A Robust Platform for the Molecular Design of Potent, Protease-Stable, Long-Acting GIP Analogues.

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid peptide hormone that regulates postprandial glucose levels. GIP binds to its cognate receptor, GIPR, and mediates metabolic physiology by improved insulin sensitivity, ß-cell proliferation, increased energy consumption, and stimulated glucagon secretion. Dipeptidyl peptidase-4 (DPP4) catalyzes the rapid inactivation of GIP within 6 min in vivo. Here, we report a molecular platform for the design of GIP analogues that are refractory to DPP4 action and exhibit differential activation of the receptor, thus offering potentially hundreds of GIP-based compounds to fine-tune pharmacology. The lead compound from our studies, which harbored a combination of N-terminal alkylation and side-chain lipidation, was equipotent and retained full efficacy at GIPR as the native peptide, while being completely refractory toward DPP4, and was resistant to trypsin. The GIP analogue identified from these studies was further evaluated in vivo and is one of the longest-acting GIPR agonists to date.

Structural insights reveal interplay between LAG-3 homodimerization, ligand binding, and function.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases.

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation Resolution.

Regulatory T (Treg) cell responses and apoptotic cell clearance (efferocytosis) represent critical arms of the inflammation resolution response. We sought to determine whether these processes might be linked through Treg-cell-mediated enhancement of efferocytosis. In zymosan-induced peritonitis and lipopolysaccharide-induced lung injury, Treg cells increased early in resolution, and Treg cell depletion decreased efferocytosis. In advanced atherosclerosis, where defective efferocytosis drives disease progression, Treg cell expansion improved efferocytosis. Mechanistic studies revealed the following sequence: (1) Treg cells secreted interleukin-13 (IL-13), which stimulated IL-10 production in macrophages; (2) autocrine-paracrine signaling by IL-10 induced Vav1 in macrophages; and (3) Vav1 activated Rac1 to promote apoptotic cell engulfment. In summary, Treg cells promote macrophage efferocytosis during inflammation resolution via a transcellular signaling pathway that enhances apoptotic cell internalization. These findings suggest an expanded role of Treg cells in inflammation resolution and provide a mechanistic basis for Treg-cell-enhancement strategies for non-resolving inflammatory diseases.