WO2019000620A1 - 一种治疗hiv感染的嵌合抗原受体的重组基因构建及其应用 - Google Patents

一种治疗hiv感染的嵌合抗原受体的重组基因构建及其应用 Download PDF

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WO2019000620A1
WO2019000620A1 PCT/CN2017/099261 CN2017099261W WO2019000620A1 WO 2019000620 A1 WO2019000620 A1 WO 2019000620A1 CN 2017099261 W CN2017099261 W CN 2017099261W WO 2019000620 A1 WO2019000620 A1 WO 2019000620A1
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cells
car
hiv
chimeric antigen
cell
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张同存
顾潮江
廖兴华
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武汉波睿达生物科技有限公司
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Priority to EP17915822.5A priority Critical patent/EP3511347A4/en
Priority to JP2019538366A priority patent/JP6890664B2/ja
Priority to US16/317,514 priority patent/US20190389940A1/en
Publication of WO2019000620A1 publication Critical patent/WO2019000620A1/zh

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Definitions

  • the invention relates to the technical field of immunotherapy of infectious diseases, in particular to a recombinant gene construction and application of a chimeric antigen receptor for treating HIV infection.
  • HIV-1 human immunodeficiency virus 1
  • HAART Highly active antiretroviral therapy
  • CAR chimeric antigen receptor
  • MHC Major histocompatibility complex
  • CD3 ⁇ can enhance the tumoricidal ability of T cells and extend It has prolonged its survival time in the body, and has achieved encouraging results in tumor immunotherapy such as leukemia and lymphoma.
  • Roberts et al. tried to treat HIV infection with CAR-T cells. They chose CD4 sequence as a single-chain antibody to bind gp120 on the surface of infected cells. Although it has some functions of killing cells and after years of efforts, Eventually ended in failure.
  • the transduction efficiency of the retroviral vector is low. In order to obtain sufficient reversible CAR-T cells, excessive in vitro expansion leads to cell death and loss of CAR molecules after reinfusion. .
  • CAR molecular design itself has defects, in which CD4 domain may cause transduced CTLs to be infected by HIV or virus-infected cells to escape the killing of CAR-T cells by down-regulating the expression of CD4 molecules.
  • the present invention provides a novel single-chain antibody ScFv capable of recognizing gp120 on the surface of HIV-infected cells and a chimeric antigen receptor (CAR) made of the single-chain antibody, For the N6-CAR molecule.
  • ScFv single-chain antibody ScFv capable of recognizing gp120 on the surface of HIV-infected cells
  • CAR chimeric antigen receptor
  • Another object of the present invention is to provide an expression vector capable of expressing the above N6-CAR and a CD8 + T lymphocyte modified with an N6-CAR vector.
  • Still another object of the present invention is to provide use of N6-CAR molecule-modified CD8 + T lymphocytes for the preparation of a medicament for combating HIV infection.
  • a single-chain antibody ScFv capable of recognizing gp120 on the surface of HIV-infected cells, which is obtained by concatenating antibody light chain and heavy chain variable regions of gp120 on the surface of HIV-infected cells as a whole CAR
  • the extracellular binding domain of the molecule has the amino acid sequence set forth in SEQ ID NO.
  • the present invention also provides a gene encoding the above-described single-chain antibody ScFv, the nucleotide sequence of which is shown in SEQ ID NO.
  • the present invention also provides a chimeric antigen receptor for treating HIV infection, which is a sequential splicing of a signal peptide through an N-terminus to a C-terminus, a single-chain antibody ScFv, CD8hinge, and a white blood cell provided by the present invention.
  • the antigenic differentiation group is obtained by the transmembrane region CD28-TM and the intracellular domain ICD, 4-1BB and the ⁇ chain CD3 of the leukocyte antigen differentiation group 3.
  • the amino acid sequence of the obtained chimeric antigen receptor is SEQ ID NO. 4 is shown.
  • the present invention also provides a coding gene encoding the chimeric antigen receptor N6-CAR described above, the nucleotide sequence of which is shown in SEQ ID NO.
  • An expression vector comprising a gene encoding an amino acid sequence of the chimeric antigen receptor N6-CAR of SEQ ID NO. 4, wherein the vector is a PTK881 vector, and the CMV promoter is replaced with EF1 ⁇ .
  • the PTK-EF1 ⁇ -N6 vector engineered after the promoter has the nucleotide sequence shown in SEQ ID NO.
  • the present invention provides a genetically modified CD8 + T lymphocyte, which is a lentiviral vector transfected into a 293T cell transfected with a PTK-EF1 ⁇ -N6 expression vector to transduce CD8 + T lymphocytes, thereby obtaining a chimeric antigen.
  • a genetically modified CD8 + T lymphocyte which is a lentiviral vector transfected into a 293T cell transfected with a PTK-EF1 ⁇ -N6 expression vector to transduce CD8 + T lymphocytes, thereby obtaining a chimeric antigen.
  • Genetically engineered T-lymphocytes The killing effect of N6-CAR-modified CD8+ cells on the expression of gp120 cell line was more obvious.
  • the present invention also provides said genetically modified N6-CAR use of CD8 + T lymphocytes, the N6-CAR gene-modified CD8 + T lymphocytes medicament is applied to the preparation of living cells against infection by HIV.
  • the genetically modified CD8 + T lymphocytes capable of expressing a chimeric antigen receptor in the present invention have been found to have significant activity for inhibiting and killing HIV virus in vitro and in vivo, and can be prepared as an active ingredient. HIV-infected drugs.
  • Figure 1 is a schematic view showing the structure of a chimeric antigen receptor for HIV infection constructed according to the present invention
  • FIG. 2 is a schematic view showing the structure of a PTK-EF1 ⁇ -N6 lentiviral vector constructed according to the present invention
  • Figure 3 is a diagram showing the expression level (A) of N6-CAR in transduced CD8 + T lymphocytes and its functional proliferative ability (B) after stimulation in the present invention
  • Figure 4 is a diagram showing the activity of killing HIV-infected cells by N6-CAR transduced CD8 + T lymphocytes in vitro;
  • Figure 5 is a graph showing the activity of N8-CAR transduced CD8 + T lymphocytes inhibiting virus in the present invention under co-culture conditions;
  • Figure 6 is a graph showing the activity of N6-CAR transduced CD8 + T lymphocytes in killing HIV-infected cells in humanized mice in the present invention.
  • the present invention provides a chimeric antigen receptor (CAR) recombinant gene for treating HIV infection and a construction method thereof, and the specific splicing method is: sequentially splicing a signal peptide, and recognizing a gp120 single-chain antibody ScFv which is infected with the surface of an HIV virus cell. , CD8hinge, leukocyte antigen differentiation group, molecular transmembrane region CD28-TM+ICD, 4-1BB and CD3 (leukocyte antigen differentiation group molecule 3) ⁇ chain, and finally get a complete chimeric antigen receptor that can treat HIV (CAR The molecule has an amino acid sequence as shown in SEQ ID NO. 4, and its structure is shown in Figure 1. The nucleotide sequence of the gene encoding the chimeric antigen receptor (CAR) is shown in SEQ ID NO.
  • SEQ ID NO. 2 The amino acid sequence of the chimeric antigen receptor-derived single-chain antibody ScFv for treating HIV infection is shown in SEQ ID NO. 2, which is a tandem antibody against the light chain and heavy chain of the antibody against the surface of the HIV virus cell gp120.
  • the variable region is obtained, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.
  • the structural design of the CAR molecule of the present invention is described in detail by the chimeric antigen receptor (CAR) molecule shown in SEQ ID NO.
  • the sequence nitrogen end of the CAR molecule is a CAR-derived ScFv sequence that specifically recognizes gp120 on the surface of HIV-infected cells; the sequence carbon end of the CAR molecule is based on three generations of CAR structure, including CD8hinge, CD28TM+ICD, 4-1BB and
  • the intracellular domain of CD3 ⁇ is composed in tandem, and the ScFv and intracellular signaling molecules are linked by the transmembrane domain of the CD28 molecule.
  • each fragment can function as follows: a signal peptide can secrete CAR into the extracellular, CD28TM+ICD binds the CAR anchor of the present invention to the cell membrane; ScFv specifically recognizes gp120 which recognizes the surface of the infected HIV virus cell; CD3 ⁇ is an intracellular signal-activating sequence that activates the CD3 ⁇ activation signal after ScFv binds to the antigen, initiating lymphocyte killing activity.
  • the CAR was synthesized according to the sequence shown in SEQ ID NO. 4, and the full-length CAR coding gene was inserted into the expression vector of interest by a seamless recombinant cloning technique (see Fig. 2).
  • the preferred plasmid vector is a PTK-EF1 ⁇ -N6 vector transformed with the PTK881 vector and replaced with the EF1 ⁇ promoter (the nucleotide sequence is shown in SEQ ID NO. 5).
  • a recombinant plasmid PTK-EF1 ⁇ -N6 vector inserted into the CAR gene and capable of expressing CAR was obtained, and the nucleotide sequence thereof is shown in SEQ ID NO.
  • the virus packaging steps are as follows:
  • the lentiviral liquid supernatant was filtered through a 0.22 ⁇ m filter, dispensed into a 250 ml centrifuge bottle, and centrifuged at 30,000 g for 2.5 hours at 4 ° C. After centrifugation, the centrifuge bottle was carefully transferred to a biological safety cabinet and evacuated with a vacuum pump. Clear, leave a precipitate, add T cells culture medium 500 ⁇ l / centrifuge bottle, blow the precipitate with a gun to mix and mix, that is, get the lentiviral vector containing N6-CAR molecule, immediately use or dispense and store at -80 °C.
  • Example 3 Preparation of CD8 + T cells (CAR-T cells) capable of expressing a chimeric antigen receptor
  • Step 1 Isolation of patient PBMC cells
  • the centrifuge tube appears to be clearly layered from bottom to top: red blood cell layer, granule cell layer, Ficoll layer, mononuclear cell layer and plasma layer. Pipette the plasma layer to a distance of about 5 mm from the white film layer. Carefully pipet all the liquid above the red blood cell layer into the centrifuge tube, dilute with PBS, and the volume ratio to the cell suspension should be greater than 1:3, and mix.
  • Step 2 Sort CD8 + T cells
  • the PBMC cells in step 1 were resuspended in 30 ml of physiological saline and sampled and counted (after sampling, add 50 ml to mix, 500 g, 10 min, 18 ° C, ascending, descending rapidly, and removing the supernatant). After counting, mix every 10 7 /80 ⁇ L buffer (if the supernatant is not cleaned, it is recommended not to add buffer), add resuspension per 10 7 /20 ⁇ L CD8Microbeads, and incubate at 4-8 °C for 15min.
  • CD8 + T cells were washed out, they were resuspended to 30 ml with physiological saline, sampled, and centrifuged at 500 g, 10 min, and 18 ° C to obtain a cell pellet, which was used for culture.
  • step 2 The CD8 + T cells in step 2 were counted, added to the culture flask at a density of 2 ⁇ 10 6 /ml, and mixed and placed in a CO 2 incubator for 2 hours.
  • Step 4 CD8 + T cells are transduced with N6-CAR molecules to prepare CAR-T cells
  • CD8 + T cell suspension After adding the magnetic beads for 12 hours, an appropriate amount of the CD8 + T cell suspension in a good condition in the step 3 was placed, and centrifuged at 300 g for 5 minutes in a centrifuge tube. The supernatant was discarded, and the chimeric antigen receptor (CAR) virus vector was added at a ratio of 1 x 10 6 /ml of cells while adding Polybrene at a final concentration of 4 ⁇ g/ml, and mixed. The cell suspension was incubated in a small volume at 37 °C. After incubating for 4 hours, an appropriate amount of T cell complete medium was added for culture.
  • CAR chimeric antigen receptor
  • Example 4 Detection of activity of CAR-T cells in killing HIV-infected cells in vitro
  • N6-CAR-T cells were mixed with two HIV-1 infected cell lines H9-NL4-3 and H9-NDK, respectively, in a 96-well plate with a U-bottom. Perform cell killing experiments. First, the HIV-infected cell line H9 and the negative control cells were labeled with Calcein-AM, 100 ⁇ l (containing the target cell number 10 4 ) in a 96-well plate, and 100 ⁇ l of the gradient-diluted CAR-T cells were added to the corresponding 96-well plates to ensure The effective target ratio ranged from 5:1 to 10:1 with a final volume of 200 ⁇ l per well.
  • N6-CAR-T cells significantly killed the HIV-infected target cell lines of the two strains in a dose-dependent manner from 5:1 to 10:1 in the target-to-interval range, but there was no significant difference to the control target cells.
  • the killing effect indicates that the N6-CAR-T cell killing target cell is specific for HIV-gp120.
  • Example 5 Detection of inhibition of viral replication activity by CAR-T cells in vitro under co-culture conditions
  • N6-CAR-T cell group could eliminate 99.5% of HIV-1 infected cells with the CD8+ T cell group modified without CAR molecule, showing significant killing effect and fully demonstrating N6- The specificity and efficiency of CAR-T cells.
  • Example 6 Activity detection of CAR-T lymphocytes in killing HIV-infected cells in vivo
  • mice were infected, PBMC were isolated from healthy volunteers, CD8 + T cells were isolated, N6-CAR lentivirus was transduced, amplified in vitro for 10 days, counted and resuspended in 500 ⁇ l PBS, 1x10 7 CD8+T/kg The dose is intravenously returned. Two weeks later, the spleens were collected from the mice and placed in an embedding agent to prepare frozen sections.
  • Frozen sections of 10 ⁇ m thickness greater than 20 sheets were prepared, photographed under a fluorescence confocal microscope, and photographs were quantified using Velocity 5.0 software (Fig. 6A).
  • a single cell suspension was prepared from the collected spleen cells, 5 ⁇ 10 6 cells were extracted from DNAzol to extract genomic DNA, and the number of copies of the provirus was quantified by Nested-QPCR to estimate the number of all HIV-infected cells in the body ( FIG. 6B ).

