**4. Discussion**

**Figure 4.** Dot blot after anti gp90 labelling

508 Advances in Biomaterials Science and Biomedical Applications

**Figure 5.** Dot blot after anti HSP70 labelling

This experiment showed the feasibility of this protocol as an autologous vaccine for cancer in dogs in veterinary practice. We have also developed an *in vitro* test to assess the functionality of purified heat shock proteins. This test checks in a few minutes the vaccine's ability to stimulate antigen-presenting cells (APCs) through toll-like receptor (TLR) activation. Even if the sample size is small, the dogs' overall survival (210 days) was much higher than expected, as the average OS for this type of pathology is two months. In larger series of stage Vb lymphomas associating chemotherapy and the same vaccine protocol, we could demonstrate that after vaccination the dogs showed a delayed cutaneous hypersensibility after their own tumor antigen injection in the derm (Marconato, unpublished results).

High grade lymphomas in dogs are an interesting model for cancer vaccines because survival is very short, so the effect on OS is very easy to measure. The clinical efficiency of vaccine is not always related to the vaccine's effect on biological parameters. Models with a short life expectancy are thus of interest. Furthermore vaccination without the use of other drugs is of particular interest.

The amount of proteins used in this vaccine was justified by the different experiments published about autovaccines using HSPs in animals and humans. Furthermore we checked that the amount of proteins could activate 20000 APCs *in vitro.* The presence of the tumor proteins at the surface of the HA-particles allows the activation of the TLRs. It indicates that the proteins are immobilized at the surface of the particles and their adsorption does not denaturize these proteins.

Cancer vaccines are the focus of great interest. Although cancer cells synthesise abnormal proteins, they are are not recognised by the immune system. Different immunosuppression mechanisms by cancerous tumours have been described [15]. The use of multiantigen vaccines could reduce the cancer cell's "invisibility" to the immune system compared to monoproteic vaccines. Different kinds of immune therapy are under investigation. Cell engineering immunotherapy protocols have been tested, including activation of dendritic cells *in vitro* by tumour antigens before being reinjected into the patient [16]. Other trials concern amplification of the intratumoural lymphocytes (TILs) *in vitro* [17] before being reinjected into the donor. Recently, different types of antibodies were approved for the oncology field, in particular antibodies against VEGF (Vascular endothelial growth factor) to inhibit tumour vasculariza‐ tion [18, 19].

Heat Shock Proteins (HSPs) have proved to be of therapeutic interest in human medicine for some applications. In order to be useful as a cancer vaccination, these HSPs must be made available to APCs. HA-powder is a good material for use as a vector of HSPs to APCs. It has been shown that when injected in the dermis or subcutaneous tissue, it triggers a foreign body reaction and that the cells of this foreign body reaction could be transfected with a DNA molecule carried by the particles [20]. It suggests that, further to DNA vectorisation, the particles could help in the transfection of HSPs and their associated peptides in APCs.

Although gp96 has been described as able to stimulate TLR4, it is not sure that these proteins are the only proteins responsible for TLRs stimulation in this case. There are contaminating proteins in various bands of the SDS which could interfere with the TLRs. The non stimulation of the TLR2 indicates that the TLR4 is not activated by contaminating endotoxins.

Hydroxyapatite has been used as an adjuvant for various infectious vaccines such as diphtheria and tetanus [21]. The Hydroxyapatite lattice is a hexagonal structure which allows numerous substitutions. Ions and small amino acids can thus be trapped in the HA lattice. Consequently, HA powder has been used to purify DNA, proteins or even viruses from biological solutions [22]. In this case, the surface properties of the particles allowed both the purification of HSPs and their use as a vector to APCs and as an adjuvant.

The HA-powder characteristics seems well suited for its role of cancer vaccine adjuvant. The granulometry range allows the phagocytosis by the APCs as it was demonstrated previously [14]. Furthermore, the grain boundaries in each particle is degraded by the cells making the particles fragmented and thus decreasing the real granulometry a few days after injection. It was also demonstrated that the phagocytosis of these particles by the APCs induced the synthesis of various cytokines and lymphokines necessary for the cross-priming of the CD8. The HA-adjuvant effect does not seem to be due to TLR activation as HA-particles alone do not trigger TLR2 and 4 activation. Other mineral adjuvants such as aluminum oxide have been demonstrated to activate the inflamasome. The inflamasome is a multi-protein complex involved in the production of mature IL-1β. The alumn-induced release of IL-1β in macro‐ phages is done under activation of the NLRP3 [23]. It is suggested from unpublished results that the adjuvancy effect of the HA-particles is also due to the inflamasome activation.

Tumor associated antigens show a very poor antigenicity. Thus the presence of an adjuvant like calcium phosphate particles is essential in order to increase the visibility of these antigens by the patient immune system.

This method was used in a short pilot study in humans and proved to be very safe, as only local effects (erythema) were reported in some patients (24). Although it was not the goal of the pilot study, some remission in the extent of the tumour was observed and constituted a good proof of concept. Gp96 has already been used in human medicine to treat a series of patients with indolent non Hodgkin's lymphoma [23]. The results are difficult to compare, as the patients could have been treated by chemo- or radiotherapy more than six weeks before the vaccination protocol. However, at three months most of the patients were rated SD, including those who were resistant to previous therapy. No patients suffered side effects.
