Background

In addition to supplying oxygen, red blood cells also function naturally as collectors of foreign materials in the circulation, forming immune complexes on their surface (referred to as opsonization). Cells of the reticuloendothelial system (e.g., macrophages in the liver and spleen) scan and collect red blood cell-bound immune complexes for degradation, disposal, and also to present them very effectively to the immune system for protection in the event of recurrence. Earlier reports indicate that immunogens bound to red blood cells by various chemical reactions outside the body generate greatly increased immune responses. The Augmenta technology causes vaccine immunogens and anti-toxin antibodies to self-target to the immune system via red blood cells, but without the need to withdraw blood.

Technology

A red blood cell immunotargeting fusion protein has been constructed for the purpose of potentiating vaccine-induced immune responses (see results). The concept has been reduced to practice in the mouse studies summarized below.

The mouse-directed fusion protein is being developed for various immediately accessible applications (monoclonal antibody production for research, diagnostics and therapeutics produced in normal and “humanized” mice), all of which will be sources of near-term revenue.

Analogous fusion proteins will be suitable for immunizing other species of animals for the production of monoclonal and polyclonal antibodies, as well as for animal health vaccines. Finally, prophylactic and therapeutic vaccines will be improved with the Augmenta technology, with applications in infectious, malignant and other disease areas amenable to vaccine intervention.

    

 

Immunization

Augmenta’s immunotargeting strategy uses a fusion protein (FP) comprised of a fragment derived from an anti-red blood cell monoclonal antibody (scFv) and streptavidin (StAv), a molecule that binds biotin very tightly. The test vaccine immunogen is M2e, a peptide found on the surface of all strain A influenza viruses, including seasonal flu, H5N1 or "bird flu", and H1N1 or "swine flue". The M2e peptide bearing a biotin group was combined with the FP ex vivo and injected into mice using a conventional immunization schedule. Serum was analyzed by ELISA for the production of anti-M2e antibodies. Mice immunized with M2e peptide alone or peptide in combination with the adjuvant alum generated a modest IgG response. By comparison, much smaller amounts of M2e coupled to the FP ex vivo generated a potent immune response. In fact, 37 ng of FP-borne M2e peptide elicited a more potent immune response than 30µg of unconjugated peptide. The total immunogenicity increase imparted by the FP was 2,000-fold greater than for peptide alone or with alum adjuvant.

This result demonstrates that routing immunogens to the surface of red blood cells greatly augments their immunogenicity in the absence of exogenous immune adjuvants. The strategy has immediate and long-term applicability to a variety of immunization strategies.

These findings have been demonstrated for intravenous, sub-cutaneous and intra-muscular routes of administration. They have also been corroborated by several independent groups.


Toxin clearance results

An independent evaluation of the fusion protein (FP) was recently conducted in collaboration with the lab of Dr. Scott Dessain at the Lankenau Institute for Medical Research in Wynnewood, PA. Instead of immunization, the FP was used to help clear botulinum neurotoxin from the bloodstream of mice using the lab's high affinity human monoclonal antibodies (HuMabs, 6A and 4LCA in the figure) against the toxin, each of which neutralizes a certain dose of toxin. When these antibodies bind botulinum toxin, toxin-HuMab immune complexes form and circulate in the blood for some time. This binding by HuMabs prevents toxicity. However, unless rapidly cleared from the circulation, some toxin can detach from the circulating complex and is free to bind neurons, causing lethal paralysis. In recent work, these HuMabs were biotinylated and coupled to Augmenta's FP which led to the complete healthy survival of mice against up to 5,000 times the lethal toxin dose. This represents up to 600-fold increase in the neutralizing potency of the antitoxin antibodies (see accompanying figure). The interpretation is that the fusion protein transports the Mab-toxin complexes to the RBC and thence to the liver and spleen where they are rapidly degraded, preventing toxin from release. This provides added support to the proposed mechanism of action of the FP in a non-immunization setting. It also introduces a second major application for this technology in toxin clearance as a potential countermeasure against bioterror toxins or other pathogens.


Intellectual Property

Augmenta Biologicals’ technology is protected by pending patent applications (e.g., WO2007150020)