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Abstract

提供了一种能够识别感染HIV病毒细胞表面的gp120的单链抗体ScFv，所述抗体通过串联针对感染HIV病毒细胞表面的gp120的抗体轻链、重链可变区得到的；将该单链抗体制成嵌合型抗原受体(CAR)，将CAR编码基因转入质粒载体，并将转入了CAR编码基因的慢病毒载体转导到CD8 +T淋巴细胞，所得的CAR-T淋巴细胞在体外和体内实验中均具有抑制、杀灭HIV感染细胞的活性，可制备抗HIV感染的药物。

Description

一种治疗HIV感染的嵌合抗原受体的重组基因构建及其应用 技术领域
本发明涉及感染性疾病免疫治疗技术领域，具体涉及一种治疗HIV感染的嵌合抗原受体的重组基因构建及其应用。
背景技术
艾滋病是由人类免疫缺陷病毒1型(Human Immunodeficiency Virus 1，HIV-1)感染引起的一种重大威胁人类生命安全的传染病，据世界卫生组织的最新统计,自发现到2014年底已造成3900多万人死亡，目前世界上艾滋病毒感染者仍有3700万。我国艾滋病毒感染者逐年增加，其总患病人数已突破百万。目前尚无有效疫苗且现有药物不能彻底治愈。
高效抗逆转录病毒治疗(HAART)是HIV/AIDS治疗史上的第一次革命，其极大的降低了HIV/AIDS的发病率和病死率，显著延长了患者寿命，甚至降低了HIV的传播。但也面临着很多挑战：1)患者必须终生服药，需付出昂贵的经济代价；2)严重的毒副作用；3)耐药毒株的出现；4)更为重要的是cART不能彻底清除病毒，主要是因为药物仅对复制中的病毒有效，而对HIV在感染早期建立的潜伏性病毒“储藏库”(reservoir)是无效的。一旦抗逆转录病毒治疗中断，病毒储藏库中的整合前病毒再度激活，几乎所有患者体内病毒血症会迅速反弹。
艾滋病毒在其传播和繁殖过程中的高变异性使得以往有效的艾滋病药物失效，因此出现了后来的“鸡尾酒疗法”，也就是联合多种抗病毒药物来进行高效抗逆转录病毒治疗，但是鸡尾酒疗法同样有着其局限性。综合目前国际上艾滋病研究的前沿和热点问题，研究者普遍认为HIV不能治愈的主要原因是HIV在感染极早期就在机体内建立起隐秘的病毒“储藏库”，即静息记忆的CD4+T淋巴细胞(Resting memory CD4+T)。
要找到治愈艾滋病的一个重大挑战，就是如何能够重新激活潜伏的HIV从而被机体的免疫系统自发识别和清除(shock and kill)。同时，研究发现，即使在接受cART治疗的感染者体内，也出现不可逆转的免疫损伤，特别是细胞毒性T细胞(CTL)的数量减少和功能缺陷,这表明通过抗逆转录病毒治疗重建的机体免疫系统将不能有效的清除那些被激活的细胞，需要通过联合增强机体HIV特异性免疫应答的方法来增强对HIV储藏库的清除效果。因此，对HIV潜伏性病毒“储藏库”的形成及激活策略和以重建机体免疫功能的探索都是迈向治愈HIV时代极有价值的研究方向。
近几年来，基于嵌合抗原受体(chimeric antigen receptor,CAR)肿瘤免疫治疗技术创造了杀伤肿瘤细胞的全新途径。由于其具有高亲和力和MHC(Major histocompatibility complex)非依赖性等优点，特别是由两个CD28及4-1BB、CD3ζ偶联形成的3代CAR可增强T细胞的杀瘤能力并延 长了其在体内的存活时间，从而在白血病和淋巴瘤等肿瘤免疫治疗中取得了令人鼓舞的成效。其实早在1994年，Roberts等人尝试用CAR-T细胞治疗HIV感染，他们选取CD4序列作为单链抗体用于结合感染细胞表面的gp120，虽然具有部分杀感染细胞功能且经过多年的努力，但最终以失败而告终。其主要原因有以下几个方面：1.使用逆转录病毒载体转导效率较低，为了获得足够的可回输CAR-T细胞，过度的体外扩增导致回输后细胞死亡和CAR分子的丢失。2.CAR分子设计本身存在缺陷，其中CD4结构域可能引起转导的CTLs被HIV感染或者病毒感染细胞通过下调CD4分子的表达而逃脱CAR-T细胞的杀伤。
技术问题
本发明为了克服现有技术的上述不足，提供了一种新的能识别HIV病毒感染细胞表面gp120的单链抗体ScFv以及由此单链抗体制成的嵌合型抗原受体(CAR)，称为N6-CAR分子。
