U.S. patent application number 10/940691 was filed with the patent office on 2007-05-17 for methods and compositions using cellular asialodeterminants and glycoconjugates for targeting cells to tissues and organs.
This patent application is currently assigned to Department of Veterans Affairs, Rehabilitation R&D Service. Invention is credited to Catherine A. Phillips.
Application Number | 20070110734 10/940691 |
Document ID | / |
Family ID | 28041926 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070110734 |
Kind Code |
A9 |
Phillips; Catherine A. |
May 17, 2007 |
Methods and compositions using cellular asialodeterminants and
glycoconjugates for targeting cells to tissues and organs
Abstract
The present invention is directed to methods for delivering
cells to a target tissue in a mammal using glycoconjugate to
traffic the cell to a desired organ in the mammal. The methods
according to the present invention are especially applicable to
administering lymphoid cells such as natural killer (NK) cells
activated with interleukin-2 (IL-2), lymphokine-activated killer
(LAK) cells and/or tumor-infiltrating lymphocytes (TILs) and/or
cytotoxic lymphocytes (CTLs), or stem cells such as those derived
from the bone marrow or from umbilical cord tissue. The methods are
also useful for targeting a gene of interest to a tissue in a
mammal by introducing a cell containing the gene of interest and
administering a glycoconjugate to the mammal.
Inventors: |
Phillips; Catherine A.;
(Amarillo, TX) |
Correspondence
Address: |
BIOTECHNOLOGY LAW GROUP;C/O PORTFOLIOIP
PO BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Department of Veterans Affairs,
Rehabilitation R&D Service
Baltimore
MD
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20060171934 A1 |
August 3, 2006 |
|
|
Family ID: |
28041926 |
Appl. No.: |
10/940691 |
Filed: |
February 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US03/07934 |
Mar 14, 2003 |
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10940691 |
Feb 1, 2005 |
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60364498 |
Mar 15, 2002 |
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Current U.S.
Class: |
424/93.21 ;
424/93.7; 514/2.4; 514/4.4 |
Current CPC
Class: |
A61K 47/64 20170801;
A61K 35/17 20130101; A61K 35/28 20130101; A61P 35/00 20180101; A61P
37/02 20180101; A61K 38/1741 20130101; A61P 9/00 20180101; A61K
47/549 20170801; A61K 35/50 20130101; Y10S 514/885 20130101; A61K
35/34 20130101; A61P 37/00 20180101; A61K 35/51 20130101; A61P
11/00 20180101; A61K 35/28 20130101; A61K 2300/00 20130101; A61K
35/50 20130101; A61K 2300/00 20130101; A61K 35/34 20130101; A61K
2300/00 20130101; A61K 35/17 20130101; A61K 2300/00 20130101; A61K
35/51 20130101; A61K 2300/00 20130101; A61K 38/1741 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/093.21 ;
424/093.7; 514/008 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 48/00 20060101 A61K048/00; A61K 35/14 20060101
A61K035/14 |
Claims
1. A method for delivering a stem cell or lymphoid cell to a target
tissue in a mammal comprising the steps of: (a) administering a
glycoconjugate to a mammal; (b) administering the cell to the
mammal.
2. The method of claim 1, wherein the cell is a hematopoietic stem
cell.
3. The method of claim 2, wherein the stem cell is obtained from
the bone marrow, placenta, muscle, fat or an umbilical cord.
4. The method of claim 1 wherein the lymphoid cell is selected from
the group consisting of a natural killer (NK) cell, a
lymphokine-activated killer (LAK) cell, a tumor-infiltrating
lymphocyte (TIL), a cytotoxic lymphocyte (CTL), and mixtures
thereof.
5. The method of claim 1, wherein the glycoconjugate is represented
by the general formula P-(S)x-Gal, wherein P is a peptide residue
of a human serum glycoprotein and S is a sugar residue of a human
serum glycoprotein; x is an integer from 1 to 100 and Gal is
galactose residue.
6. The method of claim 1, wherein the glycoconjugate is selected
from the group consisting of an orosomucoid and an
asialoorosomucoid.
7. The method of claim 1, wherein the target tissue is a tissue of
an organ selected from the group consisting of the heart, the
liver, the lungs, and the kidneys.
8. The method of claim 1, wherein the glycoconjugate is
administered to the mammal prior to the cell.
9. The method of claim 1, wherein the glycoconjugate and the cell
are administered intravenously to the mammal.
10. A method for targeting a hematopoietic stem cell to the heart
of a mammal comprising the steps of: (a) administering an
asialo-orosomucoid to the mammal; and (b) administering the cell to
the mammal.
11. The method of claim 10, wherein the cell is administered after
the step of administering the asialo-orosomucoid.
12. The method of claim 10, wherein the asialo-orosomucoid is
administered via a vessel proximal to the heart.
13. The method of claim 12 wherein the asialo-orosomucoid is
administered via a jugular vein.
14. The method of claim 10 wherein the heart of a mammal has
suffered ischemic injury prior to administering the
asialo-orosomucoid.
15. A method for targeting a mesenchymal stem cell to the heart of
a mammal comprising the steps of: (a) administering an orosomucoid
to the mammal; and (b) administering the cell to the mammal.
16. The method of claim 15, wherein the orosomucoid is administered
via a vessel proximal to the heart.
17. The method of claim 16 wherein the orosomucoid is administered
via a jugular vein.
18. The method of claim 15 wherein the heart of a mammal has
suffered ischemic injury prior to administering the
orosomucoid.
19. The method of claim 15, wherein the cell is administered after
the step of administering the orosomucoid.
20. A method for targeting a hematopoietic stem cell to the liver
of a mammal comprising the steps of: (a) administering an
orosomucoid to the mammal; and (b) administering the cell to the
mammal.
21. The method of claim 20, wherein the cell is administered after
the step of administering the orosomucoid.
22. A method for targeting a mesenchymal stem cell to the liver of
a mammal comprising the steps of: (a) administering an
asialoorosomucoid to the mammal; arid (b) administering the cell to
the mammal.
23. The method of claim 22, wherein the cell is administered after
the step of administering the orosomucoid.
24. A method for targeting a gene of interest to a tissue in a
mammal, wherein said ene of interest comprises a transgene, said
method comprising the steps of: (1) introducing a cell comprising
the gene of interest to the mammal; and (2) administering a
glycoconjugate.
25. The method of claim 24, wherein the cell is a hematopoietic
stem cell.
26. The method of claim 24, wherein the cell is a lymphoid
cell.
27. The method of claim 26, wherein the stem cell is obtained from
the bone marrow, peripheral circulation or an umbilical cord.
28. The method of claim 24, wherein the glycoconjugate is selected
from the group consisting of an orosomucoid and an
asialoorosomucoid.
29. A method for treating a disease characterized by tissue damage
in a mammal comprising the steps of: (1) administering a stem cell
to the mammal; and (2) administering a glycoconjugate to the
mammal.
30. The method of claim 29, wherein the stem cell is obtained from
the hone marrow, peripheral circulation or an umbilical cord.
31. The method of claim 29, wherein the glycoconjugate is selected
from the group consisting of an orosomucoid and an
asialoorosomucoid.
32. The method of claim 29, wherein the disease is selected from
the group consisting of a heart disease, a lung disease, a liver
disease a neurological disease and a kidney disease.
33. The method of claim 29, wherein the disease is selected from
the group consisting of myocardial infarction, emphysema, cystic
fibrosis, hepatitis, stroke, nephritis and microalbuminuria.
34. A pharmaceutical composition comprising a lymphoid cell or a
stem cell and a glycoconjugate.
35. The pharmaceutical composition of claim 34, wherein the
glycoconjugate is selected from the group consisting of an
orosomucoid and an asialoorosomucoid.
36. The pharmaceutical composition of claim 34, wherein the cell is
a stem cell.
37. The pharmaceutical composition of claim 34, wherein the cell is
a lymphoid cell.
38. An article of manufacture, comprising packaging material and a
pharmaceutical composition contained within the packaging material,
wherein the pharmaceutical composition comprises a glycoconjugate
that is therapeutically effective for targeting a cell to a desired
organ, and wherein the packaging material comprises a label which
indicates that the pharmaceutical composition can be used for
targeting a cell to a desired organ.
39. The article of manufacture of claim 38, further comprising
additional reagents for making cell suspensions to be administered
to a mammal and printed instructions, for use in targeting
cells.
40. The article of manufacture of claim 39 further comprising a
quantity of stem cells suitable for targeting of such cells in a
mammal.
41. The article of manufacture of claim 38, wherein the
glycoconjugate is selected from the group consisting of an
orosomucoid and an asialoorosomucoid.
42. The article of manufacture of claim 40, wherein the cell is a
hematopoietic stem cell.
43. A method to improve the efficiency of an adoptive immunotherapy
using a lymphoid cell comprising modification of sialoglycoprotein
determinants on the lymphoid cell surface.
44. The method of claim 43 wherein the modification comprises
removal of sialic to generate new asialoglycoprotein
determininants.
45. The method of claim 44 wherein the modification comprises
removal of sialic acid by an enzyme.
46. The method of claim 45 wherein the modification comprises
removal of sialic acid by a neuraminidase.
47. The method of claim 43 wherein the modification comprises
addition of sialic acid by an enzyme.
48. The method of claim 43 wherein the adoptive immunotherapy is
for a liver metastasis or a primary liver tumor. regional
administration to the liver of activated lymphocytes.
49. The method of claim 6 wherein the glycoconjugate is
administered via a vessel proximal to the organ wherein the target
tissue is located.
50. The method of claim 6 wherein the organ is the liver and the
glycoconjugate is administered viavia the hepatic artery or portal
vein or peripheral vein
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/364,498, filed Mar. 15, 2002, the
entirety of which is incorporated by reference herein for all
purposes.
FIELD OF THE INVENTION
[0002] The present invention is in the field of clinical medicine
and therapy. The invention relates to methods and compositions for
targeting cells to an organ of interest, using sialo- or
asialodeterminants, particularly neoasialodeterminants, on cell
surfaces and/or on free glycoconjugates.
BACKGROUND OF THE INVENTION
[0003] Morell et al. determined that when a sialyl group of
ceruloplasmin is removed by neuraminidase, this plasma protein
rapidly disappears from serum. They disclosed that this phenomenon
is due to the uptake by the asialoglycoprotein (ASGP) receptor
present in liver cells (J. Biol. Chem., 243:155 (1968)).
Thereafter, it was reported that the ASGP receptor is present only
in liver cells (Adv. Enzymol., 41:99, (1974)). Such specific uptake
by liver cells has been identified from the fact that when
asialoceruloplasmin or asialoorosomucoid, which is experimentally
labeled with tritium, is injected into the living body, the isotope
is selectively detected only in liver cells. Scheinberg, I. H., et
al., Hepatic removal of circulating proteins, in Davidson C. S.,
ed. Problems in Liver Diseases, pp. 279-285, New York, Stratton
Company, (1979). In addition, it was also disclosed that this
receptor specifically recognizes and absorbs glycoproteins having
D-galactose or N-acetylgalactosamine as the terminal sugar group
(Ann. Rev. Biochem. 51:531, (1982)).
[0004] The cell membrane of liver cells comprises a cell structure
which combines with asialoglycoprotein terminated with galactose.
This cell structure was first named hepato-binding protein (HBP)
but is presently called asialoglycoprotein (ASGP) receptor.
Further, it has been observed that among various desialylated
glycoproteins, the desialylated alpha(1)-acid glycoprotein,
asialoorosomucoid, most rapidly disappears from the serum after
injection. Therefore, it has been determined that
asialo-alpha(1)-acid glycoprotein is both specifically and well
taken up by liver cells (J. Biol. Chem., 245:4397 (1970)). The ASGP
receptor is constituted with a single polypeptide having a
molecular weight of about 40,000 and can recognize a glycoprotein
having a galactose residue at the nonreductive terminal position of
the saccharide chain (i.e., asialoglycoprotein).
[0005] While the physiological functions of an ASGP receptor are
still uncertain, it is believed that an ASGP receptor participates
in the metabolism of glycoproteins. In fact, the increase of the
blood level of an ASGP is observed in case of hepatic diseases such
as chronic hepatitis, liver cirrhosis and hepatic cancer. Further,
the decrease of the quantity of an ASGP receptor is observed in an
experimental model of hepatic disorder induced by administration of
chemicals.
[0006] In view of these phenomena, it may be possible to diagnose
hepatic diseases through assessment of the quantity and quality of
an ASGP receptor determined by the use of an ASGP-like substance,
i.e., an ASGP receptor-directing compound. In fact,
asialoglycoconjugates have been covalently linked to other agents
as a means of targeting chemical (immunosuppressive drugs) and
biological agents (antibodies) to be taken up by the liver for
therapeutic and diagnostic purposes (see, e.g., U.S. Pat. Nos.
5,346,696, 5,679,323, and 5,089,604).
[0007] Adoptive cellular immunotherapy in general is a treatment
that employs biological reagents to effect an immune-mediated
response. Currently, most adoptive immunotherapies are
autolymphocyte therapies (ALT) directed to treatments using the
patient's own immune cells which have been processed to either
enhance the immune cell mediated response or to recognize specific
antigens or foreign substances in the body, including cancer cells.
The treatments are accomplished by removing the patient's
lymphocytes and exposing these cells in vitro to biologics and
drugs to activate the immune function of the cells. Once the
autologous cells are activated, these ex vivo activated cells are
reinfused into the patient to enhance the immune system to treat
various forms of cancer, infectious diseases, autoimmune diseases
or immune deficiency diseases.
[0008] Adoptive immunotherapies may utilize, for instance, natural
killer (NK) cells activated with interleukin-2 (IL-2),
lymphokine-activated killer (LAK) cells and/or tumor-infiltrating
lymphocytes (TILs) and/or cytotoxic lymphocytes (CTLs). LAK therapy
involves the in vitro generation of LAK cells by culturing
autologous peripheral blood leukocytes in high concentrations of
IL-2. The LAK cells are then reinfused into the cancer patient in a
treatment that may also involves infusion of IL-2. Rosenberg, et
al., "Cancer immunotherapy using interleukin-2 and interleukin-2
activated lymphocytes," Annual Review of Immunology 4:681-709
(1986). TIL therapy involves the generation of LAK cells from
mononuclear cells originally derived from the inflammatory
infiltrating cells present in and around solid tumors, obtained
from surgical resection specimens. Rosenberg, et al., "A new
approach to the adoptive immunotherapy of cancer with
tumor-infiltrating lymphocytes," Science 233:1318-1321 (1986). Many
further variations of adoptive immunotherapy have been developed in
recent years. See, e.g., U.S. Pat. No. 6,406,699, issued Jun. 18,
2002 to Wood, disclosing and claiming a composition and method of
cancer antigen immunotherapy, and methods in references disclosed
and cited therein.
[0009] In addition to cancer immunotherapies, adoptive
immunotherapy has applications for deficiency or dysfunction of T
cells associated with several diseases and conditions, including
recurrent infections by viruses such as herpesvirus (HSV, VZV,
CMV), hepatitis B virus, and papillomavirus. See, e.g., Spiegel, R.
