Note: Descriptions are shown in the official language in which they were submitted.
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RECOMBINANT HUMAN ALPHA-FETOPROTEIN AND USES THEREOF
Background of the Invention
This invention relates to the expression and
purification of cloned human alpha-fetoprotein; methods for
treating autoimmune diseases; cancer therapeutics and diagnostic
methods; and cell growth and cell culture.
Alpha-fetoprotein (AFP) is a serum protein normally
found at significant levels only in fetal blood. In adult blood
increased alpha-fetoprotein levels are associated with liver
regeneration and certain carcinomas.
Summary of the Invention
Various embodiments of this invention provide a
medicament for inhibiting a neoplasm in a mammal, wherein said
neoplasm is characterized by the expression of a receptor that
binds alpha-fetoprotein, said medicament comprising a
pharmaceutically acceptable carrier and recombinant human alpha-
fetoprotein (rHuAFP) or a biologically active fragment thereof,
conjugated or linked to a cytotoxin; wherein said biologically
active fragment of rHuAFP is selected from the group consisting
of: Domain I consisting of amino acids with a sequence of SEQ ID
NO: 6; Domain II consisting of amino acids with a sequence of SEQ
ID NO: 7; Domain III consisting of amino acids with a sequence of
SEQ ID NO: 8; Domain I+II consisting of amino acids with a
sequence of SEQ ID NO: 9; Domain II+III consisting of amino acids
with a sequence of SEQ ID NO: 10; and rHuAFP fragment I consisting
of amino acids with a sequence of SEQ ID NO: 11.
Various embodiments of this invention provide use of
recombinant human alpha-fetoprotein (rHuAFP) or a biologically
active fragment thereof, conjugated or linked to a cytotoxin in
manufacture of a medicament for inhibiting a neoplasm, wherein
said biologically active fragment of rHuAFP is selected from the
group consisting of: Domain I consisting of amino acids with a
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sequence of SEQ ID NO: 6; Domain II consisting of amino acids with
a sequence of SEQ ID.NO: 7; Domain III consisting of amino acids
with a sequence of SEQ ID NO: 8; Domain I+II consisting of amino
acids with a sequence of SEQ ID NO: 9; Domain II+III consisting of
amino acids with a sequence of SEQ ID NO: 10; and rHuAFP fragment
I consisting of amino acids with a sequence of SEQ ID NO: 11.
In the aforementioned embodiments the cytotoxin may be
linked to the rHuAFP or biologically active fragment thereof by a
non-covalent bond, a covalent bond, or both. The cytotoxin may be
cytoxic while conjugated or linked to the rHuAFP or biologically
active fragment thereof and/or the cytotoxin is cytotoxic in an
unconjugated form free from the rHuAFP or biologically active
fragment thereof.
Various embodiments of this invention provide a
composition for use in detecting a neoplasm in a mammal, wherein
said neoplasm is characterized by the expression of a receptor
that binds alpha-fetoprotein, said composition comprising a
pharmaceutically acceptable carrier and recombinant human alpha-
fetoprotein (rHuAFP) or a biologically active fragment thereof,
conjugated or linked to a detectable label; wherein said
biologically active fragment of rHuAFP is selected from the group
consisting of: Domain I consisting of amino acids with a sequence
of SEQ ID NO: 6; Domain II consisting of amino acids with a
sequence of SEQ ID NO: 7; Domain III consisting of amino acids
with a sequence of SEQ ID NO: 8; Domain I+II consisting of amino
acids with a sequence of SEQ ID NO: 9; Domain II+III consisting of
amino acids with a sequence of SEQ ID NO: 10; and rHuAFP fragment
I consisting of amino acids with a sequence of SEQ ID NO: 11.
Various embodiments of this invention provide use of
.30 recombinant human alpha-fetoprotein (rHuAFP) or a biologically
active fragment thereof, conjugated or linked to a detectable
label in manufacture of a composition for detecting a neoplasm in
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a mammal, wherein said biologically active fragment of rHuAFP is
selected from the group consisting of: Domain I consisting of
amino acids with a sequence of SEQ ID NO: 6; Domain II consisting
of amino acids with a sequence of SEQ ID NO: 7; Domain III
consisting of amino acids with a sequence of SEQ ID NO: 8; Domain
I+II consisting of amino acids with a sequence of SEQ ID NO: 9;
Domain II+III consisting of amino acids with a sequence of SEQ ID
NO: 10; and rHuAFP fragment I consisting of amino acids with a
sequence of SEQ ID NO: 11.
In general, the invention features substantially pure
biologically-active recombinant human alpha-fetoprotein including
a sequence that is substantially identical to either amino acids 1
to 389 of Fig. 1 (SEQ ID NO: 9) or a fragment thereof; amino acids
198 to 590 of Fig. 1 (SEQ ID NO: 10) or a fragment thereof; amino
acids 198 to 389 of Fig. 1 (SEQ ID NO: 7) or a fragment thereof;
amino acids 390 to 590 of Fig. 1 (SEQ ID NO: 8) or a fragment
thereof; and amino acids 266 to 590 of Fig. 1 (SEQ ID NO: 11) or a
fragment thereof.
In another related aspect, the invention features a
method for using an insect cell for producing biologically active
recombinant human alpha-fetoprotein or a fragment or analog
thereof involving
a) providing a transformed insect cell (e.g.,
Spodoptera frugiperda) including a recombinant DNA molecule
encoding the human alpha-fetoprotein or fragment or analog thereof
operably linked to an expression control element which directs the
expression of the human alpha-fetoprotein or fragment or analog
thereof;
b) culturing the transformed cell; and
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c) recovering the biologically active human alpha-
fetoprotein or fragment or analog thereof..
0
in related aspects, the invention also features
substantially pure human alpha-fetoprotein or a fragment
or analog thereof produced using any of the methods
described herein, and therapeutic compositions including
substantially pure human alpha-fetoprotein (or a fragment
or analog thereof) produced using any of the expression
systems described herein.
In another aspect, the invention features a method
of inhibiting autoreactive immune cell proliferation in a
mammal (e.g., a human patient), involving administering
to the mammal a therapeutically effective amount of
recombinant human alpha-fetoprotein or an immune cell
anti-proliferative fragment or analog thereof. Such a
method is based on my discovery that unglycosylated
recombinant human alpha-fetoprotein which is made in a
prokaryote (e.g., E. coli) is useful for inhibiting
autoreactive immune cells derived from a mammal.
Preferably, such immune cells include T cells or B cells;
and the recombinant human alpha-fetoprotein (or an immune
cell anti-proliferative fragment or analog thereof) used
in such methods is produced in a prokaryotic cell (e.g.,
E. coli) and is unglycosylated.
In another aspect, the invention features a method
of treating an autoimmune disease in a mammal (e.g., a
human patient), involving administering to the mammal a
therapeutically effective amount of recombinant human
alpha-fetoprotein or an immune cell anti-proliferative
fragment or analog thereof. Such an autoimmune disease
is multiple sclerosis; is rheumatoid arthritis; is
myasthenia gravis; is insulin-dependent diabetes
mellitus; or is systemic lupus erythematosus. In other
preferred embodiments the autoimmune disease is acquired
immune deficiency syndrome or involves a rejection of a
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transplanted organ, tissue, or cell. Preferably, the
recombinant human alpha-fetoprotein used in such methods
is produced in a prokaryotic cell (e.g., E. coli) and is
unglycosylated. In other preferred embodiments, such
methods further involve administering to the mammal an
immunosuppressive agent in an effective dose which is
lower than the standard dose when the immunosuppressive
agent is used by itself. Preferably, such an
immunosuppressive agent is cyclosporine; is a steroid; is
azathioprine; is FK-506; or is 15-deoxyspergualin. In
yet another preferred embodiment, such a method involves
administering to the mammal a tolerizing agent.
Preferably, the recombinant human alpha-fetoprotein used
in such methods is produced in a prokaryotic cell (e.g.,
E. coli) and is unglycosylated.
According to the invention, administration of
recombinant human alpha-fetoprotein ("rHuAFP") (or a
fragment or analog thereof) can be an effective means of
preventing or treating or ameliorating autoimmune
diseases in a mammal. To illustrate this, I have shown
that recombinant HuAFP produced in a prokaryotic
expression system is effective in suppressing T cell
proliferation in response to self antigens, despite the
fact that such rHuAFP is not modified in the same fashion
as naturally occurring HuAFP. The use of natural HuAFP
has heretofore been limited by its unavailability,
natural HuAFP is obtained by laborious purification from
limited supplies of umbilical cords and umbilical cord
serum. Because biologically active rHuAFP can now be
prepared in large quantities using the techniques of
recombinant DNA, the use of rHuAFP for treating
autoimmune diseases is now possible. The use of rHuAFP
is especially advantageous since there are no known
adverse side effects related to human alpha-fetoprotein
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and it is believed that relatively high doses can be
safely administered.
In still other aspects,.the invention features
compositions and methods for the protection, treatment,
and diagnosis of neoplasia, in particular, cancer. This
aspect of the invention is based on my discovery that
unglycosylated recombinant human alpha-fetoprotein made
in a prokaryote (e.g., E. coli) is useful for treating
and diagnosing mammals with neoplasms, especially
malignant tumors, such as breast or prostate carcinomas,
and other carcinomas caused by a proliferation of
malignant cells which express receptors which are
recognized by recombinant human alpha-fetoprotein.
In one aspect, the invention features a method of
inhibiting a neoplasm in a mammal (e.g., a human
patient), involving administering to the mammal a
therapeutically effective amount of recombinant human
alpha-fetoprotein or an anti-neoplasm fragment or analog
thereof. Preferably, the neoplasm is a malignant tumor
(e.g., a breast tumor or a prostate tumor); and the
recombinant human alpha-fetoprotein is produced in a
prokaryotic cell (e.g., E. coli) and is unglycosylated.
In preferred embodiments, the cells of the neoplasm
express a receptor which is recognized by the recombinant
human alpha-fetoprotein. Such a neoplasm is generally a
carcinoma such as an adenocarcinoma or a sarcoma. In
preferred embodiments, the neoplasm proliferates in
response to a hormone, e.g, estrogen or androgen.
Preferably, administration of recombinant human alpha-
fetoprotein inhibits the proliferation of cells of the
neoplasm or, alternatively, kills cells of the neoplasm
in the mammal. The method further includes administering
to the mammal a chemotherapeutic agent.
In another aspect, the invention features a method
of protecting a mammal from developing a neoplasm,
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involving administering to the mammal a therapeutically
effective amount of recombinant human alpha-fetoprotein.
Preferably, the recombinant human alpha-fetoprotein is
produced in a prokaryotic cell (e.g., E. coli) and is
5 unglycosylated.
In another aspect, the invention features a hybrid
cytotoxin including a recombinant human alpha-fetoprotein
(or a fragment or analog thereof) linked to a cytotoxic
agent. Examples of such cytotoxic agents include,
without limitation, diphtheria toxin, Pseudomonas
exotoxin A; ricin and other plant toxins such as abrin,
modeccin, volkensin, viscumin; chlorea toxin (produced by
Vibrio cholerae bacteria); the so-called "Shiga-like"
toxins (produced by E. coli and other enteric bacteria);
Salmonella heat-labile enterotoxin; and E. coli heat-
labile enterotoxin. In other preferred embodiments, the
cytotoxic agent is non-proteinaceous. Examples of such
non-proteinaceous cytotoxic agents include, without
limitation, anti-cancer agents such as doxorubicin, as
well as a-emitting radionuclides such as astatine and f3-
emitting nuclides such as yttrium. Preferably, the
cytotoxic agent of the hybrid cytotoxin is linked by a
peptide bond to the recombinant human alpha-fetoprotein,
and the hybrid toxin is produced by expression of a
genetically engineered hybrid DNA molecule. In other
preferred embodiments, the cytotoxic agent of the hybrid
cytotoxin is a protein; such a cytotoxic agent is
chemically conjugated to the recombinant human alpha-
fetoprotein.
In other aspects, the invention features a
detectably-labelled recombinant human alpha-fetoprotein
or a detectably-labelled fragment or analog thereof
capable of binding to a human neoplastic cell.
Preferably such a molecule is labelled with a
radionuclide, e.g., technetium-99m, iodine-125, iodine-
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131, or indium. Other detectable labels include, without
limitation, enzymes, fluorophores, or other moieties or
compounds which emit a detectable signal (e.g.,
radioactivity, fluorescence, color) or emit a detectable
signal after exposure of the label to its substrate or,
alternatively, the detectable signal can be an epitope
recognized by an antibody (e.g., an epitope of alpha-
fetoprotein or an epitope which is specifically
engineered into the recombinant alpha-fetoprotein such as
the HA or mvc epitopes). Preferably, the molecule
targets a malignant tumor (e.g. a breast tumor, a
prostate tumor, or a carcinoma) which expresses a
receptor which is recognized by the recombinant human
alpha-fetoprotein (or fragment or analog thereof).
Typically, such recombinant alpha-fetoprotein is produced
in a prokaryotic cell (e.g., E. coli) and is
unglycosylated.
Detectably-labelled recombinant human alpha-
fetoprotein (or is a fragment or analof thereof) is
useful for methods of imaging a neoplastic cell-
containing region in a human patient in vivo. In
general, the method involves: (a) providing a detectably-
labelled molecule of recombinant human alpha-fetoprotein
(or a fragment or analog thereof); (b) administering the
molecule to the patient; (c) allowing the labelled
molecule to bind and allowing unbound molecule to be
cleared from the region; and (d) obtaining an image of
the neoplastic cell-containing region. Preferably, the
region is the breast or is the prostate. In other
preferred embodiments, the region, without limitation, is
liver tissue, is lung tissue, is spleen tissue, is
pancreatic tissue, is brain tissue, is lymph tissue, or
is bone marrow. Preferably, the image is obtained using
dynamic gamma scintigraphy.
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Detectably-labelled recombinant human alpha-
fetoprotein (or a fragment or analog thereof) can also be
used in a method for diagnosing a neoplasm in a mammal
(e.g., a human patient). Such a method includes: (a)
contacting the biological sample with the detectably-
labelled molecule of recombinant human alpha-fetoprotein;
and (b) detecting the label bound to the sample, where
the detection of label above background levels is
indicative that the patient has a neoplasm. Preferably,
the method involves a biological sample including cells
fixed and sectioned prior to the contacting step, and the
label bound to the sample is bound to areas corresponding
to the cell membrane of the cells. In preferred
embodiments, the biological sample is from the breast or
prostate of a human patient.
Detectably-labelled recombinant human alpha-
fetoprotein (or fragment or analog thereof) can also be
used in a method for detecting a neoplasm a mammal in
vivo. Such a method includes: (a) administering a
diagnostically effective amount of the detectably-
labelled molecule of recombinant human alpha-fetoprotein;
and (b) detecting the presence of the detectable label
bound to a tissue of the mammal, where an amount of label
above background levels is indicative of the presence of
the neoplasm in the mammal.
In preferred embodiments, the method involves a human
patient suspected of having a breast cancer, and the
tissue is breast tissue. In other preferred embodiments,
the method involves a human patient suspected of having a
prostate cancer, and the tissue is prostate tissue.
Preferably, the detectably labelled recombinant human
alpha-fetoprotein is linked to a radionuclide (e.g.,
technetium-90) and the detection step is accomplished by
radioimaging (e.g., dynamic gamma scintigraphy).
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In another aspect, the invention features kits for
detecting a neoplasm or any cell expressing a receptor
which is recognized by recombinant human alpha-
fetoprotein (or a fragment or analog thereof) in vivo, in
situ or in vitro. In general, the kits include a
recombinant human alpha-fetoprotein which is recognized
by a neoplasm, and which may be detectably labeled. if
the recombinant human alpha-fetoprotein is unlabelled, a
second reagent containing a detectable label (e.g. a
radionuclide such as technetium-90, iodine-125, iodine-
131, or indium) is preferably included. Where the
detectable label is an enzyme, the kit further includes a
substrate reagent for the enzyme. The kit may also
include a reagent for linking the detectable label to the
recombinant alpha-fetoprotein. In another embodiment,
the kit for detecting a neoplasm or any unwanted cell
expressing a receptor which is recognized by recombinant
human alpha-fetoprotein (or a fragment or analog thereof)
includes a reagent containing an antibody which
specifically binds the recombinant human alpha-
fetoprotein and a reagent including a detectably labeled
recombinant human alpha-fetoprotein that is specifically
bound by the anti-alpha-fetoprotein antibody.
Preferably, the recombinant human alpha-fetoprotein of
the kit is produced in a prokaryotic cell (E. coli) and
is unglycosylated.
The use of recombinant human alpha-fetoprotein for
the treatment and diagnosis of cancer offers a number of
advantages. For example, rHuAFP can be administered
directly to a tumor site. Recombinant HuAFP can also be
chemically defined and synthesized, and prepared in large
quantities using the techniques of recombinant DNA, e.g.,
those which are described herein. Moreover, unlike
conventional cancer chemotherapies and radiotherapies,
recombinant human alpha- fetoprotein causes minimal side
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effects such as nausea, vomiting, and neurotoxicity.
Accordingly, relatively high doses of rHuAFP can be
safely administered.
The diagnostic methods of the invention are
advantageous since they allow for rapid and convenient
diagnosis of a neoplasm. For example, the use of rHuAFP
as a diagnostic agent (e.g., by radioimaging using
scintigraphy) is especially advantageous for real time
imaging of cancer for pre-surgical or intraoperative
localization and for staging of a cancer, e.g., breast
cancer, as well as during post-surgical examinations.
The use of such diagnostic procedures permits non-
invasive determination of the presence, location, or
absence of a neoplasm which is advantageous for
monitoring the condition of a patient.
In still other aspects, the invention features a
cell culture medium including recombinant human alpha-
fetoprotein or a cell-stimulating fragment or analog
thereof. This aspect of the invention is based on my
discovery that unglycosylated recombinant human alpha-
fetoprotein made in a prokaryote (e.g., E. coli) is a
cell proliferative agent, e.g., promotes the growth of
bone marrow in vitro. Preferably, such recombinant human
alpha-fetoprotein is produced in a prokaryotic cell (E.
coli) and is unglycosylated.
Accordingly, this aspect of invention features a
method of cell culture, the method including (a)
providing a cell culture medium including recombinant
human alpha-fetoprotein; (b) providing a cell; (c) and
growing the cell in the medium, where the cell
proliferates, and is maintained. Preferably, the cell is
a mammalian cell. Examples of such mammalian cells
include bone marrow cells (e.g., T cells, natural killer
cell, lymphocyte), hybridomas, or a genetically-
engineered cell line. Examples of other cells include
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hematopoietic cells such as stem cells, blast cells,
progentior cells (e.g., erythroid progenitor cells such
as burst-forming units and colony-forming units),
myeloblasts, macrophages, monocytes, macrophages,
lymphocytes, T-lymphocytes, B-lymphocytes, eosinophils,
basophils, tissue mast cells, megarkaryocytes (see e.g.,
Best and Taylor's Physiological Basis of Medical
Practice, John B. West, ed., Williams & Wilkins,
Baltimore). In other preferred embodiments the method
involves ex vivo cell culture.
In another aspect, the invention features a method
for inhibiting myelotoxcity in a mammal (e.g., a human
patient) involving administering to the mammal a
therapeutically effective amount of recombinant human
alpha-fetoprotein or a myelotoxic-inhibiting analog or
fragment thereof. Preferably, the recombinant human
alpha-fetoprotein is produced in a prokaryotic cell (E.
coli) and is unglycosylated.
In another aspect, the invention features a method
of inhibiting suppression of bone marrow cell
proliferation in a mammal, the method involving
administering to the mammal an effective amount of
recombinant alpha-fetoprotein or an anti-suppressive
fragment or analog thereof. Preferably, the recombinant
human alpha-fetoprotein is produced in a prokaryotic cell
(e.g., E. coli) and is unglycosylated.
In another aspect, the invention features a method
of promoting bone marrow cell proliferation in a mammal,
involving administering to the mammal an effective amount
of recombinant human alpha-fetoprotein or a cell-
stimulating fragment or analog thereof. Preferably, the
recombinant human alpha-fetoprotein is produced in a
prokaryotic cell (e.g., E. coli) and is unglycosylated.
In another aspect, the invention features a method
of preventing bone marrow cell transplantation rejection
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in a mammal, involving administering to the mammal an
effective amount of recombinant human alpha-fetoprotein
or an anti-rejection fragment or analog thereof.
Preferably, the recombinant human alpha-fetoprotein is
produced in a prokaryotic cell (e.g., E. coli) and is
unglycosylated. According to
the methods of the invention, administration of rHuAFP
(or a fragment or analog thereof) can be an effective
means for promoting and boosting cell growth in vitro, ex
vivo, or in vivo. Additionally, administration of the
compounds of the invention can also be an effective means
of preventing or treating or ameliorating myleotoxcemia
in a mammal.
The use of rHuAFP (or a fragment or analog
thereof) as a principal component of tissue culture media
is advantageous since there is little potential for
contamination with pathogens.
By "human alpha-fetoprotein" is meant a
polypeptide having substantially the same amino acid
sequence as the protein encoded by the human alpha-
fetoprotein gene as described by Morinaga et al., Proc.
Natl. Acad. Sci., USA 80: 4604 (1983). The method of
producing recombinant human alpha-fetoprotein in a
prokaryotic cell is described in U.S. Pat. No. 5,384,250,
and according to the methods described herein.
By "expression control element" is meant a
nucleotide sequence which includes recognition sequences
for factors that control expression of a protein coding
sequence to which it is operably linked. Accordingly, an
expression control element generally includes sequences
for controlling both transcription and translation, for
example, promoters, ribosome binding sites, repressor
binding sites, and activator binding sites.
By "substantially the same amino acid sequence" is
meant a polypeptide that exhibits at least 80% homology
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with naturally occurring amino acid sequence of human
alpha-fetoprotein, typically at least about 85% homology
with the natural human alpha-fetoprotein sequence, more
typically at least about 90% homology, usually at least
about 95% homology, and more usually at least about 97%
homology with the natural human alpha-fetoprotein
sequence. The length of comparison sequences will
generally be at least 16 amino acids, usually at least 20
amino acids, more usually at least 25 amino acids,
typically at least 30 amino acids, and preferably more
than 35 amino acids.
Homology, for polypeptides, is typically measured
using sequence analysis software (e.g., Sequence Analysis
Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, WI 53705). Protein analysis
software matches similar sequences by assigning degrees
of homology to various substitutions, deletions,
substitutions, and other modifications. Conservative
substitutions typically include substitutions within the
following groups: glycine alanine; valine, isoleucine,
leucine; aspartic acid, glutamic acid, asparagine,
glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine.
As used herein, the term "substantially pure"
describes a protein or polypeptide which has been
separated from components which naturally accompany it.
Typically, a protein of interest is substantially pure
when at least 60% to 75% of the total protein in a sample
is the protein of interest. Minor variants or chemical
modifications typically share the same polypeptide
sequence. A substantially pure protein will typically
comprise over about 85 to 90% of the protein in sample,
more usually will comprise at least about 95%, and
preferably will be over about 99% pure. Normally, purity
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is measured on a chromatography column, polyacrylamide
gel, or by HPLC analysis.
A protein is substantially free of naturally
associated components when it is separated from the
native contaminants which accompany it in its natural
state. Thus, a protein which is chemically synthesized
or produced in a cellular system different from the cell
from which it naturally originates will be substantially
free from its naturally associated components. Thus the
term can be used to describe polypeptides and nucleic
acids derived from eukaryotic organisms which have been
synthesized in E. coli and other prokaryotes.
The present invention provides for substantially
pure human alpha-fetoprotein. Various methods for the
isolation of human AFP from biological material may be
devised, based in part upon the structural and functional
properties of human alpha-fetoprotein. Alternatively,
anti-AFP antibodies may immobilized on a solid substrate
to generate a highly specific affinity column for
purification of human AFP.
Besides substantially full-length polypeptides,
the present invention provides for biologically active
recombinant fragments or analogs of human alpha-
fetoprotein. For example, fragments active in ligand
binding or immunosuppression.
The natural or synthetic DNA fragments coding for
human alpha-fetoprotein or a desired fragment thereof
will be incorporated into DNA constructs capable of
introduction into and expression in cell culture. DNA
constructs prepared for introduction into such hosts will
typically include an origin of replication which can be
utilized by the host cell, a DNA fragment encoding the
desired portion of human alpha-fetoprotein, transcription
and translational initiation regulatory sequences
operably linked to the alpha-fetoprotein encoding
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segment, and transcriptional and translational
termination regulatory sequences operably, linked to the
alpha-fetoprotein encoding segment. The transcriptional
regulatory sequences will typically include a
heterologous promoter which is recognized by the host.
The selection of an appropriate promoter will depend upon
the host, but promoters such as the trp, tac and phage
promoters, tRNA promoters and glycolytic enzyme promoters
may be used under appropriate circumstances (Sambrook et
al. eds., Molecular Cloning: Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor, NY 1989). In
some instances it may be desirable to include
appropriately positioned recognition sequences for
factors capable of regulating transcription in the host
cell (e.g., the lac repressor of E. coli). Commercially
available expression vectors, which include the
replication system and transcriptional and translational
regulatory sequences together with convenient sites for
the insertion of a DNA fragment encoding the gene to be
expressed may be used.
The various promoters, transcriptional, and
translational described above are generally referred to
as an "expression control element."
It is also possible to integrate a DNA fragment
encoding all or part of human AFP into the host cell's
chromosome.
The vectors containing the DNA segments of
interest can be transferred into the host cell by well-
known methods, which vary depending on the type of
cellular host (Sambrook et al., supra). The term
"transformed cell" is meant to also include the progeny
of a transformed cell.
Prokaryotic hosts useful for high level expression
of recombinant proteins include: various strains of E.
coli, Bacillus subtilis, and Pseudomonas.
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The method of the invention provides a means by
which to generate large quantities of human alpha-
fetoprotein having biological activity. AFP produced
according to the method of the invention has biological
activity despite the fact that it is not modified in the
same fashion as naturally occurring human AFP.
By "immune cell anti-proliferative" is meant
capable of inhibiting the growth of an undesirable immune
cell (e.g., an autoreactive T cell as measured using the
assays described herein).
By "neoplasm" is meant any unwanted growth of
cells serving no physiological function. In general, a
cell of a neoplasm has been released from its normal cell
division control, i.e., a cell whose growth is not
regulated by the ordinary biochemical and physical
influences in the cellular environment. In most cases, a
neoplastic cell proliferates to form a clone of cells
which are either benign or malignant. Examples of
neoplasms include, without limitation, transformed and
immortalized cells, tumors, and carcinomas such as breast
cell carcinomas and prostate carcinomas.
By "therapeutically effective amount" is meant
a dose of unglycosylated recombinant human alpha-
fetoprotein or an anti-neoplasm fragment or analog
thereof capable of inhibiting the proliferation of a
neoplasm or is capable of inhibiting autoreactive immune
cell proliferation or stimulating the proliferation of a
cell (e.g., a bone marrow cell).
By "diagnostically effective amount" is meant a
dose of detectably-labelled recombinant human alpha-
fetoprotein or a detectably-labelled fragment or analog
thereof that can be detected within a targeted region in
a mammal (e.g., a human patient).
By "cell-stimulating" is meant increasing cell
proliferation, increasing cell division, promoting cell
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differentiation and/or development, or promoting cell
longevity.
By "myelotoxic-inhibiting" is meant inhibiting
myeloablation.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, and from the claims.
Detailed Description
The drawings will first be described.
Drawings
Fig. 1 is the nucleotide sequence (SEQ ID NO: 4)
and deduced amino acid sequence (SEQ ID NO: 5) of the
cDNA encoding human alpha-fetoprotein.
Fig. 2 is the 10% SDS-PAGE analysis of rHuAFP
Fragment I (SEQ ID NO: 11) (Lane A, MW marker; Lane B,
natural human alpha-fetoprotein (AFP); Lane C, unpurified
rHuAFP; Lane D, rHuAFP Fragment I, and Lane E, rHuAFP
(amino acids 1- 590 of Fig. 1, SEQ ID NO: 5).
Fig. 3 is a bar graph showing the inhibition of
human AMLR by E. coli-derived rHuAFP and domain
fragments.
Fig. 4 is a series of graphs (Figs. 4A-4D) showing
the purity and biochemical characteristics of
baculovirus- and E. coli-derived rHuAFP using
polyacrylamide gel electrophoresis and column
chromatography. Fig. 4A is a 10% non-denaturing alkaline
polyacrylamide gel showing the purity of rHuAFP. Mouse
amniotic fluid proteins (transferrin, AFP and albumin)
are shown in lane 1, natural HuAFP (lane 2), baculovirus-
derived rHuAFP (lane 3), and E. coli-derived rHuAFP (lane
4). Fig. 4B is a 10% sodium dodecyl sulfate-
polyacrylamide gel showing the purity of rHuAFP produced
using baculovirus and E. coli expression systems.
Molecular weight markers are shown in lane 1, natural
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HuAFP, baculovirus- and E. coli-derived rHuAFP are shown
in lanes 2, 3, and 4, respectively. Fig. 4C is a series
of FPLC chromatograms of natural HuAFP, baculovirus-
derived and E. coli-derived rHuAFP eluted on a MonoQ
anion exchange column. The superimposed chromatograms
-identify natural HuAFP (Chromatogram 1), baculovirus- and
E. coli-derived rHuAFP (Chromatograms 2 and 3,
respectively). Fig. 4D is a series of HPLC chromatograms
obtained by passing 50 pg of natural and rHuAFP by
passing through a reverse phase Delta Pak C18 column
(Waters) and eluting with a gradient of 0-100%
acetonitrile in 0.1% TFA. The superimposed chromatograms
identify natural HuAFP (Chromatogram 1) and baculovirus-
and E. coli-derived rHuAFP (Chromatograms 2 and 3,
respectively).
Fig. 5 is a bar graph showing that monoclonal
anti-natural HuAFP antibodies block immunosuppression of
the AMLR by rHuAFP produced using baculovirus and E. coli
expression systems. Immunosuppression by rHuAFP produced
using baculovirus and E. coli expression systems was
significant (p<0.002) and blocking of rHuAFP-mediated
immunosuppression by the AMLR by monoclonal anti-natural
HuAFP (a AFP) antibodies was also significant (p<0.03).
AMLR cultures were set up with 2 X 105 responding T cells
with 2.5 X 105 irradiated autologous non-T cells in the
presence or absence of protein, harvested at 144 hours,
and autoproliferation was measured by the amount of 3H-
thymidine incorporated by autoreactive T cells. Blocking
of the autoproliferative effects of rHuAFP was carried
out by adding murine anti-human AFP monoclonal antibodies
at a dilution of 1/8 (125 jig/ml) to AMLR cultures
suppressed by 100 gg/ml baculovirus derived (diagonal
bars) and by 100 g/ml of E. coli-derived (open bars)
rHUAFP. Control cultures consisted of the AMLR in the
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presence of 1/8 dilution of anti-human AFP (a AFP)
monoclonal antibodies.
Fig. 6 is a series of bar graphs (Figs. 6A and 6B)
showing the effects of rHuAFP-mediated immunosuppression
using human AMLR (Fig. 6A) and PBL (Fig. 6B) assays.
Fig. 6A shows the results of autologous mixed lymphocyte
reaction (AMLR) prepared by co-culturing 250,000 T cells
with an equivalent amount of autologous irradiated non-T
lymphocytes. Recombinant HuAFP preparations derived from
E. coli and baculovirus expression systems and albumin
were added at a concentration of 100 g/ml at the
initiation of culture. Proliferative responses were
measured at 144 hours by 3H-thymidine incorporation.
Fig. 6B shows the results of PBLs (2 x 105) stimulated
with 1 gg/ml ConA which were cultured in RPMI medium
supplemented with only 2 mg/ml albumin for 48 hrs.
Albumin and rHuAFP derived from E. coli and baculovirus
were added to the initiation of the cultures at a
concentration of 100 gg/ml. Proliferative responses were
measured as the amount of 3H-thymidine incorporated
during DNA synthesis. The SEM were determined to
represent less than 5% of the value of the mean.
Fig. 7 is a plasmid map of pVT-PlacZ.
Fig. 8 is a series of graphs showing the
inhibitory effects of the rHuAFP on kinetics of T cell
activation (Fig. 8A) and the dose-response relationship
of rHuAFP on autoproliferating T cells (Fig. 8B). Fig.
8A is a graph showing proliferative responses over a 4
day time course of cells cultured in the absence (v) and
in the presence of 100 tLg/ml (v) rHuAFP. (=) denotes the
background proliferation of the responder cell population
cultured separately. Recombinant HuAFP-mediated
suppression on the AMLR over the time course was
significant (p<0.01). Fig. 8B is a graph showing the
inhibition of autoproliferating T cells at 144 hours with
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amounts of rHuAFP ranging from 6-100 g/ml (v). (v)
denotes the control response of the reaction in the
absence of protein. Inhibition of autoreactive T cells
by rHuAFP in the range of 12.5-100 pg/ml is significant
(p<0.005).
Fig. 9 is a bar graph showing the effect of rHuAFP
on estrogen-stimulated post-confluent growth of MCF-7
breast cancer cells.
Fig. 10 is a bar graph showing murine bone marrow
proliferation in serum-free RPMI medium in the presence
or absence of both 400 g/ml rHuAFP and 5 g/ml
transferrin.
Expression of Recombinant Human Alpha-Fetoprotein
Construction of a cDNA Library
A cDNA library was constructed with size-
fractionated cDNA (0.5-3 kb) prepared from poly(A)+ RNA
isolated from liver cells (-3 grams wet weight) of a 4.5
months old human abortus. (Alternatively, a fetal cDNA
library may be obtained from Clontech Laboratories, Inc.,
Palo Alto, CA.) Total RNA was prepared by the guanidium
thiocyanate method (Chirgwin et al., Biochemistry
18:5294, 1979), and mRNA was selected by oligo(dT)-
cellulose chromatography (Collaborative Research,
Bedford, MA) (Current Protocols in Molecular Biology,
Ausubel et al., eds., Wiley Interscience, New York,
1989). cDNA was synthesized using the Librarian II cDNA
synthesis kit (Invitrogen, San Diego, CA) and
fractionated on a 1% agarose gel. Fragments of 0.5 to 3
kb were extracted and ligated to vector pTZ18-RB
(Invitrogen), and used to transform competent E. coli
DHlaF' (Invitrogen). Colony lifts were performed with
Colony/Plaque Screen filters (DuPont, Wilmington, DE),
and the transferred bacterial colonies were lysed and
denatured by incubation in a solution of 0.5M NaOH, 1.5M
NaCl for 10 min. The filters were washed for 5 min in
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1.5M NaCl, 0.50M Tris-HC1 (pH 7.6), and air dried.
Filters were then washed 5 times in chloroform, soaked in
0.3M NaCl to remove cellular debris, and then air dried.
The DNA was fixed to the nitrocellulose by baking at 80
under vacuum for 2 hrs. The baked filters were
prehybridized for 3 hr at 37 C in 6X SSC (1X SSC = 150mM
NaCl, 15mM sodium citrate [pH 7.0]), 1X Denhardt's
solution (0.2 g/l polyvinylpyrrolidone, 0.2 g/1 BSA, 0.2
g/l Ficoll 400), 0.05% sodium pyrophosphate, 0.5 % SDS,
and 100 Ag/ml E. coli DNA. Hybridization was performed
for 18-24 hr at 37 C in the same solution without SDS,
containing 1-2 x 106 cpm/ml of two oligonucleotides 32P-
labelled by 5'-end phosphorylation (Current Protocols in
Molecular Biology, supra). The sequence of the
oligonucleotides used for probing the library: 5'-
TGTCTGCAGGATGGGGAAAAA-3' (SEQ ID NO: 1) and
5'-CATGAAATGACTCCAGTA-3' (SEQ ID NO: 2), correspond to
positions 772 to 792 and positions 1405 to 1422 of the
human AFP coding sequence respectively. Filters were
washed twice for 30 min at 37 C with 6X SSC, 0.05% sodium
pyrophosphate and once for 30 min at 48 C in the same
solution. Dried filters were exposed to Kodak XAR films
in the presence of Du Pont Cronex Lightning Plus
intensifier screens for 24-48 hr to identify positive
clones. Positive clones were isolated, amplified, and
subjected to Southern blot analysis (Current Protocols in
Molecular Biology, supra). Briefly, purified DNA was
hydrolyzed with the appropriate restriction enzymes, and
the resulting fragments were resolved on a 1% agarose
gel. The DNA was then transferred to a nitrocellulose
membrane. Hybridization conditions were as described
above except that a third 32P-labelled oligonucleotide
(5'-CATAGAAATGAATATGGA-3' (SEQ ID NO: 3), representing
positions 7 to 24 of the human AFP coding region) was
used in addition to the other two probes described above.
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Five positive clones were identified among the 3,000
colonies screened. One clone, pLHuAFP, was used in the
construction described below.
Construction of Full Length Human AFP cDNA
A construct containing a translation initiation
codon followed by the human AFP coding sequence and a
translation termination codon was created using the
following five DNA fragments.
Fragment 1: Two unphosphorylated oligonucleotides
were annealed to form a double-stranded DNA molecule
consisting of a 5'-end cohesive EcoRI recognition site,
followed by an ATG initiation codon and the first 60 bp
of the human AFP cDNA up to and including the Pstl
restriction site located at position 60 in the coding
sequence (In this scheme, nucleotide 1 is the first
nucleotide of the first codon (Thr) in the mature protein
and corresponds to nucleotide 102 of Morinaga et al.,
supra). This fragment was ligated to pUC119 (pUC19 with
the intergenic region of M13 from HgiA I at 5465 to Ahall
at 5941 inserted at the Nde I site of pUC19) linearized
with Eco RI and Pst I. The resulting DNA was amplified
in E. coli NM522 (Pharmacia, Piscataway, NJ) The EcoRI-
Pstl insert was recovered by enzymatic digestion of the
recombinant plasmid followed by electrophoretic
separation on a 5% polyacrylamide gel and isolation from
the gel.
Fragment 2: A 97 bp human AFP cDNA fragment
(positions 57 to 153) was obtained by digesting pLHuAFP
with PstI and NsiI and gel purifying as described above.
This clone contains the entire coding region of human AFP
as well as 5' and 3' untranslated sequences.
Fragment 3: A 224 bp human AFP cDNA fragment
(positions 150 to 373) was obtained by digesting pLHuAFP
with NsiI and AlwNI and purifying as described above.
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Fragment 4: A 1322 bp human AFP cDNA fragment (positions
371 to 1692) was obtained by digesting pLHuAFP with AlwNI
and Styl and purifying as described above.
Fragment 5: Two unphosphorylated oligonucleotides
were annealed to form a 86 bp double-stranded DNA
contains the human AFP sequence from position 1693 in the
Styl site to the TAA termination codon that ends the AFP
coding region at position 1773, followed by a cohesive
BamHI site. This synthetic DNA was used without any
further manipulations.
pBlueScript (StrataGene, La Jolla, CA) was
completely hydrolyzed with EcoRI and BamHI, and added to
ligation mixture containing the five purified fragments
described above. A control ligation contained only the
linearized pBluescript. Portions of these two ligation
mixtures were used to transform competent E. coli DH5a
(GIBCO/BRL, Grand Island, NY). Recombinant plasmids were
isolated from several transformants and screened by
extensive restriction enzyme analysis and DNA sequencing.
One recombinant plasmid was selected and termed pHuAFP.
It was used for subsequent insertion of the human AFP
gene into several expression vectors. pHuAFP includes a
unique EcoRI-BamHI fragment that contains the complete
coding sequence for human AFP in addition to an ATG start
codon at the 5'-end and a TAA stop codon at the 3'-end.
AFP Expression Vectors
Successful high-level synthesis of human AFP in
E. coli was achieved in three different expression
systems. The TRP system gave direct expression. The RX1
system yielded a fusion protein containing 20 amino acids
encoded by trpE and vector sequences. The MAL system
expressed AFP fused to the malE gene product, a 42 kd
maltose-binding protein.
TRP Expression System: The 1186 bp EcoRI-BamHI
AFP encoding fragment of pHuAFP was cloned into the
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expression vector pTrp4 (Olsen et al., J. Biotechnol.
9:179, 1989) downstream of the try promoter and a
modified ribosome-binding site.
Briefly, pHuAFP was digested with EcoRI and BamHI,
and the ends were filled using Klenow polymerase. The
1186 bp AFP fragment was then gel purified. pTrp4 was
Clal digested, the ends were filled using Klenow
polymerase, and the linearized vector was gel purified.
The 1186 bp AFP fragment and pTrp4 backbone were ligated
and used to transform competent E. coli of the following
strains: DH5a, BL21 (F.W. Studier, Brookhaven National
Laboratory, Upton, NY), SG927 (American Type Culture
Collection, Rockville, MD: Acc. No. 39627), SG928 (ATCC
Acc. No. 39628), and SG935 (ATCC Acc. No. 39623).
