Development of a sandwich ELISA assay for quantification of human tissue transglutaminase in cell lysates and tissue homogenates.

Tissue transglutaminase (tTG) belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. There is a strong evidence that tTG is involved in pathology, such as neurodegenerative diseases, cancer, and celiac disease. To study physiopathological implication of tTG, a sandwich immunoassay has been developed with a new monoclonal antibody for the capture and polyclonal antibody both generated in house. Using this ready to use assay, the tTG protein level can be measured in human tissue homogenates and cells extracts easily in about 4 h. The limit of detection is 1.7 ng/ml; the coefficients of intra- and inter-assay variations range from 1 to 2 % and from 7 to 10 %, respectively. The assay is specific to tTG, and no cross reactivity with TG1, TG3, TG6, TG7, or factor XIIIa was observed. Finally, in the addition to the tTG activity assay previously developed, this assay should be a valuable tool to increase our knowledge of the tTG involvement in physiological and pathological states.

Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase.

During the last few years Antibody-Drug Conjugates (ADCs) have become one of the most active and very promising therapeutic weapons. Lessons learned from the traditional chemical conjugations (via lysine or cysteine residues of the antibodies) and the clinical studies of the developed ADCs have recently paved the way to the improvement of the conjugation technologies. Use of site-specific conjugation is considered as the promising path for improving the design and development of homogeneous ADCs with controlled Drug-Antibody ratio (DAR). Moreover, some of these conjugations can be applied to antibody fragments such as Fab, scfv and VHH for which random and chemical conjugation showed significant limitations. In this study, we identified a novel small peptide substrate (Q-tag) with high affinity and specificity of bacterial transglutaminase which can be genetically fused to different formats of antibodies of interest for the development of enzymatic site-specific conjugation we named "CovIsolink" platform. We describe the synthesis of chemically defined drugs conjugation in which the site and stoichiometry of conjugation are controlled using a genetically encoded Q-tag peptide with specific amino acids which serves as a substrate of bacterial transglutaminase. This approach has enabled the generation of homogeneous conjugates with DAR 1,7 for full IgG and 0,8 drug ratio for Fab, scfv and VHH antibody fragments without the presence of significant amounts of unconjugated antibody and fragments. As a proof of concept, Q-tagged anti Her-2 (human IgG1 (Trastuzumab) and the corresponding fragments (Fab, scfv and VHH) were engineered and conjugated with different aminated-payloads. The corresponding Cov-ADCs were evaluated in series of in vitro and in vivo assays, demonstrating similar tumor cell killing potency as Trastuzumab emtansine (Kadcyla®) even with lower drug-to-antibody ratio (DAR).

Optimised methods (SDS/PAGE and LC-MS) reveal deamidation in all examined transglutaminase-mediated reactions.

Transglutaminases (TGs) are a family of structurally and functionally related enzymes that catalyse calcium-dependent post-translational modifications of proteins through protein-protein crosslinking, amine incorporation, or deamidation. For many years deamidation mediated by TGs was considered to be a side reaction, but recently substrate-specific deamidations have been reported. Here we describe an optimised SDS/PAGE assay for the easy and rapid monitoring of the TG reaction with small peptides. The relative proportion of deamidation to transamidation was evaluated by densitometric analysis and confirmed by nano-liquid chromatography-nano-electrospray ionisation MS. We further investigated the effect of reaction conditions on transamidation and deamidation of TG1, TG2 and blood coagulation factor XIII A-subunit (FXIII-A) enzymes using a panel of glutamine-containing peptide substrates. The ratio of transamidation to deamidation was enhanced at high excess of the acyl-acceptor substrate and increasing pH. In addition, it was influenced by peptide substrates as well. Whereas deamidation was favoured at low cadaverine concentrations and acidic pH, no significant effect of calcium was observed on the ratio of transamidation/deamidation. Under our experimental conditions, deamidation always occurred in vitro even at high excess of the acyl-acceptor substrate, and the reaction outcome was shifted to deamidation at neutral pH. Our results provide clear evidence of the deamidation in the TG reaction, and may serve as an important approach for in vivo analysis of deamidation to better understand the role of TGs in biological events.

Repurposing rotavirus vaccines for intratumoral immunotherapy can overcome resistance to immune checkpoint blockade.

Although immune checkpoint-targeted therapies are currently revolutionizing cancer care, only a minority of patients develop durable objective responses to anti-PD-1, PD-L1, and CTLA-4 therapy. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors and overcome the resistance to these immunotherapies. Oncolytic properties of common viruses can be exploited for the priming of antitumor immunity, and such oncolytic viruses are currently in active clinical development in combination with immune checkpoint-targeted therapies. However, the routine implementation of these therapies is limited by their manufacturing constraints, the risk of exposure of clinical staff, and the ongoing regulations on genetically modified organisms. We sought to determine whether anti-infectious disease vaccines could be used as a commercially available source of immunostimulatory agents for cancer immunotherapy. We found that rotavirus vaccines have both immunostimulatory and oncolytic properties. In vitro, they can directly kill cancer cells with features of immunogenic cell death. In vivo, intratumoral rotavirus therapy has antitumor effects that are dependent on the immune system. In several immunocompetent murine tumor models, intratumoral rotavirus overcomes resistance to and synergizes with immune checkpoint-targeted therapy. Heat- and UV-inactivated rotavirus lost their oncolytic activity but kept their synergy with immune checkpoint-targeted antibodies through the up-regulation of the double-stranded RNA receptor retinoic acid-induced gene 1 (RIG-I). Rotavirus vaccines are clinical-grade products used in pediatric and adult populations. Therefore, in situ immunization strategies with intratumoral-attenuated rotavirus could be implemented quickly in the clinic.