Lipid nanoparticle for generating CAR-T (Chimeric Antigen Receptor T) cells in vivo as well as construction method and application of lipid nanoparticle

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses lipid nanoparticles for generating CAR-T cells in vivo as well as a construction method and application of the lipid nanoparticles. The invention relates to a preparation method and application of lipid nanoparticle LNP on one hand, and relates to a method for preparing Ab-LNP based on lipid nanoparticle LNP on the other hand and application of Ab-LNP. According to the present invention, the lipid nanoparticles respectively encapsulate and encode mRNA molecules through the microfluidic mixing technology, and then the surface of the obtained LNP is modified with the T cell targeting antibody through the click chemistry technology to prepare the engineered mRNA-LNP in-vivo targeting delivery carrier, which can effectively treat multiple myeloma or B lymphoid leukemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to lipid nanoparticles for in vivo generation of CAR-T cells, a construction method thereof, and applications thereof. Background Art

[0002] CAR-T therapy is a treatment method that activates T cells isolated from the human body through genetic engineering technology and installs a CAR (chimeric antigen receptor) as a positioning and navigation device, transforming T cells into "super soldiers", namely CAR-T cells. Using CAR as the "positioning and navigation device", after being re-infused into the body, it can specifically recognize tumor cells in the body and release a large number of various effector factors to efficiently kill tumor cells, thereby achieving the purpose of treating malignant tumors. CAR-T therapy has played a significant role in refractory / relapsed hematological tumors. So far, CAR-T research has been booming all over the world, and 13 CAR-T products have been launched globally. The preparation of CAR-T cells is a labor-intensive process, involving many complex processes and extremely strict quality control.

[0003] However, the deficiencies of traditional in vitro preparation of CAR-T cells are mainly reflected in its complex and high-cost process flow, strong dependence on the state of the patient's own T cells, and potential safety risks. Currently, commercial cell products need to isolate T cells from the patient's body, perform gene modification and amplification in vitro through viral vectors, and then re-infuse them. This process usually takes 2-4 weeks and is highly dependent on the initial quality of the patient's T cells. If the patient's T cell count is insufficient or their function is exhausted due to chemotherapy or disease progression, the risk of in vitro amplification failure increases significantly. In addition, fluctuations in the transfection efficiency of viral vectors may cause uneven CAR expression, and long-term in vitro culture is likely to induce T cells to differentiate into terminal effector cells, weakening their in vivo persistence and anti-tumor activity. The characteristics of personalized production not only result in a single treatment cost of up to hundreds of thousands of dollars, but also face strict cold chain transportation and GMP facility requirements, restricting the accessibility of patients in remote areas. In terms of safety, high-dose re-infused CAR-T cells may cause severe cytokine release syndrome (CRS) or neurotoxicity, and the random integration of viral vectors into the genome poses a potential carcinogenic risk, further restricting clinical applications.

