Stimulation of hematopoietic cells in vitro

Details Stimulation of hematopoietic cells in vitro Stimulation of hematopoietic cells in vitro

1998-09-29

2001-07-10

Borin, Michael (Department: 1631)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S377000, C435S383000, C435S375000

Reexamination Certificate

active

06258597

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the use of agents which bind to dipeptidyl peptidase IV (DPIV, also known as CD26) for the stimulation of hematopoietic cells in vitro
BACKGROUND OF THE INVENTION
Bone marrow transplantation is widely used with patients undergoing high dose chemotherapy or radiation therapy. The dose limiting side effects of chemotherapy and radiation therapy are their deleterious effects on hematopoietic cells through destruction of the bone marrow cells which are the precursor cells for all hematopoietic cells. This damage to the marrow results in myelosuppression or myeloablation, rendering patients susceptible to opportunistic infections for a prolonged period of time. Bone marrow transplantation involves the infusion of early bone marrow progenitor cells that have the ability to re-establish the patients' hematopoietic system, including the immune system. Transplantation decreases the time normally required for the restoration of the immune system after chemotherapy or radiation therapy and, thus, the time of risk for opportunistic infections.
Bone marrow cells contain totipotent stem cells which give rise to hematopoietic cells of all lineages including the lymphoid, myeloid and erythroid lineages. Stem cells have the ability to renew themselves as well as to differentiate into progenitor cells of all hematopoietic lineages. Progenitor cells retain the ability to proliferate and give rise to differentiated cells of all lineages. Differentiated cells lose the ability to proliferate and exhibit morphological characteristics specific for their lineages (such as macrophages, granulocytes, platelets, red blood cells, T cells and B cells). Stem cells and progenitor cells express CD34 on their surface while differentiated cells do not. Bone marrow includes stem cells as well as progenitor cells of the lymphoid (T and B cells), myeloid (granulocytes, macrophages) and erythroid (red blood cells) lineages.
For use in bone marrow transplants, hematopoietic precursor cells can be derived either from the cancer patient (autologous transplant) or from a histocompatible donor (allogeneic donor). These cells can be isolated from bone marrow, peripheral blood or from umbilical cord blood. In all cases, cells are harvested before chemotherapy or radiation therapy. The number of progenitor cells that can be harvested at one time is small and, in many cases, is not sufficient for a successful transplant. Accordingly, several methods have been developed to expand, in vitro, bone marrow cells or progenitor cells obtained from blood aphereses or from umbilical cord blood.
The ability to expand these cells has helped advance bone marrow transplant technology as a viable adjunct therapy for cancer treatments that involve high doses of chemotherapy and/or irradiation. However, the existing methods for hematopoietic cell expansion require the addition of appropriate cytokines to permit the in vitro expansion of hematopoietic stem cells. The high cost of such growth factors has adversely affected the ability of those skilled in the art to expand hematopoietic cells in vitro for transplantation or other purposes. Accordingly, a need exists to develop new methods for expanding hematopoietic cells in vitro which do not require exogenously added cytokines to support cell growth and differentiation.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for stimulating the growth and differentiation of hematopoietic cells in vitro. Advantageously, the methods of the invention do not require the addition of exogenously added cytokines to support the stimulation of hematopoietic cells in vitro. Accordingly, the methods and compositions of the invention are useful for increasing the number of hematopoietic cells in vitro and/or causing the differentiation of early progenitor cells. Increasing the number and/or differentiation of hematopoietic cells in culture permits the characterization of such cells in culture under a variety of conditions, as well as the use of such cultured cells for the production of recombinant or naturally occurring molecules therefrom in vitro. In addition, the stimulated hematopoietic cells of the invention are useful for the treatment of disorders that are characterized by a reduced number of hematopoietic cells or their precursors in vivo. Such conditions occur frequently in patients who are immunosuppressed, for example, as a consequence of chemotherapy and/or radiation therapy for cancer.
The novelty of the invention is based, at least in part, on the discovery that inhibitors of dipeptidyl peptidase type IV (“DPIV”) are useful for stimulating the growth and differentiation of hematopoietic cells in the absence of exogenously added cytokines or other growth factors or stromal cells. This discovery contradicts the dogma in the field of hematopoietic cell stimulation which provides that the addition of cytokines or cells that produce cytokines (stromal cells) is an essential element for maintaining and stimulating the growth and differentiation of hematopoietic cells in culture. (See, e.g., PCT Intl. Application No. PCT/US93/017173, published as WO94/03055).
According to one aspect of the invention, a method for stimulating hematopoietic cells to grow and differentiate in vitro is provided. The method involves: (1) contacting the hematopoietic cells with a sufficient amount of an inhibitor of a dipeptidyl peptidase type IV to increase the number and/or differentiation of hematopoietic cells when the cells are cultured in the presence of the inhibitor relative to the number and differentiation of hematopoietic cells that are present in a control culture that is not contacted with the inhibitor but is otherwise subjected to the same culture conditions as the hematopoietic cells which are cultured in the presence of the inhibitor; (2) culturing the hematopoietic cells in the presence of the inhibitor and in the absence of exogenously added cytokine under conditions and for a time sufficient to increase the number of hematopoietic cells and/or the differentiation of such cells relative to the number of hematopoietic cells that were present in the control culture; (3) culturing the hematopoietic cells in the presence or absence of stromal cells, and (4) culturing stromal cells in the presence of the DPIV inhibitor. In general, increasing the number of hematopoietic cells refers to increasing the number of cells by at least approximately 2-fold relative to the number of hematopoietic cells that are present when the cells initially are contacted with the inhibitor. In general, the number of cells that are present in a control culture that is not contacted with the inhibitor but is otherwise identically treated is approximately the same as the initial number of cells in the culture prior to contact with the inhibitor. Preferably, the number of hematopoietic cells are increased at least approximately 4-fold, 1 0-fold, 20-fold or, most preferably, at least 100-fold relative to the number of hematopoietic cells that are present when the hematopoietic cells initially are contacted with the inhibitor.
As used herein, hematopoietic cells includes hematopoietic stem cells, primordial stem cells, early progenitor cells, CD34+ cells, early lineage cells of the mesenchymal, myeloid, lymphoid and erythroid lineages, bone marrow cells, blood cells, umbilical cord blood cells, stromal cells, and other hematopoietic precursor cells that are known to those of ordinary skill in the art.
As used herein, an inhibitor of dipeptidyl peptidase type IV (“DPIV”) generally refers to a molecule which inhibits the functional activity of the DPIV. Accordingly, the inhibitors of the invention include inhibitors of the enzymatic activity of the dipeptidyl peptidase type IV. Preferably, the inhibitors of the enzymatic activity of DPIV associate with the active site of DPIV by covalently bonding thereto or by forming an ionic interaction therewith. Such inhibitors include competitive inhibitors of DPIV, such as transition state analogs of DPIV, and

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