Structure-guided optimization of light-activated chimeric G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of GPCR function is the specific coupling of activated receptors to selected downstream signaling pathways. The ability to tune this coupling would permit the development of receptors with new capabilities. GPCRs and G-proteins have been recently resolved structurally at high resolution, but this information was in only very few cases harnessed for a rational engineering of these protein complexes. Here, we demonstrate the structure-guided optimization of coupling in chimeric light-activated GPCRs (OptoXRs). Our hypothesis was that the incorporation of structural GPCR-Gα contacts will lead to improved receptor activity. We first evaluated structure-based alignments as complements to existing sequence-based methods for generation of chimeric receptors. We then show in a prototypical light-activated β2AR that inclusion of α-helical residues forming structural contacts to Gα resulted in receptors with 7- to 20-fold increased function compared to other design strategies. In turn, elimination of GPCR-Gα contacts diminished function. Finally, the efficient receptor design served as a platform for the optimization of a further light-activated receptor and spectral tuning of the photoreceptor core domain. Our work exemplifies how increased OptoXR potency and new functionalities can be achieved through structure-based design towards targeted inputs into cells and cellular networks.

CAR T Cells: Cancer Cell Surface Receptors Are the Target for Cancer Therapy

Immunotherapy has become a prominent strategy for the treatment of cancer. A method that improves the immune system's ability to attack a tumor (Enhances antigen binding). Targeted killing of malignant cells by adoptive transfer of chimeric antigen receptor (CAR) T cells is a promising immunotherapy technique in the treatment of cancers. For this purpose, the patient's immune cells, with genetic engineering aid, are loaded with chimeric receptors that have particular antigen binding and activate cytotoxic T lymphocytes. That increases the effectiveness of immune cells and destroying cancer cells. This review discusses the basic structure and function of CAR-T cells and how antigenic targets are identified to treat different cancers and address the disadvantages of this treatment for cancer.

The American lobster genome reveals insights on longevity, neural, and immune adaptations

The American lobster, Homarus americanus, is integral to marine ecosystems and supports an important commercial fishery. This iconic species also serves as a valuable model for deciphering neural networks controlling rhythmic motor patterns and olfaction. Here, we report a high-quality draft assembly of the H. americanus genome with 25,284 predicted gene models. Analysis of the neural gene complement revealed extraordinary development of the chemosensory machinery, including a profound diversification of ligand-gated ion channels and secretory molecules. The discovery of a novel class of chimeric receptors coupling pattern recognition and neurotransmitter binding suggests a deep integration between the neural and immune systems. A robust repertoire of genes involved in innate immunity, genome stability, cell survival, chemical defense, and cuticle formation represents a diversity of defense mechanisms essential to thrive in the benthic marine environment. Together, these unique evolutionary adaptations contribute to the longevity and ecological success of this long-lived benthic predator.

210 Regulation of TIM-3 by phosphatidylserine

BackgroundImmune checkpoint blockade has proven effective in targeting exhausted T-cells to reactivate the immune system against cancer. However, the majority of patients fail to respond to currently available therapies, which primarily target PD-1. Thus, a key challenge for checkpoint blockade therapy is to identify and understand new therapeutic targets. Another immune checkpoint receptor is TIM-3, which – like PD-1 – is expressed on exhausted T-cells in the tumor microenvironment.1, 2 TIM-3 belongs to a family of phosphatidylserine (PS) receptors, including TIM-1 and TIM-4, which have well-documented roles in the engulfment of apoptotic cells by phagocytes.3 However, the role of PS in regulating TIM-3 function is less clear. We therefore investigated how TIM-3 modulates T-cell signaling and how PS influences TIM-3 activity, with the ultimate goal of improving the translation of candidate TIM-3 therapies to the clinic.MethodsSurface plasmon resonance (SPR) was used to quantify the interaction between human TIM-3 and PS. A Jurkat T-cell model was used to investigate the role of TIM-3 in T-cell receptor (TCR) signaling and to determine the role of PS in regulating TIM-3 function.ResultsTIM-3 bound PS-containing membranes with low micromolar affinity in vitro. In the Jurkat cell model system, high – but not low – surface levels of TIM-3 promoted T-cell signaling, suggesting a threshold of receptor expression needed to modulate T-cell signaling, similar to what has recently been reported for PD-1.4 However, chimeric receptors that maintained the TIM-3 cytoplasmic tail but were unable to bind PS failed to enhance T-cell signaling like the full-length TIM-3 receptor. Cells expressing mutant TIM-3, which displayed reduced PS binding as quantified by SPR, also displayed reduced T-cell signaling compared to cells expressing wild-type TIM-3. Importantly, treatment of TIM-3-expressing cells with a functional TIM-3 antibody that blocks PS binding also reduced T-cell signaling compared with untreated TIM-3-expressing cells.ConclusionsOur results support a role for PS as a ligand capable of modulating TIM-3 activity. Using chimeric receptors, TIM-3 mutants, changes in receptor expression, and a functional TIM-3 antibody, we show that preventing the interaction between TIM-3 and PS blocks TIM-3 activity. These data suggest that blocking the PS-TIM-3 interaction is a key mechanism for functional antibodies targeting TIM-3. Ultimately, this work supports the development and use of clinical antibodies that block the interaction of TIM-3 with PS and provides new mechanistic insight into how TIM-3 modulates TCR signaling.AcknowledgementsThis work was supported by the PhRMA Foundation Pre-Doctoral Fellowship in Pharmacology/Toxicology.ReferencesFourcade J, Sun Z, Benallaoua M, et al. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J Exp Med. 2010;207(10):2175–2186.Zhou Q, Munger ME, Veenstra RG, et al. Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood. 2011;117(17):4501–4510.Kobayashi N, Karisola P, Peña-Cruz V, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity. 2007;27(6):927–940.Hui E, Cheung J, Zhu J, et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017;355(6332):1428–1433.

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

ABSTRACTCancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fc gamma receptors or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. Yet how these competing signals are integrated is poorly understood mechanistically, raising questions about how to effectively titrate immune responses. Here we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand on targets over a broad range of absolute molecular densities. Using endogenous as well as chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering this ratio reduces FcγR phosphorylation due to inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα in vitro, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.

NTRK Fusions in Central Nervous System Tumors: A Rare, but Worthy Target

The neurotrophic tropomyosin receptor kinase (NTRK) genes (NTRK1, NTRK2, and NTRK3) code for three transmembrane high-affinity tyrosine-kinase receptors for nerve growth factors (TRK-A, TRK-B, and TRK-C) which are mainly involved in nervous system development. Loss of function alterations in these genes can lead to nervous system development problems; conversely, activating alterations harbor oncogenic potential, promoting cell proliferation/survival and tumorigenesis. Chromosomal rearrangements are the most clinically relevant alterations of pathological NTRK activation, leading to constitutionally active chimeric receptors. NTRK fusions have been detected with extremely variable frequencies in many pediatric and adult cancer types, including central nervous system (CNS) tumors. These alterations can be detected by different laboratory assays (e.g., immunohistochemistry, FISH, sequencing), but each of these approaches has specific advantages and limitations which must be taken into account for an appropriate use in diagnostics or research. Moreover, therapeutic targeting of this molecular marker recently showed extreme efficacy. Considering the overall lack of effective treatments for brain neoplasms, it is expected that detection of NTRK fusions will soon become a mainstay in the diagnostic assessment of CNS tumors, and thus in-depth knowledge regarding this topic is warranted.