RESUMEN
The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 1011 total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.
Asunto(s)
Vectores Genéticos , Lentivirus , Lentivirus/genética , Ligandos , Reproducibilidad de los Resultados , Terapia Genética/métodosRESUMEN
Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody-antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (VHH) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody-ligand complex and lowers its aggregation temperature to favor crystallization. The VHH crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Cristalización/métodos , Cristalografía por Rayos X/métodos , Fragmentos Fab de Inmunoglobulinas/química , Cadenas kappa de Inmunoglobulina/química , Agregado de Proteínas , Anticuerpos de Dominio Único/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Alineación de SecuenciaRESUMEN
Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example. To that end, we have previously reported the expression of a novel protein vaccine for malaria using the ExpreS2Drosophila melanogaster Schneider 2 stable cell line system, however, a very low overall process yield (typically <5% recovery of hexa-histidine-tagged protein) meant the initial purification strategy was not suitable for scale-up and clinical biomanufacture of such a vaccine. Here we describe a newly available affinity purification method that was ideally suited to purification of the same protein which encodes the P. falciparum reticulocyte-binding protein homolog 5 - currently the leading antigen for assessment in next generation vaccines aiming to prevent red blood cell invasion by the blood-stage parasite. This purification system makes use of a C-terminal tag known as 'C-tag', composed of the four amino acids, glutamic acid - proline - glutamic acid - alanine (E-P-E-A), which is selectively purified on a CaptureSelect™ affinity resin coupled to a camelid single chain antibody, called NbSyn2. The C-terminal fusion of this short C-tag to P. falciparum reticulocyte-binding protein homolog 5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated Schneider 2 cell supernatant under mild conditions. Biochemical and immunological analysis showed that the C-tagged and hexa-histidine-tagged P. falciparum reticulocyte-binding protein homolog 5 proteins are comparable. The C-tag technology has the potential to form the basis of a current good manufacturing practice-compliant platform, which could greatly improve the speed and ease with which novel protein-based products progress to clinical testing.
Asunto(s)
Proteínas Portadoras/química , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/metabolismo , Animales , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Línea Celular , Clonación Molecular , ConejosRESUMEN
Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process-related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VH H antibody fragments as "tunable" immunoaffinity ligands for separation of product-related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma-carboxylglutamic acid domain.
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Cromatografía de Afinidad/métodos , Anticuerpos/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Exotoxinas/aislamiento & purificación , Protrombina/aislamiento & purificaciónRESUMEN
INTRODUCTION: Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. AIMS: To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. METHODS AND RESULTS: The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of â¼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. CONCLUSIONS: Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin.
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Factor VIII/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Electroforesis en Gel de Poliacrilamida , Factor VIII/química , Células HEK293 , Humanos , Proteínas Recombinantes/química , Reproducibilidad de los ResultadosRESUMEN
Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of EPO in a high-throughput setting.
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Eritropoyetina/análisis , Anticuerpos de Cadena Única/química , Animales , Células CHO , Camelus , Cricetinae , Cricetulus , Eritropoyetina/inmunología , Humanos , Concentración de Iones de Hidrógeno , Inmunoensayo , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established.
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Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , Fragmentos Fab de Inmunoglobulinas , Químicos de Laboratorio , Animales , Células CHO , Cricetinae , Cricetulus , Escherichia coli , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/metabolismo , Químicos de Laboratorio/química , Químicos de Laboratorio/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Ever since the introduction of bacterial derived surface proteins like protein A that demonstrate a natural binding reactivity towards antibodies, affinity chromatography has evolved into a well-established technology for the purification of antibodies and antibody fragments. Although high selectivity is provided by these types of affinity ligands, not all antibodies or antibody fragments are covered, which then forces the use of non-affinity-based processes that are less selective and often result in lower one-step purity and yield. To fill these gaps, we here describe a novel range of CaptureSelect(™) affinity resins that enables immunoaffinity chromatography for a much broader range of antibody targets.
Asunto(s)
Anticuerpos/aislamiento & purificación , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Cromatografía de AfinidadRESUMEN
Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Camélidos del Nuevo Mundo/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Proteínas Recombinantes/inmunología , Infecciones por Rotavirus/virología , Rotavirus/inmunología , Animales , Anticuerpos Neutralizantes/uso terapéutico , Western Blotting , Diarrea Infantil/virología , Genotipo , Humanos , Inmunización , Fragmentos de Inmunoglobulinas/uso terapéutico , Lactante , Ratones , Proteínas Recombinantes/uso terapéutico , Rotavirus/genética , Rotavirus/patogenicidad , Infecciones por Rotavirus/tratamiento farmacológicoRESUMEN
Myasthenia gravis is a paralytic disorder with autoantibodies against acetylcholine receptors at the neuromuscular junction. A proportion of patients instead has antibodies against muscle-specific kinase, a protein essential for acetylcholine receptor clustering. These are generally of the immunoglobulin-G4 subclass and correlate with disease severity, suggesting specific myasthenogenic activity. However, immunoglobulin-G4 subclass antibodies are generally considered to be 'benign' and direct proof for their pathogenicity in muscle-specific kinase myasthenia gravis (or other immunoglobulin-G4-associated disorders) is lacking. Furthermore, the exact electrophysiological synaptic defects caused at neuromuscular junctions by human anti-muscle-specific kinase autoantibodies are hitherto unknown. We show that purified immunoglobulin-G4, but not immunoglobulin-G1-3, from patients with muscle-specific kinase myasthenia gravis binds to mouse neuromuscular junctions in vitro, and that injection into immunodeficient mice causes paralysis. Injected immunoglobulin-G4 caused reduced density and fragmented area of neuromuscular junction acetylcholine receptors. Detailed electrophysiological synaptic analyses revealed severe reduction of postsynaptic acetylcholine sensitivity, and exaggerated depression of presynaptic acetylcholine release during high-rate activity, together causing the (fatigable) muscle weakness. Intriguingly, compensatory transmitter release upregulation, which is the normal homeostatic response in acetylcholine receptor myasthenia gravis, was absent. This conveys extra vulnerability to neurotransmission at muscle-specific kinase myasthenia gravis neuromuscular junctions. Thus, we demonstrate that patient anti-muscle-specific kinase immunoglobulin-G4 is myasthenogenic, independent of additional immune system components, and have elucidated the underlying electrophysiological neuromuscular junction abnormalities.
Asunto(s)
Inmunoglobulina G/efectos adversos , Inmunoglobulina G/sangre , Miastenia Gravis/sangre , Enfermedades de la Unión Neuromuscular/complicaciones , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Potenciales de Acción/efectos de los fármacos , Adulto , Animales , Autoanticuerpos/sangre , Modelos Animales de Enfermedad , Electromiografía , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Placa Motora/efectos de los fármacos , Placa Motora/fisiopatología , Contracción Muscular/efectos de los fármacos , Fuerza Muscular/efectos de los fármacos , Fuerza Muscular/fisiología , Miastenia Gravis/complicaciones , Miastenia Gravis/inmunología , Miastenia Gravis/terapia , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Unión Neuromuscular/ultraestructura , Enfermedades de la Unión Neuromuscular/patología , Plasmaféresis/métodos , Adulto JovenRESUMEN
AIMS: Using genetically engineered lactobacilli, producing high avidity llama VHH domains (referred to as anti-rotavirus proteins; ARPs), to test the effect of multimeric antibody fragments as prophylaxis and therapy against rotavirus infection. METHODS: Two ARPs, ARP1 and ARP3, shown to bind to different epitopes and act synergistically against rotavirus, were displayed on the surface of Lactobacillus paracasei as monovalent or bivalent proteins (mono- or bi-specific). RESULTS: Although a nonsignificant difference was observed between lactobacilli producing bispecific ARP3-ARP1 and monomeric ARPs, lactobacilli producing bispecific ARP3-ARP1 were superior at reducing the rate of diarrhea when used for prophylactic and therapeutic intervention in a mouse model of rotavirus infection in comparison to nontreated animals. CONCLUSION: Expression of bispecific antibodies in lactobacilli resulted in slight improvement of their efficacy. Furthermore, increasing the specificity would theoretically reduce the rate of appearance of viral escape mutants and would have a broader capacity to be effective against a range of viral serotypes.
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Anticuerpos Biespecíficos/biosíntesis , Camélidos del Nuevo Mundo/inmunología , Diarrea/prevención & control , Lactobacillus/metabolismo , Infecciones por Rotavirus/prevención & control , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Camélidos del Nuevo Mundo/genética , Diarrea/virología , Inmunización Pasiva , Lactobacillus/genética , Ratones , Ratones Endogámicos BALB C , Rotavirus/inmunología , Infecciones por Rotavirus/virologíaRESUMEN
Toxic-shock syndrome is primarily caused by the Toxic-shock syndrome toxin 1 (TSST-1), which is secreted by the Gram-positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and V(beta)-specific T-cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy-chain antibodies (VHH) in the immuno capturing of TSST-1 from plasma. Data is presented that the selected VHHs are highly specific for TSST-1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST-1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4 ng/mL TSST-1 more than 96% of TSST-1 was depleted from pig plasma. These data pave the way to further explore application of high-affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans.
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Antígenos Bacterianos/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Cromatografía de Afinidad/métodos , Enterotoxinas/aislamiento & purificación , Plasma/química , Staphylococcus aureus/patogenicidad , Superantígenos/aislamiento & purificación , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Camélidos del Nuevo Mundo , Enterotoxinas/inmunología , Enterotoxinas/metabolismo , Humanos , Unión Proteica , Sensibilidad y Especificidad , Superantígenos/inmunologíaRESUMEN
Large scale, highly specific purification of valuable proteins from blood and removal of undesirable components promise to have wide therapeutic applications. Moreover, depletion of bulk proteins from blood is a prerequisite for clinical proteomics. Here we describe the development of specific, high affinity Camelid antibody fragments (VHH) derived from immune libraries for purification and depletion of the bulk protein HSA and IgG from human serum and plasma for therapeutic and research purposes. The anti-IgG VHH substantially improved depletion of IgGs from blood over the classical method based on protein A. To demonstrate the improved performance of VHH based IgG depletion, we analyzed the presence of auto-antibodies in human plasma before and after depletion from two groups of patients with auto-immune disease: Goodpasture syndrome (GP) and systemic lupus erythematosus (SLE). VHHs can be produced efficiently and cost effectively in Saccharomyces cerevisiae, a genetically regarded as safe (GRAS) microorganism. A good manufacturing process (GMP) for purification of these VHHs has also been developed. Moreover, as VHHs are single protein chains, they can be coupled relatively easily to solid matrices. These three factors are important for developing affinity purification medication.
Asunto(s)
Marcadores de Afinidad , Anticuerpos Antiidiotipos/metabolismo , Cromatografía de Afinidad , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Albúmina Sérica/inmunología , Albúmina Sérica/metabolismo , Animales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Camélidos del Nuevo Mundo , Humanos , Ligandos , Unión ProteicaRESUMEN
BACKGROUND: Rotavirus-induced diarrhea poses a worldwide medical problem in causing substantial morbidity and mortality among children in developing countries. We therefore developed a system for passive immunotherapy in which recombinant lactobacilli constitutively express neutralizing variable domain of llama heavy-chain (VHH) antibody fragments against rotavirus. METHODS: VHH were expressed in Lactobacillus paracasei, in both secreted and cell surface-anchored forms. Electron microscopy was used to investigate the binding efficacy of VHH-expressing lactobacilli. To investigate the in vivo function of VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. RESULTS: Efficient binding of the VHH antibody fragments to rotavirus was shown by enzyme-linked immunosorbent assay and scanning electron microscopy. VHH fragments expressed by lactobacilli conferred a significant reduction in infection in cell cultures. When administered orally, lactobacilli-producing surface-expressed VHH markedly shortened disease duration, severity, and viral load in a mouse model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried form. CONCLUSIONS: Transformed lactobacilli may form the basis of a novel form of prophylactic treatment against rotavirus infections and other diarrheal diseases.
Asunto(s)
Anticuerpos Antivirales/inmunología , Diarrea/prevención & control , Inmunización Pasiva/métodos , Cadenas Pesadas de Inmunoglobulina/inmunología , Lactobacillus/genética , Infecciones por Rotavirus/prevención & control , Rotavirus/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Camélidos del Nuevo Mundo/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Heces/virología , Vectores Genéticos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Lactobacillus/inmunología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Unión ProteicaRESUMEN
Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.
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Camélidos del Nuevo Mundo/inmunología , Fragmentos Fab de Inmunoglobulinas/fisiología , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/inmunología , Transducción de Señal/inmunología , Animales , Sitios de Unión de Anticuerpos , Unión Competitiva/inmunología , Células Cultivadas , Escherichia coli/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Neisseria meningitidis/inmunología , Unión Proteica/inmunologíaRESUMEN
Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.