HPV (human papillomavirus) vaccine carrying CAR (chimeric antigen receptor) structure as well as preparation method and application thereof

Abstract

The invention provides an HPV (human papillomavirus) vaccine carrying a CAR (chimeric antigen receptor) structure as well as a preparation method and application thereof, and belongs to the technical field of medical preparations. The CAR structure comprises E7 single-chain antibodies aiming at HPV16, 18, 31 and 33 subtypes and E7 single-chain antibodies aiming at HPV45, 52, 56, 58 and 59 subtypes; the nucleic acid artificial sequence of the E7 single-chain antibody aiming at the HPV16, 18, 31 and 33 subtypes is as shown in SEQ ID NO.5 in a sequence table; the nucleic acid artificial sequence of the E7 single-chain antibody aiming at the HPV45, 52, 56, 58 and 59 subtypes is shown as SEQ ID NO.8 in a sequence table. The prepared HPV vaccine carrying the CAR structure effectively captures CD8 + T cells in a body, forms CAR-T cells, effectively reduces the HPV virus titer in the body of a mouse, can resist infection of various HPV virus subtypes at the same time, and has a remarkable protection effect on the mouse.

Description

Technical Field

[0001] The present invention provides an HPV vaccine carrying a CAR structure, a preparation method and application thereof, and belongs to the technical field of medical preparations. Background Art

[0002] The main risk factor for cervical cancer is infection with human papillomavirus (HPV), especially high-risk strains such as HPV16 and HPV18. Viral infection can cause abnormal changes in cervical epithelial cells and then develop into cervical cancer. HPV is a double-stranded circular DNA virus that is approximately 8,000 base pairs long and has 8 or 9 open reading frames (ORFs).

[0003] The genome of HPV is divided into three main regions, namely the early region (E), the late region (L), and the upstream regulatory region (URR). The early region (E) contains genes necessary for encoding the viral cycle and plays an important role in cell transformation, including E1, E2, E4, E5, E6, and E7 genes; the late region (L) encodes the major coat protein L1 and the minor coat protein L2 of the virus particles; the upstream regulatory region (URR) is a non-coding region containing replication origins and transcription factor binding sites, which regulates DNA replication by controlling viral gene transcription. Existing studies have found that HPV-infected diseased cells can continuously express E6 and E7 proteins. Therefore, E6 and E7 can be used as target molecules for the immune system to identify HPV-infected cells and related tumor cells.

[0004] At present, all the HPV preventive vaccines available in foreign countries are HPV L1 virus-like particle vaccines (abbreviated as L1 VLP). The immune response induced by L1VLP can only prevent HPV infection, and has no therapeutic effect on existing infection or lesions. Therefore, it is urgent to develop an HPV vaccine for therapeutic purposes. DNA vaccines have the characteristics of easy production, stable physical properties, and low cost for population application. They have advantages in vaccine accessibility and can also induce the body to produce a comprehensive and lasting immune response, especially cellular immune response. The application of vaccines containing CAR structures in the treatment of HPV-related tumors is still in the exploratory stage. One study prepared CAR-T cells that specifically kill HPV16-positive cervical cancer cells. The third-generation CAR gene sequence was synthesized by targeting the HPV16 E711-19:HLA-A0201 complex, and it was transduced into peripheral blood T cells to prepare CAR-T cells. The results of in vitro co-culture experiments showed that when CAR-T cells were co-cultured with target cells CaSki and HeLa cells at an effector-target ratio of 20:1, the in vitro killing rates of CAR-T cells against target cells Ca Ski and HeLa were 75.2% and 49.3%, respectively.

