WO2009152186A1 - Methods for enhancing cell therapy efficacy including treatment with cd26 peptidase inhibitors - Google Patents

Abstract

Disclosed are methods, compositions of matter, and kits useful for augmentation of stem, progenitor and mature cells through the inhibition of cell surface CD26, dipeptidylpeptidase IV. The methods and compositions may be used for the treatment of a wide variety of disorders in which augmentation of cell trafficking, homing and engraftment is desired.

Description

METHODS FOR ENHANCING CELL THERAPY EFFICACY INCLUDING TREATMENT WITH CD26 PEPTIDASE INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/060,080, filed on June 9, 2008 entitled METHODS FOR ENHANCING CELL THERAPY EFFICACY INCLUDING TREATMENT WITH CD26 PEPTIDASE INHIBITORS, which is incorporated herein by reference in its entirety.

BACKGROUND

Field

[0002] The present embodiments relate generally to the field of cellular therapy. More specifically, some embodiments relate to methods of enhancing the natural process of cell migration through modulation of various processes involved in cell homing. More specifically, some embodiments relate to augmentation of the SDF-1-CXCR4 axis by treatment of cells with agents/conditions that lead to an enhanced response to SDF-I (CXCL 12) induced migration and homing.

Description of the Related Art

[0003] Cell therapy offers immense possibilities for treatment of a wide variety of medical conditions. Currently cell therapy is practiced in various applications, for example, bone marrow transplantation for treatment of hematopoietic and non-hematopoietic malignancies. The successful establishment of procedures for transplantation of donor cells into recipients whose function is malignant (leukemia) or altered (stroke, limb ischemia, etc), or insufficient (due to chemotherapy, radiotherapy, or congenital abnormality) constitutes a major medical breakthrough in the therapeutic management of these conditions. For non- hematological purposes, cellular therapy is currently in experimental investigations ranging anywhere from treatment of stroke, to post-myocardial infarction, to in-born errors of metabolism. [0004] One limiting factor of any cellular therapy is the need for cells to migrate to the targeted tissue in order to maximize their therapeutic potential. With regard to hematopoietic stem cells as a particular example, it is known that only a small percentage of these cells when administered systemically home to the bone marrow microenvironment. This is regulated in part by adhesive factors on the luminal surface of endothelial cells lining the bone marrow and in part by chemotactic gradients secreted at a constant rate by bone marrow stromal cells. In addition, for other regenerative purposes, such as treatment of myocardial infarction or stroke, only a small fraction of injected stem cells actually enter the area of tissue damage. Thus, there exists a need to administer a high number of stem cells, sometimes prohibitively too high to be obtained in an autologous and even allogeneic setting.

SUMMARY

[0005] The ability of leukocytes to interact with the endothelium has been known for decades. It has also been known that various glycosylation patterns are critical for leukocytes to "roll" on the endothelium before extravasation. Some embodiments relate to the unexpected finding that by modification of the SDF-CXCR4 axis, it is possible to enhance the efficacy of cellular therapy.

[0006] Accordingly, provided herein are methods of enhancing homing and engraftment of a therapeutical ly-provided or administered cell in a patient. In should be noted that the term "patient" is meant to broadly include any animal. For example, the animal can be a mammal, a bird, a fish, a reptile, a fish, an insect or any other animal. Some non-limiting examples of mammals may include humans and other primates, equines such as horses, bovines such as cows, mice, rats, rabbits, Guinea Pigs, pigs, and the like. It is also worth noting that the compositions and methods can be used with or applied to individual cells (for example ex vivo treatment or modification), to insect cells, etc. Also provided are cells that have been modified to enhance homing and engraftment. The embodiments provided herein are based in part on the surprising finding that by modification of molecules involved in the cell-endothelium interaction, it is possible to enhance the homing and subsequent efficacy of cellular therapy. [0007] Some embodiments relate to methods of enhancing homing and engraftment of a therapeutically-provided or administered cell in a patient comprising: selecting a patient in need of treatment with a cell population; providing cell population that has been contacted for a period of time insufficient for cell division to occur with a CD26 peptidase inhibitor in an amount effective to inhibit CD26 peptidase activity and effective to increase the migratory response to CXCL 12, resulting in a population of modified cells; and providing or administering the population of modified cells to a patient in need thereof.

[0008] Still some embodiments relate to methods of enhancing homing and engraftment of a cell, comprising a) providing cell population that has been contacted for a period of time insufficient for cell division to occur with a CD26 peptidase inhibitor in an amount effective to inhibit CD26 peptidase activity and effective to increase the migratory response to CXCL 12, resulting in a population of modified cells; and b) providing or administering said population of modified cells to a "patient" in need thereof. In some aspects the inhibitor can be contacted for a period of time to permit the inhibitor to bind to CD26 on a cell of the cell population.

[0009] In certain aspects, the CD26 inhibitor is selected from the group consisting of Diprotin A (Ile-Pro-Ile) and Valine-Pyrrolidide. In certain aspects, the CD26 inhibitor is Valine-Pyrrolidide. In certain aspects, the CD26 inhibitor is Diprotin A (Ile-Pro-Ile). In certain aspects, the CD26 inhibitor is selected from the group consisting of sitagliptin, vildagliptin, saxagliptin, alogliptin or any other class of compounds shown to exhibit potent inhibitor of either purified, soluble or cell surface (CD26) dipeptidylpeptidase. In some aspects the inhibitor can be, for example, any potent CD26 inhibitor exhibiting either reversible or irreversible inhibition. In some aspects the inhibitor can be, for example, an antibody directed the cell surface CD26. In some aspects, the CD26 expression can be down regulated by the introduction of an siRNA , or an antisense oligonucleotide, or by any other suitable means, including those within the skill of those in the art.

[0010] In certain aspects, the cell population can be contacted with said CD26 inhibitor for about 5 minutes to about 12 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for about 15 minutes to about 6 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for less than 6 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for less than 2 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for less than 1 hour.

[0011] In certain aspects, the inhibitor is provided or administered in a concentration of less than about 1 nM, about 1 μM, about 5 μM, about 10 μM, about 50 μM, about 100 μM, about 1 nM or about 5 nM. In certain aspects, the inhibitor is administered in a concentration of no less than about 5 mM.

[0012] In certain aspects, at least 1 x 10⁵ donor cells per mL are treated.

[0013] In certain aspects, the cells are provided or administered to a patient for a bone marrow transplant, optionally comprising providing or administering a CD26 inhibitor to said patient in an amount effective to inhibit the peptidase activity thereof prior to, or during said transplant.

[0014] In certain aspects, the inhibitor is provided or administered to said patient in a concentration of about 1 to about 50 μMol/kg, about 30 μMol/kg, or about 10 μMol/kg total body weight.

[0015] In certain aspects, prior to said providing or administering, said population of modified cells can have been further modified by treatment with an enzyme and appropriate substrate(s) under conditions sufficient for causing an alteration of cell surface charge. In certain aspects, the enzyme is selected from a group comprising of alpha 1,3- fucosyltransferase I, alpha 1,3-fucosyltransferase III, alpha 1,3-fucosyltransferase IV, alpha 1, 3 -fucosy transferase V, alpha 1,3-fucosyltransferase VI, alpha 1,3-fucosyltransferase VII, and alpha 1,3-fucosyltransferase IX. PCT Application No. PCT/US2009/ filed on

June 9, 2009, entitled AUGMENTATION OF CELL THERAPY EFFICACY BY SURFACE MODIFICATION OF MOLECULES INVOLVED IN ENDOTHELIAL INTERACTION INCLUDING TREATMENT WITH ALPHA 1-3 FUCOS YLTRANSFERASE, which is incorporated herein by reference in its entirety, discloses and describes methods and compositions, any of which can be used with the technology of this application in any combination.

[0016] In certain aspects, the cell population can include or consists essentially of a population of stem cells. In certain aspects, the stem cells are selected from a group consisting of: embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and mobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells or somatic nuclear transfer and side population stem cells.

[0017] In certain aspects of the above embodiments, the cell population can include or consists essentially of a population of committed progenitor cells or differentiated cells. In certain aspects, the cell population comprises or consists essentially of a population of mature blood cells. In certain aspects, the mature blood cell is selected from the group consisting of: neutrophils, macrophages and T-cells. In certain aspects, the T-cells are from a heterogeneous population of T-cells.

[0018] In certain aspects, the population of modified cells can be, for example, provided via or administered from a route selected from a group consisting of: intravenously, intraarterially, intramuscularly, subcutaneously, transdermally, intratracheal Iy, intraperitoneally, intravitreally, via direct injection, directly into the microvascular compartment of bone or into spinal fluid. The cells, compositions or other materials can be used or administered with a scaffolding support. In certain aspects, the population of modified cells is administered in or proximal to a site of injury. In certain aspects, the homing and engraftment takes place within the bone marrow of said patient in need thereof.

[0019] Also provided herein are methods of modification of the SDF-CXCR4 axis so as to increase the ability of said cells to home into an area of biological need. Said cells may be differentiated cells whose engraftment is desired, such as islets, hepatocytes, neutrophils or cells may be progenitor cells capable of differentiating into functional cells such as hepatic, renal, cardiac, or islet progenitors, or alternatively, said cells may be stem cells with multilineage differentiation ability such as embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, neural stem cells, circulating and im mobilized peripheral blood stem cells, mesenchymal stem cells, endothelial stem cells, cardiomyocyte stem cells, germinal stem cells, committed endothelial progenitor cells, committed progenitor cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, chemically, biologically, or electronically reprogrammed stem cells such as induced pluripotent stem cells or somatic nuclear transfer and side population stem cells, cancer cells or tumor cancer cells.

[0020] Another aspect relates to enhancing the ability of cells to modulate the immune system through enabling said cells to function with augmented efficiency at trafficking and homing. In another aspect, cells useful for immune therapy are "reprogrammed" ex vivo for endowment of distinct immunological properties. Surface modification may be performed before reprogramming, during reprogramming or after reprogramming. Reprogramming may be performed so as to increase immune stimulatory properties of the immune cells, or may be performed to allow said immune cells to suppress other immune cells. Reprogramming may be performed during expansion of cells, or to cells that have already been expanded.

[0021] In another aspect the interaction between a spermatozoa and an egg cell is enhanced through prior treated of spermatozoa, or egg cell, or both, with an enzyme capable of altering cell surface properties of said spermatozoa, or egg cell, or both. Said enzyme increases the interaction between the spermatozoa and egg cell through augmenting time, and/or affinity of interaction.

[0022] In another aspect a fertilized egg prior to implantation is treated with a compound or plurality of compounds in order to alter cell surface membrane properties and thereby increase potential for said fertilized egg to implant in the deciduas and achieve pregnancy.

[0023] Some embodiments relate to methods of enhancing homing and engraftment of a cell, comprising providing an isolated population of cells and a CD26 peptidase inhibitor in an amount effective to inhibit CD26 peptidase activity and effective to increase the migratory response to CXCL 12; and providing said population of cells and inhibitor to a patient in need thereof. The isolated population of cells can be one or more of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and mobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as pluirpotent stem cells and somatic nuclear transfer and side population stem cells, and the like. The inhibitor can be, for example, Diprotin A (Ile-Pro-Ile), Valine-Pyrrolidide, sitagliptin, vildagliptin, saxagliptin, alogliptin, or any other class of compounds shown to exhibit potent inhibition of either purified, soluble or cell surface (CD26) dipeptidylpeptiase.

[0024] Some embodiments relate to isolated populations of cells having increased migratory response to CXCL 12 compared to cells of the same type that have not been contacted with a CD26 inhibitor, wherein the isolated population comprises one or more of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and immobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells and somatic nuclear transfer and side population stem cells.

[0025] Also, some embodiments relate to isolated populations of cells comprising a CD26 inhibitor bound to CD26 or a population of cells that comprises CD26 inhibitor bound to CD26 wherein the amount of bound inhibitor is greater than the amount of inhibitor for the wild type cell, wherein the isolated population comprises one or more of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and immobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells and somatic nuclear transfer and side population stem cells. DETAILED DESCRIPTION

[0026] Embodiments disclosed herein relate generally to compositions and methods for enhancing cell therapy efficacy including treatment with CD26 peptidase inhibitors. Provided herein are compositions for and methods of enhancing homing and engraftment of a therapeutically-provided or administered cell in a patient. Also provided are cells that have been modified to enhance homing and engraftment. The embodiments provided herein are based in part on the surprising finding that by modification of molecules involved in the cell-endothelium interaction, it is possible to enhance the homing and subsequent efficacy of cellular therapy.

[0027] SDF-1-CXCR4 interaction constitutes a major axis in the regulation of trafficking of both normal and cancer stem cells (Kucia M et al Stem Cells 2005). The α- chemokine stromal-derived factor (SDF-I) is a multifunctional cytokine and unique amongst the more than 50 known and different chemokines in that it binds exclusively to CXCR4 and has CXCR4 as its only receptor (Nagasawa T et al Nature 1996: Ma Q, et al Immunity 1999: Bagri A et al Development 2002: Lazarini F et al Glia 2003: Zou Y et al Nature 1998: Tachibana K Nature 1998). This cytokine is constitutively expressed on bone marrow stromal cells. CXCR4, the cognate receptor for SDF-I, is a 7 -transmembrane G-protein- coupled receptor and one of 20 different chemokine receptors that have been cloned to date (Zlotnik A and Yoshie O Immunity 2000: Horuk R Cytokine Growth Factor Rev 2001). This receptor plays a critical role in cell survival, proliferation, directed migration, and engraftment (Lee Y et al 2002: Aiuti A et al Eur J Immunol 1999: Aiuti A et al J Exp Med 1997: Mohle R et al Blood 1998: Kim CH and Broxmeyer HE Blood 1998: Viardot A et al Ann Hematol 1998: Peled A et al Science 1999: Rosu-Myles M et al PNAS 2000: Lataillade JJ et al Blood 2000: Ma Q et al Immunity 1999). The references listed in this paragraph are each incorporated herein by reference in their entirety.

[0028] Present evidence suggests that SDF-I plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells and their homing and retention in the bone marrow (BM) (Kucia M et al Stem Cells 2005). Also, while the CXCR4 receptor is present on hematopoietic cells supporting interaction with SDF-I, it is also present on non hematopoietic tissue-committed stem/progenitor cells and cancer cells implying a broader role as pivotal regulator of trafficking of various types of cells in the body. The role of SDF-I in trafficking is highlighted in a study with murine knockout mouse showing that the secretion of this chemokine by bone marrow stromal cells is critical for the colonization of BM by fetal liver-derived hematopoietic stem cells during embryogenesis (Nagasawa T et al Nature 1996: Ma Q, et al Immunity 1999: Bagri A et al Development 2002: Lazarini F et al Glia 2003: Zou Y et al Nature 1998: Tachibana K Nature 1998). Even in adult life this chemokine plays a critical role in trafficking since perturbation of the SDF-1-CXCR4 axis by mobilizing agents is essential in order to cause egress of hematopoietic stem/progenitor cells from BM into peripheral blood (Petit I et al Immunol 2002: Liles WC et al Blood 2003: Devine SM et al J Clin Oncol 2004). While the role of SDF-I is well established in bone marrow migration/colonization/engraftment this chemokine also plays a role in the proper development of other organs/tissues in the body, such as heart, brain and larger vessels (Ma Q, et al Immunity 1999: Bagri A et al Development 2002: Lazarini F et al Glia 2003: Zou Y et al Nature 1998: Tachibana K Nature 1998). Thus, SDF-I appears to be one of the most important motomorphogenic factors and chemoattractants not only for hematopoietic stem cells but also for non hematopoietic CXCR4+ tissue/organ committed progenitor/stem cells. The references listed in this paragraph are each incorporated herein by reference in their entirety.

[0029] While the role of SDF-1-CXCR4 interaction was investigated initially with hemato- and lymphopoietic cells (Aiuti A et al J Exp Med 1997: Ma Q et al PNAS 1998), it has also been shown to regulate trafficking/homing of CXCR4+ hematopoietic stem and progenitor cells, pre B lymphocytes and T lymphocytes (Nagasawa T et al Nature 1996: Aiuti A et al J Exp Med 1997: Ma Q et al PNAS 19985: Kim CH and Broxmeyer HE J Leuko Biol 1999: Rosu-Myles M et al PNAS 2000), various tissue committed stem cells (TCSCs) such as neural (Bagri A et al Development 2002: Lazarini F et al Glia 2003: Zou Y et al Nature 1998), skeletal muscle satellite (Pituch-Noworolska A et al Folia Histochem Cytobiol 2003: Ratajczak MZ et al Stem Cells 2003), myocardial (Damas JK et al Cardiolvasc Res 2000: Kucia M et al Circ Res 2004), endothelium (Peichev M et al Blood 2000: Yamaguchi J et al Circulation 2003: Wojakowski W et al Circulation 2004), liver oval (Hatch HM et al Cloning Stem Cells 2002), retinal pigment epithelium TCSCs (Crane IJ et al J Immunol 2000), primordial germ cells (Ara T et al PNAS 2003) and embryonic stem cells. Other CXCR4+ tissue/organ-specific cells include: bronchial endocrine, mammary gland epithelium, neuroectodermal, renal tubular epithelium, ovarian epithelium and cervical epithelium stem cells (Kucia M et al Stem Cells 2005). The references listed in this paragraph are each incorporated herein by reference in their entirety.

[0030] Additional evidence for a pivotal role of CXCR4 in stem cell trafficking during development, tissue injury and regeneration is its diversity of regulation by several transcription factors related to organ development and factors related to stress and tissue damage. Over nine different paired-box transcription factors (PAX genes) that bind to the CXCR4 promoter (Reca R et al Blood 2004: Tomescu O et al Lab Invest 2004) have been located in different TCSCs suggesting that the various PAX proteins may regulate CXCR4 expression in a tissue specific manner. For example, PAXl is present in osteoblastic TCSCx, PAX3 in neural-crest and skeletal muscle TCSCs, PAX5 in B lymphocytic progenitors, PAX6 in endocrine pancreatic TCSCs and PAX7 in skeletal muscle satellite TCSCx. CXCR4 expression may also be positively regulated by transcription factors related to stress/hypoxia and tissue damage such as NF-κB (Helbig G et al J Biol Chem 2003), lysophosphatidylcholine (Han KH et al J Leukoc Biol 2004), TGF-βl (Franitza S et al Eur J Immunol 2002), VEGF (Bachelder RE et al Cancer 2002), IFN-α (Yonezawa A et al J Virol 2003), and several interleukins (IL-2, IL-4 and IL-7) (Iikura M J Leukoc Biol 2001 : Jourdan P et al J Immunol 2000). The references listed in this paragraph are each incorporated herein by reference in their entirety.

[0031] Several molecules/factors arise during tissue damage and chronic inflammation that chemoattract normal stem cells to initiate and support regeneration. Factors that positively affect the sensitivity/responsiveness of CXCR4+ cells to a SDV-I gradient in inflammation/tissue damage include: anaphylatoxin C3a (C3 complement protein cleavage fragment, _des-_ArgC3a (product of C3a degradation by carboxypeptidase), fibronectin, hyaluronic acid; in coagulation these factors include: fibrinogen, soluble μPAR, thrombin: in cell activation these factors include: sVCAM-1, sICAM-1, platelet-derived membrane microvesicles (Wysoczynski M et al Blood 2005: Janowska-Wieczorek A et al Blood 2001) and sphingosine-1 phosphate. Finally, priming at the molecular level can occur as a result of cholesterol content of the cell membrane leading to incorporation of CXCR4 and small GTP- as RAC-I into membrane lipid rafts (Wysoczynski M et al Blood 2005). Priming can also be affected by factors related to leukopheresis/mobilization (Wysoczynski M et al Blood 2005). Tissue damage and inflammation, with the release of multiple agents/chemicals/factors that act as chemoattractants or enhance chemoattraction along with the release of proteolytic factors, may exert pleiotropic effects on not only the trafficking of stem cells but also regenerative potential. The references listed in this paragraph are each incorporated herein by reference in their entirety.

[0032] Further support for a pivotal role of the SDF-1-CXCR4 axis in cell homing is observed with the involvement of CD26 in homing. CD26 is a cell surface peptidase (DPPIV/dipeptidylpeptidase IV) present on stem cells that removes dipeptides from the amino terminus of proteins such as SDF-I. By peptide cleavage DPPIV inactivates/degrades certain growth factors, chemokines and neuropeptides that promote cellular growth and survival. DPPIV is expressed on many hematopoietic cell populations including B and natural killer cells, activated T lymphocytes, endothelial cells, fibroblasts and epithelial cells (Vanham G et al J Acquire Immune Defic Syndr 1993, Kahne T et al Int J MoI Med 1999: Huhn J et al Immuno Lett 2000) and in various normal tissues including neural crest stem cell derived melanocytes, neurons, lung, and prostate epithelial, and cancer cells. Evidence has been presented showing that endogenous CD26 expression negatively regulates homing and engraftment. From a mechanistic perspective, CD26-mediated removal of the N-terminal amino acids results in significant changes in receptor binding and/or functional activity. To confirm an involvement in homing inhibition or deletion of CD26 resulted in an enhanced efficiency of transplantation (Christopherson KW et al Science 2004, which is incorporated by referenced herein in its entirety.) See also U.S. Patent Pubs. 2004/0247574 and 2008/0085266 to Christopherson et al., each of which is hereby incorporated by reference in its entirety. The references listed in this paragraph are each incorporated herein by reference in their entirety.

[0033] What is desirable is the generation of novel methods of enhancing cellular engraftment to areas of need in a simple and clinically applicable manner. Accordingly, the methods provided herein are based at least in part on the unexpected finding that by modification of the SDF-CXCR4 axis, it is possible to enhance the efficacy of cellular therapy with stem cells, differentiated cells, committed progenitor cells and differentiated cells.

Definitions

[0034] The following definitions are provided to facilitate an understanding of the methods provided herein:

[0035] The term "SDF", the chemokine CXCL 12 (stromal cell derived factor- 1, or "SDF-I") stimulates hematopoietic stem and progenitor cells (HSCs/HPCs) through the corresponding chemokine receptor CXCR4. SDF is thought to be important for both proper HSC homing, retention, and engraftment into the bone marrow (BM) and mobilization out of the BM. The terms "SDF-I" and CXCL 12 are used interchangeably herein.

[0036] The term "CD26" (DPPIV/dipeptidylpeptidase IV) is a membrane bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains after a proline or an alanine. "CD26 activity" or "DPPIV activity" encompasses any activity of CD26, including peptidase activity. "CD26" or "DPPIV" inhibitor or antagonist refers to any substance, chemical, biological, and so forth, which is capable of inhibiting CD26 activity. Preferably, CD26 or DPPIV inhibitors and antagonists inhibit CD26 peptidase activity, at levels sufficient to improve stem cell homing.

[0037] The term "short term exposure" refers to exposure of stem cells to a CD26 inhibitor for a short period of time, for example, for less time than is required for cell expansion to occur. Alternatively, short term exposure means for a time period of about 5 minutes to about 12 hours. Preferably, "short term exposure" is from 15 minutes to 6 hours.

[0038] The term "homing" refers to localization to a particular area, for example localization of transplanted stem cells to the bone marrow.

[0039] The term "donor" refers to the organism donating the therapeutic stem cells.

[0040] The term "recipient" is the patient receiving the therapeutic stem cells. [0041] The term "stem cell" or "hematopoietic stem cell" means a pluripotent cell of the hematopoietic system capable of differentiating into a cell of a specific lineage, such as lymphoid, or myeloid.

[0042] The term "transfected stem cell" or "transduced stem cell" describes a stem cell into which exogenous DNA or an exogenous DNA gene has been introduced, for example by retroviral infection.

[0043] The term "engrafting" or "engraftment" means the persistence of proliferating stem cells in a particular location over time.

[0044] The term "myeloablated" refers to a patient who has undergone irradiation, or other treatment, such as chemotherapeutic treatment, to cause the death of at least 50% of the bone marrow cells of the patient.

[0045] The term "non-myeloablated" refers to a patient who has not undergone irradiation, or other treatment (such as chemotherapy) to cause the death of the bone marrow cells of the mammal.

[0046] The term "autologous" describes nuclear genetic identity between donor cells or tissue and those of the recipient.

[0047] The term "multipotent" means that a cell is capable, through its progeny, of giving rise to several different cell types found in the adult animal.

[0048] The term "pluripotent" means that a cell is capable, through its progeny, of giving rise to all of the cell types which comprise the adult animal including the germ cells. Both embryonic stem and embryonic germ cells are pluripotent cells under this definition.

[0049] The term "transgenic" animal or cell refers to animals or cells whose genome has been subject to technical intervention including the addition, removal, or modification of genetic information. The term "chimeric" also refers to an animal or cell whose genome has modified.

[0050] The term "knockout mouse" refers to a mouse with a DNA sequence introduced into its germline by way of human intervention, preferably a sequence which is designed to specifically alter cognate endogenous alleles. Preferably a targeted gene has been "knocked out" to assess the biological and functional consequences of elimination of such target genes. Such mice also provide an ideal in vivo model for assessing restoration of the lost phenotype via complementation with cognate alleles from the same or different species using recombinant DNA techniques.

[0051] The term "totipotent" as used herein may refer to a cell that gives rise to a live born animal. The term "totipotent" may also refer to a cell that gives rise to all of the cells in a particular animal. A totipotent cell may give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps. Totipotent cells may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of an organ or appendage by manipulation of a homeotic gene.

[0052] The term "cultured" as used herein in reference to cells may refer to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment. An in vitro environment may be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example. Specific examples of suitable in vitro environments for cell cultures are described in the art (Culture of Animal Cells: a manual of basic techniques (3.sup.rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan John Wiley and Sons, Ltd).

[0053] The term "cell line" as used herein may refer to cultured cells that can be passaged at least one time without terminating. Some embodiments relate to cell lines that can be passaged at least 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 200 times. Cell passaging is defined hereafter.

[0054] The term "suspension" as used herein may refer to cell culture conditions in which cells are not attached to a solid support. Cells proliferating in suspension may be stirred while proliferating.

[0055] The term "monolayer" as used herein may refer to cells that are attached to a solid support while proliferating in suitable culture conditions. A small portion of cells proliferating in a monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support. Preferably less than 15% of these cells are not attached to the solid support, more preferably less than 10% of these cells are not attached to the solid support, and most preferably less than 5% of these cells are not attached to the solid support.

[0056] The term "plated" or "plating" as used herein in reference to cells may refer to establishing cell cultures in vitro. For example, cells may be diluted in cell culture media and then added to a cell culture plate, dish, or flask. Cell culture plates are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and/or cell densities.

[0057] The term "cell plating" may also extend to the term "cell passaging." Cells may be passaged using cell culture techniques well known to those skilled in the art. The term "cell passaging" may refer to a technique that involves the steps of (1) releasing cells from a solid support or substrate and disassociation of these cells, and (2) diluting the cells in media suitable for further cell proliferation. Cell passaging may also refer to removing a portion of liquid medium containing cultured cells and adding liquid medium to the original culture vessel to dilute the cells and allow further cell proliferation. In addition, cells may also be added to a new culture vessel which has been supplemented with medium suitable for further cell proliferation.

[0058] The term "proliferation" as used herein in reference to cells may refer to a group of cells that can increase in number over a period of time. The term "isolated" as used herein may refer to a cell that is mechanically separated from another group of cells. Examples of a group of cells are a developing cell mass, a cell culture, a cell line, and an animal.

[0059] The term "differentiated cell" as used herein may refer to a precursor cell that has developed from an unspecialized phenotype to a specialized phenotype.

[0060] The term "undifferentiated cell" as used herein may refer to a precursor cell that has an unspecialized phenotype and is capable of differentiating. An example of an undifferentiated cell is a stem cell.

[0061] The term "asynchronous population" as used herein may refer to cells that are not arrested at any one stage of the cell cycle. Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time. Some known stages of the cell cycle are Gl, S, G2, and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases. Cells can be arrested in the M stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by colcemid exposure. Examples of methods for arresting cells in one stage of a cell cycle are discussed in WO 97/07669, entitled "Quiescent Cell Populations for Nuclear Transfer".

[0062] The term "modified nuclear DNA" as used herein may refer to a nuclear deoxyribonucleic acid sequence of a cell, embryo, fetus, or animal that has been manipulated by one or more recombinant DNA techniques. Examples of recombinant DNA techniques are well known to a person of ordinary skill in the art, which may include (1) inserting a DNA sequence from another organism (e.g., a human organism) into target nuclear DNA, (2) deleting one or more DNA sequences from target nuclear DNA, and (3) introducing one or more base mutations (e.g., site-directed mutations) into target nuclear DNA. Cells with modified nuclear DNA may be referred to as "transgenic cells" or "chimeric cells." Transgenic cells can be useful as materials for nuclear transfer cloning techniques provided herein. The phrase "modified nuclear DNA" may also encompass "corrective nucleic acid sequence(s)" which replace a mutated nucleic acid molecule with a nucleic acid encoding a biologically active, phenotypically normal polypeptide. The constructs utilized to generate modified nuclear DNA may optionally comprise a reporter gene encoding a detectable product.

[0063] As used herein, the terms "reporter," "reporter system", "reporter gene," or "reporter gene product" shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radioimmunoassay, or by calorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like. [0064] The term "selectable marker" as used herein refers to a molecule that when expressed in cells renders those cells resistant to a selection agent. Nucleic acids encoding selectable marker may also comprise such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like. Suitable selection agents include antibiotics such as kanamycin, neomycin, and hygromycin.

[0065] Methods and tools for insertion, deletion, and mutation of nuclear DNA of mammalian cells are well-known to a person of ordinary skill in the art. See, Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press; U.S. Pat. No. 5,633,067, "Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo," DeBoer et al., issued May 27, 1997; U.S. Pat. No. 5,612,205, "Homologous Recombination in Mammalian Cells," Kay et al., issued Mar. 18, 1997; and PCT publication WO 93/22432, "Method for Identifying Transgenic Pre- Implantation Embryos"; WO 98/16630, Piedrahita & Bazer, published Apr. 23, 1998, "Methods for the Generation of Primordial Germ Cells and Transgenic Animal Species." These methods include techniques for transfecting cells with foreign DNA fragments and the proper design of the foreign DNA fragments such that they effect insertion, deletion, and/or mutation of the target DNA genome. The references listed in this paragraph are each incorporated herein by reference in their entirety.

[0066] Any of the cell types defined herein may be altered to harbor modified nuclear DNA. Examples of methods for modifying a target DNA genome by insertion, deletion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, homologous recombination, gene targeting, transposable elements, and/or any other method for introducing foreign DNA. Other modification techniques well known to a person of ordinary skill in the art include deleting DNA sequences from a genome, and/or altering nuclear DNA sequences. Examples of techniques for altering nuclear DNA sequences are site-directed mutagenesis and polymerase chain reaction procedures. Therefore, some embodiments relate in part to mammalian cells that are simultaneously totipotent and transgenic.

[0067] The term "recombinant product" as used herein may refer to the product produced from a DNA sequence that comprises at least a portion of the modified nuclear DNA. This product may be a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a polypeptide that binds to a regulatory element (a term described hereafter), a structural protein, an RNA molecule, and/or a ribozyme, for example. These products are well defined in the art.

[0068] The term "promoters" or "promoter" as used herein may refer to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably linked operatively to an adjacent DNA sequence. A promoter typically increases an amount of recombinant product expressed from a DNA sequence as compared to an amount of the expressed recombinant product when no promoter exists. A promoter from one organism may be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism. In addition, one promoter element may increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem. Hence, one promoter element may enhance the expression of one or more recombinant products. Multiple promoter elements are well-known to persons of ordinary skill in the art.

[0069] The term "enhancers" or "enhancer" as used herein may refer to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer elements are typically located upstream of a promoter element or can be located downstream of a coding DNA sequence (e.g., a DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a DNA sequence that encodes recombinant product. Enhancer elements may increase an amount of recombinant product expressed from a DNA sequence above increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art.

[0070] The terms "transfected" and "transfection" as used herein refer to methods of delivering exogenous DNA into a cell. These methods involve a variety of techniques, such as treating cells with high concentrations of salt, an electric field, liposomes, polycationic micelles, or detergent, to render a host cell outer membrane or wall permeable to nucleic acid molecules of interest. These specified methods are not limiting and various embodiments relate to any transformation technique well known to a person of ordinary skill in the art.

[0071] The term "antibiotic" as used herein may refer to any molecule that decreases growth rates of a bacterium, yeast, fungi, mold, or other contaminants in a cell culture. Antibiotics are optional components of cell culture media. Examples of antibiotics are well known in the art. (See, e.g. Sigma and DIFCO catalogs.)

Therapeutic Methods

[0072] Provided herein are methods and means of endowing enhanced trafficking/homing capabilities onto cells for use in cell therapy, said means consisting of inhibition or reduction of cell surface peptidase activity or augmentation of the availability of cell-surface binding partners on cells in order to augment ability to "roll", 'tether', 'adhere' on the endothelium. The concept of cells rolling, tethering, adhering on the endothelium is commonly known in the art and means of augmentation of this rolling process have been described strictly in the areas of hematopoietic cells, as well as tumor cells.

[0073] Thus, the methods described herein are based in part on the novel observation that inhibition of CD26 and/or increase in the levels or CXCR4 increases homing to tissue after injury and can be used to enhance cell migration to an area of need. The novel finding is that modulation of the CD26 and/or CXCR4 cell components through various means is useful for enhancing functional capabilities of a wide variety of stem, progenitor and mature cells. Accordingly, the technology described herein may be useful for treatment of a wide variety of medical conditions which are amenable to cell therapy.

[0074] In accordance with the above, some embodiments relate to methods of enhancing homing and engraftment of a therapeutically-provided or administered cell in a patient. Also provided are cells that have been modified to enhance homing and engraftment. The embodiments provided herein are based in part on the surprising finding that by modification of molecules involved in the cell-endothelium interaction, it is possible to enhance the homing and subsequent efficacy of cellular therapy.

[0075] Thus, in one method is provided method of enhancing homing and engraftment of a therapeutically-provided or administered cell in a patient comprising: selecting a patient in need of treatment with a cell population; providing a cell population that has been contacted for a period of time insufficient for cell division to occur with a CD26 peptidase inhibitor in an amount effective to inhibit CD26 peptidase activity and effective to increase the migratory response to CXCL 12, resulting in a population of modified cells; and providing or administering the population of modified cells to a patient in need thereof.

[0076] In certain aspects, the CD26 inhibitor is selected from the group consisting of Diprotin A (Ile-Pro-Ile) and Valine-Pyrrolidide. In certain aspects, the CD26 inhibitor is Valine-Pyrrolidide. In certain aspects, the CD26 inhibitor is Diprotin A (Ile-Pro-Ile). In certain aspects, the CD26 inhibitor is selected from the group consisting of sitagliptin, vildagliptin, saxagliptin, alogliptin or any other class of compounds shown to exhibit potent inhibitor of either purified, soluble or cell surface (CD26) dipeptidylpeptidase. Exemplary methods of treating stem cells with CD26 inhibitors are described in the incorporated materials of Christopherson et al. (U.S. Pub No. 2004/0247574).

[0077] In certain aspects, the cell population is contacted with said CD26 inhibitor for about 5 minutes to about 12 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for about 15 minutes to about 6 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for less than 6 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for less than 2 hours. In certain aspects, the cell population is contacted with said CD26 inhibitor for less than 1 hour.

[0078] In certain aspects, the inhibitor can be, for example, provided or administered in a concentration of less than about 1 nM, about 1 μM, about 5 μM, about 10 μM, about 50 μM, about 100 μM, about 1 nM or about 5 nM. In certain aspects, the inhibitor can be, for example, provided or administered in a concentration of no less than about 5 mM.

[0079] In certain aspects, at least 1 donor cell is treated. In selected embodiments, at least 1 x 10² , 1 x 10³ , 1 x 10⁴ , 1 x 10⁵ , or at least 1 x 10⁶ donor cells per mL are treated.

[0080] In certain aspects, the inhibitor can be, for example, provided or administered to said patient in a concentration of about 1 to about 50 μMol/kg, about 30 μMol/kg, or about 10 μMol/kg total body weight. Inhibition and Downregulation of CD26

[0081] There are various approaches enhanced homing that can be applied to cells. Two separate approaches are known to those skilled in the art. These include (1) inhibition/downregulation of CD26 (2) enhancement/upregulation of CXCR4 receptor.

[0082] In one embodiment, cells are treated with agents that are inhibitors of cell surface dipeptidylpeptidase (CD26). These agents can be reversible, long term, or irreversible inhibitors of CD26. Exemplary CD26 inhibitors may include, but are not limited to Diprotin A (Ile-Pro-Ile), Valine-Pyrrolidide, or any other molecule which inhibits or antagonizes CD26 activity. Alternatively, cells can be treated with agents or conditions that lead to downregulation of cell surface CD26. This includes siRNA or siRNA-like technology or agents that affect transcription factors located at the promoter site or direct inhibition of promoter sites regulating the production of CD26, or genetically engineered to decrease CD26 production or response to enhancers or endogenous agents.

[0083] In another embodiment, enhancers of homing can be utilized to increase the rate and extent of engraftment and can enhance homing to a broad range of organs, such as the spleen, lymph nodes, and lung amongst others. In some situations homing to another site/tissue/organ is achieved first followed then by homing to the bone marrow or another targeted tissue/organ in the body. In addition, stem/progenitor/expanded/mature/cancer cells may be treated with enhancer for varying periods (hrs to day) of time depending on features of the enhancer. Enhancers include treatment of cells under hypoxia conditions or other conditions to raise hypoxia inducible factor, incubation with stem cell factor (SCF), SCF plus IL6, SDF, SDF + IL6, hepatocyte growth factor, C3a, cAMP or analogue thereof, hylauronic acid, glucocorticoids, lysophophatidylcholine, TGF-βl, vascular endothelial growth factor, IFN-α, interleukins such as IL-2, IL-4 or IL-7, sphingosine-1 -phosphate, fibronectin, fibrinogen, thrombin, soluble PAR, sVCAM-1 or ICAM, agents that regulate Paired box transcription factor genes, positively regulate transcription factors related to stress, hypoxia and tissue damage such as NF-κB, genetically engineered to increase CXCR4 levels or response to enhances and endogenous agents, agents that activate or enhance secondary pathways associated with CXCR4 receptor activation such as calcium influx, focal adhesion components such as praline-rich kinase-2, CrK-associated substrate focal adhesion kinase (FAK), paxillin or NcK.

[0084] Thus, in certain embodiments provided herein, cells can be treated ex vivo with a kit that contains all the agents, such as buffer and inhibitor necessary to achieve inhibition or downregulation of cell surface CD26 or enhancement of the CXCR4 receptor. In another embodiment, cells such as cord blood can be pretreated with the DPPIV inhibition kit or CXCR4 receptor enhancement kit prior to freezing or storage.

Combination Treatment with Glycosylase Enzymes

[0085] In certain aspects, prior to said providing or administering, said population of modified cells can have been further modified by treatment with an enzyme and appropriate substrate(s) under conditions sufficient for causing an alteration of cell surface charge. Enyzmes which modify cell surface molecules are known in the art. Such enzymes include a purified glycosyltransferase. Glycosyltransferase include for example, fucosyltransferase, galactosyltransferase, sialytransferase and N- acetylglucosaminotransferase. The fucosyltransferase can be, for example, an alpha 1,3 fucosyltransferase such as an alpha 1,3-fucosyltransferase I, alpha 1 ,3-fucosyltransferase III, alpha 1,3-fucosyltransferase IV, alpha 1,3-fucosyltransferase V, alpha 1,3-fucosyltransferase VI, alpha 1,3-fucosyltransferase VII, and alpha 1,3-fucosyltransferase IX. PCT Application

No. PCT/US2009/ , filed on June 9, 2009, entitled AUGMENTATION OF CELL

THERAPY EFFICACY BY SURFACE MODIFICATION OF MOLECULES INVOLVED IN ENDOTHELIAL INTERACTION INCLUDING TREATMENT WITH ALPHA 1-3 FUCOSYLTRANSFERASE, which is incorporated herein by reference in its entirety, discloses and describes methods and compositions, any of which can be used with the technology of this application in any combination.

[0086] In certain aspects, the cell surface molecule is modified in the presence of a sugar donor suitable for the specific glycosyltransferase. Sugar donors for glycosyltransferases are known in the art. For example, when the glycoslytransferase is a fucosyltransferase, the donor is GDP-fucose. Whereas, when the glycosyltransferase is a siayltransferase, the donor is CMP-sialic acid. Therapeutic Cell Populations

[0087] Stem Cells. In certain aspects, the cell population comprises or consists essentially of a population of stem cells. In certain aspects, the stem cells are selected from a group consisting of: embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and mobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells or somatic nuclear transfer and side population stem cells. In certain aspects, the embryonic stem cells are totipotent.

[0088] As used herein, a "mesenchymal cell" means a cell forming a mesenchymal tissue, such as osteoblat, chondrocyte, myoblast, adipocyte, stroma cell, tendon cell, and the like, a mesenchymal stem cell capable of differentiating into these cells, and its premesenchymal stem cell. Mesenchymal cells generated during the embryo development, mesenchymal cells within an animal body, and mesenchymal cells differentiated and generated from pluripotent stem cells in vitro or in vivo are all encompassed in the term "mesenchymal cell."

[0089] As used herein, a "mesenchymal stem cell" means a mesenchymal cell possessing the ability of differentiating into mesenchymal cells of one or more types and the ability of self-replication. The mesenchymal stem cell differentiated from a pluripotent stem cell in vitro is positive for PDGFRα and negative for FLKl . Mesenchymal stem cells are able to differentiate into osteoblasts, chondrocytes, myoblasts, adipocytes, stroma cells, tendon cells, and the like, as with mesodermal cells.

[0090] As used herein, a "premesenchymal stem cell" means a mesenchymal cell possessing the ability of differentiating into mesenchymal stem cells of one or more types and the ability of self-replication. The premesenchymal stem cell differentiated from a pluripotent stem cell in vitro expresses Soxl, a neuroectodermal marker. The premesenchymal stem cell is able to differentiate into a mesenchymal stem cell which is PDGFRα-positive and FLKl- negative.

[0091] As used herein, a "neural stem cell" refers to a multipotent cell obtained from the central nervous system that can be caused to differentiate into cells that posses one or more biological activities of a neural cell type. Neural stem cells differentiate spontaneously into neurons, astrocytes, and oligodendrocytes after plating onto substrates which stimulate adhesion and differentiation, for example poly-L-ornithine orlaminin. In addition, these multipotent CNS stem cells proliferate and expand in response to epidermal growth factor ("EGF") and basic fibroblast growth factor ("bFGF") and differentiate into neurons, astrocytes and oligodendrocytes.

[0092) Committed Progenitor Cells and Differentiated Cells. In certain aspects, the cell population comprises or consists essentially of a population of committed progenitor cells or differentiated cells. In certain aspects, the the cell population is a mature blood cell population. In certain aspects, the mature blood cell is selected from the group consisting of: neutrophils, macrophages and T-cells. In certain aspects, the T-cells are from a heterogeneous population of T-cells.

Patients in Need of Treatment with Modified Cell Populations

[0093] Our findings are centered on the novel finding that inhibition of CD26 and/or increase in the levels or CXCR4 increases homing to tissue after injury and can be used to enhance cell migration to an area of need. The novel finding is that modulation of the CD26 and/or CXCR4 cell components through various means is useful for enhancing functional capabilities of a wide variety of stem, progenitor and mature cells. Accordingly, the technology described may be useful for treatment of a wide variety of medical conditions which are amenable to cell therapy.

[0094] Thus, the methods provided herein are useful for enhancing functional capabilities in a wide variety of not only hematological disorders but also non hematological disorders. Specifically, these methods may be useful for the treatment of medical conditions which are amenable to cell therapy. More specifically, these methods may be useful for the treatment of acute leukemia, chronic leukemia, myelodysplastic syndromes, stem cell disorders, myeloproliferative disorders, lymphoproliferative disorders, phagocyte disorders, histiocytic disorders, liposomal storage diseases, aging disorders, arterial and blood vessel disease and cardiovascular diseases (e.g., stroke, peripheral artery disease, etc.), congenital immune system disorders, inherited erythrocyte abnormalities, other inherited disorders, inherited platelet abnormalities, plasma cell disorder, various maligancies such as brain tumors or Ewing sarcoma, Autoimmune Diseases, and other applications such as bone marrow transplants, diabetes, heart disease, liver disease, hematopoietic rescue situations following intense chemo/radiation, limb ischemia and limb regeneration (including for example, repair, regeneration or treatment of cartilage, muscle, skin, blood vessels, and the like).

[0095] In certain aspects, the patient in need of treatment with a cell population suffers from a condition selected from the group consisting of: an acute leukemia, a chronic leukemia, a myelodysplastic syndrome, a stem cell disorder, a myeloproliferative disorder, a lymphoproliferative disorder, a phagocyte disorder, a histiocytic disorder, a lysosomal storage disease, a gongenital immune system disorder, an inherited erythrocyte abnormality, an inherited platelet abnormality, a plasma cell disorder, a tumor and an autoimmune disease. In certain aspects, the patient in need of treatment with a cell population suffers from a condition selected from the group consisting of: peripheral arterial disease, ischemic limb injury, diabetes, heart disease, liver disease, bone disease, muscular dystrophy, Alzheimer's disease, multiple schlerosis, Parkinson's disease, ALS, spinal cord injury, stroke and infertility. Further examples of the above-described conditions are set forth in Table I below.

Table I

agents |

[0096] Cellular therapy can also be desirable for treatment of diseases in which the immune system is sought to be enhanced. One particular form of cell therapy involves the expansion of T cells that possess specificity for a distinct antigen, for example a tumor antigen. In other types of cell therapy, T cells are generated, and reprogrammed ex vivo for ability to kill a plurality of cells that express a plurality of markers. Examples of such cell therapy include expansion of autologous T cells with IL-2, stimulation with tumor cell lysates, and reintroduction of said cells into the patient.

[0097] On the other hand, cell therapy may be performed in situations where suppression of an immune response is desired. In such situations expansion of cells such as CD4+ CD25+ is desirable since these cells are capable of inhibiting immune responses in an antigen-specific manner. Methods for expansion of these cells are commonly known and include use of cytokines such as TGF-b.

[0098] One issue in bone marrow homing is that the receptors on endothelial cells for the glycosylated ligands of circulating cells are constitutively expressed. These receptors, such as P and E selectins, induce numerous activities after interacting with cells, including causing apoptosis or proliferative arrest (Winkler et al., Blood 2004 103: 1685 - 1692). Accordingly, the administration of hematopoietic cells and their subsequent homing to the bone marrow is dependent on molecules that are constitutively expressed.

[0099] For the purpose of a broader application of this approach for regenerative medicine in which stem cells are administered for non-hematopoietic purposes, the directional localization of cells where desired is much more complex and involves ligands that are not constitutively expressed, but expressed as a result of inflammation or tissue damage. For example, administration of stem cells for the purpose of treating myocardial infarction depends on homing of the stem cells into areas surrounded by cytokine release which induces expression of E selectin and P selectin on the endothelium and increased levels of SDF. This upregulation of receptors and chemotaxic agents allows not only homing of various cells into areas of injury, but also migration of inflammatory cells which exacerbate the injury. Routes of Administration

[0100] Administration of the modified cells is performed in agreement with standard practices that are known to one skilled in the art. Several embodiments are possible. For example, routes of administration may include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion or inhalation), transdermal (topical), transmucosal, and rectal administration. In certain particular aspects, the population of modified cells can be administered from a route selected from a group consisting of: intravenously, intraarterially, intramuscularly, subcutaneously, transdermal Iy, intratracheally, intraperitoneally, intravitreally, via direct injection, or into the microvascular compartment of bone or into spinal fluid. In some aspects the compositions, cells or other materials can be administered or used with a scaffolding support. In certain aspects, the population of modified cells is administered in or proximal to a site of injury. In certain aspects, the homing and engraftment takes place within the bone marrow of the patient in need thereof.

[0101] Some embodiments are useful for enhancing functional capabilities in a wide variety of not only hematological disorders but also non hematological disorders. Specifically, some embodiments may be useful for the treatment of medical conditions which are amenable to cell therapy. More specifically, some embodiments relate to methods, compositions and kits (comprising any of the materials described herein in any combination) for the treatment of acute leukemias, chronic leukemias, myelodysplastic syndromes, stem cell disorders, myeloproliferative disorders, lymphoproliferative disorders, phagocyte disorders, histiocytic disorders, liposomal storage diseases, congenital immune system disorders, inherited erythrocyte abnormalities, other inherited disorders, inherited platelet abnormalities, plasma cell disorder, various maligancies such as brain tumors or Ewing sarcoma, Autoimmune Diseases, and other applications such as bone marrow transplants, diabetes, heart disease, liver disease, hematopoietic rescue situations following intense chemo/radiation, limb ischemia and limb regeneration.

[0102] Furthermore, presented herein is the finding that the general increased adhesion of cells that have been treated ex vivo as provided herein can be utilized for augmented binding to localized niche areas in absence of chemotactic gradient such as in the context of portal vein injection or pulmonary artery injection.

EXAMPLES

EXAMPLE 1 SYSTEMIC TREATMENT WITH A CD26 INHIBITOR TO ACHIEVE ENHANCED

ENGRAFTMENT OF INJECTED CELLS

[0103] Inhibition of cell surface CD26 (dipeptidylpeptidase, DPPIV) has been shown to result in enhanced engraftment of both human cord blood and mobilized peripheral blood injected in animals (Christophererson et al., Science 2004, 16:305:1000-1003; Kawai et al, Stem Cell & Development, 2007, 361-379, which is incorporated herein by reference in its entirety). While ex vivo treatment of the to-be-injected graft with a sustained or irreversible CD26 inhibitor is one approach, another approach is the systemic administration of a potent and selective DPPIV inhibitor. For this latter approach there are a number of different injection scenarios for enhancing the engraftment of the injected cells. The selection of a particular scenario can depend on the pharmacokinetic and pharmacodynamic characteristics of the inhibitor following any one of a number of different routes of administration. An inhibitor with a short half life can be injected, for example, multiple times to achieve a sustained level in the blood stream sufficient for sustained inhibition of bone marrow and plasma DPPIV activity. An inhibitor with a long half life may be injected, for example, either once or twice to achieve a sustained level in the blood stream sufficient for sustained inhibition of bone marrow and plasma DPPIV activity. In some aspects, for maximal effect on engraftment the inhibitor is injected either prior to or simultaneous with the injection of the cells. In addition, to maximize the potential for enhancing engraftment the inhibitor is injected under conditions to achieve sustained inhibition of bone marrow and plasma DPPIV for 6 - 24 hrs following the injection of the graft.

EXAMPLE 2 EX VIVO INHIBITION OF CD26 ON MESENCHYMAL STEM CELLS [0104] Inhibition of cell surface DPPIV on mesenchymal stem cells (MSC) is achieved by exposure to a specific CD26 inhibitor in vitro or administered to the patient to achieve circulating concentrations sufficient to inhibit endogenous DPPIV activity. The cell population or mixture containing the cells is exposed to 5 mM Diprotin A (Ile-Pro-Ile) or CD26 inhibitor for 5 min to one hour, which is a sufficient period of time to ensure a significant decrease in enzyme activity, but which may vary depending on the selection of cells. CD26 activity is measured in PBS buffer (pH 7.4) at 37⁰C containing serum albumin and using the chromogenic substrate, Gly-Pro-nitroanilide. Proteolytic activity is determined by measurement of the amount of p-nitroanilide (pNA) formed in the supernatant at 405 nm every two minutes. The pmoles of pNA formed is then calculated by comparison to a pNA standard curve. The results are plotted and the slope calculated at the linear portion of the curve giving a measurement of DPPIV activity expressed as pmoles/min/cells.

[0105] Migration of the cells is recorded as a functional response to CD26 inhibition following pretreatment with an inhibitor. Chemotaxis assays are performed using 96 well chemotaxis chambers. In brief, 300 μl of RPMI supplemented with 10% serum albumin and varying concentrations of the chemokine, CXCL 12, ranging from 0 to 400 ng/ml is added to the lower chamber. Cells are added to the upper side of the membrane. Total cell number in the lower well is obtained by counting using a hemocytometer after 4 hours of incubation at 37⁰C. Percent migration is calculated by dividing the number of cells in the lower well by the total cell input and multiplied by 100 and subtracting random migration to the lower chamber without chemokine present.

EXAMPLE 3 ADMINISTRATION OF MODIFIED STEM CELLS TO BONE MARROW

TRANSPLANT PATIENTS

[0106] Patients in need of a bone marrow transplant are subjected to either myeloablative or non-myeloablative conditions. Stem cells obtained from one of a number of different sources are incubated ex vivo with 5 mM Diprotin A (Ile-Pro-Ile) or CD26 inhibitor at sufficient concentrations and for a sufficient period of time to result in maximal enhancement of CXCR4 receptor to CXCL 12. Following treatment, the cell preparation is washed or directly injected into the patient. Effectiveness of this application in the patient is determined with accelerated appearance over time of neutrophils and platelets in the blood stream compared to patients injected with control untreated stem cells.

EXAMPLE 4

MODIFICATION OF IMMUNE MODULATORY CELLS

[0107] A cell population consisting of cells with immune modulatory potential are treated with so as to endow increased surface ligands for endothelium. Cells are diluted to a concentration of 10(7) per ml and treated with 5 mM Diprotin A (Ile-Pro-Ile) or CD26 inhibitor for 30 minutes at 37⁰C in a humidified atmosphere containing 5% CO2 under conditions that cause minimum toxicity to CD34+ cells as tested by propidium iodide staining measured by flow cytometry. Other modifications of this treatment procedure may be performed based on the knowledge of one skilled in the art. Said treated immune cells are subsequently assessed for CXCR4/CXCL12 binding using functional methodology (assessment of rolling on endothelium). Cells are subsequently administered to a patient for immune modulation.

EXAMPLE 5 AUGMENTING EFFICACY OF TUMOR INFILTRATING LYMPHOCYTES AFTER EX

VIVO EXPANSION

[0108] Tumor infiltrating lymphocytes are collected as described by Zhou et al The Journal of Immunology, 2005, 175: 7046-7052. Briefly, explants of small (2 mm3) tumor fragments or 1 x 106 viable cells of tumor tissue digests are used to initiate TIL culture in 2 ml of RPMI 1640-based medium (Invitrogen Life Technologies) containing 10% human serum and 6000 IU/ml IL-2 (Chiron). After 2-4 wk of culture, usually several million TIL cells are obtained and screened by IFN secretion assay for recognition of tumor cells. Antitumor TIL cultures were further expanded in AIM V medium (Invitrogen Life Technologies) supplemented with irradiated allogeneic feeder cells, anti-CD3 Ab (Ortho Biotech), and 6000 IU/ml IL-2. This expansion protocol typically resulted in 1000-fold expansions of cells by the time of administration 14-15 days after initiation of the expansions. Subsequent to expansion cells are harvested, centrifuged, diluted to a concentration of 10(7) per ml and treated with 5 mM Diprotin A (Ile-Pro-Ile) or CD26 inhibitor for 10 min - 60 min hours at 37°C in a humidified atmosphere containing 5% CO2 under conditions that cause minimum toxicity to cells as tested by propidium iodide staining measured by flow cytometry. Cells are administered on a weekly basis at a concentration of at least about 1 million cells but in some situations up to 100 million over a period of 60-120 minutes. After 4 cycles of therapy tumor regression is noted.

[0109] One skilled in the art will appreciate that these methods, compositions, and cells are and may be adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, procedures, and devices described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the claimed technology. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the technology and are defined by the scope of the disclosure. It will be apparent to one skilled in the art that varying substitutions and modifications may be made without departing from the scope and spirit of the invention. Those skilled in the art recognize that the aspects and embodiments set forth herein may be practiced separate from each other or in conjunction with each other. Therefore, combinations of separate embodiments are within the scope of the technology as disclosed herein. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the technology pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0110] The embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions indicates the exclusion of equivalents of the features shown and described or portions thereof. Thus, it should be understood that although the present technology has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this technology as defined by the disclosure.

Claims

WHAT IS CLAIMED IS:

1. A method of enhancing homing and engraftment of a cell, comprising: a) providing cell population that has been contacted for a period of time insufficient for cell division to occur with a CD26 peptidase inhibitor in an amount effective to inhibit CD26 peptidase activity and effective to increase the migratory response to CXCL 12, resulting in a population of modified cells; and b) providing said population of modified cells to a patient in need thereof.

2. The method of claim 1, wherein the CD26 inhibitor is selected from the group consisting of Diprotin A (Ile-Pro-Ile), Valine-Pyrrolidide, sitagliptin, vildagliptin, saxagliptin, alogliptin, and any other class of compounds shown to exhibit potent inhibitor of either purified, soluble or cell surface (CD26) dipeptidylpeptiase.

3. The method of claim 2, wherein the CD26 inhibitor is Valine-Pyrrolidide.

4. The method of claim 2, wherein the CD26 inhibitor is Diprotin A (Ile-Pro-Ile).

5. The method of claim 1, wherein the inhibitor is any potent CD26 inhibitor exhibiting either reversible or irreversible inhibition

6. The method of claim 1 wherein the inhibitor is an antibody directed at the cell surface CD26.

7. The method of claim 1 wherein the CD26 expression is downregulated by the introduction of an siRNA or an antisense oligonucleotide.

8. The method of claim 1, wherein the cell population is contacted with said CD26 inhibitor for about 5 minutes to about 24 hours.

9. The method of claim 1, wherein the cell population is contacted with said CD26 inhibitor for about 15 minutes to about 6 hours.

10. The method of claim 1 , wherein the cell population is contacted with said CD26 inhibitor for less than 6 hours.

11. The method of claim 1 , wherein the cell population is contacted with said CD26 inhibitor for less than 2 hours.

12. The method of claim 1, wherein the cell population is contacted with said CD26 inhibitor for less than 1 hour.

13. The method of claim 1, wherein the inhibitor is at a concentration of less than about InM.

14. The method of claim 1, wherein the inhibitor is at a concentration of less than 1 uM

15. The method of claim 1 , wherein the inhibitor is at a concentration of less than 1 mM.

16. The method of claim 1 , wherein the inhibitor is provided in a concentration of less than about 5 mM.

17. The method of claim 1, wherein at least 1 x 10⁵ donor cells per mL are treated.

18. The method of claim 1 , wherein said cells are provided to a patient for a bone marrow transplant, optionally comprising providing a CD26 inhibitor to said patient in an amount effective to inhibit the peptidase activity thereof prior to, or during said transplant.

19. The method of claim 18, wherein the inhibitor is provided to said patient in a concentration of about 1 to about 50 μMol/kg total body weight.

20. The method of claim 18, wherein the inhibitor is provided to said patient in a concentration of about 1 to about 30 μMol/kg total body weight.

21. The method of claim 18, wherein the inhibitor is provided to said patient in a concentration of about 1 to about 10 μMol/kg total body weight.

22. The method of claim 1, wherein, prior to said providing, said population of modified cells has been further modified by treatment with an enzyme and appropriate substrate(s) under conditions sufficient for causing an alteration of cell surface charge or sufficient to enhance homing and engraftment of said cell.

23. The method of claim 22, wherein said enzyme is selected from a group comprising of alpha 1,3-fucosyltransferase I, alpha 1,3-fucosyltransferase III, alpha 1,3- fucosyltransferase IV, alpha 1,3-fucosyltransferase V, alpha 1,3-fucosyltransferase VI, alpha 1,3-fucosyltransferase VII, and alpha 1,3-fucosyltransferase IX.

24. The method of any of claims 1-23, wherein the cell population comprises or consists essentially of a population of stem cells.

25. The method of claim 24, wherein said stem cells are selected from a group consisting of: embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and mobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells and somatic nuclear transfer and side population stem cells.

26. The method of claim 25, wherein said embryonic stem cells are totipotent.

27. The method of claim 25, wherein the stem cells are hematopoietic stem cells.

28. The method of claim 25, wherein the stem cells are mesenchymal stem cells.

29. The method of claim 25, wherein the stem cells are neural stem cells.

30. The method of claim 25, wherein the stem cells are cardiomyocyte stem cells.

31. The method of any of claims 1-25, wherein said cell population comprises or consists essentially of a population of committed progenitor cells or differentiated cells.

32. The method of claim 31, wherein the cell population comprises or consists essentially of a population of mature blood cells.

33. The method of claim 32, wherein said mature blood cell is selected from the group consisting of: neutrophils, macrophages and T-cells.

34. The method of claim 33, wherein said T-cells are from a heterogeneous population of T-cells.

35. The method of claim 1, wherein said population of modified cells is provided via a route selected from a group consisting of: intravenously, intraarterially, intramuscularly, subcutaneously, transdermally, intratracheally, intraperitoneally, intravitreally, into bone or into spinal fluid.

36. The method of claim 1, wherein said population of modified cells is provided in or proximal to a site of injury.

37. The method of claim 1, wherein said homing and engraftment takes place within the bone marrow of said patient in need thereof.

38. A method of enhancing homing and engraftment of a cell, comprising: a) providing an isolated population of cells and a CD26 peptidase inhibitor in an amount effective to inhibit CD26 peptidase activity and effective to increase the migratory response to CXCL 12; and b) providing said population of cells and inhibitor to a patient in need thereof.

39. The method of Claim 38, wherein the isolated population of cells is selected from the group consisting of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and mobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as pluirpotent stem cells and somatic nuclear transfer and side population stem cells.

40. The method of Claim 38, wherein the inhibitor is selected from the group consisting of Diprotin A (Ile-Pro-Ile), Valine-Pyrrolidide, sitagliptin, vildagliptin, saxagliptin, alogliptin, and any other class of compounds shown to exhibit potent inhibition of either purified, soluble or cell surface (CD26) dipeptidylpeptiase.

41. An isolated population of cells having increased migratory response to CXCL 12 compared to cells of the same type that have not been contacted with a CD26 inhibitor, wherein the isolated population comprises one or more of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and immobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells and somatic nuclear transfer and side population stem cells.

42. An isolated population of cells comprising a CD26 inhibitor bound to CD26, wherein the isolated population comprises one or more of embryonic stem cells, cord blood stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiomyocyte stem cells, circulating and immobilized peripheral blood stem cells, mesenchymal stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells and somatic nuclear transfer and side population stem cells.