CpG-based immunotherapy impairs antitumor activity of BRAF inhibitors in a B-cell-dependent manner.

Combining immunotherapy with targeted therapy has increasingly become an appealing therapeutic paradigm for cancer treatment due to its great potential for generating durable and synergistic antitumor response. In this study, however, we unexpectedly found that two types of CpG-based tumor peptide vaccine treatments consistently negated the antitumor activity of a selective BRAF inhibitor in tumors with BRAF mutation rather than showing a synergistic antitumor effect. Our further studies demonstrated that CpG alone was sufficient to dampen BRAF inhibitor-induced antitumor responses, suggesting that the impaired antitumor activity of the BRAF inhibitor observed in mice receiving CpG-based peptide vaccine is mainly dependent upon the use of CpG. Mechanistically, CpG increased the number of circulating B cells, which produced elevated amounts of tumor necrosis factor-α (TNFα) that contributed to the increased tumor resistance to BRAF inhibitors. More importantly, B-cell depletion or TNFα neutralization can restore the antitumor effect of BRAF inhibition in mice receiving CpG treatment, indicating that TNFα-secreting B cells play an indispensable role in BRAF inhibitor resistance induced by CpG. Taken together, our results strongly suggest that precautions must be implemented when designing combinatorial approaches for cancer treatment, because distinct regimens, despite their respective therapeutic benefit as monotherapy, may together provide antagonistic clinical outcomes.

Viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses.

The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8(+) T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimetic platform for cancer vaccine delivery. Simultaneous conjugation of a melanoma-associated gp100 epitope and CpG to the E2 nanoparticle (CpG-gp-E2) yielded an antigen-specific increase in the CD8(+) T cell proliferation index and IFN-γ secretion by 1.5-fold and 5-fold, respectively, compared to an unbound peptide and CpG formulation. Remarkably, a single nanoparticle immunization resulted in a 120-fold increase in the frequency of melanoma epitope-specific CD8(+) T cells in draining lymph nodes and a 30-fold increase in the spleen, relative to free peptide with free CpG. Furthermore, in the very aggressive B16 melanoma murine tumor model, prophylactic immunization with CpG-gp-E2 delayed the onset of tumor growth by approximately 5.5 days and increased animal survival time by approximately 40%, compared to PBS-treated animals. These results show that by combining optimal particle size and simultaneous co-delivery of molecular vaccine components, antigen-specific anti-tumor immune responses can be significantly increased.

Administration of sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate conjugated GP100(25-33) peptide-coupled spleen cells effectively mounts antigen-specific immune response against mouse melanoma.

It remains a top research priority to develop immunotherapeutic approaches to induce potent antigen-specific immune responses against tumors. However, in spite of some promising results, most strategies are ineffective because they generate low numbers of tumor-reactive cytotoxic T lymphocytes (CTLs). Here we designed a strategy to enhance antigen-specific immune response via administering sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-conjugated melanoma tumor antigen GP10025-33 peptide-coupled syngeneic spleen cells in a mouse model of melanoma. We found that infusion of GP10025-33 peptide-coupled spleen cells significantly attenuated the growth of melanoma in prophylactic and therapeutic immunizations. Consistent with these findings, the adoptive transfer of spleen cells from immunized mice to naïve syngeneic mice was able to transfer anti-tumor effect, suggesting that GP10025-33 peptide-specific immune response was induced. Further studies showed that, CD8+ T cell proliferation and the frequency of interferon (IFN)-γ-producing CD8+ T cells upon ex vivo stimulation by GP10025-33 were significantly increased compared to control groups. Tumor antigen, GP10025-23 specific immune response was also confirmed by ELISpot and GP100-tetramer assays. This approach is simple, easy-handled, and efficiently delivering antigens to lymphoid tissues. Our study offers an opportunity for clinically translating this approach into tumor immunotherapy.

In vivo delivery of peptides and Toll-like receptor ligands by mannose-functionalized polymeric nanoparticles induces prophylactic and therapeutic anti-tumor immune responses in a melanoma model.

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.

Induction of cytotoxic T cells as a novel independent survival factor in malignant melanoma with percutaneous peptide immunization.

