WO2020164167A1 - 一种用于通用型car-t制备的重组腺相关病毒载体及其构建方法和应用 - Google Patents

一种用于通用型car-t制备的重组腺相关病毒载体及其构建方法和应用 Download PDF

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WO2020164167A1
WO2020164167A1 PCT/CN2019/075717 CN2019075717W WO2020164167A1 WO 2020164167 A1 WO2020164167 A1 WO 2020164167A1 CN 2019075717 W CN2019075717 W CN 2019075717W WO 2020164167 A1 WO2020164167 A1 WO 2020164167A1
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sequence
associated virus
car
recombinant adeno
virus vector
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芦志华
朱滨
董明洁
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北京门罗生物科技有限公司
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/30—Mixture of cells

Definitions

  • the present invention relates to the field of biomedicine, in particular to a recombinant adeno-associated virus vector used for CAR-T preparation and its construction method and application.
  • CAR-T uses antibody fragments that can bind to specific antigens to recognize antigens on the surface of tumor cells.
  • CD19 antigen-specific CAR-T cells have been used in clinical trials for the treatment of B-cell leukemia and lymphoma, and have shown sustained disease relief effects.
  • Chimeric antigen receptors (CAR) endow T cells with the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-modified T cells to recognize a wider range of targets than the natural T cell surface receptor TCR.
  • CAR-T technology has had significant effects in the treatment of acute leukemia and non-Hodgkin’s lymphoma, and is considered to be one of the most promising tumor treatment methods.
  • CAR-T treatment is as follows: through genetic engineering modification, the T cells of cancer patients isolated and collected in vitro express chimeric antigen receptors (CAR) that recognize a single tumor antigen, and after a large number of CAR-T cells are expanded in vitro It is returned to cancer patients for cellular immunotherapy.
  • CAR as a chimeric protein expressed by genes, contains the antigen-binding domain of an antibody (eg, single-chain antibody scFv) connected to the T cell signaling domain.
  • an antibody eg, single-chain antibody scFv
  • the CAR-T cell adoptive immunotherapy system uses genetic modification of the patient's own T cells, and uses the principle of antigen-antibody binding to circumvent the MHC-restricted antigen presentation, thereby achieving precise targeting. At the same time, it overcomes the possible immune escape of tumor cells by down-regulating the expression of MHC molecules to reduce antigen presentation.
  • CAR-T cancer-associated antigen
  • CAR-T can achieve very good curative effects in the treatment of blood system diseases, it can only be autologously infused, that is, T cells are extracted from the patient, genetically modified and amplified, and then returned to the patient. Therefore, this treatment cannot be used as widely as drugs.
  • Some patients cannot undergo CAR-T reinfusion because they cannot obtain a sufficient number of T cells, thus losing the possibility of treatment.
  • Plasmid vectors express genes or DNA sequences at multiple cloning sites into proteins in cells. Plasmid vector DNA is artificially modified and contains other DNA components such as gene sequences for expressing antibiotics, promoter sequences, multiple cloning sites (MCS), etc., and it can be genetically engineered with others
  • the helper plasmid is automatically assembled in engineered cells (such as 293T cells) into a lentivirus capable of infection.
  • Such a lentivirus has the function of expressing CAR protein in cells. Adding such a lentivirus to the culture medium for culturing T cells can infect T cells, that is, enter the T cells, and then use the elements in the T cells to express CAR proteins. After these CAR proteins are expressed, they will be anchored in the T cells. surface.
  • virus-mediated gene expression technologies are usually used, such as lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV), etc.
  • lentiviruses and retroviruses are inserted into the genome randomly after entering cells, and adenoviruses and adeno-associated viruses also have a certain probability. Inserting into the genome may destroy the genes in the cell, resulting in cell abnormalities, and may even transform the cell into a tumor cell, which in turn causes tumors. Therefore, using these viral technologies to prepare CART for cellular immunotherapy has the risk of causing tumors.
  • the present invention relates to a recombinant adeno-associated virus vector
  • the adeno-associated virus vector contains the following operatively linked sequence elements: 5'-end inverted repeat sequence, 3'-end inverted repeat sequence and sequence encoding CAR gene .
  • the sequence encoding the CAR gene is CD19CAR (4-1BB).
  • the adeno-associated virus vector may further include the following operatively linked sequence elements: SA sequence, 2A sequence, polyA sequence, 5'genome homologous sequence (5'HA), 3'genome homologous sequence (3' HA).
  • the 5'HA sequence includes the sequence shown in SEQ ID NO:1;
  • the SA sequence includes the sequence shown in SEQ ID NO: 2;
  • the 2A sequence includes the sequence shown in SEQ ID NO: 3;
  • the CD19CAR (4-1BB) sequence includes the sequence shown in SEQ ID NO: 4;
  • the polyA sequence includes the sequence shown in SEQ ID NO: 5;
  • the 3’HA sequence includes the sequence shown in SEQ ID NO: 6
  • the 5'end inverted repeat sequence includes the sequence shown in SEQ ID NO: 7
  • the 3'end inverted repeat sequence includes the sequence shown in SEQ ID NO: 8.
  • the recombinant adeno-associated virus vector comprises nucleotides having at least about 70%, at least about 80%, at least about 90% sequence identity or more sequence identity with the sequence shown in SEQ ID NO: 9 sequence.
  • the present invention also relates to a method for preparing the recombinant adeno-associated virus vector.
  • the method includes the following steps: providing a packaging cell line of the aforementioned viral vector; and recovering the recombinant AAV virus from the supernatant of the packaging cell line.
  • the present invention also relates to a recombinant adeno-associated virus, which is obtained by packaging any of the aforementioned recombinant adeno-associated virus vectors.
  • the present invention also relates to a method for expressing CAR genes, the method comprising providing a nucleotide sequence comprising any of the foregoing recombinant adeno-associated virus (AAV); combining with CRISPR/cas9 gene editing technology, The AAV homologously recombines into the genome of the T cell, and the AAV expresses the CAR gene in the T cell.
  • AAV adeno-associated virus
  • the present invention also relates to the application of any of the aforementioned recombinant adeno-associated virus vectors and recombinant adeno-associated viruses in the preparation of CAR-T cells or anti-tumor drugs.
  • the present invention also relates to a method for preparing CAR-T cells of the aforementioned recombinant adeno-associated virus vector, which is characterized in that the method includes the following steps:
  • the first step is to construct any of the aforementioned recombinant adeno-associated virus vectors
  • the second step is virus packaging
  • the third step is T cell isolation, activation and amplification, CRISPR/cas9 gene editing, AAV virus-mediated gene recombination;
  • Any of the aforementioned recombinant adeno-associated virus vectors are expressed on T cells isolated and collected from the peripheral blood of cancer patients or healthy people through gene editing methods to obtain CAR-T cells.
  • the present invention also relates to CAR-T cells prepared by the above method.
  • the present invention also relates to a kit, which contains any of the foregoing recombinant adeno-associated virus vector, recombinant adeno-associated virus, or the CAR-T cell.
  • the invention also relates to the application of the CAR-T cell or the kit in the preparation of anti-tumor drugs.
  • this application uses gene editing technology, combined with gene recombination technology, from the expression
  • the structure of the AAV vector has been improved, so as to achieve the targeted and precise integration of CAR gene fragments, ensuring the continuous and stable expression of the CAR gene, and avoiding the risk of causing tumors.
  • the expression of CAR is under the control of the endogenous promoter, so that the expression of CAR is regulated by normal physiology, significantly reducing the side effects of treatment, and obtaining a physiological universal CAR-T.
  • the AAV vector with improved structure obtained in the present invention is non-pathogenic
  • the general-purpose T cells and general-purpose CAR-T cells of the invention can be applied to the treatment of malignant tumors or infectious diseases by allogeneic reinfusion, which greatly reduces the treatment cost;
  • the prepared CAR is integrated before the exon of the TCR constant region and is regulated by an endogenous promoter, ensuring that the expression of CAR is physiologically regulated and the expression is uniform. Due to this physiological and uniformity, CAR will not be overexpressed, and the expression level of CAR is consistent with the expression level of the original TCR, and the individual differences between CAR-T cells are small;
  • CAR is accurately integrated into the designated TCR constant region gene through homologous recombination, rather than randomly integrated into the genome of T cells, so there will be no triggering The risk of tumors.
  • operably linked refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • the “element” refers to a series of functional nucleic acid sequences useful for protein expression.
  • the “element” is systematically constructed to form an expression construct.
  • the sequence of the “element” may be those provided in the present invention, and also include their variants, as long as these variants basically retain the function of the "element” by inserting or deleting some bases (such as 1-50bp; preferably 1-30bp, more preferably 1-20bp, more preferably 1-10bp), or by random or site-directed mutagenesis.
  • Adeno-associated virus (adeno-associated virus, AAV) vector is a vector that can be artificially modified by genetic engineering using certain characteristics of naturally occurring adeno-associated virus.
  • Adeno-associated virus (AAV) is a virus that cannot replicate itself and has low immunogenicity.
  • serotypes of AAV There are currently about 10 serotypes of AAV, and different serotypes of AAV can selectively target different tissues.
  • the loading capacity of AAV virus vectors is limited, not exceeding 5.0 kb.
  • variants of the element described above that have been appropriately changed and still retain their original functions are also included in the present invention.
  • the full-length nucleotide sequence of the gene pointed to by each element of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences.
  • the upstream and downstream positions of the aforementioned elements in the vector may also include restriction enzyme cleavage sites, which facilitates the organic connection of the elements.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • the vector containing the above-mentioned appropriate polynucleotide sequence and appropriate promoter or control sequence can be used for virus packaging.
  • the present invention also provides a kit containing the recombinant adeno-associated virus vector expressing CAR or a virus packaged by the vector.
  • Other reagents commonly used for virus packaging, transfection, injection, etc. can also be included in the kit for the convenience of those skilled in the art.
  • the kit may also include instructions for instructing those skilled in the art to operate.
  • Figure 1- Figure 3 are schematic diagrams of the three plasmid structures, in order: AAV6-TCR-CD19CAR (4-1BB) (experimental vector), AAV6-TCR-GFP (negative control), LV-EF-1a-CD19CAT (4- 1BB)-mCherry (positive control);
  • Figure 4 A diagram of the gene-edited AAV6-TCR-CD19CAR(4-1BB) plasmid structure
  • FIG. 5 Flow cytometry results of TCR knockout of T cells from two different individuals after gene editing
  • Figure 6 Flow cytometry results of CD19CAR (4-1BB) homologous recombination mediated by AAV6;
  • Figure 7 Flow cytometric test results of tumor killing ability of AAV6-TCR-CD19CAR-T;
  • Figure 8 Detection and comparison results of cytokine concentrations at different time points, where the values of the bars from left to right in the bar chart represent Kmix, LV, AAV, LV+Kmix, AAV+Kmix, respectively;
  • Figure 9 Flow cytometric detection and comparison results of the failure markers of CAR-T cells, where the upper broken line in the broken line chart is LV CD19 CAR-T+Kmix, and the lower broken line is AAV CD19 CAR-T+Kmix;
  • Figure 10a, b CAR-T anti-tumor in vivo experimental results, where Figure 10b is the survival curve, lentivirus-transduced LV CAR-T and AAV-transduced AAV-TCR-CAR-T both significantly prolong the survival time of mice , There is no significant difference between the two groups.
  • the plasmid constructed in this example includes
  • AAV6-TCR-CD19CAR(4-1BB) (experimental carrier, structure diagram is shown in Figure 1, 4)
  • AAV6-TCR-GFP negative control, structure diagram is shown in Figure 2
  • the functional region of the plasmid LTR-EF-1a-CD19CAT(4-1BB)-mCherry-LTR for gene synthesis.
  • This sequence was cloned into the lentiviral vector plasmid.
  • the structure of the plasmid functional region is: ITR-5’HA-SA-2A-GFP-polyA-3’HA-ITR
  • GFP primers were designed, and GFP fragments were obtained by PCR.
  • Poisonous plasmids include: pSLQ5367, pCMV-dR8.91, pMD2-G, Reagent, Opti MEM.
  • the specific ratio is in accordance with the 10-cm plate in Table 1.
  • AAV virus collection virus particles exist in both packaging cells and culture supernatant.
  • PBMC peripheral blood mononuclear cells
  • step 2) Place the blood sample in step 2) on the upper layer of the solution in step 1), minimize the mixing of blood and Ficoll-PaqueTM PLUS, and centrifuge at 400g for 30 minutes at room temperature, and then slow down naturally;
  • step 3 Discard the upper plasma after centrifugation in step 3, and take the boundary layer between the plasma and Ficoll-PaqueTM PLUS solution is the peripheral blood mononuclear cells (the tube is divided into four layers after centrifugation, from top to bottom in turn are plasma, Peripheral blood mononuclear cells, Ficoll-PaqueTM PLUS fluid, red blood cell and granulocyte layer).
  • the peripheral blood mononuclear cells the tube is divided into four layers after centrifugation, from top to bottom in turn are plasma, Peripheral blood mononuclear cells, Ficoll-PaqueTM PLUS fluid, red blood cell and granulocyte layer.
  • T memory stem cells Tmsc
  • This kit is a negative selection kit.
  • T cell culture medium IL-2: OpTmizer TM CTS TM T-cell Expansion SFM+5% CTS TM Immune Cell SR+1% Penicillin-Streptomycin 100X Solution+1% L-glutamine+IL-2 200IU/mL .
  • T cell culture medium (IL-7/15): OpTmizer TM CTS TM T-cell Expansion SFM+5% CTS TM Immune Cell SR+1% Penicillin-Streptomycin 100X Solution+1% L-glutamine+IL-710ng/ ml+IL-15 10ng/mL.
  • the initial cell concentration is 1M/mL, and the height of the medium liquid level in Flask is not higher than 1cm.
  • T cell activation use Dynabeads Human T-Activator CD3/CD28 magnetic beads
  • T cell culture medium (IL-7/15) was changed to activate cell culture at a starting cell density of 1M/mL for activated T cells.
  • the medium was supplemented every 2 days and appropriate cytokines were added.
  • AAV6 virus-mediated CAR gene homologous recombination 48-72 hours after T cell activation in Example 3, AAV6 virus-mediated CAR gene homologous recombination, in situ insertion of the cut site for genetic modification, immediate transduction, AAV6 virus MOI: 2.5 ⁇ 10e5-10e6.
  • the sgRNA sequence is as follows:
  • the CAR gene homologous recombination mediated by AAV6 virus inserts the splice site in situ. Transduction immediately after electroporation, AAV6MOI: 2.5 ⁇ 10e5-10e6.
  • T cells were from two different individuals.
  • Flow cytometry detects the expression of TCR and CD3.
  • the TCR knockout rates of T cells in two different individuals were 87.2% and 68.6%, respectively.
  • FIG. 6 shows: T cells are from two different individuals. The expression of CAR by flow cytometry was 69.1% and 67.5% respectively. Compared with CAR-T transduced by lentivirus, the expression of CAR in this CAR-T cell is more uniform.
  • K562-CD19+/K562-CD19- cells are co-cultured, the initial cell number is 5 ⁇ 10e4 each, of which CD19+ cells express mCherry fluorescent protein
  • NegativeCtrl 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • PositiveCtrl 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • Test 1 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • Test 2 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1
  • Cytotoxicity test take 20ul culture medium supernatant at different time points to measure cytokines: IL-2, IFN-r, TNF-a
  • the markers of T cell failure were detected at different time points: PD-1, TIM-3, LAG-3
  • T cell failure markers PD-1, LAG-3, and TIM-3 were detected.
  • the expression of failure markers of AAV-TCR-CD19CAR-T cells was significantly lower than that of the control group.

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Abstract

提供了一种生理性通用型CAR-T制备的重组腺相关病毒载体及其构建方法和抗肿瘤应用，该重组腺相关病毒载体能够用于CAR基因的表达和CAR-T细胞的制备。

Description

一种用于通用型CAR-T制备的重组腺相关病毒载体及其构建方法和应用 技术领域
本发明涉及生物医药领域，具体地涉及用于CAR-T制备的重组腺相关病毒载体及其构建方法和应用。
背景技术
CAR-T是利用能够与特定抗原结合的抗体片段来识别肿瘤细胞表面的抗原。近年来，CD19抗原特异性CAR-T细胞用于治疗B细胞白血病和淋巴瘤临床试验中，显示出持续的疾病缓解效果。嵌合抗原受体(CAR)赋予T细胞HLA非依赖的方式识别肿瘤抗原的能力，这使得经过CAR改造的T细胞相对于天然T细胞表面受体TCR能够识别更广泛的目标。近年来，CAR-T技术在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。
CAR-T治疗的原理如下：通过基因工程修饰，使体外分离收集的癌症患者的T细胞表达识别单一肿瘤抗原的嵌合抗原受体(CAR)，并在体外大量扩增CAR-T细胞后将其输回癌症患者体内进行细胞免疫治疗。CAR作为一种基因表达的嵌合蛋白包含与T细胞信号传导结构域连接的、抗体的抗原结合结构域(如：单链抗体scFv)。CAR-T细胞过继性免疫的显著优势在于：细胞免疫治疗更具精确性。CAR-T细胞过继免疫治疗***应用基因修饰病人自体的T细胞，利用抗原抗体结合原理规避了依赖MHC限制的抗原呈递，从而具有精确的靶向性。同时克服了肿瘤细胞可能通过下调MHC分子表达以降低抗原呈递的免疫逃逸。
目前CAR-T疗法的研发主要集中在CAR的构建，通过多方面的修饰以增强CAR-T细胞的靶向性，免疫杀伤性、效用持久性及安全性。尽管CAR的构建已取得了诸多显著进展，然而，基于传统的CAR-T细胞过继性免疫治疗***始终存在以下明显缺陷：(1)只能自体输注。目前CAR-T治疗血液***疾病虽然能够取得很好的疗效，但是只能自体输注，即从患者体内抽取T细胞，进行基因修饰后扩增，然后再回输给患者。因此这种治疗方式不能像药物一样广泛应用。有的患者由于无法获得足够数量的T细胞而无法进行CAR-T回输，丧失了治疗的可能性。(2)制备繁琐：每一套靶向某单一肿瘤相关抗原的CAR-T***均需要单独构建，并通过实验检测其安全性、靶向性和有效性，应用于临床的评价周期较长，耗时费力，经济性较差。(3)在CAR-T细胞过继免疫治疗过程中，对CAR-T细胞在体内的靶向能力、生物代谢情况缺乏有效的评价和监控手段，因而常导致治疗过度或治疗无效。
在CAR蛋白在T细胞表面表达的过程中，需要借助病毒载体，通过DNA合成技术合成能够在细胞中表达CAR蛋白的DNA序列；然后通过分子克隆技术将CAR的DNA序列装进质粒载体的更长的环状DNA中。质粒载体把装在多克隆位点的基因或DNA序列在细胞中表达成蛋白质。质粒载体DNA是经过人工改造的，包含其他的DNA组件比如表达抗生素的基因序列、启动子序列、多克隆位点(multiple cloning site,MCS)等等，并且，其可以和其他一些经过基因工程改造的辅助质粒(helper plasmid)一起在工程细胞(比如293T细胞)中自动组装成有感染(infection)能力的慢病毒。这样的慢病毒就具有在细胞中表达CAR蛋白的作用。将这样的慢病毒加入培养T细胞的培养基中，其就能够感染T细胞，即进入T细胞，然后利用T细胞内的元素来表达CAR蛋白，这些CAR蛋白表达之后就锚 定在T细胞的表面。目前通常采用病毒介导的基因表达技术，比如慢病毒(lentivirus)、逆转录病毒(retrovirus)、腺病毒(adenovirus)、腺相关病毒(adeno-associated virus,AAV)等。
然而，这些病毒技术都有一定的不足，例如主要就在于慢病毒和逆转录病毒进入细胞之后，就会随机地***(insertion)到基因组中，而腺病毒和腺相关病毒也会有一定的概率***到基因组中，都可能会破坏细胞中的基因，从而导致细胞异常，甚至可能会让细胞转变成肿瘤细胞，进而引发肿瘤。因此，采用这些病毒技术来制备CART进行细胞免疫治疗会有引发肿瘤的风险。
发明内容
具体的，本发明涉及一种重组腺相关病毒载体，所述腺相关病毒载体包含如下操作性连接的序列元件：5’末端反向重复序列、3’末端反向重复序列以及编码CAR基因的序列。具体的，所述编码CAR基因的序列为CD19CAR(4-1BB)。
