The co-delivery of adenovirus-based immune checkpoint vaccine elicits a potent anti-tumor effect in renal carcinoma.

Immune-based checkpoint therapy has made significant progress in cancer treatment, but its therapeutic effect is limited. A replication-defective adenovirus (Ad) vaccine encoding tumor antigen carbonic anhydrase IX (CAIX) combined with Ad-encoding immune checkpoint PD-L1 was developed to treat renal carcinoma. Three tumor models, subcutaneous, lung metastasis and orthotopic tumor were established, and Ad vaccines were used to immunize them and evaluate the vaccine's therapeutic effect. Compared to the single Ad vaccine group, the subcutaneous tumor growth was significantly reduced in Ad-CAIX/Ad-PD-L1 combination group. Co-immunization of Ad-CAIX/Ad-PD-L1 enhanced the induction and maturation of CD11c+ or CD8+CD11c+ DCs in the spleen and tumor and promoted the strong tumor-specific CD8+ T cell immune responses. In vivo CD8 T cell deletion assay showed that the anti-tumor effect of the Ad-CAIX/Ad-PD-L1 vaccine was mainly dependent on functional CD8+ T cell immune responses. Furthermore, the Ad-CAIX/Ad-PD-L1 vaccine effectively inhibited tumor growth and lung metastasis in metastatic or orthotopic models. These results indicate that the combination strategy of the immune checkpoint vaccine shows promising potential as an approach for malignant tumor therapy.

Adenovirus-assembled DC vaccine induces dual-targeting CTLs for tumor antigen and adenovirus to eradicate tumors.

The dendritic cell (DC) vaccine is a promising cancerimmunotherapy strategy, but its efficacy in treating the solid tumor is limited. To overcome this limitation, an oncolytic adenovirus (OAV-IL-12) was developed to enhance antigen targeting ability of adenovirus-assembled DC vaccine (DCs-CD137L/CAIX) for renal carcinoma treatment. Peritumoral administration of OAV-IL-12 increased the number of tumor-infiltrating DCs and their subsets (CD8+DCs and CD103+DCs). Combining OAV-IL-12 with DCs-CD137L/CAIX significantly inhibited the growth of subcutaneous tumors by inducing potent cytotoxic T lymphocyte (CTL) effect and improving the immune infiltration in tumor lesions. Interestingly, this treatment also reduced tumor growth distal to the OAV-IL-12 injecting side via eliciting a systemic CTL response. Furthermore, OAV-IL-12 potentiated DCs-CD137L/CAIX treatment induced dual CTL responses against both CAIX and adenovirus antigens. The therapeutic benefits of this treatment approach mainly relied on multifunctional CD8+T cell immune responses, as indicated by the depletion assay. Moreover, OAV-IL-12 potentiated DCs-CD137L/CAIX treatment generated a long-lasting protective effect against tumors by inducing memory CD8+T cell immune responses. These results suggest that the effective tumor targeting of the adenovirus-based DC vaccine, boosted by OAV-IL-12, is a promising treatment approach for renal carcinoma and other solid tumors.

HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8+T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a fatal malignant tumor, but effective clinical interventions are limited. PLGA/PEI-mediated DNA vaccine encoding the dual targets of high-mobility group box 1 (HMGB1) or GPC3 was developed for HCC treatment. Compared with PLGA/PEI-GPC3 immunization, PLGA/PEI-HMGB1/GPC3 co-immunization significantly inhibited the subcutaneous tumor growth, while increasing the infiltration of CD8+T cells and DCs. Furthermore, the PLGA/PEI-HMGB1/GPC3 vaccine induced a strong CTL effect and promoted functional CD8+T cell proliferation. Intriguingly, the depletion assay proved that the therapeutic effect PLGA/PEI-HMGB1/GPC3 vaccine was dependent on antigen-specific CD8+T cell immune responses. In the rechallenge experiment, PLGA/PEI-HMGB1/GPC3 vaccine provided a long-lasting resistance to the growth of the contralateral tumor by inducing the memory CD8+T cell responses. Collectively, PLGA/PEI-HMGB1/GPC3 vaccine could induce a strong and long-lasting CTL effect and inhibit the tumor progression or re-attack. Therefore, the combined co-immunization of PLGA/PEI-HMGB1/GPC3 might be served as an effective anti-tumor strategy against HCC.

