Her2 adenovirus and use thereof

Abstract

The application discloses a HER2 adenovirus and application thereof, and belongs to the technical field of adenovirus preparation. The application provides a novel HER2 specific chimeric antigen, and the nucleotide sequence is shown as SEQ ID NO. 19. The chimeric antigen is formed by inserting a CAR structure into an MCS region of an Ad5F35 type adenovirus vector, and the CAR structure is as follows: signal peptide + C-MYC + HER2scfv + hinge region + transmembrane region + CD3Z + FCER1G + CD19. The novel HER2 adenovirus can specifically target HER2 positive tumor cells, has a wide host range, has high infection efficiency, and can be used for infecting hematopoietic stem cells, dendritic cells, T cells, NK cells, K562, U937 and other difficult-to-transfect blood cells. The infected cells can rapidly express the adenovirus, the expression stability is high, the adenovirus cannot be integrated into host chromosomes, and the safety is good.

Description

Technical Field

[0001] This invention relates to the field of adenovirus preparation technology, and in particular to a HER2 adenovirus and its applications. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, seriously endangering their lives and health. According to the International Agency for Research on Cancer (IARC) of the World Health Organization, in 2020, there were 2.26 million new cases of breast cancer globally, accounting for 11.7% of all cancer cases, surpassing lung cancer for the first time to become the "world's leading cause of cancer death." Human epidermal growth factor receptor 2 (HER2) positive breast cancer accounts for 15%-20% of all breast cancers. Compared to other breast cancer subtypes, HER2-positive breast cancer is characterized by its high invasiveness, high recurrence and metastasis rates, and poor prognosis, making it a hot research topic in the field of breast cancer. HER2 testing has become an important component of breast cancer diagnosis and treatment.

[0003] HER2 is a transmembrane protein with tyrosine kinase activity, encoded by the proto-oncogene HER2 / neu. HER2 is expressed at low levels in many normal tissues, but is overexpressed in some tumor cells. It is closely related to tumor occurrence, development, and prognosis, and can promote tumor angiogenesis, increasing the invasive and metastatic abilities of tumor cells.

[0004] In the field of molecular biology, adenovirus vectors are one of the most commonly used vectors for introducing exogenous genes into animal cells. Due to their advantages such as a wide variety of target cells, convenient preparation, high amplification efficiency, high titer, high transduction efficiency, low immunogenicity, high transduction efficiency for both proliferating and quiescent cells, non-integration into the host genome, no insertion mutations, relatively stable physicochemical properties, easy separation and purification, and ability to accommodate large target gene fragments, they are widely used in experimental and clinical research such as gene therapy, in vitro gene transfection, and gene vaccine preparation. Summary of the Invention

[0005] The purpose of this invention is to provide a HER2 adenovirus and its application, which is to provide a novel adenovirus that has higher infection efficiency in blood cells, does not rely on CAR receptors, has a wider host range, higher infection efficiency, specifically targets HER2-positive tumor cells, and has better safety.

[0006] To achieve the above objectives, the present invention provides a CAR structure, wherein the CAR structure is: signal peptide + C-MYC + HER2scfv + hinge region + transmembrane region + CD3Z + FCERT1G + CD19.

[0007] Preferably, the transmembrane region and CD3Z are connected by the sequence shown in SEQ ID NO.1; and FCER1G and CD19 are connected by the sequence shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the signal peptide is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the C-MYC is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0009] Preferably, the scfv nucleotide sequence of the HER2 is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the hinge region is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; the nucleotide sequence of the transmembrane region is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12.

[0010] Preferably, the nucleotide sequence of CD3z is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14; the nucleotide sequence of FCER1G is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16; the nucleotide sequence of CD19 is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18.

[0011] A HER2 adenovirus that integrates the aforementioned CAR structure.

[0012] Preferably, the CAR structure is directionally inserted into the MCS region of the Ad5F35 adenovirus vector through genetic engineering to obtain a recombinant HER2-targeting adenovirus vector; the nucleotide sequence of the HER2 adenovirus is shown in SEQ ID NO.19.

[0013] Application of a HER2 adenovirus as described above in gene editing.

[0014] The use of a HER2 adenovirus as described above in the preparation of targeted therapeutic drugs for HER2-positive tumors.

[0015] The application of a HER2 adenovirus as described above in vaccine preparation.

[0016] The Ad5 / F35 adenovirus vector is a chimeric gene delivery tool. Its core feature is the modification of the Ad5 vector's spike protein, integrating the receptor-binding domain of the F35 serotype into the Ad5 backbone, thereby altering the receptor type for infecting target cells. While the original Ad5 relies on the CAR receptor to infect cells, Ad5 / F35, through modification of the F35 spike protein, binds to the CD46 receptor, significantly improving infection efficiency in cells with low CAR receptor expression (such as mesenchymal stem cells and immune cells). The Ad5 / F35 adenovirus vector retains the high capacity (accommodating 8kb of foreign genes) and efficient transduction properties of the Ad5 vector, while also possessing a broader host range; safety is ensured by deleting the E1 / E3 genes, preventing integration into the host genome.

[0017] Therefore, the HER2 adenovirus and its application provided by this invention have the following specific technical effects:

[0018] (1) This invention provides a novel, HER2-specific chimeric antigen receptor with the nucleotide sequence shown in SEQ ID NO.19. It is constructed by directionally inserting the chimeric antigen receptor (CAR) structure into the MCS region of the Ad5F35 adenovirus vector. The CAR structure is: signal peptide + C-MYC + HER2scfv + hinge region + transmembrane region + CD3Z + FCER1G + CD19.

[0019] (2) The novel HER2 adenovirus provided by the present invention can specifically target HER2-positive tumor cells, has a wide host range, and high infection efficiency. It can be used to infect hematopoietic stem cells, dendritic cells, T cells, NK cells, K562, U937 and other difficult-to-transfect blood cells. After infection, it can be rapidly expressed in cells with high expression stability. It will not integrate into the host chromosome and has good safety.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a map of the recombinant vector HER2-tomato constructed in Example 1 of this invention;

[0023] Figure 2These are electrophoresis images and concentration detection results of the recombinant vector HER2-tomato extracted in Example 1 of this invention; where A is the electrophoresis image of the extracted recombinant vector HER2-tomato, and B is the plasmid concentration detection result of the extracted recombinant vector HER2-tomato.

[0024] Figure 3 This is a fluorescence image of 293 cells 24 hours after the recombinant vectors HER2-tomato and right1.4 were transfected into the cells in Example 2 of this invention.

[0025] Figure 4 These are fluorescence images of cells transfected for 14 days in Example 3 of this invention;

[0026] Figure 5 This is the flow cytometry result of the viral titer determination in Example 3 of this invention; where A is the adenovirus diluted 10... 4 After being infected with 293 cells, the percentage of positive cells was measured 24 hours later. B and C were two replicate groups.

[0027] Figure 6 This is the flow cytometry result of the infection efficiency measured after infecting macrophages in Example 4 of the present invention;

[0028] Figure 7 This is the flow cytometry result of HER2 expression efficiency measured after macrophage infection in Example 4 of this invention. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail are all conventional techniques in the field.

[0032] Example 1

[0033] A HER2 adenovirus vector was constructed, as follows:

[0034] (1) The sequence information was sent to a company to synthesize a fusion gene fragment sequence with the following structure: signal peptide + C-MYC + HER2scfv + hinge region + transmembrane region + CD3Z + FCER1G + CD19. The company was commissioned to ligate this fragment to the tdtomato sequence via P2A and insert it into the MCS region of the zap1.1 vector. The resulting recombinant vector was named HER2-tomato (Ad5 / F35 adenovirus overexpression vector). The structural diagram of the recombinant vector HER2-tomato is shown below. Figure 1 The sequence is shown in SEQ ID NO.19. The received HER2-tomato was prepared into a 100 ng / μL solution according to the accompanying instructions.