J., "The alpha interferons: Clinical overview", Seminars in
Oncology 14:1 (1987). ALT is also being evaluated in the treatment
of patients infected with HIV. O. Martinez-Maza, "HIV-Induced
Immune Dysfunction and AIDS-Associated Neoplasms," in Biological
Approaches to Cancer Treatment: Biomodulation, M. Mitchell, Editor,
McGraw-Hill, Inc., Chapter 9, pages 181-204 (1993).
[0010] A stem cell is a special kind of cell that has a unique
capacity to renew itself and to give rise to specialized cell
types. Although most cells of the body such as heart cells or skin
cells, are committed to conduct a specific function, a stem cell is
uncommitted and remains uncommitted until it receives a signal to
develop into a specialized cell. In 1998, stem cells from early
human embryos were first isolated and grown in culture. It is
recognized that these stem cells are, indeed, capable of becoming
almost all of the specialized cells of the body. In recent years,
stem cells present in adults also have been shown to have the
potential to generate replacement cells for a broad array of
tissues and organs, such as the heart, the liver, the pancreas, and
the nervous system. Thus, this class of adult human stem cell holds
the promise of being able to repair or replace cells or tissues
that are damaged or destroyed by many devastating diseases and
disabilities. It is highly useful to effect such therapies by
targeting stem cells to particular organs of the body.
[0011] In the prior art, lymphocytes and stem cells generally have
been presented to the desired organs either by injection into the
tissue or by infusion into the local circulation. However,
localization of normal bone marrow stem cells and lymphocytes to
the liver has been demonstrated upon injection of such cells into
mice. Samlowski et al., Immunol. 88:309-322 (1984); Samlowski et
al., Proc. Natl. Acad. Sci. 82:2508-2512 (1985).
[0012] It is also known that a large proportion of cells infused
into mammals adhere to the lung endothelium, independent of cell
type or physiological homing properties. It has been observed that
stem cells accumulate in the lungs when they are administered.
Morrison et al., Nature Medicine 2:1281-1282 (1996); Martino et
al., Eur. J. Immunol. 23:1023-1028 (1993); Pereira et al., Proc.
Natl. Acad. Sci. USA 92:4857-4861 (1993); and Gao et al., Cells
Tissues Organs 169:12-20 (2001).
[0013] Orosomucoid, asialo-orosomucoid and
agalacto/asialo-orosomucoid have been shown to inhibit neutrophil
activation, superoxide anion generation, and platelet activation.
Costello et al., Clin Exp Immunol 55:465-472 (1984); and Costello
et al., Nature 281:677-678 (1979). These proteins also induced
transient immunosuppression and protected against TNF challenge.
Bennett, et al., Proc. Natl. Acad. Sci. USA 77:6109-6113 (1980) and
Libert, et al., J. Exp. Med. 180:1571-1575 (1994). Orosomucoid
demonstrated specific binding to pulmonary endothelial cells, which
appeared to be independent of carbohydrate recognition sites.
Schnitzer, et al., Am. J. Physiol 263:H48-H55 (1992). Moreover,
orosomucoid was shown to bind to skin capillary endothelial cells
in a dose dependent manner, thereby maintaining normal capillary
permeability in the face of inflammatory agonists that caused
leakage in control animals. Muchitsch, et al., Arch Int Pharmacodyn
331:313-321 (1996). Similarly, infused orosomucoid bound to kidney
capillaries and restored the permselectivity of glomerular
filtration. Muchitsch, et al., Nephron 81:194-199 (1999).
[0014] Entrapment of neuraminidase-treated lymphocytes in the liver
also has been reported, including autoimmune reactions against
liver cells by syngeneic neuraminidase-treated lymphocytes, in mice
intravenously injected with lymphocytes isolated from spleen or
thymus. Kolb-Bachofen, V., et al., Immunol. 123:2830-2834 (1979).
Studies on interactions between neuraminidase-treated rat
lymphocytes and liver cells in culture have demonstrated adhesion
between cells is due to stereo-specific interactions between a
mammalian hepatic membrane lectin (i.e., the ASGP receptor) and
galactosyl residues which are exposed on the lymphocyte surface
after removal of sialic acid residues. Kolb, H., et al., Adv. Exp.
Med. Biol. 114:219-222 (1979).
[0015] In view of the above, a need exists to develop methods for
delivery of lymphocytes and stem cells through the circulation to
specific organs. Such methods would provide a means to target
non-invasively solid organs such as the liver, heart, lungs and
kidneys. In addition, very diffuse tissues, such as the lung, which
are not amenable to dosage by injection could be targeted. Such
methods would be useful in adoptive immunotherapies and
regenerative stem cell therapies involving such organs as the
liver, heart, lungs and kidneys.
[0016] The present invention addresses these and other needs.
BRIEF SUMMARY OF THE INVENTION
[0017] The present invention features a method for delivering a
cell to a target tissue in a mammal comprising the steps of
administering a carbohydrate presenting molecule (e.g., a
glycoconjugate) to a mammal and then administering the cell to the
mammal.
[0018] As used herein, the term "administering" refers to any
method of inducing an increased concentration of the cell in the
circulation of the mammal, whether by infusion from an extraneous
source or by mobilizing the cell into the circulation from a depot
within the mammal, such as the marrow. Means for mobilizing stem
cells, for instance, using GM-CSF and GCSF, for example, are well
known in the art. See, e.g., Simmons et al., The mobilization of
primitive hemopoietic progenitors into the peripheral blood. Stem
Cells, 12 Suppl 1:187-201 (1994).
[0019] The methods according to the present invention are
especially applicable to stem cells, such as those derived from the
bone marrow, peripheral blood, umbilical cord or from mesenchymal
stem cells expanded in culture. The stem cells within the scope of
the invention include any cell capable of differentiating into a
desired target tissue. Such cells include pluripotent stem cells,
embryonic stem cells, multipotent adult stem cells, and progenitor
or precursor cells.
[0020] The methods according to the present invention also are
especially applicable to immune system cells, such as natural
killer (NK) cells activated with interleukin-2 (IL-2),
lymphokine-activated killer (LAK) cells and/or activated
lymphocytes including but not limited to tumor-infiltrating
lymphocytes (TILs).
[0021] The methods of the present invention allow cells such as
normal stem or immune cells to be targeted to such target tissues
as the heart, the liver, the kidneys and the lungs, among others.
In some embodiments wherein the cell is targeted to the heart, the
methods feature administering an orosomucoid (O) or administering
an asialoorosomucoid (ASO), and administering the cell to the
mammal. In embodiments wherein the cell is targeted to the lungs,
the methods feature administering the cell to the mammal in a
saline or a serum albumin-saline solution or cell culture media
without protein/albumin. In embodiments wherein the cell is
targeted to the liver, the methods feature administering an
orosomucoid or an asialoorosomucoid and administering the cell to
the mammal. In some embodiments, the orosomucoid is administered
concurrently or prior to administering the cell to the mammal. The
methods according to the present invention are also useful for
either inhibiting or enhancing sequestration of a stem cell or
immune cell in the liver of a mammal even in the absence of
targeting the cell to a target organ.
[0022] The glycoconjugates of the present invention may be
generally represented by the general formula P-(S)x-Gal wherein P
is a peptide residue of a human serum glycoprotein and S is a sugar
residue of a human serum glycoprotein; x is an integer from 1 to
100 and Gal is galactose residue. The glycoconjugates may be
partially or completely asialylated. Especially useful
glycoconjugates include fetuins, asialofetuins, orosomucoids and
asialoorosomucoids.
[0023] The glycoconjugates may be administered to the mammal in any
time frame relative to administering the cell. They may be
administered before, after or simultaneously with the
administration of the cell. In a typical embodiment, the
glycoconjugates are administered prior to the cell. The
glycoconjugates and the cell may be administered via any suitable
route. In preferred embodiments, they are administered
parenterally, and more preferably, intravenously to the mammal.
[0024] The methods according to the present invention are also
useful for targeting a gene of interest to a tissue in a mammal by
introducing a cell naturally containing, or a cell transformed
with, the gene of interest to the mammal. Such methods are useful
for treating a disease characterized by a deficiency in a gene
product in a mammal by administering a cell comprising a functional
gene encoding the gene product into the mammal and administering a
glycoconjugate to the mammal. According to these methods, a cell
containing an exogenous functional gene of interest may be
administered and localized to a particular organ in the body where
it can function to produce a deficient gene product.
[0025] Also, the methods according to the present invention are
useful for treating a disease characterized by tissue damage in a
mammal by administering a cell and administering a glycoconjugate
to the mammal. Because stem cells have the potential to generate
replacement cells for a broad array of tissues and organs, such as
the heart, the pancreas, and the nervous system, stem cells may be
targeted to particular organs in the body to repair or replace
cells or tissues that are damaged or destroyed by many devastating
diseases and disabilities. In some embodiments, the disease may be
a heart disease, a lung disease, a kidney disease or a liver
disease, for example, myocardial infarction, emphysema, cystic
fibrosis, microalbuminuria, nephritis, stroke or hepatitis.
[0026] The methods according to the present invention are also
useful for treating a disease characterized by tissue damage in a
mammal by administering a glycoconjugate to the mammal and
administering chemicals or biopharmaceuticals that mobilize stem
cells into the circulation. The concentration of circulating
mobilized stem cells may be limited because certain organs may
sequester stem cells, thereby limiting delivery of an effective
dose to the damaged organ. By inhibiting sequestration, the
glycoconjugates of the invention increase the cell dose at the
organ; thereby increasing the potential to generate replacement
cells. The methods including agents to mobilize stem cells also can
be used for a broad array of tissues and organs, such as the heart,
the pancreas, and the nervous system. Mobilized stem cells may be
targeted to particular organs in the body to repair or replace
cells or tissues that are damaged or destroyed by many devastating
diseases and disabilities. In some embodiments wherein stem cells
are mobilized, the disease may be a heart disease, a lung disease,
a kidney disease, a neurological disease or a liver disease such
as, for example, myocardial infarction, emphysema, cystic fibrosis,
microalbuminuria, nephritis, stroke or hepatitis.
[0027] In other embodiments, the present invention provides
pharmaceutical compositions comprising a cell and a glycoconjugate,
e.g.; glycoprotein. Glycoproteins useful in the present invention
include, for example, fetuins, orosomucoids (O) and
asialoorosomucoids (ASO). In other aspects, the present invention
features an article of manufacture, comprising packaging material
and a pharmaceutical agent contained within the packaging material,
wherein the pharmaceutical agent comprises a glycoconjugate of the
invention that is therapeutically effective for targeting a cell to
a desired organ according to the present invention, and wherein the
packaging material comprises a label which indicates that the
pharmaceutical agent can be used for targeting a cell to a desired
organ according to the present invention. In some embodiments, the
article of manufacture further comprises additional reagents, such
as solutions for making cell suspensions to be administered, and/or
printed instructions, for use in targeting cells according to the
invention. Such articles include, for instance, kits for treating
tissue damage or for delivering a functional gene or gene product
to a tissue in a mammal comprising a cell and a glycoprotein.
Glycoproteins useful in the articles of manufacture of the
invention include fetuins, asialofetuins, orosomucoids and
asialoorosomucoids.
[0028] In still other embodiments, the present invention provides
methods for derivatization of stem cell or lymphoid cell
populations to generate an asialodeterminant-bearing cell
preparation to facilitate hepatic entrapment. In particular, the
invention provides derivatized, activated stem cells or lymphocytes
that have asialadeterminants on their surface that have been
generated by enzymatic or chemical means so that these cells, when
administered parenterally, circulate, are bound, and sequestered or
entrapped by the liver via the ASGP receptor. Methods for treating
whole viable cells with a sialidase, such as neuraminidase, are
known in the art. See, e.g., Neubauer, R. H. et al., Identification
of normal and transformed lymphocyte subsets of nonhuman primates
with monoclonal antibodies to human lymphocytes. J. Immunol.
130:1323-1329 (1983); Kolb-Bachofen, V., et al., 1979, supra; and
Kolb, H., et al., 1979, supra.
[0029] For instance, the invention provides a process of
derivatization of stem cells to generate neoasialadourminants on
the surface of such calls, for the purpose of directing these tells
to the liver to repair or regenerate liver functions and
structures, or for delivery of normal genes or genetically
engineered cells for the purpose of curing or ameliorating disease
states. Operative elements of this aspect of the invention are the
ability to direct the localization of the transfused stem cells
bearing artificially created neoasialoglycodeterminants, their
ability to create a micro-chimera of the recipient, and the
mechanism by which of the neodeterminants are specifically
sequestered by the liver. Thus, assimilation of these
neoasialoglycodeterminant-bearing cells would result in
microchimerism (a mixture of derivatized stem cells and the
original host cells that were genetically abnormal. The modified
stem cells would express at least the minimum required amount of
the abnormal or missing protein or regulatory function needed for
reversing or ameliorating the disease phenotype. The modified stem
cells could be derived from patient's blood, bone marrow or other
stem cell-producing organ such as adipose tissue, or may be derived
from another individual or a stem cell line.
[0030] The invention also provides methods for manipulation of in
vivo cell trafficking patterns of lymphoid cytolytic cells by
specifically facilitating the hepatic sequestration of parenterally
administered activated lymphocytes by derivitizing the cell surface
with enzymes that generate "neoasialodeterminants". Thus, activated
lymphoid populations have cell surface asialodeterminants capable
of binding to the ASGP receptor, and this binding can be further
enhanced by enzymatic treatments that generate new cell surface
asialodeterminants.
[0031] These methods may be used, for instance, to improve the
efficiency of adoptive immunotherapy for liver metastasis or
primary liver tumors by facilitating hepatic entrapment (via the
ASGP receptor) of parenterally administered cells that have been
derivatized to generate cell surface asialodeterminants. Metastasis
of various cancers to the liver are difficult to treat. For
example, elimination of breast cancer metastases to liver must be
achieved prior to harvesting of bone marrow or autologous stem cell
products for transplantation. Chemotherapy alone can take months to
achieve a complete response. It often leads to bone marrow
suppression making the harvesting of stem cells from individuals
extremely difficult. In cases where the tumor is chemotherapy
resistant, very few therapeutic options remain. Adoptive
immunotherapies do exist in which the patients own cells can be
"educated" in culture to recognize the tumor and then these cells
are transferred to the patient intravenously to find and destroy
the tumor.
[0032] If the tumor burden is primarily in the liver, it may be
useful to have several cycles of therapy directed specifically
toward the elimination of tumor from the liver. This can be
accomplished according to the present invention, by treating the
activated lymphoid populations (that have been grown or "educated"
in culture) with enzymes or other treatments that include (but are
not limited to sialidases, such as neuraminidases, that modify the
cell surface glycosylation sites to expose asialodeterminants. The
number of these determinants are thereby dramatically increased and
hence the modified cells bind to hepatic ASGP receptors more
readily and dissociate less frequently than cells bearing the
"normal" number of asialodeterminants.
[0033] Assimilation of neoasialoglycodeterminant-bearing lymphoid
cells results in microchimerism, as described for stem cells above,
a mixture of infused lymphocytes that have or have not been
genetically engineered with the original host lymphoid cells that
exist at the site. The infused lymphoid cells would augment or
enhance the immune response by dividing and entering the
circulation and recruiting other cell populations to participate in
the local immune response. The hepatic environment is ideally
suited for the development of immune responses due to the presence
of cells of the innate immune system as well as professional
antigen presenting cells in the sinusoids and vasculature,
particularly the portal system.
