Trastuzumab-MMAU Antibody-Auristatin Conjugates: Valine-Glucoserine Linker with Stabilized Maleimide Conjugation Improves In Vivo Efficacy and Tolerability.

PURPOSE: Antibody-drug conjugates (ADCs) have shown impressive clinical activity with approval of many agents in hematological and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic MMAE prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. EXPERIMENTAL DESIGN: Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo anti-tumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). RESULTS: The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared to a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of 8 and 4 respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAU DAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. CONCLUSIONS: The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in non-human primates, leading to a superior preclinical therapeutic window. The data supports potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.

N-methyl-D-aspartate receptor mechanosensitivity is governed by C terminus of NR2B subunit.

N-methyl-D-aspartate receptors (NMDARs), critical mediators of both physiologic and pathologic neurological signaling, have previously been shown to be sensitive to mechanical stretch through the loss of its native Mg(2+) block. However, the regulation of this mechanosensitivity has yet to be further explored. Furthermore, as it has become apparent that NMDAR-mediated signaling is dependent on specific NMDAR subtypes, as governed by the identity of the NR2 subunit, a crucial unanswered question is the role of subunit composition in observed NMDAR mechanosensitivity. Here, we used a recombinant system to assess the mechanosensitivity of specific subtypes and demonstrate that the mechanosensitive property is uniquely governed by the NR2B subunit. NR1/NR2B NMDARs displayed significant stretch sensitivity, whereas NR1/NR2A NMDARs did not respond to stretch. Furthermore, NR2B mechanosensitivity was regulated by PKC activity, because PKC inhibition reduced stretch responses in transfected HEK 293 cells and primary cortical neurons. Finally, using NR2B point mutations, we identified a PKC phosphorylation site, Ser-1323 on NR2B, as a unique critical regulator of stretch sensitivity. These data suggest that the selective mechanosensitivity of NR2B can significantly impact neuronal response to traumatic brain injury and illustrate that the mechanical tone of the neuron can be dynamically regulated by PKC activity.

Cathepsins B and L differentially regulate amyloid precursor protein processing.

Previous studies have shown that cathepsins control amyloid beta (Abeta) levels in chromaffin cells via a regulated secretory pathway. In the present study, this concept was extended to investigations in primary hippocampal neurons to test whether Abeta release was coregulated by cathepsins and electrical activity, proposed components of a regulated secretory pathway. Inhibition of cathepsin B (catB) activity with CA074Me or attenuation of catB expression through small interfering RNA produced decreases in Abeta release, similar to levels produced with suppression of beta-site APP-cleaving enzyme 1 (BACE1) expression. To test whether the catB-dependent release of Abeta was linked to ongoing electrical activity, neurons were treated with tetrodotoxin (TTX) and CA074Me. These comparisons demonstrated no additivity between decreases in Abeta release produced by TTX and CA074Me. In contrast, pharmacological inhibition of cathepsin L (catL) selectively elevated Abeta42 levels but not Abeta40 or total Abeta. Mechanistic studies measuring C-terminal fragments of amyloid precursor protein (APP) suggested that catL elevated alpha-secretase activity, thereby suppressing Abeta42 levels. The mechanism of catB-mediated regulation of Abeta release remains unclear but may involve elevation of beta-secretase. In summary, these studies provide evidence for a significant alternative pathway for APP processing that involves catB and activity-dependent release of Abeta in a regulated secretory pathway for primary neurons.

Calcium-permeable AMPA receptors appear in cortical neurons after traumatic mechanical injury and contribute to neuronal fate.

Traumatic brain injury (TBI) is one of the most disabling injuries in the population, with 1.5 million Americans new cases each year and 5.3 million Americans overall requiring long-term daily care as a result of their injuries. One critical aspect in developing effective treatments for TBI is determining if new, specific receptor populations emerge in the early phase after injury that can subsequently be targeted to reduce neuronal death after injury. One specific glutamate receptor subtype, the calcium-permeable AMPA receptor (CP-AMPAR), is becoming increasingly recognized for its role in physiological and pathophysiological processes. Although present in relatively low levels in the mature brain, recent studies show that CP-AMPARs can appear following ischemic brain injury or status epilepticus, and the mechanisms that regulate the appearance of these receptors include alterations in transcription, RNA editing, and receptor trafficking. In this report, we use an in vitro model of TBI to show a gradual appearance of CP-AMPARs four hours following injury to cortical neurons. Moreover, the appearance of these receptors is mediated by the phosphorylation of CaMKIIalpha following injury. Selectively blocking CP-AMPARs after mechanical injury leads to a significant reduction in the cell death that occurs 24 h following injury in untreated controls, and is similar in protection offered by broad-spectrum NMDA and AMPA receptor antagonists. These data point to a potentially new and more targeted therapeutic approach for treating TBI.

Linking impact to cellular and molecular sequelae of CNS injury: modeling in vivo complexity with in vitro simplicity.

Traumatic brain injury (TBI) represents one of most common disorders to the central nervous system (CNS). Despite significant efforts, though, an effective clinical treatment for TBI is not yet available. The complexity of human TBI is modeled with a broad group of experimental models, with each model matching some aspect of the human condition. In the past 15 years, these in vivo models were complemented with a group of in vitro models, with these in vitro models allowing investigators to more precisely identify the mechanism(s) of TBI, the different intracellular events that occur in acute period following injury, and the possible treatment of this injury in vitro. In this paper, we review the available in vitro models to study TBI, discuss their biomechanical basis for human TBI, and review the findings from these in vitro models. Finally, we synthesize the current knowledge and point out possible future directions for this group of models, especially in the effort toward developing new therapies for the traumatically brain injured patient.

