Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2000-07-05
2003-01-14
Saucier, Sandra E. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C435S002000
Reexamination Certificate
active
06506381
ABSTRACT:
BACKGROUND OF THE INVENTION
The red blood cell is a dominant presence in the circulatory system, representing approximately 98% of the formed elements which are present. Therefore, these cells can be viewed as a potentially important deployment platform for a variety of biomolecules which can be attached and displayed on the surface of the red blood cells. Barriers to some forms of such a deployment strategy include, for example, the fact that such modified red blood cells may be short-lived in circulation, thereby rendering them less effective. A strategy for the successful development of a red blood cell platform could provide a means for the treatment/and or prevention of a wide range of human disorders.
SUMMARY OF THE INVENTION
The present invention relates to modified red blood cells which function as deployment platforms for important biomolecules. Such modified red blood cells can confer, for example, in vivo protection against exposure to an otherwise lethal nerve agent.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery that red blood cells, modified as described herein, can function as a successful deployment platform for important biomolecules. More specifically, as discussed in the Exemplification section which follows, Applicant has demonstrated in vivo protection against exposure of an animal to an otherwise lethal nerve agent. Protection was provided by decorating red blood cells in the animal with an enzyme capable of degrading the nerve agent.
Thus, the present invention relates, in one aspect, to a modified red blood cell which is relatively long-lived in circulation, the modified red blood cell bearing on its surface at least one biomolecule capable of neutralizing challenge by an endogenous or exogenous agent. As used herein, the expression “long-lived in circulation” will be defined within the context of ex vivo modification and reintroduction. That is, when red blood cells are removed from an animal, modified ex vivo and reintroduced into the animal, the long-lived criteria is satisfied when at least about 70% remain in circulation 24 hours after reintroduction.
The expression “biomolecule”, as used herein, refers to any molecule which may be found in a living organism. With respect to the present invention, proteins are the most significant class of biomolecules. However, other important classes of biomolecules are included within the scope of the present invention, including, for example, carbohydrates. In general, the role of the biomolecule on the surface of the red blood cell is to either 1) act as an affinity reagent, specifically binding to another biomolecule; or 2) act as a molecular tool, modifying or degrading a biomolecule of interest.
As mentioned above, the proteins are a particularly significant class of biomolecules. The proteins include such important species as antibodies (which are useful as affinity reagents) and enzymes (which can catalyze the modification or degradation of a biomolecule of interest).
As used herein, the expression “endogenous” refers to agents (e.g., chemical or biological agents) which are typically found in the animal of interest. The expression “exogenous” refers to agents which are not typically found in the organism of interest. Issues to be considered in connection with the neutralization of endogenous versus exogenous agents using a biomolecule fixed to a red blood cell platform are not necessarily identical. The experiments described in the Exemplification section which follows relate specifically to an exogenous chemical agent.
Modified red blood cells of the type described herein are capable of neutralizing challenge by an endogenous or exogenous agent. The expression “neutralizing challenge” can not be defined precisely for all endogenous or exogenous agents. Rather, one must consider each endogenous or exogenous agent on a case by case basis, and consider the consequences of exposure or challenge by such agents to determine the meaning of the term “neutralizing”.
Consider, for example, exogenous biological agents such as bacteria or viruses. Certain pathologies are associated with bacterial or viral infection—such pathologies can be determined by reference to medical handbooks. “Neutralization”, as used herein, can refer, for example, to the prevention, elimination, mitigation, or delay in onset of such pathologies.
In the Exemplification section set forth below, an exogenous chemical agent is considered. More specifically, a toxic nerve agent is introduced into an animal carrying modified red blood cells of the present invention. In the absence of the modified red blood cells, animals exposed to the nerve agent die. Thus, in this context, “neutralization” refers to the fact that animals carrying the modified red blood cells survive.
An example which relates to an endogenous agent is LDL cholesterol. It is known that LDL cholesterol is found in vivo in both oxidized and reduced states. It is the oxidized form of LDL cholesterol that is dangerous. It is ingested by the cells of an atherosclerotic plaque which swell causing occlusion. Certain individuals apparently underexpress the enzyme responsible for maintaining LDL cholesterol in the reduced form (glutathione peroxidase). A method of therapy in such individuals is to deploy this enzyme on the surface of red blood cells thereby aiding in the maintenance of LDL cholesterol in the reduced form.
In another aspect, the present invention relates to a modified red blood cell, the surface of which is decorated with an ensemble (i.e., a plurality) of biomolecules. Such an ensemble of molecules can work in concert to achieve a desired neutralizing effect. The use of an ensemble of biomolecules is particularly important with respect to the neutralization of complex exogenous biological agents such as bacteria and viruses.
For example, red blood cells can be modified to bear an antibody or antibodies specific for a bacterium of interest. Such antibodies can bring the modified red blood cell into contact with the bacterium of interest if present in the circulation system. Other biomolecules present on the surface of the red blood cell can be provided which have the ability to breach the outer membrane/cell wall of the bacterium. These include, for example, lysozymes, bacteriocidal permeability increasing peptides and other pore forming antimicrobials. In addition, the bacterial electron transport array may be used to generate hydroxyl radicals within the bacterial inner cell membrane. Electron mediators such as hemin, derivatives of quinones, menadione or methyl viologen may be deployed on the surface of the red blood cell. Such electron mediators will produce hydroxyl radicals within the bacterial inner membrane by reducing oxygen directly. The penetration of such electron mediators will be assisted by the presence of lysozyme, which removes the peptidoglycan and allows the interaction of the electron mediator with the inner membrane. Potential synergy with bacteriocidal permeability increasing peptides for further disruption of lipo-polysaccharide or peptidoglycan layers is also likely.
The killing of bacteria by the addition of hemin has been demonstrated in relevant experiments. More specifically, this has been demonstrated in
B. subtilis
as well as
S. aureus
and other gram positive bacteria. Oxygen was required for bacterial killing. Bacteriocidal quantities of hemin did not damage bacteria in the absence of oxygen. Porphyrin without iron was also tested and a lack of bacteriocidal effect was observed due to the essential role of Fe in electron mediation. Moreover, when Zn was substituted for Fe the resulting complex demonstrated the expected reduction in bacteriocidal efficacy. It was also demonstrated that hemin, attached to polyethylene glycol tethers, does not kill bacteria with an intact peptidoglycan coat. The killing of gram negatives was achieved with hemin, provided that the lipopolysaccharide layer was first disrupted with polyethylene imine.
Deploying and ordering the bioengineered macromolecules into a multic
Bitensky Mark W.
Yoshida Tatsuro
Farrell Kevin M.
Saucier Sandra E.
Trustees of Boston University
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