PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

Abstract

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative control-transduced groups. In conclusion, a novel HLA-A2-restricted and PLAC1-specific TCR was identified. The present study demonstrated PLAC1 to be a potential target for breast cancer treatment; and the usage of PLAC1-specific TCR-engineered T cells may be a novel strategy for PLAC1-positive breast cancer treatment.

Keywords: T cell receptor; breast cancer; cancer immunotherapy; cytotoxic T cells; placenta specific 1.

Figure 1.

Generation of PLAC1-specific TCR-engineered CD8+ T cells. (A) Schematic illustration of the lentiviral vector encoding an anti-PLAC1 TCR expression cassette. TCRα- and TCRβ-chains were linked with a 2A peptide sequence. (B) CD8+ T cells, selected from peripheral blood mononuclear cells with a negative selection procedure using magnetic beads, were analyzed using flow cytometry for CD3 and CD8 expression via FITC-labeled mAb against human CD8 and PerCP-Cy5.5-labeled mAb against human CD3. (C) CD8+ T cells transduced with PLAC1-specific TCR or negative control or untransduced were stained with PE-labeled PLAC1-multimer (PE-multimer) and FITC-labeled CD8 mAb (CD8-FITC). The results are representative of three independent experiments. PLAC1, placenta-specific 1; TCR, T cell receptor; CD8+ T cell, cytotoxic T cell; CD, cluster of differentiation; FITC, fluorescein isothiocyanate; PE, phycoerythrin; mAb, monoclonal antibody.

Figure 2.

Identification of PLAC1 and HLA-A2 serotype-positive breast cancer cell lines. (A) Immunofluorescence staining with rabbit anti-PLAC1 primary antibody or a rabbit IgG and PE-conjugated secondary antibody in the human non-metastatic breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDAMB-231. The scale bar indicates 50 µm. (B) MCF-7 and MDAMB-231 cells were analyzed using flow cytometry for HLA-A2 expression using the PE-labeled HLA-A2 antibody and PE-labeled isotype control. The results are representative of three independent experiments. PLAC1, placenta-specific 1; HLA, human leukocyte antigen; PE, phycoerythrin; IgG, immunoglobulin G.

Figure 3.

Evaluation of the function of PLAC1 TCR-engineered CD8+ T cells. Human leukocyte antigen-A2-restricted and PLAC1-specific TCR-, NC-transduced or untransduced CD8+ T cells were co-cultured for 4 h with 1×10⁴ MCF-7 and MDAMB-231 cells respectively at a range of E:T ratios (5:1, 10:1 and 20:1). Concentration of (A) IFN-γ and (B) TNF-α secreted into the culture medium were measured using an enzyme-linked immunosorbent assay. (C) TCR- or NC-transduced CD8+ T cells were co-cultured for 4 h with 1×10⁴ MCF-7 and MDAMB-231 cells. Cytolysis was determined using a lactate dehydrogenase activity assay. Subsequent to background subtraction, the percentage lysis was calculated by 100% × [(experimental release-effector spontaneous release-target spontaneous release)/(target maximum release-target spontaneous release)]. *P<0.05 and ^#P<0.01 with comparisons shown by lines. PLAC1, placenta-specific 1; TCR, T cell receptor; CD8+ T cell, cytotoxic T cell; NC, negative control; E:T, effector cell to target cell; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α.

Figure 4.

PLAC1 TCR-engineered CD8+ T cells specifically recognize and kill breast cancer cells. (A) Immunofluorescence staining with rabbit anti-PLAC1 primary antibody or a rabbit IgG and PE-conjugated secondary antibody in the human breast cancer cell lines T47D. Scale bar indicates 50 µm. The results are representative of three independent experiments. (B) T47D cells were analyzed using flow cytometry for HLA-A2 expression using the PE-labeled HLA-A2 antibody and PE-labeled isotype control. The results are representative of three independent experiments. (C) Lysis of T47D breast cancer cells by the engineered CD8+ T cells was determined using a LDH activity assay. Data are expressed as the means ± SD of three independent experiments. (D) MCF-7 cells were transfected with PLAC1 siRNA or NC siRNA and then analyzed using western blot analysis for PLAC1 expression following normalization to β-actin. The results are representative of three independent experiments. Bars represent the relative protein level as compared with the MCF-7 cells alone group. ^#P<0.01 vs. MCF-7 + NC siRNA group. (E) Lysis of MCF-7-NC siRNA and MCF-7-PLAC1 siRNA cells by the engineered CD8+ T cells was determined using a LDH activity assay. Data are expressed as the means ± SD of three independent experiments. *P<0.05 and ^#P<0.01 with comparisons shown by lines. PLAC1, placenta-specific 1; PE, phycoerythrin; IgG, immunoglobulin G; E:T, effector cell to target cell; TCR, T cell receptor; NC, negative control; siRNA, small interfering RNA; CD8+ T cell, cytotoxic T cell; HLA, human leukocyte antigen; SD, standard deviation; LDH, lactate dehydrogenase.

Figure 5.

Mechanism of enhanced activation of TCR-transduced CD8+ T cells. Cell lysates of TCR- or NC-transduced CD8+ T cells were analyzed by western blot analysis for the expression of PI3Kγ, AKT, p-AKT, ERK1/2, p-ERK1/2, JNK, p-JNK, p38, p-p38, NF-κB and GAPDH. The results are representative of three independent experiments. NC, negative control; TCR, T cell receptor; CD8+ T cells, cytotoxic T cells; PI3Kγ, phosphoinositide 3-kinase γ; AKT, protein kinase B; ERK1/2, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinases; NF-κB, nuclear factor κB; p-, phosphorylated.

Figure 6.

TCR-transduced CD8+ T cells inhibit tumor growth in vivo. MCF-7 cells (5×10⁶) were inoculated subcutaneously into 15 BALB/c-nu mice to establish a subcutaneous transplant tumor model. When tumors reached a mean volume of 150 mm³, the mice underwent intravenous tail vein transplantation with either normal saline, 1×10⁷ PLAC1 TCR-transduced CD8+ T cells or NC-transduced CD8+ T cells twice with a 1-week interval. The tumors were measured once/week using electronic calipers. The longest length and width were recorded, and the tumor volume was calculated according to the formula (π/6) (length) × (width)². *P<0.05 compared with the TCR (PLAC1) group, with comparisons shown by lines. TCR, T cell receptor; CD8+ T cells, cytotoxic T cells; PLAC1, placenta-specific 1; NC, negative control.