Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor

Abstract Objectives Traditional cancer therapy and regular immunotherapy are ineffective for treating triple‐negative breast cancer (TNBC) patients. Recently, chimeric antigen receptor‐engineered natural killer cells (CAR NK) have been applied to target several hormone receptors on different cancer cells to improve the efficacy of immunotherapy. Furthermore, epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. Here, we demonstrated that EGFR‐specific CAR NK cells (EGFR‐CAR NK cells) could be potentially used to treat patients with TNBC exhibiting enhanced EGFR expression. Materials and methods We investigated the cytotoxic effects of EGFR‐CAR NK cells against TNBC cells in vitro and in vivo. The two types of EGFR‐CAR NK cells were generated by transducing lentiviral vectors containing DNA sequences encoding the single‐chain variable fragment (scFv) regions of the two anti‐EGFR antibodies. The cytotoxic and anti‐tumor effects of the two cell types were examined by performing cytokine release and cytotoxicity assays in vitro, and tumor growth assays in breast cancer cell line‐derived xenograft (CLDX) and patient‐derived xenograft (PDX) mouse models. Results Both EGFR‐CAR NK cell types were activated by TNBC cells exhibiting upregulated EGFR expression and specifically triggered the lysis of the TNBC cells in vitro. Furthermore, the two EGFR‐CAR NK cell types inhibited CLDX and PDX tumors in mice. Conclusions This study suggested that treatment with EGFR‐CAR NK cells could be a promising strategy for TNBC patients.


| INTRODUC TI ON
The prevalence of triple-negative breast cancer (TNBC) is approximately 15%-20% among all patients diagnosed with the condition. 1 TNBC tumors do not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). Hence, patients with TNBC cannot undergo regular immunotherapy or hormone therapy that targets these receptors.
Patients with this form of breast cancer undergoing chemotherapy or radiotherapy experience several side effects. [2][3][4] Furthermore, the recurrence rates of TNBC among patients who have undergone these therapies are higher than those among patients with other forms of breast cancer. 1 Therefore, there is a need to develop a specific and effective therapeutic strategy to improve the outcomes of TNBC.
Studies that analyzed the epidermal growth factor receptor (EGFR)-associated gene expression profile revealed that 45%-70% of patients with TNBC exhibited EGFR overexpression, which was associated with poor prognosis. 5,6 Additionally, EGFR plays an important role in TNBC progression and EGFR mutation rarely occurs in patients with TNBC. [7][8][9][10][11][12][13][14][15][16] Furthermore, several anti-EGFR monoclonal antibodies (mAbs) and small-molecule tyrosine kinase inhibitors (TKIs) have been tested in clinical trials for the treatment of TNBC. However, the short-term activity of these molecules limits their therapeutic efficacy. 5,17 Therefore, there is a need to develop an optimized EGFR-targeted treatment for patients with TNBC.
Chimeric antigen receptor (CAR)-engineered natural killer (NK) (CAR NK) cell therapy is one of the most promising immunotherapies for cancer. 18,19 Recently, third-generation CARs were designed, which contain an extracellular binding domain, a hinge region, a transmembrane domain, and an intracellular domain. The extracellular binding domain includes a single-chain variable fragment (scFv) derived from a tumor antigen-reactive antibody. The intracellular domain includes both the signaling domain (CD3ζ), which mediates NK cell activation, and the co-stimulatory domains (CD28 and 4-1BB), which enhance the NK cell functions, such as proliferation, resistance to apoptosis, cytokine secretion, and persistence.
Furthermore, CAR NK cell technology has been applied in several clinical or preclinical treatments for various tumors, 20,21 such as breast cancer, 22 colorectal cancer, 23 and glioblastoma. 24 Chimeric antigen receptor-engineered NK cell therapy has more advantages than the CAR-engineered T-cell (CAR T-cell) therapy. Patients undergoing CAR NK cell treatment are unlikely to suffer from graftversus-host disease (GVHD), which may occur in patients undergoing CAR T-cell therapy. 25,26 The detrimental effects of CAR NK cells are much lower than those of CAR T cells, since activation of NK cells does not result in cytokine release syndrome as may be observed by the activation of T cells. Additionally, unlike CAR T cells, CAR NK cells can be generated from various sources, such as peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells, umbilical cord blood cells, human embryonic stem cells, and NK-92 cell lines. 18,[27][28][29] In the present study, EGFR-specific CAR NK cells (EGFR-CAR NK cells) were generated by fusing the scFv of an anti-EGFR antibody to the artificially combined receptor molecules, in order to examine their anti-tumor effects on TNBC cells. The anti-EGFR scFv region recognized the wild-type EGFR on TNBC cells. After recognition, the activated NK cells exerted cytotoxic effects on the TNBC cells exhibiting upregulated EGFR expression. Further, activation of the EGFR-CAR NK cells significantly inhibited the progression of breast cancer in vitro and in vivo. The results of this study suggested that EGFR-CAR NK cell immunotherapy could be the optimal treatment strategy for patients with TNBC in the future.