RX1 Expression System: Human AFP cDNA was cloned
into the expression vector pRXl (Rimm et al., Gene
75:323, 1989) adjacent to the trP promoter and in the
translation frame of TrpE. The human AFP cDNA was
excised from pHuAFP by digestion with EcoRI and BamHI and
cloned into suitably treated pRX1 (BioRad Laboratories,
Hercules, CA). The E. coli strains described above and
CAG456 (D.W. Cleveland, Johns Hopkins University,
Baltimore, MD) were then transformed with the final
plasmid construction identified as pRX1/HuAFP.
MAL Expression System: AFP cDNA was into inserted
in the expression vector pMAL (New England Biolabs, Inc.,
Beverly, MA) under control of the tac promoter and in the
translation frame of MalE. Briefly, pHuAFP was
hydrolysed with BamHi and the ends made blunt using
Klenow polymerase. The human AFP cDNA was released from
the rest of the plasmid DNA by EcoRI digestion and then
gel purified. The purified fragment was ligated to
appropriately digested pMAL-C. A correctly oriented
recombinant plasmid, designated pMAL/HuAFP, was used to
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transform E. coli DH5a, TBI (New England Biolabs) and
SG935.
The AFP coding region used in the construction of
the three expression vectors was sequenced and found to
encode full length AFP.
Expression of AFP in E. coli
Bacterial cultures were incubated at 30 C or 37 C
with aeration. Overnight cultures of E. coli were grown
in LB medium supplemented with the appropriate
antibiotics as required (Tetracycline-HC1 was at 50
g/ml, and ampicillin-Na was at 100 gg/ml).
TRP and RX1 Expression Systems: The trb promoter
was induced under tryptophan starvation conditions.
Induction was performed in M9CA medium prepared as
follows: 1 g Casamino acids (Difco Laboratories, Detroit,
MI), 6 g Na2HPO4, 3g KH2PO41 0.5 g NaCl, 1 g NH4C1 is
added to one liter milli-Q water (Millipore Corp.,
Bedford, MA), the pH adjusted to 7.4 and the solution
autoclaved. The cooled medium is made 2mM MgSO4, 0.1mM
CaCl2, and 0.2% glucose. After a 100-fold dilution of an
overnight culture in M9CA supplemented with antibiotics,
the cells were grown at 30 C to A550 of 0.4, harvested by
centrifugation, and stored as pellets at -20 C.
MAL Expression System: The tac promoter was
induced with the gratuitous inducer IPTG. Overnight
cultures were diluted 100-fold in LB medium supplemented
with antibiotics, and the cells grown at 37 C to A550 of
0.4. IPTG was then added to a final concentration of
0.3mM, and the bacteria incubated an additional 2 hr.
The cells were then harvested by centrifugation and
stored as pellets at -20 C.
Detection of AFP Expressed in E. Coli
Analytical studies were performed to determine the
expression and behavior of recombinant AFP. Cell pellets
were either suspended in SDS-lysis solution (0.16M Tris-
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HC1 [pH 6.8], 4% w/v SDS, 0.2M DTT, 20% glycerol, 0.02%
bromophenol blue), boiled for 5 min, and used for
analysis by SDS-PAGE or suspended in a lysis buffer
consisting of 10 mM Na2HPO41 30mM NaCl, 0.25% Tween 20,
10mM EDTA, 10mM EGTA and incubated with 1 mg/ml lysozyme
at 4 C for 30 min prior to sonication in pulse mode for 3
x 1 min at 50% power (Sonics and Materials, Danbury, CT:
model VC300 sonifier). The lysate was centrifuged at
1o,000g for 20 min, and the supernatant containing
soluble protein was decanted in a separate test tube and
frozen at -20 C until used. The pellet containing
insoluble protein was resuspended in SDS-lysis buffer,
boiled for 5 min and kept at -20 C until used. Total
protein released in SDS-lysis buffer, as well as soluble
and pellet fractions were analyzed by SDS-PAGE and
immunological detection following western blot transfer.
In these studies Coomassie blue stained gels were
routinely scanned with a video densitometer (BioRad,
model 620). This allowed a qualitative assessment of the
amount of recombinant AFP produced as a percentage of
total cellular protein.
Purification of AFP Expressed in the TRP System
All procedures were carried out at 4 C, unless
otherwise stated. Each frozen cell pellet from a one
liter culture was resuspended in 25 ml of lysis buffer A,
50mM Tris-HC1 [pH 7.5], 20% sucrose, 100 gg/ml lysozyme,
10 g/ml PMSF), and incubated for 10 min. EDTA was added
to a final concentration of 35mM, and the extract allowed
to stand a further 10 min. Following the addition of 25
ml of lysis buffer B (50mM Tris-HC1 [pH 7.5], 25mM EDTA,
0.2% Triton X-100), the lysate was incubated an
additional 30 min. The cell lysate was centrifuged at
12,000g for 20 min, and the precipitate containing the
recombinant AFP was washed twice with 50 ml of wash
buffer (50mM Tris-HC1 [pH 8.0], 10mM EDTA, 0.2% Triton X-
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100), followed each time by centrifugation as above. The
precipitate was dissolved in 50 ml of denaturation buffer
(O.1M K2HPO4 [pH 8.5], 6M guanidine-HC1, 0.1M 2-
mercaptoethanol), sonicated, and then mixed on a Nutator
(Clay Adams) for 4 hr. The solubilized extract was
diluted 50-fold in 50mM Tris-HC1, 100mM NaCl, 1mM EDTA,
and the recombinant AFP protein allowed to renature for
24 hr. This 50-fold dilution step is important because
prior to dilution AFP appears to be microaggregated.
Subsequent to dilution and reconcentration, AFP is not
aggregated. The solution was concentrated 100-fold on
YM10 membranes using an Amicon filtration unit, and
clarified through a Millex 0.22 gm membrane filter
(Millipore). The recombinant AFP was further purified at
room temperature on a Mono Q column (Pharmacia)
equilibrated in 20mM Tris-HC1 (pH 8.0) with bound
proteins eluted using a linear gradient of 0-100% 1M
NaCl, 20mM Tris-HC1 (pH 8.0). Fractions were analyzed by
SDS-PAGE, APAGE, and Western blotting.
These general techniques of polypeptide expression
and purification can also be used to produce and isolate
useful human alpha-fetoprotein fragments or analogs
(described below).
Polyacrylamide Gel Electrophoresis and Western
Immunodetection Procedures
SDS-PAGE in discontinuous buffer system and
alkaline-PAGE were performed according to Hames et al.
(Gel Electrophoresis of Proteins: A Practical Approach,
IRL Press, London, 1981) using the mini-Protean
electrophoresis apparatus (BioRad). Immunological
detection of recombinant human AFP following SDS-PAGE or
APAGE was accomplished by soaking the gels in transfer
buffer (12.5mM Tris-HC1, 96mM glycine, 20% methanol [pH
8.2]) for 15 min. Individual gels were then layered with
an Immobilon PVDF membrane (Millipore) and sandwiched
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between the two electrode grids of the mini-Protean
transfer device (BioRad), with the gels adjacent to the
cathode. The system was immersed in transfer buffer, and
a 150 mA current was applied for 2 hr. Unreacted sites
on the Immobilon PVDF sheets were blocked in 20mM Tris-
HC1 (pH 7.5), 500mM NaCl, 3% gelatin for 1 hr. Rabbit
anti-human AFP antiserum and goat anti-rabbit IgG
antibodies conjugated to alkaline phosphatase (BioRad)
were used as the primary and the secondary antibodies,
respectively. The alkaline phosphatase activity was
detected using 5-brome-4-chloro-3-indolyl phosphate and
p-nitroblue=tetrazolium (Bio-Rad).
Ouantitation of AFP Expression
Recombinant human AFP was quantitated using a
human AFP ELISA kit (Abbott Laboratories, Chicago, IL).
AFP yield was estimated by scanning silver stained
gels. When SG935 cells are transformed with the AFP
encoding plasmid that employs the Trp expression system,
AFP represents 2 to 5% of total cellular E. coli protein
(approximately 3-7 mg AFP per liter of culture). As
described above, most AFP in the initial extract is
insoluble. The above-described resolubilization
procedure permits 50-60% recovery of AFP in the form of
stable, semi-purified, monomeric AFP (approximate yield
50 mg/20 1 of E. coli). This can be further purified to
yield 25 mg of pure monomeric AFP.
N-Terminal Analysis
Automatic Edman degradations were performed using
a Porton protein/peptide gas phase microsequencer with an
integrated customized microbore HPLC to optimize
sequence. Protein sequence analysis was aided by the use
of selected programs within the PC/Gene software package
(Intelligenetics).
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Cloning, Expression, and Purification of HuAFP Using A
Baculovirus Expression System
Recombinant baculovirus expressing HuAFP (or a
fragment or analog therof) is constructed according to
standard methods known in the art (see, e.g., U.S. Pat.
No. 4,745,051). This process generally involves two
steps. The gene to be expressed, e.g., rHuAFP or a
fragment or analog thereof (described infra), is first
cloned into a plasmid transfer vector downstream from a
baculovirus promoter that is flanked by baculovirus DNA
derived from a nonessential locus, e.g., the polyhedrin
gene. This plasmid is then introduced into insect cells
along with circular wild-type genomic DNA for homologous
recombination to occur. Resulting recombinant progeny
are then screened, e.g., using sequential plaque assays
to purify recombinant virus away from the nonrecombinant
parental strain. Viral amplification is also generally
necessary to obtain sufficient virus for protein
expression. Recombinant virus are plaque purified and
their DNA structure confirmed using standard methods well
known in the art.
The rHuAFP cDNA fragment was isolated from plasmid
pI18 with EcoRI/BamHI and purified using Geneclean as
described infra. The cloning and expression of the HuAFP
cDNA in baculovirus was performed using plasmid pVT-
PLacZ. Human AFP cDNA was cloned into plasmid pVT-PlacZ
(Fig. 7) in frame with the 3'-end of the insect derived
melittin signal peptide sequence. The baculovirus vector
pVT-PlacZ was modified by replacing the multiple cloning
site with the oligonucleotide 5'-GATCTAGAATTCGGATCCGGT-3'
(SEQ ID NO: 21) and its complementary fragment,
containing EcoRI and BamHI restriction sites in the 5'
and 3' direction, reducing the number of non-AFP coding
nucleotides between the melittin signal peptide cleavage
site and the location of the AFP cDNA insert at the EcoRI
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endonuclease sequence. The insert was then ligated into
the modified pVT-PLacZ vectors at the EcoRI and BamHI DNA
sequences.
The generation of recombinant baculovirus
containing rHuAFP coding sequences was performed
according to standard techniques. Accordingly, purified
recombinant baculovirus containing the coding sequences
of HuAFP were generated by co-transfection of the pVT-
PLacZ transfer vectors and wild-type baculovirus,
followed by two rounds of plaque purification. Sf9
insect cells seeded at a density of 1 x 106 cells/ml in
500 ml spinner flasks were infected with recombinant
baculovirus in serum-free Grace medium at a multiplicity
of infection of 5. The supernatant containing secreted
rHuAFP was harvested and cells were removed by
centrifuging at 200 x g. The rHuAFP containing medium
was concentrated 10-20 fold by ultrafiltration with a
YM30 Amicon membrane, dialysed overnight against PBS and
then applied to a Con A lectin column (Pharmacia). Bound
rHuAFP was eluted with 0.4M methyl a-D mannopyranoside
and was purified by elution from Mono Q resin during a
linear gradient from 0-100% 1M NaCl in 20 mM phosphate
buffer, pH 8Ø Recombinant HuAFP was characterized
according to methods well known in the art.
2 found that baculovirus produced rHuAFP
represented approximately 20% of the total proteins
secreted into serum-free medium by the Sf9 insect cells.
This AFP was also found to be monomeric as analyzed by
non-reducing alkaline PAGE. The majority of the
baculovirus-derived HuAFP bound to immobilized Con A.
This resulted in effective removal of more than 90% of
contaminating proteins which were nonadhered to lectin
columns. Final purification of the baculovirus derived
rAFP preparations was accomplished by eluting protein
with 270-310 mM NaCl from MonoQ beads, yielding a single
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polypeptide with an apparent molecular mass of
approximately 68 kD. We obtained at least lmg of
purified protein was obtained per liter of growth
culture.
The baculovirus-derived rHuAFP molecular weight is
similar to the natural human molecule (Fig. 4B). This
finding, in addition to the binding of baculovirus-
derived rHuAFP and the observed non-adherence of E. coli-
derived rHuAFP to the ConA column, indicates that the
baculovirus-derived rHuAFP is glycosylated. However, the
degree of glycosylation of the BrAFP is expected to be
less than that of the native molecule, since Sf9 cells
infected with recombinant baculovirus have been
documented to be deficient in their ability to carry out
complex glycosylation normally observed with higher
eukaryotic derived proteins. Purity of the isolated
baculovirus-derived rHuAFP was verified as a single band
on APAGE and SDS-PAGE (Figs. 4A & 4B), and as a sole peak
on FPLC and HPLC chromatograms as shown in Figures 4C and
4D, respectively. N-terminal sequencing further
confirmed the identity of pure rHuAFP. The N-terminal
sequence of the rHuAFP was as follows: Asp-Leu-Glu-Phe-
Met-Thr-Leu-His-Arg-Asn (SEQ ID NO: 22). Western blot
analysis of serum free supernatants from recombinant
baculovirus infected Sf9 cells detected a single
immunoreactive band with monospecific anti-HuAFP Ab that
was absent form the supernatant of uninfected or wild-
type virus-infected Sf9 cells.
Experiments were performed to evaluate the
biological activity of the baculovirus produced HuAFP
according to methods known in the art. For example, the
immunosuppressive activity of 100 gg/ml of baculovirus-
produced HuAFP was assessed by its ability to suppress
human AMLR as described above. As shown in Figs. 5 and
6A, baculovirus-derived rHuAFP inhibited the
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proliferative response of autoreactive lymphocytes
stimulated by autologous non-T cells at 144 hours. The
addition of an identical amount of human serum albumin
failed to diminish lymphoproliferative responses.
To demonstrate that rHuAFP was the substrate
resposnsible for the inhibition of autoproliferating T
cells, blocking of the rHuAFP-mediated suppression of the
AMLR was performed using commercial murine anti-human AFP
monoclonal antibodies (MAb). As shown in Fig. 5,
suppression of proliferating autoreactive T cells by 100
gg/ml E. coli- and baculovirus-derived rHuAFP was
completely blocked by anti-HuAFP MAb. The addition of
100 gg/ml of HSA did not diminish the AMLR response and
the presence of MAb alone in the reaction culture was
without any effect.
In addition, we tested the biological activity of
rHuAFP to suppress the mitogen induced proliferation of
peripheral blood lymphocytes (PBL) in RPMI tissue culture
media supplemented only with 2 mg/ml purified human
albumin (ICN, Mississauga, ON). As shown in Table II
(below) and Fig. 6B, suppression of ConA stimulated PBLs
occurred with 100 g/ml rHuAFP whereas the same
concentration of albumin was ineffective.
Other rHuAFPs (e.g., rHuAFP (Amino acids 1(Thr) -
590 (Val); SEQ ID NO: 5); Domain I (Amino acids 1 (Thr) -
197 (Ser), SEQ ID NO: 6; Domain II (Amino acids 198 (Ser)
- 389 (Ser), SEQ ID NO: 7); Domain III (Amino acids 390
(Gln) - 590 (Val), SEQ ID NO: 8); Domain I + II (Amino
acids 1 (Thr) - 389 (Ser), SEQ ID NO: 9); Domain II +
III, (Amino acids 198 (Ser) - 590 (Val), SEQ ID NO: 10);
rHuAFP Fragment I (Amino acids 266 (Met) - 590 (Val), SEQ
ID NO: 11) can be produced using the above described
baculovirus expression system according to standard
methods, e.g., any of the methods described herein.
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In one working example, the vector pVT-PLacZ/HuAFP
(amino acids 1 - 590) is constructed by inserting the
cDNA for HuAFP into pVT-P10, an intermediate vector in
the construction of pVT-PLacZ (Richardson et al., (1992)
Engineering Glycoproteins for secretion using the
baculovirus expression system. In: Baculovirus and
Recombinant Protein Production Processes, eds., J.M.
Viak, E.-J. Schlaeger, and A.R. Bernard, Editiones Roche,
Basel, Switzerland. pp. 67-73.). The pVT-P10 vector is
digested with BamHI, followed by incubation with mung
bean Nuclease (New England Biolabs, Mississauga, Ont.).
The vector is then further hydrolyzed with EcoRl
downstream of the blunt-end BamHl site to facilitate
directional cloning of the HuAFP cDNA. The rHuAFP cDNA
encoding amino acids 1-590 is obtained by PCR
amplification, employing the following oligonucleotide
primers: (5'-AAAAAACTCGAGATACACTGCATAGAAATGAA-3'; SEQ ID
NO: 23), containing an Xhol site and (5'-
AA.AAAAGAATTCTTAAACTCCCAAAGCAGCACG-3'; SEQ ID NO:24),
containing an EcoRl site, and plasmid p118 as the
template DNA containing the coding region of HuAFP. The
PCR reaction is performed according to standard methods,
e.g., in a reaction mixture containing 34 L H2O, 10 L 10
x reaction buffer, 20 AL dNTP, 2 L DNA template, 10 AL 10
pmol/ L 5' primer, 10 L 10 pmol/ L 3' primer, 1 AL
glycerol, 10 AL DMSO, and 1 L Pfu polymerase.
Annealing, extension, and denaturation temperatures are
also performed according to standard contions, e.g.,
50 C, 72 C, and 94 C, respectively, for 30 cycles, using
the GeneAmp PCR System 9600 (Perking Elmer Cetus). DNA
from the PCR reaction is purified using the Genaclean kit
(Bio 11 Inc., LaJolla, CA). The rHuAFP cDNA fragment is
first digested with Xhol followed by treatment with mung
bean nuclease. Next, the HuAFP cDNA is digested with
EcoRl to facilitate directional cloning into pVT-plo.
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The PCR-produced rHuAFP cDNA is ligated into the blunt
ended 5' BamHl site and into the 3' EcoRl site. The Q-
galactosidase gene containing at the 3'end a
polyadenylation site from SV40, isolated from the vector
pJV-Nhel (Vialard et al., (1990) Synthesis of the
membrane fusion and hemagglutinin proteins of Measles
Virus, using a novel baculovirus vector containing the Q-
galactosidase gene, J. Virology 64: 37-50) by using the
restriction enzyme BamHl, is then inserted into the
compatible BgIII, producing the final construct: pVT-
PLacZ/HuAFP (containing amino acids 1-590). Such a
construct is then used for expressing rHuAFP (amino acids
1-590).
Fragments and Analogs
The invention includes biologically active
fragments of rHuAFP. A biologically active fragment of
rHuAFP is one that possesses at least one of the
following activities: (a) directs a specific interaction
with a target cell, e.g., binds to a cell expressing a
receptor which is recognized by rHuAFP (e.g., the
membrane of a cancer cell such as an MCF-7 or a bone
marrow cell); (b) halts, reduces, or inhibits the growth
of a neoplasm or an autoreactive immune cell (e.g., binds
to a cell surface receptor and imparts an anti-
proliferative signal); stimulates, increases, expands, or
otherwise causes the proliferation of a cell such as a
bone marrow cell (e.g., binds to a cell surface receptor
a proliferative or stimulating-signal); or blocks or
inhibits or prevents an immunopathologic antibody
reaction. The ability of rHuAFP fragments or analogs to
bind to a receptor which is recognized rHuAFP can be
tested using any standard binding assay known in the art.
Biological activity of such fragments and analogs are
tested according to methods known in the art (e.g., those
described herein).
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In general, fragments of rHuAFP are produced
according to the techniques of polypeptide expression and
purification described supra. For example, suitable
fragments of rHuAFP can be produced by transformation of
a suitable host bacterial cell with part of an HuAFP-
encoding cDNA fragment (e.g., the cDNA described above)
in a suitable expression vehicle. Alternatively, such
fragments can be generated by standard techniques of PCR
and cloned into the expression vectors (supra).
Fragments can also be produced by chemical synthesis
(e.g., by the methods described in Solid Phase Peptide
Synthesis, 2nd ed., 1984, The Pierce Chemical Co.,
Rockford, Il). Accordingly, once a fragment of rHuAFP is
expressed, it may be isolated by various chromatographic
and/or immunological methods known in the art. Lysis and
fractionation of rHuAFP-containing cells prior to
affinity chromatography may be performed by standard
methods. The ability of a candidate rHuAFP fragment to
exhibit a biological activity of alpha-fetoprotein is
assessed by methods known to those skilled in the art
(e.g., those described herein).
