Autovaccination Against Tumors Using a Mineral/Protein Composite

FRAYSSINET P.; CUELLO CARRIÓN F. D.; LAQUERRIERE P.; GRANDJEAN-LAQUERRIERE A.; CIOCCA D. R.

Heat Shock Proteins in Biology and Medicine

Research Singpost

Lugar: Kerala.; Año: 2006; p. 1 - 14

The heat shock proteins (HSP) are a class of protein synthesized when cells are submitted to a stress and in particular to heat. They stabilize the folding of other cell proteins to which they become intimately associated. They also have other functions. In particular, some such as gp96 are involved in the process of T cell cross-priming by antigen presenting cells (APC). Heat shock protein gp96 is located in the endoplasmic reticulum where it exerts protective functions during cell stress. It can additionally exert potent stimulatory activity on both innate and adaptative immunity. As gp96 is associated to many peptides synthesized by the cell, the purification of the gp96 produced by cancer cells provides a good finger print of the cancer cell peptide synthesis. The gp96-peptide complexes bind to CD91 receptors and are taken up by APC. The peptides carried on gp96 are then processed and delivered to the cell surface in association with MHC class I antigens for recognition by antigen-specific CD8+ T cells. Thus, gp96 can be used to make some of the associated peptides recognised by the T cells. The classical purification route is long and tedious, involving several stages of precipitation, chromatography, dialysis and electrophoresis. There is a much simpler one-step method based on the adsorption of the gp96 to hydroxyapatite ceramic powder. Gp96 adsorbs to the ceramic at neutral pH and low ionic strength and desorbs in 200mM phosphate buffer at the same pH. When the HA-powder is loaded with a tumor homogenate, it is easy to wash the powder with buffers in the physico-chemical conditions needed to remove contaminant proteins while keeping the gp96at the powder surface. The release of gp96 occurs in interesting physicochemical conditions because they are close to those found inside cells. The gp96-loaded powder can then be associated to co-factors or cytokines and injected intradermically to stimulate the immune system against the peptides synthesized by the cancer cells.  We have demonstrated that it is very straightforward to adsorb a plasmid carrying a galactosidase (lac-Z) gene on the particles of the same ceramic powder. When HA-powder carrying a plasmid with a lac-Z gene was placed in contact with isolated cells, the cells were transfected and subsequently expressed the galactosidase gene. When implanted inside connective tissue, the particles triggered a mild and transient foreign body reaction. The first cells to be transfected were the cells of the foreign body reaction comprising macrophages, dendritic cells and monocytes. These cells were then able to migrate away from the site of contact indicating that the HA-powder targets the antigen presenting cells, and allow preferential delivery of the molecules carried by the particles inside by the antigen presenting cells. Furthermore, when primary cell lines of human monocytes were grown in the presence of HA-powders having different shapes, particle sizes, and surface areas, it was shown that the synthesis of TNF-á and IL-6 varied with the powder characteristics. This indicates that the activation of these cells depends on the characteristics of the powder. It was also shown that internalization of the particles was necessary to activate the monocytes. The internalization of HA-ceramics is well known. The granules are constituted by small grains of matter linked by grain boundaries. Once fragments are inside cells and tissues, the grain boundaries are degraded and grains of matter are released from the particles. These grains are phago- or endocytosed and are dissolved in the low-pH cell compartments. The ceramics are totally degraded in the tissues within a few days or weeks depending on their characteristics. This technology can be used for the purification and targeting of APC with other proteins involved in cancer vaccination. The use of the same material both for purification, APC targeting and activation allows direct availability of a customized vaccine against some tumors in humans.