# Engineered IL-7 Receptor Enhances the Therapeutic Effect of AXL-CAR-T Cells on Triple-Negative Breast Cancer

Authors: Zhenhui Zhao; Yan Li; Wei Liu; Xun Li
Journal: BioMed Research International
 (2020)

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## Abstract

Triple-negative breast cancer (TNBC) is a very aggressive malignant type of tumor that currently lacks effective targeted therapies. In hematological malignancies, chimeric antigen receptor T (CAR-T) cells have shown very significant antitumor ability; however, in solid tumors, the efficacy is poor. In order to apply CAR-T cells in the treatment of TNBC, in this study, constitutively activated IL-7 receptor (C7R) that has been reported is used to enhance the antitumor function of constructed CAR-T cells by ourselves. Usingin vitro coincubation experiments with target cells and in vivo antitumor experiments in mice, we found that the coexpressed C7R can significantly improve the activation, cell proliferation, and cytotoxicity of CAR-T cells. In addition, the in vivo experiments suggested that the enhanced CAR-T cells displayed significant antitumor activity in a TNBC subcutaneous xenograft model, in which in vivo, the survival time of CAR-T cells was prolonged. Together, these results indicated that CAR-T cells that coexpress C7R may be a novel therapeutic strategy for TNBC.


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## Body

## 1. Introduction

Breast cancer is the most common malignant tumor in women; triple-negative breast cancer (TNBC) accounts for 15%–20% of the total incidence of breast cancer [1], which is more aggressive than other breast cancer subtypes [2]. TNBC is prone to recurrence and metastasis, which are more likely to occur at 1–3 years after surgery. Most patients die within the first 5 years after treatment; therefore, the death risk of TNBC is significantly higher than that of other breast cancer subtypes [3]. Currently, the standard treatments for TNBC are still confined to surgery, chemotherapy, and radiation therapy; therefore, identifying other effective therapeutic strategies for TNBC is of utmost importance.Recently, engineered chimeric antigen receptor T (CAR-T) cells have been proven to be the most promising strategy for cancer treatment [4]. Specifically, CAR-T cells targeting CD19 have achieved significant success in the treatment of hematological malignancies, including leukemia and lymphoma [5, 6]. Presently, two CD19 CAR-T products have been approved in the United States. However, when compared with CD19-CAR-T cells, the therapeutic effects of CAR-T cells on solid tumors are poor due to poor activity and the short survival time of CAR-T cells in solid tumors [7, 8]. To solve these problems, identifying novel techniques to enhance the antitumor ability of CAR-T cells in solid tumors is urgently warranted. As the crucial components for T cell activation, proliferation, and cytotoxicity, immunostimulatory cytokines have been proven to be able to significantly enhance the antitumor activity of CAR-T cells [9]. It has previously been reported that IL-7 prolonged the survival time of tumor-specific T cells in vivo [10]. Moreover, in preclinical studies, it was demonstrated that genetically modified T cells resulted in IL-7 secretion or IL-7 receptor overexpression, thereby achieving enhanced antitumor effects [11]. In previous studies, it has been reported that a constitutively activated IL-7 receptor (C7R) could result in IL-7 signaling in the absence of a ligand or with the existence of gamma chain (γc) of a coreceptor [12]. This may be because the insertion of a transmembrane domain such as cysteine and/or proline resulted in the homodimerization of IL-7Rα [6]. Upon the formation of a homodimer, cross-phosphorylation of JAK1/JAK1 would activate STAT5 [13], thereby activating the downstream signaling of IL-7. In a recent publication, it was shown that the antitumor activity of CAR-T cells that coexpressed C7R against metastatic neuroblastoma and in situ glioblastoma was significant.Here, we hypothesized that CAR-T cells that constitutively expressed C7R presented good antitumor activity against TNBC. AXL is a receptor tyrosine kinase (RTK) that was initially discovered in patients with chronic myeloid leukemia [14]. In a number of studies, it has been shown that AXL was abnormally overexpressed in human invasive and metastatic tumors [15], and its overexpression was significantly associated with a low survival rate [16]. Researchers have found that AXL mainly existed in the membrane of human breast cancer cells, and the expression of AXL in cancer tissues was much higher than in normal breast tissues from 23 normal human breast samples and 111 breast cancer tissue samples [16, 17]. These findings suggested that AXL was a good target for cancer treatment [18, 19]. Recently, it was reported that AXL-targeting CAR-T cells displayed antitumor activity against AXL-positive TNBC cells, indicating that it would be a potential therapeutic strategy for AXL-positive TNBC patients [20]. Therefore, we designed AXL-targeting CAR-T cells that coexpressed C7R, in order to investigate the therapeutic effects of enhanced CAR-T cells on TNBC. Our results suggested that CAR-T cells with constitutive expression of C7R exhibited significant antitumor activity against TNBC cells, which was higher when compared with that of traditional AXL-CAR-T cells. Furthermore, in vivo experiments showed that CAR-T cells with constitutive expression of C7R showed prolonged survival in mice and therefore may improve therapeutic effects and reduce tumor recurrence. Taken together, our findings showed that CAR-T cells with constitutive expression of C7R had significant antitumor activity against TNBC, which overcame the limitations of traditional CAR-T cells in the treatment of solid tumors and provided a novel strategy for the treatment of TNBC.


## 2. Methods and Materials

### 2.1. Cell Lines and Culture Conditions

After gaining the approval of informed consent form by the Examination Committee of the Affiliated Tumor Hospital of Xinjiang Medical University (Xinjiang, China), fresh blood samples were collected from healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated from blood by gradient centrifugation using LymphoprepTM (Axis-Shield, Norseland), followed by enrichment of T cells through positive screening using human T cell subtype CD3+ magnetic beads (Miltenyi Biotec Inc, Auburn, CA, USA). Subsequently, isolated T cells were cultured in X-VIVO15 medium (Lonza, Basel, Switzerland), supplemented with 5% human AB serum (Valley Biomedical Inc, Winchester, VA, USA), 10 mM·N-acetyl-L-cysteine (Sigma Aldrich, St. Louis, MO, USA), and 300 U/mL Human IL-2 (PeproTech, Rocky Hill, CT, USA).TMBC cell lines MDA-MB-231 and MDA-MB-468, and the breast cancer cell line MCF-7 were obtained from the American Type Culture Collection. In brief, MDA-MB-231 cells and MDA-MB-468 cells were cultured in L-15 medium (Hyclone, Logan, UT, USA), while MCF-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Hyclone). Cell culture media were supplemented with 10% fetal calf serum, 2 mmol/L-glutamine (Gibco, Gaithersburg, MD, USA), 100 U/mL penicillin, and 100μg/mL streptomycin (Sangong Biotech, Shanghai, China).


### 2.2. Construction of Plasmids

Lentiviral vectors encoding CAR or Luciferase were constructed using the pLV-puro vector (Hanbio Biotechnology Co., LTD, Shanghai, China). Briefly, to modify T cells, the AXL-CAR plasmid possessed the following elements (5′ to 3′ end): Xho Isite, signal peptide sequence, anti-AXL scFv, hinge region and CD8α transmembrane region, 4-1BB cytoplasmic domain, CD3ζ, P2A, and GFP sequences, as well as an aXba I site. The AXL-CAR.C7R plasmid was linked to the C7R sequence through IRES after CD3ζ. To modify target cells, the Luciferase vector included the following components (5′ end to 3′ end): Xho I site, Luciferase-linked RFP sequence through IRES, and an Xba I site. The sequences of genetic elements were obtained from the National Center for Biotechnology Information (NCBI). After codon optimization, the DNA sequence was synthesized and ligated into the pLV-puro vector via Xho I and Xba I sites.


### 2.3. Lentiviral Engineering of T Cells and Target Cells

Isolated T cells were activated by the addition of human CD3/CD28 beads (Invitrogen, Carlsbad, CA, USA) to the cell culture medium at a ratio of 2 : 1 at 48 hours prior to transduction. Then, the engineered virus was added to the cell culture with a MOI of 10, followed by polybrene (Yeasen Biotech, China) at a final concentration of 5μg/mL. Cells were centrifuged at 1200 × g for 60 minutes and incubated overnight at 37°C and 5% CO2. At 5 days after the transfection, modified T cells were harvested and the expression of CAR was determined by flow cytometry and Western blot analysis.All tumor cells, including MDA-MB-231, MDA-MB-468, and MCF-7 cells, were cultured to the log phase. When the cells reached a confluency of ∼70%, fresh complete medium containing 6μg/mL polybrene and 50 μL of virus was added to each well of a 6-well plate. After incubation for 24 hours, the medium was replaced by 2 ml of fresh complete medium. After 5 days of transduction, cells expressing RFP were selected by medium containing 1.5 μg/ml puromycin (Beyotime, Shanghai, China). RFP was detected by flow cytometry and Western blot to determine whether the transfection was successful.


### 2.4. Flow Cytometry and Western Blot Analysis

For flow cytometry, cells were collected by centrifugation and washed three times with FACS wash buffer (1x PBS with 0.5% BSA, and 0.03% sodium azide). Cell surface was performed using an anti-AXL antibody (Becton Dickinson (BD), San Jose, CA, USA). Regarding the evaluation of T cell activation, T cell surface marker CD69 was detected using an APC-conjugated CD69 antibody (Biolegend, San Diego, CA, USA) after overnight incubation of effective target cells. The phenotypes of T cells were determined using a FITC-conjugated CD4 antibody (BD) and a PE-conjugated CD8 antibody (BD). After incubation for 30 minutes at 4°C in the dark, cells were washed and analyzed by flow cytometry.


### 2.5. Western Blot Analysis

A total of 2 × 106 T cells were incubated with 200 μL of RIPA buffer system (Beyotime, Shanghai, China) for 10 minutes. The cell lysate was centrifuged to collect proteins, which were separated through SDS-PAGE, after which Western blot analysis was performed. The primary antibodies for CD34, CD3ζ, and GAPDH included rabbit anti-CD34 (CST, USA), mouse anti-CD3ζ (Abeam, UK), and mouse anti-GAPDH (Beyotime). The corresponding secondary antibodies were horseradish peroxidase (HRP) labeled goat antirabbit IgG (Beyotime) and goat antimouse IgG (Beyotime).


### 2.6. Enzyme-Linked Immunosorbent Assays

Forin vitro experiments, 1 × 104 cells were mixed with effective target cells at a ratio of 2 : 1 in a U-bottom 96-well plate. After incubation for 24 h, the supernatant was collected and the released IL-2, IFN-γ was detected by ELISA assay following the manufacturer’s instructions (MultiSciences, Hangzhou, China).Forin vivo experiments, 100 μL of peripheral blood was collected from mice 24 hours after the reinfusion of T cells. Serum cytokines, including IL-2, IL-4, IL-6, IFNγ, and TNFα, were analyzed by ELISA assays following the manufacturer’s instructions (MultiSciences).


### 2.7. Quantitation of T Cell Proliferation

Tumor cells were treated with 10μg/mL of Mitomycin C (Sigma Aldrich) for 2 h, and then 5 × 104 Mitomycin C-treated tumor cells were incubated with target cells. The number of target cells was 1 × 105 in all treatment groups, and the T cell density was maintained to 5 × 105/mL. After 7 days of coincubation, the cells were replaced by freshly treated tumor cells in all groups to perform antigen-specific stimulations for three times. Prior to the third stimulation, two sets of CAR-T cells were prestained with Cell Trace Violet (Invitrogen), and then stained CAR-T cells were cultured with MDA-MB-231 cells at an effect cell to target cell ratio of 2 : 1 for 96 hours. The intensity of Cell Trace Violet in each group was determined by flow cytometry. The ratio of CD8+ to CD4+ T cells was measured on the 18th day after two antigen-specific stimulations. No exogenous cytokines were added during the entire proliferation period.


### 2.8. Cytotoxicity Assays

A total of 1 × 104 tumor cells were seeded and incubated in 100 μL T cell culture medium containing 10% FBS at an effect cell to target cell ratio of 2 : 1 for 24 h at 37°C. The cytotoxicity of CAR-T cells towards target cells was evaluated at the level of lactate dehydrogenase (LDH) using an LDH cytotoxicity assay kit (Cayman, Ann Arbor, MI, USA). The experimental group and the control group were designed according to the manufacturer’s guidelines. The LDH activity in each sample was determined by measuring the absorbance at 490 nm using a Multiskan FC plate reader (Thermo Scientific, Waltham, MA, USA). Finally, the cytotoxicity of T cells was calculated according to the following formula: specific cytotoxicity (%)  =  (mixture cell experiment − effector cell spontaneous − target cell spontaneous − medium control)/(target cell maximum − target cell spontaneous − medium control) × 100.


### 2.9. Xenograft Mouse Models and Live Cell Imaging Assays

To establish xenograft mouse models, 5–7-week-old female NOD-SCID IL-2 receptor gamma null (NSG) mice were housed in the experimental animal research center of the Xinjiang Medical University (Xinjiang, China). All animal experiments were approved by the medical Ethics committee of Xinjiang Medical University (Xinjiang, China) and were in accordance with the national institutes of health regulations for the care and use of animals in the research. NSG mice were purchased from Shanghai Runnuo Biotechnology Co., Ltd. (Shanghai, China), housed in a sterile room, and were daily monitored. Xenograft models were established by subcutaneous injection with 1 × 107 MDA-MB-231-luc cells or MCF-7-luc cells mixed with Matrigel. At day 7 after injection, 100 μL of PBS, 1 × 107 control T cells, AXL-CAR-T cells, or AXL-CAR was given via caudal vein. However, for the MCF-7 group, only 100 μL of PBS or AXL-CAR.C7R T cells was injected. Bioluminescence was measured by a Xenogen IVIS Spectrum System (Life Technologies, Waltham, USA). Measurements were performed once a week. The dying mice were euthanized.


### 2.10. Quantitation of T Cell Counts

In the same animal experiment as described above, 100μL of blood was collected from the mice on the 27th day after tumor cell injection, in order to measure T cell proliferation in vivo. The total number of T cells in the collected blood sample was counted, and the number of CD8+ and CD4+ T cells was measured.


### 2.11. Immunohistochemistry Assay

To assess the persistence of human T cell and Ki67 expression in treated xenograft models, formalin-fixed paraffin-embedded tumor tissue sections were prepared and stained using mouse monoclonal anti-CD3 antibody (1 : 150, Thermo Scientific). Other tumor sections were incubated overnight with rabbit polyclonal anti-Ki67 antibody (1 : 100, Invitrogen). For the detection of human granzyme B, tumor tissue was stained with rabbit antihuman granzyme B polyclonal antibody (1 : 100, Abcam) for 45 minutes, and then the glass slide was costained with 3,3′-diaminobenzidine (DAB) and hematoxylin. Images were taken to quantify the number of CD3-positive and Ki67-positive cells, and granzyme B staining was analyzed by integral optical density (IDO) measurement using Image-Pro PLUS v6.0 software for data quantification.


### 2.12. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6.0. Differences between groups were assessed by Student’st-test, and P<0.05 was considered statistically significant.


