Introduction
Acute myeloid leukaemia (AML) is a prevalent type of acute leukaemia. It is a highly heterogeneous malignant clonal disease that originates from the haematopoietic tissue. Patients have a high mortality rate, low long-term survival rate, and are prone to relapse, and thus, this disease greatly endangers human health and life [
1,
2]. AML is a heterogeneous group of diseases and that there are several subgroups according to various classifications including immunophenotype, cytogenetics, the immunophenotype on the basis of which AML is divided because the classification is very complex and the treatment depends on it and various treatment protocols are applied [
3,
4]. For decades, there has been little change in the treatment of AML, which still comprises chemotherapy and haematopoietic stem cell transplantation. Anthracyclines combined with cytarabine remain the classic chemotherapy regimen. Despite a relatively high remission rate, patients are at increased risk because of drug resistance and relapse [
5‐
9].
Chimeric antigen receptor-T (CAR-T) cellular immunotherapy induces specific activation of T cells through antibodies that can recognize antigens on tumour cell surfaces, thereby avoiding the limitations of the major histocompatibility complex. Modified T cells can better recognize and destroy tumours than natural T cells. In recent years, CAR-T therapy has made great progress in cancer treatment and is considered one of the most promising tumour treatment methods. At present, CAR-T therapy has the greatest effect in acute lymphoblastic leukaemia (ALL) treatment [
10‐
12], in which it is most widely used. CD19-targeting CAR-T treatment has achieved noteworthy results in the treatment of adult and child ALL [
13,
14], with a remission rate of up to 90% [
15]. CAR-T treatment of AML with antigens CD123 and CD33 also demonstrated good results. However, these two antigens are expressed in normal myeloid cells, which will inevitably result in toxic side effects in patients [
16‐
22]. Therefore, identifying relatively safe and efficacious therapeutic targets is essential.
B7-H3, also known as CD276, was discovered in 2001. Studies have shown that B7-H3 can stimulate the expansion and destruction of T cells, and may selectively stimulate signal receptors on T cells [
23]. B7-H3 is a tumour-associated antigen that plays an important role in tumour progression and metastasis. The survival rate of patients with negative B7-H3 protein expression is higher than that in patients with positive expression [
24‐
27]. Several studies have shown that B7-H3 is abnormally expressed in human malignancies, such as melanoma, leukaemia, and breast and prostate cancer. The vast majority of cancer patients exhibited abnormally high expression (60–93%) of B7-H3 in cancer tissue, whereas B7-H3 expression in normal healthy tissue was low [
28‐
32]. B7-H3 was highly expressed in AML, with the highest expression observed in the M3 and M5 subtypes [
33].
Statistics show that approximately 40% of patients with AML express B7-H3 [
34]. Moreover, some studies have shown B7-H3 expression in > 50% of cells in 31 pancreatic tumour specimens, but not in normal pancreatic tissue specimens [
28]. B7-H3 expression is regulated by RNA transcription and is a surface immunoregulatory glycoprotein that inhibits natural killer cells and T cells. Although B7-H3 transcripts are widely expressed in human solid tumours and normal tissues, the B7-H3 protein is preferentially expressed in tumour tissue [
29]. B7-H3 is expressed in only a few tissues and cells, including activated lymphocytes and tumour cells [
35]. Therefore, although B7-H3 is expressed in part of AML, it is an excellent therapeutic target for the disease because it does not cause toxicity to the hematopoietic system.
It has been reported that B7-H3 is not expressed in immune cells [
36]. In addition, most literature regarding B7-H3 has shown that B7-H3 is related to tumour progression and metastasis. More importantly, B7-H3 is highly expressed in tumour-related stromal cell fibroblasts and is associated with tumour neovascularization. Therefore, treatment targeting B7-H3 is expected to disrupt drug inhibition in the tumour microenvironment. This new target has attracted more attention in recent years [
25]. Currently, several research groups have constructed B7-H3-CAR-T cells, which have shown encouraging tumour cell destruction in preclinical studies of various solid tumours such as pancreatic cancer, childhood neuroblastoma, and osteosarcoma [
37‐
40]. In the current study, we constructed B7-H3-CAR-T cells with 4-1BB as a co-stimulatory domain carrying a safety switch, a truncated EGFR molecule.
