Generation of highly activated, antigen-specific tumor-infiltrating CD8+ T cells induced by a novel T cell-targeted immunotherapy.

The induction of tumor-targeted, cytotoxic T lymphocytes has been recognized as a key component to successful immunotherapy. DPX-based treatment was previously shown to effectively recruit activated CD8+ T cells to the tumor. Herein, we analyze the unique phenotype of the CD8+ T cells recruited into the tumor in response to DPX-based therapy, and how combination with checkpoint inhibitors impacts T cell response. C3-tumor-bearing mice were treated with cyclophosphamide (CPA) for seven continuous days every other week, followed by DPX treatment along with anti-CTLA-4 and/or anti-PD-1. Efficacy, immunogenicity, and CD8+ T cells tumor infiltration were assessed. The expression of various markers, including checkpoint markers, peptide specificity, and proliferation and activation markers, was determined by flow cytometry. tSNE analysis of the flow data revealed a resident phenotype of CD8+ T cells (PD-1+TIM-3+CTLA-4+) within untreated tumors, whereas DPX/CPA treatment induced recruitment of a novel population of CD8+ T cells (PD-1+TIM-3+CTLA-4-) within tumors. Combination of anti-CTLA-4 (ipilimumab) with DPX/CPA versus DPX/CPA alone significantly increased survival and inhibition of tumor growth, without changing overall systemic immunogenicity. Addition of checkpoint inhibitors did not significantly change the phenotype of the newly recruited cells induced by DPX/CPA. Yet, anti-CTLA-4 treatment in combination with DPX/CPA enhanced a non-antigen specific response within the tumor. Finally, the tumor-recruited CD8+ T cells induced by DPX/CPA were highly activated, antigen-specific, and proliferative, while resident phenotype CD8+ T cells, seemingly initially exhausted, were reactivated with combination treatment. This study supports the potential of combining DPX/CPA with ipilimumab to further enhance survival clinically.

Novel Peptide-Based PD1 Immunomodulators Demonstrate Efficacy in Infectious Disease Vaccines and Therapeutics.

Many pathogens use the same immune evasion mechanisms as cancer cells. Patients with chronic infections have elevated levels of checkpoint receptors (e.g., programed cell death 1, PD1) on T cells. Monoclonal antibody (mAb)-based inhibitors to checkpoint receptors have also been shown to enhance T-cell responses in models of chronic infection. Therefore, inhibitors have the potential to act as a vaccine "adjuvant" by facilitating the expansion of vaccine antigen-specific T-cell repertoires. Here, we report the discovery and characterization of a peptide-based class of PD1 checkpoint inhibitors, which have a potent adaptive immunity adjuvant capability for vaccines against infectious diseases. Briefly, after identifying peptides that bind to the recombinant human PD1, we screened for in vitro efficacy in reporter assays and human peripheral blood mononuclear cells (PBMC) readouts. We first found the baseline in vivo performance of the peptides in a standard mouse oncology model that demonstrated equivalent efficacy compared to mAbs against the PD1 checkpoint. Subsequently, two strategies were used to demonstrate the utility of our peptides in infectious disease indications: (1) as a therapeutic in a bacteria-induced lethal sepsis model in which our peptides were found to increase survival with enhanced bacterial clearance and increased macrophage function; and (2) as an adjuvant in combination with a prophylactic malaria vaccine in which our peptides increased T-cell immunogenicity and the protective efficacy of the vaccine. Therefore, our peptides are promising as both a therapeutic agent and a vaccine adjuvant for infectious disease with a potentially safer and more cost-effective target product profile compared to mAbs. These findings are essential for deploying a new immunomodulatory regimen in infectious disease primary and clinical care settings.

Type III hypersensitivity reactions to a B cell epitope antigen are abrogated using a depot forming vaccine platform.

Peptide antigens are combined with an adjuvant in order to increase immunogenicity in vivo. The immunogenicity and safety of a RSV vaccine formulated in a novel oil-based platform, DepoVax™ (DPX), was compared to an alum formulation. A peptide B cell epitope derived from RSV small hydrophobic ectodomain (SHe) served as the antigen. Both vaccines induced SHe-specific antibodies after immunization of mice. A single dose of the DPX-based formulation resulted in anti-SHe titres for up to 20 weeks. Boosting with Alum-SHe, but not with DPX-SHe, led to unexpected clinical signs such as decreased activity, cyanosis and drop in body temperature in mice but not in rabbits. The severity of adverse reactions correlated with magnitude of SHe-specific IgG immune responses and decreased complement component 3 plasma levels, indicating a type III hypersensitivity reaction. By RP-HPLC analysis, we found that only 8-20% of the antigen was found to be adsorbed to alum in vitro, indicating that this antigen is likely released systemically upon injection in vivo. Clinical signs were not observed in rabbits, indicating the response correlates with peptide dose relative to size of animal. These results suggest that peptide antigens targeted to produce B cell mediated response may result in increased incidence of type III hypersensitivity reactions when delivered in non-depot forming vaccines. The DPX formulation induced strong antibody titres to the antigen without causing adverse events, likely due to the strength of the depot in vivo, and demonstrates the potential safety and immunogenicity of this platform for B cell peptide antigens.