ENGRAFTMENT OF HEMATOPOIETIC CELLS FROM A PRIMATE DONOR TO A PRIMATE RECIPIENT

Abstract: A method for the replacement of the hematopoietic system of one primate with a hematopoietic system derived from another primate of a different species.

Description: ENGRAFTMENT OF HEMATOPOIETIC CELLS FROM A PRIMATE DONOR TO A PRIMATE RECIPIENT FIELD OF THE INVENTION The present invention is generally in the field of transplantation and more specifically concerns engraftment of hematopoietic cells from a primate donor of one species to a primate recipient of another species. The present invention also concerns non-human chimeric primates, having stably engrafted xenogeneic hematopoietic systems from another primate of a different species. The present invention further concerns various uses of the above method and non-human primates. PRIOR ART The references listed below are considered relevant for the purpose of giving an appreciation of the state of the art prior to the invention. The acknowledgement of this art herein does not amount to an admission that these references are in any way relevant regarding the patentability of the invention as defined herein. The list of prior art referred to herein consists of the following references: 1. Gale, R.P. and Reisner, Y. (19S6), Graft rejection and graft-versus- host-disease; mirror images, Lancet i, 1468. 2. Reisner, Y-, Kapoor. N., Good, R.A. and O'Reilly, R.J. (1984), Allogeneic bone marrow transplantation in mouse, monkey and man using lectin-separated grafts. In Tolerance in Bone Marrow and Organ Transplantation (Slavin, S. ( eds.), pp. 293-308. 3. Reisner, Y. (1990), Engraπment of T-cεll-depleted bcne marrow in murine models for allogeneic bone marrow transplantation. In Bone Marrow Transplantation (Champlin, R., eds.), pp. 9-25, Kluwer Academic Publishers. 4. O'Reilly, R.J., Keever, C.A., Small, T.N. and Brochstein, J. (1989). The use of HLA non-identical T-cell-depleted marrow transplants for correction of severe combined immunodeficiency disease. Imm nodefic. Ref. I, 273-309. 5. O'Reilly, R.J., Collins, N.H., Keman, N., Brochstein, J., Dinsmore, R., Kilpatrick, D., Siena, S., Keever, C, Shank, B., Wolf, L.,Dupont, B. and Reisner, Y. (1985) Transplantation of marrow-depleted of T-cells by soybean lectin agglutination and E-rosette depletion: major histocompatibility complex-related graft resistance in leukemic transplant recipients. Transplant. Proc, 12:455-459. 6. Vallera, D.A. and Blazar, B.R. (1989) T-cell depletion for graft- versus-host disease (GVHD) prophylaxis: a perspective on engraftment in mice and humans. Transplantation, 47:751-760. 7. Van-Bekkum, D.W. and Lowenberg, B. (1985), Bone Marrow Transplantation: Biological Mechanisms and Clinical Practice, Marcel Dekker, New York. 8. Lapidot, T., Terenzi, A., Singer, T.S., Salomon, O. and Reisner, Y. (1989), Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts, Blood, 73:2025. 9. European Patent Application, publication No. 438053, (1991). 10. Andrews, R.G., Bryant, E.M., Bartelmez, S.H., Muirhead, D.Y., Knitter, G.H., Bensinger, W., Strong, D.M., and Bernstein, I.D., (1992), CD34 4 Marrow Cells, Devoid of T and B Lymphocytes, Reconstitute Stable Lymphopoiesis and Myelopoiesis in Lethally Irradiated Allogeneic Baboons, Blood, 7:1693-1701. 11. Reisner, Y., Friedrich, W. and Fabian, 1. (1986), A shorter procedure for preparation of E-rosette-depleted bone marrow for transplantation. Transplantation, 42:312-315. 12. Sheridan, W.P., Begley, C.G., Juttner, C.A., Szer, J., To, L.B., Maher, D., McGrath, K.M., Morstyn, G. and Fox, R.M. (1992), Effect of peripheral-blood progenitor cells mobilized by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy, Lancet i, 640-644. In the following text, the above references will be acknowledged by indicating their number from the above list. BACKGROUND OF THE INVENTION Bone marrow transplantation (BMT) while being in principle a promising therapy in various clinical situations, is usually hindered by immune-related complications including graft rejection and graft-versus- host diseases (GVHD) 1 . GVHD can be reduced by using a T-cell- depleted bone marrow and indeed mismatched T-cell-depleted marrow has been successfully used for the treatment of children with severe combined immune deficiency (SCID)-" 4 . In such transplanted SCID patients, donor- type T-cells which are tolerant to the host are formed and there is thus a reduction in the GVHD. The success in the treatment of SCID patients lead to the extension of such treatment to leukemia patients for whom matched sibling donors were not available or to individuals who had matched sibling donors but were nevertheless at high risk for GVHD. However, the experience with leukemia patients was disappointing due to a high rate of graft rejection or graft failure and the recurrence of leukemia. In recipients of HLA-identical T-cell-depleted bone marrow, the incidence of graft rejection