As is discussed above, a rHuAFP fragment may also
be expressed as a fusion protein with maltose binding
protein produced in E. coli. Using the maltose binding
protein fusion and purification system (New England
Biolabs, Beverly, MA), the cloned human cDNA sequence can
be inserted downstream and in frame of the gene encoding
maltose binding protein (malE), and the malE fusion
protein can then be overexpressed. In the absence of
convenient restriction sites in the human cDNA sequence,
PCR can be used to introduce restriction sites compatible
with the vector at the 5' and 3' end of the cDNA fragment
to facilitate insertion of the cDNA fragment into the
vector. Following expression of the fusion protein,
it can be purified by affinity chromatography. For
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example, the fusion protein can be purified by virtue of
the ability of the maltose binding protein portion of the
fusion protein to bind to amylose immobilized on a
column.
To facilitate protein purification, the pMalE
plasmid contains a factor Xa cleavage site upstream of
the site into which the cDNA is inserted into the vector.
Thus, the fusion protein purified as described above can
then be cleaved with factor Xa to separate the maltose
binding protein from a fragment of the recombinant human
cDNA gene product. The cleavage products can be
subjected to further chromatography to purify rHuAFP from
the maltose binding protein. Alternatively, a fragment
of rHuAFP may be expressed as a fusion protein containing
a polyhistidine tag can be produced. Such an alpha-
fetoprotein fusion protein may then be isolated by
binding of the polyhistidine tag to an affinity column
having a nickel moiety which binds the polyhistidine
region with high affinity. The fusion protein may then
be eluted by shifting the pH within the affinity column.
The rHuAFP can be released from the polyhistidine
sequences present in the resultant fusion protein by
cleavage of the fusion protein with specific proteases.
Recombinant HuAFP fragment expression products
(e.g., produced by any of the prokaryotic systems
described supra) may be assayed by immunological
procedures, such as Western blot, immunoprecipitation
analysis of recombinant cell extracts, or
immunofluorescence (using, e.g., the methods described in
Ausubel et al., Current Protocols in Molecular Biology,
Greene Publishing Associates and Wiley Interscience (John
Wiley & Sons), New York, 1994).
if desired, the purified recombinant gene product
or fragment thereof can then be used to raise polyclonal
or monoclonal antibodies against the human recombinant
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alpha-fetoprotein using well-known methods (see Coligan
et al., eds., Current Protocols in immunology, 1992,
Greene Publishing Associates and Wiley-Interscience). To
generate monoclonal antibodies, a mouse can be immunized
with the recombinant protein, and antibody-secreting B
cells isolated and immortalized with anon-secretory
myeloma cell fusion partner. Hybridomas are then
screened for production of recombinant human alpha-
fetoprotein (or a fragment or analog thereof)-specific
antibodies and cloned to obtain a homogenous cell
population which produces monoclonal antibodies.
As used herein, the term "fragment," as applied to
a rHuAFP polypeptide, is preferably at least 20
contiguous amino acids, preferably at least 50 contiguous
amino acids, more preferably at least 100 contiguous
amino acids, and most preferably at least 200 to 400 or
more contiguous amino acids in length. Fragments of
rHuAFP molecules can be generated by methods known to
those skilled in the art, e.g., proteolytic cleavage or
expression of recombinant peptides, or may result from
normal protein processing (e.g., removal of amino acids
from nascent polypeptide that are not required for
biological activity).
Recombinant HuAFP fragments of interest include,
but are not limited to, Domain I (amino acids 1 (Thr) -
197 (Ser), see Fig. 1, SEQ ID NO: 6), Domain II (amino
acids 198 (Ser) - 389 (Ser), see Fig. 1, SEQ ID NO: 7),
Domain III (amino acids 390 (Gln) - 590 (Val), see Fig.
1, SEQ ID NO: ), Domain I+II (amino acids 1 (Thr) - 389
(Ser), see Fig. 1, SEQ ID NO: 9), Domain II+III (amino
acids 198 (Ser) - 590 (Val), see Fig. 1, SEQ ID NO: 10),
and rHuAFP Fragment I (amino acids 266 (Met) - 590 (Val),
see Fig. 1, SEQ ID NO: 11). Activity of a fragment is
evaluated experimentally using conventional techniques
and assays, e.g., the assays described herein.
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The invention further includes analogs of full-
length rHuAFP or fragments thereof. Analogs can differ
from rHuAFP by amino acid sequence differences, or by
modifications (e.g., post-translational modifications)
which do not affect sequence, or by both. Analogs of the
invention will generally exhibit at least 80%, more
preferably 85%, and most preferably 90% or even 99%
amino acid identity with all or part of a rHuAFP amino
acid sequence. Modifications (which do not normally
alter primary sequence) include in vivo, or in vitro
chemical derivatization of polypeptides, e.g.,
acetylation, or carboxylation; such modifications may
occur during polypeptide synthesis or processing or
following treatment with isolated modifying enzymes.
Analogs can also differ from the naturally occurring
rHuAFP by alterations in primary sequence, for example,
substitution of one amino acid for another with similar
characteristics (e.g., valine for glycine, arginine for
lysine, etc.) or by one or more non-conservative amino
acid substitutions, deletions, or insertions which do not
abolish the polypeptide's biological activity. These
include genetic variants, both natural and induced (for
example, resulting from random mutagenesis by irradiation
or exposure to ethanemethylsulfate or by site-specific
mutagenesis as described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Press, 1989, or Ausubel et al., supra)). Also included
are cyclized peptide molecules and analogs which contain
residues other than L-amino acids, e.g., D-amino acids or
non-naturally occurring or synthetic amino acids, e.g.,
or y amino acids, or L-amino acids with non-natural side
chains (see e.g., Noren et al., Science 244:182, 1989).
Methods for site-specific incorporation of non-natural
amino acids into the protein backbone of proteins is
described, e.g., in Ellman et al., Science 255:197, 1992.
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Also included are chemically synthesized polypeptides or
peptides with modified peptide bonds (e.g., non-peptide
bonds as described in U.S. Pat. No. 4,897,445 and U.S.
Pat. No. 5,059,653) or modified side chains to obtain the
desired pharmaceutical properties as described herein.
Useful mutants and analogs are identified using
conventional methods, e.g., those described herein.
Recombinant HuAFP As An Immunosuppressive Agent
Immunosuppressive attributes of rHuAFP (or a
fragment or analog thereof) are evaluated by any standard
assay for analysis of immunoregulatory activity in vivo
or in vitro. As discussed infra, the art provides a
number of animal systems for in vivo testing of
immunosuppressive characteristics of rHuAFP (or a
fragment or analog thereof) on an autoimmune disease,
e.g., the nonobese diabetic (NOD) mouse. Furthermore, a
wide variety of in vitro systems are also available for
testing immunosuppressive aspects of rHuAFP e.g., one
such in vitro assay evaluates the inhibition of
autoantigen-induced proliferation of T cells in an
autologous mixed lymphocyte reaction (AMLR).
The following examples demonstrate that
unglycosylated rHuAFP and a fragment of rHuAFP inhibit T
cell autoproliferation in response to self antigens.
These examples are provided to illustrate, not limit, the
invention.
EXPERIMENTAL
MATERIALS AND METHODS
Gel Electrophoresis, Immunoblotting and
Purification
The purity and characterization of rHuAFP was
evaluated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and nondenaturing alkaline
PAGE (APAGE) according to standard methods. Gels were
subsequently analyzed either by staining with Coomassie
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brilliant blue or by transferring electrophoretically
separated polypeptides to Immobilon PVDF membranes
(Millipore, Mississauga, ON) for immunoblotting analysis.
Recombinant HuAFP-monospecific rabbit anti-natural HuAFP
polyclonal antibody complexes were identified by
alkaline-phosphatase-conjugated goat anti-rabbit IgG and
the immunoreactive bands were detected with the BCIP/NBT
color development solution (Bio-Rad Laboratories,
Mississauga, ON) according to the manufacturer's
instructions.
Column chromatography was performed according to
standard methods.
Polymerase Chain Reaction (PCR) rHuAFP Fragments
Plasmid constructs encoding fragments of human
alpha-fetoprotein were prepared using polymerase chain
reaction (PCR) techniques known to those skilled in the
art of molecular biology, using oligonucleotide primers
designed to amplify specific portions of the human alpha-
fetoprotein gene (see e.g., PCR Technology, H.A. Erlich,
ed., Stockton Press, New York, 1989; PCR Protocols: A
Guide to Methods and Applications, M.A. Innis, David H.
Gelfand, John J. Sninsky, and Thomas J. White, eds.,
Academic Press, Inc., New York, 1990, and Ausubel et.
al., supra).
The following six rHuAFP fragments were prepared
to evaluate their biological activity (e.g., according to
the -methods disclosed herein):
Domain I Amino acids 1 (Thr) - 197 (Ser), (Fig. 1, SEQ ID NO: 6)
Domain II Amino acids 198 (Ser) - 389 (Ser), (Fig. 1, SEQ ID NO: 7)
Domain III Amino acids 390 (Gin) - 590 (Vat), (Fig. 1, SEQ ID NO: 8)
Domain I + II Amino acids 1 (Thr) - 389 (Ser), (Fig. 1, SEQ ID NO: 9)
Domain II + III Amino acids 198 (Ser) - 590 (Vat), (Fig. 1, SEQ ID NO: 10)
rHuAFP Fragment I Amino acids 266 (Met) - 590 (Vat), (Fig. 1, SEQ ID NO: 11)
Amino acids sequences were deduced from those shown for
human alpha-fetoprotein (1(Thr) - 590 (Val); SEQ ID NO: 5
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in Fig. 1. Fragments of rHuAFP designated Domain I,
Domain II, Domain III, Domain I+II, Domain II+III and
rHuAFP Fragment I were synthesized using standard PCR
reaction conditions in 100 L reactions containing 34 gL
H2O, 10 L 10X reaction buffer, 20 gL 1 mM dNTP, 2 L DNA
template (HuAFP cloned in pI18), appropriate 5' and 3'
oligonucleotide primers (10 gL 10 pmol/ L 5' primer, 10
L 10 pmol/ L 3' primer), 1 gL glycerol, 10 L DMSO, and
1 gL Pfu polymerase (Stratagene, LaJolla, CA). Primers
used for PCR amplifications were:
Dom125 5'-AAAAAAGGTACCACACTGCATAGAAATGAA-3' (SEQ ID NO: 14)
DomI3 5'-AAAAAAGGATCCTTAGCTTTCTCTTAATTCTTT-3' (SEQ ID NO: 15)
DomII5 5-'AAAAAAATCGATATGAGCTTGTTAAATCAACAT-3' (SEQ ID NO: 16)
DomII3 51-AAAAAAGGATCCTTAGCTCTCCTGGATGTATTT-3' (SEQ ID NO: 17)
DomIII5 5'-AAAAAAATCGATATGCAAGCATTGGCAAAGCGA-3' (SEQ ID NO: 18)
DomIII3 5'-AAAAAAGGATCCTTAAACTCCCAAAGCAGCACG-3' (SEQ ID NO: 19)
5'rHuAFP Fragment I 51-AAAAAAATCGATATGTCCTACATATGTTCTCAA-3' (SEQ ID NO: 20)
Accordingly, primer pairs Dom125 and DomI3, DomII5 and
DomII3, DomIII5 and DomIII3, 5'rHuAFP Fragment I and
DomIII3, Dom125 and DomII3, and DomII5 and DomIII3 were
used to isolate cDNA sequences of Domain I, Domain II,
Domain III, rHuAFP Fragment I, Domain I+II, and Domain
II+III, respectively, of rHuAFP. Annealing, extension,
and denaturation temperatures were 50 C, 72 C, and 94 C,
respectively, for 30 cycles. PCR products were purified
according to standard methods. Purified PCR products
encoding Domain I and Domain I+II were digested
individually with KpnI and BamHI and cloned separately
into KpnI/BamHI- treated pTrp4. Purified PCR products
encoding Domain II, Domain III, Domain II+III, and rHuAFP
Fragment I were digested individually with Bsp106I and
BamHI and were cloned separately into Bspl06I/BamHI-
treated pTrp4. Each plasmid construct was subsequently
transformed into competent E. coli cells. Since the
expression product will begin with the amino acid
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sequence encoded by the translation start signal
methionine, it is expected that such signal will be
removed, or in any event, not affect the bioactivity of
the ultimate expression product.
Autoloctous Mixed Lymphocyte Reactions (AMLR)
Isolation of human peripheral blood mononuclear
cells (PBMC), their fractionation into non-T cell
populations, and the AMLR, were performed according to
standard procedures. Responder T cells were isolated by
passing 1.5 x 108 PMBC over a commercial Ig-anti-Ig
affinity column (Biotek Laboratories) and 2 X 105
responder cells were subsequently cultured with 2 x 105
autologous 137Cs-irradiated (2500 rads) non-T stimulator
cells from a single donor. The medium employed consisted
of RPMI-1640 supplemented with 20 mM HEPES (Gibco), 5 x
10-5 M 2-mercaptoethanol (BDH, Montreal, QC), 4 mM L-
glutamine (Gibco), 100 U/ml penicillin (Gibco) and 100
/cg/ml streptomycin sulfate, with the addition of 10%
fresh human serum autologous to the responder T cell
donor for the AMLR. Varying concentrations of purified
rHuAFP, human serum albumin (HSA), anti-HuAFP monoclonal
antibodies clone #164 (125 pg/ml final concentration in
culture) (Leinco Technologies, St. Louis, MO) were added
at the initiation of cultures. AMLR cultures were
incubated for 4 to 7 days, at 37 C in 95% air and 5% CO2.
At the indicated intervals, DNA synthesis was assayed by
a 6 hour pulse with 1 ACi of 3H-thymidine (specific
activity 56 to 80 Ci/mmole, ICN). The cultures were
harvested on a multiple sample harvester (Skatron,
Sterling, VA), and the incorporation of 3H-TdR was
measured in a Packard 2500 TR liquid scintillation
counter. Results are expressed as mean cpm the
standard error of the mean of triplicate or quadruplicate
cultures.
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Peripheral Blood Lymphocyte (PBL) Assays
Heparinized blood from normal donors was diluted
1:1 with PBS and human peripheral blood lymphocytes (PBL)
were separated from red blood cells by density
centrifugation on Ficoll-Hypaque (Sigma, St. Louis, MO).
They were washed at least 3 times with PBS and verified
for cell viability by the Trypan Blue exclusion method.
Human PBL (2.5 x 105 cells) were cultured according to
standard methods. Results are expressed as the mean cpm
thymidine incorporation SEM of triplicate cultures.
RESULTS
Expression and Purification
Purity of isolated rHuAFP expressed in E. coli was
verified as a single band on Coomassie stained APAGE and
SDS-PAGE are shown in Fig. 1A-1B, respectively. Soluble
monomeric rHuAFP derived from E. coli was obtained by
eluting a protein fraction containing rHuAFP employing Q-
sepharose chromatography. Approximately 1 mg of pure
rHuAFP per liter of bacterial culture was recovered as a
single homogeneous peak by FPLC Mono-Q anion exchange
with 220-230 mM NaCl and migrated at approximately 65 kD
on SDS-PAGE (Fig. 1B). Recombinant HuAFP exhibits a
lower molecular weight on SDS-PAGE than natural HuAFP,
since prokaryotic expression systems lack the enzymatic
machinery required for glycosylation of proteins.
Rechromatographed samples of pure rHuAFP on FPLC and HPLC
yielded a single peak as shown in Fig. 1C and Fig. 1D,
confirming the purity of the rHuAFP preparation. In
addition, N-terminal sequencing data correspond to the
expected amino acid sequence at the N-terminus of rHuAFP.
In addition, E. coli containing the expression
plasmid encoding rHuAFP were cultured as described above.
Fig. 2 (lane D) shows the SDS-PAGE profile of purified
rHuAFP Fragment I. N-terminal amino acid sequence
analysis showed that rHuAFP Fragment I possessed the
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amino acid sequence Ser267-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-
Thr275 (SEQ ID NO: 13) which corresponds to the expected
N-terminal amino acid sequence of rHuAFP Fragment I (see
Fig. 1, SEQ ID NO: 11) where the initiating methionine is
cleaved intracellularly.
Inhibition of the Autologous Mixed Lymphocyte
Reactions (AMLR)
The immunosuppressive activity of rHuAFP was
assessed by its ability to suppress human autologous
mixed lymphocyte reactions (AMLR). As shown in Fig. 8A,
rHuAFP inhibited the proliferative response of
autoreactive lymphocytes stimulated by autologous non-T
cells, throughout the 4 to 7 day time course measuring
autoproliferation. Results from dose-response studies
performed at the peak of T cell autoproliferation, as
shown in Fig. 8B, demonstrate that the addition of rHuAFP
at the initiation of cultures suppressed the AMLR in a
dose-dependent manner. Furthermore, parallel viability
studies established that the inhibitory activity of
rHuAFP on human autoreactive T cells was not due to non-
specific cytotoxic effects.
To further substantiate that rHuAFP was the agent
responsible for the inhibition of autoproliferating T
cells, blocking of rHuAFP-mediated suppression of the
AMLR was performed using commercial murine anti-human AFP
monoclonal antibodies (MAb). As illustrated in Fig. 5,
suppression of proliferating autoreactive T cells by 100
pg/ml of rHuAFP was completely blocked by anti-HuAFP MAb.
The addition of 100 pg/ml of HSA did not diminish the
AMLR response and the presence of MAb alone in the
reaction culture was without any effect.
Recombinant polypeptides produced in prokaryotic
expression systems are at risk for contamination with
host cell lipopolysaccharide (LPS) during their isolation
from bacteria. It has been demonstrated that small
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amounts of LPS can antagonize the biological activities
of cytokines, thereby impairing the immune responsiveness
of macrophages. Accordingly, the effect of endotoxin on
various rHuAFP preparations was evaluated by performing
AMLR experiments with recombinant protein depleted of
endotoxin by passage over Detoxi-gel (Pierce) versus that
of rHuAFP which was untreated. Results of these
experiments showed that both preparations had equivalent
levels of immunosuppressive activity.
As shown in Figure 8A and Figure 8B, the results
of this study also demonstrate that rHuAFP suppresses the
proliferation of autoreaction T cells with a potency
equivalent to glycosylated nHuAFP by eliciting inhibitory
effects on autoproliferating T cells throughout the in
vitro reactions, with highly significant inhibition being
achieved with rHuAFP concentrations ranging from 5 gg/ml
to 100 gg/ml. In addition, as shown in Table I and Fig.
3, rHuAFP Fragment I inhibited the proliferative response
of autoreactive lymphocytes stimulated by autologous non-
T cells at 144 hours. Moreover, as shown in Fig. 3,
Domains I and III also inhibited the AMLR.
Table I
Reaction Setup Thymidine Incorporation
(CPM)
T Cells 7118 964
AMLR 83103 6480
AMLR + rHuAFP Fragment I 29692 2963
(100 g/ml) 1 11
Inhibition of the Peripheral Blood Lymphocyte
Reactions (PBL)
The immunosuppressive activity of 100 gg/ml rHuAFP
Fragment I, and E. coli- and baculovirus-derived rHuAFP
(described below) was also assessed for the ability to
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suppress human peripheral blood lymphocyte reactions
(PBL). As shown in Table II, E. coli- and baculovirus-
derived rHuAFP and Fragment I (produced as described
above) were found to inhibit the proliferative responses
of Con A stimulated human peripheral blood lymphocytes.
Table II
3H-Thymidine incorporation (CPM SE)
No protein 102,353 t 5,566 91,502 4,333 99,700 4,464
100 g/ml Hunan Albumin 89,860 t 5,800 82,924 11,085 94,123 t 1,633
100 g/ml rHuAFP-E. soli 33,641 t 3,893 -
100 g/ml rHuAFP-Baculovirus - 31,331 6,303
104g/ml HuAFP Fragment I - - 39,019 t 161
Autoimmune Disease
As is discussed above, autoimmune diseases are
characterized by a loss of tolerance to self antigens,
causing cells of the immune systems, e.g., T or B cells
(or both), to react against self tissue antigens.
Autoimmune diseases may involve any organ system,
although some are affected more commonly than others.
Examples of tissues affected by autoimmune conditions
include: the white matter of the brain and spinal cord in
multiple sclerosis; the lining of the joints in
rheumatoid arthritis; and the insulin secreting Q islet
cells of the pancreas in insulin-dependent diabetes
mellitus. Other forms of autoimmune disease destroy the
connections between nerve and muscle in myasthenia gravis
or destroy the kidneys and other organs in systemic lupus
erythematosus. Examples of other autoimmune diseases
include, without limitation, Addison's disease, Crohn's
disease, Graves' disease, psoriasis, scieroderma, and
ulcerative colitis.
