Development and Optimization of a Platform for Next-Generation Monoclonal Antibodies Against Powassan Virus and Other Neuroinvasive Tick-Borne Viruses

Background: Powassan virus (POWV) is a tick-borne flavivirus that causes encephalitis and has a 12% case- fatality rate. There are no Food and Drug Administration (FDA)-approved therapeutics or vaccines against POWV, and supportive care is the standard treatment. This proposal directly responds to the Tick-Borne Disease Research Program's (TBDRP) program announcement and addresses the program's vision to prevent and resolve or minimize the impact of tick-borne illnesses through therapeutics and/or diagnostics. Despite recent advances in the development of POWV-specific monoclonal antibodies (mAbs), their delivery across the blood-brain barrier (BBB) and safety parameters have yet to be described. Engagement of cell surface Fc-gamma receptors (Fc-gamma-R) by antibody Fc-domains activates a subset of immune effector cells, which may provoke phagocytosis of infected cells, production of inflammatory cytokines, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Currently, it is unknown if Fc-mediated effector functions induced by immunotherapies are beneficial or harmful in the treatment of neuroinvasive viral disease. Hypothesis: We hypothesize that transport across the BBB and inhibition of Fc-mediated effector functions will increase the therapeutic window of protection for POWV-specific mAbs and prevent antibody-dependent cytotoxicity in the brain, respectively. To test this hypothesis we will, (1) generate novel BBB-targeting ultralong CDR3s (UL-CDR3s) that can be integrated in a plug-and-play manner into the scaffold of any mAb; (2) we will employ the state-of-the art Orthogonal Dual-Interacting Nanotherapeutic (ODIN) platform to generate bispecific mAbs against POWV using an already existing POWV-specific mAb, m158.25; and (3) we will characterize, for the first time, Fc-mediated cytotoxicity of immunotherapies during neuroinvasive infection. Specific Aims and Study Design Specific Aim 1: Engineer novel UL-CDR3s that bind BBB receptors and are easily deployable in a plug-and-play mechanism. First, we will develop novel BBB-targeting UL-CDR3s through immunization of cattle with various receptor proteins involved in receptor mediated transcytosis (RMT) across the BBB. UL-CDR3s have benefits over other mAbs formats, including their exceptionally small size (approximately seven kDa) , which makes them ideal for transport across the BBB, and their "stalk and knob" configuration that gives them more flexibility and breadth when binding epitopes. Newly identified UL-CDR3s will be integrated into the framework of a recently described POWV-neutralizing mAb, m158.25. Currently, the safety profile and therapeutic benefit of immunotherapy Fc-effector functions during a neurotropic viral infection is unknown. Consequently, Aim 1 will use scFvm158.25-UL-CDR3s, which have no Fc-domain. ODINs will be tested for affinity toward BBB receptors and efficacy against POWV in vitro and in vivo. Specific Aim 2: Characterize the role of Fc-mediated effector functions during POWV infection to help guide the design of neurotropic mAbs. Here, we propose to characterize the safety parameters and the immune response that is induced by the Fc-domains of neuro-targeting antiviral mAbs. Aims 1 and 2 will be performed in parallel. Generation of BBB-targeting UL-CDR3s in Aim 1 will take approximately six months; therefore, in Aim 2, we will use a previously described TfR binding scFv, 8D3, to target transport of m158.25 to the brain. We will generate 8D3-m158.25 Fc-variants with increased, decreased, or ablated FcR binding abilities to interrogate their efficacy, safety, and induced immune response in mice. Brain samples will be collected from infected and treated animals and used for pathology analyses and bulk transcriptomics. Impact: Because there are no FDA-approved therapeutics or vaccines against POWV, supportive care is the standard treatment. This proposal directly responds to the TBDRP's announcement and addresses its vision. This work is innovative because we will generate, for the first time, BBB-targeting UL-CDR3s, employ state of the art in vitro and in vivo models to test their efficacy, and characterize neurological immune responses that are mediated by Fc domains of immunotherapies during a POWV infection. Moreover, it will provide a set of parameters and tools that will be easily deployable in a plug-and-play manner, not only against neuroinvasive tick-borne viruses, but also against encephalitic viruses that pose a threat to public and Warfighter health. Career Development: Dr. Catalina Florez seeks to apply her antibody development experience to the field of tick- borne flaviviruses and develop BBB-targeting bispecific monoclonal antibodies mAbs with longer and safer therapeutic windows. This award will provide Dr. Florez with the opportunity to secure time and additional resources to expand her expertise to the field of tick-borne neurotropic viral diseases. She will accomplish this objective with the support of a skilled team and the mentorship of Dr. Andrew Herbert and Dr. Jonathan Lai. Less