A nanoparticle for generating CAR-T in vivo and its application

Abstract

The present invention provides a nanoparticle for generating CAR-T in vivo and its application, belonging to the technical field of medical preparations. The nanoparticle is a nanoparticle encapsulating mRNA coupled to CD7 antibody; the mRNA is transcribed from a target gene; the target gene includes a TROP2 single-chain antibody; the nucleotide sequence of the TROP2 single-chain antibody is shown in SEQ ID NO: 1. The present invention optimizes the nucleotide sequence of the TROP2 single-chain antibody, constructs a CAR containing a TROP2 single-chain antibody, then transcribes to obtain mRNA, prepares an LNP encapsulating mRNA, and then uses the CD7 antibody after amino acid sequence optimization to couple the LNP encapsulating mRNA, which has high transfection efficiency in T cells and can be injected into a mouse tumor model to prolong the survival of mice; the transfected T cells have high killing efficiency against HELA cells and LOVO cells.

Description

Technical Field

[0001] The present invention provides a nanoparticle for generating CAR-T in vivo and application thereof, belonging to the technical field of medical preparations. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T cell) therapy has become a breakthrough immunotherapy in cancer treatment. In order to overcome the complexity and high manufacturing cost of current in vitro CAR-T cell therapy products, alternative strategies for producing CAR-T cells directly in vivo have been developed in recent years. This strategy involves directly infusing the CAR gene through engineered nanoparticles or viral vectors to produce CAR-T cells in vivo.

[0003] TROP2 is a 36kDa transmembrane glycoprotein that is mainly expressed in epithelial cells and was first discovered in human trophoblast cells. TROP2 affects the tight junctions of the epithelial barrier by binding to related targets, increases tumor proliferation, activates Raf and NF-kappa, and inhibits IGF-1R signaling. TROP2 expression is minimal or absent in adult tissues. In contrast, TROP2 is overexpressed in a variety of cancers, and its expression is associated with tumor aggressive behavior, cancer progression, and poor prognosis. Therefore, it is necessary to target cancer cells expressing TROP2 for treatment.

[0004] Studies have shown that IL-15 can enhance stem cell-like memory CAR-T cell populations, leading to superior tumor killing ability and reduced PD-1 expression, while improving the persistence and anti-tumor activity of CAR-T cells. Increased secretion of related chemokines in the tumor microenvironment is associated with improved trafficking of CAR-T cells to tumor sites.

[0005] There is no report in the prior art on preparing TROP2 single-chain antibody into nanoparticles and then coupling them with CD7 antibody. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a nanoparticle for generating CAR-T in vivo and an application thereof. LNP is used to encapsulate mRNA containing TROP2 single-chain antibody and coupled with CD7 antibody to prepare LNP nanoparticles coupled with CD7 antibody, thereby achieving the following invention objectives: the LNP nanoparticles coupled with CD7 antibody form CAR-T cells in vivo and exert an in vivo anti-tumor effect; the T cells transfected with the LNP nanoparticles coupled with CD7 antibody have a high in vitro killing efficiency against cervical cancer HELA cells and colorectal cancer LOVO cells.

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

[0008] A nanoparticle for generating CAR-T in vivo, wherein the nanoparticle is an mRNA-encapsulated nanoparticle coupled to a CD7 antibody; the mRNA is transcribed from a target gene; the target gene includes a TROP2 single-chain antibody; the nucleotide sequence of the TROP2 single-chain antibody is shown in SEQ ID NO: 1.

[0009] The nucleotide sequence of the target gene is shown in SEQ ID NO:2.

[0010] The heavy chain amino acid sequence of the CD7 antibody is shown in SEQ ID NO: 5, and the light chain amino acid sequence is shown in SEQ ID NO: 6.

[0011] The nanoparticles are nanoparticles in which mRNA is encapsulated by LNP.

[0012] The concentration of mRNA in the nanoparticles was 36-44 μg / mL.

[0013] The nanoparticles are used in the preparation of medicines for treating cervical cancer and / or colorectal cancer.

