CD5 antibody-coupled mRNA lipid nanoparticle and its application

Abstract

The present invention provides a CD5 antibody-coupled mRNA lipid nanoparticle and its application, belonging to the technical field of medical preparations, wherein the mRNA is transcribed from a target gene; the target gene includes a GPC3 single-chain antibody; the nucleic acid artificial sequence of the GPC3 single-chain antibody is shown in SEQ ID NO.2 or SEQ ID NO.3 in the sequence table. The present invention improves the efficiency of LNP / mRNA-CAR targeting and binding to T cells and the killing ability of tumor cells by modifying LNP / mRNA-CAR with CD5 antibodies, and LNP / mRNA-CAR can significantly improve the reprogramming efficiency of T cells in mice by coupling CD5 antibodies, and can produce more GPC3-CAR-T cells in vivo. Compared with commercially available CD5 antibodies, the sequence-optimized CD5 antibody of the present invention has a higher efficiency in directly targeting and binding to CD5+T cells.

Description

Technical Field

[0001] The invention relates to CD5 antibody-coupled mRNA lipid nanoparticles and application thereof, belonging to the technical field of medical preparations. Background Art

[0002] In recent years, tumor immunotherapy has developed rapidly and has become a hot spot and breakthrough in the field of tumor treatment. Tumor immunotherapy includes immune cell therapy, immune checkpoint inhibitors, cytokines, cell vaccines, etc. Among them, chimeric antigen receptor T cell (CAR-T cell) therapy is a new type of cell therapy that uses T cells modified in vitro to recognize target cells through specific CAR sequences, and kills and eliminates them. It can achieve targeted elimination of harmful cells in vivo using the function of endogenous cells. However, CAR-T therapy also brings a variety of side effects, including cytokine release syndrome caused by the reinfusion of a large number of T cells, neurotoxicity, and possible organ failure. In addition, the method of extracting and culturing from circulating blood and reinfusion after genetic modification is expensive and not conducive to scale expansion.

[0003] Recent scientific studies have shown that messenger RNA (mRNA) can be used to manufacture CAR-T cells, and lipid nanoparticles (LNPs) can improve the delivery of mRNA encoding engineered CAR molecules. In addition, the combination of LNP, mRNA, and CAR-T technology has been proposed for the treatment of hematological and solid tumors, and these new technologies have the potential to improve the efficacy and safety of CAR-T cell therapy for cancer.

[0004] Using LNP / mRNA-CAR to transform and reprogram cells in vivo can avoid a series of complex in vitro processes of traditional in vitro CAR cell therapy, greatly reducing the difficulty and cost of preparation. At the same time, because mRNA will not enter the cell nucleus and will not edit the DNA of T cells, the transformed CAR cells are only aggressive for a few days and have potential controllable safety and tolerance.

[0005] Patent CN116515873A discloses the design and application of LNP-mRNA delivery system for constructing TAC-T cells in vivo. The patent discloses a method of delivering TAC-T mRNA targeted to activated myocardial fibroblasts in vivo by lipid nanoparticles (LNP) and modifying CD5 antibodies, which is used to produce TAC-T mRNA targeting activated myocardial fibroblasts in vivo after injection into the body, thereby reducing the number of activated myocardial fibroblasts in the heart of heart failure and being applied to the relief and treatment of heart failure.

[0006] Patent CN117210479A discloses the design and application of a CD5 antibody-modified LNP-mRNA delivery system for in vivo production of CAR-T. The patent provides a method for encapsulating mRNA encoding a CAR sequence in a targeted LNP and coupling it with a CD5 antibody, which can produce CAR-T cells in vivo, which can be used to reduce the number of activated myocardial fibroblasts in the heart of heart failure and be applied to the relief and treatment of heart failure.

[0007] The mRNA of the above patent can produce a CAR sequence that targets and recognizes FAP protein, thereby producing CAR-T cells to kill activated myocardial fibroblasts for the treatment of heart failure. The CD5 antibody disclosed in the above patent has a low efficiency in targeting and binding T cells, and has a low killing rate on target cells.

