Tumor cells engineered to codisplay on their surface 4-1BBL and LIGHT costimulatory proteins as a novel vaccine approach for cancer immunotherapy

Abstract

Primary tumor cells genetically modified to express a collection of immunological ligands on their surface may have the utility as therapeutic autologous cancer vaccines. However, genetic modification of primary tumor cells is not only cost, labor and time intensive, but also has safety repercussions. As an alternative, we developed the ProtEx technology that involves generation of immunological ligands with core streptavidin (SA) and their display on biotinylated cells in a rapid and efficient manner. We herein demonstrate that TC-1 tumor cells can be rapidly and efficiently engineered to codisplay on their surface two costimulatory proteins, SA-4-1BBL and SA-LIGHT, simultaneously. Vaccination with irradiated TC-1 cells codisplaying both chimeric proteins showed 100% efficacy in a prophylactic and >55% efficacy in a therapeutic tumor setting. In contrast, vaccination with TC-1 cells engineered with either protein alone showed significantly reduced efficacy in the prophylactic setting. Vaccine efficacy was associated with the generation of primary and memory T-cell and antibody responses against the tumor without detectable signs of autoimmunity. Engineering tumor cells in a rapid and effective manner to simultaneously display on their surface a collection of immunostimulatory proteins with additive/synergistic functions presents a novel alternative approach to gene therapy with considerable potential for cancer immunotherapy.

Figure 1

Engineering TC-1 tumor cells to simultaneously display on their surface SA-4-1BBL and LIGHT proteins. (a) Flow cytometry analysis of engineered TC-1 cells. The TC-1 cells were biotinylated using 15 μM EZ-Link Sulfo-NHS-SS-biotin followed by incubation with 0.2 μg of proteins either individually or in combination per 10⁶ cells. The presence of SA-4-1BBL and SA-LIGHT on the cell surface was assessed using antibodies against 4-1BBL and SA, respectively. (b) The TC-1 cells were biotinylated using 50 μM EZ-Link Sulfo-NHS-SS-biotin followed by incubation with 2 μg of proteins either individually or in combination per 10⁶ cells. (c) Confocal microscope analysis of TC-1 tumor cells for the cell surface presence of chimeric proteins. TC-1 cells were engineered with SA-4-1BBL, SA-LIGHT, or the two proteins in combination with the conditions stated in (b) and stained using Abs against 4-1BBL and SA. Fluorochrome-labeled SA was used as a third protein to assess the presence of free biotin remaining on the cell surface after engineering with the chimeric proteins.

Figure 2

Vaccination with TC-1 cells engineered to display both SA-LIGHT and SA-4-1BBL generates potent in vivo killing responses. Two million of irradiated TC-1 cells engineered with SA-4-1BBL, SA-LIGHT, or both molecules simultaneously were used to vaccinate C57BL/6 (CD45.1) mice s.c. on days 7 and 3 prior to i.v. injection of target syngeneic splenocytes (CD45.2) labeled with CFSE and pulsed with the E7_49–57 CD8⁺ T cell dominant epitope. Two days after target injection, total splenocytes were harvested and analyzed for specific in vivo killing using multiparameter flow cytometry by gating on CD45.2 cells. TC-1 cells engineered with control core streptavidin (SA) served as control. (a) Shows a representative in vivo killing histogram and (b) Tabulation of killing response for three animals per group.

Figure 3

Vaccination with TC-1 cells engineered with both SA-LIGHT and SA-4-1BBL prevents tumor growth in a prophylactic setting. (a) Naïve C57BL/6 mice were vaccinated twice with 2 × 10⁶ irradiated TC-1 cells engineered to display on their surface control SA, SA-4-1BBL, SA-LIGHT, or both SA-4-1BBL and SA-LIGHT proteins together 7 and 3 days prior to challenge with live 1 × 10⁵ TC-1 cells. One million of TC-1 cells engineered with the individual molecules were mixed and used for vaccination to test if the presence of both molecules on the same cell affects vaccine efficacy. (b) The long term (over 60 days) tumor free animals from (a) were boosted with another dose of the same vaccine formulation and in vivo E7_49–57 peptide-specific killing was assed one week later. Tabulation of killing response for three animals per group.

Figure 4

Vaccination with TC-1 cells engineered with both SA-LIGHT and SA-4-1BBL proteins prevents tumor growth in a therapeutic setting. (a) Naïve C57BL/6 mice were challenged with 1 × 10⁵ live TC-1 cells followed by vaccination s.c. with 2 × 10⁶ irradiated TC-1 cells engineered using 50 μM biotin and 2 μg/protein/10⁶ cells on the same day. Mice without vaccine or those vaccinated with TC-1 cells engineered with equimolar SA or syngeneic splenocytes codisplaying both molecules were used as controls (p <0.001). (b) Mice vaccinated with TC-1 tumors engineered with costimulatory proteins that failed immunotherapy had higher number of intratumoral CD8⁺ T cells as compared with controls.

Figure 5

Tumor immunotherapy is associated with robust long-term T cell responses. (a) Long term tumor free animals have tumor specific killing responses. Tumor-free mice were vaccinated s.c. with one dose of TC-1 cells engineered with both SA-4-1BBL and SA-LIGHT proteins on day 60 post tumor challenge. Naïve C57BL/6 mice vaccinated with the same regimen were used as control. E7_49–57 peptide-specific in vivo killing response was assessed one week later. (b) Long term tumor free animals retain tumor specific T cell proliferative responses. Splenocytes from vaccinated mice in (a) were pulsed with a recombinant E7 protein in a 5-day thymidine incorporation assay to assess T cell proliferation. (c) Long term tumor free animals have higher percentages of CD8⁺ T cells expressing IFN-γ. Draining lymph nodes from boosted long term tumor free animals and naïve mice were treated with PMA/ionomycine for 6 hr and analyzed for intracellular cytokines using multiparameter flow cytometry.

Figure 6

Long term tumor free mice retain higher CD44^high memory T cell pool. Splenocytes from the boosted long term tumor free animals were stained with various cell surface markers and analyzed in multiparameter flow cytometry to assess memory CD4⁺ and CD8⁺ T cells. (a) A representative flow cytometry histogram and (b) Tabulated data for a minimum of 3 animals per group.

Figure 7

Tumor immunotherapy is associated with higher titers of serum Abs against E7 protein. Serum from the boosted long term tumor free animals and naïve mice were harvested and tested in a recombinant E7 protein ELISA.