Research ArticleExperimental Studies
Open Access

T Cells Expressing CAR Equipped With Extracellular Domain of BTLA Are Effective Against HVEM-over-expressing Melanoma Cell Lines

YEONGRIN KIM, JI EUN KIM, DA YEON LEE, JI U CHOI, JIN SONG PARK, HEUNG KYOUNG LEE, SANG UN CHOI, SIMON PARK and CHI HOON PARK
Anticancer Research August 2023, 43 (8) 3419-3427; DOI: https://doi.org/10.21873/anticanres.16517

Abstract

Background/Aim: Several chimeric antigen receptor (CAR) T cells have been used to treat melanoma but have not shown favorable results. This study investigated whether Herpes virus entry mediator (HVEM), which is overexpressed in melanoma, is a potential novel antigen for CAR T cell therapy. Materials and Methods: A CAR construct, composed of the BTLA extracellular domain for HVEM recognition (BTLA-28z), was developed and tested. Results: Jurkat cells transduced with BTLA-28z exhibited enhanced IL-2 secretion when incubated with HVEM-over-expressing melanoma cells. KHYG-1 cells transduced with BTLA-28z also lysed melanoma cell lines. Using primary T cells, we generated CAR T cells targeting HVEM. BTLA-28z CAR T cells exhibited excellent lytic activities against melanoma cell lines. Conclusion: HVEM-targeting CAR T cells may be useful for the treatment of melanoma.

Key Words:

Chimeric antigen receptor (CAR) T cell therapy has revolutionized recent cancer therapies. It has shown overwhelming clinical efficacy in haemato-oncology. Anti-CD19 or BCMA CAR T cells are already approved by the U.S. Food and Drug Administration (FDA) and are in clinical use based on their outstanding effects (1, 2). CAR T cells targeting CD20, CD22, or CD7 are in clinical trials showing promising results in B or T cell malignancies (3-5). However, clinical results for solid tumors are limited (6, 7). Several CAR T cells have been used to treat melanoma in clinical trials but have not shown favorable results. Many targets, including high molecular weight melanoma-associated antigen, GD2 ganglioside, HER2, chondroitin sulfate proteoglycan 4, human endogenous retrovirus (HERV-K) envelope protein, CD70, B7-H3, DNAX accessory molecule-1, and αvβ3 integrin, were studied in CAR T cells against melanoma (7-13). Although they have shown excellent anti-melanoma effects in preclinical models, they have failed to show promising results in clinical trials (14). Herpes virus entry mediator (HVEM), also known as TNFRSF14, is overexpressed in various tumors, such as colorectal cancer, gastric cancer, glioblastoma, and lung cancer (15-18). Especially, more than 90% of melanoma patients express HVEM in tumors as shown using immunohistochemistry (19). HVEM binding to CD160 or BTLA triggers an inhibitory signal in T lymphocytes, similar to the PD1-PDL1 interaction. Here, we developed a unique melanoma-targeting CAR construct using HVEM-binding proteins including BTLA and CD160 as HVEM recognition domains. Our data demonstrate that T cells reengineered with this construct show outstanding activity against melanoma cell lines.

Materials and Methods

Cell line. 293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Cytiva, Marlborough, MA, USA; #SH30243.01) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA; #16000-044). KHYG-1 cells were cultured in RPMI1640 medium (Cytiva, #SH30027.01) supplemented with 10% FBS and 200 IU/ml of Human IL-2 (R&D Systems, Minneapolis, MN, USA; #202-IL). Jurkat and melanoma cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS. T-cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 200 IU/ml of Human IL-2. All the cells were incubated in a humidified incubator at 37°C with 5% CO2.

Plasmid construction. The BTLA-3z CAR is based on the first-generation CAR. This construct encodes for a fusion protein consisting of the extracellular domain of human BTLA and the cytoplasmic domain of human CD3ζ (CD3z). The CD3z domain contributes to T cell activation. BTLA-28z, FITC-28z, and CD160-28z CARs have an extracellular domain (BTLA, CD160, or anti-FITC scFv region), a human CD28 transmembrane domain, and a cytoplasmic domain consisting of the cytoplasmic domains of human CD28 and human CD3z. The CD28 and CD3z domains are co-stimulatory and activating domains, respectively. BTLA-3z, BTLA-28z, and CD160-28z CAR constructs were prepared using gene cloning, and the FITC-28z CAR construct was synthesized by Macrogene (Seoul, Republic of Korea). BTLA (OriGene, Rockville, MD, USA #RC219458) and CD160 (OriGene, #RC204886) DNA were purchased from OriGene.

Lentivirus and retrovirus production. For generating the CAR lentivirus, 293T cells were transfected with three plasmids: packaging, envelope, and transfer vectors. The packaging vector was psPAX2 (Addgene, Watertown, MA, USA; #12260), the envelope vector was pMD2.G (Addgene, #12259), and the transfer vector was pLVX-CD160-28z-puro, pLVX-BTLA-3z-puro, pLVX-BTLA-28z-puro, or pLVX-FITC-28z-puro. After 24 hours, the culture medium was replaced with fresh, complete DMEM. The supernatant containing the lentivirus was harvested every 24 hours for two days. The harvested lentivirus supernatant was filtered through a 0.45 μm polyethersulfone (PES) membrane filter (Merck Millipore, Burlington, MA, USA; #SLHP033RB). For retrovirus production, 293T cells were transfected with the three retroviral plasmids. The retroviral packaging vector was pEQ-PAM-3E, the retroviral envelope vector was pLTR-RD114A (Addgene, #17576), and the transfer vector was MSCV-BTLA-28z. The BTLA-28z CAR DNA was inserted into MSCV-IRES-GFP (Addgene, #20672). The production protocol of the retroviruses was the same as that of the lentiviruses.

