Porcine protein and uses thereof

Details Porcine protein and uses thereof Porcine protein and uses thereof

1999-03-26

2001-06-12

Saunders, David (Department: 1644)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C424S184100, C424S185100, C530S300000

Reexamination Certificate

active

06245890

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel porcine protein that is involved in xenograft rejection in humans. Specifically, the present invention relates to a porcine protein, found in porcine red blood cells, that binds to human serum antibodies involved in xenograft rejection, but which does not bind to human serum antibodies against the dominant xenograft rejection antigen, namely, the &agr;Gal epitope. The present invention also relates to methods for the detection, isolation and use of the porcine red blood cell protein, as well as methods for the detection, isolation and use of human serum antibodies that bind to the protein. Finally, pharmaceutical compositions and methods of treatment are also provided.
BACKGROUND OF THE INVENTION
Organ transplantation has become the treatment of choice for various diseases associated with organ failure (Platt J L (1998)
Nature
392:11-17; Auchincloss H Jr, and Sachs D H (1998)
Ann Rev Immunol
16:433-70). However, the supply of organs from organ donors falls far short of meeting the rapidly increasing demand (Hammer C, Linke R, Wagner F, and Diefenbeck M (1998)
Int Arch Allergy Immunol
116:5-21). In the United States, only 5% of the patients on various waiting lists for organ transplants ever receive the appropriate organs. One attractive approach to overcoming this shortage is to use animals as a source of organs for transplantation (Greenstein J L and Sachs D H (1997)
Nat Biotechnol
15:235-238). The transplantation of organs or cells between members of different species is called xenotransplantation. Non-human primates are the closest biological relatives of human beings; therefore, their organs are most similar to humans, anatomically, physiologically and biochemically. In fact, organs from chimpanzees and baboons have been shown in clinical studies to exhibit extended xenograft survival following rather simple immunosuppression procedures (Bailey L L, Nehlsen-Cannarella S L, Concepcion W, and Jolley W B (1985)
JAMA
254(23):3321-3329; Starzl T, Marchioro T L, and Peters G (1964)
Transplantation
2:752-776). However, the large-scale production of these endangered non-human primates for the purpose of securing organs for transplantation is considered unethical and socially unacceptable (Cortesini R (1998)
Transplant Proc
30(5):2463-2464). Furthermore, the transmission of pathogens from primates to humans is well documented, and pathogen-free primates are extremely difficult to raise (Allan J S (1995)
Nat Medicine
2(1):18-21). In addition, a significant obstacle to the widespread adoption of xenotransplantation is the immunological incompatibility between nonprimate animals and humans, which results in strong host rejection responses to the xenotransplanted organ. Overcoming these host rejection responses is essential to enable widespread use of nonprimate animal organs in xenotransplantation.
The first immune barrier to xenograft survival is hyperacute rejection, which may occur within minutes after revascularization of an organ (Rosenberg J C, Hawkins E, and Rector F (1971)
Transplantation
11(2):151-157). The rejection is induced by the activation of the host (recipient) complement cascade upon the binding of recipient xenoreactive natural antibodies to the xenograft. The major xenograft antigen (or “xenoantigen”) responsible for the hyperacute rejection response has been identified as a carbohydrate epitope, Gal&agr;1,3Gal&bgr;1,4GlcNAc (referred to herein as “the &agr;Gal epitope”). The &agr;Gal epitope forms glycoconjugates on the cell surface of animal organs and cells (Thall A, and Galili U (1990)
Biochemistry
29: 3959-3965; Good A H, Cooper D K C, Malcolm A J, Ippolito R M, Koren E, Neething F A, Ye Y, Zuhdi N and Lamontagne L R (1992)
Transplantation Proceedings