is about 10-15%, whereas in recipients of HLA-nonidentical T- cell-depleted marrow, the rate of rejection is about 50%*. Bone marrow allograπ rejection has been documented extensively in different animal models 3,6,7 . In the mouse, such rejection can be overcome by increasing the marrow inoculum. Such inoculum increase allowed the production of long-term immunologicaliy vigorous chimera with a high rate of success 3 . One of the suggestions brought forward in the art to increase the marrow inoculum size, was by the use of frozen HLA-nonidentical marrow from cadavers 8 . It was furthermore suggested to increase the number of the donor" stem cells by in vitro treatment with hormones, such as GM-CSF, prior to transplantation 3 . EP-438,053 9 , discloses the engraftment of hematopoietic lineages from one mammal to another. In accordance with this European patent application, a non-human mammal Ml is treated to substantially suppress or destroy its hematopoietic cells and is then transplanted with hematopoietic cells from at least two sources, one of which are cells originating from a mammal M3, of a species other than Ml, preferably human and the second of these sources are hematopoietic cells originating from a mammal M2, of a species other than M3 having an immune deficiency, e.g. SCID. Consequently, a chimeric mammal M4 is obtained having long-term and stable xenogeneic hematopoietic cells. The specific and preferred mammal which was disclosed in the aforementioned European application was a mouse. Engraftment of hematopoietic CD34 *" cells from one baboon to a mixed lymphocyte culture (MLC) non-reactive baboon was also reported 10 . _ r _ 16850 GLOSSARY The following are the meanings of some terms which will be used at times in the description below: "Xenografting" - to denote the transplantation of tissue or organ from one species to a different species; "Xenograft(s)" - to denote the xenografted tissue or organ; "Xenografted primate" - a primate which is a recipient of a xeπograft; "Human-primate hematopoietic xenografting" - the grafting of human hematopoietic cells into a non-human primate recipient; "Primate -human hematopoietic xenografting" - to denote the xenografting of hematopoietic cells from a non-human primate donor to a human recipient. The hematopoietic cells in such xenografting may be from a normal primate donor or may be human hematopoietic cells from a human- primate hematopoietic xenograft, Other scientific terms which will be used herein have meanings which are generally acceptable in the art. GENERAL DESCRIPTION OF THE INVENTION The present invention has, as one of its objects, the provision of a method for replacement of the hematopoietic system of one primate with a hematopoietic system derived from another primate of a different species. It is a further object of the invention to provide non-human xenografted primate, the hematopoietic system of which has been replaced with that of another primate of a different species, preferably human. It is : a further object of the invention to provide a transplantation therapy for the treatment of various diseases or disorders in the hematopoi¬ etic system of primates, particularly human. It is furthermore an object of the invention to provide a novel method of xenografting of non-hematopoietic cells or organs between primates of different species, e.g between a non-human primate donor to a human recipient. It is a further object of the present invention to provide a primate model for the studying of the pathology of various human diseases of or associated with the hematopoietic system as well as for the development of therapies for such diseases. It is furthermore an object of the invention to provide a method for obtaining a supply of human hematopoietic cells. It is furthermore an object of the invention to provide novel means for the preparation of human monoclonal or polyclonal antibodies as well as specific cytotoxic T-cells. The present invention is based on the understanding that a major cause of failure of xenografting of hematopoietic cells in primates in addition to GVHD lies in that the recipient primate usually retains some stem cells which af t er a certain period of time prevail over the xenografted marrow cells, which even t ually results in a graft rejection. This is so even after treatment intended to destroy all the hematopoietic lineage in the recipient primate, since some of the stem cells manage to escape such treatment. In accordance with the invention it has thus been realized that in order to overcome this deficiency and obtain long-term stable xenografts of hematopoietic cells in primates, the recipient primate should be xenografted with a hematopoietic stem cell preparation from the donor, in a total inoculum of stem cells which is in large excess to the stem cells remaining 5 in the recipient. In accordance with the invention it has further been realized that human-primate