Preclinical evaluation of the efficacy, pharmacokinetics and immunogenicity of JS-001, a programmed cell death protein-1 (PD-1) monoclonal antibody.

JS-001 is the first monoclonal antibody (mAb) against programmed cell death protein-1 (PD-1) approved by the China Food and Drug Administration (CFDA) into the clinical trails. To date, however, no pre-clinical pharmacological and pharmacokinetic (PK) data are available. In this study, we investigated the efficacy of JS-001 and conducted a preclinical PK study, including the monitoring of anti-drug antibodies (ADAs). We found that JS-001 specifically bound to PD-1 antigen with an EC50 of 21 nmol/L, and competently blocked the binding of PD-1 antigen to PD-L1 and PD-L2 with IC50 of 3.0 and 3.1 nmol/L, respectively. Furthermore, JS-001 displayed distinct species cross-reactivity: it could bind to the PD-1 antigen on the peripheral blood mononuclear cells (PBMCs) of humans and cynomolgus monkeys, but not to those of mice and woodchucks; the Kd values for the interaction between JS-001 and PD-1 antigens on CD8+ T cells of human and cynomolgus monkey were 2.1 nmol/L and 1.2 nmol/L, respectively. In vitro, treatment with JS-001 (0.01-10 µg/mL) dose-dependently stimulated human T cell proliferation, as well as IFN-γ and TNF-α secretion. In HBsAg-vaccinated cynomolgus monkeys, the expression of PD-1+/CD4+ and PD-1+/CD8+ was significantly elevated, intramuscular injection of JS-001 (1 and 10 mg/kg) resulted in dramatic decreases in PD-1+/CD4+ and PD-1+/CD8+ expression in a dose-dependent manner, which was supported by PD-1 receptor occupancy (RO) results. In the PK study, 18 cynomolgus monkeys treated with single, ascending doses of 1, 10, and 75 mg/kg, and another 6 cynomolgus monkeys received 10 mg/kg successive administration. The plasma clearance of JS-001 followed a linear PK profile with single administration in the 1 and 10 mg/kg groups and a non-linear PK profile in the 75 mg/kg group. In the successive 10 mg/kg administration group, no drug accumulation was observed. But the AUC from the last exposure was lower than that of the first administration, which was probably due to the production of ADAs, as demonstrated in immunogenicity study. These non-clinical data are encouraging and provide a basis for the efficacy and safety of JS-001 in clinical trials.

Early Quantitative Gamma-Band EEG Marker is Associated with Outcomes After Cardiac Arrest and Targeted Temperature Management.

BACKGROUND: Brain recovery after cardiac arrest (CA) is sensitive to temperature. Yet the effect of temperature management on different EEG frequency bands has not been elucidated. A novel quantitative EEG algorithm, sub-band information quantity (SIQ), was applied to evaluate EEG recovery and outcomes after CA. METHODS: Twenty-four Wistar rats undergoing 7-min CA were randomly assigned to immediate hypothermia (32-34 °C), normothermia (36.5-37.5 °C), or hyperthermia (38.5-39.5 °C) (n = 8). EEG was recorded continuously for the first 8 h and then for serial 30-min epochs daily. The neurologic deficit score (NDS) at 72-h was the primary functional outcome. Another four rats without brain injury were added as a control. RESULTS: Better recovery of gamma-band SIQ was found in the hypothermia group (0.60 ± 0.03) compared with the normothermia group (0.40 ± 0.03) (p < 0.01) and in the normothermia group compared with the hyperthermia group (0.34 ± 0.03) (p < 0.05). The NDS was also improved in the lower temperature groups: hypothermia [median (25th, 75th), 74 (61, 74)] versus normothermia [49 (47, 61)] versus hyperthermia [43 (0, 50)] (p < 0.01). Throughout the 72-h experiment, the gamma-band SIQ showed the strongest correlation at every time point (ranging 0.520-0.788 from 30-min to 72-h post-resuscitation, all p < 0.05) whereas the delta-band SIQ had poor correlation with the 72-h NDS. No significant difference of sub-band EEG was found with temperature manipulation alone. CONCLUSIONS: Recovery of gamma-band SIQ-qEEG was strongly associated with functional outcomes after CA. Induced hypothermia was associated with faster recovery of gamma-band SIQ and improved functional outcomes. Targeted temperature management primarily affected gamma frequency oscillations but not delta rhythm.

Electrophysiological Monitoring of Brain Injury and Recovery after Cardiac Arrest.

Reliable prognostic methods for cerebral functional outcome of post cardiac-arrest (CA) patients are necessary, especially since therapeutic hypothermia (TH) as a standard treatment. Traditional neurophysiological prognostic indicators, such as clinical examination and chemical biomarkers, may result in indecisive outcome predictions and do not directly reflect neuronal activity, though they have remained the mainstay of clinical prognosis. The most recent advances in electrophysiological methods--electroencephalography (EEG) pattern, evoked potential (EP) and cellular electrophysiological measurement--were developed to complement these deficiencies, and will be examined in this review article. EEG pattern (reactivity and continuity) provides real-time and accurate information for early-stage (particularly in the first 24 h) hypoxic-ischemic (HI) brain injury patients with high sensitivity. However, the signal is easily affected by external stimuli, thus the measurements of EP should be combined with EEG background to validate the predicted neurologic functional result. Cellular electrophysiology, such as multi-unit activity (MUA) and local field potentials (LFP), has strong potential for improving prognostication and therapy by offering additional neurophysiologic information to understand the underlying mechanisms of therapeutic methods. Electrophysiology provides reliable and precise prognostication on both global and cellular levels secondary to cerebral injury in cardiac arrest patients treated with TH.

New bone formation in a true bone ceramic scaffold loaded with desferrioxamine in the treatment of segmental bone defect: a preliminary study.

BACKGROUND: Desferrioxamine (DFO), an iron chelator, can stimulate osteogenesis and angiogenesis by stabilizing hypoxia-inducible factor 1α. We postulate that a bone graft substitute combined with DFO is beneficial to the reconstruction of bone defects. METHODS: We implanted pure true bone ceramic (TBC) and DFO-loaded TBC (DFO/TBC) scaffolds into 15-mm rabbit radial defects for 8 weeks. The bone segments were examined with X-ray, micro-CT and histology. RESULTS: Radiographs showed that the DFO/TBC scaffold became radiopaque, and the gaps between the scaffold and radial cut ends were often invisible. Variables from micro-CT, including the bone volume fraction (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N), were significantly increased in pure TBC and DFO/TBC scaffolds that had been implanted for 8 weeks compared to unimplanted TBC scaffolds (p values <0.05-0.001). Between the former two groups, BV/TV and Tb.Th were significantly increased in DFO/TBC scaffolds (p < 0.001), but Tb.N did not show significant differences. Histological examinations showed considerably increased new bone and decreased TBC trabecular remnants in DFO/TBC scaffolds compared to pure TBC scaffolds. Many cavities in the new bone area in DFO/TBC scaffolds were occupied by bone marrow elements and blood vessels. Percent of new bone with tetracycline labeling was significantly greater in DFO/TBC scaffolds than in pure TBC scaffolds (p < 0.001). CONCLUSION: This preliminary study reveals that DFO can effectively induce new bone growing into TBC scaffolds, suggesting that the DFO/TBC composite is a promising bone graft substitute for the treatment of bone defects.

Periosteal CD68+ F4/80+ Macrophages Are Mechanosensitive for Cortical Bone Formation by Secretion and Activation of TGF-ß1.

Mechanical force regulates bone density, modeling, and homeostasis. Substantial periosteal bone formation is generated by external mechanical stimuli, yet its mechanism is poorly understood. Here, it is shown that myeloid-lineage cells differentiate into subgroups and regulate periosteal bone formation in response to mechanical loading. Mechanical loading on tibiae significantly increases the number of periosteal myeloid-lineage cells and the levels of active transforming growth factor ß (TGF-ß), resulting in cortical bone formation. Knockout of Tgfb1 in myeloid-lineage cells attenuates mechanical loading-induced periosteal bone formation in mice. Moreover, CD68+ F4/80+ macrophages, a subtype of myeloid-lineage cells, express and activate TGF-ß1 for recruitment of osteoprogenitors. Particularly, mechanical loading induces the differentiation of periosteal CD68+ F4/80- myeloid-lineage cells to the CD68+ F4/80+ macrophages via signaling of piezo-type mechanosensitive ion channel component 1 (Piezo1) for TGF-ß1 secretion. Importantly, CD68+ F4/80+ macrophages activate TGF-ß1 by expression and secretion of thrombospondin-1 (Thbs1). Administration of Thbs1 inhibitor significantly impairs loading-induced TGF-ß activation and recruitment of osteoprogenitors in the periosteum. The results suggest that periosteal myeloid-lineage cells respond to mechanical forces and consequently produce and activate TGF-ß1 for periosteal bone formation.

Inhibition of Integrin αvß6 Activation of TGF-ß Attenuates Tendinopathy.

Tendinopathy is a common tendon disorder that causes pain and impairs function. It is the most common reason for consultation with musculoskeletal specialists. The available therapies for tendinopathy are limited in number and efficacy and have unclear cellular and molecular mechanisms. Here it is shown that transforming growth factor-beta (TGF-ß) activated by integrin αvß6 promotes tendinopathy in mice. Excessive active TGF-ß is found during tendinopathy progression, which led to tenocytes' phenotype transition to chondrocytes. Transgenic expression of active TGF-ß in tendons induced spontaneous tendinopathy, whereas systemic injection of a TGF-ß neutralizing antibody attenuated tendinopathy. Inducible knockout of the TGF-ß type 2 receptor gene (Tgfbr2) in tenocytes inhibited tendinopathy progression in mice. Moreover, it is found that integrin αvß6 induces TGF-ß activation in response to mechanical load in tendons. Conditional knockout of the integrin αv gene in tendons prevented tendinopathy in mice. The study suggests that integrin αvß6 activation of TGF-ß is the mechanism of tendinopathy, and that integrin αvß6 may be a therapeutic target in tendinopathy.

An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion.

Osteoporosis (OP) is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures. Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed. We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation. We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment. The secretion of platelet-derived growth factor-BB (PDGF-BB), the number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells, and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice. The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP+ cells. These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP+ mononuclear cells. To test the therapeutic potential of the Siglec-15 neutralizing antibody, we injected the antibody in an ovariectomy-induced osteoporotic mouse model, which mimics postmenopausal osteoporosis in women, and in two fracture healing models because fracture is the most serious health consequence of osteoporosis. The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis. Of note, the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models. Thus, the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.

Sensory nerves regulate mesenchymal stromal cell lineage commitment by tuning sympathetic tones.

The sensory nerve was recently identified as being involved in regulation of bone mass accrual. We previously discovered that prostaglandin E2 (PGE2) secreted by osteoblasts could activate sensory nerve EP4 receptor to promote bone formation by inhibiting sympathetic activity. However, the fundamental units of bone formation are active osteoblasts, which originate from mesenchymal stromal/stem cells (MSCs). Here, we found that after sensory denervation, knockout of the EP4 receptor in sensory nerves, or knockout of COX-2 in osteoblasts, could significantly promote adipogenesis and inhibit osteogenesis in adult mice. Furthermore, injection of SW033291 (a small molecule that locally increases the PGE2 level) or propranolol (a beta blocker) significantly promoted osteogenesis and inhibited adipogenesis. This effect of SW033291, but not propranolol, was abolished in conditional EP4-KO mice under normal conditions or in the bone repair process. We conclude that the PGE2/EP4 sensory nerve axis could regulate MSC differentiation in bone marrow of adult mice.

Author Correction: Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Real-time quantitative monitoring of cerebral blood flow by laser speckle contrast imaging after cardiac arrest with targeted temperature management.

Brain injury is the main cause of mortality and morbidity after cardiac arrest (CA). Changes in cerebral blood flow (CBF) after reperfusion are associated with brain injury and recovery. To characterize the relative CBF (rCBF) after CA, 14 rats underwent 7 min asphyxia-CA and were randomly treated with 6 h post-resuscitation normothermic (36.5-37.5℃) or hypothermic- (32-34℃) targeted temperature management (TTM) (N = 7). rCBF was monitored by a laser speckle contrast imaging (LSCI) technique. Brain recovery was evaluated by neurologic deficit score (NDS) and quantitative EEG - information quantity (qEEG-IQ). There were regional differences in rCBF among veins of distinct cerebral areas and heterogeneous responses among the three components of the vascular system. Hypothermia immediately following return of spontaneous circulation led to a longer hyperemia duration (19.7 ± 1.8 vs. 12.7 ± 0.8 min, p < 0.01), a lower rCBF (0.73 ± 0.01 vs. 0.79 ± 0.01; p < 0.001) at the hypoperfusion phase, a better NDS (median [25th-75th], 74 [61-77] vs. 49 [40-77], p < 0.01), and a higher qEEG-IQ (0.94 ± 0.02 vs. 0.77 ± 0.02, p < 0.001) compared with normothermic TTM. High resolution LSCI technique demonstrated hypothermic TTM extends hyperemia duration, delays onset of hypoperfusion phase and lowered rCBF, which is associated with early restoration of electrophysiological recovery and improved functional outcome after CA.

Macrophage-lineage TRAP+ cells recruit periosteum-derived cells for periosteal osteogenesis and regeneration.

The periosteum, a thin tissue that covers almost the entire bone surface, accounts for more than 80% of human bone mass and is essential for bone regeneration. Its osteogenic and bone regenerative abilities are well studied, but much is unknown about the periosteum. In this study, we found that macrophage-lineage cells recruit periosteum-derived cells (PDCs) for cortical bone formation. Knockout of colony stimulating factor-1 eliminated macrophage-lineage cells and resulted in loss of PDCs with impaired periosteal bone formation. Moreover, macrophage-lineage TRAP+ cells induced transcriptional expression of periostin and recruitment of PDCs to the periosteal surface through secretion of platelet-derived growth factor-BB (PDGF-BB), where the recruited PDCs underwent osteoblast differentiation coupled with type H vessel formation. We also found that subsets of Nestin+ and LepR+ PDCs possess multipotent and self-renewal abilities and contribute to cortical bone formation. Nestin+ PDCs are found primarily during bone development, whereas LepR+ PDCs are essential for bone homeostasis in adult mice. Importantly, conditional knockout of Pdgfrß (platelet-derived growth factor receptor beta) in LepR+ cells impaired periosteal bone formation and regeneration. These findings uncover the essential role of periosteal macrophage-lineage cells in regulating periosteum homeostasis and regeneration.

Prostaglandin E2 mediates sensory nerve regulation of bone homeostasis.

Whether sensory nerve can sense bone density or metabolic activity to control bone homeostasis is unknown. Here we found prostaglandin E2 (PGE2) secreted by osteoblastic cells activates PGE2 receptor 4 (EP4) in sensory nerves to regulate bone formation by inhibiting sympathetic activity through the central nervous system. PGE2 secreted by osteoblasts increases when bone density decreases as demonstrated in osteoporotic animal models. Ablation of sensory nerves erodes the skeletal integrity. Specifically, knockout of the EP4 gene in the sensory nerves or cyclooxygenase-2 (COX2) in the osteoblastic cells significantly reduces bone volume in adult mice. Sympathetic tone is increased in sensory denervation models, and propranolol, a ß2-adrenergic antagonist, rescues bone loss. Furthermore, injection of SW033291, a small molecule to increase PGE2 level locally, significantly boostes bone formation, whereas the effect is obstructed in EP4 knockout mice. Thus, we show that PGE2 mediates sensory nerve to control bone homeostasis and promote regeneration.

Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.

Spinal pain is a major clinical problem, however, its origins and underlying mechanisms remain unclear. Here we report that in mice, osteoclasts induce sensory innervation in the porous endplates which contributes to spinal hypersensitivity in mice. Sensory innervation of the porous areas of sclerotic endplates in mice was confirmed. Lumbar spine instability (LSI), or aging, induces spinal hypersensitivity in mice. In these conditions, we show that there are elevated levels of PGE2 which activate sensory nerves, leading to sodium influx through Nav 1.8 channels. We show that knockout of PGE2 receptor 4 in sensory nerves significantly reduces spinal hypersensitivity. Inhibition of osteoclast formation by knockout Rankl in the osteocytes significantly inhibits LSI-induced porosity of endplates, sensory innervation, and spinal hypersensitivity. Knockout of Netrin-1 in osteoclasts abrogates sensory innervation into porous endplates and spinal hypersensitivity. These findings suggest that osteoclast-initiated porosity of endplates and sensory innervation are potential therapeutic targets for spinal pain.

Aberrant TGF-ß activation in bone tendon insertion induces enthesopathy-like disease.

Enthesopathy is a disorder of bone, tendon, or ligament insertion. It represents one-fourth of all tendon-ligament diseases and is one of the most difficult tendon-ligament disorders to treat. Despite its high prevalence, the exact pathogenesis of this condition remains unknown. Here, we show that TGF-ß was activated in both a semi-Achilles tendon transection (SMTS) mouse model and in a dorsiflexion immobilization (DI) mouse model of enthesopathy. High concentrations of active TGF-ß recruited mesenchymal stromal stem cells (MSCs) and led to excessive vessel formation, bone deterioration, and fibrocartilage calcification. Transgenic expression of active TGF-ß1 in bone also induced enthesopathy with a phenotype similar to that observed in SMTS and DI mice. Systemic inhibition of TGF-ß activity by injection of 1D11, a TGF-ß-neutralizing antibody, but not a vehicle antibody, attenuated the excessive vessel formation and restored uncoupled bone remodeling in SMTS mice. 1D11-treated SMTS fibrocartilage had increased proteoglycan and decreased collagen X and matrix metalloproteinase 13 expression relative to control antibody treatment. Notably, inducible knockout of the TGF-ß type II receptor in mouse MSCs preserved the bone microarchitecture and fibrocartilage composition after SMTS relative to the WT littermate controls. Thus, elevated levels of active TGF-ß in the enthesis bone marrow induce the initial pathological changes of enthesopathy, indicating that TGF-ß inhibition could be a potential therapeutic strategy.

Inhibition of overactive TGF-ß attenuates progression of heterotopic ossification in mice.

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-ß initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-ß that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-ß in tendon induces spontaneous HO, whereas systemic injection of a TGF-ß neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-ß type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-ß as inducers and promoters of ectopic bone formation, and suggest that TGF-ß might be a therapeutic target in HO.

Ciliary parathyroid hormone signaling activates transforming growth factor-ß to maintain intervertebral disc homeostasis during aging.

Degenerative disc disease (DDD) is associated with intervertebral disc degeneration of spinal instability. Here, we report that the cilia of nucleus pulposus (NP) cells mediate mechanotransduction to maintain anabolic activity in the discs. We found that mechanical stress promotes transport of parathyroid hormone 1 receptor (PTH1R) to the cilia and enhances parathyroid hormone (PTH) signaling in NP cells. PTH induces transcription of integrin αvß6 to activate the transforming growth factor (TGF)-ß-connective tissue growth factor (CCN2)-matrix proteins signaling cascade. Intermittent injection of PTH (iPTH) effectively attenuates disc degeneration of aged mice by direct signaling through NP cells, specifically improving intervertebral disc height and volume by increasing levels of TGF-ß activity, CCN2, and aggrecan. PTH1R is expressed in both mouse and human NP cells. Importantly, knockout PTH1R or cilia in the NP cells results in significant disc degeneration and blunts the effect of PTH on attenuation of aged discs. Thus, mechanical stress-induced transport of PTH1R to the cilia enhances PTH signaling, which helps maintain intervertebral disc homeostasis, particularly during aging, indicating therapeutic potential of iPTH for DDD.

Quantitative EEG markers in severe post-resuscitation brain injury with therapeutic hypothermia.

Therapeutic hypothermia has been regarded as one of the most effective post-cardiac arrest (CA) treatments to improve survival and functional recovery. However, many clinical prognostic markers after resuscitation have become less reliable under hypothermia. In this study, we applied and compared two developed quantitative measures - information quantity (IQ) and sub-band IQ (SIQ) - to evaluate the accuracy of EEG markers on predicting cortical recovery under therapeutic hypothermia. A total of 14 rats under 9-min asphyxial-CA, leading to severe brain injury, were randomly divided into two groups: hypothermia (32°C-34°C) and normothermia (36.5-37.5°C) (n=7 per group). For each rat, EEG and temperature were continuously recorded for the first 15 hrs. EEG was then recorded for serial 30 mins at 24, 48 and 72 hrs. The neurologic deficit score was evaluated daily to assess the neurologic recovery. Early SIQ and IQ were both significantly correlated with the 72-hr NDS, when the rats remained comatose. Both IQ and SIQ were able to discriminate the animals with good and bad functional outcomes starting from 1 hr after resuscitation. There was no significant difference in 72-hr NDS results (hypothermia (median (25th, 75th), 65 (52, 67)) versus normothermia (53.5 (52.25, 66.75))) (p>0.05) due to the high mortality rate (5/14) with severe brain injury. Contrary to IQ recovery but similarly to NDS scores, the SIQ recovery was not significantly different between the hypothermia (0.66±0.04) and normothermia (0.64±0.04) groups (p>0.05). IQ could identify the presence of high-frequency oscillations during the recovery from severe brain injury. We demonstrated that while SIQ was able to provide additional sub-band EEG information related to the recovery of different brain functions, both early IQ and SIQ markers are able to accurately predict neurologic outcome after CA.