Drug for in-vivo engineered CAR-T and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a medicine for in-vivo engineering CAR-T and a preparation method thereof. The drug provided by the invention comprises a first RNA and a second RNA which are used for coding a chimeric antigen receptor in a specific molar ratio, the second RNA is used for coding at least two cytokines of CCL19, IL-17, IL-15, IL-2 or IL-24, by adopting the drug, the gene delivery efficiency and targeting of in-vivo engineered CAR-T can be improved, better T cell transformation and CAR + T cell generation effects are realized, the CAR-T cell depletion is reduced, and the drug has a good application prospect. According to the present invention, the CAR-T gene is constructed, such that the CAR-T gene can survive for a long time in the tumor microenvironment and continuously provide the treatment effect, has the excellent anti-tumor treatment effect, has the excellent application prospect in the in-vivo engineering CAR-T treatment, and has the important significance on the promotion of the efficient, safe and economical development of the CAR-T treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a drug for in vivo engineered CAR-T and its preparation method. Background Technology

[0002] In vivo engineered CAR-T refers to a tumor treatment method that directly modifies T cells in the patient's body using a gene delivery system to express chimeric antigen receptors (CARs), ultimately forming CAR-T cells with tumor-killing functions. Compared to in vitro engineered CAR-T, in vivo engineered CAR-T does not require the collection of T cells; it only requires the delivery of the CAR gene into the in vivo T cells via a vector to achieve in situ modification of the T cells. This has the potential to simplify the process, reduce costs, and expand the scope of applications.

[0003] However, T cells are widely distributed in tissues such as blood, lymph nodes, and spleen, and most of them are in a resting or low-activity state. It is difficult for vectors to target T cells accurately and efficiently. Moreover, compared with in vitro modification methods that can screen for depletion subtypes, in vivo modified T cells are difficult to survive for a long time in the hypoxic and metabolically disordered tumor microenvironment and to exert a sustained therapeutic effect. They have problems such as low gene delivery efficiency, poor targeting, and difficulty in maintaining CAR-T cell function, which have significant limitations.

[0004] Therefore, obtaining a CAR-T cell therapy with excellent gene delivery efficiency, T cell targeting, and the ability to improve the functional maintenance of CAR-T cells in the tumor microenvironment is of great significance for promoting the development of CAR-T therapy towards a more efficient, safe, and inclusive direction. Summary of the Invention

[0005] The primary objective of this invention is to address the problems of low gene delivery efficiency, poor targeting, and difficulty in maintaining CAR-T cell function in existing in vivo engineered CAR-T cells, and to provide a drug for in vivo engineered CAR-T cells.

[0006] A second objective of this invention is to provide a method for preparing a drug for in vivo engineered CAR-T.

[0007] Specifically, the drug for in vivo engineered CAR-T provided by the present invention includes a first RNA and a second RNA in a molar ratio of 1:(0.2~3); wherein the first RNA encodes a chimeric antigen receptor; and the second RNA includes at least two of the following: CCL19-RNA, which encodes CCL19; IL-7-RNA, which encodes IL-7; IL-15-RNA, which encodes IL-15; IL-2-RNA, which encodes IL-2; and IL-24-RNA, which encodes IL-24.

[0008] Further, in the first RNA, the target sites of the chimeric antigen receptor include one or more of BCMA, CD19, CD20, CD22, CD30, CD123, CD38, CD5, Claudin 18.2, MUC1, NKG2D ligand, human epidermal growth factor receptor 2, prostate-specific membrane antigen, EGFRvIII, mesothelin, carcinoembryonic antigen, disialotetrahexosylganglioside, and prostate-specific membrane antigen.

[0009] Furthermore, the first RNA comprises a nucleotide fragment with the sequence shown in SEQ ID NO:1.

[0010] Further, the drug has at least one of the following characteristics: (i) the second RNA comprises CCL19-RNA and IL-7-RNA, and the molar ratio of the first RNA, CCL19-RNA, and IL-7-RNA is 1:(0.2~1):(0.2~1); (ii) the second RNA comprises CCL19-RNA, IL-7-RNA, and IL-15-RNA, and the molar ratio of the first RNA, CCL19-RNA, IL-7-RNA, and IL-15-RNA is 1:(0.2~1):(0.2~1). (iii) The second RNA comprises CCL19-RNA and IL-2-RNA, and the molar ratio of the first RNA, CCL19-RNA and IL-2-RNA is 1:(0.2~1):(0.2~1); (iv) The second RNA comprises CCL19-RNA and IL-24-RNA, and the molar ratio of the first RNA, CCL19-RNA and IL-24-RNA is 1:(0.2~1):(0.2~1).

[0011] Further, the drug has at least one of the following characteristics: (i) the CCL19-RNA comprises a nucleotide sequence as shown in SEQ ID NO:2; (ii) the IL-7-RNA comprises a nucleotide sequence as shown in SEQ ID NO:3; (iii) the IL-15-RNA comprises a nucleotide sequence as shown in SEQ ID NO:4; (iv) the IL-2-RNA comprises a nucleotide sequence as shown in SEQ ID NO:5; and (v) the IL-24-RNA comprises a nucleotide sequence as shown in SEQ ID NO:6.

