was gradually upregulated, and after the removal of CoCl2, the expression of the CD133-CD3ζ structure was gradually downregulated (such as Figure 8 BC), indicating that the expression of dualCAR-ODD in this study is controlled by hypoxia. Based on these data, this study selected 150μM CoCl2 treatment for 24h to induce hypoxia in CAR-NK92MI cells in vitro (such as Figure 8 D), it can be seen that under normoxic conditions, both dualCAR and dualCAR-ODD can detect Myc signals, but only dual CAR can detect Flag, and dualCAR-ODD cannot detect Flag, indicating that CD133 is degraded and cannot be assembled into a dual-target CAR. Figure 8 B, C explored the experimental conditions and found that after inducing chemical hypoxia by treating with CoCl2 at a specific concentration for a specific time, the Flag expression of dualCAR and dualCAR-ODD was basically the same, indicating that the CD133 CAR of dualCAR-ODD was no longer degraded under hypoxic conditions.

[0086] For CAR-Jurkat cells, after treatment with different concentrations of CoCl2 for 24 h, flow cytometry with 7-AAD was used to detect cell death. It can be seen that CoCl2 concentrations not exceeding 200 μM are non-toxic to CAR-Jurkat cells (e.g. Fig. 9 A), and then set different CoCl2 concentration gradients to detect the expression of the CD133-CD3ζ structure connected to the ODD. It was found that with the increase of CoCl2 concentration, the expression of the CD133-CD3ζ structure showed a trend of first upregulation and then downregulation (such as Fig. 9 B) indicates that the expression of dualCAR-ODD of the present invention in Jurkat cells is also controlled by hypoxia conditions.

[0087] Based on these data, this study selected 100 μM CoCl2 treatment for 24 h to induce hypoxia in CAR-Jurkat cells in vitro (e.g. Fig. 9 C).

[0088] 8. Luciferase chemiluminescence assay to determine in vitro tumor killing effect

[0089] 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;

[0090] 2) Remove the target cell culture medium, add 100 μL of different CAR-NK92MI or CAR-T 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);

[0091] 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 in a dark place. All subsequent operations must be kept away from light.

[0092] 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;

[0093] 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 in vitro killing efficiency was calculated according to the following formula: (1-co-culture group reading / negative control group reading)×100%.

[0094] The results are as follows Fig.10The results show that the dualCAR-NK92MI and dualCAR-ODD-NK92MI cells of the present invention have a killing effect on different target cells under normoxic or hypoxic conditions. For EGFR and CD133 double-positive target cells Huh7, MCF-7 and MDA-MB-231, dualCAR-NK92MI mediated significant killing under normoxic conditions, while dualCAR-ODD-NK92MI had almost no killing function (e.g. Fig.10 A); Under hypoxic conditions, dualCAR-NK92MI and dualCAR-ODD-NK92MI exerted similar killing effects (e.g. Fig.10 B) This result proves that the dualCAR-ODD-NK92MI of the present invention has no killing function under normoxic conditions, but has complete killing ability under hypoxic conditions. Fig.11 The results showed the killing effect of dualCAR-ODD-T cells on target cells under normoxic conditions. It can be seen that compared with dualCAR-T, dualCAR-ODD-T has almost no killing function, proving that the dualCAR-ODD-T of the present invention has no killing function under normoxic conditions.

[0095] 9. CBA method to determine cytokine secretion

[0096] 1) MDA-MB-231EGFR+CD133+ and Huh7EGFR+CD133dim 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;

[0097] 2) Collect and adjust the density of CAR-NK92MI and CAR-T to 2×10 5 cells / mL;

[0098] 3) Remove the target cell culture medium from the ELISA plate, add 100 μL of different CAR-NK92MI or CAR-T cells (efficacy-target ratio 2:1) to each well, and culture in a CO2 incubator, with 3 replicate wells per group;

[0099] 4) After 24 hours, the culture medium of each well was collected and centrifuged at 1000 g for 5 min to remove cells and debris. TM Detect cytokine concentrations in the culture medium.

[0100] The results are as follows Fig.12The cytokine secretion levels of dualCAR-NK92MI and dualCAR-ODD-NK92MI co-cultured with target cells under normoxic or hypoxic conditions in the present invention are shown. It can be seen that under normoxic conditions, dualCAR-NK92MI secreted a large amount of cytokines, while dualCAR-ODD-NK92MI only secreted low levels of cytokines (such as Fig.12 A); however, under hypoxic conditions, the levels of cytokines secreted by dualCAR-ODD-NK92MI were significantly upregulated (e.g. Fig.12 B) This result demonstrates that dualCAR-ODD-NK92MI has a hypoxia-responsive activation mode.

[0101] The results are as follows Fig.13 The cytokine secretion levels of dualCAR-T and dualCAR-ODD-T co-cultured with target cells under normoxic or hypoxic conditions in the present invention are shown. It can be seen that under normoxic conditions, dualCAR-T secreted a large amount of cytokines, while dualCAR-ODD-T only secreted low levels of cytokines. This result proves that the dualCAR-ODD-T in the present invention cannot be activated under normoxic conditions.

