Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.

Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and ß2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

Functional characterization of vasopressin type 2 receptor substitutions (R137H/C/L) leading to nephrogenic diabetes insipidus and nephrogenic syndrome of inappropriate antidiuresis: implications for treatments.

Substitution of arginine-137 of the vasopressin type 2 receptor (V2R) for histidine (R137H-V2R) leads to nephrogenic diabetes insipidus (NDI), whereas substitution of the same residue to cysteine or leucine (R137C/L-V2R) causes the nephrogenic syndrome of inappropriate antidiuresis (NSIAD). These two diseases have opposite clinical outcomes. Still, the three mutant receptors were shown to share constitutive beta-arrestin recruitment and endocytosis, resistance to vasopressin-stimulated cAMP production and mitogen-activated protein kinase activation, and compromised cell surface targeting, raising questions about the contribution of these phenomenons to the diseases and their potential treatments. Blocking endocytosis exacerbated the elevated basal cAMP levels promoted by R137C/L-V2R but not the cAMP production elicited by R137H-V2R, demonstrating that substitution of Arg137 to Cys/Leu, but not His, leads to constitutive V2R-stimulated cAMP accumulation that most likely underlies NSIAD. The constitutively elevated endocytosis of R137C/L-V2R attenuates the signaling and most likely reduces the severity of NSIAD, whereas the elevated endocytosis of R137H-V2R probably contributes to NDI. The constitutive signaling of R137C/L-V2R was not inhibited by treatment with the V2R inverse agonist satavaptan (SR121463). In contrast, owing to its pharmacological chaperone property, SR121463 increased the R137C/L-V2R maturation and cell surface targeting, leading to a further increase in basal cAMP production, thus disqualifying it as a potential treatment for patients with R137C/L-V2R-induced NSIAD. However, vasopressin was found to promote beta-arrestin/AP-2-dependent internalization of R137H/C/L-V2R beyond their already elevated endocytosis levels, raising the possibility that vasopressin could have a therapeutic value for patients with R137C/L-V2R-induced NSIAD by reducing steady-state surface receptor levels, thus lowering basal cAMP production.

Dendritic cells dictate responses to PD-L1 blockade cancer immunotherapy.

PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non-small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell immunity.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies.

Purpose: Despite promising clinical activity, T-cell-engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell-binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785-97. ©2018 AACR See related commentary by Prakash and Diefenbach, p. 4631.

Engineered protein connectivity to actin mimics PDZ-dependent recycling of G protein-coupled receptors but not its regulation by Hrs.

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the beta2-adrenergic receptor (beta2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the delta-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.