AIMM Therapeutics (Netherlands)

Molecular glues, compounds that bind cooperatively atprotein–proteininterfaces (PPIs), are revolutionizing chemical biology and drug discovery,allowing the modulation of traditional “undruggable”targets. Here, we focus on a native regulatory PPI between the scaffoldingprotein 14-3-3 and C-RAF, a key component of the MAPK signaling pathway.Extensive drug discovery efforts have focused on the MAPK pathwaydue to its central role in oncology and developmental disorders (RASopathies).However, the modulation of its protein complexes is underexplored.C-RAF activity is regulated on multiple levels including dimerization,phosphorylation, and complex formation with 14-3-3, which preventsC-RAF activation by binding to a C-RAF sequence centered on phospho-serine259. We used a fragment-merging approach to design molecular gluesthat bound to the composite surface of this 14-3-3/C-RAFpS259 complex.Molecular glues stabilized the inhibitory complex up to 300-fold;their glue-based mechanism of action was confirmed by crystallographyand biophysical studies. Selectivity among the other RAF isoformsand other RAF phosphorylation sites was evaluated. The best compoundsshowed excellent selectivity among a broad panel of 80 14-3-3 clients.Cellular assays demonstrated on-target engagement, enhanced phosphorylationlevels of C-RAFpS259, and reduced levels of RAF dimerization and ERKphosphorylation. Overall, this approach enabled chemical biology studiesfor a C-RAF site that was intrinsically disordered prior to 14-3-3binding and had not been targeted previously. These molecular glueswill be useful chemical probes and starting points for drug discoveryefforts to modulate native PPI stabilization in the MAPK pathway withapplications in oncology and RASopathies.

The emergence of SARS-CoV-2 variants that are more resistant to antibody-mediated neutralization pose a new hurdle in combating the COVID-19 pandemic. Although vaccines based on the original Wuhan sequence have been shown to be effective at preventing COVID-19, their efficacy is likely to be decreased against more neutralization-resistant variants-of-concern (VOC), in particular, the Beta variant originating in South Africa. We assessed, in mice, rabbits, and non-human primates, whether a third vaccination with experimental Wuhan-based Spike vaccines could alleviate this problem. Our data show that a third immunization improves neutralizing antibody titers against the variants-of-concern, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). After three vaccinations, the level of neutralization against Beta was similar to the level of neutralization against the original strain after two vaccinations, suggesting that simply providing a third immunization could nullify the reduced activity of current vaccines against VOC.

IntroductionT‐cell antigen receptor (TCR) interaction with cognate peptide:MHC complexes trigger clustering of TCR:CD3 complexes and signal transduction. Triggered TCR:CD3 complexes are rapidly internalized and degraded in a process called ligand‐induced TCR downregulation. Classic studies in immortalized T‐cell lines have revealed a major role for the Src family kinase Lck in TCR downregulation. However, to what extent a similar mechanism operates in primary human T cells remains unclear.MethodsHere, we developed an anti‐CD3‐mediated TCR downregulation assay, in which T‐cell gene expression in primary human T cells can be knocked down by microRNA constructs. In parallel, we used CRISPR/Cas9‐mediated knockout in Jurkat cells for validation experiments.ResultsWe efficiently knocked down the expression of tyrosine kinases Lck, Fyn, and ZAP70, and found that, whereas this impaired T cell activation and effector function, TCR downregulation was not affected. Although TCR downregulation was marginally inhibited by the simultaneous knockdown of Lck and Fyn, its full abrogation required broad‐acting tyrosine kinase inhibitors.ConclusionsThese data suggest that there is substantial redundancy in the contribution of individual tyrosine kinases to TCR downregulation in primary human T cells. Our results highlight that TCR downregulation and T cell activation are controlled by different signaling events and illustrate the need for further research to untangle these processes.