Therapeutic Adenovirus Vaccine Combined Immunization with IL-12 Induces Potent CD8+ T Cell Anti-Tumor Immunity in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the cancers with the highest morbidity and mortality in the world. However, clinical progress in the treatment of HCC has not shown a satisfactory therapeutic effect. Here, we have developed a novel strategy to treat HCC with an adenovirus (Ad)-based vaccine, which contains a specific antigen glypican-3 (GPC3) and an immunostimulatory cytokine IL-12. In the subcutaneous tumor model, Ad-IL-12/GPC3 vaccine was injected into muscles three times to evaluate its therapeutic effect. Compared with the control immunization group, the Ad-IL-12/GPC3 immunization group showed a significant tumor growth inhibition effect, which was confirmed by the reduced tumor volume and the increased tumor inhibition. Ad-IL-12/GPC3 co-immunization promoted the induction and maturation of CD11c+ or CD8+CD11c+ DCs and increased the number of tumor-infiltrating CD8+ T cells. Furthermore, in the Ad-IL-12/GPC3 group, the proliferation of CD8+ T cells, the induction of multifunctional CD8+ T cells, and CTL activity were significantly increased. Interestingly, the deletion of CD8+ T cells abolished tumor growth inhibition by Ad-IL-12/GPC3 treatment, suggesting that CD8+ T cell immune responses were required to eliminate the tumor. Likewise, Ad-IL-12/GPC3 vaccine also effectively inhibited lung tumor growth or metastasis by enhancing CD8+ DCs-mediated multifunctional CD8+ T cell immune responses in the lung metastasis model. Therefore, these results indicate that IL-12 combined with Ad-GPC3 vaccine co-immunization might provide a promising therapeutic strategy for HCC patients.

The performance and perspectives of dendritic cell vaccines modified by immune checkpoint inhibitors or stimulants.

Therapeutic dendritic cell (DC) vaccines stimulate the elimination of tumor cells by the immune system. However, while antigen-specific T cell responses induced by DC vaccines are commonly observed, the clinical response rate is relatively poor, necessitating vaccine optimization. There is evidence that the suppression of DC function by immune checkpoints hinders the anti-tumor immune responses mediated by DC vaccines, ultimately leading to the immune escape of the tumor cells. The use of immune checkpoint inhibitors (ICIs) and immune checkpoint activators (ICAs) has extended the immunotherapeutic range. It is known that both inhibitory and stimulatory checkpoint molecules are expressed by most DC subsets and can thus be used to manipulate the effectiveness of DC vaccines. Such manipulation has been investigated using strategies such as chemotherapy, agonistic or antagonistic antibodies, siRNA, shRNA, CRISPR-Cas9, soluble antibodies, lentiviruses, and adenoviruses to maximize the efficacy of DC vaccines. Thus, a deeper understanding of immune checkpoints may assist in the development of improved DC vaccines. Here, we review the actions of various ICIs or ICAs shown by preclinical studies, as well as their potential application in DC vaccines. New therapeutic interventional strategies for blocking and stimulating immune checkpoint molecules in DCs are also described in detail.

IFI35 Promotes Renal Cancer Progression by Inhibiting pSTAT1/pSTAT6-Dependent Autophagy.

Interferon-induced protein 35 (IFI35), is currently acknowledged to govern the virus-related immune inflammatory responses. However, the biological significance and function of IFI35 in renal cell cancer (RCC) is still not well understood. Here, IFI35 expression and function were investigated in RCC tissues, renal cancer cells, and animal models. The results showed that IFI35 expression was significantly increased in 200 specimens of RCC patients. We found that higher IFI35 levels were significantly correlated with poor RCC prognosis. In human cell lines, the knockdown of IFI35 suppressed the malignant behavior of renal cancer cells. Similarly, the IFI35 knockdown resulted in significant inhibition of tumor progression in the subcutaneous or lung metastasis mouse model. Furthermore, the knockdown of IFI35 promoted the induction of autophagy by enhancing the autophagy-related gene expression (LC3-II, Beclin-1, and ATG-5). Additionally, blockade of STAT1/STAT6 phosphorylation (pSTAT1/pSTAT6) abrogated the induced autophagy by IFI35 knockdown in renal cancer cells. The autophagy inhibitor 3-MA also abolished the prevention of tumor growth by deleting IFI35 in renal cancer models. The above results suggest that the knockdown of IFI35 suppressed tumor progression of renal cancer by pSTAT1/pSTAT6-dependent autophagy. Our research revealed that IFI35 may serve as a potential diagnosis and therapeutic target for RCC.

