WO2024060422A1 - 靶向间皮素和nkg2d配体的双靶向car-t细胞及其应用 - Google Patents

靶向间皮素和nkg2d配体的双靶向car-t细胞及其应用 Download PDF

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WO2024060422A1
WO2024060422A1 PCT/CN2022/138155 CN2022138155W WO2024060422A1 WO 2024060422 A1 WO2024060422 A1 WO 2024060422A1 CN 2022138155 W CN2022138155 W CN 2022138155W WO 2024060422 A1 WO2024060422 A1 WO 2024060422A1
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targeting
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mesothelin
nkg2d
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万晓春
刘茂玄
陈有海
王琪
许晨光
刘骏晨
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Shenzhen Institute of Advanced Technology of CAS
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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
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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

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to dual-targeting CAR-T cells targeting mesothelin and NKG2D ligands and their applications.
  • docetaxel belongs to the taxane class of anti-tumor drugs, which can promote microtubule polymerization in the M phase and inhibit the mitosis process of tumor cells, thereby achieving the purpose of anti-tumor.
  • tumor cells generally have resistance to chemotherapy drugs. Primary or secondary DTX resistance in tumor cells will lead to chemotherapy failure and affect patient prognosis.
  • Other reported anthracyclines, mTOR inhibitors, Src tyrosine kinase inhibitors, and vascular endothelial growth factor inhibitors have been reported to have certain efficacy on some solid tumors, but there is no conclusion on their impact on patients' long-term survival. Currently, many solid tumors still lack clear treatments and effective drugs.
  • CAR-T targeted anti-tumor cellular immunity technology includes antibodies that recognize cancer-specific targets, hinge regions, transmembrane regions, intracellular signaling regions (immunoreceptor tyrosine activation motifs, ITAM) and intracellular
  • ITAM immunoglobulin activation motifs
  • the chimeric antigen receptor of the signaling domain co-stimulatory molecule CD28 and CD137 (4-1BB) conduction domain is expressed in a lentiviral vector, and the vector is transfected into autologous T cells, so that the modified CAR-T cells have targeted It can specifically recognize and kill cells expressing specific antigens, and can also proliferate and activate in the body.
  • CAR-T studies in the treatment of solid tumors. For example, Li et al.
  • EphA2-targeting CAR-T cells can lyse EphA2-positive lung cancer cells.
  • Louis et al. used GD2-targeting CAR-T cells to treat 19 cases of neuroblastoma, and 3 patients achieved complete remission without serious adverse reactions.
  • Beatty et al also reported that some patients with refractory metastatic pancreatic cancer received mesothelin-targeted CAR-T cell therapy.
  • Gianpietro successfully screened B7-H3, a suitable target for CAR-T cells in the treatment of solid tumors.
  • the team successfully constructed B7-H3.CAR-T cells that co-activate 4-1BB.
  • the Mesothelin target is specifically highly expressed in triple-negative breast cancer, and some researchers have reported that mesothelin-targeted CAR-T cells have a certain effect on triple-negative breast cancer.
  • CAR-T cell therapy has achieved great success in the treatment of blood cancers, it is not effective against solid tumors.
  • One of the main reasons is the antigen heterogeneity of solid tumors, which causes single-target CAR-T to easily cause antigen escape, making the efficacy less than ideal.
  • the present invention aims to provide dual-targeting CAR-T cells targeting mesothelin and NKG2D ligands and their applications.
  • Mesothelin is a tumor-associated antigen that is overexpressed in most malignant pleural mesotheliomas, pancreatic cancer, ovarian cancer, and some lung cancers. Although mesothelin has limited expression in normal tissues, it is expressed at low levels on the surface of normal peritoneal, pleural, and pericardial mesothelial cells. In addition, mesothelin is a target of endogenous immune responses in pleural mesothelioma, ovarian cancer, and pancreatic cancer, indicating that this target has good immunogenicity.
  • NKG2D protein is a C-type lectin-like glycoprotein that is widely expressed on the surface of human CD8 + T cells, NK cells, ⁇ T cells and CD4 + T cell subsets.
  • NKG2D is an activating receptor that plays an important role in the activation of NK cells. However, it cannot directly activate T cells, but only provides costimulatory signals to participate in the activation of T cells.
  • the NKG2D receptor can recognize ligand proteins of various structures, which are mainly divided into six types of MHC-I molecule-related molecules A/B, namely MICA/MICB ligand proteins and MHC-I class molecule-related molecules, UL16-binding proteins, namely ULBP1 -6 ligand proteins.
  • NKG2D ligands Since the two major types of NKG2D ligands are generally barely expressed or very lowly expressed on normal cells, but are mainly expressed on the surface of various tumor cells or the expression regulation increases during virus infection, they can be considered as a kind of stress response of the body. Performance. When the NKG2D protein on immune cells in the body combines with cells expressing related NKG2D ligands, it will quickly activate the immune cells and trigger a series of effector reactions, thereby exerting a cytotoxic effect and ultimately killing the cells.
  • the present invention combines the dual-target chimeric antigen receptors of tumors targeting mesothelin and NKG2D to find new treatment methods and optimal treatment targets for tumors, thereby improving the efficacy of current CAR-T cell therapy for tumors.
  • the invention provides a chimeric antigen receptor targeting mesothelin and NKG2D ligand.
  • the chimeric antigen receptor includes a nanobody targeting mesothelin and a protein targeting NKG2D ligand.
  • the mesothelin nanobody and the protein targeting NKG2D ligand are connected by a linker peptide.
  • the mesothelin-targeting Nanobody comprises the amino acid sequence shown in SEQ ID NO. 1;
  • the protein targeting NKG2D ligand includes the amino acid sequence shown in SEQ ID NO. 3.
  • the chimeric antigen receptor further comprises a leader peptide, an extracellular hinge region, a transmembrane region, and an intracellular signaling region;
  • the amino acid sequence of the chimeric antigen receptor targeting mesothelin and NKG2D ligand includes a leader peptide connected sequentially from the amino terminus to the carboxyl terminus, a nanobody targeting mesothelin, a connecting peptide, and a ligand targeting NKG2D.
  • the amino acid sequence of the chimeric antigen receptor targeting mesothelin and NKG2D ligand includes a leader peptide connected sequentially from the amino terminus to the carboxyl terminus, a protein targeting NKG2D ligand, a connecting peptide, and a targeting mesothelin ligand.
  • the leader peptide is selected from one or more combinations of CD8 ⁇ signal peptide, GM-CSF signal peptide, CD28 signal peptide, CD4 signal peptide, CD5 signal peptide, CD134 signal peptide and CD137 signal peptide;
  • the extracellular hinge region is selected from one or more combinations of CD8 ⁇ hinge region, CD28 hinge region, CD4 hinge region, CD5 hinge region, CD134 hinge region, CD137 hinge region and ICOS hinge region;
  • the transmembrane region is selected from one or more combinations of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region and CD28 transmembrane region;
  • the intracellular signal region is selected from the group consisting of 4-1BB signal region, CD3 ⁇ signal region, ICOS signal region, CD27 signal region, OX40 signal region, CD28 signal region, IL1R1 signal region, CD70 signal region and TNFRSF19L signal region.
  • 4-1BB signal region CD3 ⁇ signal region
  • ICOS signal region CD27 signal region
  • OX40 signal region CD28 signal region
  • IL1R1 signal region CD70 signal region
  • TNFRSF19L signal region TNFRSF19L signal region.
  • the present invention also provides nucleotide sequences encoding the chimeric antigen receptors targeting mesothelin and NKG2D ligands.
  • Another aspect of the invention provides expression vectors containing said nucleotide sequences.
  • the expression vector is selected from a lentiviral expression vector, a retroviral expression vector or an adenoviral expression vector.
  • Another aspect of the present invention provides a dual-targeted immune cell targeting mesothelin and NKG2D ligand, where the cell is an immune cell expressing the chimeric antigen receptor targeting mesothelin and NKG2D ligand.
  • the immune cells are selected from T cells, NK cells or NKT cells;
  • the immune cells are selected from T cells.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the expression vector or the dual-targeted immune cells targeting mesothelin and NKG2D ligand.
  • Another aspect of the present invention provides the use of the dual-targeted immune cells targeting mesothelin and NKG2D ligand in the preparation of tumor therapeutic drugs.
  • the tumor is a tumor with high mesothelin expression
  • the tumor is a solid tumor
  • the tumor includes pleural mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, and breast cancer.
  • the present invention provides a dual-target chimeric antigen receptor targeting mesothelin (Mesothelin) and NKG2D ligand (NKG2DL), which includes a VHH nanobody that specifically recognizes Mesothelin and an antibody that specifically recognizes NKG2D, using modern genetic engineering.
  • Technology expresses Mesothelin VHH and NKG2D on human killer T cells to prepare mesothelin and NKG2D ligand dual-targeting CAR-T cells, NKG2D
  • CAR can also further identify tumor vascular endothelial cells in the tumor microenvironment.
  • Infusing the constructed cells back into the patient can avoid problems such as antigen escape of tumor cells and reduce the risk of CAR-T cell treatment of tumors.
  • the recurrence rate, efficient and specific killing of cancer cells can be used to improve the current therapeutic effect of immune cell therapy on tumors.
  • Figure 1 Schematic representation of lentiviral plasmid construction.
  • FIG. 1 Two dual-target CAR-T cell preparation processes.
  • Figure 4 Schematic diagram of two types of dual-target CAR-T targeting tumors.
  • This embodiment provides a chimeric antigen receptor targeting mesothelin and NKG2D ligand, which includes a mesothelin-targeting Nanobody and a protein targeting NKG2D ligand.
  • the mesothelin-targeting Nanobody The antibody and protein targeting the NKG2D ligand are linked by a linker peptide.
  • the chimeric antigen receptor also includes a leader peptide, an extracellular hinge region, a transmembrane region, an intracellular costimulatory region and an intracellular signal transduction region.
  • the chimeric antigen receptor consists of a leader peptide (Leader), a protein targeting NKG2D ligand (NKG2D), a connecting peptide (Link), a nanobody targeting mesothelin (MSLN VHH), The extracellular hinge region (hinge), transmembrane region (TM), intracellular costimulatory region (4-1BB) and intracellular signal transduction region (CD3 ⁇ ) are connected in sequence from the amino terminus to the carboxyl terminus.
