Susan P. Fuller

Selection of Tumor-binding Ligands in Cancer Patients with Phage Display Libraries

Phage display has been used extensively in vitro and in animal models to generate ligands and to identify cancer-relevant targets. We report here the use of phage-display libraries in cancer patients to identify tumor-targeting ligands. Eight patients with stage IV cancer, including breast, melanoma, and pancreas, had phage-displayed random peptide or scFv library (1.6 x 10(8)-1 x 10(11) transducing units/kg) administered i.v.; tumors were excised after 30 minutes; and tumor-homing phage were recovered. In three patients, repeat panning was possible using phage recovered and amplified from that same patients tumor. No serious side effects, including allergic reactions, were observed with up to three infusions. Patients developed antiphage antibodies that reached a submaximal level within the 10-day protocol window for serial phage administration. Tumor phage were recoverable from all the patients. Using a filter-based ELISA, several clones from a subset of the patients were identified that bound to a tumor from the same patient in which clones were recovered. The clone-binding to tumor was confirmed by immunostaining, bioassay, and real-time PCR-based methods. Binding studies with noncancer and cancer cell lines of the same histology showed specificity of the tumor-binding clones. Analysis of insert sequences of tumor-homing peptide clones showed several motifs, indicating nonrandom accumulation of clones in human tumors. This is the first reported series of cancer patients to receive phage library for serial panning of tumor targeting ligands. The lack of toxicity and the ability to recover clones with favorable characteristics are a first step for further research with this technology in cancer patients.

Identification of a small peptide that inhibits the phosphorylation of ErbB2 and proliferation of ErbB2 overexpressing breast cancer cells.

ErbB2 is overexpressed in approximately 30% of breast cancer patients with a correlation to poor prognosis. ErbB2 has been identified as a useful receptor for molecular targeting. A cyclic 20 amino acid phage display random peptide library was constructed using the fUSE5 gene III system. The library was panned against 2 different purified forms of the external domain of ErbB2. This resulted in the identification of several ErbB2‐binding phage clones with variable binding to different ErbB2 preparations. One clone (EC‐1) bound all preparations of ErbB2 including live cells and fresh frozen human breast cancer specimens. The synthetic peptide based on the deduced sequence of the EC‐1 clone and its biotin‐conjugated form retained binding affinity for purified ErbB2 and ErbB2 overexpressing cell lysates. EC‐1 peptide was able to effectively inhibit the phosphorylation of ErbB2 on residues Y1248 and Y877 in a dose‐ and time‐dependent manner. Furthermore, EC‐1 peptide selectively inhibits the proliferation of ErbB2 overexpressing breast cancer cells. The linear portion of the cyclic EC‐1 peptide was shown to be essential for binding ErbB2. In addition, 4 biased phage libraries were constructed allowing 4 different regions of the EC‐1 peptide to have random sequence. Screening these EC‐1 biased libraries did not result in higher affinity peptides but did demonstrate the importance of amino acids at position 1–4 on the N‐terminal flanking arm and 11–15 within the cyclic ring. Interestingly, EC‐1 contains homologous motifs with known ErbB receptor family ligands. We have identified a small peptide that binds to the extracellular domain of ErbB2, inhibits ErbB2 autophosphorylation and inhibits the proliferation of ErbB2 overexpressing cells. This supports the notion that small peptides can bind to targets important in cancer therapy even if a target does not have a natural ligand. Continuing research with this peptide includes increasing its affinity to ErbB2, evaluation of pharmacokinetics and evaluation of anti‐proliferative effects with conjugate anti‐cancer agents.

Complement depletion does not reduce brain injury in a rabbit model of thromboembolic stroke

The contribution of the complement system to cerebral ischemic and ischemia/reperfusion injury was examined in a rabbit model of thromboembolic stroke by delivery of an autologous clot embolus to the intracranial circulation via the internal carotid artery. A two-by-two factorial design was employed to study the impact of complement depletion via pretreatment with cobra venom factor (CVF, 100 U/kg i.v.) in the setting of permanent (without tissue plasminogen activator; t-PA) and transient (with t-PA) cerebral ischemia. Thirty-two New Zealand white rabbits were assigned to one of four groups (n=8, each group): control without t-PA, control with t-PA, CVF without t-PA and CVF with t-PA. In the complement intact animals, t-PA administration resulted in an approximate 30% reduction in infarct size when compared to the group not receiving t-PA (20.4+/-6.6% of hemisphere area vs. 30.1+/-7.2%; mean+/-SEM). However, infarct sizes in the complement depleted rabbits, with (30.7+/-8.2%) or without (30.2+/-7.9%) t-PA, were no different from the control group receiving no therapy. Similarly, no difference in regional cerebral blood flow or final intracranial pressure values was noted between any of the four groups. Complement activation does not appear to be a primary contributor to brain injury in acute thromboembolic stroke.

HUMANIZED ANTI-L-SELECTIN MONOCLONAL ANTIBODY DREG200 THERAPY IN ACUTE THROMBOEMBOLIC STROKE

Strategies directed against activated neutrophils have reduced ischemia-induced brain injury. However, therapies targeted specially against the neutrophil adhesion protein L-selectin have not yet been examined in stroke. This study therefore examined the effects of a monoclonal antibody directed against L-selectin in a rabbit model of thromboembolic stroke with (n = 16) or without (n = 10) concomitant t-PA therapy. Rabbits received either the humanized monoclonal antibody DREG200 directed against the L-selectin receptor or humanized control monoclonal antibody HuDREG55 which does not bind to rabbit L-selectin in addition to t-PA therapy (n = 8, each group). HuDREG200 (2 mg kg-1 i.v.) was given as a bolus 3 h following clot embolization, followed immediately by a 2 h intravenous infusion of t-PA (6.3 mg kg-1. Without t-PA therapy rabbits received HuDREG200 (2 mgkg-1, i.v.; n = 5) or HuDREG55 (n = 5) 1 h following clot embolization. The group receiving HuDREG200 in addition to t-PA demonstrated a moderate improvement in brain infarct size (8.4 +/- 2.4 vs. 13.5 +/- 3.5, %hemisphere, mean +/- sem), ICP (final reading 10.0 +/- 1.6 vs. 12.4 +/- 3.0 torr) and restoration in regional cerebral blood flow (30.2 +/- 7.8 vs. 21.6 +/- 10.9 cc 100 g-1 min-1) when compared to t-PA therapy alone although statistical significance was not achieved. No efficacy was demonstrated in the group receiving HUDREG200 without concomitant t-PA therapy. The results suggest the addition of a humanized anti-L-selectin monoclonal antibody HuDREG200 in combination with t-PA may further improve outcome in acute thromboembolic stroke, although future studies are necessary to support these findings.

TGF-β1 post-treatment in a rabbit model of cerebral ischaemia

AbstractTransforming growth factor-β1 (TGF-β1), suggested in some studies to suppress astrocyte and neutrophil function, has also reduced ischaemic brain injury when administered immediately prior to clot embolization in models of thromboembolic stroke. The effect of TGF-β1 as a post-treatment paradigm was investigated in a rabbit model of thromboembolic stroke. Following clot embolization, regional cerebral blood flow fell to<10 cc 100 g–1 min–1 in all animals. TCF-β1 (10 jug) or vehicle (n = 5 each group) was infused via the contralateral carotid artery. TGF-β1 administration resulted in a rapid and selective reduction in the peripheral neutrophil count as compared to a significant (p<0.05) increase in control values (2336 ± 817 vs 4320 ± 928 neutrophils mm , mean ± SEM). Neutrophil aggregation was increased within 30 min of TGF-β1 infusion when compared to control (2.07 ± 0.70 vs 1.09 ± 0.17 ohms, p<0.05); neutrophil chemiluminescence, an index of the oxygen respiratory burst was not significantly affe...

16(R)-hydroxyeicosatetraenoic acid, a novel cytochrome P450 product of arachidonic acid, suppresses activation of human polymorphonuclear leukocytes and reduces intracranial pressure in a rabbit model of thromboembolic stroke

