U.S. patent application number 14/188531 was filed with the patent office on 2015-01-29 for treatment of autoimmune conditions with copolymer 1 and related copolymers.
This patent application is currently assigned to PRESIDENT AND FELLOWS OF HARVARD COLLEGE. The applicant listed for this patent is Rina Aharoni, Ruth Arnon, Masha Fridkis-Hareli, Michael Sela, Jack L. Strominger, Dvora Teitelbaum. Invention is credited to Rina Aharoni, Ruth Arnon, Masha Fridkis-Hareli, Michael Sela, Jack L. Strominger, Dvora Teitelbaum.
Application Number | 20150030560 14/188531 |
Document ID | / |
Family ID | 46326165 |
Filed Date | 2015-01-29 |
United States Patent
Application |
20150030560 |
Kind Code |
A1 |
Aharoni; Rina ; et
al. |
January 29, 2015 |
TREATMENT OF AUTOIMMUNE CONDITIONS WITH COPOLYMER 1 AND RELATED
COPOLYMERS
Abstract
The present invention is directed to polypeptides containing at
least three amino acids randomly joined in a linear array; wherein
at least one of the three amino acids is an aromatic amino acid, at
least one of the three amino acids is a charged amino acid and at
least one amino acid is an aliphatic amino acid. In a preferred
embodiment the polypeptide contains three or four of the following
amino acids: tyrosine, alanine, glutamic acid or lysine. According
to the present invention, the present polypeptides bind to antigen
presenting cells, purified human lymphocyte antigens (HLA) and/or
Copolymer 1-specific T cells. Moreover, according to the present
invention, these polypeptides can be formulated into pharmaceutical
compositions for treating autoimmune disease. The present invention
further contemplates methods of treating an autoimmune disease in a
mammal by administering a pharmaceutically effective amount of any
one of the present polypeptides to the mammal.
Inventors: |
Aharoni; Rina; (Rehovot,
IL) ; Teitelbaum; Dvora; (Rehovot, IL) ;
Arnon; Ruth; (Rehovot, IL) ; Sela; Michael;
(Rehovot, IL) ; Fridkis-Hareli; Masha; (Cambridge,
MA) ; Strominger; Jack L.; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aharoni; Rina
Teitelbaum; Dvora
Arnon; Ruth
Sela; Michael
Fridkis-Hareli; Masha
Strominger; Jack L. |
Rehovot
Rehovot
Rehovot
Rehovot
Cambridge
Lexington |
MA
MA |
IL
IL
IL
IL
US
US |
|
|
Assignee: |
PRESIDENT AND FELLOWS OF HARVARD
COLLEGE
Cambridge
MA
YEDA RESEARCH AND DEVELOPMENT CO., LTD.
Rehovot
|
Family ID: |
46326165 |
Appl. No.: |
14/188531 |
Filed: |
February 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12231292 |
Aug 29, 2008 |
|
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14188531 |
|
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Current U.S.
Class: |
424/78.37 ;
528/208 |
Current CPC
Class: |
A61P 29/00 20180101;
Y10S 514/886 20130101; A61P 25/28 20180101; A61P 19/02 20180101;
G01N 33/6842 20130101; A61K 38/16 20130101; C08G 73/02 20130101;
A61K 31/785 20130101; Y10S 514/866 20130101; Y10S 514/885 20130101;
Y10S 424/81 20130101; A61P 37/00 20180101; Y10S 514/903
20130101 |
Class at
Publication: |
424/78.37 ;
528/208 |
International
Class: |
A61K 31/785 20060101
A61K031/785; C08G 73/02 20060101 C08G073/02 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made in part with government support
under grant CA47554 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A terpolymer consisting essentially of tyrosine, alanine and
lysine randomly polymerized into a polypeptide.
2.-15. (canceled)
16. A pharmaceutical composition for the treatment of an autoimmune
disease, comprising a therapeutically effective amount of a
terpolymer comprising three different amino acids randomly
polymerized into a polypeptide, and a pharmaceutically acceptable
carrier, wherein the three different amino acids are selected from
the group of tyrosine, glutamic acid, alanine and lysine.
17.-44. (canceled)
45. A method for treating an autoimmune disease which comprises
administering a therapeutically effective amount of a polypeptide
consisting essentially of amino acids tyrosine, glutamic acid,
alanine and lysine, wherein said autoimmune disease is not multiple
sclerosis.
46.-176. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Ser. No.
12/231,292, filed Aug. 29, 2008 which is a continuation of U.S.
Ser. No. 11/528,894, filed Sep. 27, 2006 which is a divisional 10
of U.S. Ser. No. 09/768,872, filed Jan. 23, 2001 which is a
continuation of PCT/US99/16747, filed Jul. 23, 1999, which claims
the benefit of provisional applications 60/093,859 filed Jul. 23,
1998, 60/101,825, filed Sep. 25, 1998, 60/102,960, filed Oct. 2,
1998, 60/106,350, filed Oct. 30, 1998, and 60/108,184, filed Nov.
12, 1998, all of which are incorporated by reference herein.
INTRODUCTION
[0003] The present invention provides compositions and methods for
treating autoimmune diseases using therapeutically effective
amounts of a polypeptide related to Copolymer 1. Copolymer 1 is a
heterogeneous mixture of synthetic random linear copolymers of
tyrosine, alanine, glutamic acid and lysine and, in appropriate
therapeutic amounts and average molecular sizes, is used to treat
multiple sclerosis. When such mixtures of synthetic random linear
copolymers consist essentially of the three of the four amino acids
found in Copolymer 1, they are referred to as Terpolymers. The
present invention relates in part to Terpolymers. Preferably, the
Terpolymers are composed of tyrosine, alanine and lysine, or of
glutamic acid, tyrosine and lysine, or of glutamic acid, alanine
and lysine. Surprisingly, the Terpolymers have efficacy for
treating a variety of autoimmune diseases and bind to Class II
major histocompatibility complex (MHC) molecules as well as to
antigen presenting cells.
BACKGROUND OF THE INVENTION
[0004] Autoimmune diseases occur when an organism's immune system
fails to recognize some of the organism's own tissues as "self" and
attacks them as "foreign." Normally, self-tolerance is developed
early by developmental events within the immune system that prevent
the organism's own T cells and B cells from reacting with the
organism's own tissues. MHC cell surface proteins help regulate
these early immune responses by binding to and presenting processed
peptides to T cells.
[0005] This self-tolerance process breaks down when autoimmune
diseases develop. Now the organism's own tissues and proteins are
recognized as "autoantigens" and are attacked by the organism's
immune system. For example, multiple sclerosis is believed to be an
autoimmune disease occurring when the immune system attacks the
myelin sheath, whose function is to insulate and protect nerves. It
is a progressive disease characterized by demyelination, followed
by neuronal and motor function loss. Rheumatoid arthritis ("RA") is
also believed to be an autoimmune disease which involves chronic
inflammation of the synovial joints and infiltration by activated T
cells, macrophages and plasma cells, leading to a progressive
destruction of the articular cartilage. It is the most severe form
of joint disease. The nature of the autoantigen(s) attacked in
rheumatoid arthritis is poorly understood, although collagen type
II is a candidate.
[0006] A tendency to develop multiple sclerosis and rheumatoid
arthritis is inherited--these diseases occur more frequently in
individuals carrying one or more characteristic MHC class II
alleles. For example, inherited susceptibility for rheumatoid
arthritis is strongly associated with the MHC class II DRB1 *0401,
DRB 1 *0404, or DRB 1*0405 or the DRB1*0101 alleles. The
histocompatibility locus antigens (HLA) are found on the surface of
cells and help determine the individuality of tissues from
different persons. Genes for histocompatibility locus antigens are
located in the same region of chromosome 6 as the major
histocompatibility complex (MHC). The MHC region expresses a number
of distinctive classes of molecules in various cells of the body,
the genes being, in order of sequence along the chromosome, the
Class I, II and III MHC genes. The Class I genes consist of HLA
genes, which are further subdivided into A, B and C subregions. The
Class II genes are subdivided into the DR, DQ and DP subregions.
