U.S. patent application number 12/036796 was filed with the patent office on 2009-02-26 for recombinant human alpha-fetoprotein as an immunosuppressive agent.
This patent application is currently assigned to Merrimack Pharmaceuticals, Inc.. Invention is credited to Robert A. Murgita.
Application Number | 20090053254 12/036796 |
Document ID | / |
Family ID | 23488581 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090053254 |
Kind Code |
A1 |
Murgita; Robert A. |
February 26, 2009 |
RECOMBINANT HUMAN ALPHA-FETOPROTEIN AS AN IMMUNOSUPPRESSIVE
AGENT
Abstract
Disclosed are methods of inhibiting autoreactive immune cell
proliferation in a mammal, involving administering to the mammal a
therapeutically effective amount of recombinant human
alpha-fetoprotein or an immune cell anti-proliferative fragment or
analog thereof.
Inventors: |
Murgita; Robert A.;
(Montreal, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Merrimack Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
23488581 |
Appl. No.: |
12/036796 |
Filed: |
February 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10838476 |
May 4, 2004 |
7423024 |
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12036796 |
|
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|
09940308 |
Aug 27, 2001 |
6774108 |
|
|
10838476 |
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|
09186723 |
Nov 5, 1998 |
6288034 |
|
|
09940308 |
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08377309 |
Jan 24, 1995 |
5965528 |
|
|
09186723 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
C12N 2799/026 20130101;
A61K 2300/00 20130101; A61K 38/1709 20130101; A61K 47/62 20170801;
C07K 2319/00 20130101; C07K 14/4715 20130101; A61K 38/1709
20130101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1. A method of treating autoimmune deficiency syndrome (AIDS) in a
mammal in need thereof, said method comprising administering to
said mammal recombinant human alpha-fetoprotein or a
biologically-active fragment thereof.
2-12. (canceled)
13. The method of claim 1, wherein said recombinant human
alpha-fetoprotein or fragment thereof is glycosylated.
14. The method of claim 1, wherein said recombinant human
alpha-fetoprotein or fragment thereof is unglycosylated.
15. The method of claim 1, wherein said biologically-active
fragment is selected from Domain I, Domain II, Domain III, Domain
I+II, Domain II+III, or Fragment I.
16. The method of claim 1, wherein said mammal is a human.
17. The method of claim 1, wherein said method comprises
administering a therapeutically effective amount of said
recombinant human alpha-fetoprotein or biologically-active fragment
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
10/838,476, filed on May 4, 2004, allowed, which is a continuation
of U.S. Ser. No. 09/940,308, filed on Aug. 27, 2001, now U.S. Pat.
No. 6,774,108, which is a continuation-in-part of U.S. Ser. No.
09/186,723, filed on Nov. 5, 1998, now U.S. Pat. No. 6,288,034,
which is a continuation of U.S. Ser. No. 08/377,309, filed Jan. 24,
1995, now U.S. Pat. No. 5,965,528, each of which is hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to methods for treating autoimmune
diseases.
[0003] Responses of the immune system initiate the destruction and
elimination of invading organisms and toxic molecules produced by
them. Because these immune reactions are destructive, it is
essential that they be made in response only to molecules that are
foreign to the host and not to those of the host itself. The
ability to distinguish foreign molecules from self molecules is a
fundamental feature of the immune system. Occasionally the immune
system fails to make this distinction and reacts destructively
against the host's own molecules; such autoimmune diseases can be
fatal. Thus, tolerance to self antigens breaks down, causing the
components of the immune system such as T or B cells (or both) to
react against their own tissue antigens. Multiple sclerosis,
rheumatoid arthritis, myasthenia gravis, insulin-dependent diabetes
mellitus, and systemic lupus erythematosus are a few examples of
such autoimmune diseases.
SUMMARY OF THE INVENTION
[0004] I have discovered that recombinant human alpha-fetoprotein
made in a prokaryote (e.g., E. coli or baculovirus) or eukaryote is
useful for inhibiting autoreactive immune cells derived from a
mammal. Accordingly, the invention features a method of inhibiting
transplant rejection 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. Preferably, such
immune cells include T cells or B cells; and the recombinant human
alpha-fetoprotein used in such methods is produced in a prokaryotic
cell (e.g., E. coli or baculovirus) or eukaryotic (e.g., transgenic
animal) and is glycosylated or unglycosylated.
[0005] In another aspect, the invention features a method of
inhibiting graft-versus-host 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.
Preferably, the recombinant human alpha-fetoprotein used in such
methods is produced in a prokaryotic cell (e.g., E. coli or
baculovirus) or eukaryotic (e.g., transgenic animal) and is
glycosylated or unglycosylated.
[0006] In yet another aspect, the invention features a method of
mitigating the side effects in a mammal (e.g. a human patient)
undergoing chemotherapy, involving administering to the mammal a
therapeutically effective amount of recombinant human
alpha-fetoprotein or an immune cell anti-proliferative fragment or
analog thereof. Preferably, the recombinant human alpha-fetoprotein
used in such methods is produced in a prokaryotic cell (e.g., E.
coli or baculovirus) or eukaryotic (e.g., transgenic animal) and is
glycosylated or unglycosylated.
[0007] In an additional aspect, the invention features a method of
mitigating the side effects in a mammal (e.g., a human patient)
undergoing irradiation therapy, involving administering to the
mammal a therapeutically effective amount of recombinant human
alpha-fetoprotein or an immune cell anti-proliferative fragment or
analog thereof. Preferably, the recombinant human alpha-fetoprotein
used in such methods is produced in a prokaryotic cell (e.g., E.
coli or baculovirus) or eukaryote (e.g., transgenic animal) and is
glycosylated or unglycosylated. In other preferred embodiments,
such methods further involve administering to the mammal an
immunosuppressive agent in an effective dose that 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 or baculovirus) or
eukaryote (e.g., transgenic animal) and is glycosylated or
unglycosylated.
[0008] 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).
[0009] By "therapeutically effective amount" is meant a dose of
unglycosylated recombinant human alpha-fetoprotein (or a fragment
or analog thereof) capable of inhibiting autoreactive immune cell
proliferation.
[0010] By "recombinant 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. Ser. No. 08/133,773
issuing as U.S. Pat. No. 5,384,250.
[0011] 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 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 and it is believed that
relatively high doses can be safely administered.
[0012] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION
[0013] The drawings will first be described.
DRAWINGS
[0014] FIG. 1A is an autoradiography showing the purity of
recombinant AFP produced in E. coli. (ErAFP) on a 10%
non-denaturing alkaline polyacrylamide gel. Mouse amniotic fluid
proteins (transferrin, AFP and albumin) are shown in lane 1,
natural human AFP (HuAFP) and ErAFP are shown in lane 2 and lane 3,
respectively.
[0015] FIG. 1B is an autoradiography showing the purity of ErAFP
produced in E. coli on a 10% sodium dodecyl sulfate-polyacrylamide
gel. Molecular weight markers are shown in lane 1, HuAFP and ErAFP
are shown in lane 2 and lane 3, respectively.
[0016] FIG. 1C is series of FPLC chromatograms showing the elution
profile of HuAFP and ErAFP from a MonoQ anion exchange column. The
superimposed chromatograms identify HuAFP (Chromatogram 1) and
ErAFP (Chromatogram 2).
[0017] FIG. 1D is a series of HPLC chromatograms showing the
elution profile of HuAFP and ErAFP obtained by passing 50 .mu.g of
HuAFP and ErAFP 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 HuAFP (Chromatogram 1)
and ErAFP (Chromatogram 2).
[0018] FIG. 2A is an autoradiograph showing the purity of
recombinant AFP produced in baculovirus (BrAFP) on a 10%
non-denaturing alkaline polyacrylamide gel. Protein samples are
HuAFP (lane 2), BrAFP (lane 3), and ErAFP (lane 4). Molecular
weight markers and mouse amniotic fluid are shown in lane 1.
[0019] FIG. 2B is an autoradiograph showing the purity of BrAFP on
10% SDS-acrylamide gel. Protein samples are HuAFP (lane 2), BrAFP
(lane 3), and ErAFP (lane 4). Molecular weight markers and mouse
amniotic fluid are shown in lane 1.
[0020] FIG. 2C is a series of FPLC chromatograms showing the
elution profile of HuAFP, BrAFP, and ErAFP from a MonoQ anion
exchange column. The superimposed chromatograms identify HuAFP
(Chromatogram 1), BrAFP (Chromatogram 2), and ErAFP (Chromatogram
3).
[0021] FIG. 2D is a series of HPLC chromatograms showing the
elution profile of HuAFP, BrAFP, and ErAFP obtained by passing 50
.mu.g of HuAFP, BrAFP, and ErAFP 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), BrAFP (Chromatogram 2), and ErAFP
(Chromatogram 3).
[0022] FIG. 3A is a graph showing the inhibitory effect of the
ErAFP on the kinetics of T cell activation. The proliferative
responses were measured over a 4 day time course of cells cultured
in the absence (V) and in the presence of 100 .mu.g/ml () ErAFP. (
) denotes the background proliferation of the responder cell
population cultured separately. ErAFP-mediated suppression on the
AMLR over the time course was significant (p<0.01).
[0023] FIG. 3B is a graph showing the dose-response relationship of
ErAFP on autoproliferating T cells. The inhibition of
autoproliferating T cells was determined at 144 hours with amounts
of ErAFP ranging from 6-100 .mu.g/ml (). (.gradient.) denotes the
control response of the reaction in the absence of protein.
Inhibition of autoreactive T cells by ErAFP in the range of
12.5-100 .mu.g/ml is significant (p<0.005).
[0024] FIG. 4 is a bar graph showing that monoclonal anti-HuAFP
antibodies (aAFP) block immunosuppression of the autologous mixed
lymphocyte reactions (AMLR) by ErAFP (E. coli AFP).
Immunosuppression by ErAFP was significant (p<0.002) and
blocking of ErAFP-mediated immunosuppression by monoclonal
anti-HuAFP antibodies was also significant (p<0.03).
[0025] FIG. 5 is a chart showing that monoclonal antibodies that
recognize HuAFP block immunosuppression of AMLR by BrAFP and
ErAFP.
[0026] FIG. 6 is a chart showing the immunosuppressive effects of
BrAFP, ErAFP, and the AFP fragment of amino acids 1-22
(.DELTA.(1-266)) on mitogen stimulated peripheral blood
lymphocytes.
[0027] FIG. 7A is a bar graph showing the immunosuppressive effect
of human derived full-length HuAFP (HuAFP) versus human domain III
AFP (HuDomIII) in AMLR. The gel insert confirms the size of the
various recombinant AFP used in the AMLR assays: molecular weight
markers (MW), 1 .mu.g HuAFP (lane 1), and 1 .mu.g HuDom III (lane
2).
[0028] FIG. 7B is a graph showing the time course of HuAFP and
HuDom III on immunosuppression of AMLR.
[0029] FIG. 8 is a schematic showing the nucleotide sequence (SEQ
ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the
cDNA encoding human alpha-fetoprotein.
[0030] FIG. 9 is an autoradiograph showing the sizes of various AFP
and AFP fragments (Lane A, MW marker; Lane B, HuAFP, Lane C,
unpurified HuAFP and HuAFP Fragment I, Lane D, purified HuAFP
Fragment I, and Lane E, purified full-length HuAFP).
[0031] FIG. 10A is a series of histograms showing an increased
percentage of bone marrow cells (BM) that express the major
histocompatibilty class I protein (MHC I) in presence of
rHuAFP.
[0032] FIG. 10B is a bar graph showing an increased number of BM
cells that express MHC I in the presence of rHuAFP.
[0033] FIG. 11A is a series of histograms showing an increased
percentage of BM cells that express the major histocompatibilty
class II protein (MHC II) in presence of rHuAFP.
[0034] FIG. 11B is a bar graph showing an increased number of BM
cells that express MHC II in the presence of rHuAFP.
[0035] FIG. 12A is a series of FACS histogram patterns showing
increased expression of the MHCI protein H-2K.sup.K and increased
expression of the MHCII protein I-2A.sup.k in BM cells cultured in
the presence of rHuAFP.
[0036] FIG. 12B is a series of bar graphs showing an increase in
the percent of BM cells that express H-2K.sup.K+ and I-2A.sup.K+ in
either medium alone (control), rHuAFP, human albumin (HuAlb), or
mouse albumin (MoAlb).
[0037] FIG. 13 is a bar graph showing the viability of BM cell
cultures treated with 1% FCS in the presence or absence of IL-3,
IL-7, or rHuAFP.
[0038] FIG. 14A is a bar graph showing the density of
non-irradiated BM cells cultured in the presence of rHuAFP or
Il-7.
[0039] FIG. 14B is a bar graph showing the density of irradiated BM
cells cultured in the presence of rHuAFP or Il-7.
[0040] FIG. 15A is a series of bar graphs showing enhanced
expression of MHCI and enhanced density of MCHI-expressing BM in
the presences of rHuAFP after irradiation.
[0041] FIG. 15B is a series of bar graphs showing enhanced
expression of MHCII and enhanced density of MCHII-expressing BM in
the presences of rHuAFP after irradiation.
PRODUCTION OF RECOMBINANT HUMAN ALPHA-FETOPROTEIN
[0042] As summarized above, the invention includes therapies for
the prevention and treatment of autoimmune diseases involving
recombinant human alpha-fetoprotein ("rHuAFP") or fragments or
analogs thereof.
Production of Recombinant E. coli Alpha-Fetoprotein
[0043] Methods for producing such rHuAFP in a prokaryotic cell are
described in U.S. Ser. No. 08/133,773 and in U.S. Pat. No.
5,384,250, issued Jan. 24, 1995, hereby incorporated by
reference.
Expression, Purification, and Characterization of Recombinant AFP
Produced in Baculovirus (BrAFP)
[0044] One eukaryotic expression system that is widely employed for
the overexpression of heterologous genes is the baculovirus
expression system. There are several advantages to generating
recombinant protein in baculovirus infected insect cells, including
the ability of this expression system to produce high levels of
soluble, secreted, and post-translationally modified proteins (see,
O'Reilly, et al. Baculovirus Expression Vectors. A Laboratory
Manual. W.H. Freeman and Company, New York, 1980). To investigate
whether post-translational modifications play a role in mediating
AFP immunomodulation, we compared the biological activities of a
eukaryotic protein with that produced by a prokaryotic organism. E.
coli was selected as the prokaryotic host in which to express AFP
because it offered advantages such as ease and simplicity in
cloning and expressing a heterologous gene (see, Balbas &
Bolivar. Gene Expression Technology, Goeddel, D. V. ed. New York
Academic Press, 1990), simple fusion protein strategies which
ensures good translation initiation that may also permit one to
overcome the instability problems that can be encountered with
small peptides, and rapid generation of biomass due to high rates
of cell growth (Id.).
[0045] 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
from the supernatant of uninfected or wild-type virus-infected Sf9
cells. Passage of the supernatant containing secreted BrAFP over
ConA lectin chromatography resulted in the binding of the
recombinant protein and elution in the flow through of more than
90% of the contaminating proteins. Methyl .alpha.-D mannopyranoside
was used to elute BrAFP from the lectin column. Final purification
of the BrAFP preparation was achieved by Mono Q FPLC
chromatography, yielding a single polypeptide with an apparent
molecular mass of 67 kD (FIG. 2B, lane 3). The BrAFP molecular
weight is similar to that observed for the natural human molecule
(FIG. 2B, lane 2). This result, in addition to the binding of BrAFP
to the ConA column, indicated that BrAFP was post-translationally
modified via glycosylation. However, the pattern of glycosylation
of the BrAFP is expected to differ from that of the native
molecule, since Sf9 cells infected with recombinant baculovirus
have been reported to be deficient in their ability to carry out
complex glycosylation normally observed with higher eukaryotic
derived proteins (O'Reilly, supra; James, et al. Biotechnology
13:592-596, 1995). Purity of the isolated BrAFP was verified by
APAGE and SDS-PAGE (FIGS. 2A & 2B, lane 2, respectively), and
is illustrated by a single symmetrical peal on FPLC and HPLC
chromatograms as shown in FIG. 2C, graph 2, and FIG. 2D, graph 2,
respectively. N-terminal analysis revealed that the melittin signal
peptide was cleaved from the mature recombinant human AFP
polypeptide as predicted.
[0046] Recombinant AFP expressed in E. coli represented
approximately 10% of total cell protein as determined by
densitometric analysis of Coomassie blue stained SDS-PAGE gels.
Alkaline washes of lysed E. Coli pellets removed major
contaminating proteins resulting in a 4-fold enrichment of ErAFP.
The recombinant protein was solubulized by dissolving the pellet in
a buffer containing guanidine and .beta.-mercaptoethanol, and
subsequently refolded by rapid dilution of the denaturant and
reducing agent. Monomer BrAFP and ErAFP was efficiently separated
from micro aggregates by employing Q-Sparse chromatography. Pure
ErAFP was subsequently recovered as a single homogenous peak by
FPLC Mono-Q anion exchange chromatography. The final product
migrated at 65 kD on SDS-PAGE (FIG. 2B, lane 4). Rechromatographed
samples of pure ErAFP on FPLC and HPLC yielded a single peak as
shown in FIG. 2C, graph 3 and FIG. 2D, graph 3 respectively,
confirming the purity of the BrAFP and ErAFP preparation.
[0047] Baculovirus transfer vector pVT-PlacZ was modified by
replacing the MCS with the oligonucleotide
5'-GATCTAGAATTCGGATCCGGT-3' (SEQ ID NO: 17) and its complementary
fragment, containing EcoR I and BamH I restrictions sites in the 5'
to 3' direction. The rHuAFP cDNA fragment was isolated as above,
inserted into the vector at the EcoR I and BamH I restriction sties
and transformations were verified for the presence and correct
orientation of the rHuAFP cDNA fragment under the control of the
polyhedrin promoter by using restriction enzyme analysis.
[0048] Four mg of transfer vector pVT-PLacZ/HuAFP and 1 mg of
linearized wild-type AcMNPV baculovirus DNA (Invitrogen, San Diego,
Calif.) were co-transfected in the presence of 50 ml of Lipofectin
Reagent (Gibco) into Spodoptera frigiperda (Sf9) cells. After 4-6
days of incubation, the transfection mixture was screened by a
.beta.-galactosidase assay and DNA slot-blot hybridizations for
recombinant viruses containing both the .beta.-galactosidase and
rAFP cDNA's. Sf9 cells seeded at a density of 1.times.10.sup.6
cells/ml in 500 ml spinner flasks were infected with BrAFP
baculovirus in serum-free Grace medium at a multiplicity of
infection of 5. The supernatant was harvested at 72 hours
post-infection by pelleting the Sf9 cells at 200.times.g for 10
minutes and the resultant media was concentrated 10-20 fold by
ultrafiltration with a YM30 Amicon filter membrane (Amicon).
[0049] Concentrated Grace media containing baculovirus produced AFP
was dialyzed against PBS overnight and then applied to a ConA
Lectin column (Pharmacia) where all rAFP was bound to the column.
Recombinant AFP (BrAFP) was eluted with 0.4M methyl a-D
mannopyranoside and this fraction was further purified by
recovering protein from a FPLC anion-exchange MonoQ column
(Pharmacia) using a linear NaCl gradient from 0-100% 1 M NaCl in 20
mM phosphate buffer pH 8.0. Purified BrAFP protein preparations
were dialyzed against 1.times.PBS and stored at -20.degree. C.
Expression of Alpha-Fetoprotein in Eukaryotes
[0050] Recombinant alpha-fetoprotein can be expressed in transgenic
animals. Transgenic animals may be prepared using methods well
known to the skilled artisan. For example, to prepare transgenic
rodents such as mice, methods such as those set forth by Hogan et
al., eds. (Manipulating The Mouse Embryo: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1986])
may be employed. Additionally, other animals that are suitable for
expression of an alpha-fetoprotein transgene include goat, sheep,
and cow.
[0051] Recombinant alpha-fetoprotein transgene expression can be
directed to the milk of the transgenic animal. The
alpha-fetoprotein transgene is generated in association with a
mammary promoter to direct expression of the protein to the
animal's milk; is introduced into the pronucleus of a mammalian
zygote (usually by microinjection into one of the two nuclei
(usually the male nucleus) in the pronucleus); and thereafter
implanted into a foster mother. A proportion of the animals
produced by the foster mother will carry and express the introduced
gene that has integrated into a chromosome. Usually the integrated
gene is passed on to offspring by conventional breeding thus
allowing ready expansion of stock. Preferably the alpha-fetoprotein
is simply harvested from the milk of female transgenic animals.
Procedures for directing expression of a gene to the animal's milk
are found in the following publications: Simons et al. (1988),
Bio/Technology 6:179-183; Wright et al. (1991) Bio/Technology
9:830-834; U.S. Pat. No. 4,873,191 and; U.S. Pat. No. 5,322,775.
Manipulation of mouse embryos is described in Hogan et al,
"Manipulating the Mouse Embryo; A Laboratory Manual", Cold Spring
Harbor Laboratory 1986.
[0052] Mammalian cells (for example, CHO, COS, and myeloma cells)
can be used as host for the expression of alpha-fetoprotein cDNAs
and fragments thereof to produce the corresponding proteins and
peptides. For expression of constructs leading to direct expression
of active COS or CHO cell expression systems are preferred. The
alpha-fetoprotein cDNAs can be introduced to plasmids and allowed
to integrate into chromosomal DNA especially for CHO cells or
allowed to replicate to very high copy number especially in COS
cells. The plasmids generally require a selectable marker for
maintenance in transfected hosts, an efficient eukaryotic promoter
to allow a high level of transcription from the cDNAs, convenient
restriction enzyme sites for cloning and polyadenylation, and
transcription termination signals for message stability. Several
such vectors have been described in the literature (Bebbington, C.
et al, 1992, Bio/Technology, vol 10, p 169-175, and Wright, A.,
1991, Methods, vol 2, p 125-135) and there are commercially
available vectors, (such as pRc/CMV, Invitrogen Corp.) which are
suitable.
Fragments and Analogs
[0053] The invention includes biologically active fragments of AFP
from 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 an autoreactive immune cell); or (b) halts, reduces, or
inhibits the growth of an autoreactive immune cell (e.g., binds to
a cell surface receptor and imparts an anti-proliferative signal);
or (c) blocks, inhibits, or prevents an immunopathologic antibody
reaction. The ability of rHuAFP fragments or analogs to bind to a
receptor which is recognized by rHuAFP can be tested using any
standard binding assay known in the art. Methods for assaying the
biological activity or rHuAFP fragments and analogs are also known
in the art, e.g., those described herein. Accordingly, a rHuAFP
fragment, like the full-length rHuAFP molecule, can be used inhibit
autoreactive immune cell proliferation.
[0054] In general, fragments of rHuAFP are produced according to
the techniques of polypeptide expression and purification described
in U.S. Ser. No. 08/133,773 (U.S. Pat. No. 5,384,250). For example,
suitable fragments of rHuAFP can be produced by transformation of a
suitable host bacterial cell with part of a 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).
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. Once isolated, the recombinant protein can, if desired, be
further purified, e.g., by high performance liquid chromatography
(see, e.g., Fisher, Laboratory Techniques In Biochemistry And
Molecular Biology, Work and Burdon, eds., Elsevier, 1980).
[0055] 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), 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.
[0056] Following expression of the fusion protein, it can be
purified by affinity chromatography. For 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.
[0057] 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.
[0058] Recombinant HuAFP fragment expression products (e.g.,
produced by any of the prokaryotic systems described in U.S. No.
Ser. No. 08/133,773 (U.S. Pat. No. 5,384,250)) 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).
[0059] Once a fragment of rHuAFP is expressed, it is isolated using
the methods described supra. Once isolated, the fragment of rHuAFP
can, if desired, be further purified by using the techniques
described 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, Ill.).
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).
[0060] The purified recombinant gene product or fragment thereof
can then be used to raise polyclonal or monoclonal antibodies
against the human recombinant 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 a non-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 that
produces monoclonal antibodies.
[0061] 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).
[0062] Recombinant HuAFP fragments of interest include, but are not
limited to, Domain I (amino acids 1 (Thr)-197 (Ser), see FIG. 4,
SEQ ID NO: 3), Domain II (amino acids 198 (Ser)-389 (Ser), see FIG.
4, SEQ ID NO: 4), Domain III (amino acids 390 (Gln)-590 (Val), see
FIG. 4, SEQ ID NO: 5), Domain I+II (amino acids 1 (Thr)-389 (Ser),
see FIG. 4, SEQ ID NO: 6), Domain II+III (amino acids 198 (Ser)-590
(Val), see FIG. 4, SEQ ID NO: 7), and rHuAFP Fragment I (amino
acids 266 (Met)-590 (Val), see FIG. 4, SEQ ID NO: 8). Activity of a
fragment is evaluated experimentally using conventional techniques
and assays, e.g., the assays described herein.
[0063] 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., .beta. or
.gamma. 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. 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.
[0064] The cloning, expression, isolation and characterization of
exemplary rHuAFP fragments now follows.
These examples are provided to illustrate, not limit, the
invention.
[0065] Experimental
Materials and Methods
[0066] Polymerase Chain Reaction (PCR) rHuAFP Fragments
[0067] 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).
[0068] 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. 4, SEQ ID
NO: 3) Domain II Amino acids 198 (Ser)-389 (Ser), (FIG. 4, SEQ ID
NO: 4) Domain III Amino acids 390 (Gln)-590 (Val), (FIG. 4, SEQ ID
NO: 5) Domain I+II Amino acids 1 (Thr)-389 (Ser), (FIG. 4, SEQ ID
NO: 6) Domain II+III Amino acids 198 (Ser)-590 (Val), (FIG. 4, SEQ
ID NO: 7) rHuAFP Fragment I Amino acids 266 (Met)-590 (Val), (FIG.
4, SEQ ID NO: 8) Amino acid sequences were deduced from those for
human alpha-fetoprotein (1 (Thr)-590 (Val); SEQ ID NO: 2) shown in
FIG. 4. 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 .mu.L
reactions containing 34 .mu.L H.sub.2O, 10 .mu.L 10.times. reaction
buffer, 20 .mu.L 1 mM dNTP, 2 .mu.L DNA template (HuAFP cloned in
pI18), appropriate 5' and 3' oligonucleotide primers (10 .mu.L 10
pmol/.mu.L 5' primer, 10 .mu.L 10 pmol/.mu.L 3' primer), 1 .mu.L
glycerol, 10 .mu.L DMSO, and 1 .mu.L Pfu polymerase (Stratagene,
LaJolla, Calif.). Primers used for PCR amplifications were:
TABLE-US-00001 DomI25 (SEQ ID NO: 9)
5'-AAAAAGGTACCACACTGCATAGAAATGAA-3' DomI3 (SEQ ID NO: 10)
5'-AAAAAAGGATCCTTAGCTTTCTCTTAATTCTTT-3' DomII5 (SEQ ID NO: 11)
5-'AAAAAAATCGATATGAGCTTGTTAAATCAACAT-3' DomII3 (SEQ ID NO: 12)
5'-AAAAAAGGATCCTTAGCTCTCCTGGATGTATTT-3' DomIII5 (SEQ ID NO: 13)
5'-AAAAAAATCGATATGCAAGCATTGGCAAAGCGA-3' DomIII3 (SEQ ID NO: 14)
5'-AAAAAAGGATCCTTAAACTCCCAAAGCAGCACG-3' 5'rHuAFP Fragment I (SEQ ID
NO: 15) 5'-AAAAAAATCGATATGTCCTACATATGTTCTCAA-3'
Accordingly, primer pairs DomI25 and DomI3, DomII5 and DomII3,
DomIII5 and DomIII3, 5'rHuAFP Fragment I and DomIII3, DomI25 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.degree. C., 72.degree. C.,
and 94.degree. 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
Bsp106I/BamHI-treated pTrp4. Each plasmid construct was
subsequently transformed into competent E. coli cells. Since the
expression product will begin with the amino acid 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.
[0069] Autologous Mixed Lymphocyte Reactions (AMLR)
[0070] AMLR assays were performed as described below.
Results
[0071] Expression and Purification
[0072] E. coli containing the expression plasmid encoding rHuAFP
Fragment I was cultured and purified. FIG. 9 (lane D) shows the
SDS-PAGE profile of the purified rHuAFP Fragment I. N-terminal
amino acid sequence analysis showed that rHuAFP Fragment I
possessed the amino acid sequence
Ser.sub.267-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr.sub.275 (SEQ ID NO: 16)
which corresponds to the expected N-terminal amino acid sequence of
rHuAFP Fragment I (see FIG. 8, SEQ ID NO: 2) where the initiating
methionine is cleaved intracellularly.
[0073] Inhibition of the Autologous Mixed Lymphocyte Reactions
(AMLR)
[0074] The immunosuppressive activity of 100 .mu.g/ml rHuAFP
Fragment I was assessed by its ability to suppress human autologous
mixed lymphocyte reactions (AMLR). As shown in Table I, rHuAFP
Fragment I inhibited the proliferative response of autoreactive
lymphocytes stimulated by autologous non-T cells at 144 hours.
TABLE-US-00002 TABLE I Thymidine Incorporation Reaction Setup (CPM)
T Cells 7118 .+-. 964 AMLR 83103 .+-. 6480 AMLR + rHuAFP Fragment I
29692 .+-. 2963 (100 .mu.g/ml)
Recombinant HuAFP as an Immunosuppressive Agent
[0075] Immunosuppressive attributes of rHuAFP (or a fragment or
analog thereof) were 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).
[0076] The following examples demonstrate that unglycosylated
rHuAFP inhibits T cell autoproliferation in response to self
antigens. These examples are provided to illustrate, not limit, the
invention.
Experimental
Materials and Methods
[0077] Gel Electrophoresis, Immunoblotting and Purification
[0078] 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 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.
[0079] Column chromatography was performed according to standard
methods.
[0080] Cell Preparation
[0081] Human peripheral blood mononuclear cells (PBMC) were
isolated from heparin-treated whole blood of normal adult donors by
venipuncture. Blood was diluted 1:1 with PBS, layered on
Ficoll-Hypaque (Sigma, St. Louis, Mo.) and centrifuged at
400.times.g for 30 min at 25.degree. C. Cells were removed from the
interface, washed 3 times in PBS and examined under microscope for
viability using the Trypan Blue exclusion method. Cell preparations
which were less than 95% viable were discarded. At this stage, the
cell preparations were ready to be cultured for the Con A mitogen
assay. For the autologous mixed lymphocyte reaction (AMLR), PBMC
were fractionated into T cell and non T cell populations. Responder
T cells for the AMLR were prepared by passing 1.5.times.10.sup.8
PBMC over a commercial Ig-anti-Ig affinity column (Biotex
Laboratories, Edmonton, AB), washing 3 times in PBS, and
resuspending them in RPMI-1640 medium. The separation of non-T from
T cell populations in PBMC was based on the capacity of T cells to
form E-rossettes with sheep red blood cells (SBRC) (Mendas, et al.
J. Immunol. 111:860-867, 1973). One ml of pelleted SRBC was treated
with 1 U of neuraminidase (Sigma) for 1 hour at 37.degree. C.,
washed 3 times, and resuspended in 50 ml of RPMI media to yield a
2% SRBC solution. This procedure enhanced cellular interactions
between T-cells and SRBC. PBMC (5-9.times.10.sup.7) were then
incubated in a ratio of 1.times.10.sup.7 PBMC: 2 ml SRBC solution:
2 ml heat inactivated FCS at 37.degree. C. for 10 min. This was
followed by 5 min of centrifugation at 200.times.g prior to a
second incubation for 60 min at 4.degree. C. The cell mixtures were
then gently resuspended and the rossetted T cells separated from
the non-T cells by density centrifugation on Ficoll-Hypaque for 30
min at 400.times.g. The non-T cells were collected from the
interface, washed three times and resuspended in RPMI-1640
media.
[0082] Autologous Mixed Lymphocyte Reactions (AMLR)
[0083] Isolation of human PBMC, their fractionation into non-T cell
populations, and the AMLR, were performed according by the standard
procedure described above. Responder T cells were isolated by
passing 1.5.times.10.sup.8 PMBC over a commercial Ig-anti-Ig
affinity column (Biotek Laboratories) and 2.times.10.sup.5
responder cells were subsequently cultured with 2.times.10.sup.5
autologous .sup.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.times.10.sup.-5 M
2-mercaptoethanol (BDH, Montreal, QC), 4 mM L-glutamine (Gibco),
100 U/ml penicillin (Gibco) and 100 .mu.g/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 .mu.g/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.degree. C. in 95% air and 5% CO.sub.2. At the indicated
intervals, DNA synthesis was assayed by a 6 hour pulse with 1
.mu.Ci of .sup.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 .sup.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.
[0084] Mitogen-Stimulated Lymphocyte Assays
[0085] Mitogen cultures consisted of 2.5.times.10.sup.5 PBMC
stimulated with 1 .mu.g/ml of Con A (Pharmacia). The media employed
consisted of RPMI-1640 supplemented with 20 mM HEPES (Gibco),
5.times.10.sup.-5 M 2-mercaptoethanol (BDH, Montreal, QC), 4 mM
L-glutamine (Gibco), 100 U/ml penicillin (Gibco) and 100 mg/ml
streptomycin sulfate and 2 mg/ml of low endotoxin human serum
albumin (HSA) (ICN Biomedials Canada, Mississauga, ON). Purified
rAFP from both recombinant sources were added to the cultures at a
concentration of 100 .mu.g/ml. Mitogen reactions were cultured for
48 hours at 37.degree. C. in 95% air and 5% CO.sub.2 and assayed
for proliferative responses as described for the AMLR.
Results
[0086] Expression and Purification of Human Alpha-Fetoprotein
[0087] 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.
[0088] Inhibition of the AMLR
[0089] The immunosuppressive activity of rHuAFP was assessed by its
ability to suppress human AMLR. As shown in FIG. 3A, 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. 3B, 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.
[0090] 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. 4, suppression of proliferating autoreactive
T cells by 100 .mu.g/ml of rHuAFP was completely blocked by
anti-HuAFP MAb. The addition of 100 .mu.g/ml of HSA did not
diminish the AMLR response and the presence of MAb alone in the
reaction culture was without any effect.
[0091] 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 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.
[0092] As shown in FIG. 3A and FIG. 3B, the results of this study
also demonstrate that rHuAFP suppresses the proliferation of
autoreaction T cells with a potency equivalent to glycosylated
rHuAFP 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
.mu.g/ml to 100 .mu.g/ml.
Suppression of Autoproliferating and Mitogen Responsive Lymphocytes
by BrAFP
[0093] In order to address in a definitive manner whether
post-synthetic alterations play a role in mediating the
immunosuppressive properties of AFP, we assessed the ability of
BrAFP and ErAFP, which is not post-translationally modified, to
suppress the proliferative response of autoreactive T cells in the
AMLR. In FIG. 5, 100 .mu.g/ml of BrAFP and ErAFP added at the
initiation of the AMLR suppressed the lymphoproliferative response
by 57% and 58%, respectively. Moreover, BrAFP and ErAFP
anti-proliferative activity was blocked by the addition of
anti-human AFP Mab. An equivalent amount of HSA augmented the
reaction. The possibility that AFP might be causing a shift in the
kinetics of the AMLR was eliminated, when rHuAFP, at a
concentration of 100 .mu.g/ml, was shown to inhibit autoreactive T
lymphocytes from proliferating in response to autologous non-T
cells throughout the autoproliferation stages of the time course
from 96 to 168 hours (FIG. 3A).
[0094] We next examined the effects of various concentrations of
rAFP on DNA synthesis in autoproliferating T cells. A
representative experiment (FIG. 3B) demonstrates a marked
dose-dependent inhibition of .sup.3H-thymidine incorporation, with
significant anti-proliferative effects still observed at 12
.mu.g/ml. Viability studies established that the inhibitory
activity of rAFP on human autoreactive T cells was not due to
non-specific cytotoxic effects.
[0095] We carried out experiments in serum-free media to control
for the possibility that exogenous serum factors may interact with
the genetically engineered protein and mediate the
anti-proliferative activity of recombinant human AFP. As shown in
FIG. 6, experiments 1 and 2 demonstrate that the addition of 100
.mu.g/ml of either BrAFP or ErAFP to in vitro cultures containing
mitogen stimulated PBMC in RPMI media supplemented with 2 mg/ml HSA
reduced lymphoproliferation by more than 60%. The addition of 100
.mu.g/ml HSA also reduced lymphoproliferation by more than 60%. The
addition of HSA at 100 .mu.g/ml had no effect on the Con A assay.
These results demonstrate that neither post-translational
modifications nor exogenous serum factors mediate AFP
immunosuppression.
Endotoxin does not Influence AFP-Mediated Immunosuppression
[0096] Recombinant polypeptides produced in prokaryotic expression
systems are at risk for contamination with host cell
lipopolysaccharides (endotoxin) during their isolation from
bacteria. It has been demonstrated that small amounts of LPS can
antagonize the biological activities of cytokines, thereby
impairing the immune responsiveness of macrophages (Bogdan, et al.
J. Immunol. 151:301-331, 1993). We therefore evaluated the effect
of endotoxin on various ErAFP preparations by performing AMLR
experiments with recombinant protein that had been treated to
remove endogenous endotoxin by passage over Detoxi-gel (Pierce)
versus that ErAFP which was not subjected to the affinity resin. As
shown in FIG. 5, Exp 3, a five fold reduction in the amount of
endotoxin to levels that are below those that stimulate the release
of interleukin 1 from human monocytes (Duff, et al. J Immunol.
Methods 52:323-331, 1982) did not alter the immunosuppressive
activity of the recombinant protein.
Immunosuppression by a 35 kD Fragment Corresponding to an
NH.sub.2-Terminus Deletion of Full-Length ErAFP
[0097] An immunoblot analysis of whole bacterial cell extracts
containing ErAFP identified, in addition to the 67 kD whole AFP
molecule, an immunoreactive protein band with an approximate
molecular weight of 35 kD. This protein was purified on MonoQ FPLC.
Amino terminal sequencing of the 35 kD fragment revealed that this
polypeptide corresponded to the COOH two-thirds of full-length AFP,
beginning at amino acid position 267:
TABLE-US-00003 (SEQ ID NO: 18) 1 5 10
Ser-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr-Leu-
Consequently, we wanted to determine whether this truncated AFP
fragment termed AFP .DELTA.(1-226) retained the immunosuppressive
activity that is observed with the intact molecule. For comparison,
the 25 kD fragment was evaluated in parallel with the complete
ErAFP molecule for its ability to down regulate in vitro T cell
proliferative reactions. It was observed that the AFP
.DELTA.(1-226) polypeptide was similar to full-length rAFP with
respect to mediating immunoregulation, suppressing the AMLR
throughout the kinetics of autoproliferation (FIG. 3) and
inhibiting mitogen induced PBL proliferation by 61% (FIG. 6, Exp.
3). This finding indicates that the first 266 amino acids of AFP
are not required for immunoregulation.
[0098] Generation of a Bioactive AFP Fragment Corresponding to
Domain 3
[0099] The previous study indicated that immunoregulatory active
sites are present within the last two thirds of Domain 2 and intact
Domain 3. Thus, a gene segment corresponding to the third domain of
AFP (Morinaga, et al. Proc. Natl. Acad. Sci. USA 80:4604, 1983) was
cloned by PCR into E. coli. The protein was identified by
immunoblot employing anti-human AFP polyclonal antibodies and was
subsequently purified by Q-sepharose and Mono Q anion exchange
chromatography. The inhibitory activity of Domain 3 on
autoproliferating and mitogen induced proliferating T lymphocytes
was performed in parallel with full-length rAFP. As shown in the
representative experiment in FIG. 7A, the truncated AFP segment
suppressed Con A stimulated PBL's by 60% and inhibited the AMLR by
79%, whereas full-length rAFP downregulated the same in vitro
responses by 50% and 58% respectively. These results demonstrate
that active sites for immunoregulation exist in the latter third of
the AFP molecule.
Autoimmune Disease
[0100] 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
.beta. 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, scleroderma,
and ulcerative colitis.
[0101] 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, Conn., 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 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.
[0102] 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.
[0103] Multiple Sclerosis
[0104] 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.
[0105] 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.)
[0106] 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 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.
[0107] Rheumatoid Arthritis
[0108] 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.
[0109] 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.
[0110] 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 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.
[0111] Myasthenia Gravis
[0112] 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.
[0113] Experimental autoimmune MG (EAMG) has been studied in a
variety of animals including rabbits, monkeys, Lewis 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.
[0114] To evaluate rHuAFP or a fragment or analog thereof, EAMG is
induced in an appropriate laboratory animal, e.g., the C57BL/6
strain (H-2.sup.B) 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 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.
[0115] Insulin-Dependent Diabetes Mellitus
[0116] 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 .beta. 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. Engl. 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).
[0117] 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.
[0118] The BB rat spontaneously develops a disease similar to IDDM,
with insulitis (infiltration of mononuclear cells into the
pancreatic islets) and 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).
[0119] 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.
[0120] Chemically induced diabetes is accomplished using multiple
injections of small doses of streptozotocin, a drug toxic for
pancreatic .beta. cells, which causes severe insulitis and diabetes
(see e.g., Kikutani et al., Adv. Immunol. 51:285, 1992).
[0121] 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).
[0122] 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 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 .beta. 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.
[0123] Systemic Lupus Erythematosus
[0124] 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)
[0125] A number of animal models of human SLE, e.g., inbred mouse
strains including NZB mice and their F.sub.1 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 F.sub.1
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).
[0126] 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 F.sub.1
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. 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
[0127] 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.
[0128] 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 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.
[0129] 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., Il-15) or an interferon (.alpha.-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.
[0130] 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.
[0131] The method(s) of the invention can also be used to treat
non-human mammals, for example, domestic pets, or livestock.
OTHER EMBODIMENTS
[0132] 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., 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.
[0133] 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., .beta. 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.
[0134] In one working example, the immunosuppressive effect of
rHuAFP on clinical transplantation, i.e., the 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, 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 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 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 et al., supra; Bancroft and Stevens, supra) of kidney
tissue is performed and tissue samples 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 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 transplant
rejection is considered useful in the invention.
Graft-Versus-Host Disease
[0135] Studies of neonatal mice grafted with allogeneic T cells
indicates that treatment with rHuAFP has an inhibitory effect on
GVHD. SCID mice at 3 days of age were used as recipients because of
their lack of functional B and T cells. In this experimental system
the grafted T cells can react against the host whereas the
recipient mice cannot mount an effective response against the
graft. A SCID mouse (3 days post-natal) was injected i.p. with 100
.mu.l PBS and 4 hours later with 5.times.10.sup.6 spleen T cells
from C57Bl/6 in 100 .mu.l PBS on day 0. This mouse was thereafter
injected with 100 .mu.l PBS day 1 and 3. The results from this
mouse are presented in the B6 column. A SCID mouse (3 days
post-natal) was injected i.p. with 200 .mu.g rHuAFP in 100 .mu.l
PBP (100 .mu.g rHuAFP per gram body weight) and 4 hours later with
5.times.10.sup.6 spleen T cells from C56B1/6 in 100 .mu.l PBS on
day 0. This mouse was thereafter injected with 50 .mu.g rHuAFP per
gram body weight in 100 .mu.l PBS on day 1 and 3. The results from
this mouse are presented in the B6+AFP column.
[0136] In this experimental system loss of weight and decreased
cell numbers in the lymphoid organs are cardinal signs of severe
GVHD. Differences could be detected in weight, as the mouse inj.
B6+AFP gained more weight than the one inj. B6 (Table 2). The
weight of the AFP treated mouse was close to that of age-matched
untreated SCID mice at our animal facilities.
TABLE-US-00004 TABLE 2 Weight (g) inj inj Day B6 B6 + AFP 0 1.6 1.9
3 2.8 3.2 5 3.2 4.1
[0137] Moreover, at 5 days p.i. the total cell numbers in spleen,
bone marrow, and liver were lower in the mouse inj. B6 when
compared to the mouse inj. B6+AFP (Table 3). A SCID mouse received
an allogenic T cell graft in the absence (inj. B6) or presence
(inj. B6+AFP) of rHuAFP as described for FIG. 5. At 5 days
post-injection, organ size was determined an presented by the
number cells in each tissue (cells.times.10.sup.-6).
TABLE-US-00005 TABLE 3 Organ sizes (cells .times. 10.sup.-6) Organ
inj. B6 inj. B6 + AFP Spleen 12 32 Bone Marrow 6.0 9.2 Liver 0.56
1.0
[0138] At day 5 p.i. the mouse injected with C57Bl/6 only had a
higher percentage of exogenous T cells in spleen, bone marrow, and
liver than the mouse treated with AFP as determined in FACS. Also,
at this time-point the amount of cells expressing CD19 and CD117
(c-kit) was lower in the liver of the mouse that was not treated
with AFP both when counted in percentage and total cell numbers
which indicates a more severe GVHD. When sera from the mice were
analyzed for interferon-.gamma. content, an interleukin that is
elevated during acute GVHD, the concentration in mice inj. B6 (6.5
ng/ml) was twice as high as for mice inj. B6+AFP (3.1 ng/ml). Thus,
these data from GVHD studies indicates an inhibitory effect of AFP
on the allogeneic T cells and suppress GVHD.
Transplant rejection
[0139] Results
[0140] Enhanced MHC Expression on BM Cells Cultured in the Presence
of rHuAFP.
[0141] BM cells from adult C.B-17 mice cultured for three days in
the presence of optimal doses of IL-3 (2000 U/ml), IL-7 (1%), or
rHuAFP (100 .mu.g/ml) were analyzed for expression of MHC class I
(H-2K.sup.d) and MHC class II (I-A.sup.d). Bone marrow cells
(2.5.times.10.sup.6/ml) from C.B.-17 mice were cultured in 2 ml of
f-DMEM medium containing 1% FCS in the presence or absence of IL-3,
IL-7, or rHuAFP. At 3 days of culture, cells were harvested and
stained with anti-H-2K.sup.d and analyzed in FACS. The figure shows
percent of cells with high expression of H-2K.sup.d in medium
control, IL-3, IL-7, and rHuAFP cultures in a representative
experiment. As demonstrated in a representative experiment, a
substantial increase in the intensity of MHC class I (MHC I)
expression was seen on BM cells cultured in the presence of rHuAFP
where 90% of the cells were MHC I.sup.high compared to 61%, 40% and
33% for cells cultured with IL-3, IL-7 and with medium only
respectively (FIGS. 10A and 10B). In the individual experiments,
rHuAFP induced high intensity of MHC I expression on between 88%
and 98% of the cells and interestingly, similar percentages were
seen even after a 10-fold decrease in the concentration of rHuAFP,
i.e. to 10 .mu.g/ml.
[0142] Despite the fact that the total cell numbers were not higher
in cultures supplemented with AFP than with IL-3 or IL-7 (FIG. 13),
also the absolute numbers of cells with high expression of MHC I
were significantly higher in the presence of AFP (FIG. 2B).
(Schneider). Bone marrow cells (10.sup.6 to 2.5.times.10.sup.6/ml)
from C.B-17 mice were cultured in 2 ml of f-DMEM medium containing
1% FCS in the presence or absence of IL-3, IL-7, or rHuAFP. At 3
days of culture, cells were harvested and cell viability was
determined with the trypan blue dye exclusion test. The figure
shows mean percent standard deviation of viable cells at 3 days of
culture out of total number of seeded cells at day 0 as counted
from 4 separate experiments where the total number of seeded cells
varied between 2.times.10.sup.6 to 5.times.10.sup.6 per well. The
cells were cultured and treated as described in FIG. 1A. The figure
shows absolute numbers of cells with high expression of H-2K.sup.d
in medium control, IL-3, IL-7, and rHuAFP cultures in a
representative experiment.
[0143] An increased frequency of MHC class II (MHC II) positive
cells was also seen in the presence of rHuAFP. Bone marrow cells
from C.B-17 mice were cultured as described in FIGS. 11A and 11B.
At 3 days of culture, cells were harvested and stained with
anti-I-A.sup.d and analyzed in FACS. The figure shows percent of
cells positive for I-A.sup.d in medium control, IL-3, IL-7, and
rHuAFP cultures in representative experiment. Flow cytometry
analysis showed that 55% of the BM cells were MHC II.sup.+ after
three days of culture with rHuAFP (FIG. 11B) with was nearly twice
the percentage at day 0 (data not shown) and more than in the
medium control and in the presence of IL-3 and IL-7.
[0144] The absolute numbers of MHC II positive cells were similar
in IL-7 and AFP cultures but significantly higher than in the
medium control and IL-3 cultures (FIG. 11B). (Schneider) Bone
marrow cells from C.B-17 mice were cultured as described in FIG. 1A
and harvested and analyzed as described in FIG. 11A. The figures
shows absolute numbers of cells positive for I-A.sup.d in medium
control, IL-3, IL-7, and rHuAFP cultures in representative
experiment.
[0145] There are numerous studies which have shown that AFP can
exert growth regulatory effects of MHC class II expressing cells
such as monocytes and thyroid epithelia cells (Wang, et al.
Hepatology 22:921-928, 1995). We wanted to ascertain whether
I-A.sup.k expressing cells within whole bone marrow would be
modulated upon co-culturing with 100 .mu.g/ml rHuAFP.
Simultaneously, we investigated the effects of rHuAFP on MHC class
I expressing cells within the adult BM. This was performed
employing fluorescein-conjugated anti-H-2K.sup.k antibodies. As
illustrated in FIG. 12A, only a minority of cells in normal BM
exhibit a high expression level of MHC molecules. When BM cells
were cultured in the presence of rHuAFP, there is a distinct
pattern of staining intensity. AFP increased the proportion of
I-A.sup.k expressing cells to 40% versus 12% in control cultures of
media alone or containing equivalent amounts of either mouse or
human albumin. Cells defined as H-2K.sup.k, high represented
approximately 80% of the total BM analyzed in cultures containing
rHuAFP, illustrating a 15 fold increase over control cultures with
mouse and human albumin additions or no protein additions.
[0146] The Effects of AFP on BM Cell Cultures Cannot be Reproduced
with Human or Mouse Albumin.
[0147] AFP share many physio-chemical properties with albumin, such
as the overall structures, including the three-domain structure,
and comparable binding properties. Therefore we determined whether
the effects in BM cells observed for AFP could also be ascribed to
mouse or human albumin. For this purpose BM cells from CBA/J mice
were cultured in the presence of 100 .mu.g/ml of rHuAFP, human
albumin, or mouse albumin. As demonstrated in FIG. 12B neither
human or mouse albumin shared the properties of AFP considering
increase in MHC I or II intensity or enhanced frequencies of DN T
cells and IgM positive B lineage cells. Instead, human and mouse
albumin cultures were comparable to the medium control in these
experiments.
Experimental
[0148] Materials and Methods
[0149] Mice. C.B-17 (H-2.sup.d) and CBA/J (H-2.sup.k) mouse strains
were obtained from Bomholtsgaard, Denmark and wee then bred and
maintained in our own animal facilities.
[0150] Preparation of hone marrow cells. Femurs and tibias were
removed aseptically from mice and flushed with PBS using a syringe.
Single cell suspensions were then washed three times in PBS. Cell
viability was determined by the trypan blue dye exclusion test.
[0151] Membrane labeling of bone marrow cells with PKH467-Gl. Bone
marrow cells were labeled using the PKH67 Green Fluorescent Cell
Linker Kit (PKH67-GL, Sigma Biosciences, St. Louis, Mo., USA).
Briefly, cells were diluted in Diluent C (2.times.10.sup.7
cells/ml) and then mixed with an equal volume of 2.times.10.sup.-6
M PKH67 dye in Diluent C to a final concentration of 10.sup.-6 M
dye and 10.sup.7 cells/ml. After 2 minutes incubation at 25.degree.
C. the reaction was stopped by adding an equal volume of complete
f-DMEM medium containing 10% FCS. Cells were washed and analyzed on
a FACScan.RTM. flow cytometer (Becton Dickinson, San Jose, Calif.)
to determine labeling intensity.
[0152] In Vitro Cultures and Cell Proliferation. BM cells were
cultured in 37.degree. C. in a humidified atmosphere of 7.5%
CO.sub.2 in an incubator (Biocenter 2001, Salvis AG, Reussbuhl,
Switzerland) in flat-bottomed 24 well plates (A/S Nunc, Roskilde,
Denmark) or round-bottomed 96 well plates (Corning Costar, Acton,
Mass., USA) in f-DMEM medium supplemented with 2 mM L-glutamine,
5.times.10.sup.-5 M 2-mercaptoethanol, and 10 .mu.g/ml gentamicin.
The cultures were complemented with recombinant IL-3 (Karasuyama
& Melchers, Eur. J. Immunol. 18:97-104, 1998; kindly provided
by Prof Jan Andersson, Basel Institute for Immunology,
Switzerland), crude supernatant from the IL-7 producing hybridoma
JM-IL-7 (kindly provided by Dr. Jan Andersson), recombinant human
alpha-fetoprotein (Boismenu, et al. Adv. Exp. Med. Biol.
383:255-269, 1995), human albumin, or mouse albumin and
supplemented with 5 .mu.g/ml transferrin, 1% fetal calf serum
(FCS), or 0.5% autologous normal mouse serum (NMS). Cells from
24-well plates were harvested for flow cytometry analysis and
determined for cell viability by the trypan blue dye exclusion
test. Cell proliferation was determined by incubating triplicate
cultures in round-bottomed 96 well plates with 1 .mu.Ci/culture of
.sup.3H-thymidine (Amersham International plc, Amersham, UK; spec.
act., 925 Gbq/mmol) for 4 h prior to harvest onto glassfiber
filters in a multiple cell harvester (1295-004 Betaplate.RTM.
Pharmacia LKB, Uppsala, Sweden). Radioactivity on dried filters was
measured by scintillation counting in a beta counter (1205
Betaplate.RTM., Pharmacia LKB, Uppsala, Sweden).
[0153] Flow cytometry analysis. If not otherwise stated, the mAbs
were obtained from PharMingen, San Diego, Calif. All steps were
carried out at 4.degree. C. Cells (10.sup.5 to 10.sup.6 per sample)
were pre-incubated for 30 minutes with 50 .mu.l crude supernatant
from 2.4 G2 hybridomas, washed once in 250 .mu.l PBS and then
stained for 30 minutes with pretitered concentrations of the
following mABs (obtained from Pharmigen) in 50 .mu.l of PBS:
FITC-labeled anti-H-2K.sup.d (SF1-1.1), biotinylated anti-I-A.sup.d
(AMS-32.1), FITC-labeled anti-H-2K.sup.k (AF2-12.1), and
biotinylated anti-I-A.sup.k (11-5.2). The cells were then washed
once in 250% PBS and cells stained with biotinylated mAb were
incubated for 25 min were streptavidin-PE (Becton Dickinson, San
Jose, Calif.) followed by three washes with 250 .mu.l of PBS. The
samples were diluted to a final volume of 0.5 ml in PBS containing
1 .mu.g/ml of propidium iodide and analyzed on a FACScan.RTM. flow
cytometer (Becton Dickinson, San Jose, Calif.). An amount of
5-20.times.10.sup.3 cells were collected per sample using a FSC vs.
SSC live gate to ignore erthrocytes and an FL3 vs. FL2 live gate to
exclude dead propidium iodide stained cells.
[0154] Treatment of BM cells with albumin. Bone marrow cells
(2.times.10.sup.6/ml) from CBA/J mice were cultured in 2 ml of
f-DMEM medium containing 1% FCS in the presence or absence of
rHuAFP, human albumin (HuAlb), or mouse albumin (MoAlb). At 4 days
of culture, cells were harvested, stained with anti-H-2K.sup.k, and
analyzed in FACS. FIG. 12B shows percent cells that were
H-2K.sup.k,high in medium without added AFP or supplemented with
either rHuAFP, HuAlb, or MoAlb. FIG. 12B shows percent cells that
were 1-A.sup.k+ in medium without added AFP or supplemented with
rHuAFP, HuAlb, or MoAlb.
Bone Marrow Reconstitution in the Presence of AFP In Vitro and In
Vivo
[0155] Enhanced BM Cell Recovery in the Presence of rHuAFP.
[0156] The impact of AFP to enhance BM cell reconstitution
following sublethal gamma irradiation was analyzed by irradiating
BM cells from C.B-17 mice with a dose of 600 rad. Thereafter,
irradiated and non-irradiated cells were cultured in the absence or
presence of IL-7 or rHuAFP. After 3 days of culture cells were
harvested and cell densities were determined by the trypan blue dye
exclusion test. Consistent with the data presented in FIG. 13 the
highest cell density for non-irradiated cells was observed in the
IL-7 cultures (FIG. 14A). BM cells from C.B-17 mice were irradiated
with 600 rad. Thereafter, the irradiated and non-irradiated cells
were cultured in the absence or presence of IL-7 or rHuAFP. After 3
days of culture, cells were harvested and cell densities were
determined by the trypan blue day exclusion test. Conversely,
following irradiation there was a significant enhancement in cell
recovery among cells cultured in the presence of 100 .mu.g/ml of
rHuAFP (FIG. 14B).
[0157] All publications, manufacturer's instructions, patents, and
patent applications mentioned in this specification are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
Sequence CWU 1
1
1812027DNAHomo sapiens 1atattgtgct tccaccactg ccaataacaa aataactagc
aaccatgaag tgggtggaat 60caattttttt aattttccta ctaaatttta ctgaatccag
aacactgcat agaaatgaat 120atggaatagc ttccatattg gattcttacc
aatgtactgc agagataagt ttagctgacc 180tggctaccat attttttgcc
cagtttgttc aagaagccac ttacaaggaa gtaagcaaaa 240tggtgaaaga
tgcattgact gcaattgaga aacccactgg agatgaacag tcttcagggt
300gtttagaaaa ccagctacct gcctttctgg aagaactttg ccatgagaaa
gaaattttgg 360agaagtacgg acattcagac tgctgcagcc aaagtgaaga
gggaagacat aactgttttc 420ttgcacacaa aaagcccact gcagcatgga
tcccactttt ccaagttcca gaacctgtca 480caagctgtga agcatatgaa
gaagacaggg agacattcat gaacaaattc atttatgaga 540tagcaagaag
gcatcccttc ctgtatgcac ctacaattct tctttcggct gctgggtatg
600agaaaataat tccatcttgc tgcaaagctg aaaatgcagt tgaatgcttc
caaacaaagg 660cagcaacagt tacaaaagaa ttaagagaaa gcagcttgtt
aaatcaacat gcatgtccag 720taatgaaaaa ttttgggacc cgaactttcc
aagccataac tgttactaaa ctgagtcaga 780agtttaccaa agttaatttt
actgaaatcc agaaactagt cctggatgtg gcccatgtac 840atgagcactg
ttgcagagca gatgtgctgg attgtctgca ggatggggaa aaaatcatgt
900cctacatatg ttctcaacaa gacactctgt caaacaaaat aacagaatgc
tgcaaactga 960ccacgctgga acgtggtcaa tgtataattc atgcagaaaa
tgatgaaaaa cctgaaggtc 1020tatctccaaa tctaaacagg tttttaggag
atagagattt taaccaattt tcttcagggg 1080aaaaaaatat cttcttggca
agttttgttc atgaatattc aagaagacat cctcagcttg 1140ctgtctcagt
aattctaaga gttgctaaag gataccagga gttattggag aagtgtttcc
1200agactgaaaa ccctcttgaa tgccaagata aaggagaaga agaattacag
aaatacatcc 1260aggagagcca agcattggca aagcgaagct gcggcctctt
ccagaaacta ggagaatatt 1320acttacaaaa tgagtttctc gttgcttaca
caaagaaagc cccccagctg acctcgtcgg 1380agctgatggc catcaccaga
aaaatggcag ccacagcagc cacttgttgc caactcagtg 1440aggacaaact
attggcctgt ggcgagggag cggctgacat tattatcgga cacttatgta
1500tcagacatga aatgactcca gtaaaccctg gtgttggcca gtgctgcact
tcttcatatg 1560ccaacaggag gccatgcttc agcagcttgg tggtggatga
aacatatgtc cctcctgcat 1620tctctgatga caagttcatt ttccataagg
atctgtgcca agctcagggt gtagcgctgc 1680aaaggatgaa gcaagagttt
ctcattaacc ttgtgaagca aaagccacaa ataacagagg 1740aacaacttga
ggctctcatt gcagatttct caggcctgtt ggagaaatgc tgccaaggcc
1800aggaacagga agtctgcttt gctgaagagg gacaaaaact gatttcaaaa
actggtgctg 1860ctttgggagt ttaaattact tcaggggaag agaagacaaa
acgagtcttt cattcggtgt 1920gaacttttct ctttaatttt aactgattta
acactttttg tgaattaatg aaatgataaa 1980gacttttatg tgagatttcc
ttatcacaga aataaaatat ctccaaa 20272590PRTHomo sapiens 2Thr Leu His
Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr1 5 10 15Gln Cys
Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe 20 25 30Ala
Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val 35 40
45Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser
50 55 60Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu
Cys65 70 75 80His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp
Cys Cys Ser 85 90 95Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala
His Lys Lys Pro 100 105 110Thr Ala Ala Trp Ile Pro Leu Phe Gln Val
Pro Glu Pro Val Thr Ser 115 120 125Cys Glu Ala Tyr Glu Glu Asp Arg
Glu Thr Phe Met Asn Lys Phe Ile 130 135 140Tyr Glu Ile Ala Arg Arg
His Pro Phe Leu Tyr Ala Pro Thr Ile Leu145 150 155 160Leu Ser Ala
Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala 165 170 175Glu
Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys 180 185
190Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Pro Val Met
195 200 205Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr
Lys Leu 210 215 220Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile
Gln Lys Leu Val225 230 235 240Leu Asp Val Ala His Val His Glu His
Cys Cys Arg Ala Asp Val Leu 245 250 255Asp Cys Leu Gln Asp Gly Glu
Lys Ile Met Ser Tyr Ile Cys Ser Gln 260 265 270Gln Asp Thr Leu Ser
Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr 275 280 285Leu Glu Arg
Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro 290 295 300Glu
Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe305 310
315 320Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe
Val 325 330 335His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser
Val Ile Leu 340 345 350Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu
Lys Cys Phe Gln Thr 355 360 365Glu Asn Pro Leu Glu Cys Gln Asp Lys
Gly Glu Glu Glu Leu Gln Lys 370 375 380Tyr Ile Gln Glu Ser Gln Ala
Leu Ala Lys Arg Ser Cys Gly Leu Phe385 390 395 400Gln Lys Leu Gly
Glu Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr 405 410 415Thr Lys
Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr 420 425
430Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp
435 440 445Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile Ile
Gly His 450 455 460Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro
Gly Val Gly Gln465 470 475 480Cys Cys Thr Ser Ser Tyr Ala Asn Arg
Arg Pro Cys Phe Ser Ser Leu 485 490 495Val Val Asp Glu Thr Tyr Val
Pro Pro Ala Phe Ser Asp Asp Lys Phe 500 505 510Ile Phe His Lys Asp
Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Arg 515 520 525Met Lys Gln
Glu Phe Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile 530 535 540Thr
Glu Glu Gln Leu Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu545 550
555 560Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe Ala Glu
Glu 565 570 575Gly Gln Lys Leu Ile Ser Lys Thr Gly Ala Ala Leu Gly
Val 580 585 5903197PRTHomo sapiens 3Thr Leu His Arg Asn Glu Tyr Gly
Ile Ala Ser Ile Leu Asp Ser Tyr1 5 10 15Gln Cys Thr Ala Glu Ile Ser
Leu Ala Asp Leu Ala Thr Ile Phe Phe 20 25 30Ala Gln Phe Val Gln Glu
Ala Thr Tyr Lys Glu Val Ser Lys Met Val 35 40 45Lys Asp Ala Leu Thr
Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser 50 55 60Ser Gly Cys Leu
Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys65 70 75 80His Glu
Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser 85 90 95Gln
Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro 100 105
110Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser
115 120 125Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys
Phe Ile 130 135 140Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala
Pro Thr Ile Leu145 150 155 160Leu Ser Ala Ala Gly Tyr Glu Lys Ile
Ile Pro Ser Cys Cys Lys Ala 165 170 175Glu Asn Ala Val Glu Cys Phe
Gln Thr Lys Ala Ala Thr Val Thr Lys 180 185 190Glu Leu Arg Glu Ser
1954192PRTHomo sapiens 4Ser Leu Leu Asn Gln His Ala Cys Pro Val Met
Lys Asn Phe Gly Thr1 5 10 15Arg Thr Phe Gln Ala Ile Thr Val Thr Lys
Leu Ser Gln Lys Phe Thr 20 25 30Lys Val Asn Phe Thr Glu Ile Gln Lys
Leu Val Leu Asp Val Ala His 35 40 45Val His Glu His Cys Cys Arg Ala
Asp Val Leu Asp Cys Leu Gln Asp 50 55 60Gly Glu Lys Ile Met Ser Tyr
Ile Cys Ser Gln Gln Asp Thr Leu Ser65 70 75 80Asn Lys Ile Thr Glu
Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln 85 90 95Cys Ile Ile His
Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro 100 105 110Asn Leu
Asn Arg Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser 115 120
125Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg
130 135 140Arg His Pro Gln Leu Ala Val Ser Val Ile Leu Arg Val Ala
Lys Gly145 150 155 160Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr
Glu Asn Pro Leu Glu 165 170 175Cys Gln Asp Lys Gly Glu Glu Glu Leu
Gln Lys Tyr Ile Gln Glu Ser 180 185 1905201PRTHomo sapiens 5Gln Ala
Leu Ala Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu1 5 10 15Tyr
Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro 20 25
30Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala
35 40 45Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala
Cys 50 55 60Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His Leu Cys Ile
Arg His65 70 75 80Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln Cys
Cys Thr Ser Ser 85 90 95Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu
Val Val Asp Glu Thr 100 105 110Tyr Val Pro Pro Ala Phe Ser Asp Asp
Lys Phe Ile Phe His Lys Asp 115 120 125Leu Cys Gln Ala Gln Gly Val
Ala Leu Gln Arg Met Lys Gln Glu Phe 130 135 140Leu Ile Asn Leu Val
Lys Gln Lys Pro Gln Ile Thr Glu Glu Gln Leu145 150 155 160Glu Ala
Leu Ile Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln 165 170
175Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile
180 185 190Ser Lys Thr Gly Ala Ala Leu Gly Val 195 2006389PRTHomo
sapiens 6Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp
Ser Tyr1 5 10 15Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr
Ile Phe Phe 20 25 30Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val
Ser Lys Met Val 35 40 45Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr
Gly Asp Glu Gln Ser 50 55 60Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala
Phe Leu Glu Glu Leu Cys65 70 75 80His Glu Lys Glu Ile Leu Glu Lys
Tyr Gly His Ser Asp Cys Cys Ser 85 90 95Gln Ser Glu Glu Gly Arg His
Asn Cys Phe Leu Ala His Lys Lys Pro 100 105 110Thr Ala Ala Trp Ile
Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser 115 120 125Cys Glu Ala
Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile 130 135 140Tyr
Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu145 150
155 160Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys
Ala 165 170 175Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr
Val Thr Lys 180 185 190Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His
Ala Cys Pro Val Met 195 200 205Lys Asn Phe Gly Thr Arg Thr Phe Gln
Ala Ile Thr Val Thr Lys Leu 210 215 220Ser Gln Lys Phe Thr Lys Val
Asn Phe Thr Glu Ile Gln Lys Leu Val225 230 235 240Leu Asp Val Ala
His Val His Glu His Cys Cys Arg Ala Asp Val Leu 245 250 255Asp Cys
Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln 260 265
270Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr
275 280 285Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu
Lys Pro 290 295 300Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly
Asp Arg Asp Phe305 310 315 320Asn Gln Phe Ser Ser Gly Glu Lys Asn
Ile Phe Leu Ala Ser Phe Val 325 330 335His Glu Tyr Ser Arg Arg His
Pro Gln Leu Ala Val Ser Val Ile Leu 340 345 350Arg Val Ala Lys Gly
Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr 355 360 365Glu Asn Pro
Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys 370 375 380Tyr
Ile Gln Glu Ser3857393PRTHomo sapiens 7Ser Leu Leu Asn Gln His Ala
Cys Pro Val Met Lys Asn Phe Gly Thr1 5 10 15Arg Thr Phe Gln Ala Ile
Thr Val Thr Lys Leu Ser Gln Lys Phe Thr 20 25 30Lys Val Asn Phe Thr
Glu Ile Gln Lys Leu Val Leu Asp Val Ala His 35 40 45Val His Glu His
Cys Cys Arg Ala Asp Val Leu Asp Cys Leu Gln Asp 50 55 60Gly Glu Lys
Ile Met Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu Ser65 70 75 80Asn
Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln 85 90
95Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro
100 105 110Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe
Ser Ser 115 120 125Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val His
Glu Tyr Ser Arg 130 135 140Arg His Pro Gln Leu Ala Val Ser Val Ile
Leu Arg Val Ala Lys Gly145 150 155 160Tyr Gln Glu Leu Leu Glu Lys
Cys Phe Gln Thr Glu Asn Pro Leu Glu 165 170 175Cys Gln Asp Lys Gly
Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser 180 185 190Gln Ala Leu
Ala Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu 195 200 205Tyr
Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro 210 215
220Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala
Ala225 230 235 240Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys
Leu Leu Ala Cys 245 250 255Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly
His Leu Cys Ile Arg His 260 265 270Glu Met Thr Pro Val Asn Pro Gly
Val Gly Gln Cys Cys Thr Ser Ser 275 280 285Tyr Ala Asn Arg Arg Pro
Cys Phe Ser Ser Leu Val Val Asp Glu Thr 290 295 300Tyr Val Pro Pro
Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp305 310 315 320Leu
Cys Gln Ala Gln Gly Val Ala Leu Gln Arg Met Lys Gln Glu Phe 325 330
335Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile Thr Glu Glu Gln Leu
340 345 350Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys
Cys Gln 355 360 365Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu Gly
Gln Lys Leu Ile 370 375 380Ser Lys Thr Gly Ala Ala Leu Gly Val385
3908325PRTHomo sapiens 8Met Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu
Ser Asn Lys Ile Thr1 5 10 15Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg
Gly Gln Cys Ile Ile His 20 25 30Ala Glu Asn Asp Glu Lys Pro Glu Gly
Leu Ser Pro Asn Leu Asn Arg 35 40 45Phe Leu Gly Asp Arg Asp Phe Asn
Gln Phe Ser Ser Gly Glu Lys Asn 50 55 60Ile Phe Leu Ala Ser Phe Val
His Glu Tyr Ser Arg Arg His Pro Gln65 70 75 80Leu Ala Val Ser Val
Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu 85 90 95Leu Glu Lys Cys
Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys 100 105 110Gly Glu
Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala 115 120
125Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln
130 135 140Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu
Thr Ser145 150 155 160Ser Glu Leu Met Ala Ile Thr
Arg Lys Met Ala Ala Thr Ala Ala Thr 165 170 175Cys Cys Gln Leu Ser
Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala 180 185 190Ala Asp Ile
Ile Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro 195 200 205Val
Asn Pro Gly Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg 210 215
220Arg Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro
Pro225 230 235 240Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp
Leu Cys Gln Ala 245 250 255Gln Gly Val Ala Leu Gln Arg Met Lys Gln
Glu Phe Leu Ile Asn Leu 260 265 270Val Lys Gln Lys Pro Gln Ile Thr
Glu Glu Gln Leu Glu Ala Leu Ile 275 280 285Ala Asp Phe Ser Gly Leu
Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln 290 295 300Glu Val Cys Phe
Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Gly305 310 315 320Ala
Ala Leu Gly Val 325930DNAHomo sapiens 9aaaaaaggta ccacactgca
tagaaatgaa 301033DNAHomo sapiens 10aaaaaaggat ccttagcttt ctcttaattc
ttt 331133DNAHomo sapiens 11aaaaaaatcg atatgagctt gttaaatcaa cat
331233DNAHomo sapiens 12aaaaaaggat ccttagctct cctggatgta ttt
331333DNAHomo sapiens 13aaaaaaatcg atatgcaagc attggcaaag cga
331433DNAHomo sapiens 14aaaaaaggat ccttaaactc ccaaagcagc acg
331533DNAHomo sapiens 15aaaaaaatcg atatgtccta catatgttct caa
33169PRTHomo sapiens 16Ser Tyr Ile Cys Ser Gln Gln Asp Thr1
51721DNAHomo sapiens 17gatctagaat tcggatccgg t 211810PRTHomo
sapiens 18Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu1 5 10
* * * * *