WO2022120942A1 - 靶向cll1嵌合抗原受体及其应用 - Google Patents

靶向cll1嵌合抗原受体及其应用 Download PDF

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WO2022120942A1
WO2022120942A1 PCT/CN2020/138257 CN2020138257W WO2022120942A1 WO 2022120942 A1 WO2022120942 A1 WO 2022120942A1 CN 2020138257 W CN2020138257 W CN 2020138257W WO 2022120942 A1 WO2022120942 A1 WO 2022120942A1
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Prior art keywords
seq
cll1
antigen receptor
chimeric antigen
targeting
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PCT/CN2020/138257
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English (en)
French (fr)
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李光超
罗敏
丁雯
周兆
王学俊
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广州百暨基因科技有限公司
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Priority to GB2207722.6A priority Critical patent/GB2606869A/en
Priority to JP2022529577A priority patent/JP2023510465A/ja
Priority to AU2020480789A priority patent/AU2020480789A1/en
Priority to KR1020227011658A priority patent/KR20220084040A/ko
Priority to EP20958917.5A priority patent/EP4039712A1/en
Priority to US17/612,212 priority patent/US20220354890A1/en
Publication of WO2022120942A1 publication Critical patent/WO2022120942A1/zh

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Definitions

  • the present application belongs to the technical field of biomedicine, and relates to targeting CLL1 chimeric antigen receptor and application thereof.
  • CLL1 C-type lectin-like molecule 1
  • CLEC12A C-type lectin domain family 12 member A
  • CLL1 is restricted to hematopoietic cells, including mainly myeloid-derived cells in peripheral blood and bone marrow, such as monocytes, dendritic cells, granulocytes, and most acute myeloid leukemia (AML) cells. It is worth noting that although CLL1 is abundantly expressed on myeloid cells in peripheral blood and bone marrow, it is not expressed on myeloid-derived cells in peripheral tissues, such as tissue macrophages and tissue dendritic cells. .
  • CLL1 is expressed on AML stem cells (CD34+/CD38-) and a small subset of hematopoietic progenitor cells (CD34+/CD38+ or CD34+/CD33+), but not on normal hematopoietic stem cells (CD34+/CD38- or CD34+/CD33-) not express.
  • CLL1 is expected to be a potential diagnostic and therapeutic target for AML.
  • immunotherapy targeting CLL1 there are few reports of immunotherapy targeting CLL1.
  • the present application provides a targeting CLL1 chimeric antigen receptor and application thereof.
  • the targeting CLL1 chimeric antigen receptor adopts an anti-CLL1 antibody with binding ability to CLL1 as an antigen binding domain, which can not only bind to purified or free
  • the CLL1 protein can also bind to the CLL1 protein on the cell surface, and the immune cells expressing the CLL1-targeting chimeric antigen receptor have important application prospects in the field of tumor treatment.
  • the present application provides a targeting CLL1 chimeric antigen receptor comprising an antigen binding domain, a hinge region, a transmembrane domain and a signaling domain;
  • antigen binding domain is an anti-CLL1 antibody.
  • an anti-CLL1 antibody with binding ability to CLL1 is used as the antigen-binding domain of the chimeric antigen receptor, so that the chimeric antigen receptor can specifically bind to CLL1-positive tumor cells and achieve specific targets for CLL1-positive tumors. to the effect.
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, wherein SEQ ID NO: 1 and SEQ ID NO: 2 are linked by a linker peptide to form anti-CLL1 Antibody 19C1; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, wherein SEQ ID NO: 3 and SEQ ID NO: 4 are linked by a linker peptide to form anti-CLL1 Antibody 23D7; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, wherein SEQ ID NO:5 and SEQ ID NO:6 are linked by a linker peptide to form anti-CLL1 Antibody 27H4; wherein
  • the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in one of SEQ ID NO: 8-10, wherein SEQ ID NO: 7 and SEQ ID NO : 8 is linked by a linker peptide to form anti-CLL1 antibody H27H4L1, SEQ ID NO:7 and SEQ ID NO:9 are linked by a linker peptide to form anti-CLL1 antibody H27H4L2, or SEQ ID NO:7 and SEQ ID NO:10 are linked by a linker peptide to form Anti-CLL1 antibody H27H4L3; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, wherein SEQ ID NO: 11 and SEQ ID NO: 12 are linked by a linker peptide to form anti-CLL1 Antibody 1075.7; wherein
  • the hinge region comprises the CD8 ⁇ hinge region.
  • the transmembrane domain comprises a CD8 ⁇ transmembrane domain and/or a CD28 transmembrane domain, preferably a CD8 ⁇ transmembrane domain.
  • the signaling domain comprises CD3 ⁇ .
  • the signaling domain further comprises any one or a combination of at least two of 4-1BB, CD28 intracellular domain, DAP10 or OX40.
  • the targeting CLL1 chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide comprises CD8 ⁇ signal peptide and/or IgG ⁇ light chain signal peptide.
  • the targeting CLL1 chimeric antigen receptor includes signal peptide, anti-CLL1 antibody, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ .
  • the targeting CLL1 chimeric antigen receptor is 19C1-CAR, which includes the amino acid sequence set forth in SEQ ID NO: 13; wherein
  • the targeting CLL1 chimeric antigen receptor is 23D7-CAR, which comprises the amino acid sequence shown in SEQ ID NO: 14; wherein
  • the targeting CLL1 chimeric antigen receptor is 27H4-CAR, which comprises the amino acid sequence set forth in SEQ ID NO: 15; wherein
  • the targeting CLL1 chimeric antigen receptor is H27H4-CAR comprising the amino acid sequence shown in SEQ ID NO: 16; wherein
  • the chimeric antigen receptor targeting CLL1 is 1075.7-CAR, which comprises the amino acid sequence set forth in SEQ ID NO: 17; wherein
  • the present application provides a nucleic acid molecule comprising the encoding gene targeting the CLL1 chimeric antigen receptor described in the first aspect.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 18, which is the encoding gene of 19C1-CAR;
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 19, which is the encoding gene of 23D7-CAR;
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 20, which is the encoding gene of 27H4-CAR;
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 21, which is the encoding gene of H27H4-CAR;
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 22, which is the encoding gene of 1075.7-CAR;
  • the present application provides an expression vector, the expression vector comprising the nucleic acid molecule described in the second aspect.
