Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5.

Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.

Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.

Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety.

Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor gene family known to induce apoptosis in a number of cancer cell lines and may have broad-spectrum activity against human malignancies. These studies have evaluated the potency of recombinant soluble human Apo2L/TRAIL in a mouse xenograft model and the disposition and safety of Apo2L/TRAIL in rodents and nonhuman primates. Mice with established COLO205 tumors were given daily i.v. injections of Apo2L/TRAIL (30-120 mg/kg/day). Control tumors doubled in size every 2 to 3 days, while time to tumor doubling in the treatment groups was significantly longer and related to dose (14-21 days). For pharmacokinetic studies, Apo2L/TRAIL was given as an i.v. bolus to mice (10 mg/kg), rats (10 mg/kg), cynomolgus monkeys (1, 5, and 50 mg/kg), and chimpanzees (1 and 5 mg/kg). Apo2L/TRAIL was rapidly eliminated from the serum of all species studied. Half-lives were approximately 3 to 5 min in rodents and approximately 23 to 31 min in nonhuman primates. Allometric scaling provided estimates of Apo2L/TRAIL kinetics in humans, suggesting that on a milligram per kilogram basis, doses significantly lower than those used in xenograft studies could be effective in humans. Apo2L/TRAIL clearance was highly correlated with glomerular filtration rate across species, indicating that the kidneys play a critical role in the elimination of this molecule. Safety evaluations in cynomolgus monkeys and chimpanzees revealed no abnormalities associated with Apo2L/TRAIL exposure. In conclusion, these studies have characterized the disposition of Apo2L/TRAIL in rodents and primates and provide information that will be used to predict the pharmacokinetics of Apo2L/TRAIL in humans.

Effect of IFN-gamma on the killing of S. aureus in human whole blood. Assessment of bacterial viability by CFU determination and by a new method using alamarBlue.

Given the increasing incidence of methicillin resistant Staphylococcus aureus (MRSA) and the recent emergence of MRSA with a reduced susceptibility to vancomycin, alternative approaches to the treatment of infection are of increasing relevance. The purpose of these studies was to evaluate the effect of IFN-gamma on the ability of white blood cells to kill S. aureus and to develop a simpler, higher throughput bacterial killing assay. Using a methicillin sensitive clinical isolate of S. aureus, a clinical isolate of MRSA, and a commercially available strain of MRSA, studies were conducted using a killing assay in which the bacteria were added directly into whole blood. The viability of the bacteria in samples harvested at various time points was then evaluated both by the classic CFU assay and by a new assay using alamarBlue. In the latter method, serially diluted samples and a standard curve containing known concentrations of bacteria were placed on 96-well plates, and alamarBlue was added. Fluorescence readings were taken, and the viability of the bacteria in the samples was calculated using the standard curve. The results of these studies demonstrated that the CFU and alamarBlue methods yielded equivalent detection of bacteria diluted in buffer. For samples incubated in whole blood, however, the alamarBlue method tended to yield lower viabilities than the CFU method due to the emergence of a slower growing subpopulation of S. aureus upon incubation in the blood matrix. A significant increase in bacterial killing was observed upon pretreatment of whole blood for 24 h with 5 or 25 ng/ml IFN-gamma. This increase in killing was detected equivalently by the CFU and alamarBlue methods. In summary, these studies describe a method that allows for the higher throughput analysis of the effects of immunomodulators on bacterial killing.

A neutralizing monoclonal antibody specific for the dimer interface region of IL-8.

We have generated two mAbs, 6G4.2.5 and A5.12.14, that are similarly capable of neutralizing the biologic activity of wild-type IL-8. To characterize these antibodies further, their reactivity against a series of engineered IL-8 monomer and dimer variants was examined using a neutrophil degranulation assay. While 6G4.2.5 was found to block effectively the biologic activity of all variants regardless of their dimerization status, the results for A5.12.14 differed dramatically. A5.12.14 fully inhibited the agonist activity of one of the monomer variants, partially blocked the activity of another, and had no effect on the activity of two other variants. These results suggested that the binding epitope of A5.12.14 was being affected by the particular amino acid substitutions introduced into the dimer interface region of the variants to disfavor dimerization. If A5.12.14 indeed binds to the dimer interface region of IL-8, it could be predicted that this mAb would be unable to inhibit the activity of dimeric IL-8. This was confirmed in studies which showed that A5.12.14 had no demonstrable effect on the activity of a constitutively dimeric IL-8 variant. These studies represent the first example of a mAb specific for the dimerization status of IL-8.

