WO2022127401A1 - 靶向cd123的嵌合抗原受体及其用途 - Google Patents

靶向cd123的嵌合抗原受体及其用途 Download PDF

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WO2022127401A1
WO2022127401A1 PCT/CN2021/127505 CN2021127505W WO2022127401A1 WO 2022127401 A1 WO2022127401 A1 WO 2022127401A1 CN 2021127505 W CN2021127505 W CN 2021127505W WO 2022127401 A1 WO2022127401 A1 WO 2022127401A1
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chain variable
variable domain
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amino acid
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何霆
齐菲菲
鲁薪安
丁艳萍
梁梦梦
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Shandong Jinsai Co Ltd
Beijing Immunochina Pharmaceuticals Co Ltd
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Shandong Jinsai Co Ltd
Beijing Immunochina Pharmaceuticals Co Ltd
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Priority to JP2023537626A priority patent/JP7610234B2/ja
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Definitions

  • the present invention relates to scFv sequences targeting CD123 with different antigen recognition domains, chimeric antigen receptor (CAR) and CAR-T cells designed based on these scFv sequences, and their use in the treatment of CD123-expressing tumors, especially acute myeloid The use of leukemia.
  • CAR chimeric antigen receptor
  • AML Acute myeloid leukemia
  • AHCT allogeneic hematopoietic cell transplantation
  • CAR-T cells chimeric antigen receptor T cells
  • the molecular structure of chimeric antigen receptor (CAR) mainly includes antibody single-chain variable fragment (scFv), intracellular signaling domain, hinge region and transmembrane region connecting scFv and intracellular signaling domain.
  • scFv antibody single-chain variable fragment
  • CAR-T cells anti-CD19 and anti-CD20 chimeric antigen receptor T cells
  • the CAR-T targets for AML treatment include CD123, CD33, CLL1, FLT3, etc.
  • CAR-T targeting CD123 is the most studied.
  • CD123 is the IL-3 receptor alpha chain (IL-3Ra).
  • CD123 and CD131 together constitute a high-affinity IL-3 receptor, and after binding to IL-3, it can promote cell proliferation and differentiation, while inhibiting hematopoietic cell apoptosis [9, 10].
  • the high expression of CD123 is associated with the activation of signal transducer and activator of transcription 5 (STAT5) signaling pathway. STAT5 activation pathway is involved in a variety of hematological malignancies [11].
  • STAT5 activation pathway is involved in a variety of hematological malignancies [11].
  • CD123 is mainly expressed in the hematopoietic system, and CD123 is low or not expressed in normal CD34+CD38- cells [12].
  • CD123 is highly expressed in myeloid progenitor cells and B lymphoid progenitor cells, while low or no expression is found in erythroid progenitor cells and progenitor cells with multi-directional differentiation potential [13, 14]. CD123 is highly expressed in more than 60%-90% of AML blasts [15-18]. Bras AE et al [19] examined 455 AML patients, of which about 55% of the patients' AML cells expressed CD123, and the positive rate was over 80%.
  • CAR-T cells have achieved good results in tumor cell therapy, there are still major problems in the efficacy and safety of CAR-T, mainly including off-target effects, cytokine storm, and neurotoxicity [20].
  • the optimal efficacy and safety of CAR-T cells depend on many factors, among which the effective activation of CAR-T cells plays an important role.
  • the degree of activation of CAR-T cells is affected by various factors such as the affinity of the CAR molecule scFv, the length of the hinge region, the costimulatory signaling domain, and the density of target cell surface antigens.
  • the scFv of the CAR molecule acts as a region that directly binds to the target cell surface antigen, and its affinity is of great significance to the activation of the overall CAR-T cell.
  • the scFv affinity of the CAR molecule is low, the CAR-T cells cannot obtain sufficient antigen recognition, so that the optimal activation state cannot be achieved; when the scFv affinity of the CAR molecule is high, the CAR-T cells may recognize tumor cells as well as Normal cells with relatively low antigen density produce severe off-target effects.
  • the present invention provides an antibody that specifically binds to CD123, comprising a heavy chain variable domain VH and a light chain variable domain VL, the heavy chain variable domain comprising SEQ ID NO: 1 ( VH-CDR1 amino acid sequence of GYX 1 X 2 X 3 X 4 (D) YX 5 X 6 X 7 ), SEQ ID NO: 2 (X 8 X 9 X 10 (X 11 ) X 12 X 13 X 14 X 15 X 16 X 17 X 18 NX 19 X 20 X 21 KX 22 X 23 X 24 ) VH-CDR2 amino acid sequence and SEQ ID NO: 3 (AX 25 X 26 X 27 X 28 X 29 X 30 X 31 (YD) X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 ) the VH-CDR3 amino acid sequence; wherein X 1 is selected from S, T; X 2 is selected from F
  • the present invention provides a CD123-targeting chimeric antigen receptor, comprising: the antibody of the first aspect; a hinge region; a transmembrane domain; a costimulatory domain; and a CD3 ⁇ intracellular signal transduction guide domain.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the first aspect or the chimeric antigen receptor of the second aspect.
  • the present invention provides a vector comprising the nucleic acid molecule of the third aspect.
  • the present invention provides a cell transfected with the vector of the fourth aspect, thereby comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first aspect, the chimeric antigen receptor of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect or the cell of the fifth aspect , and a pharmaceutically acceptable carrier.
  • the present invention provides the antibody of the first aspect, the chimeric antigen receptor of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect or the cell of the fifth aspect for preparing and treating tumors expressing CD123 the use of drugs.
  • the present invention obtains the optimal scFv sequence of the target CD123 by performing affinity screening on the scFv of the CAR molecule of the target CD123 and comparison of the killing effect of the CAR-T cell, so that the CAR-T cell has better safety and efficacy.
  • the present invention can effectively improve the killing efficiency of CAR-T cells targeting CD123 to tumor cell lines and primary tumor cells, and improve the cell proliferation ability.
  • Figure 1 shows the transduction efficiencies of CD123 CAR molecules of different scFvs in T cells.
  • Figure 2 shows the total cell proliferation of CD123 CAR-T during culture.
  • Figure 3 shows the effects of target cells AML primary cells and THP-1 cells on the differentiation of CD123 CAR-T cells, respectively.
  • Figure 4 shows the killing efficiency of CD123 CAR-T on CD123 positive cell line (THP-1) and CD123 negative cell line (K562).
  • Figure 5 shows the killing efficiency of CD123 CAR-T on AML primary cells.
  • Figure 6 shows the cytokine expression of CD123 CAR-T in AML primary cells after 5 hours of stimulation.