其中，所述腺相关病毒载体还可进一步包含如下操作性连接的序列元件：SA序列、2A序列、polyA序列、5’基因组同源序列(5’HA)、3’基因组同源序列(3’HA)。
所述的5’HA序列包含SEQ ID NO:1所示的序列；
所述的SA序列包含SEQ ID NO:2所示的序列；
所述的2A序列包含SEQ ID NO:3所示的序列；
所述的CD19CAR(4-1BB)序列包含SEQ ID NO:4所示的序列；
所述的polyA序列包含SEQ ID NO:5所示的序列；
所述的3’HA序列包含SEQ ID NO:6所示的序列
所述的5’末端反向重复序列包含SEQ ID NO:7所示的序列
所述的3’末端反向重复序列包含SEQ ID NO:8所示的序列。
进一步的，所述重组腺相关病毒载体包含与SEQ ID NO:9所示的序列具有至少约70％、至少约80％、至少约90％序列同一性或更多的序列同一性的核苷酸序列。
本发明还涉及所述的重组腺相关病毒载体的制备方法，所述方法包括以下步骤：提供前述病毒载体的包装细胞系；和从所述包装细胞系的上清液中回收重组AAV病毒。
本发明还涉及一种重组腺相关病毒，所述病毒由前述任意的重组腺相关病毒载体包装获得。
另一方面，本发明还涉及一种用于表达CAR基因的方法，所述方法包括提供包括编码前述任意的重组腺相关病毒(AAV)的核苷酸序列；结合CRISPR/cas9基因编辑技术，将所述AAV同源重组到T细胞的基因组内，所述AAV在T细胞中表达所述CAR基因。
另一方面，本发明还涉及前述任意的重组腺相关病毒载体、重组腺相关病毒在制备CAR-T细胞或抗肿瘤药物中的应用。
另一方面，本发明还涉及前述一种重组腺相关病毒载体的CAR-T细胞制备方法，其特征在于：该方法包括如下步骤：
第一步，构建前述任意的重组腺相关病毒载体；
第二步，病毒包装；
第三步，T细胞分离、激活与扩增，CRISPR/cas9基因编辑，AAV病毒介导的基因重组；
前述任意的重组腺相关病毒载体是通过基因编辑手段，将其表达于癌症患者或健康人外周血中分离收集的T细胞以获得CAR-T细胞。
因此，本发明还涉及由上述方法制备获得的CAR-T细胞。
本发明还涉及一种试剂盒，所述试剂盒中包含前述任意的重组腺相关病毒载体、重组腺相关病毒，或所述CAR-T细胞。
本发明还涉及所述CAR-T细胞或所述试剂盒在制备抗肿瘤药物中的应用。
本发明人经过深入的研究，鉴于CAR-T免疫疗法的高成本，治疗效果个体差异大，安全性低，有引发肿瘤的风险等问题，本申请利用基因编辑技术，结合基因重组技术，从表达CAR基因的载体入手，对AAV载体的结构进行了改进，从而实现CAR的基因片段定点精确整合，确保了CAR基因的持续稳定表达，避免了引发肿瘤的风险。同时还将CAR的表达至于内源性启动子的控制之下，使得CAR的表达受正常生理调控，显著减少治疗副作用，获得了一种生理性的通用型CAR-T。
总的来说，本发明的有益效果在于：
1、本发明所述的获得的结构改进的AAV载体制备的无致病性；
2、发明所述的通用型T细胞和通用型CAR-T细胞可以通过同种异体回输的方式应用于恶性肿瘤或感染性疾病的治疗，极大地降低了治疗成本；
3、制备获得的CAR整合在TCR恒定区外显子之前，并且受到内源性启动子的调控，保证了CAR的表达是受生理调控的，且表达均一。由于这种生理性及均一性，CAR不会出现过度的表达，且CAR的表达水平与原来TCR的表达水平一致，各CAR-T细胞之间的个体差异性小；
4、安全性更高，由于CAR的表达是在生理范围内的，当CAR-T细胞激活后，释放的细胞因子也在生理范围内，不会出现过度的释放，从而避免细胞因子风暴的产生。
5、不存在引发肿瘤的风险，利用基因编辑技术，将CAR通过同源重组的方式精确的整合到指定的TCR恒定区基因上，而非随机整合到T细胞的基因组上，因此不会出现引发肿瘤的风险。
术语
如本文所用，所述的“操作性连接”或“可操作性相连”是指两个或多个核酸区域或核酸序列的功能性的空间排列。
所述的“元件”是指一些对于蛋白的表达有用的一系列功能性的核酸序列，本发明中，所述的“元件”被***地构建以形成一种表达构建体。所述的“元件”的序列可以是本发明中所提供的那些，也包括它们的变体，只要这些变体基本上保留了所述“元件”的功能，其通过***或删除一些碱基(如1-50bp；较佳地1-30bp，更佳地1-20bp，更佳地1-10bp)，或进行随机或定点突变等来获得。
质粒
腺相关病毒(adeno-associated virus，AAV)载体是利用天然存在的腺相关病毒某些特性经过基因工程改造后产生的一种可供人工转基因的载体。腺相关病毒(Adeno-associated virus，AAV)是一种不能自我复制的病毒，具有较低的免疫原性。目前有约10种血清型AAV，不同血清型的AAV能够选择性地靶向不同组织。但是AAV病毒载体装载容量有限，不超过5.0kb。
