Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

Abstract

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.

Immortalizing Mesenchymal Stromal Cells from Aged Donors While Keeping Their Essential Features

“…After incubation at 32°C, the supernatant was collected and filtered through a 0.45 μm pore size membrane filter (Millipore, Burlington, Massachusetts, USA). 8 μg of HDMB (Sigma-Aldrich Química S.A.) was added per ml of retroviral supernatants [28,46], and this mixture was employed for the infection of MSCs at 70% confluence in 6-well adherent culture dishes (Costar Corning Incorporated) ( Figure 1). No viruses were added to one well of each dish, which was used as a negative control for antibiotic selection.…”

“…Therefore, strategies to enhance infection efficiency should be employed. One of these strategies is spinoculation, which has been used during decades to improve viral infection of several types of cells in vitro [21][22][23][24][25][26][27][28][29][30][31][32][33][34] (Table 1), although the process responsible for spinoculation-induced enhancement of infection has not been discovered yet [33]. However, it is known that the enhancement of infection induced by spinoculation is cell type-dependent [20,25] and also related to the speed of centrifugation [33] in a cell type-dependent manner [23].…”

“…Therefore, spinoculation parameters must be optimized for each transduction system (virus and target cell type). Since spinoculation-induced enhancement of infection is also related to virus concentration, it could be possible to increase it by prolonging the posttransfection incubation of packaging cells before harvesting [22,28,32,33]. As virus half-life at 37°C is shorter than at 32°C, packaging cell incubation and centrifugal infection may need to be performed at 32°C [21,[27][28][29].…”

CAR T cells in solid tumors: challenges and opportunities

“…Other studies suggested that HRG1β-based CAR-T cells successfully inhibit breast cancer proliferation through HER family receptors and able to deliver an attractive therapeutic approach to defeat cancer resistance against HER2-based targeted therapy [62]. In parallel, Munisvaradass et al found that human anti-HER2 CAR T cells showed desirable targeting and triggered cell death in HER2 overexpressing breast cancer cells [63]. Moreover, mesothelin recognition by special CAR T cells has been described as a promising immunotherapy goal for breast cancer treatment [64].…”

CAR-T Therapy for Solid Tumors: Development of New Strategies

“…The scFv redirects CAR-T cells to recognize cancer cells in an HLA (human leukocyte antigen)-independent manner and the TCR intracellular domain induces T-cell dependent cancer killing [14,15] . After the remarkable demonstration of efficiency by the first engineered T cells pioneered by Eshhar and coworkers [16] , variability in T cell functionalities have emerged.…”