Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
1991-11-22
2002-03-05
Clark, Deborah J. R. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C435S455000, C424S577000
Reexamination Certificate
active
06353150
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for engrafting human hematopoietic cells in a non-human mammal by bone marrow transplantation. More particularly, the present invention relates to chimeric mammals with human hematopoietic cells, including progenitor cells and mature cells of the myeloid, lymphoid and erythroid lineages, a process for engrafting human hematopoietic cells to create a chimeric mammal, and a process for creating hybridoma cells capable of producing a human monoclonal antibody.
BACKGROUND OF THE INVENTION
The mammalian hematopoietic system is arranged as a hierarchy consisting of a wide array of cells ranging from large numbers of mature differentiated cells to rare pluripotent stem cells capable of self-renewal and differentiation. Much of our knowledge concerning organization and regulation of mammalian hematopoietic systems is derived from studies with murine hematopoietic systems. Conversely, our understanding of specifics of the human hematopoietic system is much less complete due to a lack of a reliable model for human hematopoiesis. Therefore, there is a need in the art for a human-specific hematopoiesis model that includes human stem cells.
Various investigators have attempted to transplant human hematopoietic cells into a murine bone marrow microenvironment with varying degrees of success. Xenogeneic bone marrow transplantation is possible between closely related species, such as rat and mouse. However, xenogeneic bone marrow transplantation has not enabled the stable transplant of human hematopoietic cells of multiple lineages into more distantly related mammalian species, such as mice or rats. For example, engraftment of human cells in severe combined immune deficiency (SCID) mice, permitted infection of mice with human immunodeficiency virus (HIV) (Namikawa et al.,
J. Exp. Med.
172:1055, 1990). However, detectable human cells were limited to a human T cell lineage and survived for only two to three months. Further, the T cells were detected only in blood and not in any other organ. Thus, transplantation of human hematopoietic cells into SCID mice was limited to the transient survival of only one lineage of human hematopoietic cells.
McCune et al. (
Science
241:1632, 1988) attempted to facilitate engraftment of human hematopoietic cells (from human fetal liver) into a SCID mouse by further transplanting a graft of human fetal thymic epithelium under the kidney capsule. Control SCID mice that were tansplanted with human hematopoietic cells but not fetal thymic epithelium did not engraft or differentiate into human T cells. No other human cell lineage except human T cells were detected in the mouse tissues. Thus, it appears that transplanted human hematopoiesis observed by McCune et al. occurred only in implanted human thymic tissue and that no engraftment of human cells in murine tissue actually occurred.
EP-A438053 describes an attempt to create a mouse-human chimeric animal. Bone marrow cells from SCID mice and human bone marrow cells were added to another strain of mouse that was lethally irradiated. SCID mouse bone marrow was used because T and B lymphocytic lineages in SCID mice cannot develop from SCID pluripotent stem cells due to a deficiency in rearrangement of antigen receptor genes (Shuler et al.,
Cell
46:963, 1986). The resulting chimeric mice had a small number of human cells in the thymus with a predominant CD3
+
CD4
+
CD8
+
phenotype. Lubin et al.
Science
252:427, 1991 cotransplanted a T cell depleted human bone marrow with SCID bone marrow into lethally irradiated Balb/c mice. Lubin et al. found only T cells and B cells in blood over several months. Therefore, this procedure did not produce multilineage engraftment of human hematopoietic cells.
Kamel-Reid et al. (
Science
242:1706, 1988) report the engraftment of human bone marrow cells into SCID mice without implantation of fetal tissue. However, observation periods were limited to thirty days, so long term engraftment was not determined.
Therefore, recent progress to establish a stable chimeric animal model to characterize normal development of human hematopoietic stem cells has been limited to transient or incidental engraftment of one or two human lineages. A chimeric mouse with a high proportion (i.e., predominance) of human hematopoietic cells has not yet been achieved. Such a chimeric mammal would be useful as an experimental model for studying a variety of human and veterinary diseases. Such a chimeric mammal would also be useful for studying normal human hematopoiesis, human stem cells and all of their progeny and those growth and inhibitory factors that govern hematopoiesis. Further, a chimeric mammal with functioning human immune cells would be useful to produce human monoclonal antibodies. The present invention was made to improve techniques used to produce chimeric mammals with human hematopoietic cells.
SUMMARY OF THE INVENTION
The present invention provides a chimeric mammal having a stable bone marrow graft of human hematopoietic cells capable of differentiating into multiple lineages of mature human cells, wherein at least 30% of the hematopoietic cells in the mammal's bone marrow are of human origin. The chimeric mammal is preferably a mouse, and most preferably a SCID mouse. Further, the graft consists essentially of human hematopoietic progenitor cells, multipotent human progenitor cells and mature human hematopoietic cells of lymphoid, myeloid and erythroid lineages.
The inventive method comprises sublethally irradiating an immunodeficient mammal, infusing human hematopoietic cells into the mammal and administering an effective amount of human mast cell growth factor (MGF) and a human granulocyte macrophage colony stimulating factor/interleukin-3 fusion protein (GM-CSF/IL-3 FP) to promote engraftment of human hematopoietic cells. Preferably, GM-CSF/IL-3 FP is a specific fusion protein called PIXY321.
The present invention further provides a chimeric mammal having a stable bone marrow graft of lineage-specific human hematopoietic cells, wherein the lineage-specific human hematopoietic cells are selected from the group consisting of erythroid cells, myeloid cells, lymphoid cells, multipotent progenitor cells and combinations thereof. A process for obtaining a chimeric mammal having a stable bone marrow graft of lineage-specific human hematopoietic cells comprises, sublethally irradiating an immunodeficient mammal, infusing human hematopoietic cells into the immunodeficient mammal, and administering MGF, a GM-CSF/IL-3 FP and an additional, lineage-specific human growth factor which is effective to promote differentiation and proliferation of the desired lineage of human hematopoietic cells. Additional, lineage-specific human growth factors are selected from the group consisting of erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), interleukins-2, -4, -5, -6, -7, -9, -10, -11, and -12, and combinations thereof.
Further still, the present invention provides a human hybridoma cell capable of producing a human monoclonal antibody (hMAb) specific for a selected antigen. The human hybridoma cell is made by a process comprising immunizing a chimeric mammal made according to the inventive process, fusing spleen cells from the chimeric mammal with myeloma cells to make hybridoma cells, and selecting for hybridoma cells which secrete hMAbs that bind to the antigen.
DETAILED DESCRIPTION OF THE INVENTION
The present invention involves various methods of using MGF and a GM-CSF/IL3 FP, such as PIXY321, to promote engraftment of transplanted human hematopoietic cells into various mammalian species. We have significantly improved upon previous methods to create a human-chimeric mammal having at least 30% human cells in the chimeric mammal's bone marrow. Quite often, the proportion of human origin cells versus host mammal hematopoietic cells in the chimeric mammal bone marrow often exceeds
Dick John E.
Lapidot Tsvee
Williams Douglas E.
Clark Deborah J. R.
Howe Stuart D.
HSC Research and Development Limited Partnership
Paras, Jr. Peter
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