U.S. patent application number 11/081969 was filed with the patent office on 2005-08-18 for progenitor cell preservation factors and methods for and products of their use.
This patent application is currently assigned to Regents of the University of California. Invention is credited to Chrispeels, Maarten J., Colucci, M. Gabriella, Moore, Jeffrey G..
Application Number | 20050181997 11/081969 |
Document ID | / |
Family ID | 34840996 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181997 |
Kind Code |
A1 |
Colucci, M. Gabriella ; et
al. |
August 18, 2005 |
Progenitor cell preservation factors and methods for and products
of their use
Abstract
Disclosed is the FRIL family of progenitor cell preservation
factors and nucleic acids encoding the same. FRIL family members
preserve progenitor cells both in vivo and ex vivo. FRIL family
members find use as therapeutics for alleviating and/or reducing
the hematopoietic progenitor cell-depleting activity of many cancer
therapeutics. FRIL family members are also useful for isolating
rare, primitive progenitor cells.
Inventors: |
Colucci, M. Gabriella;
(Naples, IT) ; Chrispeels, Maarten J.; (La Jolla,
CA) ; Moore, Jeffrey G.; (Brookline, MA) |
Correspondence
Address: |
WILMER CUTLER PICKERING HALE AND DORR LLP
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Regents of the University of
California
La Jolla
CA
ImClone Systems Incorporated
New York
NY
|
Family ID: |
34840996 |
Appl. No.: |
11/081969 |
Filed: |
March 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11081969 |
Mar 16, 2005 |
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09476485 |
Dec 30, 1999 |
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09476485 |
Dec 30, 1999 |
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08881189 |
Jun 24, 1997 |
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6310195 |
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Current U.S.
Class: |
424/184.1 ;
435/372; 435/419; 435/468; 435/6.11; 435/69.1; 514/19.3; 514/7.9;
530/370; 536/23.6 |
Current CPC
Class: |
A61K 2035/124 20130101;
C12N 2501/59 20130101; C12N 5/0647 20130101; C12N 15/90 20130101;
C12N 2501/26 20130101; C07K 14/42 20130101 |
Class at
Publication: |
514/012 ;
530/370; 435/006; 435/069.1; 435/419; 435/468; 536/023.6;
435/372 |
International
Class: |
A61K 038/16; C12Q
001/68; C07H 021/04; C07K 014/415; C12N 005/04; C12N 005/08 |
Claims
1-61. (canceled)
62. A method for alleviating or reducing the hematopoietic
progenitor cell-depleting activity of a therapeutic treatment in a
patient, comprising administering to the patient a therapeutically
effective amount of a pharmaceutical composition prior to
administration of the therapeutic treatment to the patient, wherein
the pharmaceutical formulation comprises: (a) a pharmaceutically
acceptable carrier; and (b) a protein that: (1) binds to a normally
glycosylated FLT3 receptor; (2) has at least 95% amino acid
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO:8; and (3)
preserves hematopoietic progenitor cells.
63. The method of claim 62, wherein the patient is human.
64. The method of claim 63, wherein the patient has cancer.
65. The method of claim 62, wherein the therapeutic treatment is
selected from the group consisting of a radiotherapeutic, a
chemotherapeutic, or a combination of a radiotherapeutic and a
chemotherapeutic.
66. The method of claim 65, wherein the chemotherapeutic is
selected from the group consisting of cytarabine, doxorubicin, and
5-fluorouracil.
67. The method of claim 62, wherein the protein is a recombinant
protein.
68. A method for preserving progenitor cells ex vivo, comprising
contacting a population of cells comprising at least one progenitor
cell with an effective amount of a pharmaceutical composition for
an effective period of time, wherein the progenitor cells in the
population are rendered quiescent, wherein the pharmaceutical
formulation comprises: (a) a pharmaceutically acceptable carrier;
and (b) a protein that: (1) binds to a normally glycosylated FLT3
receptor; (2) has at least 95% amino acid sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NO: 2, SEQ ID NO: 6, and SEQ ID NO:8; and (3) preserves
hematopoietic progenitor cells.
69. The method of claim 68, wherein the progenitor cells are from a
human.
70. The method of claim 68, wherein the population of cells is bone
marrow cells.
71. The method of claim 68, wherein the non-progenitor cells in the
population of cells differentiate or die.
72. The method of claim 68, wherein the population of cells is
removed from a cancer patient prior to treatment of the cancer
patient with a therapeutic treatment having a hematopoietic
progenitor cell-depleting activity.
73. The method of claim 72, wherein the therapeutic treatment is
selected from the group consisting of a radiotherapeutic, a
chemotherapeutic, or a combination of a radiotherapeutic and a
chemotherapeutic.
74. The method of claim 73, wherein the chemotherapeutic is
selected from the group consisting of cytarabine, doxorubicin, and
5-fluorouracil.
75. The method of claim 68, wherein the protein is a recombinant
protein.
76. A method for preserving progenitor cells in vivo, comprising
administering to a patient an effective amount of a pharmaceutical
composition for an effective period of time, wherein the progenitor
cells in the patient are rendered quiescent, wherein the
pharmaceutical formulation comprises: (a) a pharmaceutically
acceptable carrier; and (b) a protein that: (1) binds to a normally
glycosylated FLT3 receptor; (2) has at least 95% amino acid
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO:8; and (3)
preserves hematopoietic progenitor cells.
77. The method of claim 76, wherein the patient is human.
78. The method of claim 77, wherein the patient is a cancer
patient.
79. The method of claim 76, wherein the effective amount of the
pharmaceutical composition is administered prior to the treatment
of the patient with a therapeutic treatment having a hematopoietic
progenitor cell-depleting activity.
80. A method for identifying a progenitor cell, comprising
contacting a candidate cell with a FRIL family member molecule,
wherein binding of the candidate cell to the FRIL family member
molecule identifies the candidate cell as a progenitor cell,
wherein the FRIL family member molecule comprises a protein that:
(1) binds to a normally glycosylated FLT3 receptor; (2) has at
least 95% amino acid sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6,
and SEQ ID NO:8; and (3) preserves hematopoietic progenitor
cells.
81. The method of claim 80, wherein the candidate cell is in a
population of cells.
82. The method of claim 80,wherein the candidate cell is from a
human.
83. A progenitor cell identified by the method of claim 80.
84. A method for identifying a member of the FRIL family of
progenitor cell preservation factors, comprising contacting a
candidate compound with a glycosylated extracellular domain of an
FLT3 receptor, wherein the glycosylation pattern of the
extracellular domain of the FLT3 receptor is the same as the
glycosylation pattern of an extracellular domain of a normally
glycosylated FLT3 receptor, wherein a candidate compound that binds
the glycosylated extracellular domain of the FLT3 receptor is
identified as a FRIL family member.
85. The method of claim 84, wherein the candidate compound is a
lectin.
86. The method of claim 85, wherein the lectin is synthetic.
87. The method of claim 85, wherein the lectin is from a
legume.
88. A method for isolating a population of progenitor cells,
comprising contacting a population of cells with a plurality of
FRIL family member molecules, and separating the unbound cells,
wherein the cells bound to the FRIL family member molecules are an
isolated population of progenitor cells, wherein the FRIL family
member molecules comprise a protein that: (1) binds to a normally
glycosylated FLT3 receptor; (2) has at least 95% amino acid
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO:8; and (3)
preserves hematopoietic progenitor cells.
89. The method of claim 88, wherein the isolated population of
progenitor cells is from a human.
90. The method of claim 88, wherein the FRIL family member
molecules are detectably labeled.
91. The method of claim 88, wherein the FRIL family member
molecules are immobilized on a solid support.
92. The method of claim 91, wherein the solid support is a
bead.
93. The method of claim 92, wherein the bead is magnetic.
94. The method of claim 88, wherein the unbound cells are separated
by applying a magnet to the population of cells contacted with the
FRIL family member molecules immobilized on the magnetic bead.
95. The method of claim 93, wherein the population of cells bound
to the FRIL family member molecules immobilized on a magnetic bead
are rinsed with a physiologically acceptable solution while the
magnet is applied.
96. The method of claim 91, wherein the solid support is the bottom
of a tissue culture plate.
97. The method of claim 88, wherein the isolated population of
progenitor cells is a population of hematopoietic progenitor
cells.
98. The method of claim 88, wherein the population of cells is
selected from the group consisting of whole blood, umbilical cord
blood, bone marrow cells, and fetal liver cells.
99. The method of claim 88, wherein the population of cells is a
sorted population of cells, wherein a cell of the sorted population
does not express a cell surface molecule selected from the group
consisting of CD11b, CD11c, and CD38.
100. The method of claim 99, wherein the sorted population of cells
is sorted by flow cytometry or by magnetic bead selection.
101. An isolated population of progenitor cells isolated by the
method of claim 88.
102. The isolated population of progenitor cells of claim 101,
wherein the isolated population of progenitor cells is from a
human.
103. The cells of claim 101, wherein the cells of the isolated
population of progenitor cells do not express CD34.
104. The cells of claim 101, wherein the cells of the isolated
population of progenitor cells express a receptor tyrosine kinase
selected from the group consisting of from FLK1, FLT1, FLT3, FLT4,
and Kit.
105. The cells of claim 101, wherein the cells of the isolated
population of progenitor cells express a cell surface molecule
selected from the group consisting of CD11b and CD11c.
106. The isolated population of progenitor cells of claim 101,
wherein the cells of the isolated population of progenitor cells
express FLT3.
107. The isolated population of progenitor cells of claim 101,
wherein the cells of the isolated population of progenitor cells
are selected from the group consisting of hemangioblasts, a
messenchymal stem cells, bone progenitor cells, hepatic progenitor
cells, endothelial progenitor cells, hematopoietic progenitor
cells, embryonal stem cells, brain progenitor cell, and dendritic
progenitor cells.
108. The isolated population of progenitor cells of claim 101,
wherein the cells of the isolated population of progenitor cells
are hematopoietic progenitor cells.
109. The isolated population of progenitor cells of claim 101,
wherein transplantation of isolated population of progenitor cells
into an animal lacking a population of hematopoietic progenitor
cells sufficient to enable survival of the animal reconstitutes the
animal, wherein the transplanted animal survives.
Description
[0001] This application is a continuation in part of U.S. Ser. No.
08/881,189, filed Jun. 24, 1997, the entire contents of which are
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to the preservation of progenitor
cells. More specifically, the invention relates to the in vivo or
ex vivo preservation of progenitor cells, such as hematopoietic
progenitor cells.
[0004] 2. Summary of the Related Art
[0005] The wide variety of functionally and phenotypically
different types of cells in a multi-cellular eukaryotic organism
results in part from the proliferation and differentiation of rare
and mostly quiescent populations of progenitor cells. For example,
hematopoiesis involves the process of producing a balanced supply
of different blood cells from such progenitor cells found in the
adult bone marrow. The development of other cell types also depends
upon production of the differentiated cells from such progenitor
cells.
[0006] Progenitor cells are activated by signals, such as cell-cell
contact or soluble regulators, to generate daughter cells that are
identical to the parent (i.e., self-renewal of the parent) and/or
to generate daughter cells that are more differentiated than the
parent, thus beginning an irreversible process that ends with the
production of differentiated, functional cells. In the process of
hematopoiesis, differentiation is coupled to proliferation as a
progenitor cell gives rise to more differentiated daughter cells
that progressively become committed to producing only one blood
cell type. The enormous activation of hematopoietic progenitor
cells needed to meet the body's daily requirement for hundreds of
billions of new mature blood cells is directed by potent soluble
regulators (e.g., colony stimulating factors and cytokines) acting
upon the hematopoietic progenitor cells themselves, and their more
differentiated daughter cells.
[0007] Although progenitor cells eventually produce so many of the
mature cells of the body, they occur only rarely. Moreover,
typically the more primitive (i.e., undifferentiated) the
progenitor cell, the more rare the progenitor cell. For example,
the currently believed most primitive of the hematopoietic
progenitor cells, which are called hematopoietic stem cells, occur
at a frequency of only from about 1 in 10,000 to about 1 in 100,000
of the cells in the bone marrow. Hematopoietic stem cells have the
capacity to generate more than 10.sup.13 mature blood cells of all
lineages, including other progenitor cells which, although more
differentiated than hematopoietic stem cells, are themselves
capable of giving rise to several different types of mature blood
cells.
[0008] Hematopoietic stem cells are responsible for sustaining
blood cell production over the life of an animal. The small
population of hematopoietic stem cells is sufficient to produce all
the mature blood cells in a healthy individuals; however, some
unhealthy individuals suffer from a lack of a sufficient number of
progenitor cells and/or mature blood cells. For example, cancer
patients receiving chemotherapeutic or radiotherapy treatments
designed to kill the rapidly dividing cancer cells also suffer from
the depletion of white blood cells and platelets, thus exposing
these patients to life threatening opportunistic infections and
bleeding episodes. Indeed, this hematopoietic progenitor
cell-depleting activity is the dose-limiting factor for most of
these chemotherapeutic and radiotherapeutic agents.
[0009] Many cancer patients are routinely treated with cytokines,
including G-CSF, GM-CSF, SCF, Erythropoietin, and IL-11, to
accelerate restoration of hematopoiesis following chemotherapy
(Moore, M. A., Blood 78: 1-19, 1991). However, these cytokines lead
to the irreversible differentiation of hematopoietic progenitor
cells, including hematopoietic stem cells, into more differentiated
daughter cells. Thus, better protection of hematopoietic progenitor
cells is needed during chemotherapy.
[0010] Workers in the field have attempted to use cytokines in mice
to protect progenitors from the toxicity of chemotherapy (Neta et
al., J. Immunol. 136: 2483-2485, 1986; Neta et al., J. Immunol.
140: 108-111, 1988; Neta et al., J. Exp. Med. 173: 1177-1182, 1991;
de Haan et al., Blood 87: 4581-4588, 1996; Lyman and Jacobsen,
Blood 91: 1101-1134, 1998; Dalmau et al., Bone Marrow Transplant.
12: 551-563, 1993; Grzegorzewski et al., J. Exp. Med. 180:
1046-1057, 1994; Grzegorzewski et al., Blood 94:1066a
(Abstr.)1999). Marshall et al. (Euro. J. Cancer 34: 1023-1029,
1998) and Gilmore et al. (Exp. Hematol. 27: 195-202, 1999) describe
the use in clinical trials of a chemokine that allegedly inhibits
progenitor cell proliferation, MIP1-.alpha., as a chemoprotectant.
Marshall (Marshall, A., Nat.. Biotechnol. 16: 129, 1999) describes
the use of MPIF-1, a chemokine that allegedly inhibits progenitor
cell proliferation, in clinical trials as a chemoprotectant.
[0011] There are several drawbacks to using chemokines, cytokines,
and other immunoregulators as chemoprotectants during the
chemotherapeutic or radiotherapeutic treatment of cancer patient.
These drawbacks include the cost of production and toxicity to the
patient.
[0012] Therefore, there is a need for improved reagents that are
non-toxic and inexpensive to produce for use in preserving
progenitor cells.
BRIEF SUMMARY OF THE INVENTION
[0013] The invention provides a family of factors that preserve
progenitor cells. Members of this family, the FRIL family, are
non-toxic, inexpensively produced reagents that preserve progenitor
cells. The invention provides compositions comprising at least one
member of the FRIL family, as well as methods for using members of
the FRIL family to preserve progenitor cells both in vivo and ex
vivo.
[0014] Accordingly, in a first aspect, the invention provides an
essentially pure composition of one or more members of the FRIL
family of progenitor cell preservation factors.
[0015] In certain embodiments of the first aspect of the invention,
the FRIL family member is from a legume. In some embodiments, the
legume is Dolichos lab lab. In some embodiments, the legume is
Phaseolus vulgaris. In some embodiments, the legume is Sphenostylis
stenocarpa.
[0016] In certain embodiments of the first aspect of the invention,
the FRIL family member is a mutant derived from a second member of
the FRIL family, wherein the mutant is selected from the group
consisting of a substitution mutant, a deletion mutant, an addition
mutant, or a combination thereof.
[0017] In certain embodiments of the first aspect of the invention,
the FRIL family member is a fusion protein comprising a first
portion and a second portion, wherein the first portion is derived
from a second member of the FRIL family.
[0018] In a second aspect, the invention provides a recombinant
nucleic acid molecule encoding a composition of a member of the
FRIL family of progenitor cell preservation factors.
[0019] In a third aspect, the invention provides a pharmaceutical
formulation comprising an essentially pure composition of one or
more members of the FRIL family of progenitor cell preservation
factors and a pharmaceutically acceptable carrier.
[0020] In certain embodiments of the third aspect of the invention,
administration of a therapeutically effective amount of the
formulation to a patient suffering from a condition whereby the
patient's hematopoietic progenitor cells are depleted alleviates
and/or reduces the condition in the patient.
[0021] In certain embodiments of the third aspect-of the invention,
administration of a therapeutically effective amount of the
formulation to a patient prior to treatment of the patient with a
therapeutic treatment having a hematopoietic progenitor
cell-depleting activity alleviates and/or reduces the hematopoietic
progenitor cell-depleting activity of the therapeutic treatment in
the patient. In certain embodiments, the patient is a human or is a
domesticated mammal. In some embodiments, the patient has cancer.
In some embodiments, the therapeutic treatment is a
radiotherapeutic or a chemotherapeutic treatment, including,
without limitation, cytarabine (Ara-C), doxorubicin (Dox), or
5-fluorouracil (5-FU), or a combination of a radiotherapeutic and a
chemotherapeutic.
[0022] In a fourth aspect, the invention provides a method for
alleviating or reducing the hematopoietic progenitor cell-depleting
activity of a therapeutic treatment in a patient, comprising
administering to the animal a therapeutically effective amount of a
composition of a FRIL family member prior to administration of the
therapeutic treatment to the patient.
[0023] In certain embodiments of the fourth aspect, the patient is
a human or is a domesticated mammal. In some embodiments, the
patient has cancer. In some embodiments, the therapeutic treatment
is a radiotherapeutic or a chemotherapeutic treatment, including,
without limitation, cytarabine (Ara-C), doxorubicin (Dox), or
5-fluorouracil (5-FU), or a combination of a radiotherapeutic and a
chemotherapeutic.
[0024] In a fifth aspect, the invention provides a method for
isolating a population of progenitor cells, comprising contacting a
population of cells with a plurality of FRIL family member
molecules, and separating the unbound cells, wherein the cells
bound to the FRIL family member molecules are an isolated
population of progenitor cells. Preferably, the isolated population
of progenitor cells is from a human.
[0025] In certain embodiments of the fifth aspect of the invention,
the FRIL family member molecules are detectably labeled. In certain
embodiments, the detectably labeled FRIL family member molecules
are labeled with a chromophore. In certain embodiments, the unbound
cells are separated by using a flow cytometry cell sorter to sort
the population of cells contacted with the FRIL family member
molecules detectably labeled with a chromophore.
[0026] In certain embodiments of the fifth aspect, the FRIL family
member molecules is immobilized on a solid support. In some
embodiments, the solid support is a bead, such as a magnetic bead.
In some embodiments, the unbound cells are separated by applying a
magnet to the population of cells contacted with the FRIL family
member molecules immobilized on the magnetic bead. In further
embodiments, the population of cells bound to the FRIL family
member molecules immobilized on a magnetic bead is rinsed with a
physiologically acceptable solution while the magnet is
applied.
[0027] In certain embodiments of the fifth aspect, the solid
support is the bottom of a tissue culture plate. In some
embodiments, the unbound cells are separated by rinsing the
population of cells contacted with the FRIL family member
immobilized on the bottom of a tissue culture plate with a
physiologically acceptable solution.
[0028] In preferred embodiments of the fifth aspect of the
invention, the isolated population of progenitor cells is a
population of hematopoietic progenitor cell. In various
embodiments, the population of cells is whole blood, umbilical cord
blood, bone marrow cells, or fetal liver cells.
[0029] In certain embodiments of the fifth aspect of the invention,
the population of cells is a sorted population of cells, wherein a
cell of the sorted population does not express a cell surface
molecule selected from the group consisting of CD11b, CD11c, and
CD38. In certain embodiments, the sorted population of cells is
sorted by flow cytometry or by magnetic bead selection.
[0030] In a sixth aspect, invention provides an isolated population
of progenitor cells isolated by a method comprising contacting a
population of cells with a plurality of FRIL family member
molecules, and separating the unbound cells, wherein the cells
bound to the FRIL family member molecules are an isolated
population of progenitor cell. Preferably, the progenitor cell is
from a human.
[0031] In certain embodiments of the sixth aspect, the cells of the
isolated population do not express CD34. In certain embodiments,
the cells of the isolated population express a receptor tyrosine
kinase selected from the group consisting of from FLK1, FLT1, FLT3,
FLT4, and Kit. In some embodiments, the cells of the isolated
population express a cell surface molecule selected from the group
consisting of CD11b and CD11c. In preferred embodiments, the cells
of the isolated population express FLT3.
[0032] In various embodiments of the sixth aspect of the invention,
the cells of the isolated population are hemangioblasts,
messenchymal stem cells, bone progenitor cells, hepatic progenitor
cells, endothelial progenitor cells, hematopoietic progenitor
cells, embryonal stem cells, brain progenitor cells, or dendritic
progenitor cells. Preferably, the cells of the isolated population
are hematopoietic progenitor cells.
[0033] In certain embodiments of the sixth aspect of the invention,
where the cells of the isolated population are hematopoietic
progenitor cells, transplantation of the isolated population into
an animal lacking a population of hematopoietic progenitor cells
sufficient to enable survival of the animal reconstitutes the
animal, wherein the transplanted animal survives. In certain
embodiments, the hematopoietic progenitor cells are from a human or
a mouse and wherein the animal is a mouse. In some embodiments, the
mouse is a SCID mouse or the mouse is sublethally irradiated or the
mouse is treated with a sublethal dose of a chemotherapeutic. In
certain embodiments, the hematopoietic progenitor cells are from a
human and the animal is a human. In some embodiments, the human is
a cancer patient receiving a treatment that depletes the patient's
hematopoietic progenitor cells. In some embodiments, the treatment
is a radiotherapeutic or a chemotherapeutic treatment, induding,
without limitation, cytarabine (Ara-C), doxorubicin (Dox), or
5-fluorouracil (5-FU), or a combination of a radiotherapeutic and a
chemotherapeutic.
[0034] In a seventh aspect, the invention provides a method for
preserving progenitor cells ex vivo comprising contacting a
population of cells comprising at least one progenitor cell with an
effective amount of a composition of a FRIL family member for an
effective period of time, wherein the progenitor cells in the
population are rendered quiescent.
[0035] In certain embodiments of the seventh aspect, the progenitor
cells are from a human. In certain embodiments, the population of
cells is bone marrow cells. In some embodiments, the non-progenitor
cells in the population of cells differentiate or die.
[0036] In certain embodiments of the seventh aspect, the population
of cells is removed from a cancer patient prior to treatment of the
cancer patient with a therapeutic treatment having a hematopoietic
progenitor cell-depleting activity. In some embodiments, the
therapeutic treatment is a radiotherapeutic or a chemotherapeutic
treatment, including, without limitation, cytarabine (Ara-C),
doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combination of a
radiotherapeutic and a chemotherapeutic.
[0037] In an eighth aspect, the invention provides a method for
preserving progenitor cells in vivo, comprising administering to a
patient an effective amount of a composition of a FRIL family
member for an effective period of time, wherein the progenitor
cells in the patient are rendered quiescent. Preferably, the
patient is a human or a domesticated animal.
[0038] In certain embodiments of the eighth aspect of the
invention, the patient is a cancer patient. In some embodiments,
the effective amount of the composition of a FRIL family member is
administered prior to the treatment of the patient with a
therapeutic treatment having a hematopoietic progenitor
cell-depleting activity. In some embodiments, the therapeutic
treatment is a radiotherapeutic or a chemotherapeutic treatment,
including, without limitation, cytarabine (Ara-C), doxorubicin
(Dox), or 5-fluorouracil (5-FU), or a combination of a
radiotherapeutic and a chemotherapeutic.
[0039] In a ninth aspect, the invention provides a method for
identifying a progenitor cell, comprising contacting a candidate
cell with a FRIL family member molecule, wherein binding of the
candidate cell to the FRIL family member molecule identifies the
candidate cell as a progenitor cell.
[0040] In certain embodiments of the ninth aspect, the candidate
cell is in a population of cells. In certain embodiments, the
candidate cell is from a human.
[0041] In a tenth aspect, the invention provides a progenitor cell
identified by a method comprising contacting a candidate cell with
a FRIL family member molecule, wherein binding of the candidate
cell to the FRIL family member identifies the candidate cell as a
progenitor cell.
[0042] In an eleventh aspect, the invention provides a method for
identifying a composition of a member of the FRIL family of
progenitor cell preservation factors, comprising contacting a
candidate compound with a glycosylated extracellular domain of an
FLT3 receptor, wherein the glycosylation pattern of the
extracellular domain of the FLT3 receptor is the same as the
glycosylation pattern of an extracellular domain of a normally
glycosylated FLT3 receptor, wherein a candidate compound that binds
the glycosylated extracellular domain of the FLT3 receptor is
identified as a composition of a FRIL family member.
[0043] In certain embodiments of the eleventh aspect, the candidate
compound is a lectin. In certain embodiments, the lectin is
synthetic. In certain embodiments, the lectin is from a legume.
[0044] In a twelfth aspect, the invention provides an essentially
pure composition of a FRIL family member identified by the method
comprising contacting a candidate compound with a glycosylated
extracellular domain of an FLT3 receptor, wherein the glycosylation
pattern of the extracellular domain of the FLT3 receptor is the
same as the glycosylation pattern of an extracellular domain of a
normally glycosylated FLT3 receptor, wherein a candidate compound
that binds the glycosylated extracellular domain of the FLT3
receptor is identified as a member of the FRIL family.
[0045] According to the invention, compositions of a FRIL family
member may be used as therapeutic agents to preserve progenitor
cells in patients, such as cancer patients receiving chemotherapy,
who have suffer from a condition that diminishes their progenitor
cells. For example, compositions of a FRIL family member may be
administered with a pharmaceutically-acceptable carrier (e.g.,
physiological sterile saline solution) via any route of
administration to a cancer patient receiving chemotherapy in an
attempt to reduce the progenitor cell-depleting effects of the
chemotherapeutic so that the patient can receive a higher dose of
the chemotherapeutic and, preferably, recover from cancer.
Pharmaceutically-acceptable carriers and their formulations are
well-known and generally described in, for example, Remington's
Pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack
Publishing Co., Easton, Pa., 1990).
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a map of a cloning vector pCR2.1-DLA manufactured
by ligating a cDNA according to the invention in the EcoRI site of
the cloning vector pCR2.1.
[0047] FIG. 2 shows a direct amino acid sequence comparison of the
mannose lectin described by Gowda et al. (J Biol Chem
269:18789-18793, 1994) and the derived amino acid sequence of
Dl-FRIL, a representative, non-limiting FRIL family member of the
invention, encoded by a representative, non-limiting nucleic acid
of the invention.
[0048] FIG. 3 is a map of a cloning vector pCR2.1-DLA(D)
manufactured by ligating a mutated cDNA in the EcoRI site of the
cloning vector pCR2.1.
[0049] FIG. 4 is a map of a cloning vector pBS-SpDLA manufactured
by ligating a recombinant fragment in the EcoRI site of the cloning
vector pBluescript SK+.
[0050] FIG. 5 is a map of a cloning vector pCR2.1-SpM1(D)
manufactured by ligating a mutated recombinant clone in the EcoRI
site of the cloning vector pCR2.1.
[0051] FIG. 6 is a map of a recombinant expression vector
pBIN-VicPro manufactured by subcloning the vicilin promoter
obtained from the pCW66 vector in the EcoRI/ClaI site of the plant
binary vector pBIN19 for Agrobacterium-mediated transformation.
[0052] FIG. 7 is a map of a recombinant expression vector
pBINVicPro-SpDLA manufactured by ligating a recombinant fragment in
the EcoRI/SacI site of the pBINVicPro vector.
[0053] FIG. 8 is a map of a recombinant expression vector
pBINVicPro-SpDLA(D) manufactured by ligating a mutated recombinant
clone in the EcoRI site of the pBINVicPro vector.
[0054] FIG. 9 is a map of a recombinant expression vector
pGEX4T-1-DLA manufactured by ligating a wild-type cDNA clone in the
EcoRI/SalI site of the E. coli expression vector pGEX4T-1.
[0055] FIG. 10 is a map of a recombinant expression vector
pGEX4T-1-DLA(D) manufactured by ligating a mutant cDNA clone in the
EcoRI/XhoI site of the E. coli expression vector pGEX4T-1.
[0056] FIG. 11 is a representation of an electrophoretogram of a
Southern blot of total protein extracts of E. coli cells
transformed with the recombinant expression vectors pGEX4T-1-DLA
and pGEX4T-1-DLA(D).
[0057] FIG. 12 is a representation of an electrophoretogram of a
Western blot of purified GST-fusion proteins with and without
cleavage by thrombin.
[0058] FIG. 13A is a representation of a graph showing that a crude
extract of an E. coli culture containing expressed Dl-FRIL, a
representative, non-limiting FRIL family member of the invention,
specifically stimulates hFLT3 3T3 cells; FIG. 13B is a graph
showing that the same extract does not stimulate untransfected 3T3
cells.
[0059] FIG. 14A is a representation of a histogram showing that
purified Dl-FRIL, a representative, non-limiting FRIL family member
of the invention, preserves cord blood mononudear cells in a
dose-responsive manner.
[0060] FIG. 14B is a representation of a histogram showing that
purified Dl-FRIL, a representative, non-limiting FRIL family member
of the invention, preserves hematopoietic progenitors in a
dose-responsive manner.
[0061] FIG. 15A is a representation of a photograph of colonies
derived from human cord blood mononuclear cells cultured in 40
ng/mL Dl-FRIL, a representative, non-limiting FRIL family member of
the invention, for 3 weeks, and then replated in methylcellulose
colony assay medium.
[0062] FIG. 15B is a representation of a photograph of colonies
derived from human cord blood mononuclear cells cultured in 40
ng/mL Dl-FRIL, a representative, non-limiting FRIL family member of
the invention, for 4 weeks, and then replated into methylcellulose
colony assay medium.
[0063] FIG. 16 is a schematic diagram showing the serial replating
of progenitor cells cultured in Dl-FRIL, a representative,
non-limiting FRIL family member of the invention, or Dl-FRIL+recFL
(i.e., recombinant FLT3-Ligand). The human cord mononuclear cells
were first cultured in supensiion in 40 ng/mL Dl-FRIL of 40 ng/mL
D1-FRIL+40 ng/mL recFL (solid black box). The cells were then
harvested and assessed for progenitor activity by being replated
into methylcellulose colony assay medium for 6 weeks (middle
striped box). Then the cells were harvested from the colony assay
and again replated into methylcellulose colony assay medium for an
additional 4 weeks (far right striped box). Progenitor frequencies
were determined for cells after 3 weeks of suspension culture in
Dl-FRIL or Dl-FRIL+recFL, and after an additional 6 weeks of
methylcellulose culture (absent Dl-FRIL and/or recFL).
[0064] FIG. 17 is a representation of a photograph of colonies
derived from human cord blood mononuclear cells initially cultured
in 40 ng/mL Dl-FRIL, a representative, non-limiting FRIL family
member of the invention, for 3 weeks, then replated into
methylcellulose colony assay medium for 6 weeks, and then replated
again into methylcellulose colony assay medium for an additional 4
weeks.
[0065] FIG. 18 is a representation of a bar graph showing that a
representative, non-limiting FRIL family member of the invention,
Dl-FRIL, encoded by a representative, non-limiting nucleic acid of
the invention is sufficient to preserve progenitor cells ex vivo,
whereas a cytokine cocktail fails to preserve such cells.
[0066] FIGS. 19A and 19B are representations of line graphs showing
the biological specificity of receptor-transfected 3T3 cells. FIG.
19A shows that rhM-CSF specifically stimulated Fms 3T3 (solid
circles) but not either mFlt3/Fms 3T3 (open circles) or parent 3T3
cells (solid squares) in biological screening assay in a
dose-dependent manner. FIG. 19B shows that PHA-LCM (reciprocal
dilution) stimulated mFlt3/Fms 3T3 (solid circles) and Stk 3T3
(open circles) but not parent untransfected 3T3 cells (solid
squares).
[0067] FIGS. 20A-20D are representations of the detected biological
activities of PHA-LCM fractionated by anion exchange
chromatography. FIG. 20A shows the number of viable cells cord
blood cells observed microscopically. FIG. 20B shows the
stimulation of mFlt3/Fms 3T3 cells by anion exchange column
fractions. FIG. 20C shows the stimulation of Stk 3T3 cells by anion
exchange column fractions. FIG. 20D shows the stimulation of Fms
3T3 cells by anion exchange column fractions.
[0068] FIGS. 21A and 21B are representations of line graphs showing
the co-factors required in the Flt3 3T3 assay during purification
of Pv-FRIL, a representative, non-limiting FRIL family member of
the invention. FIG. 21A shows that the plateau stimulation of Flt3
3T3 cells decreased during purification from crude, 10-fold
concentrated PHA-LCM (solid circles) to partially purified (open
circles), and highly purified (solid squares). Medium control is
shown is open squares. FIG. 21B shows that the decreased plateau
stimulation of Flt3 3T3 cells (solid circles) was restored by
addition of sub-optimal concentrations (1:200) of crude PHA-LCM
(solid squares). Corresponding medium controls are shown in open
symbols.
[0069] FIGS. 22A-22D are representations of a series of flow
cytometry histograms showing the re-analysis of CD34 expression of
cord blood CD34.sup.+ cells after two weeks in suspension culture
with pooled AQS affinity column fractions. CD34 expression was
re-analyzed by flow cytometry on pooled AQS affinity column
fractions 1-5 (FIG. 22A), fractions 6-10 (FIG. 22B), fractions
11-15 (FIG. 22C), and fractions 16-20 (FIG. 22D). Fluorescence
intensity on the abcissa and frequency of events on the ordinate in
FIGS. 22A-22D.
[0070] FIG. 23 is a representation of a line graph showing the
IL1-dependent response of mFlt3/Fms 3T3 cells to FRIL isolated from
commercial red kidney bean extract. mFlt3/Fms 3T3 cells respond to
FRIL in the presence of rhIL1 (solid circles) but not the absence
of IL1 (solid squares). Corresponding medium only controls are
shown with open symbols.
[0071] FIG. 24A is a map of a cloning vector pCR2.1-Pv-FRIL
manufactured by ligating a cDNA according to the invention in the
EcoRI site of the cloning vector pCR2.1.
[0072] FIG. 24B shows a direct amino acid sequence comparison of
Pv-FRIL, a representative, non-limiting FRIL family member of the
invention, with Dl-FRIL, another representative, non-limiting FRIL
family member of the invention, and the PHA lectin, PHA-E.
[0073] FIG. 25 is a map of a cloning vector pCR2.1-SpPv-FRIL
manufactured by ligating a cDNA according to the invention in the
Xhol site of the cloning vector pCR2.1.
[0074] FIG. 26 is a map of a cloning vector pM-SpPv-FRIL
manufactured by ligating a cDNA according to the invention in the
Bg1II/Xhol sites of the cloning vector SpPv-FRIL.
[0075] FIGS. 27A-27B are representations of line graphs showing the
total cell numbers and progenitor levels in the presence of
Dl-FRIL,a representative, non-limiting FRIL family member of the
invention, or cytokines. Enriched CB CD34.sup.+ cells were cultured
for 3, 6, 10, or 13 days in the presence of Dl-FRIL (solid symbols)
or cytokines (open symbols). Colonies were scored on day 14 and
progenitor levels were calculated based on total cell numbers.
Values shown represent the mean.+-.SEM of data from up to 10
experiments. FIG. 27A shows the total cell numbers over time. FIG.
27B shows the progenitor levels in cultures over time.
[0076] FIGS. 28A-28D are representations of line and bar graphs
showing the total cell numbers and progenitor levels first in the
presence of Dl-FRIL, a representative, non-limiting FRIL family
member of the invention, and second in presence of cytokines. FIG.
28A shows the total numbers of cells cultured with Dl-FRIL for
entire 10 days (solid symbols) or for 6 days followed by 4 days of
cytokine stimulation (open symbols). FIG. 28B shows the progenitor
levels in cells cultured with Dl-FRIL for entire 10 days (solid
symbols) or for 6 days followed by 4 days of cytokine stimulation
(open symbols). FIG. 28C shows the total numbers of cells cultured
with Dl-FRIL for 13 days (solid symbols) or for 10 days followed by
3 days of cytokine stimulation (open symbols). FIG. 28D shows the
progenitor levels in cells cultured with Dl-FRIL for 13 days (solid
symbols) or for 10 days followed by 3 days of cytokine stimulation
(open symbols).
[0077] FIGS. 29A-29D are representations of representative Southern
blot analyses showing the quantitative analysis of SRC after ex
vivo cultures with Dl-FRIL,a representative, non-limiting FRIL
family member of the invention, or cytokines and after
transplantation into mice. FIG. 29A is a representation of a
representative Southern blot showing human DNA in the marrow of
mice transplanted with cells that were cultured with Dl-FRIL for 6
days (lane 1), Dl-FRIL for 10 days (lane 2), or with Dl-FRIL for 6
days followed by 4 days with cytokine stimulation (lane 3). FIG.
29B is a representation of a representative Southern blot showing
human DNA in the marrow of mice transplanted with cells cultured
with Dl-FRIL for 10 days (lanes 1-2), or with Dl-FRIL for 6 days
followed by 4 days with cytokine stimulation (lanes 34). FIG. 29C
is a representation of a representative Southern blot showing human
DNA in the marrow of mice transplanted with the original cells
prior to seeding (lane 1), or with cells cultured with Dl-FRIL for
13 days (lane 2). FIG. 29D is a representation of a representative
Southern blot showing human DNA in the marrow of mice transplanted
with cells cultured with FRIL for 10 days (lane 1), Dl-FRIL for 6
days followed by 4 days of cytokine stimulation (lane 2), Dl-FRIL
for 13 days (lane 3), or with Dl-FRIL for 10 days followed by 3
days of cytokine stimulation (lane 4).
[0078] FIG. 29E is a representation of a Southern blot analysis
showing the detection of a 0%; 0.1%, 1%, and 10% human DNA per
murine DNA.
[0079] FIG. 30 is a representation of a survival chart summarizing
the levels of human cell engraftment in the marrow of mice
transplanted with CD34.sup.+ cells cultured in either Dl-FRIL, a
representative, non-limiting FRIL family member of the invention,
for ten days (left panel), or in the presence of Dl-FRIL for 6 days
followed by culture in the presence of cytokines for four days.
[0080] FIG. 31 is a representation of a representative Southern
blotting analysis showing the levels of levels of human cell
engraftment in the BM of NOD/SCID B2M.sup.null transplanted with
CD34.sup.+CD38.sup.-/low cells cultured in the presence of Dl-FRIL,
a representative, non-limiting FRIL family member of the invention.
Sorted cells (2.times.10.sup.5 initial cells/treatment) were
cultured in the presence of Dl-FRIL for 6 days followed by
additional 4 days exposure to cytokines, or with Dl-FRIL alone for
10 days. After 10 days, 3.6.times.10.sup.5 cells harvested from
cytokine culture were divided and transplanted into 3 mice (lanes
1-3), while 3.5.times.10.sup.4 cells harvested from Dl-FRIL alone
culture were transplanted to one mouse (lane 4). DNA was harvested
from the bone marrow of transplanted mice and subjected to Southern
blotting analysis with radiolabeled human chromosome 17-specific
.alpha.-satellite probe (p17H8). A representative experiment out of
4 is shown.
[0081] FIG. 32 is a representation of a bar graph showing the
average fold increase of engraftment levels obtained with
CD34.sup.+CD38.sup.-/low cells that were cultured with Dl-FRIL, a
representative, non-limiting FRIL family member of the invention,
for 6 days and with cytokines for 4 days (right bar), as compared
to 10 days with Dl-FRIL alone (left bar). Data represent mean.+-.SE
from 4 experiments.
[0082] FIGS. 33A-33F are representations of a bar graph (FIG. 33A)
and representative flow cytometry histograms (FIG. 33B-33F) showing
the multilineage differentiation of SRC cultured with Dl-FRIL, a
representative, non-limiting FRIL family member of the invention,
in the BM of transplanted mice. FIG. 33A shows the number of
colonies per 2.times.10.sup.5 cells, where the cells treated as
indicated prior to transplantation into mice were recovered from
the murine BM, and seeded into semisolid media selective for human
colonies. Progenitor levels were calculated based on the total
human cell numbers (2.times.10.sup.5 cells) in the marrow of
transplanted mice. Values shown represent the mean.+-.SEM of data
from 3 experiments, 9 mice/treatment. FIG. 33B shows a
representative flow cytometry analysis showing nonspecific labeling
where mouse IgG was used as isotype control. FIGS. 33C and 33D show
representative flow cytometry analyses of BM cells from mice that
were transplanted with CD34.sup.+ cells (FIG. 33C) or
CD34.sup.+CD38.sup.-/low cells (FIG. 33D) precultured with Dl-FRIL
for 10 days, where the harvested BM cells were stained with anti
human CD45 and anti-human CD19. FIGS. 33E and 33F show
representative flow cytometry analyses of BM cells from mice that
were engrafted by CD34.sup.+ cells (FIG. 33E) or
CD34.sup.+CD38.sup.-/low cells (FIG. 33F) precultured with Dl-FRIL
for 10 days, where the harvested BM cells were subsequently
cultured with SCF+IL-15 for 10 days, and then stained with human
specific monoclonal anti-CD45 and anti-CD56.
[0083] FIGS. 34A-34B are representations of bar graphs showing the
growth effect of Dl-FRIL, a representative, non-limiting FRIL
family member of the invention, on CD34.sup.+ cells and progenitors
compared to Flt3 ligand and cytokine combinations. (+) indicates
co-culture of factors for the entire 10 days while (.fwdarw.)
indicates substitution of the first factor after 6 days with
cytokines, as indicated under the x axis. FIG. 34A shows the total
cell numbers. FIG. 34B shows the percentage of CFU-GEMM out of
total colonies. Values shown are per 2.times.10.sup.5 seeded cells,
and represent the mean.+-.SEM of data from 5 experiments.
[0084] FIGS. 35A-35G are representatives of flow cytometry
histograms showing the cell cycle analyses of CD34.sup.+ cells
cultured with Dl-FRIL, a representative, non-limiting FRIL family
member of the invention, or various cytokines, or with combinations
thereof. DNA content was determined by flow cytometry with
propidium iodide staining. Enriched CD34.sup.+ cells were cultured
for 3 days with no treatment (FIG. 35A), Dl-FRIL (FIG. 35B), Flt3-L
(FIG. 35C), Dl-FRIL+Flt3-L (FIG. 35D), SCF+G-CSF+IL-3+IL-6 (SG36)
(FIG. 35E), SG36+Dl-FRIL (FIG. 35F), and SG36+Flt3-L (FIG.
35G).
[0085] FIGS. 36A-36C are representations of line graphs showing the
dose response of CB mnc chemotherapeutic agents in the presence and
absence of Dl-FRIL, a representative, non-limiting FRIL family
member of the invention. Chemotherapy agents were assayed over a
5-log dose range on CB MNC (2.times.10.sup.5 cells/0.1 mL) in AIMV
(Life Technologies) containing Agar-SCM (StemCell Technologies).
Viable cells were determined after 5 days of culture by XTT. Solid
squares indicate chemotherapy drug with no Dl-FRIL; solid triangles
indicate cultures containing Dl-FRIL at 10 ng/ml in all wells; and
open circles indicate Dl-FRIL in all wells at 100 ng/ml. FIG. 36A
shows the dose response to Ara-C; FIG. 36B shows the dose response
to doxorubicin, and FIG. 36C shows the dose response to 5-FU.
[0086] FIG. 37 is a representation of a photograph of purified
Dl-FRIL, a representative, non-limiting FRIL family member of the
invention, resolved by SDS-PAGE analysis. Five discrete bands
appeared which corresponded to the .alpha. and .beta. subunits,
each of which was subjected to amino-terminal sequencing. The amino
acid sequences of the five bands are as indicated.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0087] The invention relates the preservation of progenitor cells
by members of the FRIL family of progenitor cell preservation
factors. More specifically, the invention relates to the in vivo or
ex vivo preservation of progenitor cells, such as hematopoietic
progenitor cells, using members of the FRIL family of progenitor
cell preservation factors.
[0088] The invention provides a family of factors that preserve
progenitor cells. Members of this family, the FRIL family, are
non-toxic, inexpensively produced reagents that preserve progenitor
cells. The invention provides compositions comprising at least one
member of the FRIL family, as well as methods for using members of
the FRIL family to preserve progenitor cells both in vivo and ex
vivo.
[0089] All of the patents and publications cited herein reflect the
knowledge in the art and are hereby incorporated by reference in
entirety to the same extent as if each were specifically stated to
be incorporated by reference. Any inconsistency between these
patents and publications and the present disclosure shall be
resolved in favor of the present disclosure.
[0090] In a first aspect, the invention provides an essentially
pure composition of a member of the FRIL family of progenitor cell
preservation factors. The term, "FRIL family of progenitor cell
preservation factors" is used to mean a family of lectins, wherein
each FRIL family member molecule binds to a normally glycosylated
FLT3 receptor, wherein each FRIL family member molecule preserves
progenitor cells, and wherein one FRIL family member molecule that
is isolated from a hyacinth bean (i.e., Dolichos lab lab) has an
amino acid sequence which comprises the following eight amino acid
sequence: TNNVLQXT (SEQ ID NO: 24). By "FRIL family member" or FRIL
family member molecule" is meant one or more molecules of the FRIL
family of progenitor cell preservation factors.
[0091] In accordance with the first aspect of the invention, a
composition of a FRIL family member, which includes a mutant of
another FRIL family member molecule or a fusion protein comprising
a portion derived from a FRIL family member molecule or mutant
thereof, wherein each FRIL family member molecule binds to a
normally glycosylated FLT3 receptor has at least about 45% amino
acid sequence identity with the amino acid sequence of another
member of the FRIL family, preferably at least about 50% identity,
even more preferably at least about 55% identity, still more
preferably at least about 60% identity, and still more preferably
at least about 65% identity with the sequence of the second
protein. In the case of proteins having high sequence identity, the
amino acid sequence of the first protein shares at least about 75%
sequence identity, preferably at least about 85% identity, and more
preferably at least about 95% identity, with the amino acid
sequence of another member of the FRIL family.
[0092] Both amino acid sequence identity and nucleic acid sequence
identity between two proteins or two nucleic add molecules can be
measured according to standard methods. For example, in order to
compare a first amino acid sequence to a second amino acid sequence
or a first nucleic acid sequence to a second nucleic acid sequence
for the purpose of determining percentage identity between the two
sequences, the sequences are aligned so as to maximize the number
of identical amino acid or nucleic acid residues. The sequences of
proteins sharing at least 50% amino add sequence identity or the
sequences of nucleic acids sharing at least 45% nucleic acid
sequence identity can usually be aligned by visual inspection. If
visual inspection is insufficient, the proteins or nucleic acids
may be aligned in accordance with the FASTA method in accordance
with Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444-2448,
1988), or, preferably, any of the methods described by George, D.
G. et al., in Macromolecular Sequencing and Synthesis, Selected
Methods and Applications, pages 127-149, Alan R. Liss, Inc. (1988),
such as formula 4 at page 137 using a match score of 1, a mismatch
score of 0, and a gap penalty of -1. From this method, percentage
of sequence identity between the first and second amino acid
sequences or between the first and second nucleic acid can be
determined.
[0093] Other methods for determining amino acid or nucleic acid
sequence identity are described in Feng and Doolittle (Journal of
Molecular Evolution 25: 351-360, 1987) and Higgins and Sharp
(CABIOS 5: 151-153,1989).
[0094] Another method for determining amino acid or nucleic acid
sequence identity between two proteins or nucleic acids is by using
sequence analysis software with the default parameters specified
therein. Various software packages exist including Sequence
Analysis Software Package of the Genetics Computer Group
(University of Wisconsin Biotechnology Center, Madison, Wis.), and
the various BLAST programs of the National Center for Biotechnology
(National Library of Medicine, Bethesda, Md.).
[0095] Unless otherwise specified, percentage of amino acid
sequence identity or percentage of nucleic acid sequence identity
is determined using the basic BLAST program of the National Center
for Biotechnology (National Library of Medicine, Bethesda, Md.),
using the default settings defined therein.
[0096] Another test for percentage identity of two nucleic acid
sequences is whether they hybridize under normal hybridization
conditions, preferably under stringent hybridization conditions.
Thus, also included in the invention are proteins that are encoded
by nucleic acid molecules that hybridize under high stringent
conditions to a sequence complementary to SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, and/or SEQ ID NO: 7. The term "stringent
conditions," as used herein, is equivalent to "high stringent
conditions" and "high stringency." These terms are used
interchangeably in the art.
[0097] Stringent conditions are defined in a number of ways. In one
definition, stringent conditions are selected to be about
50.degree. C. lower than the thermal melting point (T.sub.m) for a
specific sequence at a defined ionic strength and pH. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched
sequence. Typical stringent conditions are those in which the salt
concentration is at least about 0.02 M at pH 7 and the temperature
is at least about 60.degree. C. "Stringent conditions," in
referring to percentage identity (e.g., homology) or substantial
similarity in the hybridization context, can be combined conditions
of salt, temperature, organic solvents or other parameters that are
typically known to control hybridization reactions. The combination
of parameters is more important than the measure of any single
parameter. If incompletely complementary sequences recognize each
other under high stringency conditions, then these sequences
hybridize under conditions of high stringency (see U.S. Pat. No.
5,786,210; Wetmur and Davidson, J. Mol. Biol. 31, 349-370, 1968).
Control of hybridization conditions, and the relationships between
hybridization conditions and degree of homology are understood by
those skilled in the art. See, e.g., Sambrook et al., Molecular
Cloning. A Laboratory Manual, 2ed., Cold Spring Harbor Laboratory,
Cold Spring Harbor, 1989. Further examples of stringent conditions
can be found in Goeddel et al., U.S. Pat. No. 5,789,550.
[0098] In a non-limiting example, "stringent conditions" can be
provided in a variety of ways such as overnight incubation at
42.degree. C. in a solution comprising: 20% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6),5.times.Denhardt's solution, 10% dextran sulfate, and 20
.mu.g/ml denatured, sheared salmon sperm DNA. Alternatively, the
stringent conditions are characterized by a hybridization buffer
comprising 30% formamide in 5.times.SSPE (0.18 M NaCl, 0.01 M
NaPO.sub.4, pH 7.7, 0.0001 M EDTA) buffer at a temperature of
42.degree. C., and subsequent washing at 42.degree. C. with
0.2.times.SSPE. Preferably, stringent conditions involve the use of
a hybridization buffer comprising 50% formamide in 5.times.SSPE at
a temperature of 42.degree. C. and washing at the same temperature
with 0.2.times.SSPE.
[0099] As used herein, by "preserves progenitor cells" is meant an
ability of a FRIL family member molecule (or mutant thereof or
fusion protein comprising a FRIL family member molecule or mutant
thereof) to retain (i.e., preserve) progenitor cells in an
undifferentiated state, which can be determined using the assays
described below (e.g., the SCID mouse reconstituting cell assay and
the methylcellulose or other semi-solid medium based hematopoietic
progenitor cell assay). In accordance with the invention,
"progenitor cell" refers to any normal somatic cell that has the
capacity to generate fully differentiated, functional progeny by
differentiation and proliferation. Progenitor cells include
progenitors from any tissue or organ system, including, but not
limited to, blood, mesenchymal, embryonic, nerve, muscle, skin,
gut, bone, kidney, liver, pancreas, thymus, brain and the like.
Progenitor cells are distinguished from "differentiated cells," the
latter being defined as those cells that may or may not have the
capacity to proliferate, i.e., self-replicate, but that are unable
to undergo further differentiation to a different cell type under
normal physiological conditions. Moreover, progenitor cells are
further distinguished from abnormal cells such as neoplastic cells,
as defined herein. For example, leukemia cells proliferate
(self-replicate), but generally do not further differentiate,
despite appearing to be immature or undifferentiated.
[0100] Progenitor cells include all the cells in a lineage of
differentiation and proliferation prior to the most differentiated
or the fully mature cell. Thus, for example, progenitors include
the skin progenitor in the mature individual. The skin progenitor
is capable of differentiation to only one type of cell, but is
itself not fully mature or fully differentiated.
[0101] By "hematopoiesis" is meant the development of mature,
functional blood cells. The progenitor cells that give rise to
mature, functional blood cells are called hematopoietic progenitor
cells. The most primitive, undifferentiated hematopoietic
progenitor cell is called a hematopoietic stem cell. Hematopoietic
stem cells typically reside in the bone marrow primarily in a
quiescent state, and may form identical daughter cells through a
process called "self-renewal."
[0102] Production of some mature, functional blood cells results
from proliferation and differentiation of "unipotential
progenitors," i.e., those progenitors that have the capacity to
make only one type of blood cell. For red blood cell (erythrocyte)
production, a unipotential progenitor called a "CFU-E" (colony
forming unit-erythroid) has the capacity to generate two to 32
mature progeny cells. Various other hematopoietic progenitors have
been characterized. For example, hematopoietic progenitor cells
include those cells that are capable of successive cycles of
differentiating and proliferating to yield up to eight different
mature hematopoietic cell lineages.
[0103] Uncommitted progenitor cells, such as hematopoietic stem
cells, can be described as being "totipotent," i.e., both necessary
and sufficient for generating all types of mature cells. Progenitor
cells that retain a capacity to generate all cell lineages, but
that can not self-renew, are termed "pluripotent." Cells that can
produce some but not all blood lineages and can not self-renew are
termed "multipotent."
[0104] Progenitor cells can be defined by mRNA levels of genes that
either specifically regulate progenitors or serve as markers of
lineage commitment. For example, genes induced in primitive human
hematopoietic progenitor cells include those encoding the shared
beta subunits of the IL3, IL5, and/or granulocyte-macrophage
colony-stimulating factor (GM-CSF) receptors, termed the beta
common chain (McClanahan et al., Blood 81:3903-2915, 1993); CD34
genes; and/or the receptors for Kit (Turner et al., Blood
88:3383-3390, 1996), FLT1, FLT4 (Galland et al., Oncogene
8:3233-1240, 1993), FLK1 (Broxmeyer et al., Int. J. Hematol.
62:303-215, 1995), and FLT3 (Lyman and Jacobsen, Blood
91:3101-1134., 1998). Those genes for intermediate progenitors
include the c-Fms, G-CSF receptor, and/or CD34 genes; and the IL-7
receptor gene, a gene induced for B lymphopoiesis.
[0105] Murine primitive progenitor populations include receptors
for interleukin-1 alpha (IL-1.alpha.), IL-3, IL-6, granulocyte
colony-stimulating factor (G-CSF), and/or FLK1-1 (the murine
homologue of human KDR which binds VEGF) (Broxmeyer, supra), but
lack receptors for macrophage colony-stimulating factor (M-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), and
leukemia inhibitory factor (LIF). Cells within the intermediate
progenitor cell population include receptors for GM-CSF, G-CSF,
IL-6, and/or IL-1.alpha..
[0106] In accordance with the first aspect of the invention, the
terms "bind," "binds," or "bound" are used interchangeably to mean
that a FRIL family member molecule of the invention binds to a
normally glycosylated FLT3 receptor with an affinity higher than
the affinity with which the FLT3-Ligand binds the FLT3 receptor.
Preferably, a FRIL family member molecule binds to a normally
glycosylated FLT3 receptor with an affinity that is at least as
high as the affinity with which an antibody binds its specific
ligand. Even more preferably, a FRIL family member molecule of the
invention binds to a normally glycosylated FLT3 receptor with an
affinity that is higher than the affinity with which an antibody
binds its specific ligand. Still more preferably, a FRIL family
member molecule of the invention binds to a normally glycosylated
FLT3 receptor with a dissociation constant (K.sub.D) of at least
10.sup.-7 M, more preferably 10.sup.-8 M, even more preferably
10.sup.-9 M, still more preferably, at least 10.sup.-10 M, and most
preferably, a FRIL family member molecule of the invention binds to
a normally glycosylated FLT3 receptor with a dissociation constant
(K.sub.D) of at least 10.sup.-11 M. Standard methods for
determining binding and binding affinity are known.
[0107] In accordance with the invention, by "normally glycosylated
FLT3 receptor" is meant an FLT3 receptor that has a glycosylation
pattern of an FLT3 cell glycosylated by a normal cell. By "normal
cell," as used herein in accordance with all aspects of the present
invention, is meant a cell that is not neoplastic. As used herein,
by "neoplastic cell" is meant a cell that shows aberrant
proliferation, particularly increased proliferation, that is not
regulated by such factors as cell-cell contact inhibition and
soluble regulators (e.g., cytokines or hormones), and that
abnormally glycosylates the FLT3 receptor such that the
glycosylation pattern on the FLT3 receptor on the neoplastic cells
is abnormal and such that the FLT3 receptor on the neoplastic cell
is not bound by a FRIL family member molecule.
[0108] In accordance with the first aspect of the invention, by
"essentially pure" means a molecule, such as a nucleic acid or
protein (e.g., a FRIL family member molecule), or composition of a
molecule that is more free from other organic molecules (e.g.,
carbohydrates, nucleic acids, proteins, and lipids) that naturally
occur with an impure molecule, and is substantially free as well of
materials used during the purification process. For example, a
protein or nucleic acid molecule is considered to be essentially
pure if it is at least approximately 60%, preferably at least
approximately 75%, more preferably approximately at least 85%, most
preferably approximately at least 90%, and optimally approximately
at least 95% pure, i.e., free from other organic molecules with
which it naturally occurs and free from materials used during the
purification process. Methods for purifying proteins are known in
the art and include, without limitation, HPLC, SDS-PAGE,
immunoprecipitation, recombinant protein production, affinity
chromatography using specific antibodies, ion-exchange,
size-exclusion, and hydrophobic interaction chromatography, or a
combination of any of these methods. These and other suitable
methods are described, e.g., in Marston, "The purification of
eukaryotic proteins expressed in E. coli," in DNA Cloning, Glover
D. M., ed., Volume III, IRL Press Ltd., Oxford, 1987; Marston and
Hartley, "Solubilization of protein aggregates," pp. 266-267 in
Guide to Protein Purification, Deutscher M P, ed., Academic Press,
San Diego, 1990; Laemmli, U. K., Nature 227:680-685, 1970. A FRIL
family member can also be purified by binding to a mannose, which
may be coupled on a sold support (e.g., a sepharose bead).
[0109] Methods for purifying nucleic adds are known in the art and
include, without limitation, Guanidine-HCl extraction, polymerase
chain reaction, CsCl gradient fractionation, phenol: chloroform
extraction, ethanol precipitation, and standard recombinant DNA
methodologies. Standard methods for purifying both proteins and
nucleic acid molecules are provided in, e.g., Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y., 1994; Sambrook et al., supra.
[0110] In accordance with the first aspect of the invention, a FRIL
family member molecule may be purified from a natural source by
methods well known in the art. For example, the purification of
Dl-FRIL from Dolichos lab lab is described below in Example 1. The
purification of Pv-FRIL from Phaseolus vulgaris as described below
in Example 5. The purification of YamFRIL from Sphenostylis
stenocarpa is described below in Example 22. Such methods also
include, for example, those described by Moore in PCT application
PCT/US97/22486 and by Gowda et al., supra. A suitable natural
source from which to purify a FRIL family member molecule includes
plants, especially legume plants. Legumes, such as the garden pea
or the common bean, are plants ("leguminous plants") from a family
(Leguminosae) of dicotyledonous herbs, shrubs, and trees bearing
(nitrogen-fixing bacteria) nodules on their roots. These plants are
commonly associated with their seeds (e.g., the garden pea or the
common bean)
[0111] More specifically, a FRIL family member molecule according
to the first aspect of the invention can be purified from members
of the tribe Phaseoleae. For example, a FRIL family member molecule
can be purified from Dolichos lab lab (e.g., hyacinth beans, which
is also known by other common names throughout the world).
Alternatively, a FRIL family member molecule can be purified from
varieties of the common bean (Phaseolus vulgaris) (e.g., red kidney
beans and white kidney beans), from yam bean (Sphenostylis
stenocarpa) or from Vigna sinensis, commonly known as the
black-eyed pea.
[0112] As demonstrated in the examples below, purification of a
FRIL family member molecule from a legume is rapid and inexpensive,
and results in a large amount of essentially pure lectin. A native
FRIL family member molecule can be easily purified from legumes,
such as hyacinth beans (pesticide-free), by mannose-affinity
chromatography or ovalbumin affinity chromatography, and is more
than 100 times cheaper to produce than recombinant cytokines. In
accordance with the first aspect of the invention, by "lectin" is
meant a protein that binds sugar residues with high affinity. Most
preferably, a FRIL family member molecule is a
mannose/glucose-specific legume lectin.
[0113] As demonstrated in the examples below, FRIL family member
molecules, and compositions of FRIL family member molecules, have
many attributes as reagents to either alleviate the progenitor
cell-depleting activity of a therapeutic (e.g., a chemotherapeutic)
or to alleviate the symptoms of a condition where the patient's
progenitor cells are depleted. For example, FRIL family members
have unique properties and are the first soluble regulators
reported to preserve hematopoietic stem cells and progenitors in a
dormant state for extended periods, even in the presence of potent
stimulators of proliferation and differentiation. Moreover, because
mice tolerate very high levels of compositions of FRIL family
members, this may permit more effective protection of stem cells
and progenitors by preventing their recruitment during aggressive
dose intensification regimens aimed at increasing frequency and
dosage levels of chemotherapy. While the biological activity of
Dl-FRIL is similar to cytokines (ng/ml range), as demonstrated in
the examples below, mice tolerated up to a 1,000-fold more Dl-FRIL
than cytokines.
[0114] In addition, by preserving hematopoietic stem cells and
other progenitor cells in a dormant state, one or more members of
the FRIL family allows for the administration of a broad range of
cell cycle active chemotherapy drugs with greater frequency and
higher dose. Thus, administration of a composition of one or more
FRIL family members may permit more aggressive dose-intensification
chemotherapy regimens for a broad range of chemotherapy drugs.
Administration of a composition of a FRIL family member also
provides for a larger reservoir of progenitor cells which could
rapidly respond to stimulatory signals after completing
chemotherapy.
[0115] In certain embodiment of the first aspect of the invention,
the FRIL family member of the invention is from a legume (e.g., a
bean plant).
[0116] In some embodiments, the FRIL family member molecule of the
invention is from a hyacinth bean (i.e., Dolichos lab lab), and has
an amino add sequence comprising the sequence of SEQ ID NO: 24.
Preferably, the FRIL family member molecule of the invention
isolated from a hyacinth bean has the amino add sequence of SEQ ID
NO: 2 or comprises a signal sequence having the amino add sequence
of SEQ ID NO: 4, and, even more preferably, is encoded by a nucleic
add having the nucleic add sequence of SEQ ID NO: 1 or the nucleic
add sequence of SEQ ID NO: 3.
[0117] In some embodiments, the FRIL family member molecule of the
invention is from a red kidney bean (i.e., Phaseolus vulgaris).
Preferably, the FRIL family member of the invention isolated from a
red kidney bean has the amino acid sequence of SEQ ID NO: 6, and,
even more preferably, is encoded by a nucleic add having the
nucleic add sequence of SEQ ID NO: 5.
[0118] In some embodiments, the FRIL family member of the invention
is from a yam bean (i.e., Sphenostylis stenocarpa). Preferably, the
FRIL family member of the invention isolated from a yam bean has
the amino acid sequence comprising the amino acid sequence of SEQ
ID NO: 8, more preferably has a .beta. subunit having an amino acid
sequence which comprises the amino acid sequence of SEQ ID NO: 9,
even more preferably has an .alpha. subunit having an amino acid
sequence which comprises the amino acid sequence of SEQ ID NO: 10,
and, even more preferably, is encoded by a nucleic acid having a
nucleic acid sequence which comprises the nucleic acid sequence of
SEQ ID NO: 7.
[0119] In certain embodiments of the first aspect of the invention,
the FRIL family member molecule is a mutant derived from a second
member of the FRIL family, wherein the mutant is selected from the
group consisting of a substitution mutant, a deletion mutant, an
addition mutant, or a combination thereof (e.g., a mutant of
Dl-FRIL or Pv-FRIL described below). For example, it is preferred
to substitute amino acids in a sequence with equivalent amino
acids. Groups of amino acids known normally to be equivalent are:
(1) Ala(A), Ser(S), Thr(T), Pro(P), and Gly(G); (2) Asn(N), Asp(D),
Glu(E), Gln(Q); (3) His(H), Arg(R), Lys(K); (4) Met(M), Leu(L),
Ile(I), Val(V); and (5) Phe(F), Tyr(Y), Trp(W). Substitutions,
additions, and/or deletions in an amino acid sequence can be made
as long as the mutant FRIL family member molecule continues to
satisfy the functional criteria described herein. An amino acid
sequence that is substantially the same as another sequence, but
that differs from the other sequence by means of one or more
substitutions, additions, and/or deletions, is considered to be an
equivalent sequence. Preferably, less than 50%, more preferably
less than 25%, and still more preferably less than 10%, of the
number of amino acid residues in a sequence are substituted for,
added to, or deleted from the FRIL family member molecule upon
which the mutant FRIL family member was derived.
[0120] In certain embodiments of the first aspect of the invention,
the FRIL family member molecule is a fusion protein comprising a
first portion and a second portion, wherein the first portion is
derived from a second member of the FRIL family. By "fusion
protein" is meant a molecule comprising at least two proteins or
polypeptide fragments thereof joined together, wherein the proteins
or polypeptide fragments thereof are not joined together in the
naturally-occurring organism from which the proteins or polypeptide
fragments thereof were derived. The two proteins or polypeptide
fragments thereof of a fusion protein may be joined by any means,
including, without limitation, a chemical linker, a peptide bond,
or a non-covalent bond, such as an ionic bond. By "protein" or
"polypeptide" is meant a chain of two or more amino acid residues
joined with a peptide bond regardless of length or
post-translational modification such as acetylation, glycosylation,
lipidation, acetylation, or phosphorylation.
[0121] A FRIL family member molecule that is a fusion protein may
comprise a first portion derived from a FRIL family member and a
second portion derived from a protein or other molecule not related
to the FRIL family (eg., the heavy chain of an antibody).
[0122] An additional FRIL family member molecule that is a fusion
protein is FRIL family member comprising the .alpha. subunit from a
first FRIL family member and a .beta. subunit from a second FRIL
family member. Such a fusion protein may be generated, for example,
by joining a nucleic acid sequence encoding the .alpha. subunit of
the first FRIL family member in frame with a nucleic acid sequence
encoding the .beta. subunit of the second FRIL family member. The
nucleic add encoding such a fusion protein can be engineered to
encode an enzyme-specific cleavage site between the portion
encoding the .alpha. subunit of the first FRIL family member and
the portion encoding the .beta. subunit of the second FRIL family
member.
[0123] Where a FRIL family member is a fusion protein, identity of
the fusion protein as a FRIL family member is determined by the
sequence identity between the FRIL family member-derived portion of
the fusion protein and a second FRIL family member, where the FRIL
family member-derived portion of the fusion protein and the second
FRIL family member share at least about 45% amino acid sequence
identity, even more preferably at least about 50% identity, even
more preferably at least about 55% identity, still more preferably
at least about 60% identity, still more preferably at least about
65% identity yet more preferably at least about 75% sequence
identity, still more preferably at least about 85% identity, and
most preferably at least about 95% identity, with the amino acid
sequence of a second member of the FRIL family.
[0124] A FRIL family member in accordance with the first aspect of
the invention can also be a recombinant protein made by expressing
a recombinant nucleic acid that encodes FRIL in a suitable host.
Thus, in a second aspect, the invention features an essentially
pure nucleic acid molecule encoding a member of the FRIL family of
progenitor cell preservation factors. Exemplary purifications of
the nucleic acid molecules of the invention from Dolichos lab lab
and Phaseolus vulgaris are described below. As is well known, if an
amino acid sequence (primary structure) is known, a family of
nucleic acids can then be constructed, each having a sequence that
differs from the others by at least one nucleotide, but where each
different nucleic acid still encodes the same protein. For example,
if a protein has been sequenced but its corresponding gene has not
been identified, the gene can be acquired through amplification of
genomic DNA using a set of degenerate primers that specify all
possible sequences encoding the protein. Thus, a nucleic acid in
accordance to this aspect of the invention need not have a
naturally occurring sequence, but need only encode a FRIL family
member according to the first aspect of the invention.
[0125] In accordance with the second aspect of the invention, a
"member of the FRIL family of progenitor cell preservation factors"
is a described above the first aspect of the invention.
"Essentially pure" is used as described for the first aspect of the
invention.
[0126] By a "recombinant nucleic acid" is meant a nucleic acid
which encodes a FRIL family member molecule, or a portion encoding
at least 15 contiguous amino acids thereof, or a mutant thereof, or
a fusion protein comprising the molecule, portion thereof or mutant
thereof or is capable of expressing an antisense molecule
specifically complementary thereto, or a sense molecule that shares
nucleic acid sequence identity thereto wherein the recombinant
nucleic acid may be in the form of linear DNA or RNA, covalently
closed circular DNA or RNA, or as part of a chromosome, provided
however that it cannot be the native chromosomal locus for a FRIL
family member molecule. Preferred recombinant nucleic acids of the
invention are vectors, which may include an origin of replication
and are thus replicatable in one or more cell type. Certain
preferred recombinant nucleic acids are expression vectors, and
further comprise at least a promoter and passive terminator,
thereby allowing transcription of the recombinant nucleic acid in a
bacterial, fungal, plant, insect or mammalian cell. By "nucleic
acid" or "nucleic acid molecule" as used herein, means any
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including,
without limitation, complementary DNA (cDNA), genomic DNA, RNA,
hnRNA, messenger RNA (mRNA), DNA/RNA hybrids, or synthetic nucleic
acids (e.g., an oligonucleotide) comprising ribonucleic and/or
deoxyribonucleic acids or synthetic variants thereof. The nucleic
acid of the invention includes, without limitation, an
oligonucleotide or a polynucleotide. The nucleic acid can be single
stranded, or partially or completely double stranded (duplex).
Duplex nucleic acids can be homoduplex or heteroduplex.
[0127] In accordance with the second aspect of the invention, a
nucleic acid encoding a FRIL family member has at least about 50%
nudeic acid sequence identity with a nucleic acid encoding another
member of the FRIL family, preferably at least about 555% nucleic
acid sequence identity, more preferably at least about 60% nucleic
acid sequence identity, more preferably at least about 65% nucleic
acid sequence identity, still more preferably at least about 75%
nudeic acid sequence identity, still more preferably at least about
85% nucleic acid sequence identity, and most preferably at least
about 95% nucleic acid sequence identity with a nucleic acid
encoding another member of the FRIL family. Percentage nudeic acid
sequence identity can be determined as described for the first
aspect of the invention.
[0128] A recombinant nucleic acid according to the second aspect of
the invention can also be chemically synthesized by methods known
in the art. For example, recombinant DNA can be synthesized
chemically from the four nucleotides in whole or in part by methods
known in the art. Such methods include those described in
Caruthers, M. H., Science 230(4723):281-285, 1985). DNA can also be
synthesized by preparing overlapping double-stranded
oligonucleotides, filling in the gaps, and ligating the ends
together. See, generally, Sambrook et al., supra, and Glover and
Hames, eds., DNA Cloning, 2d ed., Vols. 14, IRL Press, Oxford,
1995.
[0129] A recombinant nucleic acid molecule of the invention
encoding a mutant FRIL family member can be prepared from wild-type
DNA by site-directed mutagenesis (see, for example, Zoller and
Smith, Nucleic. Acids. Res. 10:6487-6500, 1982; Zoller, M. J.,
Methods Enzymol. 100:468-500, 1983; Zoller, M. J., DNA
3(6):479-488, 1984.; and McPherson, M. J., ed., Directed
Mutagenesis: A Practical Approach, IRL Press, Oxford, 1991.
[0130] A recombinant nucleic acid of the second aspect of the
invention can be amplified by methods known in the art. One
suitable method is the polymerase chain reaction (PCR) method
described in Saiki et al., Science 239:487, 1988, Mullis et al.,
U.S. Pat. No. 4,683,195, and Sambrook et al., supra. It is
convenient to amplify the clones in the lambda-gt10 or lambda-gt11
vectors using lambda-gt10- or lambda-gt11-specific oligomers as the
amplimers (available from Clontech, Palo Alto, Calif.). Larger
synthetic nucleic acid structures can also be manufactured having
specific and recognizable utilities according to the invention. For
example, vectors (e.g., recombinant expression vectors) are known
which permit the incorporation of recombinant nucleic acids of
interest for cloning and transformation of other cells. Thus, the
invention further includes vectors (e.g., plasmids, phages, and
cosmids) which incorporate a nucleotide sequence of the invention,
especially vectors which include the recombinant nucleic acid
molecule of the invention for expression of a FRIL family
member.
[0131] A recombinant nucleic acid of the invention can be
replicated and used to express a FRIL family member following
insertion into a wide variety of host cells in a wide variety of
cloning and expression vectors. The host can be prokaryotic or
eukaryotic. The nucleic acid can be obtained from natural sources
and, optionally, modified. The genes can also be synthesized in
whole or in part.
[0132] Cloning vectors can comprise segments of chromosomal,
non-chromosomal and synthetic DNA sequences. Some suitable
prokaryotic cloning vectors include plasmids from E. coli, such as
colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors
also include derivatives of phage DNA such as M13fd, and other
filamentous single-stranded DNA phages.
[0133] Vectors for expressing proteins in bacteria, especially E.
coli, are also known. Such vectors include the pK233 (or any of the
tac family of plasmids), T7, and lambda P.sub.L. Examples of
vectors that express fusion proteins are PATH vectors described in
Dieckmann and Tzagoloff (J. Biol. Chem. 260(3):1513-1520, 1985).
These vectors contain DNA sequences that encode anthranilate
synthetase (TrpE) followed by a polylinker at the carboxy terminus.
Other expression vector systems are based on beta-galactosidase
(pEX); maltose binding protein (pMAL); glutathione S-transferase
(pGST) (see, e.g., Smith, D. B., Gene 67:31-40, 1988 and Abath, F.
G., Peptide Research 3(4):167-168, 1990). Vectors useful for
cloning and expression in yeast are also available. A suitable
example is the 2 .mu. circle plasmid.
[0134] Suitable cloning/expression vectors for use in mammalian
cells are also known. Such vectors include well-known derivatives
of SV-40, adenovirus, cytomegalovirus (CMV) retrovirus-derived DNA
sequences. Any such vectors, when coupled with vectors derived from
a combination of plasmids and phage DNA, i.e., shuttle vectors,
allow for the isolation and identification of protein coding
sequences in prokaryotes.
[0135] Further eukaryotic expression vectors are known in the art
(e.g., Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982;
Subramani et al., Mol. Cell. Biol. 1:854-864, 1981; Kaufmann and
Sharp, J. Mol. Biol. 159:601-621, 1982; Kaufmann and Sharp, Mol.
Cell. Biol. 159:601-664, 1982; Scahill et al., Proc. Natl. Acad.
Sci. USA 80:4654-4659, 1983; Urlaub and Chasin, Proc. Natl. Acad.
Sci. USA 77:4216-4220, 1980).
[0136] The expression vectors useful in the present invention
contain at least one expression control sequence that is
operatively linked to the recombinant nucleic acid molecule or
fragment thereof to be expressed. The control sequence is inserted
in the vector in order to control and to regulate the expression of
the recombinant nucleic acid of the invention. Examples of useful
expression control sequences are the lac system, the trp system,
the tac system, the trc system, major operator and promoter regions
of phage lambda, the control region of fd coat protein, the
glycolytic promoters of yeast, e.g., the promoter for
3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,
e.g., Pho5, the promoters of the yeast alpha-mating factors, and
promoters derived from polyoma, adenovirus, retrovirus, and simian
virus, e.g., the early and late promoters or SV40, and other
sequences known to control the expression of genes of prokaryotic
or eukaryotic cells and their viruses or combinations thereof.
[0137] Useful expression hosts for expressing the recombinant
nucleic acids of the invention include well-known prokaryotic and
eukaryotic cells. Some suitable prokaryotic hosts include, for
example, E. coli, such as E. coli SG-936, E. coli HB101, E. coli
W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRCl,
Pseudomonas, Bacillus, such as B. subtilis, and Streptomyces.
Suitable eukaryotic cells include yeasts and other fungi, insect,
animal cells, such as COS cells and CHO cells, human cells and
plant cells in tissue culture.
[0138] Given the recombinant nucleic acid sequences disclosed
herein, the artisan can further design recombinant nucleic acids
having particular functions in various types of applications. For
example, the artisan can construct oligonucleotides or
polynucleotides for use as primers in nucleic acid amplification
procedures, such as the polymerase chain reaction (PCR), ligase
chain reaction (LCR), Repair Chain Reaction (RCR), PCR
oligonucleotide ligation assay (PCR-OLA), and the like.
Oligonucleotides useful as probes in hybridization studies, such as
in situ hybridization, can be constructed. Numerous methods for
labeling such probes with radioisotopes, fluorescent tags, enzymes,
binding moieties (e.g., biotin), and the like are known, so that
the probes of the invention can be adapted for easy
detectability.
[0139] Oligonucleotides can also be designed and manufactured for
other purposes. For example, the invention enables the artisan to
design antisense oligonucleotides, and triplex-forming
oligonucleotides, and the like, for use in the study of
structure/function relationships. Homologous recombination can be
implemented by adaptation of the nucleic acid of the invention for
use as targeting means.
[0140] Recombinant nucleic acids of the invention produced as
described above can further be modified to alter biophysical or
biological properties by means of techniques known in the art. For
example, the recombinant nucleic acid can be modified to increase
its stability against nucleases (e.g., "end-capping"), or to modify
its lipophilicity, solubility, or binding affinity to complementary
sequences. Methods for modifying nucleic acids to achieve specific
purposes are disclosed in the art, for example, in Sambrook et al.,
supra. Moreover, the recombinant nucleic acid of the invention can
include one or more portions of nucleotide sequence that are
non-coding for a FRIL family member.
[0141] In a third aspect, the invention provides a pharmaceutical
formulation comprising an essentially pure composition of one or
more members of the FRIL family of progenitor cell preservation
factors and a pharmaceutically acceptable carrier. By
"pharmaceutically acceptable carrier" is meant any inert carrier
that is non-toxic to the animal to which it is administered and
that retains the therapeutic properties of the compound with which
it is administered (i.e., the FRIL family member). Pharmaceutically
acceptable carriers and their formulations are well-known and
generally described in, for example, Remington's Pharmaceutical
Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co.,
Easton, Pa., 1990). One exemplary pharmaceutically acceptable
carrier is physiological saline. Pharmaceutical formulations of the
invention may employ any pharmaceutically acceptable carrier,
depending upon the route of administration of the composition.
[0142] Compositions of FRIL family members may be used safely and
efficaciously as a therapeutics. The gastrointestinal tracts of
animals come in constant contact with lectins, such as FRIL family
members, in raw and/or cooked vegetables and fruits. Many lectins
pass through the gastrointestinal tract biologically intact
(Pusztai, A., Eur. J. Clin. Nutr. 47: 691-699, 1993). Some lectins
interact with the gut and are transported into the peripheral blood
circulation. For example, a recent study found peanut agglutinin
(PNA) in the blood of humans at levels of 1-5 .mu.g/ml an hour
after ingesting 200 g of raw peanuts (Wang et al., Lancet 352:
1831-1832, 1998). Antibodies to dietary lectins are commonly found
in people at levels of .about.1 .mu.g/ml (Tchernychev and Wilchek,
FEBS Lett. 397: 139-142, 1996). These circulating antibodies do not
block carbohydrate binding of the lectins.
[0143] In certain embodiments of the third aspect of the invention,
administration of a therapeutically effective amount of the
pharmaceutical formulation to a patient suffering from a condition
whereby the patient's hematopoietic progenitor cells are depleted
alleviates and/or reduces the condition in the patient.
[0144] In accordance with the third aspect of the invention, by
"therapeutically effective amount" is meant a dosage of a
composition of a FRIL family member or pharmaceutical formulation
comprising a composition of a FRIL family member that is effective
to alleviate and/or reduce either a condition whereby the patient's
hematopoietic progenitor cells are depleted or a hematopoietic
progenitor cell-depleting activity of a therapeutic (e.g., a
chemotherapeutic). Preferably, such administration is systematic
(e.g., by intravenous injection). When administered systemically, a
therapeutically effective amount is an amount of between about 500
ng of the FRIL family member/kg total body weight and about 5 mg/kg
total body weight per day. Preferably, a therapeutically effective
amount is between about 500 ng/kg and 500 .mu.g/kg total body
weight of the FRIL family member per day. Still more preferably, a
therapeutically effective amount is between about 5 .mu.g/kg and 50
.mu.g/kg total body weight of the FRIL family member per day. Most
preferably, a therapeutically effective amount is an amount that
delivers about 50 .mu.g/kg total body weight of the FRIL family
member per day.
[0145] A composition of a FRIL family member of the invention and
pharmaceutical formulation comprising a composition of a FRIL
family member of the invention may be administered patients having,
or predisposed to developing, a condition whereby the patient's
hematopoietic progenitor cells are depleted. Such a condition may
be congenital. For example, the patient may have severe combined
immunodeficiency or aplastic anemia.
[0146] The condition may also be induced by a drug. Thus, in
certain embodiments of the third aspect of the invention,
administration of a therapeutically effective amount of the
pharmaceutical formulation to a patient prior to treatment of the
patient with a therapeutic treatment having a hematopoietic
progenitor cell-depleting activity alleviates the hematopoietic
progenitor cell-depleting activity of the therapeutic in the
patient. For example, cancer patients are often treated with
radiotherapeutics or chemotherapeutics that have hematopoietic
progenitor cell-depleting activity. By "hematopoietic progenitor
cell-depleting activity" is meant an activity of a therapeutic
treatment whereby the hematopoietic progenitor cells in the patient
being treated with the therapeutic treatment are depleted, either
by killing the progenitor cells or by inducing the progenitor cells
to undergo irreversible differentiation. Non-limiting examples of
therapeutic treatments having hematopoietic progenitor
cell-depleting activity are the chemotherapeutic agents cytarabine
(Ara-C), doxorubicin (Dox), daunorubicin, and 5-fluorouracil
(5-FU).
[0147] In certain embodiments, administration of the pharmaceutical
formulation of the invention to a patient prior to the treatment of
the patient with a therapeutic treatment having a hematopoietic
progenitor cell-depleting activity enables treatment of the patient
with a higher dosage of the therapeutic treatment. The higher
dosage of the therapeutic treatment may be accomplished by either
an increased dose of the therapeutic treatment and/or an increased
duration of treatment with the therapeutic treatment. For example,
a child diagnosed with childhood Acute Myelogenous Leukemia (AML)
is typically initially treated for the first seven days with
daunorubicin at 45 mg/m.sup.2 on Days 1-3 plus Ara-C at 100
mg/m.sup.2 for 7 days plus GTG at 100 mg/m.sup.2 for 7 days. The
same child pretreated with a composition in accordance with this
aspect of the invention may be able to tolerate a higher dosage
(i.e., higher dose and/or prolonged treatment period) of any or all
of these chemotherapeutics. Such an increase in dosage tolerance of
a therapeutic treatment(e.g., a chemotherapeutic) having a
hematopoietic progenitor cell-depleting activity in a cancer
patient is desirable since a higher dosage may result in the
destruction of more cancerous cells.
[0148] The pharmaceutical formulations and/or compositions of the
invention may be administered by any appropriate means. For
example, the pharmaceutical formulations and/or compositions of the
invention may be administered to an mammal within a
pharmaceutically-acceptable diluent, carrier, or excipient, in unit
dosage form according to conventional pharmaceutical practice.
Administration may begin before the mammal is symptomatic for a
condition whereby the patient's hematopoietic progenitor cells are
depleted. For example, administration of the pharmaceutical
formulations of the third aspect of the invention to a cancer
patient may begin before the patient receives radiotherapy and/or
chemotherapy treatment.
[0149] Any appropriate route of administration of a pharmaceutical
formulation and/or composition of the invention may be employed,
including, without limitation, parenteral intravenous,
intra-arterial, subcutaneous, sublingual, transdermal, topical,
intrapulmonary, intramuscular, intraperitoneal, by inhalation,
intranasal, aerosol, intrarectal, intravaginal, or by oral
administration. Pharmaceutical formulations and/or compositions of
the invention may be in the form of liquid solutions or
suspensions; for oral administration, formulations may be in the
form of tablets or capsules; and for intranasal formulations, in
the form of powders, nasal drops, or aerosols. The pharmaceutical
formulations and/or compositions may be administered locally to the
area affected by a condition whereby the patient's hematopoietic
progenitor cells are depleted. For example, the pharmaceutical
formulations and/or compositions of the invention may be
administered directly into the patient's bone marrow. The
pharmaceutical formulations and/or compositions of the invention
may be administered systemically.
[0150] In certain embodiments of the third aspect of the invention,
the patient is human or a domesticated animal. By "domesticated
animal" is meant an animal domesticated by humans, including,
without limitation, a cat, dog, elephants, horse, sheep, cow, pig,
and goat. In some embodiments, the patient has cancer.
[0151] In some embodiments of the third aspect, the treatment is a
radiotherapeutic or a chemotherapeutic treatment, including,
without limitation, cytarabine (Ara-C), doxorubicin (Dox), or
5-fluorouracil (5-FU), or a combination of a radiotherapeutic and a
chemotherapeutic.
[0152] In a fourth aspect, the invention provides a method for a
method for alleviating and/or reducing the hematopoietic progenitor
cell-depleting activity of a therapeutic treatment in a patient,
comprising administering to the animal a therapeutically effective
amount of a composition of a FRIL family member prior to
administration of the therapeutic treatment to the patient.
"Hematopoietic progenitor cell-depleting activity" is as described
for the third aspect of the invention. Routes of administration of
a composition of a FRIL family member of this aspect of the
invention are as described for the administration of the
pharmaceutical formulation of the third aspect of the invention.
"Therapeutically effective amount" is as described for the third
aspect of the invention.
[0153] In certain embodiments of the fourth aspect, the patient is
a human or a domesticated animal. "Domesticated animal" is as
described for the third aspect of the invention. In certain
embodiments, the patient has cancer. In some embodiments, the
therapeutic treatment is a radiotherapeutic or a chemotherapeutic
treatment, including, without limitation, cytarabine (Ara-C),
doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combination of a
radiotherapeutic and a chemotherapeutic.
[0154] In a fifth aspect, the invention provides a method for
isolating a population of progenitor cells, comprising contacting a
population of cells with a plurality of FRIL family member
molecules, and separating the unbound cells, wherein the cells
bound to the FRIL family member molecules are an isolated
population of progenitor cells. "FRIL family member molecule" and
"progenitor cell" are as described for the first aspect of the
invention. By "unbound cell" is meant a cell that does not bind to
a FRIL family member. "Bind" is a described for the first aspect of
the invention.
[0155] By "isolated" is meant a population of progenitor cells that
is separated from a larger population of cells, wherein the
percentage of progenitor cells in the isolated population is at
least two fold greater than the percentage of progenitor cells in
the larger population. Preferably, the percentage of progenitor
cells in the isolated population is at least four fold greater than
the percentage of progenitor cells in the larger population. A
non-limiting example of the increased percentage of progenitor
cells in an isolated population of progenitor cells of the
invention is shown below in Table 11. Preferably, the isolated
population of progenitor cells of the invention is at most 2% of
the total population of umbilical cord blood mononudear cells (CB
mnc). Preferably, the population of progenitor cells of the
invention is at most 1% of the total population of umbilical cord
blood mononuclear cells (CB mnc). Preferably, an isolated
population of progenitor cells binds to a normally glycosylated
FLT3 receptor. "Normally glycosylated FLT3 receptor" is as defined
above.
[0156] In preferred embodiments, the isolated population of
progenitor cells is from a human or is from a domesticated
animal.
[0157] In certain embodiments of the fifth aspect of the invention,
the FRIL family member molecules are detectably labeled. By
"detectably labeled" is meant that the FRIL family member is
attached to a label that is detectable visually or instrumentally.
Detectable labels such as enzymes and chromophoric molecules can be
conjugated to the FRIL family member molecules by means of coupling
agents, such as dialdehydes, carbodiimides, and dimaleimides.
Numerous methods of labeling proteins are known in the art. The
label can also be directly attached through a functional group on
the FRIL family member. Such a functional group may be present on
the FRIL family member molecule to be detectably labeled;
alternatively, the FRIL family member molecules can be modified
using standard techniques to contain a functional group. Some
examples of suitable functional groups include, without limitation,
amino, carboxyl, sulfhydryl, maleimide, isocyanate,
isothiocyanate.
[0158] In certain embodiments, the detectable label is radioactive
or non-radioactive. Some examples of useful radioactive labels
include .sup.32P, .sup.125I, .sup.131I, and .sup.3H. Use of
radioactive labels have been described in U.K. patent document
2,034,323, and U.S. Pat. Nos. 4,358,535, and 4,302,204. Some
examples of non-radioactive labels include enzymes, chromophores,
atoms and molecules detectable by electron microscopy, and metal
ions detectable by their magnetic properties.
[0159] In certain embodiments of the fifth aspect of the invention,
the detectable label is an enzymatic label. Some useful enzymatic
labels include enzymes that cause a detectable change in a
substrate. Some useful enzymes and their substrates include, for
example, horseradish peroxidase (pyrogallol and
o-phenylenediamine), beta-galactosidase (fluorescein
beta-D-galactopyranoside), and alkaline phosphatase
(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The
use of enzymatic labels have been described in U.K. 2,019,404, and
EP 63,879, each incorporated herein by reference, and by Rotman,
Proc. Natl. Acad. Sci. USA 47:1981-1991, 1961.
[0160] In certain embodiments of this aspect of the invention, the
detectably labeled FRIL family member molecules are labeled by
being specifically bound by an antibody that is detectably
labeled.
[0161] In certain embodiments of this aspect of the invention, the
FRIL family member molecules are conjugated to a receptor (or
ligand) and is detectably labeled by binding the receptor (or
ligand) with the ligand (or receptor), wherein the ligand (or
receptor) is detectably labeled. Any of the known ligand-receptor
combinations is suitable. Some suitable ligand-receptor pairs
include, for example, biotin-avidin or -streptavidin, and
antibody-antigen. In certain embodiments, biotin-avidin combination
is preferred.
[0162] In certain embodiments, the detectable label is a
chromophore. Useful chromophores include, for example, fluorescent,
chemiluminescent, and bioluminescent molecules, as well as dyes.
Some specific chromophores useful in the present invention include,
for example, fluorescein, rhodamine, Texas red, phycoerythrin,
umbelliferone, luminol.
[0163] In certain embodiments of this aspect of the invention,
where the FRIL family member molecules are detectably labeled to a
chromophore, the unbound cells are separated by using a flow
cytometry cell sorter to sort the population of cells contacted
with the FRIL family member molecules detectably labeled with a
fluorescent marker.
[0164] In certain embodiments of the fifth aspect of the invention,
the FRIL family member molecules are immobilized on a solid
support. "Solid support" includes any surface, including, without
limitation, the surface of a sepharose bead, a gel, a matrix, a
magnetic bead, and a plastic surface (e.g., the bottom of a tissue
culture dish or flask).
[0165] In some embodiments, the solid support is a bead, for
example, a magnetic bead. In some embodiments, where the solid
support is a magnetic bead, the unbound cells are separated by
applying a magnet to the population of cells contacted with the
FRIL family member molecules immobilized on the magnetic bead. In
further embodiments, the population of cells bound to the FRIL
family member molecules immobilized on a magnetic bead is rinsed
with a physiologically acceptable solution while the magnet is
applied. By "physiologically acceptable solution" is meant an inert
solution, such as sterile saline solution or tissue culture medium,
which is non-toxic to the cells.
[0166] Methods for isolating cells that bind FRIL family member
molecule-coated magnetic beads are described below in Example 16.
Magnetic beads are commerically available (e.g., from Dynabeads
Tosylactivated, Lake Success, N.Y.; or from Miltenyi Biotec,
Auburn, Calif.). Since the FRIL family member is protein, it can be
conjugated to a magnetic bead via amino- or sulfhydryl-groups of
the protein. A FRIL family member molecule can also immobilized on
magnetic beads by a biotin-strepavidin interaction.
[0167] Preferred magnetic beads are the MACS super-paramagnetic
MicroBeads (from Miltenyi Biotec) which are extremely small,
approximately 50 nm in diameter (MACS beads are about one million
times smaller in volume than eukaryotic cells). Because MACS beads
react like magnetic antibodies, magnetic labeling is achieved
within minutes. MACS MicroBeads form a stable colloidal suspension
and do not precipitate or aggregate in magnetic fields. Because of
their size and composition (iron oxide and polysaccharide) make the
MACS biodegradable, so labeled cells retain their physiological
function. This property of MACS beads is particularly useful for
bead-sorted FRIL family member-binding cells, which bind the FRIL
family member with such high affinity that it is difficult to
remove the beads.
[0168] In certain embodiments of the fifth aspect, the solid
support is the bottom of a tissue culture plate. In some
embodiments, the unbound cells are separated by rinsing the
population of cells contacted with the FRIL family member molecules
immobilized on the bottom of a tissue culture plate with a
physiologically acceptable solution.
[0169] FRIL family member molecules may be directly or indirectly
attached to the bottom of a tissue culture plate. Following a
standard "panning" protocol (see, e.g., Stengelin et al., EMBO J.
7(4):1053-1059, 1988; Aruffo and Seed, Proc. Natl. Acad. Sci. USA
84(23): 8573-8577, 1987), a population of cells suspected of
containing FRIL family member-binding progenitor cells is incubated
on the plate. The plate is then gently rinsed with a
physiologically acceptable solution, thereby removing the unbound
cells while leaving the FRIL family member-binding population of
cells attached to the FRIL family member-coated plate.
[0170] In preferred embodiments of the fifth aspect of the
invention, the isolated population of progenitor cell is a
population of hematopoietic progenitor cells. In various
embodiments, the population of cells is whole blood, umbilical cord
blood, bone marrow cells, or fetal liver cells.
[0171] In certain embodiments of the fifth aspect of the invention,
the population of cells is a sorted population of cells, wherein a
cell of the sorted population does not express CD11b, CD11c, or
CD38. Because more primitive progenitor cell stypically expresses
few cell surface molecules, prior to isolating a population of
progenitor cells that binds to a FRIL family member, the population
of cells is preferably sorted to first remove cells that express
one or more of the following cell surface molecules: CD11b, CD11c,
and CD38. Following this negative sort (i.e., a sort, wherein the
cells retained do not express CD11b, CD11c, and/or CD38), the
sorted population is positively sorted for an ability to bind a
FRIL family member.
[0172] In certain embodiments, the sorted population of cells is
sorted by flow cytometry or by magnetic bead selection. Thus, a
population of cells (e.g., human umbilical cord blood cells) may be
first contacted with chromophore-labeled antibodies (or other
molecule such as a ligand) which specifically bind CD11b, CD11c,
and/or CD38. Following binding, the population of cells is then
negatively sorted by flow cytometry, where the cells which are not
bound by the antibodies (and so do not express CD11b, CD11c, and/or
CD38) are retained and further sorted for an ability to bind a FRIL
family member, wherein the population of cells that binds a FRIL
family member is an isolated population of progenitor cells
according to the invention.
[0173] A population of cells may also be contacted with a molecule,
such as an antibody, which specifically binds CD11b, CD11c, and/or
CD38, wherein the molecule is attached to a solid support, such as
a magnetic bead. The population is then negatively sorted by
applying a magnet to the beads, and retaining the cells that do not
bind the beads and so are not attracted to the magnet. These sorted
cells are then further sorted for an ability to bind a FRIL family
member, wherein the population of cells that binds a FRIL family
member is an isolated population of progenitor cells according to
the invention.
[0174] In a sixth aspect, invention provides an isolated population
of progenitor cells isolated by a method comprising contacting a
population of cells with a plurality of FRIL family member
molecules, and separating the unbound cells, wherein the cells
bound to the FRIL family member are an isolated population of
progenitor cells. "FRIL family member molecule," "progenitor cell,"
"unbound cell," and "bind" are as described above.
[0175] In preferred embodiments, the isolated population of
progenitor cells is from a human or a domesticated animal.
[0176] In certain embodiments of the sixth aspect, the cells of the
isolated population do not express CD34 on their cell surface. In
certain embodiments, the cells of the isolated population express
the FLK1, FLT1, FLT3, FLT4, or Kit receptor tyrosine kinases. In
some embodiments, the cells of the isolated population express the
CD11b or CD11c cell surface molecules. In preferred embodiments,
the cells of the isolated population express FLT3.
[0177] In various embodiments of the sixth aspect of the invention,
the cells of the isolated population are hemangioblasts,
messenchymal stem cells, bone progenitor cells, hepatic progenitor
cells, endothelial progenitor cells, hematopoietic progenitor
cells, embryonal stem cells, brain progenitor cells, or dendritic
progenitor cells. By "hemangioblast" is meant a cell that is a
progenitor cell for both hemaopoietic and endothelial lineages.
Preferably, the cells of the isolated population are hematopoietic
progenitor cells. By a "messenchymal stem cells" is meant the
population of cells that is the progenitor for bone marrow stromal
cells, including, without limitation, adipose tissue cells,
cartilage-producing cells, muscle cells, and bone cells. Such cells
of this aspect of the invention can be used, for example, for
tissue repair.
[0178] Determination of what type of cells a progenitor cell of the
invention will give rise to is made by the various progenitor cell
assays described below. For example, if a progenitor cell is
suspected of being an endothelial progenitor cell, the cell may be
cultured in a methylcellulose progenitor cell assay in the presence
of vascular endothelial growth factor. Should cells arising from
such a culture have endothelial cell markers, the progenitor cell
of the invention is a endothelial progenitor cell or perhaps a
hemangioblast.
[0179] In certain embodiments of the sixth aspect of the invention,
where the cells of the isolated population are hematopoietic
progenitor cells, transplantation of the cell into an animal
lacking a population of hematopoietic progenitor cells sufficient
to enable survival of the animal reconstitutes the animal, wherein
the transplanted animal survives. Determination of the ability of a
hematopoietic progenitor cell to reconstitute a animal lacking a
population of hematopoietic progenitor cells sufficient to enable
survival of the animal may be made using the methods described
below for the NOD-SCID mouse.
[0180] In certain embodiments, the hematopoietic progenitor cells
are from a mouse or a human and the animal is a mouse. In some
embodiments, the mouse is a severe combined immunodeficient (SCID)
mouse (e.g., the NOD-SCID mouse described below) or a mouse that
has been exposed to a sublethal dose of radiation and/or
chemotherapy
[0181] In certain embodiments of the sixth aspect of the invention,
the hematopoietic progenitor cells are from a human and the animal
is a human. In some embodiments, the human is a cancer patient
receiving a treatment that depletes the patient's hematopoietic
progenitor cells. In some embodiments, the treatment is a
radiotherapeutic or a chemotherapeutic treatment, induding, without
limitation, cytarabine (Ara-C), doxorubicin (Dox), or
5-fluorouracil (5-FU), or a combination of a radiotherapeutic and a
chemotherapeutic.
[0182] In a seventh aspect, the invention provides a method for
preserving progenitor cells ex vivo, comprising contacting a
population of cells comprising at least one progenitor cell with an
effective amount of a composition of a FRIL family member for an
effective period of time, wherein the progenitor cell in the
population are rendered quiescent. "FRIL family member" and
"progenitor cell" are as described above for the first aspect of
the invention.
[0183] In accordance with this aspect of the invention, the terms
"effective amount" and "effective period of time" are used to
denote known treatments at dosages and for periods of time
effective to preserve progenitor cells. Where administered to a
patient, preferably, such administration is systemic (e.g., by
intravenous injection). Effective amounts and effective periods of
time can be determined using the models and assays described
herein. For example, the Examples below describe the preservation
of progenitor cells that have SCID-reconstituting ability. In
accordance with the invention, an effective amount may range from
about 0.1 ng/mL to about 1 .mu.g/mL of a FRIL family member,
preferably about 1.0 ng/mL to to about 1.0 .mu.g/mL, more
preferably about 1.0 ng/mL to about 100 ng/mL, even more preferably
about 10 ng/mL to about 50 ng/mL, and most preferably about 50
ng/mL of a FRIL family member in culture. In accordance with the
invention, an effective period of time includes culturing the cells
in the presence of a FRIL family member for between would include
from about 2 hours to 5 days, more preferably from about 12 hours
to about 3 days, and most preferably for about 24 hours.
[0184] In certain embodiments of the seventh aspect, the progenitor
cells are from a human or from a domesticated animal. In certain
embodiments, the population of cells is bone marrow cells.
[0185] In some embodiments, the non-progenitor cells in the
population of cells differentiate or die. Thus, although the
progenitor cells in the culture do not actually expand in number,
they are enriched relative to the number of cells in the
culture.
[0186] In certain embodiments of the seventh aspect, the population
of cells is removed from a cancer patient prior to treatment of the
cancer patient with a therapeutic treatment having a hematopoietic
progenitor cell-depleting activity. In some embodiments, the
therapeutic treatment is a radiotherapeutic or a chemotherapeutic
treatment, including, without limitation, cytarabine (Ara-C),
doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combination of a
radiotherapeutic and a chemotherapeutic.
[0187] In an eighth aspect, the invention provides a method for
preserving progenitor cells in vivo, comprising administering to a
patient a therapeutically effective amount of a composition of a
FRIL family member for a therapeutically effective period of time,
wherein the progenitor cells in the patient are rendered quiescent.
"FRIL family member" and "progenitor cell" are as described above
for the first aspect of the invention. "Therapeutically effective
amount" is as described for the third aspect of the invention.
[0188] By "therapeutically effective period of time" is meant
treatment for a period of time effective to preserve progenitor
cells. Where administered to a patient, preferably, such
administration is systemic (e.g., by intravenous injection).
Effective amounts and effective periods of time can be determined
using the models and assays described herein. For example, the
examples below describe the preservation of progenitor cells that
have SCID-reconstituting ability. In accordance with the invention,
a therapeutically effective period of time is injecting a
therapeutically effective amount of a composition and/or
pharmaceutical formulation of a FRIL family member between about 5
days before the patient receives treatment with a therapeutic
treatment (e.g., a chemotherapeutic) having a progenitor
cell-depleting activity to about 2 hours prior to treatment with
the therapeutic treatment, wherein the therapeutically effective
amount of a composition and/or pharmaceutical formulation of the
FRIL family member is administered daily. In accordance with the
invention, a preferred therapeutically effective period of time is
injecting a patient (e.g., a cancer patient) with a therapeutically
effective amount of a composition and/or pharmaceutical formulation
of a FRIL family member between about 2 days before the patient
receives treatment with a therapeutic treatment (e.g., a
chemotherapeutic) having a progenitor cell-depleting activity to
about 1 day prior to treatment with the therapeutic treatment,
wherein the therapeutically effective amount of a composition
and/or pharmaceutical formulation of a FRIL is administered daily.
It will be understood that once the patient starts to receive
treatment of a therapeutic treatment having a progenitor
cell-depleting activity, the therapeutically effective amount of a
composition and/or pharmaceutical formulation of a FRIL family
member may be different from the therapeutically effective amount
of the a composition and/or pharmaceutical formulation of a FRIL
family member that the patient received prior to receiving
treatment with the therapeutic treatment.
[0189] Preferably, the patient is a human or a domesticated animal.
"Domesticated animal" is as described for the third aspect of the
invention.
[0190] In certain embodiments of the eighth aspect of the
invention, the patient is a cancer patient. In some embodiments,
the effective amount of the composition of the FRIL family member
is administered prior to the treatment of the patient with a
therapeutic treatment having a hematopoietic progenitor
cell-depleting activity. In some embodiments, the therapeutic
treatment is a radiotherapeutic or a chemotherapeutic treatment,
including, without limitation, cytarabine (Ara-C), doxorubidn
(Dox), or 5-fluorouracil (5-FU), or a combination of a
radiotherapeutic and a chemotherapeutic.
[0191] In a ninth aspect, the invention provides a method for
identifying a progenitor cell, comprising contacting a candidate
cell with a FRIL family member molecule, wherein binding of the
candidate cell to the FRIL family member molecule identifies the
candidate cell as a progenitor cell. "FRIL family member molecule"
and "progenitor cell" are as described above for the first aspect
of the invention.
[0192] In certain embodiments of the ninth aspect, the candidate
cell is in a population of cells. In certain embodiments, the
candidate cell is from a human.
[0193] In a tenth aspect, the invention provides a progenitor cell
identified by a method comprising contacting a candidate cell with
a FRIL family member molecule, wherein binding of the candidate
cell to the FRIL family member molecule identifies the candidate
cell as a progenitor cell. "FRIL family member" and "progenitor
cell" are as described above for the first aspect of the
invention.
[0194] In an eleventh aspect, the invention provides a method for
identifying a composition of a member of the FRIL family of
progenitor cell preservation factors, comprising contacting a
candidate compound with a glycosylated extracellular domain of an
FLT3 receptor, wherein the glycosylation pattern of the
extracellular domain of the FLT3 receptor is the same as the
glycosylation pattern of an extracellular domain of a normally
glycosylated FLT3 receptor, wherein a candidate compound that binds
the glycosylated extracellular domain of the FLT3 receptor is
identified as a composition of a FRIL family member. "FRIL family
member," "progenitor cell," and "normally glycosylated FLT3
receptor" are as described above for the first aspect of the
invention.
[0195] In accordance with the eleventh aspect of the invention, the
normally glycosylated extracellular domain of the FLT3 receptor may
expressed on a cell surface (e.g., on the surface of an NIH 3T3
cell, as described in the examples below). Any normal cell may be
transfected with a nucleic acid sequence encoding the extracellular
domain of the FLT3 receptor (from, e.g., a human or a mouse)
provided that the cell normally glycosylates the FLT3 receptor it
expresses on its cell surface. It should be noted that the cell
need not be transfected with a nucleic acid sequence encoding the
entire FLT3 receptor. For example, the cell may be transfected with
a nucleic acid molecule encoding a fusion protein comprising the
intracellular domain of a non-FLT3 receptor (e.g., the Fms
receptor) and the extracellular domain of the FLT3 receptor. A
candidate compound according to this aspect of the invention is
then incubated with the cell expressing the normally glycosylated
extracellular domain of the FLT3 receptor, and binding of the
candidate compound to the cell (as can be measured, as described
below, by survival of the cell) identifies the candidate compound
as a FRIL family member.
[0196] Alternatively, the normally glycosylated extracellular
domain of the FLT3 receptor need not be expressed by a cell.
Instead, the normally glycosylated extracellular domain of the FLT3
receptor may be detectably labeled or immobilized on a solid
support (e.g., a magnetic bead). For example, the normally
glycosylated extracellular domain of the FLT3 receptor can be bound
to the surface of a 96 well microtiter plate. Compositions
comprising different candidate compounds (or pools of such
compounds) can be detectably labeled (e.g., with biotin), and added
to the wells. After incubation for an amount of time required for
binding of a positive control (e.g., a composition of a known FRIL
family member) the plate is washed and detectably labeled avidin is
added to each well. The plate can then be read on a standard
microtiter plate reader to determine binding of a composition of a
candidate compound to the plate-bound normally glycosylated
extracellular domain of the FLT3 receptor, wherein binding (as
measured by detection of the bound detectably labeled avidin)
identifies the candidate compound in the composition as a FRIL
family member.
[0197] In certain embodiments of the eleventh aspect, the candidate
compound is a lectin. In certain embodiments, the lectin is
synthetic. In certain embodiments, the lectin is from a legume.
[0198] In a twelfth aspect, the invention provides an essentially
pure composition of a FRIL family member identified by the method
comprising contacting a candidate compound with a glycosylated
extracellular domain of an FLT3 receptor, wherein the glycosylation
pattern of the extracellular domain of the FLT3 receptor is the
same as the glycosylation pattern of an extracellular domain of a
normally glycosylated FLT3 receptor, wherein a candidate compound
that binds the glycosylated extracellular domain of the FLT3
receptor is identified as an essentially pure composition of a
member of the FRIL family. "FRIL family member," "progenitor cell,"
and "normally glycosylated FLT3 receptor," and "essentially pure"
are as described above for the first aspect of the invention.
[0199] The following examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature. Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific substances and procedures described
herein. Such equivalents are considered to be within the scope of
this invention, and are covered by the following claims.
EXAMPLE 1
Purification and Cloning of a FRIL Family Member, Dl-FRIL
[0200] Purification of Dl-FRIL from Dolichos lab lab
[0201] Seeds from the hyacinth beans (Dolichos lab lab) were
purchased from Stokes Seeds (Buffalo, N.Y.) and grown in a
greenhouse. Dry seeds were ground in a coffee mill and the power
was extracted in 5 volumes of 50 mM Tris/HCl containing 1 nM each
of MgCl.sub.2 and CaCl.sub.2 for 4 hours at 4.degree. C. Bean
solids were pelleted by centrifugation at 10,000.times.g for 20
min. The pH of the supernatant was acidified to pH 4.0 with acetic
acid, followed by a second centrifugation to clarify the
supernatant, and finally the pH was readjusted to 8.0 with sodium
hydroxide. This crude extract was stored at -20.degree. C.
[0202] Single-step purification of the FRIL family member was
achieved by binding to a mannose-Sepharose matrix (Sigma). The gel
(i.e., matrix) was tumbled with the thawed crude extract for 4-12
hours at 4.degree. C., carefully washed several times with 50 mM
Tris/HCl containing 1 nM each of MgCl.sub.2 and CaCl.sub.2, and
then eluted with 20 mM .alpha.-methyl .alpha.-D-mannoside. Because
this FRIL family member was isolated from Dolichos lab lab, it is
referred to herein as Dl-FRIL.
[0203] RNA Isolation and cDNA Synthesis of Dl-FRIL
[0204] Total RNA was prepared from mid-maturation Dolichos lab lab
seeds stored at -70.degree. C. following the procedure of Pawloski
et al. Mol. Plant Biol. Manual 5: 1-13,1994. Poly (A.sup.+) RNA was
obtained from this total RNA using the PolyATract mRNA Isolation
System (Promega) according to the manufacturer's instructions.
Avian myeloblastosis virus reverse transcriptase (Promega) was used
to generate cDNA from 0.5 .mu.g poly(A.sup.+)RNA, or from 3.0 .mu.g
of total RNA, using 1 .mu.g of oligo(dT) in standard reaction
conditions (Sambrook et al., Molecular Cloning. A Laboratory
Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
1989).
[0205] Polymerase Chain Reaction and cDNA Cloning of Dl-FRIL
[0206] Based on the amino acid sequence published by Gowda et al.,
J. Biol. Chem. 269:18789-18793, 1994, two degenerate
oligonucleotide primers were designed using Phaseolus codon usage
(Devereux et al., Nucleic Acids Res. 12:387-394, 1984):
1 (SEQ ID NO:_) MLA AA(AG)TT(TC)GA(TC)CC(AT)AA(TC)CA(AG)GA(- AG)GA
(SEQ ID NO:_) MLZ TT(AT)CC(AG)TT(TC)TGCCA(A- G)TCCCA
[0207] A 500+bp product was amplified from cDNA prepared as
described above, by 30 cycles of polymerase chain reaction (PCR),
each cycle comprising 40 seconds at 94.degree. C., 40 seconds at
50.degree. C., 60 seconds at 72.degree. C., followed by an
extension step at 72.degree. C. for 10 min. Reactions were
performed in 50 .mu.L containing 30 pmol of each primer, 0.2 mM
deoxyribonucleotides, and 0.5 unit of AmpliTaq polymerase (Perkin
Elmer, Norwalk, Conn.) in the corresponding buffer.
[0208] The 500 bp product obtained by PCR was cloned in the cloning
vector, pCR2.1 (Invitrogen, Carlsbad, Calif.), and sequenced by
sequenase dideoxy chain termination (United States Biochemicals)
using the following primers:
2 GTACCGAGCTCGGAT (SEQ ID NO:_) TCTAGATGCATGCTCGAG. (SEQ ID
NO:_)
[0209] This sequence was designated "Dl-FRILa," as relating to the
gene encoding the protein of interest, designated "Dl-FRIL" as
noted above.
[0210] Based on the sequence of the Dl-FRILa amplified product, a
specific primer was prepared:
3 MLX GTTGGACGTCAATTCCGATGTG (SEQ ID NO:_)
[0211] A degenerate primer corresponding to the first five amino
acids of the sequence published by Gowda et al., J. Biol. Chem.
269:18789-18793, 1994 was also prepared:
4 MLI GC(TC)CA(AG)TC(TC)CT(TC)TC(TC)TT (SEQ ID NO:_)
[0212] The MLX and MLI primers were used in combination to amplify
a 480 bp product from cDNA prepared as described above, through 30
PCR cycles using the same conditions described above. This
secondary amplified fragment was cloned in the pCR2.1 vector and
sequenced as described above, and was designated "Dl-FRILb."
[0213] The 3' end of DL-FRIL was obtained through rapid
amplification of cDNA ends by polymerase chain reaction (RACE-PCR)
(see, e.g., Frohman "RACE: Rapid amplification of cDNA ends," pp.
28-38 in PCR Protocols: A Guide to Methods and Applications, Innis
M A, Gelfand D H, Sninsky J J, and White T J, eds., Academic Press,
San Diego, 1990) using the 5'/3' RACEKIT (Boehringer Mannheim,
Indianapolis, Ind.) according to the manufacturer's instructions.
In the cDNA synthesis for the 3' RACE, an oligo(dT) anchor primer
("AP") supplied with the kit was used, at a concentration of 32.5
.mu.M, using the standard conditions described earlier in this
Example.
5 AP GACCACGCGTATCGATGTCGAC (SEQ ID NO:_)
[0214] Nested PCR amplifications were performed using the AP anchor
primer in combination with a specific primer having the following
sequence:
6 MLB AAGTTAGACAGTGCAGGAAAC. (SEQ ID NO:_)
[0215] The amplification conditions were again 30 cycles of 40
seconds at 94.degree. C., 40 seconds at 55.degree. C., 60 seconds
at 72.degree. C. each, with an extension step at 72.degree. C. for
10 min A 900+ bp product was obtained, which was subcloned in
pCR2.1 and sequenced as described above, and was designated
"Dl-FRILc" (SEQ ID NO:1).
[0216] To obtain the full length cDNA clone, the anchor primer AP
was used in combination with a specific primer corresponding to the
first 5 amino acids encoded at the 5'-terminus:
7 MLII GCACAGTCATTGTCATTTAG. (SEQ ID NO:_)
[0217] The full length cDNA was obtained through 30 cycles of PCR,
each cycle comprising 60 seconds at 94.degree. C., 60 seconds at
58.degree. C., 90 seconds at 72.degree. C., with an extension step
at 72.degree. C. for 10 min. The reaction was performed in 100
.mu.L containing 30 pmol of each primer, 0.2 mM
deoxyribonucleotide, 1.0 unit of Pfu polymerase (Stratagene, La
Jolla, Calif.). The MLII and AP primers were designed to generate
an EcoRI site at each end (3' and 5') of the polynucleotide
sequence. The full length cDNA was ligated into the EcoRI site of
the cloning vector pCR2.1, resulting in the final product
"pCR2.1-DLA" illustrated schematically in FIG. 1.
[0218] The Nucleotide Sequence of Dl-FRIL
[0219] The Dl-FRILc done was sequenced completely using the dideoxy
chain termination method. The nucleotide sequence of the
full-length cDNA was determined to be:
8 1 GCACAGTCAT TGTCATTTAG TTTCACCAAG TTTGATCCTA ACCAAGAGGA (SEQ ID
NO:1) 51 TCTTATCTTC CAAGGTCATG CCACTTCTAC AAACAATGTC TTACAAGTCA 101
CCAAGTTAGA CAGTGCAGGA AACCCTGTGA GTTCTAGTGC GGGAAGAGTG 151
TTATATTCTG CACCATTGCG CCTTTGGGAA GACTCTGCGG TATTGACAAG 201
CTTTGACACC ATTATCAACT TTGAAATCTC AACACCTTAC ACTTCTCGTA 251
TAGCTGATGG CTTGGCCTTC TTCATTGCAC CACCTGACTC TGTCATCAGT 301
TATCATGGTG GTTTTCTTGG ACTCTTTCCC AACGCAAACA CTCTCAACAA 351
CTCTTCCACC TCTGAAAACC AAACCACCAC TAAGGCTGCA TCAAGCAACG 401
TTGTTGCTGT TGAATTTGAC ACCTATCTTA ATCCCGATTA TGGTGATCCA 451
AACTACATAC ACATCGGAAT TGACGTCAAC TCTATTAGAT CCAAGGTAAC 501
TGCTAAGTGG GACTGGCAAA ATGGGAAAAT AGCCACTGCA CACATTAGCT 551
ATAACTCTGT CTCTAAAAGA CTATCTGTTA CTAGTTATTA TGCTGGGAGT 601
AAACCTGCGA CTCTCTCCTA TGATATTGAG TTACATACAG TGCTTCCTGA 651
ATGGGTCAGA GTAGGGTTAT CTGCTTCAAC TGGACAAGAT AAAGAAAGAA 701
ATACCGTTCA CTCATGGTCT TTCACTTCAA GCTTGTGGAC CAATGTGGCG 751
AAGAAGGAGA ATGAAAACAA GTATATTACA AGAGGCGTTC TGTGATGATA 801
TATGTGTATC AATGATTTTC TATGTTATAA GCATGTAATG TGCGATGAGT 851
CAATAATCAC AAGTACAGTG TAGTACTTGT ATGTTGTTTG TGTAAGAGTC 901
AGTTTGCTTT TAATAATAAC AAGTGCAGTT AGTACTTGT
[0220] The Dl-FRIL nucleotide sequence enabled inference of the
following derived amino acid sequence for the Dl-FRIL protein:
9 AQSLSFSFTK FDPNQEDLIF QGHATSTNNV LQVTKLDSAG NPVSSSAGRV (SEQ ID
NO:2) LYSAPLRLWE DSAVLTSFDT IINFEISTPY TSRIADGLAF FIAPPDSVIS
YHGGFLGLFP NANTLNNSST SENQTTTKAA SSNVVAVEFD TYLNPDYGDP NYIHIGIDVM
SIRSKVTAKW DWQNGKIATA HISYNSVSKR LSVTSYYAGS KPATLSYDIE LHTVLPEWVR
VGLSASTGQD KERNTVHSWS FTSSLWTNVA KKENENKYIT RGVL
[0221] The naturally-occurring signal sequence from the FRIL family
member isolated from Dolichos lab lab (i.e., Dl-FRIL) has the
following sequence:
10 MASSNLLTLA LFLVLLTHAN SA (SEQ ID NO: 4)
[0222] This sequence is located directly N-terminal to the first
amino acid of SEQ ID NO: 2. The nucleic add sequence of the
naturally-occurring Dl-FRIL protein is provided below.
11 1 ATGGCTTCCT CCAACTTACT CACCCTAGCC CTCTTCCTTG TGCTTCTCAC (SEQ ID
NO: 3) 51 CCACGCAAAC TCAGCCGCAC AGTCATTGTC ATTTAGTTTC ACCAAGTTTG
101 ATCCTAACCA AGAGGATCTT ATCTTCCAAG GTCATGCCAC TTCTACAAAC 151
AATGTCTTAC AAGTCACCAA GTTAGACAGT GCAGGAAACC CTGTGAGTTC 201
TAGTGCGGGA AGAGTGTTAT ATTCTGCACC ATTGCGCCTT TGGGAAGACT 251
CTGCGGTATT GACAAGCTTT GACACCATTA TCAACTTTGA AATCTCAACA 301
CCTTACACTT CTCGTATAGC TGATGGCTTG GCCTTCTTCA TTGCACCACC 351
TGACTCTGTC ATCAGTTATC ATGGTGGTTT TCTTGGACTC TTTCCCAACG 401
CAAACACTCT CAACAACTCT TCCACCTCTG AAAACCAAAC CACCACTAAG 451
GCTGCATCAA GCAACGTTGT TGCTGTTGAA TTTGACACCT ATCTTAATCC 501
CGATTATGGT GATCCAAACT ACATACACAT CGGAATTGAC GTCAACTCTA 551
TTAGATCCAA GGTAACTGCT AAGTGGGACT GGCAAAATGG GAAAATAGCC 601
ACTGCACACA TTAGCTATAA CTCTGTCTCT AAAAGACTAT CTGTTACTAG 651
TTATTATGCT GGGAGTAAAC CTGCGACTCT CTCCTATGAT ATTGAGTTAC 701
ATACAGTGCT TCCTGAATGG GTCAGAGTAG GGTTATCTGC TTCAACTGGA 751
CAAGATAAAG AAAGAAATAC CGTTCACTCA TGGTCTTTCA CTTCAAGCTT 801
GTGGACCAAT GTGGCGAAGA AGGAGAATGA AAACAAGTAT ATTACAAGAG 851
GCGTTCTGTG ATGATATATG TGTATCAATG ATTTTCTATG TTATAAGCAT 901
GTAATGTGCG ATGAGTCAAT AATCACAAGT ACAGTGTAGT ACTTGTATGT 951
TGTTTGTGTA AGAGTCAGTT TGCTTTTAAT AATAACAAGT GCAGTTAGTA 1001
CTTGT
[0223] A comparative illustration of the derived Dl-FRIL amino acid
sequence with the reported amino acid sequence of the mannose
lectin as determined by Gowda et al. (J. Biol. Chem.
269:18789-18793, 1994) is shown in FIG. 2. The single sequence
derived for Dl-FRIL protein comprises domains that correspond
directly and with substantial homology to the a subunit (SEQ ID NO:
______) and .beta. subunit (SEQ ID NO: ______) of the protein
described by Gowda et al., supra. When the .beta. subunit of the
Gowda et al. (supra) protein is assigned to the N-terminal domain
and is followed linearly by the .alpha. subunit, the arrangement of
the polypeptides shows homology to other legume lectins.
[0224] The derived Dl-FRIL amino acid sequence, however, comprises
an additional of seven amino acid residues (aa27-34) that does not
occur in the amino acid sequence described Gowda et al., supra.
Several other differences between the amino acid sequences of
Dl-FRIL and the amino acid sequence described by Gowda et al.,
supra, are also readily discernible from FIG. 2.
[0225] Site-Specific Mutagenesis
[0226] To establish functionality of homologs of the protein
encoded by the Dl-FRIL cDNA, a mutation was made in the Dl-FRIL
cDNA done. The domains of the derived protein and the pea lectin
that include the mutation site are shown below:
12 D1-FRIL .YLNPDYG.DPNYIHIGIDV (SEQ ID NO: _) Pea
FY.NAAWDPSNRDRHIGIDV (SEQ ID NO: _)
[0227] It is known that the asparagine residue (the highlighted
"N") in the pea lectin is involved in binding to its saccharide
ligand. The corresponding asparagine in Dl-FRIL (position 141 of
the amino acid sequence of SEQ ID NO: 2) was mutated to aspartic
acid ("D"). This mutation was designated "N141D" for
convenience.
[0228] To introduce the mutation, recombinant PCR was performed
(see, e.g., Higuchi, R., PCR Protocols: A Guide to Methods and
Applications, Innis M. A., Gelfand D. H., Sninsky J. J., and White
T J, eds., Academic Press, San Diego, 1990). Two PCR reactions were
carried out separately on the full length cDNA using two primers
that contain the same mutation and produce two products with an
overlapping region:
13 MutI CCATAATCGGGATCAAGATAGGTG (SEQ ID NO: _) MutII
CACCTATCTTGATCCCGATTATGG (SEQ ID NO: _)
[0229] The primary PCR products were purified with the QIAquick PCR
Purification kit (QIAGEN, Valencia, Calif.), according to the
manufacturer's instructions. The overlapping primary products were
then combined and amplified together in a single second reaction
using flanking primers:
14 M1 Forw AACTCAGCCGCACAGTCATTGTCA (SEQ ID NO: _) APEcoRI
GAATTCGACCACGCGTATCGATGTCGAC (SEQ ID NO: _)
[0230] Both the primary and the secondary PCR reactions were
performed in 100 .mu.L containing 50 pmol of each primer, 0.4 mM
deoxyribonucleotide and 1.0 unit Pfu polymerase (Stratagene) in the
corresponding buffer. The primary PCR reaction amplified the two
separate fragments in 30 cycles, each cycle comprising 40 seconds
at 94.degree. C., 40 seconds at 50.degree. C., 60 seconds at
72.degree. C., with an extension step at 72.degree. C. for 10 min.
The second PCR reaction amplified the recombinant fragment in 12
cycles using the same conditions described above.
[0231] The resulting full-length fragment contained the mutation.
The recombinant mutated product was cloned in the EcoRI site of the
cloning vector pCR2.1, as illustrated schematically in FIG. 3, and
sequenced as described above. This plasmid is referred to as
"pCR2.1-DLA(D)."
[0232] Construction of Dl-FRIL-Expressing Plant Expression Vectors
and Nicotiana tabacum Transformation
[0233] Recombinant PCR was used to modify the 5' ends of both the
wild-type and the mutant Dl-FRIL clones, to introduce a signal
peptide for entry of the protein into the endoplasmic reticulum.
Following the procedure of Higuchi, supra, the sequence encoding
the signal peptide and the full-length cDNA clones were amplified
in two separate primary PCR reactions. The signal peptide sequence
was obtained from the amplification of the binary vector pTA4,
harboring the complete sequence of the .alpha.-amylase inhibitor
gene (Hoffman et al., Nucleic Acids Res. 10:7819-7828, 1982; Moreno
et al., Proc. Natl. Acad. Sci. USA 86:7885-7889, 1989).
[0234] The following primers were used for amplification of the
signal peptide sequence:
15 Sigforw GAATTCATGGCTTCCTCCAAC (SEQ ID NO: _) Sigrev
TGACTGTGCGGCTGAGTTTGCGTGGGTG (SEQ ID NO: _)
[0235] The primers M1Forw (SEQ ID NO: ______) and AP EcoRI (SEQ ID
NO: ______) used for amplification of the Dl-FRIL cDNA described
above, were again used to amplify the Dl-FRIL cDNA.
[0236] The primers used for the secondary reactions were Sigforw
and AP EcoRI, which were designed to generate EcoRI sites at the 5'
and the 3' ends. Both the primary and the secondary PCR reactions
were performed as discussed above for the site-directed
mutagenesis.
[0237] The wild-type recombinant product SpDLA was cloned in the
EcoRI site of the pbluescript SK+ cloning vector (Stratagene) to
give the vector pBS-SpDLA, as shown in FIG. 4. The mutant SpDLA(D)
was cloned in the same site of the cloning vector pCR2.1 to give
the vector pCR2.1-SpM1, as shown in FIG. 5. The nucleotide sequence
of each PCR product was determined as described above to verify the
correct attachment of the signal peptide. The nucleotide sequence
of SpDLA is defined by SEQ ID NO:22, and the derived amino acid
sequence is defined by SEQ ID NO:23.
[0238] The sequences of SEQ ID NO: 22 and SEQ ID NO: 23 are as
follows:
16 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ATGGCTTCCT CCAACTTACT
CACCCTAGCC CTCTTCCTTG TGCTTCTCAC CCACGCAAAC 60 TCAGCCGCAC
AGTCATTGTC ATTTAGTTTC ACCAAGTTTG ATCCTAACCA AGAGGATCTT 120
ATCTTCCAAG GTCATGCCAC TTCTACAAAC AATGTCTTAC AAGTCACCAA GTTAGACAGT
180 GCAGGAAACC CTGTGAGTTC TAGTGCGGGA AGAGTGTTAT ATTCTGCACC
ATTGCGCCTT 240 TGGGAAGACT CTGCGGTATT GACAAGCTTT GACACCATTA
TCAACTTTGA AATCTCAACA 300 CCTTACACTT CTCGTATAGC TGATGGCTTG
GCCTTCTTCA TTGCACCACC TGACTCTGTC 360 ATCAGTTATC ATGGTGGTTT
TCTTGGACTC TTTCCCAACG CAAACACTCT CAACAACTCT 420 TCCACCTCTG
AAAACCAAAC CACCACTAAG GCTGCATCAA GCAACGTTGT TGCTGTTGAA 480
TTTGACACCT ATCTTAATCC CGATTATGGT GATCCAAACT ACATACACAT CGGAATTGAC
540 GTCAACTCTA TTAGATCCAA GGTAACTGCT AAGTGGGACT GGCAAAATGG
GAAAATAGCC 600 ACTGCACACA TTAGCTATAA CTCTGTCTCT AAAAGACTAT
CTGTTACTAG TTATTATGCT 660 GGGAGTAAAC CTGCGACTCT CTCCTATGAT
ATTGAGTTAC ATACAGTGCT TCCTGAATGG 720 GTCAGAGTAG GGTTATCTGC
TTCAACTGGA CAAGATAAAG AAAGAAATAC CGTTCACTCA 780 TGGTCTTTCA
CTTCAAGCTT GTGGACCAAT GTGGCGAAGA AGGAGAATGA AAACAAGTAT 840
ATTACAAGAG GCGTTCTGTG ATGATATATG TGTATCAATG ATTTTCTATG TTATAAGCAT
900 GTAATGTGCG ATGAGTCAAT AATCACAAGT ACAGTGTAGT ACTTGTATGT
TGTTTGTGTA 960 AGAGTCAGTT TGCTTTTAAT AATAACAAGT GCAGTTAGTA CTTGT
1005 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Met Ala Ser Ser Asn
Leu Leu Thr Leu Ala Leu Phe Leu Val Leu Leu 5 10 15 Thr His Ala Asn
Ser Ala Ala Gln Ser Leu Ser Phe Ser Phe Thr Lys 20 25 30 Phe Asp
Pro Asn Gln Glu Asp Leu Ile Phe Gln Gly His Ala Thr Ser 35 40 45
Thr Asn Asn Val Leu Gln Val Thr Lys Leu Asp Ser Ala Gly Asn Pro 50
55 60 Val Ser Ser Ser Ala Gly Arg Val Leu Tyr Ser Ala Pro Leu Arg
Leu 65 70 75 80 Trp Glu Asp Ser Ala Val Leu Thr Ser Phe Asp Thr Ile
Ile Asn Phe 85 90 95 Glu Ile Ser Thr Pro Tyr Thr Ser Arg Ile Ala
Asp Gly Leu Ala Phe 100 105 110 Phe Ile Ala Pro Pro Asp Ser Val Ile
Ser Tyr His Gly Gly Phe Leu 115 120 125 Gly Leu Phe Pro Asn Ala Asn
Thr Leu Asn Asn Ser Ser Thr Ser Glu 130 135 140 Asn Gln Thr Thr Thr
Lys Ala Ala Ser Ser Asn Val Val Ala Val Glu 145 150 155 160 Phe Asp
Thr Tyr Leu Asn Pro Asp Tyr Gly Asp Pro Asn Tyr Ile His 165 170 175
Ile Gly Ile Asp Val Asn Ser Ile Arg Ser Lys Val Thr Ala Lys Trp 180
185 190 Asp Trp Gln Asn Gly Lys Ile Ala Thr Ala His Ile Ser Tyr Asn
Ser 195 200 205 Val Ser Lys Arg Leu Ser Val Thr Ser Tyr Tyr Ala Gly
Ser Lys Pro 210 215 220 Ala Thr Leu Ser Tyr Asp Ile Glu Leu His Thr
Val Leu Pro Glu Trp 225 230 235 240 Val Arg Val Gly Leu Ser Ala Ser
Thr Gly Gln Asp Lys Glu Arg Asn 245 250 255 Thr Val His Ser Trp Ser
Phe Thr Ser Ser Leu Trp Thr Asn Val Ala 260 265 270 Lys Lys Glu Asn
Glu Asn Lys Tyr Ile Thr Arg Gly Val Leu 275 280 285
[0239] A Plant Expression Vector Encoding Recombinant Dl-FRIL
[0240] A binary vector was constructed for seed-specific expression
of Dl-FRIL. For seed expression, the vicilin promoter obtained from
the pCW66 (Higgins et al., Plant Mol. Biol. 11:683-695, 1988) was
cloned in EcoRI/KpnI sites of the plant expression vector pBIN19,
to form pBINVicPro, as illustrated in FIG. 6. Downstream of the
vicilin promoter, the SpDLA cDNA sequence was ligated into the
EcoRI/SacI site, giving rise to the pBINVicPro-SpDLA, which is
illustrated in FIG. 7. The mutated cDNA done SpDLA(D) was ligated
in EcoRI site of the pBINVicPro vector to yield
pBINVicPro-SpDLA(D), which is illustrated in FIG. 8. No additional
termination sequences were added, relying instead on the stop
codons and the polyadenylation site of the DLA and DLA(D) cDNA
clones. Both vectors were transferred into Agrobacterium
tumefaciens strain LBA4404 according to the freeze-thaw procedure
reported by An et al., "Binary vectors," in Plant Molecular Biology
Manual, Vol. A3, Gelvin S B, Schilperoort R A, and Verma D P S,
eds., Kluwer Academic Publisher, Dordrecht, The Netherlands, pp.
1-19, 1988).
[0241] Agrobacterium-mediated transformation of Nicotiana tabacum
leaf disks was carried out and assayed as described (Horsch et al.,
Science 227:1229-1231, 1985) using LBA4404 harboring the
seed-specific expression vector pBINVicPro-SpDLA (FIG. 9).
Kanamycin-resistant plants (resistance being conferred by
transformation with the pBIN19-based vectors that carry the gene)
were scored for their ability to form roots in two consecutive
steps of propagation in Murashige-Skoog medium containing 3% of
sucrose and kanamycin sulfate (Sigma, St. Louis, Mo.) at 100
mg/mL.
[0242] Recombinant Dl-FRIL fusion proteins were cleaved by the
transformed plant cells in vivo. Thus, the transformed plant cells
produced mature Dl-FRIL which, when purified, had a molecular mass
of 60 kDa and comprised four subunits, two alpha subunits and two
beta subunits (i.e., an .alpha..sub.2.beta..sub.2 heterodimer),
where each subunit is about 15-18 kDa.
[0243] Expression of Recombinant Dl-FRIL in E. coli
[0244] The Dl-FRIL wild-type cDNA and mutant clones (without signal
peptides), were ligated into the EcoRI/SalI and EcoRI/XhoI of the
expression vector pGEX 4T-1 (Pharmacia Biotech, Uppsala, Sweden),
to form the expression constructs pGEX-M1 and pGEX M1(D),
respectively illustrated in FIGS. 9 and 10. The host E. coli
strain, BL21(D3), was purchased from Novagen (Madison, Wis., and
transformed with the above construct using the calcium chloride
method (see Sambrook et al., supra; Gelvin and Schilperoort, Plant
Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht,
The Netherlands, 1988; Altabella et al., Plant Physiol 93:805-810,
1990; and Pueyo et al., Planta 196:586-596, 1995). The induction of
the tac promoter (Ptac) was achieved by adding IPTG
(isopropyl-.beta.-D-thiogalactopyranoside) (Sigma) at a 1.0 mM
final concentration when the cells reached an optical density of
0.4-0.6 at 600 nm. The cultures were allowed to grow for 12 hours
at 37.degree. C. after the addition of IPTG. Control non-induced
cultures were maintained under similar conditions. The cells were
lysed by treatment with 4 mg/mL lysozyme in phosphate-buffered
saline containing 1% TRITON.RTM. X-100.
[0245] Total cellular protein was extracted from transformed E.
coli cells and analyzed on SDS-PAGE on a 15% gel using a standard
procedure (Sambrook et al., supra). The cells from 1 mL of E. coli
culture were suspended in the same volume of loading buffer (50 mM
Tris HCl pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol
blue) and vortexed. Following transfer to a nitrocellulose
membrane, protein was stained with Coomassie Brilliant Blue R250. A
representative separation is shown in FIG. 11, with the lanes
identified in Table 1, below.
17TABLE 1 Key to FIG. 11 Lane No. Content 1 Molecular Mass Marker
(Bio-Rad) 2 Total Protein Extract from Non-Induced BL21(D3) pGEX-M1
3 Total Protein Extract from Induced BL21(D3) pGEX-M1 4 Total
Protein Extract from Non-Induced BL21(D3) pGEX- M1(D) 5 Total
Protein Extract from Induced BL21(D3) pGEX-M1(D)
[0246] The separation of proteins in FIG. 11 shows that the induced
cells (lanes 3, 5) both produced an abundant polypeptide having a
molecular mass of about 60 kDa (indicated by arrow). The
non-induced cells failed to produce any significant amount of this
protein (FIG. 11, lanes 2, 4).
[0247] Purification of Recombinant Dl-FRIL from Transformed E.
coli
[0248] Induced E. coli cells (200 mL) as described above were
harvested after 12 hour induction at 37.degree. C. by
centrifugation at 5000 g for 10 min. The pellet was washed with 50
mM Tris-HCl pH 8.0, 2 mM EDTA, and resuspended in {fraction (1/10)}
vol of 1% TRITON surfactant in TBS (20 mM Tris pH 7.5, 500 mM
NaCl). The cells were lysed by adding 4 mg/mL of lysozyme and
incubating at room temperature for 30-60 min. After centrifugation
at 5000 g, the supernatant containing the total soluble proteins
was discarded and the resulting pellet, comprising the inclusion
bodies and containing the accumulated the recombinant fusion
protein, was extracted with 8 M guanidine-HCl (Martson and Hartley,
"Solubilization of Protein Aggregates" in Guide to Protein
Purification vol. 182, eds. Deutscher, M. P., pp 266-267,
1993).
[0249] The recombinant fusion protein solubilized by guanidine-HCl
was purified on GST-Sepharose beads (Pharmacia Biotech, Uppsala,
Sweden) according the manufacturer's instructions and eluted in 1
mL of reduced glutathione (Sigma). Samples of the purified fusion
proteins were cleaved with thrombin (Novagen) using 5 cleavage
units/mL purified fusion protein.
[0250] For immunoblot analysis (Western blot), the purified
proteins were separated by SDS-PAGE in general accordance with the
method described above. The gel was S equilibrated in transfer
buffer (25 mM Tris pH 8.3, 192 mM Glycine, 20% MeOH) and blotted
onto nitrocellulose (Bio-Rad, Hercules, Calif.) for 1 hour at 100 V
using a Bio-Rad electrotransfer apparatus. Non-specific binding was
blocked by incubating the blots for at least 1 hour in 1.times.TBS
(20 mM Tris pH 7.5,500 mM NaCl) containing 3% gelatin. Blotting was
followed by incubation with a primary antibody (a polyclonal rabbit
serum raised against the N-terminal peptide of the .beta.-subunit
of Phaseolus vulgaris FRIL (i.e., Pv-FRIL), 1:100 dilution, 3 hour;
described below in Example 5), followed by incubation with a
secondary antibody (goat anti-rabbit IgG conjugated to horseradish
peroxidase at 1:1000 dilution for 1 hour). The blots were washed
and the color developed with the color development reagent
(Bio-Rad). A representative result is shown in FIG. 12, with the
lanes identified in Table 2, below.
18TABLE 2 Key to FIG. 12 Lane No. Content 1 Purified Fusion Protein
M1 2 Purified Fusion Protein M1(D) 3 Control 4 Purified Fusion
Protein M1 After Cleavage with Thrombin 5 Purified Fusion Protein
M1(D) After Cleavage with Thrombin 6 Control
[0251] The separation shown in FIG. 12 demonstrates that the two
forms of fusion protein have similar molecular masses of about 60
kDa, and that thrombin cleaved both types of fusion protein to
produce a new polypeptide of molecular mass 30 kDa.
EXAMPLE 2
Recombinant Dl-FRIL Specifically Stimulates Proliferation of 3T3
Cells Expressing the FLT3 Receptor
[0252] Dl-FRIL interacts with the mammalian FLK2/FLT3 tyrosine
kinase receptor. A specific and quantitative biological assay using
NIH 3T3 fibroblasts transfected either with a chimeric receptor
having the extracellular portion of the murine FLT3 receptor
combined with the intracellular portion of the human Fms receptor
(Dosil et al., Mol. Cell. Biol. 13(10):6572-6585 1993) or with the
full length human receptor (Small et al., Proc. Natl. Acad. Sci.
USA 91:459-463, 1994) can be used to evaluate lectin biological
activity during purification. Serial two-fold dilutions of lectin
samples across rows of a 96 well plate allowed for greater than a
thousand-fold range to access FLT3 3T3 biological activity. Either
the murine or human FLT3 ligand (FL) (Lyman et al., Cell
75:1157-1167, 1993; Hannum et al., Nature 368: 643-648, 1994) or
the FRIL was found to rescue FLT3-transfected cells from death in
this assay.
[0253] Specifically, 3T3 cells cultured in tissue culture plates
(Becton Dickinson Labware, Lincoln Park, N.J.) were removed from
the plates by washing cells twice in Hank's buffered saline
solution (HBSS; Gibco Laboratories, Grand Island, N.Y.).
Non-enzymatic cell dissociation buffer (Gibco) was added for 15
minutes at room temperature. The resulting cells wee washed in
medium. FLT3 3T3 cells were cultured at a final concentration of
3,000 cells per well in a volume of 100 .mu.L of serum-defined
medium containing 10 mg/mL rhIL1-.alpha., 10% AIMV (Gibco, Grand
Island, N.Y.) and 90% Dulbecco's modification of Eagle's medium
(DMEM; Gibco) in 96 well plates. Under these assay conditions,
cells die after two to four days of culture in a humidified
incubator at 37.degree. C. and 5% CO.sub.2 unless exogenously added
ligand rescues cells from death. Each 96 well plate contained wells
of cells containing calf serum, which stimulates all 3T3 cells, as
a positive controls and wells of cells containing medium only as a
negative control ("blank"). Full-length Fms-transfected 3T3 cells
(biological response shown in Tessler et al., J. Biol. Chem.,
269:12456-12461, 1994) served as receptor-transfected control
target cells, and parent 3T3 cells served as untransfected control
cells. Proliferation and cell survival was quantitated by addition
of XTT (2,3-bis[Methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5
carboxanilide inner salt) (Diagnostic Chemicals Ltd, Charlottetown,
Prince Edward Island, Canada), which is a tetraformazan salt
cleaved by actively respiring cells (Roehm et al., J. Immunol.
Methods 142: 257-265, 1991). Proliferation and cell survival was
quantitated spectrophotometrically using a Vmax kinetic plate
reader (Molecular Devices Corp., Mountain View, Calif.), and
recorded as either relative activity (units/mL) or as specific
activity (units/mg). One unit of biological activity was defined as
the reciprocal dilution at which half-maximal stimulation of cells
is detected.
[0254] The crude protein extract from the E. coli cultures
described in Example 1, above, was tested to determine whether
expressed recombinant Dl-FRIL possessed any capacity to stimulate
FLT3 3T3 cells using this assay. The data from this experiment are
summarized in FIGS. 13A and 13B. Specifically, FIG. 13A is a graph
showing that the crude extract of the E. coli culture containing
expressed Dl-FRIL specifically stimulates hFLT3 cells; FIG. 13B is
a graph showing that the same extract does not stimulate
untransfected 3T3 cells. In FIGS. 13A and 13B, medium control is
represented by a solid line. The ordinate (absorbance) indicates
cell viability measured by XTT at three days; the abscissa shows
the reciprocal dilution of the extract sample. The apparent
inhibition of proliferation observed at higher concentrations (FIG.
13A) is not understood, but may relate to toxic components in the
crude E. coli extract or the consequences of dose-related
preservation of the 3T3 fibroblasts.
EXAMPLE 3
Recombinant Dl-FRIL Preserves Mononuclear Cells and Progenitors in
Liquid Culture
[0255] The recombinant Dl-FRIL protein preserved functional
progenitors for at least four weeks in liquid culture. FIGS. 14A
and 14B, and Table 3 illustrate the results of experiments in which
recombinant Dl-FRIL was shown to act in a dose-responsive manner to
preserve human cord blood progenitors.
[0256] To do this, umbilical cord blood from healthy donors was
collected in 100 units/ml of heparin. Cord blood mononuclear cells
(CB mnc) were isolated within 4 hours of collection by
FICOLL-PAQUE.RTM. separation (Pharmacia Biotech, Piscataway, N.J.)
following manufacturer's instruction and washed in X-VIVO 10 medium
(BioWhittaker, Walkersville, Md.). CB mnc were then cultured in six
well tissue culture plates (Corning Inc., Corning, N.Y.) at a
concentration of 200,000 cells/mL in a volume of 4 mL of X-VIVO 10
(i.e., 800,000 cells total per well). Dl-FRIL and/or recombinant E.
coli Flt3-L (recFL; BioSource International, Camarillo, Calif.)
were added at a concentration of 40 ng/ml at the outset (with no
addition as a control). Cultures were incubated in humidified
chambers without medium changes for up to 29 days.
[0257] After incubation, the cultured CB mnc cells were harvested
by washing in X-VIVO 10 (i.e., harvested cells were pelleted and
resuspended in X-VIVO 10) to remove the Dl-FRIL and/or recFL, and
then determining viable cell number by trypan blue (Sigma)
exclusion. These results are shown in FIG. 14A. The progenitor
number and capacity of harvested cells were assessed by plating the
washed cells in triplicate in fetal bovine serum-free,
methylcellulose colony assay medium containing IL-2,
granulocyte-macrophage CSF, and kit ligand (StemCell Technologies,
Vancouver, BC, Canada). After two weeks, the resultant colonies
from each of the triplicate wells were scored and the results are
shown in FIG. 14B. Thus, FIGS. 14A and 14B show that recombinant
Dl-FRIL preserved cord blood mononuclear cells and progenitors in a
dose-responsive manner in liquid culture.
[0258] Table 3 shows the resulting colonies after 15, 21, or 29
days of incubation, demonstrating that Dl-FRIL, but not recFL,
preserved progenitors in suspension culture.
19TABLE 3 Day Medium Myeloid* Erythroid* Mix* Blast* 15 Dl-FRIL
1,033 .+-. 12 67 .+-. 12 7 .+-. 12 0 recFL 40 .+-. 69 0 0 0 Dl-FRIL
+ 933 .+-. 250 167 .+-. 95 0 0 recFL No Addition 0 0 0 0 21 Dl-FRIL
387 .+-. 83 7 .+-. 12 0 167 .+-. 64 recFL 0 0 0 0 Dl-FRIL + 473
.+-. 133 53 .+-. 42 0 300 .+-. 34 recFL No Addition 0 0 0 0 29
Dl-FRIL 0 0 0 80 .+-. 72 recFL 0 0 0 0 Dl-FRIL + 0 0 0 40 .+-. 20
recFL No Addition 0 0 0 0 *Data reported as .+-.SD of the three
values from the triplicate methylcellulose colony assay. The
reported experiment is a representative of four experiments.
[0259] In FIGS. 14A and 14B and in Table 3, "blast" refers to
colonies consisting of primitive, morphologically undifferentiated
cells; "mix" refers to colonies consisting of myeloid and erythroid
cells; "erythroid" refers to colonies consisting of erythroid
cells; and "myeloid" refers to colonies consisting of myeloid
cells. In FIGS. 14A and 14B, cell number (FIG. 14A) or colony
number (FIG. 14B) is shown on the ordinate; the abscissa shows the
reciprocal dilution of the sample.
[0260] As shown in Table 3, colonies derived from mature myeloid
and erythroid progenitors formed from cells cultured for 15 days in
either FRIL or FRIL plus recFL; 24-fold fewer mature colonies
formed from cells cultured in recFL alone; and no colonies appeared
if neither was present. After 21 days in culture, Table 3 shows
that myeloid and erythroid colonies formed only from cells exposed
to Dl-FRIL. The frequency of myeloid colonies in Dl-FRIL-only
cultures (based on the initial number of CB mnc) decreased by 2.7
fold from 1 in 774 after 2 weeks in culture to 1 in 2,067 after 3
weeks; erythroid colonies decreased in frequency by 9.6 fold from 1
in 11,940 to 1 in 114,287 (see Table 3).
[0261] The colonies from this assay were photographed at various
time points. As shown in FIG. 15A, in addition to myeloid and
erythroid colonies, day 21 cultures contained small colonies
consisting of undifferentiated cells. FIG. 15B shows that only
blast-like colonies were observed when the cells were cultured in
Dl-FRIL for 29 days (see also Table 3).
[0262] The progenitor capacity of the blast-like colonies was
examined further for cells initially cultured for 3 weeks in either
Dl-FRIL, recFL, no addition, or Dl-FRIL+recFL, and then without
these regulators for an additional 6 weeks in a methylcellulose
colony assay. No colonies were detected from the cells cultured for
21 days in either recFL alone or medium control. Viable cells were
harvested from the cells initially cultured in Dl-FRIL alone and
replated in an colony assay (in methylcellulose colony assay
medium) for an additional 4 weeks. A schematic diagram of this
experiment is shown in FIG. 16. As schematically diagramed in FIG.
16, the frequency of blast-like colonies cultured in Dl-FRIL alone
decreased by 2.1 fold from day 21 to day 29, from 1 in 4,790 to 1
in 10,000, and 7.5 fold in Dl-FRIL+recFL cultures from 1 in 2,667
to 1 in 20,000 of the initial CB mnc cells cultures. Following the
protocol schematically diagrammed in FIG. 16, small, diffuse,
blast-like colonies were detected at a frequency of 1 in 67 (900
colonies/600,000 CB mnc) exclusively in dishes of cells initially
cultured in Dl-FRIL alone, and at a frequency of 1 in 132 (990
colonies/131,000 CB mnc) for cells cultured in Dl-FRIL+recFL (see
examplary colonies in FIG. 17).
EXAMPLE 4
Recombinant Dl-FRIL Acts Directly on Progenitor Cells
[0263] To assess whether recombinant Dl-FRIL acts directly or
indirectly through accessory cells to preserve progenitor cells,
cord blood mononuclear cells were first enriched for progenitors
expressing the CD34 antigen by immunomagnetic bead isolation (Dynal
Corp., Lake Success, N.Y.). Five hundred CD34.sup.+ cells were
placed into wells containing 100 .mu.L of serum-free medium
(BIT9500, StemCell Technologies, Vancouver, BC, Canada) either in
the presence of recFL (PeproTech, Princeton, N.J.) or a cytokine
cocktail of recombinant human interleukin 3 (rhIL3)+recombinant
human interleukin 6 (rhIL6)+recombinant human interleukin 11
(rhIL11)+rhTpo Thrombopoietin+FL (FLT3-Ligand) (BioSource
International, Camarillo, Calif.) in 96-well plates and cultured
for four weeks without medium changes. The numbers of functional
progenitors from these cultures were assessed by plating cells in
complete serum-free methylcellulose colony assay medium (StemCell
Technologies). After two weeks, the resultant colonies were scored
and the results are shown in FIG. 18 (solid bars=recombinant
Dl-FRIL; open bars=cytokine cocktail). Clearly, progenitors were
preserved only in the recombinant Dl-FRIL-containing cultures (FIG.
18). Thus, purified recombinant Dl-FRIL acts directly on primitive
hematopoietic progenitors.
EXAMPLE 5
Identification and Cloning of Pv-FRIL, a Second FRIL Family Member
FRIL Activity in PHA-LCM Media
[0264] A biological screening assay to search for novel stimulators
of the Flt3 receptor was developed using NIH 3T3 cells transfected
with expression vectors containing cDNA of murine and human Flt3
and the related Fms receptor. The mFlt3/Fms 3T3 cell line is a 3T3
cell line transfected with nucleic acid encoding a fusion protein
consisting of the murine extracellular domain of the Flt3 receptor
fused to the transmembrane and intracellular domains of the human
Fms (provided by Dr. Ihor Lemischka, Princeton University,
Princeton, N.J.). The Stk 3T3 cell line is a 3T3 cell line
transfected with the full-length human Flt3 receptor (provided by
Dr. Donald Small, Johns Hopkins University, Baltimore, Md.). The
human FMS 3T3 cell line is a 3T3 cell line transfected with the
full-length human Fms receptor (provided by Dr. Charles Sherr,
Saint Jude Children's Research Hospital, Memphis, Tenn. Parent 3T3
cells were purchased from American Type Culture Collection ("ATCC";
Manassas, Va.). Receptor-transfected cells contained Neo resistance
genes and were maintained in medium containing 750 g/ml of G418
(Life Technologies, Rockville, Md.).
[0265] To create factor-dependence for receptor-transfected 3T3
cells, growth conditions were compromised to permit only ligands to
rescue cells from death. To do this, 3T3 cell lines were assayed in
96 well plates (Becton Dickinson Labware, Lincoln Park, N.J.)
containing 3,000 cells in 100 .mu.L of serum-free medium consisting
of 10% AIMV (Life Technologies) and 90% DMEM. In each experiment,
samples were serially diluted two-fold across rows starting at a
1:10 dilution. Viable cells were quantitated after 3-5 days by XTT
(2,3-bis[Methoxy-4-nitro-5sulfophenyl]-2H-tetrazoli- um-5
carboxanilide inner salt) (Sigma, St. Louis, Mo.) (Roehm et al.,
supra). Relative activity (units/ml) and specific activity
(units/mg) are defined as the reciprocal dilution at which
half-maximal stimulation of cells was detected.
[0266] The FMS 3T3 cells (transfected with cDNA encoding the human
Fms tyrosine kinase receptor) and its ligand, human M-CSF, served
as a model system. Recombinant human M-CSF stock of 1 .mu.g/mL was
serially diluted (where the first dilution, 1:20, was 50 ng/mL) was
used to stimulate Fms3T3 cells. As shown in FIG. 19A, Fms 3T3 cells
responded to M-CSF in a dose-responsive manner. Neither the
mFlt3/Fms 3T3 cells nor the parent untransfected 3T3 cells
responded to M-CSF (FIG. 19A).
[0267] Various sources of conditioned medium were screened for the
presence of Flt3 3T3 stimulatory activity. The most potent source
was conditioned medium harvested from human peripheral blood cells
activated to secrete high levels and a broad range of cytokines by
the mitogenic legume lectin, phytohemagglutinin (PHA), which is
derived from impure red kidney bean extracts. This source, commonly
called PHA leukocyte-conditioned medium (PHA-LCM), has been used as
a standard positive control in hematopoietic colony assays for over
two decades (Sharon and Lis, Science 246: 227, 1989). To make
PHA-LCM, leukopherized blood from normal volunteers was purchased
from North American Biologicals Inc., Miami, Fla. Mononuclear cells
were isolated by FICOLL-PAQUE.RTM., washed in AIMV, and cultured at
a concentration of 2.times.10.sup.6 cells/ml in AIMV containing a
1% volume of crude red kidney bean extract containing PHA from Life
Technologies (catalog number 10576-015) in either T150 flasks
(Becton Dickinson Labware, Lincoln Park, N.J.) or roller bottles
(Becton Dickinson Labware) for one week. Cells and debris were
removed by centrifugation and conditioned medium was stored at
-20.degree. C.
[0268] PHA-LCM induced proliferation of mFlt3/Fms 3T3 cells and Stk
3T3 (expressing the human Flt3 receptor) in an indistinguishable
manner at approximately 200 units/mL (FIG. 19B). Untransfected 3T3
cells did not respond to PHA-LCM (FIG. 19B) and Fms 3T3 cells
responded weakly (data not shown).
[0269] Purification of Pv-FRIL from PHA-stimulated Leukocyte
Conditioned Media
[0270] Each batch of PHA-LCM was generated in serum-free medium
with cells from individual normal donors. To start purification,
the PHA-LCM with Flt3 3T3 activity was pooled in 30 liter lots with
approximately 10.sup.7 Flt3 3T3 units. Twenty-five liters of
PHA-LCM was diafiltered into 50 mM Tris-HCl, pH 7.6, 50 mM NaCl and
then concentrated to 2-2.5 liters by tangential flow
ultrafiltration on a 10 kDa molecular weight cutoff membrane
(Pellicon, Millipore, Bedford, Mass.). A Blue-sepharose FF
(Pharmacia Biotech) column (10 cm.times.15 cm) was equilibrated
with 50 mM Tris-HCl, pH 7.4, 50 mM NaCl. To remove the human serum
albumin, concentrated PHA-LCM was applied to the column at 25
ml/min by pump and the flow through was collected. The flow-through
fraction from Blue-sepharose was subjected to anion exchange
chromatography by being applied to a Q-sepharose FF (Pharmacia
Biotech) column (5 cm.times.5 cm) equilibrated with 10 mM Tris-HCl,
pH 7.6. The column was washed with equilibration buffer and then
eluted at 12 ml/min with a continuous gradient of 0-0.7 M NaCl in
10 mM Tris. Fractions were collected (6 ml/fraction) and an aliquot
of each fraction was tested for Flt3 3T3 activity (i.e., an ability
to stimulate mFlt3/fms 3T3 cells and/or Stk 3T3 cells) in 96 well
plates.
[0271] After validation that a fraction had Flt3 T3 activity, a
phenyl-sepharose HP (Pharmacia Biotech) column (1.6 cm.times.10 cm)
was equilibrated with 20 mM phosphate, pH 7, 1.5 M
NH.sub.4SO.sub.4. The pooled sample from Q-sepharose was adjusted
to 1.5 M NH.sub.4SO.sub.4 and applied to the phenyl-sepharose
column. The column was washed with equilibration buffer and eluted
at 1 ml/min with a gradient of 1.5-0.1 M NH.sub.4SO.sub.4 in 20 mM
phosphate, pH 7. Fractions (1 ml) were collected and tested for
Flt3 3T3 activity. Fractions with Flt3 3T3 activity were pooled and
dialyzed against 50 mM Tris-HCl, pH 7.2, 100 mM NaCl and
concentrated by vacuum centrifugation.
[0272] A Superdex 75 (Pharmacia Biotech) column (1.6 cm.times.60
cm) was equilibrated with 50 mM Tris-HCl, pH 7.4, 100 mM NaCl. The
pooled sample from phenyl-sepharose was applied to the Superdex 75
column and eluted at 0.6 ml/min. Fractions (1.8 ml) were collected
and tested for Flt3 3T3 activity. The active fractions were
dialyzed against Tris-HCl, pH 7.2 and concentrated by vacuum
centrifugation.
[0273] A C4 reverse-phase column (4.6 mm.times.100 mm, Vydac,
Hesperia, Calif.) was equilibrated with 0.1% trifluoroacetic acid
(TFA) in HPLC grade H.sub.2O. Pooled and concentrated sample from
Superdex 75 chromatography was applied to the C4 column and the
column was eluted with a gradient of 10-55% acetonitrile, 0.1% TFA
over 70 min at 0.5 ml/min. Fractions of 0.5 mL were collected and
evaporated by vacuum centrifugation.
[0274] Analysis by ELISA for cytokines (IL1-.alpha., IL1-.beta.,
IL2, IL3, IL4, IL6, GM-CSF, G-CSF and SCF) during purification of
Pv-FRIL were performed using kits purchased from R&D Systems
(Minneapolis, Minn.).
[0275] Pv-FRIL Specific Rabbit Anti-Serum
[0276] Throughout purification of Pv-FRIL a New Zealand White
rabbit (HRP, Denver, Pa.) was immunized with crude PHA-LCM, boosted
with increasingly purified samples containing Flt3 3T3 activity,
and finally immunized with a peptide corresponding to Pv-FRIL
(AQSLSF[N, C, S]FTKFDLD), referred to as the AQS-peptide. Samples
were glutaraldehyde conjugated to keyhole limpet hemocyanin (KLH,
Sigma). The rabbit was immunized with KLH-AQS peptide-containing
samples using either Complete Freund's Adjuvant (Sigma) or Hunter's
Titermax (Vaxcel, Inc., Norcross, Ga.). Antiserum demonstrated a
1:5,000 titer to the AQS peptide in an ELISA (data not shown).
Since the antiserum contained reactivities to other proteins,
further enrichment for AQS peptide-specific antibodies was achieved
by either depletion of cross-reactive antibodies or by affinity
purification using a AQS peptide covalently linked to an agarose
support (AminoLink coupling gel, Pierce).
[0277] An anti-AQS affinity column was prepared either by purifying
IgG from high titer rabbit antiserum by protein A affinity
chromatography (ImmunoPure kit, Pierce) or antibody isolated from
the AQS peptide column and then covalently linking antibody to an
activated agarose support (AminoLink coupling gel, Pierce).
[0278] Activity of Purified Pv-FRIL
[0279] To relate the observations of Pv-FRIL's activity on
receptor-transfected 3T3 cells to Flt3 receptor-expressing
hematopoietic progenitors, a suspension culture of human cord blood
cells enriched for Flt3.sup.+ progenitors by CD34 immunomagnetic
bead selection was adapted to a 96 well plate format. To do this,
umbilical cord blood from healthy donors was collected in 100
units/ml of heparin (Fujisawa Healthcare, Deerfield, Ill.).
Mononuclear cells were isolated by FICOLL-PAQUE.RTM. (Pharmacia
Biotech, Piscataway, N.J.), washed in HBSS, and resuspended in
serum-defined medium, either AIMV or XVIVO-10 (BioWhittaker,
Walkersville, Md.). Mononudear cells were enriched for Flt3.sup.+
progenitors by CD34 immunomagnetic bead selection (Dynal
Corporation, Lake Success, N.Y.). CD34.sup.+ cells were cultured in
6 well plates (Becton Dickinson Labware) in at a concentration of
10.sup.5 cells in 1 mL of DMEM containing 10 ng/mL recombinant
human IL3 (BioSource International, Camarillo, Calif.) and 10%
fetal calf serum. The number of refractive cells present in culture
wells was scored microscopically.
[0280] Culture medium always contained IL-3 since early-acting
cytokines require additional co-factor(s) for survival and
proliferation. FIG. 20A shows that cord blood cells responded to
column fractions in two regions of the material eluted from an
anion exchange column. The first region of activity corresponded
with Flt3 3T3 stimulatory activity (FIGS. 20B and 20C); the second
associated with an activity detected with Fms 3T3 cells (FIG. 20D);
no response was detected in untransfected 3T3 cells (data not
shown). The active material, corresponding to Flt3 3T3 activity
(peak one in FIG. 20A), was further characterized and purified on
different chromatographic matrices including a cation exchange
resin, heparin sepharose, hydroxyappatite, ConA-sepharose, phenyl
sepharose, and gel filtration (Superdex 75) (data not shown).
[0281] The further purified Pv-FRIL was used in the Flt3 3T3 assay
described above. Plateau stimulation of Flt3 3T3 cells decreased
with sequential purification steps (see FIG. 21A), suggesting
removal of essential co-factor(s). Addition of suboptimal levels of
crude PHA-LCM to Pv-FRIL obtained in the later stages of
purification, restored activity of this partially purified Pv-FRIL
to maximal plateau levels (FIG. 21B).
[0282] Analysis by ELISA for cytokines that act on hematopoietic
progenitors (interleukin 1-.alpha. (IL1-.alpha.), interleukin
1-.beta. (IL1-.beta.), interleukin 2 (IL2), interleukin 3 (IL3),
interleukin 4 (IL4), interleukin 6 (IL6), granulocyte-macrophage
colony stimulating factor (GM-CSF), G-CSF (granulocyte colony
stimulating factor), and stem cell factor (SCF)) in fractions
containing Pv-FRIL purified to near homogeneity revealed that
IL1-.alpha. had remained with Pv-FRIL through every step of
purification (data not shown).
[0283] Flt3 3T3 activity was depleted but not eliminated either by
adding neutralizing antibodies to IL1 or by removing IL1 by
antibody affinity chromatography (data not shown). However, at the
levels (<1 ng/mL) found in fractions containing purified
Pv-FRIL, exogenous recombinant hIL1-.alpha. by itself had no
stimulatory activity (data not shown). This observation suggested
the possibility that IL1-.alpha. may act as a necessary co-factor
to obtain maximal stimulation by Pv-FRIL. In subsequent experiments
when testing purified Pv-FRIL, the co-factor requirement was met by
the addition of either IL1-.alpha. or PHA-LCM added at a
concentration that did not stimulate Flt3 3T3 cells by itself.
[0284] Using this modified Flt3 3T3 assay with the addition of
sub-stimulatory amounts of either IL1-.alpha. or PHA-LCM, the
active protein was purified to near homogeneity in three
independent experiments. Table 4 summarizes the results of one such
experiment in which a 1% recovery was accompanied by an 80,000-fold
purification and resulted in a fraction with a specific activity of
244,500 units/mg.
20TABLE 4 Total Total Specific Protein Activity Activity Fold
Recovery Purification Step (mg) (units) (u/mg) Purification (%)
PHA-LCM 231,774 7,500,000 3 1 100 Blue Sepharose FF 1,294 2,600,000
670 35 Q-Sepharose FF 347 1,400,000 4,035 1,345 19 Phenyl-Sepharose
HP 14 1,200,000 85,714 28,571 16 Superdex 75 0.12 29,340 244,500
81,500 1 Pv-FRIL was purified by its ability to stimulate
Flt3-expressing 3T3 cells using four different chromatographic
media. This resulted in a 80,000-fold purification with a 1%
yield.
[0285] Purified Pv-FRIL
[0286] SDS-PAGE showed the purified material to contain a limited
number of polypeptides and the molecular size of the active
material was determined by eluting the protein from SDS-PAGE gel
slices run under non-reducing conditions and assaying the activity
of the eluted material. Flt3 3T3 activity was always found in a gel
slice that contained 14-22 kDa polypeptides and sometimes in a gel
slice containing 32-43 kDa polypeptides (data not shown).
[0287] The polypeptide(s) in the active fraction corresponding to
the 14-22 kDa range were subjected to aminoterminal sequencing by
Edman degradation. To do this, an 18 kDa species from C4
reverse-phase chromatography was resolved by SDS-PAGE,
electroblotted onto polyvinylidene difluoride (PVDF) membrane
(Immobilon-P, Millipore) and stained with Ponceau S (Bio-Rad
Laboratories, Inc., Hercules, Calif.). The 18 kDa band was cut from
the PVDF membrane and N-terminal sequence was determined by
automated Edman degradation on an ABI model 477A protein sequencer
(PE Applied Biosystems, Foster, Calif.). The derived peptide
sequences were compared against the SwissProt protein sequence
database.
[0288] In each of three experiments, the sequence AQSLSFXFTKDALD
was obtained from a polypeptide of 18 kDa (where X is an unknown
amino acid). For the material at the dye front (14 kDa and below)
the aminoterminal sequence of TDSRVVAVEFDXFP was found twice. The
amino terminus of a smooth muscle protein (SM22-.alpha.) was found
twice and the amino terminus of myoglobin identified once. Since
the sequence starting with AQS was the only sequence identified in
each experiment, this polypeptide was concluded to be responsible
for Flt3 3T3 activity.
[0289] Further purification of Pv-FRIL was obtained by
immunoaffinity chromatography using a rabbit antiserum described
above that was generated against a synthetic peptide of 13 amino
acids corresponding to the N-terminus obtained for the 18 kDa
polypeptide (referred to as anti-AQS). Crude PHA-LCM was applied to
a rabbit anti-AQS affinity column, and after washing, bound protein
was eluted under acidic conditions. Four pools of fractions were
assayed for activity in the two different assay systems. The Flt3
3T3 cells responded weakly (<100 u/mL) to the pooled fractions
(data not shown). FIGS. 22A-22D shows results of an experiment
where the pooled fractions were assayed on cord blood cells in the
presence of IL3. After two weeks of suspension culture, the number
of viable cells and status of CD34 expression was evaluated. A
representative of three AQS affinity chromatography experiments is
shown in FIGS. 22A-22D. Cell cultures supplemented with the two
early column fraction pools contained approximately four-fold more
cells (426,000 cells and 466,250 cells, respectively) than at the
100,000 cells seeded and no appreciable CD34 staining (FIGS. 22A
and 22B). The increase in cell number and loss of CD34 expression
is attributed to the expected consequences of IL3-induced
proliferation and differentiation. In contrast, cell cultures
treated with the late-eluting fraction pool (FIG. 22D) contained
less than 10,000 cells, or a tenth of input cells, and a uniform
population of cells expressing CD34. The late-eluting AQS affinity
pool did not show the potent effects of IL3 (high cell numbers and
exhausted medium); instead the persistence of viable cells
expression CD34 after two weeks in suspension culture suggested
that the active component might preserve CD34.sup.+Flt3.sup.+
progenitors.
[0290] Pv-FRIL Is Derived From Phaseolus vulgaris
[0291] Because PHA is derived form red kidney bean extract and
because a FRIL family member, Dl-FRIL, was isolated from another
legume, namely Dolichos lab lab, mannose-binding lectins were
isolated from red kidney bean (Phaseolus vulgaris) extract using
standard methods, such as the procedure of Rudiger, H., Isolation
of Plant Lectins, H.-J. Gabius and S. Gabius, eds., pp. 31-46,
Berlin, 1993). The kidney bean mannose-binding lectin consisted of
polypeptides with molecular weights of 18 kDa and 15 kDa, and the
aminotermini of these two polypeptides started with AQSLSFXFKFDPN
AND TDSRVVAVEDF, respectively (where X is an unknown amino
acid).
[0292] Pv-FRIL isolated from Phaseolus vulgaris was tested for
activity in the Flt3 3T3 cell assay. As shown in FIG. 23, Flt3 3T3
cells responded in a dose dependent manner to Pv-FRIL, while parent
untransfected 3T3 cells did not.
[0293] Purification of Pv-FRIL from Phaseolus vulgaris
[0294] Dry seeds from the red and white kidney beans (Phaseolus
vulgaris) were purchased from W. Atlee Burpee & Company,
Warminster, Pa. Lectins were eluted using a standard protocol.
Briefly, beans were pulverized in a home coffee grinder and added
to buffer of 50 mM Tris/HCl, pH. 8.0, 1 nM each of MgCl.sub.2 and
CaCl.sub.2 for 4 hours at 4.degree. C. with constant mixing. Bean
solids were pelleted by centrifugation at 10,000.times.g for 20
min. The pH of the supernatant was modified to pH 4.0 with acetic
acid and constant mixing to remove contaminating storage proteins,
followed by centrifugation to clarify the supernatant, and finally
the pH was readjusted to 8.0 with sodium hydroxide before storing
at -20.degree. C.
[0295] Specific binding of Pv-FRIL to mannose enabled a single-step
purification of the lectin from the supernatant of the bean
extract. 50 .mu.L extracts of either red or white kidney beans were
incubated in a conical tube with 1 mL of mannose covalently bound
to agarose beans (Sigma) at 4.degree. C. with constant mixing for 4
hours to overnight. Beans were washed gently by centrifugation (300
g, 5 min) in lectin binding buffer. Mannose binding protein was
eluted from the mannose beans after washing by incubation either
with 200 mM .alpha.-methyl mannoside (Sigma) or 100 mM glycine, pH
2.8.
[0296] DNA Isolation and PCR amplification of Pv-FRIL-Encoding
Nucleic Acid
[0297] Total genomic DNA was isolated from young Phaseolus vulgaris
shoots according to the procedure of Dellaporta ("Plant DNA
miniprep and microprep: Versions 2.1-2.3,".Freeling, M. and V.
Walbot (Ed.). The Maize Handbook. XXVI+759 p. Springer-Verlag New
York, Inc.: New York, N.Y., USA; Berlin, Germany.), and stored at
-20.degree. C. Based on the determined N-terminal amino acid
sequences of Pv-FRIL, four degenerate oligonucleotides (PVbeta1,
PVbeta2, PValfa1, PValfa2) were designed using Phaseolus vulgaris
codon usage. The sequences of the primers is as follows:
21 PVBeta1: TTY ACY AAR TTY GAY YTN GA PVBeta2: ATY TTY CAR GGW GAY
GC PVAlfa1: TTR ACR TCR ATW CCR ATR TG PVAlfa2: TAR TTW GGR TCR ATR
TTR GCR TT
[0298] Two sequential polymerase chain reactions (PCR) were
performed. In the first reaction, 10 ng of bean genomic DNA was
amplified by 30 cycles of PCR, each cycle comprising 40 seconds at
94.degree. C., 40 seconds at 50.degree. C., 60 seconds at
72.degree. C., and an extension step at 72.degree. C. for 10 min.
The reactions were performed in 50 .mu.l containing 30 pmol of each
primer, PVbeta 1 and PValfa1, 0.2 mM deoxyribonucleotides and 0.5
unit of Ampli-Taq polymerase (Perkin Elmer) in the corresponding
buffer. One microliter of the PCR product was amplified by 30 PCR
cycles using the same conditions as described above. The reaction
was performed in 50 .mu.L containing 30 pmol of the two primers,
PVbeta 2 and PV alfa2, using 0.2 mM deoxyribonucleotides and 0.5
unit of Ampli-Taq polymerase (Perkin Elmer) in the corresponding
buffer. The 460 bp fragment obtained was cloned in a T/A plasmid,
pCR2.1 (Invitrogen) and sequenced by sequenase dideoxy chain
termination (United States Biochemicals).
[0299] RNA Isolation and cDNA synthesis of Pv-FRIL-Encoding Nucleic
Acid
[0300] Total RNA was prepared from mid-maturation Phaseolus
vulgaris seeds stored at -70.degree. C. following the procedure
reported by Pawloski et al. (Mol. Plant Biol. Manual 5:1-13, 1994).
The 5'/3' RACEKIT (Boehringer Mannheim) was used to generate cDNA
from 5.0 .mu.g total RNA used according to the manufacturer's
instructions. In the cDNA synthesis for the 3' RACE, the oligo(dT)
anchor primer was at the concentration of 32.5 .mu.M,.in the
standard conditions. For the 5' RACE, a specific primer (SPV1) was
used at the concentration of 32.5 .mu.M. The cDNA purification and
the subsequent tailing reaction was performed according to the
manufacturer's instructions.
[0301] Polymerase Chain Reaction and cDNA cloning of
Pv-FRIL-Encoding Nucleic Acid
[0302] The 3' end of Pv-FRIL was obtained through rapid
amplification of cDNA ends by polymerase chain reaction (RACE-PCR)
using the 5'/3' RACEKIT (Boehringer Mannheim) used according to the
manufacturer's instructions. Nested PCR amplifications were
performed using the PCR-Anchor primer with the specific primers
(PV3 and PV4) in two successive amplification reactions. The
sequences of these primers is as follows:
22 PV3: CAA TGT CTT ACA ACT CAC TAA G PV4: AGT GTG GGA AGA GTG TTA
TTC
[0303] The amplification conditions were 30 cycles of 40 seconds at
94.degree. C., 40 seconds at 55.degree. C., 60 seconds at
72.degree. C. each and an extension step at 72.degree. C. for 10
min. The reactions were performed in 50 .mu.L containing 30 pmol of
each primer, PV3 and PCR-Anchor primer in the first, and PV4 and
PCR-Anchor primer in the second, 0.2 mM deoxyribonucleotides and
0.5 units of Ampli-Taq polymerase (Perkin Elmer) in the
corresponding buffer. The 831 bp product obtained was sub-cloned in
pCR2.1 and sequenced as above reported.
[0304] For the 5'RACE, again nested PCR reactions were performed
using in combination with the Anchor Primer the specific primers
SPV2 and SPV3. The sequences of these primers is as follows:
23 SPV2: ACC AAA GCT TTG GTT TTC AGA SPV3: TCT GAA AAC GTT TGA GTA
GAG
[0305] The amplification conditions for both reactions were 30
cycles of 40 seconds at 94.degree. C., 40 seconds at 50.degree. C.,
60 seconds at 72.degree. C. each and an extension step at
72.degree. C. for 10 min. The reactions were performed in 50 .mu.L
containing 30 pmol of each primer, SPV2 and PCR-Anchor primer in
the first, and SPV3 and PCR-Anchor primer in the second, 0.2 mM
deoxyribonucleotides and 0.5 unit of Ampli-Taq polymerase (Perkin
Elmer) in the corresponding buffer.
[0306] To obtain the full-length cDNA done, recombinant PCR was
performed (Higuchi R., supra). Two PCR reactions were carried out
separately, one on the 5' fragment and the other on the 3' RACE
product using primers with an overlapping region. The overlapping
primary products were subsequently re-amplified using the flanking
primers resulting in a full-length fragment.
[0307] The primary PCR products were purified with the QIAquick PCR
Purification kit (QIAGEN) used according to the manufacturer's
instructions, and amplified together in a single second reaction.
For the second PCR reaction, the primers PVEcoRI and the APEcoRI
were used. The sequences of these primers is as follows:
24 PVEcoRI TAC ATG AAT TCG CTC AGT CAT TAT CTT TTA AC APEcoRI: GAA
TTC GAC CAC GCG TAT CGA TGT CGAC
[0308] Both primary and secondary PCR reactions were performed in
100 .mu.L containing 50 pmol of each primer, 0.4 mM
deoxyribonucleotide and 1.0 unit Pfu polymerase (Stratagene) in the
corresponding buffer. The primary PCR reaction amplified the two
separate fragments by 30 cycles, each cycle comprising 40 seconds
at 94.degree. C., 40 seconds at 50.degree. C., 60 seconds at
72.degree. C. and an extension step at 72.degree. C. for 10 min.
The second PCR reaction amplified the recombinant fragment in 12
cycles using the same conditions reported above. The full lengthed
product was cloned in the EcoRI site of the cloning vector pCR2.1
(FIG. 24A) and sequenced as noted above. This plasmid is referred
to as pCR2.1-Pv-FRIL.
[0309] The nucleic acid sequence of the Pv-FRIL cDNA is as
follows:
25 1 GCTCAGTCAT TATCTTTTAA CTTTACCAAG (SEQ ID NO: 5) TTTGATCTTG
ACCAAAAAGA 51 TCTTATCTTC CAAGGTGATG CCACTTCTAC AAACAATGTC
TTACAACTCA 101 CTAAGTTAGA CAGTGGAGGA AACCCTGTGG GTGCTAGTGT
GGGAAGAGTG 151 TTATTCTCTG CACCATTTCA TCTTTGGGAA AACTCTATGG
CAGTGTCAAG 201 CTTTGAAACT AATCTCACCA TTCAAATCTC AACACCTCAC
CCTTATTATG 251 CAGCTGATGG CTTTGCCTTC TTCCTTGCAC CACATGACAC
TGTCATCCCT 301 CCAAATTCTT GGGGCAAATT CCTTGGACTC TACTCAAACG
TTTTCAGAAA 351 CTCCCCCACC TCTGAAAACC AAAGCTTTGG TGATGTCAAT
ACTGACTCAA 401 GAGTTGTTGC TGTCGAATTT GACACCTTCC CTAATGCCAA
TATTGATCCA 451 AATTACAGAC ACATTGGAAT CGATGTGAAC TCTATTAAGT
CCAAGGAAAC 501 TGCTAGGTGG GAGTGGCAAA ATGGGAAAAC GGCCACTGCA
CGCATCAGCT 551 ATAACTCTGC CTCTAAAAAA TCAACTGTTA CTACGTTTTA
TCCTGGGATG 601 GAAGTTGTGG CTCTCTCCCA TGATGTTGAC TTACATGCAG
AGCTTCCTGA 651 ATGGGTTAGA GTAGGGTTAT CTGCTTCAAC TGGAGAGGAG
AAACAAAAAA 701 ATACCATTAT CTCATGGTCT TTCACTTCAA GCTTGAAGAA
CAACGAGGTG 751 AAGGAGCCGA AAGAAGACAT GTATATTGCA AACGTTGTGC
GATCATATAC 801 ATGGATCAAT GACGTTCTAT CTTATATAAG CAATAAATAA
ATGTATGATG 851 CACTCAATAA TAATCACAAG TACGTACGGT GTAGTACTTG
TATGTTGTTT 901 ATGAAAAAAA AAAA
[0310] The amino acid sequence of Pv-FRIL is as follows:
26
AQSLSFNFTKFDLDQKDLIFQGDATSTNNVLQLTKLDSGGNPVGASVGRVLFSAPFHLWENSMA- V
(SEQ ID NO: 6) SSFETNLTIQISTPHPYYAADGFAFFLAPHDTVIPPNSWG-
KFLGLYSNVFRNSPTSENQSFGDVN TDSRVVAVEFDTFPNANIDPNYRHIGIDVNSI-
KSKETARWEWQNGKTATARISYNSASKKSTVTT FYPGMEVVALSHDVDLHAELPEWV-
RVGLSASTGEEKQKNTIISWSFTSSLKNNEVKEPKEDMYIA
NVVRSYTWINDVLSYISNK*MYDALNNNHKYVRCSTCMLFMKKK
[0311] The amino acid sequence of Pv-FRIL was compared to the amino
acid sequences of Dl-FRIL and of the PHA-E lectin. This comparison
is shown on FIG. 24B.
[0312] Pv-FRIL-Encoding Plant Expression Vectors and Nicotiana
tabacum Transformation
[0313] Recombinant PCR was used to introduce a signal peptide for
entry of Pv-FRIL into the endoplasmic reticulum at the 5' end of
the Pv-FRIL cDNA clone. Following the procedure of Higuchi (supra)
the signal peptide and the full length cDNA clone were amplified in
two separate primary PCR reactions. The signal peptide was obtained
from the amplification of the binary vector pTA4, harboring the
complete sequence of the bean .alpha.-amylase inhibitor gene
(Hoffman et al., L. M., Y. Ma and R. F. Barker, Nucleic Acid Res.
10: 7819-7828, 1982; Moreno and Chrispeels, Proc. Natl. Acad. Sci.
USA 86: 7885-7889, 1989).
[0314] The primers used for the two primary reactions are the
following: Amplification of the Signal Peptide
27 Sigforw AGA TCT ATG GCT TCC TCC AAC BglII: Sigrew: AAA GAT AAT
GAC TGA GCG GCT GAG TTT GCG TG
[0315] Amplification of the mannose lectin cDNA:
28 SpMlforw: CAC GCA AAC TCA GCC GCT CAG TCA TTA TCT TT APXhoI: CTC
GAG GAC CAC GCG TAT CGA TGT CGA
[0316] The primers used for the secondary reactions, Sigforw and
AP, were designed to generate BglII sites at the 5' and XhoI at the
3' ends. Both, primary and secondary PCR reactions were performed
as discussed above. The recombinant product SpPv-FRIL was incubated
for 10 min. at 72.degree. C. with 0.5 units of Ampli-Taq polymerase
(Perkin Elmer) and cloned in the cloning vector pCR2.1 (FIG. 25).
The nucleotide sequence of the PCR product was determined as
described above to verify the correct attachment of the signal
peptide.
[0317] A binary vector was constructed for constitutive expression
of Pv-FRIL in tobacco plants. The recombinant SpPv-FRIL was cloned
in BglII/XhoI sites of a plant expression vector resulting in the
formation of pM-SpPv-FRIL (FIG. 26).
[0318] The binary vector was transferred in Agrobacterium
tumefaciens strain C58 according to the freeze-thaw procedure
reported of An et al., supra. Agrobacterium-mediated transformation
of Nicotiana tabacum leaf disks was carried out as described by
Horsh et al. (Science 227: 1229-1231, 1985) using C58 harboring the
expression vector pM-SpPv-FRIL. Kanamycin resistant plants were
scored for their ability to form roots in two consecutive steps of
propagation in Murashige-Skoog medium containing 3% sucrose and
kanamycin sulfate (Sigma) at 100 mg/L.
[0319] Tobacco plants transformed with this construct were grown in
a growth room under controlled conditions. The leaves (20 grams) of
young plants were harvested and frozen in liquid nitrogen and
powdered in a mortar with a pestle. The powder was stirred in a
buffer mixture consisting of 1.times.phosphate buffered saline
containing 1 mM CaCl.sub.2 with a cocktail of protease inhibitors
(PMSF, Pepstatin and Leupeptin). This slurry was centrifuged at
2000 rpm and the supernatant was centrifuged at 40,000 rpm in a
Beckman ultracentrifuge. The clear supernatant was tumbled with 1
ml of ovalbumin-Sepharose for 3 hours. The beads were washed with
the same buffer and tumbled overnight with 200 mM trehalose in
{fraction (1/10)} phosphate buffered saline containing the cocktail
of protease inhibitors. Coomassie blue stained gel showed this
preparation to be pure Pv-FRIL. An immunoblot showed the presence
of both alpha and beta subunits, thus, the single polypeptide chain
encoding both subunits was cleaved by the transformed cells in
vivo. Binding to the ovalbumin-sepharose and release by trehalose
shows that the product of the transgene is an active lectin.
EXAMPLE 6
Dl-FRIL Supports Prolonged ex vivo Maintenance of Ouiescent Human
CD34+,CD38-/SCID-Repopulating Cells
[0320] To further characterize the progenitor cell preservation
activity of Dl-FRIL, a functional in vivo assay for primitive human
hematopoietic cells was used to determine the cells' ability to
home to and repopulate the marrow of sublethally irradiated C.B-17
and NOD/LtSz mice homozygous for the severe combined
immunodeficiency Prkdc.sup.scid mutation (Lapidot et al., Science
255:1137, 1992; Larochelle et al., Nat. Med. 2:1329-1337, 1996). To
do this, the following methods were used:
[0321] Preparation of Human Cells
[0322] Human umbilical cord blood (CB) samples were obtained from
full term deliveries. The blood samples were diluted 1:1 in
phosphate-buffered-saline (PBS) without Mg.sup.+2/Ca.sup.+2,
supplemented with 10% fetal bovine serum (FBS). Low density
mononuclear cells were collected after standard separation on
Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden), and washed in
RPMI with 10% FBS. Some samples were frozen in 10% DMSO, while the
others were used fresh. Enrichment of CD34.sup.+ cells was
performed with mini MACS separation kit (Miltenyi Biotec, Bergisch
Gladbach, Germany) according to the manufacturer's instructions.
The purity of the enriched CD34.sup.+ cells was 60-80% using one
column. CD34.sup.+CD38.sup.-/low cells were purified by FACS
sorting (FACStar.sup.+, Becton Dickinson, San Jose, Calif.) after
staining CD34.sup.+ enriched cells with mAb anti human CD34-FITC
(Becton Dickinson) and anti human CD38 PE (Coulter, Miami Fla. USA)
(Purity>99%).
[0323] Mice
[0324] Eight week old NOD/LtSz-Prkdc.sup.scid/Prkdc.sup.scid
(NOD-SCID) mice and NOD/SCID .beta.2 microglobulin knockout mice,
hereafter termed NOD/SCID B2M.sup.null (Christianson et al., J.
Immunol. 158:3578-3586, 1997), bred and maintained under defined
flora conditions in sterile micro isolator cages, were irradiated
with a sublethal dose of 375 cGy at 67 cGy/min. from a cobalt
(.sup.60Co) source prior to transplantation.
[0325] Human cells were injected into the tail vein of irradiated
mice in 0.5 mL of RPMI with 10% FBS. In some experiments (as
indicated) non engrafting irradiated (1500 cGy) CD34.sup.- cells
served as carrier cells, and were cotransplanted with cultured
cells at a final concentration of 0.5.times.10.sup.6 cells/mouse.
Mice were sacrificed 1 month post transplantation, and bone marrow
(BM) cells were flushed from the 8 bones of each mouse (femurs,
tibias, humeri, and pelvis).
[0326] Ex vivo Cultures
[0327] Human CD34.sup.+ enriched cells were cultured in 24 well
plates (2-4.times.10.sup.5 cells in 0.5 mL), containing RPMI
supplemented with 10% FBS+1% BSA. Ex vivo cultures contained the
following cytokine combination: Stem cell factor (SCF)-100 ng/mL
and Flt3 ligand (Flt3-L)-100 ng/mL (R&D Systems Inc.
Minneapolis, Minn., USA), rhIL-6-50 ng/mL and sIL6R-1280 ng/mL,
(InterPharm Laboratories, Ares-Serono Group, Ness Ziona, Israel) or
Dl-FRIL-10 ng/mL (ImClone Systems Inc., NY, USA). In some
experiments where indicated, the growth factor cocktail included
300 ng/mL SCF, 300 ng/mL Flt3-L, 50 ng/mL G-CSF, 10 ng/mL IL-3
(R&D Systems), and 10 ng/mL IL-6. The cultures were incubated
at 37.degree. C. in a humidified atmosphere containing 5% CO.sub.2.
After 3, 6, 10 and 13 days, the cells were, collected, counted,
analyzed by FACS, seeded in to semisolid cultures and transplanted
into NOD-SCID mice. For CD56.sup.+ NK cell development, BM cells
from engrafted mice were cultured with 100 ng/mL of SCF and IL-15
(R&D) for 10-14 days.
[0328] Preparation of Dl-FRIL
[0329] Dl-FRIL was isolated from the seeds of hyacinth beans
(Dolichos lab lab) using the protocol described above in Example
1.
[0330] CFU Assay.
[0331] Semisolid cultures were performed in order to detect the
levels of human progenitors in ex vivo cultures, and in the marrow
of transplanted mice. The cells were plated (4.times.10.sup.3
cells/mL) in 0.9% methylcellulose (Sigma, St. Louis, Mo., USA), 30%
FBS, 5.times.10.sup.-5M 2ME, 50 ng/mL SCF, 5 ng/mL IL-3, 5 ng/mL
GM-CSF (R&D), and 2 u/mL Erythropoietin (Ortho Bio Tech, Don
Mills, ON, Canada). Human cells from the BM of engrafted mice were
plated (2.times.10.sup.5 cells/mL) in 15% FBS+15% human plasma,
selective for human colonies only. The cultures were incubated at
37.degree. C. in a humidified atmosphere containing 5% CO.sub.2 and
scored 14 days later with an inverted microscope for myeloid,
erythroid, and mixed colonies by morphologic criteria.
[0332] Cell Cycle Analysis.
[0333] Cells were analyzed for their DNA content by staining with
propidium iodide (Sigma). The cells were cultured in ex vivo
cultures as described, for 3, 6, 10, and 13 days as indicated. At
each time point, the cells were collected, resuspended to a final
concentration of 0.1-1.times.10.sup.6 cells/mL, and incubated with
0.1% Triton.times.100 (Sigma) for 20 minutes on ice. 50 mg/mL
propidium iodide (PI) were added before analysis. Flow cytometeric
analyses were performed using FACSort (Becton Dickinson, San Jose,
Calif.).
[0334] Flow Cytometry Analyses.
[0335] Human and mouse Fc receptors on BM cells from transplanted
mice were blocked by using human plasma (1:50) and anti mouse Fc
receptor blockers (anti mouse CD16/CD32 mAb, Pharmingen, San Diego,
Calif., USA). Isotype control mAb were used in order to exclude
false positive cells (Coulter, Miami Fla. USA). The purity of
enriched subpopulations after magnetic bead separation, were
analyzed by two color staining, using anti-human CD34 FITC (Becton
Dickinson, San Jose, Calif., USA) and anti-human CD38 PE (Coulter).
The levels of human cells and lymphoid lineages in the marrow of
engrafted mice were detected by double staining with anti-human
CD45 FITC (Immuno Quality Products, Groningen, The Netherlands)
together with anti-human CD19 PE (Coulter) for detection of pre B
cells, or with anti-human CD56 PE (Coulter) for detection of NK
cells. Cells were washed with PBS supplemented with 1% FBS and
0.02% azide, suspended to a volume of 0.1-1.times.10.sup.6
cells/mL, stained with direct-labeled mAb and incubated for 25
minutes on ice. After staining, cells were washed once in the same
buffer and analyzed on a FACSort (Becton Dickinson). Analysis was
performed using CELLquest software (Becton Dickinson).
[0336] Human Cell Engraftment Analysis.
[0337] The levels of human cell engraftment were determined by both
flow cytometry for analysis of human myeloid CD45.sup.+ and
lymphoid CD45.sup.+CD19.sup.+ pre B cells and quantification of
human DNA as previously described (Lapidot et al., Science
255:1137, 1992; Larochelle et al., Nat. Med. 2:1329-1337, 1996;
Peled et al., Science 283:845-848, 1999). Briefly, high molecular
weight DNA was obtained from the BM of transplanted mice by
phenol/chloroform extraction. DNA (5 g) was digested with EcoRI,
subjected to electrophoresis on 0.6% agarose gel, blotted onto a
nylon membrane, and hybridized with a human chromosome 17-specific
.alpha.-satellite probe (p17H8) labeled with .sup.32P (Lapidot et
al., supra). The intensity of the bands in the samples were
compared to artificial human/mouse DNA mixtures (0%, 0.1%, 1%, and
10% human DNA) to quantify the human DNA (lanes to the right of
lane 4 in FIG. 31). Multiple exposures of the autoradiographs were
taken to ensure sensitivity down to 0.01% human DNA. A transplanted
mouse was scored positive when both human myeloid and lymphoid
cells and human DNA were detected in its BM.
[0338] From these experiments, the following results were
obtained:
[0339] Dl-FRIL Maintains but does not Expand CB CD34.sup.+
Progenitors in Suspension Culture
[0340] As shown above, Dl-FRIL by itself preserves immature CB
progenitor cells up to a month in suspension culture without medium
changes (see, e.g., FIGS. 14A and 14B, and Table 3). To compare
Dl-FRIL's progenitor-preserving properties to a combination of
cytokines (SCF, Flt3-L, IL-6, and sIL6-R) shown to maintain CB
progenitor cells in suspension culture (Sui et al., Proc. Natl.
Acad. Sci. USA 92:2859, 1995; Ebihara et al., Blood 90:4363-4368,
1997), enriched CB CD34.sup.+ cells were cultured with either
Dl-FRIL or cytokines for 3,6, 10, or 13 days in medium containing
10% FBS and 1% BSA in RPMI. Fresh media and Dl-FRIL and/or
cytokines were added on day 6. Cells harvested at each time point
were counted and assayed for clonogenic progenitor cells by plating
(i.e., seeding) in semisolid media.
[0341] As shown in FIG. 27A, the total number of cells in Dl-FRIL
cultures gradually declined over time from 2.times.10.sup.5 cells
initially seeded to 1.26.times.10.sup.5 cells at day 13, in
contrast to the expected 14.3-fold increase to 2.8.times.10.sup.6
cells in cytokine cultures at day 13. Similarly, as shown in FIG.
27B, the levels of progenitor cells in Dl-FRIL cultures also
remained relatively constant until day 10, after which they
declined to 9% of the starting population (from 24.7.times.10.sup.3
colonies on day 0 to 2.3.times.10.sup.3 colonies on day 13).
Progenitor levels increased in cytokine cultures by 10-fold from
the outset to day 13 of culture (FIG. 27B).
[0342] Dl-FRIL Maintains the Expansion Capacity of CD34.sup.+
Progenitors up to 2 Weeks in ex vivo Culture
[0343] To characterize the expansion capacity of Dl-FRIL-preserved
progenitor cells in suspension culture, CD34.sup.+ cells cultured
for 6 days with Dl-FRIL were washed and exposed to cytokines
(without Dl-FRIL) for an additional 4 days. Total cell counts and
clonogenic progenitor assays were performed and the results were
compared to cells cultured for 10 days with Dl-FRIL. Fresh media
and Dl-FRIL were added on day 6. Interestingly, the
Dl-FRIL-cultured cells proliferated in response to cytokine
stimulation, resulting in a 3.4-fold increase in cell numbers (FIG.
28A) and 13-fold increase in progenitor levels (FIG. 28B).
[0344] Further experiments tested Dl-FRIL's ability to preserve
cytokine-responsive CD34.sup.+ progenitor cells cultured with
either Dl-FRIL for 10 days followed by 3 days of cytokine
stimulation or only with Dl-FRIL for the entire 13 days. Fresh
media and Dl-FRIL were added on day 6. A 2.9-fold increase in total
cell numbers (FIG. 28C) and a 5.5-fold increase in progenitor
levels (FIG. 28D) were observed for cells cultured with Dl-FRIL for
10 days followed by an additional 3 days of cytokine stimulation
compared to cultures with Dl-FRIL alone for 13 days. These results
provide evidence that Dl-FRIL alone maintains the proliferative
capacity of human progenitor cells up to 13 days in suspension
culture and that subsequent stimulation of cells cultured for 10
days with Dl-FRIL still respond to the proliferative signals of the
cytokines.
[0345] Dl-FRIL Maintains SCID Repopulating Stem Cells (SRC) in ex
vivo Cultures
[0346] At each time point during culture, the human CB CD34.sup.+
cells were collected and the content of each well was assayed for
progenitor levels (described above) and 2.times.10.sup.5 of the
remaining cells were transplanted into one mouse. One month later,
mice were sacrificed and their bone marrows were harvested and
assayed for the presence of human myeloid and lymphoid cells.
Before assaying, human DNA levels in the bone marrow of individual
transplanted mice was quantitated by Southern blotting analysis. A
representative Southern blot analysis showing the detection of a
0%, 0.1%, 1%, and 10% human DNA per murine DNA is provided in FIG.
29E.
[0347] FIGS. 29A-29D show representative Southern blot analyses of
the BM of mice transplanted with ex vivo cultured cells. FIG. 29A
is a representative Southern blot showing human DNA in the marrow
of mice transplanted with cells cultured with FRIL for 6 days (lane
1), FRIL for 10 days (lane 2), or with FRIL for 6 days followed by
4 days with cytokine stimulation (lane 3). In a typical experiment,
the levels of engraftment by cells cultured with Dl-FRIL for 10
days decreased by approximately 10-fold from cells cultured with
Dl-FRIL for 6 days (FIG. 29A, lanes 1 and 2). However, when cells
cultured with Dl-FRIL for 6 days were subsequently stimulated with
cytokines for 4 days, there was an approximate 10-fold increase in
the level of engraftment (FIG. 29A, lane 3) compared to Dl-FRIL
alone for 10 days (lane 2). Similar results were obtained when
cells from other donors were transplanted as shown in FIG. 29B,
lanes 3-4 (cells cultured with Dl-FRIL for 6 days followed by 4
days of cytokine stimulation) compared to lanes 1-2 (cells cultured
with Dl-FRIL alone for 10 days) prior to transplantation. Dl-FRIL
cultures could be prolonged even to 13 days, as shown in FIGS.
28C-28D, prior to transplantation into mice. A modest increase in
the levels of engraftment was seen comparing the marrow of mice
transplanted with the original cells, which were not cultured with
Dl-FRIL, prior to seeding (FIG. 29C, lane 1) with cells cultured
with Dl-FRIL for 13 days (FIG. 29C, lane 2). FIG. 28D shows the
results of another experiment, where the difference between
engraftment levels obtained by cells cultured with Dl-FRIL for 10
days (lane 1) or with Dl-FRIL for 6 days followed by 4 days of
cytokine stimulation (lane 2) are minor. Nevertheless, a 10-fold
increase was observed for cells cultured either with Dl-FRIL for 13
days (FIG. 29D, lane 3) or with Dl-FRIL for 10 days followed by 3
days of cytokine stimulation (FIG. 29D, lane 4) when compared to
cells cultured for 10 days (FIG. 29D, lanes 1 and 2).
[0348] Levels of human engraftment in the marrow of mice
transplanted with CD34.sup.+ cells cultured with either Dl-FRIL for
10 days (n=12) or Dl-FRIL for 6 days (n=33) followed by cytokine
stimulation for 4 days are summarized in FIG. 30. Cells cultured
with Dl-FRIL followed by cytokines, engrafted at levels 7.4-fold
greater than cells cultured with Dl-FRIL alone for 10 days (FIG.
30, p=0.05).
[0349] To test the effect of Dl-FRIL on CB populations highly
enriched for primitive human SRC, CD34.sup.+ cells isolated by
immunomagnetic beads were further sorted by flow cytometry based on
the absence of CD38 expression. CD34.sup.+CD38.sup.-/low cells (99%
purity) cultured with either Dl-FRIL alone or Dl-FRIL followed by
cytokines showed patterns of engraftment in NOD/SCID B2M.sup.null
mice (Christianson et al., supra), similar to those observed for
CD34.sup.+ cells. These mice have been used successfully for
secondary human stem cell transplantation (Peled et al., supra),
have less innate immunity due to lack of NK activity and thus
require fewer (10 fold) human cells for engraftment compared to
NOD/SCID mice.
[0350] FIG. 31 is a representative Southern blot (1 out of 4
experiments) showing the relative level of engraftment of cells
that were cultured with Dl-FRIL for 10 days (lane 4) compared to 6
days with Dl-FRIL followed by 4 days cytokine stimulation (lanes
1-3) prior to transplantation into mice. High molecular weight DNA
was obtained from the BM of transplanted mice and subjected to
Southern blotting analysis for the presence of human DNA.
[0351] Based on these results, an increased level of engraftment in
cells exposed to cytokines after Dl-FRIL was expected.
Consequently, the same initial cell dose transplanted into one
mouse in lane 4 was divided into 3 mice in lanes 1-3, indicating an
expansion of Dl-FRIL cultured SRC when they were subsequently
exposed to cytokine stimulation. The level of engraftment in mice
transplanted with cells that were treated with Dl-FRIL followed by
cytokines (FIG. 31, lanes 1-3) was about 10-fold higher compared to
the mouse transplanted with cells cultured with Dl-FRIL alone (FIG.
31, lane 4). Since the initial dose of cells for the Dl-FRIL
+cytokines sample was split into three mice, a 30-fold increase in
the level of engraftment is observed for CD34.sup.+CD38.sup.-/low
cells stimulated by cytokines after Dl-FRIL compared to the culture
with Dl-FRIL alone. In addition, this result represents about a
3-fold greater level of expansion of SRC for
CD34.sup.+CD38.sup.-/low cells than for total CD34.sup.+ cells.
FIG. 32 summarizes the fold increase observed in the engraftment
levels of CD34 .sup.+CD38.sup.-/low cells cultured with Dl-FRIL
alone compared to cells subsequently stimulated with cytokines in 6
experiments (3 experiments with NOD/SCID B2M.sup.null mice and 3
experiments with NOD/SCID mice, gave similar results). These data
indicated a 30-fold expansion of SRC for CD34.sup.+CD38.sup.-/low
cells and a 3-fold greater level than that of total CD34.sup.+
cells in the corresponding experiments.
[0352] Further studies have demonstrated that Dl-FRIL preserves
long-term repopulating stem cells. Bone marrows harvested from
NOD/SCID mice that were initially transplanted with CD34+ cord
blood cells cultured in Dl-FRIL were transplanted into a second set
of sublethaly irradiated NOD/SCID mouse recipients. After one
month, bone marrows from the second set of recipients were analyzed
for the presence of human hematopoiesis, as determined using the
assays described above. Multilineage engraftment was observed in
the second set sublethaly irradiated NOD/SCID mouse recipients.
This observed persistence of repopulating cells in this serial
transplantation study indicated the presence of long-term
repopulating stem cells.
[0353] Dl-FRIL Preserves SRC Potential of Multilineage
Differentiation in the Murine BM
[0354] Southern blot analysis detected and quantified the levels of
human DNA in the marrow of transplanted mice without specifically
indicating the differentiation status of human cells recovered from
the murine BM. Since SCID repopulating stem cells (SRC) are defined
by multilineage differentiation of myeloid and lymphoid cells in
NOD/SCID mice, the in vivo differentiation processes of
Dl-FRIL-cultured CB CD34.sup.+ cells in the murine BM were further
studied. To do this, CD34.sup.+ or sorted CD34.sup.+CD38.sup.-/low
cells were cultured either with FRIL for 10 days or with FRIL for 6
days followed by cytokine stimulation for 4 days prior to
transplantation. BM of transplanted mice was collected 1 month
later and the cells were either seeded in semisolid media selective
for human colonies (results shown in FIG. 33A) or were analyzed for
lineage specific markers by flow cytometry (results shown in FIGS.
33B-33F).
[0355] The progenitor capacity of human CB CD34.sup.+ cells
cultured for 10 days with Dl-FRIL and harvested one month after
transplantation into NOD/SCID mice was evaluated in semi
solid-colony assays that selectively promoted growth of human
progenitors. FIG. 33A shows that the levels of human progenitors in
the bone marrow of transplanted mice increased 2.3-fold when SRC
were stimulated with cytokines for 4 days after 6 days of Dl-FRIL
incubation compared to Dl-FRIL alone for 10 days prior to
transplantation. Moreover, both myeloid and erythroid colonies
formed in the colony assays (data not shown) as well as human B
cell differentiation that was determined by flow cytometry (FIGS.
33C and 33D), indicating that incubation with Dl-FRIL maintained
the multilineage differentiation potential of SRC. A representative
flow cytometry analysis demonstrated the presence of human
CD45.sup.+CD19.sup.+ pre-B cells in the marrow of mice transplanted
with either CD34.sup.+ cells (FIG. 33C) or CD34.sup.+CD38.sup.-/low
cells (FIG. 33D) cultured with Dl-FRIL for 10 days (1% or 10%
CD19.sup.+ cells, respectively).
[0356] To determine whether human lymphoid precursors in the marrow
of transplanted mice have the potential to differentiate in
addition to myeloid cells also into lymphoid NK cells, cells from
transplanted murine bone marrow were also cultured with SCF and
IL-15 for 10 days. Cells harvested from cultures were analyzed for
human NK cells by flow cytometry using the human-specific
monoclonal antibodies to CD45 and the NK cell antigen, CD56. Cells
from mice transplanted with CD34 .sup.+ or CD34.sup.+CD38.sup.-/low
cells are shown (1% or 7.7% of CD56.sup.+ cells; FIGS. 33E and 33F,
respectively).
[0357] ex vivo Preservation of Early Progenitors with Dl-FRIL
Compared to Flt3 Ligand
[0358] Dl-FRIL was identified by its ability to stimulate
proliferation of NIH 3T3 cells transfected with Flt3 and not by
untransfected cells or cells transfected with the related Fms
tyrosine kinase receptor (Moore et al., Blood 90 supp. 1:308,
1997). Although Dl-FRIL and Flt3-L both stimulate proliferation of
Flt3 3T3 cells, they exert different activities on CB progenitor
cells. Flt3-L induces quiescent primitive cells into cyde (Lyman
and Jacobsen, Blood 91:1101-11345, 1998). In contrast, Dl-FRIL by
itself maintains progenitors in serum-defined medium from 15-29
days in culture without medium changes (Colucci et al., Proc. Natl.
Acad. Sci. USA 96(2):646-50, 1999). The abilities of Dl-FRIL or
Flt3-L to maintain CB CD34 .sup.+ progenitors in suspension
cultures consisting of 10% FBS and 1% BSA in RPMI for 10 days were
compared in the presence and absence of cytokines. The cells were
then were seeded for CFU assay.
[0359] As shown in FIG. 34A, the number of total cells harvested
after 10 days of suspension culture in Dl-FRIL increased minimally
by 1.9-fold, whereas cells cultured in either Flt3-L alone or in
combination with Dl-FRIL increased by 4.5-fold and 5.4-fold,
respectively (p<0.05). Cultures containing CB CD34.sup.+ cells
with cytokines, either alone or with Dl-FRIL and Flt3-L, led to
10.9-12.5 fold increase in cell numbers; however cells cultured
with Dl-FRIL alone followed by cytokines, increased only by
4.6-fold (FIG. 34A, p<0.05).
[0360] Interestingly, cells cultured with Dl-FRIL alone or followed
by cytokines, selectively maintained a higher number and proportion
of the primitive granulocyte-erythroid-macrophage-megakaryocyte
colony forming-units (CFU-GEMM) compared to other treatments (FIG.
34B, p<0.05). About 40% of the progenitor cells maintained by
Dl-FRIL were CFU-GEMM, a relative 2.6- to 3.7-fold increase with
Dl-FRIL cultures compared to other combinations (FIG. 34B,
p<0.05). In contrast to Flt3-L, exposure of cells first cultured
with Dl-FRIL to cytokines, maintained the high percentage of
CFU-GEMM, 18.2% versus 38.6%, respectively (FIG. 34B p<0.05).
These results demonstrate that Dl-FRIL and Flt3-L may act
differently on primitive progenitor cells in culture.
[0361] Dl-FRIL Maintains Higher Levels of CD34.sup.+ Cells in
G.sub.0/G.sub.1 Phase of Cell Cycle Compared to Cytokine Treated
Cells
[0362] In vivo, immature CD34.sup.+ cells are predominantly
quiescent, non-cycling cells (Young et al., Blood 87:545, 1996;
Movassagh et al., Stem Cells 15:214-222, 1997; Ladd et al., Blood
90:658-668, 1997). Since Dl-FRIL maintains relatively constant
levels of progenitor cells over two weeks in suspension culture
that can subsequently respond to cytokine stimulation (see FIG.
27), the cell cycle status of CD34.sup.+ cells incubated with
Dl-FRIL was investigated and compared to cultures with cytokine
stimulation. DNA content of CB CD34.sup.+ cells was analyzed by
flow cytometry immediately after isolation of cells. A mean value
of 96.6% of cells were observed in the G.sub.0/G.sub.1 phase of
cell cycle (FIG. 35A).
[0363] The cell cycle status of CB CD34.sup.+ cells was analyzed in
5 indepenent experiments with cells cultured for 3-13 days with
either Dl-FRIL or a cytokine combination (SCF, Flt3-L, G-CSF, IL-3,
IL-6) (Bhatia et al., J. Exp. Med. 186:619-624, 1997; Conneally et
al., Proc. Natl. Acad. Sci. USA 94:9836-9841, 1997). The percentage
of cycling cells in SG.sub.2M phase in both cultures decreased by
half from day 3 to day 13, from 17.7% to 9.1% for Dl-FRIL and from
50.9% to 23.2% for cytokines (Table 1) (3.4% of cells initially
seeded were in SG.sub.2M).
29TABLE 5 Percentage and numbers of cycling CD34.sup..+-. cells (in
SG.sub.2M phase) cultured with Dl-FRIL or cytokines for 3, 6, 10 or
13 days Cyto- Fold Cyto- Dl-FRIL kines increase Days in Dl-FRIL
kines (cells .times. (cells .times. induced by culture (%) (%)
10.sup.3) 10.sup.3) cytokines day 3 17.7 .+-. 1.9 50.8 .+-. 1.4 35
212 6 day 6 20 .+-. 2 26 43.6 389.5 9 day 10 10 .+-. 1.9 29.7 .+-.
1 26.4 553 21 day 13 9.1 .+-. 4.1 23.14 11.4 664.6 60 CD34.sup.+
cells were cultured (2 .times. 10.sup.5 cells/0.5 ml) with Dl-FRIL
or SCF + Flt3 ligand + IL-3 + G-CSF + IL-6. Cell cycle analysis was
performed with propidium iodide staining. Cell numbers were
calculated by multiplying percent of cycling cells by total cell
numbers. Data represents mean .+-. SE values, from 5
experiments.
[0364] More dramatically, the number of cells in SG.sub.2M phase
during culture differed from the 6.8.times.10.sup.3 cells in
SG.sub.2M phase initially seeded. The average number of cycling
cells in Dl-FRIL cultures remained relatively constant from
35.times.10.sup.3 cells at day 3 and 43.6.times.10.sup.3 cells at
day 6 to a reduced level of 26.4.times.10.sup.3 cells at day 10 and
11.4.times.10.sup.3 cells at day 13. This pattern of cells in cycle
explains the low levels of total cell numbers and progenitor cells
(see FIG. 27). In contrast, the expected number of cycling cells in
cytokine cultures increased dramatically. The average number of
cells in SG.sub.2M phase in cytokine cultures increased by 31-fold
to 212.times.10.sup.3 cells at day 3, by 57-fold to
389.5.times.10.sup.3 cells at day 6, by 81-fold to
553.times.10.sup.3 cells at day 10, and by 97.7-fold to
664.6.times.10.sup.3 cycling cells at day 13, compared to
6.8.times.10.sup.3 cells in SG.sub.2M seeded in culture (Table 1).
Taken together, these results support the notion that Dl-FRIL
preserves more immature progenitor cells in a quiescent state up to
2 weeks in culture compared to cytokine treated cultures.
[0365] To test whether Dl-FRIL acts in a dominant manner to prevent
cytokines from inducing the quiescent progenitor population into
SG.sub.2M phase, the cell cycle status of cells cultured with
either Dl-FRIL or Flt3-L, in the presence and absence of cytokines
was analyzed, after 3 days of suspension culture as indicated in
FIGS. 35A-35G and Table 6. FIGS. 35A-35G show representative cell
cycle histograms of CD34 .sup.+ cells cultured with no addition
(FIG. 35A), Dl-FRIL alone (FIG. 35B) or various cytokines or
combinations thereof (FIGS. 35C-35G) for 3 days, and the mean
percentage of cells in each cell cycle phase is summarized in Table
6.
30TABLE 6 Cell cycle status of CD34+ cells, ex vivo cultured for 3
days Culture conditions Sub G.sub.0/G.sub.1 G.sub.0/G.sub.1
SG.sub.2M Dl-FRIL 9.4 .+-. 2.8 72.9 .+-. 2.8 17.7 .+-. 1.9 Flt3-L
9.4 .+-. 3.3 68.4 .+-. 2.1* 22.2 .+-. 5.4* Dl-FRIL + Flt3-L 8.6
.+-. 3.9 67.1 .+-. 1.8* 24.3 .+-. 5.65* Cytokines 2.6 .+-. 1.7 46.5
.+-. 4.5* 50.9 .+-. 1.4* Cytokines + Dl-FRIL 2.5 .+-. 0.6 47.5 .+-.
2.2* 50.0 .+-. 2.7* Cytokines + Flt3-L 2.1 .+-. 0.5 47.7 .+-. 2.4*
50.2 .+-. 2.9* Data shown is mean percentage .+-. SE, from 4
independent experiments. p values = (vs. Dl-FRIL): *p <
0.05.
[0366] As was also shown in Table 5, Dl-FRIL maintains a
significantly higher percentage of quiescent cells and lower
percentage of cycling cells (FIG. 35B and Table 6) compared to
stimulation with cytokines (FIGS. 35C-35G and Table 6). Cultures
with Flt3-L alone led to a modest, although significant, decrease
percentage of cells in G.sub.0/G.sub.1, (p<0.05) and to a
corresponding increase in SG.sub.2M phase, compared to cells
cultured with Dl-FRIL alone (Table 6). Cultures containing Dl-FRIL
and Flt3-L together slightly increased the percentage of cells in
SG.sub.2M phase, from 17.7% (with Dl-FRIL alone) to 24.3%
(p<0.05, Table 6). As shown in Table 5, exposure of CB
CD34.sup.+ cells to cytokines for 3 days induced a substantially
greater proportion of cells into SG.sub.2M phase compared to
culture with Dl-FRIL alone (p<0.05). The percentage of cells
undergoing apoptosis, as determined by subG.sub.0/G.sub.1
population, when cultured in Dl-FRIL was slightly higher than under
cytokine conditions. Neither Dl-FRIL nor Flt3-L, under these
culture conditions, effected cytokine induction into SG.sub.2M
phase of the mostly quiescent CD34.sup.+ cell population (Table
6).
EXAMPLE 7
Dl-FRIL Preserves CB Progenitors in a Dormant State in the Presence
of Potent Stimulators
[0367] Dl-FRIL was purified from Dolichos lab lab as described
above in Example 1. Dl-FRIL was assayed over a 5-log dose range (10
ng/mL-1,000 ng/mL) on human CB MNC cultured in serum-defined medium
containing agar-leukocyte conditioned medium, a potent source of a
broad range of stimulators. The number of viable MNC and
progenitors were evaluated after 5 days in culture. Results from 1
of 3 representative experiments are shown in Table 7 below. The
number of Viable MNC at the end of culture was reduced by 1.7- to
5-fold in cultures containing 10-1,000 ng/ml of Dl-FRIL. The
frequencies of myeloid and erythroid progenitors in these cultures
increased from 1.4- to 2.4-fold over the same dose range.
31 TABLE 7 Progenitor frequency Dl-FRIL MNC (Colonies/10.sup.-5
MNC) (ng/ml) (.times.10.sup.-4) Myeloid Erythroid 1,000 20 90 +/-
85 150 +/- 42 100 55 44 +/- 15 85 +/- 12 10 70 77 +/- 12 103 +/- 6
1 120 16 +/- 2 38 +/- 7 0.1 115 21 +/- 7 50 +/- 7 0 120 38 +/- 7 63
+/- 32 Culture of CB MNC in DI-FRIL results in fewer MNC and a
greater frequency of progenitors. 4 .times. 10.sup.6 CB MNC were
cultured in 2 mL of AIMV (Life Technologies) containing Agar-SCM
(StemCell Technologies) and varying concentrations of Dl-FRIL. #
Cultures were harvested after 5 days and the number of viable MNC
and progenitors were assessed. The colony data shown were
normalized as the frequency of progenitors per 10.sup.5 MNC.
[0368] A reduction in cell number and a corresponding increase in
the frequency of progenitors in cultures containing Dl-FRIL in the
presence of potent stimulators are consistent with our hypothesis
that Dl-FRIL can prevent cytokine-induced proliferation and
differentiation of progenitors. Table 8 below shows the relative
decrease of MNC in Dl-FRIL-containing cultures compared to controls
and the relative increase in progenitor frequency of progenitors.
The total number of progenitors was reduced as expected
(progenitors are at varying stages of differentiation no direct
correlation between progenitor number and MNC would be
expected).
32 TABLE 8 Fold Fold INCREASE Dl-FRIL DECREASE Progenitor frequency
(ng/ml) MNC Myeloid Erythroid 1,000 5.0 2.4 2.4 100 2.0 1.2 1.4 10
1.7 2.1 1.6 1 1.0 0.4 0.6 0.1 1.0 0.6 0.8 0 1.0 1.0 1.0 Reduction
in MNC correlates with the relative increase in frequency of
progenitors. The relative reduction in MNC of Dl-FRIL cultures is
consistent with increased progenitor frequency in corresponding
samples.
EXAMPLE 8
Dl-FRIL Protects CB MNC from the Toxicity of Chemotherapy Drugs
[0369] Experiments showing that Dl-FRIL can prevent proliferation
and differentiation of CB progenitors in cultures containing potent
stimulators indicated that Dl-FRIL protects progenitors from the
toxicity of cell cycle-active chemotherapy drugs. The culture
system described above was adapted to a 96 well plate format and
the widely used chemotherapeutics, cytarabine (Ara-C) (FIG. 36A),
doxorubicin (Dox) (FIG. 36B), and 5-fluorouracil (5-FU) (FIG. 36C)
over a 5-log dose range were assayed on CB MNC cultured in the
presence and absence of Dl-FRIL (see FIGS. 36A-36C). Dl-FRIL was
purified from Dolichos lab lab as described above in Example 1.
Cultures containing Dl-FRIL (either at 10 ng/ml or 100 ng/ml)
resulted in a 2-3 log dose shift of CB MNC to Ara-C (FIG. 36A and
Dox (FIG. 36B). As shown in FIG. 36C, the presence of Dl-FRIL in
5-FU cultures increased viability over a large dose range.
Differences between the dose shift of Ara-C and Dox by Dl-FRIL
compared to 5-FU may be explained by recent reports demonstrating
that 5-FU acts via an RNA mechanism rather than as a DNA-specific
drug (Bunz et al., J. Clin. Invest. 104:263-269, 1999).
EXAMPLE 9
Mice Tolerate High Levels of Dl-FRIL
[0370] The in vivo toxicity of Dl-FRIL was determined in mice.
Initially, Dl-FRIL was administered intravenously to mice over a
3-log dose range of 0.006-1 mg/kg (0.32-20 .mu.g/mouse). Dl-FRIL
was well tolerated and these mice have subsequently received 2
monthly challenges of Dl-FRIL without any observable short- or
long-term adverse effects.
[0371] Protocols to test chemoprotective properties of cytokines in
mice typically involve daily pre-treatment (bolus or continuous
delivery) regimens of 4-10 days before starting chemotherapy. Using
this framework as a starting point, Dl-FRIL was injected at 5 mg/kg
(100 .mu.g/mouse) intravenously daily for 4 days. No gross adverse
effects have been observed in over 150 mice treated with this dose
regimen. Dl-FRIL was purified from Dolichos lab lab as described
above in Example 1.
[0372] The upper limits of Dl-FRIL toxicity were next explored by
injecting a single bolus intraperitoneal injection of Dl-FRIL (to
accommodate 1 mL volume) at 500 mg/kg and monitored the survival of
mice for 48 hours. Of the four BALB/c mice receiving this treatment
(2 males and 2 females, aged 5 months), only 1 mouse (a male)
survived 48 hours. The surviving mouse's weight decreased by
approximately 15% in the first 2 day and returned to normal by day
4. The surviving mouse's blood counts were in the normal range 3
days after injection of FRIL. The results demonstrate that even a
very large dose of Dl-FRIL is not completely toxic.
[0373] Additional dose range finding studies are performed to
determine toxicity in mice receiving high doses of Dl-FRIL
administered by various routes (intravenous, intraperitoneal,
subcutaneous, and oral).
EXAMPLE 10
in vivo Modulation of Progenitors by Dl-FRIL in Mice
[0374] Using the initial planned dose regimen of Dl-FRIL (5
mg/kg.times.4 days) for testing chemoprotection, hematopoietic
parameters were examined in mice 3, 5, and 7 days after completing
Dl-FRIL treatment. Dl-FRIL was purified from Dolichos lab lab as
described above in Example 1. Weight-matched BALB/c mice (females,
aged 8 weeks) were injected intravenously with either 0.2 ml of
Dl-FRIL (500 mg/ml) or 0.2 ml of HBSS daily for 4 days. Two mice
from each group were evaluated at 3 days, 5 days, and 7 days after
completing Dl-FRIL treatment. Blood was collected in heparinized
tubes by eye bleeds prior to sacrificing mice by CO.sub.2. Two
femurs and a spleen were harvested from each mouse and the samples
were processed within 1 hour. Progenitors wee accessed using
standard hematopoietic colony assays (StemCell Technologies). The
results from this study are shown below in Table 9.
33TABLE 9 Hematopoietic parameters 3, 5, and 7 days after Dl-FRIL
treatment (5 mg/kg .times. 4 days) Cellularities Days RBC WBC BM
Spleen 3 0.58 0.34 0.59 1.01 5 1.26 0.98 0.75 0.72 7 0.95 0.82 0.91
1.12 CFU-C CFU-E BFU-E + Mix Days Freq. Total Freq Total Freq Total
Bone marrow progenitors 3 1.85 1.10 1.63 1.01 1.61 1.01 5 1.17 0.87
0.86 0.63 1.03 0.80 7 1.81 1.69 0.61 0.57 2.59 2.47 Spleen
progenitors 3 -- -- 2.83 3.35 -- -- 5 2.41 1.68 0.95 0.63 2.12 1.42
7 -- -- 1.74 1.78 -- -- Dl-FRIL data are reported as relative to
control values.
[0375] As shown in Table 9, the peripheral blood counts (red blood
cell (RBC) and white blood cell (WBC)) of Dl-FRIL-treated mice were
found to be reduced by 1.7- and 2.9-fold, respectively, at 3 days,
and returned to normal by day 5. Bone marrow (BM) cellularity was
also reduced by 2.5-fold at day 3, and returned to normal after 7
days. The spleen cellularity was lower at day 5 but normal at day 3
and day 7.
[0376] As shown in Table 9, the frequency of progenitors was
slightly increased in bone marrow, by 1.6- to 1.85-fold at day 3,
but the total number of progenitors in the bone marrow remained
unchanged. In the spleen, a compensatory organ during hematopoiesis
stress, a 2.83-fold higher frequency and 3.35-fold higher number of
erythroid progenitors were observed in the spleen at day 3 (see
Table 9). The frequencies and total number of progenitors in the
bone marrow appeared normal at day 5 but fewer mature erythroid
progenitors (CFU-E) and more primitive progenitors (BFU-E/mix) were
observed at day 7. A similar reduction in CFU-E was observed in
spleens at day 5; otherwise, the frequencies and total numbers of
progenitors increased from days 3-7.
EXAMPLE 11
DL-FRIL protects mice from 5-FU induced death in the critical first
week
[0377] A dose regimen was established to determine whether FRIL
protects mice from death resulting from hematopoietic toxicity of
Ara-C and Dox. This murine 5-FU chemoprotection model is based on
studies showing that a single dose of 5-FU (150 mg/kg) resulted in
>90% reduction of bone marrow cellularity but had limited
cytotoxic effect on stem cells (Lerner and Harrison, Exp. Hematol.
18:114-118, 1990). This finding was consistent with the
understanding that stem cells reside in the bone marrow in
predominantly a quiescent state. Bone marrow cellularity in these
mice was restored after 2 weeks by the recruitment of the dormant
progenitors and responsive stem cells that escaped toxicity of
5-FU. Administration of a second dose of 5-FU (also at 150 mg/kg) 3
7 days after the initial dose killed those stem cells and
progenitors recruited into S-phase in response to the first
treatment of 5-FU (Lerner and Harrison, supra).
[0378] de Haan et al. (Blood 87:4581-4588., 1996) applied this
model to test whether prophylactic treatment of mice with
hematopoietic regulators, pegylated SCF+IL11, could expand the stem
cell and primitive progenitor compartments and better protect mice
from death by 5-FU induced hematopoietic toxicity. These
experiments demonstrated that although SCF+IL11 pretreatment could
accelerate hematopoietic recovery after it was underway by 4 days 5
days when compared to controls (from approximately 11 days to 7
days for 40% survival, the SCF+IL11 cytokine pretreatment strategy
did not rescue mice from death when the second dose of 5-FU was
administered in the critical first week when stem cells are ablated
(de Haan et al. supra).
[0379] In this study described below, the pretreatment dose regimen
for Dl-FRIL described above (5 mg/kg.times.4 days) was selected
based on the requirement of treating animals with cytokines for
several days prior to starting chemotherapy and because
Dl-FRIL-treated mice easily tolerated doses (5 mg/kg) that were 10-
to 100- fold greater than that used for cytokines. Since FRIL and
cytokines act on progenitors in the same concentration range
(ng/ml), this pretreatment dose regimen was used to test whether
Dl-FRIL can protect mice from 5-FU administered at 2 intervals in
the first week: 5-FU (150 mg/kg) was injected at day 0 and then a
second dose (also at 150 mg/kg) was injected either on day 3 (dO/3
dose interval) or day 5 (dO/5 dose interval). No survival on the
day 3 interval was observed by de Haan et al. (supra) at day 3.
[0380] Dl-FRIL was purified from Dolichos lab lab as described
above in Example 1.
[0381] Weight-matched BALB/c mice (10 mice/group) were injected
intravenously with either with 0.2 mL of Dl-FRIL (500 mg/mL) or 0.2
mL of HBSS daily for 4 days. Two hours after the final treatment of
Dl-FRIL, mice were injected intraperitoneally with 5-FU (150
mg/kg). Groups of mice received a second dose of 5-FU (150 mg/kg)
at either day 3 or day 5. No mice died from a single dose of
5-FU.
[0382] As shown in Table 10, Dl-FRIL pretreatment improved survival
of mice in two separate experiments.
34 TABLE 10 5-FU Dose Interval D0/3 D0/5 Exp. Mice FRIL HBSS FRIL
HBSS 1 Males, 16 wk 3/10 0/10 N.T. N.T. 2 Females, 8 wk 0/10 0/10
4/10 1/10 Improved survival of mice pretreated with FRIL (5 mg/kg
.times. 4 days) prior to undergoing 5-FU dose intervals of d0/3 and
d0/5.
[0383] In the first experiment, 3 of 10 mice survived a d0/3 dose
interval of 5-FU compared to no mice in the HBSS pretreatment
control. In the second experiment, 4 of 10 mice pretreated with
Dl-FRIL survived a d0/5 dose interval of 5-FU compared to 1 of 10
for HBSS pretreated mice.
EXAMPLE 12
Optimization of the Dose Regiment of a FRIL Family Member to
Protect Mice from 5-Fu Induced Death
[0384] FRIL family members are relatively abundant in legumes. For
example, Dl-FRIL accounts for approximately 0.02% of the mass of
hyacinth beans.
[0385] Dl-FRIL is purified by carbohydrate affinity chromatography
as described in Example 1, and is evaluated for purity by SDS-PAGE
(5 discrete bands are visualized on an overloaded gel; see FIG.
37); is analyzed for mass and composition by amino acid analysis;
and is assayed in the cord blood progenitor assay described
above.
[0386] The murine 5-FU chemoprotection model (Lemer and Harrison,
supra; de Haan, supra) is used to empirically derive the optimal
dose regimen of Dl-FRIL to protect mice from death. Dl-FRIL is
administered intravenously to mice over a 3-log dose range (5-5,000
mg/kg) under various regimens that include Dl-FRIL treatment prior
to chemotherapy (daily from 3 days to 2 hour before initiation of
chemotherapy) and during chemotherapy.
[0387] The mice used in these studies are BALB/c female mice, 8-10
weeks at outset of experiments Jackson Laboratory, Bar Harbor,
Me.), weight matched each for experiment, where there are 10 mice
per group. Organs from mice receiving a dose of 5,000 mg/kg of FRIL
(with no 5-FU treatment) are collected for toxicity studies
[0388] The five doses of Dl-FRIL are 0, 5, 50, 500, and 5,000
.mu.g/kg. The four dose regimens of Dl-FRIL will be -2 hour; -1 day
and -2 hour; -2 day, -1 day, and -2 hour; -3 day, -2 day, -1 day,
and -2 hour prior to 5-FU treatment. The two maintenance regimens
are either daily.times.7 day (-2hour to day 7) or every other d
(days 0, 2, 4, 6). Thus, one group of mice will receive a dose of
Dl-FRIL daily for 7 days; while the second group will receive a
dose of Dl-FRIL every other day for 7 days.
[0389] The 5-FU dose intervals of 150 mg/kg are at dose intervals
of d0/3, d0/5, and d0/7.
EXAMPLE 13
A FRIL Family Member has Chemoprotective Properties with Widely
used Cell Cycle-Active Chemotherapeutics
[0390] After establishing the optimal dose regimen of a FRIL family
member, the FRIL family member's ability to protect mice from death
by cytarabine (Ara-C) and doxorubicin.
[0391] Initial dose regimens of cytarabine (Ara-C) and doxorubicin
are as follows: Doxorubicin 14 mg/kg as single bolus i.p. injection
(Grzegorzewski et al., J.Exp.Med. 180:1047-1057, 1994); Ara-C-300
mg/kg at time as an i.p. injection at 0 and 12 hours (Paukovits et
al., Blood 77:1313-1319, 1991). Further studies are based on
targeted clinical indication
EXAMPLE 14
Characterization of the Hematopoietic Status of Mice During Optimal
Dose Regimen of a FRIL Family Member to Protect Mice from 5-FU
Induced Death
[0392] Peripheral blood counts and the status of hematopoietic
progenitors (frequency, total number, and cycling status) are
characterized in mice during and after receiving the optimal dose
regimen of a FRIL family member.
[0393] To do this, mice injected with a dose regimen of a FRIL
family member with no 5-FU treatment are evaluated daily during and
for one week after treatment with the FRIL family member. The mice
are evaluated for the following hematopoietic parameters: WBC and
RBC counts; bone marrow and spleen cellularities; and progenitor
status, which includes hematopoietic colony assays (StemCell
Technologies). The progenitors assayed are myeloid (CFU-C),
erythroid (CFU-E), and primitive, multipotential (BFU-E/Mix). The
frequencies and total numbers are determined, as well as the
cycling status of these cells, as measured by .sup.3H-thymidine
suicide assay (Moore et al., Exp. Hematol. 14:222-229, 1986).
EXAMPLE 15
Analysis of Pharmacology and Toxicology of FRIL in Mice
[0394] The clearance of a FRIL family member from the circulation
and its accumulation in the body is determined by preliminary
pharmacokinetics.
[0395] For these studies, .sup.125I-FRIL is injected into mice
(dosage of FRIL based on optimization results). Clearance of FRIL
from the blood is evaluated at the following timepoints after
injection: 5 min., 15 min., 30 min., 1 hour, 2 hours, 4 hours, 8
hours, 12 hours, 36 hours, 48 hours. Two mice are evaluated at each
timepoint. Following this study, the animals are sacrificed and the
organs collected and evaluated.
[0396] Dose-range finding experiments determine the maximal
tolerated dose of a FRIL family member by various routes of
administration (intravenous, intraperitoneal, subcutaneous, and
oral). To do this dose range finding study, four routes at four
doses are used. The routes of administration are intravenous,
intraperitoneal, subcutaneous, oral. The highest dose of a FRIL
family member is 1 g/kg. The dosage of the FRIL family member is
reduced by 2-fold until mice survive. Survival is measured at 48
hours after treatment. Other clinical observations are made,
including behavorial, lethargy, vocalization, diarrhea. CBC
analyses are made. The animals are evaluated for any necropsy
(i.e., gross lesions). Following this study, the animals are
sacrificed and the organs collected and evaluated.
[0397] Acute dose toxicity studies allow identify target organs
that may develop lesions after exposure to a FRIL family member.
For these acute dose toxicity studies, a dose is selected, and a
FRIL family member is injected daily for 7 days. Acute toxicity is
evaluated at 7 days, and recovery from acute toxicity is evaluated
at 21 days. Blood chemistries, target organs, bone marrow and
blood, and other health indicator are evaluated.
[0398] Hypersensitivity studies in guinea pigs are performed to
test for any adverse immunologic reactions. To do this, fifteen
guinea pigs (5 FRIL, 5 DNCB positive control, 5 saline negative
control) are used. A FRIL family member is intradermally injected
at 0.1 mL. Daily clinical observations at site for redness and
edema are compared to the DNCB positive control. The FRIL guinea
pigs are challenged at at 2 weeks with 0.05 mL of the FRIL family
member, and daily clinical observations are made.
[0399] A determination of development of mouse anti-FRIL family
member antibodies in mice receiving treatment with a FRIL family
member is made to determine the extent and nature of the body's
response to a FRIL family member. To do this, a FRIL family member
is attached to Dynal's tosylactivated magnetic beads. The FRIL
family member-coated beads are incubated with plasma (pooled or
individual) from a mouse who has received treatment with the FRIL
family member. Using rabbit and rat antiserum to FRIL used as
positive control, the presence of FRIL family member-specific
antibodies is evaluated by SDS-PAGE and Western blot analysis
(horseradish peroxidase or chemiluminesce). Lastly, a determination
is made as to whether sugar blocks antibody binding
(a-D-mannopyranoside and negative control).
EXAMPLE 16
Purification of Progenitor Cells using Dl-FRIL-Coated Magnetic
Beads
[0400] Using magnetic beads coated with a non-limiting FRIL family
member, Dl-FRIL, a population of progenitor cells was isolated and
characterized. To do this, the following methods were used.
[0401] Preparation of FRIL-Beads for Cell Isolation
[0402] Dl-FRIL was purified from Dolichos lab lab seeds as
described in Example 1. Dl-FRIL can be immobilized on magnetic
beads (M-280 Dynabeads Tosylactivated, Lake Success, N.Y.) via
amino- and sulfhydryl-groups of the lectin according to the
manufacturer's directions. Dl-FRIL can also immobilized on magnetic
beads by a biotin-strepavidin interaction.
[0403] In this example, Dl-FRIL was immobilized on magnetic beads
by a biotin-strepavidin interaction. Biotinylation of Dl-FRIL via
primary amine-groups (EZ-Link Sulfo-NHS-LC-LC-Biotin, Pierce
Chemical Company, Rockford, Ill.) was carried out according to the
manufacturer's directions. Biotinylated Dl-FRIL was incubated with
strepavidin-labeled magnetic beads (Dynal or Miltenyi Biotec,
Auburn, Calif.) according to the manufacturer's directions.
[0404] Preparation of Cells
[0405] Human cord blood (CB), peripheral blood, and bone marrow,
collected in sterile receptacles containing anticoagulant (e.g.,
heparin, EDTA), was processed to isolate mononuclear cells (mnc)
within six hours of collection by density centrifugation on
Ficoll-Paque PLUS (Pharmacia Biotech, Piscataway, N.J.) according
to the manufacturer's directions. Mononudear cells harvested at the
interface of plasma and Ficoll-Paque were washed and resuspended in
serum-defined medium (e.g., XVIVO-10, Biowhittaker, Walkerville,
Md. or AIM-V, Life Technologies, Rockville, Md.).
[0406] Dl-FRIL-Bead Cell Isolation
[0407] Dl-FRIL-coated beads specifically bound a minor mnc
population found in CB, peripheral blood, and bone marrow. A
ten-fold excess of Dl-FRIL-beads was incubated with the cells. For
CB, where Dl-FRIL-beads captured approximately 1% of mnc, the ratio
of beads to cells was 1:10, or 10-fold greater number of beads for
every target cell. The ratio for other FLT3-expressing cell
populations, was hematopoietic and non-hematopoietic, was
experimentally determined by serial exposure of cells to fresh
Dl-FRIL-beads.
[0408] Dl-FRIL-beads were washed twice in serum-defined medium
prior to use. An aliquot of Dl-FRIL-beads was added to 10 mL of
serum-defined medium in a 15 mL conical centrifuge tube (Falcon,
Becton-Dickinson, Lincoln, N.J.), mixed, and placed in a magnet
(Dynal or Miltenyi Biotec, depending on source of magnetic beads)
for ten minutes. Medium was aspirated with a 10 mL pipette without
disturbing beads bound to walls of centrifuge tube by the magnet
charge from the magnet. After washing, 0.5 mL of serum-defined
medium was added to the tubes to wash the beads from the walls to
the bottom of the conical tube. Medium was added to beads in a
small volume (<2 mL) and the centrifuge tube was tumbled on a
rocker in a cold room (i.e., at approximately 4.degree. C.) for one
hour. After incubation, serum-defined medium was added to a final
volume of 10 mL and the tube was placed in the magnet for ten
minutes. Medium was removed by aspiration without disturbing cells
bound to Dl-FRIL-beads on the walls of the centrifuge tube via the
magnetic charge. Cells were washed a second time by removing the
conical tube from the magnet, adding 10 mL of serum-defined medium,
mixing cells, and placing the conical tube back onto the magnet.
Following aspiration of the medium, the final volume was adjusted
to 2 mL.
[0409] Detachment of Dl-FRILbeads from Cells
[0410] For some applications, detachment of Dl-FRIL-beads from
cells is preferred. About half of Dl-FRIL-beads detached from CB
mnc after overnight incubation on a rocker in the cold room.
Although binding studies have demonstrated that excess mannose and
.alpha.-methyl .alpha.-D-mannoside prevent Dl-FRIL from binding to
Flt3, neither sugar released tightly bound Dl-FRIL-beads from CB
mnc. To remove Dl-FRIL-beads from this subpopulation of cells, the
cells were incubated in 100 mM trehalose (Sigma, St. Louis, Mo.)
for one hour on a rocker in the cold room.
[0411] It should be noted that since Miltenyi beads are very small
(approximately 50 nm) as compared to Dynal beads (approximately 10
.mu.m), when Miltenyi beads were used to purify Dl-FRIL-binding
progenitor cells, the beads were allowed to remain attached to the
purified progenitor cells.
[0412] Receptor Tyrosine Kinase Gene Expression
[0413] Receptor tyrosine kinase gene expression was characterized
by RT-PCR.
[0414] Using these methods, the following results were
obtained:
[0415] Functional Properties of Dl-FRIL Bead-Selected CB mnc
[0416] The progenitor capacity of Dl-FRIL-selected CB mnc was
tested in a methylcellulose colony assay under conditions to
promote proliferation and differentiation along either the
hematopoietic or endothelial lineages (as described above. Table 11
shows the number of hematopoietic colonies (myeloid, erythroid, and
mix) and endothelial colonies (other) that formed after culture of
unselected cells (CB mnc), Dl-FRIL-selected cells (Dl-FRIL.sup.+)
and CB mnc that did not bind to Dl-FRIL-beads (Dl-FRIL.sup.-).
35TABLE 11 Response of Dl-FRIL-selected cells to hematopoietic and
endothelial stimuli Stimulator Myeloid Erythroid Mix Other Total
+CSFs CB mnc 1 3 2 0 6 Dl-FRIL.sup.+ 14 10 4 0 28 Dl-FRIL.sup.- 1 1
2 0 4 +VEGF CB mnc 0 0 0 2 2 Dl-FRIL.sup.+ 0 0 0 19 19
Dl-FRIL.sup.- 0 0 0 5 5 Cord blood mononuclear cells were isolated
by Ficoll-Paque, bead selected, and plated in
MethoCult.quadrature., StemCell Technologies, Vancouver, BC,
Canada).
[0417] As shown in Table 11, Dl-FRIL-selection increased the number
of hematopoietic colonies (stimulated with colony stimulating
factors (CSFs) by 14-fold for myeloid colonies, 3.3- to 10-fold for
erythroid colonies (CB mnc and Dl-FRIL- cells, respectively), and
by 2-fold for mixed colonies. Similar levels of Dl-FRIL-bead
enrichment was observed for endothelial colonies (stimulated with
vascular endothelial growth factor (VEGF)): 9.5-fold over CB mnc
and 3.8-fold for Dl-FRIL.sup.- cells.
[0418] Cell Surface Phenotypic Properties of Dl-FRIL Bead-Selected
CB mnc
[0419] The cell surface phenotypic properties of Dl-FRIL
bead-selected CB mnc was characterized by flow cytometry. Table 12
shows the phenotypes (by percentage of cells expressing the
indicated cell surface phenotype marker) of the three CB cell
populations: (1) cells not selected by Dl-FRIL-beads
(Dl-FRIL.sup.-); (2) cells that detached from Dl-FRIL-beads after
overnight incubation in the coldroom on a rocker (Dl-FRIL.sup.+);
and (3) cells that retained Dl-FRIL-beads after overnight
incubation (Dl-FRIL.sup.++). The two Dl-FRIL-binding cell
populations were analyzed separately to see whether tightness of
binding (avidity) related to type of cells selected. Isotype
control levels were set at 2%; all values of 2% represent no
reactivity with test antibody.
36TABLE 12 Flow cytometric analysis of Dl-FRIL-selected CB mnc
Dl-FRIL.sup.- Dl-FRIL.sup.+ Dl-FRIL.sup.++ Antigen Cell Type (%)
(%) (%) CD3 Mature T 26 35 6 CD11b Mac-1, CR3 19 35 67 CD11c
LeuCAMc 10 22 32 CD13 Pan myeloid, CFU-GM 5 <2 <2 CD19 Pan B
4 5 12 CD32 Low affinity IgG Fc.gamma.-R 5 19 26 CD33 Myeloid
progenitors 3 2 8 CD34 Pan progenitors <2 <2 <2 CD38
Activated T 88 96 93 CD42a Platelet gpIX 5 2 7 CD69 Early
activation ag 6 8 14 (EA-1) CDw90 Thy-1, progenitor subset 8 14 13
CD117 c-kit, progenitors 4 2 2
[0420] As shown in Table 12, Dl-FRIL-beads did not capture CB mnc
that express CD34, the hallmark marker of hematopoietic stem cells
and progenitors. This observation was unexpected because
Dl-FRIL-selected cells enrich for progenitors (see Table 11).
Although CB CD34.sup.+ cells uniformly express FLT3, only 70% of
Flt3.sup.+ CB mnc also express CD34 (Rappold et al., Blood
90:111-125, 1997). Consequently, 30% of CB mnc expressed the
phenotype of CD34.sup.-Flt3.sup.+. Dl-FRIL-beads appeared to
capture this latter population of cells.
[0421] Cells expressing dendritic cell (DC) markers, CD11b and
CD11c, were enriched approximately 2-fold in the Dl-FRIL.sup.+ cell
population and over 3-fold in the Dl-FRIL.sup.++ cell population
(Table 12). This observation is consistent with reports that Flt3
is involved in dendritic cell proliferation and maturation in mice
(Pulendran et al., J. Immunol. 159:2222-2231, 1997) and humans
(Miller et al., Blood 93:96-106., 1999). The rare hematopoietic
dendritic cell population is useful in inducing tumor regression
and for the treatment of AIDS.
[0422] Differences of the cell surface phenotypes were observed
between Dl-FRIL.sup.+ and Dl-FRIL.sup.++ CB cells (see Table 11).
The percentage of CD3 T cells decreased from 35% for Dl-FRIL.sup.+
cells to 6% for Dl-FRIL.sup.++ cells. Conversely, the percentage of
CD11b.sup.+ cells and CD11c.sup.+ cells increased from 35% to 67%
and from 22% to 32% for Dl-FRIL.sup.+ and Dl-FRIL.sup.++ cell
populations, respectively.
[0423] Cells that retained Dl-FRIL-beads after overnight incubation
on a rocker in the cold room (Dl-FRIL.sup.++ cells) were observed
as single cells or as clumps of bead-bound cells. These clumps
could not be disrupted either by mechanical means or by elution
with competing sugars, mannose or mannose derivatives (data not
shown). From studies to characterize the carbohydrate-binding
properties of Dl-FRIL, .alpha.,.alpha.-trehalose demonstrated a
3.6-fold greater potency than mannose and a 1.6- to 2.1-fold
greater potency than .alpha.-methyl .alpha.-D-mannoside derivatives
that were tested (Mo et al., Glycobiology 9:173-179,1999).
Incubation of clumped Dl-FRIL-bead bound cells with 100 mM
Trehalose effectively disrupted the clumped cells and removed most
of the Dl-FRIL-beads from cells.
[0424] Two populations of trehalose-disrupted Dl-FRIL.sup.++ cells
were analyzed by flow cytometry: cells that no longer bound beads
(Dl-FRIL.sup.++) and cells that still retained beads following
incubation with trehalose (Dl-FRIL.sup.+++). The results of one
experiment are shown in Table 13.
37TABLE 13 Flow cytometric analysis of Dl-FRIL-selected CB mnc
after exposure to trehalose Dl-FRIL.sup.++ Antigen Cell Type (%)
Dl-FRIL.sup.+++ (%) CD3 Mature T 6 3 CD11b Mac-1, CR3 52 76 CD11c
LeuCAMc 16 43 CD13 Pan myeloid, CFU-GM 72 46 CD19 Pan B 5 9 CD32
Low affinity IgG Fc-.gamma. 43 19 CD33 Myeloid progenitors 56 15
CD34 Pan progenitors 2 2 CD117 c-kit, progenitors 2 12 CD135 Flt3,
progenitors 2 5
[0425] The difference in cell surface phenotypes between
Dl-FRIL.sup.++ cells and Dl-FRIL.sup.+++ cells in Table 13 was
greater than those observed for Dl-FRIL.sup.+ cells and
Dl-FRIL.sup.++ cells in Table 12. In Table 13, the percentage of
CD3, CD13, CD32, and CD33 cells decreased by 1.6- to 3.7-fold in
Dl-FRIL.sup.+++ cells compared to Dl-FRIL.sup.++ cells. Conversely,
the percentage of CD11b, CD11c, CD19, CD117, and CD135 cells
increased by 1.5- to 6-fold in Dl-FRIL.sup.+++cells compared to
Dl-FRIL.sup.++ cells. Again no CD34 was observed in either cell
population.
[0426] The increase in percentage of cells that express CD117 and
CD135, two tyrosine kinase receptors central to hematopoietic stem
cell and progenitor function (Lyman and Jacobson, Blood
91:1101-1134, 1998), suggested that avidity of Dl-FRIL-bead binding
of cells might correspond to the primitive status of the cells. The
studies described herein which characterize the carbohydrate
binding properties of Dl-FRIL supported this notion. Dl-FRIL has
neither an extending carbohydrate combining-binding site nor a
hydrophobic binding site adjacent to it (Mo et al., Glycobiology
9:173-179, 1999). Dl-FRIL binds most tightly to a trimannoysl
structure that is the basis for N-linked glycosylation in mammals.
Consequently, Dl-FRIL may bind cells that have undergone less
processing of glycosylation, which is consistent with more
primitive cells. This property of Dl-FRIL-binding may provide a
unique method to isolate primitive cells not currently possible by
antibodies to CD34. Thus, Dl-FRIL binds the normally glycosylated
FLT3 receptor more tightly than the FLT3-Ligand binds to normally
glycosylated FLT3. Dl-FRIL binds normally glycosylated FLT3
receptor more tightly than the typical antibody binds its specific
ligand.
[0427] Receptor Tyrosine Kinase Gene Expression in Dl-FRIL-Selected
CB Cells
[0428] The number of cell surface receptors and markers increases
as pluripotent hematopoietic stem cells proliferate and
differentiate. The number of functional receptors on the most
primitive cells is probably less than ten. The levels of detection
for flow cytometry are probably in the range of several hundred
cell surface molecules. Consequently, analysis of primitive cell
populations cannot be analyzed by flow cytometry.
[0429] The presence or absence of functional tyrosine kinase
receptors on primitive cells was further characterized by RT-PCR.
Expression of Flt3, Kit, Fms, Flk1, Flt1, and Flt4 mRNA was
determined for CB mnc, CD34-selected cells, and Dl-FRIL-selected
cells. For CB mnc, the PCR products for the receptors were either
faint or not detectable. The pattern of gene expression for cells
selected by CD34-beads or Dl-FRIL-beads was the same; all tyrosine
kinase receptors showed stronger PCR signals. These data suggest
that receptors associated with stem cells (Flt3 and Kit) and
primitive endothelial cells (FLk1, FIt1, and Flt4) are also
detected in Dl-FRIL selected cells.
EXAMPLE 17
Use of Beads Coated with a FRIL Family Member to Isolate CD34.sup.-
Primitive Stem Cells
[0430] A rare human stem cell population with the phenotype of
CD34.sup.-CD38.sup.-Lin.sup.- has been identified by its ability to
establish multilineage engraftment in NOD/SCID mice (Bhatia et al.,
Nat. Med. 4:1038-1045, 1998). These repopulating cells give rise to
stem cells that express the hallmark CD34 marker. Isolating
CD34.sup.-CD38.sup.-Lin.- sup.- cells is labor-intensive methods of
negative selection that includes the use of immunomagnetic beads
and flow cytometry to deplete cells that express CD34, CD38, and
lineage markers. Rapid, efficient, positive selection of
CD34.sup.-CD38.sup.-Lin.sup.- cells would be preferable for
clinical uses. However, the absence of the highly expressed CD34
marker and low number of functional receptors on this rare
population of cells will prevent use of antibodies for cell
isolation.
[0431] FRIL attached to magnetic beads are used in a unique method
to isolate the rare CD34.sup.-CD38.sup.-Lin.sup.- cell population
by binding primitive cells that express this phenotype. Isolation
of CD34.sup.-CD38.sup.-Lin.sup.- is achieved by a single-step cell
isolation. However, since FRIL-beads also recognize cells that
express CD11b, CD11c, and CD38, optimal isolation of
CD34.sup.-CD38.sup.-Lin.sup.- - cells is improved by first
negatively selecting unwanted cells by immunomagnetic beads that
bind to CD11b, CD11c, and/or CD38.
EXAMPLE 18
Use of Beads Coated with a FRIL Family Member to Isolate Normal
Stem Cells from Patients with Leukemia
[0432] A majority of leukemias express the phenotype of
CD34.sup.+Flt3.sup.+ (Carow et al., Blood 87:1089-1096, 1996).
Consequently, methods that rely on CD34 expression cannot
distinguish normal stem cells and progenitors from leukemic cells
in the bone marrow and peripheral blood of patients.
[0433] FRIL does not interact with two cell leukemic lines tested
with the CD34 .sup.+Flt3.sup.+ phenotype (KG.sub.1-A and ML-1).
FRIL neither effects growth of these leukemic cell lines nor do
FRIL-beads capture appreciable numbers of cells (data not shown).
Since FRIL-beads select normal progenitors with the phenotype of
CD34.sup.-Flt3.sup.+, FRIL-beads provide a unique method that
distinguishes between normal and leukemic cells.
[0434] A FRIL family member attached to magnetic beads is used to
isolate normal hematopoietic stem cells and progenitors from the
bone marrow and peripheral blood of leukemia patients. This is
accomplished using a method similar to leukopheresis, where blood
is passed through a device that retains cells of interest. In this
example, FRIL-beads binds CD34.sup.-Flt3.sup.+ normal cells. Since
FRIL also interacts with Flt3-expressing CD11b and CD11c cells,
prior exposure and removal of the cells that immunomagnetic beads
that bind to CD11b and/or CD11c (i.e., negative selection) may
permit enrichment of primitive cells.
EXAMPLE 19
Use of FRIL-Beads to Isolate Dendritic Progenitors and Mature Cells
from Normal Individuals
[0435] Dendritic cells (DC) are immune cells that capture antigens
and initiate T cell-mediated immune responses (Banchereau and
Steinman, Nature 392:245-252, 1998). DC act as first lines of
defense in the skin, gut, and lymphoid organs. Antigens on DC can
activate naive and quiescent T cells and small numbers of DC pulsed
with lose dosages of antigens stimulate strong T cell responses.
Under certain circumstances, DC also induce T cell tolerance.
Consequently, the unique properties of DC has generated significant
interest to use these cells to treat cancer and AIDS.
[0436] DC are derived from CD34.sup.+ progenitors in the bone
marrow of humans. The cytokines GM-CSF, TNF-.alpha., and Flt3
ligand (FL) influence DC development (Banchereau and Steinman,
supra; Pulendran et al., J. Immunol. 159:2222-2231, 1997).
Injection of FLT3-Ligand in mice dramatically increases the number
of DC (Pulendran et al., supra). FRIL interacts with the Flt3
receptor on DC, and FRIL-beads capture cells with the dendritic
phenotype of CD11b and CD11c. Selecting dendritic cells with
FRIL-beads from human bone marrow, peripheral blood, or cord blood
allows the efficient and effective isolation of DC for clinical
use.
EXAMPLE 20
Use of FRIL-Beads to Isolate Endothelial Stem Cells and
Progenitors
[0437] Endothelial stem cells and progenitors give rise to cells
that form blood vessels in a process called angiogenesis. During
strokes and heart attacks, new blood vessels are needed to repair
damage. Activation of endothelial stem cells and progenitors to
produce more mature cells is mediated by the cytokines that
activate the Flk1/KDR, Flt1, and Flt4 tyrosine kinase receptors.
Flt3 is expressed on very primitive endothelial progenitors.
FRIL-beads are used to capture a population of cells from cord
blood that express all of these receptors.
EXAMPLE 21
Use of FRIL Family Members and Non-FRIL Family Member Lectins to
Alter Signal Transduction and Other Cellular Pathways
[0438] Drugs designed to alter signal transduction pathways need to
specifically distinguish target cells. FRIL is used as a targeting
vehicle to deliver small molecules to Flt3-expressing cells such as
stem cells, progenitors, and dendritic cells. FRIL has several
advantages for drug delivery: 1) FRIL specific for Flt3; 2) FRIL is
stable in the cytoplasm; 3) FRIL is capable of undergoing
conjugation with small molecules; 4) FRIL can be delivered in
dose-responsive manner; and 5) FRIL provides specificity for
overlapping pathways of signal transduction.
[0439] Other legume- or bulb-derived lectins can also delivery
small molecule drugs to specific cell populations. For example, the
lectins PHA and ConA both bind to CD3-T cell receptor complex and
the FC-gamma receptor (CD32) (Leca et al. Scand. J. Immunol.
23:535-544, 1986); UDA binds the V.beta. domain of the T cell
receptor (Galelli et al., J. Immunol. 151:1821-1831, 1993).
[0440] Standard methods of conjugation are used to attach small
molecules, oligos, or enzymes to plant lectins.
EXAMPLE 22
Purification and Cloning of YamFRIL from Sphenostylis
stenocarpa
[0441] Dry seeds of Yam bean (Sphenostylis stenocarpa) were ground
in a coffee mill and the powder was extracted with 5 volumes of 10
mM Na Acetate buffer, pH 5.2, containing 1 mM CaCl.sub.2 for 1 hour
at 4.degree. C. After centrifugation, the clear supernatant was
neutalized with Tris-HCl pH 8.0.
[0442] YamFril purification was achieved through and absorption on
a ovalbumin gel affinity column (Sigma) and was eluted with 200 mM
trehalose.
[0443] The resulting protein was fractionated into 2 polypeptides
that were submitted to N-terminal amino acid sequences.
38 Beta band: AQSVSFTFTKFDSDQ (SEQ ID NO: 9) Alpha band:
AASNNVVAVEFDTXPN (SEQ ID NO: 10)
[0444] Reverse-transcriptase PCR was performed on total RNA
obtained from International Institute of Tropical Agriculture
(Ibadan, Oyo State, Nigeria), using degenerate primers based on the
alpha and beta N-terminus sequences (i.e., SEQ ID NO: 10 and SEQ ID
NO: 9, respectively).
[0445] 3' RACE PCR was performed on total and polyA+RNA using gene
specific primers with an Anchor Primer.
[0446] Partial cDNA clones were obtained and the following
sequences deduced.
39 YamFril: partial mRNA sequence ACGAAGTTCGACAGCGACCAAAAG-
GATCTTATGTTCCAAGGTCATACCATTTCTAGCAGC (SEQ ID NO: 7)
AATGTCATACAACTCACCAAGTTAGACAGTAATGGAAACCCTGTGAGTACCAGTGTGGGA
AGAGTGTTATACTCTGCACCATTGCGCCTTTGGGAAAGCTCTACAGTAGTGTCAACCTTT
GAGACCACTTTCACCTTTCAAATCTCAACACCTTACACTAGTCCTCCTGGTGATGGGCTC
GCCTTCTTCCTTGCACCATATGACACTGTCATCCCTCCAAATTCTGCTGGCAATCTTCTT
GGACTCTTTCCTAACTTAAATGCTTTAAGAAACTCCACCACCAGTAAAGAAACCACTATT
GATGTCAATGCTGCATCTAACAACGTTGTTGCCGTTGAATTTGACACCTACCCTAAC- GAC
AATATTGGTGATCCAAGATACAAACACATTGGAATCGATGTCAACTCTATCAGG- TCCAAG
GCAACTGTTGCGTGGGACTGGCAAAATGGGAAAACAGCCACTGCACACATC- AGCTATAAC
TCTGCCTCTAAAAGACTATCTGTTACTACTTTTTATCCTGGGGGTAAA- GCTGTGAGTCTT
TCCCATGACGTTGAGCTCACTCAAGTGCTTCCTCAATGGATTAGA- GTAGGGTTCTCTGCT
TCAACAGGATTAGAGAAA YamFril: deduced amino acid sequence
AQSVSFTFTKFDSDQKDLMFQGHTISSSN- VIQLTKLDSNGNPVSTSVGRVLYSAPLRLWE (SEQ
ID NO: 8)
SSTVVSTFETTFTFQISTPYTSPPGDGLAFFLAPYDTVIPPNSAGNLLGLFPNLNALRNS
TTSKETTIDVNAASNNVVAVEFDTYPNDNIGDPRYKHIGIDVNSIRSKATVAWDWQNGKT
ATAHISYNSASKRLSVTTFYPGGKAVSLSHDVELTQVLPQWIRVGFSASTGLEK
[0447]
Sequence CWU 1
1
61 1 939 DNA Artificial Sequence D1-FRIL. 1 gcacagtcat tgtcatttag
tttcaccaag tttgatccta accaagagga tcttatcttc 60 caaggtcatg
ccacttctac aaacaatgtc ttacaagtca ccaagttaga cagtgcagga 120
aaccctgtga gttctagtgc gggaagagtg ttatattctg caccattgcg cctttgggaa
180 gactctgcgg tattgacaag ctttgacacc attatcaact ttgaaatctc
aacaccttac 240 acttctcgta tagctgatgg cttggccttc ttcattgcac
cacctgactc tgtcatcagt 300 tatcatggtg gttttcttgg actctttccc
aacgcaaaca ctctcaacaa ctcttccacc 360 tctgaaaacc aaaccaccac
taaggctgca tcaagcaacg ttgttgctgt tgaatttgac 420 acctatctta
atcccgatta tggtgatcca aactacatac acatcggaat tgacgtcaac 480
tctattagat ccaaggtaac tgctaagtgg gactggcaaa atgggaaaat agccactgca
540 cacattagct ataactctgt ctctaaaaga ctatctgtta ctagttatta
tgctgggagt 600 aaacctgcga ctctctccta tgatattgag ttacatacag
tgcttcctga atgggtcaga 660 gtagggttat ctgcttcaac tggacaagat
aaagaaagaa ataccgttca ctcatggtct 720 ttcacttcaa gcttgtggac
caatgtggcg aagaaggaga atgaaaacaa gtatattaca 780 agaggcgttc
tgtgatgata tatgtgtatc aatgattttc tatgttataa gcatgtaatg 840
tgcgatgagt caataatcac aagtacagtg tagtacttgt atgttgtttg tgtaagagtc
900 agtttgcttt taataataac aagtgcagtt agtacttgt 939 2 264 PRT
Artificial Sequence D1-FRIL. 2 Ala Gln Ser Leu Ser Phe Ser Phe Thr
Lys Phe Asp Pro Asn Gln Glu 1 5 10 15 Asp Leu Ile Phe Gln Gly His
Ala Thr Ser Thr Asn Asn Val Leu Gln 20 25 30 Val Thr Lys Leu Asp
Ser Ala Gly Asn Pro Val Ser Ser Ser Ala Gly 35 40 45 Arg Val Leu
Tyr Ser Ala Pro Leu Arg Leu Trp Glu Asp Ser Ala Val 50 55 60 Leu
Thr Ser Phe Asp Thr Ile Ile Asn Phe Glu Ile Ser Thr Pro Tyr 65 70
75 80 Thr Ser Arg Ile Ala Asp Gly Leu Ala Phe Phe Ile Ala Pro Pro
Asp 85 90 95 Ser Val Ile Ser Tyr His Gly Gly Phe Leu Gly Leu Phe
Pro Asn Ala 100 105 110 Asn Thr Leu Asn Asn Ser Ser Thr Ser Glu Asn
Gln Thr Thr Thr Lys 115 120 125 Ala Ala Ser Ser Asn Val Val Ala Val
Glu Phe Asp Thr Tyr Leu Asn 130 135 140 Pro Asp Tyr Gly Asp Pro Asn
Tyr Ile His Ile Gly Ile Asp Val Asn 145 150 155 160 Ser Ile Arg Ser
Lys Val Thr Ala Lys Trp Asp Trp Gln Asn Gly Lys 165 170 175 Ile Ala
Thr Ala His Ile Ser Tyr Asn Ser Val Ser Lys Arg Leu Ser 180 185 190
Val Thr Ser Tyr Tyr Ala Gly Ser Lys Pro Ala Thr Leu Ser Tyr Asp 195
200 205 Ile Glu Leu His Thr Val Leu Pro Glu Trp Val Arg Val Gly Leu
Ser 210 215 220 Ala Ser Thr Gly Gln Asp Lys Glu Arg Asn Thr Val His
Ser Trp Ser 225 230 235 240 Phe Thr Ser Ser Leu Trp Thr Asn Val Ala
Lys Lys Glu Asn Glu Asn 245 250 255 Lys Tyr Ile Thr Arg Gly Val Leu
260 3 1005 DNA Artificial Sequence Nucleic acid sequence of the
naturally-occurring D1-FRIL protein. 3 atggcttcct ccaacttact
caccctagcc ctcttccttg tgcttctcac ccacgcaaac 60 tcagccgcac
agtcattgtc atttagtttc accaagtttg atcctaacca agaggatctt 120
atcttccaag gtcatgccac ttctacaaac aatgtcttac aagtcaccaa gttagacagt
180 gcaggaaacc ctgtgagttc tagtgcggga agagtgttat attctgcacc
attgcgcctt 240 tgggaagact ctgcggtatt gacaagcttt gacaccatta
tcaactttga aatctcaaca 300 ccttacactt ctcgtatagc tgatggcttg
gccttcttca ttgcaccacc tgactctgtc 360 atcagttatc atggtggttt
tcttggactc tttcccaacg caaacactct caacaactct 420 tccacctctg
aaaaccaaac caccactaag gctgcatcaa gcaacgttgt tgctgttgaa 480
tttgacacct atcttaatcc cgattatggt gatccaaact acatacacat cggaattgac
540 gtcaactcta ttagatccaa ggtaactgct aagtgggact ggcaaaatgg
gaaaatagcc 600 actgcacaca ttagctataa ctctgtctct aaaagactat
ctgttactag ttattatgct 660 gggagtaaac ctgcgactct ctcctatgat
attgagttac atacagtgct tcctgaatgg 720 gtcagagtag ggttatctgc
ttcaactgga caagataaag aaagaaatac cgttcactca 780 tggtctttca
cttcaagctt gtggaccaat gtggcgaaga aggagaatga aaacaagtat 840
attacaagag gcgttctgtg atgatatatg tgtatcaatg attttctatg ttataagcat
900 gtaatgtgcg atgagtcaat aatcacaagt acagtgtagt acttgtatgt
tgtttgtgta 960 agagtcagtt tgcttttaat aataacaagt gcagttagta cttgt
1005 4 22 PRT Artificial Sequence Signal sequence from the FRIL
family isolated from Dolichos lab lab 4 Met Ala Ser Ser Asn Leu Leu
Thr Leu Ala Leu Phe Leu Val Leu Leu 1 5 10 15 Thr His Ala Asn Ser
Ala 20 5 914 DNA Artificial Sequence Pv-FRIL. 5 gctcagtcat
tatcttttaa ctttaccaag tttgatcttg accaaaaaga tcttatcttc 60
caaggtgatg ccacttctac aaacaatgtc ttacaactca ctaagttaga cagtggagga
120 aaccctgtgg gtgctagtgt gggaagagtg ttattctctg caccatttca
tctttgggaa 180 aactctatgg cagtgtcaag ctttgaaact aatctcacca
ttcaaatctc aacacctcac 240 ccttattatg cagctgatgg ctttgccttc
ttccttgcac cacatgacac tgtcatccct 300 ccaaattctt ggggcaaatt
ccttggactc tactcaaacg ttttcagaaa ctcccccacc 360 tctgaaaacc
aaagctttgg tgatgtcaat actgactcaa gagttgttgc tgtcgaattt 420
gacaccttcc ctaatgccaa tattgatcca aattacagac acattggaat cgatgtgaac
480 tctattaagt ccaaggaaac tgctaggtgg gagtggcaaa atgggaaaac
ggccactgca 540 cgcatcagct ataactctgc ctctaaaaaa tcaactgtta
ctacgtttta tcctgggatg 600 gaagttgtgg ctctctccca tgatgttgac
ttacatgcag agcttcctga atgggttaga 660 gtagggttat ctgcttcaac
tggagaggag aaacaaaaaa ataccattat ctcatggtct 720 ttcacttcaa
gcttgaagaa caacgaggtg aaggagccga aagaagacat gtatattgca 780
aacgttgtgc gatcatatac atggatcaat gacgttctat cttatataag caataaataa
840 atgtatgatg cactcaataa taatcacaag tacgtacggt gtagtacttg
tatgttgttt 900 atgaaaaaaa aaaa 914 6 279 PRT Artificial Sequence
Pv-FRIL. 6 Ala Gln Ser Leu Ser Phe Asn Phe Thr Lys Phe Asp Leu Asp
Gln Lys 1 5 10 15 Asp Leu Ile Phe Gln Gly Asp Ala Thr Ser Thr Asn
Asn Val Leu Gln 20 25 30 Leu Thr Lys Leu Asp Ser Gly Gly Asn Pro
Val Gly Ala Ser Val Gly 35 40 45 Arg Val Leu Phe Ser Ala Pro Phe
His Leu Trp Glu Asn Ser Met Ala 50 55 60 Val Ser Ser Phe Glu Thr
Asn Leu Thr Ile Gln Ile Ser Thr Pro His 65 70 75 80 Pro Tyr Tyr Ala
Ala Asp Gly Phe Ala Phe Phe Leu Ala Pro His Asp 85 90 95 Thr Val
Ile Pro Pro Asn Ser Trp Gly Lys Phe Leu Gly Leu Tyr Ser 100 105 110
Asn Val Phe Arg Asn Ser Pro Thr Ser Glu Asn Gln Ser Phe Gly Asp 115
120 125 Val Asn Thr Asp Ser Arg Val Val Ala Val Glu Phe Asp Thr Phe
Pro 130 135 140 Asn Ala Asn Ile Asp Pro Asn Tyr Arg His Ile Gly Ile
Asp Val Asn 145 150 155 160 Ser Ile Lys Ser Lys Glu Thr Ala Arg Trp
Glu Trp Gln Asn Gly Lys 165 170 175 Thr Ala Thr Ala Arg Ile Ser Tyr
Asn Ser Ala Ser Lys Lys Ser Thr 180 185 190 Val Thr Thr Phe Tyr Pro
Gly Met Glu Val Val Ala Leu Ser His Asp 195 200 205 Val Asp Leu His
Ala Glu Leu Pro Glu Trp Val Arg Val Gly Leu Ser 210 215 220 Ala Ser
Thr Gly Glu Glu Lys Gln Lys Asn Thr Ile Ile Ser Trp Ser 225 230 235
240 Phe Thr Ser Ser Leu Lys Asn Asn Glu Val Lys Glu Pro Lys Glu Asp
245 250 255 Met Tyr Ile Ala Asn Val Val Arg Ser Tyr Thr Trp Ile Asn
Asp Val 260 265 270 Leu Ser Tyr Ile Ser Asn Lys 275 7 678 DNA
Artificial Sequence YamFril partial mRNA sequence. 7 acgaagttcg
acagcgacca aaaggatctt atgttccaag gtcataccat ttctagcagc 60
aatgtcatac aactcaccaa gttagacagt aatggaaacc ctgtgagtac cagtgtggga
120 agagtgttat actctgcacc attgcgcctt tgggaaagct ctacagtagt
gtcaaccttt 180 gagaccactt tcacctttca aatctcaaca ccttacacta
gtcctcctgg tgatgggctc 240 gccttcttcc ttgcaccata tgacactgtc
atccctccaa attctgctgg caatcttctt 300 ggactctttc ctaacttaaa
tgctttaaga aactccacca ccagtaaaga aaccactatt 360 gatgtcaatg
ctgcatctaa caacgttgtt gccgttgaat ttgacaccta ccctaacgac 420
aatattggtg atccaagata caaacacatt ggaatcgatg tcaactctat caggtccaag
480 gcaactgttg cgtgggactg gcaaaatggg aaaacagcca ctgcacacat
cagctataac 540 tctgcctcta aaagactatc tgttactact ttttatcctg
ggggtaaagc tgtgagtctt 600 tcccatgacg ttgagctcac tcaagtgctt
cctcaatgga ttagagtagg gttctctgct 660 tcaacaggat tagagaaa 678 8 234
PRT Artificial Sequence YamFril deduced amino acid squence. 8 Ala
Gln Ser Val Ser Phe Thr Phe Thr Lys Phe Asp Ser Asp Gln Lys 1 5 10
15 Asp Leu Met Phe Gln Gly His Thr Ile Ser Ser Ser Asn Val Ile Gln
20 25 30 Leu Thr Lys Leu Asp Ser Asn Gly Asn Pro Val Ser Thr Ser
Val Gly 35 40 45 Arg Val Leu Tyr Ser Ala Pro Leu Arg Leu Trp Glu
Ser Ser Thr Val 50 55 60 Val Ser Thr Phe Glu Thr Thr Phe Thr Phe
Gln Ile Ser Thr Pro Tyr 65 70 75 80 Thr Ser Pro Pro Gly Asp Gly Leu
Ala Phe Phe Leu Ala Pro Tyr Asp 85 90 95 Thr Val Ile Pro Pro Asn
Ser Ala Gly Asn Leu Leu Gly Leu Phe Pro 100 105 110 Asn Leu Asn Ala
Leu Arg Asn Ser Thr Thr Ser Lys Glu Thr Thr Ile 115 120 125 Asp Val
Asn Ala Ala Ser Asn Asn Val Val Ala Val Glu Phe Asp Thr 130 135 140
Tyr Pro Asn Asp Asn Ile Gly Asp Pro Arg Tyr Lys His Ile Gly Ile 145
150 155 160 Asp Val Asn Ser Ile Arg Ser Lys Ala Thr Val Ala Trp Asp
Trp Gln 165 170 175 Asn Gly Lys Thr Ala Thr Ala His Ile Ser Tyr Asn
Ser Ala Ser Lys 180 185 190 Arg Leu Ser Val Thr Thr Phe Tyr Pro Gly
Gly Lys Ala Val Ser Leu 195 200 205 Ser His Asp Val Glu Leu Thr Gln
Val Leu Pro Gln Trp Ile Arg Val 210 215 220 Gly Phe Ser Ala Ser Thr
Gly Leu Glu Lys 225 230 9 15 PRT Artificial Sequence Beta band
polypeptide. 9 Ala Gln Ser Val Ser Phe Thr Phe Thr Lys Phe Asp Ser
Asp Gln 1 5 10 15 10 16 PRT Artificial Sequence Alpha band
polypeptide. 10 Ala Ala Ser Asn Asn Val Val Ala Val Glu Phe Asp Thr
Xaa Pro Asn 1 5 10 15 11 23 DNA Artificial Sequence MLA degenerate
oligonucleotide primer. 11 aanttnganc cnaancanga nga 23 12 20 DNA
Artificial Sequence MLZ degenerate oligonucleotide primer. 12
ttnccnttnt gccantccca 20 13 15 DNA Artificial Sequence primer. 13
gtaccgagct cggat 15 14 18 DNA Artificial Sequence primer. 14
tctagatgca tgctcgag 18 15 22 DNA Artificial Sequence MLX primer. 15
gttggacgtc aattccgatg tg 22 16 17 DNA Artificial Sequence MLI
degenerate primer. 16 gcncantcnc tntcntt 17 17 22 DNA Artificial
Sequence Oligo(dT) anchor primer. 17 gaccacgcgt atcgatgtcg ac 22 18
21 DNA Artificial Sequence MLB primer. 18 aagttagaca gtgcaggaaa c
21 19 20 DNA Artificial Sequence MLII primer. 19 gcacagtcat
tgtcatttag 20 20 18 PRT Artificial Sequence D1-FRIL. 20 Tyr Leu Asn
Pro Asp Tyr Gly Asp Pro Asn Tyr Ile His Ile Gly Ile 1 5 10 15 Asp
Val 21 19 PRT Artificial Sequence Pea. 21 Phe Tyr Asn Ala Ala Trp
Asp Pro Ser Asn Arg Asp Arg His Ile Gly 1 5 10 15 Ile Asp Val 22
1005 DNA Artificial Sequence SpDLA. 22 atggcttcct ccaacttact
caccctagcc ctcttccttg tgcttctcac ccacgcaaac 60 tcagccgcac
agtcattgtc atttagtttc accaagtttg atcctaacca agaggatctt 120
atcttccaag gtcatgccac ttctacaaac aatgtcttac aagtcaccaa gttagacagt
180 gcaggaaacc ctgtgagttc tagtgcggga agagtgttat attctgcacc
attgcgcctt 240 tgggaagact ctgcggtatt gacaagcttt gacaccatta
tcaactttga aatctcaaca 300 ccttacactt ctcgtatagc tgatggcttg
gccttcttca ttgcaccacc tgactctgtc 360 atcagttatc atggtggttt
tcttggactc tttcccaacg caaacactct caacaactct 420 tccacctctg
aaaaccaaac caccactaag gctgcatcaa gcaacgttgt tgctgttgaa 480
tttgacacct atcttaatcc cgattatggt gatccaaact acatacacat cggaattgac
540 gtcaactcta ttagatccaa ggtaactgct aagtgggact ggcaaaatgg
gaaaatagcc 600 actgcacaca ttagctataa ctctgtctct aaaagactat
ctgttactag ttattatgct 660 gggagtaaac ctgcgactct ctcctatgat
attgagttac atacagtgct tcctgaatgg 720 gtcagagtag ggttatctgc
ttcaactgga caagataaag aaagaaatac cgttcactca 780 tggtctttca
cttcaagctt gtggaccaat gtggcgaaga aggagaatga aaacaagtat 840
attacaagag gcgttctgtg atgatatatg tgtatcaatg attttctatg ttataagcat
900 gtaatgtgcg atgagtcaat aatcacaagt acagtgtagt acttgtatgt
tgtttgtgta 960 agagtcagtt tgcttttaat aataacaagt gcagttagta cttgt
1005 23 286 PRT Artificial Sequence SpDLA. 23 Met Ala Ser Ser Asn
Leu Leu Thr Leu Ala Leu Phe Leu Val Leu Leu 1 5 10 15 Thr His Ala
Asn Ser Ala Ala Gln Ser Leu Ser Phe Ser Phe Thr Lys 20 25 30 Phe
Asp Pro Asn Gln Glu Asp Leu Ile Phe Gln Gly His Ala Thr Ser 35 40
45 Thr Asn Asn Val Leu Gln Val Thr Lys Leu Asp Ser Ala Gly Asn Pro
50 55 60 Val Ser Ser Ser Ala Gly Arg Val Leu Tyr Ser Ala Pro Leu
Arg Leu 65 70 75 80 Trp Glu Asp Ser Ala Val Leu Thr Ser Phe Asp Thr
Ile Ile Asn Phe 85 90 95 Glu Ile Ser Thr Pro Tyr Thr Ser Arg Ile
Ala Asp Gly Leu Ala Phe 100 105 110 Phe Ile Ala Pro Pro Asp Ser Val
Ile Ser Tyr His Gly Gly Phe Leu 115 120 125 Gly Leu Phe Pro Asn Ala
Asn Thr Leu Asn Asn Ser Ser Thr Ser Glu 130 135 140 Asn Gln Thr Thr
Thr Lys Ala Ala Ser Ser Asn Val Val Ala Val Glu 145 150 155 160 Phe
Asp Thr Tyr Leu Asn Pro Asp Tyr Gly Asp Pro Asn Tyr Ile His 165 170
175 Ile Gly Ile Asp Val Asn Ser Ile Arg Ser Lys Val Thr Ala Lys Trp
180 185 190 Asp Trp Gln Asn Gly Lys Ile Ala Thr Ala His Ile Ser Tyr
Asn Ser 195 200 205 Val Ser Lys Arg Leu Ser Val Thr Ser Tyr Tyr Ala
Gly Ser Lys Pro 210 215 220 Ala Thr Leu Ser Tyr Asp Ile Glu Leu His
Thr Val Leu Pro Glu Trp 225 230 235 240 Val Arg Val Gly Leu Ser Ala
Ser Thr Gly Gln Asp Lys Glu Arg Asn 245 250 255 Thr Val His Ser Trp
Ser Phe Thr Ser Ser Leu Trp Thr Asn Val Ala 260 265 270 Lys Lys Glu
Asn Glu Asn Lys Tyr Ile Thr Arg Gly Val Leu 275 280 285 24 8 PRT
Dolichos lablab MOD_RES (7) any amino acid. 24 Thr Asn Asn Val Leu
Gln Xaa Thr 1 5 25 24 DNA Artificial Sequence MutI primer. 25
ccataatcgg gatcaagata ggtg 24 26 24 DNA Artificial Sequence MutII
primer. 26 cacctatctt gatcccgatt atgg 24 27 24 DNA Artificial
Sequence M1 Forw primer. 27 aactcagccg cacagtcatt gtca 24 28 28 DNA
Artificial Sequence APEcoRI primer. 28 gaattcgacc acgcgtatcg
atgtcgac 28 29 21 DNA Artificial Sequence Sigforw primer. 29
gaattcatgg cttcctccaa c 21 30 28 DNA Artificial Sequence Sigrev
primer. 30 tgactgtgcg gctgagtttg cgtgggtg 28 31 14 PRT Artificial
Sequence Peptide corresponding to Pv-FRIL. 31 Ala Gln Ser Leu Ser
Phe Xaa Phe Thr Lys Phe Asp Leu Asp 1 5 10 32 14 PRT Artificial
Sequence Polypeptide of 18 kDa. 32 Ala Gln Ser Leu Ser Phe Xaa Phe
Thr Lys Asp Ala Leu Asp 1 5 10 33 14 PRT Artificial Sequence
Aminoterminal sequence. 33 Thr Asp Ser Arg Val Val Ala Val Glu Phe
Asp Xaa Phe Pro 1 5 10 34 13 PRT Artificial Sequence Aminoterminal
polypeptide. 34 Ala Gln Ser Leu Ser Phe Xaa Phe Lys Phe Asp Pro Asn
1 5 10 35 11 PRT Artificial Sequence Aminoterminal polypeptide. 35
Thr Asp Ser Arg Val Val Ala Val Glu Asp Phe 1 5 10 36 20 DNA
Artificial Sequence Degenerate oligonucleotide PVBeta1. 36
ttyacyaart tygayytnga 20 37 17 DNA Artificial Sequence Degenerate
oligonucleotide PVBeta2. 37 atyttycarg gwgaygc 17 38 20 DNA
Artificial Sequence Degenerate oligonucleotide PVAlfa1. 38
ttracrtcra twccratrtg 20 39 23 DNA Artificial
Sequence Degenerate oligonucleotide PVAlfa2. 39 tarttwggrt
cratrttrgc rtt 23 40 22 DNA Artificial Sequence PV3 PCR-Anchor
primer. 40 caatgtctta caactcacta ag 22 41 21 DNA Artificial
Sequence PV4 PCR-Anchor primer. 41 agtgtgggaa gagtgttatt c 21 42 21
DNA Artificial Sequence SPV2 Anchor primer. 42 accaaagctt
tggttttcag a 21 43 21 DNA Artificial Sequence SPV3 Anchor primer.
43 tctgaaaacg tttgagtaga g 21 44 32 DNA Artificial Sequence PVEcoRI
primer. 44 tacatgaatt cgctcagtca ttatctttta ac 32 45 21 DNA
Artificial Sequence Sigfor Bg1II primer. 45 agatctatgg cttcctccaa c
21 46 32 DNA Artificial Sequence Sigrev primer. 46 aaagataatg
actgagcggc tgagtttgcg tg 32 47 32 DNA Artificial Sequence SpM1forw
primer. 47 cacgcaaact cagccgctca gtcattatct tt 32 48 27 DNA
Artificial Sequence APXhoI primer. 48 ctcgaggacc acgcgtatcg atgtcga
27 49 105 PRT Artificial Sequence Beta-subunit of the mannose
lectin of Gowda et al. 49 Ala Gln Ser Leu Ser Phe Ser Phe Thr Lys
Phe Asp Pro Asn Gln 1 5 10 15 Glu Asp Leu Ile Phe Gln Gly Thr Ala
Thr Ser Lys Leu Asp Ser Ala 20 25 30 Gly Asn Pro Val Ser Ser Ser
Ala Gly Arg Val Leu Tyr Ser Ala Pro 35 40 45 Leu Arg Leu Trp Glu
Asp Ser Ala Val Leu Thr Ser Phe Asp Pro Thr 50 55 60 Ile Tyr Ile
Phe Thr Asn Tyr Thr Ser Arg Ile Ala Asp Gly Leu Ala 65 70 75 Phe
Ile Ala Pro Pro Asp Ser Val Ile Ser Tyr His Gly Gly Phe Leu 80 85
90 95 Gly Leu Phe Pro Asn Ala Ala Glu Ser Gly 100 105 50 123 PRT
Artificial Sequence Beta-subunit of D1-FRIL. 50 Ala Gln Ser Leu Ser
Phe Ser Phe Thr Lys Phe Asp Pro Asn Gln Glu 1 5 10 15 Asp Leu Ile
Phe Gln Gly His Ala Thr Ser Thr Asn Asn Val Leu Gln 20 25 30 Val
Thr Lys Leu Asp Ser Ala Gly Asn Pro Val Ser Ser Ser Ala Gly 35 40
45 Arg Val Leu Tyr Ser Ala Pro Leu Arg Leu Trp Glu Asp Ser Ala Val
50 55 60 Leu Thr Ser Phe Asp Thr Ile Ile Asn Phe Glu Ile Ser Thr
Pro Tyr 65 70 75 80 Thr Ser Arg Ile Ala Asp Gly Leu Ala Phe Phe Ile
Ala Pro Pro Asp 85 90 95 Ser Val Ile Ser Tyr His Gly Gly Phe Leu
Gly Leu Phe Pro Asn Ala 100 105 110 Asn Thr Leu Asn Asn Ser Ser Thr
Ser Glu Asn 115 120 51 132 PRT Artificial Sequence Alpha-subunit of
the mannose lectin of Gowda et al. 51 Ile Ala Glu Ser Asn Val Val
Ala Val Glu Phe Asp Thr Asp Tyr Leu 1 5 10 15 Asn Pro Asp Tyr Gly
Asp Pro Asn Tyr Ile His Ile Gly Ile Asp Val 20 25 30 Asn Ser Ile
Arg Ser Lys Val Thr Ala Ser Trp Asp Trp Gln Asn Gly 35 40 45 Lys
Ile Ala Thr Ala His Ile Ser Tyr Asn Ser Val Ser Lys Arg Leu 50 55
60 Ser Val Thr Thr Tyr Tyr Pro Gly Arg Gly Lys Pro Ala Thr Ser Tyr
65 70 75 80 Asp Ile Glu Leu His Thr Val Leu Pro Glu Trp Val Arg Val
Gly Leu 85 90 95 Ser Ala Ser Thr Gly Gln Asn Ile Glu Arg Asn Thr
Val His Ser Trp 100 105 110 Ser Phe Thr Ser Ser Leu Trp Thr Asn Val
Ala Lys Val Gly Val Ala 115 120 125 Ser Ile Ser Gly 130 52 141 PRT
Artificial Sequence Alpha-subunit of D1-FRIL. 52 Gln Thr Thr Thr
Lys Ala Ala Ser Ser Asn Val Val Ala Val Glu Phe 1 5 10 15 Asp Thr
Tyr Leu Asn Pro Asp Tyr Gly Asp Pro Asn Tyr Ile His Ile 20 25 30
Gly Ile Asp Val Asn Ser Ile Arg Ser Lys Val Thr Ala Lys Trp Asp 35
40 45 Trp Gln Asn Gly Lys Ile Ala Thr Ala His Ile Ser Tyr Asn Ser
Val 50 55 60 Ser Lys Arg Leu Ser Val Thr Ser Tyr Tyr Ala Gly Ser
Lys Pro Ala 65 70 75 80 Thr Leu Ser Tyr Asp Ile Glu Leu His Thr Val
Leu Pro Glu Trp Val 85 90 95 Arg Val Gly Leu Ser Ala Ser Thr Gly
Gln Asp Lys Glu Arg Asn Thr 100 105 110 Val His Ser Trp Ser Phe Thr
Ser Ser Leu Trp Thr Asn Val Ala Lys 115 120 125 Lys Glu Asn Glu Asn
Lys Tyr Ile Thr Arg Gly Val Leu 130 135 140 53 64 DNA Artificial
Sequence Recombinant expression vector. 53 ctggttccgc gtggatcccc
ggaattcatg cccggttcga ctcgagcggc cgcatcgtga 60 ctga 64 54 54 DNA
Artificial Sequence Recombinant expression vector. 54 ctggttccgc
gtggatcccc ggaattcatg ctcgagcggc cgcatcgtga ctga 54 55 237 PRT
Artificial Sequence Mannose lectin of Gowda et al. 55 Ala Gln Ser
Leu Ser Phe Ser Phe Thr Lys Phe Asp Pro Asn Gln Glu 1 5 10 15 Asp
Leu Ile Phe Gln Gly Thr Ala Thr Ser Lys Leu Asp Ser Ala Gly 20 25
30 Asn Pro Val Ser Ser Ser Ala Gly Arg Val Leu Tyr Ser Ala Pro Leu
35 40 45 Arg Leu Trp Glu Asp Ser Ala Val Leu Thr Ser Phe Asp Pro
Thr Ile 50 55 60 Tyr Ile Phe Thr Asn Tyr Thr Ser Arg Ile Ala Asp
Gly Leu Ala Phe 65 70 75 80 Ile Ala Pro Pro Asp Ser Val Ile Ser Tyr
His Gly Gly Phe Leu Gly 85 90 95 Leu Phe Pro Asn Ala Ala Glu Ser
Gly Ile Ala Glu Ser Asn Val Val 100 105 110 Ala Val Glu Phe Asp Thr
Asp Tyr Leu Asn Pro Asp Tyr Gly Asp Pro 115 120 125 Asn Tyr Ile His
Ile Gly Ile Asp Val Asn Ser Ile Arg Ser Lys Val 130 135 140 Thr Ala
Ser Trp Asp Trp Gln Asn Gly Lys Ile Ala Thr Ala His Ile 145 150 155
160 Ser Tyr Asn Ser Val Ser Lys Arg Leu Ser Val Thr Thr Tyr Tyr Pro
165 170 175 Gly Arg Gly Lys Pro Ala Thr Ser Tyr Asp Leu Glu Leu His
Thr Val 180 185 190 Leu Pro Glu Trp Val Arg Val Gly Leu Ser Ala Ser
Thr Gly Gln Asn 195 200 205 Ile Glu Arg Asn Thr Val His Ser Trp Ser
Phe Thr Ser Ser Leu Trp 210 215 220 Thr Asn Val Ala Lys Val Gly Val
Ala Ser Ile Ser Gly 225 230 235 56 279 PRT Artificial Sequence
PvFRIL. 56 Ala Gln Ser Leu Ser Phe Asn Phe Thr Lys Phe Asp Leu Asp
Gln Lys 1 5 10 15 Asp Leu Ile Phe Gln Gly Asp Ala Thr Ser Thr Asn
Asn Val Leu Gln 20 25 30 Leu Thr Lys Leu Asp Ser Gly Gly Asn Pro
Val Gly Ala Ser Val Gly 35 40 45 Arg Val Leu Phe Ser Ala Pro Phe
His Leu Trp Glu Asn Ser Met Ala 50 55 60 Val Ser Ser Phe Glu Thr
Asn Leu Thr Ile Gln Ile Ser Thr Pro His 65 70 75 80 Pro Tyr Tyr Ala
Ala Asp Gly Phe Ala Phe Phe Leu Ala Pro His Asp 85 90 95 Thr Val
Ile Pro Pro Asn Ser Trp Gly Lys Phe Leu Gly Leu Tyr Ser 100 105 110
Asn Val Phe Arg Asn Ser Pro Thr Ser Glu Asn Gln Ser Phe Gly Asp 115
120 125 Val Asn Thr Asp Ser Arg Val Val Ala Val Glu Phe Asp Thr Phe
Pro 130 135 140 Asn Ala Asn Ile Asp Pro Asn Tyr Arg His Ile Gly Ile
Asp Val Asn 145 150 155 160 Ser Ile Lys Ser Lys Glu Thr Ala Arg Trp
Glu Trp Gln Asn Gly Lys 165 170 175 Thr Ala Thr Ala Arg Ile Ser Tyr
Asn Ser Ala Ser Lys Lys Ser Thr 180 185 190 Val Thr Thr Phe Tyr Pro
Gly Met Glu Val Val Ala Leu Ser His Asp 195 200 205 Val Asp Leu His
Ala Glu Leu Pro Glu Trp Val Arg Val Gly Leu Ser 210 215 220 Ala Ser
Thr Gly Glu Glu Lys Gln Lys Asn Thr Ile Ile Ser Trp Ser 225 230 235
240 Phe Thr Ser Ser Leu Lys Asn Asn Glu Val Lys Glu Pro Lys Glu Asp
245 250 255 Met Tyr Ile Ala Asn Val Val Arg Ser Tyr Thr Trp Ile Asn
Asp Val 260 265 270 Leu Ser Tyr Ile Ser Asn Lys 275 57 254 PRT
Artificial Sequence PHA-E. 57 Ala Ser Gln Thr Ser Phe Ser Phe Gln
Arg Phe Asn Glu Thr Asn Leu 1 5 10 15 Ile Leu Gln Arg Asp Ala Thr
Val Ser Ser Lys Gly Gln Leu Arg Leu 20 25 30 Thr Asn Val Asn Asp
Asn Gly Glu Pro Thr Leu Ser Ser Leu Gly Arg 35 40 45 Ala Phe Tyr
Ser Ala Pro Ile Gln Ile Trp Asp Asn Thr Thr Gly Ala 50 55 60 Val
Ala Ala Ser Pro Thr Ser Phe Thr Phe Asn Ile Asp Val Pro Asn 65 70
75 80 Asn Ser Gly Pro Ala Asp Gly Leu Ala Phe Val Leu Leu Pro Val
Gly 85 90 95 Ser Gln Pro Lys Asp Lys Gly Gly Leu Leu Gly Leu Phe
Asn Asn Tyr 100 105 110 Lys Tyr Asp Ser Asn Ala His Thr Val Ala Val
Glu Phe Asp Thr Leu 115 120 125 Tyr Asn Val His Trp Asp Pro Lys Pro
Arg His Ile Gly Ile Asp Val 130 135 140 Asn Ser Ile Lys Ser Ile Lys
Thr Thr Thr Trp Asp Phe Val Lys Gly 145 150 155 160 Glu Asn Ala Glu
Val Leu Ile Thr Tyr Asp Ser Ser Thr Lys Leu Leu 165 170 175 Val Ala
Ser Leu Val Tyr Pro Ser Leu Lys Thr Ser Phe Ile Val Ser 180 185 190
Asp Thr Val Asp Leu Lys Ser Val Leu Pro Glu Trp Val Ile Val Gly 195
200 205 Phe Thr Ala Thr Thr Gly Ile Thr Lys Gly Asn Val Glu Thr Asn
Asp 210 215 220 Ile Leu Ser Trp Ser Phe Ala Ser Lys Leu Ser Asp Gly
Thr Thr Ser 225 230 235 240 Glu Ala Leu Asn Leu Ala Asn Phe Ala Leu
Asn Gln Ile Leu 245 250 58 20 PRT Artificial Sequence Synthetic
Peptide 58 Asp Ser Ser Thr Ser Glu Xaa Gln Thr Thr Thr Lys Ala Ala
Ser Ser 1 5 10 15 Asn Val Val Ala 20 59 13 PRT Artificial Sequence
Synthetic Peptide 59 Asp Ser Ser Thr Ser Glu Xaa Gln Thr Thr Thr
Lys Ala 1 5 10 60 20 PRT Artificial Sequence Synthetic Peptide 60
Thr Thr Thr Lys Ala Ala Ser Ser Asn Val Val Ala Val Glu Phe Lys 1 5
10 15 Thr Tyr Leu Asn 20 61 30 PRT Artificial Sequence Synthetic
Peptide 61 Ala Gln Ser Leu Ser Phe Phe Ser Phe Thr Lys Phe Asp Pro
Asn Gln 1 5 10 15 Glu Asp Leu Ile Phe Gln His Ala Thr Ser Thr Asn
Asn Val 20 25 30
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