[0005] The anti-HPV CAR-T cells prepared in the prior art are all targeted at one HPV virus subtype, and have not yet targeted multiple HPV virus subtypes; at the same time, there are no reports in the prior art on the generation of CAR-T cells in patients to kill HPV viruses. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides an HPV vaccine carrying a CAR structure, a preparation method and an application thereof, to achieve the following invention objectives: the T cells transfected with the HPV vaccine target multiple HPV virus subtypes and have a high in vitro killing efficiency of target cells; the HPV vaccine can effectively capture CD8+T cells in the body and improve the body's anti-HPV virus effect.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: A HPV vaccine carrying a CAR structure, the CAR structure comprising E7 single-chain antibodies against HPV16, 18, 31, and 33 subtypes and E7 single-chain antibodies against HPV45, 52, 56, 58, and 59 subtypes; the artificial nucleic acid sequence of the E7 single-chain antibodies against HPV16, 18, 31, and 33 subtypes is shown in SEQ ID NO.5 in the sequence listing; the artificial nucleic acid sequence of the E7 single-chain antibodies against HPV45, 52, 56, 58, and 59 subtypes is shown in SEQ ID NO.8 in the sequence listing.

[0008] The CAR structure is composed of the following modules connected in series: a guide, an E7 single-chain antibody targeting HPV16, 18, 31, and 33 subtypes, a linker, an E7 single-chain antibody targeting HPV45, 52, 56, 58, and 59 subtypes, a CD8 Hinge region, a CD28 transmembrane region, a CD28 / 4-1BB co-stimulatory region, and a CD3ζ intracellular region.

[0009] The artificial nucleic acid sequence of the CAR structure is shown in SEQ ID NO.2 in the sequence list.

[0010] The HPV vaccine is a lipid nanoparticle coupled to a CD8 antibody, the heavy chain amino acid sequence of the CD8 antibody is shown in SEQ ID NO.13 in the sequence list, and the light chain amino acid sequence is shown in SEQ ID NO.14 in the sequence list.

[0011] In the HPV vaccine, the DNA concentration is 50-54 ng / μL.

[0012] The preparation method of the HPV vaccine comprises constructing a CAR structure to obtain a recombinant plasmid, encapsulating the recombinant plasmid with LNP, and then coupling with a CD8 antibody to obtain the HPV vaccine.

[0013] The HPV vaccine is used in the preparation of medicines for treating cervical cancer caused by HPV virus.

[0014] The application of the HPV vaccine-modified T cells in the preparation of medicines for treating cervical cancer caused by HPV virus.

[0015] Compared with the prior art, the present invention achieves the following beneficial effects: In the present application, the nucleotide sequences of the single-chain antibody of the HPV16 / 18 / 31 / 33 E7 protein and the single-chain antibody of the HPV45 / 52 / 56 / 58 / 59 E7 protein are optimized respectively, and then connected to prepare a CAR structure containing dual targets, and then LNP coupled with CD8 antibody is prepared, that is, an HPV vaccine carrying a CAR structure is obtained. The vaccine can effectively capture CD8+T cells in the body, eliminate the complicated process of in vitro preparation, directly produce CAR-T cells in the body, and effectively resist infection by multiple HPV virus subtypes.

[0016] The HPV vaccine-modified T cells carrying the CAR structure prepared by the present invention have high killing efficiency against Ca Ski and HeLa target cells. When the efficacy-target ratio is 20:1, the killing rates against Ca Ski and HeLa target cells are 93.4% and 91.5% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow cytometric graph of the infection rate of AntiCD8-LNP-HPV-1 on activated CD8+T cells; Figure 2 Flow cytometric graph of the infection rate of AntiCD8-LNP-HPV-2 on activated CD8+T cells; Figure 3 It is a line graph of the killing efficiency of two types of AntiCD8-LNP-HPV-transfected T cells on tumor cells; A is a line graph showing the killing efficiency of two types of AntiCD8-LNP-HPV-transfected T cells on Ca Ski cells, and B is a line graph showing the killing efficiency of two types of AntiCD8-LNP-HPV-transfected T cells on HeLa cells. DETAILED DESCRIPTION