  • the present invention provides an antibody that specifically binds to CD123, comprising a heavy chain variable domain VH and a light chain variable domain VL, the heavy chain variable domain comprising VH-CDR1, VH-CDR2 and VH- CDR3, the light chain variable domain comprises VL-CDR1, VL-CDR2 and VL-CDR3.
  • the VH-CDR1 is selected from the amino acid sequence of the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12; the VH-CDR2 is selected from the group consisting of SEQ ID NOs: 14, 15, The amino acid sequence in the group that 16,17,18 is formed; Described VH-CDR3 is selected from the amino acid sequence in the group that is formed by SEQ ID NO:20,21,22,23,24; Described VL-CDR1 is selected from SEQ ID The amino acid sequence in the group consisting of NO: 26, 27, 28, 29, 30; the VL-CDR2 is selected from the amino acid sequence in the group consisting of SEQ ID NO: 32, 33, 34, 35, 36; The VL - CDR3 amino acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42.
  • antibodies of the present invention that specifically bind to CD123 include:
  • VH-CDR1 as shown in SEQ ID NO:8, VH-CDR2 as shown in SEQ ID NO:14 and VH-CDR3 as shown in SEQ ID NO:20; and A light chain variable domain comprising VL-CDR1 as set forth in SEQ ID NO:26, VL-CDR2 as set forth in SEQ ID NO:32 and VL-CDR3 as set forth in SEQ ID NO:38; and/or
  • VH-CDR1 as shown in SEQ ID NO:9
  • VH-CDR2 as shown in SEQ ID NO:15
  • VH-CDR3 as shown in SEQ ID NO:21
  • a light chain variable domain comprising VL-CDR1 as set forth in SEQ ID NO:27, VL-CDR2 as set forth in SEQ ID NO:33, and VL-CDR3 as set forth in SEQ ID NO:39;
  • VH-CDR1 as shown in SEQ ID NO:10
  • VH-CDR2 as shown in SEQ ID NO:16
  • VH-CDR3 as shown in SEQ ID NO:22
  • a light chain variable domain comprising VL-CDR1 as set forth in SEQ ID NO:28, VL-CDR2 as set forth in SEQ ID NO:34, and VL-CDR3 as set forth in SEQ ID NO:40;
  • VH-CDR1 as shown in SEQ ID NO:11
  • VH-CDR2 as shown in SEQ ID NO:17
  • VH-CDR3 as shown in SEQ ID NO:23
  • a light chain variable domain comprising VL-CDR1 as set forth in SEQ ID NO:29, VL-CDR2 as set forth in SEQ ID NO:35, and VL-CDR3 as set forth in SEQ ID NO:41;
  • VH-CDR1 as shown in SEQ ID NO:12
  • VH-CDR2 as shown in SEQ ID NO:18
  • VH-CDR3 as shown in SEQ ID NO:24
  • a light chain variable domain comprising VL-CDR1 as set forth in SEQ ID NO:30, VL-CDR2 as set forth in SEQ ID NO:36, and VL-CDR3 as set forth in SEQ ID NO:42.
  • said heavy chain variable domain in an antibody of the invention comprises or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47 and 48 an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 99% identity; and/or the light chain variable domain comprises a group selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53 and 54 An amino acid sequence in a group, or an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 99% identity thereto.
  • the present invention provides an antibody that specifically binds to CD123, comprising:
  • the present invention provides an antibody that specifically binds CD123, comprising: an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 59, 60, and 61, or An amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99% identical to it.
  • the antibody of the present invention is a single chain antibody scFv; more preferably, the heavy chain variable domain and light chain variable domain of the single chain antibody scFv are connected by a linker peptide chain rich in glycine and serine, And the order of the heavy chain variable domains and the light chain variable domains may be interchanged.
  • the present invention also provides a chimeric antigen receptor targeting CD123, comprising: the antibody of the present invention; a hinge region; a transmembrane domain; a costimulatory domain; and a CD3 ⁇ intracellular signal transduction domain.
  • the costimulatory domain is selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, B7-H3 or any combination thereof; preferably, the costimulatory domain is selected from 4-1BB or CD28, the 4-1BB comprising the amino acid sequence as set forth in SEQ ID NO:62.
  • the CD3 ⁇ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:63.
  • the hinge region and transmembrane domain are selected from the group consisting of IgG1, IgG4, CD8 ⁇ , CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD34 Hinge and transmembrane domains.
  • the chimeric antigen receptor comprises, or has at least 85%, at least 90%, at least 95% amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 66, 67, 68, 69 and 70. %, amino acid sequences that are at least 99% identical.
  • the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or chimeric antigen receptor of the present invention.
  • the present invention also provides a vector comprising the nucleic acid molecule of the present invention; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
  • the present invention also provides a cell transfected with the vector of the present invention, thereby comprising the nucleic acid molecule or the vector of the present invention; preferably, the cell is a T cell.
  • the present invention also provides the use of the antibody, chimeric antigen receptor, nucleic acid molecule, vector or cell of the present invention for preparing a medicament for treating a tumor expressing CD123; preferably, the tumor expressing CD123 is acute myeloid leukemia ( AML).
  • AML acute myeloid leukemia
  • Example 1 CD123 CAR molecular design and CAR-T preparation
  • This example takes the design of CD123 CAR molecules with different antigen recognition domains and the preparation of CAR-T cells as an example.
  • CAR-T-1 is a control group, and the scFv sequence of CD123 is reported from the University of Pennsylvania [21], which is named scFv-01 in this paper.
  • CAR-T-2-7 are modified groups, and their scFv is derived from the monoclonal antibody sequence obtained by immunizing mouse CD123 extracellular region fusion protein, which is named scFv-02-07 in this paper.
  • the nucleotide sequences of CD123 scFv with different antigen recognition domains were synthesized by gene synthesis, and then connected with CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ intracellular region sequences by homologous recombination method to form a complete CAR molecules, namely CAR 1-7.
  • the CAR nucleic acid fragment was constructed into lentiviral transfer plasmid pLenti6.3/V5 (Thermo Fisher, Waltham, MA, USA) by restriction endonuclease digestion and ligase ligation.
  • the lentiviral packaging plasmids pLP/VSVG, pLP1/MDK, pLP2/RSK (Thermo Fisher, Waltham, MA, USA) and the transfer plasmid obtained above were transfected into HEK293T with Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA)
  • the medium was collected after 48 hours, centrifuged at 300 g to remove cell debris, and centrifuged at 25,000 rpm for 3 hours with an ultracentrifuge. Dissolve the pellet with 1 mL of physiological saline, which is the desired lentiviral vector.
  • the cells in the culture system were repeatedly pipetted with a pipette, collected into a centrifuge tube, and placed on a magnetic stand to remove CD3/CD28 beads.