  • the expression vector is a viral vector or a non-viral vector containing the nucleic acid molecule described in the second aspect.
  • the viral vector includes any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector.
  • the non-viral vector comprises any one of Piggybac transposon system, Sleeping Beauty transposon system or nano-carrier.
  • the present application provides a recombinant lentivirus prepared from a mammalian cell transfected with the expression vector and the helper plasmid described in the third aspect.
  • the present application provides a chimeric antigen receptor immune cell, and the chimeric antigen receptor immune cell expresses the CLL1-targeting chimeric antigen receptor described in the first aspect.
  • the immune cells expressing the chimeric antigen receptor targeting CLL1 use the antigen binding domain of the chimeric antigen receptor to target CLL1 positive tumor cells, exert the killing function of immune cells and secrete the cytokine IFN- ⁇ .
  • the killing effect on CLL1 tumor was achieved under the target ratio.
  • the nucleic acid molecule of the second aspect is integrated into the genome of the chimeric antigen receptor immune cell.
  • the chimeric antigen receptor immune cells comprise the expression vector described in the third aspect and/or the recombinant lentivirus described in the fourth aspect.
  • the immune cells include any one of T cells, NK cells or macrophages.
  • the T cells include ⁇ T cells and/or ⁇ T cells.
  • the present application provides a pharmaceutical composition comprising the chimeric antigen receptor immune cells described in the fifth aspect.
  • the pharmaceutical composition further comprises any one or a combination of at least two pharmaceutically acceptable carriers, excipients or diluents.
  • the present application provides the targeting CLL1 chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, and the recombinant lentivirus described in the fourth aspect.
  • the malignancy comprises acute myeloid leukemia.
  • the present application provides a method for treating cancer, the method comprising administering to a patient an effective dose of the chimeric antigen receptor immune cells of the fifth aspect or the pharmaceutical composition of the sixth aspect, and One or more antineoplastic drugs are administered simultaneously, separately or sequentially.
  • the cancer comprises acute myeloid leukemia.
  • This application uses anti-CLL1 antibody as the antigen-binding domain to construct CAR molecules, and T cells expressing targeting CLL1 CAR rely on CAR elements to play a killing effect on CLL1-positive tumor cells under different effector-target ratios.
  • H27H4-CAR -T has the best killing function
  • FIG 1 shows the expression of CLL1 in different tumor cells
  • Fig. 2 is the structural representation of the chimeric antigen receptor targeting CLL1;
  • Figure 3A is the map of the lentiviral expression vector containing the H27H4CAR gene, and Figure 3B is the location of the H27H4CAR in the lentiviral expression vector;
  • Figure 4 shows the positive rate of CAR expression of different CAR-T cells
  • Figure 5 shows the killing rate of Raji-CLL1 cells by CAR-T targeting CLL1
  • Figure 6 shows the secretion of IFN- ⁇ by CAR-T targeting CLL1 to kill target cells.
  • anti-CLL1 antibodies 19C1, 23D7, 27H4, humanized 27H4 (H27H4) and 1075.7 were selected as antigen-binding domains for the construction of CAR molecules, wherein 19C1 includes SEQ ID NOs: 1-2
  • the variable region, 23D7 includes the variable region shown in SEQ ID NO: 3-4
  • 27H4 includes the variable region shown in SEQ ID NO: 5-6
  • H27H4 includes the variable region shown in SEQ ID NO: 7-8
  • the variable region, 1075.7 includes the variable regions shown in SEQ ID NOs: 11-12.
  • FITC anti-human CD371 (CLL1) antibody (biolegend) was used to co-incubate with target cells Jurkat, KG-1a, and CLL1 expression by flow cytometry.
  • THP-1, U937 and HL-60 were CLL1 positive cells, and Jurkat and KG-1a were CLL1 negative cells.
  • a chimeric antigen receptor targeting CLL1 is designed.
  • the schematic structure is shown in Figure 2, including CD8 ⁇ signal peptide, single-chain antibody (Anti-CLL1scFv) that specifically binds to CLL1 antigen, CD8 ⁇ hinge region (Hinge) and transmembrane Region (Transmembrane), 4-1BB costimulatory domain and CD3 ⁇ signaling domain, the specific CAR molecules are:
  • 119C1-CAR CD8 ⁇ signal peptide, Anti-CLL1scFv(19C1), CD8 ⁇ Hinge+TM, 4-1BB and CD3 ⁇ ;
  • 223D7-CAR CD8 ⁇ signal peptide, Anti-CLL1scFv(23D7), CD8 ⁇ Hinge+TM, 4-1BB and CD3 ⁇ ;
  • 327H4-CAR CD8 ⁇ signal peptide, Anti-CLL1scFv(27H4), CD8 ⁇ Hinge+TM, 4-1BB and CD3 ⁇ ;
  • 4H27H4-CAR IgG ⁇ light chain signal peptide, Anti-CLL1scFv (H27H4), CD8 ⁇ Hinge+TM, 4-1BB and CD3 ⁇ ;
  • CD8 ⁇ signal peptide CD8 ⁇ signal peptide, Anti-CLL1scFv(1075.7), CD8 ⁇ Hinge+TM, 4-1BB and CD3 ⁇ ;
  • the amino acid sequence of CD8 ⁇ signal peptide is shown in SEQ ID NO:23
  • the nucleic acid sequence is shown in SEQ ID NO:24
  • the amino acid sequence of IgG ⁇ light chain signal peptide is shown in SEQ ID NO:25
  • the nucleic acid sequence is shown in SEQ ID NO:25 NO:26
  • the amino acid sequence of CD8 ⁇ Hinge is shown in SEQ ID NO:27
  • the nucleic acid sequence is shown in SEQ ID NO:28
  • the amino acid sequence of CD8 ⁇ TM is shown in SEQ ID NO:29
  • the nucleic acid sequence is shown in SEQ ID NO:29:
  • the amino acid sequence of 4-1BB is as shown in SEQ ID NO:31
  • the nucleic acid sequence is as shown in SEQ ID NO:32
  • the amino acid sequence of CD3 ⁇ is as shown in SEQ ID NO:33
  • the nucleic acid sequence is as shown in SEQ ID NO:34 shown;
  • the CAR-encoding gene is codon-optimized to promote its high-efficiency expression in human cells, and the CAR-encoding gene is fully synthesized (Guangzhou Aiki Biotechnology Co., Ltd.);
  • the constructed lentiviral expression vector pBG-27H4 containing the 27H4 CAR gene is shown in Figure 3A and Figure 3B.
