A Novel Affinity Engineered Anti-CD47 Antibody With Improved Therapeutic Index That Preserves Erythrocytes and Normal Immune Cells.

Therapeutic blockade of the CD47/SIRPα axis by small molecules or monoclonal antibodies (mAbs) is a proven strategy to enhance macrophages-mediated anti-tumor activity. However, this strategy has been hampered by elevated on-target toxicities and rapid clearance due to the extensive CD47 expression on normal cells ("antigen sink") such as red blood cells (RBCs). To address these hurdles, we report on the development of STI-6643, an affinity-engineered fully human anti-CD47 IgG antibody with negligible binding to normal cells.

The Anti-ROR1 Monoclonal Antibody Zilovertamab Inhibits the Proliferation of Ovarian and Endometrial Cancer Cells.

The non-canonical Wnt signalling receptor ROR1 is aberrantly expressed in numerous cancers, including ovarian and endometrial cancer. We previously reported that silencing ROR1 could inhibit the proliferation and metastatic potential of ovarian and endometrial cancer cells in vitro. Zilovertamab is an ROR1-targeting humanised monoclonal antibody, with demonstrated safety and efficacy in clinical trials of several ROR1-related malignancies.

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial -acyl homoserine lactones.

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Here, we report the use of photoactive probes to identify MVP as a target of the -(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including A treatment of normal and cancer cells with C12 or other -acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death.

PSMA-targeted bispecific Fab conjugates that engage T cells.

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage.

Chemically generated IgG2 bispecific antibodies through disulfide bridging.

Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture.

Glycation Reactivity of a Quorum-Sensing Signaling Molecule.

Reported herein is that (4S)-4,5-dihydroxy-2,3-pentanedione (DPD) can undergo a previously undocumented non-enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidin S resulted in significant biochemical alterations. A protein-labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system.

Targeting Heat-Shock Protein 90 (HSP90) as a Complementary Strategy to Immune Checkpoint Blockade for Cancer Therapy.

The demonstration that immune checkpoint blockade can meaningfully improve outcomes for cancer patients has revolutionized the field of immuno-oncology. New biologic agents targeting specific checkpoints have shown remarkable durability in terms of patient response and, importantly, exhibit clinical activity across a range of human malignancies, including many that have traditionally proven refractory to other immunotherapies. In this rapidly evolving area, a key consideration relates to the identification of novel combinatorial strategies that exploit existing or investigational cancer therapies in order to optimize patient outcomes and the proportion of individuals able to derive benefit from this approach.

Functional role of asparaginyl endopeptidase ubiquitination by TRAF6 in tumor invasion and metastasis.

Background: Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive.

Methods: We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays.

A chemical biology approach to interrogate quorum-sensing regulated behaviors at the molecular and cellular level.

Small molecule probes have been used extensively to explore biologic systems and elucidate cellular signaling pathways. In this study, we use an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering unrecognized processes regulated by AI-2-based quorum-sensing (QS), a mechanism of bacterial intercellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S.

Quo vadis quorum quenching?

With the emergence of microbial pathogens increasingly resistant against commonly used antibiotics, new treatment strategies are desperately needed. Bacterial quorum sensing has attracted a lot of attention over the last decade as a potential new target for antimicrobial therapy. Interference with quorum sensing signaling, or quorum quenching, might offer new avenues to prevent and/or treat bacterial infections via inhibition of virulence factor expression and biofilm formation.

Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation.

In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported.

Toward implementation of quorum sensing autoinducers as biomarkers for infectious disease states.

The opportunistic bacterial pathogen Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis (CF) patients. Importantly, virulence factor expression and biofilm formation in P. aeruginosa is coordinated by quorum sensing (QS) and one of the key QS signaling molecules is 3-oxo-C12-HSL.

Immunomodulation and the quorum sensing molecule 3-oxo-C12-homoserine lactone: the importance of chemical scaffolding for probe development.

As a guide for chemical probe design, focused analogue synthetic studies were undertaken upon the lactone ring of 3-oxo-C(12)-homoserine lactone. We have concluded that hydrolytic instability of the heterocyclic ring is pivotal for its ability to modulate immune signaling and probe preparation was aligned with these findings.

Bacterial inhibition of inflammatory responses via TLR-independent mechanisms.

Identification of cellular processes modulated by microbial organisms that undermine and disarm mammalian host defences against bacterial invaders has been the focus of significant biomedical research. In this microreview we will illustrate the role of bacterial N-acyl homoserine lactones (AHL) as a strategy utilized by Gram-negative bacterial pathogens to enable colonization of the host through AHL-mediated inhibition of inflammation induced via innate immune receptor mechanisms. We will also highlight some of the signalling pathways in which the study of AHL-mediated effects on mammalian cells might lead to the discovery of global underlying principles linking inflammation and immunity to many chronic human diseases, including cancer and obesity.

C4-alkoxy-HPD: a potent class of synthetic modulators surpassing nature in AI-2 quorum sensing.

Bacteria have developed cell-to-cell communication mechanisms, termed quorum sensing (QS), that regulate bacterial gene expression in a cell population-dependent manner. Autoinducer-2 (AI-2), a class of QS signaling molecules derived from (4S)-4,5-dihydroxy-2,3-pentanedione (DPD), has been identified in both Gram-negative and Gram-positive bacteria. Despite considerable interest in the AI-2 QS system, the biomolecular communication used by distinct bacterial species still remains shrouded.

The use of small molecule probes to study spatially separated stimulus-induced signaling pathways.

Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein complexes at the cytoplasmic domains of membrane-associated receptors in a stimulus-specific manner. Here, using the paradigm of cellular activation through cytokine and innate immune receptors, we demonstrate the proof-of-principle application of small molecule probes for the dissection of receptor-proximal signaling processes, such as activation of the transcription factor NF-κB and the protein kinase p38.

Exploitation of host signaling pathways by microbial quorum sensing signals.

Environmental and commensal microbes that live within, on and around us have an enormous impact on human health. Recent progress in studies of prokaryotic interplay as well as host-bacteria interactions suggests that secreted microbial products, including quorum sensing signals (QSS), are important mediators of these intrakingdom and interkingdom relations. Reports have assigned QSS diverse and sometimes seemingly contradictory effects on mammalian cell physiology ranging from either blunting of the immune response or exerting pro-inflammatory activities to inducing cellular stress pathways and ultimately apoptosis.

A multivalent probe for AI-2 quorum-sensing receptors.

Multivalency is a common principle in the recognition of cellular receptors, and multivalent agonists and antagonists have played a major role in understanding mammalian cell receptor biology. The study of bacterial cell receptors using similar approaches, however, has lagged behind. Herein we describe our efforts toward the development of a dendrimer-based multivalent probe for studying AI-2 quorum-sensing receptors.

Structural basis for ligand recognition and discrimination of a quorum-quenching antibody.

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S.

Synthesis of 'clickable' acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation.

Alkynyl- and azido-tagged 3-oxo-C(12)-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C(12)-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative.