CROSS-REFERENCE TO RELATED APPLICATIONS
This application a continuation of Ser. No. 12/897,297, filed Oct. 4, 2010, pending, which is a continuation of Ser. No. 10/181,896 filed Nov. 7, 2002, which issued Oct. 5, 2010 as U.S. Pat. No. 7,807,463, which is a national phase of PCT/US01/02342, filed Jan. 25, 2001, which claims priority to U.S. Provisional Application No. 60/178,347, now expired, filed Jan. 25, 2000. Each of these applications is incorporated herein by reference in its entirety.
This invention was made with government support under Research Grant No.R43 DK50737 awarded by the U.S. government. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
I. Field of the Invention
The field of the present invention relates to the transplantation of organs and tissues, and particularly to the conditioning of a xenograft organ while still in the donor to resist rejection antibodies in the graft recipient (accommodation). The invention also relates to methods for assessing accommodation within the donor animal.
A major barrier to the transplantation of organs from one mammalian species to another is rejection of the xenografts. Much of the rejection is not related to tissue-specific antigens but results from the recipient being sensitized to the donor animal. For example, humans and Old World monkeys have circulating antibodies to the alpha galactosyl oligosaccharide expressed on tissues in other animals, including pigs. The antibodies bind to any transplanted pig xenograft, bind complement, and destroy the graft within an hour. This rapid reaction is referred to as hyper-acute rejection (HAR). The graft is rapidly destroyed by the binding of preformed natural antibodies to endothelial cells and fixation of the complement. Most of the preformed antibodies in humans and old world apes (>80%) are against Gal(alpha)1-3Gal epitopes (alphaGal).
Acute vascular xenograft rejection occurs at three to eight days post transplant. Induced and recurrent anti-donor antibodies bind to the endothelium, leading to endothelial activation, small vessel thrombosis, and recruitment of macrophages and NK cells. Acute xenograft rejection is also mediated by cellular rejection. In contrast to cellular allograft rejection, CD4+ cytotoxic lymphocytes contribute to the graft injury.
The current methods for prevention of HAR target the binding of antibody or the fixation of complement. Anti-donor antibodies or complement can be depleted from the blood of the recipient. Hyper-acute rejection was prevented in ABO mismatched cardiac allografts performed in baboons by infusion of soluble trisaccharides of the A and B antigen to neutralize the antibodies. Though the circulating antibodies persisted after discontinuing the oligosaccharides, some grafts showed prolonged survival. Cooper D. K., et al, Transplantation 56: 769-77 (1993). Transgenic pigs which express human complement inhibitors or with reduced expression of alphaGal have been created.
These technologies are useful on a short-term basis; however, they are not completely effective. Antibodies return rapidly after their removal and the procedure must be repeated frequently. Transgenic pigs are variable in the level of expression of the transgene. With both methodologies, episodes of HAR and acute vascular rejection are common. Efforts to suppress acute xenograft rejection using conventional chemotherapy have been only partially successful. In particular, the antibody response to pig xenografts has proved resistant to suppression.
To reliably achieve long-term survival of xenografts, immune tolerance or graft accommodation will be necessary. Immune tolerance involves programming the recipient's immune system to be specifically unresponsive to the graft. Accommodation refers to adaptation of the graft to be resistant to an existing immune response.
Partial immune tolerance to pig xenografts has been induced by ablating the recipient immune system and reconstituting it in the presence of porcine hematopoietic cells. Porcine hematopoietic cells are detectable a year later. This approach has three basic limitations. First, tolerance would not resolve the problem caused by pre-existing natural antibodies. Additional efforts, such as removal of pre-existing antibodies by immune adsorption would be required. Second, the recipient is subject to a prolonged period of immune deficiency, putting it at risk for opportunistic and zoonotic infections. Third, the tolerance would be to antigens expressed on the hematopoietic cells only. Tolerance would not be induced to the tissue-associated antigens. Pig heart and kidney xenografts were fulminantly rejected in baboons using this protocol.
The transplantation of pig thymi into immune ablated recipients enhances tolerance as the recipient pre-thymocytes mature in the porcine environment. Using this approach, porcine skin graft survival is markedly prolonged in mice and modestly prolonged in primates. The basic limitations described above with mixed chimerism would still be a problem.
Patent application No. PCT/US94/05844 teaches the induction of immune tolerance of recipient lymphocytes to xenografts by infusing lymphocytes into immune deficient donor animals. The tolerant cells are later harvested and transferred back to the recipient, conveying tolerance to the recipient. However, the preexisting immune response would limit the usefulness of that approach.
The mechanism of accommodation is unknown. It is not due to the depletion of antibodies or to replacement of donor endothelium with host endothelium within the graft. Immunohistochemistry of long term cardiac xenografts (hamster-to-rat) shows deposition of IgG, IgM, C3, and C6 on the endothelium, but minimal fibrin formation.
The possibility has been explored that accommodated endothelial cells have reduced expression of antigen. Though some reduction in antigens such as alpha gal was observed with accommodation, it was not thought to be sufficient to protect the graft. Parker W. et al., Am. J. Pathol. 152: 829-39 (1998).
It is known that accommodated grafts can be adoptively transplanted to a second recipient. The factors responsible for accommodation are present within the graft and do not require circulating regulatory cells or factors. Miyatake showed that if rejection of a hamster heart graft can be prevented in a rat recipient, the graft will also resist rejection when re-transplanted into a second recipient identical with the first recipient. T. Miyatake, N. Koyamada, W. W. Hancock, M. P. Soares, and F. H. Bach. Survival of accommodated cardiac xenografts upon re-transplantation into cyclosporine-treated recipients. Transplantation 65: 1563-1569, (1998). While the observation is of scientific value, it is not clinically useful. To apply this observation would require two identical subjects, such as human recipients, one who would host the donor organ until accommodation is achieved. The organ would then be procured and transplanted into the second subject. The very limited number of potential recipients with identical twins and the ethically unacceptable complications to the first recipient make this approach unfeasible.
Some success in achieving accommodation in cultured endothelial cells has been reported. Dorling A., et al., Xenotransplantation, 5: 84-92 (1998); and Dorling A., et al., Transplantation, 62: 1127-1136 (1996). Dorling et al. demonstrated that prolonged exposure in culture of porcine endothelial cells to normal human immunoglobulins produced endothelial cell changes suggestive of accommodation.
Apparent confirmation of these studies was provided by Shah et al., Fifth Congress of the International Xenotransplantation Association, Abstract 199 (1999). Minimal resistance to complement mediated cytotoxicity was achieved with 72 hours of culture. Better resistance was observed with 120 to 144 hours of incubation. On the other hand, others. were unable to confirm these studies using primary endothelial cell cultures. McKane W. et al. Fifth Congress of the International Xenotransplantation Association, Abstract 200 (1999). They suggested that the apparent resistance reported by others may be an artifact related to the use of immortalized endothelial cells, which constitutively express anti-apoptotic genes.
In vitro culture is unlikely to have significant clinical utility. Accommodated endothelial cells would not have significant utility by themselves. Furthermore, accommodation of cultured and transformed endothelial cells required a minimum of 72 hours and preferably 120 hours of culture. See Dorling et al. (1996), supra. If the observation were to be extended to ex vivo culture of whole organs maintained in culture, the organs would significantly deteriorate during this period.
Achieving accommodation within the recipient is very difficult, costly, and often ends in failure, with rejection of the graft.
Therefore, a need exists for a method of xenograft transplantation that avoids the high costs, the complications, and the high risk of failure associated with accommodation of the xenograft organ within the recipient after transplantation.
SUMMARY OF THE INVENTION
An objective of the invention is to provide a tissue or a graft accommodated prior to transplantation of the tissue or graft.
A second objective of the invention is to provide a method for accommodation of the donor graft prior to transplantation.
Another objective of the invention is to provide a method for development of improved in-donor accommodation technology.
In accordance with one embodiment of the invention, a method to produce a tissue or organ accommodated in a donor mammal to resist rejection in a recipient mammal, is provided. The method comprises:
infusing a donor mammal at least one time with sub-lethal levels of at least one accommodation-inducing factor; allowing prolonged exposure to said accommodation inducing factor; and
harvesting one or more of the tissues or organs which are accommodated.
