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Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death in the United States and Europe. Although the outcome of NHL patients has improved over the last years with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic molecules and approaches.Histone deacetylase inhibitors (HDACis) have emerged as a powerful new class of anti-cancer drugs for human therapy. To investigate HDACi antitumor properties on canine diffuse large B-cell lymphoma, a panel of seven HDACi compounds (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) was screened on CLBL-1 canine B-cell lymphoma cell line. Our results demonstrated that all HDACis tested exhibited dose-dependent inhibitory effects on proliferation of CLBL-1 cells, while promoting increased H3 histone acetylation. Amongst all HDACis studied, panobinostat proved to be the most promising compound and was selected for further in vitro and in vivo evaluation. Panobinostat cytotoxicity was linked to H3 histone and α-tubulin acetylation, and to apoptosis induction. Importantly, panobinostat efficiently inhibited CLBL-1 xenograft tumor growth, and strongly induced acetylation of H3 histone and apoptosis in vivo. In conclusion, these results provide new data validating HDACis and, especially, panobinostat as a novel anti-cancer therapy for veterinary applications, while contributing to comparative oncology.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.

Legume lectins comprise a structurally related, Ca/Mn-dependent, widespread, abundant and well characterized lectin family when compared to the large number of lectins from other sources described in the literature. Strangely enough, no specific function has been assigned to them aside from a possible role in storage and/or defense. Using a recent and fine-tuned methodology capable of specific lectin identification, β-conglutin, Vicia faba vicilin and β-lathyrin, the vicilin storage globulins from Lupinus albus, V. faba and Lathyrus sativus, respectively, were shown to be capable of affinity binding to thoroughly washed erythrocyte membranes and of specific elution with appropriate sugars. Based on this evidence and on sparse data published in the literature, a second family of legume lectins is proposed: the 7S family of storage proteins from leguminous seeds, or family II of legume lectins. These lectins are also structurally related, widespread and well characterized. In addition, they self-aggregate in a Ca/Mg, electrostatic dependent manner and are even more abundant than the family I of legume lectins. Using the same evidence, reserve and defense roles may be attributed to family II of legume lectins.