Rodney W. Lappe

Targeting the ANGPT-TIE2 pathway in malignancy.

Angiopoietins (ANGPTs) are ligands of the endothelial cell receptor TIE2 and have crucial roles in the tumour angiogenic switch. Increased expression of ANGPT2 relative to ANGPT1 in tumours correlates with poor prognosis. The biological effects of the ANGPT–TIE system are context dependent, which brings into question what the best strategy is to target this pathway. This Review presents an encompassing picture of what we know about this important axis in tumour biology. The various options for therapeutic intervention are discussed to identify the best path forwards.

Specifically Targeting Angiopoietin-2 Inhibits Angiogenesis, Tie2-Expressing Monocyte Infiltration, and Tumor Growth

Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1s function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold. Experimental Design: Various linker tethering sites on peptides were examined for their effect on CovX-Body in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC50s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration. Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2–Tie2 interaction (IC50 < 1 nmol/L) with dramatically improved pharmacokinetics (T½ > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%–63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2+CD11b+ cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy (∼80% TGI, P < 0.01). Conclusion: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic–pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents. Clin Cancer Res; 17(5); 1001–11. ©2011 AACR.

Chemical generation of bispecific antibodies

Bispecific antibodies (BsAbs) are regarded as promising therapeutic agents due to their ability to simultaneously bind two different antigens. Several bispecific modalities have been developed, but their utility is limited due to problems with stability and manufacturing complexity. Here we report a versatile technology, based on a scaffold antibody and pharmacophore peptide heterodimers, that enables rapid generation and chemical optimization of bispecific antibodies, which are termed bispecific CovX-Bodies. Two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. Whereas the pharmacophores are responsible for functional activities, the antibody scaffold imparts long half-life and Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. As a prototype, we developed a bispecific antibody that binds both vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) simultaneously, inhibits their function, shows efficacy in tumor xenograft studies, and greatly augments the antitumor effects of standard chemotherapy. This unique antiangiogenic bispecific antibody is in phase-1 clinical trials.

Pharmacologic characterization of an endothelinA (ETA) receptor antagonist in conscious rats.

Summary: The present experiments describe the endothelin-1 (ET-1) antagonist activity of BQ123 (cyclic D-Asp–L-Pro–D-Val–L-Leu–D-Trp) in conscious Sprague–Dawley (SD) rats, and we also examined the effect blockade of ETA receptors had on blood pressure in four experimental models of hypertension. Rats were anesthetized with methoxyflurane and instrumented with femoral arterial and venous catheters. In SD rats, BQ123 (0.1–10.0 mg/kg i.v.) administered 5 or 60 min prior to ET-1 inhibited both the magnitude and duration of the ET-1 (0.25 nmol/kg i.v.) pressor response. In addition, BQ123 (10.0 mg/kg) inhibited the pressor response evoked by administration of the ET-1 precursor, proendothelin-1 (1.0 nmol/kg). However, BQ123 (10.0 mg/kg) had no effect on the pressor response evoked by ET-3 (0.75 nmol/kg). In Wistar–Kyoto rats, BQ123 (10.0 mg/kg) reversed the hypertension produced by an infusion of ET-1 (0.01 nmol/kg/min). Administration of BQ123 produced a mild antihypertensive effect in normal- to low-renin models of hypertension, but no blood pressure lowering was observed in high-renin models of hypertension. These studies demonstrated the selectivity of the ETA receptor antagonist, BQ123 for ET-1, but not ET-3-induced pressor responses. Furthermore, ET-1 does not appear to be a major contributing factor to the maintenance of elevated levels of blood pressure in four experimental models of hypertension.

