U.S. patent application number 09/352466 was filed with the patent office on 2002-02-14 for monoclonal antibodies to stem cell factor receptors.
Invention is credited to BROUDY, VIRGINIA C, LIN, NANCY.
Application Number | 20020018775 09/352466 |
Document ID | / |
Family ID | 24734435 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020018775 |
Kind Code |
A1 |
BROUDY, VIRGINIA C ; et
al. |
February 14, 2002 |
MONOCLONAL ANTIBODIES TO STEM CELL FACTOR RECEPTORS
Abstract
The present invention relates to monoclonal antibodies specific
for a cell receptor specific for human stem cell factor (hSCF) as
well as pharmaceutical compositions containing such monoclonal
antibodies and uses of such monoclonal antibodies.
Inventors: |
BROUDY, VIRGINIA C;
(SEATTLE, WA) ; LIN, NANCY; (SEATTLE, WA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Family ID: |
24734435 |
Appl. No.: |
09/352466 |
Filed: |
July 13, 1999 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09352466 |
Jul 13, 1999 |
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08255193 |
Jun 7, 1994 |
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5922847 |
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08255193 |
Jun 7, 1994 |
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08011078 |
Jan 29, 1993 |
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5489516 |
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08011078 |
Jan 29, 1993 |
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07681245 |
Apr 5, 1991 |
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Current U.S.
Class: |
424/130.1 ;
424/141.1; 424/144.1; 424/153.1; 424/156.1; 424/178.1; 435/1.1;
435/2; 435/7.1; 530/387.1 |
Current CPC
Class: |
A61K 48/00 20130101;
A61P 35/04 20180101; C07K 16/2863 20130101; A61P 35/02 20180101;
C07K 16/2803 20130101; A61K 38/00 20130101; C12N 5/0647 20130101;
A61K 47/6849 20170801; A61K 35/28 20130101; C07K 2319/55 20130101;
A61P 35/00 20180101; C07K 2319/00 20130101 |
Class at
Publication: |
424/130.1 ;
435/1.1; 435/7.1; 435/2; 424/141.1; 424/144.1; 424/153.1;
424/156.1; 424/178.1; 530/387.1 |
International
Class: |
A01N 001/00; A01N
001/02; G01N 033/53; C12P 013/14; A61K 039/395; A61K 039/40; A61K
039/42; A61K 039/44; C07K 016/00 |
Claims
What is claimed is:
1. A monoclonal antibody comprising a monoclonal antibody having an
ability to bind to an SCF receptor.
2. A monoclonal antibody according to claim 1 wherein said SCF
receptor is a human SCF receptor.
3. A monoclonal antibody according to claim 2 wherein said
monoclonal antibody is SR-1.
4. A monoclonal antibody according to claim 1 further comprising an
ability to inhibit binding of a SCF molecule to said SCF
receptor.
5. A monoclonal antibody according to claim 4 wherein said SCF
molecule is a human SCF molecule.
6. A monoclonal antibody according to claim 5 wherein said SCF
receptor is a human SCF receptor.
7. A method of purifying hematopoietic cells comprising the steps
of: (a) exposing a mixture of cells to a monoclonal antibody
according to claim 1; (b) separating cells that bind to said
monoclonal antibody from cells that do not bind to said monoclonal
antibody.
8. A method of purifying hematopoietic cells according to claim 7
wherein said separating is by column chromatography.
9. A method of purifying hematopoietic cells according to claim 7
wherein said separating is by fluorescence-activated cell
sorting.
10. A method of purifying hematopoietic cells according to claim 7
wherein said separating is by direct immune adherence.
11. A method of reconstituting hematopoietic cells comprising bone
marrow transplantation with hematopoietic cells purified according
to the method of claim 7.
12. A method of gene therapy comprising retrovirally-mediated gene
transfer into cells purified according to claim 7.
13. A method of separating normal cells from neoplastic leukemia
cells comprising the steps of: (a) exposing a mixture of cells
comprising normal cells and neoplastic leukemia cells to a
monoclonal antibody according to claim 1; (b) separating normal
cells from neoplastic leukemia cells based upon a differential in
numbers of SCF receptors on normal cells and neoplastic leukemia
cells.
14. A method of treating leukemia cells comprising administration
of a therapeutically effective amount of a leukemia therapeutic
agent conjugated to a monoclonal antibody according to claim 1.
15. A method of treating leukemia cells comprising administration
of a therapeutically effective amount of a leukemia therapeutic
agent conjugated to a binding fragment of a monoclonal antibody
according to claim 1.
16. A method of treating solid tumors comprising administration of
a therapeutically effective amount of a solid tumor therapeutic
agent conjugated to a monoclonal antibody according to claim 1.
17. A method of treating solid tumors comprising administration of
a therapeutically effective amount of a solid tumor therapeutic
agent conjugated to a binding fragment of a monoclonal antibody
according to claim 1.
18. A method of determining the presence of SCF receptors in a cell
sample comprising the steps of: (a) exposing a cell sample to a
monoclonal antibody according to claim 1; (b) detecting the binding
of said monoclonal antibody to SCF receptors.
19. A method according to claim 18 wherein said detecting is
accomplished by using a labelled monoclonal antibody.
20. A method according to claim 18 wherein said cell sample is
selected from the group consisting of normal cells, leukemia cells
and solid tumor cells.
21. A method of modifying sensitivity to cell cycle-specific
chemotherapeutic agents comprising administration of a SCF
inhibiting amount of a monoclonal antibody according to claim
1.
22. A monoclonal antibody according to claim 1 wherein said
monoclonal antibody is a murine-human hybrid antibody.
23. A monoclonal antibody according to claim 1 wherein said
antibody is of the IgG2a isotype.
24. A hybridoma capable of producing a monoclonal antibody
according to claim 1.
25. A hybridoma according to claim 24 wherein said hybridomas is
capable of producing the monoclonal antibody SR-1.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to monoclonal antibodies
specific for a cell receptor that binds human stem cell factor
(hSCF), as well as pharmaceutical compositions containing such
monoclonal antibodies and uses of such monoclonal antibodies.
[0002] Stem Cell Factor (SCF) is a growth factor that stimulates
the proliferation of pluripotent hematopoietic progenitor cells. It
has been produced recombinantly in E. coli and various mammalian
cells [Zsebo et al., Cell 63:195-212 (1990); and co-pending U.S.
patent application Ser. Nos. 07/589,701, 07/573,616, and
07/537,198, filed Oct. 1, 1990, Aug. 24, 1990, and Jun. 11, 1990,
respectively].
[0003] The proto-oncogene c-kit has recently been identified as the
receptor for SCF [Zsebo et al., Cell 63:213-224 (1990)]. Prior to
identification of c-kit as the ligand for SCF, the c-kit receptor
was known to exist [Yarden et al., EMBO J. 6:3341-3351 (1987); Qiu
et al., EMBO J. 7:1003-1011 (1988); Flanagan and Leder, Cell
63:185-194 (1990)].
[0004] Polyclonal antibodies directed against the murine c-kit have
been reported [Cellular Biology 8:4896-4903 (1988)], but it is not
known whether these antibodies will cross react with the human
c-kit, whether they will block binding of SCF to its receptor, or
whether they will affect cell growth. A polyclonal antibody raised
against a human c-kit carboxy terminal peptide has also been
reported [EMBO J. 6:3341-3351 (1987)], but these antibodies would
not block SCF binding to the receptor. A monoclonal antibody that
recognizes human SCF receptors has been reported [Lerner et al.,
Blood 76 (Suppl): 295a, (1990); Ashman et al., Leukemia Res.
