Improved Brain Expression of Anti-Amyloid ß scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer.

BACKGROUND: The ever-increasing number of people living with Alzheimer's disease urges to develop more effective therapies. Despite considerable success, anti-Alzheimer immunotherapy still faces the challenge of intracerebral and intracellular delivery. This work introduces in situ production of anti-amyloid beta (Aß) antibody after intracerebral injection of PEG-PAsp(DET)/mRNA polyplexes as a novel immunotherapy approach and a safer alternative compared to high systemic antibodies doses or administration of adenovirus encoding anti- Aß antibodies. METHODS: We used mRNA encoding three different Aß-specific scFV with a secretion signal for passive immunotherapy. scFv contained a 6xHis-tag for immuno-detection. The secretion signal from IL2 (IL2ss) was added to allow extracellular engagement of senile plaques. Aß affinity of scFv was measured by surface plasmon resonance. To allow intracellular delivery, scFv were administered as polyplexes formed with our smart copolymer polyethylene glycol-poly[N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PEG-PAsp (DET)]. We evaluated scFv expression in cellulo by Western blot and ELISA, their ability to disaggregate amyloid aggregates by thioflavine T assay. Moreover, in vivo expression and therapeutic activity were evaluated in a murine amyloidosis model, by anti-6xHis-tag ELISA and anti- Aß ELISA, respectively. RESULTS: The selected anti-amyloid beta scFv showed affinity towards Aß and disaggregated Aß fibers in vitro. Whereas both DNA and mRNA transfection led to scFV expression in cancer cells, only mRNA led to detectable scFv expression in primary neurons. In addition, the use of IL2ss increased by 3.4-fold scFv secretion by primary neurons over mRNA polyplexes devoid of secretion signal. In vivo, a 3 to 11- fold of intracranial scFv levels was measured for mRNA compared to DNA polyplexes and higher in vivo scFv levels were obtained with mRNA containing IL2ss over non-secreted mRNA. Intracranial injection of anti-Aß mRNA polyplexes with IL2ss resulted in 40 % Aß decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aß decrease was detected in a more challenging transgenic model of Alzheimer's disease. CONCLUSION: Our results introduce a concerted approach not only for Alzheimer's disease treatment but also for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial delivery of anti-Aß scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aß in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer, IL2ss) were integrated into a single formulation.

Immunodominance of conformation-dependent B-cell epitopes of protein antigens.

Immunodominance of conformational epitopes over linear ones in four proteins was quantified making use of the B-cell hybridoma technology. The proteins were immunized in their native forms into BALB/c mice, and clonal frequencies of B-cell hybridomas that produce antibodies to the native and denatured forms were determined, using ELISA and immunoblotting. All 16 monoclonal antibodies (mAbs) to Porphyromonas gingivalis fimbria were suggested to recognize conformational epitopes expressed by the oligomer. Ten out of 14 mAbs to Serratia marcescens fimbria and 13 of 15 mAbs to hen lysozyme were also specific to their conformational epitopes. In contrast, all 18 mAbs to a surface protein of Streptococcus mutans, termed PAc, reacted to both the native and denatured forms, thereby indicating the immunodominance of linear epitopes in this protein. The results suggest that B-cell epitopes of proteins possessing stable tertiary or quaternary structures are predominantly expressed by the higher-order structures.