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).

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.

In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer.

The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92(CD16)) or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92(CAR)). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92(CD16) was always inferior to that observed after direct recognition by NK-92(CAR). In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92(CD16) + trastuzumab but not of NK-92(CAR) induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92(CAR) in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

TWIST1 is a direct transcriptional target of MYCN and MYC in neuroblastoma.

In neuroblastoma, MYCN amplification is associated with a worse prognosis and is a criterion used in the clinic to provide intensive treatments to children even with localized disease. In correlation with MYCN amplification, upregulation of TWIST1, a transcription factor playing a crucial role in inhibition of apoptosis and differentiation, was previously reported. Clinical data set analysis of MYCN, MYC and TWIST1 expression permits us to confirm that TWIST1 expression is upregulated in MYCN amplified neuroblastoma but also in a subset of neuroblastoma harboring high expression of MYCN or MYC without gene amplification. In silico analyses reveal the presence of several MYC regulatory motifs (E-Boxes and INR) within the TWIST1 promoter. Using gel shift assay and reporter activity assays, we demonstrate that both N-Myc and c-Myc proteins can bind and activate the TWIST1 promoter. Therefore, we propose TWIST1 as a direct MYC transcriptional target.

Upstream ORF affects MYCN translation depending on exon 1b alternative splicing.

BACKGROUND: The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNDelta1b) mRNA. But nothing is known about their respective ability to translate the MYCN protein. METHODS: Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two MYCN transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two MYCN mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the MYCNDelta1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein. RESULTS: Both are translated, but higher levels of protein were seen with MYCNDelta1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCNDelta1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCNDelta1b mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with MYCN mRNA. Here, we showed that MYCNOT: MYCN Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of MYCNDelta1b mRNA. CONCLUSIONS: Existence of upstream ORF in MYCN transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction.

Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells.

N-Myc oncogene amplification is a frequent event in neuroblastoma and is strongly correlated with advanced disease stage and treatment failure. Similarly to c-Myc oncogenic activation, N-Myc deregulation promotes both cell proliferation and p53-dependent apoptosis by sensitizing cells to a variety of insults. Intriguingly, p53 mutations are uncommon in neuroblastomas, strongly suggesting that an alternative cooperating event circumvents this safeguard against oncogene-driven neoplasia. By performing a pangenomic cDNA microarray analysis, we demonstrate that human Twist is constantly overexpressed in N-Myc-amplified neuroblastomas. H-Twist overexpression is responsible for the inhibition of the ARF/p53 pathway involved in the Myc-dependent apoptotic response. This oncogenic cooperation of two key regulators of embryogenesis causes cell transformation and malignant outgrowth.