Modulating pharmacokinetics of an anti-interleukin-8 F(ab')(2) by amine-specific PEGylation with preserved bioactivity.

By covalently attaching biocompatible polyethylene-glycol (PEG) groups to epsilon-amino groups of the F(ab')(2) form of a humanized anti-interleukin-8 (anti-IL-8) antibody, we sought to decrease the in vivo clearance rate to give a potentially more clinically acceptable therapeutic. The in vivo clearance was modulated by changing the hydrodynamic size of the PEGylated antibody fragments. To achieve significant increases in the hydrodynamic size with minimal loss in bioactivity, high molecular weight linear or branched PEG molecules were used. Modification involved N-hydroxy-succinamide reaction of the PEGs with primary amines (lysines and/or the N-terminus) of the anti-IL-8 F(ab')(2). The process of adding up to four linear 20 kDa PEG, or up to two branched 40 kDa PEG, gave reproducible distribution of products. The components with uniform size (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were purified by a single-step ion-exchange high-performance liquid chromatography and showed no significant loss of biological activity in ligand binding and cell-based assays. Addition of a single branched 40 kDa PEG to a F(ab')(2) (molecular weight (MW)=1.6 million Da) or up to two 40 kDa branched PEG (MW=1.9 million Da) increased the serum half-life to 48 h as compared with the unPEGylated F(ab')(2) with a half-life of 8.5 h. This study shows that by attaching high molecular weight PEGs at a one or two sites, bioactive antibody fragments can be made reproducibly with sizes tailored to achieve the desired pharmacokinetics.

Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

IL-8 single-chain homodimers and heterodimers: interactions with chemokine receptors CXCR1, CXCR2, and DARC.

Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or beta-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is the single-chain variants.

Exchanging interleukin-8 and melanoma growth-stimulating activity receptor binding specificities.

Interleukin-8 (IL-8), a CXC chemokine, is known to bring about chemotaxis and activation of neutrophils through high affinity binding to at least two distinct receptors, receptor-A and receptor-B. The IL-8 homolog melanoma growth stimulating activity (MGSA) is also active toward neutrophils. In contrast to IL-8, MGSA binds receptor-B with high affinity and binds receptor-A with approximately 400-fold lower affinity. Using the structure of IL-8 (Clore et al.(1990) Biochemistry, 29, 1689-1696; Baldwin et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 502-506) and the NMR-determined structure of MGSA (Fairbrother et al. (1994) J. Mol. Biol. 242, 252-270), we designed variants of both IL-8 and MGSA to investigate the basis of specificity for binding of these chemokines to the IL-8 receptors. The most outstanding structural difference between IL-8 and MGSA lies in the loop preceding the first beta-strand. When the corresponding (shorter) loop from MGSA was swapped into IL-8, both receptor-A and receptor-B binding affinities were significantly (>300-fold) reduced. However, with additional mutations that affect packing interactions, an IL-8 variant specific for receptor-B binding was produced. Conversely, when the same loop from IL-8 was swapped into MGSA, receptor-B binding was maintained with only a approximately 30-fold reduction in receptor-A affinity. Again, mutations affecting packing of the loop yielded a MGSA variant with high affinity for both receptors, like IL-8. Finally, we show, through point mutations in a monomeric IL-8 framework, that individual side chain substitutions can affect receptor specificity.

Immunological priming attenuates the in vivo pathophysiological response to lipopolysaccharide. Comparison of cytokine production, tissue injury, and lethality in complete Freund's adjuvant-primed mice and in unprimed mice.

Previous reports have indicated that immunological priming of animals will result in increased cytokine production and enhanced susceptibility to the toxicity of cytokines. We primed mice with complete Freund's adjuvant and challenged 2 weeks later with 1 mg/mouse of lipopolysaccharide. Primed mice produced less tumor necrosis factor than naive mice (35 +/- 8 vs 108 +/- 20 ng/ml) and also less interleukin-6 (182 +/- 37 vs 6.39 +/- 155 ng/ml). Leukopenia developed only in the naive mice. Although neutropenia and lymphocytosis developed in both groups, the alterations manifested themselves more quickly in primed mice. Primed mice had substantially greater pulmonary neutrophil sequestration determined both enzymatically and histologically but no lung damage. However, primed mice had significantly less small bowel damage than naive mice. Mortality was substantially reduced in primed mice compared with unprimed mice. These results demonstrate that immunological priming in vivo decreases cytokine production in response to lipopolysaccharide challenge, decreases organ injury, and reduces mortality.

