The preclinical pharmacology of the high affinity anti-IL-6R Nanobody (R) ALX-0061 supports its clinical development in rheumatoid arthritis

Introduction: The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody (R) with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology. Methods: ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. Results: ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. Conclusions: ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA

Real-time in vivo photoacoustic and ultrasound imaging

A real-time photoacoustic imaging system is designed and built. This system is based on a commercially available ultrasound imaging system. It can achieve a frame rate of 8 frames/sec. Vasculature in the hand of a human volunteer is imaged, and the resulting photoacoustic image is combined with the ultrasound image. The real-time photo acoustic imaging system with a hybrid ultrasound probe is demonstrated by imaging the branching of subcutaneous blood vessels in the hand. (C) 2008 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3005421

Real-time in vivo photoacoustic and ultrasound imaging\ud

A real-time photoacoustic imaging system is designed and built. This system is based on a commercially available ultrasound imaging system. It can achieve a frame rate of 8 frames/sec. Vasculature in the hand of a human volunteer is imaged, and the resulting photoacoustic image is combined with the ultrasound image. The real-time photo acoustic imaging system with a hybrid ultrasound probe is demonstrated by imaging the branching of subcutaneous blood vessels in the han

Photoacoustic determination of blood vessel diameter

A double-ring sensor was applied in photoacoustic tomographic imaging of artificial blood vessels as well as blood vessels in a rabbit ear. The peak-topeak time (τ pp) of the laser (1064 nm) induced pressure transient was used to estimate the axial vessel diameter. Comparison with the actual vessel diameter showed that the diameter could be approximated by 2cτ pp, with c the speed of sound in blood. Using this relation, the lateral diameter could also precisely be determined. In vivo imaging and monitoring of changes in vessel diameters was feasible. Finally, acoustic time traces were recorded while flushing a vessel in the rabbit ear with saline, which proved that the main contribution to the laser-induced pressure transient is caused by blood inside the vessel and that the vessel wall gives only a minor contribution

Pulsed-laser Doppler flowmetry provides basis for deep perfusion probing

A setup for pulsed-laser Doppler flowmetry ~LDF! measurements has been built and tested. Measurements were carried out comparing continuous-wave and pulsed LDF. With pulsed LDF a higher peak power can be injected into the tissue without exceeding the safety limits. This enables a much larger spacing between the locations of illumination and detection. Thus, the penetration depth, and thus the measurement volume, can be enlarged using the pulsed-LDF method. This method will allow, e.g., monitoring of the cerebral perfusion

Photoacoustic imaging of tumor angiogenesis

Photoacoustic imaging is a hybrid imaging modality that is based on the detection of acoustic waves generated by absorption of pulsed light by tissue chromophores such as hemoglobin in blood. Serial photoacoustic imaging has been performed over a 10-day period after subcutaneous inoculation of pancreatic tumor cells in a rat. The images were obtained from ultrasound generated by absorption in hemoglobin of short laser pulses at a wavelength of 1064 nm. The ultrasound signals were measured in reflection mode using a double-ring photoacoustic detector. A correction algorithm has been developed to correct for scanning and movement artifacts during the measurements. Three-dimensional data visualize the development and quantify the extent of individual blood vessels around the growing tumor, blood concentration changes inside the tumor and growth in depth of the neovascularized region