Hydroxyapatite as a tool for fast purification of hsps and carrier for targeting antigen-presenting cells in cancer immunology

FRAYSSINET P.; CUELLO CARRIÓN F. D.; CIOCCA D. R.

Concepción, Chile

Workshop; 5th International Workshop on the Molecular Biology of Stress Responses; 2006

Cell Stress Society International

The heat shock proteins (HSPs) are a class of proteins synthesized when the cells are submitted to a stress and in particular to heat. They stabilize other cell protein folding and thus are intimately associated to these late proteins. They also have other functions. In particular, some of them a such as gp96 are involved in the process of T-cell cross-priming by antigen presenting cells. The heat shock protein gp96 is located in the endoplasmic reticulum where it exerts protective functions during cell stress. It can additionally exerts potent stimulatory activity on both innate and adaptative immunity. As the gp96 is associated to many peptides synthesized by the cell, the purification of the gp96 produced by the cancer cells allows to get a good finger print of the cancer cell peptide synthesis. The gp96-peptide complexes bind to CD91 receptors and are taken up by antigen presenting cells. The peptides carried on gp96 are then processed and delivered to the cell surface in association with MHC class 1 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 way is long and tedious and is constituted by several stages of precipitation, chromatography, diaIysis and electrophoresis. There is a simpler way in one step based on the adsorption of the gp96 on hydroxyapatite ceramic powder. gp96 adsorbs on the ceramic at neutral pH and low ionic strength and desorbs from the powder in a 200 mM 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 physico-chemical conditions needed to remove the contaminant proteins and keep the gp96 at the powder surface. The gp96 release is done at interesting physico-chemical conditions because they are close to that 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 was very easy 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 in contact with isolated cells, the cells were transfected and 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 at remote distance. It thus indicates that the HA-powder targets the antigen presenting cells, and allows a preferential delivery of the molecules carried by the particles inside by the antigen presenting cells. Furthermore, when primary celllines of human monocytes were grown in the presence of HA powders having different shapes, granulometry, and surface area, it was shown that the synthesis of TNF, IL-1 and -6 varied with the powder characterístics. It indicates that the activation of these cells depends on the characteristics of the powder. It was also showed that the internalisation of the particles was necessary to activate the monocytes. The internalization process of the 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 cell low pH 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 APCs with other proteins involved in cancer vaccination. The use of the same material both for purification, APCs targeting and activation allows a very fast availability of a costumized vaccine against  some tumors in humans.