Dual-targeting vaccine of FGL1/CAIX exhibits potent anti-tumor activity by activating DC-mediated multi-functional CD8 T cell immunity.

Tumor DNA vaccine as an effective therapeutic approach can induce systemic immunity against malignant tumors, but its therapeutic effect is still not satisfactory in advanced renal cancer. Herein, a novel DNA vaccine containing dual antigens of fibrinogen-like protein 1 (FGL1) and carbonic anhydrase IX (CAIX) was developed and intramuscularly delivered by PLGA/PEI nanoparticles for renal cancer therapy. Compared with PLGA/PEI-pCAIX immunization, PLGA/PEI-pFGL1/pCAIX co-immunization significantly inhibited the subcutaneous tumor growth and promoted the differentiation and maturation of CD11c+ DCs and CD11c+CD11b+ DCs subset. Likewise, the increased capabilities of CD8 T cell proliferation, CTL responses, and multi-functional CD8+ T cell immune responses were observed in PLGA/PEI-pFGL1/pCAIX vaccine group. Interestingly, depletion of CD8+ T cells by using CD8 mAb resulted in a loss of anti-tumor function of PLGA/PEI-pFGL1/pCAIX vaccine, suggesting that the anti-tumor activity of the vaccine was dependent on CD8+ T cell immune responses. Furthermore, PLGA/PEI-pFGL1/pCAIX co-immunization also suppressed the lung metastasis of tumor mice by enhancing the multi-functional CD8+ T cell responses. Therefore, these results indicate that PLGA/PEI-pFGL1/pCAIX vaccine could provide an effective protective effect for renal cancer by enhanced DC-mediated multi-functional CD8+ T cell immune responses. This vaccine strategy offers a potential approach for solid or metastatic tumor treatment.

Adenovirus vaccine therapy with CD137L promotes CD8+ DCs-mediated multifunctional CD8+ T cell immunity and elicits potent anti-tumor activity.

Renal carcinoma progresses aggressively in patients with metastatic disease while curative strategies are limited. Here, we constructed a recombinant non-replicating adenovirus (Ad) vaccine encoding an immune activator, CD137L, and a tumor antigen, CAIX, for treating renal carcinoma. In a subcutaneous tumor model, tumor growth was significantly suppressed in the Ad-CD137L/CAIX vaccine group compared with the single vaccine group. The induction and maturity of CD11C+ and CD8+CD11C+ dendritic cell (DC) subsets were promoted in Ad-CD137L/CAIX co-immunized mice. Furthermore, the Ad-CD137L/CAIX vaccine elicited stronger tumor-specific multifunctional CD8+ T cell immune responses as demonstrated by increased proliferation and cytolytic function of CD8+ T cells. Notably, depletion of CD8+ T cells greatly compromised the effective protection provided by Ad-CD137L/CAIX vaccine, suggesting an irreplaceable role of CD8+ T cells for the immunopotency of the vaccine. In both lung metastatic and orthotopic models, Ad-CD137L/CAIX vaccine treatment significantly decreased tumor metastasis and progression and increased the induction of tumor-specific multifunctional CD8+ T cells, in contrast to treatment with the Ad-CAIX vaccine alone. The Ad-CD137L/CAIX vaccine also augmented the tumor-specific multifunctional CD8+ T cell immune response in both orthotopic and metastatic models. These results indicated that Ad-CD137L/CAIX vaccine elicited a potent anti-tumor activity by inducing CD8+DC-mediated multifunctional CD8+ T cell immune responses. The potential strategy of CD137L-based vaccine might be served as a novel treatment for renal carcinoma or other malignant tumors.

Co-immunization with L-Myc enhances CD8+ or CD103+ DCs mediated tumor-specific multi-functional CD8+ T cell responses.