[0035] The transmembrane region and CD3Z are linked by the sequence shown in SEQ ID NO.1, and FCER1G and CD19 are linked by the sequence shown in SEQ ID NO.2. The nucleotide sequence of the signal peptide is shown in SEQ ID NO.3; the amino acid sequence is shown in SEQ ID NO.4. The nucleotide sequence of C-MYC is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6. The scfv nucleotide sequence of HER2 is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8. The nucleotide sequence of the hinge region is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10. The nucleotide sequence of the transmembrane region is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12. The nucleotide sequence of CD3z is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14. The nucleotide sequence of FCER1G is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16. The nucleotide sequence of CD19 is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18.

[0036] SEQ ID NO.1: GGATCC

[0037] SEQ ID NO.2: GCTAGC

[0038] SEQ ID NO.3:

[0039] ATGGCGCTCCCTGTCACCGCACTGCTTCTTCCGCTGGCACTGCTGCTGCACGCTGCACGGCCT

[0040] SEQ ID NO.4: MALPPTALL PLALLLHAARP

[0041] SEQ ID NO.5: GAGCAAAAACTTATCTCTGAAGAGGACCTC

[0042] SEQ ID NO.6: EQKLISEEDL

[0043] SEQ ID NO.7:

[0044] GATATTCAAATGACCCAGAGTCCATCCAGCCTGAGTGCCAGTGTTGGCGACAGAGTCACCATTACTTGTCGAGCCAGCCAGGATGTGAACACCGCGGTGGCTTGGTATCAACAGAAGCCTGGAAAGGCTCCCAAGCTCCTGATCTATTCAGCTTCATTTCTCTACTCTGGGGTGTCTTCTAGGTTTTCAGGCAGTCGATCCGGCACGGACTTTACTCTGACGATTTCCAGTCTTCAACCCGAAGACTTCGCGACATACTACTGTCAACAGCATTATACCACCCCACCAACCTTTGGGCTGGGCACAAAGGTCGAGATAAAAAGAACTGGCAGTACTAGCGGGAGCGGCAAACCAGGATCCGGTGAGGGCAGCGAGGTTCAGCCTGTTGAGAGTGGCGGCGGCCTCGTGCAGCCTGGCGGACGCTTGAGGCTTTCATGCGCGGCCAGCGGGTTTAATATCAAGGAGACTTATATCCATTGGGTCAGACAAGCTCCCGGAAAAGGACTCGAGTGCGTGGCCCGGATCTACCCAACTAACGGATACACCCGGTATGCTGACTCCGTCAAGGGCAGGTTTACCATCTCCGCCGATACAAGTAAAAACACCGCCTACCTGCAGATGAATTCACTGCGGGCAGAAGATACAGCCGTGTACTACTGCTCAAGGTGGGGGGGAGATGGGTTTTACGCTATGGACGTCTGGGGGCAGGGGACCCTCGTAACAGTTAGCAGC

[0045] SEQ ID NO.8:

[0046] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVSSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGLGTKVEIKRTGSTSGSGKPGSGEGSEVQPVESGGGLVQPGGRLRLSCAASGFNIKETYIHWVRQAPGKGLECVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS

[0047] SEQ ID NO.9:

[0048] ACAACTACGCCGGCGCCACGACCACCTACTCCAGCTCCAACGATCGCTTCTCAACCACTTAGTTTGAGACCAGAGGCTTGCAGGCCGGCAGCGGGCGGGGCTGTGCATACTCGCGGGCTGGATTTTGCTTGTGAC

[0049] SEQ ID NO.10:

[0050] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0051] SEQ ID NO.11:

[0052] ATCTACATTTGGGCACCATTGGCCGGCACTTGTGGGGTTATCTTGCTCTCCCTGGTAATCACTCTTTACTGT

[0053] SEQ ID NO.12: IYIWAPLAGTCGVILLSLVITLYC

[0054] SEQ ID NO.13:

[0055] AGAGTAAAGTTTTCTCGGTCAGCCGATGCGCCTGCATACAAGCAGGGCCAAAATCAGCTTTACAACGAATTGAACCTTGGCAGAAGAGAAGAGTACGATGTACTGGACAAACGGAGGGGCCGAGACCCTGAAATGGGCGGGAAACCGCGGCGGAAAAATCCTCAGGAAGGCCTCTACAATGAGTTGCAAAAGGACAAGATGGCAGAGGCCTATTCCGAGATCGGTATGAAGGGTGAAAGAAGGAGGGGAAAAGGACACGACGGCCTCTACCAGGGACTCTCTACGGCTACAAAGGACACGTACGACGCTCTTCATATGCAAGCGCTCCCACCAAGG

[0056] SEQ ID NO.14:

[0057] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0058] SEQ ID NO.15:

[0059] CGTCTGAAGATCCAAGTGCGAAAGGCAGCTATAACCAGCTATGAGAAATCAGATGGTGTTTACACGGGCCTGAGCACCAGGAACCAGGAGACTTACGAGACTCTGAAGCATGAGAAACCACCACAG

[0060] SEQ ID NO.16:

[0061] RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ

[0062] SEQ ID NO.17:

[0063] CAGTCCTATGAGGATATGAGAGGAATCCTGTATGCAGCCCCCCAGCTCCGCTCCATTCGGGGCCAGCCTGGACCCAATCATGAGGAAGATGCAGACTCTTATGAGAACATGGATAATCCCGATGGGCCAGACCCAGCCTGGACTAGT

[0064] SEQ ID NO.18:

[0065] QSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWTS

[0066] HER2-tomato was transformed into E. coli Stbl3, and after amplification, the HER2-tomato plasmid was extracted using the PureLink™ HiPure plasmid miniprep kit. The extracted HER2-tomato plasmid was subjected to agarose gel electrophoresis (results are shown in Figure 1). Figure 2 As shown in A) and the concentration was detected (the detection results are as follows). Figure 2 (As shown in B). The plasmid was then double-digested with restriction endonucleases Pac1 and Sfi1, following the enzyme digestion system and conditions specified in the enzyme instructions. The digestion products were subjected to 1% agarose gel electrophoresis, and the 5858 bp DNA fragment was recovered using a gel recovery kit according to the instructions. This fragment contained part of the genes of AD5F35 adenovirus, as well as the car and tomato fluorescent genes.

[0067] SEQ ID NO.19:

[0068]

[0069] Example 2

[0070] The packaging details for HER2-Ad5F35 adenovirus are as follows:

[0071] (1) Cell line 293Cells, low passage (purchased from the Cell Bank of Chinese Academy of Sciences) was seeded in a six-well plate containing DMEM + 10% fetal bovine serum (FBS) and cultured at 37°C and 5% CO2 until the adhesion rate reached 70%-80% before being used for adenovirus transfection.

[0072] (2) The DNA fragment recovered in Example 1 was ligated with Right1.4 (the backbone gene of AD5F35 adenovirus) using T4 ligase. The ligation product was then transfected into 293Cells, low passage cells cultured in (1) at 70%-80% adhesion using Lipofectamine™ 3000 transfection reagent. After transfection at 37°C and 5% CO2 for 24 hours, the cells were observed under an inverted fluorescence microscope. Figure 3 As shown, red fluorescent protein expression was observed in the cells.

[0073] Example 3

[0074] HER2-Ad5F35 adenovirus amplification and viral titer detection are detailed below:

[0075] (1) In Example 2, cells transfected with the ligation product were cultured into 293Cells, lowpassage cells with an adhesion rate of 70%-80%. After 14 days of culture at 37°C and 5% CO2, the cells floated over a large area (e.g. Figure 4 (As shown), cells and supernatant were collected, and the cells were repeatedly frozen and thawed three times, labeled as P0 generation adenovirus. Cell line 293Cells, low passage was seeded in T75 medium containing DMEM + 10% FBS and cultured at 37℃ and 5% CO2 until the adhesion rate reached 80%-90%. The medium was then replaced with DMEM containing 3% FBS, and P0 generation cells were seeded in it. On the third day, when the cells were about 90% floating, the cells were collected, resuspended in PBS, and then purified using the Adenovirus Purification Miniprep Kit according to the accompanying instructions.