| Cell lines and culture
Human breast cancer cell lines (MDA-MB-231, MDA-MB-468, HS578T, and MCF7) were purchased from the American Type Culture Collection (ATCC). The cell lines were used for the experiments within 6 months. All the cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution (Gibco) in a humidified incubator at 5% CO 2 and 37°C.

| Generation of EGFR-CAR NK cells
The lentiviral vector containing third-generation CAR (Con-CAR) was purchased from iCARTab BioMed. The two DNA fragments of the anti-EGFR scFv were cloned from the monoclonal hybridoma, which was previously established in the laboratory (Patent number: The fused anti-EGFR-specific scFv (1 or 2) was cloned into the third-generation CAR (Lenti-EF1a-scFv-3rd-CAR) with CD8 hinge, CD28 transmembrane domain, and intracellular signaling domains of 4-1BB and CD3ζ. Con-CAR was used as the control.
Peripheral blood mononuclear cells were isolated from the whole blood of healthy donors by Ficoll density gradient centrifugation. NK cells were obtained by stimulating the PBMCs in the NK cell-specific medium (Dakewe) with 5% human serum (Sigma) for 14 days. The cells were incubated under saturated humidity conditions at 5% CO2 and 37°C. The 293T/17 cells were co-transfected with CAR lentiviral plasmids along with pMD2.G and psPAX2 using the Lipofectamine 2000 reagent (Invitrogen). The lentiviral supernatant was collected after 2 days. Subsequently, the NK cells were transduced with lentiviral vectors carrying the DNA sequence that encoded the EGFRspecific third-generation CARs. The transfected cells were cultured under NK cell-specific conditions for 3 days to obtain the EGFR-CAR NK cells.

| Real-time polymerase chain reaction (PCR)
Total RNA was extracted using the RNeasy kit (OMEGA). The extracted RNA was subjected to reverse transcription using the PrimeScript RT reagent kit (Takara), following the manufacturer's instructions. The cDNA was subjected to real-time PCR using SYBR Premix Ex Taq (Takara). The primer sequences used for real-time PCR analysis are listed in Table 1.

| Small interfering RNA (siRNA)
The EGFR-targeting and negative control siRNAs were purchased from GenePharma (Suzhou). The siRNA sequences are listed in Table 2. The siRNA (50 mmol/L) was transfected into the cells using Lipofectamine RNAi MAX (Invitrogen).

| Western blotting
The anti-CD3ζ, anti-EGFR, and anti-β-actin antibodies were purchased from Abcam, Cell Signal Technology, and Sigma, respectively. The anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology. The Western blotting analysis was performed according to the standard procedure. 30,31

| Flow cytometry
The breast cancer cells and EGFR-CAR NK cells were quantitated or isolated by flow cytometry using several fluorescence-conjugated antibodies, following the manufacturer's instructions. The following reagents were used for this analysis: anti-human CD3-PE-Cy7, anti-human CD56-PE, anti-human CD69-APC-Cy7, mouse control PE, mouse control APC-Cy7, mouse control PE-Cy7, and Human TruStain FcX™ blocking solution purchased from BioLegend; anti-EGFR antibody purchased from Cell Signal Technology; and goat anti-rabbit IgG purchased from Abcam. The flow cytometric analyses were performed in a BD™ flow cytometer. The data were analyzed using FlowJo v10 software.

| Cytotoxicity assay
The human breast cancer cells (1 × 10 4 ; HS578T, MDA-MB-468, MDA-MB-231, and MCF7) were co-cultured with the optimized number of CAR NK cells in each well of the 96-well flat-bottom plates.
The media containing dying and dead cells were collected for further analysis. The LDH cytotoxicity assay kit (Beyotime) and YOYO™-3 Iodide (ThermoFisher) were used to measure the cytotoxic activity of the CAR NK cells, following the manufacturer's instructions. The cytotoxic activities were analyzed using an enzyme-labeled instrument and a live cell imaging system.

