WO2022022719A1 - IL7Rα的截短体及其在制备治疗肿瘤的药物中的用途 - Google Patents

IL7Rα的截短体及其在制备治疗肿瘤的药物中的用途 Download PDF

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WO2022022719A1
WO2022022719A1 PCT/CN2021/109864 CN2021109864W WO2022022719A1 WO 2022022719 A1 WO2022022719 A1 WO 2022022719A1 CN 2021109864 W CN2021109864 W CN 2021109864W WO 2022022719 A1 WO2022022719 A1 WO 2022022719A1
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nucleic acid
il7rα
seq
cells
fusion protein
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张伟
江涛
翟优
李冠璋
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Beijing Neurosurgical Institute
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Beijing Neurosurgical Institute
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Definitions

  • the present disclosure relates to the field of medical biotechnology, in particular, to a truncation of IL7R ⁇ , its encoding nucleic acid, expression vectors, cells, pharmaceutical compositions and uses thereof.
  • Chimeric antigen receptor is a synthetic T cell receptor composed of an antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • Antigen-binding domains are located outside the T cell membrane and include single-chain antibodies or ligands for specific binding to target antigens.
  • the intracellular signaling domain is located in the T cell membrane and is used to transmit signals into the T cell to stimulate the T cell to generate an immune response.
  • T cells expressing CAR can target and recognize target antigens on the surface of tumor cells, therefore, T cells expressing CAR can be used to target and kill tumor cells.
  • the existing T cells expressing chimeric antigen receptor CAR are still weak in killing tumor cells.
  • the purpose of the present disclosure is to overcome the problem that the existing T cells expressing chimeric antigen receptor CAR still have weak killing ability to tumor cells, and to provide a truncated body of IL7R ⁇ .
  • the present disclosure provides a truncation of IL7R ⁇ , and the amino acid sequence of the truncation of IL7R ⁇ includes the sequence shown in SEQ ID NO.1.
  • the present disclosure provides a nucleic acid encoding the truncation of IL7R ⁇ described in the first aspect; preferably, the nucleic acid has the nucleotide sequence shown in SEQ ID NO.2.
  • the present disclosure provides a fusion protein comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain connected in sequence; the amino acid sequence of the intracellular signaling domain includes the first
  • the amino acid sequence of the truncated body of IL7R ⁇ preferably, the antigen binding domain includes an anti-CD44 single-chain antibody and/or an anti-CD133 single-chain antibody, and the intracellular signaling domain includes IL7R ⁇
  • the fusion protein has the amino acid sequence shown in SEQ ID NO.3.
  • the present disclosure provides a fusion nucleic acid encoding the fusion protein of the third aspect; preferably, the fusion nucleic acid has the nucleotide sequence shown in SEQ ID NO.4.
  • the present disclosure provides an expression vector into which an expression cassette is inserted, the expression cassette comprising a first nucleic acid fragment encoding an antigen-binding molecule and a second nucleic acid fragment encoding an intracellular signaling molecule, the The intracellular signaling molecule contains the truncation of IL7R ⁇ described in the first aspect, and an IRES element or a 2A peptide coding sequence is inserted between the first nucleic acid fragment and the second nucleic acid fragment; preferably, the expression Frame is the fusion nucleic acid of the fourth aspect.
  • the present disclosure provides a chimeric antigen receptor-expressing cell obtained by transfecting a host cell with the expression vector described in the fifth aspect, the chimeric antigen receptor-expressing cell contains the truncated body of IL7R ⁇ described in the first aspect.
  • the host cells are T cells.
  • the present disclosure provides the truncation of IL7R ⁇ according to the first aspect, the nucleic acid according to the second aspect, the fusion protein according to the third aspect, the fusion nucleic acid according to the fourth aspect, and the fifth aspect.
  • the tumor is a brain glioma.