12:923-928 (1988); Cambaseri et al., Leukemia Res. 12:929-939
(1988); Gadd and Ashman, Leukemia Res. 11:1329-1336 (1985)].
[0005] Thus, until the existence of the present invention, the
prior art has not been able to obtain a monoclonal antibody to the
c-kit receptor with any expectation that such a monoclonal antibody
would possess the ability to block the binding of the c-kit ligand,
SCF.
[0006] This research is partially funded by the United Sates
Government through National Institute of Health Grant P01-DK-31232
and the American Cancer Society Grant JFRA217.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a monoclonal antibody
comprising a monoclonal antibody having an ability to bind to an
SCF receptor. Preferably, the binding of the monoclonal antibody to
the SCF receptor will also inhibit binding of an SCF molecule to
said SCF receptor. Preferably, the SCF and the SCF receptor will be
of human origin.
[0008] In another aspect of the present invention, the SCF receptor
monoclonal antibodies are used in a method of purifying
hematopoietic cells comprising the steps of:
[0009] (a) exposing a mixture of cells to such monoclonal
antibodies;
[0010] (b) separating cells that bind to said monoclonal antibodies
from cells that do not bind to said monoclonal antibodies.
[0011] In another aspect of the present invention, the
hematopoietic cells purified with the SCF receptor monoclonal
antibodies are used in a method of reconstituting hematopoietic
cells comprising bone marrow transplantation.
[0012] In another aspect of the present invention, the
hematopoietic cells purified with the SCF receptor monoclonal
antibodies are used in a method of gene therapy comprising
retrovirally-mediated gene transfer into the purified cells.
[0013] Another aspect of the present invention relates to a method
of separating normal cells from neoplastic cells comprising the
steps of:
[0014] (a) exposing a mixture of cells comprising normal cells and
neoplastic cells to a monoclonal antibody according to the present
invention;
[0015] (b) separating normal cells from neoplastic leukemia cells
based upon a differential in numbers of SCF receptors on normal
cells and neoplastic leukemia cells.
[0016] Another aspect of the present invention relates to use of
the SCF receptor monoclonal antibodies for treating neoplastic
cells by administration of a therapeutically effective amount of an
anti-neoplastic therapeutic agent conjugated to such a monoclonal
antibody.
[0017] The present invention also relates to a method of treating
neoplastic cells comprising administration of a therapeutically
effective amount of a neoplastic therapeutic agent conjugated to a
binding fragment of a monoclonal antibody of the present
invention.
[0018] Another aspect of the present invention relates to a method
of determining the presence of SCF receptors in a cell sample
comprising the steps of:
[0019] (a) exposing a cell sample to a monoclonal antibody of the
present invention;
[0020] (b) detecting the binding of said monoclonal antibody to SCF
receptors.
[0021] The monoclonal antibodies of the present invention are also
useful as a method of modifying sensitivity to cell cycle-specific
chemotherapeutic agents comprising administration of a
SCF-inhibiting amount of a monoclonal antibody of the present
invention.
DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1: Scatchard analysis of .sup.125IhSCF binding to human
fetal liver cells. 0.9.times.10.sup.6 fetal liver cells were
incubated with .sup.125IhSCF (5 picomolar to 2 nanomolar) and 100
fold excess unlabelled hSCF for 4 hours at 15.degree. C.
[0023] FIG. 2: Effect of recombinant human SCF (rHuSCF) on the
growth of acute nonlymphocytic leukemia cells when administered
alone or in combination with other growth factors such as
interleukin-3.
[0024] FIG. 3: Scatchard plot of .sup.125IhSCF binding to blasts
from a patient with acute nonlymphocytic leukemia (ANLL).
[0025] FIG. 4: Scatchard analysis of .sup.125IhSCF binding to human
small cell lung cancer cells. 0.2.times.10.sup.6 small cell lung
cancer cells (H69 cell line) were incubated with .sup.125IhSCF (5
picomolar to 2 nanomolar) and 100 fold excess unlabelled hSCF for 4
hours at 15.degree. C.
[0026] FIG. 5: Scatchard plot of .sup.125IhSCF binding to OCIM1
cells.
[0027] FIG. 6: Indirect immunofluorescence analysis of SCF binding
to normal human bone marrow. The bone marrow cells were
simultaneously labelled with anti-CD34 monoclonal antibody and with
either SR-1 (FIG. 7A) or with an isotype matched control monoclonal
antibody (anti-Thy 1.1), (FIG. 7B).
[0028] FIG. 7: Recognition of the SCF receptor, c-kit, by
monoclonal antibody SR-1. COS-1 cells were transfected with V19.8
or V19.8 containing human c-kit. The COS cell membranes were
incubated with 1 nanomolar .sup.125IhSCF with or without cold SCF
or SR-1 ascities (diluted 1:1000) and bound labelled SCF was
measured.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention relates to a monoclonal antibody
comprising a monoclonal antibody having an ability to bind to an
SCF receptor. Preferably, the binding of the monoclonal antibody to
the SCF receptor will also inhibit binding of an SCF molecule to
said SCF receptor. Preferably, the SCF and the SCF receptor will be
of human origin. More preferably, the monoclonal antibody will be
of the IgG2a isotype.
[0030] Such a monoclonal antibody can be obtained by general
methods, including the steps of immunizing or sensitizing an animal
with an antigen or immunogen, obtaining the antibody-producing
cells resulting therefrom, fusing such antibody producing cells to
a stable and long living cell line (an immortal cell line) to
produce hybridomas, screening the hybridomas to select a colony
consisting of cells that produce the desired antibody, and
isolating the resulting monoclonal antibody from such cells.
[0031] Sensitization can be accomplished by injecting the antigen
into an antibody producing species. Preferably the injection will
be into a mammal and more preferably into mice. Usually an initial
injection is given followed by subsequent booster injections to
maximize the response. Optimally, the injection regime is in
multiple doses given to Balb/C mice, e.g., one injection
intraperitoneally per week for three consecutive weeks. The amount
of antigen injected must be adequate to elicit a sufficient amount
of antibody to be detectable. Preferred amounts of antigen to be
injected are 10.sup.4 to 10.sup.8 cells containing SCF receptors,
preferably 10.sup.5 to 10.sup.7 cells containing SCF receptors,
most preferably about 10.sup.6 cells containing SCF receptors.
[0032] In addition, the generation of human monoclonal antibodies
can be performed using in vitro immunization techniques [Ho et al.,
J. Immunol. 135:3831 (1985)]. The variable region of the mouse
monoclonal antibody can also be genetically engineered onto the
constant region of a human immunoglobulin which may be preferable
for use in humans to prevent problems of immunogenicity often
associated with administration of foreign proteins to humans.
[0033] These so called "chimeric antibodies" can be obtained by
splicing genes encoding the variable antigen-binding regions of a
human antibody molecule to the constant regions of a human antibody
molecule [Sahagan et al., J. Immunol. 137:106601074 (1986); Beidler
et al., J. Immunol. 141:4053-4060 (1988); Morrison et al., Ann.
N.Y. Acad. Sci. 507:187-198 (1988)].