  • the chimeric antigen is The designation is NKG2D-MSLN CAR.
  • the chimeric antigen receptor can also be composed of a leader peptide (Leader), a mesothelin-targeting Nanobody (MSLN VHH), a linker peptide (Linker), and a protein targeting NKG2D ligand (NKG2D ), extracellular hinge region (hinge), transmembrane region (TM), intracellular costimulatory region (4-1BB) and intracellular signal transduction region (CD3 ⁇ ) are connected sequentially from the amino terminus to the carboxyl terminus.
  • MSLN-NKG2D CAR a leader peptide
  • MSLN VHH mesothelin-targeting Nanobody
  • Linker linker peptide
  • NKG2D ligand NKG2D ligand
  • TM extracellular hinge region
  • TM transmembrane region
  • 4-1BB intracellular costimulatory region
  • CD3 ⁇ intracellular signal transduction region
  • CD3 ⁇ and 41BB signaling regions are activated, thereby promoting the expansion of T cells in the patient's body and exerting a killing effect.
  • amino acid sequences and nucleotide sequences of the encoding genes involved in the above CAR-NKG2D-MSLN and CAR-MSLN-NKG2D are as follows:
  • Nanobody targeting mesothelin (MSLN VHH) (amino acid sequence) SEQ ID NO.1:
  • Nanobody targeting mesothelin (MSLN VHH) (nucleotide sequence) SEQ ID NO.2:
  • NKG2D NKG2D ligand
  • NKG2D Protein targeting NKG2D ligand (NKG2D) (nucleotide sequence) SEQ ID NO.4:
  • Link peptide (Link) (nucleotide sequence) SEQ ID NO.6:
  • Extracellular hinge region (hinge) (nucleotide sequence) SEQ ID NO.7:
  • Transmembrane region (nucleotide sequence) SEQ ID NO.8:
  • Intracellular costimulatory region (4-1BB) (nucleotide sequence) SEQ ID NO.9:
  • Intracellular signal transduction region (nucleotide sequence) SEQ ID NO.10:
  • This embodiment further provides a method for constructing CAR-NKG2D-MSLN and CAR-MSLN-NKG2D lentiviral plasmids.
  • the structure of lentiviral plasmids is shown in Figure 1.
  • CAR-NKG2D-MSLN and CAR-MSLN-NKG2D lentiviruses The plasmid vector was derived from the HIV NL4-3 clone. The internal structure of HIV has been destroyed to the greatest extent to remove its pathogenicity, so the vector only retains a part of the conserved regions of HIV.
  • the MSLN-NKG2D recognition region structure was incorporated into the SIN vector, plus elongation factor 1 ⁇ (EF1 ⁇ ) as a promoter.
  • the virus is packaged using the vesicular stomatitis virus glycoprotein (VSV-G) capsid.
  • VSV-G DNA and vector DNA use different plasmids and are co-transfected into HEK293T cells only when producing viral vectors.
  • the VSV-G capsid assists in the attachment of lentiviral vectors to the cell membrane and maintains lentiviral infectivity. Specific steps are as follows:
  • the above-mentioned CAR encoding gene was inserted into the pCDH vector and located behind the elongation factor 1 ⁇ (EF1 ⁇ ) of the vector.
  • a start codon such as ATG
  • a stop codon such as TAA
  • EAA elongation factor 1 ⁇
  • the pCDH-CAR recombinant plasmid obtained above, the envelope plasmid pMD2G, and the packaging plasmid (pMDLg/pRRE and pRSV-REV) were co-transfected into the cultured HEK293T cells using the lipofectamine transfection reagent Lipofectamine 3000, and centrifuged after 48 hours. Harvest recombinant lentivirus.
  • lentiviral vectors, retroviral vectors, adenoviral vectors, etc. that express chimeric antigen receptors can be used to transfect T cells.
  • transfection methods such as liposomes and calcium phosphate can be used.
  • the preparation of NKG2D-MSLN CAR-T and MSLN-NKG2D CAR-T cells using the recombinant lentivirus constructed above will be described below. The preparation process is shown in Figure 2, which specifically includes the steps:
  • PBMC Peripheral Blood Mononuclear Cells
  • PBMC are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, etc. It is best to come from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.
  • the patient's blood is extracted and the sample is sent to the blood separation chamber; peripheral blood mononuclear cells are collected, centrifuged by Ficoll and the middle layer cells are taken; after washing with PBS, PBMC are obtained.
  • PBMC blood pressure
  • CD3/CD28 immunomagnetic beads according to the ratio of magnetic beads to cells: 1:1, and incubate at room temperature for 1-2 hours; use a magnet to incubate.
  • Cells using magnetic beads were screened; after washing with PBS and removing the immunomagnetic beads, CD3-positive T lymphocytes were obtained.
  • step (3) Use the recombinant lentivirus in step (2) above to co-culture the CD3-positive T lymphocytes obtained by immunomagnetic bead separation in step b).