The MHC-DR molecules are the best known: these occur on the
surfaces of antigen presenting cells such as macrophages, dendritic
cells of lymphoid tissue and epidermal cells. The Class III MHC
products are expressed in various components of the complement
system, as well as in some non-immune related cells.
[0007] A number of therapeutic agents have been developed to treat
autoimmune diseases, including steroidal and non-steroidal
anti-inflammatory drugs, for example, methotrexate; various
interferons; and certain inhibitors of prostaglandin synthesis.
However, these agents can be toxic when used for more than short
periods of time or cause undesirable side effects. Other
therapeutic agents bind to and/or inhibit the inflammatory activity
of tumor necrosis factor (TNF), for example, anti-TNF specific
antibodies or antibody fragments, or a soluble form of the TNF
receptor. These agents target a protein on the surface of a T cell
and generally prevent interaction with an antigen presenting cell
(APC). However, therapeutic compositions containing natural folded
proteins are often difficult to produce, formulate, store, and
deliver. Moreover, the innate heterogeneity of the immune system
can limit the effectiveness of drugs and complicate long-term
treatment of autoimmune diseases.
[0008] Thus in order to effectively treat autoimmune diseases and
other immune conditions, new drugs are needed that do not have the
side effects of the present therapeutic agents and which adequately
address to the innate heterogeneity of the immune system.
REFERENCES
[0009] Aharoni, et al., 58 Immunology Letters 79 (1997). [0010]
Allison, in IMMUNOSUPPRESSION AND ANTI-INFLAMMATORY DRUGS, ANNALS
OF THE NEW YORK ACADEMY OF SCIENCE 696:xi (1993). [0011] Ben-Nun A
et al., 243 J NEUROL (Suppl 1) S14-S22 (1996). [0012] Dorling et
al., 6 CUR. OPINIONS IMMUNOL. 765 (1994). [0013] Ferrara et al.,
324 NEW ENGLAND J. OF MEDICINE 667 (1991). [0014] Fridkis-Hareli,
et al., 63 J. NEUROCHEM. 63 (Suppl. 1) S61 (1994). [0015]
Fridkis-Hareli, et al., 163 CELL. IMMUNOL. 229. (1995). [0016]
Fridkis-Hareli, et al., 160 J. IMMUNOL. 4386 (1998). [0017]
Johnson, 1 NEUROLOGY 65-70 (1995). [0018] Kay et al., 22
TRANSPLANTATION PROCEEDINGS 96 (1990). [0019] Kelemen, et al., 102
INT ARCH ALLERGY IMMUNOL. 309 (1993). [0020] Mengle-Gaw, The Major
Histocompatibility Complex (MHC), in the ENCYCLOPEDIA OF MOLECULAR
BIOLOGY 602-06 (Oxford: Blackwell Science Ltd., 1994). [0021]
Rothbard, J. B., et al., 9 ANNU. REV. IMMUNOL. 527 (1991). [0022]
Schlegel, et al., 84 BLOOD 2802 (1994). [0023] Sela M et al., 88
BULL INST PASTEUR 303-14 (1990). [0024] Stall, 22 TRANSPLANTATION
PROCEEDINGS 5 (1990). [0025] Sykes. 10 THE FASEB JOURNAL 721
(1996). [0026] Teitelbaum et al., 1 EUR. J. IMMUNOL. 242-48 (1971).
[0027] Teitelbaum et al., 3 EUR. J. IMMUNOL. 273-79 (1973). [0028]
Teitelbaum et al., 64 J. NEUROIMMUNOL. 209-17 (1996). [0029]
Thomson, 10 IMMUNOLOGY TODAY 6 (1988). [0030] Van Den Bogaerde, et
al., 52 TRANSPLANTATION 15 (1991). [0031] Webb et al. 13
IMMUNOCHEM. 333 (1976).
SUMMARY OF THE INVENTION
[0032] Surprisingly, Copolymer 1 and the Terpolymers of the present
invention can be used to treat a variety of autoimmune diseases in
a heterogeneous patient population. These Terpolymers can inhibit
some of the physiological responses of T cells that attack
self-antigens as these diseases progress. Moreover, Copolymer 1 and
the Terpolymers of the present invention bind with high affinity to
antigen presenting cells from different genetic backgrounds and to
several class II MHC molecules to block immune cell recognition and
attack. In addition, the Terpolymers can stimulate the growth and
functioning of Copolymer 1-specific T cells to further treat and
prevent various autoimmune diseases.