[0034] A further refinement envisioned within the present invention
is production of chimeric antibodies containing a murine
hypervariable region coupled to human constant and framework
variable regions [Reichman et al., Nature 332:323-327 (1988)]. Most
antigen specificity resides in defined segments of the V regions
(hypervariable regions) or CDR regions (complementary-determining
regions). Antigen-combining sites are formed by CDR loops extending
from the remaining framework portions of the V regions. Host immune
responses may be generated against the less variable rodent
framework V regions of chimeric antibodies. Chimeric antibodies
containing human framework V regions retain the antigen binding
specificity conferred by the murine CDR regions but are unlikely to
elicit a host immune response. Total gene synthesis is the most
practical method of preparing CDR-replaced variants in which CDRs
from a rodent antibody are transplanted into a human framework.
Following sequencing of the desired V region, the sequence is
chemically synthesized, cloned, and then inserted into an
appropriate expression vector.
[0035] The antigens that are useful in producing the monoclonal
antibodies of the present invention are any cell line that displays
an SCF receptor on its surface. Such cell lines include the human
erythroleukemia cell lines OCIM1 [Papayannopoulou et al., Blood
72:1029-1038(1988)], K562 (ATCC CCL 243); the myeloid or monocytic
cell lines KG1 (ATCC CCL 246), KG1.alpha. (ATCC CCL 246.1), AML-193
[Santoli et al., J. Immunology 139:3348, (1987)], U937 (ATCC CRL
1593); the lymphoid cell lines Daudi (ATCC CCL 213), IM-9 (ATCC
CCL159); mast cell line HMC-1, [Butterfield et al., Leukemia
Research 12:345 (1988)]; bladder carcinoma cell lines 5637 (ATCC
HTB9), COS (ATCC CRL 1650), BHK (ATCC CCL 10); the gastric
carcinoma cell line KAT03 (ATCC HTB103); the small cell carcinoma
lines H69 (ATCC HTB 119), H128 (ATCC HTB 120); and the breast
carcinoma cell line DU4475 (ATCC HTB-123), which have been
deposited with the American Type Culture Collection, Rockville, Md.
Preferred antigens are the human erythroleukemia cell line
OCIM1.
[0036] As a result of the sensitization process, the sensitized
animal will produce B-cells that produce and secrete antibodies
specific for the antigen. Such cells can be isolated for further
use by removing the spleen of the immunized mouse.
[0037] The antibody producing cells thus obtained can then be fused
to a suitable stable and long living cell line (immortal cell line)
using techniques that are known in the art [Kohler and Milstein,
Nature 256:495-497 (1975)]. Suitable cell lines for fusion to the
antibody producing cells are any cell line that lacks the ability
to synthesize antibodies, preferably also lacking in the ability to
grow on medium containing a selection agent, most preferably
possessing the mutant hypoxanthine-guanidine phosphoribosyl
transferase gene (HGPRT- gene), which cannot produce the active
hypoxanthine-guanidine phosphoribosyl transferase protein.
Hypoxanthine-guanidine phosphoribosyl transferase is necessary to
grow on a medium containing aminopterin. Such cell lines that are
preferred include myeloma cells, more preferably the NS-1 murine
myeloma cell line [ATCC T1B 18; Nowinski et al., Virology
93:111-126 (1979)]. Recently, there has even been success in using
human cell lines as fusion partners [Banchereau et al., Science
251:70-72 (1991)].
[0038] The resulting fusion partners can then be screened to select
a colony consisting of cells that produce the desired antibody.
Screening techniques are known in the art, and usually involve the
growing of the fused cells on a medium containing a selection agent
that (1) would lead to the death of the unfused immortal cells when
such immortal cells lack the ability to circumvent the selection
agent but (2) allow growth of cells containing genetic material
from the antibody producing cell when such genetic material
contains the potential to circumvent the selection agent. A
preferred immortal cell line contains the HGPRT- gene and a
preferred medium contains aminopterin, more preferably the medium
hypoxanthine aminopterin thymidine (HAT). As a result, only the
fusion cells having both the HGPRT+ gene from the antibody
producing cell line and the characteristic of immortality from the
immortal cell line would survive and grow in the medium.
[0039] The successful fusion cells, or hybridomas, can then be
screened to determine if they have the ability to produce
antibodies to the antigen used for sensitization. In the case of
SCF, such screening can be by the ability of the hybridoma products
to bind to SCF receptors, the ability of the hybridoma products to
inhibit binding of SCF to SCF receptors, or by standard
immunological techniques (e.g., immunoprecipitation of
radiolabelled purified SCF receptor) or by ability of the hybridoma
products to recognize purified SCF receptor in an ELISA assay.
Preferably, the hybridomas can be screened by the ability of
hybridoma products to block binding of SCF to SCF receptors.
[0040] Stem cell factors (SCFs) useful in these assays include any
of the SCFs from various species. Such SCFs are usually in solution
with a suitable adjuvant, which adjuvant may contain buffers,
salts, etc. Preferably, the SCF will be a human SCF (HuSCF), more
preferably a recombinant human SCF (rHuSCF), and most preferably a
rHuSCF produced in E. coli. Such SCFs can be obtained as previously
described [Zsebo et al., Cell 63:195-212 (1990); and co-pending
U.S. patent application Ser. Nos. 07/589,701, 07/573,616, and
07/537,198, filed Oct. 1, 1990, Aug. 24, 1990, and Jun. 11, 1990,
respectively, all of which are hereby incorporated by reference for
their relevant teachings].
[0041] Those hybridomas that are positive for secretion of
antibodies to the SCF receptor can then be subcloned and
essentially maintained indefinitely. Such selected hybridomas can
also be cultured for the production of the monoclonal antibodies
that they secrete. The desired monoclonal antibody can be isolated
from a culture of such hybridomas using techniques that are known
in the art, including protein A-sepharose column chromatography [Ey
et al., Immunochemistry 15:429 (1978)].
[0042] The preferred monoclonal antibodies of the present invention
are those designated SR-1, deposited as BA7.3C.9 with the American
Type Culture Collection, Rockville, Md., USA on April 1991, and
given the Accession Number ______.
[0043] The monoclonal antibodies of the present invention can be
used in a method of purifying hematopoietic cells comprising the
steps of:
[0044] (a) exposing a mixture of cells to such monoclonal
antibodies;
[0045] (b) separating cells that bind to said monoclonal antibodies
from cells that do not bind to said monoclonal antibodies.
[0046] The exposure of a cell mixture to such monoclonal antibodies
can be in solution, as is the case with fluorescence-activated cell
sorting, or it can be with the monoclonal antibody immobilized on a
solid support, such as is the case with column chromatography or
direct immune adherence. In addition, a combination of soluble and
solid support monoclonal antibodies can be used to expose the cell
mixture to such monoclonal antibodies, as has been the case with
anti-CD34 antibody and a biotinylated second antibody put through
an avidin column to remove breast cancer cells in human transplants
[Bensinger et al., J. Clin. Apheresis 5:74-76 (1990); Berenson et
al., Blood 76:509-515 (1986)].
[0047] The mixture of cells that is to be exposed to the monoclonal
antibody can be any solution of bone marrow cells, blood cells or
tissue cells. Preferably, the cell mixture is from mammalian bone
marrow, circulating blood, or suspected tumor tissue. After
exposure of the cell mixture to the monoclonal antibody, those
cells with SCF receptors will bind to the monoclonal antibody to
form an antibody-SCF-receptor-cell complex. Such SCF receptor cell
complexes can then be separated from noncomplexed cells by methods
that are known in the art. Preferred methods of separation include
column chromatography, fluorescence-activated cell sorting,
magnetic bead separation, and direct immune adherence.
