Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factorb-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the deubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS).In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.

Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.

A scalably manufacturable oral antibody technology that can interfere with gastrointestinal (GI) targets is needed. Contrary to the complex native secretory IgA, we achieve this using a single-gene encoded monomeric-IgA-like antibody, composed of camelid VHH fused to IgA Fc (mVHH-IgA). This can be produced in soybean seeds or secreted from Pichia pastoris yeast, freeze-or spray-dried, and when delivered in food prevents enterotoxigenic Escherichia coli (F4-ETEC) infection in piglets.

Yeast has advanced as an alternative for mammalian cell culture for the production of recombinant therapeutic glycoproteins. Engineered yeast strains not only allow to mimic the human N-glycosylation pathway but also specific types of human O-glycosylation. This is of great value for therapeutic protein production and indispensable to determine the structure-function relationships of glycans on recombinant proteins. However, as the technology matures, some limitations have come up that may hamper biomedical applications and must be considered to exploit the full potential of the unprecedented glycan homogeneity obtained on relevant biopharmaceuticals. In this special report, we focus on the recent developments in N- and O-glycosylation engineering in yeasts of industrial importance, to produce recombinant therapeutics with customized glycans.

Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.

SummaryVHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH-Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH-Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH-Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N-glycans were expected for KDEL-tagged VHH-Fcs, several VHH-Fcs with an intact KDEL-tag carried complex-type N-glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH-Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.