Market and patient access to new oncology products in Europe: a current, multidisciplinary perspective.

To air challenging issues related to patient and market access to new anticancer agents, the Biotherapy Development Association--an international group focused on developing targeted cancer therapies using biological agents--convened a meeting on 29 November 2007 in Brussels, Belgium. The meeting provided a forum for representatives of pharmaceutical companies and academia to interact with European regulatory and postregulatory agencies. The goal was to increase all parties' understanding of their counterparts' roles in the development, licensure, and appraisal of new agents for cancer treatment, events guided by an understanding that cancer patients should have rapid and equitable access to life-prolonging treatments. Among the outcomes of the meeting were a greater understanding of the barriers facing drug developers in an evolving postregulatory world, clarity about what regulatory and postregulatory bodies expect to see in dossiers of new anticancer agents as they contemplate licensure and reimbursement, and several sets of recommendations to optimize patients' access to innovative, safe, effective, and fairly priced cancer treatments.

New perspectives on the design of cytokines and growth factors.

A combination of molecular modelling, conventional epitope scanning and combinatorial techniques, such as phage display and DNA shuffling, has greatly improved our understanding of ligand-receptor interactions. It has therefore been possible to develop powerful cytokine-growth factor antagonists and new designer cytokines, with altered receptor specificities or with greatly enhanced biological activity. Recently, small circular peptides that mimic or block the effects of natural cytokines and growth factors have been developed; such small peptides are likely to open new avenues in therapeutics.

Weekly oxaliplatin, high-dose infusional 5-fluorouracil and folinic acid as palliative third-line therapy of advanced colorectal carcinoma.

The efficacy of oxaliplatin combined with high-dose 5-fluorouracil (5-FU) and folinic acid (FA) as an outpatient salvage treatment for patients with metastasized colorectal cancer was retrospectively analyzed in one center. Tumor progression had occurred for the majority of patients during two regimens (n = 11) otherwise during one (n = 1) regimen of prior 5-FU-based chemotherapy, which had been applied in a standardized sequential fashion. As third-line therapy oxaliplatin was infused intravenously over 2 h at a dose of 60 mg/m2 prior to a 2-h infusion of FA (500 mg/m2). 5-FU (2,600 mg/m2) was subsequently given over 24 h. A favorable response was observed in 9/12 (75%) of the heavily pretreated patients, including partial remissions in 3/12, minor responses in 2/12 and stable disease in 4/12 patients. The median progression free time was 23 weeks (interquartile range i. r. 0-28) for all patients, the median survival time from start of third-line therapy 55 weeks (i. r. 40-86). The median survival time from the beginning of first-line palliative chemotherapy was 34 months (i. r. 25-45 months). The highest toxicity was WHO grade III and was observed in six patients: Nausea (2), diarrhea (3), vomiting (2) and peripheral neuropathy (1). The quality of life was not adversely affected by the oxaliplatin/5-FU/FA-regimen as assessed by the EORTC QLQ-C30 questionnaire. Thus, the results show the efficiency and low toxicity of oxaliplatin/high-dose 5-FU/FA as palliative third-line therapy of patients with metastasized colorectal cancer and emphasize that sequential palliative chemotherapy may lead to extended survival of these patients.

IL-6 type cytokine receptor complexes: hexamer, tetramer or both?

The typical protein fold of most cytokines is a bundle of four antiparallel helices. This 'four-helical bundle fold' seems to be unique to cytokines and has not been detected in other proteins. Cytokine receptors, however, can be classified as a subfamily of the immunoglobulin superfamily. Cytokines using the same receptor subunits are grouped into cytokine families. The interleukin-6 (IL-6) type cytokine family comprises six members. IL-6 type cytokines may interact with three receptor subunits instead of the usual two subunits. A tetramer would be the simplest model to describe such a receptor complex, but present orthodoxy describes the active complexes of IL-6 and ciliary neurotrophic factor (CNTF) as hexamers. Here, we summarize the structural and biochemical information on IL-6 type cytokines and discuss interactions between cytokine and individual receptor subunits at alternative positions. Contradictory results regarding the stoichiometry and assembly of signaling receptor complexes are rationalized by a new, unique model. The model stipulates that a ligand-induced transition from an active tetrameric to an inactive hexameric complex serves as a molecular switch that turns off cytokine signals in the presence of supraoptimal cytokine concentrations.