本发明的另一目的是提供一种能表达上述N6-CAR的表达载体以及N6-CAR载体基因修饰的CD8+T淋巴细胞。
本发明的再一个目的是提供N6-CAR分子修饰的CD8+T淋巴细胞在制备抗HIV感染药物方面的用途。
技术解决方案
为了实现上述目的，本发明是通过以下技术方案实现的：
提供了一种单链抗体ScFv，该单链抗体能够识别HIV病毒感染细胞表面的gp120，是通过串联针对HIV病毒感染细胞表面的gp120的抗体轻链、重链可变区而得，作为整个CAR分子的胞外结合结构域，其氨基酸序列如SEQ ID NO.2所示。
本发明也提供了编码上述单链抗体ScFv的编码基因，该基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种治疗HIV感染的嵌合型抗原受体，该嵌合型抗原受体是通过N端到C端顺次拼接信号肽、本发明提供的单链抗体ScFv、CD8hinge、白细胞抗原分化群分子跨膜区CD28-TM及细胞内结构域ICD、4-1BB和白细胞抗原分化群3的ζ链CD3得到的，所得到的嵌合型抗原受体的氨基酸序列如SEQ ID NO.4所示。
本发明同时提供了编码上述的嵌合型抗原受体N6-CAR的编码基因，该基因的核苷酸序列如SEQ ID NO.3所示。
一种含有并能表达氨基酸序列为SEQ ID NO.4所示的嵌合型抗原受体N6-CAR的编码基因的表达载体，所述载体为以PTK881载体为骨架，将CMV启动子替换成EF1α启动子后改造成的PTK-EF1α-N6载体，其核苷酸序列如SEQ ID NO.5所示。
本发明提供了一种基因修饰的CD8+T淋巴细胞，是将PTK-EF1α-N6表达载体转染293T细胞所的慢病毒载体转导CD8+T淋巴细胞，从而获得能表达嵌合型抗原受体的基因工程化的T-淋巴细胞。N6-CAR改造的CD8+细胞对表达gp120细胞系的杀伤效果更加明显。
进一步地，所述的基因修饰的CD8+T淋巴细胞是由以下方法制备得到：(1)从外周血分离PBMC后再用磁珠阳选获得CD8+T细胞，经anti-CD3/28磁珠(细胞与磁珠比例为1:3)刺激12小时后，加入重组有N6-CAR分子的慢病毒感染4小时后补液(MOI＝5)。从病毒感染后第三天开始，细胞计数并根据细胞状态和增殖情况补加培养基，细胞浓度调整至0.6x106/ml，并补充IL-2100U/mL。进一步扩增细胞直到满足回输的细胞数。
本发明同时提供了一种上述N6-CAR基因修饰的CD8+T淋巴细胞的用途，所述N6-CAR基因修饰的CD8+T淋巴细胞是应用于制备抗HIV感染的活细胞药物。
有益效果
本发明的有益效果：(1)本发明中克服早期设计的缺陷，利用能与病毒蛋白Gp120高度特异性结合的广谱中和抗体作为ScFv，能与98％的HIV-1的病毒株结合，增加该CAR-T细胞的广谱性；(2)本发明中使用含有SIN(Self-inactivating)结构的PTK质粒来生产慢病毒载体增加安全性的同时，改造CAR分子细胞内为双刺激分子以其提高N6-CAR-T细胞的扩增和存活特性，增加临床有效性和安全性。(3)本发明中的能表达嵌合型抗原受体的基因修饰CD8+T淋巴细胞在体外实验和体内实验中均发现具有显著的抑制、杀灭HIV病毒的活性，能够作为活性成份制备抗HIV感染药物。
附图说明
图1为本发明构建的抗HIV感染的嵌合型抗原受体的结构示意图；
图2为本发明构建的PTK-EF1α-N6慢病毒载体结构示意图；
图3为本发明中N6-CAR在被转导的CD8+T淋巴细胞中的表达水平(A)及其刺激后功能性增殖能力(B)检测；
图4为本发明中N6-CAR转导的CD8+T淋巴细胞体外杀灭HIV感染细胞活性检测；
图5为共培养条件下本发明中N6-CAR转导的CD8+T淋巴细胞抑制病毒的活性检测；
图6为本发明中N6-CAR转导的CD8+T淋巴细胞杀灭人源化小鼠体内的HIV感染细胞活性检测。
本发明的实施方式
展示一下实例来具体说明本发明的某些实施例，且不应解释为限制本发明的范围。对本发明公开的内容可以同时从材料、方法和反应条件进行改进，所有这些改进，均应落入本发明的的精神和 范围之内。
实施例1：
本发明提供了一种治疗HIV感染的嵌合抗原受体(CAR)重组基因及其构建方法，具体的拼接方法为：顺次拼接信号肽、能识别感染HIV病毒细胞表面的gp120单链抗体ScFv、CD8hinge、白细胞抗原分化群分子跨膜区CD28-TM+ICD、4-1BB和CD3(白细胞抗原分化群分子3)的ζ链，最后得到完整的能治疗HIV的嵌合型抗原受体(CAR)分子，其氨基酸序列见SEQ ID NO.4，其结构如图1所示；编码该嵌合型抗原受体(CAR)的基因的核苷酸序列如SEQ ID NO.3所示。
治疗HIV感染的嵌合型抗原受体来源的单链抗体ScFv的氨基酸序列如SEQ ID NO.2所示，该单链抗体ScFv是通过串联针对感染HIV病毒细胞表面gp120的抗体轻链、重链可变区而得，其编码基因的核苷酸序列如SEQ ID NO.1所示。
以SEQ ID NO.4所示的嵌合型抗原受体(CAR)分子详细介绍本发明CAR分子的结构设计。CAR分子的序列氮端是CAR来源的ScFv序列，可特异性识别感染HIV病毒细胞表面的gp120；CAR分子的序列碳端是以三代CAR结构为基础，包括CD8hinge、CD28TM+ICD、4-1BB和CD3ζ胞内结构域串联组成，通过CD28分子的跨膜结构域将ScFv和胞内信号分子相连。在上述的结构下各个片段能发挥如下的功能：信号肽可以将CAR分泌到胞外，CD28TM+ICD将本发明的CAR锚钉在细胞膜上；ScFv特异性识别识别感染HIV病毒细胞表面的gp120；CD3ζ为胞内信号激活序列，在ScFv结合抗原后CD3ζ激活信号，启动淋巴细胞的杀伤活性。
实施例2：CAR分子重组构建PTK-EF-1α-N6质粒表达载体
按照SEQ ID NO.4所示序列合成CAR，将全长CAR的编码基因通过基于无缝重组克隆技术插入目的表达载体(见图2)。经过多次试验，首选的质粒载体为以PTK881载体为骨架，将CMV启动子替换成EF1α启动子后改造成的PTK-EF1α-N6载体(其核苷酸序列如SEQ ID NO.5所示)，最终获得了插入CAR基因并能表达CAR的重组质粒PTK-EF1α-N6载体，其核苷酸序列如SEQ ID NO.6所示。
病毒包装步骤如下：
1)取两个均装有16ml DMEM培养液的离心管，向其中一管加入960μg PEI，另一管加入320μg预混的PTK881载体质粒，漩涡震荡,室温平衡10分钟。
2)取一个10ml的移液管将混有PEI的培养基吹起来，将混有质粒的培养基一滴一滴地加入PEI中，室温温育30分钟。
3)取一个T175瓶，向其中加入3ml胎牛血清，将和PEI混合的PTK-EF1α-N6载体质粒加入其中，然后将多层细胞培养瓶中的培养基倒入到T175瓶，上下左右颠倒与质粒混匀，最后将T175 瓶中的培养基倒回到多层细胞培养瓶中。37℃，5％CO2培养箱培养3天，收获上清。收集的上清4000rpm(3000g)，30min离心去除293T细胞碎片。
4)将慢病毒液上清用0.22μm滤膜过滤后，分装到250ml的离心瓶中，于4℃，30000g离心2.5小时，离心后将离心瓶小心转移至生物安全柜，用真空泵去上清，留沉淀，加入T细胞培养基500μl/离心瓶，用枪将沉淀吹散混匀，即得到含N6-CAR分子的慢病毒载体，立即使用或分装后于-80℃保存。
实施例3：制备能表达嵌合型抗原受体的CD8+T细胞(CAR-T细胞)
步骤1：分离患者PBMC细胞
(1)采集人外周血样本60-80ml，边采集边摇晃使得外周血与抗凝剂充分混合；
(2)将外周血转入50ml离心管中，用DPBS缓冲液按1:1稀释外周血，混匀。将稀释好的血样缓慢加入室温的15ml人淋巴细胞分离液的离心管中。方法如下：用10ml移液管吸取血样，伸至分离液液面上方0.5cm处，血样自然滑落铺至分离液面上，然后轻轻加入血样，注意不要冲破液面；
(3)配平离心30min，慢升慢降；
(4)离心完成后，离心管出现明显分层由下至上：红细胞层，粒细胞层，Ficoll层、单个核细胞层和血浆层。吸取血浆层至距白膜层5mm左右处弃之。小心吸取红细胞层以上的所有液体至离心管中，PBS稀释，与细胞悬液体积比应大于1:3，混匀。
(5)离心(1600r/min)5min，PBS重悬细胞混匀，取少量细胞计数。
(6)离心300g(1200r/min)5min，上清液送检无菌检测。
步骤2：分选CD8+T细胞
