Comparative characterization of two monoclonal antibodies targeting canine PD-1

Monoclonal antibodies targeting immune checkpoints have revolutionized oncology. Yet, the effectiveness of these treatments varies significantly among patients, and they are associated with unexpected adverse events, including hyperprogression. The murine research model used in drug development fails to recapitulate both the functional human immune system and the population heterogeneity. Hence, a novel model is urgently needed to study the consequences of immune checkpoint blockade. Dogs appear to be uniquely suited for this role. Approximately 1 in 4 companion dogs dies from cancer, yet no antibodies are commercially available for use in veterinary oncology. Here we characterize two novel antibodies that bind canine PD-1 with sub-nanomolar affinity as measured by SPR. Both antibodies block the clinically crucial PD-1/PD-L1 interaction in a competitive ELISA assay. Additionally, the antibodies were tested with a broad range of assays including Western Blot, ELISA, flow cytometry, immunofluorescence and immunohistochemistry. The antibodies appear to bind two distinct epitopes as predicted by molecular modeling and peptide phage display. Our study provides new tools for canine oncology research and a potential veterinary therapeutic

DNA and RNA Binding Proteins : From Motifs to Roles in Cancer

DNA and RNA binding proteins (DRBPs) are a broad class of molecules that regulate numerous cellular processes across all living organisms, creating intricate dynamic multilevel networks to control nucleotide metabolism and gene expression. These interactions are highly regulated, and dysregulation contributes to the development of a variety of diseases, including cancer. An increasing number of proteins with DNA and/or RNA binding activities have been identified in recent years, and it is important to understand how their activities are related to the molecular mechanisms of cancer. In addition, many of these proteins have overlapping functions, and it is therefore essential to analyze not only the loss of function of individual factors, but also to group abnormalities into specific types of activities in regard to particular cancer types. In this review, we summarize the classes of DNA-binding, RNA-binding, and DRBPs, drawing particular attention to the similarities and differences between these protein classes. We also perform a cross-search analysis of relevant protein databases, together with our own pipeline, to identify DRBPs involved in cancer. We discuss the most common DRBPs and how they are related to specific cancers, reviewing their biochemical, molecular biological, and cellular properties to highlight their functions and potential as targets for treatment

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy : can we overcome them?

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine

Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition

The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-β treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway