**2. Materials and methods**

#### **2.1. Dogs to be treated**

**•** Synthesis of TNF-α (tumor necrosis factor alpha) and an oxygen intermediate such as nitric

**•** Adsorption of an antibody against tumor cell antigens targeting macrophages and cytotoxic

**•** Activation of CD8+ T lymphocytes by an MHC(major histocompatibility complex) mediated cell contact mechanism between antigen-presenting cells (APCs) and CD8 cells Several kinds of immunotherapy protocol are available both in human and veterinary medicine [4, 5, 6, 7]. Heat shock proteins (HSPs) such as gp96 or HSPs70 which are synthesized by the cells submitted to stress are advantageous vaccination adjuvants due to their chaperone properties and their role in antigen presentation [8]. As chaperone molecules, almost all the cell peptides are associated with these proteins and HSP purification provides a fingerprint of the cell's protein synthesis [9]. This is particularly useful for cancer cells which synthesize numerous abnormal proteins during their natural progress [10]. These cells being genetically unstable, their abnormal protein synthesis differs from patient to patient and during the course of the disease. The HSPs and their associated peptides (AAPs) have special receptors (CD91) on dendritic cells which allow the internalization of the AAPs and their modification in order to be expressed at the surface of class I HLA proteins on the cell membrane, triggering

Cancer cells are stressed by the mechanical and metabolic characteristics of the tumour. They synthesize many HSPs [12]. We thus isolated these proteins in order to make an autologous vaccine against the tumour. The HSPs were purified using a hydroxylapatite powder (HA) column. The powder carrying the HSPs was then injected subcutaneously to stimulate the

Purification of HSPs using classical way is long and tedious. The use of hydroxylapatite powder allows a much faster purification process. Hydroxylapatite chromatography has been described by Tiselius in the early seventies. Hydroxylapatite chromatography is an adsorption chromatography. The adsorption mechanism is very poorly understood. The surface of the

groups of the proteins. However, post synthesis treatment such as sintering or spray- drying process modify the physico-chemical properties of the material surface. Furthermore, the interaction of the material surface with biological fluids triggers epitaxial growth of carbonated

It was decided that the proteins purified will be injected carried by the particles for several reasons: the hydroxylapatite particles have been described as vaccine adjuvant, they are phagocytosed by the APCs and can deliver the proteins directly in the APCs, they trigger an afflux of APCs at the injection site [14]. Most of the adjuvants used in antiinfectious vaccines

The aims of this study was to check the feasibility of this protocol using HA-particles with dogs suffering of high grade lymphoma and to know if secondary effects were detected.

. These ions are supposed to interact with the chemical


oxide by macrophages activated by IFN-γ (interferon gamma)

**•** Activation of NK (natural killer) cells by IL-2 (interleukin 2)

502 Advances in Biomaterials Science and Biomedical Applications

activation of the CD8 T cells if they are abnormal [11].

immune system's response to the tumour.

material is occupied bu Ca++ and PO<sup>4</sup>

apatite at the surface of the material [13].

are nano or microparticular.

T lymphocytes.

Two dogs (Poodle, 6 years, Jagdterrier, 8 years ) suffered from polyadenopathy without any sign of immune deficiency. One dog (mixed breed, 10 years) had a liver metastasis. The last one had a cutaneous form. Their general condition remained reasonable, with no real weight loss or fever. One dog (Poodle) had a splenomegaly. The blood numeration revealed that the white cells and calcaemia for both dogs were in the normal range. Node biopsies were performed. One was sent to the pathologist and the other was frozen (-20°C) and used to manufacture the vaccine. Disease staging was performed using the WHO-staging criteria for canine lymphoma.

A chemotherapy treatment was proposed by the physician but rejected by the dog's owners due to side effects and an informed consent was signed.

After vaccination, the dogs were subjected to a clinical examination whenever they came for the following injection and side effects noted. The volume of a control node was externally measured.