## 2.1. Cell Lines and Culture Conditions

After gaining the approval of informed consent form by the Examination Committee of the Affiliated Tumor Hospital of Xinjiang Medical University (Xinjiang, China), fresh blood samples were collected from healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated from blood by gradient centrifugation using LymphoprepTM (Axis-Shield, Norseland), followed by enrichment of T cells through positive screening using human T cell subtype CD3+ magnetic beads (Miltenyi Biotec Inc, Auburn, CA, USA). Subsequently, isolated T cells were cultured in X-VIVO15 medium (Lonza, Basel, Switzerland), supplemented with 5% human AB serum (Valley Biomedical Inc, Winchester, VA, USA), 10 mM·N-acetyl-L-cysteine (Sigma Aldrich, St. Louis, MO, USA), and 300 U/mL Human IL-2 (PeproTech, Rocky Hill, CT, USA).TMBC cell lines MDA-MB-231 and MDA-MB-468, and the breast cancer cell line MCF-7 were obtained from the American Type Culture Collection. In brief, MDA-MB-231 cells and MDA-MB-468 cells were cultured in L-15 medium (Hyclone, Logan, UT, USA), while MCF-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Hyclone). Cell culture media were supplemented with 10% fetal calf serum, 2 mmol/L-glutamine (Gibco, Gaithersburg, MD, USA), 100 U/mL penicillin, and 100μg/mL streptomycin (Sangong Biotech, Shanghai, China).


## 2.2. Construction of Plasmids

Lentiviral vectors encoding CAR or Luciferase were constructed using the pLV-puro vector (Hanbio Biotechnology Co., LTD, Shanghai, China). Briefly, to modify T cells, the AXL-CAR plasmid possessed the following elements (5′ to 3′ end): Xho Isite, signal peptide sequence, anti-AXL scFv, hinge region and CD8α transmembrane region, 4-1BB cytoplasmic domain, CD3ζ, P2A, and GFP sequences, as well as an aXba I site. The AXL-CAR.C7R plasmid was linked to the C7R sequence through IRES after CD3ζ. To modify target cells, the Luciferase vector included the following components (5′ end to 3′ end): Xho I site, Luciferase-linked RFP sequence through IRES, and an Xba I site. The sequences of genetic elements were obtained from the National Center for Biotechnology Information (NCBI). After codon optimization, the DNA sequence was synthesized and ligated into the pLV-puro vector via Xho I and Xba I sites.


## 2.3. Lentiviral Engineering of T Cells and Target Cells

Isolated T cells were activated by the addition of human CD3/CD28 beads (Invitrogen, Carlsbad, CA, USA) to the cell culture medium at a ratio of 2 : 1 at 48 hours prior to transduction. Then, the engineered virus was added to the cell culture with a MOI of 10, followed by polybrene (Yeasen Biotech, China) at a final concentration of 5μg/mL. Cells were centrifuged at 1200 × g for 60 minutes and incubated overnight at 37°C and 5% CO2. At 5 days after the transfection, modified T cells were harvested and the expression of CAR was determined by flow cytometry and Western blot analysis.All tumor cells, including MDA-MB-231, MDA-MB-468, and MCF-7 cells, were cultured to the log phase. When the cells reached a confluency of ∼70%, fresh complete medium containing 6μg/mL polybrene and 50 μL of virus was added to each well of a 6-well plate. After incubation for 24 hours, the medium was replaced by 2 ml of fresh complete medium. After 5 days of transduction, cells expressing RFP were selected by medium containing 1.5 μg/ml puromycin (Beyotime, Shanghai, China). RFP was detected by flow cytometry and Western blot to determine whether the transfection was successful.


## 2.4. Flow Cytometry and Western Blot Analysis

For flow cytometry, cells were collected by centrifugation and washed three times with FACS wash buffer (1x PBS with 0.5% BSA, and 0.03% sodium azide). Cell surface was performed using an anti-AXL antibody (Becton Dickinson (BD), San Jose, CA, USA). Regarding the evaluation of T cell activation, T cell surface marker CD69 was detected using an APC-conjugated CD69 antibody (Biolegend, San Diego, CA, USA) after overnight incubation of effective target cells. The phenotypes of T cells were determined using a FITC-conjugated CD4 antibody (BD) and a PE-conjugated CD8 antibody (BD). After incubation for 30 minutes at 4°C in the dark, cells were washed and analyzed by flow cytometry.


## 2.5. Western Blot Analysis

A total of 2 × 106 T cells were incubated with 200 μL of RIPA buffer system (Beyotime, Shanghai, China) for 10 minutes. The cell lysate was centrifuged to collect proteins, which were separated through SDS-PAGE, after which Western blot analysis was performed. The primary antibodies for CD34, CD3ζ, and GAPDH included rabbit anti-CD34 (CST, USA), mouse anti-CD3ζ (Abeam, UK), and mouse anti-GAPDH (Beyotime). The corresponding secondary antibodies were horseradish peroxidase (HRP) labeled goat antirabbit IgG (Beyotime) and goat antimouse IgG (Beyotime).


## 2.6. Enzyme-Linked Immunosorbent Assays

Forin vitro experiments, 1 × 104 cells were mixed with effective target cells at a ratio of 2 : 1 in a U-bottom 96-well plate. After incubation for 24 h, the supernatant was collected and the released IL-2, IFN-γ was detected by ELISA assay following the manufacturer’s instructions (MultiSciences, Hangzhou, China).Forin vivo experiments, 100 μL of peripheral blood was collected from mice 24 hours after the reinfusion of T cells. Serum cytokines, including IL-2, IL-4, IL-6, IFNγ, and TNFα, were analyzed by ELISA assays following the manufacturer’s instructions (MultiSciences).


## 2.7. Quantitation of T Cell Proliferation

Tumor cells were treated with 10μg/mL of Mitomycin C (Sigma Aldrich) for 2 h, and then 5 × 104 Mitomycin C-treated tumor cells were incubated with target cells. The number of target cells was 1 × 105 in all treatment groups, and the T cell density was maintained to 5 × 105/mL. After 7 days of coincubation, the cells were replaced by freshly treated tumor cells in all groups to perform antigen-specific stimulations for three times. Prior to the third stimulation, two sets of CAR-T cells were prestained with Cell Trace Violet (Invitrogen), and then stained CAR-T cells were cultured with MDA-MB-231 cells at an effect cell to target cell ratio of 2 : 1 for 96 hours. The intensity of Cell Trace Violet in each group was determined by flow cytometry. The ratio of CD8+ to CD4+ T cells was measured on the 18th day after two antigen-specific stimulations. No exogenous cytokines were added during the entire proliferation period.


## 2.8. Cytotoxicity Assays

A total of 1 × 104 tumor cells were seeded and incubated in 100 μL T cell culture medium containing 10% FBS at an effect cell to target cell ratio of 2 : 1 for 24 h at 37°C. The cytotoxicity of CAR-T cells towards target cells was evaluated at the level of lactate dehydrogenase (LDH) using an LDH cytotoxicity assay kit (Cayman, Ann Arbor, MI, USA). The experimental group and the control group were designed according to the manufacturer’s guidelines. The LDH activity in each sample was determined by measuring the absorbance at 490 nm using a Multiskan FC plate reader (Thermo Scientific, Waltham, MA, USA). Finally, the cytotoxicity of T cells was calculated according to the following formula: specific cytotoxicity (%)  =  (mixture cell experiment − effector cell spontaneous − target cell spontaneous − medium control)/(target cell maximum − target cell spontaneous − medium control) × 100.


