Phenotypic knockouts of selected metabolic pathways by targeting enzymes with camel-derived nanobodies (V(HH)s).

Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditional shutdown of enzymatic activities once the corresponding proteins have been produced. We show that given biochemical functions can be entirely suppressed in vivo with camel antibodies (VHHs, nanobodies) that target active sites of cognate enzymes expressed in the cytoplasm. As a proof of principle, we raised VHHs against 2,5-dihydroxypyridine dioxygenase (NicX) of Pseudomonas putida, involved in nicotinic acid metabolism. Once fused to a thioredoxin domain, the corresponding nanobodies inhibited the enzyme both in Escherichia coli and in P. putida cells, which then accumulated the metabolic substrate of NicX. VHHs were further engineered to track the antigen in vivo by C-terminal fusion to a fluorescent protein. Conditional expression of the resulting VHHs allows simultaneously to track and target proteins of interest and enables the design of transient phenotypes without mutating the genetic complement of the bacteria under study.

Immune response with long-term memory triggered by amorphous aggregates of misfolded anti-EGFR VHH-7D12 is directed against the native VHH-7D12 as well as the framework of the analogous VHH-9G8.

We previously demonstrated that amorphous aggregates of misfolded VHH-7D12 antibodies (VHH-Mis), a potential anti-EGFR drug, can generate a robust serum IgG response. Here we investigate the immunogenic nature, especially the specificity of the immune response induced by VHH-Mis. To this end, we used two natively folded and 77% identical anti-EGFR VHHs (VHH-7D12 and VHH-9G8) that possess a common framework but distinct complementarity determining regions (CDRs). In 60% of mice immunized with VHH-Mis, the anti-VHH-7D12 IgG titer was stronger than the anti-VHH-9G8 titer (Group-1). In the remaining mice (40%; Group-2), the anti-VHH-7D12 and anti-VHH-9G8 titer were almost identical. We rationalized these results by hypothesizing that mice in Group-1 produced IgG mostly against the VHH-7D12's CDRs, whereas in Group-2 mice, they targeted the VHH's framework. The IgG specificity against VHH-7D12 and VHH-9G8 was essentially unchanged over 17 weeks in both groups. Further, in all mice (Group-1&2) re-immunized with native VHH-7D12, the IgG titer against VHH-7D12 increased sharply but not against VHH-9G8. On the other hand, none of the three Group-1 mice re-immunized with native VHH-9G8 showed immunogenicity against VHH-7D12 nor VHH-9G8. Whereas, in Group-2 mice (three/three) re-immunized with VHH-9G8, the IgG titers against both VHHs increased but slowly. Flow-cytometric studies showed that VHH-Mis immunized mice generated a higher number of effector and central memory T-cells. Overall, these observations indicate that amorphous aggregates made of a misfolded VHH can induce serum IgG against its natively folded self and analogous VHHs having a similar framework but distinct CDRs. Furthermore, a robust long-term immune response with memory was established against its natively folded self but with a nil-to-moderate immune response against natively folded VHH analogs.

Nanobodies® as inhaled biotherapeutics for lung diseases.

Local pulmonary delivery of biotherapeutics may offer advantages for the treatment of lung diseases. Delivery of the therapeutic entity directly to the lung has the potential for a rapid onset of action, reduced systemic exposure and the need for a lower dose, as well as needleless administration. However, formulation of a protein for inhaled delivery is challenging and requires proteins with favorable biophysical properties suitable to withstand the forces associated with formulation, delivery, and inhalation devices. Nanobodies are the smallest functional fragments derived from a naturally occurring heavy chain-only immunoglobulin. They are highly soluble, stable, and show biophysical characteristics that are particularly well suited for pulmonary delivery. This paper highlights a number of clinical and preclinical studies on antibodies delivered via the pulmonary route and describes the advantages of using Nanobodies for inhaled delivery to the lung. The latter is illustrated by the specific example of ALX-0171, a Nanobody in clinical development for the treatment of respiratory syncytial virus (RSV) infections.