Induction of tumor-specific T-cell responses by vaccination with tumor lysate-loaded dendritic cells in colorectal cancer patients with carcinoembryonic-antigen positive tumors

Background Dendritic cells (DCs) are the most effective antigen-presenting cells. In the last decade, the use of DCs for immunotherapy of cancer patients has been vastly increased. High endocytic capacity together with a unique capability of initiating primary T-cell responses have made DCs the most potent candidates for this purpose. Although DC vaccination occasionally leads to tumor regression, clinical efficacy, and immunogenicity of DCs in clinical trials has not been yet clarified. The present study evaluated the safety and effectiveness of tumor-lysate loaded DC vaccines in advanced colorectal cancer (CRC) patients with carcinoembryonic antigen (CEA) positive tumors. Results Six patients HLA-A*0201-positive were vaccinated with autologous DCs loaded with tumor lysates (TL) together with tetanus toxoid antigen, hepatitis B, and influenza matrix peptides. Two additional patients were injected with DCs that were generated from their sibling or parent with one haplotype mismatch. All patients received the vaccines every 2 weeks, with a total of three intra-nodal injections per patient. The results indicated that DC vaccination was safe and well tolerated by the patients. Specific immune responses were detected and in some patients, transient stabilization or even reduction of CEA levels were observed. The injection of haplotype mismatched HLA-A*0201-positive DCs resulted in some enhancement of the anti-tumor response in vitro and led to stabilization/reduction of CEA levels in the serum, compared to the use of autologous DCs. Conclusion Altogether, these results suggest that TL-pulsed DCs may be an effective vaccine method in CRC patients. Elimination of regulatory mechanisms as well as adjustment of the vaccination protocol may improve the efficacy of DC vaccination. An erratum to this article can be found at     

New developments in dendritic cell–based vaccinations: RNA translated into clinics

Dendritic cells (DCs) are the most powerful antigen-presenting cells that induce and maintain primary immune responses in vitro and in vivo. The development of protocols for the ex vivo generation of DCs provided a rationale for designing and developing DC-based vaccination studies for the treatment of infectious and malignant diseases. Recently, it was shown that DCs transfected with ribonucleic acid (RNA) coding for a tumour-associated antigen or whole tumour RNA are able to induce potent antigen and tumour-specific T-cell responses directed against multiple epitopes. The first RNA-transfected-DC-based clinical studies have shown that this form of vaccination is feasible and safe. In some cases, clinical responses were observed, but the preliminary data require further extensive investigations that should address the technical and biological problems of manipulating human DCs, as well as the development of standardised protocols and definitions of clinical settings.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

Vaccination with p53-peptide–pulsed dendritic cells,of patients with advanced breast cancer: report from a phase I study

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2⁺, p53⁺ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2⁺ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.     

Computational modeling of the immune response to tumor antigens

Vaccination protocols designed to elicit anti-cancer immune responses have, many times, failed in producing tumor eradication and in prolonging patient survival. Usually in cancer vaccination, epitopes from one organism are included in the genome or linked with some protein of another in the hope that the immunogenic properties of the latter will boost an immune response to the former. However, recent results have demonstrated that injections of two different vectors encoding the same recombinant antigen generate high levels of specific immunity. Systematic comparison of the efficacy of different vaccination protocols has been hampered by technical limitations, and clear evidence that the use of multiple vectors has advantages over single carrier injections is lacking. We used a computational model to investigate the dynamics of the immune response to different anti-cancer vaccines based on randomly generated antigen/carrier compounds. The computer model was adapted for simulations to this new area in immunology research and carefully validated to the purpose. As a matter of fact, it reproduces a relevant number of experimental observations. The model shows that when priming and boosting with the same construct, competition rather than cooperation develops amongst T cell clones of different specificities. Moreover, from the simulations, it appears that the sequential use of multiple carriers may generate more robust anti-tumor immune responses and may lead to effective tumor eradication in a higher percentage of cases. Our results provide a rational background for the design of novel strategies for the achievement of immune control of cancer.     

Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity

Dendritic cell (DC) therapies are currently being evaluated for the treatment of cancer. The majority of ongoing clinical trials use DCs loaded with defined antigenic peptides or proteins, or tumor-derived products, such as lysates or apoptotic cells, as sources of Ag. Although several theoretical considerations suggest that DCs expressing transgenic protein Ags may be more effective immunogens than protein-loaded cells, methods for efficiently transfecting DCs are only now being developed. In this study we directly compare the immunogenicity of peptide/protein-pulsed DCs with lentiviral vector-transduced DCs, and their comparative efficacy in tumor immunotherapy. Maturing, bone marrow-derived DCs can be efficiently transduced with lentiviral vectors, and transduction does not affect DC maturation, plasticity, or Ag presentation function. Transduced DCs efficiently process and present both MHC class I- and II-restricted epitopes from the expressed transgenic Ag OVA. Compared with peptide- or protein-pulsed DCs, lentiviral vector-transduced DCs elicit stronger and longer-lasting T cell responses in vivo, as measured by both in vivo killing assays and intracellular production of IFN-gamma by Ag-specific T cells. In the B16-OVA tumor therapy model, the growth of established tumors was significantly inhibited by a single immunization using lentiviral vector-transduced DCs, resulting in significantly longer survival of immunized animals. These results suggest that compared with Ag-pulsed DCs, vaccination with lentiviral vector-transduced DCs may achieve more potent antitumor immunity. These data support the further development of lentiviral vectors to transduce DCs with genes encoding Ags or immunomodulatory adjuvants to generate and control systemic immune responses.     

Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination.     

The immune response to breast cancer, and the case for DC immunotherapy

The long-held belief that breast cancer is a weakly immunogenic tumor and a poor candidate for immunotherapy should be reappraised. There is ample evidence for the existence of an immune response, which is, however, attenuated by multiple inhibitory factors. Many tumor-associated antigens (TAA) have been identified in breast cancer, some of which appear to play a critical role in tumorigenesis and may be attractive targets for immunotherapy. There is evidence for DC recruitment and activation within breast cancers, and the presence of intratumoral activated DCs impacts favorably upon survival. Furthermore, there is a striking paucity of activated DCs within the primary draining or sentinel lymph nodes of breast cancers. Tumor infiltrating lymphocytes (TIL) are often documented, however, their function is impaired by inhibitory cytokines, increased regulatory T lymphocyte activity, tumor cell MHC molecule alterations, and aberrant Fas ligand expression, amongst others. DCs are recognized as one of the critical interfaces between a cancer and the immune system, and have emerged as a promising platform for cancer vaccination via ex vivo immunomodulation. Clinical evaluation of DC vaccination in breast cancer is still relatively limited, although evolving. This article details evidence for the immune response in breast cancer and its many failings, and reviews the clinical trials and significant preclinical data which, taken together, validate the concept of DC vaccination in breast cancer.     

Phase I/II randomized trial of dendritic cell vaccination with or without cyclophosphamide for consolidation therapy of advanced ovarian cancer in first or second remission

Purpose

In spite of increased rates of complete response to initial chemotherapy, most patients with advanced ovarian cancer relapse and succumb to progressive disease. Immunotherapy may have potential for consolidation therapy.

Experimental design

This randomized open-label phase I/II trial evaluated responses of patients with advanced ovarian cancer in remission for vaccination with monocyte-derived dendritic cells (DC) loaded with Her2/neu, hTERT, and PADRE peptides, with or without low-dose intravenous cyclophosphamide. All patients also received pneumococcal vaccine and were randomized to cyclophosphamide 2?days prior to first vaccination. Blood samples were analyzed by ELISPOT and flow cytometry.

Results

Of 11 patients, 2 recurred during vaccination. Nine received all 4 doses: 3 patients recurred at 6, 17, and 26?months, respectively, and 6 have no evidence of disease at 36?months. No grade 3/4 vaccine-related toxicities were noted. The 3-year overall survival was 90%. Patients receiving cyclophosphamide showed a non-significant improvement in survival over controls. Patients receiving cyclophosphamide had a transient reduction in neutrophils, but no change in total lymphocytes or regulatory T cells. Modest T-cell responses to Her2/neu and hTERT were seen post-vaccine by IFN-γ ELISPOT. Patients demonstrated below normal responses to the diphtheria conjugate protein CRM197, a component of the pneumococcal vaccine.

Conclusions

In this setting, peptide-loaded DC vaccination elicits modest immune responses, but survival is promising. Pneumococcal vaccination revealed substantial immune suppression, even in patients in remission. Rational design of consolidative strategies for ovarian cancer will need to overcome tolerance and immunosuppression.     

Antigen loading of DCs with irradiated apoptotic tumor cells induces improved anti-tumor immunity compared to other approaches

Dendritic cells (DCs) serve as central regulators of adaptive immunity by presenting antigens and providing necessary co-signals. Environmental information received by the DCs determines the co-signals delivered to the responding adaptive cells and, ultimately, the outcome of the interaction. DCs loaded with relevant antigens have been used as therapeutic cellular vaccines, but the optimal antigen loading method has not been determined. We compared different methods to load class I and class II epitopes from the male antigenic complex, HY, onto DCs for the potency of the immune response induced in vivo. Co-incubation of female DCs with HY peptides, RNA or cell lysate from HY expressing tumor induced immune responses equivalent to male DCs. In contrast, female DCs incubated with irradiated, apoptotic HY expressing tumor cells (or male B cells) generated a stronger immune response than male DCs or female DCs loaded using any of the other methods. DC loading with apoptotic tumor resulted in complete protection against high dose HY-expressing tumor challenge whereas 100% lethality was observed in groups receiving DCs that were loaded with peptides, RNA, or lysate. We conclude that signals provided to the DCs by apoptotic cells substantially augment the potency of DC vaccines.     