Generation of CAR Jurkat and KHYG-1 cells. Effector Jurkat cells expressing the CAR protein were generated using electroporation of a plasmid vector containing CAR DNA into Jurkat cells. The CAR-containing vectors were pLVX-BTLA-3z-puro, pLVX-BTLA-28z-puro, and pLVX-CD160-28z-puro. KHYG-1 cells expressing BTLA-28z CAR proteins were generated using lentivirus transduction. The lentivirus supernatant was mixed with KHYG-1 cells and 8 μg/ml of polybrene (Sigma-Aldrich, Saint Louis, MO, USA; #H9268). The mixture was centrifuged at 1,000 × g, for 90 min. Cells were treated for a few weeks with 0.5 μg/ml puromycin to select BTLA-28z CAR KHYG-1 cells.

Generation of CAR T cells. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood, which was purchased from Korean Red Cross Blood Services, using density gradient centrifugation. Fifteen milliliters of Lymphoprep (STEMCELL, Vancouver, Canada; #07851) was added to a 50 ml conical tube, and 30 ml of diluted whole blood was carefully poured. Whole blood was diluted in an equal volume of Phosphate-buffered saline (PBS) supplemented with 2% FBS. The layered solution was centrifuged at 800 × g for 20 min without a break and the middle mononuclear cell layer was removed and the mononuclear cells were transferred into a new conical tube. The cells were washed twice by centrifugation at 300×g for 8 min with PBS supplemented with 2% FBS. Mononuclear cells were counted and resuspended in 90% FBS containing 10% Dimethyl Sulfoxide (DMSO). The cells were cryopreserved in liquid nitrogen tanks (1-3×107 cells/vial). To selectively activate T cells, cryopreserved PBMCs were thawed and cultured in a complete RPMI1640 medium containing 10% FBS and CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA; #11132D) and 200 IU/ml human IL-2. One week later, stimulated T cells were infected with CAR lentivirus or CAR retrovirus supernatant containing 8 μg/ml of polybrene by spinoculation (1,500-1,800 × g, for 90 min). After centrifugation, the viral supernatant was replaced with fresh RPMI medium containing 10% FBS supplemented with 200 IU/ml human IL-2.

Cytotoxicity assay. The cytotoxic activity of the effector CAR KHYG-1 or CAR T cells was determined using the Bright-Glo luciferase assay system (Promega, Madison, WI, USA; #E2650). Melanoma cells were transduced with a luciferase lentivirus and selected using puromycin. Melanoma cells expressing luciferase were co-cultured with Effector CAR KHYG-1 or CAR T cells at an E: T ratio of 10:1. The luminescence signals were measured after 4 and 24 h of co-incubation. To detect the luminescent signal, BrightGlo reagent was added to each well of a 96-well plate in an amount equal to that of the mixture of target and effector cells. The cells were lysed by shaking in the 96-well plate at room temperature for 5 min. The luminescence was measured using an EnVision reader (Perkin Elmer, San Diego, CA, USA). Cytotoxic activity was calculated using the following formula:

Embedded Image

IL-2 release assay. To measure the amount of IL-2 secreted by effector cells, melanoma cells were co-cultured with CAR Jurkat or CAR T cells at an E: T ratio of 10:1 in 96-well plates. After 24 h, the cell culture medium containing IL-2 was harvested by centrifugation at 16,000 × g for 10 min. The IL-2 levels were determined using a human IL-2 ELISA kit (BioLegend, San Diego, CA, USA; #431801).

Evaluation of HVEM protein expression level on the cell surface. 1×106 cells were washed with cold 1×PBS wash buffer and resuspended in resuspension buffer (1×PBS supplemented with 10% FBS and 0.02% sodium azide). The cells were incubated on ice for 30 min with 2.5 μg/ml of anti-human HVEM antibody (Enzo life science, Farmingdale, NY, USA; #ALX-804-814-C100) or normal rat IgG (Santa Cruz Biotechnology, Dallas, TX, USA; #SC-2026). The treated cells were washed with cold 1×PBS wash buffer and incubated with Alexa-488 anti-rat IgG (Invitrogen, #A11006) for 30 min on ice. The cells were washed with 1×PBS wash buffer, resuspended in resuspension buffer containing Hoechst 33342 (Thermo Fisher Scientific, #H3570), and incubated at 37°C for 15 min. Human HVEM expression was analyzed using a NucleoCounter NC-3000 (ChemoMetec, Allerod, Denmark).

Statistical analysis. GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA) was used for data analysis. Statistical significance was evaluated using unpaired t-test (ns: non-significant, *p<0.05, **p<0.01, ***p<0.001; ****p<0.0001) (two-tailed p-value).

Results

HVEM is over-expressed in melanoma cell lines. The surface expression of HVEM was confirmed in the melanoma cell lines SK-MEL-28, CAKI-1, M14, M19-MEL, and MALME-3M. To verify the data, 293T(luciferase) and 293T(HVEM-T2A-luciferase) cells were generated by lentivirus infection. 293T(luciferase) cells expressed luciferase, and 293T(HVEM-T2A-luciferase) cells expressed both HVEM and luciferase. 293T(luciferase) did not show HVEM expression. In contrast, 293T(HVEM-T2A-luciferase) showed strong HVEM expression (Figure 1A). The SK-MEL-28, M14, M19-MEL and MALME-3M melanoma cells expressed HVEM. In particular, the HVEM was the most expressed in SK-MEL-28 cells (Figure 1A). The level of HVEM expression on the cell surface was evaluated using Nucleocounter NC-3000. The cell lines were stained with an anti-human HVEM antibody (red line) or a matched isotype control antibody (black line).

Figure 1.
Figure 1.

BTLA extracellular domain recognizes HVEM and CD28 domain boosts IL-2 secretion. (A) The expression level of HVEM in melanoma cell lines. 293T cells were infected with luciferase or HVEM-T2A-luciferase lentivirus. The expression level of HVEM on the cell surface was evaluated using Nucleocounter NC-3000. (B) Jurkat, BTLA-28z, or CD160-28z CAR Jurkat cells were incubated with HVEM-positive or negative cells. To measure IL-2 secretion from effector Jurkat cells, supernatants were harvested after 24 h of co-incubation. BTLA-28z CAR Jurkat cells secreted a higher amount of IL-2 than CD160-28z CAR Jurkat cells. (C) Western blot analysis of CD160-28z and BTLA-28z CAR protein expression in Jurkat cells. Each CAR DNA was transferred into Jurkat cells by electroporation. (D) Jurkat cells expressing BTLA-28z or BTLA-3z CAR were co-cultured with HVEM-positive or negative cells. After 24 h of co-culture, a cell culture medium was harvested to measure IL-2 secretion from Jurkat cells. BTLA-28z CAR Jurkat cells secreted higher amounts of IL-2 than either BTLA-3z CAR Jurkat or Jurkat cells when co-cultured with HVEM-positive cells. (E) Western blot analysis of BTLA-3z and BTLA-28z CAR protein expression in Jurkat cells. Electroporation was used to transfer CAR DNA into Jurkat cells. The amount of IL-2 secretion that could not be calculated is represented by 0 in the (B) and (D).

Chimeric antigen receptor engineered with BTLA extracellular domain recognizes HVEM. To target HVEM, we used the BTLA and CD160 extracellular domain, which bind to HVEM, as the antigen recognition domain, and combined the CD28 and CD3z domains as the signaling domains of the chimeric antigen receptors (BTLA-28z and CD160-28z). To determine whether BTLA-28z and CD160-28z bind HVEM, we electroporated the BTLA-28z or CD160-28z gene into the Jurkat cell line. Jurkat cells, a T-lymphocyte cell line, secrete IL-2 when activated by antigen recognition. The amount of IL-2 secreted from the Jurkat cells indicates the level of activation of CAR Jurkat cells. CAR Jurkat cells were incubated with various cell lines. 293T(HVEM-T2A-luciferase) and SK-MEL-28(luciferase) are HVEM-positive cell lines, and 293T(luciferase) and CAKI-1(luciferase) cells are HVEM-negative cell lines. BTLA-28z CAR Jurkat cells secreted higher amounts of IL-2 when co-cultured with HVEM-positive cell lines than negative cell lines. However, CD160 CAR Jurkat cells secreted higher amounts of IL-2 against HVEM-negative CAKI-1 (luciferase) than against HVEM-positive SK-MEL-28 (luciferase) (Figure 1B). These data indicate that BTLA-28z CAR Jurkat cells selectively and more effectively recognize HVEM than CD160-28a CAR Jurkat cells. Therefore, we performed further studies using the BTLA-28z CAR. CAR protein expression was analyzed using western blotting (Figure 1C).

CD28 boosts IL-2 secretion. We compared the BTLA-28z CAR and BTLA-3z CAR to find out which CAR construction is better between BTLA-28z and BTLA-3z. BTLA-28z and BTLA-3z CAR Jurkat cells (1×105 cells) were incubated with various cell lines (1.5×104 cells). SK-MEL-28(luciferase), M14(luciferase), M19-MEL(luciferase), MALME-3M(luciferase) and 293T(HVEM-T2A-luciferase) are HVEM-positive cell lines, while 293T(luciferase) is a HVEM-negative cell line. As shown in Figure 1, both CAR Jurkat cells showed higher activation when they were co-cultured with HVEM-positive cells than HVEM-negative cells. Interestingly, BTLA-28z CAR Jurkat cells secreted more IL-2 than BTLA-3z CAR Jurkat cells when incubated with HVEM-positive cells (Figure 1D). These results indicated that CD28 enhanced IL-2 secretion by CAR Jurkat cells. CAR expression was analyzed using western blotting (Figure 1E).

BTLA-28z KHYG-1 lysed HVEM-positive cells. Antigen-specific cytotoxicity assays were performed using BTLA-28z CAR KHYG-1 cells. The KHYG-1 cell line, which originates from NK lymphocytes, exhibits cytotoxic activity against target cells. Since KHYG-1 cells are straightforward to culture and readily transduced using lentivirus particles, we performed a cytotoxicity assay with KHYG-1 cells before performing it with primary T cells. The mock KHYG-1 cells [KHYG-1(puro)] and BTLA-28z CAR KHYG-1 cells [KHYG-1(BTLA-28z)] were generated by lentivirus infection. SK-MEL-28(luciferase), CAKI-1(luciferase), or 293T(HVEM-T2A-luciferase) cells were incubated with KHYG-1(puro) or KHYG-1(BTLA-28z) cells at an E: T ratio of 10:1 for 4 h. As shown in Figure 2A, KHYG-1(BTLA-28z) specifically lysed the HVEM-expressing melanoma cell lines, SK-MEL-28(luciferase) (75.40±2.76%), and HVEM-over-expressing 293T cells (53.59±0.66%). CAKI-1(luciferase), an HVEM-negative cell line, was not lysed effectively by KHYG-1(BTLA-28z) cells (−4.78±0.60%) (Figure 2A).

Figure 2.
Figure 2.

BTLA-28z KHYG-1 cells lysed the HVEM-positive cells and BTLA-28z CAR T cells secreted IL-2. (A) The mock KHYG-1 cells [KHYG-1(puro)] or BTLA-28z CAR KHYG-1 cells [KHYG-1(BTLA-28z)] were co-cultured with SK-MEL-28(luciferase), 293T(HVEM-T2A-luciferase) or CAKI-1(luciferase) at E: T ratio of 10:1 for 4 h. KHYG-1(BTLA-28z) had higher cell cytotoxicity against HVEM positive cancer cells than the KHYG-1(puro) cells. The cell cytotoxic activity was assessed using the luciferase assay. (B) The T cells expressing FITC-28z [T cell(LV-FITC-28z)] or BTLA-28z CAR [T cell(LV-BTLA-28z)] (1×105 cells) were incubated with SK-MEL-28(luciferase), 293T(HVEM-T2A-luciferase), or 293T(luciferase) cells (3×104 cells). One day after, supernatants were recovered to evaluate the amount of IL-2 secretion of each CAR T cell using ELISA. The amount of IL-2 secretion that cannot be calculated is represented by 0 in the (B). Statistical significance was evaluated using unpaired t-test (ns: non-significant, *p<0.05, **p<0.01, ***p<0.001; ****p<0.0001) (two-tailed p-value).