Abstract Current high-throughput single-cell multi-omics methods cannot concurrently map changes in (phospho)protein levels and the associated gene expression profiles. We present QuRIE-seq (Quantification of RNA and Intracellular Epitopes by sequencing) and use multi-factor omics analysis (MOFA+) to map signal transduction over multiple timescales. We demonstrate that QuRIE-seq can trace the activation of the B-cell receptor pathway at the minute and hour time-scale and provide insight into the mechanism of action of an inhibitory drug, Ibrutinib.

SummaryThe SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

BackgroundAdaptive immunity to cancer cells forms a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether tumor-targeting antibodies could be isolated from a patient that cured (now 13 years tumor-free) metastatic melanoma following adoptive transfer of ex vivo expanded autologous T cells.MethodsPatient‘s peripheral blood B cells were isolated and tested for the presence of tumor-reactive B cells using AIMM’s immmortalisation technology. Antibody AT1412 was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 binds the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities in cancer and induces ADCC and ADCP by effector cells.ResultsSpontaneous immune rejection of tumors was observed in human immune system (HIS) mouse models implanted with CD9 genetically-disrupted A375 melanoma (A375-CD9KO) tumor cells, while A375wt cells were not cleared. Most notably, no tumor rejection of A375-CD9KO tumors was observed in NSG mice, indicating that blockade of CD9 makes tumor cells susceptible to immune rejection.CD9 has been described to regulate integrin signaling, e.g. LFA-1, VLA-4, VCAM-1 and ICAM-1. AT1412 was shown to modulate CD9 function by enhancing adhesion and transmigration of T cells to endothelial (HUVEC) cells. AT1412 was most potently enhancing transendothelial T-cell migration, in contrast to a high affinity version of AT1412 or other high affinity anti-CD9 reference antibodies (e.g. ALB6). Enhanced immune cell infiltration is also observed in immunodeficient mice harbouring a human immune system (HIS). AT1412 strongly enhanced CD8 T-cell and macrophage infiltration resulting in tumor rejection (A375 melanoma). PD-1 checkpoint blockade is further sustaining this effect. In a second melanoma model carrying a PD-1 resistant and highly aggressive tumor (SK-MEL5) AT1412 together with nivolumab was inducing full tumor rejection, while either one of the antibodies alone did not.ConclusionsThe safety of AT1412 has been assessed in preclinical development and is well tolerated up to 10 mg/kg (highest dose tested) by non human primates. AT1412 demonstrated a half-life of 8.5 days, supporting 2–3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. First in Human clinical study is planned to start early 2021.Ethics ApprovalStudy protocols were approved by the Medical Ethical Committee of the Leiden University Medical Center (Leiden, Netherlands).ConsentBlood was obtained after written informed consent by the patient.

Traditionally, natural medicines have been administered as plant extracts, which are composed of a mixture of molecules. The individual molecular species in this mixture may or may not contribute to the overall medicinal effects and some may even oppose the beneficial activity of others. To better control therapeutic effects, studies that characterized specific molecules and describe their individual activity that have been performed over the past decades. These studies appear to underline that natural products are particularly effective as antioxidants and anti-inflammatory agents. In this systematic review we aimed to identify potent anti-inflammatory natural products and relate their efficacy to their chemical structure and physicochemical properties. To identify these compounds, we performed a comprehensive literature search to find those studies, in which a dose-response description and a positive control reference compound was used to benchmark the observed activity. Of the analyzed papers, 7% of initially selected studies met these requirements and were subjected to further analysis. This analysis revealed that most selected natural products indeed appeared to possess anti-inflammatory activities, in particular anti-oxidative properties. In addition, 14% of the natural products outperformed the remaining natural products in all tested assays and are attractive candidates as new anti-inflammatory agents.

Knob-in-hole antibodies can be utilized to introduce a single tag for chemo-enzymatic functionalization. By either introducing a single C-terminal sortase tag (sortase-tag expressed protein ligation) or tyrosine tag (G4Y), mono-functionalization of the monoclonal antibody trastuzumab was achieved rapidly and in high yields. This method was applied to selectively and efficiently introduce a single fluorescent tag, cytokine or single-chain variable fragment, as well as produce clean homo dimers of trastuzumab.