Autologous whole tumor cell-based vaccines have the potential to generate effective immune responses by presenting a diverse repertoire of TAAs to T cells either in a direct fashion serving as antigen-presenting cells or indirectly via cross-priming.36–38
The diverse pool of TAAs in cell based vaccines also have the potential to overcome antigenic drift of dominant TAA epitopes in response to immunological pressure, which is an important limitation of subunit vaccines based on TAAs.38–40
Autologous whole tumor cell based vaccines, however, suffer from inefficacy plausibly because of the lack of immunostimulatory, such as costimulatory, molecules expressed by tumor cells. Consistent with this notion are a series of published studies demonstrating that tumor cells genetically modified to express costimulatory molecules serve effective vaccines against cancer.5,36,39,41
However, the application of genetically modified tumor cell-based vaccines to the clinic suffers from various limitations. These include most often lack of sufficient tumor mass for genetic modification, difficulties associated with the transfer of foreign DNA into primary tumor cells using various approaches, the inability to expand the genetically modified tumor cells for vaccine preparation, and safety concerns associated with the introduction of foreign genetic material into the patient.
As an alternative to gene transfer approaches for immunomodulation, we recently established a novel approach designated as ProtEx™
that allows for the generation of chimeric immunomodulatory molecules with core streptavidin and the display of these molecules on cell membrane that has been derivitized with biotin.12,17,18,42
The rationale for this approach is that the most critical immune decisions are the end result of cell surface receptor and ligand interactions and these interactions are short and transient in nature. Therefore, the transient display of exogenous proteins representing immunological ligands, such as costimulatory molecules, on the cell membrane in an effective and rapid manner under physiological conditions may have utility as a practical and safe approach to gene therapy-based immunomodulation. As a proof-of-principle study, we have recently shown that tumor cells engineered with a chimeric CD80-SA protein were effective as prophylactic vaccine in a mouse B cell lymphoma model.18
Based on this observation, we hypothesized that the display of more than one costimulatory molecule with distinct functions on tumor cells may improve their efficacy as whole cell-based autologous tumor vaccine.
We herein tested if tumor cells can be engineered to effectively display two costimulatory molecules, SA-4-1BBL and SA-LIGHT, on their surface and if such engineered cells have improved efficacy as cancer vaccine using HPV E7 expressing the TC-1 cervical cancer mouse model. Modification of tumor cells with 15 μM biotin and engineering with equal amounts (0.2 μg protein/1 × 106 cells) of SA-4-1BBL and SA-LIGHT proteins was sufficient for the codisplay of both proteins at similar levels on almost all the targeted cells. There was a direct correlation between the concentrations of biotin and proteins used to engineer the TC-1 cells and the levels of proteins displayed on the cell surface. For example, at 50 μM biotinylation conditions, a tenfold increase in each protein concentration resulted in almost a ten-fold increase in protein levels displayed on the cell surface. Importantly, under these engineering conditions there were sufficient free biotin molecules available on the cell surface that allowed for the co-display of streptavidin at similar levels to SA-LIGHT and SA-4-1BBL. Taken together, these data demonstrate the versatility and flexibility of this platform technology for the display of a single or a combination of proteins on the cell surface at desired levels for therapeutic purposes.
