Chimeric Antigen Receptors Targeting Human Cytomegalovirus

Abstract

Human cytomegalovirus (CMV) is a ubiquitous pathogen that causes significant morbidity in some vulnerable populations. Individualized adoptive transfer of ex vivo expanded CMV-specific CD8+ T cells has provided proof-of-concept that immunotherapy can be highly effective, but a chimeric antigen receptor (CAR) approach would provide a feasible method for broad application. We created 8 novel CARs using anti-CMV neutralizing antibody sequences, which were transduced via lentiviral vector into primary CD8+ T cells. All CARs were expressed. Activity against CMV-infected target cells was assessed by release of cytokines (interferon-γ and tumor necrosis factor-α), upregulation of surface CD107a, proliferation, cytolysis of infected cells, and suppression of viral replication. While some CARs showed varying functional activity across these assays, 1 CAR based on antibody 21E9 was consistently superior in all measures. These results support development of a CMV-specific CAR for therapeutic use against CMV and potentially other applications harnessing CMV-driven immunotherapies.

Keywords: cellular immunity; chimeric antigen receptor; human cytomegalovirus.

Figure 1.

Schematic of chimeric antigen receptor structure. A targeting domain consisting of a single chain antibody against cytomegalovirus (Table 1) is fused to an IgG₄ (hinge, CH2, and CH3 domains of the heavy chain constant region)–based spacer, a CD8 transmembrane domain, and cytoplasmic signaling domains of 4-1BB and CD3ζ.

Figure 2.

Chimeric antigen receptor (CAR) expression in transduced primary CD8+ T cells by flow cytometry. Primary CD8⁺ T cells were transduced with lentiviral vectors delivering genes for the indicated CARs. The cells were then stained for cell surface human antibody expression and analyzed by flow cytometry. Not shown: 62-11.

Figure 3.

Intracellular cytokine and cell surface CD107a expression by chimeric antigen receptor (CAR)–transduced primary CD8+ T cells upon exposure to cytomegalovirus (CMV)–infected cells. A, Representative dot plots are shown for production of both intracellular interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) by untransduced (top row) or CAR-transduced (bottom row) CD8⁺ T cells exposed to uninfected (left column) or acutely CMV TR-infected (right column) ARPE-19 cells. B, Representative histograms are shown for cell surface expression of CD107a on untransduced (top) or CAR-transduced (bottom) CD8⁺ T cells exposed to uninfected (red histogram) or CMV TR-infected (blue histogram) ARPE-19 cells. C, Net percentages of untransduced or CAR-transduced CD8⁺ T cells producing both intracellular IFN-γ and TNF-α in response to CMV-infected target cells (after subtraction of the response to uninfected target cells) are plotted. D, Net percentages of untransduced or CAR-transduced CD8⁺ T cells expressing cell surface CD107a after exposure to acutely CMV-infected ARPE-19 cells are plotted. Similar results were seen with CMV TB40/E-infected ARPE-19 cells (not shown). These results are representative of 3 experiments with 3 different CD8⁺ T-cell donors; the other 4 CARs demonstrated minimal activity in 2 other experiments (not shown).

Figure 4.

Proliferation of chimeric antigen receptor (CAR)–transduced primary CD8+ T cells upon exposure to cytomegalovirus (CMV)–infected cells. Primary CD8⁺ T cells transduced with the indicated CAR were labeled with CellTrace Violet dye and co-cultured for 6 days with uninfected (open gray histograms) or CMV TB40/E-infected (filled black histograms) ARPE-19 cells and analyzed by flow cytometry for dye expression after 7 days. These results are representative of 2 independent experiments with 2 different CD8⁺ T-cell donors, each performed in biological duplicates.

Figure 5.

Killing of cytomegalovirus (CMV)–infected target cells by chimeric antigen receptor (CAR)–transduced primary CD8+ T cells. Background-specific lysis of uninfected cells (< 6%, except for 2–80 that had background levels of 20% and 32% at effector to target ratios of 20:1 and 40:1, respectively) was subtracted from specific lysis of CMV TB40/E-infected cells. In 3 independent experiments with 3 different CD8⁺ T-cell donors, only 21E9 exhibited consistent targeted killing of CMV-infected cells.

Figure 6.

Suppression of cytomegalovirus (CMV) replication in cell culture by chimeric antigen receptor (CAR)–transduced primary CD8+ T cells. ARPE-19 cells were acutely infected with CMV TR (green fluorescent protein [GFP]-expressing) for 4 days, and then co-cultured with no cells, untransduced primary CD8+ T cells, or CAR-transduced CD8⁺ T cells (at a ratio of 10 CD8⁺ T cells per target cell), followed by imaging 8 hours later. Similar results were obtained with CMV strain TB40/E (not shown). These results are representative of 3 independent experiments with 3 different CD8⁺ T-cell donors.