## 2.9. Xenograft Mouse Models and Live Cell Imaging Assays

To establish xenograft mouse models, 5–7-week-old female NOD-SCID IL-2 receptor gamma null (NSG) mice were housed in the experimental animal research center of the Xinjiang Medical University (Xinjiang, China). All animal experiments were approved by the medical Ethics committee of Xinjiang Medical University (Xinjiang, China) and were in accordance with the national institutes of health regulations for the care and use of animals in the research. NSG mice were purchased from Shanghai Runnuo Biotechnology Co., Ltd. (Shanghai, China), housed in a sterile room, and were daily monitored. Xenograft models were established by subcutaneous injection with 1 × 107 MDA-MB-231-luc cells or MCF-7-luc cells mixed with Matrigel. At day 7 after injection, 100 μL of PBS, 1 × 107 control T cells, AXL-CAR-T cells, or AXL-CAR was given via caudal vein. However, for the MCF-7 group, only 100 μL of PBS or AXL-CAR.C7R T cells was injected. Bioluminescence was measured by a Xenogen IVIS Spectrum System (Life Technologies, Waltham, USA). Measurements were performed once a week. The dying mice were euthanized.


## 2.10. Quantitation of T Cell Counts

In the same animal experiment as described above, 100μL of blood was collected from the mice on the 27th day after tumor cell injection, in order to measure T cell proliferation in vivo. The total number of T cells in the collected blood sample was counted, and the number of CD8+ and CD4+ T cells was measured.


## 2.11. Immunohistochemistry Assay

To assess the persistence of human T cell and Ki67 expression in treated xenograft models, formalin-fixed paraffin-embedded tumor tissue sections were prepared and stained using mouse monoclonal anti-CD3 antibody (1 : 150, Thermo Scientific). Other tumor sections were incubated overnight with rabbit polyclonal anti-Ki67 antibody (1 : 100, Invitrogen). For the detection of human granzyme B, tumor tissue was stained with rabbit antihuman granzyme B polyclonal antibody (1 : 100, Abcam) for 45 minutes, and then the glass slide was costained with 3,3′-diaminobenzidine (DAB) and hematoxylin. Images were taken to quantify the number of CD3-positive and Ki67-positive cells, and granzyme B staining was analyzed by integral optical density (IDO) measurement using Image-Pro PLUS v6.0 software for data quantification.


## 2.12. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6.0. Differences between groups were assessed by Student’st-test, and P<0.05 was considered statistically significant.


## 3. Results

### 3.1. Construction of Antigen-Specific Cells and Target Cells

In this study, the AXL expression of triple-negative breast cancer cells MDA-MB-231, MDA-MB-436, MDA-MB-453, and MDA-MB-468 were detected by flow cytometry. These cells all highly expressed AXL, and we selected MDA-MB-231 and MDA-MB-468 as target cells. Moreover, the AXL-negative breast cancer cell line, MCF-7, was selected as the control (Figure1(a)). For cellular and animal experiments, lentiviral transfection was employed to overexpress all tumor target cells with an RFP-tagged Luciferase. The cell lines MDA-MB-231-luc, MDA-MB-468-luc, and MCF-7-luc that overexpressed the red fluorescent protein were obtained by antibiotic screening.Figure 1
Screening of target cells and the construction of engineered T cells. (a) The expression levels of AXL in target cells detected by flow cytometry. (b) Schematic diagram of C7R, AXL-CAR, and AXL-CAR.C7R. (c) Working principle diagram of C7R. (d) CD3ζ and CD34 were detected by immunoblot assay. Exogenous CD3ζ was presented at 70 kDa only in the lanes of CAR-T cells, and endogenous CD3ζ was presented at 17 kDa in the lanes of control T cells and CAR-T cells. CD34 is presented in cells containing C7R. GAPDH is used as an internal standard. (e) The transfection efficiency of engineered T cells was determined by GFP expression in T cells using flow cytometry.
(a)
(b)
(c)
(d)
(e)To obtain effective T cells, a second-generation CAR sequence targeting AXL was first constructed, and T cells were activated through CD3ζ mediation with 4-1BB as the costimulatory signal. The preparation of the constitutively activated IL-7 receptor was performed as described previously [13]. Specifically, to avoid additional activation of the paired receptor in the extracellular domain of the IL-7 receptor, the natural extracellular domain of the receptor was substituted by the extracellular domain of CD34 to construct a recombinant receptor for CD34-IL7R∗, which was denoted as C7R (Figure 1(c)). Subsequently, based on the AXL-specific second-generation CAR, an enhanced CAR sequence that coexpressed C7R was achieved. Furthermore, a sequence that only expressed C7R was constructed as a control. All sequences were inserted into the sequence of fluorescent reporter protein GFP to help detect expression of the CAR in the modified T cells (Figure 1(b)). After lentivirus transfection, the expression of CD34 and exogenous CD3ζ was detected by WB to determine the transfection of CAR (Figure 1(d)), and the expression of GFP was detected by flow cytometry to further verify whether CAR-T cells were successfully constructed (Figure 1(e)).


### 3.2. Determination of the Optimal T Cells to Target Cell Ratio of AXL-CAR-T Cells

To evaluate the activity of AXL-CAR-T cells, the ratio between T cell and the target cell was optimized by cytotoxicity experiments. Based on previous experiments [21] and to visualize the antitumor activity of the enhanced CAR-T cells, effector cell: target cell ratio of 2 : 1, 1 : 1, and 1 : 2, respectively, were tested. After overnight incubation of effector cells and target cells, supernatants were collected from each well and the LDH concentration was measured to evaluate the antitumor activity of CAR-T cells at different ratios with target cells. The results showed that for AXL-positive TNBC cells, MDA-MB-231 and MDA-MB-468, AXL-CAR-T cells presented a higher level of cytotoxicity at effector cell: target cell ratio of 2 : 1 (Figure 2(a)). Therefore, subsequent experiments were conducted at T cell : target cell ratio of 2 : 1.Figure 2
The effect of C7R on the activation of AXL-CAR-T cells. (a) Determination of the optimal target ratio. A total of 1 × 104 target cells were incubated with T cells at different ratios. The concentrations of LDH released in the supernatant were measured 24 hours later. (b) A total of 1 × 104 target cells were incubated with T cells for 24 h at a ratio of 2 : 1. The expression level of CD69 in T cells was measured by flow cytometry. (c) A total of 1 × 104 target cells were incubated with T cells for 24 h at a ratio of 2 : 1, levels of IL-2 secretion were measured (n = 3, ∗P<0.05, ∗∗P<0.01). (d) The concentrations of secreted IFNγ (n = 3, ∗P<0.05, ∗∗P<0.01).
(a)
(b)
(c)
(d)


### 3.3. C7R Significantly Enhanced the Activation of AXL-CAR-T CellsIn Vitro

To evaluate the effects of C7R on the activation of AXL-CAR-T cells, three experimental groups were set up, including C7R transfected T cells (C7R group), AXL-CAR-T cells (AXL-CAR-T group), and a C7R transfected AXL-CAR-T cells (AXL-CAR-T.C7R group). The target cells were AXL-positive TNBC cells, MDA-MB-231, and MDA-MB-468, and AXL-negative breast cancer cells, MCF-7. After incubation for 24 h at effector cell : target cell ratio of 2 : 1, the expression level of activation marker CD69 on the surface of T cells was detected (Figure2(b)), and the concentrations of cytokines (IL-2, IFN-γ) in the supernatant were measured (Figures 2(c) and 2(d)).The expression of activated molecules on the T cell surface was significantly increased after activation of the T cell. In our study, when the target cells were AXL-positive MDA-MB-231 and MDA-MB-468, the expression level of CD69 was significantly higher in the AXL-CAR.C7R group compared to that in the AXL-CAR-T group. However, the expression level of CD69 was not significantly changed, when AXL-negative MCF-7 cells were used as the target cells. In addition, when T cells were activated, a large number of cytokines, such as IL-2 and IFN-γ were released. Our results indicated that compared to AXL-negative MCF-7 cells, the release of cytokines was significantly promoted in the AXL-CAR-T and AXL-CAR-T.C7R groups, when the target cells were AXL-positive TNBC cells, MDA-MB-231 cells and MDA-MB-468 cells. Furthermore, the cytokine levels were higher in the AXL-CAR-T.C7R group compared to the AXL-CAR-T group. Briefly, in the AXL-negative target cell group, AXL-CAR-T cells were not activated and the cytokine levels released by T cells were not significantly different from those of the C7R control group. However, in the AXL-positive target cell groups, due to the activation of AXL-CAR-T cells, the levels of released cytokines were significantly increased when compared with those of the control group. In addition, the expression of C7R could further enhance the release of cytokines in AXL-CAR-T cells. Taken together, our results suggested that the expression of C7R could effectively activate AXL-CAR-T cells in vitro.


