Pre- and postexposure efficacy of fully human antibodies against Spike protein in a novel humanized mouse model of MERS-CoV infection

© 2015, National Academy of Sciences. All rights reserved. Traditional approaches to antimicrobial drug development are poorly suited to combatting the emergence of novel pathogens. Additionally, the lack of small animal models for these infections hinders the in vivo testing of potential therapeutics. Here we demonstrate the use of the VelocImmune technology (a mouse that expresses human antibody-variable heavy chains and κ light chains) alongside the VelociGene technology (which allows for rapid engineering of the mouse genome) to quickly develop and evaluate antibodies against an emerging viral disease. Specifically, we show the rapid generation of fully human neutralizing antibodies against the recently emerged Middle East Respiratory Syndrome coronavirus (MERS-CoV) and development of a humanized mouse model for MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of the isolated antibodies. The VelocImmune and VelociGene technologies are powerful platforms that can be used to rapidly respond to emerging epidemics

Monoclonal antibodies against GFRα3 are efficacious against evoked hyperalgesic and allodynic responses in mouse join pain models but, one of these, REGN5069, was not effective against pain in a randomized, placebo-controlled clinical trial in patients with osteoarthritis pain

The artemin-GFRα3 signaling pathway has been implicated in various painful conditions including migraine, cold allodynia, hyperalgesia, inflammatory bone pain, and mouse knees contain GFRα3-immunoreactive nerve endings. We developed high affinity mouse (REGN1967) and human (REGN5069) GFRα3-blocking monoclonal antibodies and, following in vivo evaluations in mouse models of chronic joint pain (osteoarthritic-like and inflammatory), conducted a first-in-human phase 1 pharmacokinetics (PK) and safety trial of REGN5069 (NCT03645746) in healthy volunteers, and a phase 2 randomized placebo-controlled efficacy and safety trial of REGN5069 (NCT03956550) in patients with knee osteoarthritis (OA) pain. In three commonly used mouse models of chronic joint pain (destabilization of the medial meniscus, intra-articular monoiodoacetate, or Complete Freund’s Adjuvant), REGN1967 and REGN5069 attenuated evoked behaviors including tactile allodynia and thermal hyperalgesia without discernably impacting joint pathology or inflammation, prompting us to further evaluate REGN5069 in humans. In the phase 1 study in healthy subjects, the safety profiles of single doses of REGN5069 up to 3000 mg (intravenous) or 600 mg (subcutaneous) were comparable to placebo; PK were consistent with a monoclonal antibody exhibiting target-mediated disposition. In the phase 2 study in patients with OA knee pain, two doses of REGN5069 (100 mg or 1000 mg intravenous every 4 weeks) for 8 weeks failed to achieve the 12-week primary and secondary efficacy endpoints relative to placebo. In addition to possible differences in GFRα3 biology between mice and humans, we highlight here differences in experimental parameters that could have contributed to a different profile of efficacy in mouse models versus human OA pain. Additional research is required to more fully evaluate any potential role of GFRα3 in human pain

MARC1 pLOF variants affect plasma lipids and Apoprotein B levels but have no effect on coronary artery disease or type 2 diabetes.

(A) Serum lipids, glucose, body mass index and blood pressure, (B) type 2 diabetes and coronary artery disease. (TIF)</p

Marc1 gene deletion has no effect on circulating or liver lipids in male mice fed choline-deficient L-amino acid defined high fat diet (CDAA-HFD).

Nine weeks old male mice (11 WT and 10 Marc1 KO) were fed with CDAA-HFD for 18 weeks. (A) Body mass gain was measured every 2 weeks, (B) body composition (Micro CT) of mice were assessed at week 6 of experiment. (C) Serum, (D) liver lipids, (E) selected liver gene expression (mRNA by qRT-PCR) were performed at end of experiment. Each point represents individual mouse, mean ± s.e.m. are shown in all graphs, *p (TIF)</p

Reagents listed in the study.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.</div

Feeding Marc1 KO male mice with different NASH/NAFLD-causing diets revealed minimal difference in liver lipids and plasma metabolites compared to Marc1 WT littermates.

Mice fed with chow diet were sacrificed at age 13 weeks. For other diets—8–10 weeks old WT and Marc1−/− male mice were fed with HFHFD for 35 weeks or CDAA-HFD for 18 weeks or high fat diet (HFD) for 11 weeks or high sucrose diet (HSD) for 9 weeks, respectively. At the end of experiments mice were sacrificed, liver and serum collected and indicated measurements done as described in Methods. Each group contains 7–12 mice. All mice were sacrificed after ad lib feeding state (non-fasted). Mean ± s.e.m. are shown for all measurements. In bold–all measurement with *p (TIF)</p

Accelerated degradation of human MARC1 p.165T variant is not mediated by proteasomal or lysosomal pathways in HuH-7 cells.

(A) Plasmids encoding empty vector (EV), wild-type (WT) and common variants (165T) of full length human MARC1 (with C terminal FLAG tag) were transfected into HuH-7 cells. 48 hours after transfection, cell media containing DMSO only (vehicle) or 10 μM MG-132 in DMSO (proteasomal inhibitor) or 10ug/ml chloroquine in water (lysosomal inhibitor) plus DMSO separately were added and cells were incubated for additional 8 hours. After that, cells were washed, collected and solubilized in RIPA buffer, proteins separated on SDS-PAGE (4–20%) gels and blotted with corresponding primary antibodies. (B) Protein densitometry was performed on blots (A) and ratio between MARC1 variants and Calnexin were calculated. (C) Cells were prepared and transfected with WT or A165T variant human MARC1 similar as in (A), 48 h after transfection cells were incubated with DMSO or cycloheximide (CHX; 300 μg/ml) in DMSO or CHX (300 μg/ml) plus 10 μM MG-132 in DMSO or CHX (300 μg/ml) plus 10ug/ml chloroquine in DMSO for 0, 3, 6 and 9 hours. After incubation cells were collected and processed as in (A). (D) Protein densitometry was performed on (C) blots and ratios between MARC1 variants and β-actin were calculated, the value of 0 timepoint for each treatment condition were set as 1 and the relative changes in the protein levels were calculated over time. Ubiquitin was used as proteasomal inhibitor control, LC3B-I/II as lysosomal inhibitor control. CANX (calnexin) and β-actin–as protein loading controls. The experiments was repeated once with the same results. (TIF)</p

MARC1 A165T substitution leads to reduction in MARC1 protein expression in HepG2 cells.

(A) Plasmids with empty vector (Vector), wild-type and common variants of human MARC1 (CMV promoter, C terminal 1x Flag tag) were expressed in HepG2 cells. 48 hours after transfection, cells were split into two parts. First part of cells were solubilized in RIPA buffer, proteins separated on SDS-PAGE (4–20%) gels and blotted with corresponding antibodies, (B) immunoblot quantitation was performed. (C) Second part of cells was used to measure MARC1 mRNA levels using Real-Time PCR. Mean ± s.e.m. are shown in all graphs, ****p (TIF)</p