[0102] 9. Flow cytometry detection of CAR-NK92MI degranulation marker CD107a expression

[0103] 1) Adjust the concentration of target cells and CAR-NK92MI cells to 10 6 cells / mL to obtain a 1:1 effector-target ratio.

[0104] 2) Three groups were set up: basic degranulation level group (100uL CAR-NK92MI + 100uL culture medium), co-culture group (100uL CAR-NK92MI + 100uL target cells), positive control group (100uL CAR-NK92MI + 100uL culture medium containing 100ng / mL PMA and 2μg / mL ionomycin).

[0105] 3) According to the three groups, culture medium, target cells, and positive control culture medium were added to 96-well U-shaped plates, 100uL / well.

[0106] 4) Take a sufficient volume of CAR-NK cells, add anti-human CD107a antibody at 2uL / mL, mix well by pipetting, and add 100uL / well to the corresponding wells. At this time, the total volume of each well is 200uL, and incubate in an incubator.

[0107] 5) After incubation for 1 hour, 2 μL of GolgiStop was added to 75 μL of culture medium to prepare a monensin solution, and then 5 μL / well of the solution was added to a 96-well plate and mixed by pipetting.

[0108] 6) After incubation for another 3 hours, the cells were collected and stained with CD56 antibody, and the ratio of CD107a+ in CD56+ was detected by flow cytometer.

[0109] The results are as follows Fig.14 The expression level of CD107a in the dualCAR-NK92MI and dualCAR-ODD-NK92MI co-cultured with target cells under normoxic or hypoxic conditions is shown. It can be seen that under normoxic conditions, CD107a is significantly upregulated after dualCAR-NK92MI is co-cultured with target cells, while dualCAR-ODD-NK92MI has no CD107a expression at all (e.g. Fig.14 A); however, under hypoxic conditions, the CD107a levels of dualCAR-NK92MI and dualCAR-ODD-NK92MI were almost the same (e.g. Fig.14 B) This result also demonstrates that dualCAR-ODD-NK92MI has a hypoxia-responsive activation mode.

[0110] 11. Flow cytometry detection of CAR-NK92MI and CAR-Jurkat activation marker CD69 expression

[0111] 2 × 10 5 A CAR-NK92MI (or CAR-Jurkat) was co-cultured with target cells at an effector-target ratio of 1:1. After 24 hours of co-culture, the cells were collected and stained with CD56 (or CD3) and CD69, and then the proportion of CD69+ in the CD56+ (or CD3+) cell population was detected by an instrument.

[0112] The results are as follows Fig.15 The CD69 expression levels of dualCAR-NK92MI and dualCAR-ODD-NK92MI co-cultured with target cells under normoxic or hypoxic conditions are shown. It can be seen that under normoxic conditions, CD69 is significantly upregulated after dualCAR-NK92MI is co-cultured with target cells, while dualCAR-ODD-NK92MI has no CD69 expression at all (e.g. Fig.15 A); however, under hypoxic conditions, the CD69 level of dualCAR-ODD-NK92MI was significantly upregulated (e.g. Fig.15 B) This result also demonstrates that dualCAR-ODD-NK92MI has a hypoxia-responsive activation mode.

[0113] The results are as follows Fig.16The CD69 expression level of dualCAR-ODD-Jurkat co-cultured with target cells under normoxic or hypoxic conditions is shown. It can be seen that under normoxic conditions, dualCAR-ODD-Jurkat has no CD69 expression at all, while under hypoxic conditions, CD69 expression is significantly upregulated. This result proves that the dualCAR-ODD structure also has an activation mode of hypoxia response on the T cell line Jurkat.

[0114] Example 2: Verification of the in vivo anti-tumor effect of CAR-NK92MI in a mouse solid tumor model

[0115] For the MDA-MB-231 mouse solid tumor model, 6-8 week-old female NSG mice were orthotopically inoculated with MDA-MB-231EGFR+CD133+ cells (2×10 6 cells / mouse, 50 μL, containing Matrigel), and 1×10 7 CAR-NK92MI cells were transfused, and IL-2 (20000U / mouse) was injected through the tail vein 24h after the transfusion of CAR-NK92MI cells. Treatment was performed every 3-4 days until the maximum tumor volume reached 2000cm 3 The mice were then sacrificed and the tumor weights were measured. Fig.17 The in vivo anti-tumor effects of dualCAR-NK92MI and dualCAR-ODD-NK92MI on the MDA-MB-231EGFR+CD133+ model were demonstrated, indicating that dualCAR-NK92MI and dualCAR-ODD-NK92MI had the same effect of inhibiting the growth of MDA-MB-231EGFR+CD133+ tumors in vivo.