[0034] For example, several studies have shown that responses to
metastatic cutaneous melanoma, for instance, can be achieved using
regional administration to the liver of activated lymphocytes. See,
e.g., Keilhoiz, U. et al., Regional adoptive imunotherapy with
interleukin-2 and lymphokine-activated killer (LAK) cells for liver
metastasis, Eur. J. Cancer 3OA:103-105 (1994). The invention
methods of using activated lymphocytes that have been modified to
generate additional cell surface asialodeterminants permits the
delivery of activated lymphocytes to the liver regionally, via the
hepatic artery or portal vein or peripheral vein, without the use
of invasive procedures to deliver these cells to a primary hepatic
or non-hepatic tumor, or to metastatic lesions distant from a
primary hepatic or non-hepatic cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 provides a schematic of liver entrapment of bone
marrow stem cells and lymphocytes in the liver.
Asialoglycodeterminants on the surface of cells react with ASGP
receptors on the surface of hepatocytes resulting in the
localization of the bone marrow stem cells and the lymphocytes in
the liver. Glycoconjugates including asialoglycoconjugates block
such interactions between asialoglycodeterminants on the surface of
cells with ASGP receptors on the surface of hepatocytes.
[0036] FIG. 2 shows the carbohydrate structure on two exemplary
glycoproteins of the invention.
[0037] FIG. 3 shows the relative binding affinities of different
carbohydrates for the ASGP receptor.
[0038] FIG. 4 shows the relative binding affinities of different
carbohydrates for the ASGP receptor.
[0039] FIG. 5 shows a schematic of an experimental system for
studying adherence of NK/LAK cells to monolayer cultures of (1) a
human hepatoma cell line (HEP2G), an asialoglyprotein repetor
positive (ASGPR+) cell line that exhibits minimal deviation from
cells in human liver tissue, and (2) a human renal cell carcinoma
cell line (CAKI-2), an ASGPR- cell line.
[0040] FIG. 6 shows a plot of results of testing effects of
asialofetuin (ASF) and fetuin (F) on adherence of NK/LAK cells (as
represented by NK/LAK activity) to HEP2G monolayers at 4.degree. C.
LAK activity (50%) adheres to human minimal deviation hepatoma,
HEPG2, at 4.degree. C., in the presence of the control fully
sialylated protein, fetuin, F (LAK-NA/F). LAK activity does not
adhere to the HEPG2 monolayer in the presence of asialofetuin, ASF
(LAK-NA/ASF). LAK cells were incubated in the presence of ASF
alone, i.e., no adherence to monolayer not performed (LAK/ASF).
CONTROL cells did not kill RAJI targets (CONTROL). **This is
representative of three different donors.
[0041] FIG. 7 shows results of testing effects of asialofetuin
(ASF) and fetuin (F) on adherence of NK/LAK cells to HEP2G and
CAKI-2 cells at 23.degree. C. The effector cell populations were:
an untreated 3-day old LAK preparation (LAK) and the same
population treated with Vibrio cholera neuraminidase (LAK/NS). LAK
adherence to HEPG2 (ASGPR+) and CAKI-2 (ASGPR-) in the presence of
either ASF or F assayed on K562. (LAK=3 day LAK; K5=K562 targets;
FET=fetuin; ASF=asialofetujn; LAK/CAKI/ASF/K5=LAK, adherence on
CAKI pretreated with ASF, assayed on K562).
[0042] FIG. 8 shows additional results of testing effects of
asialofetuin (ASF) and fetuin (F) on adherence of NK/LAK cells to
HEP2G and CAKI-2 cells at 23.degree. C., as in FIG. 7. Adherence of
neuraminidase-treated LAK to HEPG2 (ASGPR+) and CAKI-2 (ASGPR-) in
the presence of ASF or F. (LAK/NS=neuraminidase-treated LAK;
EXAMPLE-LAK/CAK/NS/ASF/K5=neuraminidase-treated LAK, adherence on
CAKI pretreated with ASF, assayed on K562).
[0043] FIG. 9 shows additional results of testing effects of
asialofetuin (ASF) and fetuin (F) on adherence of NK/LAK cells to
HEP2G and CAKI-2 cells at 23.degree. C., as in FIG. 7. LAK
adherence to HEPG2 (ASGPR+) and CAK.I-2 (ASGPR-) in the presence of
either ASF or F assayed on RAJI. (LAK=3 day LAK; R=RAJI targets;
FET=fetuin; ASF=asialofetujn; LAK/CAKI/ASF/R=LAK, adherence on CAKI
pretreated with ASF, assayed on RAJI).
[0044] FIG. 10 shows additional results of testing effects of
asialofetuin (ASF) and fetuin (F) on adherence of NK/LAK cells to
HEP2G and CAKI-2 cells at 23.degree. C., as in FIG. 7. Adherence of
neuraminidase-treated LAK to HEPG2 (ASGPR+) and CAKI-2(ASGPR-) in
the presence of ASF or F.(LAK/NS=neuraminidase-treated LAK;
EXAMPLE-LAK/CAKI/NS/ASF/R=neuraminidase-treated LAK, adherence on
CAKI pretreated with ASF, assayed on RAJI).
[0045] FIG. 11 shows results of testing effects of cell surface
modifications on adherence of NK/LAK cells to HEP2G cell
monolayers. Cytotoxic activity of 5-day LAK, Neuramindase-treated
LAK, and Control (no IL-2), assayed on K562.
[0046] FIG. 12 shows additional results of testing effects of cell
surface modifications on adherence of NK/LAK cells to HEP2G cell
monolayers, as in FIG. 11. Cytotoxic activity of 5-day LAK,
Neuramindase-treated LAK, and Control (no IL-2), assayed on RAJI
cells.
[0047] FIG. 13 shows additional results of testing effects of cell
surface modifications on adherence of NK/LAK cells to HEP2G cell
monolayers, as in FIG. 11. Adherence of LAK activity to HEPG2
(ASGPR+) after cell surface modification with neuraminidase, 2,3-
or 2,6-sialyltransferases. (EXAMPLE:
LAK/HEP/NASE/K5=neuraminidase-treated LAK adhered to HEPG2 assayed
on K562).
[0048] FIG. 14 shows additional results of testing effects of cell
surface modifications on adherence of NK/LAK cells to HEP2G and
CAKI-2 cell monolayers, as in FIG. 11. Adherence of LAK activity to
CAKI-2 (ASGPR-) after cell surface modification with neuraminidase,
2,3- or 2,6-sialyltransferases. (Dotted lines in both FIGS. 13 and
14 are the same controls.)
[0049] FIG. 15 shows additional results of testing effects of cell
surface modifications on adherence of NK/LAK cells to cell HEP2G
and CAKI-2 monolayers, as in FIG. 11. Adherence of LAK activity to
HEPG2 (ASGPR+) after cell surface modification with neuraminidase,
2,3- or 2,6-sialyltransferases, assayed on RAJI cells.
[0050] FIG. 16 shows additional results of testing effects of cell
surface modifications on adherence of NK/LAK cells to cell HEP2G
and CAKI-2 monolayers, as in FIG. 11. Adherence of LAK activity to
CAKI-2 (ASGPR-) after cell surface modification with neuraminidase,
2,3- or 2,6-sialyltransferases, assayed on RAJI cells.
DETAILED DESCRIPTION OF THE INVENTION
[0051] A. Introduction
[0052] The present invention is directed to methods for delivering
a cell to a target tissue in a mammal. The methods comprise the
steps of administering, either simultaneously or sequentially, a
carbohydrate presenting molecule (e.g., glycoconjugate) and a cell
to the mammal. In the methods of the present invention,
glycoconjugates, especially asialoglycoconjugates, including asialo
plasma proteins such as asialoorosomucoid (asialo alpha-(1)-acid
glycoprotein), are thought to transiently bind the hepatic ASGP
receptor and thereby competitively inhibit attachment of cells
bearing asialodeterminants from these receptors. Without wishing to
be bound by theory, hyposialylated and desialylated
proteins/glycoconjugates (also called asialoglycoconjugates) and
cells which bear similar determinants are bound or "trapped" in the
liver as a consequence of binding to the hepatic ASGP receptors
(see, FIG. 1). Occupation of the receptor by the
asialoglycoconjugate inhibits sequestration of the cells bearing
similar determinants of interest in the liver.
[0053] In addition, the present disclosure shows that
glycoconjugates of the invention prevent infused cells from
concentrating in the alveolar vasculature. This finding suggests
that lung sequestration of the cells may be related to expression
of inflammatory receptors on endothelial cells, analogous to the
reperfusion syndrome (see, e.g. Kilgore et al. Cardiovasc Res
28:437-444 (1994) and Eror et al., Clin Immunol 90:266-275 (1999).
This is supported by reports that orosomucoid, ASO and
agalacto/asialo-orosomucoid inhibit neutrophil activation
superoxide anion generation, as well as platelet activation as
noted above.
[0054] The present invention further demonstrates that the
glycoproteins may be used to traffic or target cells to particular
organs of the body by altering the particular glycoconjugate
administered. The present methods are useful to improve the
efficacy of bone marrow and stem cell transplants, tissue repair,
gene therapy or adoptive immunotherapies.
[0055] In embodiments wherein the cell is targeted to the lungs,
the methods feature administering the cell to the mammal in a
saline or serum albumin-saline solution. In some embodiments
wherein the hematopoietic stem cell is targeted to the heart, the
methods feature administering an asialoorosomucoid, and
administering the cell to the mammal. In other embodiments wherein
the mesenchymal stem cell is targeted to the heart, the methods
feature administering an orosomucoid, and administering the cell to
the mammal. In embodiments wherein the hematopoietic stem cell is
targeted to the liver, the methods feature administering an
orosomucoid and administering the cell to the mammal. In other
embodiments wherein the mesenchymal stem cell is targeted to the
liver, the methods feature administering an asialoorosomucoid and
administering the cell to the to the mammal. In some embodiments,
the orosomucoid or asialoorosomucoid is administered in at least
two infusions prior to administering the cell to the mammal. The
methods according to the present invention are also useful for
inhibiting sequestration of a cell in the liver of a mammal even in
the absence of targeting the cell to a target organ.
[0056] Asialoglycoconjugates, for example, asialofetuin and other
asialo plasma proteins, are able to bind to the hepatic parenchyma
and Kupffer cell ASGP receptors. Blocking these receptors from
binding and trapping cells bearing asialodeterminants, such as bone
marrow cells, facilitates and increases the interval of their
systemic circulation. In the case of bone marrow stem cells, the
administration of these compounds prevents the loss and destruction
of bone marrow stem cells and increases the efficiency of
engraftment. Bone marrow cells have cell surface asialodeterminants
capable of binding to the ASGP receptor, and this binding can be
inhibited by the application of ASGPs.
[0057] The present invention takes advantage of the observation
that when human peripheral hematopoietic stem (CD34+) cells or
mesenchymal stem cells are infused into the jugular vein of
immunodeficient mice, they localize predominantly in the lungs.
When the cells are preceded by an infusion of asialoorosomucoid,
the hematopoietic stem cells predominantly localize in the heart,
whereas the mesenchymal stem cells localize in the liver.
Alternately, when the cells are preceded by an infusion of
orosomucoid (O), the hematopoietic stem cells localize in the
liver, whereas the mesenchymal stem cells predominantly localize in
the heart.
[0058] These protein infusions cause a more quantitative
localization into the specific organs than occurs without them.
Furthermore, hematopoietic stem cells that localize in the heart
due to the influence of asialoorosomucoid leave the vascular space
and are observed among the cardiac muscle cells by one hour after
infusion. Moreover, once in the tissue, these cells lose their CD34
antigen, indicating that they are in the process of differentiating
into cardiomyocytes or heart components (e.g., blood vessels).
Additionally, at one hour CD34+ cells have been demonstrated to
move from the vasculature into lung tissue. In an
orosomucoid-treated mouse, clusters of stem cells are found in the
liver parenchyma and are also demonstrated to lose their CD34
antigen, again suggesting differentiation into hepatocytes/hepatic
or liver parenchyma.
[0059] The present invention demonstrates the ability to direct
high concentrations of stem cells to a specific organ in an
atraumatic manner. This enhances the probability and the rate at
which stem cells migrate into a target tissue and differentiate
into the desired cell type. The present invention utilizes the
observation that delivery of orosomucoid or ASO to the vessel
proximal to the heart causes transfused stem cells to accumulate in
the heart. Without wishing to be bound by theory, the effect may be
caused by the glycoprotein infusion sensitizing the endothelium
directly downstream from the infusion site, which causes the
endothelial cells to bind stem cells and enhance their migration
across the endothelium into the tissue.
[0060] The present findings with glycoconjugates indicate that the
majority of a stem cell transfusion can be concentrated in the
target organ, thereby providing the means to deliver an effective
regimen of cell doses. This offers an opportunity to non-invasively
target solid organs such as the heart, thereby competing with
invasive direct injection. Perhaps more importantly,
glycoconjugates provide the means to target very diffuse tissues,
such as the liver and the kidney, which are not amenable to dosage
by injection.
[0061] It is recognized that hematopoietic stem cells (HSC)
recovered from the marrow, peripheral blood or umbilical cord blood
and mesenchymal stem cells (MSC) recovered as marrow stromal cells,
stromal cells from liposuction fat, or proliferated from stationary
stromal progenitor cells in cord blood-depleted expelled placentas
appear to be almost interchangeable in their differentiation
ability, and act as multipotent stem cells.
[0062] Such cells have been shown to differentiate into functional
cells when localized in specific organs and tissues: hepatocytes
and cholangiocytes in the liver, cardiac muscle cells and arterial
smooth muscle cells and endothelial cells in the heart, pneumocytes
I & II in alveoli and bronchial epithelium in the lungs,
chondrocytes for cartilage restoration, and intestinal mucosal
cells, small, medium and large blood vessels in the heart, etc.
[0063] B. Stem Cells
[0064] Stem cells may hold the key to replacing cells lost in many
devastating diseases such as Parkinson's disease, diabetes, acute
and chronic heart disease, end-stage kidney disease, liver failure,
and cancer. For many diseases, there are no effective treatments
but the goal is to find a way to replace what natural processes
have taken away.
[0065] To date, published scientific papers indicate that adult
stem cells have been identified in brain, bone marrow, peripheral
blood, blood vessels, skeletal muscle, epithelia of the skin and
digestive system, cornea, dental pulp of the tooth, retina, liver,
and pancreas. Thus, adult stem cells have been found in tissues
that develop from all three embryonic germ layers.
[0066] By way of definition, the following terms are understood in
the art:
[0067] A "stem cell" is a cell from the embryo, fetus, or adult
that has, under certain conditions, the ability to reproduce itself
for long periods or, in the case of adult stem cells, throughout
the life of the organism. It also can give rise to specialized
cells that make up the tissues and organs of the body.
[0068] A "pluripotent stem cell" has the ability to give rise to
types of cells that develop from the three germ layers (mesoderm,
endoderm, and ectoderm) from which all the cells of the body arise.
The only known sources of human pluripotent stem cells are those
isolated and cultured from early human embryos and from fetal
tissue that was destined to be part of the gonads.