  • the present embodiment adopts the four-plasmid system to carry out lentiviral packaging of the lentiviral expression vector constructed in Example 4, and the specific steps are as follows:
  • PBMCs were washed with physiological saline for several times, transferred to X-VIVO medium (containing 50ng/mL OKT3, 300IU/mL IL-2) for culture, after PBMCs were separated, used X-VIVO (containing 50ng/mL OKT3, 300IU/mL) mL IL-2) for activation, and after 2 days, the medium was replaced with X-VIVO containing 300 IU/mL for expanded culture; then counted every two days, and replaced with fresh X-VIVO medium containing 300 IU/mL, The cell concentration was maintained at (0.5-1) ⁇ 10 6 cells/mL, and the cells were continuously observed for 10 days.
  • X-VIVO medium containing 50ng/mL OKT3, 300IU/mL IL-2
  • the present embodiment utilizes RetroNectin to improve the infection efficiency of lentivirus on T cells, and the steps are as follows:
  • RetroNectin in a 6-well plate and place it in a 37°C cell incubator for 2 h; aspirate RetroNectin, seal the coated 6-well plate with Hank's solution containing 2.5% BSA, and place it in a 37°C cell incubator for 0.5 h; aspirate the blocking solution, wash the 6-well plate with Hank's solution containing 2% Hepes, add X-VIVO medium, add an appropriate amount of lentivirus solution, centrifuge at 2000g for 2h, discard the supernatant; add 1 ⁇ 10 6 T cells ( CD3 positive >90%), centrifuged at 1000g for 10 min, placed in a cell incubator at 37°C, 5% CO 2 and a certain humidity, and repeated the above steps on the second day.
  • the amount of lentivirus added is shown in Table 2.
  • CLL1-Fc fusion protein (Acrobiosystems) was used to detect CAR expression
  • FITC-Labeled anti-human Fc was used as the secondary antibody
  • T cells that were not transfected with CAR T mock
  • the xCELLigence real-time label-free dynamic analysis technology (Real Time Cell Analysis, RTCA) is used to automatically detect the cell killing effect throughout the entire process.
  • the real-time label-free cell analyzer (RTCA) is based on microelectronic impedance technology, and a large number of micro-gold electrodes are integrated at the bottom of the E-plate. When adherent cells adhere to the micro-gold electrodes, the number, diameter and adherence ability of the cells will all be affected. It affects the current conduction between the micro-gold electrodes, thereby causing the change of the impedance value. This change is extremely delicate and sensitive. Under the negative effect of toxicity, the cells directly or indirectly affect the impedance value. Thus, xCELLigence can monitor cytotoxic effects caused by molecular targets.
  • effector cell suspensions ie, CAR-T cells
  • negative control cell suspensions ie, untransfected T cells
  • 50 ⁇ L of the diluted effector cell suspension to each well.
  • negative control cell suspension blank control group add 50 ⁇ L medium
  • positive control group add 50 ⁇ L 1 ⁇ Cytolysis Solution, incubate at room temperature for 30min, so that effector cells are evenly distributed on the fixed target cells;
  • the killing ability of 27H4-CAR-T is similar to that of H27H4-CAR-T, followed by 23D7-CAR-T, followed by 19C1-CAR-T and 1075.7-CAR-T.
  • the killing ability is weak, and T mock without CAR cannot kill Raji-CLL1, indicating that the killing activity of anti-CLL1 CAR-T depends on the CAR element.
  • Human IFN- ⁇ ELISA kit (Xinbosheng) was used to detect the concentration of IFN- ⁇ cytokines released by CAR-T cells, and to analyze the amount of IFN- ⁇ secreted by CAR-T after co-culture with target cells.
  • CLL1 positive cells Raji-CLL1, HL60, U937 were used as positive target cells
  • CLL1 negative cells Raji were used as negative target cells.
  • the CAR-T cells were incubated with different target cells for 16 hours according to the effect-target ratio of 1:1, and the supernatant was collected to detect the secretion of IFN- ⁇ in the supernatant by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the detection principle adopts the double-antibody sandwich ELISA method, and the anti-human IFN- ⁇ antibody is coated on the ELISA plate.
  • the human IFN- ⁇ in the sample or standard is combined with the coated antibody, and the free components are washed away;
  • biotinylated anti-human IFN- ⁇ antibody and horseradish peroxidase-labeled avidin anti-human IFN- ⁇ antibody binds to human IFN- ⁇ bound to the coated antibody, biotin is specific to avidin
  • the chromogenic substrate (TMB) is added, which turns blue under the catalysis of horseradish peroxidase, and turns yellow after adding the stop solution.