Renal carcinoma shows a high risk of invasion and metastasis without effective treatment. Herein, we developed a chitosan (CS) nanoparticle-mediated DNA vaccine containing an activated factor L-Myc and a tumor-specific antigen CAIX for renal carcinoma treatment. The subcutaneous tumor models were intramuscularly immunized with CS-pL-Myc/pCAIX or control vaccine, respectively. Compared with single immunization group, the tumor growth was significantly suppressed in CS-pL-Myc/pCAIX co-immunization group. The increased proportion and mature of CD11c+ DCs, CD8+ CD11c+ DCs and CD103+ CD11c+ DCs were observed in the splenocytes from CS-pL-Myc/pCAIX co-immunized mice. Furthermore, the enhanced antigen-specific CD8+ T lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and multi-functional CD8+ T cell induction were detected in CS-pL-Myc/pCAIX co-immunization group compared with CS-pCAIX immunization group. Of note, the depletion of CD8 T cells resulted in the reduction of CD8+ T cells or CD8+ CD11c+ DCs and the loss of anti-tumor efficacy induced by CS-pL-Myc/pCAIX vaccine, suggesting the therapeutic efficacy of the vaccine was required for CD8+ DCs and CD103+ DCs mediated CD8+ T cells responses. Likewise, CS-pL-Myc/pCAIX co-immunization also significantly inhibited the lung metastasis of renal carcinoma models accompanied with the increased induction of multi-functional CD8+ T cell responses. Therefore, these results indicated that CS-pL-Myc/pCAIX vaccine could effectively induce CD8+ DCs and CD103+ DCs mediated tumor-specific multi-functional CD8+ T cell responses and exert the anti-tumor efficacy. This vaccine strategy offers a potential and promising approach for solid or metastatic tumor treatment.

CAIX-specific CAR-T Cells and Sunitinib Show Synergistic Effects Against Metastatic Renal Cancer Models.

Treatment with chimeric antigen receptor-modified T cell (CAR-T) has demonstrated promising therapeutic efficacy in hematologic malignancies. However, the therapeutic efficacy is still very limited for solid tumors. An immunosuppressive microenvironment is one of the main reasons for the limited efficacy. Some chemotherapeutic agents exhibit immune microenvironment modulation. Therefore, combination with chemotherapeutic agents may be one of the promising strategies to enhance the therapeutic efficacy of CAR-T against solid tumors. Sunitinib modulates the antitumor immune response by improving T-cell infiltration and function while reducing immunosuppressive factors. The authors constructed a second-generation CAR targeting human renal cell carcinoma (RCC)-specific antigen carbonic anhydrase IX (CAIX) with the costimulatory domain of 4-1BB. The results of cytokine releasing and cell killing assays showed that the CAIX-CAR-T cells have specific effector functions against CAIX renal cancer cells in vitro. Combination therapy with CAIX-CAR-T and sunitinib showed synergistic efficacy against a mouse lung metastasis model of human RCC. CAIX-CAR-T cells in the mice of the combination therapy group showed stronger proliferation and tumor infiltration than that in the mice of the CAIX-CAR-T monotherapy group. The possible mechanisms of the synergistic efficacy are: (1) sunitinib caused upregulation of CAIX in tumor cells; (2) sunitinib decreased frequency of myeloid-derived suppressor cells in the tumor microenvironment. Our study supplied an innovative immunotherapeutic approach whereby combining CAIX-CAR-T with sunitinib induces a potent antitumor response in an experimental model of metastatic RCC. The combination strategy should be considered as a potential approach to augment adoptive CAR-T cell immunotherapy.

Akt inhibition at the initial stage of CAR-T preparation enhances the CAR-positive expression rate, memory phenotype and in vivo efficacy.

The adoptive transfer of chimeric antigen receptor-modified T (CAR-T) cells is a novel cancer treatment that has led to encouraging breakthroughs in the treatment of haematological malignancies. The efficacy of infused CAR-T cells is associated with a high CAR-positive expression rate, a strong proliferative response and the persistence of CAR-T cells in vivo. Manufacturing CAR-T cells is a process usually associated with the decreased CAR-positive expression rate and terminal differentiation of the infused CAR-T cells, which causes decreased proliferation and persistence of CAR-T cells in vivo. Therefore, the preparation of a high CAR-positive expression rate and few differentiated CAR-T cells is particularly important for clinical cancer treatment. In this study, we transduced and expanded CAR-T cells targeting the epithelial cell adhesion molecule (EpCAM) in the presence of an Akt inhibitor (MK2206) during the initial stage of CAR-T cell preparation. We show that the Akt inhibitor did not suppress the proliferation or effector function of the EpCAM-CAR-T cells but increased the CAR-positive expression rate and decreased the number of terminally differentiated EpCAM-CAR-T cells. Furthermore, EpCAM-CAR-T cells prepared using this protocol appeared to have enhanced antitumor activity in vivo. Taken together, these findings suggest that Akt inhibition during the initial stage of CAR-T cell preparation could improve the performance of CAR-T cells.

Lenvatinib promotes antitumor immunity by enhancing the tumor infiltration and activation of NK cells.

Based on previous reports, the efficacy of lenvatinib against cancer is mainly attributed to its antiangiogenic activity and its ability to suppress tumor proliferation, which are mediated by targeting receptor tyrosine kinases (RTKs). However, the effects of lenvatinib on tumor immune modulation have rarely been explored. Here, we show that lenvatinib effectively inhibited murine melanoma and renal cancer, and this inhibition was associated with enhanced tumor infiltration by natural killer (NK) cells. Critically, lenvatinib-induced tumor growth inhibition was attenuated by antibody-mediated NK cell depletion or the blockade of NK cell chemotaxis with an anti-CXCR3 blocking antibody. In addition, the expression of natural cytotoxicity receptors (NCRs) by tumor-infiltrating NK cells and the expression of cytotoxic cytokines in the tumor tissue were also augmented by lenvatinib. These data thus suggest that lenvatinib may be used not only as a direct cytotoxic drug against tumor angiogenesis and proliferation but also as a potent adjunct for enhancing the efficacy of immune-based cancer therapies by enhancing the tumor infiltration and activation of NK cells.

Corrigendum to "Combination Therapy with EpCAM-CAR-NK-92 Cells and Regorafenib against Human Colorectal Cancer Models".

[This corrects the article DOI: 10.1155/2018/4263520.].

Combination Therapy with EpCAM-CAR-NK-92 Cells and Regorafenib against Human Colorectal Cancer Models.

Adoptive chimeric antigen receptor-modified T or NK cells (CAR-T or CAR-NK) offer new options for cancer treatment. CAR-T therapy has achieved encouraging breakthroughs in the treatment of hematological malignancies. However, their therapeutic efficacy against solid tumors is limited. New regimens, including combinations with chemical drugs, need to be studied to enhance the therapeutic efficacy of CAR-T or NK cells for solid tumors. An epithelial cell adhesion molecule- (EpCAM-) specific second-generation CAR was constructed and transduced into NK-92 cells by lentiviral vectors. Immune effects, including cytokine release and cytotoxicity of the CAR-NK-92 cells against EpCAM-positive colon cancer cells, were evaluated in vitro. Synergistic effects of regorafenib and CAR-NK-92 cells were analyzed in a mouse model with human colorectal cancer xenografts. The CAR-NK-92 cells can specifically recognize EpCAM-positive colorectal cancer cells and release cytokines, including IFN-γ, perforin, and granzyme B, and show specific cytotoxicity in vitro. The growth suppression efficacy of combination therapy with regorafenib and CAR-NK-92 cells on established EpCAM-positive tumor xenografts was more significant than that of monotherapy with CAR-NK-92 cells or regorafenib. Our results provided a novel strategy to treat colorectal cancer and enhance the therapeutic efficacy of CAR-modified immune effector cells for solid tumors.

Bortezomib improves adoptive carbonic anhydrase IX­specific chimeric antigen receptor­modified NK92 cell therapy in mouse models of human renal cell carcinoma.

Adoptive chimeric antigen receptor (CAR) T or NK cells offer new options for cancer treatment. Clinical results indicate that CAR­modified T cell (CAR­T) therapy has curative therapeutic efficacy for hematological malignancies. However, the efficacy of the therapy in most solid tumors, including advanced renal cell carcinoma (RCC), remains highly limited. New regimens, including combination of CAR­T cells with chemical drugs, must be studied to enhance the therapeutic efficacy of CAR­T or NK cells for solid tumors. In the present study, a carbonic anhydrase IX (CAIX)­specific third­generation CAR was transduced into NK92 cells by lentiviral vectors. The immune effects, including cytokine release and cytotoxicity, of the CAR­NK92 cells against CAIX­positive RCC cells were evaluated in vitro. Combination therapeutic effects of bortezomib and CAR­NK92 cells were analyzed in a mouse model with human RCC xenografts. The results revealed that CAIX­specific CAR­NK92 cells specifically recognized in vitro cultured CAIX­positive RCC cells and released cytokines, including IFN­Î³, perforin and granzyme B, and exhibited specific cytotoxicity. The cytotoxicity of the CAR­NK92 cells was enhanced after treating RCC cells with bortezomib in vitro. The suppressive efficacy of bortezomib combined with CAR­NK92 cells against established CAIX­positive tumor xenografts was more significant than that of the monotherapy with either CAR­NK92 cells or bortezomib. Therefore, bortezomib can enhance the effects of the CAR­NK92 cells against RCC in vitro and in vivo. This study provided an experimental basis for the novel clinical regimen of CAIX­specific CAR­modified NK or T cells for the treatment of RCC.