A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus

Abstract

Hepatocellular carcinoma (HCC) cells often have hepatitis B virus (HBV)-DNA integration and can be targeted by HBV-specific T cells. The use of viral vectors to introduce exogenous HBV-specific T-cell receptors (TCR) on T cells to redirect their specificity is complex and expensive to implement in clinical trials. Moreover, it raises safety concerns related to insertional mutagenesis and potential toxicity of long-lived HBV-specific T cells in patients with persistent infection. To develop a more practical and safer approach to cell therapy of HCC, we used electroporation of mRNA encoding anti-HBV TCR. Approximately 80% of CD8(+) T cells expressed functional HBV TCR 24 hours postelectroporation, an expression efficiency much higher than that obtained by retroviral transduction (~18%). Antigen-specific cytokine production of electroporated T cells was efficient within 72-hour period, after which the redirected T cells lost their HBV-specific function. Despite this transient functionality, the TCR-electroporated T cells efficiently prevented tumor seeding and suppressed the growth of established tumors in a xenograft model of HCC. Finally, we established a method for large-scale TCR mRNA electroporation that yielded large numbers of highly functional clinical-grade anti-HBV T cells. This method represents a practical approach to cell therapy of HCC and its inherently self-limiting toxicity suggests potential for application in other HBV-related pathologies.Molecular Therapy-Nucleic Acids (2013) 2, e114; doi:10.1038/mtna.2013.43; published online 13 August 2013.

Figure 1

High level of TCR expression and polyfunctionality of mRNA electroporated T cells. (a) Dot plots from a representative HLA-A2-HBs183-191 pentamer staining in HBV s183-TCR mRNA electroporated T cells at 6, 24, and 72 hours postelectroporation and retrovirally transduced T cells at 72 hours. T cells that were mock-electroporated or electroporated with an irrelevant CMV pp65-TCR mRNA and mock-transduced served as negative controls. The percentages of pentamer⁺ cells out of CD8⁺ or CD8⁻ cells are indicated. (b) Expression of TCR on electroporated CD8⁺ T cells and frequency of IFN-γ-producing CD8⁺ T cells after overnight coculture with s183 peptide-loaded T2 cells were determined at several time points as indicated. Results expressed as mean + SD (n = 5). (c) Dot plots from a representative activated T cells electroporated or retrovirally transduced with s183-TCR, after overnight coculture with s183 peptide-loaded T2 cells and stained for CD8 and IFN-γ (top row), TNF-α (middle row) and IL-2 (bottom row). Mock- and CMV pp65-TCR mRNA electroporated T cells cocultured with s183 peptide-loaded T2 cells served as negative controls. The percentages of cytokine-producing cells out of total T cells are indicated. (d) TNF-α and IL-2 production by IFN-γ⁺ T cells demonstrate polyfunctionality of electroporated T cells. (e) Cytokine co-expression subsets expressed as a percentage of total cytokine-producing electroporated or retrovirally transduced T cells. Mean for each group is shown. Single producers (blue slice), IFN-γ⁺, TNF-α⁺ or IL-2⁺; double producers (red slice), IFN-γ⁺ TNF-α⁺, IFN-γ⁺ IL-2⁺ or IL-2⁺ TNF-α⁺; triple producers (green slice), IFN-γ⁺ IL-2⁺ TNF-α⁺.

Figure 2

Similar signaling capacity and cytotocixity of mRNA electroporated and retrovirally transduced T cells. (a) Sensitivity of T-cell activation using T cells produced by mRNA electroporation compared with retroviral transduction. Results are displayed as percentage of maximum IFN-γ response obtained from intracellular cytokine staining. (b) Dose dependent lysis of HepG2-env targets (solid symbols and line) or HepG2-core targets (open symbols and dotted line) by electroporated T cells (triangle) compared with retrovirally transduced T cells (circle). Results are displayed as mean of triplicate measurements + SD. (c) Expression of perforin (left panel) and granzyme (right panel) on a representative activated T cells electroporated (red histograms) or retrovirally transduced (blue histograms) with s183-TCR, after 5 hours coculture with peptide-loaded T2 cells. Coculture with unpulsed T2 cells served as negative control (gray histograms). MFI of perforin and granzyme are indicated. (d) mRNA electroporated T cells have T_CM-like phenotype. Phenotype of total lymphocytes (left panel), CD8⁺ pentamer⁺ T cells (middle panel) and CD4⁺ Vb3⁺ T cells (right panel) in s183-TCR mRNA electroporated T cells (red shaded area) compared with retrovirally transduced T cells (blue shaded area). The percentages of CD45RA^+/- and CD62L^+/- cells within total lymphocytes or the gated CD8⁺ pentamer⁺ or CD4⁺ Vb3⁺ populations were determined by FACS. Cells were classified into different subsets: naive (CD45RA⁺CD62L⁺), T_CM (CD45RA⁻CD62L⁺), T_EM (CD45RA⁻CD62L⁻) and terminally differentiated EM (CD45RA⁺CD62L⁻). Results expressed as mean + SD (n = 3).