  • the present disclosure provides a pharmaceutical composition, the active ingredient of which comprises the cells expressing the chimeric antigen receptor containing IL7R ⁇ truncated body according to the sixth aspect.
  • the T cells expressing the chimeric antigen receptor containing the above-mentioned IL7R ⁇ truncated body provided by the present disclosure can effectively kill tumor cells.
  • Fig. 1 is a flow cytometer detection result diagram of CAR-T 1 cells provided in the embodiment of the present disclosure
  • Fig. 2 is a flow cytometer detection result diagram of CAR-T 2 cells provided in the embodiment of the present disclosure
  • Fig. 3 is a flow cytometer detection result diagram of CAR-T 3 cells provided in the embodiment of the present disclosure
  • Fig. 4 is a flow cytometer detection result diagram of CAR-T 4 cells provided in the embodiment of the present disclosure.
  • the first aspect of the present disclosure provides a truncated body of IL7R ⁇ , and the amino acid sequence of the truncated body of IL7R ⁇ includes the sequence shown in SEQ ID NO.1.
  • SEQ ID NO.1 the sequence shown in SEQ ID NO.1 is as follows:
  • T cells expressing the chimeric antigen receptor containing the above-mentioned truncation of IL7R ⁇ have a longer survival period and can effectively kill tumor cells.
  • the second aspect of the present disclosure provides a nucleic acid encoding the truncation of IL7R ⁇ described in the first aspect; preferably, the nucleic acid has the nucleotide sequence shown in SEQ ID NO.2.
  • nucleotide sequence shown in SEQ ID NO.2 is as follows:
  • a third aspect of the present disclosure provides a fusion protein comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain connected in sequence; the amino acid sequence of the intracellular signaling domain includes the first In one aspect, the amino acid sequence of the truncated form of IL7R ⁇ .
  • the antigen binding domain comprises an anti-CD44 single-chain antibody and/or an anti-CD133 single-chain antibody
  • the intracellular signaling domain comprises a truncation of IL7R ⁇ . More preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO.3.
  • the fusion protein shown in SEQ ID NO.3 is composed of anti-CD44 single-chain antibody, anti-CD133 single-chain antibody, CD28, truncation of IL7R ⁇ and CD3.
  • amino acid sequence shown in SEQ ID NO.3 is as follows:
  • a fourth aspect of the present disclosure provides a fusion nucleic acid encoding the fusion protein of the third aspect.
  • the fusion nucleic acid has the nucleotide sequence shown in SEQ ID NO.4.
  • nucleotide sequence shown in SEQ ID NO.4 is used to encode the amino acid sequence shown in SEQ ID NO.3.
  • nucleotide sequence shown in SEQ ID NO.4 is as follows:
  • a fifth aspect of the present disclosure provides an expression vector into which an expression cassette is inserted, the expression cassette comprising a first nucleic acid fragment encoding an antigen binding molecule and a second nucleic acid fragment encoding an intracellular signaling molecule, the The intracellular signaling molecule contains the truncation of IL7R ⁇ described in the first aspect, and an IRES element or a 2A peptide coding sequence is inserted between the first nucleic acid fragment and the second nucleic acid fragment; preferably, the expression Frame is the fusion nucleic acid of the fourth aspect.
  • a sixth aspect of the present disclosure provides a chimeric antigen receptor-expressing cell obtained by transfecting a host cell with the expression vector described in the fifth aspect, the chimeric antigen receptor-expressing cell contains the truncated body of IL7R ⁇ described in the first aspect.
  • the host cells are T cells.
  • the seventh aspect of the present disclosure provides the truncation of IL7R ⁇ according to the first aspect, the nucleic acid according to the second aspect, the fusion protein according to the third aspect, the fusion nucleic acid according to the fourth aspect, and the fifth aspect.
  • the tumor is a brain glioma.
  • the eighth aspect of the present disclosure provides a pharmaceutical composition, the active ingredient of which comprises the cells expressing the chimeric antigen receptor containing IL7R ⁇ truncated body according to the sixth aspect.
  • This example is used to illustrate the construction of the expression vector.
  • the nucleotide sequences shown in SEQ ID NO. 4, SEQ ID NO.5 and SEQ ID NO.6 were synthesized respectively by using the full sequence synthesis method.
  • the nucleotide sequence shown in SEQ ID NO.4 is used to encode the fusion protein shown in SEQ ID NO.3.
  • the nucleotide sequence shown in SEQ ID NO.5 is used to encode the fusion protein shown in SEQ ID NO.7.
  • the nucleotide sequence shown in SEQ ID NO.6 is used to encode the fusion protein shown in SEQ ID NO.8.
  • the composition of the fusion protein shown in SEQ ID NO.3, SEQ ID NO.7 and SEQ ID NO.8 is as follows:
  • G1 The fusion protein shown in SEQ ID NO.3 is composed of anti-CD44 single-chain antibody, anti-CD133 single-chain antibody, CD28, IL7R ⁇ truncated body and CD3;
  • G2 The fusion protein shown in SEQ ID NO.7 is composed of anti-CD44 single-chain antibody, anti-CD133 single-chain antibody, CD28 and CD3;
  • G3 The fusion protein shown in SEQ ID NO.8 is composed of anti-CD44 single-chain antibody, anti-CD133 single-chain antibody, CD28, IL7R ⁇ and CD3.
  • amino acid sequence of the fusion protein shown in SEQ ID NO.7 is as follows:
  • the nucleotide sequence shown in SEQ ID NO.5 is as follows:
  • amino acid sequence of the fusion protein shown in SEQ ID NO.8 is as follows:
  • the nucleotide sequence shown in SEQ ID NO.6 is as follows:
  • step (1) (2) Using pLVX-IRES- ⁇ NGFR (purchased from Clontech Company, the product number is 631982) as the vector, the three kinds of nucleotide sequences synthesized in step (1) were respectively inserted by conventional methods to obtain the three kinds of lentiviruses of this example Expression vector.
  • This example is used to illustrate the preparation and detection of T cells expressing chimeric antigen receptors.
  • Example 1 The three lentiviral expression vectors constructed in Example 1 and the pLVX-IRES- ⁇ NGFR empty vector were separately packaged with lentivirus, and then T cells were cultured, transfected and amplified in vitro according to the following methods.
  • CAR-T 1 is transfected with the nucleotide sequence shown in SEQ ID NO.4, and can express the fusion protein shown in SEQ ID NO.3
  • CAR-T 2 is transfected with the nucleotide sequence shown in SEQ ID NO.5 Nucleotide sequence, capable of expressing the fusion protein shown in SEQ ID NO.7
  • CAR-T 3 transfected with the nucleotide sequence shown in SEQ ID NO.6, capable of expressing the fusion protein shown in SEQ ID NO.8
  • CAR-T4 was transfected with an empty vector.
  • the detection method is as follows: centrifuge to collect the T cells to be detected after transfection, wash with PBS once, discard the supernatant, add the corresponding detection amount of monoclonal antibody according to the antibody instructions, protect from light for 30 minutes, wash and resuspend with PBS, and pass through the membrane. Flow cytometry was used for sandwich detection, and the antibody used in the detection was a mixture of His-tag-labeled CD44 and PE-labeled anti-His-tag antibody. The results are shown in Figures 1-4.
  • Fig. 1 is a flow cytometer detection result diagram of CAR-T 1 cells provided in the embodiment of the present disclosure
  • Fig. 2 is a flow cytometer detection result diagram of CAR-T 2 cells provided in the embodiment of the present disclosure
  • Fig. 3 is a flow cytometer detection result diagram of CAR-T 3 cells provided in the embodiment of the present disclosure
  • Fig. 4 is a flow cytometer detection result diagram of CAR-T 4 cells provided in the embodiment of the present disclosure.
  • This example is used to verify the expansion ability and survival period of the CAR-T cells constructed in Example 2.
  • the mixed cells were placed in a 24-well plate for culture, wherein each well contained 105 glioma stem cells, and the reaction system per well was 1 mL.
  • Culture conditions include: 37°C, 5% CO2, saturated humidity incubator incubation. After 0, 4, 8, 12, 18, and 26 days, different CAR-T cells were counted with a cytometer to explore the expansion of different CAR-T cells under the stimulation of target cells.
  • Example 2 Take the CAR-T 1 cells transfected and cultured in Example 2, and the traditional second-generation CAR-T cells (EGFR vIII-CD28-CD3) targeting the classic tumor target EGFR vIII, which are respectively CD44-positive and CD133-positive.
  • the glioma stem cells of GSC20 were mixed according to different effector-target ratios (number of T cells: number of glioma stem cells).
  • the mixed cells were placed in a 96-well plate for culture, wherein each well contained 4 ⁇ 104 glioma stem cells, and the reaction system per well was 200 ⁇ L.
  • Culture conditions include: 37°C, 5% CO2, saturated humidity incubator for 4 hours.
  • Lysis rate % (OD in experimental group - OD spontaneously released by glioma stem cells - OD naturally released by effector cells)/(maximum OD released by glioma stem cells - OD spontaneously released by glioma stem cells)

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Abstract

本发明提供了一种IL7Rα的截短体，该IL7Rα的截短体的氨基酸序列包含SEQ ID NO.1所示的序列；表达含有上述IL7Rα截短体的嵌合抗原受体的T细胞，能够有效杀伤肿瘤细胞。

Description

IL7Rα的截短体及其在制备治疗肿瘤的药物中的用途 技术领域
本公开涉及医药生物技术领域，具体地，涉及一种IL7Rα的截短体、其编码核酸、表达载体、细胞、药物组合物和它们的用途。
背景技术
嵌合抗原受体(Chimeric antigen receptor，CAR)是人工合成的T细胞受体，由抗原结合结构域、跨膜结构域和胞内信号传导结构域组成。抗原结合结构域位于T细胞膜外，包括单链抗体或配体，用于特异性地结合靶抗原。胞内信号传导结构域位于T细胞膜内，用于向T细胞内传导信号以刺激T细胞产生免疫反应。