BTLA-28z CAR T cells secreted IL-2 and lysed HVEM-positive cells. After confirming the effectiveness of the BTLA-28z CAR construct in Jurkat and KHYG-1 cells, we generated BTLA-28z CAR T cells using primary T cells. Primary T cells were isolated from PBMCs of healthy donors and the T cells were infected with the lentivirus by spinoculation (1,500-1,800 × g, 90 min). To determine whether BTLA-28z CAR T cells were activated following co-culture with HVEM-expressing cells, we performed IL-2 release assays. As shown in Figure 2, BTLA-28z CAR T cells [T cell(LV-BTLA-28z)] selectively secreted IL-2 following co-culture with HVEM-positive cells, which were SK-MEL-28 (luciferase) and 293T (HVEM-T2A-luciferase). The amounts of secreted IL-2 were 154.21 pg/ml and 31.35 pg/ml, respectively. In contrast, FITC-28z CAR T cells [T cell(LV-FITC-28z)], which express fluorescein isothiocyanate (FITC) and was used as a negative control, secreted less amount of IL-2 than BTLA-28z CAR T cells against HVEM positive cells SK-MEL-28(luciferase) and 293T(HVEM-T2A-luciferase). The amounts of secreted IL-2 were 1.73 pg/ml and 0.00 pg/ml, respectively (Figure 2B). In addition, as shown in Figure 3, we tested the cytotoxicity of BTLA-28z CAR T cells against melanoma cell lines. Non-transduced T cells (NTD T cell), ZsGreen expressing T cells [T cell(LV-ZsGreen)], or BTLA-CAR T cells [T cell(BTLA-28z)] (5×104 cells) were co-cultured with SK-MEL-28(luciferase), MALME-3M(luciferase), or 293T(luciferase) cells (5×103 cells) for 4 or 24 h. The effector T cells were transduced with the lentivirus. The T cell(LV-BTLA-28z) selectively lysed HVEM-positive target cells, and the cytolytic effect was higher at 24 hours. (SK-MEL-28(luciferase): 36.68±7.46%, MALME-3M(luciferase): 47.12±2.18%) (Figure 3). In addition, we examined whether the cytotoxicity of CAR T cells could be affected by the type of virus that infects T cells. We performed a cell cytotoxicity assay using two different CAR T cells. One BTLA CAR T cell was generated by lentivirus infection, and the other was generated by retrovirus infection. Non-transduced T cells (NTD T cell), lentivirus version BTLA-CAR T cells [T cell(LV-BTLA-28z)], and retrovirus version BTLA-CAR T cells [T cell(RV-BTLA-28z)] (1×105 cells) were co-cultured with SK-MEL-28(luciferase) or 293T(luciferase) cells (1×104 cells) for 4 or 24 h. As shown in Figure 4, T cell(BV-BTLA-28z) showed superior cytotoxicity compared to other effector T cells against HVEM-expressing SK-MEL-28(luciferase) cells at both 4 and 24 h incubation time. (NTD T cell: 19.97±3.75%, T cell(LV-BTLA-28z): 36.57±3.59%, T cell(RV-BTLA-28z): 83.33±0.57%, incubation time is 4 h), (NTD T cell: 30.61±3.83%, T cell(LV-BTLA-28z): 68.96±5.42%, T cell(RV-BTLA-28z): 98.52±0.77%, incubation time is 24 h) (Figure 4).

Figure 3.
Figure 3.

BTLA-28z CAR T cells lysed HVEM-positive cells. (A) The Non-transduced T cells (NTD T cell), ZsGreen-expressing T cells [T cell(LV-ZsGreen)], or BTLA-28z CAR T cells [T cell(LV-BTLA-28z)] (5×104 cells) were co-cultured with SK-MEL-28(luciferase), MALME-3M(luciferase) or 293T(luciferase) (5×103 cells) for 4 or 24 h. BTLA-28z CAR T cells had higher cytotoxicity than NTD T cell and T cell(LV-ZsGreen) against HVEM-positive cancer cells. We determined the cytotoxicity of CAR T cells by measuring the luminescence signal in living target cells expressing luciferase. The T cell(LV-ZsGreen) and T cell(LV-BTLA-28z) were generated by lentivirus infection. Statistical significance was evaluated using unpaired t-test (ns: non-significant, *p<0.05, **p<0.01, ***p<0.001; ****p<0.0001) (two-tailed p-value).

Figure 4.
Figure 4.

RV-BTLA-28z CAR T cells lysed HVEM-positive cells best. (A) The Non-transduced T cells (NTD T cell), LV-BTLA-28z [T cell(LV-BTLA-28z)], or RV-BTLA-28z CAR T cells [T cell(RV-BTLA-28z)] (1×105 cells) were incubated with SK-MEL-28(luciferase) or 293T(luciferase) (1×104 cells) for 4 or 24 h. The T cell(RV-BTLA-28z) cells had the highest cytotoxic activity against HVEM-positive cancer cells [SK-MEL-28(luciferase)]. T cell(RV-BTLA-28z) cells were infected by a retrovirus and T cell(LV-BTLA-28z) cells were transduced by lentivirus infection. Statistical significance was evaluated using unpaired t-test (ns: non-significant, *p<0.05, **p<0.01, ***p<0.001; ****p<0.0001) (two-tailed p-value).

