A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Bioassay Development for Bispecific Antibodies-Challenges and Opportunities.

Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules.

How ATP-Competitive Inhibitors Allosterically Modulate Tyrosine Kinases That Contain a Src-like Regulatory Architecture.

Small molecule kinase inhibitors that stabilize distinct ATP binding site conformations can differentially modulate the global conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects generalize to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src's regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix αC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src's helix αC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Our study highlights that an ATP-competitive inhibitor's interactions with helix αC can have a major influence on the global conformation of some tyrosine kinases.

Development of a bioassay to detect T-cell-activating impurities for T-cell-dependent bispecific antibodies.

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit a patient's T cells to kill cancer cells. There are increasing numbers of TBDs in clinical trials, demonstrating their widely recognized therapeutic potential. Due to the fact that TDBs engage and activate T cells via an anti-CD3 (aCD3) arm, aCD3 homodimer (aCD3 HD) and high-molecular-weight species (HMWS) are product-related impurities that pose a potential safety risk by triggering off-target T-cell activation through bivalent engagement and dimerization of T-cell receptors (TCRs). To monitor and control the level of unspecific T-cell activation, we developed a sensitive and quantitative T-cell-activation assay, which can detect aCD3 HD in TDB drug product by exploiting its ability to activate T cells in the absence of target cells. This assay provides in-vivo-relevant off-target T-cell-activation readout. Furthermore, we have demonstrated that this assay can serve as a platform assay for detecting T-cell-activating impurities across a broad spectrum of aCD3 bispecific molecules. It therefore has the potential to significantly benefit many T-cell-recruiting bispecific programs.

Allosteric Modulation of Src Family Kinases with ATP-Competitive Inhibitors.

The Src family kinases (SFKs) are an important family of tyrosine kinases that are allosterically regulated by their SH2 and SH3 domains. Engagement of SFK SH2 and SH3 domains with their intramolecular ligands leads to reduced kinase activity by stabilizing an inactive ATP-binding site conformation. Disruption of these intramolecular interactions stabilizes a more active ATP-binding site conformation and restores SFK activity. Interestingly, this allosteric relationship is bidirectional in that ATP-competitive ligands that stabilize distinct active site conformations can divergently modulate the abilities of the regulatory SH2 and SH3 domains to participate in intermolecular interactions. Here, we describe a series of assays that profile the bidirectional relationship between the ATP-binding sites and regulatory domains of SFKs. These methods can be used to discover ATP-competitive inhibitors that are selective for distinct ATP-binding site conformations of SFKs and for characterizing the effects that ATP-competitive inhibitors of SFKs have on domains that are distal to their site of interaction.

Targeting ABL-IRE1α Signaling Spares ER-Stressed Pancreatic ß Cells to Reverse Autoimmune Diabetes.

In cells experiencing unrelieved endoplasmic reticulum (ER) stress, the ER transmembrane kinase/endoribonuclease (RNase)-IRE1α-endonucleolytically degrades ER-localized mRNAs to promote apoptosis. Here we find that the ABL family of tyrosine kinases rheostatically enhances IRE1α's enzymatic activities, thereby potentiating ER stress-induced apoptosis. During ER stress, cytosolic ABL kinases localize to the ER membrane, where they bind, scaffold, and hyperactivate IRE1α's RNase. Imatinib-an anti-cancer tyrosine kinase inhibitor-antagonizes the ABL-IRE1α interaction, blunts IRE1α RNase hyperactivity, reduces pancreatic ß cell apoptosis, and reverses type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. A mono-selective kinase inhibitor that allosterically attenuates IRE1α's RNase-KIRA8-also efficaciously reverses established diabetes in NOD mice by sparing ß cells and preserving their physiological function. Our data support a model wherein ER-stressed ß cells contribute to their own demise during T1D pathogenesis and implicate the ABL-IRE1α axis as a drug target for the treatment of an autoimmune disease.