Nanobodies have recently emerged as alternatives to classical antibodies in therapeutic and diagnostic contexts from parasites to bacteria to viruses, promising improved stability and simpler manufacturing. To improve nanobody discovery efficiency, we developed an integrated experimental and computational pipeline for detailed characterization of the target binding properties of complete alpaca immune repertoires using our custom Nanobody Meta-clustering Analysis Platform (NanoMAP). We tested our pipeline on three distinct pools of targets, immunizing two alpacas with each pool and generating cDNA and phage display libraries from their immune repertoires. We then panned the phage libraries on each target. To produce more detailed binding information, we performed panning variations using subunits, natural variants, intact pathogens, and binding site competitors. Deep sequencing reads from nanobody libraries before and after each panning were pooled and analyzed with NanoMAP to identify nanobody clonal families and assess their levels of enrichment from the library in each panning, reflecting their affinities. NanoMAP outperformed standard clustering methods, producing clonal families that are coherent in sequence and function and detecting rare but high affinity families. By aggregating sequencing data within clonal families, NanoMAP produced reliable and rich data on nanobody repertoire binding phenotypes for each antigen, enhancing nanobody discovery capabilities.

Background. Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the gut and causes enteritis through bacterial attachment-effacement (A/E), leading to mucosal damage. This facilitates systemic uptake of Stx, which can often lead to hemolytic-uremic syndrome (HUS) and acute renal failure, particularly in children <5 years of age. STEC infections expressing Stx2 variants are a major risk factor for HUS. Beyond patients receiving supportive care, there is no treatment for STEC-mediated HUS, and antibiotics are contraindicated. The 3- to 7-day prodromal interval between onset of STEC diarrhea and HUS offers a window for intervention for diarrheic patients, contacts thereof, or individuals exposed to the source of infection. Methods. Citrobacter rodentium (Cr), a mouse pathogen that exhibits mucosal bacterial A/E, was genetically constructed to express Stx2d (Cr-Stx2d). This, along with a Cr parent strain carrying the kanamycin gene, were used to model STEC in mice. Mice were treated intraperitoneally with a broad subtype-specific, Stx-neutralizing nanobody multimer fused to the human IgG1 Fc domain (VNA2-Stx/hFc). Results. When a single treatment was administered as late as 4 days after Cr-Stx2d challenge, likely within the human STEC-HUS window of intervention, complete protection of mice was achieved. Conclusions. This Cr-Stx2d model, which mimics elements of human STEC infections by causing mucosal gut lesions and systemic Stx2d-mediated kidney damage, will help evaluate specific treatments against STEC-HUS, as demonstrated here with VNA2-Stx/hFc-treated mice given a single injection well after bacterial challenge. This product should be safe, simple, and economical to manufacture.

Safe and efficient nucleic acid delivery to targeted cell populations remains a challenge in the fields of cell and gene therapy. Toward this end, we attempted to utilize the "DogTag-DogCatcher" system to target adenoviral vectors. "DogTag" is a short peptide that forms a spontaneous isopeptide bond upon mixing with its partner protein, "DogCatcher." We genetically incorporated the DogTag peptide into the protein responsible for initial binding of the virus to its target cell, the fiber. This allowed permanent linking of DogCatcher-fused single-domain or single-chain antibodies at the fiber. This modification allowed simple, effective, and exclusive targeting of the vector to cells bound by the linked antibody. This enhanced gene transfer into primary B and T cells by up to 60-fold in vitro and 2- to 3-fold in vivo in mice without other alterations to vector tropism. Although the system's in vivo performance is currently suboptimal and additional engineering is needed prior to further use, these studies form the basis of a novel method for targeting adenovirus that can be combined with additional well-characterized adenovirus modifications toward applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

Safe and efficient nucleic acid delivery to targeted cell populations remains a significant unmet need in the fields of cell and gene therapy. Towards this end, we pursued Adenoviral vectors genetically modified with the "DogTag" molecular glue peptide, which forms a spontaneous covalent bond with its partner protein, "DogCatcher". Genetic fusion of DogCatcher to single-domain or single-chain antibodies allowed covalent tethering of the antibody at defined locales on the vector capsid. This modification allowed simple, effective and exclusive targeting of the vector to cells bound by the linked antibody. This dramatically enhanced gene transfer into primary B and T cells in vitro and in vivo in mice. These studies form the basis of a novel method for targeting Adenovirus that is functional in stringent in vivo contexts and can be combined with additional well characterized Adenovirus modifications towards applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

The application of chimeric antigen receptor (CAR) T cell therapy in solid tumors is hindered by life-threatening toxicities resulting from on-target, off-tumor killing of nonmalignant cells that express low levels of the target antigen. Mesothelin (MSLN) has been identified as a target antigen for CAR T cell treatment of mesothelioma, lung, ovarian, and other cancers because of its high expression on tumor cells and limited expression on mesothelial cells. However, fatal off-tumor toxicity of high-affinity MSLN-targeting CAR T cells has been reported in multiple clinical trials. In this study, we constructed CARs using mutant variants of a single-domain nanobody that bind both human and mouse MSLN with a wide range of affinities and examined tumor responses and their toxicities from on-target, off-tumor interactions in mouse models. CAR T cells with low nanomolar affinity (equilibrium dissociation constant, KD) exhibited profound systemic expansion with no apparent infiltration into the tumor. With a gradual reduction of CAR affinity toward the micromolar KD, the expansion of CAR T cells became more restricted to tumors. Our preclinical studies demonstrated that high-affinity MSLN CARs were associated with fatal on-target, off-tumor toxicity and that affinity-tuned CARs rendered T cells more selective for MSLN-high tumors.

The remarkable capacity of bacteria to adapt in response to selective pressures drives antimicrobial resistance. Pseudomonas aeruginosa illustrates this point, establishing chronic infections during which it evolves to survive antimicrobials and evade host defenses. Many adaptive changes occur on the P. aeruginosa cell surface but methods to identify these are limited. Here we combine phage display with high-throughput DNA sequencing to create a high throughput, multiplexed technology for surveying bacterial cell surfaces, Phage-seq. By applying phage display panning to hundreds of bacterial genotypes and analyzing the dynamics of the phage display selection process, we capture important biological information about cell surfaces. This approach also yields camelid single-domain antibodies that recognize key P. aeruginosa virulence factors on live cells. These antibodies have numerous potential applications in diagnostics and therapeutics. We propose that Phage-seq establishes a powerful paradigm for studying the bacterial cell surface by identifying and profiling many surface features in parallel.

Botulinum neurotoxins are some of the most potent natural toxins known; they cause flaccid paralysis by inhibiting synaptic vesicle release. Some serotypes, notably serotype A and B, can cause persistent paralysis lasting for several months. Because of their potency and persistence, botulinum neurotoxins are now used to manage several clinical conditions, and there is interest in expanding their clinical applications using engineered toxins with novel substrate specificities. It will also be beneficial to engineer toxins with tunable persistence. We have investigated the potential use of small-molecule proteolysis-targeting chimeras (PROTACs) to vary the persistence of modified recombinant botulinum neurotoxins. We also describe a complementary approach that has potential relevance for botulism treatment. This second approach uses a camelid heavy chain antibody directed against botulinum neurotoxin that is modified to bind the PROTAC. These strategies provide proof of principle for the use of two different approaches to fine tune the persistence of botulinum neurotoxins by selectively targeting their catalytic light chains for proteasomal degradation.

Schistosomiasis is a globally prevalent, debilitating disease that is poorly controlled by chemotherapy and for which no vaccine exists. While partial resistance in people may develop over time with repeated infections and treatments, some animals, including the brown rat (Rattus norvegicus), are only semi-permissive and have natural protection. To understand the basis of this protection, we explored the nature of the immune response in the brown rat to infection by Schistosoma mansoni. Infection leads to production of IgG to parasite glycoproteins with complex-type N-glycans that contain a non-mammalian-type modification by core α2-Xylose and core α3-Fucose (core Xyl/Fuc). These epitopes are expressed on the surfaces of schistosomula and adult worms. Importantly, IgG to these epitopes can kill schistosomula by a complement-dependent process in vitro. Additionally, sera from both infected rhesus monkey and infected brown rat were capable of killing schistosomula in a manner inhibited by glycopeptides containing core Xyl/Fuc. These results demonstrate that protective antibodies to schistosome infections in brown rats and rhesus monkeys include IgG responses to the core Xyl/Fuc epitopes in surface-expressed N-glycans, and raise the potential of novel glyco-based vaccines that might be developed to combat this disease.

New drug platforms are needed which enable the directed delivery of therapeutics to sites of disease to maximize efficacy and limit off-target effects. Here, we report the development of PROT3EcT, commensal Escherichia coli engineered for the direct secretion of proteins into their surroundings. PROT3EcT are composed of four modular components: an E. coli chassis, a modified bacterial protein secretion system, a regulatable transcriptional activator, and a secretable therapeutic payload. PROT3EcT that secrete functional single-domain antibodies, nanobodies (Nb), stably colonize and maintain a functional secretion system within the intestines of mice. A single prophylactic dose of PROT3EcT that secretes a tumor necrosis factor alpha (TNFα) neutralizing Nb is sufficient to ablate TNF levels and prevent the development of injury and inflammation in a chemically-induced model of inflammatory bowel disease. This work lays the foundation for the development of PROT3EcT as a therapeutic platform for the treatment of at least gastrointestinal-based diseases.

Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.

ABSTRACT Bacillus anthracis , the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors.

Abstract Ricin belongs to the A-B toxin family that also includes Shiga and cholera toxins. While immunity to ricin is antibody (Ab)-mediated, it is unclear what functional qualities of Abs are associated with immunity to ricin, considering that Abs with potent toxin-neutralizing activity (TNA) in vitro are not always protective in vivo. We recently produced a collection of alpaca heavy-chain only Abs (VHHs) specific for ricin’s enzymatic (RTA) and binding (RTB) subunits. To better define the attributes associated with immunity to ricin, we engineered VHH homodimers and heterodimers and characterized them in vitro and in vivo. Dimeric VHHs had 10-fold improved binding affinities for ricin and had 2-3 times increased TNA as compared to their monomeric constituents. However, neither improved affinity nor increased TNA correlated with a VHH’s ability to protect in vivo. Instead, immunity was associated with heterodimers composed of an RTA and an RTB monomer in which at least one had TNA. Analytical ultracentrifugation showed that all five protective heterodimers identified in this study promoted ricin aggregation in solution. The same five VHHs also prevented ricin from binding to cell surfaces in vitro. We propose that toxin aggregation and/or inhibition of attachment are likely key determinants of antibody-mediated protective immunity to ricin and possibly other A-B family members.

ABSTRACT Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of influenza virus-encoded proteins. We generated a panel of camelid-derived single-domain antibody fragments (VHHs) against influenza virus nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza virus genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (αNP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the viral ribonucleoprotein (vRNP) complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle. IMPORTANCE Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function and determining how to disrupt said function through pharmaceutical intervention are difficult. We present a novel method based on single-domain-antibody technology that permits specific targeting and disruption of an essential influenza virus protein in the absence of genetic manipulation of influenza virus itself. Characterization of such interactions may help identify new targets for pharmaceutical intervention. This approach can be extended to study proteins encoded by other viral pathogens.