[0018] Example 1 Design of target gene and construction of recombinant plasmid The modules of the CAR structure and their artificial nucleic acid sequences are as follows: (1) A leader, whose artificial nucleic acid sequence is shown as SEQ ID NO.3 in the sequence listing; (2) Single-chain antibody scFv-E7 (HPV16, 18, 31, 33), the artificial nucleic acid sequence before optimization is shown in SEQ ID NO.4 in the sequence listing; the artificial nucleic acid sequence after optimization is shown in SEQ ID NO.5 in the sequence listing; (3) a linker, whose artificial nucleic acid sequence is shown in SEQ ID NO.6 in the sequence listing; (4) Single-chain antibody scFv-E7 (HPV45, 52, 56, 58, 59), the artificial nucleic acid sequence before optimization is shown in SEQ ID NO.7 in the sequence listing; the artificial nucleic acid sequence after optimization is shown in SEQ ID NO.8 in the sequence listing; (5) CD8 Hinge region, the artificial nucleic acid sequence of which is shown in SEQ ID NO.9 in the sequence listing; (6) CD28 transmembrane region, whose artificial nucleic acid sequence is shown as SEQ ID NO.10 in the sequence list; (7) CD28 / 4-1BB co-stimulatory region, whose artificial nucleic acid sequence is shown in SEQ ID NO.11 in the sequence list; (8) CD3ζ intracellular region, whose artificial nucleic acid sequence is shown in SEQ ID NO.12 in the sequence list; The artificial nucleic acid sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 were connected in sequence, and the connected sequence was shown in SEQ ID NO.1 in the sequence table. Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the entire expression frame and insert it into the pUC57 vector to obtain the pUC-CAR vector. The pUC-CAR-1 vector and the PVAX1 vector were double-digested with Fast Digest BamH I (purchased from Thermo Fisher) and Fast Digest Not I (purchased from Thermo Fisher), and the linearized DNA fragments were obtained by gel cutting and recovery, and the PVAX1-CAR-HPV expression vector was formed before optimization at 16°C overnight. The above-mentioned PVAX1-CAR-HPV pre-optimization expression vector was transformed into E. coli (DH5α), and the positive clones were picked for PCR identification. Then the plasmid was extracted and sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing. If the sequencing identification was correct, the PVAX1-CAR-HPV pre-optimization expression vector was successfully constructed. The PVAX1-CAR-HPV pre-optimization recombinant plasmid was extracted from the positive clone, named PVAX1-CAR-HPV-1, diluted to 2 μg / μL, and stored at -80°C for use.

[0019] Using the same method, the artificial nucleic acid sequences of SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, 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. The connected sequence is shown in SEQ ID NO.2 in the sequence table. The PVAX1-CAR-HPV optimized expression vector was constructed according to the above method, and the PVAX1-CAR-HPV optimized recombinant plasmid was extracted from the positive clone, named PVAX1-CAR-HPV-2, diluted to 2 μg / μL, and stored at -80°C for use.

[0020] Example 2 LNP-encapsulated recombinant plasmid (1) Take 15 μL of the PVAX1-CAR-HPV-1 recombinant plasmid prepared in Example 1 (concentration 2.0 μg / μL), dilute to 300 μL using a citric acid buffer with a pH of 4 and a concentration of 10 mM to obtain a recombinant plasmid mixture; (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; (3) The recombinant plasmid mixture and the mixed lipid solution were added to the microfluidic device. The mass ratio of DNA in the recombinant plasmid mixture and the ionizable lipid DLin-MC3-DMA in the mixed lipid solution was 1:10. LNP nanoparticles encapsulating the new antigens of various HPV subtypes were prepared by the microfluidic method.

[0021] The obtained LNP nanoparticles were dialyzed in 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 the recombinant plasmid, named LNP-HPV-1.

[0022] The LNPs carrying the recombinant plasmid PVAX1-CAR-HPV-2 were prepared by the same method as above and named LNP-HPV-2.

[0023] The particle sizes of LNP-HPV-1 and LNP-HPV-2 were measured using a dynamic light scattering particle size potential meter, and the encapsulation efficiency was determined using the Quant-iT 1X dsDNA kit (Invitrogen).

[0024] The results are shown in Table 1. The encapsulation efficiency of LNP-HPV-1 was 84.6±3.59%, and the particle size was 110.6±4.0 nm; the encapsulation efficiency of LNP-HPV-2 was 86.7±2.68%, and the particle size was 107.3±5.3 nm.

[0025] Table 1 Encapsulation efficiency and particle size of LNP-HPV-1 and LNP-HPV-2

[0026] Example 3 Coupling CD8 Antibody The heavy chain amino acid sequence of the CD8 antibody is shown in SEQ ID NO.13 in the sequence list, and the light chain amino acid sequence is shown in SEQ ID NO.14 in the sequence list. The CD8 antibody was prepared according to a conventional method, and the CD8 antibody was placed in PBS containing 1mM DTT and 5mM EDTA at room temperature for 1h to reduce it, and then the DTT was removed by buffer exchange into PBS containing 5mM EDTA using a 7K Zeba desalting centrifugal column (purchased from ThermoFisher Scientific). After the buffer exchange, the reduced CD8 antibody was immediately added to LNP-HPV-1 (0.67mg of CD8 antibody was added per ml of LNP-HPV-1), and the mixture was gently shaken and incubated at room temperature for 2 hours, and then incubated at 4°C overnight. On the next day, CD8 antibody-coupled LNP-HPV-1 was separated from free antibody on a sepharose CL-4B filter column using PBS as the mobile phase, and concentrated to the initial volume of LNP-HPV-1 using a 100K Millipore ultrafiltration centrifuge tube (purchased from Solebol) to obtain CD8 antibody-coupled LNP-HPV-1, named AntiCD8-LNP-HPV-1.

[0027] LNP-HPV-2 was coupled to CD8 antibody in the same way to obtain AntiCD8-LNP-HPV-2.

[0028] The AntiCD8-LNP-HPV-1 and AntiCD8-LNP-HPV-2 were lysed by Triton X-100, and the amount of DNA was measured using the Qubit™ dsDNA BR Assay Kit. The results showed that the concentration of DNA (recombinant plasmid) in AntiCD8-LNP-HPV-1 was 52 ng / μL, and the final concentration was adjusted to 50 ng / μL. The concentration of DNA (recombinant plasmid) in AntiCD8-LNP-HPV-2 was 54 ng / μL, and the final concentration was adjusted to 50 ng / μL, and they were stored for later use.

[0029] Example 4 AntiCD8-LNP-HPV-1 and AntiCD8-LNP-HPV-2 transfected CD8+ T cells (1) Preparation of T cells 75 mL of the patient's autologous peripheral blood was collected and peripheral blood mononuclear cells were separated using Ficoll-Paque lymphocyte separation solution. The separated cells were sorted out with CD8+ sorting reagent provided by BD to sort out CD8+ T cells, and the cells were counted and counted according to 1×10 6 Cells / mL were inoculated, and KBM551 cell culture medium (purchased from Corning, catalog number: 88-551-CM) containing a final concentration of 1500 IU / mL IL-2 was added. Then, the same number of CD3CD28 magnetic beads as the cells were added to activate T cells, and the magnetic beads were removed after 24 hours of activation to obtain activated CD8+ T cells.

[0030] (2) Transfection of CD8+T cells Adjust the number of activated CD8+ T cells to 1×10 7 / mL, a cell suspension was obtained, 0.1mL of the cell suspension was inoculated in a 6-well plate, placed in a 37°C, 5% CO2, saturated humidity incubator for overnight culture, and then 100ng of AntiCD8-LNP-HPV-1 prepared in Example 3 was added. After incubation at 37°C for 48h, AntiCD8-LNP-HPV-1-CAR-T cells were collected by centrifugation, referred to as CAR-T-1. AntiCD8-LNP-HPV-2-CAR-T cells, referred to as CAR-T-2, were prepared in the same way.