Interleukin 8 in serum in granulocytopenic patients with infections.

Serum levels of interleukin 8 (IL-8) were examined in eight patients with acute myeloid leukaemia during 16 courses of chemotherapy. The patients experienced 14 episodes of fever which occurred in periods with granulocyte counts < 0.5 x 10(9)/l. Febrile episodes were classified as bacteriologically defined infection (n = 6), clinically defined infection (n = 2), and unexplained fever (n = 6). IL-8 was detected in 18/25 (72%), 2/3 (67%) and 3/7 (43%) of the serum samples in the respective groups. In contrast, IL-8 was detected in 22/90 (24%) of the samples taken when no fever was present (P < 0.00003 versus bacteriologically defined infection). The median concentration of IL-8 in samples taken during febrile episodes was 194 ng/ml (range 0-6358 ng/ml) and 0 (range 0-5392 ng/ml) on days without fever (not significant). In three patients with infections caused by, respectively, Streptococcus sanguis, Acinetobacter calcoanitratus and Candida albicans, IL-8 rose to a peak levels and declined during recovery. We conclude that IL-I is released systemically during infections with gram-positive and gram-negative bacteria and Candida albicans in patients with acute myeloid leukaemia and peripheral granulocytopenia due to chemotherapy. However, IL-8 can also be detected when no sign of infection is present.

Regulation of interleukin 8 gene expression by oxidant stress.

Interleukin 8 (IL-8) is a recently described cytokine that functions as a potent neutrophil chemoattractant and activator. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) and the regulation of IL-8 gene expression to specifically test the hypothesis that ROI would induce production of IL-8 mRNA and protein. In lipopolysaccharide-stimulated human whole blood, the OH radical scavenger dimethyl sulfoxide (Me2SO) dramatically inhibited (approximately 90%) IL-8 production, but had minimal effects on the production of tumor necrosis factor, interleukin 1 beta (IL-1), and IL-6. To determine whether NADPH-oxidase-generated free radicals were critical in the regulation of IL-8, studies were performed using blood from patients with chronic granulomatous disease. In both normal individuals and patients with chronic granulomatous disease, production of IL-8 could be initiated with lipopolysaccharide, phytohemagglutinin, or aggregated immune complexes, and this production could be inhibited by Me2SO (1% v/v). To examine if oxidant stress represents a ubiquitous mechanism for the induction of IL-8, experiments were performed in cultured cell lines. In the human hepatoma cell line Hep-G2, Me2SO dose-dependently inhibited tumor necrosis factor-stimulated IL-8 production, with a 74 +/- 1% reduction observed at a Me2SO concentration of 1%. Direct exposure to ROI demonstrated that H2O2 stimulated IL-8 production in a dose-dependent manner in Hep-G2 cells, A549 pulmonary type II epithelial cells, and human skin fibroblasts; this induction could be prevented by addition of catalase. The production of IL-8 appeared to be specific to an oxidant stress since exposure of the cells to heat shock or chemical stress did not induce expression of IL-8. These studies demonstrate that oxidant stress is an important regulator of IL-8 gene expression and support the hypothesis that low levels of ROI may serve to initiate IL-8 production which then serves to recruit neutrophils to sites of inflammation.

Differential expression of tumor necrosis factor and interleukin-6 by peritoneal macrophages in vivo and in culture.

To investigate the differences in cytokine regulation in vitro as compared to in vivo, we examined the synthesis of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by peritoneal macrophages in response to lipopolysaccharide (LPS). Mice (CBA/J) were primed with an intraperitoneal injection of complete Freund's adjuvant and after 2 weeks, peritoneal cells were harvested for culture or mice were injected intraperitoneally with LPS for in vivo studies. In ascites fluid, TNF-alpha peaked 1 hour after LPS and returned to baseline levels by 4 hours. In contrast, TNF-alpha in the media reached maximum at 7 hours. Expression of TNF-alpha messenger (m)RNA in vivo was rapid but transient, as levels peaked at 15 minutes and returned to baseline 1 hour after LPS. In contrast, TNF-alpha mRNA in vitro became maximal at 1 hour, but remained elevated to 5 hours after LPS. In vivo, IL-6 in ascites fluid peaked at 2 hours, whereas in vitro, IL-6 continued increasing to 24 hours. In vivo, IL-6 mRNA reached maximum at 30 minutes, but fell below baseline by 1.5 hours after LPS. In contrast, IL-6 mRNA in vitro was sustained at maximal expression between 5 to 9 hours after LPS. These results demonstrate that both TNF-alpha and IL-6 synthesis is more rapid in vivo than in vitro. The rapid kinetics of cytokine expression in vivo must considered when designing strategies to inhibit cytokine action in vivo.

