Method of Enhancing Hematopoietic Cell Transplantation
The invention relates to a method for enhancing the transplantation of hematopoietic cells to supplement or fully reconstitute the hematopoietic system, such as in myeloablated patients or patients otherwise deficient in hematopoietic cells. The method involves administering CD34+ cells having enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 at levels that provide desirable therapeutically effective amounts of self-renewal of the administered cells and desirable therapeutically effective amounts of differentiation of the administered cells into the various progeny cells of the hematopoietic system (i.e., therapeutically effective amounts of hematopoietic reconstitution). To provide such cells to a subject, the invention relates to detecting such cells prior to or during treatment to ascertain whether such cells are present in clinically-relevant amounts. It may also relate to treating a subject so as to provide clinically-relevant numbers of such cells, as with specific mobilization agents.
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The invention relates to a method for enhancing the transplantation of hematopoietic cells to supplement or fully reconstitute the hematopoietic system, such as, in myeloablated patients or patients otherwise deficient in hematopoietic cells. The method involves administering CD34+ cells co-expressing one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 at enhanced levels to provide self-renewal of the administered cells and/or differentiation of the administered cells into the various progeny cells of the hematopoietic system (i.e., therapeutically effective amounts of hematopoietic reconstitution). To provide such cells to a subject, the invention relates to detecting such cells prior to or during treatment to ascertain whether such cells are present in clinically-relevant amounts. It may also relate to treating a subject so as to provide clinically-relevant numbers of such cells, as with specific mobilization agents. It may also relate to treating a subject with umbilical cord blood cells or with cells that have been cultured to be expanded in numbers or cultured to be enhanced in potency for hematopoietic reconstitution. The invention also relates to compositions containing the cells.
BACKGROUND OF THE INVENTIONThe hematopoietic system can be reconstituted by cells that are the progenitor/stem cells for all blood cells. These stem/progenitor cells can be designated, as in this application, “hematopoietic-reconstituting cells” or “HRC.” Hematopoietic-reconstituting cells are capable of self-renewal and of differentiating into any cell in the hematopoietic system, including lymphocytes, monocytes, platelets, erythrocytes and myeloid cells. Hematopoietic-reconstituting cells have therapeutic potential as a result of their capacity to restore blood and immune cell function.
Transplantation of CD34+ hematopoietic-reconstituting cells is an important treatment modality for malignant and nonmalignant disorders. Most commonly, hematopoietic-reconstituting cells from bone marrow are mobilized into the peripheral blood by pharmacological treatment, thereby facilitating collection. The number of CD34+ cells in mobilized blood samples is used to indicate the appropriateness of transplantation although it does not necessarily distinguish between two necessary functions for hematopoietic reconstitution, specifically, long-term reconstitution, mediated by cells with self-renewing proliferation, and short-term hematopoietic differentiation, mediated by progenitor cells.
Transplantation of hematopoietic-reconstituting cells from bone marrow, mobilized peripheral blood and umbilical cord blood has been used to treat hematopoietic cancers such as leukemia and lymphomas, and to aid hematopoietic system recovery from high-dose chemotherapy. Myelosuppression and myeloablation often result from high-dose chemotherapy. Prior to treatment with high-dose chemotherapy, bone marrow hematopoietic progenitor/stem cells can be mobilized into the peripheral blood so that peripheral blood can be harvested and stored for later use as a source of hematopoietic-reconstituting cells. The transplantation of the stored hematopoietic-reconstituting cells can rescue hematopoietic functions after high-dose chemotherapy. Allogeneic or autologous hematopoietic-reconstituting cells can be used to mediate hematopoietic reconstitution.
It would be desirable if hematopoietic-reconstituting cells could be definitively identified in a heterogeneous mixture of cells by assessing the cells for the expression of markers associated with hematopoietic-reconstituting function. The CD34+ cell number has been used as a marker for the progenitor/stem cell quantity. However, the CD34 molecule is not associated with the two critical hematopoietic-reconstituting cell functions: the capacity for self-renewing proliferation and short-term differentiation into hematopoietic cells. See Suzuki, A. et al., Blood (1996) 87:3550-3562. Although the number of CD34+ cells can be determined, there remains a large variability in predicting hematopoietic reconstitution. It would be desirable if hematopoietic-reconstituting cells could be evaluated for their potency in mediating hematopoietic-reconstituting function by assessing the expression levels of molecules involved in mediating this function.
There are two sources of HRC that have been shown to be superior to bone marrow CD34+ cells in terms of their functional potency on a cell-by-cell basis. They are granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ cells obtained from the peripheral circulation and umbilical cord blood CD34+ cells. For instance, at the Fred Hutchinson Cancer Research Center in Seattle, G-CSF-mobilized cells demonstrate 5-7 days faster reconstitution compared to bone marrow cells even when similar doses of CD34+ cells were used (Heimfeld, S. Leukemia (2003) 17:856-858.). Enhancement in the recovery of neutrophils (7 days) and platelets (8 days) after transplantation of similar numbers of mobilized peripheral blood CD34+cells versus bone marrow CD34+ cells was also observed in a Norwegian study (Heldal D, et al. Bone Marrow Transplant (2000) 25:1129-1136.). These results are consistent with the greater number of granulocyte-macrophage colony-forming units per CD34+ cell for G-CSF mobilized peripheral blood CD34+ cells compared to bone marrow resident CD34+ cells (Pavletic Z S, et al. J Clin Oncol (1997) 15:1608-1616.).
Similarly, umbilical cord blood HRC have been shown to have a higher cloning efficiency, to proliferate more rapidly in response to cytokine stimulations, and to generate about 7-fold more progeny than HRC from the adult bone marrow (Hao Q-L, et al. Blood (1995) 86:3745-3753.). Another group of investigators found that cultures of cord blood cells produced a significantly greater increase in granulocyte-macrophage colony-forming units and granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming units than cultures of bone marrow cells (Broxmeyer H E, et al. Proc. Natl. Acad. Sci. USA (1992) 89:4109-4113.). A third group found similar findings in comparing umbilical cord blood CD34+ cells with bone marrow CD34+ cells (Cardoso A A, et al. Proc. Natl. Acad. Sci. USA (1993) 90:8707-8711.). It is also important to note that approximately 10-fold less umbilical cord blood CD34+ cells are used for transplantation than the bone marrow CD34 cells.
SUMMARY OF THE INVENTIONThe inventor has discovered that one can predict the potency of a sample of CD34+ cells to reconstitute the hematopoietic system by assessing the expression levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 in the CD34+ cells in the sample. The inventor assessed the expression levels of these molecules in CD34+ cells from three different subject groups: bone marrow from healthy subjects, umbilical cord blood, and mobilized blood from healthy subjects. It is known and accepted that CD34+ cells from either umbilical cord blood or from healthy subjects pharmacologically treated to mobilize their cells from the bone marrow are superior in hematopoietic-reconstituting function to CD34+ cells from the bone marrow of healthy persons. The inventor discovered that the expression levels of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 are enhanced in CD34+ cells from at least 1 of the sources associated with greater functional potency compared to the CD34+ cells from the source of CD34+with lesser potency (i.e. bone marrow from healthy subjects).
Accordingly, enhanced expression levels of one or more of the molecules in CD34+ cells (i.e., Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47) can be used to recognize potency of a sample in terms of hematopoietic reconstitution. Furthermore, based on these findings, CD34+ cells in a subject can be manipulated by the addition of specific mobilization agents or by culture with or without specific agents that increase the expression of these genes in the CD34+ cells. Thus, therapeutically-effective amounts of cells with enhanced expression of one or more of the molecules can be recognized. Enhanced expression is expression compared to the expression from the un-mobilized bone marrow of healthy persons. These more potent samples now can be obtained and administered to a subject to improve or completely reconstitute the hematopoietic system.
The invention is also directed to a method to identify a molecule, the expression of which is correlated with hematopoietic reconstituting function, the method comprising assessing expression of the molecule in individual CD34+ cells in samples having different levels of potency and identifying molecules, the expression of which correlates with potency, by correlating differences in expression of such molecules with the potency of the different samples.
In that method greater potency can be associated with an increase in expression.
In that method greater potency can be associated with a decrease in expression.
In that method the samples that are compared can be bone marrow, mobilized peripheral blood from healthy subjects, and umbilical cord blood.
In that method the expression that is assayed can be selected from groups consisting of RNA, protein, and post-translational modification.
That method could be used to assess expression of molecules and pathways associated with HRC function, which can include the following: Notch pathway, nucleoside salvage pathway, OTT-1, MEIS1, Ap2a2, Lin28b, Wnt signaling pathway, MetAP2 (methionine aminopeptidase 2), Pot1b, Evi1, Smad signaling, Erg (E-26-related gene), PCNA (proliferating cell nuclear antigen associated factor), Rac1/Rac2/Rac3, Prdm16, APC, Rho GTPase, p190-B, Fbw7, Gli1, Ldb1, NKAP, cyclin C, Irgm1, HoxA9, NA10HD, Fbxw7alpha, IRF8, NUP98, MycN, DDX10, ANGPT1, REN, HEY1, Sox4, Stat5, Slug, p53, prostaglandin E2, Zfx, Calcineurin, NFAT, cyclin E2, SHIP, NF-Y, Hedgehog pathway, Dmtf1, Nrf2, ANKRD28, GNA15, UGP2, Skp2, Mdm2, Sox7, Ikaros, TET2, SCL, TAL1, Jumonji, Lyl1, Foxo3a, Gimap5, ADAR1, Menin, Wnt3a, PSF1, ABCG2, Tie1/2, cMpl, CD117, mTORCl, c-Cb1, Rb, Pbx1, EWS, PU.1, Chk1, Necdin, SHP2, PUMA, FUS, WASP, NOD2, Mef2c, GABP, Angptls, SIRT1, 12/15-lipoxygenase-dependent fatty acid metabolism pathway, angiopoietin-1, angiopoietin-2, Cited2, SIMPL, p300, Heine oxygenase-1, p16ink4A, p18ink4c, p21cip1, Survivin. Frizzled-related protein 1, Rheb2, aldehyde dehydrogenase 1a1, CD130, CD123
“A” or “an” means herein one or more than one; at least one. Where the plural form is used herein, it generally includes the singular.
The term “Atg7” is understood to refer to Autophagy-related protein 7, a protein essential in the cellular function of autophagy, encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NM 006395.2. The sequence can be found at the following site: http://www.ncbi.nlm.nih.gov/nuccore/NM—006395.2, incorporated by reference for the sequence. There are two other variants of the gene. The one referenced is the longest variant. This gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
The term. “Bmi-1” refers to “a component of the Polycomb group multiprotein PRC-1 like complex, a complex class required to maintain the transcriptionally repressive state of many genes throughout development”. Bmi-1 is required for the maintenance of adult self-renewing hematopoietic stem cells. Nature 2003 May 15 423:302-305 Park et al. The sequence can be found at the following site: http://www.uniprot.org/uniprot/P35226, incorporated by reference.
A “cell bank” is industry nomenclature for cells that have been grown and stored for future use. Cells may be stored in aliquots. They can be used directly out of storage or may be expanded after storage. This is a convenience so that there are “off the shelf” cells available for administration. The cells may already be stored in a pharmaceutically-acceptable excipient so they may be directly administered or they may be mixed with an appropriate excipient when they are released from storage. Cells may be frozen or otherwise stored in a form to preserve viability. In one embodiment of the invention, cell banks are created in which the cells have been selected for enhanced potency to achieve the effects described in this application. Following release from storage, and prior to administration to the subject, it may be preferable to again assay the cells for potency. This can be done using any of the assays, direct or indirect, described in this application or otherwise known in the art. Then cells having the desired potency can then be administered to the subject for treatment. Banks can be made using cells derived from the individual to be treated (from their pre-natal tissues such as placenta, umbilical cord blood, or umbilical cord matrix or expanded from the individual at any time after birth). Or banks can contain cells for allogeneic uses.
The term “cMyc” is understood to refer to a mammalian homolog of a viral oncogene, v-Myc. It is a basic helix-loop-helix transcription factor that functions to activate a large number of genes. It is encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NM—002467.4, incorporated by reference for the sequence. However, this gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
“Co-administer” means to administer in conjunction with one another, together, coordinately, including simultaneous or sequential administration of two or more agents. In the context of the invention, the two types of CD34+ cells can be administered with these alternative regimens.
“Comprised of” is a synonym of “comprising”.
“Comprising” means, without other limitation, including the referent, necessarily, without any qualification or exclusion on what else may be included. For example, “a composition comprising x and y” encompasses any composition that contains x and y, no matter what other components may be present in the composition. Likewise, “a method comprising the step of x” encompasses any method in which x is carried out, whether x is the only step in the method or it is only one of the steps, no matter how many other steps there may be and no matter how simple or complex x is in comparison to them. “Comprised of” and similar phrases using words of the root “comprise” are used herein as synonyms of “comprising” and have the same meaning.
“Decrease” or “reduce” means to prevent entirely as well as to lower.
The term “E47” is understood to refer to a basic helix-loop-helix transcription factor that has been shown to regulate hematopoietic-reconstituting cell maintenance and proliferation. It is encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NM—001136139.2, incorporated by reference for the sequence. However, this gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, faun animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
“Effective amount” generally means an amount which provides the desired effect. For example, an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, injury, and/or disease or injury being treated, and amount of time since the injury occurred or the disease began. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art. As used herein, “effective dose” means the same as “effective amount.” In the context of the invention, effective amounts are amounts of those CD34+ cells with enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 that provide clinically-significant hematopoietic reconstitution (i.e., potency). “Effective expression” refers to expression that provides for that clinically-significant reconstitution.
“Effective route” generally means a route which provides for delivery of an agent to a desired compartment, system, or location. For example, an effective route is one through which an agent can be administered to provide at the desired site of action an amount of the agent sufficient to effectuate a beneficial or desired clinical result.
The term “enhanced”, as it is applied to the invention, means expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 that is greater than the mean expression of those molecules (RNA and/or protein) in untreated bone marrow (i.e., non-mobilized).
The term. “GATA2” refers to a member of the Zinc finger transcription factor family; it plays an essential role in regulating transcription of genes involved in the development and proliferation of hematopoietic cell lineages. The sequence can be found at the following site: http://www.ncbi.nlm.nih.gov/sene/2624, incorporated by reference. The site for a reference is: http://www.ncbi.nlm.nih.gov/nuccore/NG—029334.1?report=genbank&from=5001&to=18766, incorporated by reference.
The term “hematopoietic-reconstituting cell” or “HRC”, as used herein, refers to a progenitor and/or stem cell that can reconstitute all of the hematopoietic cells in a subject. These include, but are not limited to, lymphocytes, platelets, erythrocytes and myeloid cells, including, T cells, B cells (plasma cells), natural killer cells, dendritic cells, monocytes (macrophages), neutrophils, eosinophils, basophils (mast cells), megakaryocytes (platelets), and erythroblasts (erythrocytes). These cells are also capable, in addition to differentiation, of self-renewal, so as to proliferate the stem-progenitor population that is capable of differentiation.
The term “hematopoietic-reconstituting cell” or “HRC” generally refers to the functions of the cells that provide their ability to reconstitute the hematopoietic system to provide a clinically-relevant effect. Technically, the reconstitution function can be broken down into two functions that may be represented by two sets of cells: (1) CD34+ self-renewing hematopoietic-reconstituting cells and (2) CD34+hematopoietic-reconstituting cells that differentiate into hematopoietic cell progeny. See pending U.S. patent application Ser. No. 13/490,000, incorporated by reference for disclosure of these cells.
The term “HoxB4” is understood to refer to a transcription factor encoded by a gene having, in humans, the sequence shown in, for example, Acampora et al., Nucl. Acids. Res. 17: 10385-10402 (1989). Also see NCBI Reference, Sequence: NM—204015.4, incorporated by reference for the sequence. This gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the capacity for HoxB4 function. The gene also includes, for non-human uses, such as veterinary uses, HoxB4 orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc. See also http://www.uniprot.org/uniprot/P17483, incorporated by reference.
Use of the term “includes” is not intended to be limiting.
“Increase” or “increasing” means to induce entirely, where there was no pre-existing effect, as well as to increase the degree.
The term “isolated” refers to a cell or cells which are not associated with one or more cells or one or more cellular components that are associated with the cell or cells in vivo. An “enriched population” means a relative increase in numbers of a desired cell relative to one or more other cell types in vivo or in primary culture.
However, as used herein, the term “isolated” does not indicate the presence of only hematopoietic-reconstituting cells. Rather, the term “isolated” indicates that the cells are removed from their natural tissue environment and are present at a higher concentration as compared to the normal tissue environment. Accordingly, an “isolated” cell population may further include cell types in addition to hematopoietic-reconstituting cells and may include additional tissue components. This also can be expressed in terms of cell doublings, for example. A cell may have undergone 10, 20, 30, 40 or more doublings in vitro or ex vivo so that it is enriched compared to its original numbers in vivo or in its original tissue environment (for example, bone marrow, peripheral blood, umbilical cord blood, etc.).
The term “Mcl-I” is understood to refer to an apoptotic pathway molecule that protects cells from cell death. Mcl-1 has also been found to be a functional regulator of HRC self-renewing proliferation (Campbell C J V, et al. Blood (2010) 116:1433-1442.), encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NG—029146.1, incorporated by reference for the sequence. However, this gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
The term “Musashi2” is understood to refer to an RNA binding protein and translational inhibitor that has been shown to regulate hematopoiesis (Kharas M G, et al. (2010) Musashi2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia. Nature Medicine 16:903-908, (2010) and Andres-Aguayo L, et al. Musashi 2 is a regulator of the HSC compartment identified by a retroviral insertion screen and knockout mice. Blood 118:554-564 (2011), encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NM—138962.2, incorporated by reference for the sequence. However, this gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
“Pharmaceutically-acceptable carrier” is any pharmaceutically-acceptable medium for the cells used in the present invention. Such a medium may retain isotonicity, cell metabolism, pH, and the like. It is compatible with administration to a subject in vivo, and can be used, therefore, for cell delivery and treatment.
