Mechanism of Action of an Autovaccine Against Canine DLBCL Lymphoma Leading to a Long Overall Survival
Authors: Frayssinet Patrick , Rouquet Nicole , Marconato Laura , Rossi Federica 
Published: October 2019

Abstract
An autovaccine against canine DLBCL lymphomas associated to a CHOP protocol has proven to greatly improve the overall survival and the time to progression of a series of dogs against another series only treated by the CHOP protocol (1). This vaccine was obtained by purifying specific proteins and peptides from a node biopsy. In order that the vaccine matches the heterogeneity of the tumor, one vaccine was produced for each dog.
The biopsy was homogenised then the cell debris were removed by centrifugation. The proteins in suspension in the supernatant were precipitated by a high salt concentration. Once the proteins were suspended in buffer, the solution was passed through a column of HA (hydroxylapatite)- particles. The particles were then washed by different concentrations of phosphate buffer in order to concentrate heat shock proteins at the surface of the HA-particles and eliminate contaminating proteins. The vaccine was thus made of HA- particles used as carrier and adjuvant and proteins and peptides enriched in heat shock proteins (gp96 and HSP70). The preparation resulted in 1500 µg of proteins and 40 mg of HA for each dose. Vaccination protocol consisted in 4 injections within four weeks and one injection a month for four months. In order to establish the mechanism of action, vaccines were tested for their ability to cross prime the CD8 and to be link the CD91 and TLR2 and 4. We also checked that the HA-particles could activate the inflammasome.
Materials and methods, and results:
CD8 cross-priming:
Murine epithelial cells (4T1) were used to induce a tumor in isogenic mice (Balb/c). A vaccine was prepared from a tumor and was used to stimulate in vitrodendritic cells of the same mouse lineage. These late cells are then incubated with CTLs of a mouse already in contact with the 4T1. It appeared that the CTLs synthesize more IFN-gamma than the negative control indicating a cross priming of the CD8 lymphocytes by the vaccine particles in a dose dependent manner.
TLR2 and 4 labelling:
The vaccine proteins (from vaccines used for the vaccinated group of DLBCL dogs) were removed from their HA carrier by washing the HA- particles with a 0.5 M KHPO4solution. They were then introduced in a culture of monocytes overexpressing TLR2 or TLR4. TLR2 and 4 were co transfected with reporter gene of SEAP which is fused with NF-κβ and AP-1. It appeared that the HA-powder alone did not activate the TLR receptors while the proteins released from the powder were ligand of both TLR2 and 4.
CD91 labelling:
Proteins released from the HA carriers were associated to a peroxydase and then introduced in a culture of a monocyte line expressing CD91 (RAW264) with or without anti- CD91. The cells were labelled by the peroxydase /proteins complexes. Furthermore, it appeared that only the cells grown without the antibody were labelled.
Inflammasome activation by the HA-particles:
HA-powders having different characteristics, surface area, granulometry, sintering temperature, shape, were introduced in culture of THP-1 cells and the IL-1B excretion was measured and compared to positive contols known to activate the inflammasome. It was demonstrated that depending on the concentration, some powders activated the inflammasome more than the positive control.
Discussion:
It is generally accepted that CD8 cross- priming against cancer antigens is essential for the elimination of cancer cells by the immune system. CD91 labelling by the vaccine proteins suggest that the heat shock proteins play a key-role in the cross-priming. CD91 are specific receptors for HSPs found at the surface of antigen presenting cells. HSPs are essential molecules in the antigen processing for their expression at the APCs surface. It is also known that these proteins are ligand of the TLR2 and TLR4.
Conclusions:
It is thus possible to purify specific proteins from nodes of dogs with DLBCL lymphoma and associate them to an adjuvant material to obtain a CD8 cross-priming against the tumor cells. These biological results could be related to a better clinical outcome for vaccinated dogs
(1) Laura Marconato, Patrick Frayssinet, Nicole Rouquet, Stefano Comazzi, Vito Ferdinando Leone, Paola Laganga, Federica Rossi, Lorenzo Pezzoli, Luca Aresu, Randomized, placebo-controlled, double-blinded chemo-immunotherapy clinical trial in a Pet Dog model of Diffuse Large B-cell Lymphoma: a successful combination. Clin Cancer Res. 2014 Feb 1;20(3):668-77

Disclosures
Frayssinet: Urodelia: Employment. Rouquet:Urodelia: Employment. Marconato:Urodelia: Research Funding. Rossi:Urodelia: Research Funding.