Attenuation of rejected cells transformed with adenovirus E3

Details Attenuation of rejected cells transformed with adenovirus E3 Attenuation of rejected cells transformed with adenovirus E3

2000-08-01

2002-12-10

Crouch, Deborah (Department: 1632)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093200, C514S04400A, C435S325000, C435S455000, C435S456000

Reexamination Certificate

active

06491909

ABSTRACT:

1. FIELD OF THE INVENTION
This invention concerns an improvement in the art of the transplantation of tissue for medical purposes. Specifically it concerns methods, vectors and compounds useful in preventing the expression of certain transplantation antigens on the surface of cells to be transplanted. More specifically it concerns the blockage of the cell surface expression of one particular class of proteins the genes for which are located in the major histocompatibility complex (MHC).
2. BACKGROUND OF THE INVENTION
In cases of extreme and life threatening conditions such as renal, liver and cardiac failure, physicians have successfully transplanted tissues between genetically distinct individuals. Even when care is taken to reduce the genetic differences between the host and donor, the recipients of these grafts must usually be given drugs that reduce the activity of their immune system so that the graft is not rejected. Such immuno-suppression entails substantial risks. Even when such immuno-suppression is well tolerated, there are considerable difficulties attendant in minimizing the antigenic differences (matching) between the donor and the recipient that increases the costs and reduces the availability of this mode of therapy. Furthermore, not all tissues can be successfully transplanted between genetically distinct persons.
In principle, for example, diabetes mellitus, one of the most common and, in the longterm, one of the most debilitating chronic diseases, could be “cured” by a successful transplant of the tissue that secrete insulin, the islets of Langerhans. Despite the magnitude of the problem, the availability of islets from cadaveric donors and the successful experience in other situations, e.g., renal, cardiac and hepatic transplantation, there is presently no practical protocol that routinely provides for the survival of histoincompatible islet cells.
The probable causes for this absence may be several. In diabetes mellitus, the disease itself makes immuno-suppression especially hazardous, as diabetics are highly susceptible to infection. Also it appears that some immuno-suppressive agents, e.g., cyclosporine, are directly toxic to islets in high doses and can adversely affect graft survival, while others, e.g., glucocorticoids, are known to increase the subject's insulin requirements and may indirectly jeopardize both the survival and the beneficial effects of the graft. Lastly, in many instances diabetes results from autoimmunity directed toward the islets. To the extent such individuals are immunologically “primed” towards non-allelic islet antigens, their graft rejection would be accelerated. Thus, as an alternative to the present methods of promoting graft survival, which reduce the host's immunity, there is a need in the transplantation art for a method to specifically render the graft invisible to the host's immune system or resistant to its effects.
2.1 The Mechanism of Graft Rejection
Except between genetically identical individuals, e.g., identical. twins, the cells of each person are recognized by the immune system of others as foreign, i.e., the immune system responds to the graft just as it would respond to a parasite or virus. This so called allograft reaction, unless treated by immune-suppression, leads to the rejection and loss of the graft. The allograft reaction is caused by the recognition of histocompatibility antigens on the surface of the cells of the graft by the lymphocytes of the recipient individual. With rare exceptions, e.g., the ABO blood groups in transfusions, the host lymphocytes of greatest importance to an allograft reaction are of the type that recognize antigen directly on cell surfaces, so-called thyme lymphocytes (T-lymphocytes).
From the fact that only grafts between genetically identical individuals are truly stable, one may conclude that. almost any protein can become a histocompatibility antigen when the host's and recipient's genes encoding it are different. This surmise has been experimentally verified. However, the rapidity with which an allograft is rejected varies greatly depending upon the nature of the histoincompatibility between the host and graft. In particular, every species of higher vertebrates contains a closely linked complex of multiple genes at which genetic differences are found to cause the most rapid and severe allograft reactions. This complex of genes is called the Major Histocompatibility Complex (MHC) of the species. Several types of proteins are encoded within the MHC, including two classes of cell surface proteins, conventionally called class I and class II MHC products. In humans the class I products are also referred to as HLA-A, HLA-B and. HLA-C, and the class II products are termed HLA-DR. Many cell types, such as &bgr;-cells, express only MHC class 1 products.
Class I and class II MHC products are not only histocompatibility antigens, they are also central to antigen recognition by T-lymphocytes. Although the different T-lymphocytes from an individual can recognize and distinguish among perhaps millions of potential antigens, each of that individuals T-lymphocytes can recognize its particular antigen only when the antigen is either an MHC class I or class II product or is physically complexed with one. These observations suggest that the life of an allograft could be extended indefinitely were some mechanism available to prevent the expression of MHC products altogether by the graft.
2.2 The Viral Products That overcome Host Immunity
The problem of avoiding a host's immune reaction is of interest not only to transplant surgeons but also to parasites such as viruses. Viruses, of course, are able to reproduce only within a viable cell of the infected host. Viruses frequently kill the infected cell when they redirect the cellular machinery towards the production of viral particles (virions). Surprisingly then, one of the major host defenses against viruses are cytotoxic T-lymphocytes (CTL) which kill the infected cells. The host apparently prevents, to some significant degree, the further release of virions by killing the infected cell when viral antigens can be detected. In response, certain viruses have evolved mechanisms to reduce or avoid the host's immune attack on the host's cells in which the virus is replicating (reviewed, Gooding, L. R., 1992, CELL 71:5-7) by blocking the presentation of newly synthesized antigens. Some of these mechanisms apparently specifically prevent the maturation of MHC class I products onto the cell surface. Because the complex of MHC product and antigen is formed as the product matures, the display of newly synthesized viral antigens is blocked.
Researchers have recognized that adenovirus infected cells are poorly recognized and lysed by CTL. To determine which viral products are particularly involved, the isolated E3 region of adenovirus was co-transfected, along with a selectable marker, into the 293 embryonic kidney cell line, which had been transformed with adenovirus and contained adenovirus early genes products, that rendered the adenovirus E3 promoter active.
Stable transfectants were isolated by selection of the marker gene and a single clone expressing high levels of gp19 was selected for further study. This transfected clone was found to have undergone an about 5-10 fold reduction in the amount of HLA-A and somewhat smaller reduction in total MHC class 1 products. There were no studies of the functional consequences of this reduction. Burgert, H-G & Kvist, S., 1985, CELL 41:987-97.
Subsequently, these authors introduced a murine MHC class I product (K
d
) into the selected clone and showed that this clone was resistant to K
d
-specific CTL, Burgert, H-G, 1987, PROC.NATL.ACAD.SCI. 84:1356-60. Studies of murine immune responses to adenovirus antigens, using varying deletion mutants spanning the E3 region and anti-MHC monoclonal antibodies, establish that the gp19 protein (gp19) both binds murine MHC product and is necessary and sufficient for the inhibition of its expression on the surface of the embryonic kid

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