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6.

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.

Immunoadhesins of interleukin-6 and the IL-6/soluble IL-6R fusion protein hyper-IL-6.

Signal transduction in response to interleukin-6 (IL-6) results from homodimerization of gp130. This dimerization occurs after binding of IL-6 to its surface receptor (IL-6R) and can also be triggered by the complex of soluble IL-6R and IL-6. We fused IL-6 to the constant region of a human IgG1 heavy chain (Fc). IL-6Fc was expressed in COS-7 cells and purified via Protein A Sepharose. Using three different assays we found that the biological activity of this dimeric IL-6 protein is comparable with monomeric IL-6. Recently, we described the designer cytokine Hyper-IL-6 (H-IL-6) in which soluble IL-6R and IL-6 are connected via a flexible peptide linker. This molecule turned out to be 100-1000 times more effective than unlinked IL-6 and soluble IL-6R. Hyper-IL-6 acts on cells only expressing gp130 and is a potent stimulator of in vitro expansion of early hematopoietic precursors. Here we show that a Fc fusion protein of H-IL-6 (H-IL-6Fc) has the same biological activity on BAF/gp130 cells as H-IL-6. Furthermore, both H-IL-6 forms have a similar ability to induce the synthesis of acute phase proteins in human hepatoma cells HepG2 and in mice in vivo. The introduction of a thrombin cleavage site between H-IL-6 and the Fc portion of H-IL-6Fc made it possible to specifically recover biologically active monomeric H-IL-6 by limited proteolysis of the fusion protein. A more general use of cleavable immunoadhesins expressed in mammalian cells is discussed.

New developments in IL-6 dependent biology and therapy: where do we stand and what are the options?

Interleukin-6 (IL-6) is a four-helical protein which, on target cells, binds to a specific IL-6-receptor and two molecules of the promiscuous signal transducing protein gp130. Structure-function analysis defined three molecular contact sites between IL-6 and its receptor subunits. Using this information, competitive antagonistic proteins as well as hyperagonistic proteins were developed. Possible therapeutic applications of IL-6 antagonists are in IL-6 dependent haematological disorders (Castleman's disease, POEMS syndrome, multiple myeloma) and bone diseases (Paget's disease, osteoporosis). Designer IL-6 antagonists could suppress inflammatory activity in rheumatic and autoimmune diseases and could prevent secondary amyloidosis. IL-6 antagonists could also prove advantageous in myocardial infarction and unstable angina pectoris. IL-6 antagonists might slow down development of (mesangioproliferative) glomerulonephritis. On the other hand, hyperagonistic variants of IL-6 have a potential in ex vivo expansion of bone marrow stem cells and as thrombopoietic agents. They might also be developed into drugs to support liver regeneration in vivo and to treat stress-induced cardiac insufficiency.

A new type of cytokine receptor antagonist directly targeting gp130.

The interleukin-6-type family of cytokines bind to receptor complexes that share gp130 as a common signal-transducing subunit. So far, receptor antagonists for interleukin-6-type cytokines have been constructed that still bind to the specific ligand binding subunit of the receptor complex, but have lost the ability to stimulate gp130. Such receptor antagonists compete for a specific receptor of a member of the cytokine family. Interleukin-6 only binds to gp130 when complexed with the interleukin-6 receptor that exists as a membrane bound and soluble molecule. Here we have constructed fusion proteins that consist of the soluble form of the human interleukin-6 receptor covalently linked to interleukin-6 receptor antagonists. These fusion proteins directly bind to gp130. Moreover, at concentrations of 10-50 nM they completely neutralize not only the biological activity of interleukin-6 but also of other cytokines of the interleukin-6-type family that act via gp130 homodimers or gp130/LIF-R heterodimers. Therefore, these gp130 targeting cytokine antagonists might be useful therapeutic tools in disease states that are related to cytokines of the interleukin-6 family.

The therapeutic potential of interleukin-6 hyperagonists and antagonists.

Interleukin-6 (IL-6) is a 4-helical protein that binds to a specific IL-6 receptor on target cells and to two molecules of the promiscuous signal transducing protein, glycoprotein 130 (gp130). Structure-function analysis has led to the definition of molecular contacts between IL-6 and its receptor subunits. This knowledge has led to the design of competitive antagonistic proteins that retain their receptor binding capability, but fail to stimulate one or both gp130 proteins; the properties of such recombinant antagonistic proteins are compared with traditional neutralising monoclonal antibodies targeted at IL-6 or receptor subunits. Furthermore, several strategies have been employed to construct molecules with increased bioactivity. Possible therapeutic applications in putative IL-6 dependent haematologic disorders, e.g., Castleman's disease (CD), POEMS syndrome, multiple myeloma, and bone diseases, e.g., Paget's disease, osteoporosis, are outlined. IL-6 antagonists could also, in theory, suppress inflammatory activity in rheumatic and autoimmune diseases and could prevent secondary amyloidosis. This principle may prove advantageous in myocardial infarction (MI) and unstable angina pectoris. More generally, IL-6 antagonists could improve the wasting and microcytic anaemia of chronic diseases. IL-6 antagonists might slow down development of mesangio-proliferative glomerulonephritis (MPGN). Hyperagonistic variants of IL-6 have a potential use in the ex vivo expansion of haematopoietic progenitor cells and as thrombopoietic agents. They might well be the first drugs to aid liver regeneration in vivo.

I. A bioactive designer cytokine for human hematopoietic progenitor cell expansion.

Efficient expansion of hematopoietic progenitor cells requires, at least, the simultaneous stimulation of the receptors c-kit and gp130. While c-kit is activated by SCF; gp130, in cells which do not express sufficient amounts of IL-6R, can be activated by the complex of soluble IL-6R (sIL-6R) and IL-6. The therapeutic use of IL-6/sIL-6R, however, has been hampered by the high concentrations of the sIL-6R protein required. We have designed a fusion protein of sIL-6R and IL-6, linked by a flexible peptide chain, that was expressed to high levels. On gp130 expressing cells the fusion protein turned out to be fully active at 100 to 1,000-fold lower concentration than the combination of unlinked IL-6 and IL-6R. The fusion protein was used to effectively expand human hematopoietic progenitor cells ex vivo in a dose dependent fashion.

Dissection of the internal carotid artery after chiropractic manipulation of the neck.

A 29-year-old woman died from a right hemispheric infarction caused by dissection and subsequent thrombosis of the internal carotid artery after chiropractic manipulations of the neck. Pathologic study of several arteries of muscular and elastic type revealed a mediolytic arteriopathy with widespread mucoid degeneration and cystic transformation of the vessel wall caused by segmental degeneration of smooth muscle cells of the tunica media. We hypothesize that mediolytic arteriopathy was a predisposing factor for the dissection of the internal carotid artery after chiropractic manipulations in our patient.

Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes. A comparison with the binding to leukemia cells.

In the presence of albumin Merocyanine 540 (MC540) exhibits a very limited binding to the outer surface of the membrane of normal erythrocytes, whereas pronounced binding is observed to leukemia cells. To find out whether this difference is due to differences in the composition or structural organization of the cell membrane we analyzed effects of a number of covalent and non-covalent perturbations of the red cell membrane on the binding and fluorescence characteristics of membrane-bound MC540. It is shown that exposure of the cells to cationic chlorpromazine, neuraminidase or photodynamic treatment with AlPcS4 as sensitizer caused a limited increase (30-50%) of MC540 binding, together with a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield of membrane-bound MC540. Other forms of perturbation of the membrane structure, like hyperthermia (48 degrees C) and treatments that produce a decrease of phospholipid asymmetry in addition to accelerated flip-flop, did not result in increased MC540 binding, but did cause a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield. These changes in fluorescence properties indicate a penetration of the dye into more hydrophobic regions in the membrane. MC540, bound to Brown Norway myelocytic leukemia cells, exhibited a red shift of the fluorescence emission maximum and an increased relative fluorescence quantum yield as compared to MC540 bound to untreated erythrocytes. These changes were of the same order of magnitude as in photodynamically treated red blood cells. Dye binding per surface area, however, was about 3-times higher with these leukemia cells than with photodynamically treated red blood cells. This demonstrates that certain perturbations of the erythrocyte membrane evoked a MC540 binding that became qualitatively comparable to the dye binding to leukemia cells, although dye binding per surface area was still significantly lower.

Is plasma membrane lipid composition defined in the exocytic or the endocytic pathway?

Compared with intracellular membranes, the plasma membrane is rich in cholesterol and sphingomyelin. How does this distinct composition arise? Here David Allan and Karl-Josef Kallen take a critical view of the belief that these lipids arrive at the plasma membrane via vesicular traffic from the Golgi complex and propose instead that they may be accreted in the endocytic recycling pathway.

Synthesis of surface sphingomyelin in the plasma membrane recycling pathway of BHK cells.

Sphingomyelin, which has been degraded at the BHK cell surface by exogenous sphingomyelinase, is converted back into sphingomyelin with kinetics similar to those of plasma membrane recycling. Resynthesis of sphingomyelin under these conditions proceeds at a rate about 4-fold higher than normal biosynthesis of sphingomyelin. Neither resynthesis of sphingomyelin nor its return to the surface is inhibited by brefeldin A (BFA), which is a potent blocker of vesicular transport through the Golgi but has no effect on plasma membrane recycling. However, resynthesis of plasma membrane sphingomyelin is greatly decreased in cells undergoing mitosis or energy depletion, where endocytosis is inhibited. We conclude that the main site of surface sphingomyelin synthesis in BHK cells could be in recycling endosomes and not in the Golgi apparatus as proposed previously. We also suggest a model pathway by which cholesterol may reach the plasma membrane via recycling endosomes.

Monensin inhibits synthesis of plasma membrane sphingomyelin by blocking transport of ceramide through the Golgi: evidence for two sites of sphingomyelin synthesis in BHK cells.

The monovalent cationophore monensin, which is known to interfere with vesicular transport through the Golgi apparatus, inhibits synthesis of sphingomyelin in BHK cells by up to 40%. The monensin-sensitive component of sphingomyelin synthesis appears to be the pool which normally reaches the cell surface since treatment of cells with exogenous sphingomyelinase causes an almost identical loss of sphingomyelin. Monensin causes increases in ceramide and glucosylceramide labelling which together are equivalent to the decrease in sphingomyelin labelling. Monensin also increases synthesis of cholesterol ester, probably due to the decreased delivery of sphingomyelin to the plasma membrane. However, monensin has no effect on resynthesis of plasma membrane sphingomyelin which has been degraded by extracellular sphingomyelinase. The results support the idea that synthesis of sphingomyelin destined for the plasma membrane does not occur in the cis- or medial-Golgi but depends on vesicular transport of ceramide to a second synthesis site which is distal to the medial-Golgi.

Effects of brefeldin A on sphingomyelin transport and lipid synthesis in BHK21 cells.

1. Addition of brefeldin A (BFA) to BHK cells incubated for 4 h with [3H]acetate led to a 3-4-fold increase in incorporation of label into sphingomyelin, monoglucosylceramide and cholesterol ester compared with untreated controls. There was a similar increase in incorporation of [3H]choline into sphingomyelin. The level of cholesterol ester increased 3-fold when BFA was added to cells labelled to equilibrium with [3H]acetate, but no statistically significant changes in the levels of other lipids were seen. 2. BFA appeared to act by diverting incorporation of acetate into sphingolipids and cholesterol ester at the expense of phosphatidylcholine (decreased by up to 15%), cholesterol (decreased by 30-40%) and triacylglycerol (decreased by 35-50%). 3. Forskolin (100 microM) prevented the changes in labelling induced by 0.25 micrograms of BFA/ml, but in the presence of 1 micrograms of BFA/ml it had no effect on sphingomyelin and triacylglycerol labelling and only partly blocked the effects of BFA on labelling of cholesterol and cholesterol ester. 4. None of the labelled sphingomyelin was degraded in BFA-treated cells which were subsequently exposed to an extracellular sphingomyelinase, showing that all the newly synthesized sphingomyelin remained inside the cells. Determinations of phospholipid phosphorus in unlabelled cells confirmed that, in the presence of BFA, no newly synthesized sphingomyelin was able to reach the cell surface, supporting the idea that sphingomyelin normally depends on vesicular transport for its passage to the plasma membrane. 5. The results are consistent with the hypothesis that cholesterol synthesis and esterification processes in BHK cells are sensitive to the plasma-membrane deficit of sphingomyelin caused by BFA.