Note: Descriptions are shown in the official language in which they were submitted.
wo gl/18615 2~ 7S PCr/US91/03306
HUMAN PERIPHERAL BLOOD CELLS
IN AN IMMUNOCONPROMISED HOST
INTRODUCTION
Technical Field
The field of this invention is the production
of human hematopoietic cells.
Background
The blood cell system comprising the diverse
cells emanating from a single stem cell is essential
to the survival and well being of mammals. The
cells serve to carry oxygen, cleanse the body of
debris, inhibit the proliferation of pathogens,
monitor for neoplastic cells, and provide a wide
variety of factors essential for their own and the
growth of other cells. Recently, chimeric mice were
developed, where human fetal tissue was introduced
into CB-17 s d/scid mice to provide the chimeric
SCID-hu mouse. The mice were shown to maintain for
extended periods of time, human fetal lymph node and
thymus and provide for peripheral blood cells, when
supplied with a source of hematopoietic stem cells -
from fetal liver. While in some instances, the
maintenance of human peripheral blood cells could be
extended for long periods of time, in all instances `~
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the human cells were only a very small proportion of
the total number of peripheral blood cells, `-
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frequently substantially fewer~than one percent.
Furthermore, the circulating cells in these animals
were shown to be T Cells.
In many instances, it would be desirable to `
have a relatively high proportion of the peripheral
blood cells as human cells and desirably have a
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WO91/186l5 PCT/US91/03306
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significant proportion of the total number of
hematopoietic cells being human cells. Further, it
would be desirable to have circulatory
myelomonocytic cells and red blood cells, besides
lymphoid cells.
For many applications, it would be of
substantial advantage to have a high absolute number
of human cells. These include the opportunity for
immunization to obtain a strong immune response from
the h~man cells, studies of various diseases and
their effect on the various hematopoietic cells and
studies of drugs against various diseases and their
effect on the hematopoietic cells. There is,
therefore, substantial interest in providing for
ways in which a non-human mammal may be provided
with human blood cells for testing, research, and as
a source of blood cells of the various lineages.
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Relevant Literature
EPA 0 322 240 describes the introduction of ~
human ~etal tissue in a C~-17 scid/scid mouse. See ~ ;
references cited therein.
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SUMMARY OF THE INVENTION
Xenogeneic bone marrow is introduced into an
endogenous bone marrow depleted genetically ;~;~
immunocompromised mammalian host, where the bone ~ ;
marrow may be present as bone tissue or dispersed j:
bone marrow, particularly from a fetal source. The
fetal bone tissue may be conveniently introduced
into the peritoneal cavity or the bone marrow may be
injectèd into the long bone of~the immunocompromised
host.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Non-human mammals, particularly small mammals
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WO91/18615 2~ ~. 5 PCT/US91/03306
under 25kg, are provided having human peripheral
blood cells present as at least two percent (2%) of
the total peripheral blood cells, preferably at
least five percent (5%) of the total peripheral
blood cells and more preferably at least about
fifteen percent (15%). The enhanced level of human
cells may be achieved by depletion of endogenous
bone marrow, particularly stem cells, conveniently
by irradiating the host with at least a sublethal
dosage of radiation, generally being at least about
290 rad and varying with the size and nature of the
host. The blood cells are then reconstituted with
human bone marrow, conveniently as human bone or
bone marrow cell suspensions, particularly from a ~ ;
fetal source. The human bone marrow source may be ~-
introduced before and after ablation to enhance the
survival rate of the host upon ablation, ; ,
particularly by irradiation.
Various immunocompromised hosts may be
employed, where the host being immunocompromised is
as a result of a natural mutation, breeding,
transformation of embryonic cells to provide a
transgenia mouse, or the like. Of particular
interest are hosts which lack a functional lymphoid ;
~5 lineage. The lack of the immune system may be as a
result of a lack of a particular organ, a genetic
defect, such as an incompetent recombinase or
regulatory gene regulating the expression of the
recombinase, transport of sIg and T cell receptors
to the surface, non-functional major
- -histocompatibility antigensj or the like.- Of
particular interest are CB-17 scid/scid mice and
- mice derived therefrom which may have further
enhanced immunodeficiency, e.g. lack of natural ~ ;
killer (NK) cells.
The host is irradiated or treated in any
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WO91/18615 2~ ~5 ~CT/US91/03306
convenient manner to substantially ablate the
endogenous bone marrow. Conveniently, X-ir~adiation
may be employed where the level of radiation will be
selected to provide at least substantially complete
ahlation of the hematopoietic cell system, while
maintaining a reasonable, preferably a highl .
proportion of viable hosts. With each type of host,
the particular level of irradiation may be selected
by screening for the level of remaining viable
hematopoietic cells for the efficiency of the
ablation. Other techniques for bone marrow ablation -~
which may be employed by themselves or in
combination include cytotoxic drugs, immunotoxins,
antibodies to lymphokines and growth factors, ;:.
antibodies to natural killer cells, etc. The ;
selection of cytotoxic drugs would be based on their
ability to clear quickly from the lymphatic and
blood circulatory systems, so as to.rapidly be
reduced to a non-cytotoxic level, prior to or .
shortly after the administration of the human bone :~.
marrow. :~
The human bone marrow will normally be fetal ;
bone marrow and may be employed as bone marrow
slices, fragments, chunks, or the like, coming from ..
various parts of the fetus, such as the femur,
tibia, humerus or rib. The longest dimensions of
human bone will generally be in the size range of
about 8mm to 2cm, where the bone may be fragmented :. :.
along.the long axis, usually in not more than about ,~
four parts. Usually, the total volume of fetal.bone .
which is;introduced into the-host will vary with the ~:
.- size of the host. For mice, the.volume will
: generally be in the range of about ~Omm3 to 1,200mm3.
Alternatively, human fetal bone marrow cell ~ .
suspensions may be injected into long bones, such as
the femur or tibia. Generally the volume of the - ,
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WO91/18615 ~ ~5 ` PCT/US91/03306
bone marrow suspension will be in the range of about
lOml to 50ml for a mouse.
The age of the host will generally he at least
neonatal and usually not more than about six months,
more usually not more than three months. The
particular age is not critical to this invention,
but younger animals are more convenient to handle
and are more economical.
The site of introduction of the fetal bone
portions may be interperitoneal, subcutaneous,
mammary fat pad, or other convenient site.
After introduction of the bone marrow, the host -
is grown in accordance with conventional ways, `
feeding the host as appropriate, either with or
without the presence of antibiotics in the feed.
Conveniently, about 0.2% to 1.2% of
sulfamethoxazole/trimethoprim (Septra) may be
provided in the feed. As appropriate, additional
introductions of the human fetal bone marrow may be
made, usually not more than about two additional
explants, generally at four to twenty week
intervals.
The hos~ may receive a variety of growth
factors and cytokines, either native to the host or
human. Interleukins, colony stimulating factors,
hormones, or the like may be added, such as IL-l,
IL-3, IL-6, and G-CSF. Generally, the amount of the
additive will vary, depending upon the particular
additive, the host, and the like. For example, in
the case of IL-3 0.5 - 5 ~g/day may be administered.
In the subject host, it is found that the bone
:- tissue shows variable cellularity from~lO to 80%.
In addition,-the human cells comprise a significant
proportion of the blood cells in circulation in the
peripheral blood. The cells may include members of
the lymphoid, myelomonocytic and erythroid lineages.
WO91~1861S ~ ~ S PCT/US91/~3306
The cells may be detected by employing a wide
varlety of antibodies which are commercially
available for the detection of human markers on
blood cells, such as CD-3, -4, -8, -l0, -13,
-15, -19, -20, -33, -41, -45, -59, ~LA, or the like.
Conveniently, a blood sample may be taken and
assayed employing a fluorescence activated cell ;~
sorter. Alternatively, the cells may be lysed and a
Western blot employed. Other techniques which may ~ -
find use for the quantitative determination of a ~ ~-
particular cell type include the polymerase chain -~
reaction, gel electrophoresis, HPLC, or the like.
In addition to the bone marrow, other human
organs may also be present, particularly thymus,
lymph node, skin, liver, pancreas, tonsil, appendix, -
epithelium, kidney, etc. One or more mesenteric or
peripheral lymph nodes may be introduced. The
various organs may be introduced conveniently in the
kidney capsule, mammary fat pad, particularly the
fourth mammary fat pad for a mouse, the popliteal ~`
fossa, or other convenient site where the organ may ~ ;~
be vascularized, lymphatic vessels connected and ; ;
maintained by the host for a reasonable period of
time, usually at least four weeks, preferably at ~-
least six weeks. Thus, the non-human mammal may `
provide ~or a significant portion of the human
hematopoietic system with the continued regeneration
of stem cells and generation of more than fifty -
percent (50%) of the different types of committed or
mature cells. In this manner cells of the
.~ myelomonocytic lineage, such as macrophages, mast
cells, neutrophils-~PMNs), eosinophils, basophils,
--monocytes, megakaryocytes, etc. or the lymphoid -
lineage, B lymphocytes, T lymphocytes, NX cells,
ADCC cells, LAK cells, TIL cells, etc. may be
- propagated, studied or used for various purposes. ~ ~
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WO91/18fil~ ~?~3~5 PCT/US91/03306
The subject host may find a wide variety of
uses by virtue of the significant presence of
circulating human blood cells in the non-human host - ;
The host may be employed for the production of human
monoclonal antibodies or immortalized T lymphocytes.
The host may be immunized in accordance with i
conventional ways with an appropriate immunoqen,
which may be injected intravascularly at an
appropriate site. Of particular interest is
iniection at a site which is drained by a human
lymph node introduced into the host. The immunized
host may receive one or more booster shots, followed
by removal of a lymphoid organ, e.g. lymph node, ~ `
followed by immortalization, conveniently by fusion ;
with an appropriate myeloid cell or transfection
with Epstein-Barr virus. Alternatively, the cells
may be cloned by any convenient means, at li~iting
dilution to provide for individual clones, and the
supernatants of the individual clones may be
screened for the binding affinity of the antibodies J ',~ "
present in the host. ~ ;
Alternatively, the host may be used for ;
studying T and B lymphocyte interactions, in ;
detarmining the manner of stimulation of T and B
lymphocyte~s and the mechanism for the immune
reaction. Individual lymphocytes may be cloned to
evaluate their role in disease protection against
disease, identify sIg or T cell receptor variable
regions, isolate the DNA encoding the variable
regions, or the like. Various immunogens may be
screened for their abilities as vaccinesi:in
producing;antlbodies which may-be effective in
neutralizing pathogens and-the cells or DNA provide
a source of the variable regions associated with the ;
response. -
Pathogens of interest are those particularly ~-
WO9l/18615 PCT/US91/03306_
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associated with humans, such as HIV, HTLV-I, and - ~
II, human papilloma virus, cytomegalovirus, Epstein- -'
Barr virus, hepatitis B virus, non-A, non-B - '
hepatitis, chlamydia, malaria (Falciparum) etc. '
Also, the subject system may be used in the study of - -'
'cancer in attempting to develop cytotoxic cells '
specific for a particular cancer type. - '
The following examples are offered by way of ''~'
illustration and not by way of limitation. ;~ .
EXPERIMENTAL
Bone pieces were transplanted into either -~ -
untreated mice (6-10 weeks old) or mice (6-10 weeks ~ -'
old) pre-treated with radiation from a cesium 137
source. The mice are treated with either whole body
irradiation or irradiation of the long bones. Nice
treated with whole body irradiation receive 200 to '-
400 rads on a single dose. Alternatively, the mice
are treated with 600 rads after shielding of the
thorax and abdomen with a lead shield. The mice are -
anesthetized with Nembutol prior to shielded
irradiation. The mice were C8-17.5 scid!scid mice.
In addition, some of the mice were treated with
exogenous human IL-3 in two l~g doses per day. The ~ `
fetal bone pieces are prepared from fetuses of 18-22
week gestation by first removing all of the soft
tissues and cartilagenous portions from the bone. '~
The bones are then cross sectioned at 2 -'5 mm ' l '~
intervals. The mice are prepared by in~ection ip of
Nembutol.-'Once-the mice are fully ànesthetized, a 1
-'' cm incision'is'`madë in the skin as'wèll as the
' underlying peritoneum. The bone fragments are then
- placed~into the peritoneal cavity randomly.
Approximately 25mm to l.5cm of a femur, tibia or
humerus is implanted into each mouse. The mice
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which are irradiated are irradiated within 24 hours
of the subsequent implantation of the fetal bone.
After two to six weeks, the percentage of human
cells in the peripheral blood is determined by
employing monoclonal antibodies specific for human
hematopoietic cell markers. In un-irradiated mice a
low level of human cells is observed in the
peripheral blood by FACScan. The positive cells
vary from about O - l.6 percent. Furthermore,
injection of IL-3 does not significantly affect the
percentage of positive cells. In contrast, the mice
irradiated before marrow transplantation showed
between l.5 and 30 percent human cells in the
peripheral blood. These cells appear to be of the
myelomonocytic lineage. They show no staining with ~;
T or B cell markers (CD 3, 4, 8, l9), although they
do stain for the myeloid cell marker (CD33). ;
Furthermore, the human cells have a high side
scatter profile indicating that they are of the
myelomonocytic lineage. In addition, two of the
~our mice which were irradiated before implantation
show low but significant levels of serum
immunoglobulin after six weeks.
The above results demonstrate that one can
obtain high levels of human cells in an
immunocompromised mouse at least partially ablated
of endogenous bone marrow, by implanting relatively
large amounts of human bone marrow, particularly
with the stromal bone tissue. The human bone marrow ~-
implanted after irradiation provides a mouse which
results in substantial numbers of cells of hùman
myelomonocytic~lineage and can provide for cells of
other lineages as well, such as lymphoid. By ~ -
appropriate combinations of tissue in conjunction
with the bone marrow one can provide inside the
immunocompromised mouse a hematopoietic system
WO91/1~61S PCT/US91/03306
2~ S
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mimicXing the human hematopoietic system. Thus, one
may study various processes, compounds, drugs, or
the like, and their effect on the human
hematopoietic system by cytologic or histologic
5 analysis, selection, staining, or the like.
All publications and patent applications
mentioned in this specification are indicative of
the level of skill of those skilled in the art to
which this invention pertains. All publications and
patent applications are herein incorporaited by
reference to the same extent as if each individual
publication or patent application was specifically
and individually indicated to be incorporated by
reference.
Although the foregoing invention has been ;~-
described in some detail by way of illustration and
example for purposes of clarity of understanding, it
will be obvious that certain changes and ~-
modi~ications may be practiced within the scope of
the appended claims.
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