CAR能够靶向识别肿瘤细胞表面的靶抗原，因此，表达CAR的T细胞能够用于靶向杀伤肿瘤细胞。然而，现有的表达嵌合抗原受体CAR的T细胞对肿瘤细胞的杀伤能力仍然较弱。
发明内容
本公开的目的是克服现有的表达嵌合抗原受体CAR的T细胞对肿瘤细胞的杀伤能力仍然较弱的问题，提供一种IL7Rα的截短体。
为了实现上述目的，第一方面，本公开提供一种IL7Rα的截短体，该IL7Rα的截短体的氨基酸序列包括SEQ ID NO.1所示的序列。
第二方面，本公开提供一种核酸，该核酸编码第一方面所述的IL7Rα的截短体；优选地，所述核酸具有SEQ ID NO.2所示的核苷酸序列。
第三方面，本公开提供一种融合蛋白，所述融合蛋白含有依次连接的抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域的氨基酸序列包括第一方面所述的IL7Rα的截短体的氨基酸序列；优选地，所述抗原结合结构域包括抗CD44的单链抗体和/或抗CD133的单链抗体，所述胞内信号传导结构域包括IL7Rα的截短体；更优选地，所述融合蛋白具有SEQ ID NO.3所示的氨基酸序列。
第四方面，本公开提供一种融合核酸，所述融合核酸编码第三方面所述的融合蛋白；优选地，所述融合核酸具有SEQ ID NO.4所示的核苷酸序列。
第五方面，本公开提供一种表达载体，所述表达载体插入有表达框，所述表达框 包括编码抗原结合分子的第一核酸片段和编码胞内信号传导分子的第二核酸片段，所述胞内信号传导分子中含有第一方面所述的IL7Rα的截短体，所述第一核酸片段与所述第二核酸片段之间插入有IRES元件或2A肽编码序列；优选地，所述表达框为第四方面所述的融合核酸。
第六方面，本公开提供一种表达嵌合抗原受体的细胞，该表达嵌合抗原受体的细胞由宿主细胞转染第五方面所述的表达载体后得到，所述嵌合抗原受体中含有第一方面所述的IL7Rα的截短体。
可选地，所述宿主细胞为T细胞。
第七方面，本公开提供第一方面所述的IL7Rα的截短体、第二方面所述的核酸、第三方面所述的融合蛋白、第四方面所述的融合核酸、第五方面所述的表达载体、第六方面所述的表达嵌合抗原受体的细胞在制备治疗肿瘤的药物中的用途。
可选地，所述肿瘤为脑胶质瘤。
第八方面，本公开提供一种药物组合物，该药物组合物的有效成分包括第六方面所述的表达含有IL7Rα截短体的嵌合抗原受体的细胞。
通过上述技术方案，本公开提供的表达含有上述IL7Rα截短体的嵌合抗原受体的T细胞，能够有效杀伤肿瘤细胞。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本公开的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本公开，但并不构成对本公开的限制。在附图中：
图1是本公开实施例提供的CAR-T 1细胞的流式细胞仪检测结果图；
图2是本公开实施例提供的CAR-T 2细胞的流式细胞仪检测结果图；
图3是本公开实施例提供的CAR-T 3细胞的流式细胞仪检测结果图；
图4是本公开实施例提供的CAR-T 4细胞的流式细胞仪检测结果图。
具体实施方式
以下结合附图对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
本公开的第一方面提供一种IL7Rα的截短体，该IL7Rα的截短体的氨基酸序列包括SEQ ID NO.1所示的序列。
其中，SEQ ID NO.1所示的序列如下所示：
Figure PCTCN2021109864-appb-000001
本公开发明人发现，上述IL7Rα的截短体能够有效增加活化后T细胞的存活期，因此，表达含有上述IL7Rα截短体的嵌合抗原受体的T细胞的存活期较长，能够有效杀伤肿瘤细胞。
本公开的第二方面提供一种核酸，该核酸编码第一方面所述的IL7Rα的截短体；优选地，所述核酸具有SEQ ID NO.2所示的核苷酸序列。
其中，SEQ ID NO.2所示的核苷酸序列如下所示：
Figure PCTCN2021109864-appb-000002