[0048] The hematopoietic cells thus purified can be employed in a
method of reconstituting hematopoietic cells comprising bone marrow
transplantation. Methods of bone marrow transplantation are known
in the art [Hill et al., Bone Marrow Transplant. 4:69-74 (1989)],
but heretofore it has not been possible to use such a homogeneous
population of cells having SCF receptors as the material
transplanted. Such cells are responsible for long term engraftment
in a bone marrow transplant and can be separated from contaminating
tumor cells that may be present in the bone marrow using the
methods described above. Moreover, the cells having SCF receptors
purified by the purification method of the present invention can be
further subfractionated to obtain even more homogeneous cell
populations. For example, a population of SCF-receptor-containing
cells can be sequentially exposed to monoclonal antibodies specific
for other cell surface proteins that occur on only certain
subpopulations of the SCF-receptor-containing cells. Examples of
other monoclonal antibodies that can be used in such a sequential
method of purification include monoclonal antibodies to the CD34
antigen which is also expressed on hematopoietic stem cells
[Andrews et al., J. Exp. Med. 169:1721-1731 (1989); Civin, U.S.
Pat. No. 4,965,204, issued Oct. 23, 1990; Civin, European Patent
Application 395355, published Oct. 31, 1990].
[0049] Hematopoietic cells purified according to the present
invention can also be used in a method of gene therapy comprising
retrovirally-mediated gene transfer into the purified cells.
Methods of retrovirally-mediated gene transfer are known in the art
[Bodine et al., Proc. Natl. Acad. Sci. USA 86:8897-8901 (1989)],
but heretofore it has not been possible to use such a homogeneous
population of cells having SCF receptors as the cells transfected.
Such transfected cells can then be used in bone marrow
transplantation.
[0050] The present invention also relates to a method of separating
normal cells from neoplastic cells comprising the steps of:
[0051] (a) exposing a mixture of cells comprising normal cells and
neoplastic cells to a monoclonal antibody according to the present
invention;
[0052] (b) separating normal cells from neoplastic leukemia cells
based upon a differential in numbers of SCF receptors on normal
cells and neoplastic leukemia cells. The bone marrow cells can be
labelled with the monoclonal antibodies of the present invention,
then with a biotinylated goat anti-mouse antibody and passed
through an avidin column. This approach can be used to positively
or negatively select cells; cells with higher numbers of SCF
receptors will be retained by the column while cells with lower SCF
receptor display will pass through the column. Alternatively, a
method for separating cells with high and low SCF receptor display
is direct immune adherence and fluorescence-activated cell sorting.
With fluorescence-activated cell sorting (FACS), cells displaying
SCF receptors can be mixed with monoclonal antibodies specific for
SCF receptors. The monoclonal antibodies are (1) conjugated with a
fluorescence agent such as fluoresceine isothiocyanate (FITC) or
phycoerythrin (PE); (2) conjugated with a first biological molecule
(e.g., biotin) and mixed with a second biological molecule that
specifically binds to the first biological molecule (e.g., avidin
or streptavidin), the second biological molecule being conjugated
with a fluorescent agent such as FITC or PE; or (3) further mixed
with a second antibody specific for the species of antibody of the
anti-SCF receptor monoclonal antibody (e.g., a goat or sheep
anti-mouse antibody), the second antibody being conjugated with a
fluorescent agent such as FITC or PE. Such fluorescently labelled
cells can then be sorted using standard technology according to the
level of fluorescence exhibited by the cells.
[0053] The monoclonal antibodies of the present invention can also
be useful in treating neoplastic cells by administration of a
therapeutically effective amount of an anti-neoplastic therapeutic
agent conjugated to such a monoclonal antibody. A therapeutically
effective amount of a neoplastic therapeutic agent is any amount of
a compound that will cause inhibition of growth and/or development
of neoplastic cells, preferably causing death of the cell and a
decrease in the total number of neoplastic cells in an organism.
Examples of such neoplastic therapeutic agents include antibodies
coupled to the radioisotope .sup.125I [Press et al., J. Clin.
Oncol. 7:1027-1038 (1989)] or to toxin conjugates such as ricin
[Uhr et al., Prog. Clin. Biol. Res. 288:403-412 (1989)] and the
diptheria toxin [Moolten, J. Natl. Con. Inst. 55:473-477
(1975)].
[0054] Conjugation of the leukemia therapeutic agent to the
monoclonal antibody can be accomplished using known techniques as
described above [Press et al., J. Clin. Oncol. 7:1027-1038 (1989);
Uhr et al., Prog. Clin. Biol. Res. 288:403-412 (1989)]. Preferably,
the conjugation site on the monoclonal antibody is at a location
distinct from the binding site for the monoclonal antibody to the
SCF receptor. It is also preferred that the conjugation site on the
neoplastic therapeutic agent be at a functional group distinct from
the active site of the therapeutic agent. More preferably, the
conjugation site will also be situated so as to minimize
conformational changes of the monoclonal antibody or the neoplastic
therapeutic agent.
[0055] The present invention also relates to a method of treating
neoplastic cells comprising administration of a therapeutically
effective amount of a neoplastic therapeutic agent conjugated to a
binding fragment of a monoclonal antibody of the present invention.
Suitable binding fragments are those fragments that retain
sufficient size and structure to allow binding of the fragment to
the SCF receptor. Such fragments can be prepared by numerous
methods, including proteolytic digestion [Garvey et al., Methods in
Immunology, Chapter 31, W. A. Benjamin, Reading, Mass. (1977)]. The
prepared binding fragments can be assayed for ability to bind to
the SCF receptor using the binding assays previously described.
[0056] Another use of the monoclonal antibodies of the present
invention relates to a method of determining the presence of SCF
receptors in a cell sample comprising the steps of:
[0057] (a) exposing a cell sample to a monoclonal antibody of the
present invention;
[0058] (b) detecting the binding of said monoclonal antibody to SCF
receptors.
[0059] The exposure of a cell mixture to such monoclonal antibodies
can be in solution, as is the case for fluorescence-activated cell
sorting, or it can be on solid tissue specimens such as biopsy
material, or it can be with the monoclonal antibody immobilized on
a solid support, as is the case with column chromatography or
direct immune adherence. The mixture of cells that is to be exposed
to the monoclonal antibody can be any solution of blood cells or
tissue cells. Preferably, the cell mixture is from normal mammalian
cells, mammalian bone marrow, circulating blood, or suspected tumor
tissue, more preferably normal cells, leukemia cells and solid
tumor cells. After exposure of the cell mixture to the monoclonal
antibody, those cells with SCF receptors will bind to the
monoclonal antibody to form an antibody-SCF-receptor complex. The
presence of the antibody-SCF-receptor complex, and therefore SCF
receptors, can be detected by methods known in the art. These
methods include ELISA, immunohistochemistry, and autoradiography
using .sup.125C-labelled Staph Protein A.
[0060] The monoclonal antibodies of the present invention are also
useful as a method of modifying sensitivity to cell cycle-specific
chemotherapeutic agents comprising administration of a
SCF-inhibiting amount of a monoclonal antibody of the present
invention. An SCF-inhibiting amount of a monoclonal antibody is
sufficient quantities of monoclonal antibody to significantly
inhibit the binding of SCF to its receptor or to significantly
decrease the growth and development of cells containing the SCF
receptor, e.g., early pluripotent hematopoietic progenitors,
leukemia cells, solid tumor cells, bone marrow cells. Generally, a
significant inhibition is inhibition that is larger than the
variance due to error expected with a given method of measuring the
inhibition. Preferably, the inhibition will decrease binding of SCF
to its receptor by at least 50%, more preferably by at least 75%,
more preferably by at least 90%, and most preferably inhibition
will decrease binding of SCF to its receptor essentially entirely.