  • step (3) On the third day of culture, perform cell counting and medium replacement, and adjust the cell concentration to 0.5 ⁇ 10 6 cells/mL, inoculate, and culture; on the 5th day of culture, observe the cell status. If the cell density increases, dilute the cell concentration to 0.5 ⁇ 10 6 cells/mL, detect cell viability, and continue culturing. After expansion and culture to 9-11 days, the cells were collected, and the expression of chimeric antigen receptor CAR targeting mesothelin was detected by flow cytometry. The results are shown in Figure 3.
  • NKG2D-MSLN CAR-T MSLN-NKG2D
  • the schematic diagram of CAR-T targeted tumor killing is shown in Figure 4.
  • NKG2D-MSLN After the in vitro expansion of CAR-T and MSLN-NKG2D CAR-T cells is completed, a preliminary efficacy experiment is conducted on them.
  • the specific method of the RTCA killing experiment is as follows:

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Abstract

本发明公开了靶向间皮素和NKG2D配体的双靶向CAR-T细胞及其应用，具体公开一种嵌合抗原受体，其包含靶向间皮素的纳米抗体、靶向NKG2D配体的蛋白，所述靶向间皮素的纳米抗体和靶向NKG2D配体的蛋白由连接肽连接，并利用现代基因工程技术在人体杀伤性T细胞上表达间皮素和NKG2D，制备间皮素和NKG2D配体双靶向CAR-T细胞。本发明靶向间皮素和NKG2D配体的双靶向CAR-T细胞可以避免肿瘤细胞发生抗原逃逸等问题，降低CAR-T细胞治疗肿瘤的复发率，高效且特异性的杀伤癌细胞，可用于改善目前免疫细胞疗法对肿瘤的治疗效果。

Description

靶向间皮素和NKG2D配体的双靶向CAR-T细胞及其应用 技术领域
本发明属于生物医药领域，具体涉及靶向间皮素和NKG2D配体的双靶向CAR-T细胞及其应用。
 背景技术
许多实体肿瘤，如三阴性乳腺癌，具有高侵袭性、易转移复发、预后差等特点。目前外科手术治疗具有较大局限性，它仅仅是将目标肿瘤块切除，从而达到治愈的效果。但是对于已扩散的肿瘤来说，手术切除并不能很好地解决这个问题，应结合其他方法互相配合治疗，才有可能取得最佳疗效。更多的肿瘤治疗是依靠放化疗，目前并无合适的有效治疗靶点和治疗方案。而由于当前实体瘤癌尚无合适的靶向药物治疗，只能采取全身化疗的手段。例如多西他赛（DTX）属于紫杉烷类抗肿瘤药物，能够通过在M期促进微管聚合，抑制肿瘤细胞有丝分裂过程，从而达到抗肿瘤的目的。然而，肿瘤细胞普遍存在对化疗药物的耐药作用，肿瘤细胞产生原发性或继发性DTX耐药会导致化疗失败，影响患者预后。其余报道的蒽环类药物、mTOR抑制剂、Src络氨酸激酶抑制剂和血管内皮生长因子抑制剂均报道对一些实体瘤有一定疗效，但对患者的远期生存影响尚无结论。目前，许多实体瘤依旧缺少明确的治疗方法和有效药物。
CAR-T靶向性抗肿瘤细胞免疫技术是将包括识别癌症特异性靶点的抗体、铰链区、跨膜区、胞内信号区（免疫受体酪氨酸活化基序，ITAM) 和胞内信号区共刺激分子CD28及CD137 (4-1BB)传导结构域的嵌合抗原受体表达于慢病毒载体，并将该载体转染至自体T细胞，使修饰后的CAR-T细胞具有靶向性，能特异性地识别并杀伤表达特异性抗原的细胞，还能在体内增殖活化。目前有多种CAR-T研究应用实体瘤的治疗中。例如，Li等发表过一篇报道，他们的体外实验证明了靶向EphA2的CAR-T细胞可以使EphA2阳性的肺癌细胞溶解。Louis等用靶向GD2的CAR-T细胞治疗了19例神经母细胞瘤，其中有3例患者获得了完全缓解，并且没有严重的不良反应。2018年Beatty等也报道了一些难治性转移性胰腺癌患者接受了靶向间皮素的CAR-T细胞治疗。最近，Gianpietro带领的团队成功地为CAR-T细胞在实体瘤治疗中筛选出一个合适的靶点—B7-H3，该团队成功构建共激活4-1BB的B7-H3.CAR-T细胞，在小鼠实验中证明了该构建能提高T细胞的活性，以及肿瘤免疫治疗的效率；另外研究者发现该T细胞还能降低细胞内PD-1的表达量，对表达PD-（L）1阳性的肿瘤治疗效果更好。在后续的小鼠实验中也并无检测到副作用，在安全性上有一定保障。此外也有多种CAR-T在临床治疗于三阴乳腺癌，例如靶向MUC1、c-Met、ROR1、Mesothelin和NKG2D等的CAR-T。NKG2D CAR-T有被用于三阴乳腺癌研究的报道，数据显示NKG2D的配体在三阴乳腺癌细胞系上广泛表达，NKG2D CAR-T对三阴乳腺癌细胞系在体外实验和小鼠体内实验都表现出了良好杀伤效果。