[0033] Accordingly, the present invention provides a composition
having a polypeptide comprising three different amino acids
selected from the group of amino acids comprising Copolymer 1, that
is, glutamic acid, alanine, lysine, and tyrosine, in the
approximate relative molar ratios found in Copolymer 1.
[0034] The present invention is also directed to pharmaceutical
compositions which include a therapeutically effective amount of a
Terpolymer consisting essentially of amino acids tyrosine, alanine
and lysine, in the molar ratio of from about 0.005 to about 0.25
tyrosine, from about 0.3 to about 0.6 alanine, and from about 0.1
to about 0.5 lysine, and a pharmaceutically acceptable carrier.
This Terpolymer is preferably substantially free of glutamic
acid.
[0035] The present invention further provides a pharmaceutical
composition which includes a therapeutically effective amount of a
Terpolymer consisting essentially of glutamic acid, tyrosine and
lysine, in the molar ratio of from about 0.005 to about 0.300
glutamic acid, from about 0.005 to about 0.250 tyrosine; and from
about 0.3 to about 0.7 lysine, and a pharmaceutically acceptable
carrier. The Terpolymer is preferably substantially free of
alanine.
[0036] The present invention is also directed to pharmaceutical
compositions which include a therapeutically effective amount of a
Terpolymer consisting essentially of amino acids tyrosine, glutamic
acid and alanine in the molar ratio of from about 0.005 to about
0.25 tyrosine, from about 0.005 to about 0.3 glutamic acid, and
from about 0.005 to about 0.8 alanine and a pharmaceutically
acceptable carrier. This Terpolymer is preferably substantially
free of lysine.
[0037] The present invention also provides a pharmaceutical
composition which includes a therapeutically effective amount of a
Terpolymer consisting essentially of glutamic acid, alanine and
lysine, in the molar ratio of from about 0.005 to about 0.3
glutamic acid, from about 0.005 to about 0.6 alanine: and from
about 0.2 to about 0.7 lysine and a pharmaceutically acceptable
carrier. This Terpolymer is preferably substantially free of
tyrosine.
[0038] The present invention also provides a pharmaceutical
composition to treat autoimmune diseases which includes a
therapeutically effective amount of Copolymer 1 or a polypeptide
consisting essentially of glutamic acid, alanine, tyrosine and
lysine, and a pharmaceutically acceptable carrier.
[0039] The present invention further provides methods for treating
and preventing autoimmune diseases in a mammal which include
administering a therapeutically effective amount of a composition
comprising Copolymer 1 or a Terpolymer. In another embodiment, the
method for treating autoimmune diseases in a mammal further
involves inhibiting proliferation of T cells involved in the immune
attack. In another embodiment, the method for treating autoimmune
diseases in a mammal involves binding the Terpolymer to an antigen
presenting cell. In yet another embodiment, the method for treating
autoimmune disease in a mammal involves binding the Terpolymer to a
major histocompatibility complex class II protein which is
associated with autoimmune diseases.
[0040] Autoimmune diseases contemplated by the present invention
include arthritic conditions, demyelinating diseases and
inflammatory diseases. For example, autoimmune diseases which can
be treated by the present compositions include multiple sclerosis,
autoimmune hemolytic anemia, autoimmune oophoritis, autoimmune
thyroiditis, autoimmune uveoretinitis, Crone's disease, chronic
immune thrombocytopenic purpura, colitis, contact sensitivity
disease, diabetes mellitus, Graves disease, Guillain-Barre's
syndrome, Hashimoto's disease, idiopathic myxedema, myasthenia
gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis, or
systemic lupus erythematosus. The present compositions can be used
to treat one or more of these diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] As used below [0042] "GAL" is a random terpolymer of
glutamic acid, alanine and lysine; [0043] "TGA" or "YEA" is a
random terpolymer of tyrosine, glutamic acid, and alanine; [0044]
"TAL" or "YAK" is a random terpolymer of tyrosine, alanine, and
lysine; [0045] "GTL" or "YEK" is a random terpolymer of glutamic
acid, tyrosine, and lysine; and [0046] "YEAK" is Copolymer 1.