根据上述所提供的元件的信息，进行了适当的变化且仍然保留其原有功能的上述元件的变异体也包括在本发明中。例如，在严格条件下与本发明限定的序列杂交且具有相同功能的序列变异体。
本发明的各元件所指向的基因的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。
所述的载体中上述元件的上游以及下游的位置，还可包括限制性的酶切位点，这样有利于各元件的有机连接。
本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状。
包含上述的适当多核苷酸序列以及适当启动子或者控制序列的载体，可以用于进行病毒的包装。
试剂盒
本发明还提供了包含有所述表达CAR的重组腺相关病毒载体或由该载体包装而成的病毒的试剂盒。其它常用于进行病毒包装、转染、注射等的试剂也可被包含在所述的试剂盒中，以方便本领域技术人员使用。此外，所述试剂盒中还可包含有指导本领域技术人员操作的使用说明书。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
附图说明
图1-图3为三个质粒结构示意图，依次为：AAV6-TCR-CD19CAR(4-1BB)(实验载体)，AAV6-TCR-GFP(阴性对照)，LV-EF-1a-CD19CAT(4-1BB)-mCherry(阳性对照)；
图4：基因编辑的AAV6-TCR-CD19CAR(4-1BB)质粒结构图；
图5：经基因编辑后两个不同个体的T细胞TCR敲除流式检测结果；
图6：AAV6介导的CD19CAR(4-1BB)同源重组的流式检测结果；
图7：AAV6-TCR-CD19CAR-T的肿瘤杀伤能力流式细胞检测结果；
图8：不同时间点细胞因子浓度的检测及对比结果，其中，柱形图中每天从左到右的柱的数值分别代表Kmix、LV、AAV、LV+Kmix、AAV+Kmix；
图9：CAR-T细胞的衰竭标志物的流式检测及对比结果，其中，折线图中位于上方的折线为LV CD19 CAR-T+Kmix，位于下方的折线为AAV CD19 CAR-T+Kmix；
图10a、b：CAR-T抗肿瘤体内实验结果，其中图10b为生存曲线，慢病毒转导的LV CAR-T与AAV转导的AAV-TCR-CAR-T均显著延长小鼠的生存期，两组效果无显著差异。
具体实施方式
实施例1 质粒的构建
1、本实施例构建的质粒包括
LV-EF-1a-CD19CAR(4-1BB)-mCherry(阳性对照，结构示意图见图3)
AAV6-TCR-CD19CAR(4-1BB)(实验载体，结构示意图见图1、4)
AAV6-TCR-GFP(阴性对照，结构示意图见图2)
2、质粒构建及病毒包装过程
2.1 LV-EF-1a-CD19CAT(4-1BB)-mCherry质粒构建
序列为SEQ ID NO：10所示。：
1)获得LV-EF-1a-CD19CAR(4-1BB)-mCherry质粒
先在ApE或Snapgene软件上构建该质粒图谱。
质粒的功能区域：LTR-EF-1a-CD19CAT(4-1BB)-mCherry-LTR进行基因合成。
将该段序列克隆入慢病毒载体质粒。
质粒转化、涂板、挑克隆、提取质粒(小提)，测序确认无误，再次提取质粒(大提，去内毒素)，质粒待包病毒使用。
2.2 AAV6-TCR-CD19CAR(4-1BB)质粒构建
先在ApE或Snapgene软件上构建该质粒的图谱。
质粒功能区域的结构：ITR-5’HA-SA-2A-CD19CAR(4-1BB)-polyA-3’HA-ITR
序列为SEQ ID NO：11所示。
首先合成基因片段：5’HA-SA-2A-CD19CAR(4-1BB)-polyA-3’HA
将上述基因片段克隆入AAV6病毒载体质粒
质粒转化、涂板、挑克隆、提取质粒(小提)，测序确认无误，再次提取质粒(大提，去内毒素)，质粒待包病毒使用。
2.3 AAV6-TCR-GFP质粒构建
先在ApE或Snapgene软件上构建该质粒的图谱。
质粒功能区域的结构为：ITR-5’HA-SA-2A-GFP-polyA-3’HA-ITR
序列为SEQ ID NO：12所示。
按照Infusion原理，设计GFP引物，PCR获得GFP片段。
以上述2.2质粒为模板，按照Infusion原理，设计引物，PCR获得AAV6载体。
Infusion连接，质粒转化、涂板、挑克隆、提取质粒(小提)，测序确认无误，再次提取质粒(大提，去内毒素)，质粒待包病毒使用。
实施例2 病毒包装过程
2.1 LV包装
准备DMEM+10％FBS培养基+抗生素
复苏293T细胞，传代培养，判断细胞状态良好
包毒前一日，分6个10cm培养皿，各seed 2.5M细胞，24h后达到70％汇聚即可包毒。
包毒质粒包括：pSLQ5367，pCMV-dR8.91，pMD2-G,
Figure PCTCN2019075717-appb-000001
Reagent，Opti MEM.
具体配比依照表1中的10-cm盘。
表1 DNA转染的条件配比
Figure PCTCN2019075717-appb-000002
Figure PCTCN2019075717-appb-000003
2.2.AAV6病毒包装
以T75flask为例，前一天下午传代，细胞数量9M-10M
Figure PCTCN2019075717-appb-000004
OptiMEM 1.5ml
TanslT-VirusGen：45ul
4.AAV病毒收毒：病毒颗粒同时存在于包装细胞和培养上清中。
5.AAV病毒浓缩与纯化。
实施例3 T细胞分选
一、T细胞分选
1、获得健康捐献者的血液样品(至少50mL)。经过如下疾病的检测(不仅局限于这些检测)，合格的患者。包括：甲肝，乙肝，丙肝，艾滋病，梅毒抗体，结核，遗传性疾病等。
2、获得外周血单个核细胞(PBMC)
1)将Ficoll-PaqueTM PLUS(#07957)混合均匀后置于空管中；
2)用PBS+2％FBS稀释血液样品(2X)；
3)将步骤2)中的血液样品置于步骤1)溶液的上层，尽量减少血液与Ficoll-PaqueTM PLUS的混合，室温下400g离心30分钟后自然降速；
4)移弃步骤3)离心后的上层血浆，取血浆与Ficoll-PaqueTM PLUS液交界层即为外周血单个核细胞(其中，离心后管中分为四层，从上到下依次为血浆、外周血单个核细胞、Ficoll-PaqueTM PLUS液、红细胞与粒细胞层)。
5)用PBS+2％FBS清洗外周血单个核细胞，待用。
3、获得T细胞(T memory stem cells，Tmsc)
使用Miltenyi的Pan T cell isolation Kit human获得总T细胞。此试剂盒为阴选试剂盒。
二、T细胞培养
1)T细胞培养基(IL-2)：OpTmizer TMCTS TMT-cell Expansion SFM+5％CTS TM Immune Cell SR+1％Penicillin-Streptomycin 100X Solution+1％L-glutamine+IL-2 200IU/mL。
2)T细胞培养基(IL-7/15)：OpTmizer TMCTS TMT-cell Expansion SFM+5％CTS TM Immune Cell SR+1％Penicillin-Streptomycin 100X Solution+1％L-glutamine+IL-710ng/ml+IL-15 10ng/mL。起始细胞浓度为1M/mL，在Flask里培养基液面高度不高于1cm。
三、T细胞激活
1)T细胞激活使用：Dynabeads Human T-Activator CD3/CD28磁珠
2)洗磁珠用Beads Wash Buffer：PBS+1％BSA+2mMEDTA，pH＝7.4。
3)按等量1:1比例将T细胞与磁珠混合。
4)T25 Flask或6孔板里培养。
四、T细胞扩增
T细胞激活48-72小时后，去除Beads，进行基因转导。之后更换T细胞培养基(IL-7/15)将激活的T细胞以1M/mL为起始细胞密度，进行细胞培养，每2天补充培养基并添加适量细胞因子。
实施例4 T细胞的CAR基因转导
实施例3中的T细胞激活48-72小时以后，AAV6病毒介导的CAR基因同源重组，原位***剪切位点进行基因修饰，即刻转导，AAV6病毒MOI：2.5×10e5-10e6。
1、TCR基因敲除
1.1采用CRISPR/cas9***
1.2电转体系：
T细胞：3M
cas9蛋白 10ug(2ul)
sgRNA 2.5ug(5ul)
总体积：Buffer T 100μL
电转条件：1600V，10ms，3pulses
其中，所述的sgRNA序列如下：
CTGGATATCTGTGGGACAAGAGG(SEQ ID NO:13)
1.3 CAR基因转导
AAV6病毒介导的CAR基因同源重组，原位***剪切位点。电转后即刻转导，AAV6MOI：2.5×10e5-10e6。
1.4 TCR-/CAR+T细胞的分选
4天后，采用Miltenyi的CD3 Biotin Microbeads分选TCR阴性的T细胞。
结果如图5，T细胞分别来自两个不同个体。流式细胞仪检测TCR，CD3的表达情况。两个不同个体的T细胞TCR敲除率分别为87.2％和68.6％。
图6所示：T细胞分别来自两个不同个体。流式细胞仪检测CAR的表达情况，分别为69.1％和67.5％。与慢病毒转导的CAR-T相比，本CAR-T细胞CAR的表达更为均一。
实施例5 CAR-T细胞的功能，表型的检测
1.细胞毒性的检测
K562-CD19+/K562-CD19-细胞共培养，起始细胞数量各5×10e4,其中CD19+细胞表达mCherry荧光蛋白
再与CAR-T细胞共培养，E/T比例8:1，4:1，2:1，1:1，0.5:1，0:1
各设三个复孔
NegativeCtrl：8:1，4:1，2:1，1:1，0.5:1，0:1
PositiveCtrl：8:1，4:1，2:1，1:1，0.5:1，0:1
Test 1：8:1，4:1，2:1，1:1，0.5:1，0:1
Test 2：8:1，4:1，2:1，1:1，0.5:1，0:1
Blank k562-CD19+：0:1,
Blank k562-CD19-：0:1,
Test1+CD19-：8:1，4:1