Uptake and presentation of malignant glioma tumor cell lysates by monocyte-derived dendritic cells

Malignant glioma of the CNS is a tumor with a very bad prognosis. Development of adjuvant immunotherapy is hampered by interindividual and intratumoral antigenic heterogeneity of gliomas. To evaluate feasibility of tumor vaccination with (autologous) tumor cells, we have studied uptake of tumor cell lysates by dendritic cells (DCs), and the T-cell stimulatory capacity of the loaded DCs. DCs are professional antigen-presenting cells, which have already been used as natural adjuvants to initiate immune responses in human cancer. An efficacious uptake of tumor cell proteins, followed by processing and presentation of tumor-associated antigens by the DCs, is indeed one of the prerequisites for a potent and specific stimulation of T lymphocytes. Human monocytes were differentiated in vitro to immature DCs, and these were loaded with FITC-labeled tumor cell proteins. Uptake of the tumor cell proteins and presentation of antigens in the context of both MHC class I and II could be demonstrated using FACS analysis and confocal microscopy. After further maturation, the loaded DCs had the capacity to induce specific T-cell cytotoxic activity against tumor cells. We conclude that DCs loaded with crude tumor lysate are efficacious antigen-presenting cells able to initiate a T-cell response against malignant glioma tumor cells.     

Recent developments in cancer vaccines

The adoptive transfer of cancer Ag-specific effector T cells in patients can result in tumor rejection, thereby illustrating the immune system potential for cancer therapy. Ideally, one would like to directly induce efficient tumor-specific effector and memory T cells through vaccination. Therapeutic vaccines have two objectives: priming Ag-specific T cells and reprogramming memory T cells (i.e., a transformation from one type of immunity to another, for example, regulatory to cytotoxic). Recent successful phase III clinical trials showing benefit to the patients revived cancer vaccines. Dendritic cells (DCs) are essential in generation of immune responses, and as such represent targets and vectors for vaccination. We have learned that different DC subsets elicit different T cells. Similarly, different activation methods result in DCs able to elicit distinct T cells. We contend that a careful manipulation of activated DCs will allow cancer immunotherapists to produce the next generation of highly efficient cancer vaccines.     

Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8⁺ T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.     

Induction of alpha-fetoprotein-specific CD4- and CD8-mediated T-cell response using RNA-transfected dendritic cells

alpha-Fetoprotein (AFP) may be a possible target for a hepatocellular carcinoma (HCC)-specific vaccination. But some studies have demonstrated that dendritic cells (DCs) treated with AFP become dysfunctional. So in this study, we try to transfect AFP mRNA into DCs and observe the ability of DCs to induce AFP-specific CD4(+) and CD8(+) T cells. We hope that AFP can be processed and presented by DCs directly, rather than released to the cultures. So there will be no AFP negative effect on the function of DCs. In the study, immature DCs generated from peripheral blood mononuclear cells (PBMCs) of HLA-A2(+) HCC patients were transfected with AFP mRNA. Then the transfected, matured DCs were used to stimulate autologous T cells. The results showed that the expressions of membrane molecules of DCs after transfection were increased dramatically, and interleukin-12 (IL-12) p70 release in the supernatant was elevated significantly. There was only a minority of AFP release in the supernatants of transfected DCs. CTLs induced by the transfected DCs recognized HLA-matched AFP positive HepG2 cell line specifically and the AFP-specific proliferative T-cell responses could also be induced. These findings indicate that this AFP mRNA transfection strategy could generate fully functional DCs, which could induce specific T cells to recognize AFP(+) HCC cells.     

Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting

The realization that dendritic cells (DCs) orchestrate innate and adaptive immune responses has stimulated research on harnessing DCs to create more effective vaccines. Early clinical trials exploring autologous DCs that were loaded with antigens ex vivo to induce T-cell responses have provided proof of principle. Here, we discuss how direct targeting of antigens to DC surface receptors in vivo might replace laborious and expensive ex vivo culturing, and facilitate large-scale application of DC-based vaccination therapies.     

Targeting apoptotic tumor cells to Fc gamma R provides efficient and versatile vaccination against tumors by dendritic cells

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcgammaRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcgammaRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.