Generally, a significant decrease of the growth and/or development
of cells containing the SCF receptor is a decrease larger than the
variance due to error expected with a given method of measuring the
growth and/or development. Preferably, decrease of the growth
and/or development of cells containing the SCF receptor is a
lowering of the growth rate of SCF-receptor-containing cells,
preferably a decrease to at least one-half, more preferably to at
least one-tenth, and most preferably to at least one-hundredth.
[0061] Administration of the monoclonal antibodies of the present
invention involves administration of an appropriate amount of a
pharmaceutical composition containing the monoclonal antibodies as
an active ingredient. In addition to the active ingredient, the
pharmaceutical composition may also include appropriate buffers,
diluents and additives. Appropriate buffers include Tris-HCl,
acetate, glycine and phosphate, preferably phosphate at pH 6.5 to
7.5. Appropriate diluents include sterile aqueous solutions
adjusted to isotonicity with NaCl, lactose or mannitol, preferably
NaCl. Appropriate additives include albumin or helatin to prevent
adsorption to surfaces, detergents (e.g., Tween 20, Tween 80,
Pluronic F68), solubilizing agents (e.g., glycerol, plyethylene
glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite)
and preservatives (e.g., Thimersol, benzyl alcohol, parabens). A
preferred additive is Tween 80.
[0062] Administration may be by any conventional means including
intravenously, subcutaneously, or intramuscularly. The preferred
route of administration is intravenous. Administration may be a
single dose or may occur in an appropriate number of divided
doses.
[0063] Preferably, the pharmaceutical preparation is in unit dosage
form. In such form, the preparation is subdivided into unit doses
containing the appropriate quantities of the active component,
e.g., an effective amount to achieve the desired purpose.
[0064] The actual dosage employed may be varied depending upon the
requirements of the patient and the severity of the condition being
treated. Determination of the proper dosage for a particular
situation is within the skill of the art. Generally, treatment is
initiated with smaller dosages which are less than the optimum dose
of the compound. Thereafter, the dosage is increased by small
increments until the optimum effect under the circumstances is
reached. For convenience, the total daily dosage may be divided and
administered essentially continuously or in portions during the day
if desired. The amount and frequency of administration will be
regulated according to the judgment of the attending clinician
considering such factors as age, condition and size of the patient
as well as severity of the disease being treated.
[0065] A typical recommended dosage regime for use in the present
invention is from about 0.1 to about 10 mg active ingredient per kg
body weight per day.
EXAMPLES
[0066] The following examples are intended to illustrate specific
embodiments of the present invention without limiting the scope
thereof. All references cited are hereby incorporated by reference
for their relevant teachings.
Example 1
Sensitization of Animals
[0067] Appropriate antigens for use in sensitization were any cell
displaying SCF receptors. The presence of SCF receptors was
determined using radiolabelled SCF. Human and rodent
SCF.sup.164-165 was obtained according to the methods of Zsebo et
al., Cell 63:195-212 (1990); and copending U.S. patent application
Ser. Nos. 07/589,701, 07/573,616, and 07/537,198, filed Oct. 1,
1990, Aug. 24, 1990, and Jun. 11, 1990, respectively. These SCFs
were labelled with .sup.125I using the chloramine-T method of
Hunter and Greenwood [Nature 194:495-496 (1962)]. The specific
activity of the .sup.125I human SCF (hSCF) varied from 2,000 to
2,500 Ci/mmol. Both .sup.125I hSCF and .sup.125I rat SCF (rSCF)
retained the ability to bind to SCF-receptor-containing cells.
Moreover, self displacement analysis [Calvo et al., Biochem. J.
212:259-264 (1983)] with .sup.125IhSCF and unlabelled hSCF
demonstrated that the binding affinity was not altered by
iodination. A number of other hematopoietic growth factors were
tested for binding to the erythroleukemia cell line OCIM1
[Papayannopoulou et al., Blood 72:1029-1038 (1988)]. Table 1 shows
that a 100-fold molar excess of unlabelled hSCF competed very
effectively for binding, while a variety of other growth factors
did not.
1TABLE 1 COMPETITION WITH .sup.125IhSCF FOR BINDING TO OCIM1
CELLS.sup.a Competitor CPM Bound NONE 1725 SCF 10 IL-3 1830 GM-CSF
1742 ERYTHROPOIETIN 1775 G-CSF 1843 IL-6 1693 .sup.aOCIM1 cells
were incubated with 200 picomolar .sup.1251 hSCF with or without a
100 fold excess of the growth factors indicated.
[0068] Numerous normal hematopoietic cells, hematopoietic cell
lines and neoplastic nonhematopoietic cells were screened for
expression of SCF receptors. Normal human marrow mononuclear cells
bind hSCF, as do human fetal liver early erythroblasts. Adult late
erythroblasts, which were obtained by culturing peripheral blood
BFU-E and plucking individual colonies after 14 days, also
displayed SCF receptors. Distribution of SCF receptors on normal
human marrow cells were determined by autoradiography [Nicola and
Metcalf, J. Cell Physiol. 124:313-321 (1985)]. Large cells with
high nuclear/cytoplasmic ratio that appeared to be blasts and
promyelocytes were densely labelled with approximately 50 to 200
grains per cell. Megakaryocytes also showed .sup.125I binding.
[0069] A number of human hematopoietic cell lines displayed SCF
receptors. The erythroleukemia cell lines OCIM1 and K562 bind SCF,
as do the myeloid of monocytic cell lines KG-1, KG1a, AML-193 and
U937. The lymphoid cell lines Daudi and IM-9 and the mast cell line
HMC-1 all bound SCF. SCF receptors were also found on
nonhematopoietic cell lines including the bladder carcinoma line
5367, COS, BHK, the gastric carcinoma cell line KATO3, the small
cell carcinoma cell lines H69 and H128, and the breast carcinoma
cell line DU475.
[0070] SCF receptors were quantitated on normal human fetal liver
cells and examined for their response to SCF in colony assays.
Human fetal liver cells (gestational age 55 to 80 days) were
obtained from therapeutic abortions. Consent was obtained for the
use of these tissues, and the studies were approved by the
Institutional Review Board at the University of Washington. The
cells were incubated with .sup.125IhSCF (5 picomolar to 2
nanomolar).+-.a 100 fold excess of unlabelled SCF for 4 hours at
15.degree. C. in the presence of metabolic inhibitors. Under these
conditions, the equilibrium binding for SCF is achieved and
internalization is minimal (<17%). At the conclusion of the
incubation period, cell-associated .sup.125IhSCF was separated from
free .sup.125IhSCF by sedimenting the cells through phthalate oil,
as described in Broudy et al., Blood 75:1622-1626 (1990). Equations
for 1 or 2 classes of receptors were fitted to the data using a
ligand program [Munson and Rodbard, Analyt. Biochem. 107:220-239
(1980)]. The human fetal liver cells were found to express 2
classes of SCF receptors as shown in FIG. 1. The high affinity
receptor had a Kd of 14 picomolar and the low affinity receptor had
a Kd of 2.7 nanomolar with approximately 1,700 receptors/cell.
[0071] Neoplastic hematopoietic cells were investigated to
determine whether they would also respond to SCF and display SCF
receptors. Marrow mononuclear cells from 20 different patients with
acute nonlymphocytic leukemia (ANLL) at first presentation and two
normal adults were studied. The cells were cultured in agar
supplemented with 15% fetal calf serum and recombinant human IL-3.
Colonies (>40 cells) and clusters (<40 cells) were counted
after 8, 15, and 21 days. SCF receptors were quantified by
equilibrium binding studies with .sup.125IhSCF.+-.a 100 fold excess
of unlabelled hSCF. The cellular distribution of SCF receptors was
examined by autoradiography. SCF stimulated colony growth from 7 of
the 20 ANLL marrows studied and from both of the normal marrows.
SCF alone had little effect on colony growth, but acted
synergistically with IL-3 to increase both the number and size of
colonies (FIG. 2). Receptors for SCF were identified on the blasts
of all 20 ANLL patients. Ten of the 20 ANLL patients exhibited 2
classes of SCF receptors on their marrow blasts. A Scatchard plot
of .sup.125IhSCF binding to the blasts from one of the ANLL
patients shows approximately 500 high affinity SCF receptors (Kd 16
picomolar) and 7000 low affinity receptors (Kd 7.6 nanomolar) per
cell as illustrated in FIG. 3. These binding affinities are similar
to those found on normal human fetal liver cells and normal human
marrow mononuclear cells. Six of the 20 patients showed a single
class of high affinity receptors, while the remaining patients
showed a single low affinity binding site. Neither the number of
receptors/cell nor the presence of 1 or 2 classes of receptors
correlated with growth response to SCF, as has been observed for
IL-3, GM-CSF and G-CSF receptors on human ANLL blasts [Park et al.,
Blood 74:56-65 (1989)].
[0072] The marrow mononuclear cells from these leukemic patients
were greater than 90% blasts while marrow mononuclear cells from
normal adults contain a much lower fraction of blasts. The vast
difference in the percentage of blasts suggests that it is not
accurate to compare the average number of receptors per cell on
normal and leukemic marrow samples. Autoradiography, which permits
analysis of binding to individual cells, can more accurately be
used to compare SCF binding to normal and leukemic blasts.
Autoradiographic analysis of .sup.125IhSCF binding to normal human
marrow mononuclear cells on 8 of the ANLL marrow samples was
carried out in a single experiment to permit direct comparison.
Grain counts indicated that the normal marrow blasts displayed
approximately 50 to 200 grains/blast, while the leukemic blasts
exhibited from 2 to 20 grains/blast. Thus binding of SCF to
leukemic blasts was substantially lower than binding of SCF to
normal blasts.
[0073] SCF receptors were also found on tumor cell lines of
non-hematopoietic origin including H69, H128, and DU475. A
Scatchard plot of .sup.125IhSCF binding to H69 cells (FIG. 4) shows
1650 high affinity receptors per cell (Kd 37 picomolar) and 22,300
receptors per cell (Kd 7.2 nanomolar).
[0074] OCIM1 is a human erythroleukemia cell line that displays
receptors for erythropoietin [Broudy et al., Proc. Natl. Acad. Sci.
USA 85:6513-6517 (1988)], GM-CSF, and IL-3. Equilibrium binding
studies with .sup.125IhSCF showed that OCIM1 cells display about
200,000 SCF receptors per cell as shown in FIG. 5. A single class
of high affinity SCF receptors (Kd 45 picomolar) is evident. hSCF
did not stimulate the growth of OCIM1 cells in suspension culture.
MB-02 is a growth factor dependent human erythroleukemia cell line
that will undergo erythroid differentiation in the presence of
erythropoietin [Perrine et al., Biochem. Biophys. Res. Comm.
164:857-862 (1989)]. MB-02 cells respond to hSCF with
proliferation, but not erythroid differentiation, and display both
high and low affinity SCF receptors.
[0075] The OCIM1 cells were used as an immunogen because of their
high SCF receptor display, although any cell displaying SCF
receptors could be used as an immunogen to elicit antibodies to the
SCF receptor.
[0076] Eight week old female Balb/C mice were injected
intraperitoneally with 10.sup.6 OCIM1 cells on three occasions at
one week intervals.
Example 2
Production of Monoclonal Antibody to SCF Receptor
[0077] Five days following the third injection, the spleen was
removed and splenic cells were fused with NS-1 murine myeloma cells
[Nowinski et al., Virology 93:111-126 (1979)]. The supernatants
from a total of 288 hybridoma wells were screened for the ability
to block binding of .sup.125IhSCF to OCIM1 cells as described in
Example 5, below. A positive hybridoma was identified, cloned and
grown as an ascites-producing tumor in pristane-primed Balb/C mice.
The antibody was identified as IgG2a and was named SR-1 (deposited
as BA7.3C.9 with the American Type Culture Collection, Rockville,
Md. USA on Apr. , 1991 and given the ATCC Accession Number ______.
screening of additional hybridomas should lead to the
identification of additional anti-SCF receptor monoclonal
antibodies at a similar frequency.
Example 3
Production of Chimeric Monoclonal Antibodies having Murine Variable
Regions and Human Constant Regions
[0078] Genomic DNA is prepared from the hybridoma cell line
producing SR-1 monoclonal antibodies, and functional exons encoding
the variable regions of heavy and light chains (V.sub.H and
V.sub..kappa., respectively) are identified by DNA restriction maps
obtained by Southern analysis. Functional V.sub..kappa. exons
result when germline V.sub..kappa. genes are rearranged and joined
to the J.sub..kappa. gene segment. Similarly, a functional V.sub.H
exon is created when a V.sub.H gene is juxtaposed to the J.sub.H
gene segment. Specific DNA probe segments are designed to identify
rearranged V-regions genes via unique restriction enzyme sites that
distinguish the rearranged genotype from the unrearranged germline
DNA sequences [Oi and Morrison, BioTechniques 4:214-221 (1986)].
Recombinant DNA techniques are used to construct a genomic DNA
library, and the desired V-genes are isolated and sequenced to
confirm the identification of rearranged expressed V.sub.H and
V.sub.L exons. The V.sub.H exon is inserted into the
pSV2.DELTA.Hgpt vector [Mulligan and Berg, Science 209:1422-1427
(1980); Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076
(1981)], which contains an ampicillin resistance gene to maintain
the plasmid in E. coli, and a mycophenolic acid resistance gene to
permit selection in mammalian cells growing in medium containing
hypoxanthine, mycophenolic acid and xanthine. The desired
V.sub..kappa. exon is inserted into the pSV184.DELTA.Hneo vector,
which is derived from the pACYC184 plasmid [Chang and Cohen, J.
Bacteriol. 143:1141-1156 (1978)]. This vector contains a
chloramphenicol resistance gene used to maintain the plasmid in E.
coli and a gentamycin resistance gene used to select for mammalian
cells transfected with this plasmid vector. Transcription of the
gentamycin resistance gene (neo) is directed by the SV40 early
region promoter. Known DNA regulatory sequences for the
immunoglobulin heavy and light chains are also included in these
transfection vectors [Calame, Annual Rev. Immunol. 3:159-196
(1985); Morrison and Oi, Annual Rev. Immunol. 2:239-256 (1984)].
Both plasmids are maintained in E. coli HB101 [Boyer and
Roulland-Dussoix, J. Mol. Biol. 41:459 (1969)] grown in
chloramphenicol and ampicillin selection medium.