[0116] For the Huh7 mouse solid tumor model, 6-8 week-old male NSG mice were inoculated with Huh7EGFR+CD133dim cells (2×10 6 cells / mouse, 100 μL, containing Matrigel), and 1×10 7 CAR-NK92MI cells were transfused, and IL-2 (20000U / mouse) was injected through the tail vein 24h after the transfusion of CAR-NK92MI cells. Treatment was performed every 3-4 days until the maximum tumor volume reached 2000cm 3 The mice were then sacrificed and the tumor weights were measured. Fig.18 It shows that dualCAR-NK92MI and dualCAR-ODD-NK92MI have

[0117] The in vivo anti-tumor effect of the MDA-MB-231EGFR+CD133+ model showed that dualCAR-NK92MI and dualCAR-ODD-NK92MI had the same effect of inhibiting the growth of Huh7EGFR+CD133dim tumors in vivo.

[0118] Example 3: Mouse hematological tumor model verifies the tumor recognition ability of CAR-NK92MI

[0119] Hematologic tumor cells Nalm6 were used to simulate normal cells expressing antigens under normoxic conditions in vivo. Male NSG mice aged 6-8 weeks were inoculated with Nalm6EGFR+CD133+ cells (2×10 6 cells / mouse, 100 μL), and 1×10 7 CAR-NK92MI cells were transfused into the mice, and IL-2 (20000U / mouse) was injected through the tail vein 24h after the transfusion of CAR-NK92MI cells. The mice were treated every 3-4 days, and the survival time of the mice was recorded. Fig.19 The in vivo anti-tumor effects of dualCAR-NK92MI and dualCAR-ODD-NK92MI on the Nalm6EGFR+CD133+ model were demonstrated. It can be seen that the dualCAR-NK92MI treatment group can significantly prolong the survival of tumor-bearing mice, while the dualCAR-ODD-NK92MI group mice have the same survival time as the PBS and Mock groups. This result proves that under normoxic conditions, the dualCAR-ODD-NK92MI of the invention will not kill antigen-positive cells, that is, normal cells that partially express antigens, indicating that the dualCAR-ODD design improves the accurate recognition of solid tumors.

[0120] Conclusion: In in vitro and in vivo experiments, the dualCAR-ODD design of the present invention exhibits a hypoxia-restricted activation mode on both NK and T cells, that is, it can kill antigen-positive target cells only under hypoxic conditions and has no killing function under normoxic conditions. Due to the unique hypoxic microenvironment in solid tumor tissues, the dualCAR-ODD design disclosed in the present invention can improve the accurate identification of solid tumors and avoid the side effects caused by the "on-target / offtumor" effect.

Claims

1. A chimeric antigen receptor for enhancing tumor recognition specificity, characterized in that: The chimeric antigen receptor comprises a CD8α signal peptide, a first antigen binding domain, a tag, a CD28 Hinge, a CD28 TM, a CD28 co-stimulatory domain, a P2A cleavage peptide, a CD8α signal peptide, a tag, a second antigen binding domain, a CD8α Hinge, a CD8α TM, CD3ζ, and an ODD connected in series in sequence; The nucleic acid sequence of the ODD is shown in SEQ ID NO.15; The first antigen binding domain is different from the second antigen binding domain; Preferably, the first antigen binding domain and the second antigen binding domain are one of anti EGFR scFv and anti CD133 scFv.

2. A chimeric antigen receptor for enhancing tumor recognition specificity according to claim 1, characterized in that: The nucleic acid sequence of the CD8αSP is shown in SEQ ID NO.3; The nucleic acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.4; The nucleic acid sequence of the CD28 Hinge is shown in SEQ ID NO.6; The nucleic acid sequence of the CD28 TM is shown in SEQ ID NO.7; The nucleic acid sequence of the CD28 costimulatory domain is shown in SEQ ID NO.8; The nucleic acid sequence of the P2A cleavage peptide is shown in SEQ ID NO.9; The nucleic acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.11; The nucleic acid sequence of the CD8αHinge is shown in SEQ ID NO.12; The nucleic acid sequence of the CD8αTM is shown in SEQ ID NO.13; The nucleic acid sequence of CD3ζ is shown in SEQ ID NO.

14.

3. A nucleic acid encoding the chimeric antigen receptor according to claim 1.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid sequence is shown in SEQ ID NO.

2.

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, a retrovirus, a lentivirus, an adenovirus or an adeno-associated virus containing the nucleic acid.

7. A hypoxia-responsive dual-target genetically engineered immune cell, characterized in that: The genetically engineered immune cell transduction is the vector as claimed in claim 5.

8. The hypoxia-responsive dual-target genetically engineered immune cell according to claim 7, characterized in that: The immune cells are T cells, NK cells or NKT cells.

9. Use of the hypoxia-responsive dual-target genetically engineered immune cell 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 hypoxia-responsive dual-target genetically engineered immune cells as described in claim 7.

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