Intratumoral expression of IL-12 from lentiviral or RNA vectors acts synergistically with TLR4 agonist (GLA) to generate anti-tumor immunological memory.

Systemic interleukin-12 (IL12) anti-tumor therapy is highly potent but has had limited utility in the clinic due to severe toxicity. Here, we present two IL12-expressing vector platforms, both of which can overcome the deficiencies of previous systemic IL12 therapies: 1) an integrating lentiviral vector, and 2) a self-replicating messenger RNA formulated with polyethyleneimine. Intratumoral administration of either IL12 vector platform resulted in recruitment of immune cells, including effector T cells and dendritic cells, and the complete remission of established tumors in multiple murine models. Furthermore, concurrent intratumoral administration of the synthetic TLR4 agonist glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE) induced systemic memory T cell responses that mediated complete protection against tumor rechallenge in all survivor mice (8/8 rechallenged mice), whereas only 2/6 total rechallenged mice treated with intratrumoral IL12 monotherapy rejected the rechallenge. Taken together, expression of vectorized IL12 in combination with a TLR4 agonist represents a varied approach to broaden the applicability of intratumoral immune therapies of solid tumors.

ZVex™, a dendritic-cell-tropic lentivector, primes protective antitumor T cell responses that are significantly boosted using heterologous vaccine modalities.

Therapeutic cancer vaccines must induce high levels of tumor-specific cytotoxic CD8 T cells to be effective. We show here that tumor-antigen specific effector and memory T cell responses primed with a non-integrating, dendritic-cell targeted lentiviral vector (ZVex™) could be boosted significantly by either adjuvanted recombinant protein, adenoviral vectors, or self-replicating RNA. These heterologous prime-boost regimens also provided significantly better protection in murine tumor models. In contrast, homologous prime-boost regimens, or using the lentiviral vector as a boost, resulted in lower T cell responses with limited therapeutic efficacy. Heterologous prime-boost regimens that utilize ZVex as the prime may be attractive modalities for therapeutic cancer vaccines.

Biophysical Attributes of CpG Presentation Control TLR9 Signaling to Differentially Polarize Systemic Immune Responses.

It is currently unknown whether and how mammalian pathogen recognition receptors (PRRs) respond to biophysical patterns of pathogen-associated molecular danger signals. Using synthetic pathogen-like particles (PLPs) that mimic physical properties of bacteria or large viruses, we have discovered that the quality and quantity of Toll-like receptor 9 (TLR9) signaling by CpG in mouse dendritic cells (mDCs) are uniquely dependent on biophysical attributes; specifically, the surface density of CpG and size of the presenting PLP. These physical patterns control DC programming by regulating the kinetics and magnitude of MyD88-IRAK4 signaling, NF-κB-driven responses, and STAT3 phosphorylation, which, in turn, controls differential T cell responses and in vivo immune polarization, especially T helper 1 (Th1) versus T helper 2 (Th2) antibody responses. Our findings suggest that innate immune cells can sense and respond not only to molecular but also pathogen-associated physical patterns (PAPPs), broadening the tools for modulating immunity and helping to better understand innate response mechanisms to pathogens and develop improved vaccines.

Surface-Presentation of CpG and Protein-Antigen on Pathogen-Like Polymer Particles Generate Strong Prophylactic and Therapeutic Antitumor Protection.

Despite significant efforts, development of clinically relevant prophylactic and therapeutic cancer vaccines has proven challenging. Cancer-associated antigens, which are often self-antigens, do not activate innate immune cells sufficiently, underscoring the need for codelivery of appropriate immune-stimulatory adjuvants. Recent research has underscored the need for biomaterial-based carriers for vaccine delivery, not only to target antigens and adjuvants to antigen-presenting cells or to create "depot" like systems but also to avoid acute systemic toxicity of molecular adjuvants that occurs when adjuvants are delivered in their "naked" form. The work presented here focuses on surface-presentation of both antigens and adjuvants on a pathogen-like particle (PLP) platform and understanding how PLP-induced antitumor responses differ when protein antigens and adjuvants, specifically the TLR9 agonist CpG, are delivered on the surface of the same particle (dual-loaded) versus being codelivered on separate particles. Surface-presentation allows easier access of antigens and adjuvants to intracellular targets (e.g., to TLR9 in the phagosomal compartments) and also allows controlled multivalent presentation. Our results show that, surface presentation, as opposed to soluble molecules, was more efficient in activating dendritic cells (DCs) and polarizing them toward generating a stronger cytotoxic T cell response. Signaling and DC polarization between separate and dual-loaded particles were similar, although NF-kB signaling at higher doses was stronger in dual-loaded PLPs. In vivo, dual loaded PLPs performed better than separately loaded PLPs in a prophylactic tumor model of melanoma and were comparable to immunization using incomplete Freud's adjuvant (IFA). In contrast both PLP-based delivery modalities performed similarly in a therapeutic melanoma-vaccine model and significantly outperformed IFA-based vaccination. These results indicate that surface-presentation of antigens and adjuvants on polymer-particles is a promising modality for efficient anticancer vaccines.

The effect of combined IL10 siRNA and CpG ODN as pathogen-mimicking microparticles on Th1/Th2 cytokine balance in dendritic cells and protective immunity against B cell lymphoma.

Success of an immunotherapy for cancer often depends on the critical balance of T helper 1 (Th1) and T helper 2 (Th2) responses driven by antigen presenting cells, specifically dendritic cells (DCs). Th1-driven cytotoxic T cell (CTL) responses are key to eliminating tumor cells. It is well established that CpG oligonucleotides (ODN), a widely studied Toll-like receptor 9 (TLR9) agonist, used to enhance Th1 response, also induces high levels of the anti-inflammatory, Th2-promoting cytokine IL10, which could dampen the resulting Th1 response. Biomaterials-based immunomodulatory strategies that can reduce IL10 production while maintaining IL12 levels during CpG delivery could further enhance the Th1/Th2 cytokine balance and improve anti-tumor immune response. Here we report that dual-delivery of IL10-silencing siRNA along with CpG ODN to the same DCs using pathogen-mimicking microparticles (PMPs), significantly enhances their Th1/Th2 cytokine ratio through concurrent inhibition of CpG-induced IL10 production. Co-delivery of poly(I:C), a TLR3 agonist had only minor effects on IL10 levels. Further, simultaneous immunotherapy with CpG ODN and IL10 siRNA enhanced immune protection of an idiotype DNA vaccine in a prophylactic murine model of B cell lymphoma whereas co-delivery of poly(I:C) and CpG did not enhance protection. These results suggest that PMPs can be used to precisely modulate TLR ligand-mediated immune-stimulation in DCs, through co-delivery of cytokine-silencing siRNAs and thereby boost antitumor immunity.