[0014] Compared with the prior art, the present invention achieves the following beneficial effects:

[0015] The present invention optimizes the nucleotide sequence of the TROP2 single-chain antibody, constructs a CAR comprising the TROP2 single-chain antibody, then transcribes to obtain mRNA, prepares LNPs encapsulating the mRNA, and then uses the CD7 antibody with optimized amino acid sequence to couple the LNPs encapsulating the mRNA. The LNPs have high transfection efficiency in T cells and can prolong the survival of mice when injected into a mouse tumor model. The transfected T cells have high killing efficiency for HELA cells and LOVO cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the construction of CAR structure;

[0017] in Figure 1 A is a schematic diagram of the construction of CAR-TROP2; B is a schematic diagram of the construction of CAR-TROP2 before optimization;

[0018] Figure 2 It is a bar graph of the expression rate of TROP2 in T cells transfected with different LNPs;

[0019] Figure 3 The bar graph shows the killing efficiency of T cells transfected with different LNPs on HELA cells and LOVO cells;

[0020] Figure 4 Survival curves of colorectal cancer mice treated with different LNPs. DETAILED DESCRIPTION

[0021] Example 1 Construction of CAR-TROP2 structure and preparation of mRNA

[0022] The present invention is based on the TROP2 single-chain antibody sequence in US2022 / 0153862A1 patent, and the heavy chain and light chain are codon optimized to obtain the TROP2 single-chain antibody of the present invention (the nucleotide sequence is shown in SEQ ID NO: 1), and then according to Figure 1 The CAR structure was constructed as shown in A.

[0023] The CAR structure consists of the following modules: CD8 signal peptide, TROP2 single-chain antibody, CD8 hinge region, CD28 transmembrane region, CD28 and 4-1BB co-stimulatory region, CD3ζ intracellular region, F2A, IL-15, P2A, and CXCL10.

[0024] The CAR structure constructed according to the above method is labeled as CAR-TROP2, and its nucleotide sequence is shown in SEQ ID NO:2.

[0025] The TROP2 single-chain antibody sequence in US2022 / 0153862A1 (nucleotide sequence as shown in SEQ ID NO: 3) was used as the TROP2 single-chain antibody sequence before optimization. Figure 1 The CAR structure was constructed as shown in B, marked as CAR-TROP2-before optimization, and its nucleotide sequence is shown in SEQ ID NO: 4.

[0026] The two CAR structures were used as DNA templates, respectively, and transcription was completed under the action of T7 polymerase to synthesize two mRNAs. Commercial reagents were used for capping and tailing. The mRNA was purified using the MEGAclear kit, and the concentration of the purified mRNA was adjusted to 1 μg / μL.

[0027] Example 2 Preparation and Characterization of mRNA-LNP

[0028] (1) 30 μL of purified mRNA (1 μg / μL) was diluted to 300 μL using 10 mM citrate buffer at pH 4 to obtain an mRNA mixture.

[0029] (2) Dissolve 3 mg of ionizable lipid DLin-MC3-DMA, 1.37 mg of cholesterol, 0.35 mg of DMG-PEG2000, 0.74 mg of DSPC, and 0.16 mg of DSPE-PEG-maleimide in 1 mL of ethanol to obtain a mixed lipid solution; the molar ratio of DLin-MC3-DMA: cholesterol: DMG-PEG2000: DSPC: DSPE-PEG-maleimide is 50:38:1.5:10:0.5.

[0030] (3) Adding an mRNA mixture and a mixed lipid solution into a microfluidic device, wherein the mass ratio of the mRNA in the mRNA mixture to the ionizable lipid DLin-MC3-DMA in the mixed lipid solution is 1:10, and LNP nanoparticles encoding targeting TROP2 are prepared by a microfluidic method.

[0031] The obtained LNP nanoparticles were dialyzed in a PBS solution (pH 7.4) for 24 h, concentrated using an Amicon ultracentrifugal filter, and filtered through a 0.22 μm filter membrane twice to obtain LNPs encapsulating mRNA.

[0032] The two purified mRNAs described in Example 1 were used in the above-mentioned manner to prepare LNPs encapsulating mRNA, which were labeled as TROP2-LNP and TROP2-pre-optimization-LNP.

[0033] The encapsulation efficiency of TROP2-LNP and TROP2-pre-optimized-LNP was determined using the Quant-iT™ RiboGreen® RNA Reagent and Kit (purchased from Invitorgen), and the particle size was determined using a Malvern particle size analyzer. The results are shown in Table 1.

[0034] As shown in Table 1, the encapsulation efficiency of TROP2-LNP is 87.3%, the particle size is 108 nm, the particle size is uniform, and the PDI is 0.16; the encapsulation efficiency of TROP2-pre-optimization-LNP is 84.9%, the particle size is 112 nm, and the PDI is 0.15.

[0035] Table 1 Physicochemical properties of two mRNA-encapsulated LNPs

[0036]

[0037] Example 3 Coupling CD7 Antibody

[0038] Two kinds of CD7 antibodies were used for experiments in the present invention, one of which was a sequence-optimized CD7 antibody, whose heavy chain amino acid sequence was shown in SEQ ID NO: 5 and whose light chain amino acid sequence was SEQ ID NO: 6. The CD7 antibody was prepared according to conventional methods; the other was a CD7 antibody purchased from Wuhan Elerite Biotechnology Co., Ltd.

