Combination therapy with remdesivir and monoclonal antibodies protects nonhuman primates against advanced Sudan virus disease.

A major challenge in managing acute viral infections is ameliorating disease when treatment is delayed. Previously, we reported the success of a 2-pronged mAb and antiviral remdesivir therapeutic approach to treat advanced illness in rhesus monkeys infected with Marburg virus (MARV). Here, we explored the benefit of a similar combination therapy for Sudan ebolavirus (Sudan virus; SUDV) infection. Importantly, no licensed anti-SUDV therapeutics currently exist, and infection of rhesus macaques with SUDV results in a rapid disease course similar to MARV with a mean time to death of 8.3 days. When initiation of therapy with either remdesivir or a pan-ebolavirus mAb cocktail (MBP431) was delayed until 6 days after inoculation, only 20% of macaques survived. In contrast, when remdesivir and MBP431 treatment were combined beginning 6 days after inoculation, significant protection (80%) was achieved. Our results suggest that combination therapy may be a viable treatment for patients with advanced filovirus disease that warrants further clinical testing in future outbreaks.

Therapy for Argentine hemorrhagic fever in nonhuman primates with a humanized monoclonal antibody.

The COVID-19 pandemic has reemphasized the need to identify safe and scalable therapeutics to slow or reverse symptoms of disease caused by newly emerging and reemerging viral pathogens. Recent clinical successes of monoclonal antibodies (mAbs) in therapy for viral infections demonstrate that mAbs offer a solution for these emerging biothreats. We have explored this with respect to Junin virus (JUNV), an arenavirus classified as a category A high-priority agent and the causative agent of Argentine hemorrhagic fever (AHF). There are currently no Food and Drug Administration-approved drugs available for preventing or treating AHF, although immune plasma from convalescent patients is used routinely to treat active infections. However, immune plasma is severely limited in quantity, highly variable in quality, and poses significant safety risks including the transmission of transfusion-borne diseases. mAbs offer a highly specific and consistently potent alternative to immune plasma that can be manufactured at large scale. We previously described a chimeric mAb, cJ199, that provided protection in a guinea pig model of AHF. To adapt this mAb to a format more suitable for clinical use, we humanized the mAb (hu199) and evaluated it in a cynomolgus monkey model of AHF with two JUNV isolates, Romero and Espindola. While untreated control animals experienced 100% lethality, all animals treated with hu199 at 6 d postinoculation (dpi) survived, and 50% of animals treated at 8 dpi survived. mAbs like hu199 may offer a safer, scalable, and more reproducible alternative to immune plasma for rare viral diseases that have epidemic potential.

Combination therapy protects macaques against advanced Marburg virus disease.

Monoclonal antibodies (mAbs) and remdesivir, a small-molecule antiviral, are promising monotherapies for many viruses, including members of the genera Marburgvirus and Ebolavirus (family Filoviridae), and more recently, SARS-CoV-2. One of the major challenges of acute viral infections is the treatment of advanced disease. Thus, extending the window of therapeutic intervention is critical. Here, we explore the benefit of combination therapy with a mAb and remdesivir in a non-human primate model of Marburg virus (MARV) disease. While rhesus monkeys are protected against lethal infection when treatment with either a human mAb (MR186-YTE; 100%), or remdesivir (80%), is initiated 5 days post-inoculation (dpi) with MARV, no animals survive when either treatment is initiated alone beginning 6 dpi. However, by combining MR186-YTE with remdesivir beginning 6 dpi, significant protection (80%) is achieved, thereby extending the therapeutic window. These results suggest value in exploring combination therapy in patients presenting with advanced filovirus disease.

Passive immunization with an extended half-life monoclonal antibody protects Rhesus macaques against aerosolized ricin toxin.

Inhalation of ricin toxin (RT), a Category B biothreat agent, provokes an acute respiratory distress syndrome marked by pro-inflammatory cytokine and chemokine production, neutrophilic exudate, and pulmonary edema. The severity of RT exposure is attributed to the tropism of the toxin's B subunit (RTB) for alveolar macrophages and airway epithelial cells, coupled with the extraordinarily potent ribosome-inactivating properties of the toxin's enzymatic subunit (RTA). While there are currently no vaccines or treatments approved to prevent RT intoxication, we recently described a humanized anti-RTA IgG1 MAb, huPB10, that was able to rescue non-human primates (NHPs) from lethal dose RT aerosol challenge if administered by intravenous (IV) infusion within hours of toxin exposure. We have now engineered an extended serum half-life variant of that MAb, huPB10-LS, and evaluated it as a pre-exposure prophylactic. Five Rhesus macaques that received a single intravenous infusion (25 mg/kg) of huPB10-LS survived a lethal dose aerosol RT challenge 28 days later, whereas three control animals succumbed to RT intoxication within 48 h. The huPB10-LS treated animals remained clinically normal in the hours and days following toxin insult, suggesting that pre-existing antibody levels were sufficient to neutralize RT locally. Moreover, pro-inflammatory markers in sera and BAL fluids collected 24 h following RT challenge were significantly dampened in huPB10-LS treated animals, as compared to controls. Finally, we found that all five surviving animals, within days after RT exposure, had anti-RT serum IgG titers against epitopes other than huPB10-LS, indicative of active immunization by residual RT and/or RT-immune complexes.

The Effect of Molecular Weight, PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs.

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)2 (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)2 versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)2 with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.

Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 µM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a ß phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h ß half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

Decreased recognition of SUMO-sensitive target genes following modification of SF-1 (NR5A1).

SUMO modification of nuclear receptors, including the constitutively active receptor steroidogenic factor 1 (SF-1; NR5A1), is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines (Lys119 and Lys194) that reside adjacent to the DNA-binding domain (DBD) and ligand-binding domain (LBD), respectively. Surprisingly, while previous loss-of-function studies predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein was either unchanged or modestly impaired. Sumoylation at Lys194 also modestly reduced Ser203 phosphorylation. In contrast to these findings, sumoylation of the DBD at Lys119 resulted in a marked and selective loss of DNA binding to noncanonical SF-1 targets, such as inhibinalpha; this binding deficit was extended to all sites when the sumoylated human mutant (R92Q) protein, which exhibits lower activity, was used. Consistent with this result, the K119R mutant, compared to wild-type SF-1, was selectively recruited to a "SUMO-sensitive" site in the endogenous inhibinalpha promoter, leading to increased transcription. DNA binding and sumoylation of Lys119 appeared to be mutually exclusive, suggesting that once SF-1 is bound to DNA, sumoylation may be less important in regulating SF-1 activity. We propose that sumoylation of nuclear receptors imposes an active posttranslational mark that dampens recognition of SUMO-sensitive target genes to restrain their expression.