[0012] Furthermore, the drug has at least one of the following characteristics: (i) the first RNA is selected from one or more of linear mRNA, circular RNA, and self-amplifying RNA; (ii) the second RNA is selected from one or more of linear mRNA, circular RNA, and self-amplifying RNA.

[0013] Further, the drug has at least one of the following characteristics: (i) the first RNA is a linear RNA, the first RNA comprising a cap structure, a 5'-untranslated region, a coding region, a 3'-untranslated region and a polyA tail connected sequentially along the 5' to 3' direction; (ii) the second RNA is a linear RNA, the second RNA comprising a cap structure, a 5'-untranslated region, a coding region, a 3'-terminal untranslated region and a polyA tail connected sequentially along the 5' to 3' direction.

[0014] Furthermore, the drug includes a carrier on which the first RNA and the second RNA are mounted, the carrier being selected from one or more of lipid nanoparticles, inorganic nanoparticles, protein nanoparticles, and virus-like particles.

[0015] Furthermore, when the carrier is a lipid nanoparticle, the total loading amount of the first RNA and the second RNA on the carrier is 1 μg to 100 μg.

[0016] The present invention provides a method for preparing a drug for in vivo engineered CAR-T, comprising: S1, mixing a lipid composition with ethanol to obtain an organic phase solution; S2, mixing a first RNA, a second RNA, and water to obtain an aqueous phase solution; S3, mixing the organic phase solution and the aqueous phase solution to obtain the lipid nanoparticles; wherein the lipid composition comprises 100 to 400 parts by mass of ionizable lipids, 20 to 80 parts by mass of auxiliary lipids, 80 to 120 parts by mass of cholesterol, and polyethylene glycol lipids at a concentration of 1 to 10 parts by mass; the first RNA is the aforementioned first RNA; the second RNA is the aforementioned second RNA; and the molar ratio of the first RNA to the second RNA is 1:(0.2 to 3).

[0017] Further, the preparation method includes one or more of the following technical features: (i) the ionizable lipid includes trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl[2,3-(dioleenyloxy)propyl]ammonium chloride, 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl], 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester, 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl ester, and 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxy) (ii) The auxiliary lipids include one or more of dioleoylphosphatidylethanolamine, distearate phosphatidylcholine, and sterols; (iii) The polyethylene glycol lipids include one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, distearate phosphatidylethanolamine-polyethylene glycol, and dioleoylphosphatidylethanolamine-polyethylene glycol; (iv) In step S1, the concentration of the lipid composition in the organic phase solution is 10 mg / mL to 50 mg / mL; (v) In step S2, the total concentration of the first RNA and the second RNA in the aqueous phase solution is 0.1 mg / mL to 1 mg / mL of nucleic acid molecules; (vi) In step S3, the mixing flow rate ratio of the organic phase solution and the aqueous phase solution is 1:(2~5).

[0018] Beneficial effects: The drug provided by this invention comprises a first RNA and a second RNA in a specific molar ratio; wherein the first RNA encodes a chimeric antigen receptor, and the second RNA includes at least two RNAs capable of encoding CCL19, IL-17, IL-15, IL-2, or IL-24. Using this drug for in vivo CAR-T cell engineering can improve gene delivery efficiency and targeting, achieving better T cell modification effects, and enhancing the CD8+ expression of tumor tissue. + and CD4 + The significantly increased proportion of CAR-positive T cells, improved CAR-T cell tolerance, and reduced CAR-T cell depletion enabled them to survive long-term in the tumor microenvironment and continue to exert therapeutic effects, resulting in superior anti-tumor therapeutic efficacy. This has excellent application prospects in realizing in vivo engineered CAR-T therapy and is of great significance for promoting the development of CAR-T therapy towards a more efficient, safe, and inclusive direction.

[0019] The reasons for the aforementioned effects of the drug are speculated to include: the expression of at least two of the RNAs expressing CCL19, IL-17, IL-15, IL-2, or IL-24 in T cells and non-T cells in vivo have a synergistic effect, enabling the enrichment of T cells by utilizing the expression of cytokines in target and non-target cells without triggering a cytokine storm, thereby improving gene delivery efficiency and targeting. At the same time, it promotes the migration and residence of immune cells such as T cells, NK cells, and DCs in tumor tissue, enhances vascular permeability, alters the expression of immune-related factors in tumor tissue, synergistically amplifies the cytokine network, forms a positive feedback loop of inflammation-immune activation, thereby improving the tumor immune microenvironment and enhancing the metabolic adaptability of T cells, enabling them to survive for a long time and exert a sustained therapeutic effect in the hypoxic and metabolically disordered tumor microenvironment, thus achieving excellent anti-tumor effects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of the mRNAs in this invention (CAR-mRNA, CCL19-mRNA, IL-7-mRNA, IL-15-mRNA, IL-2-mRNA and IL-24-mRNA). Figure 2 The figure shows the experimental results of mouse survival rate provided in the embodiments of the present invention; Figure 3 The figure shows the experimental results of mouse tumor volume provided in the embodiments of the present invention; Figure 4 CD4 in mouse tumor tissue provided in the embodiments of the present invention + Figure showing the experimental results of the percentage of CAR-positive cells in T cells; Figure 5 CD8 of mouse tumor tissue provided in the embodiments of the present invention + Figure showing the experimental results of the percentage of CAR-positive cells in T cells; Figure 6 CD8 in mouse tumor cells provided in the embodiments of the present invention + / CD4 + A diagram showing the experimental results of the proportions. Detailed Implementation

[0021] The specific sequences involved in this invention are shown in Tables 1-1 to 1-3.