Tumor cells engineered with either single or a combination of both proteins generated effective E7-specific in vivo killing responses when used as vaccine in naive mice. However, vaccine efficacy was significantly enhanced when both proteins were co displayed on the surface of the same TC-1 cells as compared with individual proteins displayed on different cells, providing evidence for the utility of this combinatorial vaccine approach. Enhanced vaccine efficacy of TC-1 cells engineered with both proteins may be due to the distinct functions of these two molecules on the immune system. Unlike 4-1BBL that primarily enhances the function of antigen-activated T cells due to the lack of 4-1BB receptor on naive T cells,43
LIGHT was shown to activate naïve T cells independent of CD28 costimulation by engaging constitutively expressed HVEM receptor on their surface.35,44
Therefore, SA-LIGHT on tumor cells is anticipated to activate antigen-specific naive T cells which can then upregulate 4-1BB receptor and become responsive to SA-4-1BBL-mediated modulation, leading to improved immune responses as compared with individual molecules. In addition, LIGHT may also affect the innate arm of the immune system via interaction with LTβR receptor expressed on monocytes and HVEM receptor on DCs,35,45,46
further contributing to the T cell responses. This notion is consistent with the augmented efficacy of LIGHT only engineered TC-1 cells than those engineered with SA-4-1BBL in generating in vivo killing responses as well as preventing tumor growth. Importantly, LIGHT was shown to regulate the CD40 signaling on DCs for the generation of a CTL response,46
suggesting that similar cooperation/synergy may exists between LIGHT and 4-1BBL with respect to the activation of DCs and generation of killing responses.
The immune efficacy of TC-1 cells codisplaying both proteins on their surface was much more pronounced in a prophylactic tumor setting. Vaccination of mice with TC-1 cells co-displaying both SA-4-1BL and SA-LIGHT proteins on their surface resulted in the prevention of tumor growth in 100% of mice in a prophylactic setting and this effect was superior to vaccinations with tumor cells displaying the individual proteins. Mice vaccinated with TC-1 cells engineered with SA-4-1BBL alone although had retarded tumor growth; all animals succumbed to death within 60 days due to tumor burden. Vaccination with SA-LIGHT-engineered TC-1 cells had a efficacy than SA-4-1BBL-engineerd cells as the kinetics of tumor growth in these mice were substantially retarded with over twenty five percent of animals not developing detectable tumors over a 60-day observation period. Importantly, the simultaneous display of both molecules on the same tumor cell appeared to be critical for the maximum efficacy of the vaccine as vaccination of mice with a mixture of TC-1 cells displaying individual SA-4-1BBL and SA-LIGHT proteins was only effective in preventing tumor growth in ~65% of mice. The efficacy of vaccination with SA-LIGHT-engineered TC-1 tumor cells in the prophylactic tumor setting is consistent with our in vivo killing data. This may be due to the ability of LIGHT to directly activate DCs and monocytes and stimulate T cells in a CD28-independent fashion.47,48
Consistent with this notion, forced expression of LIGHT within tumor were shown to recruit APCs and naïve T cells into the tumor, intratumoral activation of naive T cells, leading to the eradication of established tumors as well as prevention of spontaneous metastases arising following surgical removal of the tumor.35,49
The efficacy of both proteins-engineered TC-1 cells was further confirmed in a therapeutic setting where a single or two injection of the vaccine resulted in prolonged survival in all mice with > 55 % tumor-free survival. However, vaccine efficacy required the display of higher levels of both proteins on the surface of TC-1 cells under the tested conditions. This observation is consistent with a recent study demonstrating a dose-depend efficacy in a therapeutic tumor model using an Ig.4-1BBL fusion protein for immunomodulation.50
The immune efficacy of the TC-1 cells engineered to codisplay both SA-LIGHT and SA-4-1BBL proteins on their surface as compared with cells engineered to display the individual proteins is consistent with several published studies using tumor cells genetically modified to coexpress two or more costimulatory molecules as vaccine. DNA vaccines encoding rat Her-2/neu as TAA given in combination with both 4-1BBL and either CD80 or CD86 costimulatory molecules generated cellular and humoral immune responses with antitumor efficacy.51
Vaccination with neuroblastoma cells transiently transfected to simultaneously express the co-stimulatory molecules CD54, CD80, CD86, and CD137L was effective in generating antitumor immunity that had efficacy in preventing tumor growth in a prophylactic setting in mice.37