### 3.4. Effect of C7R on the Proliferation Ability of AXL-CAR-T CellsIn Vitro

To evaluate whether the expression of C7R affected the proliferation of AXL-CAR-T cells, Mitomycin C-treated MTA-MB-231 cells were used to stimulate T cells weekly in the absence of exogenous cytokines. On day 22, Cell Trace Violet staining was used to evaluate the proliferation level of T cells. The results showed that the AXL-CAR-T.C7R group displayed higher cell proliferation ability than the AXL-CAR-T group after repeated antigen stimulation (Figure3(a)). Previous studies have shown that a high CD8 : CD4 ratio was an effective indicator for adoptive T cell immunotherapy to perform better anticancer effects [22]. Therefore, on day 18 after repeated antigen stimulation, the CD8 : CD4 ratio was determined. The results demonstrated that no significant difference was observed in the CD8 : CD4 ratio between AXL-CAR-T and AXL-CAR-T.C7R groups before antigen stimulation, whereas after repeated antigen stimulation, the CD8 : CD4 ratio in the AXL-CAR-T.C7R group was significantly higher compared to that of the AXL-CAR-T group (Figure 3(b)). According to these findings, it was found that C7R enhanced the proliferation of AXL-CAR-T cells and increased the CD8 : CD4 ratio, thereby leading to better therapeutic effects.Figure 3
The effect of C7R on the proliferation and cytotoxicity of AXL-CAR-T cells. (a) A total of 1 × 105 Mitomycin C-treated tumor cells were incubated with 2 × 105 T cells. The Mitomycin C-treated tumor cells were refreshed every week. After three antigen stimulations, the proliferation of two sets of CAR-T cells was compared. Prior to the third stimulation, the two sets of CAR-T cells were prestained with Cell Trace Violet, and stained CAR-T cells were cultured with MDA-MB-231 at a ratio of 2 : 1 for 96 hours. The intensity of Cell Trace Violet in each group was measured by flow cytometry. (b) After two antigen stimulations, the ratio of CD8+ to CD4+ T cells was measured on day 18 (n = 3, ns, P>0.05, ∗P<0.05). (c) A total of 1 × 104 tumor cells were incubated with T cells at a ratio of 2 : 1, and levels of LDH in the supernatant were measured 24 hours later to evaluate the cytotoxicity of T cells (n = 3, ∗P<0.05).
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### 3.5. C7R Increased the Killing Effect of AXL-CAR-T Cells on TNBC Cells

In ourin vitro studies, CAR-T cells were found to specifically target and kill tumor cells. Therefore, we explored whether C7R could increase the cytotoxicity of AXL-CAR-T cells to TNBC cells. The cytotoxicity of AXL-CAR-T cells was evaluated by measuring the amount of LDH in the supernatant when AXL-CAR-T cells were incubated with target cells. Three experimental groups (C7R group, AXL-CAR-T group, and AXL-CAR-T.C7R group) were incubated with three different target cells including MDA-MB-231, MDA-MB-468, and MCF-7 cells. After overnight incubation, the concentrations of LDH in the supernatant were compared, and it was found that AXL-CAR-T cells presented obvious cytotoxicity to the AXL-positive target cells. Moreover, the AXL-CAR-T.C7R group had a stronger killing ability against AXL-positive target cells compared with the AXL-CAR-T group. In the other groups, no significant release of LDH was observed (Figure 3(c)). Through cellular toxicity experiments, it was demonstrated that AXL-CAR-T cells were cytotoxic to AXL-positive cells, and that C7R could significantly enhance the antitumor activity of AXL-CAR-T cells to TNBC cells.


### 3.6. C7R Enhanced the Antitumor Activity of AXL-CAR-T Cells in Heterologous Tumor Models

To further compare thein vivo cytotoxic activity of AXL-CAR-T and AXL-CAR.C7R T cells, NOD/SCID mice models bearing subcutaneous MDA-MB-231-luc or MCF-7-luc xenograft tumors were established. The fluorescence intensity of tumor cells was monitored through in vivo imaging to assess tumor regression in mice (Figures 1(a) and 1(b)). The results of mean fluorescence intensity in vivo imaging (Figures 4(c) and 4(d)) showed that AXL-CAR-T and AXL-CAR.C7R T cells were effective in reducing the tumor sizes in mice bearing MDA-MB-231 xenograft tumors. On day 7 after T cell injection through the caudal vein, the tumor sizes in the AXL-CAR.C7R group were smaller compared to those in the AXL-CAR-T group. To assess the nonspecific cytotoxicity of AXL-CAR-T cells, we established mouse models with AXL-negative MCF-7 xenograft tumor as controls. Our results showed that AXL-CAR.C7R cell-treated mice showed no inhibition in tumor growth when compared with the PBS group. In addition, no significant changes in mouse body weight were observed during treatment (Figures 4(e) and 4(f)). In summary, the animal experiments were consistent with those obtained from the in vitro experiments, indicating that AXL-CAR-T cells coexpressing C7R and AXL-CAR-T cells both had significant antitumor effects on TNBC.Figure 4
The tumor elimination in mice mediated by engineered CAR-T cells. Bioluminescencein vivo was collected by in vivo imaging starting from the time when mice were injected with tumor cells. (a) An image of the MDA-MB-231-luc allograft tumor in mice. (b) An image of the MCF-7-luc allograft tumor in mice. (c) Fluorescence intensity changes of MDA-MB-231-luc heterologous tumors in mice. (d) Changes in fluorescence intensity of MCF-7-luc heterologous tumor in mice. (e) Body weight changes of mice with MDA-MB-231-luc allogeneic tumors after tumor cell injection and T cell treatment. (f) Body weight changes of mice with MCF-7-luc allogeneic tumors after tumor cell injection and T cell treatment (n = 4, bar value represents dispersion degree).
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### 3.7. Release of CytokinesIn Vivo and Proliferation of AXL-CAR-T Cells

In general, CAR-T cells would kill tumor cells accompanied with the release of cytokines, including IL-2, IL-4, IL-6, IFNγ, and TNFα. In this study, the concentrations of cytokines in the serum of mice treated with AXL-CAR-T cells were significantly elevated in mice bearing AXL-positive tumors. Furthermore, the increase in IL-2, IL-4, IL-6, IFNγ, and TNFα was higher in mice treated with AXL-CAR-T cells expressing C7R compared to mice treated with AXL-CAR-T cells. The cytokine levels released in serum were consistent with the level of tumor elimination observed in each treatment group. In addition, there was no significant increase of cytokines observed in AXL-negative MCF-7 tumor-bearing mice (Figures 5(a)–5(e)).Figure 5
In vivo cytokine release and the proliferation of T cells in mice. (a)–(e) The concentrations of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ) in peripheral blood of mice, which was collected at 24 hours after the administration of T cells (n = 4, ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001). Measurements were performed with 100 μL peripheral blood using ELISA. (b) On day 27, 100 μL of peripheral blood was sampled from each mouse and the number of circulating human CD4+ and CD8+ T cells was determined by flow cytometry (n = 4, ∗P<0.05, ∗∗P<0.01).
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(f)The survival of AXL-CAR-T cells was also monitored during tumor elimination. On day 7 after treatment, peripheral blood was collected from mice for AXL-CAR-T cell count and phenotypic analysis. The results showed that in MDA-MB-231 tumor-bearing mice, AXL-CAR-T cells that coexpressed C7R presented higher proliferation levels than AXL-CAR-T cells, and the CD8 : CD4 ratio was significantly higher (Figure5(f)). These findings were consistent with the tumor elimination in mice.


### 3.8. Infiltration of AXL-CAR-T Cells and Expression of Ki67 and Granzyme B

In previous studies, it has been shown that the degree of infiltration of CAR-T cells into tumor tissues highly correlated with the therapeutic effects of CAR-T cells [22]. Therefore, immunohistochemical analysis of mouse tumor was performed. The analysis showed that the number of infiltrating T cells in tumor tissues from the AXL-CAR.C7R group was higher than that from the AXL-CAR-T group (Figures 6(a) and 6(b)). This was consistent with previous measurements of circulating T cells in mouse peripheral blood. Next, we measured the intensity of Ki67 staining in tumor tissues, and the results indicated that the group treated with AXL-CAR-T cell with coexpressing C7R had dramatically lower intensity of Ki67staining than the other two groups (Figures 6(c) and 6(d)). Furthermore, among three groups, the highest level of granzyme B expression was found in the group treated with AXL-CAR-T cell expressing C7R (Figures 6(e) and 6(f)). These results suggested that the expression of C7R in AXL-CAR-T cells could prolong the persistence of AXL-CAR-T cells and improve their antitumor activity in vivo.Figure 6
Immunohistochemistry of CD3, Ki67, and granzyme B in mouse tumor tissue. (a) Detection of CD3 on tumor tissue from the MDA-MB-231 xenograft mouse model. (b) Determination of the number of CD3-positive cells by cell counting. (c) Detection of Ki67 on tumor tissue. (d) Determination of the number of Ki67-positive cells by cell counting. (e) Detection of granzyme B on tumor tissue. (f) The positivity of granzyme B staining was analyzed by integrated optical density (IDO) using Image-Pro PLUS v6.0 software. The image was taken at a magnification of ×400. Data were from three independent experiments (n = 4, bar values represent dispersion degrees; ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001).
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(f)Together, the above experiments showed that the expression of C7R could improve the antitumor activity of AXL-CAR-T cells against TNBC bothin vitro and in vivo and could partly overcome the inhibition of CAR-T cell activity in tumor microenvironment. Thus, enhanced CAR-T cell therapy could be an effective strategy for the treatment of TNBC.


## 3.1. Construction of Antigen-Specific Cells and Target Cells

In this study, the AXL expression of triple-negative breast cancer cells MDA-MB-231, MDA-MB-436, MDA-MB-453, and MDA-MB-468 were detected by flow cytometry. These cells all highly expressed AXL, and we selected MDA-MB-231 and MDA-MB-468 as target cells. Moreover, the AXL-negative breast cancer cell line, MCF-7, was selected as the control (Figure1(a)). For cellular and animal experiments, lentiviral transfection was employed to overexpress all tumor target cells with an RFP-tagged Luciferase. The cell lines MDA-MB-231-luc, MDA-MB-468-luc, and MCF-7-luc that overexpressed the red fluorescent protein were obtained by antibiotic screening.Figure 1
Screening of target cells and the construction of engineered T cells. (a) The expression levels of AXL in target cells detected by flow cytometry. (b) Schematic diagram of C7R, AXL-CAR, and AXL-CAR.C7R. (c) Working principle diagram of C7R. (d) CD3ζ and CD34 were detected by immunoblot assay. Exogenous CD3ζ was presented at 70 kDa only in the lanes of CAR-T cells, and endogenous CD3ζ was presented at 17 kDa in the lanes of control T cells and CAR-T cells. CD34 is presented in cells containing C7R. GAPDH is used as an internal standard. (e) The transfection efficiency of engineered T cells was determined by GFP expression in T cells using flow cytometry.
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(e)To obtain effective T cells, a second-generation CAR sequence targeting AXL was first constructed, and T cells were activated through CD3ζ mediation with 4-1BB as the costimulatory signal. The preparation of the constitutively activated IL-7 receptor was performed as described previously [13]. Specifically, to avoid additional activation of the paired receptor in the extracellular domain of the IL-7 receptor, the natural extracellular domain of the receptor was substituted by the extracellular domain of CD34 to construct a recombinant receptor for CD34-IL7R∗, which was denoted as C7R (Figure 1(c)). Subsequently, based on the AXL-specific second-generation CAR, an enhanced CAR sequence that coexpressed C7R was achieved. Furthermore, a sequence that only expressed C7R was constructed as a control. All sequences were inserted into the sequence of fluorescent reporter protein GFP to help detect expression of the CAR in the modified T cells (Figure 1(b)). After lentivirus transfection, the expression of CD34 and exogenous CD3ζ was detected by WB to determine the transfection of CAR (Figure 1(d)), and the expression of GFP was detected by flow cytometry to further verify whether CAR-T cells were successfully constructed (Figure 1(e)).


## 3.2. Determination of the Optimal T Cells to Target Cell Ratio of AXL-CAR-T Cells

To evaluate the activity of AXL-CAR-T cells, the ratio between T cell and the target cell was optimized by cytotoxicity experiments. Based on previous experiments [21] and to visualize the antitumor activity of the enhanced CAR-T cells, effector cell: target cell ratio of 2 : 1, 1 : 1, and 1 : 2, respectively, were tested. After overnight incubation of effector cells and target cells, supernatants were collected from each well and the LDH concentration was measured to evaluate the antitumor activity of CAR-T cells at different ratios with target cells. The results showed that for AXL-positive TNBC cells, MDA-MB-231 and MDA-MB-468, AXL-CAR-T cells presented a higher level of cytotoxicity at effector cell: target cell ratio of 2 : 1 (Figure 2(a)). Therefore, subsequent experiments were conducted at T cell : target cell ratio of 2 : 1.Figure 2
The effect of C7R on the activation of AXL-CAR-T cells. (a) Determination of the optimal target ratio. A total of 1 × 104 target cells were incubated with T cells at different ratios. The concentrations of LDH released in the supernatant were measured 24 hours later. (b) A total of 1 × 104 target cells were incubated with T cells for 24 h at a ratio of 2 : 1. The expression level of CD69 in T cells was measured by flow cytometry. (c) A total of 1 × 104 target cells were incubated with T cells for 24 h at a ratio of 2 : 1, levels of IL-2 secretion were measured (n = 3, ∗P<0.05, ∗∗P<0.01). (d) The concentrations of secreted IFNγ (n = 3, ∗P<0.05, ∗∗P<0.01).
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## 3.3. C7R Significantly Enhanced the Activation of AXL-CAR-T CellsIn Vitro

To evaluate the effects of C7R on the activation of AXL-CAR-T cells, three experimental groups were set up, including C7R transfected T cells (C7R group), AXL-CAR-T cells (AXL-CAR-T group), and a C7R transfected AXL-CAR-T cells (AXL-CAR-T.C7R group). The target cells were AXL-positive TNBC cells, MDA-MB-231, and MDA-MB-468, and AXL-negative breast cancer cells, MCF-7. After incubation for 24 h at effector cell : target cell ratio of 2 : 1, the expression level of activation marker CD69 on the surface of T cells was detected (Figure2(b)), and the concentrations of cytokines (IL-2, IFN-γ) in the supernatant were measured (Figures 2(c) and 2(d)).The expression of activated molecules on the T cell surface was significantly increased after activation of the T cell. In our study, when the target cells were AXL-positive MDA-MB-231 and MDA-MB-468, the expression level of CD69 was significantly higher in the AXL-CAR.C7R group compared to that in the AXL-CAR-T group. However, the expression level of CD69 was not significantly changed, when AXL-negative MCF-7 cells were used as the target cells. In addition, when T cells were activated, a large number of cytokines, such as IL-2 and IFN-γ were released. Our results indicated that compared to AXL-negative MCF-7 cells, the release of cytokines was significantly promoted in the AXL-CAR-T and AXL-CAR-T.C7R groups, when the target cells were AXL-positive TNBC cells, MDA-MB-231 cells and MDA-MB-468 cells. Furthermore, the cytokine levels were higher in the AXL-CAR-T.C7R group compared to the AXL-CAR-T group. Briefly, in the AXL-negative target cell group, AXL-CAR-T cells were not activated and the cytokine levels released by T cells were not significantly different from those of the C7R control group. However, in the AXL-positive target cell groups, due to the activation of AXL-CAR-T cells, the levels of released cytokines were significantly increased when compared with those of the control group. In addition, the expression of C7R could further enhance the release of cytokines in AXL-CAR-T cells. Taken together, our results suggested that the expression of C7R could effectively activate AXL-CAR-T cells in vitro.


## 3.4. Effect of C7R on the Proliferation Ability of AXL-CAR-T CellsIn Vitro

To evaluate whether the expression of C7R affected the proliferation of AXL-CAR-T cells, Mitomycin C-treated MTA-MB-231 cells were used to stimulate T cells weekly in the absence of exogenous cytokines. On day 22, Cell Trace Violet staining was used to evaluate the proliferation level of T cells. The results showed that the AXL-CAR-T.C7R group displayed higher cell proliferation ability than the AXL-CAR-T group after repeated antigen stimulation (Figure3(a)). Previous studies have shown that a high CD8 : CD4 ratio was an effective indicator for adoptive T cell immunotherapy to perform better anticancer effects [22]. Therefore, on day 18 after repeated antigen stimulation, the CD8 : CD4 ratio was determined. The results demonstrated that no significant difference was observed in the CD8 : CD4 ratio between AXL-CAR-T and AXL-CAR-T.C7R groups before antigen stimulation, whereas after repeated antigen stimulation, the CD8 : CD4 ratio in the AXL-CAR-T.C7R group was significantly higher compared to that of the AXL-CAR-T group (Figure 3(b)). According to these findings, it was found that C7R enhanced the proliferation of AXL-CAR-T cells and increased the CD8 : CD4 ratio, thereby leading to better therapeutic effects.Figure 3
The effect of C7R on the proliferation and cytotoxicity of AXL-CAR-T cells. (a) A total of 1 × 105 Mitomycin C-treated tumor cells were incubated with 2 × 105 T cells. The Mitomycin C-treated tumor cells were refreshed every week. After three antigen stimulations, the proliferation of two sets of CAR-T cells was compared. Prior to the third stimulation, the two sets of CAR-T cells were prestained with Cell Trace Violet, and stained CAR-T cells were cultured with MDA-MB-231 at a ratio of 2 : 1 for 96 hours. The intensity of Cell Trace Violet in each group was measured by flow cytometry. (b) After two antigen stimulations, the ratio of CD8+ to CD4+ T cells was measured on day 18 (n = 3, ns, P>0.05, ∗P<0.05). (c) A total of 1 × 104 tumor cells were incubated with T cells at a ratio of 2 : 1, and levels of LDH in the supernatant were measured 24 hours later to evaluate the cytotoxicity of T cells (n = 3, ∗P<0.05).
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## 3.5. C7R Increased the Killing Effect of AXL-CAR-T Cells on TNBC Cells

In ourin vitro studies, CAR-T cells were found to specifically target and kill tumor cells. Therefore, we explored whether C7R could increase the cytotoxicity of AXL-CAR-T cells to TNBC cells. The cytotoxicity of AXL-CAR-T cells was evaluated by measuring the amount of LDH in the supernatant when AXL-CAR-T cells were incubated with target cells. Three experimental groups (C7R group, AXL-CAR-T group, and AXL-CAR-T.C7R group) were incubated with three different target cells including MDA-MB-231, MDA-MB-468, and MCF-7 cells. After overnight incubation, the concentrations of LDH in the supernatant were compared, and it was found that AXL-CAR-T cells presented obvious cytotoxicity to the AXL-positive target cells. Moreover, the AXL-CAR-T.C7R group had a stronger killing ability against AXL-positive target cells compared with the AXL-CAR-T group. In the other groups, no significant release of LDH was observed (Figure 3(c)). Through cellular toxicity experiments, it was demonstrated that AXL-CAR-T cells were cytotoxic to AXL-positive cells, and that C7R could significantly enhance the antitumor activity of AXL-CAR-T cells to TNBC cells.


## 3.6. C7R Enhanced the Antitumor Activity of AXL-CAR-T Cells in Heterologous Tumor Models

To further compare thein vivo cytotoxic activity of AXL-CAR-T and AXL-CAR.C7R T cells, NOD/SCID mice models bearing subcutaneous MDA-MB-231-luc or MCF-7-luc xenograft tumors were established. The fluorescence intensity of tumor cells was monitored through in vivo imaging to assess tumor regression in mice (Figures 1(a) and 1(b)). The results of mean fluorescence intensity in vivo imaging (Figures 4(c) and 4(d)) showed that AXL-CAR-T and AXL-CAR.C7R T cells were effective in reducing the tumor sizes in mice bearing MDA-MB-231 xenograft tumors. On day 7 after T cell injection through the caudal vein, the tumor sizes in the AXL-CAR.C7R group were smaller compared to those in the AXL-CAR-T group. To assess the nonspecific cytotoxicity of AXL-CAR-T cells, we established mouse models with AXL-negative MCF-7 xenograft tumor as controls. Our results showed that AXL-CAR.C7R cell-treated mice showed no inhibition in tumor growth when compared with the PBS group. In addition, no significant changes in mouse body weight were observed during treatment (Figures 4(e) and 4(f)). In summary, the animal experiments were consistent with those obtained from the in vitro experiments, indicating that AXL-CAR-T cells coexpressing C7R and AXL-CAR-T cells both had significant antitumor effects on TNBC.Figure 4
The tumor elimination in mice mediated by engineered CAR-T cells. Bioluminescencein vivo was collected by in vivo imaging starting from the time when mice were injected with tumor cells. (a) An image of the MDA-MB-231-luc allograft tumor in mice. (b) An image of the MCF-7-luc allograft tumor in mice. (c) Fluorescence intensity changes of MDA-MB-231-luc heterologous tumors in mice. (d) Changes in fluorescence intensity of MCF-7-luc heterologous tumor in mice. (e) Body weight changes of mice with MDA-MB-231-luc allogeneic tumors after tumor cell injection and T cell treatment. (f) Body weight changes of mice with MCF-7-luc allogeneic tumors after tumor cell injection and T cell treatment (n = 4, bar value represents dispersion degree).
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## 3.7. Release of CytokinesIn Vivo and Proliferation of AXL-CAR-T Cells

In general, CAR-T cells would kill tumor cells accompanied with the release of cytokines, including IL-2, IL-4, IL-6, IFNγ, and TNFα. In this study, the concentrations of cytokines in the serum of mice treated with AXL-CAR-T cells were significantly elevated in mice bearing AXL-positive tumors. Furthermore, the increase in IL-2, IL-4, IL-6, IFNγ, and TNFα was higher in mice treated with AXL-CAR-T cells expressing C7R compared to mice treated with AXL-CAR-T cells. The cytokine levels released in serum were consistent with the level of tumor elimination observed in each treatment group. In addition, there was no significant increase of cytokines observed in AXL-negative MCF-7 tumor-bearing mice (Figures 5(a)–5(e)).Figure 5
In vivo cytokine release and the proliferation of T cells in mice. (a)–(e) The concentrations of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ) in peripheral blood of mice, which was collected at 24 hours after the administration of T cells (n = 4, ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001). Measurements were performed with 100 μL peripheral blood using ELISA. (b) On day 27, 100 μL of peripheral blood was sampled from each mouse and the number of circulating human CD4+ and CD8+ T cells was determined by flow cytometry (n = 4, ∗P<0.05, ∗∗P<0.01).
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(f)The survival of AXL-CAR-T cells was also monitored during tumor elimination. On day 7 after treatment, peripheral blood was collected from mice for AXL-CAR-T cell count and phenotypic analysis. The results showed that in MDA-MB-231 tumor-bearing mice, AXL-CAR-T cells that coexpressed C7R presented higher proliferation levels than AXL-CAR-T cells, and the CD8 : CD4 ratio was significantly higher (Figure5(f)). These findings were consistent with the tumor elimination in mice.


## 3.8. Infiltration of AXL-CAR-T Cells and Expression of Ki67 and Granzyme B

In previous studies, it has been shown that the degree of infiltration of CAR-T cells into tumor tissues highly correlated with the therapeutic effects of CAR-T cells [22]. Therefore, immunohistochemical analysis of mouse tumor was performed. The analysis showed that the number of infiltrating T cells in tumor tissues from the AXL-CAR.C7R group was higher than that from the AXL-CAR-T group (Figures 6(a) and 6(b)). This was consistent with previous measurements of circulating T cells in mouse peripheral blood. Next, we measured the intensity of Ki67 staining in tumor tissues, and the results indicated that the group treated with AXL-CAR-T cell with coexpressing C7R had dramatically lower intensity of Ki67staining than the other two groups (Figures 6(c) and 6(d)). Furthermore, among three groups, the highest level of granzyme B expression was found in the group treated with AXL-CAR-T cell expressing C7R (Figures 6(e) and 6(f)). These results suggested that the expression of C7R in AXL-CAR-T cells could prolong the persistence of AXL-CAR-T cells and improve their antitumor activity in vivo.Figure 6
Immunohistochemistry of CD3, Ki67, and granzyme B in mouse tumor tissue. (a) Detection of CD3 on tumor tissue from the MDA-MB-231 xenograft mouse model. (b) Determination of the number of CD3-positive cells by cell counting. (c) Detection of Ki67 on tumor tissue. (d) Determination of the number of Ki67-positive cells by cell counting. (e) Detection of granzyme B on tumor tissue. (f) The positivity of granzyme B staining was analyzed by integrated optical density (IDO) using Image-Pro PLUS v6.0 software. The image was taken at a magnification of ×400. Data were from three independent experiments (n = 4, bar values represent dispersion degrees; ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001).
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(f)Together, the above experiments showed that the expression of C7R could improve the antitumor activity of AXL-CAR-T cells against TNBC bothin vitro and in vivo and could partly overcome the inhibition of CAR-T cell activity in tumor microenvironment. Thus, enhanced CAR-T cell therapy could be an effective strategy for the treatment of TNBC.


## 4. Discussion

Breast cancer is the most common type of cancer in women and is the second fatal type of cancer, after lung cancer [23]. TNBC is a special subtype of breast cancer, which is aggressive, prone to recurrence and metastasis, and poor in prognosis when compared to other types of breast cancer [24]. Currently, chemotherapy is still the only option for treating TNBC, but treatment outcome is far from satisfying [25]. Therefore, novel and effective therapeutic methods for the treatment of TNBC are urgently needed. CAR-T cell therapy induced significant antitumor effects in patients with hematological malignancies [26]; however this therapy is limited in treating solid tumors, due to poor target and low antitumor activity. Recently, it has been reported that next to the first (antigen recognition) and the second (costimulatory) signal pathways, the third signal (cytokine) pathway of CAR-T cells is also required for the antitumor activity of CAR-T cells [27]. Therefore, in several studies, CAR-T cells were constructed that express C7R, to dramatically improve the antitumor activity of CAR-T cells by activating IL-7 signal pathway and extending the survival time of CAR-T cells [13]. In addition, C7R activates engineered CAR-T cells through the third signal pathway without activating other T cells and thereby avoids the cytotoxicity of cytokines to untargeted cells. Thus, using engineered CAR-T cells could be an effective method in treating solid tumors.In previous studies, it has been shown that due to the abnormal overexpression of AXL in tumor cells [28], AXL could be used as a target of solid tumors for CAR-T cells. In our study, we found that TNBC cells overexpressed AXL; thus we constructed second-generation AXL-CAR-T cells and AXL-CAR-T cells expressing C7R to investigate whether engineered CAR-T cells had antitumor activity on TNBC. We found that AXL-CAR-T cells could effectively kill AXL-positive TNBC cells both in vitro and in vivo, and that C7R could greatly activate AXL-CAR-T cells so as to increase the cytotoxicity of AXL-CAR-T cells towards AXL-positive tumor cells. Using a mouse subcutaneous xenograft model, we found that AXL-CAR-T cells could mediate elimination of the tumor, and that the expression of C7R in AXL-CAR-T cells could extend their survival time in mice and increase their infiltration and cytokine release. In addition, AXL-negative MCF-7 breast cancer cells were selected as control cells in our study. In both in vitro and in vivo experiments, AXL-CAR-T cells were not effectively activated and therefore were not able to kill MCF-7 cells, suggesting that AXL would be a promising tumor-associated antigen with the potential to be used as a target for TNBC in CAR-T cell treatment.A7-CAR-T cells coexpressing C7R displayed remarkable antitumor activity to TNBC, which provided a novel therapeutic strategy. Hence, some potential targets were selected in TNBC for broadening the application of engineered CAR-T cells in the treatment of TNBC. In fact, several potential targets might be applied in the treatment of engineering CAR-T cells against TNBC, including NKG2D ligand (NKG2DL). NKG2DL is expressed in various tumor cells and immunosuppressive cells [29, 30], and CAR-T cells that target NKG2DL also exhibited antitumor activity on TNBC [31]. Furthermore, TEM8 is a breast cancer stem cell marker [32, 33], and TEM8-targeted CAR-T cells have proven to have significant potential in the treatment of TNBC [34]. Given the existence of these potential targets, CAR-T cells expressing C7R would be a very promising strategy in the treatment of TNBC.In summary, through a number of experiments, we demonstrated that AXL-CAR-T cells expressing C7R had good antitumor activity. However, our study has some limitations. Inin vivo experiments, there was no significant difference in the therapeutic effect between the AXL-CART cell treatment group and the enhanced AXL-CART cells. Preliminary inference was that the mice were treated in the early stage of tumor growth, which led to the complete elimination of tumors in the AXL-CART cell group, thus the enhanced AXL-CART cells failed to show significantly enhanced antitumor activity. The therapeutic effect of enhanced AXL-CART cells on solid tumors with larger tumor volume will be explored in our future research. In addition, CAR-T cells that target a single AXL antigen may have a risk at off-targeting. Therefore, we planned to construct dual-receptor or CAR-T cells against TNBC to reduce the toxicity of off-targeting. In addition, we established a subcutaneous allograft model in mice but did not establish a primary tumor model. Furthermore, creating a survival curve of mouse models at the long term has not been performed. In the future, we will work on these studies to further improve our study. In conclusion, through a series of cellular and animal experiments, we have demonstrated that AXL-CAR-T cells expressing C7R had significant antitumor effects on TNBC, which were better than the second-generation AXL-CAR-T cells because the cells were more active and showed increased survival. Thus, engineered CAR-T cells may provide a novel solution for the treatment of TNBC.



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*Source: 4795171-2020-01-08.xml*