  • the OD value was measured at a wavelength of 450 nm, and the concentration of IFN- ⁇ was proportional to OD450.
  • the concentration of IFN- ⁇ in the sample was calculated by drawing a standard curve.
  • the CLL1 CAR-T cells targeted in this application have a significant killing effect on CLL1-positive tumor cells under different effector-target ratios. After co-culture with tumor cells, they secrete a large amount of cytokine IFN- ⁇ . It has application prospects in the field of tumor therapy.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the present application must rely on the above-mentioned detailed method to implement.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

提供了靶向CLL1嵌合抗原受体及其应用，所述靶向CLL1嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；所述抗原结合结构域为抗CLL1抗体。采用抗CLL1抗体作为抗原结合结构域构建嵌合抗原受体分子，靶向CLL1嵌合抗原受体对CLL1阳性肿瘤细胞具有特异性靶向作用，表达靶向CLL1嵌合抗原受体的免疫细胞体内外杀伤作用显著，与CLL1阳性肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，对CLL1阳性肿瘤细胞具有特异性清除作用。

Description

靶向CLL1嵌合抗原受体及其应用 技术领域
本申请属于生物医药技术领域，涉及靶向CLL1嵌合抗原受体及其应用。
背景技术
C型凝集素样分子1(CLL1)，又称C型凝集素域家族12成员A(CLEC12A)，是一种II型跨膜蛋白。研究表明，CLL1限制性地表达于造血细胞上，主要包括外周血和骨髓中髓系来源的细胞，比如单核细胞、树突状细胞、粒细胞以及大多数急性髓细胞白血病(AML)细胞。值得注意的是，虽然CLL1大量表达于外周血和骨髓中的髓细胞上，但是在外周组织中的髓系来源的细胞上不表达，比如组织巨噬细胞和组织树突状细胞均不表达CLL1。研究还发现，CLL1表达于AML干细胞(CD34+/CD38-)以及一小部分造血祖细胞(CD34+/CD38+或者CD34+/CD33+)上，但是在正常的造血干细胞(CD34+/CD38-或者CD34+/CD33-)上不表达。
由于这种特殊的表达模式，CLL1有望成为潜在的诊断和治疗AML的靶点。但目前鲜有靶向CLL1的免疫疗法报道。
发明内容
本申请提供了靶向CLL1嵌合抗原受体及其应用，所述靶向CLL1嵌合抗原受体采用对CLL1具有结合能力的抗CLL1抗体为抗原结合结构域，不仅可以结合纯化的或游离的CLL1蛋白，还可以结合细胞表面的CLL1蛋白，表达所述靶向CLL1嵌合抗原受体的免疫细胞在肿瘤治疗领域具有重要应用前景。
第一方面，本申请提供了靶向CLL1嵌合抗原受体，所述靶向CLL1嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；
其中所述抗原结合结构域为抗CLL1抗体。
本申请中，采用对CLL1具有结合能力的抗CLL1抗体作为嵌合抗原受体的抗原结合结构域，使得嵌合抗原受体可以特异性结合CLL1阳性肿瘤细胞，实现对CLL1阳性肿瘤的特异性靶向作用。
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列，其中SEQ ID NO:1和SEQ ID NO:2通过连接肽连接形成抗CLL1抗体19C1；其中
SEQ ID NO:1：
Figure PCTCN2020138257-appb-000001
SEQ ID NO:2：
Figure PCTCN2020138257-appb-000002
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列，其中SEQ ID NO:3和SEQ ID NO:4通过连接肽连接形成抗CLL1抗体23D7；其中
SEQ ID NO:3：
Figure PCTCN2020138257-appb-000003
SEQ ID NO:4：
Figure PCTCN2020138257-appb-000004
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，其中SEQ ID NO:5和SEQ ID NO:6通过连接肽连接形成抗CLL1抗体27H4；其中
SEQ ID NO:5：
Figure PCTCN2020138257-appb-000005
SEQ ID NO:6：
Figure PCTCN2020138257-appb-000006
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:7所示的氨基酸序列和SEQ ID NO:8～10之一所示的氨基酸序列，其中SEQ ID NO:7和SEQ ID NO:8通过连接肽连接形成抗CLL1抗体H27H4L1，SEQ ID NO:7和SEQ ID NO:9通过连接肽连接形成抗CLL1抗体H27H4L2，或者SEQ ID NO:7和SEQ ID NO:10通过连接肽连接形成抗CLL1抗体H27H4L3；其中
SEQ ID NO:7：
Figure PCTCN2020138257-appb-000007
SEQ ID NO:8：
Figure PCTCN2020138257-appb-000008
SEQ ID NO:9：
Figure PCTCN2020138257-appb-000009
SEQ ID NO:10：
Figure PCTCN2020138257-appb-000010
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列，其中SEQ ID NO:11和SEQ ID NO:12通过连接肽连接形成抗CLL1抗体1075.7；其中