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The art provides a wide variety of experimental
animal systems, transgenic and non-transgenic, for
testing therapies for human illness involving autoimmune
diseases (see e.g., Paul, W.E., Fundamental Immunology,
2nd ed., Raven Press, New York, 1989; and Kandel et al.
Principles of Neural Science, 3rd ed., Appleton and
Lange, Norwalk, CT, 1991; and Current Protocols In
Immunology, Coligan, J.E., Kruisbeek, A.M., Margulies,
D.H., Shevach, E.M., and Strober, eds., Green Publishing
Associates (John Wiley & Sons), New York, 1992). Based
on the above-described experimental results showing
immunosuppressive activity of unglycosylated rHuAFP, it
is reasonable to believe that other autoimmune diseases
can be treated by administration of such rHuAFP (or a
fragment or analog thereof) produced in a prokaryotic
system. Accordingly, the invention provides the use of
rHuAFP (or a fragment or analog thereof) for treatment
(i.e., prevention or suppression or amelioration or
promotion of remission) of any autoimmune disease.
There now follow examples of animal systems useful
for evaluating the efficacy of recombinant human alpha-
fetoprotein or an immune cell anti-proliferative fragment
or analog thereof in treating autoimmune diseases. These
examples are provided for the purpose of illustrating,
not limiting, the invention.
Multiple Sclerosis
Multiple sclerosis (MS) is a demyelinating disease
involving scattered areas of the white matter of the
central nervous system. In MS, myelin basic protein and
proteolipid protein are the major targets of an
autoimmune response involving T lymphocytes, among other
immune system components. Loss of the myelin sheath of
nerve cells (demyelination) occurs, resulting in
neurological symptoms that culminate in coma or
paralysis.
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Experimental autoimmune encephalomyelitis (EAE) is
a primary model used in the art to examine and assess the
effectiveness of therapeutic agents for treating MS. EAE
is an inflammatory autoimmune demyelinating disease
induced in laboratory animals by immunization with
central nervous system tissue. When animals (e.g., mice,
rats, guinea pigs, rabbits, monkeys, etc.) are injected
with adjuvant, e.g., complete Freund's adjuvant, plus
myelin basic protein or proteolipid protein, EAE is
induced, which is similar, pathologically to MS (see
e.g., Alvord et al., Experimental Allergic
Encephalomyelitis-A Useful Model for Multiple Sclerosis,
Liss, New York, 1984; Swanborg, Meth. Enzymol. 162:413,
1988; and McCarron et al., J. Immunol., 147: 3296, 1991.)
To evaluate rHuAFP or a fragment or analog
thereof, EAE is induced in an appropriate laboratory
animal, e.g., a mouse or rabbit, according to methods
known in the art. To evaluate the compound's
immunosuppressive effect on EAE, i.e., its ability to
prevent or ameliorate EAE, the compound is administered
according to standard methods, e.g., intravenously or
intraperitoneal, at an appropriate dosage on a daily
basis. Generally, administration is initiated prior to
inducing EAE and/or after the clinical appearance of EAE.
Control animals receive a placebo, e.g., human serum
albumin, similarly administered as for rHuAFP or related
molecules. The effect of the test molecules on EAE is
monitored according to any standard method. For example,
weight loss and muscle paralysis in EAE-induced animals
is monitored on a daily basis. If desired, histological
inspection (e.g., by using any standard histochemical or
immunohistochemical procedure, see e.g., Ausubel et al.,
Current Protocols In Molecular Biology, Greene Publishing
Associates (John Wiley & Son), New York, 1994; Bancroft
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and Stevens, Theory and Practice of Histochemical
Techniques, Churchill Livingstone, 1982) of brain and
spinal cord tissues is performed and tissue samples
examined microscopically for evidence of EAE, e.g.,
evidence of perivascular cellular infiltrates.
Comparative studies between treated and control animals
are used to determine the relative efficacy of the-test
molecules in preventing or ameliorating EAE. A molecule
which prevents or ameliorates (decreases or suppresses or
relieves or promotes remission of) the symptoms of EAE is
considered useful in the invention.
Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a common chronic
illness in which the synovial membrane of multiple joints
becomes inflamed, causing damage to cartilage and bone.
RA is associated with human lymphocyte antigen (HLA)-DR4
and considered to be an autoimmune disorder involving T
cells, see e.g., Sewell et al., Lancet 341: 283, 1993.
RA results from a complex interaction of synovial cells
with various cellular elements (and their soluble
products) that infiltrate from the circulation into the
synovial lining of joints. A series of biological events
occur which ultimately lead to a lesion which invades and
erodes collagen and the cartilage matrix of the joint.
A number of animal models of RA, e.g., the MRL-
lpr/lpr mouse, are known in the art which develop a form
of arthritis resembling the human disease (see e.g.,
Fundamental Immunology, supra). Alternatively,
autoimmune collagen arthritis (ACA) and adjuvant
arthritis (AA) can be induced in an appropriate animal
according to standard methods.
To evaluate rHuAFP or a fragment or analog thereof
on immunosuppressive on RA, i.e., the compound's ability
to prevent or ameliorate RA, the test molecule is
administered to a MRL-lpr/lpr mouse according to standard
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methods, e.g., intravenously or intraperitoneally, at an
appropriate dosage on a daily basis. Generally,
administration is initiated prior to the onset of RA
and/or after the clinical appearance of RA. Control
animals receive a placebo, e.g., human serum albumin,
similarly administered as for rHuAFP or related
molecules. The effect of the test molecule on RA is
monitored according to standard methods. For example,
analysis of the cellular component(s) of a synovial joint
are monitored on a daily basis. If desired, histological
inspection (e.g., by using any standard histochemical or
immunohistochemical procedure, see e.g., Ausubel et al.,
supra; Bancroft and Stevens, supra) of the synovial joint
is performed and tissue samples examined microscopically
for evidence of RA, e.g., evidence of erosion of collagen
and cartilage matrix in a joint. Comparative studies
between treated and control animals are used to determine
the relative efficacy of the test molecule in preventing
or ameliorating RA. A test molecule which prevents or
ameliorates (decreases or suppresses or relieves or
promotes remission of) the symptoms of RA is considered
useful in the invention.
Myasthenia Gravis
Myasthenia gravis (MG) is a disorder of
neuromuscular transmission in which there are
autoantibodies against acetylcholine receptors of
neuromuscular junctions. Antibodies attack the junction,
causing weakness and paralysis. Females are afflicted
twice as often as males, typically during the third
decade of life. Muscular weakness is the predominant
feature of the disease. Clinical signs include drooping
of the eyelids and double vision. There is an
association between MG and hyperthyroidism.
Experimental autoimmune MG (EAMG) has been studied
in a variety of animals including rabbits, monkeys, Lewis
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rats and inbred strains of mice (see e.g., Principles of
Neural Science, supra), the symptoms of EAMG resemble the
essential characteristics of the human disease. A single
injection of acetylcholine receptor, e.g., purified from
the electric organs of the eel Torpedo californica, along
with adjuvants, causes an acute phase of weakness within
8 to 12 days and then chronic weakness after about 30
days. The response to the eel receptor is T cell
dependent. The C57BL/6 strain (H-2B) is a high responder
to Torpedo receptor and highly susceptible to EAMG.
To evaluate rHuAFP or a fragment or analog
thereof, EAMG is induced in an appropriate laboratory
animal, e.g., the C57BL/6 strain (H-2B) mouse, according
to methods known in the art. To evaluate the compound's
immunosuppressive effect on EAMG, i.e., its ability to
prevent or ameliorate EAMG, the compound is administered
according to standard methods, e.g., intravenously or
intraperitoneally, at an appropriate dosage on a daily
basis. Generally, administration is initiated prior to
inducing EAMG and/or after the clinical appearance of
EAMG. Control animals receive a placebo, e.g., human
serum albumin, similarly administered as for rHuAFP or
related molecules. The effect of the test molecules on
EAMG is monitored according to standard methods. For
example, nerve stimulation in an electromyographic muscle
assay (e.g., according to the methods of Pachner et al.,
Ann. Neurol. 11:48, 1982) in EAMG-induced animals can be
assayed. If desired, histological inspection (e.g., by
using any standard histochemical or immunohistochemical
procedure, see e.g., Ausubel et al., supra; Bancroft and
Stevens, supra) of tissue samples is performed and tissue
samples examined microscopically for evidence of EAMG,
e.g., evidence of monocyte infiltration and/or
autoantibody localization at acetylcholine receptors of
neuromuscular junctions. Comparative studies between
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treated and control animals are used to determine the
relative efficacy of the test molecules.in preventing or
ameliorating EAMG. A molecule which prevents or
ameliorates (decreases or suppresses or relieves or
promotes remission of) the symptoms of EAMG is considered
useful in the invention.
Insulin-Dependent Diabetes Mellitus
Diabetes is a disorder of glucose metabolism.
Insulin-dependent diabetes mellitus (IDDM), also known as
Type I diabetes, is an autoimmune disease characterized
by T-cell mediated destruction of pancreatic $ cells in
the islets of Langerhans, accompanied by an immune
response to a diversity of self peptides leading to
hyperglycemia, among other pathological events. IDDM
patients depend on exogenous insulin to maintain normal
glucose metabolism. Humans at risk for developing IDDM
can be identified prior to onset of hyperglycemia by the
abnormal occurrence of autoantibodies to insulin, islet
cells, glutamic acid carboxylase, as well as other
autologous proteins (see e.g., Baekkeskov et al., J.
Clin. Invest. 79:926, 1987; Dean et al., Diabetologia 29:
339, 1986; Rossini et al., Annu. Rev. Immunol. 3:289,
1985; Srikanta et al., N. Encrl. J. Med. 308:322, 1983).
Autoantibody patterns, in general, are predictive for the
eventual disease progression and/or risk for developing
the disease (see e.g., Keller et al., Lancet 341:927,
1993).
Examples of animal models which spontaneously
develop IDDM resembling the human disease include the
Bio-Breeding (BB) rat and nonobese diabetic (NOD) mouse.
Diabetes is also experimentally induced by
streptozotocin.
The BB rat spontaneously develops a disease
similar to IDDM, with insulitis (infiltration of
mononuclear cells into the pancreatic islets) and
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autoantibodies against self cells and insulin (see e.g.,
Baekkeskov et al., J. Clin. Invest. 79:926, 1987; Rossini
et al, supra; Nakhooda et al., Diabetes 26: 100, 1977;
Dean et al., Clin. Exp. Immunol. 69: 308, 1987).
NOD mice typically develop insulitis between 5 and
8 weeks of age, and by 7 months 70% of the females and
40% of the males become diabetic. T cells transferred
from diabetic mice to young nondiabetic NOD mice induce
diabetes within 2 to 3 weeks (see e.g., Bendelac et al.,
J. Exp. Med. 166:823, 1987). NOD mice usually die within
1 to 2 months after the onset of diabetes unless they
receive insulin therapy.
Chemically induced diabetes is accomplished using
multiple injections of small doses of streptozotocin, a
drug toxic for pancreatic 6 cells, which causes severe
insulitis and diabetes (see e.g., Kikutani et al., Adv.
Immunol. 51:285, 1992).
Accordingly, the art provides a variety animal
models resembling human IDDM which can be used to examine
and assess approaches for the prevention or amelioration
of diabetes involving rHuAFP (or a fragment or analog
thereof).
To evaluate the immunosuppressive effect of rHuAFP
or a fragment or analog thereof on the development of
diabetes mouse, i.e., the compound's ability to treat or
prevent insulitis and diabetes, the test compound is
administered to an appropriate test animal, e.g, a NOD
mouse, according to standard methods, e.g., intravenously
or intraperitoneally, at an appropriate dosage on a daily
basis. Generally, administration is initiated prior to
the onset of insulitis and diabetes and/or after the
clinical appearance of diabetic characteristics. Control
animals receive a placebo, e.g., human serum albumin,
similarly administered as for rHuAFP or related
molecules. The effect of test molecules on insulitis and
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diabetes is monitored according to standard methods. For
example, weight loss, ketone body formation, and blood '
glucose concentration is monitored on a daily basis. If
desired, histological inspection (e.g., by using any
standard histochemical or immunohistochemical procedure,
see e.g., Ausubel et al., supra; Bancroft and Stevens,
supra) of pancreatic islet cells is performed and tissue
samples examined microscopically for evidence of
insulitis and (3 cell destruction. Comparative studies
between treated and control animals are used to determine
the relative efficacy of the test molecules in preventing
or ameliorating the diabetic condition. A molecule which
prevents or ameliorates (decreases or suppresses or
relieves or promotes remission of) the symptoms of
diabetes, e.g., IDDM, is considered useful in the
invention.
Systemic Lupus Erythematosus
Systemic lupus erythematosus (SLE) is a severe
systemic autoimmune disease. About 90% of patients with
this disease are young women. This marked preponderance
of females is not seen before puberty or after menopause.
The illness generally begins in young adulthood when a
characteristic skin rash appears over cheekbones and
forehead. Hair loss is common, as is severe kidney
damage, arthritis, accumulation of fluid around the heart
and inflammation of the lining of the lungs. In nearly
half of the patients the blood vessels of the brain also
become inflamed, leading to paralysis and convulsions.
The activity of the disease, like other autoimmune
diseases, can fluctuate: long quiescent periods of good
health can terminate abruptly and inexplicably with the
onset of a new attack. A large number of different
autoantibodies are known to occur in SLE, e.g.,
autoantibodies against DNA, RNA and histones (see, e.g.,
Fundamental immunology, supra)
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A number of animal models of human SLE, e.g.,
inbred mouse strains including NZB mice and their F1
hybrids, MRL mice, and BXSB mice, are known in the art
(see e.g., Bielschowsky et al. Proc. Univ. Otago Med.
Sch. 37:9, 1959; Braverman et al., J. Invest. Derm. 50:
483, 1968; Howie et al. Adv. Immunol. 9:215, 1968;
Genetic Control of Autoimmune Disease, Rose, M., Bigazzi,
P.E., and Warner, N.L. eds., Elsevier, Amsterdam, 1979;
and Current Protocols In Immunology, supra). For
example, the NZBxNZW F1 mouse is an excellent model of
human SLE, female mice develop high levels of anti-double-
and single-stranded DNA autoantibodies, other anti-
nuclear antibodies, and renal disease; death usually
occurs at approximately 8 months (see e.g.,
Theofilopoulos et al., Adv. Immunol. 37:269, 1985).
To evaluate the immunosuppressive effect of rHuAFP
or a fragment or analog thereof on SLE, i.e., the
compound's ability of rHuAFP to prevent or ameliorate
SLE, test compounds are administered to an appropriate
animal, e.g., the NZBxNZW F1 mouse, according to standard
methods, e.g., intravenously or intraperitoneally, at an
appropriate dosage on a daily basis. Generally,
administration is initiated prior to the onset of SLE
and/or after the clinical appearance of SLE. Control
animals receive a placebo, e.g., human serum albumin,
similarly administered as for rHuAFP or related
molecules. The effect of the test compound on SLE is
monitored according to standard methods. For example,
analysis of autoantibodies, e.g., anti-DNA antibodies can
be monitored. If desired, histological inspection (e.g.,
by using any standard histochemical or
immunohistochemical procedure, see e.g., Ausubel et al.,
supra; Bancroft and Stevens, supra) of kidney tissue is
performed and tissue samples examined microscopically for
evidence of SLE, e.g., evidence of lupus nephritis.
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Comparative studies between treated and control animals
are used to determine the relative efficacy of the test'
compounds in preventing or ameliorating SLE. A molecule
which prevents or ameliorates (decreases or suppresses or
relieves or promotes remission of) the symptoms of SLE is
considered useful in the invention.
Therapeutic Administration
As demonstrated above, recombinant alpha-
fetoprotein, e.g., rHuAFP (or a fragment or analog
thereof) is effective in inhibiting proliferation of
autoimmune cells and accordingly is useful for the
prevention or amelioration of autoimmune diseases
including, but not limited to, multiple sclerosis,
rheumatoid arthritis, diabetes mellitus, systemic=lupus
erythematosus, and myasthenia gravis. Accordingly,
recombinant human alpha-fetoprotein (or a fragment or
analog thereof) can be formulated according to known
methods to prepare pharmaceutically useful compositions.
Recombinant alpha-fetoprotein, e.g., rHuAFP (or a
fragment or analog thereof), is preferably administered
to the patient in an amount which is effective in
preventing or ameliorating the symptoms of an autoimmune
disease. Generally, a dosage of 0.1 ng/kg to 10 g/kg body
is adequate. If desired, administration is performed on
a daily basis. Because there are no known adverse side
effects related to recombinant human alpha-fetoprotein,
it is believed that relatively high dosages can be safely
administered. For example, treatment of human patients
will be carried out using a therapeutically effective
amount of rHuAFP (or a fragment or analog thereof) in a
physiologically acceptable carrier. Suitable carriers
and their formulation are described for example in
Remington's Pharmaceutical Sciences by E.W. Martin. The
amount of rHuAFP to be administered will vary depending
upon the manner of administration, the age and body
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weight of the patient, and with the type of disease, and
size of the patient predisposed to or suffering from the
disease. Preferable routes of administration include,
for example, subcutaneous, intravenous, intramuscular, or
intradermal injections which provide continuous,
sustained levels of the drug in the patient. In other
preferred routes of administration, rHuAFP can be given
to a patient by injection or implantation of a slow
release preparation, for example, in a slowly
dissociating polymeric or crystalline form; this sort of
sustained administration can follow an initial delivery
of the drug by more conventional routes (for example,
those described above). Alternatively, rHuAFP can be
administered using an infusion pump (e.g., an external or
implantable infusion pump), thus allowing a precise
degree of control over the rate of drug release, or
through installation of rHuAFP in the nasal passages in a
similar fashion to that used to promote absorption of
insulin. As an alternative to nasal transmucosal
absorption, rHuAFP can be delivered by aerosol deposition
of the powder or solution into the lungs.
Furthermore, the method(s) of the invention can
also employ combination therapy in which rHuAFP is
administered either simultaneously or sequentially with a
therapeutic agent such as a general or specific
tolerizing agent, e.g., an anti-idiotypic agent (e.g., a
monoclonal) or a therapeutic vaccine or an oral agent
(e.g., insulin, collagen or myelin basic protein) or a
cytokine (e.g., I1-15) or an interferon (c-interferon) or
an immunosuppressive agent. Preferably, an
immunosuppressive agent is administered in an effective
dose which is lower than the standard dose when the
immunosuppressive agent is used by itself. Preferred
immunosuppressive agents are cyclosporine, FK-506,
steroids, azathioprine, or 15-deoxyspergualin.
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Treatment is started generally with the diagnosis
or suspicion of an autoimmune disease and is generally
repeated on a daily basis. Protection or prevention from
the development (or progression or exacerbation) of an
autoimmune disease is also achieved by administration of
rHuAFP prior to the onset of the disease. If desired,
the efficacy of the treatment or protection regimens is
assessed with the methods of monitoring or diagnosing
patients for autoimmune disease.
Recombinant Human AFP in the Treatment and Diagnosis of
Cancer
CYTOTOXIC AGENTS
A hybrid cytotoxin of rHuAFP is prepared by
conjugating a full-length rHuAFP or a fragment or analog
thereof to any number of known toxic entities using
conventional techniques. Such toxins are useful for
inhibiting the development of a neoplasm (as described
infra). Useful cytotoxins are preferably significantly
cytotoxic only when present intracellularly and are
substantially excluded from any given cell in the absence
of a targeting domain. As described below, peptide
toxins fulfill both of these criteria and are readily
incorporated into hybrid molecules. If desired, a mixed
cytotoxin (i.e., a cytotoxin composed of all or part of
two or more toxins) can also be used. Several useful
toxins are described in more detail below.
Toxin molecules useful in the method of the
invention are preferably toxins, such as peptide toxins,
which are significantly cytotoxic only when present
intracellularly. Of course, under these circumstances
the molecule must be able to enter a cell bearing the
targeted receptor. This ability depends on the nature of
the molecule and the nature of the cell receptor. For
example, cell receptors which naturally allow uptake of a
ligand are likely to provide a means for a molecule which
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includes a toxin to enter a cell bearing that receptor.
As is discussed below, the peptide toxin, useful in the
methods of the invention is fused to a rHuAFP (or
fragment or analog thereof) binding domain by producing a
recombinant DNA molecule which encodes a hybrid protein
molecule.
Many peptide toxins have a generalized eukaryotic
receptor binding domain; in these instances the toxin
must be modified to prevent intoxication of non-receptor
bearing cells. Any such modifications must be made in a
manner which preserves the cytotoxic functions of the
molecule (.see U.S. Department of Health and Human
Services, U.S. Serial No. 401,412). Potentially useful
toxins include, but are not limited to: cholera toxin,
ricin, 0-Shiga-like toxin (SLT-I, SLT-II, SLT IIV), LT
toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus
toxin, Pseudomonas exotoxin, saporin, modeccin, and
gelanin.
The cytotoxic portion of some molecules useful in
the invention, if desired, can be provided by a mixed
toxin molecule. A mixed toxin molecule is a molecule
derived from two different polypeptide toxins.
Generally, as discussed above, polypeptide toxins have,
in addition to the domain responsible for generalized
eukaryotic cell binding, an enzymatically active domain
and a translocation domain. The binding and
translocation domains are required for cell recognition
and toxin entry respectively. The enzymatically active
domain is the domain responsible for cytotoxic activity
once the molecule is inside a cell.
Naturally-occurring proteins which are known to
have a translocation domain include diphtheria toxin,
Pseudomonas exotoxin A, and possibly other peptide
toxins. The translocation domains of diphtheria toxin
and Pseudomonas exotoxin A are well characterized (see,
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e.g., Hoch et al., Proc. Natl. Acad. Sci. USA 82:1692,
1985; Colombatti et al., J. Biol. Chem. 261:3030, 1986;
and Deleers et al., FEBS Lett. 160:82, 1983), and the
existence and location of such a domain in other
molecules may be determined by methods such as those
employed by Hwang et al. Cell 48:129, 1987); and Gray et
al. Proc. Natl. Acad. Sci. USA 81:2645, 1984).
For example, one useful rHuAFP/mixed toxin hybrid
molecule is formed by fusing the enzymatically active A
subunit of E. coli Shiga-like toxin (see, e.g.,
Calderwood et al., Proc. Natl. Acad. Sci. USA 84:4364,
1987) to the translocation domain (amino acid residues
202 through 460) of diphtheria toxin, and to rHuAFP. The
rHuAFP portion of the three-part hybrid causes the
molecule to attach specifically to cells bearing
receptors which is recognized by rHuAFP, and the
diphtheria toxin translocation portion acts to insert the
enzymatically active A subunit of the Shiga-like toxin
into the targeted cell. The enzymatically active portion
of Shiga-like toxin, like diphtheria toxin, acts on the
protein synthesis machinery of the cell to prevent
protein synthesis, thus killing the cell.
Functional components of the hybrid cytotoxins of
the invention are linked together via a non-covalent or
covalent bond, or both. Non-covalent interactions can be
ionic, hydrophobic, or hydrophilic, such as interactions
involved in a leucine-zipper or antibody-protein G
interaction (see, e.g., Derrick et al., Nature 359:752,
1992). An example of a covalent linkage is a disulfide
bond.
A hybrid cytotoxin is prepared by chemically
conjugating rHuAFP (or fragment or analog) to a any
number of known toxic entities, e.g., those described
above. Such reactions are carried out by standard
techniques known to those skilled in the art. A typical
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way of conjugating a protein to a protein toxin
(including, e.g., bacterial toxins such as diphtheria
toxin or Pseudomonas exotoxin A, or plant toxins such as
ricin) is by crosslinking through a disulfide bond (see,
e.g., Chang et al., J. Biol. Chem. 252:1515, 1977) or a
heterobifunctional molecule (see, e.g., Cawley et al.
Cell 22:563, 1980). See also Stevens et al., U.S. Pat.
No. 4,894,227.
Alternatively, the hybrid cytotoxin is prepared by
expression of a hybrid DNA engineered to encode both the
rHuAFP (or a fragment or analog thereof) and the toxin
(or a toxic portion thereof), using technology available
to those of ordinary skill in the art of making such
hybrids (see, e.g., Murphy, U.S. Pat. No. 4,675,382, and
Chadhary et al., Proc. Natl. Acad. Sci. USA 84:4538,
1987). For example, a recombinant fusion protein of
rHuAFP and a cytotoxic agent is made according to methods
known in the art (see, e.g., Murphy supra and Huston et
al., Meth. Enzymol. 203:46, 1991). If the hybrid
cytotoxin is produced by expression of a fused gene, a
peptide bond serves as the link between the cytotoxic
agent and the targeting ligand. Another method useful
for conjugating a protein or polypeptide to a protein
toxin employs the polymer, monomethoxy-polyethylene
glycol (mPEG), as described in Maiti et al., Int. J.
Cancer Suppl. 3:17, 1988.
If desired, following its synthesis, the hybrid
cytotoxin is affinity purified according to standard
methods using antibodies against the targeting portion of
the molecule, e.g., antibodies against human alpha-
fetoprotein. Similarly, antibodies directed against the
cytotoxic agent are also useful for purifying the hybrid
cytotoxin molecule by standard immunological techniques.
The resulting hybrid cytotoxin is then formulated for use
as an agent against unwanted cells, e.g. cancer cells,
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following procedures standard in the field of
pharmacology.
Molecules of the invention can be screened for the
ability to decrease viability of cells bearing the
targeted receptor by means of assays known in the art,
e.g., those methods described herein.
Because hybrid cytotoxins of the invention are
potent cytotoxic agents for cells bearing the a receptor
which is recognized by rHuAFP, rHuAFP is useful in the
treatment of diseases involving unwanted alpha-
fetoprotein receptor-positive cells, e.g., cancer cells.
DIAGNOSTIC AGENTS
Recombinant rHuAFP or a fragment or analog thereof
can be attached to a detectable label to produce an agent
useful for detecting and localizing a neoplasm in vivo,
in situ, or in vitro . Methods for attaching such labels
to proteins are known in the art. For example, a
detectable label is attached by chemical conjugation, but
where the label is a polypeptide, it could alternatively
be attached by genetic engineering techniques.
Detectable labels are generally selected from a
variety of such labels known in the art, but are normally
radioisotopes, flurophores, enzymes (e.g., horseradish
peroxidase), or other moieties or compounds which emit a
detectable signal (e.g., radioactivity, fluorescence,
color) or emit a detectable signal after exposure of the
label to its substrate. Various detectable
label/substrate pairs (e.g., horseradish
peroxidase/diaminobenzidine, avidin/streptavidin,
luciferase/luciferin, /3-galactosidase/X-gal(5-Bromo-4-
Chloro-3-Indoyl-D-Galactopyranoside), and methods for
labelling proteins for such detection purposes are known
in the art. The usefulness of such an agent can be
assayed, for example, by implanting a tumor cell line,
e.g, MCF-7, into a host, e.g., a mouse, and determining
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whether the agent of the invention detectably labels the
tumor produced by such implanted cells, e.g., by
radioimaging using scintigraphy. Such an agent can also
be used to assay for the presence of any unwanted cell
bearing an alpha-fetoprotein receptor, e.g., by using
Western blot analysis or histochemical staining of a
tissue sample, according to known methods.
Recombinant HuAFP as an Anti-Cancer Agent
Anti-cancer agents of the invention (e.g., rHuAFP
or a fragment or analog thereof; or a hybrid cytotoxin of
rHuAFP) are useful for inhibiting a neoplasm, e.g.,
breast or, prostate carcinomas. Those skilled in the art
will understand that any number of methods, both in vitro
and in vivo, are used to determine the efficacy of anti-
cancer agents useful in the methods of the invention.
For example, the reduction of tumor growth can be
monitored in a mouse or rat growing a prostate cancer
(e.g., tumor xenografts of LNCaP androgen receptor-
positive human prostate cancer cell line) following the
administration of the test compound. In a working
example, a human tumor cell lines (e.g., cell lines such
as MCF-7(ATCC HTB 22), T-47D (ATCC HTB 133), MDA-MB-231
(ATCC HTB 26), BT-20 (ATCC HTB 19), NIH:OV-CAR-3 (ATCC
HTB 161), LnCaP.FGC (ATCC CRL 1740), and Du-145 (ATCC HTB
81) growing in culture is released from monolayer by
trypsinization, diluted into single-cell suspension and
then solidified by centrifugation into a pellet which IS
subsequently exposed to 15 Al fibrinogen (50 mg/ml) and
10 gl thrombin (50 units/ml) for 30 minutes at 37 C.
Fibrin clots containing tumor are then cut into pieces
approximately 1.5 mm in diameter. Each piece of tumor is
subsequently implanted under the kidney capsule of a
mouse according to standard methods. If desired, mice
can be immunosuppressed by daily subcutaneous (s.c.)
injection of 60 mg/kg cyclosporine A (Sandimmune IV)
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beginning immediately prior to tumor implantation
according to conventional methods. If necessary,
estrogen and androgen supplementation of mice is achieved
by standard methods, e.g, implantation of silastic tubing
containing estradiol or by injection of testosterone
propionate. Typically, hormone supplementation is
commenced on the day of tumor implantation. Generally,
administration of the test molecule is initiated prior to
tumor implantation and/or after tumor implantation.
Control animals receive a placebo, e.g., human serum
albumin or diluent, similarly administered as for rHuAFP
or related molecules. The effect of the test molecule on
tumor growth is monitored according to any standard
method. For example, tumor growth is monitored by weekly
measurement of tumor size by laparotomy using a
dissecting microscope equipped with an ocular micrometer.
A molecule shown experimentally to halt or reduce or
inhibit the growth of such implanted tumors is considered
useful in the invention.
Toxicity of test compounds towards cells bearing
receptors that are recognized by rHuAFP can be tested in
vitro according to any standard protocol. For example, a
cultured cancer cell line, e.g., MCF-7 estrogen-
receptor-positive human breast cancer cell line, is
maintained in plastic tissue culture flasks (Costar) in
DMEM with penicillin (100 U/mi), streptomycin (100
gg/ml), 5% fetal calf serum, insulin (10 ng/ml), L-
glutamine (2 mM) and non-essential amino acids (1%).
Cells are seeded in 96-well V-bottomed plates (Linbro-
Flow Laboratories, McLean, VA) at a concentration of 1 x
105 per well in complete medium. Putative toxins are
added to varying concentrations (10-12M to 10-6M) and the
cultures are incubated for 18 hrs. at 37 C in a 5% Coe
atmosphere. Following incubation, the plates are
centrifuged for 5 min. at 170 x g, and the medium removed
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and replaced with 100 l leucine-free medium (MEM, Gibco)
containing 8 gCi/ml (3H-leucine; New England Nuclear,
Boston, MA). After an additional 90 min. at 37 C, the
plates are centrifuged for 5 min. at 170 x g, the medium
is removed, and the cells are collected on glass fiber
filters using a cell harvester (Skatron, Sterling, VA).
Filters are washed, dried, and counted according to
standard methods. Cells cultured with medium alone serve
as the control. A test compound which reduces or halts
or inhibits cell growth compared to untreated control
cells, is detected as an indication of toxicity and is
considered useful in the invention.
Evaluation of whether a test compound confers
protection against the development of a neoplasm (e.g.,
breast or prostate cancers) generally involves using an
animal known to develop a neoplasm (e.g., the transgenic
mouse described in U.S. Pat. No. 4,736,866). An
appropriate animal is treated with the test compound
according to standard methods, and a reduced incidence of
neoplasm development, compared to untreated control
animals, is detected as an indication of protection.
As is discussed below, I have discovered that
unglycosylated rHuAFP produced in a prokaryotic
expression system is effective in treating cancer. For
example, rHuAFP has been found to be a potent inhibitor
of breast carcinoma growth in vitro.
The experimental examples described below
demonstrate the efficacy of rHuAFP as an anti-cancer
agent. These examples are provided to illustrate, not
limit, the invention.
EXPERIMENTAL
MATERIALS AND METHODS
Culture Media and Tumor Cells
Dulbecco's modified Eagle's medium (DMEM), RPMI
1640, fetal calf serum, glutamine, non-essential amino
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acids and penicillin-streptomycin mixture were obtained
from GIBCO (BRL). Donor calf serum was obtained from
Hyclone, Logan, UT, and porcine insulin was obtained from
Squibb, Inc., Princeton, NJ.
The MCF-7 estrogen-receptor-positive human breast
cancer cell line was obtained from Dr. Alberto C. Baldi,
Institute of Experimental Biology and Medicine, Buenos
Aires, Argentina. Stock cultures were maintained in
plastic tissue culture flasks (Costar) in DMEM with
penicillin (100 U/ml), streptomycin (100 pg/ml), 5% fetal
calf serum, insulin (10 ng/ml), L-glutamine (2 mM) and
non-essential amino acids (1%).
Estrogen-Stimulated Post-confluent Growth of MCF-7
Cells in Culture.
This assay is based on the finding that MCF-7
cells in estrogen-containing medium grow past confluence
and accumulate into foci; but, in the absence of
estrogen, cell proliferation stops after the cultures
establish cell-cell contact, and no foci are formed (see,
e.g., Gierthy et al., Breast Cancer Res. Treat. 12:227,
1988). 1 x 107 MCF-7 breast cancer cells were seeded in
16-mm wells contained in 24-well tissue culture plates.
Culture medium was phenol red-free DMEM supplemented with
5% donor calf serum (prescreened for absence of
detectable estrogens), L-glutamine (2 mM), non-essential
amino acids (lX, GIBCO), insulin (10 ng/ml), penicillin-
streptomycin (lX, GIBCO) and estradiol diluted to a final
concentration of 1.8 X 10-9 M. Cultures were refed at 24
hr and every 4 days thereafter with 2 ml of culture
medium containing rHuAFP and human serum albumin to yield
a final protein concentration of 100 g/ml per well.
Cells reached confluence within 5 days, and a substantial
number of foci were apparent within 10 days in wells
containing estrogen alone. Cells were fixed with
buffered formalin and stained with 1% Rhodamine B. The
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stained foci were quantitated using an Artek 870 Macro-
Micro Automated Colony Counter. Data are presented as
mean number of foci per treatment group.
RESULTS
Activity of rHuAFP Against MCF-7 Breast Cancer
cells
The results shown in Fig. 9 demonstrate that
rHuAFP inhibits estrogen-stimulated postconfluent growth
of MCF-7 breast cancer cells in vitro. Control
experiments using human albumin or no protein had no
effect on MCF-7 foci formation. These data indicate that
rHuAFP has a direct inhibitory effect on the growth of
carcinoma cell cultures.
Therapeutic Administration
As demonstrated above, rHuAFP is effective in
inhibiting a neoplasm, e.g., a breast cell carcinoma.
Accordingly, compounds of the invention can be formulated
according to known methods to prepare pharmaceutically
useful compositions. Treatment of human patients will be
carried out using a therapeutically effective amount of
an anti-cancer agent of rHuAFP in a physiologically
acceptable carrier. Suitable carriers and their
formulation are described for example in Remington's
Pharmaceutical Sciences by E.W. Martin. The amount of
the anti-cancer agent to be administered varies depending
upon the manner of administration, the age and body
weight of the patient, and with the type of disease,
extensiveness of the disease, and size of the patient
suffering from the disease. Generally amounts will be in
the range of those used for other agents used in the
treatment of cancer, although in certain instances lower
amounts will be needed because of the increased
specificity of the compound. For example, rHuAFP is
administered systemically, as described below, at a
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dosage that inhibits malignant cell proliferation,
typically in the range of 0.1 ng - 10 g/kg body weight.
Furthermore, the method of the invention can also
employ combination therapy in which rHuAFP is
administered either simultaneously or sequentially with a
chemotherapeutic agent. Typically, a chemotherapeutic
agent is administered according to standard methods
or,alternatively, in a dose which is lower than the
standard dose when the chemotherapeutic agent is used by
itself. Examples of chemotherapeutic agents include,
without limitation, mechlorethamine, cyclophosphamide,
ifosfamide, L-sarcolysin, chlorambucil,
hexamethylmelamine, thiotepa, busuifan, carmustine,
lomustine, semustine, streptozocin, dacarbazine,
methotrexate, fluorouracil, cytarabine, mercaptopurine,
thioguanine, pentostatin, vinblastine, vincristine,
etoposide, teniposide, actinomycin D, daunomycin,
doxorubicin, bleomycin, plicamycin, mitomycin, cisplatin,
mitoxantrone, hydroxyurea, procarbozine, mitotane,
aminoglutethimide, prednisone, hydroxyprogesterone,
diethylstilbestrol, tamoxifen, flutamide, or leuprolide.
Treatment is started generally with the diagnosis
or suspicion of a neoplasm and is generally repeated on a
daily basis. Protection from the development of neoplasm
is also achieved by administration of rHuAFP on a daily
basis. If desired, the efficacy of the treatment or
protection regimens is assessed with the methods of
monitoring or diagnosing patients for cancer.
Furthermore, the compounds of the invention can
also be used to treat mammals to destroy any unwanted
cells bearing alpha-fetoprotein receptors associated with
a pathological condition. The method(s) of the invention
can also be used to treat non-human mammals, for example,
domestic pets, or livestock. As described below, the
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anti-cancer agents of the invention can be administered
systemically or locally.
Systemic Administration
For use as an anti-cancer agent, the compounds of
the invention can be administered systemically, for
example, formulated in a pharmaceutically-acceptable
buffer such as physiological saline. Preferable routes
of administration include, for example, subcutaneous,
intravenous, interperitoneally, intramuscular, or
intradermal injections which provide continuous,
sustained levels of the drug in the patient. In other
preferred. routes of administration, the compounds of the
invention can be given to a patient by injection of a
slow release preparation, for example, in a slowly
dissociating polymeric or crystalline form; this sort of
sustained administration can follow an initial delivery
of the drug by more conventional routes (for example,
those described above). Alternatively, the compounds can
be administered using an infusion pump, thus allowing a
precise degree of control over the rate of drug release,
or through installation of the compounds in the nasal
passages in a similar fashion to that used to promote
absorption of insulin. As an alternative to nasal
transmucosal absorption, the compounds can be delivered
by aerosol deposition of the powder or solution into the
lungs.
Local Administration
The anti-cancer agents of the invention also can
be administered locally to treat cancer. Since the
desired action of the agent is generally upon a
circumscribed mass of tissue, for example a tumor,
delivery of the drug by means which result in high local
concentrations in the vicinity of the tumor is especially
desirable.
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Recombinant HuAFP as a Diagnostic Agent
Recombinant HuAFP (or fragment or analog thereof)
linked to a detectable label finds diagnostic use in the
detection or monitoring or assaying for the presence of a
neoplasm (e.g., breast or prostate cancers).
For example, in vivo studies can be conducted on
human patients to determine the presence of a neoplasm
using a detectably labelled rHuAFP (e.g., Tc-99m-labelled
rHuAFP). In general, the detectably labelled rHuAFP is
administered intravenously and imaging can be performed
using scanners by methods known to those skilled in the
art, e.g., by radioimaging using scintigraphy.
In another working example, a neoplasm or any cell
bearing a receptor which is recognized by rHuAFP may be
detected in a tissue sample, e.g., a biopsy, a bodily
fluid, by using rHuAFP (or fragment or analog thereof)
linked to a detectable label. After determining that a
patient should be tested for the presence of such cells,
a tissue sample, a biopsy, or a sample of bodily fluid,
preferably lymph, blood, serum, or urine, is collected
from the patient. Accordingly, the subcellular location
or presence of a receptor which is recognized by rHuAFP
is determined either in situ or in vitro using
fractionated cells by any standard biochemical or
histochemical procedure (see e.g., Ausubel et al., supra;
Bancroft and Stevens, Theory and Practice of Histological
Techniques, Churchill Livingstone, 1982). Appropriate
control samples for the assay include a tissue sample or
a bodily fluid collected from individuals who do not have
cells bearing alpha-fetoprotein (negative control), or
samples which contain a known, predetermined amount of
alpha-fetoprotein receptor (positive control).
The diagnostic assay may be performed in solution
or may use a solid (insoluble) support (e.g. polystyrene,
nitrocellulose, or beads) or in a tissue sample prepared
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for histological examination, using any standard methods.
For example, to determine whether the patient from whom'
the test sample was collected has cells bearing receptors
which is recognized by rHuAFP, the level of binding of
the detectably labelled rHuAFP in the test sample is
compared to the level of binding in the negative and/or
positive control samples. A level of binding in the test
sample greater than the level of binding in the negative
control sample, or at least equal to the level of binding
in the positive control sample, indicates that the
patient has cells bearing alpha-fetoprotein receptors.
Materials for performing the diagnostic assays
according to the methods of the invention may be provided
as a kit having instructions for use. In general, the
kit is composed in part of a rHuAFP (or fragment or
analog thereof). This kit may further include a second
reagent, e.g., a detectable label, which is used to label
rHuAFP (or a fragment or analog thereof). The kits
exemplified above are useful in, for example, detecting
the presence of a tumor in a sample of human tissue in
vitro, or for in vivo examination purposes.
The experimental examples described below
demonstrate the efficacy of rHuAFP diagnosing a neoplasm.
These examples are provided to illustrate, not limit, the
invention.
EXPERIMENTAL
MATERIALS AND METHODS
Animals
MCF-7 human breast cancer cells implanted in the
lateral thorax region of CB-17 SCID mice were grown to a
size of 1 cm diameter (approx. 5 gm) under estrogen
stimulation according to methods known in the art.
Technetium Labelling
99mTc-recombinant labelled alpha-fetoprotein was
prepared from an AFP aliquot mixed with 0.5 ml 0.9%
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sodium chloride injection solution (Baxter Healthcare
Corporation, Deerfield, IL). The solution is added to an
UitraTag RBC Reaction Vial (Mallinckrodt Medical Inc.,
St. Louis, MO 63134 Lot No. 0683040), containing stannous
chloride dihydrate, sodium citrate dihydrate, and
dextrose anhydrous, in a lyophilized form stored under
argon. The contents of the vial are mixed by gentle
swirling, and incubated at room temperature for 5
minutes. At the completion of the incubation, 0.8-1.2
Gbq Technetium 99mTc Sodium Pertechnetate Injection is
added (99mTc Generator Mallincrokt Medical, Inc. St.
Louis, MO) in a volume of 1-2 ml. The contents of the
vial are mixed by gentle swirling and incubated for 15
minutes. Dose aliquots were assayed at 0, 3, and 6 hours
after preparation. Thin-layer chromatography performed
on preparations using ITLC-SG (Gelman Instrument Co., Ann
Arbor, MI) with 0.9% NaCl showed 95-99% of the 99Tc was
bound to the recombinant alpha-fetoprotein.
Imaging
Experimental animals are sedated with Medafane. A
24 gauge, 3/4 inch catheter (Surflo IV catheter, Terumo
Medical) is then secured in a lateral tail vein. The
animal is then further anesthetized with a slow infusion
of 20-25 mg/kg body weight of pentobarbital
intravenously. Anesthesia is maintained for restraint as
required with injections of 5 mg of additional
pentobarbital.
Isotope biodistribution data is collected using a
Elscint Dymax 409 gamma camera. This data is
subsequently analyzed by a computer (Siemans Gammasonics
Microdelta). Animals are imaged in triples, being placed
in the dorsal recumbent position on a thin polyethylene
panel. To eliminate motion during imaging, the animals
are restrained as necessary, on these panels by strips of
tape over their extremities so as not to restrict
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respiration. Dynamic images obtained over 60 minutes are
used to determine the biodistribution of the labeled
protein. Typically, twelve sequential, five minute
images are obtained with low energy general purpose
collimation, and 1.5 hardware zoom into the computer
matrices having 128 by 128 picture elements.
Study animals are typically injected with 37MBq of Tc-99m
labelled protein.
RESULTS
Tracer Biodistribution and Kinetics
Following administration of 37MBq (approx. 4-6 g
Tc-99m recombinant human alpha-fetoprotein) in the tail
vein, tracer biodistribution kinetics were measured
during the initial hour after injection and at 24 hours.
Tissue uptake kinetics were measured in % injected
activity/per 100 ROI (Region of Interest) pixels (%IA).
During the first hour there is rapid renal clearance,
mild localization in the liver and little evident
activity in other tissues. At 1 hour, tumor uptake was
(mean SEM: 1.9 0.3%IA) and the tumor to heart (T/H)
region ratio was 0.84 0.23. By 24 hours, tumor uptake
was (0.8+/-0.1%IA) and T/A and tumor to background (T/B
upper chest) region ratios were 1.43 0.41 and 2.66
0.54, respectively. Studies comparing 99mTc-labelled
rHuAFP to 99mTc-labelled human serum albumin (used as a
non-specific protein control) repeated in the same
animals showed that T/B image ROI activity ration was 2.7
and 5.8 for 99mTc-labelled rHuAFP at 1 and 24 hr,
respectively and at 24 hours was 40% greater for 99mTc-
labelled rHuAFP compared to Tc-99m human serum albumin.
These results show that rHuAFP can be labelled with Tc-
99m and that this labelled agent has low non-specific
tissue uptake and rapid renal clearance from the blood.
Localization in human breast cancer xenografts is
initially rapid, increases with time, and is due to
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specific tumor uptake. These results demonstrate that
rHuAFP labelled with Tc-99m is useful as a diagnostic
agent for breast carcinoma.
Diagnostic Administration
As discussed above, rHuAFP (or fragment or analog
thereof) linked to a detectable label finds diagnostic
use in the detection or monitoring or assaying of a
neoplasm (e.g., a breast cell carcinoma). Accordingly,
patients who present with the classical symptoms of
cancer, e.g., breast cancer or prostate cancer, or have a
medical history which indicates susceptibility to such
cancer may be tested with the methods of the invention.
Other appropriate patients for such testing include those
who have a family history of breast or prostate
carcinomas. Patients who are receiving drugs or have
been exposed to toxins implicated in the induction of a
cancer should also be tested.
The diagnostic methods employing detectably
labelled rHuAFP (or a fragment or analog thereof) of the
invention may be used to detect the presence of a cancer
prior to, or after the onset of, clinical symptoms
associated with the cancer.
The method of the invention facilitates diagnosis
of a neoplasm prior to or coincident with the onset of
clinical symptoms (e.g., a palpable tumorous mass). For
example, the method of the subject invention may provide
a diagnosis of breast cancer prior to onset of clinical
symptoms. Furthermore, the method of the invention
allows the clinician to provide an accurate diagnosis of
a neoplasm such as breast or prostate cancer.
Diagnostic imaging methods of the invention will
be carried out using a diagnostically effective amount of
a diagnostic agent of rHuAFP in a physiologically
acceptable carrier. Suitable carriers and their
formulation are described for example in Remington's
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Pharmaceutical Sciences by E.W. Martin. The amount of
the diagnostic agent to be administered varies depending
upon the manner of administration, the age and body
weight of the patient, and with the type of disease,
extensiveness of the disease, and size of the patient
suffering from the disease. Generally, however, amounts
will be in the range of those used for other agents used
in the diagnosis of cancer, although in certain instances
lower amounts will be needed because of the increased
specificity of the compound. For example, a detectably
labelled rHuAFP is administered intravenously to a
patient, as is described above, at a dosage that allows
imaging of a neoplasm, e.g., by radioimaging using
scintigraphy. Typically, a dosage is in the range of 0.1
ng - 10 g/kg body weight.
cell Culture Media of the Invention
The invention furhter provides a media containing
rHuAFP (or a fragment or analog thereof) for cell
culture. While media of the invention generally does not
require the use of serum (e.g., fetal bovine serum, calf
serum, horse serum, normal mouse serum, human serum,
porcine serum, rabbit serum etc.), since such rHuAFP is
intended to replace or supplement the use of serum, those
skilled in the art will understand and recognize that
serum can be added if desired. Media formulations are
generally prepared according to methods known in the art.
Accordingly, any standard medium, e.g., RMPI-1630 Medium,
CMRL Medium, Dulbecco's Modified Eagle Medium (D-MEM),
Fischer's Medium, Iscove's Modified Dulbecco's Medium,
McCoy's Medium, Minimum Essential Medium, NCTC Medium,
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and the like can be formulated with rHuAFP (or a fragment
or analog thereof) at the desired effective
concentration. If desired, media supplements, e.g., salt
solutions (e.g., Hank's Balanced Salt Solution or Earle's
Balanced Salt Solution), antibiotics, nucleic acids,
amino acids, carbohydrates, and vitamins are added
according to known methods. If desired, growth factors,
colony-stimulating factors, cytokines and the like can
also be added to media according to standard methods.
For example, media of the invention can contain any of
the following substances, alone or in combination, with
rHuAFP (or a fragment or analog thereof): erythropoietin,
granulocyte/macrophage colony-stimulating factor (GM-
CSF), granulocyte colony-stimulating factor (G-CSF),
macrophage colony-stimulating factor (M-CSF), an
interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, etc.),
insulin-growth factor (IGF), transferrin, albumin, and
stem-cell growth factor (SCF). Media of the invention
are useful for culturing a variety of eukaryotic cells,
e.g., mammalian cells, yeast cells, amphibian cells, and
insect cells. Media can also be used for culturing any
tissue or organ. Such media can also be used in a
variety of culture conditions and for a variety of
biological applications. Examples of such culture
conditions include, without limitation, bioreactors
(e.g., continuous or hollow fiber bioreactors), cell-
suspension cultures, semisolid cultures, liquid cultures,
and long-term cell suspension cultures. Media of the
invention are also useful for industrial applications,
e.g., culturing hybridoma cells, genetically-engineered
mammalian cells, tissues or organs.
Recombinant Human Alpha-Fetoprotein As A Cell-
Proliferative Accent
Cell growth-promoting attributes of rHuAFP (or a
fragment or analog thereof) is evaluated by any standard
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assay for analysis of cell proliferation in vitro and in
vivo. As discussed infra, the art provides animal
systems for in vivo testing of cell growth promoting or
boosting characteristics of rHuAFP (or a fragment or
analog thereof). Furthermore, a wide variety of in vitro
systems are also available for testing growth-promoting
or growth-boosting aspects of rHuAFP (or a fragment or
analog thereof).
Any cell that proliferates in response to rHuAFP
(or a fragment or analog thereof) can be identified
according to standard methods known in the art. For
example, proliferation of a cell (e.g., a bone marrow
cell) can be monitored by culturing in a liquid media
containing the test compound, either alone or in
combination with other growth factors, added artificially
to a serum-free or serum-based medium. Alternatively,
such bone marrow cells can be cultured in a semisolid
matrix of dilute agar or methylcellulose, and the test
compound, alone or in combination with other growth
factors, can be added artificially to a serum-free or
serum-reduced medium. In the semisolid matrix the
progeny of an isolated precursor cell, proliferating in
response to rHuAFP or a fragment or analog thereof,
remain together as a distinguishable colony. For
example, a bone marrow cell may be seen to give rise to a
clone of a plurality of bone marrow cells, e.g., NK
cells. Such culture systems provide a facile way for
assaying whether a cell responds to rHuAFP (a fragment or
analog thereof) either alone or in combination with other
growth factors.
If desired, identification and separation of
expanded subpopulations of cells is performed according
to standard methods. For example, cells may be analyzed
by fluorescence-activated cell sorting (FACS). This
procedure generally involves labelling cells with
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antibodies coupled to a fluorescent dye and separating
the labeled cells from the unlabelled cells in a FAGS,
e.g., FACScan (Becton Dickson). Thus virtually any cell
can be identified and separated, e.g., by analyzing the
presence of cell surface antigens (see e.g., Shah et al.,
J. Immunol. 140:1861, 1988). When a population of cells
is obtained, it is then analyzed biochemically or,
alternatively, provides a starting population for
additional cell culture, allowing the action of the cells
to be evaluated under defined conditions in culture.
In one working example, the effect of rHuAFP (or a
fragment or analog thereof) on the growth of human bone
marrow cells is examined as follows. In general, human
bone marrow samples are obtained according to standard
procedures after informed consent. For example, bone
marrow is obtained from the iliac crest of a healthy
donor and the marrow cells are diluted in phosphate-
buffered saline at room temperature. Cells are then
washed and cultured in an appropriate growth medium. For
example, cultures can be set up by inoculating bone
marrow cells in 20-30 ml of McCoy's medium containing 50
U/ml penicillin, 50 U/ml streptomycin and 2 mM L-
glutamine. Cultures are incubated in the presence or
absence of the test compound alone, or in combination
with other growth factors, e.g., transferrin or GM-CSF.
The cultures are subsequently incubated at 37 C in a
humidified atmosphere containing 5% C021 5% 02, and 90% N2
for the desired time period. Cell proliferation assays
are performed according to standard methods. For
example, replicate samples cultured in the presence and
absence of the test compound are analyzed by pulsing the
cells with 1-2 jCi of 3HTdR. After an incubation period,
cultures are harvested onto glass-fiber filters and the
incorporated 3H measured by liquid scintillation.
Comparative studies between treated and control cells,
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e.g., cell cultured in the presence of rHuAFP versus
cells cultured in the absence of rHuAFP, are used to
determine the relative efficacy of the test molecule in
stimulating cell proliferation. A molecule which
stimulates cell proliferation is considered useful in the
invention.
To evaluate the proliferative effects of rHuAFP
(or a fragment or analog thereof) e.g., the effect of the
test compound on hematopoiesis in vivo, the test molecule
is administered to sublethally irradiated mice (or mice
treated with an immunosuppressive agent such as
cyclosporine or FK-506, or a chemotherapeutic agent such
as 5-fluorouracil or cyclophosphamide or any other method
known in the art to deplete bone marrow) and normal mice
according to standard methods, e.g., intravenously or
intraperitoneally, at an appropriate dosage on a daily
basis. Generally, administration of the test compound to
treated mice is initiated prior to and/or after treating
the animal, e.g., with sublethal radiation or
immunotherapy or chemotherapy. Control animals receive a
placebo, e.g., human serum albumin or diluent, similarly
administered as for rHuAFP or related molecules. The
effect of the test molecule on hematopoiesis is monitored
by standard techniques. For example, white blood cell
count in peripheral blood and spleen in both treated and
control animals are analyzed. Qualitative and
quantitative analyses of bone marrow, e.g., lymphocytic
lineage or myeloid lineage or any other cell type, can
also be determined and analyzed according to conventional
methods. Comparative data between treated and control
animals are used to determine the relative efficacy of
the test molecule in promoting cell proliferation, e.g.,
stimulates bone marrow cell production, mature B
lymphocyte, thymocyte, or peripheral T lymphocyte cell
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production. A test molecule which stimulates cell
proliferation is considered useful in the invention.
The following example demonstrates that
unglycosylated rHuAFP stimulates the growth of bone
marrow cells in vitro. This example is provided to
illustrate, not limit, the invention.
EXPERIMENTAL
MATERIALS AND METHODS
Animals
Adult male and female CBA/J mice were obtained
from the Jackson Laboratory (Bar Harbor, Maine). All
mice were. bred and maintained in our animal facility.
Animals used in this study were 12 to 20 weeks old.
Cultures
Bone marrow cells were collected by flushing the
tibias and femurs of CBA/J mice with modified Dulbecco's
phosphate-buffered saline (PBS) using a sterile syringe
and 25-gauge needle. Homogenous single-cell suspensions
were obtained by the repeated passage of cell mixtures
through a Pasteur pipet. All cells were washed twice by
centrifugation at 250g for 10 min in PBS and then
assessed for viability by trypan blue dye exclusion. A
cell viability of 95% or better was recorded in all
experiments. Cells were then adjusted to the desired
concentration prior to use. Bone marrow cells (250,000)
were cultured in 96-well round-bottom microtiter plates
(Flow Laboratories, Mississauga, Ontario, Canada). The
culture medium was serum-free RPMI plus 4 mM L-glutamine,
20 mM Hepes, 100 U/ml penicillin, 100 Ag/ml streptomycin
(GIBCO Laboratories, Burlington, Ontario, Canada), 5
gg/ml transferrin, and 5 X 10-5 2-mercaptoethanol (Eastman
Chemicals Co., Rochester N.Y.). Cells were cultured in
the presence or absence of rHuAFP at a concentration of
400 g/ml, respectively. Total volume of all cultures
was 0.2 ml. Cultures were maintained at 37 C in 95%
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humidified air and 5% CO2. Six hours prior to
harvesting, the cultures were pulsed with 1 ACi tritiated
thymidine (NEN, sp act 77.1 Ci/mmol). Cells were then
harvested on glass fiber mats (Flow Labs) with a multiple
sample harvester (Skatron, Flow Labs). Water-insoluble
tritiated thymidine incorporation was measured with an
LKB 1215 Rackbeta II using standard liquid scintillation
techniques.
RESULTS
Effects of rHuAFP on Bone Marrow Proliferation in
Serum-Free Media
The effects of purified rHuAFP on cultured murine
bone marrow was evaluated in serum-free medium. In this
experiment, 2.5 X 105 viable cells from bone marrow of
CBA/J mice were cultured for 72 hours in serum-free RPMI
media in the presence or absence of rHuAFP at a final
concentration of 400 gg/ml and transferrin at a final
concentration of 5 gg/ml. Data shown in Fig. 10 indicate
that bone marrow cells undergo a strong proliferative
response in the presence of unglycosylated rHuAFP; with a
stimulation index (SI) of 35. No such proliferation was
observed when bone marrow cells were cultured in the
absence of rHuAFP.
Therapy
As demonstrated above, rHuAFP is effective in
promoting the proliferation of cells and accordingly is
useful for therapy involving the promotion of cell
proliferation, e.g., proliferation of bone marrow cells,
and in treatment for the prevention of side effects of
immunosuppressive therapy, radiotherapy or chemotherapy,
or other therapies known to depress the immune system and
suppress bone marrow production, causing myelotoxicity.
Accordingly, rHuAFP (or a fragment or analog thereof) is
employed to treat deficiencies in hematopoietic
progenitor or stem cells, or related disorders.
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Recombinant HuAFP (or a fragment or analog thereof) may
also be employed in methods for treating cancer and other
pathological states resulting in myelotoxcity, exposure
to radiation or drugs, and including for example,
leukopenia, bacterial and viral infections, anemia, B
cell or T cell deficiencies, including immune cell or
hematopoietic cell deficiency following autologous or
non-autologous bone marrow transplantation. Recombinant
HuAFP (or a fragment or analog thereof) may also be
employed to stimulate development of megakaryocytes and
natural killer cells in vitro or in vivo.
The media, compositions, and methods of the
invention are also useful for treating cancers that are
treated by bone marrow transplants (BMT) that involve
removing bone marrow cells from the patient, maintaining
these cells in an ex vivo culture while the patient is
treated with radiation or chemotherapy, and then
transplanting these cells back into the patient after the
treatment has been completed to restore the patient's
bone marrow. Accordingly, rHuAFP may be employed for BMT
as a means for reconstituting bone marrow in ex vivo cell
culture medium and for promoting bone marrow cell
proliferation in vivo. Recombinant HuAFP (a fragment or
analog thereof) is also useful for other cell therapies,
e.g. cell expansion and/or gene therapy protocols,
therapies requiring ex vivo cell culture. Recombinant
HuAFP (a fragment or analog) is also useful in the
prevention of autologous or allogenic bone marrow
transplant rejection.
Therapeutic Administration
Recombinant HuAFP (or a fragment or analog
thereof) can be formulated according to known methods to
prepare pharmaceutically useful compositions.
Recombinant human alpha-fetoprotein, e.g., rHuAFP (or a
fragment or analog thereof), is preferably administered
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to the patient in an amount which is effective in
preventing or ameliorating the symptoms of myleotoxcity.
Generally, a dosage of 0.1 ng/kg to 10 g/kg body weight
is adequate. For example, treatment of human patients
will be carried out using a therapeutically effective
amount of rHuAFP (or a fragment or analog thereof) in a
physiologically acceptable carrier. Suitable carriers
and their formulation are described for example in
Remington's Pharmaceutical Sciences by E.W. Martin. The
amount of rHuAFP to be administered will vary depending
upon the manner of administration, the age and body
weight of.the patient, and with the type of disease, and
size of the patient predisposed to or suffering from the
disease. Preferable routes of administration include,
for example, oral, subcutaneous, intravenous,
intrperitoneally, intramuscular, transdermal or
intradermal injections which provide continuous,
sustained levels of the drug in the patient. In other
preferred routes of administration, rHuAFP can be given
to a patient by injection or implantation of a slow
release preparation, for example, in a slowly
dissociating polymeric or crystalline form; this sort of
sustained administration can follow an initial delivery
of the drug by more conventional routes (for example,
those described above). Alternatively, rHuAFP can be
administered using an external or implantable infusion
pump, thus allowing a precise degree of control over the
rate of drug release, or through installation of rHuAFP
in the nasal passages in a similar fashion to that used
to promote absorption of insulin. As an alternative to
nasal transmucosal absorption, rHuAFP can be delivered by
aerosol deposition of the powder or solution into the
lungs.
The therapeutic method(s) and compositions of the
present invention may also include co-administration with
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other human growth factors. Exemplary cytokines or
hematopoietins for such use include, without limitation,
factors such as an interleukin (e.g., IL-1), GM-CSF, G-
CSF, M-CSF, tumor necrosis factor (TNF), transferrin, and
erythropoietin. Growth factors like B cell growth
factor, B cell differentiation factor, or eosinophil
differentiation factors may also prove useful in co-
administration with rHuAFP (or a fragment or analog
thereof). The dosage recited above would be adjusted to
compensate for such additional components in the
therapeutic composition. Progress of the treated patient
can be monitored by conventional methods.
Treatment is started generally with the diagnosis
or suspicion of myelotoxcity and is generally repeated on
a regular or daily basis to ameliorate or prevent the
progression or exacerbation of the condition. Protection
or prevention from the development of a myleotoxcemic
condition is also achieved by administration of rHuAFP
prior to the onset of the disease. If desired, the
efficacy of the treatment or protection regimens is
assessed with the methods of monitoring or diagnosing
patients for myelotoxcity.
The method(s) of the invention can also be used to
treat non-human mammals, for example, domestic pets, or
livestock.
other Embodiments
In other embodiments, the invention includes the
use of rHuAFP (or fragment or analog thereof) for the
prevention or treatment of acquired immunodeficiency
syndrome (AIDS). To evaluate the immunosuppressive effect
of rHuAFP or a fragment or analog thereof on AIDS, i.e.,
the compound's ability to prevent or ameliorate an
autoimmune component of AIDS, test compounds are
administered to an appropriate animal (e.g., a human
patient), according to standard methods, e.g.,
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intravenously or intraperitoneally, at an appropriate
dosage on a daily basis as is discussed, above.
Generally, administration is initiated prior to the onset
of AIDS and/or after the clinical appearance of AIDS.
Control animals receive a placebo, e.g., human serum
albumin, similarly administered as for rHuAFP or related
molecules. The effect of the test compound on AIDS is
monitored according to standard methods. For example,
analysis of the ability of the test compound to inhibit
or prevent or ameliorate the destruction of helper T
cells can be monitored. Comparative studies between
treated and control animals are used to determine the
relative efficacy of the test compounds in preventing or
ameliorating AIDS. A molecule which prevents or
ameliorates (decreases or suppresses or relieves or
promotes remission of) the symptoms of AIDS is considered
useful in the invention.
In the invention also includes the use of a
therapeutically effective amount rHuAFP (or fragment or
analog thereof) for inhibiting the rejection of a
transplanted organ (e.g., the heart, the liver, the lung,
the pancreas, and the kidney), tissue (e.g., skin, bone
marrow, dura mater, bone, implanted collagen, an
implanted bioreactor), or cell (e.g., $ islet cells of
the pancreas, stem cells, hematopoietic cells, lymph
cells, neuroendocrine or adrenal cells) in a mammal.
Such transplanted organs, tissues, or cells may be
derived from any source, e.g., such biological material
can be allogenic, phenogenic, autologous, synthetic,
artificial or genetically-engineered. For example, the
method can also be used when the patient is the recipient
of an allograft such a heart or kidney from another
species.
In one working example, the immunosuppressive
effect of rHuAFP on clinical transplantation, i.e., the
CA 02211324 2010-04-01
ability of rHuAFP to prevent or ameliorate transplant
rejection (e.g., hyperacute rejection, acute rejection
and chronic rejection), is evaluated by administering
rHuAFP to an NIH minipig according to standard methods,
5 e.g., intravenously or intraperitoneally, at an
appropriate dosage on a daily basis. Generally,
administration of rHUAFP is initiated prior to the
transplant, e.g., transplantation of a kidney and/or
after the transplant procedure. Control animals receive
10 a placebo, e.g., human serum albumin, similarly
administered as for rHuAFP. The effect of rHuAFP on
transplant rejection is monitored according to standard
methods. one manifestation of the rejection process is
diminished function of the transplanted organ, for
15 example, analysis of urine output can be monitored. If
desired, histological inspection (e.g., by using any
standard histochemical or immunohistochemical procedure,
see e.g., Ausubel at al., supra; Bancroft and Stevens,
supra) of kidney tissue is performed and tissue samples
20 obtained by biopsy are examined microscopically for
evidence of transplant rejection, e.g., chronic
interstitial fibrosis, vascular thrombosis, or the
presence of abnormal lymphocytic infiltrates.
Comparative studies between treated and control animals
25 are used to determine the relative efficacy of rHuAFP in
preventing or ameliorating transplant rejection.
Recombinant HuAFP (a fragment or analog thereof) which
prevents or ameliorates (decreases or suppresses or
relieves or promotes remission of) the symptoms of
30 transplant rejection is considered useful in the
invention.
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application was specifically and individually indicated
to be incorporated by reference.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Murgita, Robert A.
(ii) TITLE OF INVENTION: EXPRESSION AND PURIFICATION OF CLONED
HUMAN ALPHA-FETOPROTEIN
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fish & Richardson P.C.
(B) STREET: 225 Franklin Street, Suite 3100
(C) CITY: Boston
(D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02110-2804
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US96/----
(B) FILING DATE: 24-JAN-1996
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/377,317
(B) FILING DATE: 24-JAN-1995
(C) CLASSIFICATION:
(A) APPLICATION NUMBER: 08/377,311
(B) FILING DATE: 24-JAN-1995
(C) CLASSIFICATION:
(A) APPLICATION NUMBER: 08/377,309
(B) FILING DATE: 24-JAN-1995
(C) CLASSIFICATION:
(A) APPLICATION NUMBER: 08/377,316
(B) FILING DATE: 24-JAN-1995
(C) CLASSIFICATION:
(A) APPLICATION NUMBER: 08/505,012
(B) FILING DATE: 21-JULY-1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Clark, Paul T.
(B) REGISTRATION NUMBER: 30,162
(C) REFERENCE/DOCKET NUMBER: 06727/003001
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 542-5070
(B) TELEFAX: (617) 542-8906
(C) TELEX: 200154
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(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TGTCTGCAGG ATGGGGAAAA A 21
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CATGAAATGA CTCCAGTA 18
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CATAGAAATG AATATGGA 18
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2022 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ATATTGTGCT TCCACCACTG CCAATAACAA AATAACTAGC AACCATGAAG TGGGTGGAAT 60
CAATTTTTTT AATTTTCCTA CTAAATTTTA CTGAATCCAG AACACTGCAT AGAAATGAAT 120
ATGGAATAGC TTCCATATTG GATTCTTACC AATGTACTGC AGAGATAAGT TTAGCTGACC 180
TGGCTACCAT ATTTTTTGCC CAGTTTGTTC AAGAAGCCAC TTACAAGGAA GTAAGCAAAA 240
TGGTGAAAGA TGCATTGACT GCAATTGAGA AACCCACTGG AGATGAACAG TCTTCAGGGT 300
GTTAAGAAAA CCAGCTACCT GCCTTTCTGG AAGAACTTTG CCATGAGAAA GAAATTTTGG 360
AGAAGTACGG ACATTCAGAC TGCTGCAGCC AAAGTGAAGA GGGAAGACAT AACTGTTTTC 420
TTGCACACAA AAAGCCCACT GCAGCATGGA TCCCACTTTT CCAAGTTCCA GAACCTGTCA 480
CAAGCTGTGA AGCATATGAA GAAGACAGGG AGACATTCAT GAACAAATTC ATTTATGAGA 540
TACCAAGAAG GCATCCCTTC CTGTATGCAC CTACAATTCT TCTTTCGGCT GCTGGGTATG 600
AGAAAATAAT TCCATCTTGC TGCAAAGCTG AAAATGCAGT TGAATGCTTC CAAACAAAGG 660
CAGCAACAGT TACAAAAGAA TTAAGAGAAA GCAGCTTGTT AAATCAACAT GCATGTCCAG 720
TAATGAAAAA TTTTGGGACC CGAACTTTCC AAGCCATAAC TGTTACTAAA CTGAGTCAGA 780
AGTTTACCAA AGTTAATTTT ACTGAAATCC AGAAACTAGT CCTGGATGTG GCCCATGTAC 840
ATGAGCACTG TTGCAGAGCA GATGTGCTGG ATTGTCTGCA GGATGGGGAA AAAATCATGT 900
CCTACATATG TTCTCAACAA GACACTCTGT CAAACAAAAT AACAGAATGC TGCAAACTGA 960
CCACGCTGGA ACGTGGTCAA TGTATAATTC ATGCAGAAAA TGATGAAAAA CCTGAAGGTC 1020
TATCTCCAAA TCTAAACAGG TTTTTAGGAG ATAGAGATTT TAACCAATTT TCTTCAGGGG 1080
AAAAAAATAT CTTCTTGGCA AGTTTTGTTC ATGAATATTC AAGAAGACAT CCTCAGCTTG 1140
CTGTCTCAGT AATTCTAAGA GTTGCTAAAG GATACCAGGA GTTATTGGAG AAGTGTTTCC 1200
AGACTGAAAA CCCTCTTGAA TGCCAAGATA AAGGAGAAGA AGAATTACAG AAATACATCC 1260
AGGAGAGCCA AGCATTGGCA AAGCGAAGCT GCGGCCTCTT CCAGAAACTA GGAGAATATT 1320
ACTTACAAAA TGAGTTTCTC GTTGCTTACA CAAAGAAAGC CCCCCAGCTG ACCTCGTCGG 1380
AGCTGATGGC CATCACCAGA AAAATGGCAG CCACAGCAGC CACTTGTTGC CAACTCAGTG 1440
AGGACAAACT ATTGGCCTGT GGCGAGGGAG CGGCTGACAT TATTATCGGA CACTTATGTA 1500
TCAGACATGA AATGACTCCA GTAAACCCTG GTGTTGGCCA GTGCTGCACT TCTTCATATG 1560
CCAACAGGAG GCCATGCTTC AGCAGCTTGG TGGTGGATGA AACATATGTC CCTCCTGCAT 1620
TCTCTGATGA CAAGTTCATT TTCCATAAGG ATCTGTGCCA AGCTCAGGGT GTAGCGCTGC 1680
AAAGGATGAA GCAAGAGTTT CTCATTAACC TTGTGAAGCA AAAGCCACAA ATAACAGAGG 1740
AACAACTTGA GGCTCTCATT GCAGATTTCT CAGGCCTGTT GGAGAAATGC TGCCAAGGCC 1800
AGGAACAGGA AGTCTGCTTT GCTGAAGAGG GACAAAAACT GATTTCAAAA ACTGGTGCTG 1860
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CTTTGGGAGT TTAAATTACT TCAGGGGAAG AGAAGACAAA ACGAGTCTTT CATTCGGTGT 1920
GAACTTTTCT CTTTAATTTT AACTGATTTA ACACTTTTTG TGAATTAATG ATAAAGACTT 1980
TTATGTGAGA TTTCCTTATC ACAGAAATAA AATATCTCCA AA 2022
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 590 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr
1 5 10 15
Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe
20 25 30
Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val
35 40 45
Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser
50 55 60
Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys
65 70 75 80
His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser
85 90 95
Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro
100 105 110
Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser
115 120 125
Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile
130 135 140
Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu
145 150 155 160
Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala
165 170 175
Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys
180 185 190
Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Pro Val Met
195 200 205
Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu
210 215 220
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Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gin Lys Leu Val
225 230 235 240
Leu Asp Val Ala His Val His Glu His Cys Cys Arg Ala Asp Val Leu
245 250 255
Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln
260 265 270
Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr
275 280 285
Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro
290 295 300
Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe
305 310 315 320
Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val
325 330 335
His Glu Tyr Ser Arg Arg His Pro Gin Leu Ala Val Ser Val Ile Leu
340 345 350
Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr
355 360 365
Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys
370 375 380
Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Giy Leu Phe
385 390 395 400
Gln Lys Leu Gly Glu Tyr Tyr Leu Gin Asn Glu Phe Leu Val Ala Tyr
405 410 415
Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr
420 425 430
Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp
435 440 445
Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His
450 455 460
Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln
465 470 475 480
Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu
485 490 495
Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe
500 505 510
Ile Phe His Lys Asp Leu Cys Gin Ala Gln Gly Val Ala Leu Gln Arg
515 520 525
Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gin Lys Pro Gln Ile
530 535 540
Thr Glu Glu Gln Leu Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu
545 550 555 560
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Glu Lys Cys Cys Gln Gly Gln Glu Gln Giu Val Cys Phe Ala Glu Glu
565 570 575
Gly Gln Lys Leu Ile Ser Lys Thr Gly Ala Ala Leu Gly Val
580 585 590
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr
1 5 10 15
Gln Cys Thr Ala Giu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe
20 25 30
Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val
35 40 45
Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser
50 55 60
Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Giu Giu Leu Cys
65 70 75 80
His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser
85 90 95
Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro
100 105 110
Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser
115 120 125
Cys Glu Ala Tyr Glu Glu Asp Arg Giu Thr Phe Met Asn Lys Phe Ile
130 135 140
Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu
145 150 155 160
Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala
165 170 175
Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys
180 185 190
Glu Leu Arg Glu Ser
195
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 192 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ser Leu Leu Asn Gln His Ala Cys Pro Val Met Lys Asn Phe Gly Thr
1 5 10 15
Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu Ser Gln Lys Phe Thr
20 25 30
Lys Val Asn Phe Thr Glu Ile Gin Lys Leu Val Leu Asp Val Ala His
35 40 45
Val His Glu His Cys Cys Arg Ala Asp Val Leu Asp Cys Leu Gln Asp
50 55 60
Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln Gin Asp Thr Leu Ser
65 70 75 80
Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln
85 90 95
Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro
100 105 110
Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser
115 120 125
Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg
130 135 140
Arg His Pro Gln Leu Ala Val Ser Val Ile Leu Arg Val Ala Lys Gly
145 150 155 160
Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gin Thr Glu Asn Pro Leu Glu
165 170 175
Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gin Glu Ser
180 185 190
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 201 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:B:
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Gin Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe Gin Lys Leu Gly Glu
1 5 10 15
Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro
20 25 30
Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala
35 40 45
Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys
50 55 60
Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His Leu Cys Ile Arg His
65 70 75 80
Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln Cys Cys Thr Ser Ser
85 90 95
Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr
100 105 110
Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp
115 120 125
Leu Cys Gln Ala Gln Gly Val Ala Leu Gin Arg Met Lys Gln Glu Phe
130 135 140
Leu Ile Asn Leu Val Lys Gin Lys Pro Gln Ile Thr Glu Glu Gin Leu
145 150 155 160
Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln
165 170 175
Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile
180 185 190
Ser Lys Thr Gly Ala Ala Leu Gly Val
195 200
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 389 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO;9:
Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr
1 5 10 15
Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe
20 25 30
Ala Gin Phe Val Gin Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val
35 40 45
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Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser
50 55 60
Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Giu Leu Cys
65 70 75 8O
His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser
85 90 95
Gin Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro
100 105 110
Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser
115 120 125
Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile
130 135 140
Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu
145 150 155 160
Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala
165 170 175
Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys
180 185 190
Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Pro Val Met
195 200 205
Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu
210 215 220
Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gin Lys Leu Val
225 230 235 240
Leu Asp Val Ala His Val His Glu His Cys Cys Arg Ala Asp Val Leu
245 250 255
Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln
260 265 270
Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr
275 280 285
Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro
290 295 300
Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe
305 310 315 320
Asn Gln Phe Ser Ser Giy Glu Lys Asn Ile Phe Leu Ala Ser Phe Val
325 330 335
His Glu Tyr Ser Arg Arg His Pro Gin Leu Ala Val Ser Val Ile Leu
340 345 350
Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr
355 360 365
Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys
370 375 380
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Tyr Ile Gin Glu Ser
385
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ser Lou Lou Asn Gin His Ala Cys Pro Val Met Lys Asn Phe Gly Thr
1 5 10 15
Arg Thr Phe Gin Ala Ile Thr Val Thr Lys Lou Ser Gin Lys Phe Thr
20 25 30
Lys Val Asn Phe Thr Glu Ile Gin Lys Lou Val Lou Asp Val Ala His
35 40 45
Val His Glu His Cys Cys Arg Ala Asp Val Lou Asp Cys Lou Gin Asp
50 55 60
Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gin Gin Asp Thr Lou Ser
65 70 75 80
Asn Lys Ile Thr Glu Cys Cys Lys Lou Thr Thr Lou Glu Arg Gly Gin
85 90 95
Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro Glu Gly Lou Ser Pro
100 105 110
Asn Lou Asn Arg Phe Lou Gly Asp Arg Asp Phe Asn Gin Phe Ser Ser
115 120 125
Gly Glu Lys Asn Ile Phe Lou Ala Ser Phe Val His Glu Tyr Ser Arg
130 135 140
Arg His Pro Gin Lou Ala Val Ser Val Ile Lou Arg Val Ala Lys Gly
145 150 155 160
Tyr Gin Glu Lou Lou Glu Lys Cys Phe Gin Thr Glu Asn Pro Lou Glu
165 170 175
Cys Gin Asp Lys Gly Glu Glu Glu Lou Gin Lys Tyr Ile Gin Glu Ser
180 185 190
Gln Ala Lou Ala Lys Arg Ser Cys Gly Lou Phe Gin Lys Lou Gly Glu
195 200 205
Tyr Tyr Lou Gin Asn Glu Phe Lou Val Ala Tyr Thr Lys Lys Ala Pro
210 215 220
Gin Lou Thr Ser Ser Glu Lou Met Ala Ile Thr Arg Lys Met Ala Ala
225 230 235 240
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Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys
245 250 255
Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His Leu Cys Ile Arg His
260 265 270
Glu Met Thr Pro Val Asn Pro Gly Val Gly Gin Cys Cys Thr Ser Ser
275 280 285
Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr
290 295 300
Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp
305 310 315 320
Leu Cys Gin Ala Gln Gly Val Ala Leu Gin Arg Met Lys Gln Glu Phe
325 330 335
Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile Thr Glu Giu Gln Leu
340 345 350
Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln
355 360 365
Gly Gin Glu Gln Glu Val Cys Phe Ala Giu Glu Gly Gln Lys Leu Ile
370 375 380
Ser Lys Thr Gly Ala Ala Leu Gly Val
385 390
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 325 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Ser Tyr Ile Cys Ser Gin Gln Asp Thr Leu Ser Asn Lys Ile Thr
1 5 10 15
Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His
20 25 30
Ala Glu Asn Asp Giu Lys Pro Glu Giy Leu Ser Pro Asn Leu Asn Arg
35 40 45
Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn
50 55 60
Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln
65 70 75 80
Leu Ala Val Ser Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu
85 90 95
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Leu Glu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys
100 105 110
Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser Gin Ala Leu Ala
115 120 125
Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln
130 135 140
Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser
145 150 155 160
Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr
165 170 175
Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala
180 185 190
Ala Asp Ile Ile Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro
195 200 205
Val Asn Pro Gly Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg
210 215 220
Arg Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro
225 230 235 240
Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp'Leu Cys Gln Ala
245 250 255
Gln Gly Val Ala Leu Gln Arg Met Lys Gln Glu Phe Leu Ile Asn Leu
260 265 270
Val Lys Gln Lys Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Leu Ile
275 280 285
Ala Asp Phe Ser Giy Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln
290 295 300
Glu Val Cys Phe Ala Glu Glu Giy Gln Lys Leu Ile Ser Lys Thr Gly
305 310 315 320
Ala Ala Leu Gly Val
325
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu
1 5 10 15
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Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gin Cys Ile Ile His Ala
20 25 30
Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro'Asn Leu Asn Arg Phe
35 40 45
Leu Gly Asp Arg Asp Phe Asn Gin Phe Ser Ser Giy Glu Lys Asn Ile
50 55 60
Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu
65 70 75 80
Ala Val Ser Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu
85 90 95
Glu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly
100 105 110
Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys
115 120 125
Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn
130 135 140
Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser
145 150 155 160
Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys
165 170 175
Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys Gly Giu Gly Ala Ala
180 185 190
Asp Ile Ile Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val
195 200 205
Asn Pro Gly Val Gly Gin Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg
210 215 220
Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala
225 230 235 240
Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gin
245 250 255
Giy Val Ala Leu Gin Arg Met Lys Gin Glu Phe Leu Ile Asn Leu Val
260 265 270
Lys Gin Lys Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Leu Ile Ala
275 280 285
Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Giu
290 295 300
Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Gly Ala
305 310 315 320
Ala Leu Gly Val
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Ser Tyr Ile Cys Ser Gln Gln Asp Thr
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AAAAAAGGTA CCACACTGCA TAGAAATGAA 30
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
AAAAAAGGAT CCTTAGCTTT CTCTTAATTC TTT 33
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
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AAAAAAATCG ATATGAGCTT GTTAAATCAA CAT 33
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
AAAAAAGGAT CCTTAGCTCT CCTGGATGTA TTT 33
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
AAAAAAATCG ATATGCAAGC ATTGGCAAAG CGA 33
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
AAAAAAGGAT CCTTAAACTC CCAAAGCAGC ACG 33
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AAAAAAATCG ATATGTCCTA CATATGTTCT CAA 33
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GATCTAGAAT TCGGATCCGG T 21
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Asp Leu Glu Phe Met Thr Leu His Arg Asn
1 5 10
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
AAAAAACTCG AGATACACTG CATAGAAATG AA 32
(2) INFORMATION FOR SEQ ID NO:24:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
AAAAAAGAAT TCTTAAACTC CCAAAGCAGC ACG 33
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I claim:
