Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

In situ sprayed bioresponsive immunotherapeutic gel for post-surgical cancer treatment

Cancer recurrence after surgical resection remains a significant cause of treatment failure. Here, we have developed an in situ formed immunotherapeutic bioresponsive gel that controls both local tumour recurrence after surgery and development of distant tumours. Briefly, calcium carbonate nanoparticles pre-loaded with the anti-CD47 antibody are encapsulated in the fibrin gel and scavenge H+ in the surgical wound, allowing polarization of tumour-associated macrophages to the M1-like phenotype. The released anti-CD47 antibody blocks the ‘don’t eat me’ signal in cancer cells, thereby increasing phagocytosis of cancer cells by macrophages. Macrophages can promote effective antigen presentation and initiate T cell mediated immune responses that control tumour growth. Our findings indicate that the immunotherapeutic fibrin gel ‘awakens’ the host innate and adaptive immune systems to inhibit both local tumour recurrence post surgery and potential metastatic spread

Pharmacokinetics and delivery of proteins modified with FcRn binding ligands

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve protein circulation and delivery is to fuse the Fc-domain of immunoglobulin G (IgG) to therapeutic proteins so that the resulting fusion proteins interact with the neonatal Fc receptor (FcRn). Although successful, Fc-fusion proteins significantly increase molecular weight thereby restricting tissue penetration, decrease protein function, and are limited to mammalian expression systems. As an alternative to grafting the high molecular weight Fc-domain to therapeutic proteins, we have modified their N- and/or C-terminus with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results from Mezo and coworkers [Mezo et al. (2008) PNAS 105:2337-42] who identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn binding peptide (FcBP) allows for expression of FcBP fusion proteins in E. coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of hIgG1. The FcBP fusion proteins are internalized, recycled and transcytosed across cell monolayers that express FcRn. Although FcBP fusion proteins mimic the human IgG1 interaction with human FcRn in vitro, the half-life of FcBP fusion proteins in wild type C57BL/6J and human FcRn transgenic mice is independent of FcRn binding, whereas the half-life of human and mouse IgG1 correlate with their species matched FcRn-binding affinity. These results promoted a detailed investigation into potential factors that may contribute to the lack of correlation between the in vitro and in vivo results including: serum stability, serum competition, renal clearance, and the function of the hybrid human-mouse FcRn/B2m receptor; however, no single variable explains the FcRn-independent half-life of FcBP fusion proteins. We speculate that an additional component(s), in collaboration with FcRn, regulates IgG homeostasis in vivo and is not replicated by FcBP fusion. If such a novel FcRn regulatory component exists, it would have a significant impact on FcRn biology and provide new opportunities to engineer IgG or alternative FcRn-targeted molecules for enhanced serum persistence

Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (λDE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni(2+) affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli

Fusion of a Short Peptide that Binds Immunoglobulin G to a Recombinant Protein Substantially Increases Its Plasma Half-Life in Mice

<div><p>We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) <i>Science</i> 287∶1279–1283]. IgGBP fusion increases the <i>in vivo</i> half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the <i>in vitro</i> and <i>in vivo</i> properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in <i>Escherichia coli</i> and bind specifically to human IgG with an affinity of ∼40 nM and ∼20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn <i>in vitro</i>. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.</p></div

IgGBP fusion extends mKate half-life in hFcRn Tg mice when co-administered as a 1∶1 mol mixture with hIgG1 without altering hIgG1 clearance.

<p>(<b>a</b>) Schematic of the co-administration scheme. In this experiment, human FcRn Tg mice were not pre-dosed with exogenous hIgG1. Instead mKate-IgGBP and hIgG1 were pre-mixed in a 1∶1 mol ratio and co-injected via the tail vein. (<b>b</b>) Clearance of mKate-IgGBP in hFcRn Tg mice dosed alone (blue triangles) or co-dosed at a 1∶1 mol mixture with hIgG1 (yellow triangles). The % mKate-IgGBP remaining was calculated by normalizing the fluorescent emission at all time points to the maximum value observed in the first bleed 5 min after protein injection. (<b>c</b>) Clearance of labeled human IgG1 in hFcRn Tg mice dosed as a single agent via the tail vein (blue triangles) compared to the clearance of labeled hIgG1 co-administered as a 1∶1 mol mixture with mKate-IgGBP was measured to determine if bound mKate-IgGBP alters the eliminate profile of hIgG1 (red squares). The % hIgG1 remaining was calculated by normalizing the fluorescent emission at all time points to the maximum value observed in the first bleed 5 min after protein injection. Dashed lines in each panel represent the data fit to a 2-compartment PK model in Prism and the β-phase half-life shown in the figure was calculated as described in the Methods section. The data shown in each panel are the mean (n = 3 bleeds per time point) and error bars indicate s.d.</p