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Combination of 4-1BB and DAP10 promotes proliferation and persistence of NKG2D(bbz) CAR-T cells

Chimeric antigen receptor (CAR)-T cell therapy has been shown to have considerable therapeutic effects in hematological malignancies, and NKG2D(z) CAR-T cell therapy has been verified to be safe based on clinical trials. However, due to the poor persistence of NKG2D(z) CAR-T cells, their therapeutic effect is not obvious. Here, we constructed NKG2D(bbz) CAR-T cells that can simultaneously activate 4-1BB and DAP10 costimulatory signaling. They were found to be cytotoxic to the target cells in vitro and in vivo. They exhibited low differentiation, low exhaustion, and good proliferation. Importantly, the proportions of central memory T (Tcm) and stem cell-like memory T (Tscm) cell subsets were strikingly increased. After long-term incubation with the target cells, they displayed reduced exhaustion compared to NKG2D(z) CAR-T cells. Further, in the presence of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, they exhibited reduced exhaustion and apoptosis, upregulated Bcl2 expression, and an increased proportion of Tcm cell subsets. Finally, NKG2D(bbz) CAR-T cells had better antitumor effects in vivo. In summary, the results showed that NKG2D(bbz) CAR-T cells may be valuable for cellular immunotherapy of cancer.


Introduction
Chimeric antigen receptor (CAR)-T cell therapy has achieved significant success and attracted much attention in the treatment of hematologic cancers (1,2). Since the successful US Food and Drug Administration (FDA) approval of the CAR-T cell therapy tisagenlecleucel (Kymriah) in 2017, several other CAR-T cell products for various hematological malignancies have been approved (3). The therapy involves using gene editing to allow T cells to express CARs that are not restricted by histocompatibility complex (MHC) molecules and can directly recognize and lyse tumor cells (4). The first-generation CARs had only a CD3z signaling domain, along with an extracellular antigen recognition domain. Modular construction has allowed them to be modified to create second-and third-generation CARs that connect CD28, 4-1BB, or both signaling domains to CD3z, in order to attempt to simulate the costimulation (signal 2) provided by antigen-presenting cells during T cell receptor (TCR) recognition, which is required for the activation of all physiological T cells (5)(6)(7).
CAR-T cells usually only recognize a single target, so antigen escape can easily occur due to antigen loss by cancer cells (8,9). In contrast, we use the activated receptor natural-killer group 2, member D (NKG2D), which can recognize eight ligands, as the extracellular antigen recognition domain. NKG2D is mainly expressed on the surface of NK and CD8 + T cells (10). NKG2D ligands include MICA, MICB and ULBP1-6, which are expressed on the surface of most tumor cells but almost absent from the surface of normal cells (11). NKG2D(z) CAR (composed of CD3z and full-length NKG2D) T cells have been shown to be cytotoxic to multiple myeloma, lymphoma, and ovarian cancer cells in mice (12)(13)(14). Furthermore, the safety of these cells has been demonstrated in phase I clinical trials; however, due to their limited expansion and persistence, the therapeutic effect of these low-dose CAR-T cells was poor (15, 16).
T cells expressing CAR with a CD28 or 4-1BB signaling domain can both exert effective cytotoxicity, but differ in effector function, clinical efficacy, and toxicity. These differences are thought to be caused by differences in signaling cascade activation, leading to different subsets and viability of CAR-T cells (17)(18)(19)(20). 4-1BB significantly increases the proportion of the central memory T (Tcm) cell subset and enhances the persistence and viability of CAR-T cells in vivo. DAP10 functions as a costimulatory molecule downstream of NKG2D in CD8 + T cells, thus enhancing the response of effector T cells, increasing cytokine secretion, and causing T cells to differentiate into memory precursor cells (21). Therefore, the activation of DAP10 costimulatory signaling by CARs may enable CAR-T cells to overcome immunosuppression encountered in the microenvironment of solid tumors (22-25). To enhance the persistence of NKG2D(z) CAR-T cells, we constructed an NKG2D(bbz) CAR structure involving full-length NKG2D, 4-1BB, and CD3z. NKG2D interacts with DAP10 of T cells and thereby recruits p85 protein, acting as a costimulatory molecule similar to CD28. Thus, the NKG2D(bbz) CAR-T cells can simultaneously activate 4-1BB and DAP10 costimulatory signaling.

