Antibody and nanobody-based inhalation powders for therapeutic immune response in the lung

Over the past decades, the rise of biotechnological drugs has profoundly transformed the pharmaceutical landscape, enabling the production of complex biologically active molecules with high specificity and reduced off-target effects. Despite their clinical and commercial success, biopharmaceuticals face significant formulation challenges due to their intrinsic structural instability and the need for parenteral administration, which limits patient compliance and increases logistic costs. Pulmonary delivery of proteins as dry powders represents a promising alternative, offering the advantage of non-invasive administration, local targeting of respiratory diseases, and improved stability compared to liquid formulations. This doctoral project aimed to develop stable inhalable protein-based formulations produced by spray drying for antiviral response against SARS-CoV-2, used as a model pathogen. The work was divided into two main parts. The first phase focused on formulating spray-dried powders of rat hyperimmune serum containing polyclonal anti-SARS-CoV-2 antibodies. A broad screening of sugars and amino acids, alone or in dual combinations, was performed to identify excipients capable of preserving protein stability during dehydration and thermal stress. The dual-excipient formulation combining hydroxypropyl-β-cyclodextrin (HPβCD) and L-leucine displayed suitable aerodynamic properties (MMAD ≤ 5 µm; FPF 70–80%) and high immunoglobulin activity retention (>75%), confirming the feasibility of producing stable, inhalable polyclonal antibody preparations for antiviral prophylaxis. This work also identified excipient systems enabling facile formulation of spray-dried antibodies and provided the basis for developing more complex and less stable protein formulations. In the second phase of the project, the focus shifted to the recombinant fusion protein A12-IJ, an intrinsically less stable protein, more prone to aggregation. A12-IJ is a fusion construct consisting of an anti-SARS-CoV-2 nanobody fused to a non-toxic tetanus toxin (TT) fragment via a linker. This construct enables the redirection of pre-existing circulating anti-tetanus antibodies—arising from widespread global vaccination—toward the virus, leading to pathogen neutralization, potential Fc-mediated immune activation, and extended nanobody half-life. Optimization of buffer and excipient composition was crucial to ensure stability. Potassium phosphate (KP) buffer prevented precipitation, while NaCl and Tween 20 improved monomer retention in solution. However, incorporation of Tween 20 into spray-dried powders, though beneficial for activity retention, resulted in cohesive, poorly dispersible particles with inadequate aerodynamic performance. In the absence of surfactant, reformulation using a NaCl-enriched buffer and a higher content of HPβCD yielded significant improvement: formulation consisting of protein/HPβCD/L-Leucine (weight ratio 1:3.075:9.225) demonstrated excellent post-spray-drying activity retention (~90%), acceptable aerosol performance (FPF ~33%), and remarkable stability under accelerated storage conditions. Overall, this work demonstrates the importance of rational excipient selection, buffer optimization, and formulation design in developing stable protein-based powders for pulmonary delivery. The identified HPβCD/L-leucine system proved effective for both polyclonal and recombinant proteins, with specific optimization enabling the stabilization of the labile A12-IJ construct. These results establish a formulation strategy potentially extendable to other protein therapeutics intended for inhalation and lay the groundwork for future in vivo studies to evaluate the neutralizing efficacy of A12-IJ when administered directly to the lungs in an animal model.