[0079] Protoplasts of these bacterial cells, prepared by treatment
with lyzozyme and EDTA, are fused via polyethylene glycol treatment
or electroporation) with the immunoglobulin nonproducing mouse
SP2/0 myeloma cell line (ATCC CRL 1581). Transfected SP2/0 cells
are isolated using medium containing gentamycin, hypoxanthine,
mycophelolic acid and xanthine. The resulting transfectomas are
screened for production of mouse:human chimeric SR-1 antibodies
using the techniques described in Example 2.
Example 4
Assay to Determine Binding of Monoclonal Antibody SR-1 to SCF
Receptor
[0080] COS-1 cells were transfected with a vector containing the
transmembrane and external domain of human c-kit [Zsebo et al.,
Cell 63:213-224 (1990)], or with the vector alone. Indirect
immunofluorescence analysis using SR-1 followed by FITC-conjugated
goat anti-mouse IgG showed that SR-1 recognized none of the cells
transfected with vector alone and 5 to 10% of the cells transfected
with c-kit. This demonstrates that SR-1 binds to c-kit.
[0081] Analysis by indirect immunofluorescence also showed that the
monoclonal antibody (SR-1) recognizes a surface epitope present on
both OCIM1 cells and a small percentage (2-5%) of marrow
mononuclear cells (FIG. 6A). The SR-1 antibody recognizes a portion
(50-75%) of the hematopoietic cells that display CD34 (FIG. 6B).
These results were obtained when bone marrow cells were
simultaneously labelled with anti-CD34 monoclonal antibody
(antibody 12.8, Andrews et al., J. Exp. Med. 172:355, (1990);
Civin, U.S. Pat. No. 4,965,204, issued Oct. 23, 1990; Civin,
European Patent Application 395355, published Oct. 31, 1990) and
with either SR-1 or with an isotype matched control monoclonal
antibody anti-Thy 1.1, Andrews et al., J. Exp. Med. 172:355,
(1990). These examples demonstrate that SR-1 and indirect
immunofluorescence analysis can be used to identify cells that
express c-kit. SR-1 antibodies have also been directly conjugated
to PE, and this preparation has been used to identify cells that
display c-kit. Alternative methods are biotinylation of the SR-1
antibody, with binding of this preparation detected using avidin or
streptavidin conjugated to FITC or PE.
Example 5
Assay to Determine Inhibition of SCF Binding to SCF Receptor by
Monoclonal Antibody SR-1
[0082] Cells that express the SCF receptor were incubated with
.sup.125IhSCF (100 picomolar) with or without varying quantities of
SR-1 antibody. Preferably, a dilution of 1:1000 to 1:100,000 of
SR-1 ascites is used. At the conclusion of the incubation, cell
associated .sup.125IhSCF was separated from free .sup.125IhSCF by
sedimenting the cells through phthalate oil [Broudy et al., Blood
75:1622-1626 (1990)].
[0083] The ascites blocks binding of .sup.125IhSCF to OCIM1 cells
at a 1:100,000 dilution (Table 2). This monoclonal antibody is
specific for the human SCF receptor in that it does not block
binding of .sup.125I-ratSCF to the murine MC/9 cell line.
2TABLE 2 SR-1 BLOCKS BINDING OF .sup.125I hSCF TO OCIM1 CELLS.sup.a
Addition CPM Bound 0 1548 Unlabelled SCF 89 Ascites 1:1,000 70
Ascites 1:10,000 39 Ascites 1:100,000 118 Ascites 1:1,000,000 760
Ascites 1:10,000,000 1375 .sup.aOCIM1 cells were incubated with 100
picomolar .sup.125IhSCF with dilutions of SR-1 ascites fluid.
[0084] COS-1 cells were also transfected with V19.8, Zsebo et al.,
Cell 63:213-224 (1990 or V19.8 containing human c-kit. The COS cell
membranes were incubated with 1 nanomolar .sup.125IhSCF with or
without cold SCF or SR-1 ascities (diluted 1:1000) and bound
labelled SCF measured. SR-1 blocked binding of .sup.125IhSCF to
c-kit as effectively as unlabelled SCF (FIG. 7).
Example 6
SR-1 Neutralizes the Biologic Effect of SCF
[0085] In addition to blocking the binding of .sup.125IhSCF to
cells, SR-1 blocks the biologic effects of SCF on colony growth.
SCF stimulates the growth of early erythroid colony forming cells
(BFU-E), and SR-1 blocks this effect. SCF does not alter the growth
of more mature erythroid colony forming cells (CFU-E) and SR-1 has
no effect on CFU-E growth.
[0086] Human marrow mononuclear cells were cultured in recombinant
human erythropoietin (1 unit/ml, Amgen Inc., Thousand Oaks, Calif.)
plus hSCF (50 ng/ml) with or without SR-1 ascites (1:1000 dilution)
in semisolid medium. CFU-E were counted on day 7, BFU-E were
counted on day 14. Three experiments with duplicate plates were
conducted and the results from one such experiment are presented in
Table 3.
3TABLE 3 EFFECT OF SR-1 ON HUMAN ERYTHROID COLONY GROWTH
Colonies/10.sup.5 Cells SR-1 ANTIBODY CFU-E BFU-E - 39 142 + 38
20
Example 7
Conjugation of Monoclonal Antibody SR-1 to a Therapeutic Agent
[0087] SR-1 is coupled or conjugated to a variety of agents, for
therapeutic and diagnostic use of the resulting conjugates,
Scheinberg et al., Oncology 1, 31-37 (1987).
[0088] For use in in vivo imaging of tumors and tumor masses
containing cells expressing c-kit receptor, antibody or antibody
fragments are coupled to radioisotopes such as .sup.123I,
.sup.131I, 111.sup.In, .sup.90Y, .sup.99Tc. For use in therapy of
such tumors and in therapy of dispersed malignancies such as
leukemias, antibody or antibody fragments are coupled to
radioisotopes such as .sup.32P, .sup.131I, .sup.90Y, .sup.186Re,
.sup.212Pb, .sup.212Bi [Scheinberg et al., Oncology 1, 31-37 (1987)
and Humm, J. L., J. Nuclear Medicine 27, 1490-1497 (1986)].
Conjugation of radioisotopes to antibody is accomplished by direct
attachment of radioisotopes to antibody by methods that include
pertinning techniques [Schwartz J., Nuclear Medicine 28, 721 (1987)
and Rhodes et al., J. Nuclear Medicine 21, 54 (1980)]; or by way of
bifunctional chelate linkers such as those utilizing
diethylenetriaminepentaacetic acid (DTPA) [Hnatowich et al., J.
Nuclear Medicine 26:503-509 (1985)], N.sub.2S.sub.2 [Fritzberg et
al. Proc. Natl. Acad. Sciences U.S.A. 85:4025 (1988)], or
macrocyclic chelators [Moi et al., Cancer Research (Suppl.)
50:7895-7935 (1990)], which bind both antibody and
radioisotope.
[0089] For use in therapy, a variety of other toxic agents are
attached to antibody. These include antitumor drugs and antibiotics
which are toxic by way of interaction with DNA via intercalation
(e.g., daunomycin, adriamycin, aclacinomycin) or cleavage of DNA
(e.g., esperamycin, calicheamycin, neocarzinostatin), and other
toxic cytostatic drugs such as cis-platinum, vinblastine, and
methotrexate [Scheinberg et al., Oncology 1:31-37 (1987);
Greenfield et al., Antibody, Immunoconjugates, and
Radiopharmaceuticals 4:107-119 (1991); Dillman et al., Cancer
Research 48:6097-6101 (1988); Hamann et al., Abstracts of 197th
American Chemical Society National Meeting, Dallas, Tex., U.S.A.,
Apr. 9-14, 1989, Abstract No. 71A; Y. Sugiura et al., Proc. Natl.