[0031] The expression of chimeric antigen receptors of CAR-T cells was detected by flow cytometry. Activated CD8+ T cells were used as negative controls, such as Figure 1 , 2 As shown, the infection rate of AntiCD8-LNP-HPV-1 on activated CD8+T cells was 57.6%, and the infection rate of AntiCD8-LNP-HPV-2 on activated CD8+T cells was 62.2%.

[0032] Example 5 In vitro killing experiment The concentrations of effector cells and target cells were adjusted with DMEM medium containing 10 vol% FBS and inoculated in 96-well plates with effector-target ratios of 1:1, 5:1, 10:1, and 20:1. The effector cells in the experimental group were the CAR-T-1 cells and CAR-T-2 cells prepared in Example 4, respectively. The control group was treated with an equal volume of PBS, and the target cells were luciferase-labeled Ca Ski human cervical cancer cells and HeLa human cervical cancer cells. Ca Ski human cervical cancer cells contain HPV16 sequences, and HeLa human cervical cancer cells contain HPV18 sequences.

[0033] Each target cell was divided into three groups, each with three replicates, and cultured in a 5% CO2, 37°C incubator. After 24 hours, the OD value of each group was measured by a microplate reader. The cell killing rate was calculated as follows: killing rate (%) = [1- OD value of experimental group / OD value of control group] × 100%.

[0034] The results are shown in Table 2 and Figure 3 As shown in the figure: When the effector-target ratio is 20:1, the killing rates of CAR-T-2 against Ca Ski and HeLa cells are 93.4% and 91.5%, respectively, and the killing rates of CAR-T-1 against Ca Ski and HeLa cells are 73.6% and 75.8%, respectively. Compared with the T cells transfected with AntiCD8-LNP-HPV-1, the killing efficiency of T cells transfected with AntiCD8-LNP-HPV-2 against the two tumor cells is significantly improved. This indicates that after the nucleotide sequence of the single-chain antibody scFv-E7 is optimized, the killing efficiency of T cells transfected with AntiCD8-LNP-HPV-2 against tumor cells can be significantly improved.

[0035] Table 2 Killing rate of two CAR-T cells on tumor cells

[0036] Example 6 Efficacy experiment of AntiCD8-LNP-HPV in mouse model 1. HPV pseudovirus used in this example HPV (16) Luciferase Pseudotyped Virus (GM-0220PV173) HPV (18) Luciferase Pseudotyped Virus (GM-0220PV174) HPV (31) Luciferase Pseudotyped Virus (GM-0220PV175) HPV (45) Luciferase Pseudotyped Virus (GM-0220PV176) HPV (58) Luciferase Pseudotyped Virus (GM-0220PV177) The above pseudoviruses were purchased from Jiman Biotechnology (Shanghai) Co., Ltd. 2. Establishment of a mouse model carrying HPV virus According to the above five viruses, mice were divided into five groups, with 15 mice in each group, to establish a mouse model carrying HPV virus. The specific method is as follows: 1) Dilute medroxyprogesterone acetate (component Depo-M, purchased from a pharmacy) with sterile PBS at a ratio of 1:5 to a final concentration of 30 mg / mL Depo-M solution. Each mouse was subcutaneously injected with 100 µL of Depo-M solution and cultured for 4 days.

[0037] 2) Preparation of virus-CMC mixture: 4 g of CMC powder (purchased from Nanjing Ximenuo Biotechnology Co., Ltd.) was dissolved in 100 g of deionized water to obtain a 4% CMC solution. The pseudovirus prepared in step 1 was fully mixed with the 4% CMC solution at a volume ratio of 3:1 to obtain a virus-CMC mixture.