[0008] The prior art has not disclosed mRNA lipid nanoparticles coupled with CD5 antibodies that target and recognize the tumor antigen protein GPC3. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention provides a CD5 antibody-coupled mRNA lipid nanoparticle and a preparation method thereof, to achieve the following invention objectives: the CD5 antibody-coupled mRNA lipid nanoparticle has a high efficiency in targeting and binding to T cells and a high killing rate of target cells.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] An mRNA lipid nanoparticle coupled with a CD5 antibody, wherein the mRNA is transcribed from a target gene; the target gene includes a GPC3 single-chain antibody; the artificial nucleic acid sequence of the GPC3 single-chain antibody is shown in SEQ ID NO.2 or SEQ ID NO.3 in the sequence table.

[0012] The target gene is obtained by serially connecting the following modules: CD8 signal peptide, GPC3 single-chain antibody, CD8 Hinge region, CD8 transmembrane region, CD28 co-stimulatory region, 4-1BB co-stimulatory region, CD3ζ signal transduction region, T2A, RISR-RIAD, P2A, IL-15.

[0013] The artificial nucleic acid sequence of the CD8 signal peptide is shown in SEQ ID NO.1 in the sequence listing; the artificial nucleic acid sequence of the CD8 Hinge region is shown in SEQ ID NO.4 in the sequence listing; the artificial nucleic acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.5 in the sequence listing; the artificial nucleic acid sequence of the CD28 co-stimulatory region is shown in SEQ ID NO.6 in the sequence listing; the artificial nucleic acid sequence of the 4-1BB co-stimulatory region is shown in SEQ ID NO.7 in the sequence listing; the artificial nucleic acid sequence of the CD3ζ signaling region is shown in SEQ ID NO.8 in the sequence listing; the artificial nucleic acid sequence of T2A is shown in SEQ ID NO.9 in the sequence listing; the artificial nucleic acid sequence of RISR-RIAD is shown in SEQ ID NO.10 in the sequence listing; the artificial nucleic acid sequence of P2A is shown in SEQ ID NO.11 in the sequence listing; the artificial nucleic acid sequence of IL-15 is shown in SEQ ID NO.12 in the sequence listing.

[0014] The heavy chain amino acid sequence of the CD5 antibody is shown in SEQ ID NO.14 in the sequence listing, and the light chain amino acid sequence is shown in SEQ ID NO.15 in the sequence listing.

[0015] The concentration of mRNA in the lipid nanoparticles is 50-55 μg / mL.

[0016] The mRNA lipid nanoparticles coupled with CD5 antibodies adopt LNP to encapsulate mRNA.

[0017] The application of the mRNA lipid nanoparticles coupled with CD5 antibodies in the preparation of anti-liver cancer drugs.

[0018] Compared with the prior art, the present invention achieves the following beneficial effects:

[0019] (1) The present invention improves the efficiency of LNP / mRNA-CAR targeting and binding to T cells and the ability to kill tumor cells by modifying LNP / mRNA-CAR with CD5 antibodies. In addition, LNP / mRNA-CAR can significantly improve the reprogramming efficiency of T cells in mice by coupling with CD5 antibodies, and can produce more GPC3-CAR-T cells in vivo.

[0020] (2) Compared with commercially available CD5 antibodies, the CD5 antibody of the present invention (sequence-optimized CD5 antibody) is more efficient in directly targeting and binding to CD5+T cells, thereby improving the efficiency of LNP / mRNA-CAR-guided T cells and the tumor cell killing effect; it is more efficient in reprogramming T cells in mice and has stronger anti-tumor activity.

[0021] (3) The present invention optimizes the nucleic acid sequence of GPC3 to improve the ability of CD5-LNP / mRNA-CAR transfected T cells to secrete cytokines and the killing rate of tumor cells, making them have stronger anti-tumor activity.

[0022] (4) The CD5-LNP / mRNA-CAR prepared by the present invention transforms and reprograms T cells in vivo to generate functional CAR-T cells in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a connection diagram of each module of GPC3-CAR;

[0024] Figure 2 It is a bar graph of cell viability after co-incubation of CD5-LNP / mRNA-CAR and CD5+T cells;

[0025] A is a bar graph of cell viability after co-incubation of CD5-LNP / mRNA-CAR1 and CD5+T cells;

[0026] B is a bar graph of cell viability after co-incubation of CD5-LNP / mRNA-CAR2 and CD5+T cells;

[0027] C is a bar graph of cell viability after co-incubation of CD5+T cells with CD5-LNP / mRNA-CAR1;

[0028] D is a bar graph of cell viability after co-incubation of CD5+T cells with CD5-LNP / mRNA-CAR2;

[0029] Figure 3 This is a bar graph of the transfection efficiency of CD5+T cells transfected with CD5-LNP / mRNA-CAR;

[0030] Figure 4 The figure is a bar graph of the cytokine secretion of the experimental group, comparative group and control group in Example 7;

[0031] Figure 5 The figure is a bar graph showing the killing rate of liver cancer cells in the experimental group, comparative group and control group in Example 8;

[0032] Figure 6 Flow cytometry of CD5-LNP / mRNA-CAR1 reprogramming T cells in vivo;

[0033] Figure 7 Flow cytometry of CD5-LNP / mRNA-CAR2 reprogramming T cells in vivo;

[0034] Figure 8Flow cytometry of CD5-LNP / mRNA-CAR1 reprogramming T cells in vivo;

[0035] Fig. 9 Flow cytometry of CD5-LNP / mRNA-CAR2 reprogramming T cells in vivo;

[0036] Fig.10 Flow cytometry for in vivo reprogramming of T cells by LNP / mRNA-CAR1. DETAILED DESCRIPTION

[0037] Example 1 Construction of a recombinant plasmid containing GPC3-CAR

[0038] The connection diagram of each module of GPC3-CAR is as follows Figure 1 As shown, the modules of GPC3-CAR and their artificial nucleic acid sequences are as follows:

[0039] (1) CD8 signal peptide, the artificial nucleic acid sequence of which is shown in SEQ ID NO.1 in the sequence listing;

[0040] (2) GPC3 single-chain antibody, whose artificial nucleic acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.3 in the sequence listing;

[0041] (3) CD8 Hinge region, the artificial nucleic acid sequence of which is shown in SEQ ID NO.4 in the sequence listing;

[0042] (4) CD8 transmembrane region, whose artificial nucleic acid sequence is shown in SEQ ID NO.5 in the sequence list;

[0043] (5) CD28 co-stimulatory region, whose artificial nucleic acid sequence is shown in SEQ ID NO.6 in the sequence list;

[0044] (6) 4-1BB co-stimulatory region, whose artificial nucleic acid sequence is shown in SEQ ID NO.7 in the sequence list;

[0045] (7) CD3ζ signal transduction region, whose artificial nucleic acid sequence is shown in SEQ ID NO.8 in the sequence list;

[0046] (8) T2A, whose artificial nucleic acid sequence is shown as SEQ ID NO.9 in the sequence listing;

[0047] (9) RISR-RIAD, whose artificial nucleic acid sequence is shown as SEQ ID NO.10 in the sequence listing;

[0048] (10) P2A, whose artificial nucleic acid sequence is shown as SEQ ID NO.11 in the sequence listing;

[0049] (11) IL-15, the artificial nucleic acid sequence of which is shown as SEQ ID NO.12 in the sequence listing;

[0050] The artificial nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 were connected in sequence, and Yunzhou Biotechnology Co., Ltd. was commissioned to synthesize the entire expression frame, cloned into the IVT vector (pmRVac, designed by Yunzhou Biotechnology), and transformed into E. coli (Top10). Positive clones were screened by colony PCR, and plasmid DNA was extracted from the screened positive clones for sequence verification. After the sequencing was correct, the plasmid was extracted using the plasmid extraction kit of OMEGA Company to obtain a recombinant plasmid containing GPC3-CAR, named GPC3-CAR1, and diluted to 1.0 μg / μL;

[0051] The artificial nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 were connected in sequence to obtain a recombinant plasmid containing GPC3-CAR according to the above method, named GPC3-CAR2, and diluted to 1.0 μg / μL.

[0052] Example 2 In vitro transcription into mRNA

[0053] (1) Linearization of recombinant plasmid and DNA recovery

[0054] The enzyme digestion system (50 μL in total) is as follows: 10×Buffer: 5 μL; NotI enzyme: 3 μL; recombinant plasmid: 2 μL (1 μg / μL); water: 40 μL.

[0055] The recombinant plasmids GPC3-CAR1 and GPC3-CAR2 prepared in Example 1 were subjected to single enzyme digestion reactions, and the specific operation was: digestion in a 37°C water bath for 1 hour, and then quickly transferred to a 65°C water bath for 5 minutes to terminate the digestion reaction. After the digestion reaction was completed, DNA was identified in 1% agarose gel electrophoresis, and the size of the obtained DNA fragment was in line with expectations.

[0056] The target DNA fragment was cut out and recovered using an agarose gel DNA recovery kit. The concentration of the recovered DNA fragment was measured using a spectrophotometer. The concentration of the DNA fragment was adjusted to 1 μg / μL and stored at -20°C for future use.

[0057] (2) In vitro transcription into mRNA

[0058] The two recombinant plasmids GPC3-CAR1 and GPC3-CAR2 were linearized and digested, and the DNA fragments obtained were recovered and transcribed into mRNA in vitro, respectively. The capping mMESSAGE mMACHINE T7 kit (Ambion) was used to synthesize 5′-capped mRNA in vitro, and a Poly (A) tail structure was added to the 3′ end of the mRNA to obtain mRNA with 5′-capped and 3′-tailed structures. The mRNA was then purified to obtain purified mRNA, which was named mRNA-CAR1 and mRNA-CAR2. The concentration of the purified mRNA was adjusted to 2 μg / μL and stored in a -80°C refrigerator for later use.

[0059] The purified mRNA was placed in a 70°C water bath for 10 min, placed on ice for 3 min, and electrophoresed in urea-acrylamide gel at 100V constant voltage for 30 min. The size and integrity of the mRNA were verified using a gel imager. The electrophoresis results showed that the mRNA fragments were 2289 bp and 2286 bp, respectively, which were consistent with expectations.

[0060] Example 3 Preparation of LNPs loaded with mRNA-CAR

[0061] (1) Preparation of ethanol solution of liposome mixture

[0062] Weigh 10.4 mg DLin-MC3-DMA, 2.7 mg DSPC, 4.9 mg cholesterol and 2.2 mg PEG200-DMG (molar ratio of 50:10:37.5:2.5), add 1.25 mL ethanol, stir well to dissolve, and obtain an ethanol solution of the liposome mixture.

[0063] (2) Preparation of mRNA aqueous solution

[0064] 1 μL of the purified mRNA (2 μg / μL) prepared in Example 2 was dissolved in 1.25 mL of 10 mM citric acid buffer (pH=4), and pipetted up and down 50 times; 2.5 mL of PBS was added, and the solution was vigorously pipetted 50 times to mix well to obtain an mRNA aqueous solution.

[0065] (3) Preparation of LNP particles loaded with mRNA-CAR

[0066] Using a nanoparticle synthesis system (Ignite), the ethanol solution of the liposome mixture and the mRNA aqueous solution were mixed in a ratio of 1:3 (volume ratio) to synthesize LNP particles encapsulating mRNA-CAR; the obtained LNP particles were dialyzed in a PBS solution (pH 7.4) for 24 h, concentrated to a volume of 1 mL using an Amicon ultracentrifugal filter, and filtered through a 0.22 μm filter membrane twice to obtain an LNP / mRNA-CAR solution, which was stored at -20°C for future use.

[0067] The mRNA-CAR1 and mRNA-CAR2 of Example 2 were prepared according to the above method to obtain LNP / mRNA-CAR1 solution and LNP / mRNA-CAR2 solution, respectively.

[0068] Blank control: The preparation method is the same as above, with the following changes: in step (2), an equal amount of DEPC water is used to replace the purified mRNA. The blank control is represented by LNP / blank.

[0069] (4) LNP particle characterization and analysis

[0070] Dynamic light scattering (DLS) was used to analyze the average diameter and dispersion index (PDI) of the prepared LNP / mRNA-CAR, and Quant-iT™ RiboGreen® RNA Reagent and Kit (purchased from Invitorgen) was used to measure the encapsulation efficiency of the LNP particles.

[0071] The results are shown in Table 1. The encapsulation efficiency of LNP particles in the LNP / mRNA-CAR solution was as high as over 90%, and almost all of the mRNA was encapsulated.

[0072] Table 1 Nanoparticle characterization

[0073]

[0074] Example 4 LNP / mRNA-CAR coupled with CD5 antibody

[0075] In the present invention, two CD5 antibodies are used for coupling LNP / mRNA-CAR, one is a sequence-optimized CD5 antibody, whose heavy chain amino acid sequence is SEQ ID NO.14 and light chain amino acid sequence is SEQ ID NO.15, and the CD5 antibody is prepared according to a conventional method; the other is a CD5 antibody purchased from Wuhan Elerite Biotechnology Co., Ltd.

[0076] The two CD5 antibodies were placed in PBS containing 1 mM DTT and 5 mM EDTA at room temperature for 1 hour to reduce them, and the two CD5 antibodies were coupled to LNP / mRNA-CAR according to the method for coupling CD8 antibody in Example 3 of CN118286410A.

[0077] The sequence-optimized CD5 antibody of the present invention is coupled to LNP / mRNA-CAR1 and LNP / mRNA-CAR2, respectively, to obtain CD5-LNP / mRNA-CAR1 and CD5-LNP / mRNA-CAR2; the purchased CD5 antibody is coupled to LNP / mRNA-CAR1 and LNP / mRNA-CAR2, respectively, to obtain CD5-LNP / mRNA-CAR1 and CD5-LNP / mRNA-CAR2.

[0078] The entire CD5-LNP / mRNA-CAR was lysed by Triton X-100, and the amount of mRNA was measured using the Quant-iT™ RiboGreen® RNA Reagent and Kit.

[0079] The results showed that the concentration of mRNA in CD5-LNP / mRNA-CAR1 was 55 μg / mL, the concentration of mRNA in CD5-LNP / mRNA-CAR2 was 51 μg / mL, the concentration of mRNA in CD5-LNP / mRNA-CAR1 was 53 μg / mL, and the concentration of mRNA in CD5-LNP / mRNA-CAR2 was 50 μg / mL.

[0080] Example 5 Cytotoxicity Study of CD5-LNP / mRNA-CAR

[0081] (1) Preparation of CD5+ T cells

[0082] 50 mL of peripheral blood donated by volunteers was collected and PBMC (peripheral blood mononuclear cells) was isolated using TBD sample density separation solution (purchased from Tianjin Haoyang Huake Biotechnology Co., Ltd.). The separated cells were sorted out with CD5+ sorting reagent provided by BD to sort out CD5+ T cells, and the cells were counted and counted according to 1×10 6cells / mL, added to the culture medium (purchased from Corning, catalog number: 88-551-CM) containing 1000IU / mL recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical), and cultured in a 37°C, 5% CO2 incubator for 24 hours, and then added with 1500IU / mL (final concentration) of recombinant interleukin 2 (purchased from Shenyang Sansheng Pharmaceutical) and 50ng / mL (final concentration) of CD3 monoclonal antibody (purchased from Tongli Haiyuan) and continued to culture for 24 hours to obtain activated CD5+T cells.

[0083] (2) Cytotoxicity study of CD5-LNP / mRNA-CAR

[0084] Activated CD5+ T cells were seeded in 96-well plates (5×10 3 After overnight culture, CD5-LNP / mRNA-CAR1, CD5-LNP / mRNA-CAR2, CD5-LNP / mRNA-CAR1, and CD5-LNP / mRNA-CAR2 (calculated as mRNA, the final concentration was 2 μg / mL) were added respectively. The T cell viability was detected at 0h, 6h, 12h, 18h, and 24h of co-incubation. The results are shown in Figure 2 shown.

[0085] Depend on Figure 2 It can be seen that the four types of CD5-LNP / mRNA-CAR have no obvious cytotoxicity to CD5+T cells and have good safety.