  • the T cells were centrifuged and counted, and some cells were taken using a flow cytometer (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA) to detect the transduction efficiency of CAR molecules in each group of cells.
  • Figure 1 shows the transduction efficiency of CAR molecules in each group of cells after 5 days, 7 days, 9 days and 12 days of lentivirus infection of T cells.
  • the CAR molecule transduction efficiency in the CAR-T-2, CAR-T-3, and CAR-T-7 groups was similar to that in the CAR-T-4, CAR-T-5, CAR-T-4, CAR-T-5,
  • the CAR-T-6 group was significantly higher than the control group, indicating that the CAR protein expression of CAR-T cells in each group was better.
  • This example takes the detection of the proliferation ability of CD123 CAR-T with different antigen recognition domains as an example.
  • lentivirus-infected T cells were used as the second day of culture. After that, CAR-T cells were collected and centrifuged every 2 to 3 days, and 1-2 ml of culture system was added to resuspend them. After 10 microliters of cells were diluted to a certain number, they were stained with trypan blue (Solarbio, Cat#C0040), and the total number of living cells and the total number of dead cells were counted under an inverted microscope (Ts2-FL, Nikon, Japan). cell proliferation.
  • Figure 2 shows that the total cell proliferation of CAR-T-2 and CAR-T-7 with different antigen recognition domains was significantly increased compared with the control group CAR-T-1. It shows that the scFv design of CAR-T-2 and CAR-T-7 can improve the proliferation ability of CD123 CAR-T cells.
  • Example 3 Cell differentiation of modified CD123 CAR-T under stimulation of target cells
  • This example takes the detection of CD123 CAR-T with different antigen recognition domains, and its T cell differentiation under the stimulation of two target cells, AML primary cells or THP-1 cells (both expressing CD123), as an example.
  • the CD45RA + CD62L + cell population represents the combination of the naive T cell population and the stem memory T cell population
  • the CD45RA + CD62L ⁇ cell population is the effector T cell population.
  • the proportion of CD45RA + CD62L + cell population in CAR-T-2 and CAR-T-7 with different antigen recognition domains is higher than that in control CAR-T-1, and the proportion of CD45RA + CD62L - cell population is lower than that in control CAR -T-1. It shows that the modified CD123 antigen recognition domain has better CAR-T cell persistence.
  • Example 4 In vitro killing efficiency of modified CD123 CAR-T on cell lines
  • This example takes the detection of the killing efficiency of CD123 CAR-T with different antigen recognition domains on tumor cell lines expressing CD123 and not expressing CD123 as an example.
  • the cell line THP-1 expressing CD123 and the cell line K562 not expressing CD123 were collected and centrifuged respectively, resuspended in 1 ml of normal saline (Hebei Tiancheng Pharmaceutical Co., Ltd., China), and added 5 ⁇ l of Calcein-AM (concentration 1 ⁇ g) / ⁇ L, Cat#C3100MP, ThermoFisher, USA), mix gently, and incubate in a 37°C incubator for 30 minutes to label target cells. After incubation, wash twice with normal saline, add X-VIVO 15 to resuspend and count.
  • Figure 4 shows that for the CD123 positive cell line THP-1, the killing efficiency of CAR-T-3 and CAR-T-5 in CD123 CAR-T with different antigen recognition domains was significantly higher than that of the control group CAR-T-1 .
  • the killing efficiency of CD123 CAR-T in each group and the control group CAR-T-1 on the K562 cell line that does not express CD123 was the same as that in the T cell group. It shows that each group of CD123 CAR-T cells has a certain killing effect on target cells, and has good specificity, and the scFv design of CAR-T-3 and CAR-T-5 is more conducive to CAR-T killing tumor cells.
  • Example 5 In vitro killing efficiency of modified CD123 CAR-T on AML primary cells
  • This example takes the detection of the killing efficiency of CD123 CAR-T with different antigen recognition domains on AML primary cells as an example.
  • high antigen density ie AML 1#, CD123 expression rate of 99.87%, MFI of 13298
  • medium antigen density ie AML 2#, CD123 expression rate of 75.4%, MFI of 7518
  • low antigen density ie AML 3#, CD123 expression rate of 94.58%, MFI of 5252
  • bone marrow blood samples from CD123-positive AML patients
  • the antigen density level is defined as follows, high antigen density: Median Fluorescence Intensity (MFI) greater than 10000; Antigen density: MFI greater than 6000 and less than 10000; low antigen density: MFI less than 6000), mononuclear cells were isolated from bone marrow blood samples of AML patients, and analyzed using CD34 MicroBeads (Cat#130-100-453, Miltenyi, Germany).
  • Triton-X-100 was added to lyse the positive control group, that is, 1 ⁇ 10 5 primary AML cells labeled above; Fluorescence values (excitation wavelength: 495 nm, emission wavelength: 515 nm) were detected using a multi-plate reader (Varioscan Lux, ThermoFisher).
  • Figure 5 shows that compared with the control group CAR-T-1, CAR-T-3 with different antigen recognition domains showed that 3 high (i.e. AML 1#), medium (i.e. AML 2#), low (i.e. AML 3 #) Primary cells from CD123-positive AML patients with antigen density all had significantly enhanced killing efficiency. It shows that CD123 CAR-T cells with CAR-T-3 scFv can effectively improve the killing efficiency of primary cells.
  • This example takes the detection of cytokine expression levels of CD123 CAR-T with different antigen recognition domains under the stimulation of target cells AML primary cells as an example.
  • Figure 6 shows that compared with the control group CAR-T-1, the CAR-T-6 group with different antigen recognition domains significantly increased the expression levels of IL-2, TNF, and IFN- ⁇ under the stimulation of target cells; the rest The cytokine expression of CAR-T cells in each group was similar to that of CAR-T-1 in the control group. It shows that the scFv design of CAR-T-6 is beneficial to the expression of CD123 CAR-T cytokines.
  • Tables 1 and 2 are the specific amino acid sequences involved in the present invention.
  • interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia, 2000, 14(10), 1777–1784.