Acad. Sci. U.S.A. 86:7672-7676 (1989)]. These agents are coupled in
ways that include covalent attachment upon reaction with
appropriate derivatives of the agents.
[0090] Also for use in therapy, many protein and glycoprotein
toxins are conjugated to antibody [Blttler et al., Cancer Cells
1:50-55 (1989); Immunotoxins, Edited by A. E. Frankel Kluwer
Academic Publishers, Boston (1988)]. These include bacterial toxins
such as Diphtheria toxin, Shigella toxin, and Pseudomonas exotoxin;
plant toxins such as ricin, abrin, modeccin, viscumin, pokeweed
antiviral protein, saporin, momordin, and gelonin. The toxins
contain a catalytic fragment and in some cases fragments or domains
that recognize cell surface structures or facilitate translocation
across cell membranes. Thus appropriately modified toxins or toxin
fragments are used, which permit improved specificity without loss
of potency (e.g., modified toxins which themselves lack the
capability for cell surface recognition, so that such recognition
is provided only by the antibody to which conjugation is done, but
which retain the membrane translocation capability which enhances
potency) [Hnatowich et al., J. Nuclear Medicine 26:503-509 (1985)].
Conjugation of toxins to antibody is done by heterobiofunctional
cross-linkers such as N-succinimidyl 3-(2-pyridyldithio) propionate
(SPDP) or 2-iminothiolane [Cawley et al., Cell 22:563-570 (1980);
Goff et al., Bioconjugate Chemistry 1:381-386 (1990); and Thorpe et
al., J. Natl. Cancer Inst., 79:1011]. In addition, toxins fused to
a c-kit binding component of antibody are generated by recombinant
expression of genetically-engineered elements of toxin and antibody
genes joined as a continuous genetic element.
[0091] Prior to diagnostic or therapeutic use, conjugated
antibodies are tested to judge their toxic potency, target
specificity, in vitro and in vivo stability, and other properties,
[Blttler et al., Cancer Cells 1:50-55 (1989) and Immunotoxins,
Edited by A. E. Frankel Kluwer Academic Publishers, Boston (1988)].
It is desired that the toxicity of the toxic agent, and the binding
affinity and specificity of the antibody, be minimally affected by
the coupling procedures used. Thus conjugates are tested for
binding to SCF receptor (see Example 3), and inhibition of SCF
binding to SCF receptor (see Example 4). In vitro toxicity toward
target cells such as the erythroleukemia cell line OCIM1 is tested
by measuring incorporation of labeled compounds into macromolecules
in treated versus untreated cell cultures, and more directly by
determining the number of cells in treated versus untreated
cultures that are able to grow in clonogenic and cell growth
back-extrapolation assays. In vivo stability, clearance, and
specific toxicity are judged by administration of conjugate to
appropriate animal recipients. Such recipients include normal mice
and in vivo tumor and leukemia xenograft models comprising human
neoplastic cells introduced into immunodeficient strains of mice,
such as the nude mouse or SCID mouse.
Example 8
Preparation of Pharmaceutical Composition Containing Monoclonal
Antibody SR-1
[0092] Pharmaceutical compositions of the present invention include
an effective amount of the active ingredient, SR-1, alone or in
combination with a suitable buffer, diluent and/or additive. Such
compositions are provided as sterile aqueous solutions or as
lyophilized or otherwise dried formulations. Typically, antibodies
are formulated in such vehicles at concentrations from about 1
mg/ml to 10 mg/ml.
[0093] One example of a suitable pharmaceutical composition for
injection contains monoclonal antibody SR-1 (1 mg/ml) in a buffered
solution (pH 7.0.+-.0.5) of monobasic sodium phosphate (0.45 mg/ml)
and Tween 80 (0.2mg/ml) in sterile H.sub.2O.
Example 9
Selection of Cells Containing SCF Receptors
[0094] a. Selection of Cells Containing SCF Receptors by Direct
Immune Adherence with SR-1.
[0095] Cells expressing SCF receptors were selected by direct
immune adherence, and the proliferative potential of these cells
was determined in colony assays. Monocyte-depleted normal human
marrow mononuclear cells were separated by direct immune adherence
with SR-1, and cultured in erythropoietin plus IL-3 in semisolid
medium for 14 days. The data represent the average of duplicate
plates from 1 of 3 experiments.
[0096] The results (Table 4) show a 50-fold enrichment in BFU-E in
the SR-1 adherent population of cells. The fraction of BFU-E ranged
from 4-7%, and the overall recovery of BFU-E in the SR-1 adherent
population was 70%. These results indicate that SR-1 can be used to
purify populations of progenitor cells by direct immune
adherence.
4TABLE 4 ISOLATION OF PROGENITOR CELLS WITH SR-1 Colonies/10.sup.5
Cells Cells BFU-E CFU-GM Marrow mononuclear 117 151 SR-1 Adherent
5040 840 SR-1 Non-adherent 6 24
[0097] To determine whether more primitive hematopoietic cells are
found in the SR-1 adherent population, the cells were cultured in
suspension for 12 days. At 3 day intervals, aliquots of cells were
removed and replated in methylcellulose colony assays to quantitate
progenitors. The results show that the SR-1 adherent cells
generated large numbers of BFU-E and CFU-GM (up to 3-fold above
input) throughout the 12 day suspension culture period. The number
of BFU-E and CFU-GM in the SR-1 non-adherent population did not
increase above input, and continuously declined. This indicates
that the SR-1 adherent population of cells contains more primitive
hematopoietic cells that are capable of generating progenitor
cells.
[0098] b. Selection of Cells Containing SCF Receptors by
Fluorescence-activated Cells Sorting with SR-1.
[0099] Normal human bone marrow mononuclear cells were
simultaneously labeled with anti-CD34 (monoclonal antibody 12.8
[Andrews et al., J. Exp. Med. 172-355 (1990)] and SR-1, and
separated on a FACS as illustrated in Example 10. The fraction of
progenitors was determined by colony assays in methylcellulose. The
results show that the CD34 positive, SR-1 positive population of
cells contained 80% of the BFU-E and 96% of the CFU-GM. The CD34
positive, SR-1 negative population of cells contained 20% of the
BFU-E and 4% of the CFU-GM. This indicates that SR-1 identifies a
subset of CD34 positive cells that contains the majority of myeloid
and erythroid progenitor cells. Furthermore, these progenitor cells
can be positively selected by cell sorting with SR-1.
Example 10
Fluorescence Activated Cell Sorting of Cells Displaying SCF
Receptors.
[0100] In fluorescence activated cell sorting (FACS), cells
displaying SCF receptors were mixed with the monoclonal antibody
SR-1. The SR-1 monoclonal antibody was either (1) conjugated with
the fluorescence label FITC or PE; (2) conjugated with biotin and
mixed with avidin or streptoavidin which is conjugated with FITC or
PE; or (3) further mixed with a goat anti-mouse FITC or a sheep
anti-mouse FITC.
[0101] Such directly labelled or indirectly labelled
SCF-receptor-containing cells were then separated on the basis of
the level of fluorescence using standard FACS methods.
* * * * *