| Cell line-derived xenograft (CLDX) mouse model
Female nude mice were purchased from Beijing Biocytogen Co.,Ltd and maintained under pathogen-free conditions. The TNBC cells

| Patient-derived xenograft (PDX) mouse model
The medical ethics committee of the Suzhou Institute of Biomedical Engineering and Technology (A-06) approved this method. The patients provided their signed authorization to use the human triple-negative breast tumor tissues. The cells from these tissues were engrafted into the mammary fat pad of each mouse as described previously. NK cells (1 × 10 7 ) were injected into the PDX tumors (at least >4 mm 3 ) in mice at weeks 1, 2, and 3. The tumor size was measured as described previously. 32

| Immunohistochemical assay
Primary breast tumors and PDX tumors were fixed with 4% paraformaldehyde, embedded in paraffin blocks, and micro-dissected

Gene
Primer forward Primer reverse

TA B L E 1 Primer sequences for realtime PCR
into several thin sections. The sections were subjected to deparaffinization and antigen retrieval using citric acid buffer (pH 3.5) for 15 minutes. The specimens were incubated with 1% hydrogen peroxidase for 10 minutes. Subsequently, the specimens were incubated overnight with the horseradish peroxidase (HRP)-conjugated antibodies against ER, PR, HER2, or EGFR (Cell Signaling Technology) at 4°C. The staining was performed using the HRP-IHC kit, following the manufacturer's instructions.

| Generation and characterization of EGFR-CAR NK cells
The third-generation EGFR-CAR (Lenti-EF1a-scFv-3rd-CAR) was constructed by fusing the anti-EGFR-specific scFv (1 or 2) with the CD8 hinge, CD28 transmembrane domain, and intracellular signaling domains of 4-1BB and CD3ζ ( Figure 1A,B). The constructed lentiviral vectors carrying the CARs were verified by NcoI/KpnI restriction digestion and gel electrophoresis ( Figure 1C). Human primary NK cells were activated/isolated from the PBMCs cultured with (day14) or without (day0) NK cell-specific medium. PBMC cultures was higher than the day 0 PBMCs (Figure 2A-D).
The potential NK cell population was further transduced with the lentiviral vectors that carried either of the two EGFR-specific CARs (EGFR-CAR-1 and EGFR-CAR-2) or a third-generation CAR as the control CAR (Con-CAR). The generation of EGFR-CAR or Con-CAR NK cells was verified by real-time PCR and Western blotting analyses using the primer of CD3ζ or anti-CD3ζ antibody.
The expression of CARs was analyzed in the transduced NK cells ( Figure 2E,F). The non-transduced or transduced NK cells treated with the EGFR-or IgG-FITC were subjected to flow cytometry, to further characterize whether the EGFR-CAR NK cells were able to recognize EGFR in vitro ( Figure 2G). Approximately, 75% of the EGFR-CAR-1 or EGFR-CAR-2 NK cells were labeled with EGFR-FITC ( Figure 2G). Additionally, the transduction of CARs did not reduce the rate of proliferation of the NK cells ( Figure S1). Thus, the generated EGFR-CAR NK cells could specifically recognize EGFR in vitro.

| EGFR-CAR NK cells exert cytotoxic activity against TNBC cells by inducing cell lysis in vitro
The Western blotting analysis revealed that the protein expression  Table 3). These data suggested that activation of the EGFR-CAR NK cells was likely induced by TNBC cells exhibiting upregulated EGFR expression in vitro.
Cytotoxicity assay was performed to quantify the specific lysis percentage by measuring lactate dehydrogenase (LDH) activity in the co-cultured systems with the ratio between the effector (NK

cells) and target cells (breast cancer cells) (E/T ratio). This assay was
performed to investigate the ability of the activated EGFR-CAR NK cells to specifically trigger TNBC cell death. As expected, higher E/T ratio between the EGFR-CAR NK cells and the high-EGFR-expressing TNBC cells significantly elevated the percentage of the specific lysis in the co-cultured systems ( Figure 3D-G and Table 3).  Table 3). In addition, higher E/T ratio between the EGFR-CAR

NK cells and EGFR-knockdown TNBC cells did not increase in a manner similar to that between EGFR-CAR NK cells and TNBC cells
( Figure 3D-G and Table 3). Furthermore, the results of the cell lysis assays (YOYO™-3 Iodide staining) confirmed that the EGFR-CAR NK cells triggered significantly greater lysis of TNBC cells exhibiting upregulated EGFR expression than the Con-CAR NK or non-transduced NK cells( Figure S4).
These data suggested that the activated EGFR-CAR NK cells likely triggered cell lysis or death of the TNBC cells exhibiting upregulated EGFR expression in vitro.