 技术问题
此外Mesothelin靶点特异性地在三阴性乳腺癌中高表达，并有研究者报道出靶向间皮素的CAR-T细胞对三阴性乳腺癌有一定的疗效。虽然CAR-T细胞疗法在血液癌的治疗中取得巨大成功，但对于实体瘤效果不佳。其中一个主要的原因是实体瘤的抗原异质性，导致单靶点CAR-T易引起抗原逃逸，使疗效不甚理想。
 技术解决方案
为了解决现有技术中单抗原靶点CAR-T在肿瘤治疗方面的不足，本发明旨在提供靶向间皮素和NKG2D配体的双靶向CAR-T细胞及其应用。
间皮素是肿瘤相关抗原，在大多数恶性胸膜间皮瘤，胰腺癌，卵巢癌以及一些肺癌中过表达。虽然间皮素在正常组织中具有表达有限，但它在正常的腹膜、胸膜和心包间皮表面上以低水平表达。另外，间皮素是胸膜间皮瘤，卵巢癌和胰腺癌中内源性免疫反应的靶点，表面该靶点免疫原性较好。使用基于抗体的策略靶向间皮素过表达的肿瘤的临床试验已经显示出初步的安全性和潜在的效果，其中仅仅具有浆膜炎这个被确定为剂量依赖性的靶向肿瘤外抗原的毒性，故该靶点安全性非常出色。
NKG2D蛋白是一种C型凝集素样糖蛋白，广泛表达在人CD8 +T细胞、NK细胞、γδT细胞和CD4 +T细胞亚群表面。NKG2D是一个激活型受体，在NK细胞的激活上起到了重要作用。但其不能直接激活T细胞，只是提供共刺激信号而参与T细胞的激活。NKG2D受体可识别多种结构的配体蛋白，主要分为MHC-Ⅰ类分子相关分子A/B，即MICA/MICB配体蛋白和MHC-Ⅰ类分子相关分子6种UL16结合蛋白，即ULBP1-6配体蛋白。由于NKG2D的两大类配体一般在正常细胞上几乎不表达或者极低表达，而主要是在多种肿瘤细胞表面表达或者病毒感染时表达调控会上升，可认为是一种机体应激反应的表现。当体内免疫细胞上的NKG2D蛋白与表达相关的NKG2D配体细胞相结合后，会迅速使免疫细胞激活引发系列效应反应，从而发挥细胞毒效应，最终杀死该细胞。
为了降低CAR-T细胞治疗肿瘤的复发率，提高治疗效果，本发明联合应用靶向间皮素（Mesothelin）和NKG2D的肿瘤的双靶点嵌合抗原受体，为肿瘤找到新的治疗方法和最佳治疗靶点，提高目前CAR-T细胞疗法对肿瘤的疗效。
本发明具体技术方案如下：
本发明提供一种靶向间皮素和NKG2D配体的嵌合抗原受体，所述嵌合抗原受体包含靶向间皮素的纳米抗体、靶向NKG2D配体的蛋白，所述靶向间皮素的纳米抗体和靶向NKG2D配体的蛋白由连接肽连接。
进一步地，所述靶向间皮素的纳米抗体包含如SEQ ID NO. 1所示的氨基酸序列；
所述靶向NKG2D配体的蛋白包含如SEQ ID NO. 3所示的氨基酸序列。
进一步地，所述嵌合抗原受体还包含前导肽、胞外铰链区、跨膜区、胞内信号区；
所述靶向间皮素和NKG2D配体的嵌合抗原受体的氨基酸序列包括从氨基端到羧基端顺次连接的前导肽、靶向间皮素的纳米抗体、连接肽、靶向NKG2D配体的蛋白、胞外铰链区、跨膜区、胞内信号区；
或，所述靶向间皮素和NKG2D配体的嵌合抗原受体的氨基酸序列包括从氨基端到羧基端顺次连接的前导肽、靶向NKG2D配体的蛋白、连接肽、靶向间皮素的纳米抗体、胞外铰链区、跨膜区、胞内信号区。
优选地，所述前导肽选自CD8α信号肽、GM-CSF信号肽、CD28信号肽、CD4信号肽、CD5信号肽、CD134信号肽和CD137信号肽中的一种或多种的组合；
优选地，所述胞外铰链区选自CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区和ICOS铰链区中的一种或多种的组合；
优选地，所述跨膜区选自CD3跨膜区、CD4跨膜区、CD8跨膜区和CD28跨膜区中的一种或多种的组合；
优选地，所述胞内信号区选自4-1BB信号区、CD3ζ信号区、ICOS信号区、CD27信号区、OX40信号区、CD28信号区、IL1R1信号区、CD70信号区和TNFRSF19L信号区中的一种或多种的组合。
本发明还提供编码所述靶向间皮素和NKG2D配体的嵌合抗原受体的核苷酸序列。
本发明另一方面提供含有所述核苷酸序列的表达载体。
进一步地，所述表达载体选自慢病毒表达载体、逆转病毒表达载体或腺病毒表达载体。
本发明另一方面提供一种靶向间皮素和NKG2D配体的双靶向免疫细胞，所述细胞为表达所述靶向间皮素和NKG2D配体的嵌合抗原受体的免疫细胞。
进一步地，所述免疫细胞选自T细胞、NK细胞或NKT细胞；
优选地，所述免疫细胞选自T细胞。
本发明另一方面提供一种药物组合物，其包含所述的表达载体或所述的靶向间皮素和NKG2D配体的双靶向免疫细胞。
本发明另一方面提供所述的靶向间皮素和NKG2D配体的双靶向免疫细胞在制备肿瘤治疗药物中的应用。
进一步地，所述肿瘤为间皮素高表达的肿瘤；
优选地，所述肿瘤为实体瘤；
优选地，所述肿瘤包括胸膜间皮瘤、胰腺癌、卵巢癌、肺癌、乳腺癌。
 有益效果
本发明的有益效果为：
本发明提供靶向间皮素（Mesothelin）和NKG2D配体（NKG2DL）的双靶点嵌合抗原受体，其包括特异性识别Mesothelin的VHH纳米抗体和特异性识别NKG2D的抗体，利用现代基因工程技术在人体杀伤性T细胞上表达Mesothelin VHH和NKG2D，制备间皮素和NKG2D配体双靶向CAR-T细胞，NKG2D CAR除了可以识别肿瘤细胞外，还能进一步识别肿瘤微环境中的肿瘤血管内皮细胞，将构建的细胞回输至患者体内，可以避免肿瘤细胞发生抗原逃逸等问题，降低CAR-T细胞治疗肿瘤的复发率，高效且特异性的杀伤癌细胞，可用于改善目前免疫细胞疗法对肿瘤的治疗效果。
 附图说明
图1：慢病毒质粒的构造示意。
图2：两种双靶点CAR-T细胞制备流程。
图3：两种双靶点CAR-T细胞的阳性率。
图4：两种双靶点CAR-T靶向杀伤肿瘤示意图。
图5：两种双靶点CAR-T细胞体外肿瘤杀伤效果。（A）是经过杀伤约36 h后，E:T=5：1；（B）是经过杀伤约36 h后，E:T=2：1。
 本发明的实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
1. 靶向间皮素和NKG2D配体的嵌合抗原受体的结构
本实施例提供一种靶向间皮素和NKG2D配体的嵌合抗原受体，其包含靶向间皮素的纳米抗体、靶向NKG2D配体的蛋白，所述靶向间皮素的纳米抗体和靶向NKG2D配体的蛋白由连接肽连接。进一步地，嵌合抗原受体还包含前导肽、胞外铰链区、跨膜区、胞内共刺激区和胞内信号转导区。
在一个具体的实施方案中，嵌合抗原受体由前导肽（Leader）、靶向NKG2D配体的蛋白（NKG2D）、连接肽（Link）、靶向间皮素的纳米抗体（MSLN VHH）、胞外铰链区（hinge）、跨膜区（TM）、胞内共刺激区（4-1BB）和胞内信号转导区（CD3ζ）从氨基端到羧基端顺次连接，该嵌合抗原受体记为NKG2D-MSLN CAR。
在另一个具体的实施方案中，嵌合抗原受体也可由前导肽（Leader）、靶向间皮素的纳米抗体（MSLN VHH）、连接肽（Linker）、靶向NKG2D配体的蛋白（NKG2D）、胞外铰链区（hinge）、跨膜区（TM）、胞内共刺激区（4-1BB）和胞内信号转导区（CD3ζ）从氨基端到羧基端顺次连接，该嵌合抗原受体记为MSLN-NKG2D CAR。
在CAR-NKG2D-MSLN和CAR-MSLN-NKG2D与Mesothelin或者NKG2D结合后，CD3ζ和41BB信号区域即被激活，从而促进T细胞在患者体内的扩增并发挥杀伤作用。
上述CAR-NKG2D-MSLN和CAR-MSLN-NKG2D中涉及的氨基酸序列、编码基因的核苷酸序列如下：
靶向间皮素的纳米抗体（MSLN VHH）（氨基酸序列）SEQ ID NO.1：
QVQLVQSGGGLVHPGGSLRLSCAASGIDLSLYRMRWYRQAPGKERDLVALITDDGTSYYEDSVKGRFTITRDNPSNKVFLQMNSLKPEDTAVYYCNAETPLSPVNYWGQGTQVTVS
靶向间皮素的纳米抗体（MSLN VHH）（核苷酸序列）SEQ ID NO.2：
CAAGTACAACTCGTGCAAAGTGGAGGCGGATTGGTGCATCCAGGAGGGAGCCTCAGACTGTCATGCGCTGCCAGCGGCATAGATCTTTCTTTGTACCGGATGAGATGGTACAGGCAGGCGCCAGGAAAGGAGAGAGATCTCGTCGCACTGATCACCGACGATGGGACCAGCTACTACGAAGACAGTGTCAAGGGCCGGTTCACAATCACCAGAGACAACCCCAGCAACAAGGTGTTTCTCCAAATGAACAGCCTTAAACCAGAGGACACCGCCGTGTATTATTGCAACGCAGAGACACCTCTGTCTCCTGTAAACTACTGGGGGCAGGGAACTCAGGTGACCGTGAGC
靶向NKG2D配体的蛋白（NKG2D）（氨基酸序列）SEQ ID NO.3：
VTRQMCIYTNPTSCNEIYGKFSSAYLACDGKQMEIITLLNPSLISGDEWQWSGNTPIHVLGMWHYSKVLKLLDQDEKSYVKLLSANQSMCSAQSEYWNKSEDFFQYCNNKYCIWNKPCPGCYSETLPIQVEQNFLSNLFVASWI
靶向NKG2D配体的蛋白（NKG2D）（核苷酸序列）SEQ ID NO.4：