BACKGROUND: Malignant melanoma (MM) often shows multiple chemo-resistance, leading to poor prognosis of the patients. Therapeutic anti-cancer vaccination may be a feasible way to prolong the survival of patients. We have demonstrated that application of antigenic peptides via the tape-stripped, horny layer-removed skin, known as percutaneous peptide immunization (PPI), induces tumor cell-specific cytotoxic T lymphocytes (CTLs) in rodents and humans. OBJECTIVE: To evaluate clinical significance of PPI in advanced MM patients. METHODS: We performed PPI in 59 patients undergoing advanced MM with Melan-A, tyrosinase, MAGE-2, MAGE-3 and gp-100 peptides based on HLA typing in individuals. The induction of CTLs was assessed by the tetramer or pentamer flow cytometry in 35 patients. Patients showing positive CTL responses to all antigens were defined as complete responder (n=18), and those showing negative responses to at least one applied antigen were classified as incomplete responder (n=17). The primary endpoint of the study was overall survival (OS). For statistical analysis, log-rank test, univariate and multivariate Cox proportional hazard model were used. RESULTS: OS of the complete responders was longer than that of the incomplete responders (median survival time: 55.8 vs 20.3 months, log rank P=0.089). A hazard ratio for the complete responders relative to the incomplete responders was 0.23 (95% confidence interval: 0.06-0.93, P=0.039) in a multivariate Cox proportional hazard model. CONCLUSION: The induction of CTLs was a novel independent survival factor, and the induction of peptide-specific CTLs by PPI contributes to the prolonged survival and represents an impact on therapeutic approaches in MM. Unique trial number: UMIN000005706.

Cancer vaccines: Trafficking of tumor-specific T cells to tumor after therapeutic vaccination.

Cancer vaccines can induce robust activation of tumor-specific CD8(+) T cells that can destroy tumors. Understanding the mechanism by which cancer vaccines work is essential in designing next-generation vaccines with more potent therapeutic activity. We recently reported that short peptides emulsified in poorly biodegradable, Incomplete Freund's Adjuvant (IFA) primed CD8(+) T cells that did not localize to the tumor site but accumulated at the persisting, antigen-rich vaccination site. The vaccination site eventually became a T cell graveyard where T cells responded to chronically released gp100 peptide by releasing cytokines, including interferon-γ (IFN-γ), which in turn upregulated Fas ligand (FasL) on host cells, causing apoptosis of Fas(+) T cells. T cells that escaped apoptosis rapidly became exhausted, memory formation was poor, and therapeutic impact was minimal. Replacing the non-biodegradable IFA-based formulation with water-based, short-lived formulation in the presence of immunostimulatory molecules allowed T cells to traffic to tumors, causing their regression. In this review, we discuss recent advances in immunotherapeutic approaches that could enhance vaccine-primed immune cells fitness and render the tumor microenvironment more accessible for immune cell infiltration.

Efficacy and safety of ipilimumab in metastatic melanoma patients surviving more than 2 years following treatment in a phase III trial (MDX010-20).

BACKGROUND: In a phase III trial (ClinicalTrials.gov registration ID: NCT00094653), ipilimumab significantly improved survival versus a vaccine control in pretreated patients with metastatic melanoma. Here, we characterize outcomes of those patients who survived ≥ 2 years. METHODS: Patients were randomized (3 : 1 : 1) to receive ipilimumab 3 mg/kg + gp100 vaccine, ipilimumab 3 mg/kg + placebo, or gp100 vaccine alone. Baseline demographic data, duration of survival, responses, and safety among patients with ≥ 2 years' survival were analyzed. RESULTS: Among 676 randomized patients, 474 and 259 patients had at least 2 or 3 years of potential follow-up, respectively, and were eligible for analysis. Among these, 94 (20%) and 42 (16%) survived ≥ 2 and ≥ 3 years, respectively. Survival rates at 2 and 3 years were 25% (24 of 95) and 25% (13 of 53) with ipilimumab alone and 19% (54 of 284) and 15% (24 of 156) with ipilimumab plus gp100. Safety among patients with ≥ 2 years' survival was comparable with the overall study population, with the onset of new ipilimumab-related toxic effect (all grades) reported in 6 of 78 (8%) patients. CONCLUSIONS: Ipilimumab results in survival of ≥ 2 years in one-fifth of pretreated patients with 2 years potential follow-up in a phase III trial. New onset, low-grade events starting after administration of the last dose were infrequent. TRIAL REGISTRATION ID: NCT00094653.

Vaccination with mRNA-electroporated dendritic cells induces robust tumor antigen-specific CD4+ and CD8+ T cells responses in stage III and IV melanoma patients.

PURPOSE: Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients. EXPERIMENTAL DESIGN: Twenty-six stage III HLA*02:01 melanoma patients scheduled for radical lymph node dissection (stage III) and 19 melanoma patients with irresectable locoregional or distant metastatic disease (referred to as stage IV) were included. Monocyte-derived DC, electroporated with mRNA encoding gp100 and tyrosinase, were pulsed with keyhole limpet hemocyanin and administered intranodally. TAA-specific T-cell responses were monitored in blood and skin-test infiltrating lymphocyte (SKIL) cultures. RESULTS: Comparable numbers of vaccine-induced CD8(+) and/or CD4(+) TAA-specific T-cell responses were detected in SKIL cultures; 17/26 stage III patients and 11/19 stage IV patients. Strikingly, in this population, TAA-specific CD8(+) T cells that recognize multiple epitopes and produce elevated levels of IFNγ upon antigenic challenge in vitro, were significantly more often observed in stage III patients; 15/17 versus 3/11 stage IV patients, P = 0.0033. In stage IV patients, one mixed and one partial response were documented. The presence or absence of IFNγ-producing TAA-specific CD8(+) T cells in stage IV patients was associated with marked difference in median overall survival of 24.1 months versus 11.0 months, respectively. CONCLUSION: Vaccination with mRNA-electroporated DC induces a broad repertoire of IFNγ producing TAA-specific CD8(+) and CD4(+) T-cell responses, particularly in stage III melanoma patients.

Multi-compartmental vaccine delivery system for enhanced immune response to gp100 peptide antigen in melanoma immunotherapy.

PURPOSE: To develop a multi-compartmental vaccine delivery system for safe and efficient delivery of the gp100 peptide antigen in melanoma immunotherapy. METHODS: Water-in-oil-in-water (W/O/W) multiple emulsion-based multi-compartmental vaccine delivery system containing the gp100 peptide was prepared by a two-step emulsification method. In vivo prophylactic and active immunization effectiveness of the novel squalane oil-containing gp100 vaccine was evaluated in the murine B16 melanoma model and compared with that of an incomplete Freund's adjuvant (IFA)-based vaccine. RESULTS: Morphological evaluation of the W/O/W multiple emulsions showed that the oil-droplets were homogenously dispersed with the gp100 peptide encapsulated in an inner aqueous phase. Immunization with the gp100 peptide delivered in the W/O/W multiple emulsions-based vaccine resulted in increased protection against tumor challenge compared to IFA-based vaccine (p < 0.05, n = 8) signifying induction of enhanced anti-tumor immunity. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation and local infiltration of antigen specific cytotoxic T-lymphocytes into and/or surrounding the tumor mass. Moreover, the newly developed vaccine formulation did not induce any overt systemic toxicity. CONCLUSION: Novel W/O/W multiple emulsions-based vaccine efficiently delivers the gp100 peptide antigen to induce cell-mediated anti-tumor immunity and offers an alternate, safe vaccine delivery system.

Transcutaneous immunization with hydrophilic recombinant gp100 protein induces antigen-specific cellular immune response.

The objective of this study was to evaluate the potential of transcutaneous immunization with tumor antigen to induce cell-mediated immunity. For this purpose, hydrophilic recombinant gp100 protein (HR-gp100) was topically applied on human intact skin in vitro, and used as a vaccine in a mouse model. We demonstrate that HR-gp100 permeates into human skin, and is processed and presented by human dendritic cells. In a mouse model, an HR-gp100-based vaccine triggered antigen-specific T cell responses, as shown by proliferation assays, ELISA and intracellular staining for IFN-γ. Transcutaneous antigen delivery may provide a safe, simple and effective method to elicit cell-mediated immunity.

Different adjuvanticity of incomplete freund's adjuvant derived from beef or vegetable components in melanoma patients immunized with a peptide vaccine.

Adjuvants are requisite components of many vaccines designed to elicit T-cell immunity although the exact components of commonly used adjuvants are not always fully defined. In 2006, owing to concerns of prion contamination, the formulation of Montanide ISA 51 Incomplete Freund's Adjuvant (IFA) was changed from using oleic acid isolated from beef tallow to that isolated from olives. In sequential clinical trials in the Surgery Branch, NCI patients at high risk for recurrence of melanoma were immunized with the gp100 melanoma/melanocyte antigenic peptide, gp100: 209-217 (210M), emulsified in the beef-derived IFA or the olive-derived IFA. The in vivo generation of gp100 reactive T cells was significantly less in patients receiving the olive compared with the beef IFA as assessed by both ELISPOT (P2=0.0001) and in vitro sensitization assays (P2=0.0001). Local skin reactions to the peptide emulsion were also far less severe using the olive IFA (P2=0.0003). Thus it seems likely that contaminants in the beef-derived IFA played an important role in the increased adjuvanticity of this preparation compared with the olive-derived IFA. These findings raise serious concerns related to the use of the available olive-derived IFA for immunization in clinical trials. A survey of ongoing clinical trials listed in ClinicalTrials.gov revealed 36 trials currently accruing patients that are using the olive-derived Montanide ISA 51 IFA.