Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

The PD-1/PD-L1 pathway is a key immune checkpoint that regulates T cell activation. There is strong rationale to develop PD-1 agonists as therapeutics against autoimmunity, but progress in this area has been limited. Here, we generated T cell receptor (TCR) targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the colocalization of PD-1 with the TCR complex at the target cell-T cell interface. PD-1 agonist ImmTAAI molecules specifically bound to target cells and were highly effective in activating the PD-1 receptor on interacting T cells to achieve immune suppression. Potent PD-1 antibody ImmTAAI molecules closely mimicked the mechanism of action of endogenously expressed PD-L1 in their localization to the target cell-T cell interface, inhibition of proximal TCR signaling events, and suppression of T cell function. At picomolar concentrations, these bispecifics suppressed cytokine production and inhibited CD8+ T cell-mediated cytotoxicity in vitro. Crucially, in soluble form, the PD-1 ImmTAAI molecules were inactive and, hence, could avoid systemic immunosuppression. This study outlines a promising new route to generate more effective, potent, tissue-targeted PD-1 agonists that can inhibit T cell function locally with the potential to treat autoimmune and chronic inflammatory diseases of high unmet need.

Design and optimization of selective protein kinase C θ (PKCθ) inhibitors for the treatment of autoimmune diseases.

Protein kinase C θ (PKCθ) has a central role in T cell activation and survival; however, the dependency of T cell responses to the inhibition of this enzyme appears to be dictated by the nature of the antigen and by the inflammatory environment. Studies in PKCθ-deficient mice have demonstrated that while antiviral responses are PKCθ-independent, T cell responses associated with autoimmune diseases are PKCθ-dependent. Thus, potent and selective inhibition of PKCθ is expected to block autoimmune T cell responses without compromising antiviral immunity. Herein, we describe the development of potent and selective PKCθ inhibitors, which show exceptional potency in cells and in vivo. By use of a structure based rational design approach, a 1000-fold improvement in potency and 76-fold improvement in selectivity over closely related PKC isoforms such as PKCδ were obtained from the initial HTS hit, together with a big improvement in lipophilic efficiency (LiPE).

Discovery and structure-activity relationship of 3-aminopyrid-2-ones as potent and selective interleukin-2 inducible T-cell kinase (Itk) inhibitors.

Interleukin-2 inducible T-cell kinase (Itk) plays a role in T-cell functions, and its inhibition potentially represents an attractive intervention point to treat autoimmune and allergic diseases. Herein we describe the discovery of a series of potent and selective novel inhibitors of Itk. These inhibitors were identified by structure-based design, starting from a fragment generated de novo, the 3-aminopyrid-2-one motif. Functionalization of the 3-amino group enabled rapid enhancement of the inhibitory activity against Itk, while introduction of a substituted heteroaromatic ring in position 5 of the pyridone fragment was key to achieving optimal selectivity over related kinases. A careful analysis of the hydration patterns in the kinase active site was necessary to fully explain the observed selectivity profile. The best molecule prepared in this optimization campaign, 7v, inhibits Itk with a K(i) of 7 nM and has a good selectivity profile across kinases.

Optimal chemotactic responses of leukemic T cells to stromal cell-derived factor-1 requires the activation of both class IA and IB phosphoinositide 3-kinases.

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show that expression of either kinase-dead PI3Kgamma (KD-PI3Kgamma) or Deltap85 (a construct unable to bind class I(A) p110alpha, -beta, or -delta) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kgamma markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40-60% inhibition. Interestingly, the expression of Deltap85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kgamma mutant, typically in the range of 20-40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.

Development and characterisation of tetracycline-regulated phosphoinositide 3-kinase mutants: assessing the role of multiple phosphoinositide 3-kinases in chemokine signaling.

A combination of pharmacological, biochemical, molecular and genetic evidence supports a key role for phosphoinositide 3-kinase (PI3K) and its associated signalling cascade in cell migration in response to members of the chemokine family. PI3Ks can be divided into three main classes on the basis of their in vitro lipid substrate specificity, structure and likely mode of regulation. The prototypical class I PI3Ks are heterodimers consisting of the class I(A) 85-kDa regulatory/adaptor subunit and a catalytic 110-kDa subunit and the class I(B) PI3K (PI3Kgamma), which is stimulated by G protein betagamma subunits. Whilst genetic evidence supports a key role for PI3Kgamma in mediating chemotactic responses, it is clear that other PI3K isoforms can be activated by chemokines and can potentially contribute to the chemotactic responses to chemokines. In order to get a more accurate picture of the precise role of individual PI3Ks in functional responses to chemokines, we report development of tetracycline-inducible dominant-negative constructs of the class I(A) and class I(B) PI3Ks and their expression in the leukemic T cell line Jurkat. SDF-1/CXCR4-mediated chemotaxis of Jurkat cells is strongly, but incompletely abrogated (e.g. approximately 60-70%) in clones expressing the dominant-negative PI3Kgamma construct. Interestingly, Jurkat cells expressing a dominant-negative mutant of class I(A) PI3K also exhibited marked abrogation of chemotactic responses to SDF-1, albeit to lesser extent (e.g. approximately 30-40% inhibition) than observed with the class I(B) mutant. These data suggests that both class I(A) and class I(B) isoforms can contribute to chemotactic responses, and both are required for optimal migratory responses to SDF-1. Furthermore, neither isoform alone is able to support optimal migration in the absence of the other. This may reflect an important interplay between the two different forms of PI3K that has yet to be fully elucidated. The use of inducible expression systems such as that described here will be an important approach in assessing the role of not only individual PI3Ks, but also their downstream effector proteins, in supporting actin polymerisation and cytoskeletal rearrangements as well as chemotaxis and adhesion molecule up-regulation.

Chemokine signalling: pivoting around multiple phosphoinositide 3-kinases.

The role of chemokines in mediating directional cell migration is well established, but more recently it has become evident that chemokines are able to couple to distinct signalling pathways that are involved in not only chemotaxis, but also cell growth and transcriptional activation. The signalling pathway controlled by the phosphoinositide 3-kinase (PI3K) family of lipid kinases has been the focus of much attention with respect to their role in chemokine-mediated functional responses. Indeed, there now exists convincing biochemical, pharmacological and genetic evidence that both CC and CXC chemokines stimulate PI3K-dependent chemotaxis of inflammatory cells such as eosinophils, macrophages, neutrophils and T lymphocytes. This review considers the role of individual PI3Ks (e.g. the p85/p110 heterodimer, PI3Kgamma and PI3KC2alpha) as well their downstream effector targets in mediating chemokine-stimulated cell migration.