[0086] Example 6 CD5-LNP / mRNA-CAR transfection of CD5+T cells

[0087] CD5+ T cells were seeded in 24-well plates (6×10 4 The cells were divided into 5 groups, and 24 hours later, CD5-LNP / mRNA-CAR1 (50 ng / well, calculated as mRNA), CD5-LNP / mRNA-CAR2 (50 ng / well, calculated as mRNA), CD5-LNP / mRNA-CAR1 (50 ng / well, calculated as mRNA), CD5-LNP / mRNA-CAR2 (50 ng / well, calculated as mRNA), and LNP-mRNA-CAR1 (50 ng / well, calculated as mRNA) were added respectively. After incubation for 24 hours, the cells were collected, and the expression rate of GFP was detected by flow cytometry to determine the transfection efficiency. The results are shown in Figure 3 And as shown in Table 2.

[0088] Depend on Figure 3As shown in Table 2, the expression rate of GFP-tagged protein in CD5+T cells treated with LNP / mRNA-CAR coupled with CD5 antibody was between 50.2% and 6.3.7%, while the expression rate of GFP-tagged protein in CD5+T cells treated with LNP / mRNA-CAR1 without CD5 antibody was only 21.3%, which indicates that LNP / mRNA-CAR can directly target and bind to CD5+T cells after coupling with CD5 antibody, and the transfection efficiency is significantly improved.

[0089] In addition, the expression rate of GFP label protein in CD5+T cells treated with LNP / mRNA-CAR coupled with the CD5 antibody of the present invention (sequence-optimized CD5 antibody) was 63.2-63.7%, while the expression rate of GFP label protein in CD5+T cells treated with LNP / mRNA-CAR coupled with commercially available CD5 antibody was 50.2-50.5%, indicating that the CD5 antibody of the present invention is more efficient in directly targeting and binding to CD5+T cells than the commercially available CD5 antibody, thereby improving the efficiency of LNP / mRNA-CAR in guiding T cells.

[0090] Table 2 Expression rate of GFP-tagged protein

[0091]

[0092] Example 7 Cytokine Secretion Detection

[0093] IL-2 and IFN-γ are key indicators for evaluating whether T cells produce a strong immune response. The cytokine secretion of T cells is detected by the ELSIA method.

[0094] (1) Effector cells

[0095] Activated CD5+ T cells were seeded in 24-well plates (5×10 5 24 hours later, CD5-LNP / mRNA-CAR1, CD5-LNP / mRNA-CAR2, CD5-LNP / mRNA-CAR1, CD5-LNP /

[0096] mRNA-CAR2, CD5-LNP / mRNA optimized-CAR, and LNP / mRNA-CAR1 were transfected, and the cells were collected 48 hours later, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The obtained cells were effector cells.

[0097] The preparation method of CD5-LNP / mRNA-CAR before optimization is as follows:

[0098] The recombinant plasmid was constructed according to the method of Example 1 of the present invention, with the following changes: the GPC3 single-chain antibody used the artificial nucleic acid sequence before optimization, as shown in SEQ ID NO.13 in the sequence list;

[0099] LNP / mRNA-CAR was prepared according to the method described in Example 2 and Example 3, and was named LNP / mRNA pre-optimization-CAR;

[0100] The optimized CD5 antibody of the present invention was coupled according to the method of Example 4 and named CD5-LNP / mRNA pre-optimization-CAR.

[0101] (2) Target cells

[0102] Human hepatoma cell Hep3B (purchased from Shanghai Xinyu Biotechnology) was used as the target cell and cultured in DMEM medium (containing 10 vol% FBS) to the logarithmic growth phase.

[0103] (3) Cell co-culture

[0104] Effector cells (1×10 5 / well) and target cells (1×10 5 / well) and inoculated into 96-well plates, 100 μL / well, and divided into eight groups, as follows:

[0105] Experimental group 1: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR1 transfected) and Hep3B cells;

[0106] Experimental group 2: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR2 transfection) and Hep3B cells;

[0107] Experimental group 3: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR1 transfected) and Hep3B cells;

[0108] Experimental group 4: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR2 transfected) and Hep3B cells;

[0109] Control group: CD5+T cells (CD5-LNP / mRNA optimization-CAR transfection) and Hep3B cells co-cultured;

[0110] Control group: CD5+T cells (LNP / mRNA-CAR1 transfected) and Hep3B cells co-cultured;

[0111] Three replicates were added to each group and the cells were placed in a 37°C, 5% CO2 incubator for 48 h. The culture supernatant was collected for the detection of cytokines IL-2 and IFN-γ.