(1)步骤1中的PBMC细胞，用30ml生理盐水重悬后取样计数(取样完后，补加至50ml混匀，500g、10min、18℃、升快、降快离心，去上清)，计数后按每107/80μL buffer混匀(如果上清没有去干净，建议不用加buffer)，加入按每107/20μL CD8Microbeads重悬，4-8℃孵育15min。
(2)孵育完成后，使用按每107个1～2ml buffer洗细胞，500g、10min离心。
(3)用500μL buffer重悬多达108个细胞(若细胞数目比较多，buffer使用的也会多)。
(4)将美天旎专用LS柱，置于磁力架上，使用3ml buffer冲洗LS柱后，将细胞重悬液加入到LS柱里，使之流尽。用3ml buffer洗LS柱三次，每次需要流尽。将LS柱离开磁力架，使用5ml buffer，加入到LS柱中，用活塞将标记后的细胞冲洗出来(可以冲洗两次，确保标记的细胞均可以冲洗出来)。
(5)CD8+T细胞冲洗出来后，用生理盐水重悬至30ml，取样计数，500g、10min、18℃离心，得细胞沉淀，即可用于培养。
步骤3：CD8+T细胞激活
(1)将步骤2中CD8+T细胞，计数，按密度为2x106/ml加入培养瓶中，混匀放入CO2培养箱培养2小时。
(2)取出培养瓶，轻摇，使沉降于底部的悬浮细胞浮起，移液管吸取培养基转入离心管中，少许培养基洗培养瓶将悬浮细胞全部收集，混匀计数。
(3)根据细胞计数调整细胞浓度，按1.2x106/ml的浓度接种培养瓶中(100～120ml in a T150，50～60ml in a T75，15～29ml in a T25)，加CD3/CD28磁珠，按细胞与磁珠比例为1:3(加之前磁珠用培养基洗涤3次，去除保存液)，加入IL-2 100U/mL，混匀放入CO2培养箱培养，收集细胞。
步骤4：CD8+T细胞被转导N6-CAR分子制备CAR-T细胞
加入磁珠12小时后，取适量步骤3中生长状态良好的CD8+T细胞悬液，放入离心管中300g离心5分钟。弃去上清液，以1x106/ml细胞的比例加入嵌合型抗原受体(CAR)病毒载体，同时加入终浓度4μg/ml的Polybrene，混匀。将细胞悬液在37℃条件下小体积孵育。孵育4小时后补加适量T细胞完全培养基进行培养。细胞培养的第3天，细胞计数并根据细胞状态和增殖情况补加培养基，细胞浓度调整至0.6x106/ml，并补充IL-2 100U/mL。细胞培养的第5天，细胞混匀转离心管中，在磁力架上去除磁珠，细胞计数并补加培养基，并补充IL-2 100U/mL，细胞密度调整至0.6x106/ml继续培养。并利用流式检测SCFV的表达，同时，取部分CD8+T淋巴细胞经goat anti-human Fab antibody抗体刺激并连续传代，以确定anti-gp120CAR转导的CD8+T淋巴细胞自扩增能力，结果如图3所示。
结果表明，Anti-gp120CAR病毒转导细胞培养到五天后，有40％的CD8+T细胞表达CAR分子。当抗体goat anti-human Fab antibody特异性地与CD8+T细胞表达CAR分子结合后，能有效和剂量依赖地激活细胞的增殖，随着传代次数增加，CAR分子阳性细胞种群比例也逐渐升高。
实施例4：CAR-T细胞体外杀灭HIV感染细胞活性的检测
为了进一步检测N6-CAR的功能，我们将N6-CAR-T细胞与两株HIV-1感染的细胞系H9-NL4-3和H9-NDK分别进行混合培养，在U形底的96孔板中进行细胞杀伤实验。首先利用Calcein-AM标记HIV感染细胞系H9和阴性对照细胞，取100μl(含靶细胞数量104)于96孔板中，取100μl梯度稀释的CAR-T细胞加入相应的96孔板中，确保效靶比范围为5：1到10：1，每孔终体积为200μl。室温200g离心30分钟，37℃孵育2-3小时。离心取上清测定荧光并计算出裂解百分数并用于判断N6-CAR-T细胞对HIV感染细胞的细胞毒性，实验结果如图4所示。
结果显示，从5:1到10:1效靶比区间范围内，N6-CAR-T细胞以剂量依赖的方式显著杀伤两个毒株HIV感染的靶细胞系，而对对照靶细胞没有明显的杀伤效果，说明N6-CAR-T细胞杀伤靶细胞作用是HIV-gp120特异性的。
实施例5：共培养条件下CAR-T细胞体外抑制病毒复制活性的检测