[0047] FIG. 1 illustrates the effect of Copolymer 1 and Terpolymers
on proliferation of T cells which are specific for certain myelin
basic protein (MBP) peptides. Several of the present Terpolymers
inhibit proliferation of T cell lines which are specific for myelin
basic protein antigen, MBP 84-102 (SEQ ID NO: 1). The following
symbols were used: Copolymer 1 ( ); GAL (.quadrature.); TGA
(.DELTA.); TAL (.largecircle.); GTL (X). FIG. 1A illustrates
inhibition of the proliferation of mouse T cell clone MBP-sp-1
specific to MBP 84-102 peptide (0.5 g/well). As a control,
proliferation of mouse T cell clone MBP-sp-1 stimulated by MBP
84-102 (SEQ ID NO: 1) without inhibitor was 21,145 cpm. FIG. 1B
illustrates inhibition of the response of human T cell clone GP-25
to MBP 84-102 peptide (0.125 g/well). Proliferation of human T cell
clone GP-25 stimulated by MBP 84-102 (SEQ ID NO: 1) peptide without
inhibitor was 11,442 cpm.
[0048] FIG. 2A compares binding of the present polypeptides with
the binding of Copolymer 1, to Class II major histocompatibility
molecules DR1 (top), DR2 (middle) and DR4 (bottom). Binding by
Copolymer 1 (.box-solid.), was compared to binding by the following
biotinylated Terpolymers: GAL (.quadrature.); TGA
(.tangle-solidup.); GTL (.DELTA.); and TAL (.diamond-solid.). The
amount of Class II major histocompatibility molecule was held
constant at 0.5 .mu.g/sample and the concentration of polypeptide
was varied between 0-8 .mu.M as indicated on the y-axis. Binding
was at pH 5.0 for 40 hr at 37.degree. C. Binding was detected by
capturing the polypeptide-class II complexes with an LB3.1 antibody
and detecting the amount of biotinylated polypeptide bound by
monitoring the absorbency at 410 nm after reaction with
streptavidin-conjugated alkaline phosphatase.
[0049] FIG. 2B provides Lineweaver-Burke plots of the binding data
provided in FIG. 2A.
[0050] FIG. 3 illustrates the competitive inhibition of Copolymer 1
binding to Class II major histocompatibility molecules by the
present polypeptides. Purified HLA-DR1 (top), HLA-DR2 (middle) and
HLA-DR4 (bottom) molecules were incubated with a constant amount
(1.5 .mu.M) of biotinylated Copolymer 1, either alone or in the
presence of one of the following unlabeled polypeptides: Copolymer
1 (.box-solid.); GAL (.quadrature.); TGA (.tangle-solidup.); GTL
(.DELTA.); and TAL (.diamond-solid.). Inhibition by these
polypeptides was compared to inhibition by the myelin basic protein
antigen HA 306-318 (.largecircle.) (SEC) ID NO: 2). Binding was at
pH 5.0 for 40 hr at 37.degree. C. The amount of unlabeled
polypeptide was varied between 0.1-1000 .mu.M, as indicated on the
y-axis. Specific binding is expressed as the percentage of
inhibition using equation 1:
percent inhibition = 100 - ( signal with competitor - background )
( signal without competitor - background ) .times. 100 1
##EQU00001##
[0051] FIG. 4A illustrates the competitive inhibition of TAL
binding to Class II major histocompatibility molecules by bacterial
superantigens SEA, SEB and TSST-1. Purified HLA-DR1 (top), HLA-DR2
(middle) and HLA-DR4 (bottom) molecules were incubated with a
constant amount of biotinylated TAL, in the presence of increasing
amounts of unlabeled bacterial superantigen SEA (.quadrature.); SEB
(.box-solid.); or TSST-1 ( ). Binding was at pH 5.0 for 40 hr at
37.degree. C. The amount of superantigen was varied between
0.1-1000 .mu.M, as indicated on the y-axis. Specific binding is
expressed as the percentage of inhibition using equation 1.
[0052] FIG. 48 illustrates the competitive inhibition of TGA
binding to Class II major histocompatibility molecules by bacterial
superantigens SEA, SEB and TSST-1. Purified HLA-DR1 (top), HLA-DR2
(middle) and HLA-DR4 (bottom) molecules were incubated with a
constant amount of biotinylated polypeptide TGA, in the presence of
increasing amounts of unlabeled bacterial superantigen SEA
(.quadrature.); SEB (.box-solid.); or TSST-1 ( ). Binding was at pH
5.0 for 40 hr at 37.degree. C. The amount of superantigen was
varied between 0.1-1000 .mu.M, as indicated on the y-axis. Specific
binding is expressed as the percentage of inhibition, calculated
according to equation above 1.
[0053] FIG. 4C illustrates the competitive inhibition of GAL
binding to Class II major histocompatibility molecules by bacterial
superantigens SEA, SEB and TSST-1. Purified HLA-DR1 (top), and
HLA-DR2 (bottom) molecules were incubated with a constant amount of
biotinylated GAL, in the presence of increasing amounts of
unlabeled bacterial superantigen SEA (.quadrature.); SEB
(.box-solid.); or TSST-1 ( ). Binding was at pH 5.0 for 40 hr at
37.degree. C. The amount of superantigen was varied between
0.1-1000 .mu.M, as indicated on the y-axis. Specific binding is
expressed as the percentage of inhibition using equation 1
above.
[0054] FIGS. 5(A and B) shows inhibition of binding of labeled
molecules to MHC class II purified proteins by Terpolymers of the
present invention. FIG. 5A shows inhibition of binding to the MHC
HLA-DR1 protein. FIG. 5B shows inhibition of binding to the MHC
HLA-DR4 protein. Unlabeled competitors include Terpolymers of the
present invention, influenza virus hemagglutinin (HA) peptide
306-318 (SEQ ID NO:2), and type II collagen (CII) peptide 261-273
(SEQ ID NO:3). The concentration of unlabeled competitor is
indicated on the abscissa. In each panel, inhibition by CII 261-273
(SEQ ID NO:3) is shown as open circles (.smallcircle.), inhibition
by HA 306-318 (SEQ ID NO: 2) is shown by solid circles ( ),
inhibition by one of the present terpolymers is indicated as shown
by open or solid triangles or squares. The extent of inhibition by
GTL is shown using open triangles (.DELTA.), by TAL is shown as
solid triangles (.tangle-solidup.), by TGA as open squares
(.quadrature.), and by Copolymer 1 as solid squares (.box-solid.).
Specific binding observed and shown on the ordinate was calculated
as percentage of inhibition using equation 1 above.
[0055] FIG. 6 shows inhibition of IL-2 production by
DR1-restricted-CII-specific T cell hybridomas in the presence of
different polypeptides of the present invention. Irradiated L57.23
cells (fibroblasts transfected with a gene encoding HLA-DR1) were
coincubated in duplicate with collagen peptide CII 261-273 (40
.mu.g/ml) and varying concentrations, shown on the abscissa, of one
of the present polypeptides for 2 hr at 37.degree. C., then T cells
(clone 3.19 or 19.3 as indicated) were added, and the mixtures were
further incubated for 24 hr at 37.degree. C. Supernatants (30
.mu.l) were then removed, and were assayed for activation by
IL-2-induced proliferation of IL-2-dependent cytotoxic T
lymphocytes (CTL-L). The extent of inhibition by TAL is shown as
solid circles ( ), by TGA as solid triangles (.DELTA.), by GTL as
open triangles (.quadrature.), and by Copolymer 1 as solid squares
(.box-solid.). Percent inhibition of CTL-L proliferation shown on
the ordinate was calculated according to equation 1.
[0056] FIGS. 7(A and B) shows inhibition of IL-2 production by
DR4-restricted CII-specific T cell hybridomas (3838 and D3) in the
presence of different polypeptides of the present invention. FIG.