[0038] 3) The mice were anesthetized using a gas anesthesia system (XGI-8), with the abdomen facing upward. 50 µL of 4% N9 (purchased from a pharmacy) was pipetted, and the tip of the pipette was inserted into the bottom of the mouse vagina and infused into the vagina. After 6 hours, 20 µL of the prepared virus-CMC mixture was pipetted and infused into the mouse vagina. The culture was continued for 4 days, and luciferase was injected. The fluorescence was clearly observed using a mouse in vivo imaging device, indicating that the HPV virus-carrying mouse model was successfully established.

[0039] The 15 mice in each group were further divided into three groups, A, B, and C, with 5 mice in each group, as shown in Table 3, and injected with the prepared AntiCD8-LNP-HPV-1 (Group A), AntiCD8-LNP-HPV-2 (Group B), and PBS control group (Group C). The date of mouse model establishment was day 0, and immunization began with a dose of 50 μg / mouse / time. Immunization was boosted once on the 3rd day, and the virus titer in the mice was detected on the 7th day.

[0040] The results are shown in Table 4. The virus titer in mice after immunization with the HPV vaccine prepared in the present application was significantly lower than that in the control group. The HPV vaccine prepared after sequence optimization of the single-chain antibody scFv-E7 had the lowest virus titer in mice after immunization, indicating that the HPV vaccine prepared in the present application has a significant protective effect on mice and can resist infection with multiple HPV virus subtypes.

[0041] Table 3 Experimental groups

[0042] Table 4 Virus titers in mice 7 days after immunization

Claims

1. An HPV vaccine carrying a CAR structure, characterized in that: The CAR structure includes E7 single-chain antibodies against HPV16, 18, 31, and 33 subtypes and E7 single-chain antibodies against HPV45, 52, 56, 58, and 59 subtypes; the artificial nucleic acid sequence of the E7 single-chain antibodies against HPV16, 18, 31, and 33 subtypes is shown in SEQ ID NO.5 in the sequence listing; the artificial nucleic acid sequence of the E7 single-chain antibodies against HPV45, 52, 56, 58, and 59 subtypes is shown in SEQ ID NO.8 in the sequence listing.

2. The HPV vaccine carrying a CAR structure according to claim 1, characterized in that: The CAR structure is composed of the following modules connected in series: a guide, an E7 single-chain antibody targeting HPV16, 18, 31, and 33 subtypes, a linker, an E7 single-chain antibody targeting HPV45, 52, 56, 58, and 59 subtypes, a CD8 Hinge region, a CD28 transmembrane region, a CD28 / 4-1BB co-stimulatory region, and a CD3ζ intracellular region.

3. The HPV vaccine carrying a CAR structure according to claim 1, characterized in that: The artificial nucleic acid sequence of the CAR structure is shown in SEQ ID NO.2 in the sequence list.

4. The HPV vaccine carrying a CAR structure according to claim 1, characterized in that: The HPV vaccine is a lipid nanoparticle coupled to a CD8 antibody, the heavy chain amino acid sequence of the CD8 antibody is shown in SEQ ID NO.13 in the sequence list, and the light chain amino acid sequence is shown in SEQ ID NO.14 in the sequence list.

5. The HPV vaccine carrying a CAR structure according to claim 1, characterized in that: In the HPV vaccine, the DNA concentration is 50-54 ng / μL.

6. The method for preparing the HPV vaccine according to any one of claims 1 to 5, characterized in that: The preparation method of the HPV vaccine comprises constructing a CAR structure to obtain a recombinant plasmid, encapsulating the recombinant plasmid with LNP, and then coupling with a CD8 antibody to obtain the HPV vaccine.

7. Use of the HPV vaccine according to any one of claims 1 to 5 in the preparation of medicines for treating cervical cancer caused by HPV virus.

8. Use of the HPV vaccine-modified T cells according to any one of claims 1 to 5 in the preparation of medicines for treating cervical cancer caused by HPV virus.

Images