Mechanisms and consequences of neuronal stretch injury in vitro differ with the model of trauma.

The deformation to the brain that occurs during traumatic brain injury (TBI) results in a complex strain distribution throughout the brain tissue. Recently, many in vitro models of neuronal injury have been developed to simplify the mechanics which occur during TBI. We hypothesized that the type of mechanical insult imparted onto neurons would significantly alter both the mechanism and severity of the neuronal response to injury. In this study, primary cortical neurons were cultured on an elastic substrate and subjected to graded levels (0%, 10%, 30%, 50%) of either uniaxial (cells stretched in one direction only) or biaxial (cells simultaneously stretched in two directions) stretch. We found that neurons stretched in either injury paradigm exhibited immediate increases in intracellular free calcium ([Ca2+]i), but the magnitude of the ([Ca2+]i) rise was nearly an order of magnitude higher in biaxially stretched neurons compared to uniaxially stretched neurons. Moreover, while the ([Ca2+]i) transient after uniaxial stretch was blocked with specific channel antagonists (APV, CNQX, nimodipine, TTX), a substantial ([Ca2+]i) transient persisted in biaxially stretched neurons. We theorized that the additional calcium influx after biaxial stretch entered through nonspecific pores/tears formed in the membrane, since biaxially stretched neurons exhibited significant uptake of carboxyfluorescein, a molecule typically impermeant to cell membranes. Despite the large ([Ca2+]i) transients, neither injury profile resulted in death within 24 h of injury. Interestingly, though, uniaxially stretched neurons exhibited enhanced [Ca+2]i influx following NMDA stimulation 24 h after trauma, compared to both control and biaxially stretched neurons. These data point out that the type of mechanical insult will influence the acute mechanisms of injury in vitro, can cause differences in the response to potential secondary excitotoxic injury mechanisms, and emphasizes the need to further study how these mechanical conditions can separately affect cell fate following mechanical injury.

NR2A and NR2B subunits differentially mediate MAP kinase signaling and mitochondrial morphology following excitotoxic insult.

NMDA receptors are essential for neurotransmission and key mediators of synaptic signaling, but they can also trigger deleterious degenerative processes that lead to cell death. Growing evidence suggests that selective blockade of the heterogeneous subunits that comprise the NMDA receptor may enable better control of pharmacotherapies for treating neurological diseases and injuries. We investigated the relationship between NMDAR activation, MAPK signaling, and mitochondrial shape following an excitotoxic insult. NR2A- and NR2B-containing NMDARs differentially mediated acute changes in cytosolic calcium, alterations in mitochondrial morphology, and phosphorylation of the MAPKs ERK and JNK. Activation of NR2A-containing NMDARs was associated with JNK phosphorylation that was neuroprotective in neuronal cultures subjected to excitotoxicity. In contrast, activation of NR2B-containing NMDARs triggered calcium accumulation in mitochondria that was strongly associated with mitochondrial swelling and neuronal cell death. Indeed, while blockade of NR2B-containing receptors was neuroprotective, this protection was lost when NR2A-initiated JNK phosphorylation was inhibited. Given the modest selectivity of the NR2A inhibitor, NVP-AAM077, the results highlight the significance of the relative, rather than absolute, activation of these two NMDA subtypes in modulating cell death pathways. Therefore, the balance between concurrent activation of NR2B-containing and NR2A-containing NMDARs dictates neuronal fate following excitotoxicity.

Pharmacologically induced calcium oscillations protect neurons from increases in cytosolic calcium after trauma.

Increases in cytosolic calcium ([Ca(2+)](i)) following mechanical injury are often considered a major contributing factor to the cellular sequelae in traumatic brain injury (TBI). However, very little is known on how developmental changes may affect the calcium signaling in mechanically injured neurons. One key feature in the developing brain that may directly impact its sensitivity to stretch is the reduced inhibition which results in spontaneous [Ca(2+)](i) oscillations. In this study, we examined the mechanism of stretch-induced [Ca(2+)](i) transients in 18-days in vitro (DIV) neurons exhibiting bicuculline-induced [Ca(2+)](i) oscillations. We used an in vitro model of mechanical trauma to apply a defined uniaxial strain to cultured cortical neurons and used increases in [Ca(2+)](i) as a measure of the neuronal response to the stretch insult. We found that stretch-induced increases in [Ca(2+)](i) in 18-DIV neurons were inhibited by pretreatment with either the NMDA receptor antagonist, APV [D(-)-2-Amino-5-phosphonopentanoic acid], or by depolymerizing the actin cytoskeleton prior to stretch. Blocking synaptic NMDA receptors prior to stretch significantly attenuated most of the [Ca(2+)](i) transient. In comparison, cultures with pharmacologically induced [Ca(2+)](i) oscillations showed a substantially reduced [Ca(2+)](i) peak after stretch. We provide evidence showing that a contributing factor to this mechanical desensitization from induced [Ca(2+)](i) oscillations is the PKC-mediated uncoupling of NMDA receptors (NMDARs) from spectrin, an actin-associated protein, thereby rendering neurons insensitive to stretch. These results provide novel insights into how the [Ca(2+)](i) response to stretch is initiated, and how reduced inhibition - a feature of the developing brain - may affect the sensitivity of the immature brain to trauma.