| Anti-TNBC activity of the EGFR-CAR NK cells in mouse models
Tumor growth assays were performed by inoculating the TNBC cell lines into the breast fat pad of the female nude mice that were TA B L E 3 Cytokine release and cytotoxicity assay data      mice treated with EGFR-CAR-1 or EGFR-CAR-2 NK cells were also smaller than those treated with the Con-CAR-NK cells ( Figure 5).

Non-transduced T Cell Con-CAR-T Cell EGFR-CAR-1 T Cell EGFR-CAR-2 T Cell
Additionally, immunohistochemical analyses revealed that the protein (EGFR, ER, PR, and HER2) expression patterns of the PDX tumors in mice were similar to those of the original TNBC tissues from the patients (Figure 5A,B). Immunofluorescence imaging analysis revealed that CAR NK cells (CD56+) were present in the tumor core region after being injected into the tumor for 7 days ( Figure S6). The body weights of the mice were not affected by treatment with EGFR-CAR-1, EGFR-CAR-2, or Con-CAR NK cells in both xenograft models, suggesting that the tumor sizes were not affected by the health condition of the mice ( Figure 4D,H,L,P and Figure 5F). These data indicated that the EGFR-CAR NK cells likely inhibited the growth of TNBC tumors exhibiting upregulated EGFR expression in mice. In this study, the EGFR-CAR NK cells were generated by transducing a lentiviral vector containing the sequences encoding EGFR-CARs ( Figure 1). Western blotting and flow cytometry analyses indicated that the EGFR-CAR NK cells could specifically recognize EGFR in vitro (Figures 2 and 3 and Figure S4). The activated EGFR-CAR NK cells induced cell lysis or death of the TNBC cells exhibiting upregulated EGFR expression in vitro (Figure 3 and Figure S4). Furthermore, the EGFR-CAR NK cells exerted a significant anti-tumor effect on TNBC exhibiting upregulated EGFR expression in the two TNBC xenograft models (Figures 4 and 5). In addition, the tumor-bearing mice treated with the EGFR-CAR NK cells lived longer than the mice treated with Con-CAR NK cells ( Figure S5).  [25][26][27][28] In this study, EGFR-CAR NK cells recognized EGFR more efficiently than the Con-CAR NK cells ( Figure 2G), and EGFR-CAR NK cells were activated and secreted more IFN-γ, granzyme B, and perforin when co-cultured with TNBC cells exhibiting upregulated EGFR expression in vitro ( Figure 3A-C). Additionally, the activated EGFR-CAR NK cells induced cytotoxic activity in TNBC cells exhibiting upregulated EGFR expression more dramatically than MCF7 cells in vitro, according to the data from both the LDH release and YOYO-3 labeling assays (Figure 3 and Figure S4).
These results suggested that cell lysis triggered by the EGFR-CAR NK cells might be dependent on the amount of EGFR in breast cancer cells.
First-generation antigen-specific CAR NK cell immunotherapy was reported to be less effective against solid cancers than blood cancers. 45 However, the third-generation CAR NK cells that could mediate more intracellular signaling pathways demonstrated better anti-tumor activity. 46 The findings of this study revealed that EGFR-CAR NK cells significantly inhibited TNBC exhibiting upregulated EGFR expression in the CLDX (Figure 4 and Figure S5) and PDX mouse ( Figure 5) models.
The present study demonstrated that the activated EGFR-CAR NK cells upregulated cytokine secretion, promoted cytotoxicity against the TNBC cells exhibiting upregulated EGFR expression in vitro, and inhibited tumor growth in mice without affecting mice bodyweight.
However, EGFR is also expressed in several normal tissues. 47

| CON CLUS ION
In this study, we confirmed that EGFR-CAR NK cells could effectively recognize TNBC cells exhibiting upregulated EGFR expression. Additionally, the two distinct EGFR-CAR NK cells inhibited the growth of the TNBC tumor both in vitro and in vivo. Thus, EGFR-CAR NK cells could be potentially applied in the treatment of patients with TNBC.

ACK N OWLED G EM ENTS
We thank all members of Prof. Zhang's laboratory for their valu-

CO N FLI C T O F I NTE R E S T S
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in the manuscript.