Drug – bio-affecting and body treating compositions – Conjugate or complex of monoclonal or polyclonal antibody,... – Conjugated to proteinaceous toxin or fragment thereof
Reexamination Certificate
1999-07-27
2003-09-02
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Conjugate or complex of monoclonal or polyclonal antibody,...
Conjugated to proteinaceous toxin or fragment thereof
C530S350000
Reexamination Certificate
active
06613330
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of organ and tissue xenotransplantation, and autoimmune diseases.
BACKGROUND OF THE INVENTION
The area of organ transplantation has been developed in the last 30 years to address the needs of many patients who suffer from organ failure and thus require a transplant of a functioning organ. Such human organs, termed allografts, are obtained from brain dead donors, or from family members in the case of kidney grafts. Nevertheless, more than 80% of patients in need of an organ such as heart, kidney, or liver fail to receive the allograft because of an insufficient number of organ donors. This shortage in allografts has prompted research for an alternative source of organs from other mammalian species. In particular, extensive research is being conducted on the use of tissue or organ grafts from other animal species, pigs in particular, (termed xenografts) for transplantation in humans. Transplantation of xenografts in humans or in monkeys was repeatedly found to result in rejection by a natural antibody. Galili and others have found that this rejection of xenografts is mediated by the interaction of the natural anti-Gal antibody, produced in humans, with &agr;-gal epitopes (Gal&agr;1-3Gal&bgr;1-4GlcNAc-R, or Gal&agr;1-3Gal&bgr;1-3GlcNAc-R) on pig cells (Galili and Avila, &agr;-gal and Anti-Gal, Subcellular Biochemistry Volume 32, New York: Kluwer Academic/Plenum Publishers, 1999).
Anti-Gal is a natural antibody that constitutes 1% of circulating immunoglobulins in humans, apes and Old World monkeys and can be produced by approximately 1% of B lymphocytes. By analyzing the specificity of this natural antibody, Galili et al. (Galili, et al., 1985, J. Exp. Med. 162:573; Galili, 1993, Springer Seminars in Immunopathology 15:155-171) found that anti-Gal interacts specifically with &agr;-gal epitopes (termed in the literature also &agr;-galactosyl epitopes or Gal &agr;1-3Gal epitopes) on glycolipids and glycoproteins. They also found that the &agr;-gal epitope is expressed as millions of epitopes per cell on various cells of nonprimate mammals, prosimians and New World monkeys (monkeys of South America) but not on cells of Old World Monkeys (monkeys of Asia and Africa, termed here as monkeys), apes and humans (Galili, et al.,1987 Proc. Natl. Acad. Sci (USA) 84:1369; Galili, et al., 1988, J. Biol. Chem. 263:17755). Transplantation of pig heart or kidney xenografts into monkeys was found to result in rejection within minutes. This rapid graft rejection, termed hyperacute rejection, is caused by the binding of natural anti-Gal IgM antibodies to the endothelial cells of the graft, activation of complement and lysis of these cells by activated complement components C7, C8 and C9 that form the membrane attack complex (MAC). This complex of activated complement molecules bores holes in the endothelial cell membrane (Lawson, et al., 1996, Transplantation 62:303). Concomitantly, this process induces platelet aggregation within blood vessels of the graft resulting in a rapid collapse of the vascular bed of the graft and ischemia, ultimately causing graft rejection (Lawson, et al., 1996, Transplantation 62:303).
Complement mediated hyperacute rejection of xenografts in monkeys suggested that xenograft rejection may be prevented by inhibition of complement activation. Recently grown transgenic pigs for human complement regulatory proteins, such as decay accelerating factor (DAF), have provided organs that do not activate complement upon binding of anti-Gal IgM molecules (McCurry et al., 1995, Nature Medicine 1:421.). Though no hyper acute rejection occurred with such organs, delayed rejection was observed within 24 hr (McCurry et al., 1995, Nature Medicine 1:421). This delayed xenograft rejection is largely mediated by the anti-Gal IgG molecules.