为了进一步证明N6-CAR-T细胞在清除野生型HIV-1感染的原代CD4+T细胞方面的有效性，利用野生型HIV-1NL4-3-EGFP和NDK-EGEP两个毒株分别感染健康人血液样本中分离的CD4+T淋巴细胞，感染后3小时换液。感染后的第8天，该细胞与N6-CAR改造的同源CD8+T淋巴细胞以1：4的比例混合,在24孔板中进行细胞杀伤实验。靶细胞数量为106/孔，RMPI1640完全培养基体积为500μl/孔。48小时后，通过流式细胞术检测EGFP+CD4+T淋巴细胞的比例，验证N6-CAR-T细胞的杀伤作用，实验结果如图5所示。
结果表明，以未经CAR分子改造的CD8+T细胞组为参照，N6-CAR-T细胞组能清除其中99.5％的HIV-1感染的细胞，表现出显著的杀伤效果，充分展示了N6-CAR-T细胞的特异性和高效性。
实施例6：CAR-T淋巴细胞在体内杀灭感染HIV病毒细胞的活性检测
为了进一步证明Anti-gp120CAR-T淋巴细胞能否在体内清除HIV感染的细胞，我们将带有荧光基因的NL4-3-EGFP病毒(1x106pg p24/mouse)静脉注射到人源化小鼠BLT体内，感染小鼠同时，从健康志愿者分离PBMC然后分离CD8+T细胞，转导N6-CAR慢病毒，体外扩增10天后，计数后用500μl PBS重悬，按1x107CD8+T/kg的剂量静脉回输。两周后，从小鼠体内收集脾脏，放入包埋剂中制备冷冻切片。制备大于20张的10μm厚度的冷冻切片，在荧光共聚焦显微镜下照相，将相片利用Velocity 5.0软件进行量化分析(图6A)。同时，以收集到的部分脾脏细胞制备单细胞悬液，取5x106细胞于DNAzol来提取基因组DNA，利用Nested-QPCR定量前病毒的拷贝数来评估体内所有感染HIV的细胞数目(图6B)。
结果表明，与未接收CAR-T细胞治疗的对照组相比，病毒蛋白表达水平和病毒基因组水平显著性各减少97.1％，证实了CAR-T细胞能在体内进行有效地裂解和清除HIV感染的细胞，为CAR-T细胞进行人体临床测试奠定理论基础。

Claims (8)

  1. 一种单链抗体scFv，其特征在于：该单链抗体能够识别HIV病毒感染细胞表面的gp120，是通过串联针对HIV病毒感染细胞表面的gp120的抗体轻链、重链可变区而得，作为整个CAR分子的胞外结合结构域，其氨基酸序列如SEQ ID NO.2所示。
  2. 编码权利要求1所述的单链抗体ScFv的基因，其特征在于：其核苷酸序列如SEQ ID NO.1所示。
  3. 一种治疗HIV感染的嵌合型抗原受体N6-CAR，其特征在于：该嵌合型抗原受体是通过N端到C端顺次拼接信号肽、权利要求1所述的单链抗体ScFv、CD8hinge、白细胞抗原分化群分子跨膜区CD28-TM及其细胞内结构域(ICD)、4-1BB和白细胞抗原分化群3的ζ链CD3得到的，所得到的嵌合型抗原受体的氨基酸序列如SEQ ID NO.4所示。
  4. 编码权利要求3所述的嵌合型抗原受体的基因，其特征在于：其核苷酸序列如SEQ ID NO.3所示。
  5. 一种含有并能表达氨基酸序列为SEQ ID NO.4所示的嵌合型抗原受体的编码基因的表达载体，其特征在于：所述载体为以PTK881载体为骨架，将CMV启动子替换成EF-1α启动子后改造成的PTK-EF1α-N6载体，其核苷酸序列如SEQ ID NO.5所示。
  6. 一种基因修饰的CD8+T淋巴细胞，其特征在于：是将PTK-EF1α-N6表达载体转染293T细胞所得的慢病毒载体转导CD8+T淋巴细胞，从而获得的能表达嵌合型抗原受体的基因工程化的T-淋巴细胞。
  7. 根据权利要求6所述的基因修饰的CD8+T细胞，其特征在于：由以下方法制备得到：(1)从外周血分离PBMC后再用磁珠阳选获得CD8+T细胞，经anti-CD3/28磁珠(细胞与磁珠比例为1:3)刺激12小时后，加入重组有N6-CAR分子的慢病毒感染4小时后补液(MOI＝5)；从病毒感染后第三天开始，细胞计数并根据细胞状态和增殖情况补加培养基，细胞浓度调整至0.6x106/ml，并补充IL-2 100U/mL，进一步扩增细胞直到满足回输的细胞数。
  8. 一种N6-CAR基因修饰的CD8+T淋巴细胞的用途，其特征在于：所述基因修饰的CD8+T淋巴细胞应用于制备抗HIV感染的活细胞药物。
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