7A shows the effects of coincubating irradiated 3838 or D3 Priess
cells with collagen peptide CII 261-273 (SEQ ID NO: 3) at the fixed
concentration of 40 .mu.g/ml, and with varying concentrations of
each of the present polypeptides, for 2 hr at 37.degree. C. FIG. 7B
shows the effects of incubating L cells transfected with a gene
encoding HIA-DR4 with collagen peptide CII 261-273 (SEQ ID NO:3) at
the fixed Concentration of 40 .mu.g/ml, and with varying
concentrations of each of the present polypeptides, for 2 hr at
37.degree. C. T cells were then added (clones 3838 or D3 as
indicated), samples were further incubated for 24 hr at 37.degree.
C., and supernatants were assayed as described in FIG. 6. Each
polypeptide was tested in duplicate. The concentration of the
present polypeptides is indicated on the abscissa. The same symbols
as used in FIG. 6 are used for this figure.
DETAILED DESCRIPTION OF THE INVENTION
[0057] According to the present invention, polypeptides having at
least three different amino acids randomly polymerized in a linear
configuration are useful for treating autoimmune diseases.
Autoimmune diseases occur when the immune system inappropriately
attacks certain tissues or cells. The polypeptides of the present
invention can prevent the immune system from attacking, for
example, by suppressing the proliferation or function of T or B
cells responsible for the attack, or by shielding the tissue from
attack by binding to a MHC protein on the surface of the cells that
make up the tissue.
[0058] Amino acids of the present invention include, but are not
limited to the 20 commonly occurring amino acids. Also included are
naturally occurring and synthetic derivatives, for example,
selenocysteine. Amino acids further include amino acid analogs. An
amino acid "analog" is a chemically related form of the amino acid
having a different configuration, for example, an isomer, or a
D-configuration rather than an L-configuration, or an organic
molecule with the approximate size and shape of the amino acid, or
an amino acid with modification to the atoms that are involved in
the peptide bond, so as to be protease resistant when polymerized
in a polypeptide.
[0059] The phrases "amino acid" and "amino acid sequence" as
defined here and in the claims can include one or more components
which are amino acid derivatives and/or amino acid analogs
comprising part or the entirety of the residues for any one or more
of the 20 naturally occurring amino acids indicated by that
sequence. For example, in an amino acid sequence having one or more
tyrosine residues, a portion of one or more of those residues can
be substituted with homotyrosine. Further, an amino acid sequence
having one or more non-peptide or peptidomimetic bonds between two
adjacent residues, is included within this definition.
[0060] The one letter and three letter amino acid codes (and the
amino acid that each represents) are as follows: A means ala
(alanine); C means cys (cysteine); D means asp (aspartic acid); E
means glu (glutamic acid); F means phe (phenylalanine); G means gly
(glycine); H means his (histidine); I means ile (isoleucine); K
means lys (lysine); L means leu (leucine); M means met
(methionine); N means asn (asparagine); P means pro (proline); Q
means gln (glutamine); R means arg (arginine); S means ser
(serine); T means thr (threonine); V means val (valine); W means
trp (tryptophan); and Y means tyr (tyrosine).
[0061] The term "hydrophobic" amino acid is defined here and in the
claims as including aliphatic amino acids alanine (A, or ala),
glycine (G, or gly), isoleucine (I, or ile), leucine (L, or leu),
proline (P, or pro), and valine (V, or val), the terms in
parentheses being the one letter and three letter standard code
abbreviations for each amino acid, and aromatic amino acids
tryptophan (W, or trp), phenylalanine (F or phe), and tyrosine (Y,
or tyr). The amino acids confer hydrophobicity as a function of the
length of aliphatic and size of aromatic side chains, when found as
residues within a protein.
[0062] The term "charged" amino acid is defined here and in the
claims as including amino acids aspartic acid (D, or asp), glutamic
acid (E, or glu), histidine (H, or his), arginine (R, or arg) and
lysine (K, or lys), which confer a positive (his, lys and arg) or
negative (asp and gly) charge at physiological values of pH in
aqueous solutions on proteins containing these residues.