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Abstract

本发明提供了特异性结合CD123的单链抗体scFv，其包含选自SEQ ID NO：56、57、58、59、60和61的氨基酸序列。还提供了靶向CD123的嵌合抗原受体，其包含选自SEQ ID NO：65、66、67、68、69和70的氨基酸序列。还提供了编码所述scFv或嵌合抗原受体的核酸分子，包含所述核酸分子的载体和细胞，以及所述抗体、嵌合抗原受体、核酸分子、载体或细胞用于制备治疗表达CD123的肿瘤的药物的用途。

Description

靶向CD123的嵌合抗原受体及其用途 技术领域
本发明涉及具有不同抗原识别域的靶向CD123的scFv序列，基于这些scFv序列设计的嵌合抗原受体(CAR)和CAR-T细胞，以及其用于治疗表达CD123的肿瘤、特别是急性髓系白血病的用途。
背景技术
急性髓系白血病(AML)是一种发病率不断上升的恶性肿瘤，主要影响中位年龄为66岁的老年人。美国国家癌症研究所(National Cancer Institute)估计，2018年的新病例将达到19520例，而其中可能会有10670人死于这种疾病[1]。尽管65-74岁的患者在过去连续三十年中，12个月生存率从20％提高到30％，但目前AML的治疗效果仍不令人满意，这些患者的5年生存率＜5％[2]。对于较年轻的AML患者，主要的治疗方法是强化化疗，对于复发风险较高的患者(治愈率35-40％)，采用异基因造血细胞移植(AHCT)，而老年患者的预后仍然很差[2]。复发的AML患者获得第二次缓解的几率小于50％，而在第二次抢救性化疗后，只有16％的患者能实现病情控制[3]。自20世纪70年代引入蒽环类和阿糖胞苷类联合化疗以来，它一直是主要的治疗方法[4，5]。因此，对于此疾病，需要新的治疗策略。
嵌合抗原受体T细胞(CAR-T细胞)的概念首先提出于20世纪80年代后期，其结合了抗体特异性和T细胞杀伤效应，形成过继性免疫效应[6，7]。嵌合抗原受体(CAR)分子结构主要包括抗体单链可变片段(scFv)、胞内信号域及连接scFv与胞内信号域的铰链区和跨膜区。近年来，利用抗CD19和抗CD20嵌合抗原受体T细胞(CAR-T细胞)治疗淋巴样恶性肿瘤(B细胞急性淋巴细胞白血病和非霍奇金淋巴瘤)取得了良好的效果[8]。目前，针对AML治疗的CAR-T靶点，有CD123、CD33、CLL1、FLT3等；其中，靶向CD123的CAR-T研究最多。
CD123是IL-3受体a链(IL-3Ra)。CD123与CD131一起组成高亲和力的IL-3受体，与IL-3结合后，能够促进细胞增殖分化，同时抑制造血细胞凋亡[9，10]。CD123的高表达与信号转导及转录激活因子5(STAT5)信号通路的活化相关。STAT5激活通路参与在多种恶性血液病中[11]。CD123主要在造血系统中表达，CD123在正常CD34+CD38-细胞中低表达或不表达[12]。此外，正常情况下在髓系祖细胞、B淋巴祖细胞中CD123有较高表达，而红系祖细胞和多向分化潜能祖细胞中低 表达或不表达[13，14]。CD123在60％-90％以上的AML原始细胞中高表达[15-18]。Bras AE等人[19]检测了455例AML患者，其中约55％的患者AML细胞表达CD123，且阳性率达80％以上。
虽然，CAR-T细胞在肿瘤细胞治疗中已取得了较好效果，但是CAR-T的疗效和安全性依然存在较大问题，主要有脱靶效应、细胞因子风暴、神经毒性等[20]。CAR-T细胞的最佳疗效和安全性，取决于多方面因素，其中CAR-T细胞的有效活化具有重要作用。CAR-T细胞的活化程度受CAR分子scFv的亲和力、铰链区长度、共刺激信号域、靶细胞表面抗原密度等多种因素影响。CAR分子的scFv作为与靶细胞表面抗原直接结合的区域，其亲和力对整体CAR-T细胞的活化意义重大。CAR分子scFv亲和力较低时，CAR-T细胞不能得到足够的抗原识别，从而无法达到最佳激活状态；CAR分子scFv亲和力较高时，CAR-T细胞可能会在识别肿瘤细胞的同时也能够识别抗原密度相对较低的正常细胞，产生严重脱靶效应。
因此，为了进一步提高CAR-T细胞的安全性和有效性，保证CAR-T细胞的有效活化，避免脱靶效应，仍需要筛选针对靶点具有适中亲和力的scFv。
发明内容
第一方面，本发明提供了一种特异性结合CD123的抗体，其包括重链可变结构域VH和轻链可变结构域VL，所述重链可变结构域包含SEQ ID NO：1(GYX 1X 2X 3X 4(D)YX 5X 6X 7)的VH-CDR1氨基酸序列，SEQ ID NO：2(X 8X 9X 10(X 11)X 12X 13X 14X 15X 16X 17X 18NX 19X 20X 21KX 22X 23X 24)的VH-CDR2氨基酸序列和SEQ ID NO：3(AX 25X 26X 27X 28X 29X 30X 31(YD)X 32X 33X 34X 35X 36X 37X 38X 39X 40)的VH-CDR3氨基酸序列；其中X 1选自S、T；X 2选自F、I；X 3选自T、S、M；X 4选自S、D、T、G；X 5选自W、Y、V、A、N；X 6选自M、V、I、W；X 7选自N、H；X 8选自Y、R；X 9选自I、C；X 10选自D、N、Y、S；X 11选自P、C；X 12选自Y、G；X 13选自D、N、S；X 14选自S、N、D、G；X 15选自E、A、G、S；X 16选自T、I、S；X 17选自H、S、N；X 18选自Y、S；X 19选自Q、E、P；X 20选自K、N、S；X 21选自F、L；X 22选自D、G、S；X 23选自K、R；X 24选自A、I；X 25选自R、G；X 26选自G、S、E；X 27选自E、P、R；X 28选自G、S；X 29选自N、Y、L、W；X 30选自W、Y、L；X 31选自G、D；X 32选自R、E；X 33选自Y、S、T；X 34选自D、Y、G；X 35选自G、Y；X 36选自Y、L；X 37选自A、 G、P；X 38选自M、L；X 39选自D、A；X 40选自Y、C；所述轻链可变结构域包含SEQ ID NO：4(X 41SX 42X 43X 44X 45X 46X 47X 48X 49(QK)X 50X 51X 52X 53W)的VL-CDR1氨基酸序列，SEQ ID NO：5(X 54SX 55X 56X 57X 58X 59)的VL-CDR2氨基酸序列和SEQ ID NO：6(X 60X 61X 62X 63X 64(P)X 65TF)的VL-CDR3氨基酸序列；其中X 41选自A、S；X 42选自Q、K、S；X 43选自S、D；X 44选自I、V、L；X 45选自S、N、D、L；X 46选自K、Y、S、D、N；X 47选自D、S；X 48选自G、S；X 49选自D、N；X 50选自S、N；X 51选自D、Y；X 52选自L、I、M；X 53选自A、N、H；X 54选自G、T、A；X 55选自T、N、Q；X 56选自L、S、R；X 57选自Q、E、A、I、D；X 58选自S、P；X 59选自G、E；X 60选自Q、N；X 61选自H、Y、S、F、G；X 62选自N、D、H、Y；X 63选自K、L、E、R、S；X 64选自Y、L、D、S、F、T；X 65选自Y、R、W；优选地，所述抗体是scFv。
第二方面，本发明提供了一种靶向CD123的嵌合抗原受体，其包含：第一方面所述的抗体；铰链区；跨膜结构域；共刺激结构域；和CD3ζ胞内信号转导结构域。
第三方面，本发明提供了一种核酸分子，其包含编码第一方面的抗体或第二方面的嵌合抗原受体的核苷酸序列。
第四方面，本发明提供了一种载体，其包含第三方面的核酸分子。
第五方面，本发明提供了一种细胞，其经第四方面的载体转染，从而包含第三方面的核酸分子或第四方面的载体。
第六方面，本发明提供了一种药物组合物，其包含第一方面的抗体、第二方面的嵌合抗原受体、第三方面的核酸分子、第四方面的载体或第五方面的细胞，以及可药用载体。
第七方面，本发明提供了第一方面的抗体、第二方面的嵌合抗原受体、第三方面的核酸分子、第四方面的载体或第五方面的细胞用于制备治疗表达CD123的肿瘤的药物的用途。
本发明通过对靶点CD123的CAR分子的scFv进行亲和力筛选以及CAR-T细胞杀伤效应比较，以获得靶点CD123的最佳scFv序列，使得CAR-T细胞具有更优的安全性和有效性。与现有技术相比，本发明能有效提高靶向CD123的CAR-T细胞对肿瘤细胞系及原代肿瘤细胞的杀伤效率，提高细胞增殖能力。
附图说明
图1表示不同scFv的CD123 CAR分子在T细胞中的转导效率。
图2表示CD123 CAR-T在培养过程中的总细胞增殖情况。
图3表示靶细胞AML原代细胞和THP-1细胞分别刺激下，对CD123 CAR-T细胞分化的影响。
图4表示CD123 CAR-T对CD123阳性细胞系(THP-1)及CD123阴性细胞系(K562)的杀伤效率。
图5表示CD123 CAR-T对AML原代细胞的杀伤效率。
图6表示CD123 CAR-T在AML原代细胞刺激5小时后的细胞因子表达。
具体实施方式
本发明提供了一种特异性结合CD123的抗体，其包括重链可变结构域VH和轻链可变结构域VL，所述重链可变结构域包含VH-CDR1、VH-CDR2和VH-CDR3，所述轻链可变结构域包含VL-CDR1、VL-CDR2和VL-CDR3。
在一个具体实施方案中，所述VH-CDR1选自由SEQ ID NO：8、9、10、11、12组成的组中的氨基酸序列；所述VH-CDR2选自由SEQ ID NO：14、15、16、17、18组成的组中的氨基酸序列；所述VH-CDR3选自由SEQ ID NO：20、21、22、23、24组成的组中的氨基酸序列；所述VL-CDR1选自由SEQ ID NO：26、27、28、29、30组成的组中的氨基酸序列；所述VL-CDR2选自由SEQ ID NO：32、33、34、35、36组成的组中的氨基酸序列；所述VL-CDR3选自由SEQ ID NO：38、39、40、41、42组成的组中的氨基酸序列。
特别地，本发明的特异性结合CD123的抗体包括：
1)包含如SEQ ID NO：8所示的VH-CDR1、如SEQ ID NO：14所示的VH-CDR2和如SEQ ID NO：20所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：26所示的VL-CDR1、如SEQ ID NO：32所示的VL-CDR2和如SEQ ID NO：38所示的VL-CDR3的轻链可变结构域；和/或
2)包含如SEQ ID NO：9所示的VH-CDR1、如SEQ ID NO：15所示的VH-CDR2和如SEQ ID NO：21所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：27所示的VL-CDR1、如SEQ ID NO：33所示的VL-CDR2和如SEQ ID NO：39所示的VL-CDR3的轻链可变结构域；和/或
3)包含如SEQ ID NO：10所示的VH-CDR1、如SEQ ID NO：16所示的VH-CDR2和如SEQ ID NO：22所示的VH-CDR3的重链可变结构域； 和包含如SEQ ID NO：28所示的VL-CDR1、如SEQ ID NO：34所示的VL-CDR2和如SEQ ID NO：40所示的VL-CDR3的轻链可变结构域；和/或
4)包含如SEQ ID NO：11所示的VH-CDR1、如SEQ ID NO：17所示的VH-CDR2和如SEQ ID NO：23所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：29所示的VL-CDR1、如SEQ ID NO：35所示的VL-CDR2和如SEQ ID NO：41所示的VL-CDR3的轻链可变结构域；和/或
5)包含如SEQ ID NO：12所示的VH-CDR1、如SEQ ID NO：18所示的VH-CDR2和如SEQ ID NO：24所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：30所示的VL-CDR1、如SEQ ID NO：36所示的VL-CDR2和如SEQ ID NO：42所示的VL-CDR3的轻链可变结构域。
在另一个具体实施方案中，本发明的抗体中的所述重链可变结构域包含选自由SEQ ID NO：44、45、46、47和48组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列；和/或所述轻链可变结构域包含选自由SEQ ID NO：50、51、52、53和54组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
特别地，本发明提供了一种特异性结合CD123的抗体，其包含：
1)如SEQ ID NO：44所示的重链可变结构域和如SEQ ID NO：50所示的轻链可变结构域；和/或
2)如SEQ ID NO：45所示的重链可变结构域和如SEQ ID NO：51所示的轻链可变结构域；和/或
3)如SEQ ID NO：46所示的重链可变结构域和如SEQ ID NO：52所示的轻链可变结构域；和/或
4)如SEQ ID NO：47所示的重链可变结构域和如SEQ ID NO：53所示的轻链可变结构域；和/或
5)如SEQ ID NO：48所示的重链可变结构域和如SEQ ID NO：54所示的轻链可变结构域；
在另一个具体实施方案中，本发明提供了一种特异性结合CD123的抗体，其包含：选自由SEQ ID NO：56、57、58、59、60和61组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
优选地，本发明所述抗体为单链抗体scFv；更优选地，所述单链抗体scFv的重链可变结构域和轻链可变结构域由富含甘氨酸和丝氨酸的 接头肽链相连，并且重链可变结构域和轻链可变结构域的顺序可互换。
本发明还提供了一种靶向CD123的嵌合抗原受体，其包含：本发明所述的抗体；铰链区；跨膜结构域；共刺激结构域；和CD3ζ胞内信号转导结构域。
在具体实施方案中，所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、NKG2C、B7-H3或其任意组合；优选地，所述共刺激结构域选自4-1BB或CD28，所述4-1BB包含如SEQ ID NO：62所述的氨基酸序列。
在一个具体实施方案中，所述CD3ζ胞内信号转导结构域包含如SEQ ID NO：63所述的氨基酸序列。