OBJECTIVEActivated polymorphonuclear leukocytes (PMNs) have been suggested to contribute to the development of increased intracranial pressure (ICP). We recently demonstrated that human PMNs produce a novel cytochrome P450-derived arachidonic acid metabolite, 16(R)-hydroxyeicosatetraenoic acid [16(R)-HETE], that modulates their function. It was thus of interest to examine this novel mediator in an acute stroke model. METHODS16-HETE was assessed initially in a variety of human PMN and platelet in vitro assays and subsequently in an established rabbit model of thromboembolic stroke. A total of 50 rabbits completed a randomized, blinded, four-arm study, receiving 16(R)-HETE, tissue plasminogen activator, both, or neither. Experiments were completed 7 hours after autologous clot embolization. The primary end point for efficacy was the suppression of increased ICP. RESULTSIn in vitro assays, 16(R)-HETE selectively inhibited human PMN adhesion and aggregation and leukotriene B4 synthesis. In the thromboembolic stroke model, animals that received 16(R)-HETE demonstrated significant suppression of increased ICP (7.7 ± 1.2 to 13.1 ± 2.7 mm Hg, baseline versus final 7-h time point, mean ± standard error), compared with either the vehicle-treated group (7.7 ± 0.9 to 15.8 ± 2.6 mm Hg) or the tissue plasminogen activator-treated group (7.6 ± 0.6 to 13.7 ± 2.1 mm Hg). The group that received the combination of 16(R)-HETE plus tissue plasminogen activator demonstrated no significant change in ICP for the duration of the protocol (8.6 ± 0.6 to 11.1 ± 1.2 mm Hg). CONCLUSION16(R)-HETE suppresses the development of increased ICP in a rabbit model of thromboembolic stroke and may serve as a novel therapeutic strategy in ischemic and inflammatory pathophysiological states.

Peroxynitrite augments fMLP-stimulated chemiluminescence by neutrophils in human whole blood.

The neutrophil respiratory burst was examined by the technique of luminol‐dependent chemiluminescence (LDCL) triggered by submaximal concentrations of N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) in diluted whole blood. We sought to identify the chemical species responsible for LDCL in whole blood, to examine the role of leukotriene B4 (LTB4) and other arachidonic acid metabolites as mediators of the fMLP signaling pathway, and to investigate the effect of peroxynitrite on this response. Both sodium azide and taurine significantly inhibited LDCL (93% inhibition with 100 μM azide, 52% inhibition with 10 mM taurine). More modest inhibition was seen with superoxide dismutase (SOD), catalase, the nitric oxide synthase inhibitor monomethyl‐l‐arginine (l‐NMMA), and with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (AA‐861; no effect), and cytochrome P‐450 (SKF 525‐A) pathways of arachidonic acid metabolism. The nitric oxide donor SIN‐1 (1–100 μM) and peroxynitrite (10–300 μM) also augmented fMLP‐induced LDCL. The augmentation seen with peroxynitrite and SIN‐1 was attenuated by SOD. Despite the increase in LDCL, peroxynitrite caused a dose‐related inhibition of fMLP‐stimulated LTB4 release. In summary, our results indicate that (1) LDCL elicited by fMLP in diluted whole blood appears primarily mediated by hypo‐chlorous acid derived from myeloperoxidase; (2) pretreatment with the nitric oxide donor SIN‐1 or with peroxynitrite augments LDCL; and (3) LTB4 release does not contribute to fMLP‐stimulated LDCL or in the modulation of LDCL by SIN‐1 or peroxynitrite. J. Leukoc. Biol. 60: 619–624; 1996.

Decreased protein catabolism after exercise in subjects with IDDM

SummaryWe examined whether the increased rates of protein catabolism (proteolysis and leucine oxidation) associated with moderate insulinopenia in subjects with IDDM would be accentuated by prior bicycle exercise (53% VO2max for 82 min). Insulin infusions maintained plasma glucose concentrations on one study day in “tight” control (TC: 6 mmol/l) and on a separate day in “loose” control (LC: 12 mmol/l). Elevations in serum ketone body, plasma NEFA, and whole-blood branched-chain amino acid concentrations on the loose control day during the basal period persisted throughout the post-exercise recovery period. Amino acid kinetics were estimated during a primed, constant infusion of l-[1-13C]leucine from plasma dilution of α-[1-13C]KIC and expired air 13CO2 enrichments. Loose control was associated with increased rates of whole-body leucine oxidation (LC 25±7 vs TC 21±8 μmol · kg−1 · h−1) and protein degradation (LC 127±12 vs TC 118±18 μmol · kg−1 · h−1) (both p<0.05). During the 2-h post exercise recovery period, there were significant decreases in rates of leucine oxidation (LC 21±7, TC 16±7) and protein degradation (LC 112±13, TC 107±11), compared to the basal period (both p<0.05, basal vs recovery). Rates of wholebody protein synthesis were unchanged by prior exercise. In conclusion, moderate insulinopenia is associated with significantly higher rates of protein degradation and leucine oxidation in the basal state. Following exercise, net protein catabolism is diminished due to reduced rates of protein degradation in the presence of maintained rates of protein synthesis. The significantly increased concentrations of fat-derived substrates (ketone bodies, NEFA) may have prevented the predicted increases in protein catabolism which we anticipated would follow acute exercise during periods of relative insulin deficiency.