[0004] It is reported that the FDA has required additional "black box warnings" to be added to the labels of 6 approved CAR-T cell therapy products. The agency believes that all commercially available CAR-T cell therapies targeting the BCMA or CD19 targets carry a risk of secondary T cell malignancies. As of December 31, 2023, the FDA has reported a total of 22 cases of T cell malignancies occurring after CAR-T cell therapy. These malignancies include T cell lymphoma, T cell large granular lymphocytic leukemia, peripheral T cell lymphoma, and cutaneous T cell lymphoma, involving 5 of the 6 previously marketed CAR-T products. According to a retrospective analysis in the New England Journal of Medicine, a study of 724 cancer patients who received CAR-T therapy at Stanford University School of Medicine between 2014 and 2016 showed that the 3-year cumulative incidence of secondary hematological second malignancies was 6.5%. At the same time, the British Journal of Hematology reported the results of the Italian prospective observational CART-SIE study, which included 651 lymphoma patients who received CD19 CAR-T treatment and found that the incidence of secondary second malignancies was 4.3% (28 / 651), with the most common type being hematological malignancies. In addition, the journal Nature Medicine retrospectively analyzed 449 cases of CAR-T treatment at the University of Pennsylvania School of Medicine since 2018 and found 16 cases of concurrent second malignancies, with solid tumors accounting for 15.2% and hematological tumors accounting for 84.8%. A team including Dana-Farber Cancer Institute in the United States and Georgetown University conducted a systematic investigation of a rare case of indolent lymphoma derived from CD4+ CAR-T cells. This 71-year-old elderly patient was admitted to the hospital due to persistent diarrhea and weight loss after receiving BCMA CAR-T cell (Cilta-cel) treatment. Endoscopic examination and pathological biopsy, along with flow cytometry and immunohistochemical staining, confirmed CD4+ T cell indolent lymphoma in the small intestine. Targeted mRNA sequencing revealed that the tumor cells contained CAR mRNA, and the malignant T cells showed monoclonal expansion. This clone was also detected in the post-treatment biopsy and peripheral blood specimens. Further whole-genome sequencing found that the CAR gene was inserted into the second intron of the SSU72 gene of this clone. The New England Journal of Medicine recently reported another case of a multiple myeloma patient who developed gastrointestinal T cell lymphoma after receiving the BCMA CAR-T therapy cilta-cel from Legend Biotech. A large number of CAR copies were detected in the duodenal biopsy sample of this patient, and further CAR integration site analysis revealed two major insertion sites: the first site was inserted in reverse into the first intron of the TP53 gene, and the second site was inserted forward into the first intron of the TANGO2 gene. The frequencies of these two insertion sites were 29% and 23% respectively.The results of immunohistochemistry showed that p53 was expressed in the crypt epithelial cells of the duodenal biopsy samples, while p53 expression was less or absent in 90% of T cells. Therefore, it was speculated that the occurrence of this T-cell lymphoma might be related to the integration of the lentiviral vector into the TP53 gene. In addition, this journal also reported two cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), that occurred after Cilta-cel treatment, with malignant monoclonal T-cell lymphoproliferation with CAR transgenic integration and expression, which was named CAR transgenic T-cell lymphoproliferative neoplasm (CTTLN). The common features of the two cases included skin involvement and missense mutations in the TET2 gene. In summary, the potential carcinogenic risk brought about by the insertion of the lentiviral vector into the host T-cell genome needs to be taken seriously, as this risk may lead to secondary malignant hematological tumors in patients receiving CAR-T cell therapy.

[0005] In contrast, in vivo CAR-T therapy has significant potential advantages by directly injecting the delivery vector into the patient's body to complete the genetic modification of T cells in situ in the body. Its core breakthrough lies in completely avoiding the in vitro operation process, shortening the treatment cycle to several days, and reducing costs through industrial large-scale production of universal vectors to achieve "off-the-shelf supply". The in vivo delivery system can specifically recognize T cells and efficiently transduce them. Even if the number or function of the patient's T cells is impaired, functional CAR-T cells may still be successfully generated, reducing the risk of manufacturing failure. More importantly, T cells modified in the natural microenvironment are more likely to maintain the stem cell-like memory phenotype (Tscm), thus enhancing their in vivo persistence and re-expansion ability. In addition, the gene delivery strategy based on mRNA technology does not require the mRNA molecule to enter the nucleus for expression in the cytoplasm and will not integrate into the host cell genome, so there is no risk of insertional mutagenesis and the induction of "second tumors". In summary, the technological innovation represented by in vivo engineered CAR-T based on mRNA-LNP technology is expected to promote the development of cell therapy towards a more efficient and inclusive direction, bringing breakthrough progress in the fields of malignant tumors and chronic diseases.

[0006] In previous studies by the inventors, some researchers proposed the "novel method for in vivo generation of CAR-T" described in CN116350757A. However, the method used a targeted lipid nanoparticle for packaging retroviruses, and the antibody conjugation method was a chemical reaction between thiol and Mal. Moreover, it was not verified whether this method could inhibit multiple myeloma or human B-lymphocytic leukemia, making it impossible to predict whether it could treat the corresponding diseases. Summary of the Invention

[0007] The present invention adopts the following technical solutions, mainly to solve the problem of lacking the ability to generate CAR-T in vivo and effectively treat multiple myeloma cells or human B-lymphocytic leukemia cells.

[0008] First, the lipid nanoparticle LNP is introduced. The lipid nanoparticle LNP can be used for in vivo targeted delivery and can be directly injected into the patient's body to complete the genetic modification of T cells in situ in the body. At the same time, since the mRNA molecule can be expressed in the cytoplasm without entering the nucleus and will not integrate into the host cell genome, there is no insertion mutation, reducing the risk of triggering "secondary tumors".

[0009] The preparation method of the lipid nanoparticle LNP includes the following steps:

[0010] S1. Obtain a compound lipid-ethanol solution: formed by mixing D-LIN-MC3-DMA, DSPC, DSPE-PEG2000-N3, cholesterol and ethanol; obtain an mRNA-citrate buffer solution: formed by mixing a citrate buffer solution and mRNA.

[0011] S2. Mix the compound lipid-ethanol solution with the mRNA-citrate buffer solution to obtain LNP.

[0012] In the aforementioned S1, obtaining the compound lipid-ethanol solution only means that this raw material needs to be used, but it does not mean that the compound lipid-ethanol solution needs to be prepared independently. It does not include the preparation method, but means that the compound lipid-ethanol solution is formed by mixing D-LIN-MC3-DMA, DSPC, DSPE-PEG2000-N3, cholesterol and ethanol. Similarly, the mRNA-citrate buffer solution also means that it is formed by mixing a citrate buffer solution and mRNA.

[0013] Regarding the preparation method of the lipid nanoparticle LNP, the technical features involved can also specifically adopt the following conditions, and any one of the conditions can be independently selected and implemented according to needs:

[0014] One of them, in S1, the molar ratio of D-LIN-MC3-DMA, DSPC, DSPE-PEG2000-N3, and cholesterol in the compound lipid-ethanol solution is 50±5:10±1:1.5±0.15:38.5±3.85; the mRNA concentration in the mRNA-citrate buffer solution is 55.09±5.5 ng / mL; in S2, the mixing ratio of the compound lipid-ethanol solution to the mRNA-citrate buffer solution is 1:3. The foregoing representation can fluctuate within a certain range, and those within this range or those that deviate from this range but have similar effects to the present invention should be within the scope of the present invention. Further, the specific ratio of each component in the compound lipid-ethanol solution is that the molar ratio of D-LIN-MC3-DMA, DSPC, DSPE-PEG2000-N3, and cholesterol is 50:10:1.5:38.5; the mRNA concentration in the mRNA-citrate buffer solution is 55.09 ng / mL. When introducing mRNA, the aqueous solution uses citrate buffer, and citrate buffer has a promoting effect on pH regulation, ion environment optimization, and lipid-nucleic acid interaction.

[0015] Another one, in this part, as long as LNP is obtained by the reaction, it should be within the scope of the present invention. Then, in order to directly obtain LNP, in S2: mix the compound lipid-ethanol solution with the mRNA-citrate buffer solution, and obtain LNP after dilution and dialysis; further, in S2, further concentrate after dilution and dialysis to obtain LNP. When mixing the compound lipid-ethanol solution with the mRNA-citrate buffer solution, the two phases can be mixed at 1 (ethanol phase):3 (buffer phase) at a rate of 3 ml / min:9 ml / min. Mixing the compound lipid-ethanol solution with the mRNA-citrate buffer solution can result in the formation of LNP, but at this time, LNP is still in the mixture. Then, first dilute the mixture with ethanol and perform dialysis to preliminarily remove other impurities, and then further concentrate to obtain LNP. Further, when further concentrating, the dialyzed LNP product can be added to an ultrafiltration tube with an MWCO of 30KDa and centrifuged at 3000g×20 min for further concentration.

[0016] Another one, the mRNA is CD19 CAR mRNA with the sequence of SEQ ID NO.1 or BCMA-CS1 CAR mRNA with the sequence of SEQ ID NO.2.

