Method of intracellular binding target molecules

Details Method of intracellular binding target molecules Method of intracellular binding target molecules

2000-04-21

2001-12-11

Stucker, Jeffrey (Department: 1648)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S326000, C435S238000, C435S330000, C435S339000, C424S093200, C514S04400A, C530S387300, C530S388300, C530S389300

Reexamination Certificate

active

06329173

ABSTRACT:

The present invention is directed to a method for intracellular binding of specific molecules, preferably proteins. More specifically, this method involves the intracellular expression and subsequent use of antibodies specific for a desired molecule.
Various abnormalities appear to be the result of the undesired expression of a particular molecule such as a protein. For example, many tumors are believed to be the result of the overexpression of cellular oncogenes, such as neu,myc, abl, etc. Other malignancies are believed to be the result of expression of an altered receptor. Certain illnesses are caused by the undesired cellular expression of viral proteins. For example, the human immunodeficiency virus (HIV) uses mammalian cells for the preparation of viral encoded proteins including structrual proteins and regulatory enzymes. Human T-cell Leukemia virus type 1 or 2, (HTLV-1 or 2) produce tumors in infected individuals as a result of viral expression. Such viral encoded proteins can result in the assembly of virions which can in turn infect other cells.
Therapeutic strategies have included the development of drugs to target the undesired proteins, means of intercellular blocking of such proteins, for example, soluble CD4, and the use of drugs which will selectively kill cells expressing the undesired proteins.
Another method of treatment that has been suggested is the transfer of genetic materials into cell. For example, by receptor mediated gene delivery, transkaryotic implantation and viral shuttle vectors such as retroviral gene transfer. In such methods, broadly referred to as gene therapy, cells which are either deficient in a protein or produce a dysfunctional protein are hoped to be mended by introducing into the cell DNA coding for the normal gene product.
In vivo gene expression has been reported following direct injection of non-infectious, non-oncogenic plasma DNA encapsulated in lyposomes [Nicolau, C., et al.,
Proc. Natl. Acad. Sci
. 80:1068 (1983)] immunoliposomes [Wang, C. Y., et al.,
Proc. Natl. Acad. Sci
84:7851 (1987)] and in a liposome/red blood cell membrane hybrid [Kaneda, Y., et al.
Science
243:375 (1989)]. Expression from a variety of calcium phosphate-precipitated gene sequences has been reported following direct intraperitoneal injection [Benvenitsy, N., et al.
Proc. Natl. Acad. Sci
83:9551 (1986); Felgner, P. L., et al.,
Nature
349:351 (1991)] or following transkaryotic implementation [Seldon, R. F., et al.
Science
242:714 (1987)]. In vivo gene targeting has also been accomplished by receptor mediated gene delivery in which a complex between an asialoorosomucoid/polysine conjugate and plasmid receptor genes have been used to target expression exclusively to the liver, following intravenous administration [Wu, G. Y., et al.,
J. Biol. Chem
. 263:14621 (1988)]. Retroviral gene transfer is reported to offer high efficiency of infection, stable integration and expression in most cells [Anderson, W. F.,
Science
226:401 (1984)]. In vivo gene therapy has been initiated in patients with ADA deficiency who have had reinfused into their blood, autologous lymphocytes carrying the ADA gene and in cancer patients with advanced melanoma who have had reinfused tumor infiltrating lymphocytes (TIL) which carry the gene for tumor necrosis factor (TNF) [Rosenberg, S. A., et al.,
N. Eng. J. Med
. 323:570 (1990) all of these articles are specifically incorporated herein by reference].
Gene modification of cells which continually express a viral inhibitor and result in the inhibition of viral infection have been proposed and referred to as intracellular immunization. (Baltimore, D.,
Nature
335:395-196 (1988)3. Towards this goal, several approaches have been tested including HIV-1 specific ribozymes [Sarver, N., et al.
Science
227:1222 (1990)], antisense RNA [Posnansky, M., et al.,
J. Virol
. 65:532 (1991)], tar decoys [Sullenger, B. A., et al.,
Cell
63:601 (1990); Lisziewicz, J., et al.,
VII Internat'l. Conf. AIDS
2:28 (1991)], dominant negative mutants and others. [Buonocorel, et al.,
Nature
345:625-628 (1990); Hasseloff, J., et al.
Nature
334:585-591 (1988); VanderKrol, A. R., et al.,
BioTechniques
6:958-976 (1988); Malim, M. H., et al.,
Cell
58:205-214 (1989); and Trono, D., et al.,
Cell
59:113-120 (1989)]. A major impediment to the development of effective gene inhibition protocols using such antisense RNA or ribozymes is the ability to achieve a high level of expression of the inhibitor encoding DNA template in the transformed cells and this may also be a potential problem for using dominant negative mutants because of the competitive nature of the inhibition.
It would be desirable to have a method which can be used to achieve a high level of expression of an inhibitor to the desired molecule.
It would be desirable to have a method which can specifically target these undesired molecules and which has wide applicability.
It would be desirable to have a method which does not introduce cytotoxic chemicals into a cell.
It would be desirable to have a method which provides a ready means of targeting undesired proteins.
SUMMARY OF THE INVENTION
We have now discovered a method by which one can target an undesired molecule (sometimes referred to as a target molecule or target antigen), preferably a protein. This method comprises the intracellular expression of an antibody capable of binding to the target. A DNA sequence containing a sufficient number of nucleotides coding for the portion of an antibody capable of binding to the target operably linked to a promoter that will permit expression of the antibody in the cell(s) of interest (antibody cassette) is delivered to a cell. Thereafter, the antibody is expressed intracellulary and binds to the target, thereby disrupting the target from its normal actions. In one preferred embodiment, the “antibody gene” of the antibody cassette would utilize a cDNA encoding heavy chain variable (V
H
) and light chain variable (V
L
) domains of an antibody which can be connected at the DNA level by an appropriate oligonucleotide as a bridge of the two variable domains, which on translation, form a single polypeptide (referred to as a single chain variable fragment (sFv)) capable of binding to a target such as a protein. The antibody gene does not encode an operable secretory sequence and thus the expressed antibody remains within the cell. In certain preferred embodiments, a nucleotide sequence encoding an intracellular localization leader is also used.
Preferred cell targets are retrovirally infected cells such as HIV infected cells, where the targets are the virally encoded protein. For example, one can use antibodies against structural proteins such as the envelope glycoprotein and gag protein, and/or against tat, rev, nef, vpu and/or vpx regulatory proteins. In one preferred embodiment, one would use an antibody cocktail (i.e. mixture of antibodies) to target a variety of the viral target proteins. Another preferred target includes oncogenes such as trans-membrane growth factor receptors, receptors, growth factors, membrane associated guanine nucleotide binding proteins, etc.


REFERENCES:
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patent: WO 93/07286 (1993-04-01), None
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