Background
The prognoses of patients with T cell malignancies remain poor [
1‐
4]. There is no better treatment strategy than chemotherapy, which may not benefit refractory/relapsed patients and can lead to serious toxicity. It is thus imperative that novel effective targeted therapeutic strategies are developed. In recent years, chimeric antigen receptor (CAR)-modified immune cells have shown outstanding efficacy for the treatment of B cell malignancies [
5,
6]. This indicated that using similar concepts to develop CAR-modified immune cells may help fight against T cell malignancies.
Conventional CAR immunotherapy utilizes modified T cells derived from patients to directly target and eliminate malignancies [
6]. However, malignant T cells may have the same phenotypic and functional characteristics as normal T cells. This leads to difficulties in distinguishing therapeutic CAR engineered T (CAR-T) cells from malignant T cells, causing the mutual killing of CAR-T cells and limiting the function of CAR-T cells against T cell malignancies [
7]. Mamonkin et al. constructed CAR-T cells targeting CD5
+ T malignant cells and found that the delayed initial expansion of anti-CD5 CAR-T cells was mainly due to fratricide mediated by perforin secretion. [
8]. Pinz et al. used anti-CD4 CAR-T cells to eliminate CD4
+ T cell lymphomas (TCLs) or T cell acute lymphoblastic leukemia (T-ALL), demonstrating that almost all CD4
+ CAR-T cells were also depleted. A recent study demonstrated that CD4
+ CAR-T cells might have a “helper effect”, which could enhance the persistence and cytotoxicity of CD8
+ CAR-T cells [
9]. Thus, the self-killing of CD4
+ CAR-T cells would decrease the cytotoxic ability of CAR-T cells. Moreover, circulating malignant T cells are often found in the peripheral blood of patients with T-ALL [
10,
11] and some TCLs [
12], which may lead to contamination of malignant T cells and then generate “CAR-malignant T cells” during the process of CAR-T cells preparation [
7]. Ruella et al. reported a relapse in a patient after 9 months of anti-CD19 CAR-T cell treatment. The relapsed leukemia cells were CD19 negative, but anti-CD19 CAR was aberrantly expressed. They found that the CAR gene was accidentally transduced into a single B malignant cell during the process of CAR-T cell preparation, and its product concealed the CD19 epitope on the surface of leukemic cells, masking their recognition by CAR-T cells [
13]. Similarly, the occurrence of “CAR-malignant T cells” may lead to disease relapse and adversely affect the prognosis of patient with T-ALL and TCL. Therefore, when targeting T-malignant cells, it is necessary to try other types of effector cells to circumvent the shortcomings of CAR-T cells.
Recently, another immune cell, the natural killer (NK) cell, has been used to engineer with CAR [
14,
15]. The use of NK cells for CAR-NK cell manufacturing is a promising strategy to avoid mutual killing of CAR-T cells in the abovementioned situation. NK cells are an important part of the innate immune system and have natural cytotoxic ability against malignant cells. NK cells serve as allogeneic effectors, mediating their activity independent of major histocompatibility complexes. Therefore, NK cells do not need to be collected from a certain patient or a specific human leukocyte antigen matched donor to naturally induce graft-versus-host disease [
16]. Unfortunately, NK cells from peripheral blood are difficult to transduce with CAR [
17]. In our preliminary experiments, we tried different methods to improve the transfection efficiency of NK cells from peripheral blood, including increasing the lentivirus titer, but these failed and led to proliferation inhibition and apoptosis induction in NK cells. As a NK cell line, NK-92 cells have been used as effector cells in immunotherapy, but are derived from a patient with NK cell lymphoma and need to be irradiated before being administered into patients to prevent potential carcinogenicity. Recently, several studies have revealed that NK-92 cells (not transduced with CAR) are safe and effective for the treatment of relapsed/refractory hematological malignancies [
18,
19].
CD5 is a type-I transmembrane glycosylated protein [
20] that has a role in negative regulation of T cell receptor signaling [
21,
22] and promotes the survival of normal and malignant lymphocytes [
23,
24]. CD5 is not expressed on the surface of hematopoietic stem cells but is highly expressed by malignant T cells [
25,
26]. Therefore, CD5 is currently considered one of the characteristic antigens of malignant T cells [
8]. In addition, CD5 is also expressed in some B cell malignancies [
27,
28]. Clinical trials using anti-CD5 monoclonal antibody have revealed a moderate therapeutic effect in patients with cutaneous T cell lymphomas (CTCLs) or chronic lymphocyte leukemia (CLL) [
29,
30]. Chen et al. designed a third-generation anti-CD5 CAR construct with the T-cell-associated costimulator 4-1BB and CD28 to generate anti-CD5 CAR-NK-92 cells, which showed specific cytotoxicity against CD5
+ malignant cells in vitro and in vivo [
15].
However, at least in preclinical models, it appears that CAR-T cells seem to be superior to CAR-NK-92 cells [
7]. CARs commonly contain three domains: an extracellular antigen binding domain, a transmembrane module, and an intracellular signaling transduction domain [
31]. The transmembrane module primarily anchors the CAR structure on the cell membrane and is usually driven from the transmembrane region of CD8 or CD28. The classical intracellular signal transduction domain contains a CD3ζ cytoplasmic domain and one or more intracellular domains of costimulatory molecules, such as 4-1BB, CD28, OX40, or ICOS [
32]. Different costimulatory domains endow CAR-T cells with different characteristics: a CD28 costimulatory domain stimulates more powerful cytotoxic ability of CAR-T cells, whereas the 4-1BB and ICOS costimulatory domain induces longer persistence of CAR-T cells [
9]. All of these costimulatory factors play important roles in the activation and function of T cells. Therefore, we hypothesized that NK-cell-associated costimulatory factors could be used to activate NK cells and exert their cytotoxic effects.
We speculated that the NK-cell-associated costimulatory domain used in a CAR construct might be suitable for engineered CAR-NK-92 cells. Recently, Li et al. used transmembrane domains and costimulatory domains typically expressed in NK cells to construct CARs and found that CAR with a NKG2D transmembrane domain and 2B4 costimulatory domain displayed superior anti-ovarian cancer activity [
14]. 2B4 is considered a NK-cell-specific costimulatory receptor belonging to the signaling lymphocytic activation molecule (SLAM) family, which transduces activation signals through SLAM-associated protein (SAP). SAP interacts with the intracellular domain of 2B4 and regulates 2B4-dependent NK cell activation [
33,
34].
In this study, the anti-CD5 single-chain variable fragment (scFv) domain of CAR was developed from a mouse anti-human CD5 monoclonal antibody (Clone HI211) that was previously established and validated in our institute. Two different anti-CD5-CARs with costimulators 4-1BB and 2B4 (referred to as BB.z-NK and 2B4.z-NK, respectively) were constructed. Their cytotoxic ability was evaluated, demonstrating that 2B4.z-NK cells exhibited rapid proliferation and higher anti-malignant efficacy in both malignant CD5+ cell lines and primary CD5+ malignant cells in vitro through upregulation of activation markers and cytotoxic granule release. Furthermore, the superior cytotoxic ability of 2B4.z-NK against T-ALL was confirmed in mouse xenograft models. In addition, both BB.z-NK and 2B4.z-NK have side effects on CD5+ normal T cells. To our knowledge, there has been no previous research describing such a strategy of using the 2B4 costimulatory domain to generate anti-CD5 CAR-NK cells for CD5+ malignancy treatment.
Methods
Patients and samples
Peripheral blood from healthy donors was acquired from the Tianjin Blood Center. Bone marrow samples were obtained from patients enrolled in the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, and patient samples were approved by the ethical advisory board of the Institute of Hematology and Blood Diseases Hospital. All subjects signed an informed consent in accordance with the Declaration of Helsinki.
Plasmid construction and lentivirus production
The murine anti-human CD5 scFv derived from mouse hybridoma cells (clone HI211, which was established in our institute) was cloned into a previously constructed pCDH-CAR plasmid containing the 4-1BB costimulatory domain [
35] to form a plasmid referred to as BB.z. Then, the 2B4 intercellular domain was used to replace the 4-1BB costimulatory domain of BB.z to construct a pCDH-CD5 scFv-CD8α hinge-CD8α transmembrane domain-2B4 costimulatory domain-CD3ζ plasmid (referred to as 2B4.z).
Lentiviral vectors were produced in 293T cells as previously described [
36].
Cell culture
Jurkat, MOLT-4, MAVER-1, 293T, and NK-92 cells were purchased from American Type Culture Collection. Jurkat, MOLT-4, and MAVER-1 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and glutaMAX (GIBCO, USA). NK-92 cells were grown in α-minimum essential medium supplemented with 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 200 U/ml recombinant human IL-2 (rhIL-2), 12.5% horse serum, and 12.5% FBS. MV4-11 cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS. Primary patients’ bone marrow mononuclear cells (BMMNCs) were seeded in IMDM supplemented with 15% FBS, 100 ng/ml rhFLT3-L, 100 ng/ml rhSCF, and 50 ng/ml rhTPO. Primary normal T cells were isolated and cultured as previously described [
36].
Establishment of stable cell lines
Jurkat cells were infected with lentivirus carrying pLV-luciferase-neo plasmid, which was kindly provided by Dr. Rong Xiang (Medical School of Nankai University, Tianjin, China), followed by clonal selection using 600 μg/ml G418 to generate stable polyclonal cells overexpressing firefly luciferase (Jurkat-luc2).
NK-92 cells were infected with lentivirus carrying BB.z CAR plasmid, 2B4.z CAR plasmid, or empty vector, followed by sorting of GFP and F (ab’)2-positive cells by flow cytometry to generate polyclonal cells stably expressing BB.z CAR (BB.z-NK), 2B4.z CAR (2B4.z-NK), or VEC-NK cells.
Cell proliferation assay
We seeded 1.5 × 104 NK cells in 96-well plates per well. After 24 h, 48 h, or 72 h incubation, cell activity was tested by applying Cell Counting Kit-8 (Dojindo, Japan) following the manufacturer’s instructions.
Apoptosis assay
We then harvested 5 × 105 NK cells and stained with Annexin V-Alexa Fluor® 647 and PI (Biolegend, USA) following the manufacturer’s instructions and then subjected cells to flow cytometry analysis (BD LSRFortessa, USA).
In vitro function studies of CAR-NK cells
Jurkat, MOLT-4, and MAVER-1 cells were used as CD5+ target cells and MV4-11 cells were used as CD5− target cells. Three donors’ normal T cells were used as target cells to evaluate the side effects of CAR-NK cells.
BB.z-NK and 2B4.z-NK cells were used as effector cells and VEC-NK cells as controls.
Analysis of direct cytotoxicity
BB.z-NK, 2B4.z-NK, or VEC-NK cells were incubated with target cells at E:T ratios of 4:1, 2:1, 1:1, 1:2, 1:4, or 1:8. After 6 h, the cell mixture was harvested and stained with APC-conjugated anti-human CD5 antibody and PE-Cy7-conjugated anti-human CD56 antibody (Biolegend, USA) for 30 min at 4 °C, and then washed and resuspended in PBS for flow cytometry analysis. The percentage of CD56−CD5+ cells represented the residual level of target cells.
Cytokine releasing assay
BB.z-NK, 2B4.z-NK, or VEC-NK cells were cocultured with target cells at E:T ratios of 1:1 for 12 h. The supernatant of the cocultured system was harvested. Expression levels of IFN-γ and TNF-α were detected using an ELISA kit (R&D, USA) according to the manufacturer’s instructions.
Degranulation assay
We cocultured 0.5 × 105 BB.z-NK, 2B4.z-NK, or VEC-NK cells with 1.5 × 105 target cells in 200 μl of NK-92 cultured medium with PE-conjugated anti-CD107a antibody (Biolegend, USA). After 1 h, 100 μg/ml monensin (BD Biosciences) was added to the cocultured system and incubated for 4 h, and then the cells were labeled with PE-Cy7-conjugated anti-human CD56 antibody and analyzed by flow cytometry. All CD56+ CD107a+ cells were regarded as degranulated NK cells.
Detection of NK cell activation markers
BB.z-NK, 2B4.z-NK, or VEC-NK cells were incubated with MAVER-1 cells at E:T ratios of 1:1. After 6 h, cells were harvested and stained with PE-conjugated anti-human CD69 antibody, APC-Cy7-conjugated anti-human HLA-DR antibody, and PE-conjugated anti-human NKG2D antibody (Biolegend, USA) for 30 min at 4 °C, and then washed and resuspended in PBS for flow cytometry analysis. The percentage of CD56+CD69+, CD56+HLA-DR+, or CD56+NKG2D+ cells represented the activated NK cells.
In vivo NSG murine studies
Eight-week-old NSG female mice were purchased from the Institute of Laboratory Animal Sciences (CAMS&PUMC, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College.
Twenty-four mice were intravenously inoculated with 3 × 10
6 Jurkat-luc2 cells. Nine days after transplantation, mice were randomly divided into four treatment groups according to the average radiance of bioluminescent imaging: group PBS, group VEC-NK, group BB.z-NK, and group 2B4.z-NK. Mice were intravenously administered PBS or 5 × 10
6 cells of either VEC-NK, BB.z-NK, or 2B4.z-NK cells at day 10, day 20, and day 26. Bioluminescent images were obtained using Caliper IVIS Lumina II (Caliper Life Sciences, USA), and the average radiance was calculated as described before [
37].
Statistical analyses
Values were expressed as the mean ± S.D. If not specifically mentioned, the statistical significance of data was assessed by an unpaired two-tailed t-test. A value of p < 0.05 was used as the standard for statistical significance.
Discussion
T cell malignancies are aggressive hematological tumors with limited treatment strategies and dismal prognoses. To develop a CD19 CAR-T cell strategy for B cell malignancies, we investigated whether CAR engineered immune cells could exhibit a cytotoxic ability towards CD5
+ T cell malignancies. Treatment of T cell malignancies using CD5 CAR-T cells remains limited due to the shared antigens between malignant T cells and normal T cells, causing the fratricide of CD5 CAR-T cells themselves. Recently, another important type of immune cell, the NK-92 cell, has been utilized as a CAR-modified immune cell. However, in preclinical models, CAR-T cells seem to be superior to CAR-NK-92 cells [
7]. Therefore, we modified the CAR structure to improve the cytotoxic ability of CAR-NK cells.
In studies of CAR-T cells, the costimulatory domain has been considered an important factor that strongly affects the curative effect of CAR-T cells [
9]. To date, nearly all engineered CAR-NK cells used the intracellular domain of T cell-associated costimulatory factors as the costimulatory structural module of CAR. Several studies constructed second-generation CARs with CD28 [
43,
46] or 4-1BB (Clinical trial: NCT01974479 and NCT01974479) costimulatory domains, while others used third-generation CARs with both the CD28 and 4-1BB costimulatory domain [
15,
47,
48]. At the beginning of our study, 4-1BB was used as a costimulatory domain of CAR, which has been proven effective in generating CAR-T cells to target CD20- [
36], CD33- [
35], and FLT-3-positive [
49] malignant cells. The CD5 BB.z-NK cells in our preliminary study showed a good true killing ability against target cells (E:T = 1:1, 12 h), while the degranulation of BB.z-NK cells was not very obvious and the secretion of TNF-α was very low. This may due to the unsuitable function of 4-1BB in NK cells. Wilcox et al. reported that when they treated mice with 4-1BB ligand or anti-4-1BB agonistic antibody, proliferation was induced in NK cells and IFN-γ secretion increased alongside NK cell helper function, but the cytotoxic ability of NK cells was not augmented [
50]. Several in vivo xenograft model studies have demonstrated that triggering the 4-1BB signaling of NK cells by treatment of mice with anti-4-1BB activating antibody [
51] or interaction with 4-1BBL-positive γδT cells [
52] would enhance NK-cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) through the activation of the 4-1BB downstream signaling pathway. In contrast, the 4-1BB-4-1BBL interaction can attenuate the activity of NK cells in the human leukemia micro-environment. Several studies reported that 35% (23/65) of patients with acute myelocytic leukemia (AML) [
53] and 32% (28/89) of patients with B cell chronic lymphocytic leukemia (B-CLL) [
54] expressed a high level of 4-1BB ligand, and at the same time, almost all NK cells in these patients expressed 4-1BB. When 4-1BB ligand-positive AML cells interacted with 4-1BB on allogeneic NK cells, cytotoxicity and IFN-γ release were reduced, but this could be restored by 4-1BB blocking antibody [
53]. When 4-1BB ligand-positive B-CLL cells interacted with 4-1BB on Rituximab-induced NK cells, ADCC was reduced [
54]. These results are completely in opposition to those observed in T cells, where the interaction between 4-1BB and 4-1BB ligand would enhance the cytotoxic ability of human T cells against AML cells [
55].
The different effects of 4-1BB between human NK and mouse NK cells, and between human NK and human T cells may be due to the different downstream signaling pathways induced by 4-1BB in these cells. The adaptor proteins TNF receptor-associated factor 1 (TRAF1) and TRAF2 will bind with the intracellular domain of 4-1BB (whether murine or human) after 4-1BB triggering [
56], inducing the activation of the NF-κB, JNK, and p38 signaling pathway and leading to the activation of T or NK cells [
57]. Because there are differences in several amino acids in the intracellular domain of human 4-1BB and murine 4-1BB, human 4-1BB can interact with another adaptor protein, TRAF3, whereas murine 4-1BB cannot [
58]. When TRAF3 and TRAF2 form heterotrimers, they can inhibit NF-κB activation [
59]. Therefore, the interaction between the 4-1BB costimulatory domain and the negative regulator-TRAF3 may result in the limited activation (low expression of CD107a and little releasing of TNF-α) of BB.z-NK, and thus, the downstream signaling pathway of NF-κB will be weakened.
In this study, we attempted to improve the cytotoxic ability of CD5 CAR-NK by changing the costimulatory domain of CAR. 2B4 is a member of the signaling lymphocytic activation molecule (SLAM)-related receptor family, which contain four immune-receptor tyrosine-based switch motifs (ITSMs) in their intracellular domain and perform important roles in regulating the reactivity of multiple immune cells [
60]. The ligand of 2B4 is CD48, which is a glycoprotein-I (GPI)-anchored Ig-like protein that can be found in nearly all hematologic cells including NK cells [
61]. Triggering 2B4 via interaction with CD48 can induce the phosphorylation of ITSMs, causing the recruitment of the adapter protein SLAM-associated protein (SAP) and EWS-Fli1-activated transcript 2 (EAT-2) [
62]. SAP can recruit Src-family kinase Fyn [
63], which then transduces downstream signals by phosphorylating phospholipase C-γ (PLC-γ) or Vav-1 [
64], activating ERK, inducing the cytotoxicity of NK cells and producing the pro-inflammatory factors-IFN-γ and TNF-α. EAT-2 can link 2B4 to PLC-γ and ERK to mediate the activation of NK cells and accelerate the polarization and secretion of cytotoxic granules [
65]. In one study of CAR-NK that used the 2B4 intracellular domain as the costimulatory domain, phosphorylation of PLC-γ, Vav-1, and ERK was promoted in NK cells [
14]. It was revealed that the 2B4 intracellular domain may be more suitable as a costimulatory domain in CAR-NK cells than that of 4-1BB.
Therefore, in our study, we used the 2B4 intracellular domain as the costimulatory domain to replace the 4-1BB intracellular domain in the CAR structure and compared the cytotoxic ability of BB.z-NK and 2B4.z-NK towards CD5+ T-malignant cells. The results showed that 2B4.z-NK released more cytotoxic granules, expressed higher NK cell activation markers (CD69, NKG2D, and HLA-DR), secreted more of the inflammatory factors IFN-γ and TNF-α, and exhibited stronger true cytotoxicity than BB.z-NK after coculture with CD5+ cell lines and primary hematologic malignant cells in vitro. Moreover, 2B4.z-NK cells exhibited predominant cytotoxic activity on T-ALL bearing mice in vivo and significantly prolonged the survival of mice versus BB.z-NK.
In addition, CD5 is expressed in almost all normal T cells and some mature B cells [
66]; thus, the side effects of CD5 CAR-NK were evaluated. The results showed that both BB.z-NK and 2B4.z-NK exhibited cell lysis properties, a side effect towards normal T cells (Fig.
5), while 2B4.z-NK revealed significantly higher cytotoxicity on normal T cells than BB.Z-NK cells, similar to their role in T cell malignancies. It is indicated that CD5 CARs targeting T cell malignancies will induce T cell aplasia similar to the B cell aplasia observed in patients treated with CD19 CAR-T cells. B cell aplasia is more resistant and can be relieved by immunoglobulin infusions [
67]. Long-term T cell aplasia increases the probability of infection in patients [
68]. Although 2B4.z-NK showed a stronger side effect towards normal T cells, the long-term T cell aplasia may be prevented by using short-lived CAR-NK cells or by bridging allogeneic hematopoietic stem cell transplantation after complete remission [
7]. In our treatment strategy, the NK-92 cell line was used as the effector cells. For further clinical trial studies, 2B4.z-NK cells must be irradiated before transfusion into patients to prevent potential carcinogenicity. The process of irradiation will lead to the short-survival of 2B4.z-NK in patients, which can shorten the period of T cell aplasia in patients but may reduce treatment outcomes as well. To address this duality, multiple injections may be effective at prolonging the persistence of 2B4.z-NK cells in patients and augmenting the curative effect.