GTGACCAGGCAGATGTGTATTTATACAAACCCTACTTCATGCAATGAGATTTACGGAAAATTCAGTAGCGCCTATCTCGCCTGCGATGGCAAGCAAATGGAGATCATTACCCTCTTGAACCCAAGTCTGATTTCCGGGGATGAATGGCAATGGTCAGGAAATACCCCCATTCACGTGCTGGGCATGTGGCATTACTCCAAGGTGCTTAAGCTTCTGGATCAGGACGAGAAAAGCTACGTAAAACTCTTGTCTGCTAATCAATCTATGTGTAGCGCTCAGAGCGAGTACTGGAATAAATCTGAAGATTTCTTCCAGTATTGTAACAATAAGTACTGTATTTGGAACAAACCTTGTCCCGGCTGTTATTCTGAGACCTTGCCTATTCAGGTGGAGCAGAACTTCTTGTCCAACCTGTTCGTGGCCTCCTGGATT
前导肽（Leader）（核苷酸序列）SEQ ID NO.5：
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC
连接肽（Link）（核苷酸序列）SEQ ID NO.6：
GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT
胞外铰链区（hinge）（核苷酸序列）SEQ ID NO.7：
ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATC
跨膜区（TM）（核苷酸序列）SEQ ID NO.8：
TACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGT
胞内共刺激区（4-1BB）（核苷酸序列）SEQ ID NO.9：
CGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG
胞内信号转导区（CD3ζ）（核苷酸序列）SEQ ID NO.10：
CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG
2. CAR-NKG2D-MSLN和CAR-MSLN-NKG2D慢病毒质粒的构建
本实施例进一步提供一种CAR-NKG2D-MSLN和CAR-MSLN-NKG2D慢病毒质粒的构建方法，慢病毒质粒的构造示意如图1所示，CAR-NKG2D-MSLN和CAR-MSLN-NKG2D慢病毒质粒的载体来自于HIV的NL4-3克隆。HIV内部结构已经被最大程度的破坏，以去除其致病性，因此载体只保留了HIV的一部分保守区域。NKG2D-MSLN，MSLN-NKG2D识别区域结构被并入SIN载体，外加延伸因子1α（EF1α）作为启动子。另外，病毒包装采用水疱性口炎病毒糖蛋白（VSV-G）衣壳。为保证安全性，VSV-G DNA和载体DNA采用不同的质粒，只在生产病毒载体时共转入HEK293T细胞中。VSV-G 衣壳会协助慢病毒载体向细胞膜的粘附，并保持慢病毒的感染性。具体步骤如下：
（1）构建含CAR编码基因的pCDH重组质粒
将上述CAR的编码基因插入到pCDH载体中，且位于该载体的延伸因子1α（EF1α）之后。CAR的编码基因插入到pCDH载体时，CAR-的编码基因的5’端可加入起始密码子（如ATG），3’端可加入终止密码子（如TAA）。然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，成功构建pCDH重组质粒，即重组表达质粒。
（2）重组慢病毒构建
将上述获得的pCDH-CAR重组质粒与包膜质粒pMD2G、包装质粒（pMDLg/pRRE和pRSV-REV）三者通过脂质体转染试剂Lipofectamine 3000共转染培养好的HEK293T细胞，48小时后离心收获重组慢病毒。
3. NKG2D-MSLN CAR-T 、MSLN-NKG2D CAR-T细胞的制备方法
NKG2D-MSLN CAR-T 、MSLN-NKG2D CAR-T细胞的制备，可以使用表达嵌合抗原受体的慢病毒载体、逆转录病毒载体、腺病毒载体等进行转染T细胞。表达嵌合抗原受体的慢病毒载体、逆转录病毒载体、腺病毒载体制备时，可以采用脂质体、磷酸钙等转染方法。下面以上述构建的重组慢病毒制备NKG2D-MSLN CAR-T 、MSLN-NKG2D CAR-T细胞进行说明，制备流程见图2，具体包括步骤：
a）PBMC（外周血单个核细胞）的分离
PBMC来源于自体静脉血、自体骨髓、脐带血和胎盘血等。最好是来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
抽取病人血液，送样至血液分离室；采集外周血单个核细胞，Ficoll离心分离后取中间层细胞；经PBS洗涤后，得到PBMC。
b）免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入不含血清的基础培养基，配成细胞悬液；按磁珠与细胞的比例为1:1，加入CD3/CD28免疫磁珠，室温孵1-2h；采用磁铁对孵育好磁珠的细胞进行筛选；PBS洗涤，去除免疫磁珠后，得到CD3阳性T淋巴细胞。
c）病毒转染法制备抗原特异性T淋巴细胞