The term “phospho-Akt(ser473)” is understood to refer to a serine/threonine-specific protein kinase, also known as protein kinase B, that is phosphorylated on the amino acid serine at position 473, encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NCBI Reference sequence: NM 005163.2. The url follows: http://www/ncbi.nlm.nih.gov/nuccore/NM 005163.2, incorporated by reference for the sequence. There is also an Akt2 which is closely related to Akt1. It has NCBI Reference Sequence: NM 001243027.1. The url follows: http://www/ncbi.nlm/nih.gov/nuccore/NM—001243027.1, incorporated by reference for the sequence. The antibodies used in the Examples detect both Akt1 and Akt2 phosphorylations. There are variants of both Akt1 and Akt2. This gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
The term “phospho-Akt(thr308)” is understood to refer to a serine/threonine-specific protein kinase, also known as protein kinase B, that is phosphorylated on the amino acid threonine at position 308, encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NM 0051612. The url follows: http://www.ncbi.nlm.nih.gov/nuccore/NM—0051632, incorporated by reference for the sequence. There is also an Akt2 which is closely related to Akt1. It has NCBI Reference Sequence: NM 001243027.1. The url follows: http://www.ncbi.nlm.nih.gov/nuccore/NM 001243027.1, incorporated by reference for the sequence. The antibodies used in the Examples detect both Akt1 and Akt2 phosphorylations. There are variants of both Akt1 and Akt2. This gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
The term “phospho-GSK-3β,” is understood to refer to glycogen synthase kinase-3beta, which is a serine/threonine protein kinase that is phosphorylated on the amino acid serine at position 9, encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NM 002093.3. The url follows: http://www.ncbi.nlm.nih.gov/nuccore/NM—002093.3, incorporated by reference for the sequence. Variants exist. This gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
The term “potency” refers to the degree of the ability of a cell population to provide hematopoietic-reconstituting cell effects, i.e., self-renewal and/or differentiation, sufficient to achieve a clinically-detectable result. In the context of the invention, potency refers to the numbers of CD34+ cells having enhanced expression of one or more of the genes, i.e., that provide greater potency to the sample.
The term “PTEN,” is understood to refer to a phosphatase and tensin homolog, encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: NM 000314.4. The url follows: http://www.ncbi.nlm.nih.gov/nuccor/NM—000314.4, incorporated by reference for the sequence. This gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
The term “reconstitute” implies a range of increase from a fully or partially deficient hematopoietic system. It is not limited to, for example; cases in which the entire hematopoietic system is ablated. Reduced intensity conditioning is used in HRC transplantation. Reduced intensity conditioning does not result in myeloablation and it is used in patients that are older, in patients who are in complete remission, and in patients with acquired aplastic anemia.
The term “reduce” as used herein means to prevent as well as decrease. In the context of treatment, to “reduce” is to both prevent or ameliorate one or more clinical symptoms. A clinical symptom is one (or more) that has or will have, if left untreated, a negative impact on the quality of life (health) of the subject. This also applies to the biological effects such as self-renewal and differentiation.
“Selecting” a cell with a desired level of potency can mean identifying (as by assay), isolating, and expanding a cell. This could create a population that has a higher potency than the parent cell population from which the cell was isolated.
To select a cell would include both an assay to determine if there is the desired effect and would also include obtaining that cell. The cell may naturally have the effect in that the cell was not incubated with or exposed to an agent that induces the effect. The cell may not be known to have the effect prior to conducting the assay. As the effects could depend on gene expression and/or secretion, one could also select on the basis of one or more of the genes that cause the effects.
Selection could be from cells in a tissue. For example, in this case, cells would be isolated from a desired tissue, expanded in culture, selected for a desired effect, and the selected cells further expanded.
Selection could also be from cells ex vivo, such as cells in culture. In this case, one or more of the cells in culture would be assayed for the effect and the cells obtained that have the effect could be further expanded.
Cells could also be selected for enhanced effect. In this case, the cell population from which the enhanced cell is obtained already has the effect. Enhanced effectiveness means a higher average amount of the effect per cell than in the parent population.
The parent population from which the enhanced cell is selected may be substantially homogeneous (the same cell type). One way to obtain such an enhanced cell from this population is to create single cells or cell pools and assay those cells or cell pools for the effect to obtain clones that naturally have the effect (as opposed to treating the cells with a modulator of the effect) and then expanding those cells that are naturally enhanced.
However, cells may be treated with one or more agents that will enhance the effect of endogenous cellular pathways. Thus, substantially homogeneous populations may be treated to enhance modulation.
If the population is not substantially homogeneous, then, it is preferable that the parental cell population to be treated contains at least 100 of the effective cell type in which enhanced effect is sought, more preferably at least 1,000 of the cells, and still more preferably, at least 10,000 of the cells. Following treatment, this sub-population can be recovered from the heterogeneous population by known cell selection techniques and further expanded if desired.
Thus, desired levels of the effect may be those that are higher than the levels in a given preceding population. For example, cells that are put into primary culture from a tissue and expanded and isolated by culture conditions that are not specifically designed to have the effect, may provide a parent population. Such a parent population can be treated to enhance the average effect per cell or screened for a cell or cells within the population that express higher effect. Such cells can be expanded then to provide a population with a higher (desired) effect.
Whereas the exemplified hematopoietic-reconstituting cells in this application express the genes naturally (i.e., not by recombinant means, such as by exogenous promoter/enhancer insertion into the endogenous gene, or by the addition of exogenous coding sequences), the invention could cover cells that are genetically engineered for enhanced expression of the genes (for example, by increasing the copy number, reducing the copy number, increasing transcription/translation, or decreasing expression, such as by negative regulators such as small molecules, anti-sense RNA and the like).
“Self-renewal” refers to the ability to produce replicate daughter stem cells having differentiation potential that is identical to those from which they arose. A similar term used in this context is “proliferation.”
“Stem cell” means a cell that can undergo self-renewal (i.e., progeny with the same differentiation potential) and also produce progeny cells that are more restricted in differentiation potential. In the context of the present invention, differentiation is into hematopoietic progeny, such as shown in
“Subject” means a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, dogs, cats, horses, cows, and pigs.
“Substantially homogeneous” refers to cell preparations where the cell type is of significant purity of at least 50%. The range of homogeneity may, however, be up to and including 100%. Accordingly, the range includes about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90% and about 90% to 100%. This is opposed to the use of the term “isolated”, which can refer to levels that are substantially less. However, as used herein, the term “isolated” refers to preparations in which the cells are found in numbers sufficient to exert a clinically-relevant biological effect, as described in this application (i.e., transplantation, such as hematopoietic reconstitution).
The term “therapeutically effective amount” refers to the amount of an agent determined to produce any therapeutic response in a mammal. For example, effective amounts of hematopoietic-reconstituting cells may prolong the survivability of the patient, and/or inhibit overt clinical symptoms. Treatments that are therapeutically effective within the meaning of the term as used herein, include treatments that improve a subject's quality of life even if they do not improve the disease outcome per se. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art. Thus, to “treat” means to deliver such an amount. Thus, treating can prevent or ameliorate any pathological symptoms of hematopoietic deficiency.
“Treat,” “treating,” or “treatment” are used broadly in relation to the invention and each such term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere with and/or result from a therapy.
The term “UBC-13” is understood to refer to a ubiquitin-conjugating enzyme that mediates lysine-63-specific protein ubiquitination involved in signal transduction, encoded by a gene having, in humans, the sequence shown in NCBI Reference Sequence: GenBank: BC000396.2 with the following url http://www.ncbi.nlm.nih.gov/nuccore/BC000396.2, incorporated by reference for the sequence. This gene is also known, like most other genes, to contain polymorphisms that still allow the gene to maintain the function. With respect to this application, it would be sufficient function so as to provide clinically-relevant levels of cells for hematopoietic reconstitution or other transplantation. The gene also includes, for non-human uses, such as veterinary uses, orthologs from other mammals. These include companion animals, farm animals and sport animals, for example, felines, canines, bovines, equines, porcines, ovines, etc.
“Validate” means to confirm. In the context of the invention, one confirms that a cell is an expressor with a desired potency. This is so that one can then use that cell (in treatment, banking, drug screening, etc.) with a reasonable expectation of efficacy. Accordingly, to validate means to confirm that the cells, having been originally found to have/established as having the desired activity, in fact, retain that activity. Thus, validation is a verification event in a two-event process involving the original determination and the follow-up determination. The second event is referred to herein as “validation.”
The cells of the invention can be used to treat various cancers and immune system disorders, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, childhood leukemias, myelodysplastic syndromes, multiple myeloma, lymphoma, chronic lymphocytic leukemia, solid tumors in children, breast cancer, solid tumor in adults, germ cell tumors, primary immunodeficiency diseases, Fanconi anemia, acquired aplastic anemia, acquired immunodeficiency diseases, thalessemia, sickle cell anemia, lysosomal storage disorders and autoimmune diseases. This treatment is also used for multiple sclerosis, systemic sclerosis, rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosis, and Crohn's disease which are all included under the autoimmune disease heading. Additionally, HRC transplantation (autologous) is used in the treatment of cardiovascular disease and stroke.
Embodiments of the InventionIn one embodiment the invention is directed to a method for assessing the capacity of a sample to therapeutically effect hematopoietic reconstitution in a subject, the method comprising assessing individual CD34+ cells for enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47, in the sample and determining the number of those cells.
In particular, the levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 in these CD34+ cells is assessed. The assessment is for cells that express the one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 at levels greater than the mean expression in un-treated (non-mobilized) bone marrow cells.
The number of these cells provide useful predictors of the effectiveness of a sample from any given tissue source. Accordingly, if a sample is selected from a particular source and assessed for numbers of cells with the enhanced expression and found to have numbers that are too low to be effective, this sample may be found unsuitable for transplantation.
In one embodiment the invention is directed to a method to therapeutically effect hematopoietic reconstitution in a subject, the method comprising administering to a subject an agent that increases the expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 in CD34+ cells in the subject so as to provide a therapeutically-effective amount of cells that effect therapeutic levels of reconstitution.
In one embodiment the invention is directed to a method to prepare a subject to donate blood for hematopoietic-reconstituting cell transplantation, the method comprising obtaining a blood sample containing hematopoietic cells from the subject; determining the expression levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 in individual CD34+ cells from the blood sample; and administering to the subject a mobilizing agent if it is determined that the blood sample does not contain a therapeutically-desirable amount of CD34+ cells expressing enhanced levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 for desired levels of hematopoietic reconstitution.
In particular, the agent increases expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47. Expression includes protein, RNA, or protein modification (see below).
In one embodiment the invention is directed to the methods wherein the sample is obtained from blood.
In one embodiment the blood is mobilized peripheral blood.
In one embodiment the blood is umbilical cord blood.
In one embodiment the invention is directed to the methods wherein the sample is from bone marrow.
In one embodiment protein expression is assayed. Protein expression that is assayed can be intracellular, extracellular (i.e. surface), or both.
In another embodiment gene expression is assayed via expression of RNA. RNA can be any RNA, including, messenger RNA and smaller RNA molecules, such as microRNAs.
In a further embodiment, post-translational modifications may be assayed, including phosphorylation, acetylation, nitrosylation, ubiquitination, sulfation, glycosylation, myristoylation, palmistoylation, isoprenylation, farnesylation, geranylgeranylation, alkylation, amidation, acylation, oxidation, SUMOylation, Pupylation, Neddylation, biotinylation, pegylation, succinylation, selenoylation, citrullination, deamidation, ADP-ribosylation, iodination, hydroxylation, gamma-carboxylation, carbamylation, S-nitrosylation, S-glutathionylation, and malonylation, as well as any other post-translational modification.
In one embodiment gene expression is assessed by flow cytometry. Another embodiment involves the detection of molecular expression levels in enriched cells by western blotting. Another embodiment involves the detection of molecular expression levels via reverse phase protein arrays involving purified cells. Kornblau S et al. Blood 2009: 113:154-164. Immunoassays on lysates of purified or enriched cells is another embodiment. Gene expression can also be assessed by measuring mRNA. mRNA determinations can be obtained with real-time PCR.
In another embodiment gene expression is assessed in single cells.
In another embodiment gene expression assessment is assessed by EAS.
In one embodiment the invention is directed to the above methods comprising the step of administering a mobilizing agent to the subject prior to the step of obtaining a blood sample.
In one embodiment the invention is directed to a method for transplanting hematopoietic-reconstituting cells in a subject in need thereof, the method comprising administering to the subject nucleated blood cells comprising a therapeutically-effective amount of CD34+ cells having enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47.
In one embodiment the invention is directed to the above methods wherein the subject has undergone myeloablation.
The invention is directed to the methods herein wherein the subject has a hematopoietic deficiency or malignancy.
In one embodiment the invention is directed to the above methods wherein assessing the co-expression of CD34+and one or more of Musashi-2, PTEN, phospho-GSK-3p, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 is performed by flow cytometry.
In one embodiment the invention is directed to the above methods wherein the CD34+ cells having enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 are isolated.
In one embodiment the isolated cells are expanded in culture for future administration. They may be stored as a cell bank.
In one embodiment the invention is directed to the above methods wherein the subject has a disorder treatable by hematopoietic stem cell transplantation.
In one embodiment the invention is directed to the above methods wherein the disorder is a hematopoietic deficiency or malignancy.
In one embodiment, transplantation is with autologous hematopoietic-reconstituting cells. In another embodiment, transplantation is with allogeneic hematopoietic-reconstituting cells.
Various techniques for assessing expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 in CD34+ cells that may be used include, but are not limited to, flow cytometry, flow cytometry with tyramide deposition technology (EAS®), single-cell mass cytometry, immunohistochemistry, western analysis after CD34+ cell isolation, enzyme-linked immunosorbent assays (ELISA), and nucleic acid analysis including single-cell polymerase chain reaction (PCR).
In one embodiment, the levels of gene expression are assessed by EAS®, disclosed, for example, in U.S. Pat. Nos. 6,280,961, 6,335,173, and 6,828,109, incorporated by reference for the amplification methods disclosed.
The CD34+ cells may be obtained from bone marrow, umbilical cord blood or peripheral blood. In peripheral blood, CD34+ cells occur naturally and can be mobilized from the bone marrow by pharmacological treatment.
In one embodiment, a mobilizing agent is administered to the subject if it is determined that the blood sample does not contain sufficient hematopoietic-reconstituting cells (i.e., with enhanced levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47). In another embodiment, the mobilizing agent is administered prior to assessing the level of the molecules in hematopoietic-reconstituting cells. In other embodiments, the process is iterative with assessment followed by mobilization and further assessments/mobilizations depending upon the results with the mobilizing agent.
The mobilizing agent may increase the number of hematopoietic-reconstituting cells from around 2×-2,000× or more. Ranges can be around 2×-10×, 10×-50×, 50×-100×, 100×-500×, 500×-1000×, 1000×-1500×, and 1500×-2000×.
In the case of inducers of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47, an increase of expression levels could be in the ranges of a 5% to greater than 100% increase. That includes, but is not limited to, about 5-10%; 10-20%; 20-30%; 30-40%; 40-50%; 50-60%; 60-70%; 70-80%; 80-90%; 90-100% or greater.
Different agents may be used for mobilizing hematopoietic-reconstituting cells, depending on the types of blood cell and/or expression levels desired. In addition, the timing of the collection of the blood sample may affect the types of cells and/or expression levels of the cells collected. For example, it may be possible that expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 early in a mobilization differs from that later in the mobilization.
Various compounds are known that modulate Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, or E47 expression. These are available in the literature and include, but are not limited to, the compounds disclosed in the following references, incorporated by reference for teaching these compounds. For example, Mcl-1 is decreased by antisense oligonucleotides, cisplatin, and imatinib, among various substances (Aichberger et al., Blood 105:3303; Moulding et al., Blood 92:2495; and Yang, C. et al., Am. J. Physiol. Renal Physiol. 292:F1710-F1717 (2007)). Musashi2 can be decreased or increased by gene transfer or RNA inhibition, respectively (Kharas et al., Nature Medicine 16:903).
Referring to
After harvesting the blood at step 33, a decision may be made at step 36 whether to remobilize the subject in order to obtain additional blood from the subject. If the decision is made to proceed with remobilizing the subject at step 36, the process proceeds to step 37 where the mobilizing agent is selected based on the desired characteristics of the additional blood to be drawn. The process then proceeds on to step 30. If the decision is made at step 36 not to remobilize the subject, the process ends.
The method illustrated in
According to the methods of the present invention, CD34+ cells with the desired enhanced expression levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, MeI-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 can be obtained from different mobilizations, and then administered to the patient in combination or sequentially.
In one embodiment the hematopoietic reconstituting cells that are administered to the subject are autologous. In another embodiment they are allogeneic.
In a further embodiment, the hematopoietic-reconstituting cells that are isolated from a subject for further administration are much more concentrated than they were in vivo. In fact these cells may form a substantially homogeneous population. Accordingly, the CD34+ cells expressing the desired levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 can be used to directly create a source of cells to be administered at a later date and stored without further manipulation. Alternatively, the cells may be cultured, for example, expanded prior to or after storage. Accordingly, one can create a master cell bank with these cells, aliquots of which can be thawed and used for later administration with or without further expansion.
Because the methods described herein allow the isolation and concentration of the cell types described herein, the invention is also directed to novel compositions containing these cells at various levels of purity that have not been obtained before. These include about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, and 90-100%.
While this application exemplifies and focuses on the identification of a few specific molecules in CD34+ cells, the increased expression of which is correlated with greater potency, this technology more generally can be applied to ascertain any molecule that could be used as a potency marker. Thus, the invention can be more generally applied in terms of identifying molecules whose increased or decreased (or modified) expression is correlated with greater potency. This embodiment could involve assessing expression levels (modification, etc.) of molecules, involved in HRC function, from bone marrow of healthy adults, from the peripheral blood of G-CSF-treated adults, and from umbilical cord blood, and then selecting molecules that show either increased or decreased expression (modification) that correlates with the potency of the sample. In this regard, various other molecules have been associated with HRC function. It would, therefore, be a logical extension to apply the method used in this application to any of those other known molecules (as well as molecules discovered in the future that are suspected of being involved with HRC function).
In a more general sense, the method would apply to any experimental paradigm in which greater potency for any biological function can be distinguished between two (or more) different types of biological samples. Expression of molecules that is correlated with the potency in a sample could be ascertained. Having established the correlation, samples could be assessed for potency/function in the future by the gene expression pattern of the molecule.
Cell CultureIn general, cells useful for the invention can be maintained and expanded in culture medium that is available and well-known in the art. Also contemplated is supplementation of cell culture medium with mammalian sera. Additional supplements can also be used advantageously to supply the cells with the necessary trace elements for optimal growth and expansion. Hormones can also be advantageously used in cell culture. Lipids and lipid carriers can also be used to supplement cell culture media, depending on the type of cell and the fate of the differentiated cell. Also contemplated is the use of feeder cell layers.
Cells in culture can be maintained either in suspension or attached to a solid support, such as extracellular matrix components. Stem cells often require additional factors that encourage their attachment to a solid support, such as type I and type II collagen, chondroitin sulfate, fibronectin, “superfibronectin” and fibronectin-like polymers, gelatin, poly-D and poly-L-lysine, thrombospondin and vitronectin. One embodiment of the present invention utilizes fibronectin. See, for example, Ohashi et al., Nature Medicine, 13:880-885 (2007); Matsumoto et al., J Bioscience and Bioengineering, 105:350-354 (2008); Kirouac et al., Cell Stem Cell, 3:369-381 (2008); Chua et al., Biomaterials, 26:2537-2547 (2005); Drobinskaya et al., Stem Cells, 26:2245-2256 (2008); Dvir-Ginzberg et al., EASES J, 22:1440-1449 (2008); Turner et al., J Biomed Mater Res Part B: Appl Biomater, 8213:156-168 (2007); and Miyazawa et al., Journal of Gastroenterology and Hepatology, 22:1959-1964 (2007)).
Once established in culture, cells can be used fresh or frozen and stored as frozen stocks, using, for example, DMEM with 40% FCS and 10% DMSO. Other methods for preparing frozen stocks for cultured cells are also available to those of skill in the art.
Pharmaceutical FormulationsIn certain embodiments, the cell populations are present within a composition adapted for and suitable for delivery, i.e., physiologically compatible.
In some embodiments the purity of the cells for administration to a subject is about 100% (substantially homogeneous). In other embodiments it is 95% to 100%. In some embodiments it is 85% to 95%. Particularly, in the case of admixtures with other cells, the percentage can be about 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%. Or isolation/purity can be expressed in terms of cell doublings where the cells have undergone, for example, 10-20, 20-30, 30-40, 40-50 or more cell doublings.
Of course, samples found to have sufficient potency can be administered without any purification at all. But the inventor also envisions scenarios in which cells are created in vitro with the desired expression levels or possibly purified from in vivo and then expanded in vitro.
The choice of formulation for administering the cells for a given application will depend on a variety of factors. Prominent among these will be the species of subject, the nature of the condition being treated, its state and distribution in the subject, the nature of other therapies and agents that are being administered, the optimum route for administration, survivability via the route, the dosing regimen, and other factors that will be apparent to those skilled in the art. For instance, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.
Final formulations of the aqueous suspension of cells/medium will typically involve adjusting the ionic strength of the suspension to isotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e., about pH 6.8 to 7.5). The final formulation will also typically contain a fluid lubricant.
In some embodiments, cells/medium are formulated in a unit dosage injectable form, such as a solution, suspension, or emulsion. Pharmaceutical formulations suitable for injection of cells/medium typically are sterile aqueous solutions and dispersions. Carriers for injectable formulations can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions to be administered in methods of the invention. Typically, any additives (in addition to the cells) are present in an amount of 0.001 to 50 wt % in solution, such as in phosphate buffered saline. The active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.
The dosage of the cells will vary within wide limits and will be fitted to the individual requirements in each particular case. In general, in the case of parenteral administration, it is customary to administer from about 0.01 to about 20 million cells/kg of recipient body weight. The number of cells will vary depending on the weight and condition of the recipient, the number or frequency of administrations, and other variables known to those of skill in the art. The cells can be administered by a route that is suitable for the tissue or organ. For example, they can be administered systemically, i.e., parenterally, by intravenous administration, or can be targeted to a particular tissue or organ; they can be administrated via subcutaneous administration or by administration into specific desired tissues.
1001471 The cells can be suspended in an appropriate excipient in a concentration from about 0.01 to about 5×106 cells/ml. Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the cells and with the recipient, such as buffered saline solution or other suitable excipients. The composition for administration can be formulated, produced, and stored according to standard methods complying with proper sterility and stability.
DosingDoses for humans or other mammals can be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art. The dose of cells/medium appropriate to be used in accordance with various embodiments of the invention will depend on numerous factors. The parameters that will determine optimal doses to be administered for primary and adjunctive therapy generally will include some or all of the following: the disease being treated and its stage; the species of the subject, their health, gender, age, weight, and metabolic rate; the subject's immunocompetence; other therapies being administered; and expected potential complications from the subject's history or genotype. The parameters may also include: whether the cells are syngeneic, autologous, allogeneic, or xenogeneic; their potency (specific activity); the site and/or distribution that must be targeted for the cells/medium to be effective; and such characteristics of the site such as accessibility to cells/medium and/or engraftment of cells. Additional parameters include co-administration with other factors (such as growth factors and cytokines). The optimal dose in a given situation also will take into consideration the way in which the cells/medium are formulated, the way they are administered, and the degree to which the cells/medium will be localized at the target sites following administration.
The optimal dose of cells could be in the range of doses used for autologous, mononuclear bone marrow transplantation. For fairly pure preparations of cells, optimal doses in various embodiments will range from 104 to 108 cells/kg of recipient mass per administration. In some embodiments the optimal dose per administration will be between 105 to 107 cells/kg. In many embodiments the optimal dose per administration will be 5×105 to 5×106 cells/kg. By way of reference, higher doses in the foregoing are analogous to the doses of nucleated cells used in autologous mononuclear bone marrow transplantation. Some of the lower doses are analogous to the number of CD34+ cells/kg used in autologous mononuclear bone marrow transplantation.
As an example, cell doses for umbilical cord blood or peripheral blood hematopoietic stem cell transplantation are somewhat different than bone marrow transplantation.
In various embodiments, cells may be administered in an initial dose, and thereafter maintained by further administration. Cells/medium may be administered by one method initially, and thereafter administered by the same method or one or more different methods. The levels can be maintained by the ongoing administration of the cells. Various embodiments administer the cells either initially or to maintain their level in the subject or both by intravenous injection. In a variety of embodiments, other forms of administration are used, dependent upon the patient's condition and other factors, discussed elsewhere herein.
Cells may be administered in many frequencies over a wide range of times. Generally lengths of treatment will be proportional to the length of the disease process, the effectiveness of the therapies being applied, and the condition and response of the subject being treated.
UsesAdministering the cells is useful in any number of pathologies, including, but not limited to, those listed herein.
In addition, other uses are provided by knowledge of the biological mechanisms described in this application. One of these includes drug discovery. This aspect involves screening one or more compounds for the ability to affect the cell's ability to achieve any of the effects described in this application. Accordingly, the assay may be designed to be conducted in vivo or in vitro.
Gene expression can be assessed by directly assaying protein or RNA. This can be done through any of the well-known techniques available in the art, such as by FACS and other antibody-based detection methods, such as immunoassays (e.g., ELISA or Western blot) and PCR and other hybridization-based detection methods. Indirect assays may also be used for expression, such as the effect of gene expression.
A further use for the invention is the establishment of cell banks to provide cells for clinical administration. Generally, a fundamental part of this procedure is to provide cells that have a desired potency for administration in various therapeutic clinical settings.
In a specific embodiment of the invention, the cells are selected for having a desired potency for hematopoietic reconstitution (or the self-renewal and/or differentiation components).
Any of the same assays useful for drug discovery could also be applied to selecting cells for the bank as well as from the bank for administration.
Accordingly, in a banking procedure, the cells would be assayed for the ability to achieve any of the above effects. Then, cells would be selected that have a desired potency for any of the desired effects, and these cells would form the basis for creating a cell bank.
It is also contemplated that potency can be increased by treatment with an exogenous compound, such as a compound discovered through screening the cells with large combinatorial libraries. These compound libraries may be libraries of agents that include, but are not limited to, small organic molecules, antisense nucleic acids, siRNA DNA aptamers, peptides, antibodies, non-antibody proteins, cytokines, chemokines, and chemo-attractants. For example, cells may be exposed such agents at any time during the growth and manufacturing procedure. The only requirement is that there be sufficient numbers for the desired assay to be conducted to assess whether or not the agent increases potency. Such an agent, found during the general drug discovery process described above, could more advantageously be applied during the last passage prior to banking.
A further use is to assess the efficacy of the cell to achieve any of the above results as a pre-treatment diagnostic that precedes administering the cells to a subject. Moreover, dosage can depend upon the potency of the cells that are being administered. Accordingly, a pre-treatment diagnostic assay for potency can be useful to determine the dose of the cells initially administered to the patient and, possibly, further administered during treatment based on the real-time assessment of clinical effect.
It is also to be understood that the cells of the invention can be used not only for purposes of treatment, but also research purposes, both in vivo and in vitro to understand the mechanism involved normally and in disease models. In one embodiment, assays, in vivo or in vitro, can be done in the presence of agents known to be involved in the biological process. The effect of those agents can then be assessed. These types of assays could also be used to screen for agents that have an effect on the events that are promoted by the cells of the invention. Accordingly, in one embodiment, one could screen for agents in the disease model that reverse the negative effects and/or promote positive effects. Conversely, one could screen for agents that have negative effects in a non-disease model.
CompositionsThe invention is also directed to cell populations with specific potencies (i.e., desired expression levels) for achieving any of the effects described herein. As described above, these populations are established by selecting for CD34+ cells that have desired enhanced levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47. These populations are used to make other compositions, for example, a cell bank comprising populations with specific desired potencies and pharmaceutical compositions containing a cell population with a specific desired potency. Cultures can be established from in an in vivo source of the cells. Or cells can be created in vitro, such as by increasing the copy number of the genes or inducing/increasing endogenous gene expression.
Although the exemplified embodiment and the embodiment discussed in most detail in this application is directed to hematopoietic-reconstituting cells, the invention may also apply to other stem cells that can be used in transplantation. In other words, the issue involves the identification in those cells of molecules associated with potency. Accordingly, the following cells may be used for transplantation.
A list of cells that may be characterized by enhanced levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 at expression levels follows: embryonic stem cells, induced pluripotent cells, human progenitor cells, mesenchymal stem cells, mesenchymal stromal cells, human CD133+stem cells, T lymphocytes, B lymphocytes, dendritic cells, regulatory T (Treg) cells, neural stem cells, neural progenitor cells, multipotent stem cells, pluripotent stem cells, endothelial progenitor cells, lymphocytes with chimeric antigen receptors, tumor infiltrating lymphocytes, genetically-engineered T lymphocytes, and natural killer cells.
A list of tumors that may show differences in expression of one or more of Musashi-2, PTEN, phospho-GSK-3 mel-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 follows: acute myelogenous leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, non-Hodgkin's lymphoma, plasma cell myeloma, carcinoma and sarcoma.
These tumors may have subpopulations that distinguish them from hematopoietic-reconstituting cells in regards to expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47, as opposed to having the entire tumor cells that conform to or do not conform to having the enhanced levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47.
In a further aspect of the invention, based on the results in the exemplified embodiments, the levels of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 can be used as a marker to distinguish between tumor cells and normal cells. The ratios from mobilized blood in lymphoma, myeloma, and healthy volunteers is separate and distinct. It is also separate and distinct from those ratios obtained from bone marrow, peripheral blood (non-mobilized) and cord blood.
ExampleIdentification of Hematopoietic-Reconstituting Cells to Assess Subsequent Transplantation
Transplantation of hematopoietic-reconstituting cells (HRC) is used as a therapeutic modality for a variety of malignant and nonmalignant disorders. These disorders include but are not limited to aplastic anemia, paroxysmal nocturnal hemoglobinuria, chronic myeloid leukemia, acute myeloid leukemia, juvenile myelomonocytic leukemia, acute lymphoblastie leukemia, myelodysplastic syndrome, myeloproliferative disorders, multiple myeloma, lymphoma, chronic lymphocytic leukemia, systemic light chain amyloidosis, breast cancer, germ cell tumors, renal cell carcinoma, solid tumors, neuroblastoma, human immunodeficiency virus infection, acute myocardial infarction, and autoimmune diseases (Appelbaum F R, et al. eds. Thomas' hematopoietic cell transplantation. 4th edition. Wiley-Blackwell. Hoboken, N.J. (2008) and Treleaven J, Barrett A J. Eds. Hematopoietic stem cell transplantation. Elsevier. New York, N.Y. (2009)).
HRC are transplanted in order to reconstitute hematopoiesis in a person who does not have that capacity. HRC are not clearly defined. They are included in cells that express the surface marker, CD34; however, this marker is expressed by many cells that do not have the capacity to mediate hematopoietic reconstitution. Moreover, the CD34 molecule itself is not associated with functions related to hematopoietic reconstitution such as self-renewing proliferation or short-term differentiation to hematopoietic lineages.
In order to ascertain whether CD34+ cells actually possess the capacity to reconstitute the hematopoietic system, investigators have assessed colony-forming units which involves the capacity of the CD34+ cells to differentiate into various hematopoietic lineages in a culture system with the addition of stimulating factors. Although these types of functional assays are valuable, they are subjective, expensive, time-consuming, and difficult to standardize among various laboratories. Moreover, they only measure short-tet m differentiation and not self-renewing proliferative capacity. The colony-forming assays do not provide any information at a molecular level.
The Federal Drug Administration (FDA) has prepared a white paper that includes guidance for developing potency test for cellular therapy products (Guidance for Industry. Potency tests for cellular and gene therapy products. Food and Drug Administration. Center for Biologics Evaluation and Research. 2011. http://www.fda.gov/BiologicsBloodVaccines/_GuidanceComplianceRegulatory Information/Guidances/default.htm.). It is stated in this document “Ideally, the potency assay will represent the product's mechanism of action”. And “all attempts should be made to develop potency measurements that reflect the product's relevant biological properties.” In this regard, the measurement of CD34+ cells is necessary as a potency measure but not sufficient to ascertain the capacity to mediate the mechanism of action required in successful transplantation.
Many molecules expressed in HRC have been associated with the potential for differentiation into cells of the various hematopoietic lineages and for the capacity for self-renewal including transcription factors and pathway molecules. (Sauvageau G, et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes & Dev. (1995) 9:1753-1765; Unger C, et al. Lentiviral-mediated HoxB4 in human embryonic stem cells initiates early hematopoiesis in a dose-dependent manner but does not promote myeloid differentiation. Stem Cells (2008) 26:2455-2466; Wilson A, et al., c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes & Dev. (2004) 18:2747-2763; Baena E, et al., c-Myc is essential for hematopoietic stem cell differentiation and regulates Lin-Sca-1+c-Kit. Exp Hematol. (2007) 35:1333-1343; Laurenti E, et al., Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity. Cell Stem Cell (2008) 3:611-624; Satoh Y, et al. Roles for c-Myc in self-renewal of hematopoietic stem cells. J Biol Chem. (2004) 279:24986-24993; Tsai F-Y, et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature (1994) 371:221-226; Heyworth C, et al. A GATA-2/ostrogen receptor chimera functions as a ligand-dependent negative regulator of self-renewal. Genes & Dev. (1999) 13:1847-1860; Ezoe S, et al. GATA-2/estrogen receptor chimera regulates cytokine-dependent growth of hematopoietic cells through accumulation of p21waf1 and p27kip1 proteins. Blood (2002) 100:3512-3520; Tipping A J, et al. High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle. Blood (2009) 113:2661-2672; Park, I., et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature (2003) 423, 302-305; Rizo, A., et al. Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis. Blood (2009) 114, 1498-1505; Liakhovitskaia, A., et al., Restoration of Runx1 expression in the Tie2 cell compartment rescues definitive haematopoietic stem cells and extends life of Runx1 knockout animals until birth. Stem Cells (2009) 27:1616-1624; Semerad, C. J., et al. E2A proteins maintain the hematopoietic stem cell pool and promote the maturation of myelolymphoid and myeloerythroid progenitors. Proc Natl Acad Sci USA (2009) 106:1930-1935; Zhang J, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature (2006) 441:518-522; Juntilla, M. M., et al. AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species. Blood (2010) 115:4030-4038; Reya, T, et al. A role for Wnt signaling in self-renewal of haematopoietic stem cells. Nature (2003) 423:409-414; Kim, J., et al. Identification of a stroma-mediated Wnt/β-catenin signal promoting self-renewal of hematopoietic stem cells in the stem cell niche. Stem Cells (2009) 27:1318-1329; Li, G., et al., Gab2 promotes hematopoietic stem cell maintenance and self-renewal synergistically with STAT5. PLoS One 5(2): e9152. Doi:10.1271/journal.pone.0009152; Gu, H., et al. Cloning of p97/Gab2, the major SHP2-binding protein in hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation. Mol. Cell. (1998) 2:729-740; Nishida, K, et al. Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors. Blood (1999) 93:1809-1816.
Most of these studies have been performed in murine models with knock-out genetic approaches. It has been difficult to assess the expression of these molecules in human HRC because they are expressed at low abundance which precludes quantitative information by standard methods of flow cytometry and because HRC are a small subpopulation of the cells collected making it difficult to determine by western blotting or RT-PCR.
The inventor has previously analyzed the expression of transcription factors, pathway molecules, and cell surface receptors associated with CD34+HRC from patients with multiple myeloma using our high-resolution immunophenotyping technology on a flow cytometric platform, enzymatic amplification staining (EAS®) (Kaplan D, et al. The functional duality of HoxB4 in hematopoietic reconstituting cells. Cytometry A. 83A:127-133 (2013); Lazarus, H M, et al. Spontaneous autologous graft-versus-host disease in plasma cell myeloma autograft recipients: Flow cytometric analysis of hematopoietic progenitor cell grafts. Biol Blood Marrow Transplant (2011) 17:970-978; Meyerson H J, et al. D cyclins in CD5 B-cell lymphoproliferative disorders. Cyclin D1 and cyclin D2 identify diagnostic groups and cyclin D1 correlates with ZAP-70 expression in chronic lymphocytic leukemia. Am J Clin Pathol. (2006) 125:241-250. Kaplan D. Enzymatic amplifactions staining for cell surface antigens. In Current protocols in cytometry. J P Robinson, editor. New York, N.Y.: Wiley, (2003) 6.14.1-6.14.11; Kaplan D, et al. D cyclins in lymphocytes. Cytometry (2005) 63A:1-9; Kaplan D, et al. CD5 expression by B lymphocytes and its regulation upon Epstein-Barr Virus transformation. Proc Natl Acad Sci USA (2001) 98:13850-13853; Kaplan, D, et al. Correlation between ZAP-70, pohospho-ZAP-70, and phosphor-Syk expression in leukemic cells from patients with CLL. Cytometry B (2010) 78:115-122; Kaplan D, and Smith D. Enzymatic amplification staining for flow cytometric analysis of cell surface molecules. Cytometry (2000) 40:81-85.
The inventor found that colony-forming units were significantly correlated with HoxB4 expression which was explained by the number of CD34+ cells in the specimens. However, analysis of colony-fanning units normalized to the CD34+ cell count revealed a significant negative correlation with HoxB4 expression. Thus, HoxB4 enhances CD34+ cell number via self-renewing expansion but concomitantly depreciates the capacity for short-term differentiation per cell. The findings demonstrate the translation of experimental findings into a clinical setting and suggest that the expression level of HoxB4 in CD34+ cells can be used as a measure of a sample's appropriateness for transplantation. At the same time statistically significant correlations were found among some of the molecules. Nevertheless, these correlations were relatively weak with r values less than 0.5.
The inventor then initiated an investigation of CD34+ HRC as shown in the Example. Molecules associated with HRC function were assessed. Improvements were also instituted in this study such as an improved stain for CD34+ events and the use of a singular flow cytometer for the entire study. With this new protocol molecular expression levels were determined in a way that is novel and useful.
ExampleCellular transplantation is a relatively new therapeutic modality. Various stem cells and specific immune cells have been used successfully in treating sundry clinical conditions.
It is important to develop potency measures for cells to be transplanted in order to ascertain that the cells will perform as expected. The Federal Drug Administration has proposed that the potency of cells used in therapies be characterized by relevant molecular properties (1).
Hematopoietic reconstituting cells (HRC) are transplanted in the treatment of both malignant and nonmalignant diseases. Transplantation is based on the number of CD34+ cells; however, the CD34 molecule has been shown to be nonessential for hematopoietic reconstitution (2). The number of CD34+ cells transplanted is clearly associated with engraftment potential in terms of the transplanted inoculum but it does not provide an indication of the potency at the level of the individual cell. CD34+ cells with distinct potencies for engraft would be useful for investigators in developing a functional measure of the potential for CD34+ cells to engraft.
There are 2 sources of HRC that have been shown to be superior to bone marrow CD34+ cells in terms of their functional potency on a cell-by-cell basis. They are G-CSF-mobilized CD34+ cells obtained from the peripheral circulation and umbilical cord blood CD34+ cells. For instance, at the Fred Hutchinson Cancer Research Center in Seattle, G-CSF-mobilized cells demonstrate 5-7 days faster reconstitution compared to bone marrow cells even when similar doses of CD34+ cells were used (3). Enhancement in the recovery of neutrophils (7 days) and platelets (8 days) after transplantation of similar numbers of mobilized peripheral blood CD34+cells versus bone marrow CD34+ cells was also observed in a Norwegian study (4). These results are consistent with the greater number of granulocyte-macrophage colony-forming units per CD34+ cell for G-CSF mobilized peripheral blood CD34+ cells compared to bone marrow resident CD34+ cells (5).
Similarly, umbilical cord blood HRC have been shown to have a higher cloning efficiency, to proliferate more rapidly in response to cytokine stimulations, and to generate about 7-fold more progeny than HRC from the adult bone marrow (6). Another group of investigators found that cultures of cord blood cells produced a significantly greater increase in granulocyte-macrophage colony-forming units and granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming units than cultures of bone marrow cells (7). A third group found similar findings in comparing umbilical cord blood CD34+ cells with bone marrow CD34+ cells (8). It is also important to note that approximately 10-fold less umbilical cord blood CD34+ cells are used for transplantation than the bone marrow CD34+ cells.
We have developed a potency measure for HRC based on the expression levels of molecules known to be important for HRC function. The analysis includes a signal amplification system on a flow cytometric platform so that the molecular expression levels have an expanded dynamic range and can be assigned to the specific cellular subpopulation.
ResultsTable 1 shows mononuclear cells from the bone marrow, umbilical cord blood, and peripheral blood G-CSF-treated healthy persons that were assessed for their capacity to make hematopoietic colonies in 2 different assays of colony forming units. These assays are functional measures of the capacity to differentiate into various hematopoietic lineages. The colony-forming units-granulocytes and macrophages (CFU-GM) assay measures the number of colonies of granulocytic or macrophagic cells that are counted upon culture with cytokines that stimulate the differentiation of the CD34+ cells. The burst-forming units-erythrocytes (BFU-E) assay measure the number of colonies of erythrocytic cells that are counted upon culture with cytokines that stimulate the differentiation of the CD34+ cells. The results have been normalized for the number of CD34+ cells in the samples. The findings demonstrate that the CD34+ cells from the umbilical cord blood and from the peripheral blood of healthy persons treated with a mobilizing agent, G-CSF, are more potent in terms of hematopoietic-reconstituting function than CD34+ cells from the bone marrow of healthy subjects. It should be clear that this measure of potency is on a per CD34+ cell basis.
Table 2 shows the expression levels of 16 molecules associated with hematopoietic-reconstituting function in experimental models in CD34+ cells from the bone marrow of healthy subjects and from the peripheral blood of healthy subjects treated with G-CSF in order to mobilize the CD34+ cells from the bone marrow to the peripheral blood. The expression levels shown are the means of the results from 20 subjects in each group. The mean values were compared by two-sided t tests and by the effect size indicator, Cohen's d. Both analytical methods demonstrate molecules with significant differences in molecular expression levels between the 2 groups.
Table 3 shows the expression levels of 16 molecules associated with hematopoietic-reconstituting function in experimental models in CD34+cells from the bone marrow of healthy subjects and from umbilical cord blood. The expression levels shown are the means of the results from 20 subjects in each group. The mean values were compared by two-sided t tests and by the effect size indicator, Cohen's d. Both analytical methods demonstrate molecules with significant differences in molecular expression levels between the 2 groups.
Relative Potency of HRC from 3 Sources
Mononuclear cells from the bone marrow, umbilical cord blood, and peripheral blood of G-CSF-treated persons were assessed for their capacity to make colonies in 3 different CFU assays (Table 1). The bone marrow and peripheral blood donors were healthy adults. The number of colonies was normalized to the number of CD34+ cells in the samples. The data show that the CD34+ cells from the umbilical cord blood and from peripheral blood of G-CSF-treated persons gave significantly more colonies than the CD34+ cells from the bone marrow.
Molecular Expression Levels in HRC from 3 Sources
We compared the molecular expression levels of 16 molecules known to be important for HRC function in 20 samples of HRC from the 3 sources known to have different potency. The molecules we assessed included pathway molecules (phospho-Akt(ser308), phospho-Akt(thr473), β-catenin, GAB2, PTEN, and phospho-GSK-3β) (9-17), transcription factors (HoxB4, GATA2, cMyc, Runx1, and E47) (18-29), a transcriptional repressor (Bmi-1) (30, 31), a translational regulator (Musashi-2) (32, 33), an anti-apoptotic molecule (Mcl-1) (34), a K63-specific ubiquitin-conjugating enzyme (UBC13) (35), and an autophagy protein (Atg7) (36). The involvement of these molecules in HRC function were previously determined by experimental manipulations (9-36). Representative flow cytometric results for a single sample of G-CSF-mobilized HRC and a single sample of umbilical cord blood are shown in
Table 1 shows that 12 of the 16 molecules demonstrated significantly increased expression levels (two-sided T test with p 0.05) in G-CSF-mobilized HRC compared to levels in HRC from the bone marrow. The molecules that had the greatest separation in expression, as determined by an effect size test (Cohen's d≧1), are Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), and Bmi-1.
Table 2 shows that 10 of the 16 molecules demonstrated significantly increased expression levels in HRC from UBC compared to levels in bone marrow-resident HRC. The molecules that had the greatest separation in expression, as determined by an effect size test (Cohen's d≧1), are Musashi-2, phospho-GSK-3β, PTEN, phospho-Akt(ser473), and HoxB4.
DiscussionThe impetus for this investigation was to develop a cellular measure for potency for HRC based on molecules known to be important in mediating hematopoietic reconstituting function. In order to accomplish this goal, we assessed HRC with different intrinsic functional capabilities. It is known and accepted that G-CSF mobilized HRC and UBC HRC are more potent than bone marrow resident HRC on a cell-by-cell basis. This conclusion is based on colony-forming assays presented here and in the literature as well as by clinical experience (3-8).
We analyzed more potent HRC from 2 disparate, independent sources. In 1 case the adult HRC were stimulated pharmacologically and in the other HRC at parturition were assessed without treatment. Our major assumption is that common molecular features of these disparate sources of more potent HRC that are not shared by the less potent source (bone marrow) constitute a measure of potency.
With this assumption in mind, we have developed a measure of potency for HRC function that is based on the expression levels of molecules known to be important for HRC function. First, potency is most clearly related to increased expression of Musashi-2, PTEN, phospho-GSK-3β, and phospho-Akt(ser473). Mcl-1 and HoxB4 are also enhanced in both groups of HRC with greater potency although the effect size is not as impressive for these 2 molecules. It seems reasonable that potency is related to an increased expression of molecules that have been independently associated with function.
It should be noted that our analysis involves human cells that have not been manipulated and that express the various analytes in their physiological concentrations. In studies of murine HRC, investigators experimentally alter molecular concentrations or activities by genetic means or chemical inhibition.
Materials and Methods Patient SamplesBone marrow from 20 healthy persons, peripheral blood from 20 healthy persons, and peripheral blood from 20 healthy persons who had been treated with G-CSF were obtained from AllCells (Emeryville, Calif.) and from the Hematopoietic Cell Repository at the Case Comprehensive Cancer Center, Case Western Reserve University. Peripheral blood from 20 persons with lymphoma and 20 persons with plasma cell myeloma were obtained after mobilization with G-CSF and cyclophosphamide from the Hematopoietic Cell Repository (37). Umbilical cord blood samples were obtained from AllCells. All samples were obtained with Institutional Review Board approval. The mononuclear cells from all samples were isolated by ficoll/hypaque discontinuous gradient centrifugation and cryopreserved until analysis.
Colony-Forming Unit AssaysMononuclear cells (1×105) were grown in duplicate in methylcellulose (Stem Cell Technologies; Vancouver, Canada) containing 10 ng/ml IL-3, 3 U/ml EPO, 50 ng/ml SCF, 10 ng/ml GM-CSF, and Hemin (0.1 mM; Sigma Chemicals; St. Louis, Mo.), and the cells were incubated at 37° C. and 5% CO2. After 12-14 days, colonies greater than 50 cells were identified by morphology, enumerated and expressed as total CFU-GM and BFU-E (38).
AntibodiesAntibodies specific for CD34 were obtained from BioLegend (San Diego, Calif.). Antibodies specific for c-Myc were from Invitrogen (Carlsbad, Calif.). Antibodies specific for HoxB4, GAB2, Atg7, Mcl-1, UBC13, and RUNX1 were from Epitomics (Burlingame, Calif.). Antibodies specific for GATA-2 and Bmi-1 were from R & D Systems (Minneapolis, Minn.). Antibodies specific for phosphatase tension homolog (PTEN), phospho-GSK-3β, phospho-Akt(thr308), phospho-Akt(ser473), and β-catenin were from Cell Signaling Technology (Danvers, Mass.). Antibodies specific for E47 were from BD Biosciences (Mountain View, Calif.). Antibodies specific for Musashi-2 were from Millipore (Billerica, Mass.).
Flow Cytometric AnalysisThe samples were analyzed for the expression of the 16 analytes by Pathfinder Biotech (Cleveland, Ohio) using enzymatic amplification staining (EAS™) as previously described (39-46). EAS™ is a validated, catalyzed reporter deposition technology based on the enzymatic activity of peroxidase. The events were gated with the characteristic forward and side scatter for CD34 cells. CD34 counterstaining was accomplished for all samples using an anti-CD34-biotin conjugate followed by incubation with Streptavidin-DyLight™649 (BioLegend) conjugate. The median fluorescence ratio was obtained for CD34+events by dividing the median fluorescence intensities for the specific antibodies by the median fluorescence intensity of a fluorescence minus one (FMO) control. Multiple quality control features for high-resolution immunophenotyping have been ascertained. Most importantly, analytical reproducibility has been demonstrated by staining identical frozen aliquots of Jurkat cells for a variety of intracellular analytes (43). In addition, we have used carboxylated polystyrene beads substituted with various amounts of human IgG (as an analyte) to demonstrate the linearity of detection by EAS™ at levels under the level of detection by indirect staining (40). Thus, the data we have obtained are reproducible and quantitative. It should be noted that HRC from 2 specimens of peripheral blood from healthy persons treated with G-CSF were not analyzed for Musashi-2 expression because of the absence of antibody when the samples were studied. Similarly, HRC from 2 samples of peripheral blood were not analyzed for E47 expression and HRC from 1 sample of peripheral blood was not analyzed for PTEN levels.
Statistical AnalysisStatistical analyses were performed using SPSS and Excel software. Comparisons between expression levels were made with t-tests and effect size. All tests were two-sided and p values less than 0.05 were considered statistically significant. For effect size, Cohen's d was calculated and a value greater than or equal to 1 is considered significant.
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- 43. Kaplan, D., et al. Correlation between ZAP-70, phospho-ZAP-70, and phospho-Syk expression in leukemic cells from patients with CLL. Cytometry B (2010) 78:115-122.
- 44. Kaplan, D. and Smith, D. Enzymatic amplification staining for flow cytometric analysis of cell surface molecules. Cytometry (2000) 40:81-85.
- 45. Lazarus, H. M., et al. Spontaneous autologous graft-versus-host disease in plasma cell myeloma autograft recipients: Flow cytometric analysis of hematopoietic progenitor cell grafts. Biol Blood Marrow Transplant (2011) 17:970-978.
- 46. Kaplan, D., et al. The functional duality of HoxB4 in hematopoietic reconstituting cells. Cytometry A (2013) 83A:127-133.
Claims
1. A method for assessing the capacity of a sample to therapeutically effect hematopoietic reconstitution in a subject, the method comprising: assessing CD34+ cells for enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47, in individual cells in the sample and determining the number of such cells.
2. The method of claim 1 wherein the one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 are expressed at a level greater than the mean expression level in un-mobilized bone marrow.
3. A method to prepare a subject to donate blood for hematopoietic-reconstituting cell (HRC) transplantation, the method comprising obtaining a blood sample containing hematopoietic cells from the subject; determining number of CD34+ cells having enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 in individual cells from the blood sample; and administering to the subject a mobilizing agent when the blood sample does not contain a desired therapeutically-effective amount of such CD34+ cells.
4. The method of claim 3 wherein the one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 are expressed at a level greater than the mean expression level in un-mobilized bone marrow.
5. The method of claim 3, further comprising the step of administering a mobilizing agent to the subject prior to the step of obtaining a blood sample.
6. A method for transplanting hematopoietic-reconstituting cells in a subject in need thereof, the method comprising administering to the subject nucleated blood cells comprising a therapeutically effective amount of CD34+ cells having enhanced expression of one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47.
7. The method of claim 6 wherein the one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 are expressed at a level greater than the mean expression level in un-mobilized bone marrow.
8. The method of claim 6 wherein the CD34+ cells expressing the one or more of Musashi-2, PTEN, phospho-GSK-3β, Mcl-1, Atg7, phospho-Akt(ser473), HoxB4, Bmi-1, UBC13, phospho-Akt(thr308), GATA2, cMyc, and E47 are isolated.
9. The method of claim 6 wherein the subject has a disorder treatable by hematopoietic stem cell transplantation.
10. The method of claim 9 wherein the disorder is a hematopoietic deficiency or malignancy.
11. A method to identify a molecule, the expression of which is correlated with the hematopoietic reconstituting function, the method comprising assessing expression of a molecule in individual CD34+ cells in samples having different levels of potency and identifying molecules, the expression of which correlates with potency, by correlating differences in expression of such molecules with the potency of the different samples.
12. The method of claim 11 wherein greater potency is associated with an increase in expression.
13. The method of claim 11 wherein greater potency is associated with a decrease in expression.
14. The method of claim 11 wherein the samples that are compared are un-mobilized bone marrow, mobilized peripheral blood from healthy subjects, and umbilical cord blood.
15. The method of any of claims 1-4, 6, 7, and 11 wherein the expression that is assayed is selected from the group consisting of RNA, protein, and post-translational modification.
16. A method to assess the potency of a sample for hematopoietic reconstitution function, the method comprising assessing the expression level of a molecule identified by the method in claim 11 in CD34+ cells in the sample.
Type: Application
Filed: Mar 15, 2013
Publication Date: Jan 2, 2014
Applicant: Verve, Ltd. (Pepper Pike, OH)
Inventor: David R. Kaplan (Shaker Heights, OH)
Application Number: 13/841,521
International Classification: G01N 33/569 (20060101); A61K 35/28 (20060101);
