Pyroptosis-inducing active caspase-1 as a genetic adjuvant in anti-cancer DNA vaccination.

Pyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase-1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory cytokines such as Interleukin-1 beta (IL-1ß). Here, we evaluated whether pyroptosis could be exploited in DNA vaccination by incorporating a constitutively active variant of caspase-1 to the antigen-expressing DNA. In vitro, transfection with constitutively active caspase-1 DNA induced pro-IL-1ß maturation and IL-1ß release as well as gasdermin D-dependent cell death. To test active caspase-1 as a genetic adjuvant for the induction of antigen-specific T cell responses, mice were vaccinated intradermally with a DNA vaccine consisting of the active caspase-1 plasmid together with a plasmid encoding an ovalbumin-derived CD8 T cell epitope. Active caspase-1 accelerated and amplified antigen-specific CD8 T cell responses when administered simultaneously with the DNA vaccine at an equimolar dose. Moreover, upon challenge with melanoma cells expressing ovalbumin, mice vaccinated with the antigen vaccine adjuvanted with active caspase-1 showed significantly better survival compared to the non-adjuvanted group. In conclusion, we have developed a novel genetic adjuvant that for the first time employs the pyroptosis pathway to improve DNA vaccination against cancer.

Lipid A analog CRX-527 conjugated to synthetic peptides enhances vaccination efficacy and tumor control.

Adjuvants play a determinant role in cancer vaccination by optimally activating APCs and shaping the T cell response. Bacterial-derived lipid A is one of the most potent immune-stimulators known, and is recognized via Toll-like receptor 4 (TLR4). In this study, we explore the use of the synthetic, non-toxic, lipid A analog CRX-527 as an adjuvant for peptide cancer vaccines. This well-defined adjuvant was covalently conjugated to antigenic peptides as a strategy to improve vaccine efficacy. We show that coupling of this TLR4 agonist to peptide antigens improves vaccine uptake by dendritic cells (DCs), maturation of DCs and T cell activation in vitro, and stimulates DC migration and functional T cell priming in vivo. This translates into enhanced tumor protection upon prophylactic and therapeutic vaccination via intradermal injection against B16-OVA melanoma and HPV-related TC1 tumors. These results highlight the potential of CRX-527 as an adjuvant for molecularly defined cancer vaccines, and support the design of adjuvant-peptide conjugates as a strategy to optimize vaccine formulation.

A Bispecific Antibody Antagonizes Prosurvival CD40 Signaling and Promotes Vγ9Vδ2 T cell-Mediated Antitumor Responses in Human B-cell Malignancies.

Novel T cell-based therapies for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are thought to have strong potential. Progress, however, has been hampered by low efficacy and high toxicity. Tumor targeting by Vγ9Vδ2 T cells, a conserved T-cell subset with potent intrinsic antitumor properties, mediated by a bispecific antibody represents a novel approach promising high efficacy with limited toxicity. Here, we describe the generation of a bispecific Vγ9Vδ2 T-cell engager directed against CD40, which, due to its overexpression and biological footprint in malignant B cells, represents an attractive target. The CD40-targeting moiety of the bispecific antibody was selected because it can prevent CD40L-induced prosurvival signaling and reduce CD40-mediated resistance of CLL cells to venetoclax. Selective activation of Vγ9Vδ2 T cells in the presence of CD40+ tumor cells induced potent Vγ9Vδ2 T-cell degranulation, cytotoxicity against CLL and MM cells in vitro, and in vivo control of MM in a xenograft model. The CD40-bispecific γδ T-cell engager demonstrated lysis of leukemic cells by autologous Vγ9Vδ2 T cells present in patient-derived samples. Taken together, our CD40 bispecific γδ T-cell engager increased the sensitivity of leukemic cells to apoptosis and induced a potent Vγ9Vδ2 T cell-dependent antileukemic response. It may, therefore, represent a potential candidate for the development of novel treatments for B-cell malignancies.

Lipo-Based Vaccines as an Approach to Target Dendritic Cells for Induction of T- and iNKT Cell Responses.

In this study we developed a liposome-based vaccine containing palmitoylated synthetic long peptides (SLP) and alpha galactosylceramide (αGC) to specifically target dendritic cells (DC) for activation of both innate (invariant natural killer T-cells [iNKT]) and adaptive (CD8+ T-cells) players of the immune system. Combination of model tumor specific antigens (gp100/MART-1) formulated as a SLP and αGC in one liposome results in strong activation of CD8+ and iNKT, as measured by IFNγ secretion. Moreover, addition of lipo-Lewis Y (LeY) to the liposomes for C-type lectin targeting increased not only uptake by monocyte-derived dendritic cells (moDC), dermal dendritic cells and Langerhans cells but also enhanced gp100-specific CD8+ T- and iNKT cell activation by human skin-emigrated antigen presenting cells in an ex vivo explant model. Loading of moDC with liposomes containing LeY also showed priming of MART-126-35L specific CD8+ T-cells. In conclusion, chemically linking a lipid tail to a glycan-based targeting moiety and SLP combined with αGC in one liposome allows for easy generation of vaccine formulations that target multiple skin DC subsets and induce tumor antigen specific CD8+ T- and iNKT cells. These liposomes present a new vaccination strategy against tumors.

A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response.

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.