hematopoietic xenografting may be used for the develop¬ ment of highly valuable models for studying the pathology of various diseases or disorders associated with the hema t opoietic system, as well as S95/16850 - 7 - for the development of therapies to the treatment of such diseases. Further¬ more, it has been realized that the resulting xenografted primate may serve as a tool for the production of various therapeutic agents for the treatment of a variety of human diseases or disorders. Against this, it has been realized in accordance with the invention that primate-human hematopoietic xenografting offers a novel transplantion therapy for the treatment of a variety of human diseases or disorders associated with the hematopoietic system. The present invention provides a method for the replacement of the hematopoietic system of a primate Ml by a hematopoietic system of a primate M2 of a different species, comprising: (a) treating the Ml primate so as to destroy most of its dividing hemato¬ poietic cells; and (b) inoculating the treated Ml primate with a hematopoietic cell prepara- tion from the M2 primate, the cell preparation being enriched with stem cells, the amount of stem cells in the inoculum being in a large excess to the stem cells of the Ml primate remaining after the treatment; whereby a stable primate xenograft M3 containing an M2 primate derived hematopoietic system is obtained. The amount of inoculated stem cells is typically 5 x 10 s - 2 x 10 9 cells/kg body weight. Specific embodiments of the above method concern human- primate hematopoietic xenografting on the one hand and primate-human hematopoietic xenografting on the other hand. The present invention also provides a non-human xenografted primate M3, characterized in that: (i) it is derived from a non-human primate Ml, the hematopoietic system of which has been essentially destroyed; and - 8 - (ii) it comprises a reconstituted hematopoietic system being derived entirely from a primale M2 of a different species than Ml. The preferred primate Ml, from which primate M3 is prepared is a monkey. The present invention further provides a method for obtaining a supply of human hematopoietic cells, comprising; (a) treating a non-human primate Ml so as to destroy most of its dividing hematopoietic cells; (b) inoculating the treated Ml primate with a stem cell enriched human hematopoietic cell preparation, the amount of stem cell in the inoculum in " large excess to the stem cells of the Ml primate remaining after treatment; (c) obtaining a xenografted non-human primate M3; and (d) growing said primate M3 - ' until the development therein of a human derived hematopoietic system; whereby an M3. xenografted non-human primate with a stable human derived hematopoietic system is obtained from which human hematopoietic cells can be withdrawn. Also provided by the present invention are hematopoietic cells obtained by the above method. By another of its aspects the present invention provides a method for obtaining human antibodies directed against a desired antigen, compris¬ ing immunizing a xenografted primate M3 and then preparing therefrom either polyclonal or monoclonal antibodies directed against said antigen. Polyclonal antibodies are obtained by withdrawing serum samples from said M3 primate and obtaining an anti-said antigen anti-serum therefrom. A monoclonal antibody directed against said antigen is obtained by immortalizing B-cells and selecting for those which secrete antibodies against said antigen. S95/16850 _. 0 _ By a further of its aspects the present invention provides a method for obtaining human cytotoxic T-cells reactive against a pathogenic cell or tissue. In accordance with this method, a human-primate hemato¬ poietic xenografted primate is immunized with said pathogenic cell or tissue or a component thereof capable of eliciting the development of cytotoxic T-cell immune reaction, and then recovering the cytotoxic T-cells from said primate and selecting for those cytotoxic T-cells which are reactive against said cell or tissue. An example of such cells or tissue are virus-infected cells, cancer cells, eukaryotic or procaryotic pathogenic microorganisms (protozoa, bacteria, microplasma), and the like. The present invention further provides therapeutic preparations comprising a therapeutically effective amount of the above antibodies or cytotoxic T-cells. The present invention also provides a method of treatment of the individual in 1 need by administration to the individual of said antibodies or said cytotoxic T-cells. The present invention also provides, by another of its aspects, a model for the study of the human hematopoietic system which comprises a primate-human hematopoietic xenografted primate, which is preferably a monkey. Such a model is particularly suitable for the study of the pathology of various diseases or disorders associated with the hematopoietic system such as sickle . cell