[0112] ELISA results showed (see Figure 4 and Table 3): The IL-2 and IFN-r contents in the experimental group and the control group were significantly higher than those in the control group, and there were significant differences, indicating that LNP / mRNA-CAR coupled with CD5 antibody can significantly improve the ability of CAR-T cells to secrete cytokines and enhance the function of CAR-T cells.

[0113] In the experimental groups, the ability of CAR-T cells coupled with the CD5 antibody of the present invention (experimental groups 1 and 2) to secrete cytokines was higher than that of CAR-T cells coupled with the commercially available CD5 antibody (experimental groups 3 and 4), indicating that the CD5 antibody prepared by the present invention (sequence-optimized CD5 antibody) enables CAR-T cells to have stronger cytokine secretion ability than the commercially available CD5 antibody.

[0114] In addition, compared with the control group, experimental groups 1 and 2 secreted higher levels of IL-2 and IFN-r, indicating that the codon-optimized nucleic acid sequence of GPC3 can enhance the ability of CAR-T cells to secrete cytokines.

[0115] Table 3 Cytokine secretion (pg / mL)

[0116]

[0117] Example 8 In vitro killing ability of CD5-LNP / mRNA-CAR

[0118] Effector cells and target cells were prepared according to the method described in Example 7.

[0119] Effector cells (1×10 5 / well) and target cells (1×10 5 / well) and inoculated in a 96-well plate, 100 μL / well, and divided into six groups, as follows:

[0120] Experimental group 1: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR1 transfected) and Hep3B cells;

[0121] Experimental group 2: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR2 transfection) and Hep3B cells;

[0122] Experimental group 3: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR1 transfected) and Hep3B cells;

[0123] Experimental group 4: co-culture of CD5+T cells (CD5-LNP / mRNA-CAR2 transfected) and Hep3B cells;

[0124] Control group: CD5+T cells (CD5-LNP / mRNA optimization-CAR transfection) and Hep3B cells co-cultured;

[0125] Control group: CD5+T cells (LNP / mRNA-CAR1 transfected) and Hep3B cells co-cultured;

[0126] The cells were placed in a 5% CO2, 37°C incubator for co-culture. After 24 h, 20 mL of CCK-8 was added to each well. After further incubation for 2 h, the ELISA reader was used to detect the wavelength at 450 nm, the OD value was read, and the cell killing rate was calculated.

[0127] Table 4 Killing rate of liver cancer cells

[0128]

[0129] The results are as follows Figure 5 As shown in Table 4, the killing rates of the cells in the experimental group and the control group were significantly higher than those in the control group, and there were significant differences, which indicated that the LNP / mRNA-CAR molecule coupled with the CD5 antibody could significantly improve the killing ability of CAR-T cells against tumor cells; in the experimental group, the killing rate of CAR-T cells coupled with the CD5 antibody of the present invention (experimental group 1 and experimental group 2) was higher than that of CAR-T cells coupled with the commercially available CD5 antibody (experimental group 3 and experimental group 4), which indicated that compared with the commercially available CD5 antibody, the LNP / mRNA-CAR-T cells coupled with the CD5 antibody of the present invention had a better killing effect on tumor cells.

[0130] In addition, compared with the control group (CD5-LNP / mRNA-CAR before optimization), the CD5-LNP / mRNA-CAR prepared by the present invention (experimental group 1 and experimental group 2) had a higher killing rate on tumor cells, which indicates that the nucleic acid sequence of GPC3 can improve the killing rate of CAR-T cells on tumor cells after codon optimization, making it have a stronger anti-tumor effect.

