HIGHLY EFFICIENT SINGLE-DOMAIN ANTIBODY NEUTRALIZATION OF INTERLEUKIN-6, THE FACTOR AT THE EPICENTER OF CYTOKINE STORM IN ACUTELY ILL COVID-19 PATIENTS

Background: COVID-19 is a worldwide medical emergency with a high mortality rate Cytokine storm (CS) has been attributed as the major cause of morbidity, multi-organ failure & mortality in COVID-19 patients In CS, the uncontrolled increase in pro-inflammatory cytokines especially IL6 results in an influx of immune cells leading to progressive tissue destruction Use of IL6 receptor (IL6R) inhibitors have shown only modest benefit in acutely ill COVID-19 cases due to need for high doses and associated side effects More efficacious and safer agents to targets IL6 are thus urgently needed We report generation of a potent camelid single domain antibody (sdAb) that robustly neutralizes human IL6 (hIL6) Methods: Two alpacas were immunized with purified hIL6 & a sdAb display phage library was prepared The library was enriched by panning for sdAbs recognizing hIL6 One group of closely related sdAbs was identified, expressed as soluble proteins, that displayed sub-nM affinity for hIL6 These sdAbs were found to neutralize hIL6 with low pM potencies in cell-based assays The most potent sdAb was expressed in CHO cells as a homodimer separated by a short amino acid spacer The homodimer was also encoded in an alphavirus-based replicon RNA (repRNA) & formulated with a Lipid InOrganic Nanoparticle (LION) The homodimer protein & repRNA were tested in vitro & in vivo Results: HEK293 cells transfected with Stat3-luciferase were treated for 6h with 50ng of hIL6 alone or hIL6 co-incubated 1h with 100ng of sdAb A complete abrogation of reporter activity was observed in the sdAb group To mimic CS, adult mice were challenged intraperitoneally (IP) for 30 min with 1mg of hIL6 alone or with sdAb, ranging from 0 25-4mg hIL6-induced Y705-STAT3 induction in liver was completely abolished by sdAb dimer at concentration ≥0 5mg, an effect not seen even with 500mg of IL6R Ab Mice were injected 5 days with an intramuscular injection of PBS or 40mg LION-formulated hIL6 sdAb dimer repRNA and then given 100ng of hIL6 IP for 30m LION-injected group showed complete absence of hepatic Y705-STAT3 in response to hIL6 Conclusion: The novel humanized IL6 sdAB efficiently inhibited hIL6-induced hepatic STAT3 activation and may have potential as a therapeutic for acutely ill COVID-19 patients exhibiting CS & other IL6-driven pathologies Additionally, using a similar pipeline, we are developing inhibitors of ACE2 and IL-1b, which can be combined for efficacious COVID-19 treatment