Development of a Novel Long-Acting Antidiabetic FGF21 Mimetic by Targeted Conjugation to a Scaffold Antibody

Fibroblast growth factor (FGF)21 improves insulin sensitivity, reduces body weight, and reverses hepatic steatosis in preclinical species. We generated long-acting FGF21 mimetics by site-specific conjugation of the protein to a scaffold antibody. Linking FGF21 through the C terminus decreased bioactivity, whereas bioactivity was maintained by linkage to selected internal positions. In mice, these CovX-Bodies retain efficacy while increasing half-life up to 70-fold compared with wild-type FGF21. A preferred midlinked CovX-Body, CVX-343, demonstrated enhanced in vivo stability in preclinical species, and a single injection improved glucose tolerance for 6 days in ob/ob mice. In diet-induced obese mice, weekly doses of CVX-343 reduced body weight, blood glucose, and lipids levels. In db/db mice, CVX-343 increased glucose tolerance, pancreatic β-cell mass, and proliferation. CVX-343, created by linkage of the CovX scaffold antibody to the engineered residue A129C of FGF21 protein, demonstrated superior preclinical pharmacodynamics by extending serum half-life of FGF21 while preserving full therapeutic functionality.

Differential responsiveness of conduit and resistance coronary arteries to endothelin A and B receptor stimulation in anesthetized dogs

The effects of endothelin-1 (ET-1), an ET(A)/ETB-receptor agonist, and IRL 1620, a potent and selective ETB-receptor agonist, were assessed on left circumflex coronary artery diameter (sonomicrometry) and flow (electromagnetic flow probe) in pentobarbital-anesthetized dogs. Intracoronary (i.c.) bolus injections of ET-1 (80 pmol/dose) caused large, sustained coronary diameter decreases (281 +/- 39 microns) and transient flow increases (5.6 +/- 2.6 ml/min), followed by transient (10.0 +/- 1.9 ml/min) and then sustained flow reductions (6.6 +/- 2.5 ml/min) before terminating in ventricular fibrillation after two to five doses (max delta s; n = 4 dogs). IRL 1620 boluses (5-2,000 pmol/dose i.c.; max delta s; n = 3) also dose-dependently and transiently increased (16.8 +/- 1.4 ml/min; 200 pmol), then transiently decreased (12.8 +/- 1.5 ml/min; 1,600 pmol) flow but had minimal effects on diameter (delta = -23 +/- 4 microns; 2,000 pmol). Doses of IRL 1620 beyond 400 pmol were accompanied by a slowly responding, sustained decrease in baseline flow (-9.2 +/- 2.7 ml/min) and baseline diameter (232 +/- 150 microns). In a separate group of dogs (n = 5), IRL 1620 (400 pmol i.c.) was evaluated before and after sequential inhibition of cyclooxygenase (indomethacin; 10 mg/kg i.v.) and then nitric oxide synthase (N omega-nitro-L-arginine methyl ester, L-NAME; 50 mg/kg i.v.). Indomethacin alone did not affect the flow increase to IRL 1620 (11.0 +/- 2.0 versus 11.8 +/- 1.8 ml/min) but blunted the flow decrease by 30 +/- 6% (10.6 +/- 1.4 versus 7.1 +/- 0.7 ml/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Regional vasodilator actions of calcitonin gene-related peptide in conscious SHR

In the present study the regional hemodynamic effects of CGRP were examined in conscious unrestrained spontaneously hypertensive rats (SHR). The animals were chronically instrumented with miniaturized pulsed Doppler flow probes to allow continuous measurement of renal, mesenteric and hindquarter blood flow. Bolus intravenous injection of CGRP (0.1-5 micrograms/kg) produced a dose-dependent fall in mean arterial pressure (maximal change = -48 +/- 5 mmHg) which was accompanied by a marked tachycardia (maximal change = 143 +/- 16 b/min). Depressor responses to CGRP were sustained for approximately 3-5 min. CGRP markedly reduced regional vascular resistance in all three vascular beds. No regional-selective vasodilator response was observed. These data indicate that CGRP is a potent vasodilator peptide in conscious SHR. The study suggests further that CGRP may contribute to the physiologic regulation of cardiovascular function.

Hemodynamic effects of calcitonin gene-related peptide in conscious rats

The cardiovascular effects of calcitonin gene-related peptide (CGRP) were examined in conscious, unrestrained rats. Changes in mean arterial pressure, heart rate and cardiac output were continuously monitored before and after i.v. bolus injection of CGRP (0.1-5 micrograms/kg). Injection of the peptide caused dose-dependent reductions in mean arterial pressure (-24 +/- 4 mmHg), which were accompanied by marked tachycardia. Cardiac output was significantly increased after CGRP but little change was observed in stroke volume. CGRP also reduced total peripheral resistance (-46 +/- 6%). These data indicate that the hypotensive actions of CGRP are mediated through peripheral vasodilation rather than through reductions in cardiac output. Pretreatment with propranolol significantly reduced the tachycardia responses to CGRP from 81 +/- 11 beats/min to 36 +/- 4 beats/min, but did not abolish the increase in heart rate. These data suggest that CGRP produces a tachycardia through reflex increases in cardiac sympathetic tone and through possible direct positive chronotropic effects on the heart.

Hemodynamic effects of infusion versus bolus administration of atrial natriuretic factor.

The cardiovascular responses to intravenous bolus administration of several synthetic atrial natriuretic peptides were examined in conscious spontaneously hypertensive rats and compared with the hemodynamic effects of continuous infusions of the peptides. Rats were instrumented with pulsed Doppler flow probes to allow measurement of regional blood flow in the conscious, unrestrained hypertensive rat. Bolus administration of increasing doses (0.036-18 nmol/kg) of atriopeptin II, alpha-rat atrial natriuretic peptide, Wy-47,663, or alpha-human atrial natriuretic peptide caused short-lived, dose-dependent reductions in mean arterial pressure and renal vascular resistance. A marked but transient (10-40 seconds) increase in renal blood flow was observed after administration of the peptides. Mesenteric and hindquarter vasodilation also were observed after bolus injection of high doses of the atrial peptides. Infusion of alpha-rat atrial natriuretic peptide or Wy-47,663 (0.045-1.44 nmol/kg/min) resulted in sustained reductions in mean arterial pressure. The fall in arterial pressure was accompanied by significant reductions in regional blood flow in the renal, mesenteric, and hindquarter vascular beds. Dose-dependent increases in regional vascular resistances were observed in all three vascular beds during the peptide infusions. These data indicate that the hemodynamic responses to synthetic atrial peptides are greatly dependent on the mode of administration of the peptide in conscious spontaneously hypertensive rats. Stable, sustained responses were observed only during infusion steady state conditions.

Regional hemodynamic effects of antihypertensive renomedullary lipids in conscious rats.

Renomedullary tissue has been proposed to exert an antihypertensive endocrine-like action. The antihypertensive polar renomedullary lipids (APRL) and neutral renomedullary lipids (ANRL) are potential mediators of this action. We evaluated the blood pressure and regional hemodynamic responses to APRL administered peripherally (i.v.) and to the central nervous system (CNS) in normal rats and rats with sinoaortic deafferentation (SAD) to remove baroreflex buffering. Rats were chronically instrumented with Doppler flow probes for measurement of mesenteric, renal, and hind-quarter vascular resistance, with arterial pressure and intravenous catheters, and with lateral cerebroventricular cannuli for intracerebroventricular (i.c.v.) administration. Intravenous APRL (0.01 to 1.0 micrograms) produced a dose-dependent decrease in blood pressure, tachycardia, and dilation of all vascular beds studied. The dose-response relationships were shifted to the left in SAD animals. APRL administered i.c.v. had no effect on intact or SAD rats. Pressor and regional vasoconstrictor responses to norepinephrine, angiotensin, and vasopressin were markedly reduced in SAD animals during constant infusion of APRL. In a second group of conscious SAD animals instrumented for blood pressure and heart rate measurements, intravenous ANRL (500 micrograms) decreased both arterial pressure (-45 +/- 16 mm Hg) and heart rate (-50 +/- 16 bpm). When given i.c.v., however, ANRL (10-100 micrograms) had no significant effect on resting blood pressure or heart rate. These studies suggest that APRL and ANRL produce no significant cardiovascular effects that are mediated through the CNS. However, both lipids are potent depressor agents, and APRL exhibits a strong peripheral vasodilator action and nonspecifically reduces reactivity to vasoconstrictor agents.