In accordance with a preferred embodiment, the accommodation-inducing factor is infused in a donor mammal which is in an immune deficient state. In accordance with another preferred embodiment, the accommodation-inducing factor is an antibody reactive with donor endothelium, such as pig endothelium, plasma cells, B lymphocytes, human B lymphocytes, conditionally immortalized B lymphocytes, anti-alphaGal antibody, a cell expressing an accommodation inducing factor such as an antibody, a hybridoma comprising a cell expressing an accommodation inducing factor, T cells reactive with cells in the graft tissue or organ, perforin, or Bandeiraea simplicifolia lectin. In accordance with another preferred embodiment, the method further comprises the step of: determining that accommodation of the tissue or organ has been achieved, prior to transplantation of said organ or tissue.
In accordance with another embodiment, a xenograft organ or tissue is provided. The xenograft organ or tissue is raised in a donor mammal treated with an accommodation inducing factor. In accordance with a preferred embodiment, the xenograft organ includes, but is not limited to a heart, a kidney, a liver, a lung, a pancreas, a heart-lung intestine, a spleen, or a thymus. The xenograft tissue includes but is not limited to bone, skin, hair, eye, neural tissue, smooth muscle, skeletal muscle, cardiac muscle, myocytes, pancreatic islets, hepatocytes, embryonic stem cells, progenitor cells, or hematopoietic cells. In accordance with another preferred embodiment, the treatment with an accommodation inducing factor occurred while the donor mammal was in an immune deficient state. In accordance with another preferred embodiment, the accommodation inducing factor is an antibody reactive with donor endothelium, an antibody reactive with pig endothelium, plasma cells, B lymphocytes, human B lymphocytes, conditionally immortalized B lymphocytes, anti-alphaGal antibody, a cell expressing an accommodation inducing factor, a hybridoma comprising a cell expressing an accommodation inducing factor, T cells reactive with cells in the graft tissue or organ, perforin, or Bandeiraea simplicifolia lectin.
In accordance with yet another embodiment, a method for developing accommodation factors is provided which comprises infusing a donor mammal at least one time with sub-lethal levels of at least one accommodation-inducing factor; administering a tissue or cells from a mammal other than the donor to the donor; allowing prolonged exposure to the accommodation-inducing factor; and harvesting the accommodated tissue or cells. In accordance with a preferred embodiment, the accommodation-inducing factor is from an individual who is the intended recipient of said harvested tissue or cell. In accordance with another preferred embodiment, the accommodation-inducing factor is infused in the donor mammal which is in an immune deficient state. In accordance with yet another preferred embodiment, the accommodation-inducing factor is an antibody reactive with donor endothelium, an antibody reactive with a cell or tissue from a mammal other than said donor which was administered to said donor, a cell expressing an antibody reactive with a cell or tissue from a mammal other than the donor which was administered to said donor, an antibody reactive with pig endothelium, plasma cells, B lymphocytes, human B lymphocytes, conditionally immortalized B lymphocytes, anti-alphaGal antibody, a cell expressing an accommodation inducing factor, a hybridoma comprising a cell expressing an accommodation inducing factor, T cell reactive to the tissue or organ, perforin, or Bandeiraea simplicifolia lectin. In accordance with still another preferred embodiment, the method comprises the additional step of determining that accommodation of said tissue or organ has been achieved, prior to harvesting of the tissue or cell.
In accordance with another embodiment, the xenograft tissue or cell is an osteoblast cell, an osteo clost cell, skin, a skin epithelial cell, a hair follicle cell, eye cell, neural cells, a skeletal muscle cell, a smooth muscle cell, a cardiac muscle cell, pancreatic islet, a hematocyte, a stem cell, a progenitor cell, or a hemapoietic cell.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows accommodation in chimeric pigs by a lymphocyte lysis assay. 72-5, 72-1, and 72-3 are chimeric pigs infused at pre-immune fetal stage with bone marrow. The control sample is lymphocytes from an un-infused pig. The lysis was evaluated by Trypan Blue—see Example 3 for detailed method—as a function of the concentration of sensitized sheep serum.
FIG. 2 shows accommodation of a chimeric pig (72-5) by a lymphocyte assay. The control is lymphocytes from an un-infused pig. The lysis was evaluated by Trypan Blue—see Example 3 for detailed method—as a function of the concentration of sensitized sheep serum. The sensitized sheep serum was isolated from a different sensitized sheep than the sheep in FIG. 1.
FIG. 3 shows protection of a chimeric pig heart from hyperacute rejection by human blood. The hearts of a control pig and chimeric pig (72-5) were perfused with sensitized human blood and the heart beat was monitored.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The invention provides a method for production of an accommodated organ or tissue suitable for transplantation and the accommodated organ. Accommodation is the process of conditioning an organ to resist injury in a sensitized recipient. It is also referred to as adaptation. The adaptation conveys resistance in the graft to cell death or apoptosis, procoagulation changes, and adhesion of leukocytes. In accordance with the invention, the accommodation is achieved by infusion of accommodation inducing factor into a donor, allowing the graft to be exposed to the accommodation inducing factor, and harvesting the graft, accommodated while in the donor for resistance to rejection in recipient.
An accommodation-inducing factor is any factor that causes accommodation within a donor of an xenograft organ or tissue. The factor can be an aliquot of plasma from a mammalian species other than the donor. Alternatively, the factor can be a ligand purified from plasma or expressed in an in vitro system. Preferably, the factor is an immune system component, for example a B lymphocyte or an antibody.
The accommodated organ or tissue is an organ or tissue which can resist rejection by a sensitized recipient. A sensitized recipient is any organism with preformed antibodies or memory T cells reactive with donor antigens, present in the graft-recipient prior to transplantation. Examples of sensitized organisms are humans and old world monkeys, sensitive to pig antigens. The sensitized recipient typically produces a hyper-acute and/or acute vascular xenograft immune response to donor tissue, generally. The response is mediated by performed antibodies and T cells present in the recipient organism prior to the introduction of the xenograft. The organ or tissue graft is harvested from a chimeric animal.
In accordance with the invention, a chimeric mammal or animal is any mammal wherein an infused transgenic accommodation-inducing factor resides. For example, a piglet who receives a infusion of cells from another mammal is a chimeric animal. In a preferred embodiment, a chimeric animal is a swine who received, during an immune deficient stage, an infusion of cells from another mammalian species, e.g., a human. The infusion occurs preferably when the donor is in an immune deficient state such as a pre-immune fetus.
Factors that induce accommodation include but are not limited to antibody reactive with donor tissue, antibody reactive with donor endothelium, antibody reactive with pig tissue or endothelium in the case where the donor is a pig or a member of the swine family, plasma cells, B lymphocytes, human B lymphocytes, conditionally immortalized B lymphocytes, anti-alphaGal antibody, a cell engineered to express an accommodation inducing factor, a hybridoma comprising a cell expressing an accommodation inducing factor, and Bandeiraea simplicifolia lectin. The factor can be a natural, isolated factor, or it can be a cell engineered to express a ligand or the purified engineered ligand.
The accommodation factors may be derived from a member of a species that would be a xenograft recipient, e.g., a human or another mammal. In a preferred embodiment, the accommodation inducing factors are isolated from an individual who will later become a recipient. The method of determining, isolating, and manipulating each of the accommodations inducing factors are well known to an artisan skilled in the art.
The invention also provides organ xenografts that are less susceptible to rejection by preformed and developing immune elements, particularly antibodies such as natural antibodies to alphaGal. These antibodies are present in most humans and Old World monkeys. The alphaGal antigen is expressed on endothelial cells and other cells or tissues from most other species, including pigs. Accommodation of a transplant organ can be achieved within the organ donor with prolonged exposure of the graft to at least one accommodation inducing factor. The mechanism of antibody accommodation is not well understood. Without commitment to any one mechanistic explanation of the phenomena, it is believed that endothelial and other cells exposed in the donor to accommodation factors express agents that provide protection against antibodies in the recipient, leading to resistance.
The invention also provides for transplant tissues from a species other than donor, made resistant to preformed or developing antibodies made against the tissue in a recipient. For example, human hematopoietic cells are placed into fetal pigs under conditions that expose them to sublethal concentrations of accommodation factors. These factors are produced by cells infused into the fetal pig or could be produced by the pig or gilt/sow. The hematopoietic cells, such as granulocytes would resist destruction in a human host with antibodies against the granulocytes.
The invention also provides a method and a kit for analysis and detection of the level of accommodation within potential donor animals. The assays are performed on blood and tissues obtained from the prospective donor animal prior to transplantation of the donor organ. Analyses include detection of recipient accommodation inducing factors, for example, immunoglobulins, B cells, and T cells in the donor animal. Other analyses include in-vitro tests of resistance of circulating cells and tissues taken from the prospective donor to lysis by reactive antibodies and complement, or reactive cytotoxic T cells of the recipient. Other alternative analyses include assessment of blood and tissues from the prospective donor for over-expression of protective genes typically expressed within accommodated organs, such as heme oxygenase-1, A-20, and bcl2. Bach, F. H. et al., Nature Medicine 3(2): 196-204 (1997).
The invention also provides a method to asses methods and factors for accommodation and improve the accommodation method according to the invention.
A. Transplant Organs or Tissue is Accommodated Prior to Transplantation
The present invention induces accommodation of the transplant organ or tissue prior to transplantation, while the organ or tissue is still in the donor animal. Induction prior to transplantation provides major advantages. The risk of rejection of the organ graft is significantly reduced. The potential costs and complications to the patient related to efforts at preventing rejection are significantly reduced. Multiple attempts can be made at achieving accommodation in multiple donor animals, if desired, and a donor which shows indication of best transplantation results may be selected for donation of the xenograft.
A donor animal is a mammal. The mammal can be, for example, but not limited to this example, a non-human primate, an artiodactyl, a carnivore, a rodent or a lagomorph. A pig fetus is a preferred recipient of the accommodation factor.
The accommodation factor is typically an antibody or an antibody producing cell but can include other factors that induce accommodation to include, but not be limited to: antibody reactive with donor tissue, antibody reactive with donor endothelium, antibody reactive with pig tissue or endothelium in the case where the donor is a pig or a member of the swine family, plasma cells, B lymphocytes, human B lymphocytes, conditionally immortalized B lymphocytes, anti-alphaGal antibody, a cell engineered to express an accommodation inducing factor, a hybridoma comprising a cell expressing an accommodation inducing factor, T cells reactive with cells in the graft tissue or organ, perforin, and Bandeiraea simplicifolia lectin. The factor can be a natural, isolated factor, or it can be a cell engineered to express a ligand or the purified engineered ligand.
The accommodation factors may be derived from any mammal, for example a member of a species that would be a xenograft recipient, e.g., a human or another mammal. In a preferred embodiment, the accommodation inducing factors are isolated from an individual who will later become a recipient. The method of determining, isolating, and manipulating each of the accommodations inducing factors are well known to an artisan skilled in the art.
The process of accommodation requires prolonged exposure to the accommodation inducing factors, at least 16 hours, up to 150 days. At that point, harvesting of the organ is undertaken.
In the preferred embodiment, a pig fetus is infused at an early stage of development, preferably about 45 days. However, accommodation in an adult mammal is possible. The donor does not have to be in an immune deficient state, but must be able to allow circulating accommodation inducing factors to persist in the donor for periods of time sufficient to allow accommodation of organs and tissues. However, infusion of an immune deficient mammal is preferred.
A first step in achieving an accommodated xenograft organ is introduction of accommodation inducing factor into a donor animal. Preferably the immune components are introduced into donor at a pre-immune stage of development. For example, cells are infused into donor animals that produce sub lethal levels of ligand which bind to cells in the donor animal. The ligand induces accommodation. The cells are infused under conditions which permit stable chimerism, i.e., stable presence of recipient immune system components in the xenograft donor organism. Later, the accommodated organ for transplantation is harvested and placed into transport medium under conditions suitable for transplantation into a recipient animal.
As an example, B cells programmed to produce antibodies reactive with pig endothelium are infused into pre-immune fetal pigs. The low levels of antibodies produced bind to the endothelial cells of the pig and induce accommodation or resistance to complement dependent cytotoxicity. An organ such as a heart procured from the modified donor pig is then placed in transport medium, such as, for example, the University of Wisconsin solution and transplanted into a human recipient. In spite of the presence of antibodies reactive to pig in the human recipient, including anti-alphaGal antibodies, the organ is resistant to hyper-acute rejection.
Alternative embodiments utilize one or more sources of antibody, such as plasma cells and conditionally immortalized B lymphocytes. Cells are produced which proliferate indefinitely in vitro, but are mortalized before transplantation. For example, a thermolabile SV40 transformation gene can be inserted. The cells then proliferate at 33° C., but not at 37° C. Further safeguards can be provided by other means, such as by surrounding the transformation gene with loxP sites. The transformation gene can then be removed with Cre Recombinase. Nakamura, J., et al., Transplantation 63(11): 1541-1547 (1997). The cells expressing the antibodies can be from the eventual xenograft recipient, but do not need to be from to the recipient. Accommodation does not depend on the induction of tolerance in the recipient. Rather, it may depend on the prolonged expression of anti-apoptotic genes in the donor organ cells.
Additional alternative implementations would utilize T cells that are reactive with the donor animal. Perforin, the protein complex produced by cytotoxic T cells and responsible for cytolysis of target cells is structurally quite similar to the membrane attack complex. Increased resistance to cytotoxic cells is developed by exposure to perforin.
Another implementation of the invention includes the production of tissue or cells of a species different from the donor mammal. For example, human granulocytes are produced that are accommodated prior to infusion into a human patient. They would then resist rejection and destruction by preformed or developing antibodies present in the recipient. Human hematopoietic cells (preferrably 107/pig, range 101 to 1010 per pig) would be infused into preimmune fetal pigs at 45 days gestation (range 12 days gestation to 7 days post-natal). Cells such as plasma cells, B lymphocytes, or hybridoma cells could be infused into the fetal pig that produce accommodating factors. At a later time, for example, after the birth of the pig, the human cells would be harvested and prepared in a manner appropriate for transfusion or transplantation into the recipient. The cells or the pig could be tested with assays that would predict accommodation within the human cells. For example, human cells could be tested for their resistance to cytotoxicity by antibodies or for the expression of genes known to protect cells from programmed death. The pig could be tested for the presence of antibodies reactive with the human cells.
B. Assays to Assess Donor Animals for Accommodation
The invention provides a method for assessing accommodation in the donor animal prior to transplantation of the organ. The assay allows confirmation of accommodation prior to transplantation. It also provides for a comparison of multiple donor animals infused with factors to accommodate organs, and the selection of the best candidate. The assay improves the results of transplant attempts and minimize the risk of rejection of the xenograft by preformed immune components.
To be effective, the assay needs to be predictive of accommodation and needs to preserve the organ tissue for transplantation. The ex vivo perfusion assay is considered the gold standard for predetermination of hyper-acute rejection and resistance to rejection. In accordance with the ex vivo perfusion assay, an organ, such as a heart, is perfused with serum or blood in a Langendorff apparatus. Non-accommodated organs cease functioning after a few minutes. Accommodated organs function for several hours. However, the accommodated organ is less suitable for transplantation after the procedure.
In accordance with the invention, an assay for accommodation includes a short term ex vivo perfusion of tissue other than the transplant organ. Alternatively, accommodation is gauged by testing blood cells or endothelial cells obtained from the putative donor animal for resistance to complement dependent cytotoxicity. Complement dependent cytotoxicity assays incubate the test cells with antibody known to be reactive and fresh complement. After a period of time, the viability of the cells is determined. With similar control cells, the antibody and complement kill most of the cells, while a larger proportion of accommodated cells would survive. Yet another method tests the donor animal for persistence of the infused cells, or tests the donor animal for antibodies or cells reactive with the donor animal tissue. The methods involved are well known in the art.
C. Method for Enhancement of Accommodation within the Donor Animal
The invention provides a system for testing improvements and identification of further accommodation stimulating agents. The system consists of infusion of various factors and cells into test animals and later assessment of the modified animals for accommodation. As examples of the system, multiple sources/fractions of pig reactive antibodies are compared for their potential to induce accommodation. Each experimental group would include fetal pigs infused with the particular fraction of cells. Later the donor and/or the organs would be assessed for accommodation. Examples of tests for accommodation would include the ex vivo perfusion of an organ such as a heart or kidney and heterotopic transplantation of a xenograft into non-human primates. The cell fraction leading to the most prolonged function of the heart would be considered the most optimal. Additional objectives of the invention include the testing of different strains of donor animals and of transgenic animals.
II. Methods for Production of Accommodated Organs or Tissues In the Donor Animal
The present invention provides transplant organs or tissues accommodated to be resistant to rejection by the recipient of the graft. It also provides a method for production of such accommodated organs. In accordance with the method, the organ, before harvesting, is exposed on a repeated or chronic basis to accommodating factors which bind to tissues in the donor animal but do not cause irreparable injury to the animal or the organ. Following prolonged exposure, the accommodated organ is harvested and prepared for transplantation into a recipient, such as a human.
Preferably, the ligand either needs to be infused into the donor on a repeated basis or produced by cells that are stable within the donor animal. The same or alternative or additional ligands are infused upon repeat of the infusion step. The donor animal should preferably be in a reduced immune activity state (immune deficient) in order to accept the ligand, i.e., not to immunologically reject it.
As an example of ligand infusion, human immunoglobulins are infused into pre-immune fetal pigs. The immunoglobulins are injected into individual fetal pigs by percutaneous injection using ultrasound guidance. Sub-lethal levels of the accommodating factors are infused.
The levels of factor to be infused is pre-determined in empirical fashion. For example, a serum fraction is used and increasing concentrations are administered to at least one other fetus, at similar stages of development. An LCD50 is determined and preferably used as an accommodation treatment. Any LCDn, where n≦90% of the lethal dose is useful as an accommodation factor dose.
The immunoglobulins include antibodies, including IgG and IgM, which specifically binds pig endothelial cells. Examples of such antibodies include anti-alphaGal present in humans and Old World primates. Galili, U., et al., Blood, 82(8): 2485-2493 (1993). AlphaGal is not expressed in these animals, but is expressed in most other species, including pigs. The immunoglobulins may include antibodies to other xenogeneic antigens as well.
The method of preparation of such antibodies or isolation of enriched fractions of such antibodies are well known to artisans skilled in the art. For example, serum can be passed through an immunoadsorption column containing alphaGal. After washing the column, the adherent anti-alphaGal antibodies is eluted. Active antibody fragments or active single donor antibodies can also be employed.
Another example of a ligand would be the lectin Bandeiraea simplicifolia which binds to alphaGal, including the alphaGal expressed on pig endothelial cells. Grehan, J. F., et al., Transplant. Proc. 32(5):975 (2000). The lectin is infused at low levels (less than 100 μg/ml) into putative donor pigs. It is infused on a daily basis for 3 to 30 days.
B lymphocytes committed to production of antibodies or plasma cells producing antibodies reactive with the donor tissue are infused under conditions allowing stable engraftment and providing sub lethal doses of antibodies. As an example, human B lymphocytes (2×108/kg, range 1×106/kg to 4×109/kg) would be infused by ultrasound guidance into fetal pigs at 45 days gestation (range 30 to 80 days) under sterile conditions. It is estimated that approximately 1% of the human B cells would be committed to production of antibody to alphaGal. Because the B cells are infused into pre-immune fetal pigs, the fetal pigs would accept the B cells and become tolerant to such cells.
The accommodation inducing factors, be they a ligand or a suspension containing immune cells, can be from an individual unrelated to the recipient. Accordingly, the cells may include B cells from unrelated humans or from other species. For example, human B cells from a type O individual can be infused into fetal baboons of type A or B. The low levels of anti-A or anti-B induce accommodation. The transplant organ could then be harvested for transplantation after the birth of the baboon. The infused cells may include immortalized cells such as hybridoma cells. In accordance with one embodiment, an accommodation-inducing cell, preferably a B lymphocyte would contain a suicide gene such as thymidine kinase or adenosine deaminase. Cohen, J. L., et al., Hum. Gene Ther. 11(18): 2473-2481 (2000). Prior to transplant, the cells could then be purged with the appropriate prodrug.
In a preferred embodiment, the accommodation inducing factors are from the same species as the recipient. In accordance to another preferred embodiment, the accommodation inducing factor is from the individual who will be the ultimate recipient. Because accommodation is a property of the transplant organ, it is not essential to adoptively transfer lymphocytes back to the recipient.
Following prolonged exposure (preferably more than 20 days, range 16 hours to 150 days), the donor animal is euthanized under conditions that permit procurement of the donor organ suitable for transplantation. For example, if the organ for transplant is a pig heart, the donor pig with accommodated tissues is sedated and anesthetized with isoflurane. Under sterile conditions, the chest is opened and cold Cardioplegia solution (such as Stanford solution containing mannitol, potassium chloride, sodium bicarbonate) is infused into the pig aorta and the heart is packed in iced saline. The heart is removed and perfused with transport medium, such as Eurocollins solution (Steffen, R., et al., Transpl. Int. 3(3):133-136 (1990)) or University of Wisconsin solution (Belzer, F. O. and Southard, J. H. Transplantation 45(4): 673-676 (1988)) The organ preservation solutions contain essential amino acids, carbohydrates, and electrolytes that maintain viability of the organ or tissue during transportation to the recipient.
Organs that may be accommodated are, for example, but not limited to for transplantation prior to transplantation include, but are not limited to heart, kidney, liver, lung, pancreas, heart-lung, intestine, spleen, or thymus. Tissues that may be accommodated include, but are not limited to bone, skin, hair, retired pigmented cells, neural tissue, skeletal muscle, myocytes, pancreatic islets, hepatocytes, embryonal or adult stem cells, and hematopoietic cells.
Another implementation of the invention relates to the production of accommodated tissue or cells of a species different from the donor mammal. Such tissu or cells may include, but are not be limited to, osteo blast cells, osteo clost, skin, skin epithelial cells, hair follicle cells, eye, neural cells, skeletal muscle, smooth muscle cells, cardiac muscle cells, pancreatic islets, hepatocytes, stem cells, progenitor cells, or hemapoietic cells. Such cells would be derived from an animal of a species other than the donor. Preferably, they would be from the same species as an eventual recipient, or even from the recipient himself. The donor animal could be any of the donors of the invention.
Accommodation factors in accordance with the invention could be administered prior to administration of the cells for accommodation (one hour to two days), at the same time as the cells for accommodation or shortly after the cells for accommodation are administered. In a preferred embodiment, the cells for accommodation and the accommodation inducing factors are administered at the same time, mixed together, or within minutes of each other. The accommodation inducing factors are factors in accordance with the invention, but may further include an antibody, an antibody producing cell, or a T-cell which recognizes cells from the species where the cells to be accommodated are derived.
After prolonged exposure in accordance to the invention, the state of accommodation of the cells of a species different from the donor mammal could be assessed by any of the methods of the invention, for example by looking for antibodies in the donor specific for the cells introduced for accommodation. Finally, the accommodated cells or tissue are harvested.
The cells introduced for accommodation or their progeny can be separated by positive means or by negative means from donor cells. For example, the donor can be from a species engineered with a conditionally lethal gene, for example the thymidine kinase (HSV-tk) gene. Cells harboring this gene are sensitive to ganciclovir. Moolten and Wells Cancer Res. 46: 5276-5281 (1986); Reardon J. Biol. Chem. 264:19039-44 (1989); and Patil et al., Breast Can. Res. Treat. 62: 109-115. Upon harvesting, donor cells can be killed, by exposure to ganciclovir. Alternatively, the cells to be accommodated are transformed with a positive selection, prior the being administered into the donor. They can be engineered to express a selective marker, such as drug resistance, or an antigen that will upon expression be located on the cell membrane such that an antibody specific to the antigen may fish out a cell expressing the antigen. Those and similar approaches are well known to an artisan skilled in the art.
For example, human granulocytes could be produced that are accommodated prior to infusion into a human patient. They would then resist rejection and destruction by preformed or developing antibodies present in the recipient. Human hematopoietic cells (preferred 107/pig, range 101 to 1010 per pig) would be infused into preimmune fetal pigs at 45 days gestation (range 12 days gestation to 7 days post-natal). Accommodation inducing factors such as plasma cells, B lymphocytes, or hybridoma cells could be infused into the fetal pig that produce accommodating factors. At a later time, for example, after the birth of the pig, the human cells would be harvested and prepared in a manner appropriate for transfusion or transplantation into the recipient. The cells or the pig could be tested with assays that would predict accommodation within the human cells. For example, human cells could be tested for their resistance to cytotoxicity by antibodies or for the expression of genes known to protect cells from programmed death. The pig could be tested for the presence of antibodies reactive with the human cells.
While pigs would be an ideal species donor mammal for accommodation, the invention can be readily implemented with any of many animals. Animals that can potentially be used as surrogates i.e. donors, include, but are not limited to those listed below. It is known that some animals offer advantages for select uses.
Preferred donors are mammals. From among 39 major orders of the class Mammalia, five orders appear particularly suitable as surrogate animals for human organ recipients: primates, artiodactyls, carnivores, rodents, and lagomorphs.