Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-12-30
2002-01-22
Gambel, Phillip (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387300, C530S387500, C530S388220, C530S389100, C530S391100, C530S391300, C530S391700
Reexamination Certificate
active
06340743
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, in general, to compositions and methods for the specific binding of a ligand to the stalk region of the polymeric immunoglobulin receptor for internalization into, or transport across, a cell.
BACKGROUND OF THE INVENTION
One of the most challenging problems facing the pharmaceutical and biopharmaceutical industries is delivering therapeutic agents past the various semi-permeable membranes within the body. Particularly in the case of macromolecules, the obstacle to cost effective or convenient treatment is often due to the lack of an adequate drug delivery system. In turn, this issue dictates whether production of a drug is economically feasible. Thus the search for alternative delivery systems often rivals the search for new drugs themselves.
Gene transfer methods can be viewed as a paradigm of macromolecular drug delivery. These methods can be divided into three categories: physical (e.g., electroporation, direct gene transfer, and particle bombardment), chemical (e.g., proteinoids, microemulsions, and liposomes), and biological (e.g., virus-derived vectors, and receptor-mediated uptake). Amongst biological transfer methods, receptor-mediated uptake is a particularly promising approach. Targeting a ligand to an endocytosed receptor acts as a means to ferry that ligand into the cell. However, one drawback of receptor-mediated systems has been their general reliance on intravenous administration which severely limits their use.
Mucosal epithelial cells line a number of readily accessible tissues such as those found in the upper respiratory and gastrointestinal tracts. The accessibility of these cells make them an attractive target for drug delivery. See, e.g., Ferkol et al., J. Clin. Invest. 92:2394-2400 (1993); Ferkol et al., J. Clin. Invest. 95:493-502 (1995). Retrograde transport of an antibody from the lumenal to the basolateral surface of epithelial cells has been reported, albeit at very low levels. Breitfeld et al., J.
Cell Biology
109:475-486 (1989). In that study, movement across the cell was followed by binding an antibody to the secretory component of polymeric immunoglobulin receptor (pIgR). Relative to the level of basolateral to apical transport, Breitfeld et al. reported that less than 5% of the transport was retrograde in nature. The nominal level of counter-transport minimizes the utility of secretory component as a means to deliver biologically active compositions into cells. Moreover, due to the abundance of cleaved pigR in the lumen, binding of ligand to cleaved pIgR, rather than the intact pigR of the cell surface, would diminish the utility of pIgR counter-transport as a mechanism of drug delivery.
Accordingly, what is needed in the art is a means to convey ligands into or across a cell surface with high efficiency. More particularly, what is needed in the art is a means to deliver macromolecules to, into, or across cells lining the gastrointestinal or respiratory tracts. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to a ligand that binds specifically to the stalk of a polymeric immunoglobulin receptor (pIgR) of a cell with the proviso that the ligand does not substantially bind to secretory component of pIgR under physiological conditions. Typically, the antibody specifically binds only to the stalk. In one embodiment, the ligand is an antibody, preferably a humanized antibody. In another embodiment the ligand is a recombinant single chain variable region fragment of an antibody. In yet another embodiment the ligand binds to an extracellular epitope within the first 33 amino acids that are cell membrane proximal to a cleavage site of the receptor.
The ligand may comprise a binding component for binding to the stalk, and a biologically active component such as a nucleic acid, protein, radioisotope, lipid, and carbohydrate. Biologically active components comprise anti-inflammatories, anti-infectives, anti-sense oligonucleotides, antibiotics, and anti-infectives. In one embodiment, the biologically active component is a nucleic acid encoding a wildtype cystic fibrosis transmembrane conductance regulator. In a preferred embodiment the cell is an epithelial cell, most preferably a mammalian epithelial cell.
In another aspect, the present invention is directed to a method of introducing a ligand into a cell expressing a polymeric immunoglobulin receptor by attaching the ligand to the stalk of the polymeric immunoglobulin receptor of the cell with the proviso that the ligand does not substantially bind to the secretory component of pIgR under physiological conditions. In one embodiment, the ligand is attached to the stalk at the apical surface of a cell; and in further embodiments, the ligand is transcytosed to the basolateral surface of the cell, and released from the stalk at the basolateral surface.
In a further aspect, the present invention relates to a method of attaching a ligand to a cell expressing a polymeric immunoglobulin receptor comprising the step of binding the ligand to a stalk of the receptor with the proviso that the ligand does not substantially bind to secretory component of pIgR under physiological conditions. In one embodiment, the ligand is introduced into the cell after binding. Alternative embodiments of the present invention may be had by reference to the various aspects of the present invention.
Amongst the various in vivo and in vitro utilities, the present invention may be used to transport therapeutic or diagnostic compositions to, into, or across, mucosal epithelial cells. Thus, the invention provides a highly efficient and convenient means to transfer nucleic acids or proteins into epithelial cells.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention is directed to a ligand that binds specifically to the stalk of a polymeric immunoglobulin receptor (pIgR) of a cell with the proviso that the ligand does not substantially bind to secretory component of pIgR under physiological conditions. The invention provides, inter alia, methods of attaching and introducing a ligand into a cell expressing pIgR.
After transport to the apical surface of epithelial cells, the majority of pIgR is cleaved and secretory component is released. We have discovered that, surprisingly and unexpectedly, cleavage of intact pIgR in the lumen leaves a residual extracellular region of pIgR (i.e., the “stalk”) intact; and further, the stalk remains accessible to binding despite the abundance of proteases typically present in lumenal milieu. The ability to bind, endocytose, and transcytose a ligand bound to the pIgR stalk provides, in part, the invention as disclosed and claimed herein.
The present invention has utility as a means of transporting therapeutic or diagnostic compositions to, into (endocytosis) or across (transcytosis) a cell expressing pIgR. Thus the invention can be used to transport biologically active compositions such as proteins, nucleic acids, or detectable labels specifically to cells expressing pIgR. The invention also provides a means of labeling and distinguishing epithelial cells from amongst a mixed cell population in pathological studies. Further, since pIgR expression is reduced in carcinomas relative to normal epithelium, the labeling of pIgR has utility as a diagnostic adjunct in endoscopic or radiologic procedures. Additionally, binding of therapeutic ligands to plgr has utility in extending their duration in the lumen of various passageways and increasing their effectiveness.
Definitions
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al. (1994)
Dictionary of Microbiology and Molecular Biology
, second edition, John Wiley and Sons (New York), and Hale and Marham (1991)
The Harper Collins Dictionary of Biology
, Harper Perennial, N.Y. provide one of skill with a general dictionary of many of the terms used in this invention
Mostov Keith E.
Richman-Eisenstat Janice
Gambel Phillip
Roark Jessica H.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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