[0017] The lipid nanoparticle LNP prepared by the preparation method of any of the foregoing lipid nanoparticles LNP may have slight differences according to different schemes, but should all be within the scope of the present invention, especially the lipid nanoparticle LNP obtained by the method further defined in the foregoing three paragraphs.

[0018] Use of any of the foregoing lipid nanoparticles (LNPs) in the preparation of a product for treating multiple myeloma or human B-lymphocytic leukemia. As a delivery vector, the lipid nanoparticles (LNPs) can be directly injected into the human body for targeted delivery.

[0019] Then, Ab-LNP is introduced. Ab-LNP is a product obtained by modifying the surface of lipid nanoparticles (LNPs) with an antibody targeting T cells through click chemistry technology. After being injected into the body, it can specifically recognize T cells and efficiently transduce. Even if the number or function of the patient's T cells is impaired, functional CAR-T cells may still be successfully generated. Moreover, T cells modified in the natural microenvironment are more likely to maintain the stem cell-like memory phenotype (Tscm), thereby enhancing their in vivo persistence and re-expansion ability. Since the mRNA molecule of Ab-LNP can be expressed in the cytoplasm without entering the nucleus and will not integrate into the host cell genome, there is no risk of insertional mutation and no risk of triggering "secondary tumors".

[0020] The preparation method of Ab-LNP includes the following steps:

[0021] Obtain LNP: The lipid nanoparticles (LNPs) prepared by using the preparation method of any of the foregoing lipid nanoparticles (LNPs) or the same LNPs obtained by other methods;

[0022] Prepare Ab-LNP: Mix and react LNP with the DBCO-modified antibody to obtain Ab-LNP.

[0023] Regarding the preparation method of Ab-LNP, the technical features involved therein can also be specifically adopted under the following conditions, and any one of the conditions can be independently selected and implemented according to needs:

[0024] One of them, in this part, as long as Ab-LNP is obtained by the reaction, it should be within the scope of the present invention. Of course, in order to directly obtain Ab-LNP, further extraction is required, such as centrifugation, etc. After mixing LNP with the DBCO-modified antibody, react at room temperature first and then incubate at 4°C.

[0025] Another one, the DBCO-modified antibody is any one of anti-human CD3, CD4, CD5, CD8, CD7 antibodies, and it can also be other non-strictly defined ones.

[0026] The third one, the preparation steps of the DBCO-modified antibody include: Mix DBCO-NHS with the antibody, incubate, and remove the excess free DBCO-NHS to obtain the DBCO-modified antibody. Further, the mixing of DBCO-NHS and the antibody can be carried out at a molar ratio of 10:1; furthermore, during incubation, a conventional protocol can be adopted, and incubation can be carried out at 4°C.

[0027] The Ab-LNP prepared by the preparation method of any one of the foregoing Ab-LNPs may vary slightly depending on different schemes, but all should be within the scope of the present invention. In particular, the Ab-LNP is obtained by the method further defined in the foregoing three paragraphs.

[0028] Use of any one of the foregoing Ab-LNPs in the preparation of a product for treating multiple myeloma or B-lymphocytic leukemia. The product for treating multiple myeloma or human B-lymphocytic leukemia is a product that can complete T cell modification in vivo. Therefore, the dosage form of the product for treating multiple myeloma or B-lymphocytic leukemia is an intravenous injection preparation. The Ab-LNP can be directly injected intravenously. After injection, it can effectively kill multiple myeloma cells and B-lymphocytic leukemia cells, thereby playing a role in relieving and treating tumors. The Ab-LNP can specifically recognize T cells after being injected into the body and complete in situ gene modification of T cells in the body.

[0029] In the present disclosure, there is no need to perform gene modification in vitro through a viral vector. Lipid nanoparticles encapsulate mRNA molecules encoding humanized CD19 CAR sequence and BCMA-CS1 bispecific CAR sequence respectively through a microfluidic mixing technique, and then the surface of the obtained LNP is modified with an antibody targeting T cells through click chemistry to prepare an engineered mRNA-LNP in vivo targeted delivery vector, which can effectively treat multiple myeloma or B-lymphocytic leukemia. Brief Description of the Drawings

[0030] Figure 1 It is the preparation process and ratio of T cell-targeted Ab-LNP; (A) Schematic diagram of the structure of BCMA-CS1 bispecific CAR mRNA; (B) Schematic diagram of the experiment of preparing mRNA-LNPs through a microfluidic device and modifying the targeting antibody through click chemistry.

[0031] Figure 2 It is the morphology of Ab-LNP; (A) Representative transmission electron microscope image of Ab-LNP, the scale bar is 100 nm; (B) Particle size distribution of Ab-LNP measured by dynamic light scattering (DLS).

[0032] Figure 3 Confocal laser scanning microscope images of primary human T cells incubated with 5-FAM-labeled mRNA-LNPs for 0 h and 4 h.

[0033] Figure 4In vitro effector function study results of generating CAR-T cells by Ab-LNP transfection; the killing activity of Ab-LNP engineered CAR-T cells against RPMI 8266-Luc cells was evaluated using bioluminescence imaging (A) and corresponding quantitative analysis (B); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = no significant difference.

[0034] Figure 5 To detect the positive rate of BCMA / CS1 CAR-T cells reprogrammed in situ in vivo by flow cytometry.

[0035] Figure 6 For the toxicity results of the cytotoxicity experiment. Specific implementation manners

[0036] The present invention will be further described below in combination with specific research cases. The experimental steps in the text are not detailed, and for the un-detailed parts, reference can be made to the existing technologies.

[0037] Experimental content

[0038] ① Construction and preparation of the engineered mRNA-LNP in vivo delivery vector (the preparation process of the T cell-targeting Ab-LNP is as shown in A in Figure 1 )

[0039] a. Prepare the compound lipid-ethanol solution. Calculate the required mRNA concentration (where the structure of BCMA-CS1 CAR mRNA is as shown in B in Figure 1 ) according to N / P = 8 and the flow rate ratio (FRR) of the citrate buffer solution to the compound lipid solution = 3. The formula is as follows:

[0040]

[0041]

[0042] b. Filter the compound lipid-ethanol solution and the mRNA-citrate buffer solution through a 0.22 μm filter membrane;

[0043] c. Set the cleaning mode. Add 3 ml of absolute ethanol to each of the two 5 ml syringes, exhaust the air bubbles, set the total volume to 6 ml, then reset, exhaust the air bubbles again, set the cleaning mode, and draw back the syringes to form air columns in the two syringes respectively. The purpose is to empty the ethanol to eliminate the influence of ethanol on RNA, and then reset after completion;

[0044] d. Aspirate the lipid-ethanol solution into a 1 ml syringe (at least 0.5 ml is required according to needs), aspirate the mRNA-citrate buffer solution into a 3 ml syringe (at least 1.5 ml is required), and exhaust the air in the syringes;

[0045] e. Mix with NEXSTAR C4 chips, with the two phases mixed at a ratio of 1 (ethanol phase): 3 (buffer phase) and at a flow rate of 3 ml / min: 9 ml / min to obtain LNP;

[0046] f. After sample collection, immediately dilute the ethanol concentration to less than 1% with 30 times the volume of PBS;

[0047] g. Dialysis: After preparation, place it in a dialysis cup (Slide-A-Lyzer TM MINI Dialysis Device, 10KDa MWCO, 0.5 mL), add 14 mL of PBS, and dialyze on a shaker. Change the solution after 2 hours until dialysis is completed after 18 hours;

[0048] h. Add the dialyzed LNP product to an ultrafiltration tube with a MWCO of 30KDa and centrifuge at 3000g × 20 min for further concentration;

[0049] i. React LNP with DBCO-modified antibody (which can be anti-human CD3, CD4, CD5, CD8, CD7 antibody) at room temperature for 4 h, and then incubate overnight at 4°C. After the reaction is completed, add the product to an ultrafiltration tube with a MWCO of 300KDa and centrifuge at 3000g × 20 min to remove the excess free antibody; (The preparation method of the DBCO-modified antibody is: mix DBCO-NHS with the antibody at a molar ratio of 10:1, incubate overnight at 4°C, and then add the product to an ultrafiltration tube with a MWCO of 30KDa and centrifuge at 3000g × 20 min to remove the excess free DBCO-NHS);

[0050] j. Store the final product Ab-LNP in PBS containing 10% sucrose at 4°C or -20°C.

[0051] CD19 CAR mRNA sequence (SEQ ID NO.1):

[0052]

[0053] BCMA-CS1 CAR mRNA sequence (SEQ ID NO.2):

[0054]

[0055] ②Characterization of Ab-LNP

[0056] a. Determination of hydrated particle size and potential

[0057] The hydrated particle size distribution and Zeta potential of the Ab-LNP aqueous solution were detected by a laser particle size analyzer. The above nanoparticle dispersions were placed in PBS at 4 °C for 21 days, and their particle size changes were monitored regularly;

[0058] b. Morphology observation

[0059] Ab-LNP was dropped onto a copper grid. After drying at room temperature, a drop of phosphotungstic acid (W / V 2%) was added for negative staining for 30 s. After drying, the morphology was observed by transmission electron microscopy.

[0060] As shown in A of Figure 2 , Ab-LNP presented as regular spherical shapes with a particle size of about 100 nm; as shown in B of Figure 2 , dynamic light scattering further confirmed the uniform distribution of the nanoparticles.

[0061] c. Determination of the encapsulation efficiency of Ab-LNP

[0062] Demulsification of Ab-LNP: Triton-100 was diluted to 2% with 1×TE. 100 μL was taken and added to CellCarrier-96 Ultra Microplates. 1 μL of the prepared LNP was added and treated for 5 minutes, and then 100 μL of RiboGreen working solution was added;

[0063] Determination of free nucleic acid in Ab-LNP: 100 μL of 1×TE was added to CellCarrier-96 Ultra Microplates. 1 μL of the prepared LNP was added and mixed evenly, and then 100 μL of RiboGreen working solution was added. Finally, the plate was read with a multifunctional microplate reader;

[0064] Data analysis: Direct encapsulation of mRNA made Ab-LNP have a better encapsulation efficiency, which was better than that of the common targeted lipid nanoparticles packaging retroviruses.

[0065] ③Study on cellular uptake of mRNA-LNP

[0066] For the confocal laser experiment:

[0067] mRNA molecules labeled with 5-carboxyfluorescein (5-FAM) were used as fluorescent probes to explore the cellular uptake phenomenon of Ab-LNP in human CD3+ primary T cells and the lysosomal escape behavior after endocytosis.

[0068] a. Seed human primary T cells at a cell density of 1×10 6 / mL into 12-well plates, maintaining a total cell number of 2×10 6 / well;

[0069] b. Add 5 μg / mL of 5-FAM-mRNA-LNP to the well plates and gently pipette to mix evenly;

[0070] c. Incubate the well plates in a 37 °C cell culture incubator for 0 h and 2 h respectively;

[0071] d. At the predetermined time points, collect the cells in each group, wash them 3 times with sterile PBS, and then add 1×lysosomal fluorescent probe Lyso-Tracker Red to the cell suspension;

[0072] e. Incubate in a cell culture incubator at 37 °C for 30 min, and then immediately wash 3 times with PBS;

[0073] f. Centrifuge at 400 g for 10 min to collect the cells;

[0074] g. Fix with 4% paraformaldehyde for 15 min at room temperature;

[0075] h. Centrifuge at 400 g for 10 min to collect the cells, and stain with DAPI staining solution for 15 min at room temperature;

[0076] i. Wash 3 times with PBS and centrifuge at 400 g for 10 min;

[0077] j. Resuspend the cell pellet in 1 mL of PBS, transfer it to a confocal dish, and observe the fluorescence signal and acquire images under a confocal microscope.

[0078] Figure 3 As shown, confocal laser scanning microscope images of human primary T cells after incubation with 5-FAM-labeled mRNA-LNPs for 0 h and 4 h. Among them, the cell nucleus, late endosomes and lysosomes, and mRNA are labeled with DAPI (blue), LysoTrackerRed (red), and 5-FAM (green) respectively. The results show that mRNA can be released from lysosomes into the cytoplasm and then further expressed as proteins.