Anti-Gal IgG molecules are presently considered the major obstacle for success of xenotransplantation. This is because these IgG molecules mediate destruction of the xenograft cells by a process termed “antibody dependent cell mediated cytotoxicity” (ADCC) (Galili,1993, Immunology Today 14:480). In this process, killer cells with Fc&ggr; receptors, such as granulocytes, monocytes, macrophages and natural killer (NK) cells, adhere to the Fc portion of anti-Gal IgG molecules on xenograft cells. The killer cells kill the xenograft cells by secreting their lytic enzymes into the contact area between the killer and target cell. Killing of xenograft cells by ADCC is a much slower process than complement mediated lysis of these cells. Nevertheless, this ADCC process effectively facilitates delayed and chronic xenograft rejection, i.e., rejection of the xenograft within days to weeks.
Removal of anti-Gal from the serum by plasmapheresis on immunoadsorbents can only delay xenograft rejection, since anti-Gal reappears within several days (Kozlowski et al., 1998 Xenotransplantation 5:122). Galili and coworkers (Galili et al., 1997, Xenotransplantation 4:127; Galili et al., 1995, Transplantation 59:1549; Galili et al., 1997, Transplantation, 63:646) reported that an even more serious problem for xenograft recipients is the major increase in production of high affinity anti-Gal as a result of the immune response to &agr;-gal epitopes on the xenograft. The immune system in humans or monkeys transplanted with pig xenografts reacts against &agr;-gal epitopes on the xenograft by producing large amounts of high affinity anti-Gal antibodies, primarily of the IgG isotype. These anti-Gal IgG molecules effectively induce chronic xenograft rejection, mainly by the mechanism of ADCC (Galili 1993, Immunology Today 14:480). The reason for this immune response in xenograft recipients is that B lymphocyte clones, which are capable of producing anti-Gal antibody molecules, interact via their specific B cell receptors, with the corresponding &agr;-gal epitopes on glycoproteins of xenograft cell membranes. As a result of this interaction, these B lymphocyte clones are stimulated to proliferate, undergo isotype switch (i.e., convert from cells producing anti-Gal IgM molecules into cells producing anti-Gal IgG molecules) and undergo affinity maturation by somatic mutations (i.e., the immunoglobulin genes coding for anti-Gal gain mutations which increase the affinity of the antibody molecules to &agr;-gal epitopes). Ultimately, these stimulated B lymphocytes in xenograft recipients differentiate into plasma cells producing large amounts of high affinity anti-Gal IgG antibody molecules in response to antigenic stimulation by &agr;-gal epitopes on the xenograft. Galili and colleagues further showed that prevention of this stimulation of B lymphocytes in xenograft recipients can prevent most of the immune rejection process against the xenograft (Stone et al., 1998, Transplantation, 65:1577). They transplanted monkeys with pig cartilage that was enzymatically treated with recombinant &agr;-galactosidase for the complete removal of &agr;-gal epitopes. The cells in this cartilage were dead because of the treatment. The inflammatory reaction against such xenografts was marginal, whereas monkeys transplanted with untreated pig cartilage (i.e., cartilage expressing &agr;-gal epitopes) displayed a massive inflammatory response (i.e., immune rejection) against the xenograft (Stone et al., 1998, Transplantation, 65:157).
Unfortunately, this enzymatic treatment is not applicable to xenografts containing live cells because the turn over of cell membranes results in the re-expression of &agr;-gal epitopes on the treated cells. Another difficulty is that the production of high affinity anti-Gal in xenograft recipients can not be prevented by the immune suppression regimens used for prevention of allograft rejection. Galili et al. (Galili et al., 1995, Transplantation 59:1549) showed that patients transplanted with pig pancreas islet cells and with a human kidney allograft were induced to produced high affinity anti-Gal antibody molecules despite the massive immunosuppressive treatment, which was potent enough to prevent the rejection of the ki
Carlson Karen Cochrane
Rush University
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