Significance: Vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. Currently available vaccines require cold storage and sophisticated manufacturing capacity, complicating their distribution, especially in less developed countries. We report a protein-based SARS-CoV-2 vaccine that directly and specifically targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike _RBD ) fused to a nanobody that recognizes class II major histocompatibility complex antigens (VHH _MHCII ). Our vaccine elicits robust humoral (high-titer binding and neutralizing antibodies) and cellular immunity against SARS-CoV-2 and its variants in both young and aged mice. VHH _MHCII -Spike _RBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy, desirable attributes for logistical reasons.

The distribution of Ly6C/G-positive cells in response to an infection of the mouse respiratory tract with influenza A virus was followed noninvasively over time by immuno-positron emission tomography. We converted nanobodies that recognize Ly6C and Ly6G, markers of neutrophils and other myeloid cells, as well as an influenza hemagglutinin–specific nanobody, into ⁸⁹ Zr-labeled PEGylated positron emission tomography (PET) imaging agents. The PET images showed strong accumulation of these imaging agents in the lungs of infected mice. Immunohistochemistry of influenza virus–infected mice and control mice, injected with a biotinylated and PEGylated version of the Ly6C/G-specific nanobody, showed the presence of abundant Ly6C/G-positive myeloid cells and positivity for Ly6C/G on bronchial epithelium in influenza virus–infected mice. This is consistent with focal inflammation in the lungs, a finding that correlated well with the immuno-PET results. No such signals were detected in control mice. Having shown by PET the accumulation of the Ly6C/G-specific nanobody in infected lungs, we synthesized conjugates of Ly6C/G-specific nanobodies with dexamethasone to enable targeted delivery of this immunosuppressive corticosteroid to sites of inflammation. Such conjugates reduced the weight loss that accompanies infection, while the equivalent amount of free dexamethasone was without effect. Nanobody-drug conjugates thus enable delivery of drugs to particular cell types at the appropriate anatomic site(s). By avoiding systemic exposure to free dexamethasone, this strategy minimizes its undesirable side effects because of the much lower effective dose of the nanobody-dexamethasone conjugate. The ability to selectively target inflammatory cells may find application in the treatment of other infections or other immune-mediated diseases.

Immuno–positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)–based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv–based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4⁺ T cells by immuno-PET. We tracked CD4⁺ and CD8⁺ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4⁺ and CD8⁺ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4⁺ and CD8⁺ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents. Key Points In this study, we convert an anti-CD4 Ab into an scFv-based immuno-PET tracer. This short-lived PEGylated probe can noninvasively monitor CD4⁺ T cells in mice. This approach should be applicable to any clinically useful Ig.

We describe the production and use of nanobody drug conjugates that comprise of VHH _kappa , a nanobody that recognizes mouse immunoglobulin kappa light chains, and one or more copies of the small molecule influenza virus neuraminidase (NA) inhibitor, zanamivir. Such compounds achieve half-life extension of zanamivir, while recruiting polyclonal immunoglobulins of all isotypes regardless of specificity to infected cells for antibody-dependent cell-mediated cytotoxicity and complement-dependent cellular cytotoxicity. Since the influenza A virus (IAV) NA is a tetramer, we produced VHH _kappa adducts with 1, 2, or 4 zanamivir molecules attached in a site-specific manner, to allow multivalent engagement of NA. Administration of a VHH _kappa adduct modified with 4 zanamivir molecules (VHH _kappa –Zan ₄ ) was ~10-fold more potent in protection against infection with IAV than VHH _kappa –Zan carrying only a single zanamivir molecule. VHH _kappa –Zan ₄ can be given intranasally to confer full protection against a lethal IAV challenge. The neutralizing antibody titers in the respiratory mucosa and in the circulation, as well as the serum IgG antibody response against the hemagglutinin and nucleoprotein, are higher in VHH _kappa –Zan ₄ treated mice that survived the lethal challenge than in controls infected with a sublethal dose of virus. VHH _kappa –Zan ₄ affords protection even when given intranasally weeks prior to a challenge with a lethal dose of IAV. This type of adduct can therefore be applied prophylactically and therapeutically and does not require prior immunization for protection against a lethal dose of IAV.

Oncogenic mutations in the epidermal growth factor receptor (EGFR) promote tumorigenesis by stabilizing active or pre-active receptor conformations. Most EGFR-driven cancers are characterized by kinase domain mutations that directly activate the receptor. However, brain cancers such as glioblastoma multiforme (GBM) uniquely harbor mutations in the EGFR ectodomain that allosterically activate the kinase domain. Despite significant advances in understanding the physiologic and pathogenic roles of EGFR, the conformational characteristics that define ligand-independent EGFR activation in GBM remain poorly understood. In this study, we use naïve and post-immune yeast-displayed nanobody libraries to discover four nanobody groups that with benchmark nanobodies define a total of five groups with unique binding signatures and specificities for GBM mutation-stabilized conformational states. Nanobodies in groups 1 and 2 block ligand, selectively bind the inactive, tethered conformation, and favor wild-type EGFR over GBM-stabilized conformations. In contrast, nanobodies in groups 4 and 5 do not block ligand, target active or pre-active conformations, and selectively bind GBM-stabilized conformations. Additionally, nanobodies in group 3 block ligand and appear to be conformation agnostic. We observed domain-specific bias in the nanobodies' selectivity for GBM mutations, suggesting that mutations across different ectodomain regions stabilize distinct conformations. This work advances our understanding of EGFR conformational equilibria in the context of GBM. The observed cooperativity and mutation-dependent binding of nanobodies emphasize their utility in dissecting EGFR activation mechanisms and in developing targeted therapies for EGFR-driven cancers, including GBM.