anemia, thalassemia,.AIDS, malaria, leukemia, lymphoma, etc. For the study of the pathology of such a disease or disorder, it may be xenografted with a hematopoietic stem cell preparation from a human having such disease or disorder. Alternatively, the xenografted primate may be subjected to a treatment to bring about development of said diseases or disorders in a human-derived reconstituted hematopoietic system. A particular aspect of the above model is as a system for the study of the efficacy of a therapeutic agent or the efficacy of a treatment - 10 - modality of a disease or disorder. A specific example is a mode! to test the efficacy of gene therapies targeted at hematopoietic cells, e.g. at stem cells. The present invention also provides a method of treatment of diseases or disorders by hematopoietic cell transplantation therapy, wherein the transplanted hematopoietic cells are stem cells obtained from a non- human primate, which may be a normal (i.e. non-treated) non-human primate, or may be the above xenografted primate M3. Examples of diseases or disorders which can be treated by such a therapy are various autoimmune diseases, HIV infections, lymphomas, leukemia, etc. In the case of HTV infection, for example, the xenografted hematopoietic stem cells will preferably be obtained from a normal non-human primate. By an embodiment of the ' above method, the transplanted hematopoietic cells are first manipulated by a gene therapy, e.g. transfecting these cells by conferring a multi-drug resistance gene ("MDR gene'% etc. Also provided by the invention, is a non-human primate blood cell preparation enriched with stem cells. Such a preparation may be used as a pharmaceutical preparation in the above transplantation therapy or may¬ be used as a bulk, biological preparation for the production of aliquots for such a therapy. Bulk preparations or aliquots for single uses may be kept frozen and then thawed prior to use. By an embodiment of this aspect of the invention, the stem- cell preparations are transfected with a foreign gene, e.g. an MDR gene. The invention also provides a method for the production of the above stem-cell enriched non-human primate blood cell preparation. Various other embodiments of the invention will become clear from the following description. - 11 - DETAILED DESCRIPTION OF THE INVENTION The present invention will be described in the following with reference to some specific, non-limiting embodiments: In accordance with the invention, a novel method of xenografting hematopoietic lineages from one primate to another, as well as stable non- human hematopoietic xenografted primates are provided. It should be noted that human-primate hematopoietic xenografts are advantageous over prior art available xenografts, e.g. such available from EP-438,053 10 , since the evolutionary distance between humans and primates is short as compared to that between human and non-primate mammals such as rodents. For example, cytokines in human and primates are very similar whereas the cytokines in human and rodents are often not cross-reactive. In accordance with one embodiment of the invention, hematopoi¬ etic stem cells from human donors are grafted into a non-human primate, preferably a monkey. Following treatment intended to destroy the recipient's dividing hematopoietic cells which may, for example be a split total body irradiation (TB1) treatment, the recipient primate is inoculated with a stem- cell enriched preparation from the human donor, which is essentially depleted from T-cells. Such a hematopoietic preparation may be a T-cell- depleted human bone marrow or may be a T-cell-depleted peripheral stem cell preparation (stem cells in the periphery are obtained as a result of various treatments, e.g. chemotherapy in cancer patients or following administrations, of G-CSF, etc.). In order to ensure the reconstitution of the human derived hematopoietic system in the non-human xenografted primate, the amount of transplanted stem cells should be in excess to stem cells of the recipients which remain after said treatment. For example, in the case of marmoset monkeys, the inoculum size (following T-cell depletion) effective in achieving this feat is in the range of 5 x 10 s - 2 x 10 9 cells. - 12 - After xenografting, the monkeys are kept under sterile conditions which are crucial at least until the reconstitution of the donor immune system in the recipient primate. Treatment such as irradiation, in particularly split-body irradia- tion, destroys most of the dividing hematopoietic cells, in particular stem cells. Cells of the hematopoietic system which remain in the treated animal comprise mainly red blood cells. Red blood cells (RBCs) have a life span in the range of 100 - 120 days and thus the RBCs in the xenografted primate are initially of the recipient type but are replaced over a period of time with donor-type red blood cells which are derived from the inoculated cells. In the initial phase the xenografted primate is essentially devoid of most of its hematopoietic immune cells and these are reconstituted over a period of time from the inoculated donor stem cells. A human-primate hematopoietic xenograft serves as an effective model for the study of the pathology of various diseases or disorders of the human hematopoietic system as well as a tool for the development of therapies for the treatment of such diseases or disorders. Examples of such diseases or disorders, for which no good model is yet available, are sickle cell anemia and thalassemia and chronic myeloid leukemia. In order to study such diseases, T-cell-depleted stem cells enriched fraction from a human donor having such a disease may be xenografted into a monkey recipient and the disease, as well as therapies for the treatment thereof, may be studied in this xenografted monkey model. Another example of use of the human-primate hematopoietic xenograft is as a model for the studying of AIDS pathology and for the development of therapies for the treatment of AIDS. In accordance with such an embodiment, after formation of a human-primate hematopoietic xenograft, the non-human xenograft is infected with the HIV, the causative virus of AIDS. The pathology of HIV as well as anti-HIV therapies can then be studied in this primate xenograft. Another example of an application of the model is in the study of retro-viral vectors or other modalities used in gene therapy targeted at hematopoietic cells, e.g. stem cells. A particular example of such a therapy is a gene therapy for the treatment of storage diseases (enzyme deficiencies) of hematopoietic cells, or transfection of stem cells with a multiple drug resistance gene in cancer patients, and the like. In addition to its use as a model for the study of various human hematopoietic diseases or disorders, the human-primate hematopoietic xenografted primate may have various other uses. For example, the xenografted primate may be inoculated with an antigen and then used for the development of human anti-sera against such antigens. The anti-sera may be polyclonal, or may be monoclonal, for which purpose B-cells from the primate xenograft are immortalized and polyclonal antibodies prepared therefrom as known per se. In a similar manner, also cytotoxic T-cells directed against a pathogenic cell or tissue may be prepared. For this purpose, the human- primate hematopoietic xenografted primates are immunized with a cell or tissue or a component thereof which is capable of eliciting the development of a cytotoxic T-cell immune reaction. Such a component may, for example, be a membranous preparation obtained from said cell or tissue. After the development of the immune reaction in said primate, cytotoxic T-cells are withdrawn and selected for such which are reactive with said cell or tissue. The above antibodies or cytotoxic T-cells may be used as therapeutic agents in the treatment of various human diseases or disorders in humans. Another important use of a human-primate hematopoietic xenografted primate is as a bank of stem cells for therapeutic use in human, and particularly in bone-marrow replacement therapy. There is at times a shortage of appropriate human donors of bone-marrow for transplantation, and such xenografted primates may be useful as a stem cell source. The inter-primate transplantation protocol provided by the present invention may also be used as a therapy for the treatment of various human diseases or disorders. A particular application is in the treatment of viral diseases which affect human tissue and cells but not non-human primate tissue and cells. For example, one of the possible treatments of AIDS is a bone-marrow replacement therapy. However, even after treatment intended to destroy dividing hematopoietic cells, such as split TB1, some CD4 * cells infected with HIV will likely remain in the body and these cells will bring infection of the donor cells by the HIV and hence the reoccurrence of AIDS. This may be avoided by xenografting of hematopoietic cells from a non- human primate donor insensitive to HIV into a human AIDS patient. Another possible application of the primate-human hematopoietic xenografting is in a combined therapy involving xenografting of non- hematopoietic organs, such as liver and the xenografting of hematopoietic cells. An example of such a treatment is in the case of viral infections of the liver such as hepatitis B or hepatitis C infections. In infected individuals with an acute hepatitis B or hepatitis C infection, human-human liver I ) transplant will generally be ineffective as a therapy since the HBV or HCV will infect the transplanted liver. The only therapy available for such individuals is the xenografting of liver from a non-human donor. However, in such transplantation, there is likely to occur an immune reaction which will cause rejection of the transplanted organ. Use of immunosuppressive 15 drugs will make the patient liable to encounter infections as well as cancer. In accordance with the present invention, an organ xenograft is accompanied by a primate-human hematopoietic xenograft, which typically precedes the organ transplant. During their development in the recipient, the immune - 15 - system will be "educated" to be non-responsive to the transplanted organ together with the development of lack of responsiveness to the host cells. As will be appreciated, the above described embodiments are only a limited repertoire out of many embodiments of the invention as defined herein. The present invention is not intended to be limited to the above described embodiment and encompasses all other embodiments of the invention within said scope. The invention will be illustrated in the following examples. Example 1 Human Stem Cell-Enriched Hematopoietic Cell Preparations Human hematopoietic cell preparations enriched with ' stem cells are obtained by T-cell-depletion of human bone-marrow. Such depletion may be obtained by fractionation by differential agglutination with soybean agglutinin and E-rosetting with sheep red blood cells, as described by Reisner et al. Λ . Human stem cell enriched hematopoietic cell preparations are also obtained from the peripheral blood system of individuals undergoing treatment which results in migration of stem cells to the periphery. This is the case in cancer patients undergoing chemotherapy which was shown to cause the appearance of such stem cells in the periphery, as described by Sheridan et al. n . Alternatively, stem cell enrichment in the periphery is achieved by induction in normal donors by the use of G-CSF (12 g/kg for 7 days) and the stem cells may be collected from such individuals after day 3 an onwards. Stem cells are obtained from the peripheral blood system by leucopheresis intended to enrich for CD34" cells, and following T-cell depletion as described above. - 16 - Example 2 Transplantation of Human Stem Cells in Primates Marmoset monkeys are treated with split total body irradiation (TBI) which consisted of 4 Gy, followed three days later by 10 Gy. One day after completion of the TBI, a transplant of 5 x 10 s - 2 x 10 9 T-cell- depleted bone-marrow cells or T-cell-depleted peripheral blood CD34* stem cells enriched fraction of Example 1, or a combination of the two, is inoculated to the monkeys. The monkeys are then kept in a sterile cage under a laminar flow hood, and fed with sterile food and water, and supported, as necessary, by blood transfusions from normal monkey donors (the blood transfusions were prior irradiated with 2 Gy). The monkeys are also treated by antibiotics, antimycotics and antiviral agents as needed. 20 days after irradiation, a treated monkey was shown to comprise components of the human hematopoietic system, i.e. granulocytes. Example 3 Preparation of Antibodies Xenografted monkeys of Example 2, are inoculated with various antigens and, the inoculation of the antigen is repeated several times as necessary. Following a sufficient time to allow the development of an immune reaction, anti-sera is prepared from these monkeys by means generally known per se. For the preparation of monoclonal antibodies, hybridomas are prepared by means generally known per se, whereby human monoclonal antibodies are obtained. - 17 - Exa ple 4 Preparation of Cytotoxic T-cells Xenografted monkeys of Example 4 are inoculated with pathogenic cells, such as cancer cells, virus-infected cells, etc. or with peptide or membranous preparations. After sufficient time for the develop¬ ment of an immune reaction cytotoxic T-cells arc withdrawn and selected for such which are reactive against said cells or tissues. Such cytotoxic T-cells, which may be expanded in vitro, are useful as a therapeutic agent for the treatment of a disease or disorder caused by such pathologic cells or tissue. Example 5 Human Bone-Marrow Replacement Therapy Human HIV patients are irradiated by split TBI, where essentially their hematopoietic cells are destroyed. The human, which is maintained under total sterile conditions, is then transplanted with a stem cell enriched preparation obtained from anon- human primate, particularly a monkey. The stem cell-enriched preparations are obtained from monkeys in a similar manner to that obtained from humans as described in Exam¬ ple 1. If necessary, prior to the development of the reconstituted hematopoietic system in the treated individual, he is continuously infused with sterile and irradiated RBC preparations. Furthermore, if necessary, the individual is administered with various blood factors. In order to avoid the occurrence of infections in the individual after treatment, the individual is constantly infused with antibiotics, antimycotic and antiviral agents. The individual is kept under essential sterile conditions until the development therein of the donor-derived hematopoietic system.

Claims: - 19 - CLAΪMS: 1. A method for the replacement of the hematopoietic system of a primate Ml by a hematopoietic system of a primate M2 of a different species, comprising: (a) treating the Ml primate so as to destroy most of its dividing hemato¬ poietic cells; and (b) inoculating the treated Ml primate with a hematopoietic cell prepara¬ tion from the M2 primate, the cell preparation being enriched with stem cells, the amount of stem cells in the inoculum being in a large excess to the stem cells of the Ml primate remaining after the treatment; whereby a stable primate xenograft M3 containing an M2 primate derived hematopoietic system is obtained. 2. A method according to Claim 1, wherein said cell preparation is essentially devoid of T-cells. 3. A method according to Claim 1 or 2, wherein the amount of the stem cells in the inoculum is about 5 x 10 s - 2 x 10 9 cells/kg body weight. 4. A method according to Claim 1, wherein one of primate Ml or M2 is a monkey and the other of primate M2 or M3 is human. 5. A method according to Claim 1, wherein the treatment of step (a) is a total body irradiation. 6. A method according to Claim 5, wherein the irradiation is a split total body irradiation. 7. A method according to Claim 1, wherein the hematopoietic cell preparation is a T-cell-depleted bone-marrow preparation. 8. A method according to Claim 1, wherein the hematopoietic cell preparation is a peripheral stem cell preparation. 50 - 20 - 9. A method according to Claim 8, wherein the peripheral stem cell preparation is obtained by withdrawing blood samples from an individual undergoing a treatment which leads to the migration of stem-cells to the peripheral blood system, and processing the blood sample so as to isolate the stem cells therefrom. 10. A method according to Claim 9, wherein said individual is a cancer patient undergoing chemotherapy or an individual treated with G-CSF in an amount to cause migration of stem cells to the periphery. 11. A non-human xenografted primate M3, characterized in that: (i) it is derived from a non-human primate Ml, the hematopoietic system of which has been essentially destroyed; and (ii) it comprises a reconstituted hematopoietic system being derived entirely from a primate M2 of a different species than Ml. 12. A xenografted primate M3 according to Claim 11, wherein primate M3 is a monkey. 13. A xenografted primate M3 according to Claims 11 or 12, wherein primate M2 is human. 14. A xenografted primate M3 produced by the method of Claim 1. 15. A method for obtaining a supply of human hematopoietic cells, comprising: (a) treating a non-human primate Ml so as to destroy most of its dividing hematopoietic cells; (b) inoculating the treated Ml primate with a stem cell enriched human hematopoietic cell preparation, the amount of stem cells in the inoculum being in a large excess to the stem cells of the Ml primate remaining after treatment thereby obtaining a xenografted non-human primate M3; and - 21 - (c) growing said primate M3 until the development therein of a human derived hematopoietic system; whereby an M3 xenografted non-human primate M3 with a stable human derived hematopoietic system is obtained from which human hematopoietic cells can be withdrawn. 16. A method according to Claim 15, wherein said cell preparation is essentially devoid of T-cells. 17. A method according to Claims 15 or 16, wherein the amount of the stem cells in the inoculum is about 5 x 10 8 - 2 x 10 9 cells/kg body weight. 18. A method according to Claim 15, wherein said Ml primate is a monkey. 19. Human hematopoietic cells obtained by the method of Claim 15. 20. A method for obtaining human antibodies directed against a desired antigen, comprising: (a) immunizing a xenografted primate M3 according to Claim 13 with the antigen; and (b) either withdrawing serum samples from said primate M3 and obtaining a polyclonal anti-said antibody preparation therefrom, or immortalizing B-cclls antibody producing cells from said primate M3, and selecting the immortalized cells which secrete antibodies directed against said antigen and obtaining monoclonal antibodies therefrom. 21. Antibodies obtained by the method of Claim 20. 22. An immortalized antibody producing cell obtained by the method of Claim 20. 23. A method for obtaining human cytotoxic T-cells reactive against a pathogenic cell or tissue, comprising: - 22 - (a) immunizing a xenografted primate M3 according to Claim 13, with said pathogenic cells or tissue or with a component thereof capable of eliciting the development of a cytotoxic T-cell immune reaction; and (b) recovering cytotoxic T-cells from said primate M3 and selecting for those reactive against said cell or tissue. 24. A method according to Claim 23, wherein said cells are selected from the group consisting of virus-infected cells, cancer cells, and eukaryotic or procaryotic pathogenic microorganisms. 25. Cytotoxic T-cells obtained by the method of Claims 23 or 24. 26. A method for the treatment of a disease or disorder in a human individual, comprising administering to the individual an effective amount of antibodies obtained from a xenografted primate according to Claim 1, containing a hematopoietic system derived from a human, said xenografted primate being immunized with a desired antigen, wherein polyclonal or monoclonal antibodies directed against said antigen are produced, and with an effective amount of cytotoxic T-cells obtained from a second xenografted primate according to Claim 1, containing a hematopoietic system derived from a human, said second xenografted primate being immunized with pathogenic cells or tissue or with a component thereof, capable of eliciting the development of a cytotoxic T-cell immune reaction, wherein cytotoxic T-cells reactive against said cell or tissue are produced. 27. A model for the study of the human hematopoietic system comprising a xenografted primate M3 according to Claim 13. 28. A method for the study of the pathology of a human hematopoi- etic disease or disorder, comprising: (a) obtaining a xenografted primate M3 according to Claim 13, wherein the inoculated hematopoietic stem cell preparation is obtained from a human individual having said disease or disorder; 0 - 23 - (b) evaluating the hematopoietic system of said xenografted primate and various conditions, whereby information on the pathology of said disease or disorder is obtained. 29. A method for the study of efficacy of a therapeutic agent or modality of treatment of a disease or disorder in the human hematopoietic system, comprising: (a) obtaining a xenografted primate M3 according to Claim 13, wherein the inoculated stem cell preparation is obtained from a human individual having said disease or disorder; (b) treating said primate by administration of said therapeutic agent or subjection to the treatment modality; and (c) evaluating the efficacy of said agent or modality in eliminating the disease or disorder or symptoms associated therewith. 30. A method according to Claim 29, wherein said disease or disorder is selected from the group of sickle cell anemia, thalasscmia, HIV infection, leukemia, lymphoma, malaria. 31. A method according to Claim 30, wherein said therapeutic agent is an agent used in gene therapy. 32. A method according to Claim 31, wherein said agent is a retro- viral vector, carrying a therapeutic gene. 33. A method for the study of the efficacy of gene therapy, compris¬ ing: (a) treating a non-human primate Ml so as to destroy most of its dividing hematopoietic cells; (b) obtaining a human T-cell -depicted hematopoietic stem cell enriched preparation, and transfecting stem cells in the preparation with an agent capable of delivering a therapeutic gene to the stem cells; (c) inoculating the preparation obtained in (b) into the treated primate obtained in (a), the amount of stem cells in the inoculum being in a large excess to the stem cells in the Ml primate remaining after the treatment, thereby obtaining a xenografted non-human primate _vl3; and (d) evaluating the expression of said gene in the hematopoietic system of said primate M3 or evaluating the clinical benefits of the inserted gene 34. A preparation for use in the treatment of a human disease or disorder by hematopoietic cells transplantation therapy comprising a preparation according to Claim 19. 35. A method for the treatment of a disease or disorder of the hematopoietic system of a human individual, comprising: (a) treating the individual so as to destroy its dividing hematopoietic cells; (b) inoculating the treated individual with a T-cell-depleted stem-cell enriched preparation obtained from a non-human primate donor, the amount of stem cell is the inoculum being in a large excess to the stem cells remaining in the individual after treatment; and (c) maintaining the individual under essential sterile conditions, until the reconstitution therein of the donor derived hematopoietic system. 36. A method according to Claim 35, wherein the amount of the stem cells in the inoculum is about 5 x 10 8 - 2 x 10 9 cells/kg body weight. 37. A method according to Claims 35 or 36, wherein stem cells are infected with a foreign therapeutic gene prior to inoculation. 38. For use in the method of Claims 35, 36 or 37, a non-human primate blood cell preparation enriched with stem cells. 39. A method for producing a preparation according to Claim 38, comprising: (a) administering a colony stimulating factor to a non-human primate so as to cause appearance of hematopoietic stem cells in the peripheral blood system; - 25 - (b) withdrawing blood samples from said non-human primate and leucophoresing the sample to collect stem cells, whereby a stem-cell enriched preparation is obtained. 40. A method according to Claim 39, further comprising transfecting the stem cells with a therapeutic gene,

Inventors: REISNER YAIR, REISNER, YAIR
Applicants: REISNER YAIR, RYCUS AVIGAIL, YEDA RES & DEV, REISNER, YAIR, RYCUS, AVIGAIL, YEDA RESEARCH AND DEVELOPMENT CO. LTD.
Application year: 1995
Main classification: A61K
IPC codes: A01K67, A01N1, A61K35, A61K39, A61K48, A61P7, A61P33, A61P35, A61P37, C07K16, C12N5, C12N15, C12P21