24:559-562). The &agr;-Gal epitope is universally present in the animal kingdom, with the exception of humans and Old World monkeys who lack the galactosyltransferase responsible for the epitope synthesis. Conversely, in normal human serum there are significant amounts of naturally occurring anti-&agr;Gal antibodies, which constitute approximately 1-3% of total IgG molecules and 3-5% of total IgM (Rother R P and Squinto S P (1996)
Cell
86:185-188). Recent studies suggest that anti-&agr;Gal IgM, rather than IgG, is responsible for the hyperacute rejection response observed in organ xenotransplantation (Kroshus T J, Bolman R M III, and Dalmasso A P (1996)
Transplantation
62:5-12).
The second immunological barrier to xenografts is termed delayed or vascular rejection. Although a detailed mechanism has yet to be elucidated, vascular endothelium cells are considered to be the target for immune activation through antibody-dependent cytotoxicity mediated by NK cells and macrophages (Lawson J H, and Platt J L (1996)
Transplantation
63:303-310). In contrast to hyperacute rejection, both IgG and IgM induce vascular rejection effectively, and complement is apparently not involved. These antibodies may represent xenoreactive natural antibodies whose specificities have not yet been characterized. Finally, the cellular immune response constitutes the last barrier for xenotransplantation. The mechanism involved is probably similar to that observed in allograft rejection, but with more potent responses.
Although immune responses to xenografts are divided into three stages, most research and clinical strategies thus far developed have been aimed only at the first stage, hyperacute rejection. One approach to reducing hyperacute rejection involved circulating human recipient blood over an &agr;Gal immunoadsorbent column to remove anti-&agr;Gal antibodies prior to transplantation of the xenograft (Taniguchi S, Neethling F A, Korchagina E Y, Bovin N, Ye Y, Kobayashi T, Niekrasz M, Li S, Koren E, Oriol R, Cooper D K C (1996)
Transplantation
62:1379-1384). However, while the procedure was successful in reducing the concentration of anti-&agr;Gal antibodies in the human blood, the reduction in antibody levels was transient, and was often followed by a rapid rebound within days. A more attractive alternative is to alter the &agr;Gal epitope on the donor organ by expressing or knocking out specific glycosyltransferase/glycosidase activities in organ donor animals (Osman N, McKenzie I F C, Ostenried K, Ioanou Y A, Desnick R J, and Sandrin M S (1997)
Proc Natl Acad Sci
94:14677-14682; Koike C, Kannag, R, Takuma Y, Akutsu F, Hayashi S, Hiraiwa N, Kadomatsu K, Muramatsu T, Yamakawa H, Nagai T, Kobayashi S, Okada H, Nakashima I, Uchida K, Yokoyama I, and Takagi H (1996)
Xenotransplantation
3:81-86) (see FIG.
1
). A third approach is to generate transgenic animals that express human complement regulatory proteins, such as DAF, CD46 and CD59 (Zaidi A, Schmoeckel M, Bhatti F, Waterworth P, Tolan M, Cozzi E, Chavez G, Langford G, Thiru S, Wallwork J, White D, and Friend P (1998)
Transplantation
65: 1584-1590). In this approach, although binding of xenoreactive antibodies still takes place, the presence of these regulatory proteins may prevent complement-induced cell lysis. Data from several studies seem to suggest that a combination of different approaches may be required to efficiently inhibit hyperacute rejection associated with xenograft transplantation.
In recent years, a consensus has emerged that the domestic pig may represent a good alternative to nonhuman primates as a donor of organs for transplantation. Porcine and human organs have similar sizes and cardiac output efficiencies. Pigs are relatively easy and inexpensive to raise in large numbers. Furthermore, pigs can be more easily raised in sterilized environments than nonhuman primates, and the use of pigs as organ donors produces fewer ethical concerns. The greater phylogenetic distance between pigs and humans means it is less likely that xenografts of pig organs or cells would impose any realistic risk of transmitting an infectious organism of epidemiological significance to the human population. However, the immunological and physiological incompatibility between pigs and humans rem

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