Discussion

CAR T cell therapies have shown impressively high clinical response rates in patients with B-cell acute lymphoblastic leukemia (B-ALL). Recently, the FDA approved CD19-specific CAR T cells for B-ALL and diffuse large B-cell lymphoma (20). Although CAR T cell-based immunotherapy has remarkable efficacy against hematological cancers, it exhibits limited therapeutic effects against solid tumors. There are various limitations in increasing the efficacy of CAR T cells in solid tumors, including target antigen heterogeneity, limited tumor infiltration, an immunosuppressive microenvironment, and intrinsic regulatory mechanisms of T cells (21, 22). Despite the challenges in using CAR T cells for treating solid tumors, many studies have undergone and focused on improving CAR T cells and overcoming the adverse tumor microenvironment. There are several therapeutic approaches on solid tumors, such as gastric cancer, breast cancer, and colorectal cancer, which target cancer-specific or over-expressed antigens on cancer cells, such as CEA, CD133, or HER2 (23). In this study, we focused on melanomas. Globally, many people are diagnosed with melanoma, and approximately 55,500 patients die of melanoma (24). There are many intrinsic or extrinsic factors associated with developing melanoma, such as age, genetic alterations, or UV exposure. For example, melanoma is most common in Caucasians. Mutations or activation of essential signaling pathways in melanocytes, such as the mitogen-activated protein kinase pathway, can cause melanoma, and mutations in B-Raf, N-Ras, or c-KIT can lead to the abnormal proliferation and survival of melanocytes (25). Although surgery, radiation therapy, and chemotherapy have been used to treat melanoma, cancer cells cannot be completely removed and there are limitations, such as drug resistance. Due to these problems, the importance of new treatment methods has emerged. In this study, we evaluated the expression of HVEM on the surface of melanoma cell lines and confirmed that HVEM is over-expressed in melanoma cells. Therefore, we used HVEM as a potential novel antigen. To examine the anticancer effects of anti-HVEM CAR T cells on melanoma cells, we first produced BTLA-28z, BTLA-3z, and CD160-28z CAR constructs through gene cloning. BTLA and CD160 bind to HVEM (26). We compared the performance of the BTLA-28z, CD160-28z, and BTLA-3z CAR constructs in Jurkat and KHYG-1 cells before generating CAR T cells. Both cell lines have the advantage of being easier to culture than T cells. Jurkat cells derived from CD4+ T cells and when activated secrete IL-2 whereas KHYG-1, an NK cell line, exhibits cytolytic activity when activated. We expressed the BTLA-28z and CD160-28z constructs in Jurkat cells, and the amount of secreted IL-2 was detected using IL-2 ELISA. The BTLA-28z and CD160-28z constructs were transferred into Jurkat cells by electroporation, and each CAR Jurkat cell line was co-cultured with an HVEM-positive or negative cell line. The ability of BTLA-28z CAR Jurkat cells to secrete IL-2 was superior to that of CD160-28z CAR Jurkat cells. However, expression of the CD160-28z CAR protein was much lower than that of BTLA-28z. We found that the protein expression levels affected IL-2 secretion in CAR Jurkat cells. In addition, we examined the performance of BTLA-28z and BTLA-3z. BTLA-28z Jurkat cells secrete higher amounts of IL-2 than BTLA-3z Jurkat cells when recognizing the HVEM antigen. This suggests that the CD28 domain promotes IL-2 secretion. Interestingly, BTLA-28z and BTLA-3z CAR Jurkat cells secreted significantly higher levels of cytokines when co-cultured with SK-MEL-28(luciferase) or 293T(HVEM-T2A-luciferase) cells. SK-MEL-28 is a melanoma cell line naturally expressing HVEM. 293T(HVEM-T2A-luciferase) cells were generated by infection with an HVEM-T2A-luciferase lentivirus, and HVEM proteins were over-expressed on their surface. We confirmed that the level of HVEM expression in both cell lines was much higher than that in the other cells, and this phenomenon caused the CAR Jurkat cells to secrete significantly more cytokines. Next, we performed a cytotoxicity assay using BTLA-28z CAR KHYG-1 cells against HVEM-positive cells [SK-MEL-28(luciferase), 293T(HVEM-T2A-luciferase)] or HVEM-negative cells [CAKI-1(luciferase)]. Before starting the cytotoxicity assay, the target cells were transduced with a luciferase virus. This is because the percentage of cell cytotoxicity was calculated according to the intensity of the luminescence signal from living target cells. BTLA-28z CAR KHYG-1 cells efficiently and specifically lysed HVEM-positive cells. These results imply that BTLA-28z CAR can be used for CAR T cell development. We generated BTLA-28z CAR T cells by lentivirus infection and examined their ability to secrete IL-2 using IL-2 ELISA. As expected, BTLA-28z CAR T cells secreted significant amounts of IL-2 when co-cultured with HVEM-positive cells. FITC-28z CAR T cells, used as negative controls, secreted much lower amounts of IL-2 than the BTLA-28z CAR T cells. This result indicates that BTLA-28z CAR T cells selectively recognize HVEM. Cell cytotoxicity assays were performed using BTLA-28z CAR T cells against HVEM-positive cells [SK-MEL-28(luciferase), MALME-3M(luciferase)] and HVEM-negative cells [293T(luciferase)]. Non-transduced T cells and ZsGreen-expressing T cells were used as negative controls. BTLA-28z CAR T cells effectively and selectively lysed HVEM-positive cells. The BTLA-28z CAR T cells were more cytotoxic to SK-MEL-28(luciferase) cells than to MALME-3M(luciferase) cells. We hypothesize that the difference in HVEM expression levels between the two cell lines influenced this result. The expression levels of HVEM in SK-MEL-28 cells were higher than those in MALME-3M cells. We also examined whether the cytolytic effects of CAR T cells differ depending on the type of virus used to produce them. We created two versions of CAR T cells. One was generated using a lentivirus and the other using a retrovirus. Both CAR T cells were co-cultured with HVEM-positive cells [SK-MEL-28(luciferase)] or HVEM-negative cells [293T(luciferase)]. NTD T cells were used as a negative control. Both versions of the BTLA CAR T cells showed effective and selective cytolytic activity against SK-MEL-28(luciferase) cells. However, a noteworthy finding was that the retroviral version of BTLA CAR T cells killed SK-MEL-28(luciferase) cells more effectively than the lentiviral version of BTLA CAR T cells. We hypothesized that the lentiviral version BTLA-CAR T cells were more differentiated and exhausted than the retroviral version CAR T cells because the size of the lentiviral vector used was very small and transduction into T cells worked well. As a result, the high transduction efficiency into T cells produced a tonic signal and made CAR T cells more differentiated and exhausted, regardless of the antigen (27). In further studies, we will develop a xenograft mouse model and examine whether our BTLA-CAR T cells can be as useful in reducing melanoma cells in vivo as in vitro. Although we screened whether HVEM was expressed in melanoma cells, we could not be certain whether HVEM is expressed only in melanoma cells. Therefore, there is concern that BTLA CAR T cells may induce on-target off-tumor toxicity. To solve this problem, it is necessary to control the anticancer activity of CAR T cells through genetic modifications or combination therapy (28).