Flow cytometry
A BD FACS AriaII and a BD Accuri ™ (BD Biosciences) were used to acquire data. Analysis was performed with FlowJo software (FlowJo). CAR expression was determined by surface staining with either antibody against NKG2D (APC/PE, Biolegend) or mCherry (mCherry was co-expressed in NKG2D CAR-T cells to more accurately assess the percentages of these cells).

Proliferation of NKG2D CAR-T cells in vitro
CAR-T cell labeling by carboxyfluorescein succinimidyl ester (CFSE) was used to evaluate the proliferation of NKG2D(bbz) CAR-T cells incubated with their specific ligand (soluble MICA [sMICA]-Fc fusion protein) without IL2/7/15. The cells were labeled with 2-5 mM CFSE at 4°C for 10 min followed by adding 9 mL buffer 1 to quench the reaction. The cells were washed twice with buffer 1 and then cultured with sMICA-Fc to stimulate NKG2D CAR-T cell proliferation without IL2/7/15. After 72 h, CFSE dilution, as an indicator of NKG2D CAR-T cell proliferation, was assessed by flow cytometry.

Cytotoxicity assays
A549, ABC1 and MDA-MB-231 cells were seeded in E-Plate 16 (Acea Biosciences, San Diego, CA) at 1×10 4 cells/well, and then monitored in a normal incubator overnight by use of an xCELLigence impedance-based real-time cell analysis (RTCA) system (Acea Biosciences). When the cells were in the logarithmic growth phase the next day, half of the medium was removed and replaced with normal or balanced medium containing NKG2D CAR-T cells or mock-T cells at an effector: target (E:T) ratio of 3:1. The cells were continuously monitored for several hours with the RTCA system, and the impedance was plotted over time.
Bioluminescence luciferase assays were also performed to measure the cytotoxic activity of CAR-T cells against NKG2D ligand-expressing target cells (K562, U266, MMIS, A549). The target cells (1×10 4 ) were co-cultured with NKG2D CAR-T cells at various E:T ratios for 8 h with the mock-T cells serving as the negative control.
To assess cytokine production, A549 cells were plated at 5×10 4 cells/well in 48-well plates and cultured in the presence or absence of 5×10 4 NKG2D CAR-T cells/well. After 12 and 24 h, the medium was obtained to assess IL2, IFN-g, GM-CSF, and TNF-a secretion by use of ELISA kits (LIANKE).

In vivo anticancer assays
Five-to-6-week-old female NOD SCID gamma (NSG) mice (GemPharmatech) were intraperitoneally injected with 5×10 6 A549-Luc-green fluorescent protein (GFP) cells. These cancer cells in the mice were then detected by live bioluminescent imaging. Images were collected and analyzed with a Xenogen-IVIS Imaging System. When the mean fluorescence intensity (MFI) value was >10 9 , the mice were divided into three groups, which received NKG2D(z) CAR-T cells (8×10 6 /mouse), NKG2D (bbz) CAR-T cells (8×10 6 /mouse), or mock-T cells (ethics ID number: Xmsq2021-0075). The weight of the mice and MFI values were regularly monitored. The supernatant was aspirated after centrifugation of peripheral blood from the mice (collected via the submandibular vein) and the secretion of IL6, IL2, IFN-g, and TNF-a in the peripheral blood was measured with a Th1/ Th2/Th17 kit (560484, BD Pharmingen). The percentages of NKG2D(z) and NKG2D(bbz) CAR-T cells were assessed based on mCherry fluorescence after lysis of erythrocytes.

Statistical analyses
All statistical analyses were performed using GraphPad Prism v7.0. The data are reported as mean ± SD (n≥3). Twoway analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test were used to compare more than two groups, and two-tailed paired t-tests were used to compare two groups. P<0.05 was considered to indicate a significant difference.

NKG2D ligand is highly expressed on the surface of some solid and blood cancer cells
The NKG2D ligands consist of eight cell-associated glycoproteins that belong to the MIC and ULBP families. We used flow cytometry to detect the expression of NKG2D ligands on the surface of multiple myeloma, liver cancer, breast cancer, and lung cancer cell lines. The results showed that the expression levels of NKG2D ligands on the surface of different tumor cells varied. The lung cancer cell line A549 highly expressed MICA, MICB, ULBP1, and ULBP2/5/6. The breast cancer cell line MB543 did not express ULBP1, but ULBP3 was partially expressed. The multiple myeloma cell line IM9 highly expressed MICA, MICB, and ULBP4 (Supplementary Figure 1A). To further verify the NKG2D ligands expression in solid tumor tissues, we performed immunohistochemical analysis to assess the expression in the tumor tissues of patients with gliomas, lipomas, and lung cancer. The results showed that MICA/B were highly expressed in the tumor tissues (Supplementary Figure 1B).
Development of NKG2D CAR involving full-length NKG2D, 4-1BB, and CD3z NKG2D(z) CAR-T cells have been shown to be effective and safe in phase I clinical trials. However, due to their limited expansion and persistence, the therapeutic efficacy of low-dose CAR-T cells was poor (15, 16). Based on the results of clinical trials, we constructed NKG2D(bbz) CAR structures, involving full-length NKG2D, 4-1BB, and CD3z ( Figure 1A). These cells could simultaneously activate DAP10 and 4-1BB costimulatory signaling. T cells were separated and extracted from the peripheral blood of healthy donors, activated with anti-CD3/ CD28 magnetic beads for 24-48 h, and then transduced with NKG2D(z) or NKG2D(bbz) lentiviruses. On day 5, the expression of NKG2D CAR was detected on the surface of the T cells ( Figure 1B). We found that the MFI of NKG2D on the surface of CD8 + NKG2D(bbz) CAR-T cells was 4232 ± 89 as opposed to 3485 ± 18.72 for CD4+ NKG2D(bbz) CAR-T cells (p<0.0001) ( Figure 1C). To further compare and evaluate the differences in biological functions between NKG2D(z) and High expression of NKG2D(bbz) CAR in human T cells. NKG2D(bbz) CAR-T cells, self-cleaving peptide 2A was used to co-express mCherry fluorescent protein based on both original CAR structures (Supplementary Figure 2; Figures 1D, E). We found that the ratio of CD8 to CD4 was 2.13 ± 0.56 for NKG2D (bbz) CAR-T cells and 1.62 ± 0.69 for NKG2D(z) CAR-T cells (p=0.0286) ( Figure 1F).

Cytotoxicity of NKG2D(bbz) CAR-T cells against the target cells
To verify that NKG2D(bbz) CAR-T cells can recognize NKG2D ligands on tumor cells and have cytotoxic effects, A549, ABC1, MDA-MB-231, K562, MMIS and U266 cells were selected, and both the RTCA method (Figures 2A, B) and luciferase method ( Figures 2C, D) were used. The NKG2D(bbz) CAR-T cells showed comparable cytotoxic activity to NKG2D(z) CAR-T cells in vitro.

Superior proliferation of NKG2D(bbz) CAR-T cells
To compare the persistence of NKG2D(bbz) and NKG2D(z) CAR-T cells in a nutrient-free environment, we measured the percentages and sizes of NKG2D CAR-T cells at day 7 after their initial activation (Supplementary Figure 3). After adjusting the NKG2D CAR expression levels to the equivalent extent, the culture conditions were changed so that there was no IL2/IL7/ IL15/other cytokines. After 48 h of culture, the apoptosis of NKG2D CAR-T cells was detected by flow cytometry. We found that the percentage of viable cells was more significantly increased for NKG2D(bbz) CAR-T cells compared to NKG2D (z) CAR-T cells (44.5 ± 2.781 vs 29.4 ± 6.856; p=0.0233) ( Figure 3A). Accordingly, we found that Bcl2 expression was also remarkedly upregulated in NKG2D(bbz) CAR-T cells compared to NKG2D(z) CAR-T cells (77.5 ± 3.07 vs 61.74 ± 7.647; p=0.0039) ( Figure 3B). Moreover, we labeled the NKG2D CAR-T cells with CFSE and found that the MFI on the surface of NKG2D(bbz) CAR-T was 45396 ± 74387 as opposed to 833000 ± 132300 for NKG2D(z) CAR-T cells after 72 h of culture (p=0.0302) ( Figure 3C). To further investigate the proliferation profiles of NKG2D(bbz) CAR-T cells in response to specific ligand stimulation, we co-incubated sMICA-Fc or MMIS myeloma cells (positive control) with NKG2D CAR-T cells for 24h. The results showed that 14.94 ± 5.048% of NKG2D CAR-T cells expressed CD69 after co-incubation with sMICA-Fc (p=0.0127) ( Figure 3D). We also verified that NKG2D on the surface of NKG2D CAR-T cells could be recognized by sMICA-Fc by use of flow cytometry. The sMICA-Fc could also be used to detect the percentage of NKG2D CAR-T cells ( Figure 3E). In addition, we found that NKG2D(bbz) CAR-T cells had faster proliferation in the presence of sMICA-Fc and absence of IL2/7/ 15 ( Figure 3F).