[0039] 1.5 nmol of CD7 antibody was functionalized with 10-fold molar amount of SATA (N-succinimidyl S-acetylthioacetate) and reacted at room temperature for 30 minutes to introduce thiol groups and obtain a thiol-containing CD7 antibody.

[0040] Use 0.05M N-hydroxylamine and add equal volumes to the thiol-containing CD7 antibody, incubate for 2 hours, deprotect the acetylated thiol group, and obtain a deprotected CD7 antibody solution. Pour the dextran gel G-25 swollen with deionized water into a chromatography column, and balance the chromatography column. After filtering the deprotected CD7 antibody solution with a 0.45μm filter membrane, add it to the chromatography column, elute with deionized water to remove unreacted components, and finally obtain a SATA-modified antibody.

[0041] The SATA-modified antibody was mixed with the mRNA-encapsulated LNP at a molar ratio of 1:2000, and the sulfhydryl group on the SATA-modified antibody was coupled to one end of the maleimide in the LNP through a thioether coupling reaction, and then purified using a Sepharose CL-4B gel filtration column to obtain LNP coupled with the CD7 antibody.

[0042] TROP2-LNP and TROP2-pre-optimization-LNP were coupled with two CD7 antibodies according to the above method to obtain LNPs coupled with CD7 antibodies, which were marked as CD7-TROP2-LNP, CD7-TROP2-pre-optimization-LNP, CD7-TROP2-LNP and CD7-TROP2-pre-optimization-LNP, respectively.

[0043] The mRNA content in the above CD7 antibody-coupled LNPs was determined by Quant-iT RiboGreen RNA assay (Invitrogen). The results showed that the concentration of mRNA in CD7-TROP2-LNP was 36 μg / mL, the concentration of mRNA in CD7-TROP2-pre-optimization-LNP was 41 μg / mL, the concentration of mRNA in CD7-TROP2-LNP was 40 μg / mL, and the concentration of mRNA in CD7-TROP2-pre-optimization-LNP was 44 μg / mL.

[0044] Example 4 In vitro transfection experiment of LNPs coupled to CD7 antibodies in T cells

[0045] (1) Preparation of T cells

[0046] 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 counted and the number of cells was 1×10 6 cells / mL, added to a medium (purchased from Corning, catalog number: 88-551-CM) containing 1000IU / mL recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical). Cultured in a 37°C, 5% CO2 incubator for 24 hours, added 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 T cells.

[0047] (2) In vitro transfection experiment

[0048] Activated T cells were seeded in 24-well plates (6 × 10 4 The cells were divided into 6 groups, and CD7 purchased-TROP2-LNP, CD7 purchased-TROP2-pre-optimization-LNP, CD7-pre-TROP2-LNP, CD7-pre-TROP2-LNP, CD7-pre-TROP2-pre-optimization-LNP, TROP2-LNP (all 50 ng / well, calculated as mRNA) and an equal volume of PBS solution were added respectively. After incubation for 24 h, the cells were collected and the expression rate was detected by TROP2 flow cytometry antibody (purchased from Abcam, catalog number: ab214488). The results are shown in Tables 2 and 3 Figure 2 shown.

[0049] Table 2 Expression rate of TROP2 in T cells transfected with different LNPs

[0050]

[0051] From Table 2 and Figure 2 It can be seen that after the CD7 antibody prepared by the present invention is coupled to LNP and transfected into T cells, the expression rate of TROP2 protein is between 76.3% and 85.2%, and after the purchased CD7 antibody is coupled to LNP and transfected into T cells, the expression rate of TROP2 protein is between 66.4% and 73.8%. It shows that the CD7 antibody prepared by the present invention has a higher binding efficiency with T cells than the purchased CD7 antibody, and the efficiency of CAR-T cell generation is higher.

[0052] Compared with T cells transfected with CD7-TROP2-pre-optimization-LNP, the expression rate of TROP2 in T cells transfected with CD7-TROP2-LNP was increased, indicating that optimization of the nucleotide sequence of TROP2 can increase the expression rate of TROP2 protein in T cells.

[0053] The expression rate of TROP2 protein in TROP2-LNP-transfected T cells was 35.7%, while the expression rate of TROP2 protein in LNP-transfected T cells after coupling with CD7 antibodies was significantly increased, indicating that coupling with CD7 antibodies can improve the binding efficiency with T cells and increase the generation rate of CAR-T cells.