Conclusion

We developed BTLA-CAR T cells targeting HVEM. These cells showed effective anticancer activity against melanoma cell lines. We suggest that BTLA-CAR T cell therapy could be a novel strategy for melanoma treatment.

Acknowledgements

This work was supported by a grant from Korea Drug Development Fund (HN21C0226000021), and the Korea Research Institute of Chemical Technology (No. KK2331-50) funded by the Ministry of Science and ICT (MSIT) of the Republic of South Korea.

Footnotes

  • Authors’ Contributions

    Yeongrin Kim, Ji Eun Kim, and Chi Hoon Park conceived and designed the study. Yeongrin Kim, Sang Un Choi, Simon Park, and Chi Hoon Park analyzed the data. Yeongrin Kim drafted the manuscript. Yeongrin Kim, Ji Eun Kim, Da Yeon Lee, Ji U Choi, Jin Song Park, and Heung Kyoung Lee performed experiments. Chi Hoon Park provided reagents, experimental expertise, and reviewed the manuscript. All Authors contributed to the article and approved the submitted version.

  • Conflicts of Interest

    The Authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

  • Received May 23, 2023.
  • Revision received June 20, 2023.
  • Accepted June 21, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Wang M,
    2. Munoz J,
    3. Goy A,
    4. Locke F,
    5. Jacobson C,
    6. Hill B,
    7. Timmerman J,
    8. Holmes H,
    9. Jaglowski S,
    10. Flinn I,
    11. McSweeney P,
    12. Miklos D,
    13. Pagel J,
    14. Kersten M,
    15. Bouabdallah K,
    16. Khanal R,
    17. Topp M,
    18. Houot R,
    19. Beitinjaneh A,
    20. Peng W,
    21. Fang X,
    22. Shen R,
    23. Siddiqi R,
    24. Kloos I,
    25. Reagan P
    : Three-year follow-up of KTE-X19 in patients with relapsed/refractory mantle cell lymphoma, including high-risk subgroups, in the ZUMA-2 study. J Clin Oncol 41(3): 555-567, 2023. DOI: 10.1200/JCO.21.02370
    OpenUrlCrossRef
  2. ↵
    1. Zhao W,
    2. Wang B,
    3. Chen L,
    4. Fu W,
    5. Xu J,
    6. Liu J,
    7. Jin S,
    8. Chen Y,
    9. Cao X,
    10. Yang Y,
    11. Zhang Y,
    12. Wang F,
    13. Zhang P,
    14. Lei B,
    15. Gu L,
    16. Wang J,
    17. Zhang H,
    18. Bai J,
    19. Xu Y,
    20. Zhu H,
    21. Du J,
    22. Jiang H,
    23. Fan X,
    24. Li J,
    25. Hou J,
    26. Chen Z,
    27. Zhang W,
    28. Mi J,
    29. Chen S,
    30. He A
    : Four-year follow-up of LCAR-B38M in relapsed or refractory multiple myeloma: a phase 1, single-arm, open-label, multicenter study in China (LEGEND-2). J Hematol Oncol 15(1): 86, 2022. DOI: 10.1186/s13045-022-01301-8
    OpenUrlCrossRefPubMed
  3. ↵
    1. Hu Y,
    2. Zhou Y,
    3. Zhang M,
    4. Zhao H,
    5. Wei G,
    6. Ge W,
    7. Cui Q,
    8. Mu Q,
    9. Chen G,
    10. Han L,
    11. Guo T,
    12. Cui J,
    13. Jiang X,
    14. Zheng X,
    15. Yu S,
    16. Li X,
    17. Zhang X,
    18. Chen M,
    19. Li X,
    20. Gao M,
    21. Wang K,
    22. Zu C,
    23. Zhang H,
    24. He X,
    25. Wang Y,
    26. Wang D,
    27. Ren J,
    28. Huang H
    : Genetically modified CD7-targeting allogeneic CAR-T cell therapy with enhanced efficacy for relapsed/refractory CD7-positive hematological malignancies: a phase I clinical study. Cell 2(11): 995-1007, 2022. DOI: 10.1038/s41422-022-00721-y
    OpenUrlCrossRef
    1. Liu Y,
    2. Deng B,
    3. Hu B,
    4. Zhang W,
    5. Zhu Q,
    6. Liu Y,
    7. Wang S,
    8. Zhang P,
    9. Yang Y,
    10. Yang J,
    11. Zheng Q,
    12. Yu X,
    13. Gao Z,
    14. Zhou C,
    15. Han W,
    16. Yang J,
    17. Jin L,
    18. Tong C,
    19. Chang A,
    20. Zhang Y
    : Sequential different B-cell antigen–targeted CAR T-cell therapy for pediatric refractory/relapsed Burkitt lymphoma. Blood Adv 6(3): 717-730, 2022. DOI: 10.1182/bloodadvances.2021004557
    OpenUrlCrossRefPubMed
  5. ↵
    1. Zhang W,
    2. Wang Y,
    3. Guo Y,
    4. Dai H,
    5. Yang Q,
    6. Zhang Y,
    7. Zhang Y,
    8. Chen M,
    9. Wang C,
    10. Feng K,
    11. Li S,
    12. Liu Y,
    13. Shi F,
    14. Luo C,
    15. Han W
    : Treatment of CD20-directed Chimeric Antigen Receptor-modified T cells in patients with relapsed or refractory B-cell non-Hodgkin lymphoma: an early phase IIa trial report. Signal Transduct Target Ther 1(1): 16002, 2016. DOI: 10.1038/sigtrans.2016.2
    OpenUrlCrossRefPubMed
  6. ↵
    1. Dai H,
    2. Tong C,
    3. Shi D,
    4. Chen M,
    5. Guo Y,
    6. Chen D,
    7. Han X,