在一个具体实施方案中，所述铰链区和跨膜结构域选自IgG1、IgG4、CD8α、CD28、IL-2受体、IL-7受体、IL-11受体、PD-1或CD34的铰链区和跨膜结构域。
优选地，所述的嵌合抗原受体包含选自由SEQ ID NO：65、66、67、68、69和70组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
本发明还提供了一种核酸分子，其包含编码本发明的抗体或嵌合抗原受体的核苷酸序列。
本发明还提供了一种载体，其包含本发明的核酸分子；优选地，所述载体是病毒载体；更优选地，所述载体是慢病毒载体。
本发明还提供了一种细胞，其经本发明的载体转染，从而包含本发明的核酸分子或载体；优选地，所述细胞是T细胞。
本发明还提供了本发明的抗体、嵌合抗原受体、核酸分子、载体或细胞用于制备治疗表达CD123的肿瘤的药物的用途；优选地，所述表达CD123的肿瘤是急性髓系白血病(AML)。
除非特别指明，本文所述技术方案可以任意组合。
本发明进一步通过以下实施例来说明。
实施例1：CD123 CAR分子设计及CAR-T制备
本实施例以设计具有不同抗原识别域的CD123 CAR分子，及CAR-T细胞制备为例。
抗原识别域设计原则：CAR-T-1为对照组，其CD123的scFv序列来自宾夕法尼亚大学报道[21]，在本文中命名为scFv-01。CAR-T-2～7为改造组，其scFv来源于小鼠免疫CD123胞外区融合蛋白所得单克隆抗体序列，在本文中命名为scFv-02-07。
将不同抗原识别域的CD123 scFv的核苷酸序列通过基因合成的方法合成，然后通过同源重组方法与CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ胞内区序列连接形成完整的CAR分子，即CAR 1-7。将此CAR核酸片段通过限制性内切酶酶切和连接酶连接的方式，构建到慢病毒转移质粒pLenti6.3/V5(Thermo Fisher，Waltham，MA，USA)中。将慢病毒包装质粒pLP/VSVG，pLP1/MDK，pLP2/RSK(Thermo Fisher，Waltham，MA，USA)与上述获得的转移质粒，用Lipofectamine 3000(Thermo Fisher，Waltham，MA，USA)转染至HEK293T细胞，48小时后收集培养基，300g离心去除细胞碎片后，用超速离心机25000rpm离心3小时。将沉淀用1mL生理盐水溶解，即为所需的慢病毒载体。
从健康志愿者的外周血单个核细胞中，使用CD3/CD28 beads(Thermo Fisher，Cat#40203D)，分离出T细胞。将分离纯化后的T细胞以1.0×10 6个细胞/mL，接入含有500IU/mL IL-2(山东金泰生物工程有限公司，中国)的X-VIVO 15培养基(Lonza，Switzerland)中培养。在培养48小时后，用带有CD123 CAR分子的慢病毒载体以MOI＝0.5感染T细胞，以获得CAR-T-1-7。慢病毒感染24小时后，进行细胞换液，并接入新鲜培养体系X-VIVO 15加500IU/mL IL-2中继续培养。在慢病毒感染5天后，使用移液枪反复吹打培养体系中的细胞并收集至离心管，置于磁力架上去除CD3/CD28 beads。T细胞离心并计数，取部分细胞使用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各组细胞的CAR分子转导效率。
图1所示为T细胞感染慢病毒5天、7天、9天、12天后，各组细胞的CAR分子转导效率。与对照组CAR-T-1相比，CAR-T-2、CAR-T-3、CAR-T-7组的CAR分子转导效率与其接近，CAR-T-4、CAR-T-5、CAR-T-6组明显高于对照组，说明各组CAR-T细胞的CAR蛋白表达情况均较好。
实施例2：改造后CD123 CAR-T的体外增殖能力
本实施例以检测具有不同抗原识别域的CD123 CAR-T的增殖能力为例。
在CD123 CAR-T细胞培养过程中，以慢病毒感染T细胞为培养第二天，之后每隔2至3天，收集CAR-T细胞并离心，加1-2毫升的培养体系重悬，取10微升的细胞稀释一定倍数后，用台盼蓝(Solarbio，Cat#C0040)进行染色，倒置显微镜(Ts2-FL，Nikon，日本)下分别计活细胞总数与死细胞总数，计算出各组细胞的增殖情况。
图2显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-2和CAR-T-7的总细胞增殖有明显提高。说明CAR-T-2和CAR-T-7的scFv设计能够改善CD123 CAR-T细胞增殖能力。
实施例3：改造后CD123 CAR-T在靶细胞刺激下的细胞分化情况
本实施例以检测具有不同抗原识别域的CD123 CAR-T，分别在两种靶细胞AML原代细胞或THP-1细胞(均表达CD123)的刺激下，其T细胞分化情况为例。
CAR-T细胞培养至第5天时，各组分别取两份5×10 5个细胞于12孔板中，以效靶比(E∶T)＝5∶1分别加入靶细胞AML原代细胞或THP-1细胞。在靶细胞刺激6天后，即CAR-T细胞培养至第11天时，各组分别取1×10 6个细胞并离心，100微升DPBS(HyClone，CAT#SH30028.02)重悬；然后每组CAR-T细胞分别加T细胞分化相关荧光抗体进行标记，使用全光谱流式细胞仪(N7-00008-0A，Cytek Biosciences，Inc.Fremont)进行检测。
图3的结果中，CD45RA +CD62L +细胞群表示初始T细胞群和干性记忆T细胞群的组合，CD45RA +CD62L -细胞群为效应T细胞群。具有不同抗原识别域的CAR-T-2和CAR-T-7的CD45RA +CD62L +细胞群占比高于对照组CAR-T-1，且CD45RA +CD62L -细胞群占比低于对照组CAR-T-1。说明改造后的CD123抗原识别域具有更好的CAR-T细胞持久性。
实施例4：改造后CD123 CAR-T在体外对细胞系的杀伤效率
本实施例以检测具有不同抗原识别域的CD123 CAR-T对表达CD123和不表达CD123的肿瘤细胞系的杀伤效率为例。
分别收集并离心表达CD123的细胞系THP-1，以及不表达CD123的细胞系K562，用1毫升生理盐水(河北天成药业有限公司，中国)重悬，加5微升Calcein-AM(浓度1μg/μL，Cat#C3100MP，ThermoFisher，USA)，轻轻混匀，置于37℃培养箱中孵育30分钟，以标记靶细胞。孵育结束后，使用生理盐水清洗2次，加X-VIVO 15重悬并计数。向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)中，每孔加入1×10 5个上述标记的靶细胞，并以E∶T＝10∶1的比例加入各组CAR-T细胞，置于37℃、5％CO 2培养箱中孵育5小时。孵育完成后，加2％浓度的Triton-X-100(Cat#T8787-100ML，Sigma，Germany)裂解阳性对照组，即1×10 5个上述标记的靶细胞；每孔分别取100微升杀伤体系上 清于酶标板中，使用多功能酶标仪(Varioscan Lux，ThermoFisher)检测荧光值(激发波长：495nm，发射波长：515nm)。
图4显示，对于CD123阳性细胞系THP-1，具有不同抗原识别域的CD123 CAR-T中CAR-T-3和CAR-T-5的杀伤效率，较对照组CAR-T-1显著升高。同时，各组改造后CD123 CAR-T和对照组CAR-T-1，对不表达CD123的K562细胞系的杀伤效率，与T细胞组一致。说明各组CD123 CAR-T细胞对靶细胞均有一定杀伤效应，并有较好的特异性，且CAR-T-3和CAR-T-5的scFv设计更有利于CAR-T杀伤肿瘤细胞。
实施例5：改造后CD123 CAR-T在体外对AML原代细胞的杀伤效率
本实施例以检测具有不同抗原识别域的CD123 CAR-T对AML原代细胞的杀伤效率为例。
取3个分别为高抗原密度(即AML 1#，CD123表达率为99.87％，MFI为13298)、中抗原密度(即AML 2#，CD123表达率为75.4％，MFI为7518)、低抗原密度(即AML 3#，CD123表达率为94.58％，MFI为5252)的CD123阳性AML患者骨髓血样(抗原密度水平定义如下，高抗原密度：中位荧光强度(Median Fluorescence Intensity，MFI)大于10000；中抗原密度：MFI大于6000小于10000；低抗原密度：MFI小于6000)，将AML患者的骨髓血样本分离单个核细胞，并使用CD34 MicroBeads(Cat#130-100-453，美天旎，德国)分选出CD34阳性AML原代细胞，用1毫升生理盐水重悬，加10微升Calcein-AM(浓度1μg/μL，ThermoFisher，USA)，轻轻混匀，置于37℃培养箱中孵育30分钟，以标记AML原代细胞。孵育结束后，使用生理盐水清洗2次，加X-VIVO15重悬并计数。向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)中，每孔加入1×10 5个上述标记的AML原代细胞，并以E∶T＝10∶1的比例，分别加入CD123 CAR-T细胞，置于37℃、5％CO 2培养箱中孵育5小时。孵育完成后，加2％浓度的Triton-X-100裂解阳性对照组，即1×10 5个上述标记的AML原代细胞；每孔分别取100微升杀伤体系上清于酶标板中，使用多功能酶标仪(Varioscan Lux，ThermoFisher)检测荧光值(激发波长：495nm，发射波长：515nm)。
图5显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-3对3个高(即AML 1#)、中(即AML 2#)、低(即AML 3#)抗原密度的CD123阳性AML患者的原代细胞均具有显著增强的杀伤效率。说明具有CAR-T-3 scFv的CD123 CAR-T细胞，能够有效提高对原代细胞的杀伤效率。
实施例6：改造后CD123 CAR-T在靶细胞刺激下的细胞因子表达
本实施例以检测具有不同抗原识别域的CD123 CAR-T在靶细胞AML原代细胞刺激下的细胞因子表达水平为例。
向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)中，每孔加入1×10 5个靶细胞AML原代细胞，此AML原代细胞的CD123表达率为99.87％、MFI为13298，并以E∶T＝10∶1的比例加入各组CAR-T细胞，置于37℃、5％CO 2培养箱中孵育5小时。取细胞上清，使用CBA试剂盒(BD Biosciences，San Jose，CA，USA)和流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各种细胞因子的表达水平。
图6显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-6组，在靶细胞刺激下，IL-2、TNF、IFN-γ的表达水平明显提高；其余各组CAR-T细胞的细胞因子表达，与对照组CAR-T-1相近。说明CAR-T-6的scFv设计有利于CD123 CAR-T细胞因子的表达。
以下表1和表2中为本发明中涉及的具体氨基酸序列。
表1示例性抗体的CDR和scFv序列
Figure PCTCN2021127505-appb-000001
Figure PCTCN2021127505-appb-000002
Figure PCTCN2021127505-appb-000003
表2示例性嵌合抗原受体序列
Figure PCTCN2021127505-appb-000004
Figure PCTCN2021127505-appb-000005
Figure PCTCN2021127505-appb-000006
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Claims (10)

  1. 一种特异性结合CD123的抗体，其包括重链可变结构域VH和轻链可变结构域VL，所述重链可变结构域包含SEQ ID NO：1(GYX 1X 2X 3X 4(D)YX 5X 6X 7)的VH-CDR1氨基酸序列，SEQ ID NO：2(X 8X 9X 10(X 11)X 12X 13X 14X 15X 16X 17X 18NX 19X 20X 21KX 22X 23X 24)的VH-CDR2氨基酸序列和SEQ ID NO：3(AX 25X 26X 27X 28X 29X 30X 31(YD)X 32X 33X 34X 35X 36X 37X 38X 39X 40)的VH-CDR3氨基酸序列；其中X 1选自S、T；X 2选自F、I；X 3选自T、S、M；X 4选自S、D、T、G；X 5选自W、Y、V、A、N；X 6选自M、V、I、W；X 7选自N、H；X 8选自Y、R；X 9选自I、C；X 10选自D、N、Y、S；X 11选自P、C；X 12选自Y、G；X 13选自D、N、S；X 14选自S、N、D、G；X 15选自E、A、G、S；X 16选自T、I、S；X 17选自H、S、N；X 18选自Y、S；X 19选自Q、E、P；X 20选自K、N、S；X 21选自F、L；X 22选自D、G、S；X 23选自K、R；X 24选自A、I；X 25选自R、G；X 26选自G、S、E；X 27选自E、P、R；X 28选自G、S；X 29选自N、Y、L、W；X 30选自W、Y、L；X 31选自G、D；X 32选自R、E；X 33选自Y、S、T；X 34选自D、Y、G；X 35选自G、Y；X 36选自Y、L；X 37选自A、G、P；X 38选自M、L；X 39选自D、A；X 40选自Y、C；所述轻链可变结构域包含SEQ ID NO：4(X 41SX 42X 43X 44X 45X 46X 47X 48X 49(QK)X 50X 51X 52X 53W)的VL-CDR1氨基酸序列，SEQ ID NO：5(X 54SX 55X 56X 57X 58X 59)的VL-CDR2氨基酸序列和SEQ ID NO：6(X 60X 61X 62X 63X 64(P)X 65TF)的VL-CDR3氨基酸序列；其中X 41选自A、S；X 42选自Q、K、S；X 43选自S、D；X 44选自I、V、L；X 45选自S、N、D、L；X 46选自K、Y、S、D、N；X 47选自D、S；X 48选自G、S；X 49选自D、N；X 50选自S、N；X 51选自 D、Y；X 52选自L、I、M；X 53选自A、N、H；X 54选自G、T、A；X 55选自T、N、Q；X 56选自L、S、R；X 57选自Q、E、A、I、D；X 58选自S、P；X 59选自G、E；X 60选自Q、N；X 61选自H、Y、S、F、G；X 62选自N、D、H、Y；X 63选自K、L、E、R、S；X 64选自Y、L、D、S、F、T；X 65选自Y、R、W；
