Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease.

Parkinson's disease (PD) is a fatal neurodegenerative disorder that is primarily caused by the degeneration and loss of dopaminergic neurons of the substantia nigra in the ventral midbrain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD identified to date, with G2019S being the most frequent LRRK2 mutation, which is responsible for up to 1-2% of sporadic PD and up to 6% of familial PD cases. As no treatment is available for this devastating disease, developing new therapeutic strategies is of foremost importance. Cellular models are commonly used for testing novel potential neuroprotective compounds. However, current cellular PD models either lack physiological relevance to dopaminergic neurons or are too complex and costly for scaling up the production process and for screening purposes. In order to combine biological relevance and throughput, we have developed a PD model in Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons by overexpressing wild-type (WT) and G2019S LRRK2 proteins. We show that these cells can differentiate into dopaminergic-like neurons and that expression of mutant LRRK2 causes a range of different phenotypes, including reduced nuclear eccentricity, altered mitochondrial and lysosomal morphologies, and increased dopaminergic cell death. This model could be used to elucidate G2019S LRRK2-mediated dopaminergic neural dysfunction and to identify novel molecular targets for disease intervention. In addition, our model could be applied to high-throughput and phenotypic screenings for the identification of novel PD therapeutics.

Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific.

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.

Preclinical Activity of SAR408701: A Novel Anti-CEACAM5-maytansinoid Antibody-drug Conjugate for the Treatment of CEACAM5-positive Epithelial Tumors.

PURPOSE: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycoprotein that has limited expression in normal adult tissues, but is overexpressed in carcinomas of the gastrointestinal tract, the genitourinary and respiratory systems, and breast cancer. As such, CEACAM5 is an attractive target for antibody-based therapies designed to selectively deliver cytotoxic drugs to certain epithelial tumors. Here, we describe preclinical data for a novel antibody-drug conjugate (ADC), SAR408701, which consists of an anti-CEACAM5 antibody (SAR408377) coupled to a maytansinoid agent DM4 via a cleavable linker. EXPERIMENTAL DESIGN: The specificity and binding affinity of SAR408701 to human and cynomolgus monkey CEACAM5 were tested in vitro. The cytotoxic activity of SAR408701 was assessed in CEACAM5-expressing tumor cell lines and using patient-derived xenograft mouse models of CEACAM5-positive tumors. Pharmacokinetic-pharmacodynamic and pharmacokinetic-efficacy relationships were established. SAR408701 toxicity was evaluated in cynomolgus monkey. RESULTS: SAR408701 bound selectively to human and cynomolgus monkey CEACAM5 with similar apparent Kd values (0.017 nmol/L and 0.024 nmol/L, respectively). Both in vitro and in vivo evaluations showed that SAR408701 has cytotoxic activity, leading to in vivo efficacy in single and repeated dosing. Single doses of SAR408701 induced significant increases in the tumor expression of phosphorylated histone H3, confirming the tubulin-targeting mechanism of action. The overall toxicity profile of SAR408701 in cynomolgus monkey was similar to that observed after intravenous administration of DM4 alone. CONCLUSIONS: On the basis of these preclinical data, the ADC SAR408701 is a promising candidate for development as a potential treatment for patients with CEACAM5-positive tumors.

Correction: Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues.

[This corrects the article DOI: 10.1371/journal.pone.0142708.].

Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues.

MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.