Immunomodulation using the combination of 4-1BBL and LIGHT costimulatory molecules is particularly attractive because the expression patterns of these two proteins are distinct and connected in a spatiotemporal manner. LIGHT can initiate robust innate and primary T cells responses against the tumor by interacting with its LTβR and HVEM receptor constitutively expressed on various immune cells, such as DCs, monocytes, NK cells as well as T cells. This initial activation of immune cells by LIGHT can then set the stage for 4-1BBL activity by upregulating the expression of 4-1BB receptor. The engagement of 4-1BBL with 4-1BB on activated immune cells may augment LIGHT-generated immune responses with particular effect on selective expansion of T effector cells and establishment of long-term memory required for immunosurveillance and prevention of recurrences. In addition, 4-1BB signaling may also overcome various tumor-induced immune evasion mechanisms, such as clonal anergy/ignorance14,26
T regulatory cells.14
Mice that underwent successful immunotherapy generated potent E7-specific in vivo killing responses and in vitro E7-specific proliferation responses when boosted 60 days post-vaccination as compared with naïve primed mice, providing evidence for long-term T cell memory. Consistent with this notion, long-term mice with successful immunotherapy had significantly higher percentages of peripheral CD4+
T cells with memory phenotype as compared with primed naïve mice. T cells from these animals also generated higher IFN-γ response and had significantly higher levels of anti-E7 antibody titer in their sera. The enhanced T cell memory, CTL, and IFN-γ responses are consistent with the expected functions of LIGHT and 4-1BBL on the immune system and their demonstrated efficacies in generating these responses in various tumor settings.14,29
For example, exogenous soluble LIGHT was shown to promote monocyte-derived DC maturation in vitro by the up-regulation of CD86, CD80, CD83, and HLA-DR antigen expression, resulting in increased antigen presentation and T-cell activation in patients with myelodysplastic syndromes.52
The forced expression of LIGHT in the tumor environment induces a massive infiltration of naive T lymphocytes that correlates with an upregulation of both chemokine production and expression of adhesion molecules.29
Activation of these infiltrating T cells, possibly through HVEM, leads to the rejection of established, highly progressive tumors at local and distal sites.29
Furthermore, we have recently shown that a soluble from of 4-1BBL as the immunomodulatory component of a vaccine based on the HPV E7 protein was effective in eradicating TC-1 tumors. The therapeutic efficacy of the vaccine was associated with the generation of potent primary and memory CD8+
T cell responses, enhanced infiltration of these cells into the tumor, and their secretion of IFN-γ.14
Mice that failed immunotherapy and developed tumor had significantly higher levels of tumor infiltrating CD8+
T cells as compared with unvaccinated mice or mice vaccinated with SA-engineered TC-1 cells as control. Although these data provide evidence for the efficacy of our vaccine regimen to facilitate the infiltration of CD8+
T cells into the tumor, the molecular basis of inability of such cells to eradicate the tumor is not known. One possibility is that these CD8+
T cells are not specific for tumor antigens or functionally compromised or actively regulated by tumor microenvironment. Further studies will be required to address these issues.
This report demonstrates the feasibility of engineering tumor cells in an effective manner to display on their surface more than one immunomodulatory molecules at desired levels in a rapid and effective manner. Cells engineered with two costimulatory molecules as vaccine had immune efficacy against tumor than cells engineered with individual proteins. The efficacy of this vaccine approach is not limited to the TC-1 tumor model expressing the xenogeneic E7 oncogene since a vaccine based on A20 tumor cells, which do not express xenogeneic TAAs, engineered with the CD80 costimulatory molecule had efficacy in an autologous setting.18
The rapid, flexible, and effective engineering of tumor cells under clinically applicable conditions presents a novel and new means of cell-based cancer immunotherapy. This approach is particularly suited for autologous cell based vaccines where primary tumor cells can be resected from patients, engineered to display on their surface immunological molecules of interest, and used as vaccine within the same day; therefore, obviating time, labor, and safety issues associated with genetic manipulation of primary tumor cells for vaccine purposes.