Materials and methods
Cell lines and culture conditions
KG-1, MOLM-16, and Jurkat cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). HEL, THP-1, HL-60, and AML5 cell lines were provided by Mr. Zhao Yun’s laboratory at the Tang Zhongying Hematology Research Center of Soochow University, Suzhou, China.
HEL, Jurkat, and AML5 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Hyclone,UT, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, Grand Island, NY, USA). KG-1 cells were cultured in Iscove's modified Dulbecco’s media (Hyclone, UT, USA) supplemented with 10% FBS. Peripheral blood was drawn from healthy volunteers, and peripheral blood mononuclear cells were extracted by the Ficoll method. T cells were activated by Transact and cultured with TexMacsTMGMP medium (Hyclone, UT, USA) supplemented with interleukin (IL)-7 (155 U/mL; Novoprotein, China) and IL-15 (190 U/mL; Novoprotein, China). Extracted T cells were transduced after being activated for 48 h in vitro. Toxicity experiments were performed when cells proliferation numbers were sufficient. All cells were cultured in an incubator containing 5% CO2 at 37 °C.
Construction of the B7-H3-specific CAR
The second-generation CAR was constructed as follows: the B7-H3-scFv sequence was cloned into a lentiviral vector, which contained a B7-H3-scFv, CD8 hinge and transmembrane region, a 41BB intracellular region sequence, a CD3ζ sequence, and a human EGFR sequence provided by PersonGen Bio Therapeutics (Suzhou, Jiangsu, China). Among them, EF1 represents the promoter, and 2A is a kind of self-cleavage sequence, which will be disconnected when translated into it. Proteins expressed by the same mRNA are separated before and after to produce two proteins, which are used to construct multi-cistronic carrier and realize co-translation of two proteins under the control of the same promoter. The role of 2A here is to achieve the co-expression of the front CAR protein and the truncated EGFR tagged protein behind. The promoter contains the signal peptide.
Generation of B7-H3-specific CAR-modified T/Jurkat cells
A lentivirus was prepared according to conventional laboratory methods [
41]. The CAR was constructed by gene synthesis and subcloned into a lentiviral plasmid and packaged into virus particles with infectious ability. T cells, 48 h post-activation, and Jurkat cells were placed in a 48-well plate (5 × 10
5 cells/well). Thereafter, 50 µL of the virus was added, and TexMacsTMGMP/1640 medium was added to a final volume of 100 µL. Following transduction for 15 h, 1000 µL of TexMacsTMGMP/1640 medium was added to each well for culture. When the number of cells became sufficient, they were transferred to a 24-well plate or 12-well plate for culturing, and the CAR positive rate was tested when the number of cells was sufficient after approximately 10 days.
Flow cytometry
Six AML cell lines (AML5, KG-1, HEL, HL-60, THP-1, and MOLM-16) were harvested and suspended in PBS (pH = 7.4). The cells were centrifuged at 4,000 rpm/min for 3 min, and the supernatant was discarded. An anti-B7-H3 antibody conjugated with allophycocyanin (APC) (1:1000) was added to the cells and incubated for 30 min. The cells were washed twice with PBS, and the expression of B7-H3 on the cell surface was detected by flow cytometry (Beckman Coulter, USA). Approximately 2 × 10
6 cultured B7-H3-CAR Jurkat, B7-H3-CAR T, and T cells were harvested, incubated with 100 µL of 1:1000 diluted anti-EGFR antibody at 37 °C for 20 min, and washed twice with PBS. The positive rate of B7-H3-CAR Jurkat and B7-H3-CAR-T cells was analysed by flow cytometry [
42].
Jurkat cells and B7-H3-CAR Jurkat cells were co-incubated with KG-1, HEL, and AML5 cells, and incubated with antibodies targeting CD25-APC and CD69-APC (BD Biosciences, USA) for 20 min, following which CD25 and CD69 expression levels were detected by flow cytometry.
Western blotting
Approximately 1 × 106 T cells and B7-H3-CAR-T cells were harvested. Lysates were separated using 8–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). Membranes were blocked with 5% skim milk for 1 h and incubated overnight with a mouse anti-human CD3ζ antibody (BD Biosciences, USA,) at 4 °C. Immune complexes were detected by incubating the PVDF membranes with a horseradish peroxidase-goat anti-mouse IgG antibody (Solarbio, Beijing, China) for 1 h at 37 °C. The protein bands were visualized with enhanced chemiluminescence reagents (Millipore, MA, USA).
Cytotoxicity assays
Target cells (HEL, AML5, and KG-1) were resuspended in PBS and treated with 1 µL 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) at 37 °C for 15 min. KG-1 cells served as a negative control group. Effector cells were added at a 1:1 effector:target (E:T) ratio, and cocultured with target cells for 48 h in a 24-well plate. Thereafter, cells were collected and resuspended in an equal volume of PBS. The cells were incubated with APC annexin V and 7-AAD (LK-AP105-100; Multisciences, Hangzhou, China), and the percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter, USA) [
43,
44].
Cytokine secretion assays
The ability of T and B7-H3-CAR-T cells to produce IL-2, tumour necrosis factor (TNF), interferon γ (IFN-γ), and granzyme B in response to AML cells (HEL, AML5, and KG-1) was analysed using a cytometric bead array (CBA) system. The test fluid was the supernatant of previously killed tumour cells. The Human Granzyme B CBA Flex Set D7 Kit (BD Bioscience, USA, catalogue #560304), Human TNF Flex Set D9 Kit (BD Bioscience, USA), Human IL-2 Flex Set A4 Kit (BD Bioscience, USA), and Human IFN-γ CBA Flex Set E7 Kit (BD Bioscience, USA) were obtained from BD Biosciences [
45].
AML mouse xenograft model
Mice were housed under standard specific-pathogen-free conditions and all procedures met the requirements of the National Institutes of Health and Institutional Animal Care and Use Committee. Experiments were performed in accordance with protocols that were approved and authorized by the Animal Welfare and Ethics Committee of Soochow University. Ethical approval for this study was (Approval Number/ID: 201907A487, 201909A374 and 201912A149). The order of mouse grouping and experiments was randomly assigned. At first, all the mice in the experimental group were labelled with ear numbers and injected with tumour cells for unified observation. After that, the mice were randomly selected and injected with different effector cells. 6- to 8-week-old, female NOD-Prkdcs-cidIl2rgtm1/Bcgen (B-NSG) mice (Biocytogen, China) were engrafted with HEL cells via tail vein injection. Each mouse was injected with 5 × 105 HEL-Luciferase-GFP cells, and the tumour burden was measured using an IVIS imaging system (IVIS-spectrum) after 2 or 5 days. Biofluorescence was recorded for 10 min after intraperitoneal injection of 150 mg/kg d-luciferin substrate. Mice were injected with an equal volume of effector cells in the tail vein [T cells, B7-H3-CAR-T cells, or phosphate buffered saline (PBS)]. Peripheral blood (100 μL), femoral bone marrow, and the organs which after erythrocyte lysis were incubated with anti-human CD45 antibody (BD Biosciences, USA). The cells were subsequently incubated for flow cytometry (Beckman Coulter, USA) analysis for the detection of tumour cells.
Histology
After the mice were killed, the spleen and ovarian tissues were taken and embedded in paraffin for immunohistochemical analysis. After dewaxing and rehydrating, the paraffin sections were microwaved in a 0.01 M citrate buffer solution for 5 min and sealed with a methanol solution containing 0.3% hydrogen peroxide. The sections were incubated with an antibody targeting B7-H3 (1:300) followed by a biotin-labelled secondary antibody. Finally, DAB chromogenic solution was added to observe the infiltration of tumour cells in the tissues. Haematoxylin and eosin (HE) staining was performed after the paraffin sections were dewaxed and rehydrated to observe the pathological changes in tumour tissues.
Statistical analysis
Statistical analyses were performed using GraphPad Prism5 Software (GraphPad Software Inc., La Jolla, CA, USA). Student’s t-test was used to compare values between two groups. P < 0.05 was considered to indicate a statistically significant difference. Kaplan–Meier curves were used to show survival of mice in each experimental group.
Discussion
AML is a refractory invasive hematopoietic stem cell malignancy [
48]. In the past decade, the life expectancy of patients with AML has improved due to the development of new treatment methods. However, AML remains one of the most deadly and difficult to treat malignant cancers due to its high drug resistance [
49‐
51]. Moreover, patients with AML have limited treatment options and a poor prognosis. Chemotherapy is not curative, and patients are prone to relapse. Therefore, new treatment methods are urgently needed [
52‐
54]. CAR-T therapy has made great progress in both haematologic and solid tumours, and is considered to be one of the most promising tumour treatment methods [
55,
56].
At present, the CAR-T treatment of AML requires bone marrow clearance before treatment because most of the targets are highly expressed in normal myeloid cells, which greatly complicates the treatment procedure and increases risks unnecessarily. Therefore, identifying an antigen that is selectively expressed in malignant cells is very important. B7-H3 was highly expressed in AML. Our experiments show that relying solely on T cells is not sufficient to eliminate tumour cells; However, B7-H3-CAR-T cells can effectively destroy B7-H3-expressing AML cells. CD25 and CD69 are activation markers and have been used to show cell proliferation in various models and on cells after and before treatment [
57]. B7-H3-CAR-T cells also have a tumour clearance effect in mice. In vivo imaging tests showed that tumour cells in the mice in the B7-H3-CAR-T group were significantly lower than those in the PBS and T cell groups. Moreover, in cases of AML with a particularly rapid progression, B7-H3-CAR-T cells can significantly prolong the survival of mice and reduce the proportion of tumour cells in peripheral blood, bone marrow, and tissues. Therefore, the experiment proved B7-H3-CAR-T cells can be used for the treatment of patients with refractory AML who express the B7-H3 antigen.
In addition, to verify the safety of the B7-H3-CAR-T cells, we investigated whether they could cause off-target toxicity to normal cells in mice. Routine peripheral blood tests showed that the B7-H3-CAR-T cells did not have a toxic effect on normal cells. Moreover, HE staining results showed that B7-H3-CAR-T cells did not damage important organs in mice. Therefore, the results suggested that the B7-H3-CAR-T cells developed in the present study have a good safety profile. Two recent studies on the potential of B7-H3-CAR in the treatment of AML have well demonstrated the toxicity of B7-H3-CAR in AML cell lines and primary AML cells, laying a good foundation for the treatment of AML. Compared with these two studies, we used proprietary single-chain antibody sequences that not only demonstrated that B7-H3 significantly prolonged the survival of AML in mice, but also added in vivo data from peripheral blood, bone marrow, and tissue to further demonstrate the safety of B7-H3-CAR [
58,
59].
In conclusion, we demonstrated that B7-H3-CAR-T cells can specifically remove AML tumour cells expressing the B7-H3 antigen in vitro and in vivo. Furthermore, the safety of the cells was demonstrated, laying the foundation for the clinical application of B7-H3-CART cells in AML treatment. Compared with CD33-CAR-T and CD123-CAR-T cells, which are the most frequently used cells at present, patients need to be clear myeloid cells in advance. The use of B7-H3-CAR-T cells may not only prolong the survival time of patients with relapsed refractory AML, but also simplify the application of CAR-T therapy in clinical practice and reduce risk in patients.
Future studies on CAR T may try to treat AML by combining it with hematopoietic stem cell transplantation (HSCT). The only treatment with long-term survival advantages for patients with AML is through the HSCT method, which, to date, there remains a relatively high recurrence rate and poor prognosis with it. The study found that CAR T treatment is a highly effective immune cell therapy known at this stage, therefore, it is best to combine the two. Patients can be pre-injected with CAR T cells prior to transplantation of allogeneic hematopoietic stem cells to help eliminate tumour cells in the body, thereby effectively reducing the disease burden of patients prior to transplantation, thus improving transplantation outcomes and survival rate [
60].
In recent years, the development of CAR T treatment technology has been relatively rapid. There have been many research institutions in a relatively advanced position in CAR T treatment-related research and continue to maintain the current development momentum. We believe that CAR T cell products will soon benefit most cancer patients in the future.
However, several limitations exist. Only three mice were randomly selected from each group for flow cytometry, immunohistochemistry and HE staining of tumor burden in vivo. But the in vivo imaging data were sufficient to demonstrate a significant effect in the CAR T group. The primary AML cells were not used to verify the effect of B7-H3-CAR T. Because primary cells are difficult to obtain and ethical issues have not been solved for the time being, toxicity tests on primary cells cannot be conducted at present. Later studies in our laboratory will make up for this point, focusing on the toxic effect of B7-H3-CAR T on primary tumor cells.
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