采用上述步骤（2）中的重组慢病毒对b）中经免疫磁珠分离法得到的CD3阳性T淋巴细胞进行共培养，培养的第3天，进行细胞计数和换液，调整细胞浓度为0.5×10 6个/mL，接种，培养；培养的第5天，观察细胞状态，如果细胞密度增大，则稀释细胞浓度为0.5×10 6个/mL，检测细胞活性，继续培养。扩增培养到第9-11天，收集细胞，同时经过流式细胞仪检测靶向间皮素的嵌合抗原受体CAR的表达，结果如图3所示。
实施例2
NKG2D-MSLN CAR-T、MSLN-NKG2D CAR-T靶向杀伤肿瘤示意图见图4。NKG2D-MSLN CAR-T、MSLN-NKG2D CAR-T细胞体外扩增完成后，对其进行初步效能实验，RTCA杀伤实验具体方法如下：
采用实时细胞分析仪（xCElligence RTCA SP）进行细胞杀伤实验。首先于该仪器配套的E-Plate中加入5,000个过表达Mesothelin的MDA-231细胞的完全培养基至E-Plate孔中培养。大约24小时后，按照不同效靶比（E:T）加入相应数量的阳性率调整至一致的两种双靶向CAR-T细胞、两种单靶点CAR-T和UTD细胞（只经磁珠刺激，未加病毒感染的T细胞对照），随后共培养一段时间（>24小时）。根据实时细胞分析仪的CI值对细胞杀伤效果进行分析。对过表达Mesothelin的MDA-231细胞的杀伤结果如附图5所示，两种双靶向CAR-T细胞对过表达Mesothelin的MDA-231细胞具有非常强的杀伤能力，且显著优于单靶点CAR-T（NKG2D CAR-T或MSLN CAR-T）。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。

Claims (11)

  1. 一种靶向间皮素和NKG2D配体的嵌合抗原受体，其特征在于，所述嵌合抗原受体包含靶向间皮素的纳米抗体、靶向NKG2D配体的蛋白，所述靶向间皮素的纳米抗体和靶向NKG2D配体的蛋白由连接肽连接。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述靶向间皮素的纳米抗体包含如SEQ ID NO. 1所示的氨基酸序列；
    所述靶向NKG2D配体的蛋白包含如SEQ ID NO. 3所示的氨基酸序列。
  3. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含前导肽、胞外铰链区、跨膜区、胞内信号区；
    所述靶向间皮素和NKG2D配体的嵌合抗原受体的氨基酸序列包括从氨基端到羧基端顺次连接的前导肽、靶向间皮素的纳米抗体、连接肽、靶向NKG2D配体的蛋白、胞外铰链区、跨膜区、胞内信号区；
    或，所述靶向间皮素和NKG2D配体的嵌合抗原受体的氨基酸序列包括从氨基端到羧基端顺次连接的前导肽、靶向NKG2D配体的蛋白、连接肽、靶向间皮素的纳米抗体、胞外铰链区、跨膜区、胞内信号区。
    优选地，所述前导肽选自CD8α信号肽、GM-CSF信号肽、CD28信号肽、CD4信号肽、CD5信号肽、CD134信号肽和CD137信号肽中的一种或多种的组合；
    优选地，所述胞外铰链区选自CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区和ICOS铰链区中的一种或多种的组合；
    优选地，所述跨膜区选自CD3跨膜区、CD4跨膜区、CD8跨膜区和CD28跨膜区中的一种或多种的组合；
    优选地，所述胞内信号区选自4-1BB信号区、CD3ζ信号区、ICOS信号区、CD27信号区、OX40信号区、CD28信号区、IL1R1信号区、CD70信号区和TNFRSF19L信号区中的一种或多种的组合。
  4. 编码权利要求1-3任一项所述靶向间皮素和NKG2D配体的嵌合抗原受体的核苷酸序列。
  5. 含有权利要求4所述核苷酸序列的表达载体。
  6. 根据权利要求5所述的表达载体，其特征在于，所述表达载体选自慢病毒表达载体、逆转病毒表达载体或腺病毒表达载体。
  7. 一种靶向间皮素和NKG2D配体的双靶向免疫细胞，其特征在于，所述细胞为表达权利要求1-3任一项所述靶向间皮素和NKG2D配体的嵌合抗原受体的免疫细胞。
  8. 根据权利要求7所述的靶向间皮素和NKG2D配体的双靶向免疫细胞，其特征在于，所述免疫细胞选自T细胞、NK细胞或NKT细胞；
    优选地，所述免疫细胞选自T细胞。
  9. 一种药物组合物，其特征在于，其包含权利要求5所述的表达载体或权利要求7所述的靶向间皮素和NKG2D配体的双靶向免疫细胞。
  10. 权利要求7或8所述的靶向间皮素和NKG2D配体的双靶向免疫细胞在制备肿瘤治疗药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述肿瘤为间皮素高表达的肿瘤；
    优选地，所述肿瘤为实体瘤；
    优选地，所述肿瘤包括胸膜间皮瘤、胰腺癌、卵巢癌、肺癌、乳腺癌。
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