(171a) Intracellular Delivery of Antigenic Proteins for Cancer Vaccination

Introduction

Cell-based
vaccines against cancer, composed of tumor antigen-loaded antigen presenting
cells (APCs), hold great therapeutic potential due to their ability to activate
the patient's immune system and drive a long-lasting, tumor-specific, CD8 T
cell response. These therapies, such as the recently approved Provenge^® for prostate cancer, have minimal
side-effects relative to chemotherapies and radiation treatment. However, one
of the main barriers to developing such vaccines has been achieving efficient
intracellular loading of APCs with protein antigens for presentation to CD8 T
cells. Since the mechanism of cross-presentation (a process wherein endocytosed
proteins can induce a CD8 response) within antigen presenting cells remains
elusive, one must develop a reliable method of delivering the desired antigen
directly to the cell cytoplasm to advance therapies utilizing the potent
effector CD8 response.  In this work,
our team has adapted our recently developed microfluidic delivery system (Sharei,
A. et al. PNAS, 2013.
110, 1082-2087) to induce selective MHC class I
antigen presentation by direct delivery of the target protein to the cell
cytosol. Compared to endocytotic methods, this system has demonstrated a 10-100
fold improvement in antigen-specific CD8 T cell activation and can potentially
be translated to more challenging systems, such as B cells.

Materials and Methods

This
work was performed using the ovalbumin (ova) antigen presentation model. In our
experiments, dendritic cells were derived from the bone marrow of healthy B6
mice and treated by the aforementioned microfluidic delivery system to
facilitate intracellular delivery of the ovalbumin protein. Antibody staining,
ELISA, flow cytometry, and proliferation assays using OT-I and OT-II transgenic
mice were used to assess antigen presentation efficiency and T cell cytokine
secretion.

Results and Discussion

In our studies, we
have demonstrated enhanced MHC-I presentation of the SIINFEKL epitope by
antibody staining and verified that the delivery process does not interfere
with DC maturation as measured by up-regulation of CD80 and CD86 in response to
stimulation by lipopolysaccharide. As illustrated in Fig. 1, this method has
also successfully induced the activation/proliferation of antigen-specific CD8
T cells (derived from OT-I mice) as measured by T cell proliferation and
cytokine secretion. Moreover, preliminary results indicate that this method can
induce functional antigen presentation in primary B cells ? a significant
challenge for existing technologies.

Figure
1.
a) Bone marrow derived DCs were treated by the device in the presence of
varying OVA concentrations and T cell proliferation was measured by CFSE
staining and flow cytometry. b) Secretion of IFN-γ from CD8 T cells
stimulated by treated DCs. The ?OVA Endo' condition corresponds to an
endocytosis only case.

Conclusion

The described
intracellular delivery technique has demonstrated enabling potential as a
platform for high efficiency antigen presentation in cancer vaccine
applications.