K562-Derived Whole-Cell Vaccine Enhances Antitumor Responses of CAR-Redirected Virus-Specific Cytotoxic T Lymphocytes In Vivo

Abstract

Purpose: Adoptive transfer of Epstein-Barr virus (EBV)-specific and cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) genetically modified to express a chimeric antigen receptor (CAR) induces objective tumor responses in clinical trials. In vivo expansion and persistence of these cells are crucial to achieve sustained clinical responses. We aimed to develop an off-the-shelf whole-cell vaccine to boost CAR-redirected virus-specific CTLs in vivo after adoptive transfer. As proof of principle, we validated our vaccine approach by boosting CMV-specific CTLs (CMV-CTLs) engineered with a CAR that targets the GD2 antigen.

Experimental design: We generated the whole-cell vaccine by engineering the K562 cell line to express the CMV-pp65 protein and the immune stimulatory molecules CD40L and OX40L. Single-cell-derived clones were used to stimulate CMV-CTLs in vitro and in vivo in a xenograft model. We also assessed whether the in vivo boosting of CAR-redirected CMV-CTLs with the whole-cell vaccine enhances the antitumor responses. Finally, we addressed potential safety concerns by including the inducible safety switch caspase9 (iC9) gene in the whole-cell vaccine.

Results: We found that K562-expressing CMV-pp65, CD40L, and OX40L effectively stimulate CMV-specific responses in vitro by promoting antigen cross-presentation to professional antigen-presenting cells (APCs). Vaccination also enhances antitumor effects of CAR-redirected CMV-CTLs in xenograft tumor models. Activation of the iC9 gene successfully induces growth arrest of engineered K562 implanted in mice.

Conclusions: Vaccination with a whole-cell vaccine obtained from K562 engineered to express CMV-pp65, CD40L, OX40L and iC9 can safely enhance the antitumor effects of CAR-redirected CMV-CTLs.

©2015 American Association for Cancer Research.

Figures

Figure 1. K562-based whole-cell vaccine encoding CMV-pp65 and CD40L matures monocytes and stimulates CMV-CTLs in vitro. Panel A

Expression of CD40L and OX40L in engineered K562. Striped histograms indicate wild type K562 cells. Panel B. Western blot showing the expression of CMV-pp65 in engineered K562. Panel C. Uptake of apoptotic bodies from irradiated K/pp65 and K/CD40L/pp65 by monocytes. Monocytes labeled with PKH26 red fluorescent cell linker compound were co-cultured (5:1 ratio) with irradiated K/CD40L/pp65 labeled with PKH2 green fluorescent cell linker compound. Analysis of fluorescence signals was performed after 72 hours of co-culture using a fluorescence microscope (Olympus IX70). Panel D. Expression of CD80, CD83, CD11c and HLA-DR by monocytes 72 hours after co-culture with irradiated K/pp65 (in blue) and K/CD40L/pp65 (in green). The red line represents the expression of CD80, CD83, CD11c and HLA-DR before the stimulation. Panel E. Frequency of CMV-CTLs assessed by IFNγ ELISpot using the CMV-pp65 pepmix. Data represented mean ± SD of 11 CMV-seropositive donors. Stimulation with an irrelevant pepmix was used as a negative control.

Figure 2. Co-expression of CD40L and OX40L by K562-derived whole-cell vaccine maximizes the stimulation of CMV-CTLs in vivo

Panel A. Schematic representation of the xenograft mouse model in NOG/SCID/γc−/− mice. Panel B. Engraftment of human CD45+ cells in the spleen, 14 days after vaccination. Panel C. Phenotypic analysis of human CD45+ cells engrafted in the spleen by day 14. Data represent mean ± SD of 8 mice per group. Panel D. Enumeration of the CMV-CTLs in isolated human CD45+ cells as assessed by IFNγ ELISpot in response to CMV-pp65 and irrelevant pepmixes. Data represent mean ± SD of 8 mice per group.