[0022] Table 1-1

[0023] Table 1-2

[0024] Table 1-3

[0025] To address the problems of low gene delivery efficiency, poor targeting, and difficulty in maintaining CAR-T cell function in existing in vivo engineered CAR-T cells, the inventors of this invention, after reviewing a large amount of literature, discovered that existing research has achieved this by introducing cytokines into in vitro engineered CAR-T cells, thus enabling CAR-T cells to... + T cells expressing related cytokines can achieve superior anti-tumor effects, but there are few reports on introducing cytokines into in vivo engineered CAR-T cells. Therefore, the inventors chose to introduce cytokines into in vivo engineered CAR-T cells as a technological improvement direction. They loaded RNA encoding CAR and RNA encoding cytokines onto the same vector to prepare a drug for in vivo engineered CAR-T cells. This drug was then used to treat tumor-bearing mice to verify its anti-tumor effects.

[0026] However, the in vivo environment is highly complex and uncontrollable, posing significant challenges to the application of cytokines in in vivo engineered CAR-T cells: co-expression of cytokines and CAR may lead to excessive activation of T cells and accelerate CAR degradation. + T cell depletion renders them incapable of killing, or leads to CAR by triggering local storms. + The elimination and apoptosis of T cells do not significantly enhance the anti-tumor effect. Furthermore, cytokines can be taken up and expressed by non-T cells such as macrophages and endothelial cells in vivo, leading to local or systemic cytokine accumulation. This results in side effects such as high fever, damage to normal tissues, and even death in mice, significantly reducing the survival time and rate of experimental mice. The inventors have indeed found in numerous experiments that, compared to tumor-bearing mice that do not receive in vivo CAR-T therapy or are treated with CAR-encoding RNA alone, treatment with RNA encoding cytokines such as CCL3, CCL4, IL-1, and IL-12, as well as RNA encoding CAR, significantly improves the in vivo CAR-T effect in mice. + There was no significant increase or even a decrease in T cells, and there were also phenomena such as increased tumor volume and accelerated death in mice.

[0027] Based on the problems discovered in experiments regarding the introduction of cytokines into in vivo engineered CAR-T cells, the inventors, through extensive and in-depth research and numerous experiments, creatively discovered that the combined use of at least two cytokines from CCL19, IL-7, IL-15, IL-2, and IL-24 in specific ratios with CAR can optimize the in vivo and tumor immune environment, improve gene delivery efficiency and targeting, achieve better T-cell modification effects, and significantly enhance CAR-T activity in tumor tissues. +The proportion of T cells is increased, thereby achieving better anti-tumor treatment effects. Based on this, the technical solution of the present invention was obtained.

[0028] In a first aspect, the present invention provides a drug for in vivo engineered CAR-T, the drug comprising a first RNA and a second RNA; wherein the first RNA encodes a chimeric antigen receptor; and the second RNA comprises at least two of the following: CCL19-RNA, wherein the CCL19-RNA encodes CCL19; IL-7-RNA, wherein the IL-7-RNA encodes IL-7; IL-15-RNA, wherein the IL-15-RNA encodes IL-15; IL-2-RNA, wherein the IL-2-RNA encodes IL-2; and IL-24-RNA, wherein the IL-24-RNA encodes IL-24.

[0029] In this invention, the molar ratio of the first RNA and the second RNA is specifically 1:(0.2~3), such as 1:0.2, 1:0.25, 1:0.3, 1:0.5, 1:0.8, 1:1.05, 1:1.5, 1:2, 1:2.1, 1:2.38, 1:2.5, 1:2.75, 1:3 or any value between them.

[0030] In this invention, the chimeric antigen receptor in the first RNA refers to a type of fusion protein that enables immune cells to accurately recognize specific target cells and initiate a killing response. Its structure specifically includes an antigen recognition domain, a transmembrane domain, and a signal transduction domain.

[0031] In this invention, the target of the chimeric antigen receptor can be determined according to the disease and / or target cells to be treated, and specific examples include, but are not limited to, one or more of the following: BCMA, CD19, CD20, CD22, CD30, CD123, CD38, CD5, Claudin 18.2, MUC1, NKG2D ligand, human epidermal growth factor receptor 2, prostate-specific membrane antigen, EGFRvIII, mesothelin, carcinoembryonic antigen, disialotetrahexosylganglioside, and prostate-specific membrane antigen.

[0032] In this invention, the first RNA is limited to the ability to achieve the expression of the corresponding chimeric antigen receptor in T cells. Those skilled in the art can design it according to the amino acid sequence of the corresponding chimeric antigen receptor and the application scenario. This invention does not impose any special limitations on its specific structure and sequence.

[0033] In some specific implementations, the first RNA may be, but is not limited to, one or more of the following: linear mRNA, circular RNA, and self-amplifying RNA.

[0034] In some specific implementations, when the first RNA is a linear RNA, it includes a cap structure, a 5'-untranslated region, a coding region, a 3'-untranslated region, and a polyA tail connected sequentially along the 5' to 3' direction. In this case, the first RNA exhibits better expression in T cells and provides a more desirable modification effect for T cells.

[0035] In some specific embodiments, the first RNA preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO:1.

[0036] In this invention, the second RNA preferably includes CCL19-RNA and IL-7-RNA, and the molar ratio of the first RNA, CCL19-RNA, and IL-7-RNA is specifically 1:(0.2~1):(0.2~1), such as 1:0.2:0.2, 1:0.2:0.5, 1:0.2:0.8, 1:0.5:0.2, 1:0.8:0.8, 1:1:1, or any value between them. In some specific embodiments, the molar ratio of the first RNA, CCL19-RNA, and IL-7-RNA is more preferably 1:(0.2~0.8):(0.2~0.8).

[0037] In this invention, the second RNA preferably includes CCL19-RNA, IL-7-RNA, and IL-15-RNA, and the molar ratio of the first RNA, CCL19-RNA, IL-7-RNA, and IL-15-RNA is specifically 1:(0.2~1):(0.2~1):(0.2~1), such as 1:0.2:0.2:0.2, 1:0.3:0.2:0.2, 1:0.5:0.2:0.2, 1:0.8:0.8:0.6, 1:0.8:0.8:0.8, 1:1:1:1, or any value between them. In some specific embodiments, the molar ratio of the first RNA, CCL19-RNA, IL-7-RNA, and IL-15-RNA is more preferably 1:(0.2~0.8):(0.2~0.8):(0.2~0.8).

[0038] In this invention, the second RNA preferably includes CCL19-RNA and IL-2-RNA, and the molar ratio of the first RNA, CCL19-RNA and IL-2 is specifically 1:(0.2~1):(0.2~1), such as 1:0.2:0.2, 1:0.2:0.3, 1:0.5:1, 1:1:1 or any value between them.

[0039] In this invention, the second RNA preferably includes CCL19-RNA and IL-24-RNA, and the molar ratio of the first RNA, CCL19-RNA and IL-24 is specifically 1:(0.2~1):(0.2~1), such as 1:0.2:0.2, 1:0.2:0.3, 1:0.5:1, 1:1:1 or any value between them.

[0040] In this invention, the second RNA is limited to enabling the expression of the corresponding cytokine in T cells. Those skilled in the art can design it based on the amino acid sequence of the corresponding cytokine and the application scenario; this invention does not impose any particular limitation on its specific structure and sequence. Specifically, those skilled in the art can design the CCL19-RNA based on the amino acid sequence of CCL19 and the application scenario; this invention does not impose any particular limitation on its specific structure and sequence. Similarly, those skilled in the art can design the IL-17-RNA based on the amino acid sequence of IL-17 and the application scenario; this invention does not impose any particular limitation on its specific structure and sequence. Likewise, those skilled in the art can design the IL-15-RNA based on the amino acid sequence of IL-15 and the application scenario; this invention does not impose any particular limitation on its specific structure and sequence.

[0041] In some specific implementations, the first RNA may be, but is not limited to, one or more of the following: linear mRNA, circular RNA, and self-amplifying RNA.

[0042] In some specific embodiments, when the second RNA is a linear RNA, the second RNA includes a cap structure, a 5'-untranslated region, a coding region, a 3'-terminal untranslated region, and a polyA tail connected sequentially along the 5' to 3' direction.

[0043] In this invention, the structures of the CCL19-RNA, IL-7-RNA, and IL-15-RNA, except for the protein-coding genes (CCL19, IL-17, or IL-15), can be completely identical, partially identical, or completely different.

[0044] In some specific embodiments, the CCL19-RNA preferably includes a nucleotide sequence as shown in SEQ ID NO:2.

[0045] In some specific embodiments, the IL-7-RNA preferably includes a nucleotide sequence as shown in SEQ ID NO:3.

[0046] In some specific embodiments, the IL-15-RNA preferably comprises a nucleotide sequence as shown in SEQ ID NO:4.

[0047] In some specific embodiments, the IL-2-RNA preferably includes a nucleotide sequence as shown in SEQ ID NO:5.

[0048] In some specific embodiments, the IL-24-RNA preferably comprises a nucleotide sequence as shown in SEQ ID NO:6.

[0049] In this invention, the drug includes a carrier for carrying and delivering a first RNA and a second RNA, and specific examples of the carrier include, but are not limited to, one or more of the following: lipid nanoparticles, inorganic nanoparticles, protein nanoparticles, and virus-like particles.

