TE-5300

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 (Aβ) 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- Aβ antibodies. Methods: We used mRNA encoding three different Aβ-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. Aβ 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- Aβ ELISA, respectively. Results: The selected anti-amyloid beta scFv showed affinity towards Aβ and disaggregated Aβ 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-Aβ mRNA polyplexes with IL2ss resulted in 40 % Aβ decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aβ 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-Aβ scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aβ 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.

The major route of iron uptake by cells occurs through transferrin receptor (TfR)–mediated endocytosis of diferric-charged plasma transferrin (holo-Tf). In this work, we pursued TfR antibodies as potential cancer therapeutics, characterizing human single-chain variable antibody fragments (scFv) specific for the human TfR isolated from a phage display library. We hypothesized that many of these antibodies would function as ligand mimetics because scFvs from the library were selected for binding and internalization into living cells. In support of this hypothesis, the anti-TfR scFvs identified were antagonists of TfR binding to holo-Tf, particularly two of the most potent antibodies, 3TF12 and 3GH7, which blocked the in vitro proliferation of a number of hematopoietic cancer cell lines. We optimized this activity of 3TF12 and 3GH7 by engineering 55-kDa bivalent antibody formats, namely, F12CH and H7CH, which could block cell proliferation with an IC50 of 0.1 μg/mL. We found that the mechanism of the scFv antibody cytotoxicity was unique compared with cytotoxic anti-TfR monoclonal antibodies that have been described, causing cell surface upregulation of TfR along with the inhibition of holo-Tf cell uptake and induction of cell death. In a nude mouse model of erythroleukemia, administration of F12CH reduced tumor growth. Together, our findings define a new class of fully human anti-TfR antibodies suitable for immunotherapy against tumors whose proliferation relies on high levels of TfR and iron uptake, such as acute lymphoid and myeloid leukemias. Cancer Res; 70(13); 5497–506. ©2010 AACR.