[0131] Example 9 Evaluation of CD5-LNP / mRNA-CAR in vivo T cell reprogramming

[0132] CD5-LNP / mRNA-CAR1 (10 μg), CD5-LNP / mRNA-CAR2 (10 μg), CD5-LNP / mRNA-CAR1 (10 μg), CD5-LNP / mRNA-CAR2 (10 μg) and LNP / mRNA-CAR1 (10 μg) were injected into C57BL / 6 mice through the tail vein. After 48 hours, blood was collected from the vein and the expression rate of GFP in blood cells was detected by flow cytometry to determine the in vivo reprogramming of T cells by CD5-LNP / mRNA-CAR.

[0133] The results are as follows Figure 6-10 As shown in Table 5: A cell population stably expressing GFP was found in mice injected with CD5-LNP / mRNA-CAR, and the expression rate of GFP was between 15% and 29.3%, while the expression rate of GFP in mice injected with LNP / mRNA-CAR not coupled with CD5 antibody was only 2.2%, which indicates that LNP / mRNA-CAR can significantly improve the reprogramming efficiency of T cells in mice by coupling with CD5 antibody, and can produce more GPC3-CAR-T cells in vivo.

[0134] In addition, compared with the commercially available CD5 antibody, the expression rate of GFP in mice of LNP / mRNA-CAR coupled with the CD5 antibody of the present invention was higher ( Figure 6 , Figure 7 ), indicating that the CD5-LNP / mRNA-CAR prepared by the present invention has higher efficiency in reprogramming T cells in mice and has stronger anti-tumor activity.

[0135] In summary, LNP / mRNA-CAR coupled to CD5 antibodies can be injected intravenously to reprogram T cells in vivo and produce functional T cells.

[0136] Table 5 Expression rate of GFP in T cells of mice

[0137]

Claims

1. An mRNA lipid nanoparticle coupled to a CD5 antibody, characterized in that: The mRNA is transcribed from the target gene; The target gene is obtained by serially connecting the following modules: CD8 signal peptide, GPC3 single-chain antibody, CD8 Hinge region, CD8 transmembrane region, CD28 co-stimulatory region, 4-1BB co-stimulatory region, CD3ζ signal transduction region, T2A, RISR-RIAD, P2A, IL-15; The artificial nucleic acid sequence of the CD8 signal peptide is shown in SEQ ID NO.1 in the sequence list; the artificial nucleic acid sequence of the GPC3 single-chain antibody is shown in SEQ ID NO.2 or SEQ ID NO.3 in the sequence list; the artificial nucleic acid sequence of the CD8 Hinge region is shown in SEQ ID NO.4 in the sequence list; the artificial nucleic acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.5 in the sequence list; the artificial nucleic acid sequence of the CD28 co-stimulatory region is shown in SEQ ID NO.6 in the sequence list; the artificial nucleic acid sequence of the 4-1BB co-stimulatory region is shown in SEQ ID NO.7 in the sequence list; the artificial nucleic acid sequence of the CD3ζ signaling region is shown in SEQ ID NO.8 in the sequence list; the artificial nucleic acid sequence of T2A is shown in SEQ ID NO.9 in the sequence list; the artificial nucleic acid sequence of RISR-RIAD is shown in SEQ ID NO.10 in the sequence list; the artificial nucleic acid sequence of P2A is shown in SEQ ID NO.11 in the sequence list; the artificial nucleic acid sequence of IL-15 is shown in SEQ ID NO.12 in the sequence list; The heavy chain amino acid sequence of the CD5 antibody is shown in SEQ ID NO.14 in the sequence listing, and the light chain amino acid sequence is shown in SEQ ID NO.15 in the sequence listing.

2. The mRNA lipid nanoparticle coupled to a CD5 antibody according to claim 1, characterized in that: The concentration of mRNA in the lipid nanoparticles is 50-55 μg / mL.

3. The mRNA lipid nanoparticle coupled to a CD5 antibody according to claim 1, characterized in that: The mRNA lipid nanoparticles coupled with CD5 antibodies adopt LNP to encapsulate mRNA.

4. Use of the CD5 antibody-coupled mRNA lipid nanoparticles according to any one of claims 1 to 3 in the preparation of anti-liver cancer drugs.

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