本公开的第三方面提供一种融合蛋白，所述融合蛋白含有依次连接的抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域的氨基酸序列包括第一方面所述的IL7Rα的截短体的氨基酸序列。优选地，所述抗原结合结构域包括抗CD44的单链抗体和/或抗CD133的单链抗体，所述胞内信号传导结构域包括IL7Rα的截短体。更优选地，所述融合蛋白具有SEQ ID NO.3所示的氨基酸序列。
其中，SEQ ID NO.3所示的融合蛋白由抗CD44的单链抗体、抗CD133的单链抗体、CD28、IL7Rα的截短体和CD3组成。
其中，SEQ ID NO.3所示的氨基酸序列如下所示：
Figure PCTCN2021109864-appb-000003
Figure PCTCN2021109864-appb-000004
本公开的第四方面提供一种融合核酸，所述融合核酸编码第三方面所述的融合蛋白。优选地，所述融合核酸具有SEQ ID NO.4所示的核苷酸序列。
其中，SEQ ID NO.4所示的核苷酸序列用于编码SEQ ID NO.3所示的氨基酸序列。
其中，SEQ ID NO.4所示的核苷酸序列如下所示：
Figure PCTCN2021109864-appb-000005
Figure PCTCN2021109864-appb-000006
本公开的第五方面提供一种表达载体，所述表达载体插入有表达框，所述表达框包括编码抗原结合分子的第一核酸片段和编码胞内信号传导分子的第二核酸片段，所述胞内信号传导分子中含有第一方面所述的IL7Rα的截短体，所述第一核酸片段与所述第二核酸片段之间插入有IRES元件或2A肽编码序列；优选地，所述表达框为第四方面所述的融合核酸。
本公开的第六方面提供一种表达嵌合抗原受体的细胞，该表达嵌合抗原受体的细胞由宿主细胞转染第五方面所述的表达载体后得到，所述嵌合抗原受体中含有第一方面所述的IL7Rα的截短体。
可选地，所述宿主细胞为T细胞。
本公开的第七方面提供第一方面所述的IL7Rα的截短体、第二方面所述的核酸、第三方面所述的融合蛋白、第四方面所述的融合核酸、第五方面所述的表达载体、第六方面所述的表达嵌合抗原受体的细胞在制备治疗肿瘤的药物中的用途。
可选地，所述肿瘤为脑胶质瘤。
本公开的第八方面提供一种药物组合物，该药物组合物的有效成分包括第六方面所述的表达含有IL7Rα截短体的嵌合抗原受体的细胞。
下面通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例1
本实施例用于说明表达载体的构建。
(1)采用全序列合成方法，分别合成如SEQ ID NO 4、SEQ ID NO.5和SEQ ID NO.6所示的核苷酸序列。SEQ ID NO.4所示的核苷酸序列用于编码SEQ ID NO.3所示的融合蛋白。SEQ ID NO.5所示的核苷酸序列用于编码SEQ ID NO.7所示的融合蛋白。SEQ ID NO.6所示的核苷酸序列用于编码SEQ ID NO.8所示的融合蛋白。其中，SEQ ID NO.3、SEQ ID NO.7和SEQ ID NO.8所示的融合蛋白的组成如下：
G1：SEQ ID NO.3所示的融合蛋白由抗CD44的单链抗体、抗CD133的单链抗体、CD28、IL7Rα截短体和CD3组成；
G2：SEQ ID NO.7所示的融合蛋白由抗CD44的单链抗体、抗CD133的单链抗体、CD28和CD3组成；
G3：SEQ ID NO.8所示的融合蛋白由抗CD44的单链抗体、抗CD133的单链抗体、CD28、IL7Rα和CD3组成。
其中，SEQ ID NO.7所示的融合蛋白的氨基酸序列如下所示：
Figure PCTCN2021109864-appb-000007
SEQ ID NO.5所示的核苷酸序列如下所示：
Figure PCTCN2021109864-appb-000008
Figure PCTCN2021109864-appb-000009
SEQ ID NO.8所示的融合蛋白的氨基酸序列如下所示：
Figure PCTCN2021109864-appb-000010
SEQ ID NO.6所示的核苷酸序列如下所示：
