Anti-PD1 antibody enhances the anti-tumor efficacy of MUC1-MBP fusion protein vaccine via increasing Th1, Tc1 activity and decreasing the proportion of MDSC in the B16-MUC1 melanoma mouse model.

In previous studies, we have obtained a notable anti-tumor efficacy of the recombinant MUC1-MBP vaccine in the process of mouse B16-MUC1 melanoma treatment. However, the tumor cannot be eliminated completely. We found that the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations) after more than five immunizations, indicating persistent vaccine stimulation may activate immunosuppressive factors. In the present study, we revealed that programmed cell death 1 (PD1), an inhibitory molecule suppressing T cell function, expressed on splenic and tumor-infiltrating T cells were up-regulated by the vaccine. Therefore, to optimize the anti-tumor efficacy of the vaccine, we employed combination immunotherapy with MUC1-MBP vaccine and αPD1 (anti-PD1 antibody). Results showed that combination immunotherapy induced a more remarkable anti-tumor efficacy, the tumor clearance being increased to 80% from 20% which obtain by MUC1-MBP vaccine immunizations. To investigate the possible underlying mechanism, IFN-γ secretion and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and xCELLigence real-time cell analyzer (RTCA) respectively. T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. Results showed that the proportion of splenic CD8+T cells and tumor infiltration was increased and the activity of CTL killing, T helper 1 (Th1), Type 1 CD8+T (Tc1) was enhanced, indicating that the anti-tumor efficacy enhanced by combination immunotherapy was mainly through boosting CD8+T cells mediated anti-tumor cellular immunity. Additionally, combination immunotherapy significantly decreased the splenic and tumor-infiltrating myeloid derived suppressor cells (MDSCs). These results demonstrated that combination immunotherapy with MUC1-MBP vaccine and αPD1 was capable to invoke a more potent anti-tumor immune response and provide a foundation for further research.

Development of a VLP-Based Vaccine Displaying an xCT Extracellular Domain for the Treatment of Metastatic Breast Cancer.

Metastatic breast cancer (MBC) is the leading cause of cancer death in women due to recurrence and resistance to conventional therapies. Thus, MBC represents an important unmet clinical need for new treatments. In this paper we generated a virus-like particle (VLP)-based vaccine (AX09) to inhibit de novo metastasis formation and ultimately prolong the survival of patients with MBC. To this aim, we engineered the bacteriophage MS2 VLP to display an extracellular loop of xCT, a promising therapeutic target involved in tumor progression and metastasis formation. Elevated levels of this protein are observed in a high percentage of invasive mammary ductal tumors including triple negative breast cancer (TNBC) and correlate with poor overall survival. Moreover, xCT expression is restricted to only a few normal cell types. Here, we tested AX09 in several MBC mouse models and showed that it was well-tolerated and elicited a strong antibody response against xCT. This antibody-based response resulted in the inhibition of xCT's function in vitro and reduced metastasis formation in vivo. Thus, AX09 represents a promising novel approach for MBC, and it is currently advancing to clinical development.

Peptidoglycan-treated tumor antigen-pulsed dendritic cells impart complete resistance against tumor rechallenge.

Solid tumors elicit suppressive T cell responses which impair antigen-presenting cell (APC) functions. Such immune suppression results in uncontrolled tumor growth and mortality. Addressing APC dysfunction, dendritic cell (DC)-mediated anti-tumor vaccination was extensively investigated in both mice and humans. These studies never achieved full resistance to tumor relapse. Herein, we describe a repetitive RM-1 murine tumor rechallenge model for recurrence in humans. Using this newly developed model, we show that priming with tumor antigen-pulsed, Toll-like receptor (TLR)2 ligand-activated DCs elicits a host-protective anti-tumor immune response in C57BL/6 mice. Upon stimulation with the TLR2 ligand peptidoglycan (PGN), the tumor antigen-pulsed DCs induce complete resistance to repetitive tumor challenges. Intra-tumoral injection of PGN reduces tumor growth. The tumor resistance is accompanied by increased expression of interleukin (IL)-27, T-box transcription factor TBX21 (T-bet), IL-12, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, along with heightened cytotoxic T lymphocyte (CTL) functions. Mice primed four times with PGN-stimulated tumor antigen-pulsed DCs remain entirely resistant to repeat challenges with RM-1 tumor cells, suggesting complete prevention of relapse and recurrence of tumor. Adoptive transfer of T cells from these mice, which were fully protected from RM-1 rechallenge, confers anti-tumor immunity to syngeneic naive recipient mice upon RM-1 challenge. These observations indicate that PGN-activated DCs induce robust host-protective anti-tumor T cells that completely resist tumor growth and recurrence.

Listeriolysin O as a strong immunogenic molecule for the development of new anti-tumor vaccines.

The pore-forming toxin listeriolysin O (LLO), which is produced by Listeria monocytogenes, mediates bacterial phagosomal escape and facilitates bacterial multiplication during infection. This toxin has recently gained attention because of its confirmed role in the controlled and specific modulation of the immune response. Currently, cancer immunotherapies are focused on conquering the immune tolerance induced by poorly immunogenic tumor antigens and eliciting strong, lasting immunological memory. An effective way to achieve these goals is the co-administration of potent immunomodulatory adjuvant components with vaccine vectors. LLO, a toxin that belongs to the family of cholesterol-dependent cytolysins (CDCs), exhibits potent cell type-non-specific toxicity and is a source of dominant CD4(+) and CD8(+) T cell epitopes. According to recent research, in addition to its effective cytotoxicity as a cancer immunotherapeutic drug, the non-specific adjuvant property of LLO makes it promising for the development of efficacious anti-tumor vaccines.

The Immunostimulatory Effect of B16 Freezing/thawing Anti-tumor Vaccine / 체질인류학회지

Since cancer has become the second most common cause of death, next to heart disease and approximately 20% of human population dies from cancer, it is much desired to develop therapeutic anti-tumor vaccine with safety and efficacy. Here we investigated the immunostimulatory effects of B16 freezing/thawing (F/T) anti-tumor vaccine (hereafter F/T vaccine), one of whole cell anti-tumor vaccines. To this end, we took advantage of the IL12 p40 reporter system which is designed for monitoring the induction of IL12 expression via the detection of co-expressed yellow fluorescent protein. First, we examined whether F/T vaccine can induce IL12 expression using bone marrow-derived dendritic cells (BMDCs) from IL12 p40 reporter mice. Second, we examined whether F/T vaccine can change the expression level of MHC molecules and co-stimulatory molecules during the activation of dendritic cells. Third, to dissect what component of F/T vaccines accounts for the immunostimulatory activities, we examined the effect of F/T vaccine on BMDC activation after treating it with DNase or proteinase. Lastly, we used MyD88 knockout mice to investigate whether F/T vaccine activates BMDCs in a TLRdependent manner. We found that treatment of BMDCs with F/T vaccine induced IL12 expression as well as the increase of MHC II expression and co-stimulatory molecules such as CD86. Interestingly, we also found that F/T vaccine increased CD1d expression on BMDCs, which may influence the activation of natural killer T cells known to be involved in anti-tumor immune responses. In addition, we found that treatment of F/T vaccine with proteinase but not DNase abolished its immunostimulatory effect, indicating that proteins in F/T vaccine mainly have its adjuvant activity. Furthermore, the activation of BMDCs with F/T vaccine was dependent on MyD88 adaptor molecule. Taken together, our findings in this study demonstrated that the F/T vaccine might be one of the valuable reagents to provide a new insight for underlying mechanism of whole-cell anti-tumor vaccines and an important clue for the development of better therapeutic anti-cancer vaccines.

Immune Response Induced by Dendritic Cells Pulsed with Tumor Extracts in vivo Inhibit Growth of Implanted Tumor and Reject Challenge of Tumor Cells in Nudes / 中国肿瘤生物治疗杂志

To study if the immune response in vivo induced by dendritic cells (DC) pulsed with tumor extract can inhibit the growth of implanted tumor and reject the challenge of tumor cells in nudes, we isolated and purified DC from hepato-cellcular cancer(HCC) patient with combination of granulocyte /macrophage colony stimulating factor and interleukin 4; extracted tumor-associated antigen from human hepatocellcular cancer cell line HepG2 tumor cells; initiated the T lymphocyte with DC pulsed by the TAA to product cytotoxic T lymphocyte (CTL); implanted the CTL to inhibit the growth of implanted tumor in nudes and protected the nudes against the further challenge of HepG2 tumor cells, and further found that the CTL induced by DC pulsed with TAA from HepG2 tumor cells can inhibit the growth of implanted tumor in nudes and protect the nudes against the further challenge of HepG2 tumor cells. The results suggest, as a new concept anti-tumor vaccine, DC pulsed by TAA may play an important role in therapy and prevent against tumor.