[0050] In some specific embodiments, when the carrier is a lipid nanoparticle, the total loading amount of the first RNA and the second RNA on the carrier is preferably 1 μg to 100 μg, such as 1 μg, 5 μg, 8 μg, 10 μg, 15 μg, 20 μg, 40 μg, 60 μg, 80 μg, 100 μg or any value between them; more preferably 5 μg to 20 μg.

[0051] Secondly, with the aim of obtaining the aforementioned drug for in vivo engineered CAR-T, the present invention also provides a method for preparing the drug for in vivo engineered CAR-T. This preparation method is a preferred embodiment for achieving the aforementioned preparation of the drug for in vivo engineered CAR-T, but is not limited thereto.

[0052] In this invention, the preparation method specifically includes: S1, mixing the lipid composition with ethanol to obtain an organic phase solution; S2, mixing the first RNA, the second RNA, and water to obtain an aqueous phase solution; S3, mixing the organic phase solution and the aqueous phase solution to obtain the lipid nanoparticles.

[0053] In this invention, in step S1, the lipid composition specifically includes: 100 to 400 parts by weight of an ionizable lipid, such as 100, 150, 180, 200, 250, 300, 345, 400 parts by weight, or any value between therewith; and 20 to 80 parts by weight of an auxiliary lipid, such as 20, 25, 30, 35, 40, 50 parts by weight. 60, 70, 80 parts by weight or any value therein; 80 to 120 parts by weight of cholesterol, such as 80, 85, 90, 100, 105, 110, 120 parts by weight or any value therein; and 1 to 10 parts by weight of polyethylene glycol lipids, such as 1, 2, 4, 8, 9, 10 parts by weight or any value therein.

[0054] In this invention, the ionizable lipid in the lipid composition refers to a class of lipids with ionization properties, capable of freely crossing cell membranes. This is a commonly used raw material in the preparation of existing lipid nanoparticles. This invention does not specifically limit its use; specific examples include, but are not limited to: trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl[2,3-(dioleoyloxy)propyl]ammonium chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl] (DC-Chol), 8-[(2-hydroxyethyl)[ One or more of the following: 6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-octylnonyl ester (SM-102), 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl ester (Dlin-MC3-DMA), and 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200).

[0055] In this invention, the auxiliary lipid in the lipid composition is a type of neutral lipid that improves stability and in vivo circulation. It is a type of raw material commonly used in the preparation of existing lipid nanoparticles. This invention does not specifically limit it. Specific examples include, but are not limited to, one or more of dioleoylphosphatidylethanolamine (OEA), distearate phosphatidylcholine (DSPC), and sterols.

[0056] In this invention, the polyethylene glycol lipid in the lipid composition refers to an amphiphilic polymer molecule composed of hydrophilic polyethylene glycol chains and hydrophobic alkyl chains and / or dialkyl chains. It is a type of raw material commonly used in the preparation of existing lipid nanoparticles. This invention does not particularly limit it. Specific examples include, but are not limited to, one or more of the following: 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG), distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG).

[0057] In this invention, in step S1, the concentration of the lipid composition in the organic phase solution is preferably 10 mg / mL to 50 mg / mL, such as 10 mg / mL, 12.5 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL or any value between them.

[0058] In this invention, in step S2, the first RNA is one or more of the first RNAs mentioned above, and the second RNA is one or more of the second RNAs mentioned above, which will not be elaborated here.

[0059] In this invention, in step S2, the total concentration of the first RNA and the second RNA in the aqueous solution is preferably 0.1 mg / mL to 1 mg / mL of nucleic acid molecules, such as 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL or any value between them.

[0060] In this invention, in step S3, the mixing flow rate ratio of the organic phase solution and the aqueous phase solution is preferably 1:(2~5), such as 1:2, 1:2.3, 1:2.5, 1:2.8, 1:3, 1:4, 1:5 or any value between them.

[0061] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0062] Preparation Example 1 This preparation example illustrates a method for preparing lipid nanoparticles, which include CAR-mRNA (sequence shown in SEQ ID NO:1) and CCL19-mRNA (sequence shown in SEQ ID NO:2) in a molar ratio of 1:0.8. The preparation specifically includes: S1. Take 250 parts by weight of SM-102 (Avet, catalog number O02010), 50 parts by weight of distearylphosphatidylcholine (Avet, catalog number DSPC), 100 parts by weight of cholesterol (Sigma, catalog number C8667) and 5 parts by weight of DMG-PEG2000 (Avet, catalog number O02005) and mix them thoroughly to obtain a lipid composition. Add the lipid composition and ethanol according to the final concentration of 25 mg / mL to obtain an organic phase solution. S2. Take CAR-mRNA and CCL19-mRNA with water at a total RNA concentration of 0.1 mg / mL to obtain an aqueous solution; S3. The organic phase solution and the aqueous phase solution were mixed in a microfluidic mixer at a flow rate of 3 mL / min and 9 mL / min, respectively, to obtain a suspension containing lipid nanoparticles. The suspension was dialyzed overnight using a Pur-A-Lyzer™ Midi dialysis kit (Merck, catalog number PURD35030-1KT) according to the instructions to obtain lipid nanoparticles LNP-1. Then, the mRNA in the lipid nanoparticles was quantified using Quant-iT RiboGreen RNA reagent (Thermo Fisher Scientific, catalog number R11491) according to the instructions. The total amount of mRNA loaded in the lipid nanoparticles LNP-1 was 5 μg.

[0063] The specific structure of CAR-mRNA is as follows: Figure 1 As shown, it includes a cap structure, a 5'-untranslated region, a coding region, a 3'-terminal untranslated region, and a polyA tail, connected sequentially along the 5' to 3' direction. The specific structure of CCL19-mRNA is as follows: Figure 1 As shown, it includes a cap structure, a 5'-untranslated region, an encoding region, a 3'-terminal untranslated region, and a polyA tail connected sequentially along the 5' to 3' direction.

[0064] Preparation Examples 2-5 Preparation Examples 2-5 used the method provided in Preparation Example 1 to prepare lipid nanoparticles, except that the molar ratio of CAR-mRNA to CCL19-mRNA in the lipid nanoparticles was different, as shown in Table 2. Other conditions were the same, and each lipid nanoparticle was obtained.

[0065] Table 2.

[0066] Preparation Example 6 This preparation example uses the method provided in Preparation Example 1 to prepare lipid nanoparticles. The difference is that the lipid nanoparticles include CAR-mRNA, CCL19-mRNA and IL-7-mRNA (sequence shown in SEQ ID NO:3) in a molar ratio of 1:0.8:0.8. Other conditions are the same, and lipid nanoparticles LNP-6 are obtained. The total amount of mRNA loaded in lipid nanoparticles LNP-6 is 5 μg.

[0067] The specific structure of IL-7-mRNA is as follows: Figure 1 As shown, it includes a cap structure, a 5'-untranslated region, an encoding region, a 3'-terminal untranslated region, and a polyA tail connected sequentially along the 5' to 3' direction.

[0068] Preparation Examples 7-10 Preparation Examples 7-10 used the method provided in Preparation Example 6 to prepare lipid nanoparticles, except that the molar ratio of CAR-mRNA, CCL19-mRNA and IL-7-mRNA in the lipid nanoparticles was different, as shown in Table 3. Other conditions were the same, and each lipid nanoparticle was obtained.

[0069] Table 3.

[0070] Preparation Example 11 This preparation example uses the method provided in Preparation Example 1 to prepare lipid nanoparticles. The difference is that the lipid nanoparticles include CAR-mRNA, CCL19-mRNA, IL-7-mRNA and IL-15-mRNA (sequences shown in SEQ ID NO:4) in a molar ratio of 1:0.8:0.8:0.6. Other conditions are the same, and lipid nanoparticles LNP-11 are obtained. The total amount of mRNA loaded in lipid nanoparticles LNP-11 is 5 μg.

[0071] The specific structure of IL-15-mRNA is as follows: Figure 1 As shown, it includes a cap structure, a 5'-untranslated region, an encoding region, a 3'-terminal untranslated region, and a polyA tail connected sequentially along the 5' to 3' direction.

[0072] Preparation Examples 12-15 This preparation example uses the method provided in Preparation Example 11 to prepare lipid nanoparticles. The difference is that the molar ratio of CAR-mRNA, CCL19-mRNA, IL-7-mRNA and IL-15-mRNA in the lipid nanoparticles is different, as shown in Table 4. Other conditions are the same, and each lipid nanoparticle is obtained.

[0073] Table 4.

[0074] Preparation Example 16 This preparation example uses the method provided in Preparation Example 1 to prepare lipid nanoparticles. The difference is that an equimolar amount of CAR-mRNA is used instead of CCL19-mRNA. All other conditions are the same, and lipid nanoparticles LNP-16 are obtained. The total amount of mRNA loaded in lipid nanoparticles LNP-16 is 5 μg.

[0075] Preparation Example 17 This preparation example uses the method provided in Preparation Example 6 to prepare lipid nanoparticles. The difference is that an equimolar amount of IL-2-mRNA (sequence shown in SEQ ID NO:5) is used instead of IL-7-mRNA. All other conditions are the same, and lipid nanoparticles LNP-17 are obtained. The total amount of mRNA loaded in lipid nanoparticles LNP-17 is 5 μg.

[0076] The specific structure of IL-2-mRNA is as follows: Figure 1 As shown, it includes a cap structure, a 5'-untranslated region, an encoding region, a 3'-terminal untranslated region, and a polyA tail connected sequentially along the 5' to 3' direction.

[0077] Preparation Example 18 This preparation example uses the method provided in Preparation Example 6 to prepare lipid nanoparticles. The difference is that an equimolar amount of IL-24-mRNA (sequence shown in SEQ ID NO:6) is used instead of IL-7-mRNA. All other conditions are the same, and lipid nanoparticles LNP-18 are obtained. The total amount of mRNA loaded in lipid nanoparticles LNP-18 is 5 μg.

[0078] The specific structure of IL-24-mRNA is as follows: Figure 1 As shown, it includes a cap structure, a 5'-untranslated region, an encoding region, a 3'-terminal untranslated region, and a polyA tail connected sequentially along the 5' to 3' direction.

[0079] Example This embodiment illustrates the killing ability of the lipid nanoparticles provided in the above preparation example against tumor cells in vivo. Eight-week-old female Balb / c mice were used as experimental animals. After one week of acclimatization under the same conditions, they were randomly divided into 18 groups and subjected to the following treatments: a. Blank control group (N=6): according to 5×10 6A20 cells were subcutaneously injected into the abdominal lymph nodes of mice and the mice were fed for 16 days. Then, PBS buffer (25 mM, pH=7) was administered to the mice via tail vein injection at a dosage of 200 μL / mouse. b. CAR monotherapy group (N=6): treated with 5×10 6 A20 cells were subcutaneously injected into mice and the mice were fed for 16 days. Then, LNP-16 provided in Preparation Example 16 was administered to the mice via tail vein injection at a dosage of 10 μg / mouse [injection volume was 200 μL, solvent was PBS buffer (25 mM, pH=7)]. c. CAR-cytokine combination therapy group (17 subgroups in total, N=6 in each subgroup): according to 5×10 6 A20 cells were subcutaneously injected into mice and the mice were fed for 16 days. Then, LNP-1-15, LNP-17 and LNP-18 cells from Preparation Examples 1-15 and 17, 18 were administered to the mice via tail vein injection at a dosage of 10 μg / mouse [injection volume was 200 μL, solvent was PBS buffer (0.025 M, pH=7)].

[0080] (1) After the drug administration, the mice in each group continued to be fed under the same conditions. The survival rate of the mice was counted during the feeding process. The day of A20 cell injection was defined as day 0 of feeding. The results are as follows: Figure 2 And as shown in Table 5.

[0081] Table 5.

[0082] Depend on Figure 2 As shown in Table 5, compared with the blank control group or the CAR treatment group alone, a single treatment of tumor-bearing mice with LNP-1 to LNP-15, LNP-17 and LNP-18 provided in Examples 1 to 15, 17 and 18 of this invention can effectively prolong the survival time of tumor-bearing mice.

[0083] (2) After the drug administration, mice in each group continued to be fed under the same conditions. During the feeding process, the tumor volume of the mice was measured and the survival rate of the mice was counted. The day of A20 cell injection was defined as day 0 of feeding. The results are as follows: Figure 3 And as shown in Table 6.

[0084] Table 6.

[0085] Depend on Figure 3As shown in Table 6, compared with the blank control group and the CAR monotherapy group, the single-dose treatment of tumor-bearing mice with LNP-6~LNP-10 provided in Examples 6~10 of this invention has an excellent inhibitory effect on tumor growth.

[0086] (3) Immediately after the mice died, tumor tissue was removed by dissection. The tumor tissue was treated with a tumor dissociation kit (BioLegend, catalog number BL-TDK-T-20) according to the instructions to obtain cell suspension. Anti-CD4 antibody (BioLegend, catalog number 100516, PE label), anti-CD8 antibody (BioLegend, catalog number 100706, FITC label), and anti-CAR antibody (Acro, catalog number FMC63, APC label) were respectively used to treat the cell suspension according to the instructions. Flow cytometry was used to detect CD8 in tumor cells. + and CD4 + The percentage of T cells, the results are as follows Figures 4-6 As shown in Table 7.

[0087] Table 7.

[0088] Depend on Figures 4-6 As shown in Table 7, compared with the blank control group and the CAR monotherapy group, a single administration of LNP-1~LNP-15, LNP-17 and LNP-18 provided in Examples 1-15 and 17, 18 of this invention to tumor-bearing mice can optimize the tumor immune microenvironment, thereby improving gene delivery efficiency and T cell targeting, and enhancing CD8+ in tumor tissue. + and CD4 + The proportion of CAR-positive cells in T cells increased significantly, especially in CD8 cells. + Increasing the CAR-positive rate in T cells and reducing CAR-T cell depletion allows these cells to survive long-term in the tumor microenvironment and continue to exert therapeutic effects, thus achieving better killing of tumor cells and exhibiting superior anti-tumor therapeutic efficacy.

[0089] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A medicament for in vivo engineering of CAR-T, characterized in that, The drug comprises a first RNA and a second RNA in a molar ratio of 1:(0.2~3); wherein the first RNA encodes a chimeric antigen receptor; and the second RNA comprises at least two of the following: CCL19-RNA, wherein the CCL19-RNA encodes CCL19; IL-7-RNA, which encodes IL-7; IL-15-RNA, which encodes IL-15; IL-2-RNA, which encodes IL-2; IL-24-RNA, which encodes IL-24.

2. The medicament for in vivo engineering of CAR-T according to claim 1, characterized in that, In the first RNA, the target sites of the chimeric antigen receptor include one or more of BCMA, CD19, CD20, CD22, CD30, CD123, CD38, CD5, Claudin18.2, MUC1, NKG2D ligand, human epidermal growth factor receptor 2, prostate-specific membrane antigen, EGFRvIII, mesothelin, carcinoembryonic antigen, disialotetrahexosylganglioside, and prostate-specific membrane antigen.

3. The medicament for in vivo engineering of CAR-T according to claim 1, characterized in that, The first RNA comprises a nucleotide fragment with the sequence shown in SEQ ID NO:

1.

4. The medicament for in vivo engineering of CAR-T according to claim 1, characterized in that, The drug has at least one of the following characteristics: (i) The second RNA comprises CCL19-RNA and IL-7-RNA, and the molar ratio of the first RNA, CCL19-RNA and IL-7-RNA is 1:(0.2~1):(0.2~1); (ii) The second RNA comprises CCL19-RNA, IL-7-RNA and IL-15-RNA, and the molar ratio of the first RNA, CCL19-RNA, IL-7-RNA and IL-15-RNA is 1:(0.2~1):(0.2~1):(0.2~1); (iii) The second RNA comprises CCL19-RNA and IL-2-RNA, and the molar ratio of the first RNA, CCL19-RNA and IL-2-RNA is 1:(0.2~1):(0.2~1); (iv) The second RNA comprises CCL19-RNA and IL-24-RNA, and the molar ratio of the first RNA, CCL19-RNA and IL-24-RNA is 1:(0.2~1):(0.2~1).

5. The drug for in vivo engineered CAR-T according to claim 1, characterized in that, The drug has at least one of the following characteristics: (i) The CCL19-RNA comprises a nucleotide sequence as shown in SEQ ID NO:2; (ii) The IL-7-RNA comprises a nucleotide sequence as shown in SEQ ID NO:3; (iii) The IL-15-RNA comprises a nucleotide sequence as shown in SEQ ID NO:4; (iv) The IL-2-RNA comprises a nucleotide sequence as shown in SEQ ID NO:5; (v) The IL-24-RNA comprises a nucleotide sequence as shown in SEQ ID NO:

6.

6. The drug for in vivo engineered CAR-T according to claim 1, characterized in that, The drug has at least one of the following characteristics: (i) The first RNA is selected from one or more of linear mRNA, circular RNA and self-amplifying RNA; (ii) The second RNA is selected from one or more of linear mRNA, circular RNA and self-amplifying RNA.

7. The drug for in vivo engineered CAR-T according to claim 1, characterized in that, The drug has at least one of the following characteristics: (i) The first RNA is a linear mRNA, which includes a cap structure, a 5'-untranslated region, a coding region, a 3'-untranslated region and a polyA tail connected sequentially along the 5' to 3' direction; (ii) The second RNA is a linear mRNA, which includes a cap structure, a 5'-untranslated region, a coding region, a 3'-untranslated region and a polyA tail connected sequentially along the 5' to 3' direction.

8. The drug for in vivo engineered CAR-T according to claim 1, characterized in that, The drug includes a carrier, on which the first RNA and the second RNA are mounted, and the carrier is selected from one or more of lipid nanoparticles, inorganic nanoparticles, protein nanoparticles and virus-like particles. Optionally, when the carrier is a lipid nanoparticle, the total loading amount of the first RNA and the second RNA on the carrier is 1 μg to 100 μg.

9. A method for preparing a drug for in vivo engineered CAR-T, characterized in that, The preparation method includes: S1, mixing the lipid composition with ethanol to obtain an organic phase solution; S2, mixing the first RNA, the second RNA, and water to obtain an aqueous phase solution; S3, mixing the organic phase solution and the aqueous phase solution to obtain the lipid nanoparticles; The lipid composition comprises 100 to 400 parts by weight of ionizable lipids, 20 to 80 parts by weight of auxiliary lipids, 80 to 120 parts by weight of cholesterol, and 1 to 10 parts by weight of polyethylene glycol lipids. The first RNA is the first RNA as described in any one of claims 1 to 7; the second RNA is the second RNA as described in any one of claims 1 to 7; and the molar ratio of the first RNA to the second RNA is 1:(0.2 to 3).

10. The method for preparing a drug for in vivo engineered CAR-T according to claim 9, characterized in that, The preparation method includes one or more of the following technical features: (i) The ionizable lipids include one or more of the following: trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl[2,3-(dioleenyloxy)propyl]ammonium chloride, 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl], 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester, 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl ester, and 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol); (ii) The auxiliary lipids include one or more of dioleoylphosphatidylethanolamine, distearate phosphatidylcholine and sterols; (iii) The polyethylene glycol lipids include one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, distearate phosphatidylethanolamine-polyethylene glycol, and dioleoyl phosphatidylethanolamine-polyethylene glycol; (iv) In step S1, the concentration of the lipid composition in the organic phase solution is 10 mg / mL to 50 mg / mL; (v) In step S2, the total concentration of the first RNA and the second RNA in the aqueous solution is 0.1 mg / mL to 1 mg / mL of nucleic acid molecules; (vi) In step S3, the mixing flow rate ratio of the organic phase solution and the aqueous phase solution is 1:(2~5).

Images