Figure 3. Virus-specificity of “dual specific” CAR-CMV-CTLs is stimulated by K562-derived whole-cell vaccine in vitro

In these experiments we compared the effector function of CAR-CMV-CTLs stimulated in vitro with control K/CD40L plus K/OX40L and K/CD40L/pp65 plus K/OX40L/pp65. Panel A. Enumeration of IFNγ-producing cells by ELISpot in response to the CMV-pp65 pepmix or the 1A7 Ab that cross-links the CAR-GD2. Data summarize means ± SD of 9 donors. Panel B. Detection of CAR and NLV-tetramer in CAR-CMV-CTLs by flow cytometry in a representative HLA-A2+ donor. While the CAR staining detects all CAR-CMV-CTLs, the tetramer only identifies CAR-CMV-CTLs specific for one single epitope (NLV) in the context of one haplotype (HLA-A2.01). Panel C. Cytotoxic activity (51Cr-release assay at a 20:1 effector:target ratio) against CHLA-255 neuroblastoma cells (GD2+ cells) and Raji lymphoma cells (GD2- cells). PHA blasts pulsed with CMV-pp65 or irrelevant pepmixes were also used as target cells. Data summarize mean ± SD of 4 donors. Panel D. Frequency of IFNγ-producing cells in response to CHLA-255 and Raji at 1:1 effector:target ratio. Data summarize mean ± SD of 3 donors. Panel E. Antitumor activity of CAR-CMV-CTLs in co-culture experiments against CHLA-255 (GD2+ cells) (right panels) and Raji cells (GD2- cells) (left panels). Both CHLA-255 and Raji cells were transduced with a retroviral vector encoding GFP. Tumor cells and CAR-CMV-CTLs were plated at 1:1 ratios, and CAR-CMV-CTLs (CD3+ cells) and tumor cells (GFP+ cells) were quantified by flow cytometry after 4 days of co-culture. Representative of 4 different donors.

Figure 4. Vaccination with K562-derived whole-cell vaccine expressing CMV-pp65, CD40L and OX40L enhances antitumor effects of CAR-CMV-CTLs in vivo

Panel A. NOG/SCID/γc−/− mice engrafted i.p. with the neuroblastoma cell line CHLA-255 labeled with firefly luciferase were infused i.p. with control or CAR-CMV-CTLs and vaccinated. The graph summarizes tumor bioluminescence. Summary of CMV-CTL line prepared from 4 donors: 15 mice (control CMV-CTLs plus K/CD40L/pp65 and K/OX40L/pp65), 17 mice (CAR-CMV-CTLs plus K/CD40L and K/OX40L) and 17 mice (CAR-CMV-CTLs plus K/CD40L/pp65 and K/OX40L/pp65) were used per group. Panel B. Mice euthanized were analyzed for the presence of macroscopic tumors. The graph summarizes the volume of the tumor collected in the different groups. Panel C. Enumeration of the CMV-CTLs in the isolated human CD45+ cells from the spleen as assessed by IFNγ ELISpot in response to CMV-pp65 and irrelevant pepmixes or the 1A7 Ab that cross-links the CAR-GD2. Data represent mean ± SD. Panel D. Mice were inoculated i.v. with the GD2+ lung carcinoma cell line A459 labeled with Firefly luciferase. Mice were then infused i.v. with control or CAR-CMV-CTLs and vaccinated. Tumor bioluminescence was then measured overtime. The graph is representative of one of 4 experiments using CMV-CTLs from 4 donors. Panel E. Kaplan-Meier analysis of tumor-bearing mice. Summary of CMV-CTL lines prepared from 4 donors: 15 mice (control CMV-CTLs plus K/CD40L/pp65 and K/OX40L/pp65), 13 mice (CAR-CMV-CTLs plus K/CD40L and K/OX40L), 13 mice (CAR-CMV-CTLs plus K/CD40L/pp65 and K/OX40L/pp65) and 8 mice (CAR-CMV-CTLs alone) were used per group.

Figure 5. Activation of the iC9 suicide gene eliminates engrafted K562-derived whole-cell vaccine in vivo

Panel A. Characterization of the clones by flow cytometry analysis. Gray areas indicate wild type K562 cells. Panel B. Western blot showing the expression of CMV-pp65 in the clones expressing the iC9 transgene. Panels C and D. NOG/SCID/γc−/− mice were inoculated subcutaneously with irradiated (C) or non-irradiated (D) K562-derived whole-cell vaccine expressing the iC9 gene and labeled with an enhanced firefly luciferase. Tumor growth was measured by in vivo imaging. Panel E. Effects of the administration of the chemical inducer of dimerization (CID) AP20187 on the growth of engineered vaccine.