SEQ ID NO:11：
Figure PCTCN2020138257-appb-000011
SEQ ID NO:12：
Figure PCTCN2020138257-appb-000012
优选地，所述铰链区包括CD8α铰链区。
优选地，所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区，优选为CD8α跨膜区。
优选地，所述信号传导结构域包括CD3ζ。
优选地，所述信号传导结构域还包括4-1BB、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
优选地，所述靶向CLL1嵌合抗原受体还包括信号肽。
优选地，所述信号肽包括CD8α信号肽和/或IgGκ轻链信号肽。
作为优选技术方案，所述靶向CLL1嵌合抗原受体包括信号肽、抗CLL1抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
在一些具体实施方案中，所述靶向CLL1嵌合抗原受体是19C1-CAR，其包括SEQ ID NO:13所示的氨基酸序列；其中
SEQ ID NO:13：
Figure PCTCN2020138257-appb-000013
在一些具体实施方案中，所述靶向CLL1嵌合抗原受体是23D7-CAR，其包括SEQ ID NO: 14所示的氨基酸序列；其中
SEQ ID NO:14：
Figure PCTCN2020138257-appb-000014
在一些具体实施方案中，所述靶向CLL1嵌合抗原受体是27H4-CAR，其包括SEQ ID NO:15所示的氨基酸序列；其中
SEQ ID NO:15：
Figure PCTCN2020138257-appb-000015
在一些具体实施方案中，所述靶向CLL1嵌合抗原受体是H27H4-CAR，其包括SEQ ID NO:16所示的氨基酸序列；其中
SEQ ID NO:16：
Figure PCTCN2020138257-appb-000016
在一些具体实施方案中，所述靶向CLL1嵌合抗原受体是1075.7-CAR，其包括SEQ ID NO:17所示的氨基酸序列；其中
SEQ ID NO:17：
Figure PCTCN2020138257-appb-000017
Figure PCTCN2020138257-appb-000018
第二方面，本申请提供了核酸分子，所述核酸分子包括第一方面所述的靶向CLL1嵌合抗原受体的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:18所示的核酸序列，为19C1-CAR的编码基因；
SEQ ID NO:18：
Figure PCTCN2020138257-appb-000019
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:19所示的核酸序列，为23D7-CAR的编码基因；
SEQ ID NO:19：
Figure PCTCN2020138257-appb-000020
Figure PCTCN2020138257-appb-000021
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:20所示的核酸序列，为27H4-CAR的编码基因；
SEQ ID NO:20：
Figure PCTCN2020138257-appb-000022
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:21所示的核酸序列，为H27H4-CAR的编码基因；
SEQ ID NO:21：
Figure PCTCN2020138257-appb-000023
Figure PCTCN2020138257-appb-000024
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:22所示的核酸序列，为1075.7-CAR的编码基因；
SEQ ID NO:22：
Figure PCTCN2020138257-appb-000025
第三方面，本申请提供了一种表达载体，所述表达载体包括第二方面所述的核酸分子。
优选地，所述表达载体为含有第二方面所述的核酸分子的病毒载体或非病毒载体。
优选地，所述病毒载体包括慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种。
优选地，所述非病毒载体包括Piggybac转座子系统、Sleeping Beauty转座子系统或纳米载体中的任意一种。
第四方面，本申请提供了一种重组慢病毒，所述重组慢病毒由转染有第三方面所述的表达载体和辅助质粒的哺乳细胞制备得到。
第五方面，本申请提供了一种嵌合抗原受体免疫细胞，所述嵌合抗原受体免疫细胞表达第一方面所述的靶向CLL1嵌合抗原受体。
本申请中，表达靶向CLL1嵌合抗原受体的免疫细胞利用嵌合抗原受体的抗原结合结构域靶向CLL1阳性肿瘤细胞，发挥免疫细胞的杀伤功能分泌细胞因子IFN-γ，在不同效靶比下实现对CLL1肿瘤的杀伤作用。
优选地，所述嵌合抗原受体免疫细胞的基因组中整合有第二方面所述的核酸分子。
优选地，所述嵌合抗原受体免疫细胞包括第三方面所述的表达载体和/或第四方面所述的重组慢病毒。
优选地，所述免疫细胞包括T细胞、NK细胞或巨噬细胞中的任意一种。
优选地，所述T细胞包括αβΤ细胞和/或γδΤ细胞。
第六方面，本申请提供了一种药物组合物，所述药物组合物包括第五方面所述的嵌合抗原受体免疫细胞。
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第七方面，本申请提供了第一方面所述的靶向CLL1嵌合抗原受体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组慢病毒、第五方面所述的嵌合抗原受体免疫细胞或第六方面所述的药物组合物在制备恶性肿瘤治疗药物中的应用。
优选地，所述恶性肿瘤包括急性髓系白血病。
第八方面，本申请提供了一种治疗癌症的方法，所述方法包括向患者施用有效剂量的第五方面所述的嵌合抗原受体免疫细胞或第六方面所述的药物组合物，并同时、分开或依次施用一种或多种抗肿瘤药物。
优选地，所述癌症包括急性髓系白血病。
与现有技术相比，本申请具有如下有益效果：
(1)本申请采用抗CLL1抗体作为抗原结合结构域构建CAR分子，表达靶向CLL1CAR的T细胞依赖于CAR元件在不同的效靶比下对CLL1阳性肿瘤细胞发挥杀伤作用，其中，H27H4-CAR-T具有最优的杀伤功能；
(2)本申请的靶向CLL1CAR-T细胞与CLL1阳性肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，证明了CAR-T对CLL1肿瘤细胞的特异性杀伤功效。
附图说明
图1为不同肿瘤细胞对CLL1的表达情况；
图2为靶向CLL1的嵌合抗原受体的结构示意图；
图3A为含有H27H4CAR基因的慢病毒表达载体图谱，图3B为H27H4CAR在慢病毒表达载体中的位置；
图4为不同CAR-T细胞的CAR表达阳性率；
图5为靶向CLL1的CAR-T对Raji-CLL1细胞的杀伤率；
图6为靶向CLL1的CAR-T杀伤靶细胞的IFN-γ分泌情况。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 抗CLL1抗体的来源
本实施例选择抗CLL1抗体19C1、23D7、27H4、人源化27H4(H27H4)和1075.7(专利US8536310B2)作为抗原结合结构域用于构建CAR分子，其中，19C1包括SEQ ID NO:1～2所示的可变区、23D7包括SEQ ID NO:3～4所示的可变区、27H4包括SEQ ID NO:5～6所示的可变区、H27H4包括SEQ ID NO:7～8所示的可变区、1075.7包括SEQ ID NO:11～12所示的可变区。
实施例2 肿瘤细胞对CLL1的表达
本实施例采用FITC anti-human CD371(CLL1)抗体(biolegend)与靶细胞Jurkat、KG-1a、共孵育后，流式检测靶细胞对CLL1的表达。
结果如图1所示，THP-1、U937和HL-60为CLL1阳性细胞，Jurkat和KG-1a为CLL1阴性细胞。
实施例3 嵌合抗原受体的设计
本实施例设计靶向CLL1的嵌合抗原受体，结构示意图见图2，包括CD8α信号肽、与CLL1抗原特异性结合的单链抗体(Anti-CLL1scFv)、CD8α铰链区(Hinge)和跨膜区(Transmembrane)、4-1BB共刺激结构域和CD3ζ信号传导结构域，具体CAR分子为：
①19C1-CAR：CD8α信号肽、Anti-CLL1scFv(19C1)、CD8αHinge+TM、4-1BB和CD3ζ；
②23D7-CAR：CD8α信号肽、Anti-CLL1scFv(23D7)、CD8αHinge+TM、4-1BB和CD3ζ；
③27H4-CAR：CD8α信号肽、Anti-CLL1scFv(27H4)、CD8αHinge+TM、4-1BB和CD3ζ；
④H27H4-CAR：IgGκ轻链信号肽、Anti-CLL1scFv(H27H4)、CD8αHinge+TM、4-1BB和CD3ζ；
⑤1075.7-CAR：CD8α信号肽、Anti-CLL1scFv(1075.7)、CD8αHinge+TM、4-1BB和CD3ζ；
其中，CD8α信号肽的氨基酸序列如SEQ ID NO:23所示，核酸序列如SEQ ID NO:24所示，IgGκ轻链信号肽的氨基酸序列如SEQ ID NO:25所示，核酸序列如SEQ ID NO:26所示，CD8αHinge的氨基酸序列如SEQ ID NO:27所示，核酸序列如SEQ ID NO:28所示，CD8αTM的氨基酸序列如SEQ ID NO:29所示，核酸序列如SEQ ID NO:30所示，4-1BB的氨基酸序列如SEQ ID NO:31所示，核酸序列如SEQ ID NO:32所示，CD3ζ的氨基酸序列如SEQ ID NO:33所示，核酸序列如SEQ ID NO:34所示；
SEQ ID NO:23：
Figure PCTCN2020138257-appb-000026
SEQ ID NO:24：
Figure PCTCN2020138257-appb-000027
SEQ ID NO:25：
Figure PCTCN2020138257-appb-000028
SEQ ID NO:26：
Figure PCTCN2020138257-appb-000029
SEQ ID NO:27：
Figure PCTCN2020138257-appb-000030
SEQ ID NO:28：
Figure PCTCN2020138257-appb-000031
SEQ ID NO:29：
Figure PCTCN2020138257-appb-000032
SEQ ID NO:30：
Figure PCTCN2020138257-appb-000033
SEQ ID NO:31：
Figure PCTCN2020138257-appb-000034
SEQ ID NO:32：
Figure PCTCN2020138257-appb-000035
SEQ ID NO:33：
Figure PCTCN2020138257-appb-000036
SEQ ID NO:34：
Figure PCTCN2020138257-appb-000037
实施例4 靶向CLL1的嵌合抗原受体表达载体的构建
(1)根据实施例3设计的CAR分子，对CAR编码基因进行密码子优化、促进其在人源细胞中的高效表达，全基因合成CAR编码基因(广州艾基生物技术有限公司)；
(2)用EcoRI和BamHI双酶切全长CAR基因和空载体pCDH-EF1-MCS，于37℃水浴酶切30min后，使用1.5％琼脂糖凝胶进行DNA电泳，并使用琼脂糖凝胶纯化回收试剂盒(天根)对酶切产物进行纯化回收处理；
(3)配制表1所示的连接体系，对CAR基因片段和线性化pCDH-EF1-MCS进行22℃连接1h，将连接产物直接转化Stbl3大肠杆菌感受态细胞，取200μL转化产物涂布氨苄抗性LB平板，于37℃培养箱倒置培养过夜，次日早晨随机挑选3个单克隆进行菌落PCR鉴定，并将阳性克隆进行测序鉴定；
表1
成分 添加量
线性化pCDH-EF1-MCS载体 50ng
CAR基因 150ng
T4 DNA连接缓冲液 2μL
T4 DNA连接酶(NEB) 1μL
ddH 2O 补齐至20μL
示例性地，构建的含有27H4 CAR基因的慢病毒表达载体pBG-27H4如图3A和图3B所示。
实施例5 慢病毒包装
本实施例采用四质粒系统对实施例4构建的慢病毒表达载体进行慢病毒包装，具体步骤如下：
(1)将慢病毒表达载体、辅助质粒gag/pol、Rev、VSV-G构成的四质粒系统与PEI转染试剂混合后，加至一定体积的无血清DMEM中，混匀放置15min；
(2)将上述混合液加至铺有293T细胞的T75细胞培养瓶中，轻轻混匀，于37℃、5％CO 2细胞培养箱中培养6h；
(3)6h后更换新鲜培养基，继续培养，并加入10mM丁酸钠溶液；72h后收集慢病毒培养上清进行纯化检测。
实施例6 T细胞的获取与扩增
每位志愿者采集30mL全血，将外周血与生理盐水按1:1混合稀释；向离心管中加入Ficoll淋巴细胞分离液，缓缓加入稀释的外周血，1500rpm离心30min，轻轻吸取PBMC层移入另一离心管中；
用生理盐水多次洗涤PBMC，转入X-VIVO培养基(含50ng/mL OKT3，300IU/mL IL-2)中进行培养，PBMC分离后，用X-VIVO(含50ng/mL OKT3，300IU/mL IL-2)进行激活，2日后将培养基更换为含300IU/mL的X-VIVO进行扩大培养；而后每两天进行一次计数，并更换新鲜的含300IU/mL的X-VIVO培养基，将细胞浓度维持在(0.5～1)×10 6个/mL，连续观察10天。
实施例7 CAR-T细胞的制备
本实施例利用RetroNectin提高慢病毒对T细胞的感染效率，步骤如下：
将30μg RetroNectin包被于6孔板内，置于37℃细胞培养箱保持2h；吸取RetroNectin，利用含2.5％BSA的Hank’s溶液封闭包被后的6孔板，置于37℃细胞培养箱保持0.5h；吸取 封闭液，利用含2％Hepes的Hank’s溶液洗涤6孔板，加入X-VIVO培养基，加入适量的慢病毒溶液，2000g离心2h，弃上清；加入1×10 6个T细胞(CD3阳性>90％)，1000g离心10min，置于37℃、5％CO 2和一定湿度的细胞培养箱内培养，第二日重复上述步骤。
慢病毒的加入量见表2。
表2
Figure PCTCN2020138257-appb-000038
采用流式细胞仪检测T细胞表面CAR分子的表达，利用CLL1-Fc融合蛋白(Acrobiosystems公司)检测CAR表达，二抗采用FITC-Labeled anti-human Fc，未转染CAR的T细胞(T mock)作为阴性对照。
结果如图4和表3所示，19C1-CAR-T细胞的CAR表达阳性率为13.28％，23D7-CAR-T细胞的CAR表达阳性率为37.49％，27H4-CAR-T细胞的CAR表达阳性率为39.44％，H27H4-CAR-T细胞的CAR表达阳性率为33.59％，1075.7-CAR-T细胞的CAR表达阳性率为5.09％。
表3
T细胞类型 T mock 19C1 23D7 27H4 H27H4 1075.7
CAR表达阳性率 1.23％ 13.28％ 37.49％ 39.44％ 33.59％ 5.09％
实施例8 CAR-T细胞的杀伤功能
本实施例采用xCELLigence实时无标记动态分析技术(Real Time Cell Analysis，RTCA)对细胞杀伤效应进行全程自动化检测。
Figure PCTCN2020138257-appb-000039
实时无标记细胞分析仪(RTCA)基于微电子阻抗技术，在E-plate板底整合有大量微金电极，当贴壁细胞粘附到微金电极上，细胞数量、直径以及贴壁能力均会影响微金电极间的电流传导，从而引起阻抗值的改变，这一改变极为精细且灵敏，在毒负效应下，细胞直接或间接影响阻抗值。因此，xCELLigence可以监测由分子靶标引起的细胞毒性效应。
步骤如下：
(1)用一定体积的1×Tether Buffer稀释anti-CD40，向E-Plate View 96孔板的每孔中加入50μL稀释好的anti-CD40作为包被液，室温避光孵育3h，每组设置三个复孔；
(2)弃包被液，用200μL PBS轻轻洗孔2次，向每个孔中加入50μL含2％FBS的1640培养基，将E-Plate View 96孔板放入xCELLigence仪中(仪器预先1h放入培养箱中)，37℃平衡1h，测量背景值；
(3)采用稳定过表达CLL1的Raji细胞(Raji-CLL1)作为靶细胞，制备靶细胞悬液并测定细胞密度，向每个孔中加入50000个细胞/50μL，每个孔的终体积为100μL，室温静置孵育30min，将E-Plate View 96孔板重新放回仪器中，操作软件进行数据收集，每5min检测一次电阻抗，检测时间为2h；
(4)分别制备不同效靶比的效应细胞悬液(即CAR-T细胞)和阴性对照细胞悬液(即未转染的T细胞)，向每孔中加入50μL稀释好的效应细胞悬液或阴性对照细胞悬液，空白对照组加入50μL培养基，阳性对照组加入50μL 1×Cytolysis Solution，室温静置孵育30min，使效应细胞均匀分布在固定的靶细胞上；
(5)将E-Plate View 96孔板重新放入仪器中，操作软件进行数据收集，每5min检测一次电阻抗，检测时间为16h，实验结束后保存数据并分析。
结果如图5所示，19C1-CAR-T、23D7-CAR-T、27H4-CAR-T、H27H4-CAR-T或1075.7-CAR-T在不同的效靶比(1:1、2:1、4:1)下与Raji-CLL1共孵育16h后，均能有效杀伤Raji-CLL1细胞，效靶比越大杀伤能力越强。其中，H27H4-CAR-T具有最强的杀伤能力，27H4-CAR-T的杀伤能力与H27H4-CAR-T相似，23D7-CAR-T次之，19C1-CAR-T和1075.7-CAR-T的杀伤能力偏弱，未转染CAR的T mock不能杀伤Raji-CLL1，说明anti-CLL1CAR-T的杀伤活性依赖于CAR元件。
实施例9 CAR-T细胞与肿瘤细胞共培养对IFN-γ的分泌
本实施例采用Human IFN-γELISA试剂盒(欣博盛)检测CAR-T细胞释放IFN-γ细胞因子的浓度，分析CAR-T与靶细胞共培养后对IFN-γ的分泌量，具体地，采用CLL1阳性细胞Raji-CLL1、HL60、U937作为阳性靶细胞，CLL1阴性细胞Raji作为阴性靶细胞。
将CAR-T细胞与不同的靶细胞按照效靶比为1:1共孵育16h，取上清用酶联免疫法(ELISA)检测培养液上清中IFN-γ的分泌量。检测原理采用双抗体夹心ELISA法，将抗人IFN-γ抗体包被于酶标板上，实验时样品或标准品中的人IFN-γ与包被抗体结合，游离的成分被洗去；依次加入生物素化抗人IFN-γ抗体和辣根过氧化物酶标记的亲和素，抗人IFN-γ抗体与结合在包被抗体上的人IFN-γ结合、生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去；加入显色底物(TMB)，在辣根过氧化物酶的催化下呈现蓝色，加入终止液后变成黄色，用酶标仪在450nm波长处测OD值，IFN-γ浓度与OD450之间呈正比，通过绘制标准曲线计算样品中IFN-γ的浓度。
结果如图6所示，19C1-CAR-T、23D7-CAR-T、27H4-CAR-T、H27H4-CAR-T或1075.7-CAR-T与Raji-CLL1、HL60或U937共培养后释放大量IFN-γ，而与Raji细胞共培养后未见大量IFN-γ释放，说明靶向CLL1的CAR-T细胞的杀伤具有特异性。
综上所述，本申请的靶向CLL1CAR-T细胞在不同的效靶比下对CLL1阳性肿瘤细胞具有显著的杀伤作用，与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，在CLL1阳性肿瘤治疗领域具有应用前景。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上 述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 靶向CLL1嵌合抗原受体，其包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；
    其中所述抗原结合结构域为抗CLL1抗体。
  2. 根据权利要求1所述的靶向CLL1嵌合抗原受体，其中，
    所述抗原结合结构域包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列；或者
    所述抗原结合结构域包括SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列；或者
    所述抗原结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列；或者
    所述抗原结合结构域包括SEQ ID NO:7所示的氨基酸序列和SEQ ID NO:8～10之一所示的氨基酸序列；或者
    所述抗原结合结构域包括SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列。
  3. 根据权利要求1或2所述的靶向CLL1嵌合抗原受体，其中，所述铰链区包括CD8α铰链区。
  4. 根据权利要求1-3任一项所述的靶向CLL1嵌合抗原受体，其中，所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区。
  5. 根据权利要求1-4任一项所述的靶向CLL1嵌合抗原受体，其中，所述信号传导结构域包括CD3ζ；
    优选地，所述信号传导结构域还包括4-1BB、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
  6. 根据权利要求1-5任一项所述的靶向CLL1嵌合抗原受体，其中，所述靶向CLL1嵌合抗原受体还包括信号肽；
    优选地，所述信号肽包括CD8α信号肽和/或IgGκ轻链信号肽。
  7. 根据权利要求1-6任一项所述的靶向CLL1嵌合抗原受体，其中，所述靶向CLL1嵌合抗原受体包括信号肽、抗CLL1抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
  8. 根据权利要求1-7任一项所述的靶向CLL1嵌合抗原受体，其中，
    所述靶向CLL1嵌合抗原受体包括SEQ ID NO:13所示的氨基酸序列；或者
    所述靶向CLL1嵌合抗原受体包括SEQ ID NO:14所示的氨基酸序列；或者
    所述靶向CLL1嵌合抗原受体包括SEQ ID NO:15所示的氨基酸序列；或者
    所述靶向CLL1嵌合抗原受体包括SEQ ID NO:16所示的氨基酸序列；或者
    所述靶向CLL1嵌合抗原受体包括SEQ ID NO:17所示的氨基酸序列。
  9. 核酸分子，其包括权利要求1-8任一项所述的靶向CLL1嵌合抗原受体的编码基因。
  10. 根据权利要求5所述的核酸分子，其中，
    所述核酸分子包括SEQ ID NO:18所示的核酸序列；或者
    所述核酸分子包括SEQ ID NO:19所示的核酸序列；或者
    所述核酸分子包括SEQ ID NO:20所示的核酸序列；或者
    所述核酸分子包括SEQ ID NO:21所示的核酸序列；或者
    所述核酸分子包括SEQ ID NO:22所示的核酸序列。
  11. 一种表达载体，其包括权利要求9或10所述的核酸分子；
    优选地，所述表达载体为含有权利要求9或10所述的核酸分子的病毒载体或非病毒载体；
    优选地，所述病毒载体包括慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种；
    优选地，所述非病毒载体包括Piggybac转座子系统、Sleeping Beauty转座子系统或纳米载体中的任意一种。
  12. 一种重组慢病毒，其中，所述重组慢病毒由转染有权利要求11所述的表达载体和辅助质粒的哺乳细胞制备得到。
  13. 一种嵌合抗原受体免疫细胞，其中，所述嵌合抗原受体免疫细胞表达权利要求1-8任一项所述的靶向CLL1嵌合抗原受体；
    优选地，所述嵌合抗原受体免疫细胞的基因组中整合有权利要求9或10所述的核酸分子；
    优选地，所述嵌合抗原受体免疫细胞包括权利要求11所述的表达载体和/或权利要求12所述的重组慢病毒；
    优选地，所述免疫细胞包括T细胞、NK细胞或巨噬细胞中的任意一种；
    优选地，所述T细胞包括αβΤ细胞和/或γδΤ细胞。
  14. 一种药物组合物，其包括权利要求13所述的嵌合抗原受体免疫细胞；
    任选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
  15. 权利要求1-8任一项所述的靶向CLL1嵌合抗原受体、权利要求9或10所述的核酸分子、权利要求11所述的表达载体、权利要求12所述的重组慢病毒、权利要求13所述的嵌合抗原受体免疫细胞或权利要求14所述的药物组合物在制备恶性肿瘤治疗药物中的应用；
    优选地，所述恶性肿瘤包括急性髓系白血病。
PCT/CN2020/138257 2020-12-11 2020-12-22 靶向cll1嵌合抗原受体及其应用 WO2022120942A1 (zh)

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AU2020480789A AU2020480789A1 (en) 2020-12-11 2020-12-22 CLL1-targeting chimeric antigen receptor and application thereof
KR1020227011658A KR20220084040A (ko) 2020-12-11 2020-12-22 Cll1을 표적으로 하는 키메라 항원 수용체 및 그의 응용
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CN115806625B (zh) * 2021-09-15 2023-08-04 广州百暨基因科技有限公司 T细胞限定表达的嵌合抗原受体及其应用
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