    优选地，所述VH-CDR1选自由SEQ ID NO：8、9、10、11、12组成的组中的氨基酸序列；所述VH-CDR2选自由SEQ ID NO：14、15、16、17、18组成的组中的氨基酸序列；所述VH-CDR3选自由SEQ ID NO：20、21、22、23、24组成的组中的氨基酸序列；所述VL-CDR1选自由SEQ ID NO：26、27、28、29、30组成的组中的氨基酸序列；所述VL-CDR2选自由SEQ ID NO：32、33、34、35、36组成的组中的氨基酸序列；所述VL-CDR3选自由SEQ ID NO：38、39、40、41、42组成的组中的氨基酸序列；
    更优选地，所述抗体包括：
    1)包含如SEQ ID NO：8所示的VH-CDR1、如SEQ ID NO：14所示的VH-CDR2和如SEQ ID NO：20所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：26所示的VL-CDR1、如SEQ ID NO：32所示的VL-CDR2和如SEQ ID NO：38所示的VL-CDR3的轻链可变结构域；和/或
    2)包含如SEQ ID NO：9所示的VH-CDR1、如SEQ ID NO：15所示的VH-CDR2和如SEQ ID NO：21所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：27所示的VL-CDR1、如SEQ ID NO：33所示的VL-CDR2和如SEQ ID NO：39所示的VL-CDR3的轻链可变结构域；和/或
    3)包含如SEQ ID NO：10所示的VH-CDR1、如SEQ ID NO：16所示 的VH-CDR2和如SEQ ID NO：22所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：28所示的VL-CDR1、如SEQ ID NO：34所示的VL-CDR2和如SEQ ID NO：40所示的VL-CDR3的轻链可变结构域；和/或
    4)包含如SEQ ID NO：11所示的VH-CDR1、如SEQ ID NO：17所示的VH-CDR2和如SEQ ID NO：23所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：29所示的VL-CDR1、如SEQ ID NO：35所示的VL-CDR2和如SEQ ID NO：41所示的VL-CDR3的轻链可变结构域；和/或
    5)包含如SEQ ID NO：12所示的VH-CDR1、如SEQ ID NO：18所示的VH-CDR2和如SEQ ID NO：24所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：30所示的VL-CDR1、如SEQ ID NO：36所示的VL-CDR2和如SEQ ID NO：42所示的VL-CDR3的轻链可变结构域。
  2. 权利要求1所述的抗体，其中所述重链可变结构域包含选自由SEQ ID NO：44、45、46、47和48组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列；和/或所述轻链可变结构域包含选自由SEQ ID NO：50、51、52、53和54组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列；
    优选地，所述抗体包含：
    1)如SEQ ID NO：44所示的重链可变结构域和如SEQ ID NO：50所示的轻链可变结构域；和/或
    2)如SEQ ID NO：45所示的重链可变结构域和如SEQ ID NO：51所示的轻链可变结构域；和/或
    3)如SEQ ID NO：46所示的重链可变结构域和如SEQ ID NO：52所 示的轻链可变结构域；和/或
    4)如SEQ ID NO：47所示的重链可变结构域和如SEQ ID NO：53所示的轻链可变结构域；和/或
    5)如SEQ ID NO：48所示的重链可变结构域和如SEQ ID NO：54所示的轻链可变结构域；
    更优选地，所述抗体包含：选自由SEQ ID NO：56、57、58、59、60和61组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
  3. 权利要求1或2所述的抗体，其中所述抗体为单链抗体scFv；更优选地，所述单链抗体scFv的重链可变结构域和轻链可变结构域由富含甘氨酸和丝氨酸的接头肽链相连，并且重链可变结构域和轻链可变结构域的顺序可互换。
  4. 一种靶向CD123的嵌合抗原受体，其包含：权利要求1-3中任一项所述的抗体；铰链区；跨膜结构域；共刺激结构域；和CD3ζ胞内信号转导结构域；
    优选地，所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、NKG2C、B7-H3或其任意组合；更优选地，所述共刺激结构域选自4-1BB或CD28，所述4-1BB包含如SEQ ID NO：62所述的氨基酸序列；
    优选地，所述CD3ζ胞内信号转导结构域包含如SEQ ID NO：63所述的氨基酸序列；
    优选地，所述铰链区和跨膜结构域选自IgG1、IgG4、CD8α、CD28、IL-2受体、IL-7受体、IL-11受体、PD-1或CD34的铰链区和跨膜结构域。
  5. 权利要求4所述的嵌合抗原受体，所述的嵌合抗原受体包含选 自由SEQ ID NO：65、66、67、68、69和70组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
  6. 一种核酸分子，其包含编码权利要求1-3中任一项所述的抗体或权利要求4-5中任一项所述的嵌合抗原受体的核苷酸序列。
  7. 一种载体，其包含权利要求6所述的核酸分子；优选地，所述载体是病毒载体；更优选地，所述载体是慢病毒载体。
  8. 一种细胞，其经权利要求7所述的载体转染，从而包含权利要求6所述的核酸分子或权利要求7所述的载体；优选地，所述细胞为T细胞。
  9. 一种药物组合物，其包含权利要求1-3中任一项所述的抗体、权利要求4-5中任一项所述的嵌合抗原受体、权利要求6所述的核酸分子、权利要求7所述的载体或权利要求8所述的细胞，以及可药用载体。
  10. 权利要求1-3中任一项所述的抗体、权利要求4-5中任一项所述的嵌合抗原受体、权利要求6所述的核酸分子、权利要求7所述的载体或权利要求8所述的细胞用于制备治疗表达CD123的肿瘤的药物的用途；优选地，所述表达CD123的肿瘤是急性髓系白血病(AML)。
PCT/CN2021/127505 2020-12-16 2021-10-29 靶向cd123的嵌合抗原受体及其用途 Ceased WO2022127401A1 (zh)

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