We recently developed a monocyte-based cellular vaccine platform for cancer treatment. In contrast to the traditional utilization of monocytes as precursors to generate dendritic cells (DC) for vaccination purposes, we find that freshly isolated monocytes with no differentiation process can be loaded with tumor antigens (Ag) and trigger robust antitumor cytotoxic T lymphocyte (CTL) responses. In this chapter, we describe methods to prepare, administer, and evaluate murine Ly-6Chi monocyte-based cellular vaccines for their therapeutic efficacy. This includes procedures for isolation, purity determination, Ag loading, administration of bone marrow (BM)-derived monocytes, as well as methods to determine vaccine efficacy through the examination of Ag-specific CD8+ T cell expansion and antitumor responses in murine melanoma models. As a vaccine platform, undifferentiated monocytes can be easily adapted to different tumor models with a multitude of target antigens. The method described here seeks to facilitate preclinical research of monocyte-based vaccination as a strategy for cancer immunotherapy.

Gliomas, especially glioblastomas, represent one of the most aggressive and difficult-to-treat human brain tumors. In the last few decades, clinical immunotherapy has been developed and has provided exceptional achievements in checkpoint inhibitors and vaccines for cancer treatment. Immunization with cellular vaccines has the advantage of containing specific antigens and acceptable safety to potentially improve cancer therapy. Based on T cells, dendritic cells (DC), tumor cells and natural killer cells, the safety and feasibility of cellular vaccines have been validated in clinical trials for glioma treatment. For TAA engineered T cells, therapy mainly uses chimeric antigen receptors (IL13Rα2, EGFRvIII and HER2) and DNA methylation-induced technology (CT antigen) to activate the immune response. Autologous dendritic cells/tumor antigen vaccine (ADCTA) pulsed with tumor lysate and peptides elicit antigen-specific and cytotoxic T cell responses in patients with malignant gliomas, while its pro-survival effect is biased. Vaccinations using autologous tumor cells modified with TAAs or fusion with fibroblast cells are characterized by both effective humoral and cell-mediated immunity. Even though few therapeutic effects have been observed, most of this therapy showed safety and feasibility, asking for larger cohort studies and better guidelines to optimize cellular vaccine efficiency in anti-glioma therapy.

Mismatch-repair deficiency (MMR-D) is closely linked to hypermutation and accordingly, high immunogenicity. MMR-D-related tumors thus constitute ideal vaccination targets for both therapeutic and prophylactic approaches. Herein, the prophylactic and therapeutic impact of a cellular vaccine on tumor growth and tumor-immune microenvironment was studied in a murine MLH1-/- knockout mouse model. Prophylactic application of the lysate (+/- CpG ODN 1826) delayed tumor development, accompanied by increased levels of circulating T cell numbers. Therapeutic application of the vaccine prolonged overall survival (median time: 11.5 (lysate) and 12 weeks (lysate + CpG ODN) vs. 3 weeks (control group), respectively) along with reduced tumor burden, as confirmed by PET/CT imaging and immune stimulation (increased CD3+CD8+ T - and NK cell numbers, reduced levels of TIM-3+ cells in both treatment groups). Coding microsatellite analysis of MMR-D-related target genes revealed increased mutational load upon vaccination (total mutation frequency within 28 genes: 28.6% vaccine groups vs. 14.9% control group, respectively). Reactive immune cells recognized autologous tumor cells, but also NK cells target YAC-1 in IFNγ ELISpot and, even more importantly, in functional kill assays. Assessment of tumor microenvironment revealed infiltration of CD8+ T-cells and granulocytes, but also upregulation of immune checkpoint molecules (LAG-3, PD-L1). The present study is the first reporting in vivo results on a therapeutic cellular MMR-D vaccine. Vaccination-induced prolonged survival was achieved in a clinically-relevant mouse model for MMR-D-related diseases by long-term impairment of tumor growth and this could be attributed to re-activated immune responses.

The success of active immunotherapies in the prevention of many infectious diseases over the course of over 200 years has lead scientists to wonder if the same principles could be applied to cancer. Antigen-specific active immunotherapies for the treatment of cancer have been researched for over two decades, however, the overwhelming majority of these studies have failed to stimulate robust clinical responses. It is clear that current active immunotherapy research should incorporate methods to increase the immunostimulatory capacity of these therapies. To directly address this need, we propose the addition of the immunostimulatory heat shock proteins (HSPs) to active immunotherapeutic strategies to augment their efficacy. Heat shock proteins are a family of highly conserved intracellular chaperone proteins, and are the most abundant family proteins inside cells. This ubiquity, and their robust immunostimulatory capacity, points to their importance in regulation of intracellular processes and, therefore, indicators of loss of cellular integrity if found extracellularly. Thus, we emphasize the importance of taking into consideration the location of vaccine-derived HSP/tumor-antigen complexes when designing active immunotheraputic strategies.