Armed nanobodies as anti-infectives and anti-tumor agents - Summary A nanobody that recognizes immunoglobulin light chains, conjugated to a molecular entity that recognizes a virus-infected or a cancerous cell, is an effective therapeutic: A single injection of fusion constructs comprising an anti-kappa light chain nanobody (VHHkappa) and zanamivir, a small molecule that targets influenza neuraminidase, protects mice from a lethal challenge with both A- and B-strains of influenza. In the model that established protection by VHHkappa adducts against influenza, the underlying mechanism of action involves antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but the relative contribution of each is not known. We shall therefore use FcgR common g chain-deficient mice and C3- deficient mice to assess the relative contributions of ADCC and CDC. The generation of Fc constructs of different Ig isotypes and bearing FcR-engagement disabling mutations, similarly modified with zanamivir, will be used to complement this analysis. Having established proof-of-concept for influenza and optimized parameters for elimination of influenza virus- infected cells, we will explore nanobodies that recognize other pathogens (Ebola virus, SARS-CoV-2, HIV) in combination with VHHkappa in a series of collaborative experiments. The agents to be developed may inspire novel immunomodulatory therapeutics, to be used as a stand-alone approach, or in combination with approved drugs. The possibilities of post-exposure prophylaxis against viral infections (Ebola, SARS-CoV-2, HIV) in the absence of pre-existing immunity, deserve particular emphasis. We shall further enhance the activity of the proposed VHHkappa fusions through the generation of the corresponding drug adducts, using cytotoxic drugs such as maytansinoids as compounds that have shown clinical utility. Enveloped viruses (e.g., HIV, SARS-CoV-2) export viral proteins to the surface of the infected cell during budding. Infected cells can thus be distinguished from uninfected cells based on the surface display of viral proteins. We now extend these in vivo observations to fusions of VHHkappa with anti-checkpoint (PD-L1, CTLA-4) nanobodies. We generated maytansinoid-modified VHHkappa fusions with the anti-PD-L1 and anti-CTLA-4 VHHs. Our preliminary data show enhanced anti-tumor activity in the MC38 and B16.F10 mouse tumor models in comparison with commonly used monoclonal antibodies. However, not all such fusions (examples: fusions of VHHkappa with nanobodies that recognize Class II MHC or CD8) have shown the intended depletion efficacy in vivo. This proposal seeks to establish the parameters -including biodistribution and surface expression levels of the targeted molecules- that determine success or failure of VHHkappa fusions. The availability of VHHs that recognize human kappa light chains suggest the possibility of clinical translation of this approach.