[0054] Example 5 Evaluation of in vitro tumor killing ability

[0055] The T cells transfected with LNPs coupled to CD7 antibodies in Example 4 were labeled as CD7-TROP2-LNP-T cells, CD7-TROP2-pre-optimization-LNP-T cells, CD7-TROP2-LNP-T cells, CD7-TROP2-pre-optimization-LNP-T cells, and TROP2-LNP-T cells, respectively, and inoculated into 96-well plates according to the groups, with 1×10 cells per well. 4 luciferase-labeled cervical cancer HELA cells (1×10 4 Cells / well) were added and cultured for 4 h. The cells were collected and the killing of tumor cells in each group was determined using an ELISA instrument. The killing ability of CAR-T cells on the colorectal cancer LOVO cell line was detected in the same way.

[0056] The results are as follows Figure 3 As shown in Table 3, the killing efficiency of CD7-TROP2-LNP-T cells on HELA cells and LOVO cells was significantly higher than that of CD7-TROP2-LNP-T, and there was a significant difference between the two, indicating that the T cells transfected with TROP2-LNP coupled to the CD7 antibody prepared by the present invention can significantly improve the killing efficiency of tumor cells compared with the T cells transfected with TROP2-LNP coupled to the purchased CD7 antibody.

[0057] The killing efficiency of CD7-TROP2-LNP on HELA cells and LOVO cells was 94.6% and 95.1%, respectively, and the killing efficiency of CD7-TROP2-pre-optimization-LNP on HELA cells and LOVO cells was 80.3% and 82.7%, indicating that sequence optimization of TROP2 can improve the killing efficiency of the final CAR-T cells against tumor cells.

[0058] Table 3 Killing efficiency of tumor cells by T cells transfected with different LNPs

[0059]

[0060] Example 6 In vivo efficacy evaluation of CD7-TROP2-LNP

[0061] Digest LOVO cells in the logarithmic growth phase at a concentration of 1×10 7 Cells / mL were added, and 200 μL of LOVO cells were injected subcutaneously into C57BL / 6 mice to construct a colorectal cancer mouse model. 3 The mouse model was successfully constructed.

[0062] Fourteen days after modeling, the mice were randomly divided into 6 groups (n=10) and injected with PBS, CD7-TROP2-LNP, CD7-TROP2-pre-optimized-LNP, CD7-TROP2-LNP, CD7-TROP2-pre-optimized-LNP, and TROP2-LNP (dosage of 0.3 mg / kg, calculated as mRNA) through the tail vein once a week for two times (once every other week). The survival status of the mice was monitored within 60 days after modeling, and the survival curve was drawn. The results are shown in Figure 4 shown.

[0063] Depend on Figure 4 It can be seen that CD7-TROP2-LNP showed the best survival improvement effect, with a survival rate of 50% within 60 days. The survival rate of mice in the CD7-TROP2-optimized pre-LNP group was 20% within 60 days, the survival rate of mice in the CD7-TROP2-LNP group was 30% within 60 days, the survival rate of mice in the CD7-TROP2-optimized pre-LNP group was 20% within 60 days, the survival rate of mice in the TROP2-CXCL10-LNP group was 10% within 60 days, and all mice in the PBS group died.

[0064] Under the same TROP2-LNP condition, the survival rate of mice injected with the CD8 antibody-coupled TROP2-LNP prepared by the present invention was higher than that of mice injected with the purchased CD7 antibody-coupled TROP2-LNP, indicating that the prepared CD7 antibody-coupled TROP2-LNP can improve the survival rate of tumor mice compared with the purchased CD7 antibody-coupled TROP2-LNP.

[0065] Under the same CD7 antibody, the survival rate of mice injected with CD7 antibody-conjugated TROP2-LNP was higher than that of mice injected with CD7 antibody-conjugated TROP2-pre-optimized-LNP, indicating that the survival rate of tumor-bearing mice can be improved after the sequence of TROP2 single-chain antibody is optimized.

Claims

1. A nanoparticle for generating CAR-T in vivo, characterized in that: The nanoparticles are mRNA-encapsulated nanoparticles coupled to CD7 antibodies; the mRNA is transcribed from a target gene; the target gene includes a TROP2 single-chain antibody; the nucleotide sequence of the target gene is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the CD7 antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

6.

2. The nanoparticle for generating CAR-T in vivo according to claim 1, characterized in that: The nanoparticles are nanoparticles in which mRNA is encapsulated by LNP.

3. The nanoparticle for generating CAR-T in vivo according to claim 1, characterized in that: The concentration of mRNA in the nanoparticles was 36-44 μg / mL.

4. Use of the nanoparticles according to any one of claims 1 to 3 in the preparation of medicines for treating cervical cancer and / or colorectal cancer.

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