NKG2D(bbz) CAR-T cells exhibited better antitumor effects in vivo
To further observe the antitumor ability of NKG2D(bbz) and NKG2D(z) CAR-T cells, we incubated them with A549 cells for 96 h and then assessed their cytotoxicity by use of luciferase assays. We found that there was no significant difference in cytotoxicity after long-term incubation with tumor target cells in vitro between NKG2D(bbz) and NKG2D(z) CAR-T cells ( Figure 5A). In addition, we collected NKG2D CAR-T cells after co-incubation to assess the exhaustion of CD4+ or CD8+ NKG2D CAR-T cells and found that CD4 + and CD8 + NKG2D (z) CAR-T cells were exhausted earlier than corresponding NKG2D(bbz) CAR-T cells (Figures 5B, C). Moreover, the CD8 subsets among NKG2D(z) CAR-T cells were more likely to die after 4 days of incubation ( Figure 5D). Based on these results, we constructed a A549-bearing mouse model and found that NKG2D(bbz) CAR-T-cell-treated mice exhibited lasting antitumor effects with two infusions on day 14 and day 22 (Figures 5E, F). However, there was no significant difference in B A FIGURE 4 Compared to NKG2D(z) CAR-T cells, NKG2D(bbz) CAR-T cells exhibited reduced differentiation and exhaustion, and the Tnaive cell subset was increased. (A) CD27, CD28, CD127, CD25, CD62L, and PD1 on the surface of NKG2D(z) and NKG2D(bbz) CAR-T cells were detected by flow cytometry at 9 days after initial activation. The curves represent the flow cytometry peak map, and the bars represent the statistical map of the corresponding marker. the body weight of these NKG2D CAR-T-treated mice. On day 29, we assessed the levels of IL2, IL6, IFN-gamma;, and TNF-a in the peripheral blood of the mice and found that IL6 was significantly higher in the NKG2D(z) CAR-T-treated group than in the NKG2D(bbz) CAR-T-treated group (6689 ± 1414 vs 1794 ± 395.1; p=0.0045), while the percentage of NKG2D(bbz) CAR-T cells in peripheral blood was 4.917 ± 0.6191 compared to 1.11± 0.6942 for NKG2D(z) CAR-T cells (p=0.0021) (Figures 5G, H).

Discussion
NKG2D, which is involved in innate and adaptive immunity, is a type II transmembrane activating receptor that is mainly expressed on CD8 + T cells and NK cells (28)(29)(30). The approval of CD19-directed CAR-T cell therapy for B-cell hematological malignancies provides strong clinical validation for CAR-T therapy, and thus provides an impetus for the development of CAR-T cell therapy for other cancers.
Several studies have verified the antitumor ability of NKG2D(z) CAR-T cells in mice with ovarian cancer and multiple myeloma (13,31). The safety of NKG2D(z) CAR-T cells has also been demonstrated in clinical trials of myelodysplastic syndromes/acute myeloid leukemia and relapsed/refractory multiple myeloma (15,16). However, these cells were found to have only short-term persistence in vivo during the treatment (32). In this study, we prepared NKG2D(bbz) CAR-T cells that could simultaneously activate 4-1BB and DAP10 costimulatory signaling. To minimize the interference of NKG2D expression on CD8 + T cells and better compare the differences between NKG2D(z) and NKG2D(bbz) CAR-T cells, we used self-cleaving peptide 2A to co-express mCherry fluorescent protein. NKG2D(bbz) CAR-T cells had more Tnaive and Tcm cell subsets with higher CD27 and CD62L expression in vitro. 4-1BB costimulatory signaling makes it easier for CAR-T cells to differentiate into Tcm cells, reduces cell exhaustion, and prolongs the life of CAR-T cells in vivo (33,34). NKG2D interacts with DAP10, which contains a YINM motif and thereby recruits p85 to induce PI3K signaling and Grb2 to activate Vav-SOS signaling (35,36). Similar to CD28, the NKG2D-DAP10 complex eventually leads to AP-1, NFAT, and NF-kB nuclear translocation and subsequent cell survival, proliferation, upregulated expression of effector molecules and cytokines, and the release of cell lysate particles after a series of cascade reactions (10, 37,38).
Regarding the use of CAR-T cell therapy for solid tumors, the tumor microenvironment is inhospitable to immune cell proliferation (39). The signaling domains of the co-receptor CD28 and 4-1BB affect the metabolic characteristics of human CAR-T cells, including enhancing cell persistence in the tumor microenvironment (33,40). Compared to NKG2D(z) CAR-T cells, NKG2D(bbz) CAR-T cells had faster proliferation and less apoptosis in the absence of IL2/7/15, while PD1 expression was lower under normal culture conditions. Meanwhile, we showed that NKG2D(bbz) CAR-T cells proliferate faster under the stimulation of their specific ligand sMICA-Fc. After NKG2D The appropriate concentration of a PI3K inhibitor (LY294002) can partially resist NKG2D(z) and NKG2D(bbz) CAR-T cell exhaustion caused by the PI3K pathway by upregulating Bcl2. (z) and NKG2D(bbz) CAR-T cells were co-cultured with target tumor cells for 96 h, NKG2D(bbz) CAR-T cells exhibited lower exhaustion and secreted more IL2 within 24 h than NKG2D(z) CAR-T cells. Other studies have also shown that 4-1BB could prolong the persistence of CAR-T cells in vivo and ultimately enhance their antitumor ability (41,42). In the current study, after the second infusion of NKG2D CAR-T cells, the mice in the NKG2D(z)-treated group showed significant weight loss and higher IL6, which may lead to inflammation and death of the mice. After prolonged incubation with A549 cells, the reduced proportion of CD8 + NKG2D(z) cells may be related to the fact that CD8 + NKG2D(z) CAR-T cells were more easily lost, indicating that CD4 + and CD8 + NKG2D CAR-T cells should be prepared separately in the future to study the differences in target tumor cell lysis and cytokine secretion.
Many studies have shown that PI3K inhibition can overcome target-driven "self-fratricide" of NKG2D CAR-T cells through reducing the expression of NKG2D CAR on the CAR-T cell surface (25). In our study, we found that inhibition of PI3K activation significantly increased the Tcm subset of NKG2D(bbz) CAR-T cells in vitro, suggesting that the treated NKG2D(bbz) CAR-T cells had improved capacities for long-term survival. We also found that inhibition of PI3K activation with LY294002 partially reduced the apoptosis of NKG2D(z) and NKG2D(bbz) CAR-T cells and up-regulated Bcl2 expression. PI3K could decrease the persistence and impair the function of CAR-T cells in vivo (43,44). When CAR-T cells were cultured in vitro, the addition of IL15 could reduce mTORC1 activity so as to induce anti-apoptotic properties associated with up-regulated Bcl2 expression (45). Thus, IL15 and LY294002 together may further reduce mTORC1 activity to up-regulate Bcl2 expression. In the future, NKG2D(bbz) CAR-T cells and a PI3K inhibitor may be combined in vitro and in vivo to improve their persistence and anticancer effects.
In summary, we constructed NKG2D(bbz) CAR-T cells that could simultaneously activate both 4-1BB and DAP10 costimulatory signaling. To minimize the interference caused by the expression of natural NKG2D on the surface of CD8 + T cells, we made NKG2D(z) and NKG2D(bbz) CAR-T cells co-express mCherry fluorescent protein. Compared to NKG2D(z) CAR-T cells, NKG2D(bbz) CAR-T cells exhibited low differentiation and reduced exhaustion in vitro, and also sustained anticancer activity in vivo. Moreover, we found that a PI3K inhibitor reduced apoptosis and upregulated Bcl2 expression, and increased the proportion of the Tcm cell subset. Therefore, our study provides further experimental evidence for the clinical application of NKG2D(bbz) CAR-T cells, potentially with a PI3K inhibitor.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the tab of animal experimental ethical inspection of laboratory animal center, Wenzhou Medical University.