Figure PCTCN2021109864-appb-000011
Figure PCTCN2021109864-appb-000012
(2)采用pLVX-IRES-ΔNGFR(购自Clontech公司，货号为631982)作为载体，采用常规方法分别插入步骤(1)中合成的3种核苷酸序列，得到本实施例的3种慢病毒表达载体。
实施例2
本实施例用于说明表达嵌合抗原受体的T细胞的制备和检测。
(1)将实施例1构建的3种慢病毒表达载体和pLVX-IRES-ΔNGFR空载体分别进行慢病毒包装，然后按照下述方法分别进行T细胞的体外培养、转染和扩增。
(2)按照下述方法分离血液中的T细胞：将1mL无菌PBS与1mL血液混匀，然后缓慢加入到淋巴细胞分离液Ficoll的上层，并于4℃、400g条件下离心30min，加减速分别设置为0。离心结束后，去掉上层血浆，吸取中间白膜层细胞，加入PBS重悬洗涤，并于100g条件下离心10min，加减速正常。离心结束后，去掉上层洗涤液，加入1mL的1640+10％FBS+1％双抗+1×Glutamine培养基重悬细胞，然后利用抗人CD3/CD28磁珠(购自Thermo Fisher公司)刺激扩增，其中，重悬后细胞浓度为1×106细胞/mL，磁珠加入量为100μL，再加入100IU/mL rhIL-2(Peprotech)，刺激培养2天，得到T细胞。
(3)按照下述方法进行慢病毒转染：取4份上述分离得到的T细胞，分别加入步骤(1)包装好的慢病毒，然后加入终浓度为6μg/mL的polybrene，混匀，并于32℃、800g的条件下离心100min。离心结束后放入培养箱中继续培养24h。培养结束后将培养物于1500rpm的条件下离心15min，并将离心得到的细胞以1×106/mL的密度接种于培养板内，以rhIL2-100IU/mL刺激培养，以后每2-3天换液一次，直至2-4周，得到转染后的T淋巴细胞CAR-T 1、CAR-T 2、CAR-T 3和CAR-T 4。其中，CAR-T 1转染有SEQ ID NO.4所示的核苷酸序列，能够表达SEQ ID NO.3所示的融合蛋白；CAR-T 2转染有SEQ ID NO.5所示的核苷酸序列，能够表达SEQ ID NO.7所示的融合蛋白；CAR-T 3转染有SEQ ID NO.6所示的核苷酸序列，能够表达SEQ ID NO.8所示的融合蛋白；CAR-T 4转染有空载体。
培养结束后，用PBS重悬细胞，并用流式细胞仪检测上述4种转染后的T淋巴细胞的比例及表面CAR蛋白的表达。检测方法为：分别离心收集转染后的待检测T细胞，PBS洗涤1次后弃上清，按抗体说明书加入相应检测量的单抗避光30min后，利用PBS洗涤、重悬，过膜后采用流式细胞仪进行夹心法检测，检测时使用的抗体为His-tag标记的CD44和PE标记的抗His-tag的抗体的混合物。结果如图1～4所示。
图1是本公开实施例提供的CAR-T 1细胞的流式细胞仪检测结果图；
图2是本公开实施例提供的CAR-T 2细胞的流式细胞仪检测结果图；
图3是本公开实施例提供的CAR-T 3细胞的流式细胞仪检测结果图；
图4是本公开实施例提供的CAR-T 4细胞的流式细胞仪检测结果图。
从图1～3可以看出，CAR-T 1细胞、CAR-T 2细胞和CAR-T 3细胞中均检测出 区别于正常T淋巴细胞的其它细胞；由图4可以看出，CAR-T 4细胞并未检测出区别于正常T淋巴细胞的其它细胞。由此说明本实施例中转染有融合基因的CAR-T细胞均已成功表达目标融合蛋白。
实施例3
本实施例用于验证实施例2构建的CAR-T细胞的扩增能力和生存期。
分别取实施例2中转染并培养得到的4种CAR-T细胞，分别与CD44和CD133阳性的胶质瘤干细胞GSC20按照效靶比＝10(T细胞数量：胶质瘤干细胞数量)混合。将混合后的细胞置于24孔板中进行培养，其中，每孔中含有胶质瘤干细胞105个，每孔反应体系为1mL。培养条件包括：37℃，5％CO2，饱和湿度孵箱孵育。此后0、4、8、12、18、26天用细胞计数板为不同CAR-T细胞进行计数，探究不同CAR-T在靶细胞刺激下的扩增情况
表1
Figure PCTCN2021109864-appb-000013
由表1可以看出，表达有上述IL7Rα的截短体的CAR-T细胞具备更强的扩增能力和更长的生存期。
对比例
取实施例2中转染并培养得到CAR-T 1细胞，以及靶向经典肿瘤靶点EGFR vIII的传统第二代CAR-T细胞(EGFR vIII-CD28-CD3)，分别与CD44阳性和CD133阳性的胶质瘤干细胞GSC20按照不同的效靶比(T细胞数量：胶质瘤干细胞数量)混合。将混合后的细胞置于96孔板中进行培养，其中，每孔中含有胶质瘤干细胞4×104个，每孔反应体系为200μL。培养条件包括：37℃，5％CO2，饱和湿度孵箱孵育4小时。
乳酸脱氢酶活性测定：离心结束后，每孔吸取上清液100μL置于96孔酶标板中，同时，每孔加入100μL LDH底物，室温避光反应30min。反应结束后，每孔加50μL终止液终止酶促反应。在酶标仪490nm测定光密度值(OD)。计算各组平均光密度值(OD)，按下式计算每种T细胞对胶质瘤干细胞的裂解率。结果如表1所示。
裂解率％＝(实验组OD-胶质瘤干细胞自发释放OD-效应细胞自然释放OD)/(胶质瘤干细胞最大释放OD-胶质瘤干细胞自发释放OD)
检测结果见表2。
表2
Figure PCTCN2021109864-appb-000014
由表2可以看出，靶向经典肿瘤干细胞靶点EGFR vIII的传统第二代CAR-T细胞对胶质瘤干细胞的杀伤力不如本公开提供的表达IL7Rα截短体的T细胞。
以上结合附图详细描述了本公开的优选实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (10)

  1. 一种IL7Rα的截短体，其特征在于，该IL7Rα的截短体的氨基酸序列包括SEQ ID NO.1所示的序列。
  2. 一种核酸，其特征在于，该核酸编码权利要求1所述的IL7Rα的截短体；
    优选地，所述核酸具有SEQ ID NO.2所示的核苷酸序列。
  3. 一种融合蛋白，其特征在于，所述融合蛋白含有依次连接的抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域的氨基酸序列包括权利要求1所述的IL7Rα的截短体的氨基酸序列；
    优选地，所述抗原结合结构域包括抗CD44的单链抗体和/或抗CD133的单链抗体，所述胞内信号传导结构域包括IL7Rα的截短体；
    更优选地，所述融合蛋白具有SEQ ID NO.3所示的氨基酸序列。
  4. 一种融合核酸，其特征在于，所述融合核酸编码权利要求3所述的融合蛋白；
    优选地，所述融合核酸具有SEQ ID NO.4所示的核苷酸序列。
  5. 一种表达载体，其特征在于，所述表达载体插入有表达框，所述表达框包括编码抗原结合分子的第一核酸片段和编码胞内信号传导分子的第二核酸片段，所述胞内信号传导分子中含有权利要求1所述的IL7Rα的截短体，所述第一核酸片段与所述第二核酸片段之间插入有IRES元件或2A肽编码序列；
    优选地，所述表达框为权利要求4所述的融合核酸。
  6. 一种表达嵌合抗原受体的细胞，其特征在于，该表达嵌合抗原受体的细胞由宿主细胞转染权利要求5所述的表达载体后得到，所述嵌合抗原受体中含有权利要求1所述的IL7Rα的截短体。
  7. 根据权利要求6所述的细胞，其特征在于，所述宿主细胞为T细胞。
  8. 权利要求1所述的IL7Rα的截短体、权利要求2所述的核酸、权利要求3所述的融合蛋白、权利要求4所述的融合核酸、权利要求5所述的表达载体、权利要求6或7所述的表达嵌合抗原受体的细胞在制备治疗肿瘤的药物中的用途。
  9. 根据权利要求8所述的用途，其中，所述肿瘤为脑胶质瘤。
  10. 一种药物组合物，其特征在于，该药物组合物的有效成分包括权利要求6或7所述的表达含有IL7Rα截短体的嵌合抗原受体的细胞。
PCT/CN2021/109864 2020-07-31 2021-07-30 IL7Rα的截短体及其在制备治疗肿瘤的药物中的用途 Ceased WO2022022719A1 (zh)

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