Figure 3

Efficient tumor clearance requires both CD8 cytotoxic and CD4 helper TCR-retrovirally transduced T cells. (a) One million HepG2-env tumor cells were inoculated by intrasplenic injection in NSG mice (n = 22). Ten days after tumor inoculation, mice were treated with 3 × 10⁶ CD8 + 3 × 10⁵ CD4 (n = 4), 3 × 10⁶ CD8 (n = 4), 1.5 × 10⁶ CD8 (n = 4), 1.5 × 10⁶ CD4 (n = 3), or 3 × 10⁵ CD4 (n = 4) s183-TCR transduced T cells injected i.v. Mice treated with 3 × 10⁶ mock transduced T cells served as controls (n = 3). In all experiments, tumor size was monitored by bioluminescence imaging and results are displayed as average radiance (p/s/cm²/sr) of each mouse (colored lines) and the mean of each group (in black bold line). Control group (grey shaded area) is plotted as average radiance (p/s/cm²/sr) of the mean + SD. (b) Tumors were significantly eliminated in mice treated with total (3 × 10⁶ CD8 + 3 × 10⁵ CD4, n = 10) (P < 0.05) compared with 3 × 10⁶ CD8 alone (n = 10) or 3 × 10⁵ CD4 alone (n = 6) s183-TCR transduced T cells. Results from two independent experiments. (c) TissueFAXS staining of spleen tissue from mice treated with total s183-TCR transduced T cells at day 1 after adoptive cell transfer. Figure shows DAPI staining (blue), HepG2-env tumor cells expressing GFP (green) and CD8 (red, bottom panel; isotype control, top panel). Multiple CD8 T cells were detected in the tumor (white arrows) and in multiple fields. Two representative fields shown at 40× magnification.

Figure 4

Multiple infusions of activated mRNA electroporated T cells control tumor growth and maintained stable disease. One million HepG2-env tumor cells were inoculated by intrasplenic injection in NSG mice (n = 11). Nine days after tumor inoculation, mice were treated with three doses of 3 × 10⁶ activated s183-TCR mRNA electroporated T cells per dose, (n = 4, red line) injected i.v once every 3 days. Mice treated with 3 × 10⁶ mock-electroporated T cells served as controls (n = 3, grey shaded area). Tumor size was monitored by bioluminescence imaging and plotted as average radiance (p/s/cm²/sr) of the mean + SD.

Figure 5

Prevention of HCC tumor cells seeding by mRNA electroporated T cells. One million HepG2-env tumor cells were inoculated by intrasplenic injection in NSG mice (n = 14). Four hours later, mice (n = 4 or 3 per group) were treated with graded doses (0.75, 1.5, 3 × 10⁶ pentamer⁺ CD8) of s183-TCR mRNA electroporated T cells injected i.v. Mice treated with 3 × 10⁶ mock-electroporated T cells served as controls (grey shaded area). Tumor size was monitored by bioluminescence imaging and plotted as average radiance (p/s/cm²/sr) of the mean + SD.

Figure 6

High level of TCR expression and multifunctionality of mRNA electroporated T cells produced in large-scale, clinical-grade conditions. A schematic illustrating cell numbers, efficiency, yield, and functionality of laboratory-grade (top row) vs. clinical-grade (bottom row) electroporation of T cells. Dot plot of CD8 and HLA-A2-HBs183-191 pentamer staining in s183-TCR mRNA electroporated T cells at 24 hours postelectroporation. Bar charts show the frequency of IFN-γ, TNF-α, and IL-2 producing cells out of CD8 or CD4 electroporated T cells.