Test2+CD19-：8:1，4:1
所有4:1组均设6个复孔，1用来检测细胞表型：12,24,48h；2检测CAR的表达情况
96孔板培养
48小时后流式细胞仪检测K562-CD19+细胞的mCherry荧光
计算公式为：
100％×(1-(％CD19pos/％CD19neg at notedE:T)/(％CD19pos/CD19neg at 0:1E:T))by flow cytometry
结果如图7所示，AAV6-TCR-CD19CAR-T的肿瘤杀伤能力与CAR-T细胞与靶细胞的比值成正比，CAR-T细胞：肿瘤细胞＝4:1或8:1时效果最明显。
细胞毒性试验，不同时间点取20ul培养液上清，测细胞因子：IL-2，IFN-r，TNF-a
结果如图8所示，流式细胞因子检测细胞毒性试验不同时间点，细胞培养基中细胞因子的浓度。AAV6-TCR-CD19CAR-T的三个主要细胞因子IL-2，IFN-r，TNF-a的浓度显著降低。
3. T细胞衰竭的检测
细胞毒性试验开始后，不同时间点检测T细胞衰竭的标志物：PD-1，TIM-3，LAG-3
结果如图9所示，细胞毒性试验开始后检测T细胞的衰竭标志物：PD-1，LAG-3，TIM-3。AAV-TCR-CD19CAR-T细胞的衰竭标志物表达量显著低于对照组。
4.小鼠肿瘤模型的建立及AAV-TCR-CAR-T细胞的体内功能检测
体内实验表明(图10)：本发明所制备的AAV-TCR-CAR-T表现出等效的抗肿瘤活性，能够有效地控制小鼠体内肿瘤的生长。小鼠的生存期显著延长。
本发明虽然以较佳实施例公开如上，但并不是用来限定权利要求，任何本领域技术人员在不脱离本发明构思的前提下，都可以做出若干可能的变动和修改，因此本发明的保护范围应当以本发明权利要求所界定的范围准。
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Claims (14)

  1. 一种重组腺相关病毒载体，其特征在于，所述腺相关病毒载体包含如下操作性连接的序列元件：5’末端反向重复序列、3’末端反向重复序列以及编码CAR基因的序列。
  2. 如权利要求1所述的重组腺相关病毒载体，其特征在于，所述编码CAR基因的序列为CD19CAR(4-1BB)。
  3. 如权利要求1或2所述的重组腺相关病毒载体，其特征在于，所述腺相关病毒载体进一步包含如下操作性连接的序列元件：SA序列、2A序列、polyA序列、5’基因组同源序列(5’HA)、3’基因组同源序列(3’HA)。
  4. 如权利要求3所述的重组腺相关病毒载体，其特征在于，所述的CD19CAR(4-1BB)序列包含SEQ ID NO:4所示的序列。
  5. 如权利要求1-4任意一项所述的重组腺相关病毒载体，其特征在于，所述序列元件的序列选自如下序列：所述的5’HA序列包含SEQ ID NO:1所示的序列；所述的SA序列包含SEQ ID NO:2所示的序列；所述的2A序列包含SEQ ID NO:3所示的序列；；所述的polyA序列包含SEQ ID NO:5所示的序列；所述的3’HA序列包含SEQ ID NO:6所示的序列；所述的5’末端反向重复序列包含SEQ ID NO:7所示的序列；所述的3’末端反向重复序列包含SEQ ID NO:8所示的序列。
  6. 如权利要求1-5任意一项所述的重组腺相关病毒载体，其特征在于，所述重组腺相关病毒载体包含与SEQ ID NO:9所示的序列具有至少约70％、至少约80％、至少约90％序列同一性或更多的序列同一性的核苷酸序列。
  7. 如权利要求1-6任意一项所述的重组腺相关病毒载体的制备方法，其特征在于：所述方法包括以下步骤：提供权利要求1-6任意一项所述的病毒载体的包装细胞系；和从所述包装细胞系的上清液中回收重组AAV病毒。
  8. 一种重组腺相关病毒，其特征在于，所述病毒由权利要求1或2所述的重组腺相关病毒载体包装获得。
  9. 一种用于表达CAR基因的方法，其特征在于：所述方法包括提供包括编码权利要求1-6任意一项的重组腺相关病毒(AAV)的核苷酸序列；结合CRISPR/cas9基因编辑技术，将所述AAV同源重组到T细胞的基因组内，所述AAV在T细胞中表达所述CAR基因。
  10. 如权利要求1-6任意一项所述的重组腺相关病毒载体、权利要求8所述重组腺相关病毒，在制备CAR-T细胞或抗肿瘤药物中的应用。
  11. 一种重组腺相关病毒载体的CAR-T细胞制备方法，其特征在于：该方法包括如下步骤：
    第一步，构建权利要求1-6任意一项所述的重组腺相关病毒载体；
    第二步，病毒包装；
    第三步，T细胞分离、激活与扩增，CRISPR/cas9基因编辑，AAV病毒介导的基因重组；
    制备权利要求1-6任意一项所述的重组腺相关病毒载体是通过基因编辑手段，将其表达于癌症患者或健康人外周血中分离收集的T细胞以获得CAR-T细胞。
  12. 一种由权利要求11所述方法制备获得的CAR-T细胞。
  13. 一种试剂盒，其特征在于，所述试剂盒中包含权利要求1-6任意一项所述的重组腺相关病毒载体、权利要求8所述重组腺相关病毒，或权利要求12所述CAR-T细胞。
  14. 权利要求12所述CAR-T细胞或权利要求13所述试剂盒在制备抗肿瘤药物中的应用。
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