An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma.

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)-an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma-in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.

Antigen-specific oncolytic MV-based tumor vaccines through presentation of selected tumor-associated antigens on infected cells or virus-like particles.

Recombinant vaccine strain-derived measles virus (MV) is clinically tested both as vaccine platform to protect against other pathogens and as oncolytic virus for tumor treatment. To investigate the potential synergism in anti-tumoral efficacy of oncolytic and vaccine properties, we chose Ovalbumin and an ideal tumor antigen, claudin-6, for pre-clinical proof of concept. To enhance immunogenicity, both antigens were presented by retroviral virus-like particle produced in situ during MV-infection. All recombinant MV revealed normal growths, genetic stability, and proper expression and presentation of both antigens. Potent antigen-specific humoral and cellular immunity were found in immunized MV-susceptible IFNAR-/--CD46Ge mice. These immune responses significantly inhibited metastasis formation or increased therapeutic efficacy compared to control MV in respective novel in vivo tumor models using syngeneic B16-hCD46/mCLDN6 murine melanoma cells. These data indicate the potential of MV to trigger selected tumor antigen-specific immune responses on top of direct tumor lysis for enhanced efficacy.

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy.

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells.

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.

Surface charge-modification prevents sequestration and enhances tumor-cell specificity of a recombinant granzyme B-TGFα fusion protein.

The serine protease granzyme B (GrB) plays an important role in the immune defense mediated by cytotoxic lymphocytes. Recombinant derivatives of this pro-apoptotic protein fused to tumor-targeting ligands hold promise for cancer therapy, but their applicability may be limited by promiscuous binding to nontarget tissues via electrostatic interactions. Here, we investigated cell binding and specific cytotoxicity of chimeric molecules consisting of wild-type or surface-charge-modified human GrB and the natural EGFR ligand TGFα for tumor targeting. We mutated two cationic heparin-binding motifs responsible for electrostatic interactions of GrB with cell surface structures, and genetically fused the resulting GrBcs derivative to TGFα for expression in the yeast Pichia pastoris. Purified GrBcs-TGFα (GrBcs-T) and a corresponding fusion protein employing wild-type GrB (GrB-T) displayed similar enzymatic activity and targeted cytotoxicity against EGFR-overexpressing breast carcinoma cells in the presence of an endosomolytic reagent. However, unspecific binding of the modified GrBcs-T variant to EGFR-negative cells was dramatically reduced, preventing the sequestration by nontarget cells in mixed cell cultures and increasing tumor-cell specificity. Likewise, modification of the GrB domain alleviated unspecific extracellular effects such as cell detachment indicative of extracellular matrix degradation. Our data demonstrate improved selectivity and functionality of surface-charge-modified GrBcs, suggesting this strategy as a general approach for the development of optimized GrB fusion proteins for therapeutic applications.