    8. Wang H,
    9. Wang Y,
    10. Shen P
    : Efficacy and biomarker analysis of CD133-directed CAR T cells in advanced hepatocellular carcinoma: a single-arm, open-label, phase II trial. Oncoimmunology 9(1): 1846926, 2020. DOI: 10.1080/2162402X.2020.1846926
    OpenUrlCrossRefPubMed
  7. ↵
    1. Vitanza N,
    2. Wilson A,
    3. Huang W,
    4. Seidel K,
    5. Brown C,
    6. Gustafson J,
    7. Yokoyama J,
    8. Johnson A,
    9. Baxter B,
    10. Koning R,
    11. Reid A,
    12. Meechan M,
    13. Biery M,
    14. Myers C,
    15. Rawlings-Rhea S,
    16. Albert C,
    17. Browd S,
    18. Hauptman J,
    19. Lee A,
    20. Ojemann J,
    21. Berens M,
    22. Dun M,
    23. Foster J,
    24. Crotty E,
    25. Leary S,
    26. Cole B,
    27. Perez F,
    28. Wright J,
    29. Orentas R,
    30. Chour T,
    31. Newell E,
    32. Whiteaker J,
    33. Zhao L,
    34. Paulovich A,
    35. Pinto N,
    36. Gust J,
    37. Gardner R,
    38. Jensen M,
    39. Park J
    : Intraventricular B7-H3 CAR T cells for diffuse intrinsic pontine glioma: preliminary first-in-human bioactivity and safety. Cancer Discov 13(1): 114-131, 2022. DOI: 10.1158/2159-8290.CD-22-0750
    OpenUrlCrossRef
    1. Yvon E,
    2. Del Vecchio M,
    3. Savoldo B,
    4. Hoyos V,
    5. Dutour A,
    6. Anichini A,
    7. Dotti G,
    8. Brenner M
    : Immunotherapy of metastatic melanoma using genetically engineered GD2-specific T cells. Clin Cancer Res 15(18): 5852-5860, 2009. DOI: 10.1158/1078-0432.CCR-08-3163
    OpenUrlAbstract/FREE Full Text
    1. Burns W,
    2. Zhao Y,
    3. Frankel T,
    4. Hinrichs C,
    5. Zheng Z,
    6. Xu H,
    7. Feldman S,
    8. Ferrone S,
    9. Rosenberg S,
    10. Morgan R
    : A high molecular weight melanoma-associated antigen-specific chimeric antigen receptor redirects lymphocytes to target human melanomas. Cancer Res 70(8): 3027-3033, 2010. DOI: 10.1158/0008-5472.CAN-09-2824
    OpenUrlAbstract/FREE Full Text
    1. Wu M,
    2. Zhang T,
    3. Alcon A,
    4. Sentman C
    : DNAM-1-based chimeric antigen receptors enhance T cell effector function and exhibit in vivo efficacy against melanoma. Cancer Immunol Immunother 64(4): 409-418, 2015. DOI: 10.1007/s00262-014-1648-2
    OpenUrlCrossRefPubMed
    1. Krishnamurthy J,
    2. Rabinovich B,
    3. Mi T,
    4. Switzer K,
    5. Olivares S,
    6. Maiti S,
    7. Plummer J,
    8. Singh H,
    9. Kumaresan P,
    10. Huls H,
    11. Wang-Johanning F,
    12. Cooper L
    : Genetic engineering of T cells to target HERV-K, an ancient retrovirus on melanoma. Clin Cancer Res 21(14): 3241-3251, 2015. DOI: 10.1158/1078-0432.CCR-14-3197
    OpenUrlAbstract/FREE Full Text
    1. Wiesinger M,
    2. März J,
    3. Kummer M,
    4. Schuler G,
    5. Dörrie J,
    6. Schuler-Thurner B,
    7. Schaft N
    : Clinical-scale production of CAR-T cells for the treatment of melanoma patients by mRNA transfection of a CSPG4-specific CAR under full GMP compliance. Cancers (Basel) 11(8): 1198, 2019. DOI: 10.3390/cancers11081198
    OpenUrlCrossRefPubMed
  13. ↵
    1. Wallstabe L,
    2. Mades A,
    3. Frenz S,
    4. Einsele H,
    5. Rader C,
    6. Hudecek M
    : CAR T cells targeting αvβ3integrin are effective against advanced cancer in preclinical models. Adv Cell Gene Ther 1(2): e11, 2018. DOI: 10.1002/acg2.11
    OpenUrlCrossRef
  14. ↵
    1. Simon B,
    2. Uslu U
    : CAR-T cell therapy in melanoma: A future success story? Exp Dermatol 27(12): 1315-1321, 2018. DOI: 10.1111/exd.13792
    OpenUrlCrossRefPubMed
  15. ↵
    1. Han M,
    2. Wang S,
    3. Zhao W,
    4. Ni S,
    5. Yang N,
    6. Kong Y,
    7. Huang B,
    8. Chen A,
    9. Li X,
    10. Wang J,
    11. Wang D
    : Immune checkpoint molecule herpes virus entry mediator is overexpressed and associated with poor prognosis in human glioblastoma. EBioMedicine 43: 159-170, 2019. DOI: 10.1016/j.ebiom.2019.04.002
    OpenUrlCrossRefPubMed
    1. Inoue T,
    2. Sho M,
    3. Yasuda S,
    4. Nishiwada S,
    5. Nakamura S,
    6. Ueda T,
    7. Nishigori N,
    8. Kawasaki K,
    9. Obara S,
    10. Nakamoto T,
    11. Koyama F,
    12. Fujii H,
    13. Nakajima Y
    : HVEM expression contributes to tumor progression and prognosis in human colorectal cancer. Anticancer Res 35(3): 1361-1367, 2015.
    OpenUrlAbstract/FREE Full Text
    1. Lan X,
    2. Li S,
    3. Gao H,
    4. Nanding A,
    5. Quan L,