#### **2.2. Toxicity and response assessment**

The side effects were graded every week for the first month and every month for the next five months according to the National Cancer Institute's common Toxicity Criteria (version 2.0).

The dogs were also submitted to a physical examination at the same frequency to assess their clinical response. A complete response (CR) was defined as the disappearance of all the nodes and metastasis for at least 4 weeks. A partial response (PR) was defined as a decrease by at least 50% of the product of the 2 longest lengths of all the nodes without the appearance of new lesions for at least 4 weeks. A minor response (MR) was defined as a decrease by less than 50% using the same criteria. Stable disease (SD) was defined as the same criteria unchanged for at least 4 weeks. Progressive disease (PD) was defined as an increase by >25% of the product of the 2 longest lengths of all the nodes for at least 4 weeks or when new lesions appeared.

#### **2.3. Hydroxylapatite powder characteristics**

Hydroxylapatite powders (Fig. 1) can be used for adsorption chromatography under atmos‐ pheric or high pressure. In order that the protein solution did not fill in the column and to avoid the compaction, the powder was spray-dried then sintered at 1000°C.

The HA was transformed into a ceramic powder according to the following protocol. The synthesized calcium phosphate was suspended in a slurry which is liquid, spray-dried, then sintered. The spray-dried material was heated almost to fusion temperature which favors the migration of matter between the grains and the formation of bridges. As the surface energy was smaller for large than for small grains, their size increased and the distance between the grain centers and the particle surface area decreased.


**Table 1.** Characteristics of the powder

The sintering considerably reduced the surface area making the amount of proteins adsorbed on the powder lower. It also stabilized the powder structure. The powder was submitted to dissolution/ precipitation processes when soaked inside a saline solution [13]. The modifica‐ tion occurring at the powder surface affected the adsorption properties of the powder. The reduced surface area decreased the interactions with the saline solution containing the proteins. There was a microporosity between the ceramic grains inside the same particle allowing the protein solution to diffuse inside the particles. The characteristics of the powder used in this experiment was given in table 1

As the chromatography was carried out under atmosphere, the granulometry of the powder was an important factor in order to avoid any plugging of the column. The HSPs could be eluted from the powder by 200-300 mM NaCl solutions. The powder solution in NaCl was not stable enough to be injected as it decanted too fast in the syringe. Thus to improve the injectability the powder was put in suspension in a 2% solution of carboxymethylcellulose in 20 mM NaCl.

#### **2.4. Vaccine manufacturing protocol**

The tumor tissue and all the materials used to prepare the vaccine were handled in sterile conditions under a laminar flow. The frozen tumor (200 mgr) tissue was homogenized using a bead tissue homogenizer. 1 ml of NaHCO3 (30 mM, pH 7) was added for 1 ml of homogenate. The resulting homogenate was then centrifuged at 1000 g for 15 mn at 4°C to remove all cell fragments.

The supernatant containing the cytoplasmic proteins was used for protein purification by HA column chromatography as follows: a) two precipitations with ammonium sulphate (first at 50% and then at 70%) recovering the pellets. The last pellet was resuspended in 1 ml phosphate buffer (20 mM, pH 7). The column was filled (chromatography columns, Poly-prep, Cat. 731-1550, Bio Rad) with 0.2 gr of HA (0-25 μm), equilibrated with 10 volumes of phosphate buffer (20 mM pH7). The resuspended pellet was then added. The column was then washed with 3 ml of a 100 mM NaCl solution (fig.2).

**Figure 1.** SEM of the HA-powder used for the vaccine.

**Powder characteristics**

504 Advances in Biomaterials Science and Biomedical Applications

**Table 1.** Characteristics of the powder

20 mM NaCl.

fragments.

used in this experiment was given in table 1

**2.4. Vaccine manufacturing protocol**

with 3 ml of a 100 mM NaCl solution (fig.2).

Nature of the charged groups PO4 3-, OH-

Electrocinetical potential (mV) -35 Hydrophobicity + Surface pH 7,8 Granulometry (µm) 0-25 Surface area (m2/gr) 4

Shape spherical

The sintering considerably reduced the surface area making the amount of proteins adsorbed on the powder lower. It also stabilized the powder structure. The powder was submitted to dissolution/ precipitation processes when soaked inside a saline solution [13]. The modifica‐ tion occurring at the powder surface affected the adsorption properties of the powder. The reduced surface area decreased the interactions with the saline solution containing the proteins. There was a microporosity between the ceramic grains inside the same particle allowing the protein solution to diffuse inside the particles. The characteristics of the powder

As the chromatography was carried out under atmosphere, the granulometry of the powder was an important factor in order to avoid any plugging of the column. The HSPs could be eluted from the powder by 200-300 mM NaCl solutions. The powder solution in NaCl was not stable enough to be injected as it decanted too fast in the syringe. Thus to improve the injectability the powder was put in suspension in a 2% solution of carboxymethylcellulose in

The tumor tissue and all the materials used to prepare the vaccine were handled in sterile conditions under a laminar flow. The frozen tumor (200 mgr) tissue was homogenized using a bead tissue homogenizer. 1 ml of NaHCO3 (30 mM, pH 7) was added for 1 ml of homogenate. The resulting homogenate was then centrifuged at 1000 g for 15 mn at 4°C to remove all cell

The supernatant containing the cytoplasmic proteins was used for protein purification by HA column chromatography as follows: a) two precipitations with ammonium sulphate (first at 50% and then at 70%) recovering the pellets. The last pellet was resuspended in 1 ml phosphate buffer (20 mM, pH 7). The column was filled (chromatography columns, Poly-prep, Cat. 731-1550, Bio Rad) with 0.2 gr of HA (0-25 μm), equilibrated with 10 volumes of phosphate buffer (20 mM pH7). The resuspended pellet was then added. The column was then washed

, Ca2+

The powder was then suspended in 5 ml carboxymethylcellulose (CMC) solution (2% in 20mM NaCl). 0.5 ml of this solution was used for each vaccine shot.

**Figure 2.** Autovaccine manufacturing scheme

0.2 ml of the previous solution was used to make electrophoretic control. The solution was centrifuged at 1000g 30 for seconds. The supernatant was discarded and the powder in the pellet washed with 0.1 ml of a 0.5 M NaCl solution. The solution was again centrifuged and the supernatant was used for a SDS-Page and for protein quantification using UV spectrometer. 10 μl of the solution was also used for dot blot with anti HSP70 and anti gp90 antibodies on a nitrocellulose membrane. The antibody labelling was evidenced using a westernbreeze™ (invitrogen) kit according to the manufacturer instructions.

### **2.5. Vaccination protocol**

The dogs were injected with 0.5 ml (about 40 μg of proteins) of the vaccine subcutaneously on day 0. The dose schema consisted of 0.5 ml/dose every week for one month, followed by one dose every month for four months.

#### **2.6. activation of TLR2 and 4**

Some HSPs are TLR (Toll like receptor) agonist and the TLR activation was checked using two cell lines secreting an embryonic phosphatase alkaline when the TLR 2 or 4 are stimulated.

Two cell lines were used for TLR receptor stimulation. HEK-BlueTM-hTLR2 and HEK-BlueTMhTLR4 cells (invivogen –France) were grown in DMEM supplemented with 10% fetal calf serum and 0,5% normocin (invivogen-France) to avoid mycoplasma contamination. 20000 cells were introduced in wells of 96-well plate, 4 hours later 20 μl of different concentrations of the vaccine was added and incubated at 37°C for 20 hours. Then the medium was removed and replaced by 180 μl of resuspended QUANTI- blueTM (Invivogen) and incubated for 1 hour and the positivity evaluated. The negative control was the HA- powder without any protein immobilized on its surface. The minimal concentration turning blue the medium was noted. It was estimated that the vaccine was functional when the minimal active concentration was less than the vaccine concentration.
