(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2003/0235909 A1
`(43) Pub. Date:
`Dec. 25, 2003
`Hariri et al.
`
`US 2003O235909A1
`
`(54) MODULATION OF STEM AND
`PROGENITOR CELL DIFFERENTIATION,
`ASSAYS, AND USES THEREOF
`(76) Inventors: Robert J. Hariri, Florham Park, NJ
`(US); David I. Stirling, Warren, NJ
`(US); Laure A. Moutouh-De Parseval,
`San Diego, CA (US); Kyle W.H. Chan,
`San Diego, CA (US)
`Correspondence Address:
`PENNIE AND EDMONDS
`1155 AVENUE OF THE AMERICAS
`NEW YORK, NY 100362711
`(21) Appl. No.:
`10/411,655
`(22) Filed:
`Apr. 11, 2003
`Related U.S. Application Data
`(60) Provisional application No. 60/372,348, filed on Apr.
`12, 2002. Provisional application No. 60/437,348,
`filed on Dec. 31, 2002. Provisional application No.
`60/437,350, filed on Dec. 31, 2002.
`
`Publication Classification
`
`(51) Int. Cl." ........................... C12N 5/08; A61K 31/454
`(52) U.S. Cl. ........................... 435/372; 514/323; 435/366
`
`(57)
`
`ABSTRACT
`
`The present invention relates to methods of modulating
`mammalian Stem cell and progenitor cell differentiation. The
`methods of the invention can be employed to regulate and
`control the differentiation and maturation of mammalian,
`particularly human Stem cells along Specific cell and tissue
`lineages. The methods of the invention relate to the use of
`certain Small organic molecules to modulate the differentia
`tion of Stem or progenitor cell populations along Specific cell
`and tissue lineages, and in particular, to the differentiation of
`embryonic-like Stem cells originating from a postpartum
`placenta or for the differentiation of early progenitor cells to
`a granulocytic lineage. Finally, the invention relates to the
`use of Such differentiated Stem or progenitor cells in trans
`plantation and other medical treatments.
`
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2006, Page 1
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`

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`Patent Application Publication Dec. 25, 2003 Sheet 1 of 33
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`US 2003/0235909 A1
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`
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`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2006, Page 2
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`

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`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2006, Page 3
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`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2006, Page 4
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`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
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`US 2003/0235909 A1
`
`Dec. 25, 2003
`
`MODULATION OF STEM AND PROGENITOR
`CELL DIFFERENTIATION, ASSAYS, AND USES
`THEREOF
`0001. This application claims benefit of U.S. Provisional
`Application Nos. 60/372,348, filed Apr. 12, 2002; 60/437,
`348, filed Dec. 31, 2002; and 60/437,350, filed Dec. 31,
`2002, each of which is incorporated herein in its entirety.
`
`1. INTRODUCTION
`0002 The present invention relates to methods of modu
`lating mammalian Stem and/or progenitor cell differentia
`tion. The methods of the invention can be employed to
`regulate and control the differentiation and maturation of
`mammalian, particularly human, Stem and progenitor cells
`along specific cell and tissue lineages. The methods of the
`invention relate to the use of certain Small organic molecules
`to modulate the differentiation of Stem cell populations
`along specific cell and tissue lineages, and in particular, to
`the differentiation of embryonic-like Stem cells originating
`from a postpartum placenta or the modulation of early
`hematopoietic progenitor cells along a specific differentia
`tion pathway, particularly a granulocytic differentiation
`pathway. The invention also relates to the use of these
`organic molecules to modulate the differentiation of particu
`lar lineages of progenitor cells, Such as CD34+, CD45+ and
`CD133+ progenitor cells. The invention also relates to the
`temporal aspects of progenitor cell development, and in
`Vitro models based upon these temporal aspects. The inven
`tion further relates to the use of these modulated cells in
`prophylactic and therapeutic methods, including in pharma
`ceutical compositions of Such cells and/or Small organic
`compounds. Finally, the invention relates to the use of Such
`differentiated cells in transplantation and other medical
`treatmentS.
`
`2. BACKGROUND OF THE INVENTION
`0003. There is considerable interest in the identification,
`isolation and generation of human Stem and progenitor cells.
`Stem cells are totipotential or pluripotential precursor cells
`capable of generating a variety of mature cell lineages, and
`precursor cells are cells capable of generating cells of
`Specific cell lineages. These abilities Serve as the basis for
`the cellular differentiation and Specialization necessary for
`organ and tissue development.
`0004 Recent Success at transplanting stem and progeni
`tor cells have provided new clinical tools to reconstitute
`and/or Supplement bone marrow after myeloablation due to
`disease, exposure to toxic chemical and/or radiation. Further
`evidence exists that demonstrates that Stem cells can be
`employed to repopulate many, if not all, tissues and restore
`physiologic and anatomic functionality. The application of
`Stem cells in tissue engineering, gene therapy delivery and
`cell therapeutics is also advancing rapidly.
`0005. Many different types of mammalian and progenitor
`Stem cells have been characterized. For example, embryonic
`Stem cells, embryonic germ cells, adult Stem cells or com
`mitted Stem cells or progenitor cells are known. Certain Stem
`cells have not only been isolated and characterized but have
`also been cultured under conditions to allow differentiation
`to a limited extent. However, a basic problem remains, that
`is, it has been difficult to control or regulate the differentia
`tion of Stem cells and progenitor cells, Such as hematopoietic
`
`progenitor cells. Presently, existing methods of modulating
`the differentiation of these cells are crude and unregulatable,
`Such that the cells differentiate into unwanted cell types, at
`unwanted times. Moreover, the yield of the product cells is
`typically low.
`0006 Furthermore, obtaining sufficient numbers of
`human Stem cells for therapeutic or research purposes is
`problematic. Isolation of normally occurring populations of
`Stem or progenitor cells in adult tissues has been technically
`difficult and costly, due, in part, to the limited quantity of
`Stem or progenitor cells found in blood or tissue, and the
`Significant discomfort involved in obtaining bone marrow
`aspirates. In general, harvesting of Stem or progenitor cells
`from alternative Sources in adequate amounts for therapeutic
`and research purposes is generally laborious, involving, e.g.,
`harvesting of cells or tissues from a donor Subject or patient,
`culturing and/or propagation of cells in vitro, dissection, etc.
`With respect to Stem cells in particular, procurement of these
`cells from embryos or fetal tissue, including abortuses, has
`raised religious and ethical concerns. The widely held belief
`that the human embryo and fetus constitute independent life
`has prompted governmental restrictions on the use of Such
`Sources for all purposes, including medical research. Alter
`native Sources that do not require the use of cells procured
`from embryonic or fetal tissue are therefore desired for
`further progreSS in the use of Stem cells clinically. There are,
`however, few viable alternative Sources of Stem or progeni
`tor cells, particularly human Stem or progenitor cells, and
`thus the Supply is limited.
`0007 Hu et al. (WO 00/73421 entitled “Methods of
`isolation, cryopreservation, and therapeutic use of human
`amniotic epithelial cells,” published Dec. 7, 2000) discloses
`human amniotic epithelial cells derived from placenta at
`delivery that are isolated, cultured, cryopreserved for future
`use, or induced to differentiate. According to Hu et al., a
`placenta is harvested immediately after delivery and the
`amniotic membrane Separated from the chorion, e.g., by
`dissection. Amniotic epithelial cells are isolated from the
`amniotic membrane according to Standard cell isolation
`techniques. The disclosed cells can be cultured in various
`media, expanded in culture, cryopreserved, or induced to
`differentiate. Hu et al. discloses that amniotic epithelial cells
`are multipotential (and possibly pluripotential), and can
`differentiate into epithelial tissueS Such as corneal Surface
`epithelium or vaginal epithelium. The drawback of Such
`methods, however, is that they are labor-intensive and the
`yield of stem cells is very low.
`0008 Currently available methods for the ex vivo expan
`Sion of cell populations are also labor-intensive. For
`example, Emerson et al. (Emerson et al., U.S. Pat. No.
`6,326,198 entitled “Methods and compositions for the ex
`Vivo replication of Stem cells, for the optimization of
`hematopoietic progenitor cell cultures, and for increasing
`the metabolism, GM-CSF Secretion and/or 1 L-6 secretion of
`human stromal cells”, issued Dec. 4, 2001); discloses meth
`ods, and culture media conditions for eX Vivo culturing of
`human Stem cell division and/or the optimization of human
`hematopoietic progenitor Stem cells. According to the dis
`closed methods, human Stem cells or progenitor cells
`derived from bone marrow are cultured in a liquid culture
`medium that is replaced, preferably perfused, either con
`tinuously or periodically, at a rate of 1 ml of medium per ml
`of culture per about 24 to about 48 hour period. Metabolic
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`products are removed and depleted nutrients replenished
`while maintaining the culture under physiologically accept
`able conditions.
`0009 Kraus et al. (Kraus et al., U.S. Pat. No. 6,338,942,
`entitled "Selective expansion of target cell populations,”
`issued Jan. 15, 2002) discloses that a predetermined target
`population of cells may be selectively expanded by intro
`ducing a starting Sample of cells from cord blood or periph
`eral blood into a growth medium, causing cells of the target
`cell population to divide, and contacting the cells in the
`growth medium with a Selection element comprising binding
`molecules with specific affinity (Such as a monoclonal
`antibody for CD34) for a predetermined population of cells
`(such as CD34 cells), so as to select cells of the predeter
`mined target population from other cells in the growth
`medium.
`0010 Rodgers et al. (U.S. Pat. No. 6,335,195 entitled
`"Method for promoting hematopoietic and mesenchymal
`cell proliferation and differentiation,” issued Jan. 1, 2002)
`discloses methods for eX Vivo culture of hematopoietic and
`mesenchymal Stem cells and the induction of lineage-spe
`cific cell proliferation and differentiation by growth in the
`presence of angiotensinogen, angiotensin I (AI), AI ana
`logues, AI fragments and analogues thereof, angiotensin II
`(AII), All analogues, All fragments or analogues thereof or
`All AT type 2 receptor agonists, either alone or in combi
`nation with other growth factors and cytokines. The Stem
`cells are derived from bone marrow, peripheral blood or
`umbilical cord blood. The drawback of Such methods, how
`ever, is that Such ex vivo methods for inducing proliferation
`and differentiation of Stem cells are time-consuming, as
`discussed above, and also result in low yields of Stem cells.
`0.011
`Stem and progenitor cells have the potential to be
`used in the treatment of a variety of disorders, including
`malignancies, inborn errors of metabolism, hemoglobinopa
`thies, and immunodeficiencies. One major area of use and
`research involving Stem cells from cord blood or placenta
`has been the use of Such cells to generate Small quantities of
`cells for bone marrow and other related transplantations.
`However, to date, no one has described a method of pro
`ducing Substantial numbers of Stem or progenitor cells, Such
`as human CD34" or CD133" progenitor cells. Large num
`bers of the latter cells, in particular, would facilitate treat
`ment methods using progenitor cells. The methods of the
`invention disclosed herein addresses this need.
`0012 Retinoids, such as vitamin A and retinoic acid
`(RA), have been known to affect differentiation of stem
`cells. For example, retinoic acid has been shown to inhibit
`proliferation of abnormally committed (chronic myelog
`enous leukemia) hematopoietic stem cells (Nadkarni et al.
`1984, Tumori 70:503-505) and to induce differentiation and
`loSS of Self-renewal potential in promyelocytic leukemia
`cells (Melchner et al., 1985, Blood 66(6): 1469-1472).
`Retinoic acid has also been shown to induce differentiation
`of neurons from embryonic Stem cells and to repress Spon
`taneous mesodermal differentiation (Slager et al., Dev.
`Genet. 1993;14(3):212-24, Ray et al., 1997, J. Biol. Chem.
`272(30): 18702-18708). Retinoic acid has further been
`shown to induce differentiation of transformed germ cell
`precursors (Damjanov et al., 1993, Labor. Investig.
`68(2):220-232), placental cell precursors (Yan et al., 2001,
`Devel. Biol. 235: 422-432), and endothelial cell precursors
`
`(Hatzopoulos et al., 1998, Development 125: 1457-1468).
`The effect of retinoids on differentiation, however, has yet to
`be completely understood Such that it could be used as a
`regulatable means of controlling differentiation of Stem
`cells.
`0013 The effects of folic acid analogues, such as ami
`nopterin and amethopterin (methotrexate), on the differen
`tiation of hematopoietic Stem cells has been Studied. Folic
`acid analogues are used as chemotherapeutic agents in acute
`lymphoblastic anemias and other blood proliferation disor
`derS and cancers, and have been shown to effect differen
`tiation of Stem cells by killing off certain populations of stem
`cells (DeLoia et al., 1998, Human Reproduction 13(4):1063
`1069), and thus, would not be an effective tool for regulating
`differentiation of large quantities of Stem cells for adminis
`tration to a patient.
`0014 Several cytokines, such as IL-1, IL-2, IL-3, IL-6,
`IL-7, IL-11, as well as proteins Such as erythropoietin, Kit
`ligand, M-CSF and GM-CSF have also been shown to direct
`differentiation of Stem cells into specific cell types in the
`hematopoietic lineage (Dushnik-Levinson et al., 1995, Biol.
`Neonate 67:77-83), however, these processes are not well
`understood and Still remain too crude and imprecise to allow
`for a regulatable means of controlling differentiation of Stem
`cells.
`0015 To date, no one has described the use of com
`pounds, Such as the immunomodulatory compounds dis
`cussed below, in the differentiation of Stem cells or precursor
`cells. In particular, no one has demonstrated the use of Such
`compounds to modulate the differentiation of progenitor
`cells, Such as CD34" progenitor cells, away from a dendritic
`cell lineage, a capability useful in encouraging transplant
`immune tolerance. Likewise, no one has described the use of
`the compounds described herein to expand the progenitor
`cell populations So as to produce a pharmaceutical compo
`Sition containing Such cells. Such expanded progenitor cell
`cultures would be useful in the treatment of graft-Versus
`host disease and the development of immune tolerance.
`Because control over Stem and precursor cell differentiation
`can produce cell populations that are therapeutically useful,
`there is a need for the ability to control and regulate the
`differentiation of cells of myeloid dendritic cell lineage, or
`early progenitor cells, such as human CD34" or CD133"
`progenitor cells, for the controlled production of dendritic
`cells and/or granulocytes.
`
`3. SUMMARY OF THE INVENTION
`0016. The present invention provides methods of modu
`lating mammalian, particularly human Stem cell or progeni
`tor cell differentiation. In particular, the methods of the
`invention may be employed to regulate and control the
`differentiation and maturation of human Stem cells along
`Specific cell and tissue lineages. The invention encompasses
`the use of immunomodulatory Small organic compounds,
`more preferably amino-Substituted isolindolines, particularly
`the compounds ActimidTM or Revimid TM, to effect such
`regulation and control. The invention further contemplated
`administration of these compounds to progenitor cells at
`Specific times to modulate their differentiation in Specific
`ways.
`0017. The methods of the invention encompass the regu
`lation of differentiation of a Stem cell or progenitor cell into
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`a Specific cell lineage, including, but not limited to, a
`mesenchymal, hematopoietic, adipogenic, hepatogenic, neu
`rogenic, gliogenic, chondrogenic, Vasogenic, myogenic,
`chondrogenic, or osteogenic lineage. In particular embodi
`ment, the methods of the invention encompass the regulation
`of Stem cell differentiation to a cell of a hematopoictic
`lineage.
`0.018. The invention also encompasses the modulation of
`a committed cell to a specific cell type, e.g., mesenchymal
`cell, hematopoietic cell, adipocyte, hepatocyte, neuroblast,
`glioblast, chondrocyte, endothelial cell (EC) progenitor,
`myocyte, chondrocyte, or osteoblast. In Specific embodi
`ments, the invention encompasses the modulation of a
`committed hematopoietic progenitor cell to an erythrocyte,
`a thrombocyte, or a leukocyte (white blood cell) Such as a
`neutrophil, monocyte, macrophage, eosinophil, basophil,
`mast cell, B-cell, T-cell, or plasma cell.
`0019. In another embodiment, the methods of the inven
`tion relate to modulating the differentiation of Stem cells to
`cells of a hematopoietic lineage, in particular, CD34+,
`CD133+, and CD45+ hematopoietic lineages, and methods
`of producing prophylactically or therapeutically beneficial
`pharmaceutical compositions containing Such cells. In
`another specific embodiment, the methods of the invention
`relate to modulating the differentiation of early progenitor
`cells into cells of a dendritic cell lineage or a granulocyte
`lineage, endothelial lineage, or cardiomyocyte lineage.
`0020. In another embodiment, the invention provides
`methods for regulating the differentiation of a progenitor cell
`into a hematopoietic lineage, particularly a dendritic cell or
`granulocytic lineage, endothelial lineage, neural lineage or
`cardiomyocyte lineage. In a specific embodiment, Said pro
`genitor cell is a CD34+ or CD133+ cell. Such regulation is
`accomplished by contacting the progenitor cells during
`culture with a compound of the invention. In one embodi
`ment, said compound in an inhibitor of TNF-C. activity. In a
`more Specific embodiment, Said compound is an immuno
`modulatory compound as described herein, or thalidomide
`or, more preferably, an amino-Substituted isolindoline. In an
`even more Specific embodiment, Said compound is
`Actimid TM or Revimid TM
`0021. In another specific embodiment, the methods of the
`invention encompass the Suppression of progenitor cell
`differentiation into a dendritic cell. In another specific
`embodiment, the invention provides a method for modulat
`ing the differentiation of progenitor cells during the first Six
`days of culture to produce an expanded culture of Such
`progenitor cells. In another embodiment, the methods of the
`invention encompass the promotion of early progenitor cell
`development into a granulocyte, which may be useful for
`fighting infections. The increase of granulocyte lineage
`committed progenitors (CD 15" cells) can be of potential use
`in the reduction of neutropenia and its Subsequent infectious
`complications that represent the most common dose-limiting
`toxicity of cancer chemotherapy. In another embodiment,
`the methods of the invention may be used to SuppreSS
`dendritic cell differentiation, which is useful for mitigating
`the effects of graft-Versus-host disease.
`0022. The progenitor cells of the invention, as modulated
`by a compound of the invention, are useful for transplanta
`tion (i.e., hematopoietic reconstitution), and may be used in
`regenerative medicine as a renewable Source of replacement
`
`cells and tissues (such as pancreatic, cardiac, hepatic, kid
`ney, liver, brain, lung, bladder, intestinal or muscle cells) to
`treat normal Senescence, injury or diseaseS Such as heart
`disease, Stroke, Parkinson's disease, and Alzheimer's dis
`ease. The cells will also be useful in the determination of the
`intracellular biochemical pathways that mediate the action
`of the compounds of the invention. These cells may also be
`useful for the Screening of new drugs and toxins, for
`example, to determine potential anti-cancer drugs, to under
`Stand the origins of birth defects, etc.
`0023 The methods of the invention may be used to
`SuppreSS Specifically the generation of red blood cells or
`erythropoietic colonies (BFU-E and CFU-E), while aug
`menting both the generation of leukocyte and platelet form
`ing colonies (CFU-GM) and enhancing total colony forming
`unit production. The methods of the invention may be used
`not only to regulate the differentiation of Stem cells, and
`progenitor cells Such as CD34+ progenitor cells, but may
`also be used to Stimulate the rate of colony formation,
`providing Significant benefits to hematopoietic Stem cell
`transplantation by improving the Speed of bone marrow
`engraftment.
`0024. Any mammalian stem cell can be used in accor
`dance with the methods of the invention, including but not
`limited to, Stem cells isolated from cord blood, placenta and
`other Sources. The Stem cells may be isolated from any
`mammalian Species, e.g., mouse, rat, rabbit, guinea pig, dog,
`cat, pig, sheep, cow, horse, monkey, etc., more preferably, a
`human. The Stem cells may include pluripotent cells, i.e.,
`cells that have complete differentiation versatility, that are
`Self-renewing, and can remain dormant or quiescent within
`tissue. The Stem cells may also include multipotent cells or
`committed progenitor cells. In one preferred embodiment,
`the invention utilizes Stem cells that are viable, quiescent,
`pluripotent Stem cells that exist within, or are later produced
`by, the full-term placenta, that is, Such cells can be recovered
`following Successful birth and placental expulsion, eXSan
`guination and perfusion of the placenta, resulting in the
`production and recovery of as many as one billion nucleated
`cells, which yield 50 to 100 million multipotent and pluri
`potent Stem cells. Such cells are referred to herein as human
`placental stem cells or embryonic-like Stem cells.
`0025. In one particular embodiment of the invention,
`cells, for example cells endogenous to bone marrow or to a
`postpartum perfused placenta, including, but not limited to,
`embryonic-like Stem cells, progenitor cells Such as CD34+
`or CD133+ cells, pluripotent cells and multipotent cells, are
`exposed to the compounds of the invention and induced to
`differentiate. The endogenous cells may be propagated in
`Vitro. In another embodiment, the endogenous cells may be
`collected from the placenta and culture medium and cultured
`in Vitro under conditions appropriate, and for a time Suffi
`cient, to induce differentiation to the desired cell type or
`lineage.
`0026. In another embodiment of the invention, the stem
`or progenitor cells are derived from other Sources Such as
`cord blood, peripheral blood or adult blood, and are exposed
`to the compounds of the invention and induced to differen
`tiate. In a preferred embodiment, the differentiation is con
`ducted in Vitro under conditions appropriate, and for a time
`Sufficient, to induce differentiation into the desired lineage or
`cell type. The compounds of the invention are used in the
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`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2006, Page 37
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`Dec. 25, 2003
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`differentiation/culture media by addition, in Situ generation,
`or in any other manner that permits contact of the Stem or
`progenitor cells with the compounds of the invention.
`0027. It has been discovered that the timing of the
`administration of the compounds of the invention have a
`profound impact upon the differentiation of CD34" progeni
`tor cells. Thus, in one embodiment of the invention, differ
`entiation of CD34" progenitor cells into dendritic cells is
`delayed or Suppressed by a method comprising contacting
`the progenitor cell on the first day of culture with a com
`pound of the invention. In another embodiment, the devel
`opment of CD1a"