[0069] An "embryonic stem cell" is derived from a group of cells
called the inner cell mass, which is part of the early (4- to
5-day) embryo called the blastocyst. Once removed from the
blastocyst the cells of the inner cell mass can be cultured into
embryonic stem cells. These embryonic stem cells are not themselves
embryos.
[0070] An "adult stem cell" is an undifferentiated (unspecialized)
cell that occurs in a differentiated (specialized) tissue, renews
itself, and becomes specialized to yield all of the specialized
cell types of the tissue in which it is placed when transferred to
the appropriate tissue. Adult stem cells are capable of making
identical copies of themselves for the lifetime of the organism.
This property is referred to as "self-renewal." Adult stem cells
usually divide to generate progenitor or precursor cells, which
then differentiate or develop into "mature" cell types that have
characteristic shapes and specialized functions, e.g., muscle cell
contraction or nerve cell signaling. Sources of adult stem cells
include bone marrow, blood, the cornea and the retina of the eye,
brain, skeletal muscle, dental pulp, liver, skin, the lining of the
gastrointestinal tract and pancreas.
[0071] Stem cells from the bone marrow are the most-studied type of
adult stem cells. Currently, they are used clinically to restore
various blood and immune components to the bone marrow via
transplantation. There are currently identified two major types of
stem cells found in bone marrow: hematopoietic stem cells (HSC, or
CD34+ cells) which are typically considered to form blood and
immune cells, and stromal (mesenchymal) stem cells (MSC) that are
typically considered to form bone, cartilage, muscle and fat.
However, both types of marrow-derived stem cells recently have
demonstrated extensive plasticity and multipotency in their ability
to form the same tissues.
[0072] The marrow, located in the medullary cavity of bones, is the
sole site of hematopoiesis in adult humans. It produces about six
billion cells per kilogram of body weight per day.
Hematopoietically active (red) marrow regresses after birth until
late adolescence after which time it is focused in the lower skull
vertebrae, shoulder and pelvic girdles, ribs, and sternum. Fat
cells replace hematopoietic cells in the bones of the hands, feet,
legs and arms (yellow marrow). Fat comes to occupy about fifty
percent of the space of red marrow in the adult and further fatty
metamorphosis continues slowly with aging. In very old individuals,
a gelatinous transformation of fat to a mucoid material may occur
(white marrow). Yellow marrow can revert to hematopoietically
active marrow if prolonged demand is present such as with hemolytic
anemia. Thus hematopoiesis can be expanded by increasing the volume
of red marrow and decreasing the development (transit) time from
progenitor to mature cell.
[0073] The marrow stromal consists principally of a network of
sinuses that originate at the endosteum from cortical capillaries
and terminate in collecting vessels that enter the systemic venous
circulation. The trilaminar sinus wall is composed of endothelial
cells; an underdeveloped, thin basement membrane, and adventitial
reticular cells that are fibroblasts capable of transforming into
adipocytes. The endothelium and reticular cells are sources of
hematopoietic cytokines. Hematopoiesis takes place in the
intersinus spaces and is controlled by a complex array of
stimulatory and inhibitory cytokines, cell-to-cell contacts and the
effects of extracellular matrix components on proximate cells. In
this unique environment, lymphohematopoietic stem cells
differentiate into all of the blood cell types. Mature cells are
produced and released to maintain steady state blood cell levels.
The system may meet increased demands for additional cells as a
result of blood loss, hemolysis, inflammation, immune cytopenias,
and other causes. The engraftment efficiency of bone marrow stem
cells could be improved by preventing entrapment by the liver via
the hepatic ASGP receptor.
[0074] A "progenitor or precursor" cell occurs in fetal or adult
tissues and is partially specialized; it divides and gives rise to
differentiated cells. Researchers often distinguish
precursor/progenitor cells from adult stem cells in that when a
stem cell divides, one of the two new cells is often a stem cell
capable of replicating itself again. In contrast when a
progenitor/precursor cell divides, it can form more
progenitor/precursor cells or it can form two specialized cells.
Progenitor/precursor cells can replace cells that are damaged or
dead, thus maintaining the integrity and functions of a tissue such
as liver or brain.
[0075] Means for isolating and culturing stem cells useful in the
present invention are well known. Umbilical cord blood is an
abundant source of hematopoietic stem cells. The stem cells
obtained from umbilical cord blood and those obtained from bone
marrow or peripheral blood appear to be very similar for
transplantation use. Placenta is an excellent readily available
source for mesenchymal stem cells. Moreover, mesenchymal stem cells
have been shown to be derivable from adipose tissue and bone marrow
stromal cells and speculated to be present in other tissues. While
there are dramatic qualitative and quantitative differences in the
organs from which adult stem cells can be derived, the initial
differences between the cells may be relatively superficial and
balanced by the similar range of plasticity they exhibit. For
instance, adult stem cells both hematopoietic and mesenchymal,
under the appropriate conditions can become cardiac muscle cells.
Delineation of full range of potential for adult stem cells has
just begun. Stem cells may be isolated for transduction and
differentiation using known methods. For example, in mice, bone
marrow cells are isolated by sacrificing the mouse and cutting the
leg bones with a pair of scissors. Stem cells may also be isolated
from bone marrow cells by palming the bone marrow cells with
antibodies which bind unwanted cells, such as CD4+ and CD8+ (T
cells), CD45+ (panB cells), GR-1 (granulocytes), and lad
(differentiated antigen presenting cells). For an example of this
protocol see, Izaba et al., I. Exp. Med. 176-1693 1702 (1992).
[0076] In humans, CD34+ hematopoietic stem cells can be obtained
from a variety of sources including cord blood, bone marrow, and
mobilized peripheral blood. Purification of CD34+ cells can be
accomplished by antibody affinity procedures. An affinity column
isolation procedure for isolating CD34+ cells is described by Ho et
al., Stem Cells 13 (suppl. 31: 100-105 (1995). See also, Brenner,
Journal of Hematotherapy 2: 7-17 (1993). Methods for isolating,
purifying and culturally expanding mesenchymal stem cells are
known. Specific antigens for MSC are also known (see, U.S. Pat.
Nos. 5,486,359 and 5,837,539).
[0077] C. Carbohydrate Presenting Molecule
[0078] The carbohydrate presenting molecules useful in the present
invention can be any molecule capable of presenting the appropriate
carbohydrate structure that leads to enhancing or inhibiting the
targeting of the cell of interest to a target tissue. The targeting
function can be carried out using a carbohydrate molecule such as
an oligosaccharide, polysaccharide, or the carbohydrate structure
can be bound to larger molecule or carrier, referred to here as a
glycoconjugate. Typically, the carbohydrate molecule will be linked
to either a naturally occurring carrier (e.g., as part of a
glycoprotein or glycolipid) or the carrier may be synthetic (e.g.,
an engineered polypeptide sequence). One of skill will recognize
that a number of carriers can be used to present the appropriate
structure. Examples of appropriate carrier molecules include
polypeptides, lipids, and the like. Preparation and use of targeted
compounds using asialo carbohydrate moieties is described in the
art (see, e.g., U.S. Pat. Nos. 5,679,323, 5,089,604, 5,032,678 and
5,284,646). One of skill will recognize that such compounds can
also be used as carbohydrate presenting molecules useful in the
present invention.
[0079] In cases in which the glycoconjugate is a glycoprotein it
may be generally represented by the general formula P-(S)x-Gal
wherein P is a peptide residue of a human serum glycoprotein and S
is a sugar residue of a human serum glycoprotein; x is an integer
from I to 100 and Gal is a galactose residue. Especially useful
glycoconjugates include fetuins and asialofetuins (see, FIG. 2),
orosomucoids and asialoorosomucoids and galactose-bonded
polylysine, galactose-bonded polyglucosamine, and the like.
[0080] The methods of the present invention allow cells such as
stem cells to be targeted to such target tissues as the heart, the
liver, the kidneys and the lungs, among others. Parenteral
administration of a glycoconjugate, such as asialoorosomucoid, may
be used to block the hepatic ASGP receptor and allow the cells
bearing surface asialodeterminants (for example, peanut agglutinin
(PNA)+ cells) to continue to circulate and migrate to the marrow
space. Asialoorosomucoid is one of the glycoproteins which has been
shown to bind to the hepatic ASGP receptor and has been extensively
used to characterize this receptor.
[0081] Different compounds have different binding affinities for
the ASGP receptor, depending upon the carbohydrate presented (see,
FIGS. 3 and 4). Thus, one of skill can modulate cell targeting by
using compounds that present different carbohydrate structures.
[0082] Intravenous administration of a glycoconjugate, especially
an ASGP such as asialoorosomucoid, may be used to block the hepatic
ASGP receptor and allow the cells bearing surface
asialodeterminants to continue to circulate and migrate to the
marrow space or to the organ of interest. The glycoconjugates may
be administered to the mammal in any time frame relative to the
cells, but in some embodiments, the glycoconjugates are
administered prior to administering the cell. The
asialoglycoconjugates and the cell may be administered in any
suitable route, but in some embodiments, they are administered
intravenously to the mammal, and in other embodiments, they are
administered parenterally. In embodiments wherein the cell is
targeted to the lungs, the methods feature administering the cell
to the mammal in a saline or serum albumin-saline solution. In some
embodiments wherein the hematopoietic stem cell is targeted to the
heart, the methods feature administering an asialoorosomucoid, and
administering the cell to the mammal. In other embodiments wherein
the mesenchymal stem cell is targeted to the heart, the methods
feature administering an orosomucoid, and administering the cell to
the mammal. In embodiments wherein the hematopoietic stem cell is
targeted to the liver, the methods feature administering an
orosomucoid and administering the cell to the mammal. In other
embodiments wherein the mesenchymal stem cell is targeted to the
liver, the methods feature administering an asialoorosomucoid and
administering the cell to the to the mammal. In some embodiments,
the orosomucoid or asialoorosomucoid is administered in at least
two infusions, prior to and after administering the cell to the
mammal. The methods according to the present invention are also
useful for inhibiting sequestration of a cell in the liver of a
mammal even in the absence of targeting the cell to a target
organ.
[0083] The alpha-(1)-acid glycoprotein (orosomucoid or AAG) is a
normal constituent of human plasma (650.+-.215 .mu.g ml-1) which
increases in concentration as much as fivefold in association with
acute inflammation and cancer, and thus is recognized as an acute
phase protein. Orosomucoid consists of a single polypeptide chain,
has a molecular weight of 44,100, and contains approximately 45%
carbohydrate including 12% sialic acid. It is the most negatively
charged of the plasma proteins. Certain of the biological
properties of orosomucoid are related to its sialic acid content.
Thus, clearance and immunogenicity of orosomucoid are markedly
increased on desialylation. The biological functions of orosomucoid
are largely unknown. Orosomucoid has the ability to inhibit certain
lymphocyte reactivities including blastogenesis in response to
concanavalin A, phytohaemagglutinin and allogeneic cells, and these
inhibitory effects are enhanced in association with desialylation.
It has been reported that unphysiologically large (5-15 mg/ml)
amounts of orosomucoid inhibit the platelet aggregation induced by
ADP and adrenaline, and there is evidence that a sialic
acid-deficient species of orosomucoid appears elevated in several
chronic disease states.
[0084] D. Gene Therapy
[0085] The present invention is also directed to using living cells
to deliver therapeutic genes into the body. In some embodiments,
the therapeutic gene is a transgene. For example, the delivery
cells--a type of stem cell, a lymphocyte, or a fibroblast are
removed from the body, and a therapeutic transgene is introduced
into them via vehicles well known to those skilled in the art such
as those used in direct-gene-transfer methods. While still in the
laboratory, the genetically modified cells are tested and then
allowed to grow and multiply and, finally, are infused back into
the patient. Alternatively, allogeneic cells that bear normal,
endogenous genes can reverse a deficiency in a particular target
tissue. Use of cells bearing either transgenes or normal,
endogenous genes is referred to herein as gene therapy.
[0086] Gene therapy using genetically modified cells offers several
unique advantages over direct gene transfer into the body. First
the addition of the therapeutic transgene to the delivery cells
takes place outside the patient, which allows the clinician an
important measure of control because they can select and work only
with those cells that both contain the transgene and produce the
therapeutic agent in sufficient quantity.
[0087] Of the stem cell-based gene therapy trials that have had a
therapeutic goal, approximately one-third have focused on cancers
(e.g., ovarian, brain, breast myeloma, leukemia, and lymphoma),
one-third on human immunodeficiency virus disease (HIV-1), and
one-third on so-called single-gene diseases (e.g., Gaucher's
disease, severe combined immune deficiency (SCID), Fanconi anemia,
Fabry disease, and leukocyte adherence deficiency).
[0088] In view of the foregoing, the methods according to the
present invention are useful for targeting a gene of interest
(either a transgene or an endogenous gene) to a tissue in a mammal
by introducing a cell comprising the gene of interest and
administering a glycoconjugate to the mammal. Such methods are
useful for treating a disease characterized by a deficiency in a
gene product in a mammal by administering a cell comprising a
functional gene encoding the gene product into the mammal and
administering a glycoconjugate to the mammal. Stem cells may be
used as a vehicle for delivering genes to specific tissues in the
body. Stem cell-based therapies are a major area of investigation
in cancer research.
[0089] The current invention provides localizing of transfused
cells such as stem cells to provide a functional gene to a patient
suffering from a disease caused by a lack of that gene. In many
instances of genetically based diseases, a low level production of
that gene product will effectively ameliorate or cure the disease.
By providing the gene that is deficient through transfusion of stem
cells from a normal donor into the patient, the stem cells may be
directed to localize in an organ or tissue of choice, causing a
microchimerization of that patient in that organ or tissue, from
which organ or tissue that gene product can be delivered to the
patient. Therefore, the present invention provides the ability to
direct the localization of the transfused cells such as allogeneic
stem cells that have a stable, normal gene. Such transfused cells
then create a stable micro-chimera of the recipient.
[0090] Those of skill in the art are aware of the genetic
deficiencies causative of a large array of genetically based
diseases. Exemplary genes and diseases that can be treated include
CTFR protein in cystic fibrosis and proteins associated with
coagulopathy in the liver. For example, treatment of Hemophilia A
can be accomplished using gene therapy in such embodiment, a
transfusion of such cells as umbilical cord blood hematopoietic
stem cells may be administered to deliver an intact normal Factor
VIII gene. Alternatively, transformed cells can comprise a normal,
wild-type Factor VIII gene. Such cells carrying a functional Factor
VIII gene may be directed to localize in the liver, preferably by
orosomucoid or asialoorosomucoid perfusion prior to the infusion of
the stem cells. The cells transform into hepatocytes and begin
secreting Factor VIII into the blood.
[0091] Other embodiments of gene therapy according to the present
invention include treating Hemophilia B (Factor IX deficiency), and
antithrombin III, Protein C, and Protein S deficiencies. While
these diseases all involve the blood coagulation system, gene
therapy may include treating different tissues, such as muscular
dystrophy, cystic fibrosis, and the like.
[0092] E. Introducing Transgenes into Stem Cells
[0093] Means for introducing transgenes into cells are well known.
A variety of methods for delivering and expressing a nucleic acid
within a mammalian cell are known to those of ordinary skill in the
art. Such methods include, for example viral vectors,
liposome-based gene delivery (WO 93/24640; Mannino Gould-Fogerite,
BioTechniques 6(7):682-691 (1988); U.S. Pat. No. 5,279,833; WO
91/06309; Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414
(1987); and Budker et al., Nature Biotechnology, 14(6):760-764
(1996)). Other methods known to the skilled artisan include
electroporation (U.S. Pat. Nos. 5,545,130, 4,970,154, 5,098,843,
and 5,128,257), direct gene transfer, cell fusion, precipitation
methods, particle bombardment, and receptor-mediated uptake (U.S.
Pat. Nos. 5,547,932, 5,525,503, 5,547,932, and 5,460,831). See
also, U.S. Pat. No. 5,399,346.
[0094] Widely used retroviral vectors include those based upon
murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),
Simian Immuno deficiency virus (Sly), human immunodeficiency virus
(HIV), and combinations thereof. See, e.g., Buchscher et al., J.
Virol. 66(5):2731-2739 (1992); Johann et al., J. Virol.
66(5):1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59 (1990);
Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J.
Virol. 65:2220-2224 (1991); PCT/US94/05700, and Rosenburg &
Fauci, in Fundamental Immunology, Third Edition (Paul ed.,
1993)).
[0095] AAV-based vectors are also used to transduce cells with
target nucleic acids, e.g., in the in vitro production of nucleic
acids and polypeptides, and in vivo and ex vivo gene therapy
procedures. See, West et al., Virology 160:38-47 (1987); U.S. Pat.
No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801
(1994); Muzyczka, J. Clin. Invst. 94:1351 (1994) and Samulski
(supra) for an overview of AAV vectors. Construction of recombinant
AAV vectors are described in a number of publications, including
Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell.
Biol. (1 l):3251-3260 (1985); Tratschin et al., Mol. Cell. Biol.
4:2072-2081 (1984); Hermonat & Muzyczka, Proc. Nail. Acad. Sci.
USA 81:6466-6470 (1984), and Samulski et al., J. Virol.
63:03822-3828 (1989).
[0096] Retroviral vectors are typically used for cells useful in
the present invention. Such vectors may comprise, for example, an
HIV-2 packageable nucleic acid packaged in an HIV-2 particle,
typically using a packaging cell line. Cell transduction vectors
have considerable commercial utility as a method of introducing
genes into target cells. In particular, gene therapy procedures, in
which the cell transduction vectors of the invention are used to
transduce target cells with a therapeutic nucleic acid in an in
vivo or ex vivo procedure may be used. Gene therapy provides a
method for combating chronic diseases caused by a gene deficiency,
infectious diseases such as HIV, as well as non-infectious diseases
such as cancer.
[0097] Stem cells such as CD34+ stem cells may be used in ex vivo
procedures for cell transduction and gene therapy. The present
invention utilizes the feature that stem cells differentiate into
other cell types in vitro, or can be introduced into a mammal (such
as the donor of the cells) where they will engraft in the bone
marrow unless targeted to another organ for differentiation. Hence,
the present invention extends to directing stem cells to particular
organs to regenerate tissue such as to the heart to regenerate
cardiac muscle cells, to the lung to regenerate alveoli, and to the
kidneys to regenerate tissue and to directing cells such as CD34+
stem cells to an organ to ameliorate a genetic abnormality by
providing efficacious amounts of a deficient gene product. Methods
for differentiating CD34+ cells in vitro into clinically important
immune cell types using cytokines such a GM-CSF, IFN-.gamma. and
TNF-.alpha. are known (See, Inaba et al., J. Exp. Med.
176,1693-1702 (1992), and Szabolcs et al., 154:5851-5861 (1995)).
Yu et al., PNAS 92: 699-703 (1995) describe a method of transducing
CD34+ cells from human fetal cord blood using retroviral
vectors.
[0098] F. Pharmaceutical Compositions
[0099] In other embodiments, the present invention provides
pharmaceutical compositions comprising a cell and a glycoconjugate
of the invention. Exemplary glycoproteins include orosomucoids and
asialoorosomucoids. In other aspects, the present invention
features kits for treating tissue damage or for delivering a
functional gene or gene product to a tissue in a mammal comprising
a cell and a glycoprotein. Stem cells generally have been presented
to the desired organs either by injection into the tissue, by
infusion into the local circulation, or by mobilization of
autologous stem cells from the marrow accompanied by prior removal
of stem cell-entrapping organs before mobilization when feasible,
i.e., splenectomy.
[0100] Glycoconjugates may be administered prior to, concomitantly
with, or after infusing the stem cells. In some embodiments, an
intravenous fluid bag may be used to administer the glycoconjugate
in a saline or dextrose solution with and without protein, or
serum-free media, including, but not restricted to, RPMI 1640 or
AIM-V. In such embodiments, the glycoconjugate may be mixed with
the cells in the same bag or in a "piggyback". The glycoconjugate
may also be continued after administration of the cells to permit
longer systemic circulation times or increased specific organ
accumulation. This procedure may be repeated as often as needed for
delivering a therapeutic dose of the cells to the target organ. The
preparation may be used with little concern for toxicity given data
from animal studies demonstrating no side effects at doses of 3-7
mg of glycoconjugate per ml of blood volume (up to 12
mg/mouse).
[0101] Administration of cells transduced ex vivo can be by any of
the routes normally used for introducing a cell or molecule into
ultimate contact with blood or tissue cells. The transduced cells
may be administered in any suitable manner, preferably with
pharmaceutically acceptable carriers. Suitable methods of
administering such cells in the context of the present invention to
a patient are available, and, although more than one route can be
used to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than
another route.
[0102] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention.
[0103] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. Parenteral
administration is one useful method of administration. The
formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampules and vials, and in some embodiments, can
be stored in a freeze-dried (lyophilized) condition requiring only
the addition of the sterile liquid carrier, for example, water, for
injections, immediately prior to use. These formulations may be
administered with factors that mobilize the desired class of adult
stem cells into the circulation.
[0104] Extemporaneous injection solutions and suspensions can be
prepared from sterile powders, granules, and tablets of the kind
previously described. Cells transduced by the vector as described
above in the context of ex vivo therapy can also be administered
parenterally as described above, except that lyophilization is not
generally appropriate, since cells are destroyed by
lyophilization.
[0105] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time. The dose will be
determined by the efficacy of the particular cells employed and the
condition of the patient, as well as the body weight of the patient
to be treated. The size of the dose also will be determined by the
existence, nature, and extent of any adverse side effects that
accompany the administration of a cell type in a particular
patient. In determining the effective amount of cells to be
administered in the treatment or prophyLAKis of diseases, the
physician should evaluate circulating plasma levels, and, in the
case of replacement therapy, the production of the gene product of
interest.
[0106] Transduced cells are prepared for reinfusion according to
established methods. See, Abrahamsen et al, J. Clin. Apheresis
6-48-53 (1991; Carter et al., J. Clin. Apheresis 4:113-117 (1988);
Aebersold et al., J. Immunol. Methods 112:1-7 (1988); Muul et al.,
J. Immunol. Methods 101: 171-1 81 (1987) and Carter et al.,
Transfusion 27:362-365 (1987). After a period of about 2-4 weeks in
culture, the cells may number between 1.times.10.sup.6 and
1.times.10.sup.10. In this regard, the growth characteristics of
cells vary from patient to patient and from cell type to cell type.
About 72 hours prior to reinfusion of the transduced cells, an
aliquot is taken for analysis of phenotype, and percentage of cells
expressing the therapeutic agent.
[0107] For administration, cells of the present invention can be
administered at a rate determined by the LD-50 of the cell type,
and the side effects of the cell type at various concentrations, as
applied to the mass and overall health of the patient.
Administration can be accomplished via single or divided doses.
Adult stem cells may also be mobilized using exogenously
administered factors that stimulate their production and egress
from tissues or spaces, that may include, but are not restricted
to, bone marrow or adipose tissues. The exemplary glycoconjugates
may be administered concurrently, prior to and/or following stem
cells mobilization, or at a time when the amount of cells in the
peripheral circulation is optimal for the desired therapeutic
endpoint.
[0108] G. Adoptive Immunotherapy
[0109] It has already been shown that intravenously administered
LAK cells are sequestered predominantly in the lungs and the liver
(Lotze, M. T., et al., The in vivo distribution of autologous human
and murine lymphoid cells grown in T cell growth factor
(TCGF)-Implication for the adoptive immunotherapy of tumors. J.
Immunol. 125: 1487-1493 (1980) (possibly due to interaction of
asialodeterminants on the LAK cell surface with ASGP receptors on
the surfaces of endothelial cells, Kupffer cells, and hepatocytes
(Kolb et al., 1979; supra; Kolb-Bachofen, et al., 1984, supra) and
that metastatic tumors in these organs can be dramatically reduced
by LAK therapy (Rosenberg, 1987, supra). Intravenously injected
murine bone marrow cells, neuraminidase-treated lymphocytes,
natural killer (NK), and LAK cells all share this same trafficking
pattern (Samlowski et al., 1984, supra; Samlowski et al., 1985,
supra; Kolb et al., 1979, supra; Kolb-Bachofen, et al., 1984,
supra; Rolstad. B. et al., Natural killer cell activity in the rat
V. The circulation patterns and tissue localization of peripheral
blood large granular lymphocytes (LOL), J. Immunol. 136: 2800-2808
(1986); Rosenberg, 1987, supra). Moreover, all these cells have
asialodeterminants on their surface. Kradin, R. L., et al.,
Tumor-derived interleukin-2-dependent lymphocytes in adoptive
immunotherapy of lung cancer. Cancer Immunol. Immunother 24:
76-85.(1987), have gamma-camera imaged patients that have received
.sup.111In-labeled tumor-derived interleukin-2-dependent
lymphocytes (derived from metastatic adenocarcinoma of the lung).
These T "killer" cells derived from human tumors also migrate to
the liver and lungs. Based on this preferential localization in the
liver of human LAK cells and their ability to kill hepatocellular
carcinoma, Hsieh et al., Lysis of primary hepatic tumors by
lymphokine activated killer cells. Gut 28:117-124 (1987), have
conducted Phase I trials for the treatment of this tumor; It has
also been suggested that for treatment of liver tumors that
selective administration of LAK cells with IL-2 via a catheter
inserted into the hepatic artery should be an effective means of
administration which may decrease the magnitude and scope of side
effects (Fagan. E. A., et al., Immunotherapy for Cancer: the use of
lymphokine-activated killer (LAK) cells. Gut 28:113-116 (987)).
[0110] Human, rat, and mouse liver have been shown to specifically
sequester, trap, or "clear" desialylated serum glycoproteins (eg.,
asialotransferrin) by recognition of galactose residues made
terminal by the removal of sialic acid (i.e., asialogycoproteins)
and aged desialylated erythrocytes via high affinity hepatic
asialoglycoprorein receptors (Ashwell, G. The role of cell-surface
carbohydrates in binding phenomena. In: Mammalian Cell Membranes,
Vol. 4, Butterworth, London, OX (1977); Asbwell, G., et al.,
Carbohydrate-specific receptors of the liver. Ann. Rev. Riochem.
51: 531-554 (1982); Harford et al., The hepatic receptor for
asialoglycoproteins. In: The Glycoconjugates; Vol. 4; Part B (ed.
M. I. Horowitz) Academic Press, New York, 1982). The human, rat and
rabbit ASGP receptors display virtually identical characteristics:
specificity, cation requirements, pH optimum, affinity, subunit
size, and temperature dependent internalization of the receptor and
degradation of the asialoligand (Dunn et al., Low temperature
selectivity initibits fusion between pinocytotic vesicles and
lysosomes during heterophagy of .sup.125I-asialofetuin by the
perfused rat liver. J. Biol. Chem. 225 5971-5978 (1980); Schwartz
et al., Characterization of the ASGP receptor in a continuous
hepatoma line. J. Biol. Chem 256:88 8-8881 (1981); Ashwell et al.,
1982, supra; Mueller et al., Receptor-mediated endocytosis of
asialoglycoproteins by rat hepatocytes: receptor-positive and
receptor negative endosomes. J. Cell. Biol. 102:932-947 (1986). At
5-20.degree. C. the ligand receptor complex is not internalized;
whereas, at 37.degree. C. this complex was internalized and
degraded and the receptor recycled to the cell surface undamaged
Mueller et al., 1986, supra). On average a cell containing 225,000
receptors can internalize approximately 30,000 soluble ligand
molecules per cell per minute; each functional receptor can bind
and internalize one ligand every 8 minutes (Schwartz et al., 1982,
supra). Hepatocytes share asialo- or GalNAc/Gal-specific receptors
with Kupffer cells, and liver endothelial cells (Kolb-Bachhofen et
al., 1984, supra) and the hepatoma line, HEPG2 has been well
characterized with respect to this receptor (Schwartz et al., 1981,
supra). There are 150,000 high affinity sites per HEPG2 cell and
500,000 per normal hepatocytes; the K.sub.d of about
7.times.10.sup.9 M is the same for both. Thus, using adherence to
cell line such as HEPG2 with a well-characterized
asiaioglycoprotein receptor, as an in vitro correlate of in vivo
adherence (as in the EXAMPLES, below) is a cost effective and
simple system in which to determine parameters and possible
problems that will be encountered in the in vivo trafficking
studies.
[0111] Parenteral administration of asialoglycoconjugates (e,g.,
asialofetuin) to block asialogycoprotein receptors has been shown
to increase the efficiency of bone marrow engraftment 5- to 10-fold
by blocking hepatic sequestration of these cells by blocking
hepatic ASGP receptors (Samlowski et al., 1984, supra). Given that
LAK cells have asialodeterminants on their surfaces, as shown by
the in vitro studies herein (see Examples 5-16), then they also
most likely are taken up or sequestered in the liver via the ASGP
receptors. This would result in a net loss of circulating numbers
of LAK effector cells that would be available to participate in the
reduction or lysis of tumors. Sequestered LAK cells might not reach
the tumor. According to the present invention, by blocking the
hepatic ASGP receptors hepatic sequestration can be prevented.
Ultimately, the efficacy of LAK therapy would be improved by
eliminating hepatic sequestration of these cells by the intravenous
administration of asiaioglycoconjugates or by modification of the
LAK cell surface with sialidases or sialyltransferases. This would
allow fewer LAK cells or fewer cycles of LAK therapy or even less
IL-2 to be used during the therapy, thereby reducing the toxicity
associated with LAK therapy.
[0112] In theory, LAK therapy should be one of the safest and least
toxic therapies in the treatment of cancer; however, it has not met
expectations (Rosenberg, 1987, supra; Durant, Immunotherapy of
cancer: The end of the beginning? N. EngI. J. Med. 316:939-940
(1987)). LAK cells have also been shown to kill unmodified normal
cells, including normal lymphocytes, endothelial cells, and
hepatocytes, by some investigators, but not by others. The present
invention improves the efficacy of LAK therapy by increasing the
number of circulating "killer" cells and thereby improving the
probability that these cells will encounter tumor cells located in
the periphery, instead of being primarily sequestered in the liver.
Eliminating hepatic sequestration should therefore improve the
response rate of LAK therapy for tumors located in organs other
than liver. Preventing hepatic sequestration of LAK cells should
also decrease the severe toxicity and the liver damage associated
with this therapy. In addition, this will also reduce the
possibility of permanent damage caused by the autoimmune
destruction of the liver parenchyma by trapped lymphocytes
(Kolb-Bachofen et al., 1979, supra; Anderson et al., Toxicity of
human recombinant interleukin-2 in the mouse is mediated by
interleukin-activated lymphocytes. Lab. Investigation 59:598-612
(1988).
EXAMPLES
Procedures
[0113] Intravenous cannulas were placed into the external jugular
vein of NOD-SCID mice under anesthesia (Institutional Animal Care
and Use Committee protocol #AM87046-07) to enable the efficient
delivery of .sup.111In-labeled stem cells i.v. Tylenol elixir was
administered by mouth after recovery from anesthesia. Briefly
radiolabeled CD34+ cells were taken up in 100-250 ul of 5% human
plasma albumin in saline and injected into the cannula and then
flushed with 50 ul of the albumin-saline. The mice were imaged by
nuclear medicine.
[0114] Mice: NOD-SCID, female mice (Nonobese
diabetic/LtSz-scid/scid) were obtained from the Jackson Laboratory,
Bar Harbor, Me. at 1-2 months of age. These animals were maintained
in microisolator cages in a special isolator room. The air was HEPA
filtered, and the animals were changed in a laminar flow hood
within the facility. All food, bedding, and water was sterilized.
NOD-SCID mice were ideally suited for the study of xenotransplanted
tumors and hematopoietic cells and lymphocytes because of their
immunoincompetence including greatly reduced NK activity. See, e.g.
Hogan, et al., Biology of Blood & Marrow Transplantation
3:236-246 (1997); Noort, et al., Bone Marrow Transplantation 22
Suppl 1:S58-60 (1998).
[0115] All administrations of agents or cells were done either i.v.
or i.p.
[0116] Stem Cells: CD34+ stem cells were isolated from apheresis
stem cell collection products derived from deceased cancer
patients. They were purified to 95-99% purity using antibody
conjugated to CD34 conjugated to magnetic beads (MACS separation
columns; Miltenyi Biotec, Auburn, Calif. and cryopreserved.
[0117] Human mesenchymal stem cells (hMSCs; PT-2501) obtained
through a FDA monitored paid bone marrow donor program were
purchased from Poietics Technologies, BioWhittaker (Walkerville,
Md.). The cells were thawed according to manufacturer
recommendations, resuspended, and radiolabeled in Mesenchymal Stem
Cell Basal Medium (MSCBM).
[0118] Proteins administered: Orosomucoid (alpha-1 acid
glycoprotein) and asialoorosomucoid (ASO) were administered in the
following buffer containing 0.16 mM Caprylate. 10 mM TRIS, 150 mM
NaCl, pH 7.0.
[0119] Anesthesia & analgesia: A rodent anesthesia cocktail of
0.04 ml per 20-30 g mouse i.p. (Rodent Cocktail recipe: 1.5 ml of
50 mg/ml ketamine, plus 1.5 ml of 20 mg/ml xylazine, plus 0.5 ml of
50 mg/ml acepromazine) was used. The anesthetic agent, Rodent
anesthesia cocktail, was administered i.p. as follows:
[0120] 1) for surgery--0.04 ml per 20-30 g mouse, and
[0121] 2) for imaging--0.02 ml per 20-30 g mouse.
[0122] Post-surgical Analgesia: Tylenol 60 ul/20 g mouse (6.10 mg)
was administered by mouth after anesthesia had partially worn off.
The analgesic agent was Tylenol by mouth at 60 ul (6.10 mg) per 20
g mouse immediately after surgery or at the first signs of
distress. Xylazine contained in an anesthetic formulation may also
act as an analgesic.
[0123] Surgical procedure (Standard cannula placement): After
anesthetizing the animals as previously described, the threads for
suturing a cannula filled with citrate saline were soaked in 70%
ethanol. The anesthetized animals were secured with paper tape on
the operating platform ventral side up. The area from just below
the clavicle to the ear was shaved. The shaven area was cleaned
with Betadine and rinsed with 70% ethanol. A vertical incision was
made in the skin of the right neck from the top of the rib cage to
the jaw bone to expose the stemocleidomastoid muscle with the
external jugular vein just beneath. To clearly expose the operating
field, the skin was retracted with wire hooks (secured to small
weights). Retraction should not distort the underlying tissue but
should stabilize the area for visualization and cannula insertion.
The vein was cleared of overlying fat and fascia using microscopic
forceps. The circulation in the superior vena cava was cut off
using a half a knot of 4 O silk surgical sutures. One side of the
thread was secured with a clamped hemostat. A second piece of
thread was looped around the bottom of the vein to make a half knot
without pulling it tight. This loop was used to secure the cannula
once it had been inserted into the external jugular vein. The
surface of the vein was nicked with the microscissors. The cannula
was inserted into the vein with beveled side up. The cannula was
slid down diagonally until the anchor was flush with the wall of
the vein and the lower knot tightened. The cannula was tested by
pushing saline through it. The lower knot was finished after
verifying no leakage. A full knot was tied around the cannula using
the top thread. Saline flow in the cannula was monitored. The top
thread was used to go under, catching tissues, and a knot was tied
over the cannula again with this thread. A full knot was made using
an end of the top thread and the bottom thread. This secures the
superior and inferior threads over the hub of the cannula to
prevent accidental dislodgement. The cannula was clamped off and
the syringe removed. The cannula was positioned underneath the skin
of the neck and exteriorized just below the occiput at the nape of
the neck while rotating the animal (dorsal side up). An autoclip
was used to staple the heat shrink part of the cannula in place
near the exit. The cannula was cut to a reasonable length (1.5-2.0
inches), and a wire plug was placed into it. The animal was turned
over to its original position and the neck closed with autoclip
being careful not to puncture the cannula.
[0124] Surgical procedure (Da Vinci Microport Vascular System
cannula placement): The Da Vinci Microport Vascular System (Da
Vinci Biomedical, South Lancaster, Mass.) is a closed injection
route permitting its implantation up to 2 weeks prior to
trafficking experiments without loss of patency. The essential
difference is that the port is not externalized as before. This
eliminates additional risk for contamination and damage to the
cannula caused by chewing and scratching.
[0125] The incision area was cleaned with Betadine prior to initial
cuts. The mouse was then taped (back side up) to the surgery board.
An incision 3-4 mm was made. Next, the incision was made on the
chest 4-5 mm. A tunnel was made from the back incision to the front
incision in order to feed the cannula through the back to the
chest. Heparin was pushed through the cannula. The cannula was then
pulled through using the hemostats. The skin was pulled loose from
the tissue on the back for placement of the port. The port was
sutured down to the tissue in the middle upper neck area. It was
sutured in two places using a triple knot tie. Next, the mouse was
turned on its back with its chest up. The cannula was then cut at
an angle, where at least 1 mm and at most 2 mm of cannula was
inserted in the jugular vein. The jugular vein was isolated in the
chest after some fat and tissue was pulled away. The arms of the
mice were taped down on their sides because that pushes the chest
forward and further exposes the jugular vein. Once the jugular vein
was isolated, two sutures were placed around it. The top of the
vein was tied off enough to slow the flow of blood, but not to
completely stop the flow. The lower tie was one to 2 mm from the
top, and it was not tightened. The lower tie was used later to hold
the cannula in place and to stop excessive bleeding from the
jugular vein. Next, a small cut was made in the jugular vein
between the two ties, so that the cannula could be fed into the
vein. Once the cannula was placed in the vein the lower tie was
tightened around the cannula within the vein. Next, the cannula was
checked for leaks by running heparin through the cannula. After
verifying no leaks, both incisions were closed.
[0126] .sup.111Indium Oxine Labeling Procedure: .sup.111In-oxine
labeling of adult human CD34+ or mesenchymal stem cells (hMSCs) was
performed using a modification of the Amersham Healthcare Procedure
for labeling autologous leukocytes.
[0127] Harvesting for tissues for histopathology: Tissues were
harvested after euthanasia. After the 1-hour-image, the organs were
harvested and half the organ was fixed in 10% neutral buffered
formalin and the other half was frozen in OTC for frozen sections.
The images presented herein are from fixed tissues.
[0128] Necropsy Procedure For Collection of Mouse Tissues: An
initial midline skin incision from the anterior cervical region to
the brim of the pubis was made followed by an abdominal incision
following linea alba from the sternum to the pubis with a lateral
reflection of the abdominal wall by incision following the caudal
ribs. The sternum was reflected anteriorly by cutting the ribs at
approximately the level of the costochrondral junction, incising
the diaphragm and pericardium as needed. Anteriorly, reflection of
sternum was extended to include the ventral cervical muscles to
expose the trachea. The trachea and esophagus were incised at the
mid cervical area and reflected caudally, cutting attachments as
necessary to remove the thoracic viscera in toto. Following removal
of the thoracic viscera, the entire heart was dissected free and
immersed in 10% neutral buffered formalin. After immersion, the
heart was massaged lightly with serrated tissue forceps to force
fixative into the cardiac chambers. The trachea with attached lung
was then immersed in fixative without further dissection. The
spleen was visualized, omental attachments incised, removed and
immersed whole in formalin fixative. The stomach and intestinal
tract were removed by incising the rectum and reflecting the
viscera anteriorly while cutting attachments as necessary. The
liver was removed in toto and immersed whole in formalin fixative.
The kidneys were removed and immersed whole in formalin fixative.
The pancreas was incised from the anterior duodenum and immersed in
formalin fixative.
[0129] Trimming of Tissues for Paraffin Processing and Microtomy:
The heart was placed on the trimming board with the right ventricle
on the upperside and the left ventricle on the underside next to
the trimming surface. A single upper to lower incision was made
through the right ventricle and atrium and great vessels at the
base of the heart continuing through the interventricular septum
and the left cardiac chambers to achieve two approximately equal
halves. Each half was placed into separate embedding cassettes
containing fixative saturated foam pads and labeled "heart1" and
"heart2". The entire left and right lungs were separated from
midline tissues and placed flat on fixative-saturated foam pads in
cassettes labeled left and right lung. Liver sections were taken
from the right lateral and medial liver lobes and placed into an
appropriately labeled cassette. The left lateral and medial lobes
were sectioned and handled in a similar manner. The entire spleen
was placed in an appropriately labeled embedding cassette and
oriented with one long margin down, taking advantage of the
curvature to increase initial sectional area. For one kidney, a
whole coronal section was taken from the midpoint of the kidney.
The remaining kidney was sectioned longitudinally. Both sections
were placed in a single cassette. The collected pancreas was placed
on formalin-saturated foam pad in an appropriately labeled
cassette.
[0130] Imaging procedures: Nuclear Medicine. NOD-SCID mice were
anesthetized using rodent anesthesia cocktail. Once anesthetized,
the mice were placed on a foam hemi-cylindrical mouse positioning
device (MPD) and covered with a tube sock. The MPD allows better
visual separation of the lungs and liver as compared to placing the
mouse on a flat surface. The foam on which the mouse was placed,
and the tube sock covering maintained a comfortable temperature
permitting longer imaging without additional anesthesia. The MPD
was placed on a narrow table between the dual heads of a Siemens
E.Cam Gamma Camera and imaged statically or dynamically in 2-D or
SPECT. .sup.57Co-Spot Marker is used to mark anatomic positions
(nose, tail, cannula, etc.). The data was analyzed using a Siemens
ICON system for regions of interest or percent of injected dose
(e.g. liver, spleen, heart).
[0131] CT imaging: A CT scan was performed (G. E. Medical System
High Speed Spiral Tunnel) for tumor assessment and to enable the
registration/alignment of the nuclear medicine image with that of
the CT in order to determine precise location of injected
radiolabeled stem cells using the method described by Arata L.,
Clinical Uses for Medical Image Registration: Experiences at Three
Hospitals. Proceedings of PACMEDTec Symposium in Honolulu, Hi.,
Aug. 17-21, 1998 and Nelson, et al., Electromedica 68 (2000) 45-48.
CT scans were performed during a nuclear medicine imaging session
while the animals were under anesthesia. Anesthetized animals were
transported to CT, either just prior to or immediately after, the
nuclear medicine scan. Usually only one CT was done per animal. CT
was used to precisely localize the radiolabeled materials
anatomically, by fusing the CT image with that of the nuclear
medicine SPECT images.
[0132] Gamma camera imaging using a Siemens E. Cam dual head gamma
camera monitored the in vivo trafficking patterns of all human stem
cells described in the following examples. Mice were placed on a
Mouse Positioning Device (MPD) and placed between the detectors on
the imaging platform.
Example 1
[0133] ASO Administered I.V. Directs Human CD34+ to the Heart
[0134] Asialoorosomucoid (ASO)/High Dose HSC: When an infusion of
5.75.times.10.sup.6 HSC was preceded by 3.3 mg ASO, 77.+-.1% of the
infused cells were found in the heart immediately after infusion,
75.+-.5% remained in the heart region at 1.5 hr, decreasing to
52.+-.1% at 24 hr.
[0135] 5.75.times.10.sup.6 111In-labeled human CD34+ (hCD34+)
peripheral blood stem cells were administered intravenously (i.v.)
via an external jugular vein cannula to 2 month old, NOD-SCID,
female mice (Non-obese diabetic/LtSz-scid/scid) obtained from the
Jackson Laboratory, Bar Harbor, Me. The radiolabeled CD34+ stem
cells were administered after pretreatment of the mouse with 3.3 mg
of asialoorosomucoid (ASO) i.v. The in vivo trafficking patterns
were followed by gamma camera imaging using a Siemens E.Cam dual
head gamma camera from immediately after injection up to 36 hr
postinfusion. Human CD34+ were isolated from apheresis stem cell
collection products derived from deceased cancer patients. They
were purified to 95-99% purity using antibody conjugated to CD34
conjugated to magnetics beads (MACS) separation columns; Miltenyi
Biotec, Auburn, Calif. and cryopreserved.
[0136] Radiolabeled CD34+ stem cells administered after ASO
migrated immediately to the heart. Anatomic localization was
facilitated by the use of a .sup.57Co-point source positioned at
the level of the cannula. Up to 79.2% of the injected dose was
located in the heart at 1.5 hours. These cells did not migrate to
the liver and spleen early in the postinfusion follow up images but
could be found in the liver later after 24 hours. However,
51.6-53.2% of the originally injected dose remained in the heart at
24 hours. At 36 hours imaging was conducted with the cannula in
vivo and with the cannula removed and placed next to the sacrificed
animal. These images show that the injected cells were not trapped
in the cannula but were actually in the heart.
Example 2
O Administered I.V. Enables Human CD34+ Cells to Migrate to the
Liver and Spleen but not to the Heart
[0137] Orosomucoid/High Dose HSC: When an infusion of
5.75.times.10.sup.6HSC was preceded by 5.5 mg orosomucoid, 74.+-.3%
of infused cells were found in the liver and spleen immediately
after infusion, 74.+-.4% of the cells remained in the liver region
at 1.5 hr, decreasing to 63.+-.1% at 24 hr.
[0138] The preparation and procedures set forth in Example 1 were
repeated.
[0139] 5.75.times.10.sup.6 111In-labeled human CD34+ (hCD34+)
peripheral blood stem cells were administered intravenously (i.v.)
via an external jugular vein cannula to 2 month old, NOD-SCID,
female mice (Non-obese diabetic/LtSz-scid/scid) obtained from the
Jackson Laboratory, Bar Harbor, Me. The radiolabeled CD34+ stem
cells were administered after pretreatment of the mouse with 5.5 mg
of orosomucoid (O) i.v.
[0140] Mice were imaged and the biodistribution of the radiolabeled
hCD34+ cells monitored as described in Example I. Radiolabeled
hCD34+ administered after O migrated immediately to the
liver/spleen area and remained there until 36 hours. Anatomic
localization was facilitated by the use of a .sup.57Co-point source
positioned at the level of the cannula. The localization to the
liver/spleen region ranged from 76.3% immediately postinfusion to
63.6% at 24 hours. No .sup.111In-labeled cells were found in the
region of the heart.
[0141] At 36 hours imaging was conducted with the cannula in vivo
and with the cannula removed and placed next to the sacrificed
animal. These images show that the injected cells were not trapped
in the cannula. Radioactivity was found at or below the cannula
placement, i.e., in the region of the liver/spleen.
Example 3
O Enables Hcd34+ Cells to Migrate to the Liver/Spleen Without
Significant Migration to the Heart
[0142] Orosomucoid/Low Dose HSC: When an infusion of
0.5.times.10.sup.6 HSC (one-tenth the previous cell dose) was
preceded by 11 mg orosomucoid, 43.+-.2% of infused cells were found
in the liver and spleen immediately after infusion, and 40.+-.3% of
the cells remained in the liver region at 1 hr.
[0143] The preparation and procedures set forth in Example I were
repeated. 0.5.times.10.sup.6 111In-labeled human CD34+ (hCD34+)
peripheral blood stem cells were administered intravenously (i.v.)
via an external jugular vein cannula to 2 month old, NOD-SCID,
female mice (Non-obese diabetic/LtSz-scid/scid) obtained from the
Jackson Laboratory, Bar Harbor, Me. The radiolabeled CD34+ stem
cells were administered after pretreatment of the mouse with 11.0
mg of orosomucoid (O) i.v.
[0144] Mice were imaged and the biodistribution of the radiolabeled
hCD34+ cells monitored as described above. Approximately 1 hour
after infusion, the mice were sacrificed and the organs were
harvested, and half of the organ was fixed in 10% neutral buffered
formalin. Tissue sections were examined microscopically after
immunohistochemical staining for human CD34 and in situ
hybridization for the visualization of human DNA. Nuclear medicine
monitoring for the first ten minutes and 1 hour postinfusion showed
that the radiolabeled hCD34+ cells localized to the region of the
liver/spleen.
[0145] Microscopic examination of the heart after immunohistologic
staining for CD34 demonstrated hCD34+ cells in the endocardial
blood vessel. A few hCD34+ cells could be seen in the lung in the
alveolar septum. Clusters of cells with stem cell morphology could
be seen in the hepatic sinusoid. In situ hybridization for human
DNA clearly showed that hCD34+ cells were not found in the heart
muscle or interventricular septum but were present in the lung.
Example 4
AS0 Followed by O Directs hCD34+ Cells to the Heart and Lung but
not the Region of the Liver/Spleen
[0146] Asialcorosornucoid (ASO)+Orosomucoid/Low Dose HSC. When
infused ASO caused HSC to localize in the heart, the protocol was
changed to have the ASO bolus chased with a bolus of orosomucoid,
to test whether the accumulation in the heart would be maintained.
HSC were again concentrated in the heart when an infusion of
0.5.times.10.sup.6 HSC was preceded by 3.3 mg ASO, then 5.5 mg
orosomucoid. This caused 44.+-.5% of the infused cells to
accumulate in the heart immediately after infusion. 37.+-.3% of the
infused cells remained in the heart region at 1 hr. The
localization in the heart was the major concentrated signal from
the cells, although the percent of infused was reduced from the ca.
75% seen in Example 1.
[0147] The preparation and procedures set forth in Example I were
repeated. 0.5.times.10.sup.6 111In-labeled human CD34+ (hCD34+)
peripheral blood stem cells were administered intravenously (i.v.)
via an external jugular vein cannula to 2 month old, NOD-SCID,
female mice (Non-obese diabetic/LtSz-scid/scid) obtained from the
Jackson Laboratory, Bar Harbor, Me. The radiolabeled CD34+ stem
cells were administered after pretreatment of the mouse with 3.3 mg
of ASO i.v. followed by 5.5 mg 0 i.v.
[0148] Mice were imaged and the biodistribution of the radiolabeled
hCD34+ cells monitored as described in Example 1. Nuclear medicine
monitoring for the first ten minutes and 1 h postinfusion showed
that the radiolabeled hCD34+ cells localized to the heart.
[0149] Approximately 1 hour after infusion, the mouse was
sacrificed and the organs were harvested and half the organ was
fixed in 10% neutral buffered formalin.
[0150] Microscopic examination of the heart after immunohistologic
staining for CD34 revealed clusters of hCD34+ cells in the
interventricular septum, and cells within those clusters that were
morphologically similar to the stained cells but that were CD34
negative. These images reflected the biodistribution depicted by
nuclear medicine studies. The presence of hCD34+ cells in the heart
was dramatically demonstrated by in situ hybridization. Both
immunohistochemical staining for CD34 and in situ hybridization for
human DNA demonstrated that the infused stem cells localized to the
lung and could be readily seen in the alveolar septa, blood
vessels, and other structures. Detection of human DNA revealed the
presence of many more cells in the lung and heart than would have
been predicted by CD34 staining. No hCD34+ cells or cells
morphologically resembling hCD34+ cells were found in liver, spleen
or kidney.
Example 5
HSC Administered in 5% Human Serum Albumin (Without Orosomucoid or
ASO) Migrated Predominantly to the Lungs
[0151] Plasma Albumin/High Dose HSC: When HSC were administered
through the catheter without prior protein infusion, 78.+-.3% of
infused cells were found in the lungs at 0 hr, 54.+-.10% at 1 hr,
and 50.+-.13% at 12 hr. Histological examination of lungs of mice
similarly treated, demonstrated infused cells within the alveolar
septa and the vasculature.
[0152] 2.7.times.10.sup.6 111In-labeled HSC were administered
intravenously (i.v.) via a cannula implanted in the external
jugular vein of a two-month old, female NOD-SCID mouse in 0.1 ml
saline containing 5% human serum albumin. Mice were imaged and the
biodistribution of the radiolabeled hCD34.sup.+ cells monitored as
described in Example 1.
[0153] Radiolabeled HSC, administered in saline containing 5% human
serum albumin, migrated immediately to the lungs. Anatomic
localization of the labeled cells was facilitated by the use of a
.sup.57Co-point source positioned at the level of the cannula exit
site below the scapulae and nose. Moreover, the position marker at
the cannula was verified to be at the diaphragm by CT whole body
scans, transverse and coronal sections. The clip at the cannula
exit site served as a landmark. The lungs were visualized below the
nose marker and above the cannula marker arid the liver and spleen
below the cannula marker. Up to 95.4% of the injected dose was
located in the lungs at initial imaging (Table 1). In four mice the
values for the lungs ranged from 52.6-95.4% of whole body
incorporation for the initial imaging time points. At 1 h, HSC were
located predominantly in the lungs with some counts visible in the
blood circulation. In one mouse at 1h some localization was seen
below the cannula marker, which may have been liver and spleen;
however, the outline was indistinct. At 12 hr in that mouse,
radiolabeled CD34.sup.+ stem cells were found in the liver/spleen
region. However, more than 34.7% (range 34.7-68.5%) of the
originally injected dose remained in the lungs of other animals
imaged at 12 h.
[0154] While the localization to the lungs immediately after
injection (initial or Oh time points) varied from animal to animal,
the percent of the original localization to the lungs remaining at
subsequent scans was more constant. Using the dorsal images at 1h,
72.1-75.5% of the cells initially localized in the lung were
retained in the lung region. Using the dorsal images at 12 h, 78%,
72.1% and 50.5% of the initial lung incorporation remained in the
lungs of the three mice imaged.
Example 6
Orosomucoid Directs MSC to the Heart
[0155] Orosomucoid/Low Dose MSC: When a human mesenchymal stem cell
infusion (0.56.times.10.sup.6 cells) was preceded by 11 mg
orosomucoid, 68.+-.7% of infused cells were found in the heart at 0
hr, and 61.+-.3% at 1 hr.
[0156] MSC were obtained from BioWhittaker, (Poietics Division,
cryopreserved PT-2501 >750,000 cells per ampoule) and labeled
with .sup.111In as in previous examples, except that the MSC were
labeled, washed, and injected in Basal Stem Cell Medium (Poietics)
containing 5% human serum albumin (HSA). 0.56.times.10.sup.6 111In
labeled, human mesenchymal stem cells (MSC) were administered via
an implanted Da Vinci Microport Vascular System cannula in the
external jugular vein of a two-month old, female NOD-SCID mouse in
0.21 ml of basal stem cell medium containing 5% human serum albumin
(HSA). Immediately prior to administration of MSC, 11.0 mg of
orosomucoid was administered i.v. in 0.2 ml.
[0157] Mice were imaged and the biodistribution of the radiolabeled
MSCs cells monitored as described in Example 1. Gamma camera
monitoring initially (0 hr) and at 1 hr post-infusion showed that
the radiolabeled MSC localized to the region of the heart. Region
of interest analysis of the images revealed that approximately
61.7-75.5% of the injected radioactivity initially localized to the
heart and at 1 hr approximately 58-64% of the infused cells
remained in this region. The positions of the cannula, diaphragm,
heart, lungs, and liver were verified by CT scans (coronal
sections). In situ hybridization showed human cells predominantly
in the heart, but not the liver.
Example 7
ASO Followed by Orosomucoid Directs MSC to the Liver/Spleen
[0158] MSC were obtained from BioWhittaker, (Poietics Division,
cryopreserved PT-2501 >750,000 cells per ampoule) and labeled
with .sup.111In. As in Example 6, the MSC were labeled, washed, and
injected in Basal Stem Cell Medium (Poietics) containing 5% human
serum albumin (HSA).
[0159] Asialoorosomucoid (ASO)+Orosomucoid/Low Dose MSC: This
example was designed to compare the trafficking of MSC with HSC
(Example 4) at the low cell dose, so the sequential infusion of ASO
and orosomucoid used in Example 4 was applied. A human mesenchymal
stem cell infusion (0.56.times.106 cells) was preceded by 4.3 mg
ASO followed by 5.5 mg orosomucoid. 63.+-.5% of the infused cells
were found in the liver and spleen at 0 hr, and 57.+-.7% at 1
hr.
[0160] 0.56.times.10.sup.6 "In-labeled, MSC were administered i.v.
in 0.21 ml of basal stem cell medium containing 5% human serum
albumin (HSA). Prior to administration of MSC, 0.1 ml containing
4.3 mg of ASO, followed by 0.1 ml containing 5.5 mg orosomucoid
were administered i.v. The ASO, orosomucoid and MSC were
administered via an implanted Da Vinci Microport Vascular system
cannula in the external jugular vein of a two-month old, female
NOD-SCID mouse.
[0161] Mice were imaged and the biodistribution of the radiolabeled
MSC monitored as in Example 1. Gamma camera monitoring initially
and at 1 h post-infusion showed that the radiolabeled MSC localized
to the region of the liver/spleen. Region of interest analysis of
the initial images revealed that approximately 59.2-66.7% of the
injected radioactivity localized to the liver/spleen and at 1 h
approximately 51.9-61.1% of the infused cells remained in this
region.
[0162] The positions of the cannula, diaphragm, heart, lungs, and
liver were verified by CT scans. In situ hybridization confirmed
the gamma camera biodistribution data. Cells containing human DNA
were found predominantly in the liver.
Example 8
MSC Administered in Either Saline Alone, RPMI-1640 Alone, or Saline
Containing 5% Human Serum Albumin (Without Orosomucoid or ASO)
Migrate to the Lungs and Kidneys
[0163] MSC were obtained from BioWhittaker, (Poietics Division,
cryopreserved PT-2501 >750000 cells per ampoule) and labeled
with .sup.111In. As in Example 6, the MSC were labeled, washed, and
injected in saline alone, RPMI-1640 medium (GIBCO BRL, Grand
Island, N.Y.) and saline containing 5% human serum albumin
(HSA).
[0164] Saline alone.-1.14.times.10.sup.6 111In-labeled, MSC were
administered i.v. in 0.20 ml of saline alone. MSC were administered
via an implanted DaVinci Microport Vascular system cannula in the
external jugular vein of a two-month old, female NOD-SCID
mouse.
[0165] Mice were imaged and the biodistribution of the radiolabeled
MSC monitored as in Example 1. Gamma camera monitoring initially
post-infusion showed that the radiolabeled MSC localized to the
region of the lungs. Region of interest analysis of the initial
images revealed that 95% of the injected radioactivity localized to
the lungs. At 1 hr, 87% and 4% localized to the lungs and kidneys
respectively; at 24 hr, 61% and 13% localized to lungs and kidneys
respectively; and at 48 hr, 59% and 14% localized to the lungs and
kidneys, respectively.
[0166] The positions of the cannula, diaphragm, heart, lungs, and
liver were verified by CT scans and a .sup.57Co-Spot Marker is used
to mark anatomic positions (nose, tail, cannula, etc.)
[0167] .RPMI-1640 alone. 1.14.times.10.sup.6 111In-labeled, MSC
were administered i.v. in 0.20 ml of RPMI-1640 alone.-MSC were
administered via an implanted DaVinci Microport Vascular system
cannula in the external jugular vein of a two-month old, female
NOD-SCID mouse.
[0168] Mice were imaged and the biodistribution of the radiolabeled
MSC monitored as in Example 1. Gamma camera monitoring initially
post-infusion showed that the radiolabeled MSC localized to the
region of the lungs. Region of interest analysis of the initial
images revealed that 95% of the injected radioactivity localized to
the lungs. At 1 hr,-74% and 7% localized to the lungs and kidneys,
respectively, and at 24 hr, 69% and 9% localized to lungs and
kidneys respectively.
[0169] The positions of the cannula, diaphragm, heart, lungs, and
liver were verified by CT scans and a .sup.57Co-Spot Marker is used
to mark anatomic positions (nose, tail, cannula, etc.)
[0170] Saline containing 5% human serum albumin (HSA).
1.14.times.10.sup.6 111In-labeled, MSC were administered i.v. in
0.20 ml of saline containing 5% HSA. MSC were administered via an
implanted DaVinci Microport Vascular system cannula in the external
jugular vein of a two-month old, female NOD-SCID mouse.
[0171] Mice were imaged and the biodistribution of the radiolabeled
MSC monitored as in Example 1. Gamma camera monitoring initially
post-infusion showed that the radiolabeled MSC localized to the
region of the lungs. Region of interest analysis of the initial
images revealed that 94% of the injected radioactivity localized to
the lungs. At 1 h; 87% and 2% localized to the lungs and kidneys
respectively; at 24 hr, 59% and 11% localized to lungs and kidneys
respectively; and at 48 hr, 57% and 14% localized to the lungs and
kidneys, respectively.
Results
[0172] The results of the experiments described above are
summarized in Table 1, below. TABLE-US-00001 TABLE 1 Summary of
Results of Examples 1-8 % Infused Stem Cells/ % Infused Cells %
Infused Cells % Infused Cells Cells Protein Bolus in Lungs in
Liver/Spleen in Heart in Kidney HSC/ 78 .+-. 3% at 0 hr No Protein
54 .+-. 10% at 12 hr HSC/ 74 .+-. 3% at 0 hr Orosomucoid 74 .+-. 4%
at 1.5 hr 63 .+-. 1% at 24 hr HSC/ 77 .+-. 1% at 0 hr ASO 75 .+-.
5% at 1.5 hr 52 .+-. 1% at 24 hr MSC/ 95% at 0 hr [considerable at
48 hr 4% at 1 hr No Protein 87% at 1 hr Gao et al., Cells, 13% at
24 hr 61% at 24 hr Tissues, Organs 14% at 48 hr [majority at 0 hr
169: 12-20 (2001)] Gao et al., Cells, Tissues, Organs 169: 12-20
(2001)] MSC/ 68 .+-. 7% at 0 hr Orosomucoid 61 .+-. 3% at 1 hr MSC/
63 .+-. 5% at 0 hr ASO 57 .+-. 7% at 1 hr
Example 9
[0173] The broad objectives of the following experiments was to
determine whether human LAK cell populations bind specifically to
human hepatoma cells via the ASGP receptor and, if so, how this
cell recognition system could be manipulated for lymphocyte cell
targeting. The general experimental approach uses similar
sialo-asialo-containing plasma proteins in an in vitro system
mimicking contact with liver cells bearing ASGP receptors, shown in
FIG. 5.
Adherence of NK/LAK Activity
To Human Minimal Deviation Hepatoma Monolayers
[0174] Control cells (no IL-2 treatment) or LAK cells (IL-2-treated
human peripheral blood lymphocytes cultured 10U IL-2/ml for 3 days)
were adhered to a monolayer of HEP G2 cells for 2 hours at
4.degree. C. The monolayer was pretreated either with asialofetuin
(ASF, 200 .mu.g/ml) in media or with fetuin (F, control, 200
.mu.g/ml)) in media. After the Control or LAK cells had been
incubated on the monolayer, these cells were then decanted, washed,
and tested for cytotoxic capacity in a .sup.51Cr-release assay
against the NK-resistant target, Raji. The E:T ratios were 40:1,
20:1, 10:1, and 5:1; the standard error of the means is displayed;
the E:T ratio is plotted as the LOG E:T. The results are shown
graphically in FIG. 6.
[0175] CONCLUSION: LAK activity was reduced approximately 50% by
incubating these cells on HEPG2 monolayers that had been treated
with the control (fully sialated) protein, fetuin (which does not
block the ASGP receptor). LAK activity was not removed by
incubating these cells on HEPG2 monolayer that had been preheated
with asialofetuin (to block the ASGP receptors). LAK or Control
preparations that had been incubated with either fetuin or
asialofetuin (at 200 .mu.g/ml) for 2 h at 40C had identical
activity to untreated LAK cell populations. These data support the
notion that LAK cells bind to the hepatic ASGP receptor and this
binding can be inhibited by blocking this receptor with
asialofetuin. The extension of this finding is that hepatic
sequestration of LAK cells is at least in part due to the ASGP
receptor and that the administration of an asialoglycoconjugate,
such as asialofetuin could prevent this entrapment and alter LAK
cell trafficking.
Adherence to HEPG2 (ASGP Receptor-Positive, "ASGPR+") and CAKI-2
(ASGP Receptor-Negative, "ASGPR-") at 23.degree. C.
[0176] This experiment is the same as above, except that the
adherence to monolayers was performed at 23.degree. C. and not
4.degree. C., for 2 hours. Two monolayers were used: HEPG2, an
ASGPR+ cell line, and CAKI-2 (human renal cell carcinoma), an
ASGPR- cell line. The effector cell populations that were used
were: an untreated 3-day old LAK preparation (LAK) and the same
population treated with Vibrio cholera neuraminidase (LAK/NS) (30
mU/1.times.10.sup.7 cells/200 .mu.l). The neuraminidase-treated
population was the asialopositive lymphocyte control. All cell
populations regardless of treatment were greater than 90% viable at
the time of assay. Each type of effector population was incubated
with media alone, 200 .mu.g/ml ASF or F, as controls. All effectors
were assayed on RAJI (LAK-sensitive target; NK-insensitive target)
or K562 (NK/LAK-sensitive target); the E:T ratios and the graphic
presentation are the same above.
[0177] Results. The experiments above gave the following results
(see FIGS. 6-8). For the following discussion, activity on RAJI
will be referred to "LAK" activity; activity on K562 will be
referred to as "IL-2 activated NK" activity. Some investigators
support the idea that NK and LAK recognize and kill targets (fresh
and cultured tumor cells) using the same target structures.
[0178] (1) Preincubation of effectors, either untreated (LAK) or
treated (LAK/NS), with ASF or F, does not affect the ability of the
effectors to kill either RAJI or K562 cells.
[0179] (2) Neuraminidase treatment enhances LAK activity on RAJI,
but does not enhance IL-2 activated NK on K562 (see also FIGS. 11
& 12)
[0180] (3) Adherence to HEPG2 of IL-2 activated NK, with or without
neuraminidase treatment, can be partially inhibited by ASF, but not
by F at 23.degree. C. (FIGS. 7 & 8)
[0181] (4) Adherence to HEPG2 of LAK activity could not be
inhibited with either ASF or F at 23.degree. C. (FIGS. 9 &
10)
[0182] (5) Adherence of LAK activity of the neuraminidase-treated
population to HEPG2 could only marginally be inhibited by ASF and
not F. (FIG. 10)
[0183] (6) Adherence to CAKI-2 of IL-2 activated NK or LAK activity
could not be inhibited by either ASF or F at 23.degree. C. (FIGS.
7-10)
[0184] CONCLUSIONS: LAK activity (as determined on RAJI targets)
and IL-2 activated NK activity (as determined on K562 targets)
display different adherence characteristics to HEPG2, an ASGPR+
cell line. At 23.degree. C. using ASF, LAK activity adherence to
HEPG2 cannot be inhibited; whereas, IL-2 activated NK adherence can
be partially inhibited. At 4.degree. C. virtually all LAK activity
can be inhibited from adhering to the HEPG2 monolayer by ASF.
Adherence to the CAKI-2 (ASGPR-) monolayers cannot be blocked by
ASF at 23.degree. C.
[0185] These data suggest that adherence to the HEPG2 monolayer is
in part mediated by the ASGP receptor and adherence to the CAKI-2
monolayer does not involve this receptor. A working hypothesis is
that LAK/NK cells bind to HEPG2 via at least two receptors or
recognition structures: 1) the ASGPR, which binds an
asialodeterminant on the LAK/NK population and 2) the "LAK" or "NK"
recognition structure for a target epitope. The first should be
inhibitable by ASF; the second should not be. Binding to CAKI-2
(ASGPR-) should not be inhibited by ASF and is due to a LAK or NK
recognition structure binding to the target epitope. This can be
further supported by data derived from experiments (see below) in
which 250 .mu.g/well of ASF or F were added to the
.sup.51Cr-release assay of LAK effectors against the labeled target
CAKI-2. Even at a concentration of 1 mg/ml, ASF did not inhibit the
ability of LAK to kill CAKI-2 target.
[0186] At 23.degree. C. the ASGPR recycles and at 4.degree. C. it
does not, according to Schwartz. et al. Characterization of the
ASGP receptor in a continuous hepatoma line. J. Biol. Chem 256: 88
78-(1981); Schwartz, A. L., et al., Recycling of the ASGP receptor:
biochemical and immunocytochemical evidence. Phil. Trans. R. Soc.
Lond. 300:229-235 (1982). The differences seen in the ability to
inhibit adherence may be explained by the temperature dependence of
ASGPR recycling and possibly of the LAK recognition structure on
the target. At 4.degree. C. the LAK:target binding, both by the
ASGPR and LAK recognition structure, may have the different
affinity for ligand than at 23.degree. C., or possibly at the
increased temperature other adhesion molecules are capable of
increasing the effector:target interaction. That is, at 4.degree.
C. the only receptor on HEPG2 that binds LAK with any appreciable
affinity is the ASGPR, and this static receptor at this temperature
can easily be inhibited by its ligand, ASF. At 23.degree. C. more
than the ASGPR binds the LAK cell to the target; the ASGPR is
recycling in the presence of the ligand, ASF, leaving at least the
LAK recognition structure for the target and possibly other
secondary adhesion molecules to "cement" the interaction.
[0187] These data also suggest that the LAK (as assayed on RAJI)
and IL-2 induced NK (as assayed on K562) cells have different
affinity receptors or different on/off rates for adherence to
HEPG2.
[0188] Neuraminidase treatment, in theory, should have increased
binding of the LAK cells to the HEPG2 monolayer due to the
additional number of asialodeterminants generated by this
treatment, but did not. If the number of asialodeterminants was
already sufficient to occupy the maximum number of ASGPR on HEPG2,
increasing the number of these determinants would not alter the
end-effect. It is also possible there is a specific
asialodeterminant that is involved in the binding and that
generating more, but irrelevant determinants, will not increase
adherence. This suggests the interesting possibility that LAK and
the IL-2 activated populations may differ in the ligands that
participate in this adherence to HEPG2.
LAK Cell Killing of Tumor Targets is not Blocked By ASF or F in
Pretreatment of Targets or When Added to the .sup.51CR-Release
Assay
[0189] Because asialodeterminants may play a role in both
LAK-target interaction and LAK trafficking and liver adherence, it
is important to determine whether the use of asialoglycoprotein
agents, in vivo, to alter trafficking patterns also inhibit
cytotoxic activities, rendering such manipulations
counterproductive. The preincubation of targets with the addition
of asialofetuin or fetuin to the assay, at 250 .mu.g per well, does
not block LAK killing of the tumor target, CAKI-2. The LAK
preparation was a standard 5-day preparation; however, these data
have been replicated with 3-day LAK preparation. (% SPECIFIC
RELEASE was determined from quadruplicates whose raw counts per
minute differed by less than 10 percent; the assay was a standard
4-hour incubation.) TABLE-US-00002 TABLE 2 % .sup.51Chromium
Release from Caki-2 Targets AGENT ADDED TO ASSAY 40:1 20:1 10:1 5:1
Media 48 49 23 14 F 58 42 27 16 ASF 52 41 24 13 Spontaneous release
(media alone): 2183 cpm. Spontaneous release (fetuin alone): 2267
cpm. Spontaneous release (asialofetuin alone): 2147 cpm Total
release: 30,600 cpm.
Adherence of NK/LAK Activity After Cell Surface Modification by
Neuraminidase or 2,3- and 2,6-Siayltransferases
[0190] Five-day LAK preparations (20 U/ml; 1 Dupont unit=44.5 BRMP
units) grown in AIM-V (Gibco) were treated (according to the
protocols in B. 1.2.3 & 1.2.4) with 3 OmU Vibric Cholera
neuramindase, 0.48 mU 2,3-or 10 mu 2,6-silalyltransferase per 107
cells. Some of the effectors were incubated in media, 10% PBS in
RPMJ 1640 at 23.degree. C. for 2 hours. 2.times.107 effectors from
the untreated LALK, neuramithdase-treated LAK, LAK treated with 2,3
or 2,6 were suspended in 15 ml media and placed onto either HEPG2
(ASGPR+) or CAKI-2 (ASGPR-) monolayers at 23.degree. C. for 2
hours. The flasks were rocked every 15 minutes. The nonadherent
cells from these monolayers were decanted and assayed against K562
and RAJI, in addition to the unadhered controls. The results are
presented in FIGS. 11-16. The E:T ratios used were 40, 20, 10, and
5 to 1.
[0191] Results. See Table 2, above, for Summary. Graphic
presentation of this data in FIGS. 11-16).
[0192] (1) IL-2 activated NK (killing K562 targets) is not affected
by any cell surface modifications (FIGS. 11, 12 and 13, top 4
dotted lines); whereas, LAK activity (killing of RAJI) is
significantly enhanced by neuraminidase treatment (FIGS. 12, 15
& 16), but not by 2,3-or 2,6-sialyltransferase treaments FIGS.
15 & 16).
[0193] (2) No modification of LAK cell surfaces alters adherence to
CAKI-2 as compared to untreated LAK (assayed on RAJI) (FIG. 15). In
contrast, neuraminidase treatment promotes adherence to CAKI-2 of
IL-2 activated NK activity (FIG. 14, bottom solid line; assayed on
K562) as well as 2,3-sialyltransferase treatment (FIG. 14, solid
line above neuraminidase). Treatment with 2,6-sialyltransferase has
no effect on the adherence to CAKI-2 of either LAK or IL-2
activated NK.
[0194] (3) No modification of the cell surface dramatically
modifies adherence of LAK activity to HEPG2 (FIG. 15); however,
2,6-sialyltransferase treatment significantly promotes adherence of
IL-2 activated NK (FIG. 13, bottom solid); and conversely,
2,3-sialyltransferase treatment significantly prevents adherence of
these cells to HEPG2 (FIG. 13, top solid line).
[0195] CONCLUSIONS: IL-2 activated NK killing and LAK are affected
differently by neuraminidase treatment.
[0196] IL-2 activated NK adherence to both HEPG2 were altered by
cell surface modifications; LAK adherence was not affected by these
modifications. This may be due to the amount of sialic acid that
can be added to the LAK cell surface which could be determined by
dose-response of 2,3- and 2,6-sialyltransferases.
[0197] Adherence of IL-2 activated NK to HEPG2 at 23.degree. C.
could be partially inhibited by ASF (previously reported) and by
adding sialic acid with 2,3-sialyltransferase (while
2,6-sialyltransferase treatment promoted adherence).
[0198] It is necessary to determine whether adding higher
concentrations of ASF (or another asialocompound, e.g.,
asialoGMI-sugar) as a means of compensating for ASGPR-recycling at
23.degree. C. or even at 37.degree. C. can prevent adherence of
IL-2 activated NK or LAK. Likewise, performing dose-response
experiments with 2,3- and 2,6 sialyltransferase to achieve addition
of the maximum amount of sialic acid may allow the dissection of
the adherence mechanism because each enzyme adds to different
structures: 2,3- to O-linked sugars linked to ser/thr and
2,6-sialyltransferase to N-linked sugars linked to asn. These
glycosyltranferases may be equally important in discriminating
between the populations responsible for IL-2 activated NK (killing
of K562) and those responsible for RAJI killing, LAK.
[0199] All publications, patents, patent applications, and other
documents mentioned in the specification are indicative of the
level of those skilled in the art to which this invention pertains.
All publications, patents, patent applications, and other documents
are herein incorporated herein by reference in their entirety for
all purposes to the same extent as if each individual publication,
patent, patent application, or other document was specifically and
individually indicated to be incorporated herein by reference in
its entirety for all purposes. Subheadings are included solely for
ease of review of the document and are not intended to be a
limitation on the contents of the document in any way.
[0200] 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
modifications may be practiced within the scope of the appended
claims.
* * * * *