[0076] (2) Cell line 293Cells, low passage was seeded into a six-well plate containing DMEM + 10% FBS and cultured at 37°C and 5% CO2 until the adhesion rate was 80%. Then the culture medium was replaced with DMEM medium containing 3% FBS to determine the virus titer.

[0077] The purified virus from (1) was serially diluted and inoculated into 293 Cells, lowpassage six-well plates cultured to 80% adhesion. The next day, cells were digested with trypsin, collected, and the number of cells per well was recorded using a cell counter. The percentage of PE channel-positive cells in each well was then detected by flow cytometry. Figure 5 As shown. The viral titer was calculated using Formula I, and the average of three replicate experiments was taken, resulting in a viral titer of 1.5 × 10⁻⁶. 10 IU / mL.

[0078] FCM infection units (IU) / mL = (percentage of positive cells × total number of cells in the well × dilution factor) / amount of virus inoculated per well (Formula I).

[0079] Example 4

[0080] HER2-Ad5F35 adenovirus infection of macrophages, as detailed below:

[0081] Macrophages were seeded in T25 flasks and cultured at 37°C and 5% CO2 until approximately 80% adherence was achieved. 1.5 mL of culture medium was retained in the flask, and excess medium was discarded. HER2-Ad5F35 adenovirus was added at a viral load of 600 MOI. After 6 hours, macrophage-SFM (1X) medium was added to a final volume of 5 mL. After 48 hours, cells were digested with trypsin and collected. A portion of the collected cells was analyzed by flow cytometry to determine the infection efficiency of HER2-Ad5F35 adenovirus in macrophages. The results are shown below. Figure 6 As shown, the infection efficiency was measured to be 72%. A portion of the cells were incubated with HER2 protein, and then the HER2 expression efficiency was detected by flow cytometry. The results are as follows. Figure 7 As shown, the expression efficiency of HER2 was measured to be 68%.

[0082] The experiments in this example are only initial verifications of whether HER2 is expressed. Viral infection and expression can be optimized by adjusting the viral load, adding infection-promoting reagents, adjusting the infection time, and adjusting the infection conditions to obtain the best results.

[0083] Therefore, this invention provides a novel HER2-specific chimeric antigen, the nucleotide sequence of which is shown in SEQ ID NO.19. It is formed by inserting a CAR structure into the MCS region of an Ad5F35 adenovirus vector. The CAR structure is: signal peptide + C-MYC + HER2scfv + hinge region + transmembrane region + CD3Z + FCER1G + CD19. The novel HER2 adenovirus provided can specifically target HER2-positive tumor cells, has a broad host range, and high infection efficiency. It can be used to infect hematopoietic stem cells, dendritic cells, T cells, NK cells, K562, U937 and other difficult-to-transfect blood cells. After infection, it can be rapidly expressed in cells with high expression stability and will not integrate into the host chromosome, thus having good safety.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A CAR, characterized in that, The structure of the CAR is: signal peptide + C-MYC + HER2 scfv + hinge region + transmembrane region + CD3Z + FCERT1G + CD19; the nucleotide sequence of the signal peptide is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the C-MYC is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6; The HER2 scfv nucleotide sequence is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the hinge region is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; the nucleotide sequence of the transmembrane region is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.

12. The nucleotide sequence of CD3Z is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14; the nucleotide sequence of FCER1G is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16; the nucleotide sequence of CD19 is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.

18.

2. The CAR according to claim 1, characterized in that: The transmembrane region and CD3Z are connected by the sequence shown in SEQ ID NO.1; FCER1G and CD19 are connected by the sequence shown in SEQ ID NO.

2.

3. A HER2 adenovirus, characterized in that: The HER2 adenovirus integrates the CAR structure as described in any one of claims 1 or 2.

4. The HER2 adenovirus according to claim 3, characterized in that: The CAR is directionally inserted into the MCS region of the Ad5F35 adenovirus vector through genetic engineering, thereby obtaining a recombinant HER2-targeting adenovirus vector; the nucleotide sequence of the HER2 adenovirus is shown in SEQ ID NO.19.

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