The role of the quality assessment in the determination of overall biosimilarity: A simulated case study exercise

A determination of biosimilarity is based on a thorough characterization and comparison of the quality profiles of a similar biotherapeutic product and its reference biotherapeutic product. Although the general principles on the role of the quality assessment in a biosimilar evaluation are widely understood and agreed, detailed discussions have not been published yet. We try to bridge this gap by presenting a case study exercise based on fictional but realistic data to highlight key principles of an evaluation to determine the degree of similarity at the quality level. The case study comprises three examples for biosimilar monoclonal antibody candidates. The first describes a highly similar quality profile whereas the second and third show greater differences to the reference biotherapeutic product. The aim is to discuss whether the presented examples can be qualified as similar and which additional studies may be helpful in enabling a final assessment. The case study exercise was performed at the WHO implementation workshop for the WHO guidelines on quality assessment of similar biotherapeutic products held in Xiamen, China, in May 2012. The goal was to illustrate the interpretation of the comparative results at the quality level, the role of the quality assessment in the entire biosimilarity exercise and its influence on the clinical evaluation. This paper reflects the outcome of the exercise and discussion from Xiamen.     

Death receptor agonist therapies for cancer,which is the right TRAIL?

The activation of cell-surface death receptors represents an attractive therapeutic strategy to promote apoptosis of tumor cells. Several investigational therapeutics that target this extrinsic pathway, including recombinant human Apo2L/TRAIL and monoclonal agonist antibodies directed against death receptors-4 (DR4) or -5 (DR5), have been evaluated in the clinic. Although Phase 1/1b studies provided encouraging preliminary results, findings from randomized Phase 2 studies failed to demonstrate significant clinical benefit. This has raised multiple questions as to why pre-clinical data were not predictive of clinical response. Results from clinical studies and insight into why current agents have failed to yield robust responses are discussed. In addition, new strategies for the development of next generation death receptor agonists are reviewed.     

Evaluation of the BH3-only Protein Puma as a Direct Bak Activator

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as “direct activators” that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (K_D = 26 ± 5 nm) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.     

A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death

Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G₁/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides.     

SLC17A9 Protein Functions as a Lysosomal ATP Transporter and Regulates Cell Viability

Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability.     

Release of Interleukin-1α or Interleukin-1β Depends on Mechanism of Cell Death

The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1α and IL-1β, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1β. Conversely, necroptosis caused the processing and release of IL-1α, and this was independent of IL-1β processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.     

Persephone/Sp?tzle Pathogen Sensors Mediate the Activation of Toll Receptor Signaling in Response to Endogenous Danger Signals in Apoptosis-deficient Drosophila

Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially lead to necrosis, resulting in the release of endogenous immunogenic molecules such as damage-associated molecular patterns (DAMPs) and a noninfectious inflammatory response. However, the details about how danger signals from apoptosis-deficient cells are detected and translated to an immune response are largely unknown. In this study, we found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous Toll ligand Spätzle (Spz). We speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. We demonstrated that Toll signaling activation required the action of Persephone, a CLIP domain serine protease that usually reacts to microbial proteolytic activities. Our findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic responses in apoptosis-deficient Drosophila mutants.     

Decreasing Mitochondrial Fission Prevents Cholestatic Liver Injury

Mitochondria frequently change their shape through fission and fusion in response to physiological stimuli as well as pathological insults. Disrupted mitochondrial morphology has been observed in cholestatic liver disease. However, the role of mitochondrial shape change in cholestasis is not defined. In this study, using in vitro and in vivo models of bile acid-induced liver injury, we investigated the contribution of mitochondrial morphology to the pathogenesis of cholestatic liver disease. We found that the toxic bile salt glycochenodeoxycholate (GCDC) rapidly fragmented mitochondria, both in primary mouse hepatocytes and in the bile transporter-expressing hepatic cell line McNtcp.24, leading to a significant increase in cell death. GCDC-induced mitochondrial fragmentation was associated with an increase in reactive oxygen species (ROS) levels. We found that preventing mitochondrial fragmentation in GCDC by inhibiting mitochondrial fission significantly decreased not only ROS levels but also cell death. We also induced cholestasis in mouse livers via common bile duct ligation. Using a transgenic mouse model inducibly expressing a dominant-negative fission mutant specifically in the liver, we demonstrated that decreasing mitochondrial fission substantially diminished ROS levels, liver injury, and fibrosis under cholestatic conditions. Taken together, our results provide new evidence that controlling mitochondrial fission is an effective strategy for ameliorating cholestatic liver injury.     

Bronchopulmonary Infection of Lophomonas blattarum: A Case and Literature Review

Human infections with Lophomonas blattarum are rare. However, the majority of the infections occurred in China, 94.4% (136 cases) of all cases in the world. This infection is difficult to differentiate from other pulmonary infections with similar symptoms. Here we reported a case of L. blattarum infection confirmed by bronchoalveolar lavage fluid smear on the microscopic observations. The patient was a 21-year-old female college student. The previous case which occurred in Chongqing was 20 years ago. We briefly reviewed on this infection reported in the world during the recent 20 years. The epidemiological characteristics, possible diagnostic basis, and treatment of this disease is discussed in order to provide a better understanding of recognition, diagnosis, and treatment of L. blattarum infection.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.