    6. Yang C,
    7. Ding S,
    8. Xue Y
    : Increased BTLA and HVEM in gastric cancer are associated with progression and poor prognosis. Onco Targets Ther 10: 919-926, 2017. DOI: 10.2147/OTT.S128825
    OpenUrlCrossRefPubMed
  18. ↵
    1. Ren S,
    2. Tian Q,
    3. Amar N,
    4. Yu H,
    5. Rivard C,
    6. Caldwell C,
    7. Ng T,
    8. Tu M,
    9. Liu Y,
    10. Gao D,
    11. Ellison K,
    12. Suda K,
    13. Rozeboom L,
    14. Rivalland G,
    15. Mitchell P,
    16. Zhou C,
    17. Hirsch F
    : The immune checkpoint, HVEM may contribute to immune escape in non-small cell lung cancer lacking PD-L1 expression. Lung Cancer 125: 115-120, 2018. DOI: 10.1016/j.lungcan.2018.09.004
    OpenUrlCrossRefPubMed
  19. ↵
    1. Malissen N,
    2. Macagno N,
    3. Granjeaud S,
    4. Granier C,
    5. Moutardier V,
    6. Gaudy-Marqueste C,
    7. Habel N,
    8. Mandavit M,
    9. Guillot B,
    10. Pasero C,
    11. Tartour E,
    12. Ballotti R,
    13. Grob J,
    14. Olive D
    : HVEM has a broader expression than PD-L1 and constitutes a negative prognostic marker and potential treatment target for melanoma. Oncoimmunology 8(12): e1665976, 2019. DOI: 10.1080/2162402X.2019.1665976
    OpenUrlCrossRefPubMed
  20. ↵
    1. Santomasso B,
    2. Park J,
    3. Salloum D,
    4. Riviere I,
    5. Flynn J,
    6. Mead E,
    7. Halton E,
    8. Wang X,
    9. Senechal B,
    10. Purdon T,
    11. Cross J,
    12. Liu H,
    13. Vachha B,
    14. Chen X,
    15. DeAngelis L,
    16. Li D,
    17. Bernal Y,
    18. Gonen M,
    19. Wendel H,
    20. Sadelain M,
    21. Brentjens R
    : Clinical and biological correlates of neurotoxicity associated with CAR T-cell therapy in patients with B-cell acute lymphoblastic leukemia. Cancer Discov 8(8): 958-971, 2018. DOI: 10.1158/2159-8290.CD-17-1319
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Newick K,
    2. O’Brien S,
    3. Moon E,
    4. Albelda S
    : CAR T cell therapy for solid tumors. Annual Review of Medicine 68(1): 139-152, 2017. DOI: 10.1146/annurev-med-062315-120245
    OpenUrlCrossRefPubMed
  22. ↵
    1. Sterner R,
    2. Sterner R
    : CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J 11(4): 69, 2021. DOI: 10.1038/s41408-021-00459-7
    OpenUrlCrossRefPubMed
  23. ↵
    1. Ma S,
    2. Li X,
    3. Wang X,
    4. Cheng L,
    5. Li Z,
    6. Zhang C,
    7. Ye Z,
    8. Qian Q
    : Current progress in CAR-T cell therapy for solid tumors. Int J Biol Sci 15(12): 2548-2560, 2019. DOI: 10.7150/ijbs.34213
    OpenUrlCrossRefPubMed
  24. ↵
    1. Schadendorf D,
    2. Van Akkooi A,
    3. Berking C,
    4. Griewank K,
    5. Gutzmer R,
    6. Hauschild A,
    7. Stang A,
    8. Roesch A,
    9. Ugurel S
    : Melanoma. Lancet 392(10151): 971-984, 2018. DOI: 10.1016/S0140-6736(18)31559-9
    OpenUrlCrossRefPubMed
  25. ↵
    1. Wang H,
    2. Tran T,
    3. Duong K,
    4. Nguyen T,
    5. Le U
    : Options of therapeutics and novel delivery systems of drugs for the treatment of melanoma. Mol Pharm 19(12): 4487-4505, 2022. DOI: 10.1021/acs.molpharmaceut.2c00775
    OpenUrlCrossRefPubMed
  26. ↵
    1. Ning Z,
    2. Liu K,
    3. Xiong H
    : Roles of BTLA in immunity and immune disorders. Front Immunol 12: 654960, 2021. DOI: 10.3389/fimmu.2021.654960
    OpenUrlCrossRefPubMed
  27. ↵
    1. Gomes-Silva D,
    2. Mukherjee M,
    3. Srinivasan M,
    4. Krenciute G,
    5. Dakhova O,
    6. Zheng Y,
    7. Cabral J,
    8. Rooney C,
    9. Orange J,
    10. Brenner M,
    11. Mamonkin M
    : Tonic 4-1BB costimulation in chimeric antigen receptors impedes T cell survival and is vector-dependent. Cell Rep 21(1): 17-26, 2017. DOI: 10.1016/j.celrep.2017.09.015
    OpenUrlCrossRefPubMed
  28. ↵
    1. Rodriguez-Garcia A,
    2. Palazon A,
    3. Noguera-Ortega E,
    4. Powell D,
    5. Guedan S
    : CAR-T cells hit the tumor microenvironment: strategies to overcome tumor escape. Front Immunol 11: 1109, 2020. DOI: 10.3389/fimmu.2020.01109
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
T Cells Expressing CAR Equipped With Extracellular Domain of BTLA Are Effective Against HVEM-over-expressing Melanoma Cell Lines
YEONGRIN KIM, JI EUN KIM, DA YEON LEE, JI U CHOI, JIN SONG PARK, HEUNG KYOUNG LEE, SANG UN CHOI, SIMON PARK, CHI HOON PARK
Anticancer Research Aug 2023, 43 (8) 3419-3427; DOI: 10.21873/anticanres.16517
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords