Chimeric antigen receptor T cell targeting EGFRvIII for metastatic lung cancer therapy

Abstract

Lung cancer is the most common incident cancer and the leading cause of cancer death. In recent years, the development of tumor immunotherapy especially chimeric antigen receptor T (CAR-T) cell has shown a promising future. Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation expressed in various types of tumors and has been detected in non-small cell lung cancer with a mutation rate of 10%. Thus, EGFRvIII is a potential antigen for targeted lung cancer therapy. In this study, CAR vectors were constructed and transfected into virus-packaging cells. Then, activated T cells were infected with retrovirus harvested from stable virus-producing single clone cell lines. CAR expression on the surfaces of the T cells was detected by flow cytometry and Western blot. The function of CAR-T targeting EGFRvIII was then evaluated. The EGFRvIII-CAR vector was successfully constructed and confirmed by DNA sequencing. A stable virus-producing cell line was produced from a single clone by limited dilution. The culture conditions for the cell line, including cell density, temperature, and culture medium were optimized. After infection with retrovirus, CAR was expressed on more than 90% of the T cells. The proliferation of CAR-T cells were induced by cytokine and specific antigen in vitro. More importantly, EGFRvIII-CART specifically and efficiently recognized and killed A549-EGFRvIII cells with an effector/target ratio of 10:1 by expressing and releasing cytokines, including perforin, granzyme B, IFN-g, and TNF-α. The in vivo study indicated that the metastasis of A549-EGFRvIII cells in mice were inhibited by EGFRvIII-CART cells, and the survival of the mice was significantly prolonged with no serious side effects. EGFRvIII-CART showed significantly efficient antitumor activity against lung cancer cells expressing EGFRvIII in vivo and in vitro. Therefore, CAR-T targeting EGFRvIII is a potential therapeutic strategy in preventing recurrence and metastasis of lung cancer after surgery.

Keywords

chimeric antigen receptor T cells / epidermal growth factor receptor / lung cancer / immunotherapy / tumor immunolog y

Introduction

The incidence of cancer in China has increased rapidly since the 21st century [¹,²]. In 2015, approximately 4 292 000 of cancer cases was reported in China, with mortality reaching up to 2 814 000 and five-year-overall survival rate of 36.9%. Lung cancer is the most common incident cancer and the leading cause of cancer death [³]. Despite the continuous progress in traditional treatments, including surgery, chemotherapy, and radiotherapy, the prognosis of lung cancer, especially for advanced stage, is far from satisfactory [⁴]. The metastasis in advanced stage remains a tricky problem in medical practice [⁵].

The immune system plays an important role in inhibiting tumor progression. Unfortunately, the function of the immune system is impaired during tumor progression [⁶]. Adoptive cell therapy (ACT), which was initiated by Dr. Rosenberg in 1985 [⁷], provided a new antitumor strategy focusing on reestablishing or enhancing immune response in tumor patients through the use of reinfusion immune effector cells. The most successful attempt in recent years was the chimeric antigen receptor T (CAR-T) cells, which have the specificity of antibodies and the cytotoxicity of T lymphocytes. These CAR-T cells are engineered to express synthetic receptors redirecting T cells to recognize and kill tumor cells precisely and rapidly, without the limitation of major histocompatibility complex [⁸]. CAR-T achieved remarkable success in the treatment of leukemia. Patients with acute lymphoblastic leukemia achieved complete remission through CD19-CART therapy [⁹,¹⁰]. Despite the success in hematological malignancies, CAR-T treatment for solid tumors is still under investigation and constrained by limited optimal targets [¹¹].

Epidermal growth factor receptor variant III (EGFRvIII), one of the most common mutations of epidermal growth factor receptor (EGFR), is a tumor specific mutation expressed in various types of tumor cells including neuroblastoma [¹²,¹³], breast cancer [¹⁴], and ovarian cancer [¹⁵]. EGFRvIII has been detected in non-small cell lung cancer with a mutation rate ranging from 16% to 39% [¹⁶,¹⁷]. Moreover, EGFRvIII mutation rate was about 11.1% in Chinese lung cancer patients [¹⁸]. Thus, EGFRvIII is a potential antigen for targeted-therapy in lung cancer.

In this study, we aimed to investigate the antitumor potential of CAR-T cells in lung cancer, especially in the late stage. To this end, the expression vector of the third-generation CAR specific for EGFRvIII was constructed. For further clinical application, stable virus-producing cell clone was selected and CAR-T cells were generated by infection with retrovirus. Then, in vitro and in vivo effects of CAR-T cells in lung cancer therapy were evaluated.

Materials and methods

Ethics statement

The design and protocols of this study were approved by the Institutional Ethic Review Board of Fourth Military Medical University.

Cells and animals

The human lung cancer cell line A549 and the retrovirus producing cell lines Phoenix Eco and PT67 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells overexpressing EGFRvIII (named A549-EGFRvIII) were established by infection with a retrovirus encoding EGFRvIII. Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors. The A549, Phoenix Eco and PT67 cells were cultured in DMEM medium (Invitrogen, Carlsbad, California, USA) with 10% fetal calf serum for serial subcultivation. PBMCs were cultured in AIM-V medium (Invitrogen, Carlsbad, California, USA) with 5% human AB serum and 300U/mL IL-2 (Sigma, Burlington, MA, USA).

Establishment of the stable retrovirus producing cell line and retrovirus production

EGFRvIII single chain antibody, CD8α hinge, CD28, 4-1BB, and CD3ζ (named EGFRvIII-CAR) and CD8α hinge, CD28, 4-1BB and CD3ζ (named MOCK-CAR) were subcloned into pMSCV plasmid (Addgene 20737) and transfected into the virus-packaging cell line. Briefly, Phoenix Eco cells were seeded into 10 cm² disk, and transfected with plasmids by Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) at the following ratio: 3 μg of DNA per 12 μL of lipofectamine according to the manufacturer’s instruction. After 24 h, PG13 cells were infected with the retroviruses harvested from the Phoenix Eco packaging cell line in 24-well plate. Single clone cell line, PT67, was established by limited dilution according to a previously described method [¹⁹]. The titers of the retrovirus produced by each clone cell line were quantified by real-time PCR.

Isolation of PBMCs and generation of CAR-T cells

PBMCs were isolated from the peripheral blood of healthy donors. Briefly, 50 mL of peripheral blood were collected and treated with anticoagulants. The PBMCs were harvested by density gradient centrifugation and cultured in AIM-V containing 5% human serum and 300 U/mL IL-2. The PBMCs were either cryopreserved or infected by retrovirus. Before retrovirus infection, the PBMCs were activated with 50 ng/mL OKT-3. The efficiency of the activation was evaluated by flow cytometry. Non-tissue-treated 24-well plates were coated with 100 μg/mL RetroNectin (Takara, Nojihigashi 7-4-38, Kusatsu, Shiga, Japan). The supernatant of the stable virus-producing cell line was collected and filtered. The 5 × 10⁵ activated PBMCs and 1 mL of virus supernatant were added into the 24-well plate and centrifuged according to the instruction of manufacturer of RetroNectin.

Cell proliferation

Antigen- and cytokine-dependent cell proliferation was performed in vitro. A549-EGFRvIII cells were cocultured with EGFRvIII-CART, MOCK-CART, and PBMCs at an effector/target ratio of 1:10. Cell number was counted every other day after culture. In the cytokine-dependent cell proliferation study, CAR-T cells or PBMCs were cultured in an AIM-V medium supplemented with 3000 U/mL IL-2 and without the target cells.

Intracellular staining and flow cytometry

Cell surface fluorescent antibodies against CD45, CD3, CD4, and CD8 were purchased from BD (USA), and intracellular staining antibodies targeting perforin, granzyme B, IFN-γ and TNF-α were purchased from Biolegend (San Diego, CA, USA). Cell intracellular staining was performed according to the instruction of the manufacturer of the intracellular staining kit (BD, San Jose, CA, USA). Single chain antibody against EGFRvIII expressed on the T cell surface was detected by biotin-protein L (GeneScript, Piscataway, NJ, USA) and Streptavidin-PerCp (BD, San Jose, CA, USA). Matched isotype control antibodies were used in the analysis. Flow cytometry data was analyzed by FlowJo software 7.0 version.

Lactate dehydrogenase release assay and real-time cell analysis system

The cytolytic activities of the CAR-T cells or PBMCs were measured by lactate dehydrogenase (LDH) release assay. Effective and target cells were cocultured in a 96-well plate at different E:T ratio, and the supernatants were harvested at different times. LDH level was measured with a cytotoxicity detection kit (Promega, Madison, WI, USA). Real-time cell analysis (RTCA) system DP (ACEA Biosciences, San Diego, CA, USA) was used for the plotting of the cytolytic curve of the effect cells.

Enzyme-linked immunosorbent assay

The amount of cytokine released by the effect cells was measured through enzyme-linked immunosorbent assay (ELISA). Briefly, 1 × 10⁵ CAR-T cells or PBMCs were cocultured with 1 × 10⁴ A549 or A549-EGFRvIII cells respectively for 8 h at 37 °C. The supernatants were harvested, and the levels of TNF-α, IFN-γ, IL-2, and IL-4 were measured by ELISA according to the manufacturer’s instruction (BioLegend, San Diego, CA, USA).

Lung metastasis model

Female NOD.Cg-Prkdc^scidIL2rg^tm1Wjl mice (8–12 weeks old) were purchased from Vitalstar Biotechnology (Beijing, China) and fed in sterile condition. Thirty-two tumor-bearing mice were randomly assigned to four groups: (1) mice that received A549 cells were treated with EGFRvIII-CART, (2) mice that received A549-EGFRvIII cells were treated with PBS, (3) mice that received A549-EGFRvIII cells were treated with MOCK-CART, and (4) mice that received A549-EGFRvIII cells were treated with EGFRvIII-CART. The 1 × 10⁶ A549 or A549-EGFRvIII cells were administered through tail vein at day 0, and 1 × 10⁷ EGFRvIII-CART or MOCK-CART cells were administered through the tail vein at 7, 10, and 13 days after tumor cell injection. The mice were observed for 90 days after tumor cells injection. The PBMCs of the mice were collected once a week after the last injection of the effect cells.

Statistical analysis

All data were presented as mean±SE. Statistical analysis was performed in one-way ANOVA for multiple group comparisons and P values less than 0.05 were considered statistically significant. Statistical analysis was performed in PRISM 7.0 software.

Results

Generation of third-generation CAR targeting EGFRvIII

For the evaluation of the therapeutic effect of the CAR-T cells in lung cancer, a vector expressing EGFRvIII-CAR was constructed. Single chain antibody against EGFRvIII was synthesized. The single-chain antibody against EGFRvIII and the fragments of CD8α hinge, hCD28, h4-1BB, and CD3ζ were subcloned into a pMSCV plasmid by PCR. The MOCK-CAR lacking scFv was constructed as control. The plasmid was confirmed by enzyme digestion and sequence reaction (Fig. 1). The sequence of marker for selection in the vector was deleted for further clinical therapy use, and a similar plasmid encoding EGFP was used as control for the optimization of the experimental conditions.

Optimization of stable retrovirus-producing cell line

A stable retrovirus-producing cell line was obtained by transient transfecting the CAR vector into Phoenix Eco cell by using Lipotamine 2000. Supernatant containing retrovirus harvested from Phoenix Eco cells was used to infect PT67 cells (Fig. 2A and 2B). By limited dilution culture, PT67-EGFRvIII clone C70 (RV-E70) and PT67-MOCK clone C291 were established. The retrovirus titers of RV-E70 and C291 quantified by real time PCR were 9 × 10⁹ and 7.64 × 10⁹ copies/mL, respectively. The infection efficiency of the PBMCs was improved by optimizing the culture conditions of the retrovirus-producing cell line, including the initial cell density, temperature of culture condition, and fructose concentration in the medium. As shown in Fig. 2C–2E, the retrovirus titer was significantly increased when the producing cells were cultured at an initial density of 4 × 10⁴ cells/mL at 37 °C for 72 h after the addition of 47 mmol/L fructose. More importantly, the titers of the clone remained stable after passaging for 10 generations (Fig. 2F).

Generation of CAR-T cells

CAR-T cells were generated by isolating PBMCs from a healthy donor by density gradient centrifugation. FACS analysis indicated that the PBMCs collected were mainly composed of T cells and the percentage of the CD3⁺CD8⁺ T cells increased after culture (Fig. 3A). To improve the infection efficiency, we used OKT-3, an antibody targeting CD3, before infection. The successful activation of the T cells by OKT-3 was confirmed by the increased expression of CD69 and CD25 (Fig. 3B). After infection, CAR expression on T cell was confirmed by Western blot and FACS analysis. As shown in Fig. 3C, scFv was identified nearly all the T cells in PBMCs after infection, and this result indicates the efficient generation of CAR-T cells. Western blot analysis showed that CD3ζantibody identifies not only the wild type CD3ζ (16 kDa) molecule but also the molecule fused expression with CAR (43 kDa) (Fig. 3D).

Cytotoxicity of EGFRvIII-CART cells

The proliferation of EGFRvIII-CART cells, MOCK-CART cells and PBMCs was evaluated. They all showed IL-2 dependent proliferation and no significant differences in proliferation treated with IL-2 (Fig. 4A), which indicated that CAR-T cells retain the same proliferation characteristics as the T cells. However, when cocultured with A549-EGFRvIII cells, EGFRvIII-CART cells showed increased proliferation ability compared to MOCK-CART cells and PBMCs (Fig. 4B), indicating that EGFRvIII-CART cells can induce antigen-dependent proliferation.

To further study the cytotoxicity of EGFRvIII-CART cells, we conducted LDH-release assay at E:T ratio of 20:1, 10:1, and 5:1. As shown in Fig. 5A, the three kinds of effect cells showed no significant differences in cytotoxicity on terminal time point (hour 14) at E:T ratio of 20:1, but EGFRvIII-CART can lysis the A549-EGFRvIII cells effectively at early time (hour 4 and 6). As the E:T ratio was reduced to 10:1, EGFRvIII-CART showed significantly greater cytotoxicity in treatment of A549-EGFRvIII cells compared to other groups (P<0.01). At the E:T ratio of 5:1, the cytotoxicity of EGFRvIII-CART treated with A549-EGFRvIII cells was slightly reduced. Therefore, the E:T ratio of 10:1 was considered optimization. The same result was also confirmed by RTCA system (Fig. 5B).

The released cytokines after effect cells recognizing tumor cells were then investigated. The intracellular staining showed no significant differences in IFN-γ, TNF-α, granzyme B, and perforin between PBMCs or MOCK-CART cocultured with A549 or A549-EGFRvIII cells (Fig. 6A). However, the EGFRvIII-CART produced high levels of Th1 cytokine IFN-γ, IL-2 as well as the Th 2 cytokine IL-4 in the supernatants when they cocultured with A549-EGFRvIII cells. EGFRvIII-CART cocultured with A549 cells showed lower cytokines secretion, as well as the MOCK-CART and PBMCs cocultured with tumor cells (Fig. 6B).

Antitumor response of EGFRvIII-CART in vivo

In vivo antitumor response was evaluated in metastasis model of lung cancer. For the establishment of the advanced lung metastasis model, tumor cells were injected through the tail vein. The mice were observed for 90 days after tumor cell injection. The CAR-T cells were administered 7, 10, and 13 days after the tumor cell injection (Fig. 7A). As shown in Fig. 7B-7D, in the A549-EGFRvIII cells treated with PBS, a large number of metastatic lesions formed in the lungs, and mice died because of respiratory failure (median survival 42.5 days). In A549-EGFRvIII treated with MOCK-CART and A549 treated with EGFRvIII-CART, metastatic lesions formed, and the survival rates were improved (median survival 48 and 50 days, respectively). Significantly, less metastatic lesions formed in A549-EGFRvIII treated with EGFRvIII-CART group with a survival rate of 62.5%.

We collected CAR-T cells in peripheral blood of the mice once a week to study the in vivo expansion and survival of CAR-T cells. As shown in Fig. 7E, the human CD3 positive cells significantly decreased over time, and EGFRvIII-CART cells in A549-EGFRvIII mice could remain detectable at 11th week compared with other groups.

Discussion

The specificity of the CAR-T cells was determined by the extracellular domain, mainly the single chain antibody of the tumor antigen. Cytotoxicity depended on the intracellular co-stimulator molecules [²⁰]. By using intracellular co-stimulators, three generations of CAR-T were developed. Of the first generation of CAR-Ts, CD3 ζ was the only molecule in the T cell [²¹]. Although effective antitumor immune response could be induced, the lack of effective stimulation signal resulted in the short response time, and poor proliferation in vivo. The antitumor effect of CAR-T cells can be enhanced by adding CD28 and CD137 to the second and third generations of CAR [²²,²³].

To achieve a satisfactory antitumor effect, we used the third generation CAR-T in the treatment of lung cancer. As the microenvironment of the solid tumor is more complicated than hematologic malignancy, improving the persistence and proliferation of CAR-T cells might maximize their antitumor activity. However, the abrupt release of high-level cytokines caused clinical risk representing by “cytokine storm.” [²⁴,²⁵] But in vivo data in our study showed that no mice died of severe side effects, indicating the safety of third generation CAR-T cells. The appropriate E:T ratio plays an important role in balancing the sufficient antitumor effect and low systematic toxicity. Different E:T ratios were studied in vitro. The results in different groups were compared. In the group with E:T of 5:1, the percentage of lysis of target cells in the terminal time was lower than the percentages of the other groups, and this result may indicate insufficient effector cells. In the group with E:T of 20:1, the targeted tumor cells were lysed in the initial period of experiment. However, no significant difference was observed between the terminal times of the group with E:T of 20:1 and the group with E:T of 10:1, and the abrupt lysis of the mass target cells increased the risk of cytokine storm. Therefore, we selected 10:1 E:T ratio for the in vivo study to achieve the balance between satisfactory antitumor response and number of the effector cells infused into the mice based on the in vitro study.

The antigen targeted in CAR-T therapy should be specific and widely expressed in tumor cells, and thus applying the antigens in solid tumor therapy is difficult. EGFRvIII is a tumor-specific mutation, which results from the in-frame depletion of the 2−7 exons of EGFR. The specific and immunogenic epitope of the extracellular domain is created from the junction of exon 1 and 8 [²⁶]. EGFRvIII was previously studied as a target in glioblastoma, and the results showed that EGFRvIII-CART can control tumor growth in xenogeneic subcutaneous and orthotopic models [²⁷]. More significantly, strategies using CAR-T cells, such as incorporating miR-17-92 mainly to improve T cell survival, showed satisfactory results [²⁸]. In this study, we first constructed the third-generation CAR-T cells targeting EGFRvIII, and evaluated the efficiency in treatment of metastatic lung cancer. As far as the side effects considered in our study are concerned, our data are consistent with those of previous studies [²⁷,²⁸], and no fatal side effects were observed during the in vivo study.

Gene transfection is a major technical problem in CAR-T cell generation. Strategies used in practice can be classified into transient transfection represented by electrotransfection and lipofection transfection, and stable transfection mediated by virus. In our study, we employed retroviruses to achieve the gene modification of lymphocytes. The gene carried by the virus can integrate into the randomly replicated genomes of T cells during their proliferation. CAR is stably expressed on surfaces of T cells and prolongs CAR-T persistence in vivo and improves antitumor effect. To prevent the replication of retroviruses in patients, we established a stable virus-producing cell line. The helper plasmids of the retroviruses were integrated into the packaging of the cell genome. The retroviruses used in T cell infection cannot replicate in the T cells and meet the standard for clinical use.

The proliferation and persistence of CAR-T cells in vivo had a considerable effect on antitumor therapy. The results in our study indicated that CAR-T cells showed cytokine-dependent proliferation and better antigen-dependent proliferation than PBMCs. Cytokine-dependent proliferation enabled the CAR-T cells to expand to the sufficient number before reinfusion to the patient in vitro after infection. Thus, CAR-T cells reinfused into the body proliferated after being triggered by the antigen expressed on the tumor cells. The in vivo study showed that the CAR-T cells remained detectable 11 weeks after reinfusion, indicating the long-term persistence of CAR-T cells. Several studies improved the persistence through co-expression cytokines such as IL-2, IL-15, and IL-21 [²⁹–³²]. Gattinoni et al. improved the therapeutic response by using sublethal dose of radiation or lymphocyte depletion [³³].

Although preclinical results showed EGFRvIII-CART induces the long-term regression of metastatic lung cancer, challenges remain in clinical study. A previous clinical study reported on-target activity in the brain in patients suffering glioblastoma and infused with EGFRvIII-CART intravenously. But the in situ compensatory immunosuppressive response represented by the increased and robust expression of inhibitory molecules and infiltration of regulatory T cells were observed after infusion and might have blocked the effect of CAR-T cells to some extent [³⁴]. Meanwhile, as with other treatment in solid tumor, antigen variation in lung cancer may bring obstacles in clinical practice. For these difficulties, strategies, such as combined therapy with PD-1 checkpoint blockade [³⁵] or small molecule drugs targeting IDO-1 [³⁶] bring promising future in CAR-T treatment in solid tumors.

Collectively, we constructed EGFRvIII-CART cells and demonstrated for the first time that EGFRvIII-CART kills lung cancer cells expressing the EGFRvIII mutation. Then, we induced the regression of metastatic lung cancer, which provided an experimental clue that EGFRvIII-CART cells can be used after surgery to clear the residual tumor cells and prevent recurrence. The therapeutic effect of EGFRvIII-CART cells should be tested in various cancer models, and the strategy of ACT especially CAR-T therapy combined with other therapies still need further study.

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The Author(s) 2019. This article is published with open access at link.springer.com and journal.hep.com.cn