Oxygen radical scavengers selectively inhibit interleukin 8 production in human whole blood.

The hydroxyl radical (OH.) scavenger dimethyl sulfoxide (DMSO) was found to dose-dependently inhibit interleukin 8 (IL-8) production in LPS-stimulated human whole blood. At a concentration of 1% (vol/vol), DMSO blocked IL-8 release by approximately 90% in the presence of 1 microgram/ml LPS at a 24-h time point, but did not affect cell viability or reduce the production of tumor necrosis factor (TNF), interleukin 6, or interleukin-1 beta (IL-1 beta). DMSO was found to directly inhibit IL-8 expression at the level of transcription. Furthermore, this effect was not LPS-specific, in that IL-8 production was reduced by DMSO to a similar extent upon stimulation of blood with phytohemagglutinin, aggregated immune complexes, TNF, or IL-1 beta. Other oxygen radical scavengers that have been shown to inhibit OH.-dependent reactions (dimethyl thiourea, thiourea, mannitol, and ethanol) also inhibited IL-8 production. Conversely, addition of H2O2 caused a dose-dependent stimulation of IL-8 release. These results provide evidence that reactive oxygen metabolites play an important role in the regulation of IL-8 production and suggest that reduction of IL-8 release may contribute to the beneficial effects of antioxidants in experimental models of inflammation and ischemia/reperfusion injury.

Comparison of methods for incorporating a radioiodinated residualizing cholesteryl ester analog into low density lipoprotein.

Two different methods were evaluated for incorporating [125I]cholesteryl iopanoate ([125I]CI), a non-hydrolyzable cholesteryl ester analog, into LDL. The first procedure was an organic solvent delipidation-reconstitution procedure (R[125I-CI]LDL) while the second involved incubation of detergent (Tween-20)-solubilized [125I]CI with whole plasma (D[125I-CI]LDL). R[125I-CI]LDL behaved similar to native LDL in vitro, but was markedly different in vivo, apparently due to a heterogeneity in particle size. D[125I-CI]LDL, however, was metabolized normally both in vitro and in vivo. These results, combined with the residualizing nature of [125I]CI, demonstrate that D[125I-CI]LDL is appropriate for tracing LDL uptake in vivo.

Profile of cytokines in synovial fluid specimens from patients with arthritis. Interleukin 8 (IL-8) and IL-6 correlate with inflammatory arthritides.

The synovial fluid aspirated from patients with symptomatic arthritis was analyzed for the presence of tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 8 (IL-8). All three cytokines were found in both inflammatory and non-inflammatory arthritides: IL-8 levels ranged from less than 20 to 38,990 pg/ml, IL-6 from less than 10 to 72,300 pg/ml and TNF from less than 4 to 61 pg/ml. No inhibitors of cytokine activity were found. IL-8 and IL-6 were present in significantly higher levels in patients with inflammatory arthritis compared to patients with osteoarthritis, and there was significant correlation between the IL-6 and IL-8 levels. These findings document the presence of multiple cytokines in the synovial fluid specimens of patients with arthritis, and demonstrate that higher cytokine levels accompany inflammatory arthritis.

Local production of interleukin-8 is associated with nosocomial pneumonia.

One hundred five (70%) of 151 patients hospitalized in the intensive care unit and undergoing mechanical ventilation had bronchial secretions that tested positive for interleukin-8 within 36 hours of admission. Arterial blood, mixed venous blood, and urine collected simultaneously all tested negative, except for 11 patients admitted with intra-abdominal septic foci. The presence of interleukin-8 in the pulmonary air space early in the course of hospitalization was significantly associated with patients with multiple injuries, the need for greater ventilatory support, the occurrence of pulmonary dysfunction, and a 66% incidence of nosocomial bacterial pneumonia. We conclude that the early local production of interleukin-8 in the lungs is an early marker of pulmonary injury and may be involved in the pathogenesis of nosocomial bacterial pneumonia.