Rational Design of a Bifunctional AND-Gate Ligand To Modulate Cell–Cell Interactions

Lee, Jungmin; Vernet, Andyna; Redfield, Katherine; Lu, Shulin; Ghiran, Ionita; Way, Jeffrey C.; Silver, Pamela A.

doi:10.1021/acssynbio.9b00273

Cited by 6 publications

(15 citation statements)

References 21 publications

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“…RBCs) and other cells bearing EPORs. The results of Lee et al (2020) also showed a correlation between stimulation of platelet production and stimulation of thrombosis, indicating that enhancement of platelet formation could be used as a surrogate marker for EPO-induced thrombosis in these studies. The mutations used in those studies affected the strong face of EPO and those fusion proteins are therefore expected to affect formation of EPOR homodimers and EPOR-CD131 heterodimers.…”

mentioning

confidence: 63%

“…Burrill et al (2016) demonstrated that a weakened form of EPO with a mutation in the strong face (R150A) that is also fused to an anti-GPA antibody element can specifically activate production of RBCs and not platelets. Lee et al (2020) demonstrated that such an anti-GPA/EPO(mutant) fusion protein can specifically activate RBC formation without stimulation of blood clotting, provided that the fusion protein cannot mediate adhesion of cells bearing GPA (e.g. RBCs) and other cells bearing EPORs.…”

mentioning

confidence: 99%

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Rational engineering of an erythropoietin fusion protein to treat hypoxia

Lee

Vernet²,

Gruber

et al. 2021

Preprint

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Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10−14 M, allowing an estimate of the number of receptor–ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.

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mentioning

confidence: 63%

mentioning

confidence: 99%

Rational engineering of an erythropoietin fusion protein to treat hypoxia

Lee

Vernet²,

Gruber

et al. 2021

Preprint

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“…All coding sequences were confirmed by Sanger sequencing. To allow for efficient secretion in mammalian cells, the Glycophorin A-specific IH4 nanobody was modified by a F80Y mutation and insertion of a threonine C-terminal of G117 as described previously 26 .…”

Section: Methodsmentioning

confidence: 99%

A rapid and affordable point of care test for antibodies against SARS-CoV-2 based on hemagglutination and artificial intelligence interpretation

Redecke

Tawaratsumida

Larragoite

et al. 2021

Sci Rep

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Diagnostic tests that detect antibodies (AB) against SARS-CoV-2 for evaluation of seroprevalence and guidance of health care measures are important tools for managing the COVID-19 pandemic. Current tests have certain limitations with regard to turnaround time, costs and availability, particularly in point-of-care (POC) settings. We established a hemagglutination-based AB test that is based on bi-specific proteins which contain a dromedary-derived antibody (nanobody) binding red blood cells (RBD) and a SARS-CoV-2-derived antigen, such as the receptor-binding domain of the Spike protein (Spike-RBD). While the nanobody mediates swift binding to RBC, the antigen moiety directs instantaneous, visually apparent hemagglutination in the presence of SARS-CoV-2-specific AB generated in COVID-19 patients or vaccinated individuals. Method comparison studies with assays cleared by emergency use authorization demonstrate high specificity and sensitivity. To further increase objectivity of test interpretation, we developed an image analysis tool based on digital image acquisition (via a cell phone) and a machine learning algorithm based on defined sample-training and -validation datasets. Preliminary data, including a small clinical study, provides proof of principle for test performance in a POC setting. Together, the data support the interpretation that this AB test format, which we refer to as ‘NanoSpot.ai’, is suitable for POC testing, can be manufactured at very low costs and, based on its generic mode of action, can likely be adapted to a variety of other pathogens.

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“…We want cell binding to be driven by binding to the nonsignaling targeting receptor. In practice, a minimum requirement is that either N T k on,T > N A k on,A or N T K eq,T > N A K eq,A (or preferably both), where N T is the number of targeting receptors per cell, N A is the number of activity receptors per cell, k on,T is the on rate for the targeting element to bind to its receptor, k on,A is the on rate for the activity element to bind to its receptor, K eq,T is the equilibrium binding 26 The targeting element is a nanobody that binds to glycophorin A, which is abundant on late red blood cell precursors; the K D for this interaction is 30 nM. 46 The linker is five amino acids in length, such that when fully extended into a β-strand, it is ∼2 Å long.…”

Section: A Cell Surfacementioning

confidence: 99%

Bioinspired Design of Artificial Signaling Systems

Way¹,

Burrill

Silver

2022

Biochemistry

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Natural systems use weak interactions and avidity effects to give biological systems high specificity and signal-to-noise ratios. Here we describe design principles for engineering fusion proteins that target therapeutic fusion proteins to membranebound signaling receptors by first binding to designer-chosen coreceptors on the same cell surface. The key design elements are separate protein modules, one that has no signaling activity and binds to a cell surface receptor with high affinity and a second that binds to a receptor with low or moderate affinity and carries out a desired signaling or inhibitory activity. These principles are inspired by natural cytokines such as CNTF, IL-2, and IL-4 that bind strongly to nonsignaling receptors and then signal through low-affinity receptors. Such designs take advantage of the fact that when a protein is anchored to a cell membrane, its local concentration is extremely high with respect to those of other membrane proteins, so a second-step, low-affinity binding event is favored. Protein engineers have used these principles to design treatments for cancer, anemia, hypoxia, and HIV infection.

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Rational Design of a Bifunctional AND-Gate Ligand To Modulate Cell–Cell Interactions

Cited by 6 publications

References 21 publications

Rational engineering of an erythropoietin fusion protein to treat hypoxia

Rational engineering of an erythropoietin fusion protein to treat hypoxia

A rapid and affordable point of care test for antibodies against SARS-CoV-2 based on hemagglutination and artificial intelligence interpretation

Bioinspired Design of Artificial Signaling Systems

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