Unlocking the potential of Nanobodies: Immunotagging for targeted therapy

Nanobodies, or VHHs, are single-domain antibodies derived from camelid heavy-chain antibodies, have emerged for their stability, specificity, and small size, which allows for superior tissue penetration and access to epitopes inaccessible to conventional antibodies. However, their lack of an Fc region limits their immune effector function and reduces circulation time. To address these limitations, this research introduces the ImmunoJunction (IJ) platform, which combines VHHs with an engineered ImmunoTag—a fragment of tetanus toxin capable of recruiting anti-tetanus antibodies present in over 85% of the global population. ImmunoTag facilitates immune engagement through opsonization and cellular activation, effectively redirecting pre-existing immunity to target various pathogens or tumor antigens. The modularity of the ImmunoJunction platform offers significant potential for both therapeutic and diagnostic applications. This thesis explores the development and characterization of nanobodies targeting critical proteins associated with viral and bacterial pathogenesis, specifically the Spike protein of SARS-CoV-2 and the virulence factors PhtD, PnrA, and pneumolysin of Streptococcus pneumoniae. The first part of the study centers on SARS-CoV-2, targeting its Spike protein as a key antigen. Initially, well-characterized nanobodies from the literature were expressed as recombinant proteins to assess their neutralizing capacity both independently and in conjugation with ImmunoTag. In vitro neutralization assays demonstrated enhanced viral neutralization with ImmunoJunction constructs, validating this approach. In collaboration with Preclinics, the study further advanced by generating novel anti-Spike and anti-nucleocapsid protein (N) nanobodies via a llama-derived immune cDNA library. Screening these libraries through phage display led to the isolation of promising nanobodies, which were then expressed in Escherichia coli and evaluated for their potential neutralizing capacity. The neutralization assays revealed significant differences in efficacy among the candidates, with nanobody H8 exhibiting the strongest neutralization activity against the Wuhan variant, and nanobodies A12 and G3 demonstrating effective neutralization against Omicron BA.1 and BA.4/5. These three most effective candidates were then expressed in Escherichia coli conjugated with ImmunoTag, but unfortunately only A12-IJ was expressed and purified to appreciable levels. The second part of the research focuses on Streptococcus pneumoniae, a clinically significant pathogen, particularly in the context of rising antibiotic resistance. This segment focused on creating immune libraries to identify nanobodies targeting surface proteins PhtD, PnrA, and Pneumolysin, which play critical roles in bacterial virulence and host colonization. VHH libraries against these antigens were constructed and underwent selection by phage display to identify potent binders. For selected candidates targeting PhtD and PnrA, E. coli expression protocols were optimized to increase VHH yield and solubility. Although solubility issues initially posed challenges, the use of autoinducing media and alternative E. coli strains improved the protein yield of four VHHs: A11 (PhtD), H2 (PhtD), H5 (PhtD), and D4 (PnrA). The purified proteins were then analyzed using a peptide ELISA assay to verify binding to their respective target proteins, with results indicated that H5 (PhtD) and D4 (PnrA) produced higher signals, suggesting a greater binding affinity to their respective targets. For pneumolysin, nearly 30 high-affinity nanobody candidates were isolated, with ongoing efforts directed toward sequencing, expression, and evaluation to assess their potential. In conclusion, this work exemplifies the versatility of nanobodies in targeted therapy, emphasizing their promise in tackling antibiotic-resistant bacteria and complex viral pathogens. The findings underscores the need to evaluate not only binding affinity but also functional efficacy, indicating the necessity for further studies into the neutralization mechanisms and interactions of these nanobodies. The introduction of the ImmunoJunction platform represents a novel strategy with its modular approach that harnesses widespread tetanus immunity to enhance nanobody potency and immune engagement. This innovation opens new possibilities for nanobody-based therapeutics and diagnostics.