Nanobody–Antigen Conjugates Elicit HPV-Specific Antitumor Immune Responses

Abstract

High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4 and CD8 T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHH) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8 T-cell responses against HPV tumors. Mice immunized with VHH conjugated to an H-2D-restricted immunodominant E7 epitope (E7) had more E7-specific CD8 T cells compared with those immunized with E7 peptide alone. These CD8 T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHH-E7 vaccination resulted in greater numbers of CD8 tumor-infiltrating lymphocytes compared with mice receiving E7 peptide alone in HPV tumor-bearing mice, as measured by noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers..

In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography

“…We then installed the (Gly) 3 -metal chelators 1,4,7-triazacyclononane-N,N’,N”-triacetic acid (NOTA) and desferrioxamine (DFO) for labeling with 64 Cu 2+ and 89 Zr 4+ , respectively. 22 We likewise used sortase to install a trans -cyclooctene (TCO)-functionalized peptide, followed by a ‘click’ reaction with a tetrazine-labeled- 18 F-2-deoxyfluoroglucose (FDG) produced by oxime ligation. 23 Free label was removed by size-exclusion chromatography.…”

“…Prior to in vivo application, we performed IFNγ enzyme-linked immunospot (ELISpot) assays to assess the specificity of synTacs in vitro . Mice were immunized with the HPV E7 peptide conjugated to an anti-CD11b VHH (VHH CD11b -E7), in the presence of adjuvant (polyIC and anti-CD40 Ab) 22 , or were infected with IAV to elicit antigen-specific CD8 T cell expansion. Splenocytes were then incubated with either the HPV E7 or IAV NP peptide, or with equimolar amounts of the corresponding synTacs.…”

Recognition of Class II MHC Peptide Ligands That Contain β-Amino Acids

“…We sought to overcome the negative impact of b-residue incorporation on T cell activation through directed delivery of peptide to APC. Conjugation of an MHC-binding peptide to proteins derived from the variable domain of camelid H chainonly Abs (VHHs or nanobodies) that target cell surface proteins on APCs enhances the immunogenicity of that peptide under immunostimulatory conditions (53,60,61). Site-specific and monovalent functionalization of proteins using catalysis by sortase A (sortagging) is our preferred method to generate such adducts (62).…”

“…6). To overcome this lack of activity, we made use of past observations that targeting of antigenic peptides to APC by conjugation to VHHs of appropriate specificity can enhance immunogenicity (53,60,61). To achieve this goal we needed to introduce b-amino acids in the context of a fulllength protein.…”

Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics

“…Utilizing the penetration capacity and structural simplicity of nanobodies, studies have explored the implementation of nanobody-based antigen conjugates to enhance DC-based immunity. Some target DC surface proteins such as CD11b ( 128 , 129 ), CD36 ( 128 ), and MHC-II ( 128 , 130 ), and others have been designed to block ICPs CTLA-4 ( 131 ), and PD-L1 ( 132 ) to enhance DC-mediated T cell activation. Kwon et al ( 133 ) developed a novel anti-MHC-II nanobody conjugated to cyclotides (cyclic, plant-derived peptides) that also demonstrated cyclotide-scaffold potential against constrained epitopes.…”