[0079] ④ In vitro effector function study of CAR-T cells generated based on Ab-LNP transfection

[0080] a. Seed 1×10 5 Nalm6-Luc or RPMI-8266-Luc cells into each well of a 96-well plate in the dark;

[0081] b. Add CAR-T cells prepared with CAR-mRNA-LNP according to different effector-to-target ratios (0:1, 1:2, 1:1, 2:1, 5:1, 10:1);

[0082] c. Incubate in a 37 °C incubator for 24 h;

[0083] d. Add 10 μL of D-luciferin substrate (15 mg / mL) to each well;

[0084] e. After 10 min, detect the luminescence intensity of each well with a small animal in vivo imaging system and collect pictures.

[0085] As Figure 4 shown in A–B, as the effector-to-target ratio increases, the bioluminescence intensity of the cells gradually decreases, indicating that the killing rate of CAR-T cells against target cells gradually increases, and it can effectively kill tumor cells. And at the effector-to-target ratio of 1:1, it already has a significant killing effect compared with the 0:1 group. At the effector-to-target ratio of 5:1, it already has a very significant killing effect compared with the 0:1 group. Finally, at the effector-to-target ratio of 10:1, it already has an extremely significant killing effect compared with the 0:1 group. The above all indicate that at the effector-to-target ratio of 1:1, CAR-T cells have already exerted an effective killing effect on target cells (the Nalm6-Luc group has similar results).

[0086] ⑤ Study on the effect of Ab-LNP in in situ inducing CAR-T cells in vivo

[0087] a. NOD-Prkdcscid IL2rgem1 / Smoc (M-NSG) mice, 7–8 weeks old, weighing 20 ± 2 g, were purchased from Shanghai Model Organisms Center, Inc., with a body weight of 20 ± 2 g. The animals were housed in an isolation package laboratory in a SPF-class animal house and adaptively raised in a barrier environment for at least 1 week before being used for subsequent experiments. All experimental operations were approved by the Ethics Committee of Huazhong University of Science and Technology and were strictly carried out in accordance with relevant experimental guidelines;

[0088] b. On Day-1, inject NSG mice with human peripheral blood mononuclear cells resuspended in 1×10 7 PBS via the tail vein;

[0089] c. On Day-​0, inject Ab-LNP via the tail vein at a dose of 10 μg / mouse. After 24–48 h, collect the peripheral blood of the mice and detect the CAR positive rate of T cells in the blood by flow cytometry.

[0090] As Figure 5As shown, after intravenous injection, Ab-LNP can successfully generate CAR-T cells in situ in mice, with a CAR positive rate of up to 32.4%, reaching an excellent level in the field, indicating that Ab-LNP can effectively kill tumor cells in vivo after intravenous injection.

[0091] ⑥Cytotoxicity experiment

[0092] The cytotoxicity of Ab-LNP to human CD3+ primary T cells was evaluated using a CCK-8 kit. The experimental steps are as follows:

[0093] a. Seed human CD3+ primary T cells at 2×10 4 / well in a 96-well plate;

[0094] b. Treat the cells with a series of different doses of mRNA-LNPs (where the mRNA corresponds to 0, 0.6, 1.2, 2.4, and 4.8 μg respectively), and co-incubate for 24 h in cell culture at 37°C;

[0095] c. After the treatment, take out the plate, add 10 μL of CCK-8 detection reagent to each well, and incubate in the dark at 37°C for 1 - 4 h;

[0096] d. Place the microplate on a microplate reader and measure the absorbance value of each well at a wavelength of 450 nm.

[0097] As Figure 6 shown, the survival rate of human primary CD3+ T cells (n = 4) after treatment with Ab-LNPs containing different doses of mRNA remained at a relatively high level as detected by the CCK8 method. At each dose condition, human primary T cells maintained good cell viability, indicating that Ab-LNP has no toxicity to human CD3+ primary T cells.

[0098] Summary and analysis

[0099] The preparation of traditional CAR-T has a long time (2 - 4 weeks), high cost (hundreds of thousands of US dollars), depends on the quality of the patient's own T cells, and viral vectors may cause secondary tumors (citing FDA data and multiple research cases). The invention avoids in vitro operations through in vivo delivery, uses mRNA-LNP technology to avoid genome integration, shortens the production time, reduces the cost, and improves safety. The invention has the advantages of non-integration, delivery efficiency, shortened production cycle, and T cell phenotype of mRNA-LNP. In terms of economic effects, compared with traditional costs, the cost reduction of large-scale production is prominent. In terms of social effects, it improves the accessibility of treatment, especially for patients in remote areas, and avoids the long-term medical burden brought by the risk of secondary tumors.

[0100] Those skilled in the art can clearly understand that various modifications to the above embodiments can be made without departing from the general spirit and concept of the present invention. All of them fall within the protection scope of the present invention. The protection scope of the present invention shall be subject to the appended claims of the present invention.

Claims

1. A method for preparing lipid nanoparticles (LNP), characterized in that, It includes the following steps: S1. Obtain a compound lipid-ethanol solution, which is formed by mixing D-LIN-MC3-DMA, DSPC, DSPE-PEG2000-N3, cholesterol and ethanol; obtain an mRNA-citrate buffer solution, which is formed by mixing a citrate buffer solution and mRNA; S2. Mix the compound lipid-ethanol solution with the mRNA-citrate buffer solution to prepare LNP.

2. The preparation method of the lipid nanoparticle LNP according to claim 1, characterized in that, In S1, the molar ratio of D-LIN-MC3-DMA, DSPC, DSPE-PEG2000-N3, cholesterol in the compound lipid-ethanol solution is 50±5:10±1:1.5±0.15:38.5±3.85; the concentration of mRNA in the mRNA-citrate buffer solution is 55.09±5.5 ng / mL; preferably, the molar ratio of D-LIN-MC3-DMA, DSPC, DSPE-PEG2000-N3, cholesterol in the compound lipid-ethanol solution is 50:10:1.5:38.5, and the concentration of mRNA in the mRNA-citrate buffer solution is 55.09 ng / mL; in S2, the mixing ratio of the compound lipid-ethanol solution to the mRNA-citrate buffer solution is 1:3; The mRNA is CD19 CAR mRNA with the sequence of SEQ ID NO.1 or BCMA-CS1 CAR mRNA with the sequence of SEQ ID NO.

2.

3. The preparation method of the lipid nanoparticle LNP according to claim 1, wherein, In S2, mix the compound lipid-ethanol solution with the mRNA-citrate buffer solution, and obtain LNP after dilution and dialysis; preferably, in S2, obtain LNP after further concentration after dilution and dialysis.

4. Lipid nanoparticle LNP prepared by the preparation method of any one of the lipid nanoparticles LNP in claims 1 to 3.

5. Use of the lipid nanoparticle LNP in claim 4 in the preparation of a product for treating multiple myeloma or human B-lymphocytic leukemia.

6. Preparation method of Ab-LNP, characterized in that, It includes the following steps: Obtain LNP: Obtain the lipid nanoparticle LNP in claim 4; Ab-LNP preparation: Mix and react the LNP with an antibody modified with DBCO to obtain Ab-LNP.

7. The preparation method of the Ab-LNP according to claim 6, wherein, The antibody modified with DBCO is any one of anti-human CD3, CD4, CD5, CD8, CD7 antibodies; preferably, the preparation steps of the antibody modified with DBCO include: mixing DBCO-NHS with the antibody, incubating, and removing the excess free DBCO-NHS to obtain the antibody modified with DBCO.

8. Ab-LNP prepared by the preparation method of any one of the Ab-LNPs in claims 6 to 7.

9. Use of the Ab-LNP in claim 8 in the preparation of a product for treating multiple myeloma or human B-lymphocytic leukemia.

10. The application according to claim 9, wherein, The dosage form of the product for treating multiple myeloma or B-lymphocytic leukemia is an intravenous injection preparation.

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