Polypeptide Compositions Contemplated by the Invention
[0063] The polypeptides of the present invention comprise Copolymer
1 and Terpolymers consisting essentially of three of the four amino
acids of Copolymer 1, namely tyrosine, glutamic acid, alanine and
lysine. However, one of skill in the art can readily substitute
structurally-related and/or charge-related amino acids without
deviating from the spirit of the invention. Thus, the present
invention further contemplates conservative amino acid
substitutions for tyrosine, glutamic acid, alanine and lysine in
the present polypeptides. Such conservative substitutions are
structurally-related amino acid substitutions, including those
amino acids which have about the same charge, hydrophobicity and
size as tyrosine, glutamic acid, alanine or lysine. For example,
lysine is structurally-related to arginine and histidine; glutamic
acid is structurally-related to aspartic acid; tyrosine is
structurally-related to serine, threonine, phenylalanine and
tryptophan; and alanine is structurally-related to valine, leucine
and isoleucine. These and other conservative substitutions, such as
structurally-related synthetic amino acids, are contemplated by the
present invention.
[0064] Moreover, the Terpolymers can be composed of l- or d-amino
acids. As is known by one of skill in the art, l-amino acids occur
in most natural proteins. However, d-amino acids are commercially
available and can be substituted for some or all of the amino acids
used to make the Terpolymers. The present invention contemplates
Terpolymers formed from mixtures of d- and l-amino acids, as well
as Terpolymers consisting essentially of either l- or d-amino
acids.
[0065] The average molecular weight and the average molar fraction
of the amino acids in the Terpolymers can vary. However, an average
molecular weight range of about 2,000 to about 40,000 daltons is
contemplated. A preferred average molecular weight range is from
about 4,000 to about 12,000 daltons. Preferred average molecular
weight ranges and processes of making the Terpolymers are described
in U.S. Pat. No. 5,800,808, which is hereby incorporated by
reference in its entirety.
[0066] In one embodiment, the present invention provides
Terpolymers containing tyrosine, alanine and lysine. The average
molar fraction of the amino acids in these Terpolymers can vary,
for example, tyrosine can be present in a mole fraction of about
0.005 to about 0.250; alanine can be present in a mole fraction of
about 0.3 to about 0.6; and lysine can be present in a mole
fraction of about 0.1 to about 0.5. The average molecular weight is
between 2,000 to about 40.000 daltons and preferably between about
3,000 to about 35,000 daltons. In a more preferred embodiment, the
average molecular weight is about 5,000 to about 25,000
daltons.
[0067] In another embodiment, the present invention provides
Terpolymers containing tyrosine, glutamic acid and lysine. The
average molar fraction of the amino acids in these polypeptides can
also vary, for example, glutamic acid can be present in a mole
fraction of about 0.005 to about 0.300, tyrosine can be present in
a mole fraction of about 0.005 to about 0.250; lysine can be
present in a mole fraction of about 0.3 to about 0.7. The average
molecular weight is between 2,000 to about 40,000 daltons and
preferably between about 3,000 to about 35,000 daltons. In a more
preferred embodiment, the average molecular weight is about 5,000
to about 25,000 daltons.
[0068] In another embodiment, the present invention provides
Terpolymers containing glutamic acid, alanine and lysine. The
average molar fraction of the amino acids in these polypeptides can
also vary, for example, glutamic acid can be present in a mole
fraction of about 0.005 to about 0.300, alanine can be present in a
mole fraction of about 0.005 to about 0.600; lysine can be present
in a mole fraction of about 0.2 to about 0.7. The average molecular
weight is between 2,000 to about 40,000 daltons and preferably
between about 3,000 to about 35,000 daltons. In a more preferred
embodiment, the average molecular weight is about 5,000 to about
25,000 daltons.
[0069] In another embodiment, the present invention provides
Terpolymers containing tyrosine, glutamic acid and alanine. The
average molar fraction of the amino acids in these polypeptides can
also vary, for example, tyrosine can be present in a mole fraction
of about 0.005 to about 0.250; glutamic acid can be present in a
mole fraction of about 0.005 to about 0.300; and alanine can be
present in a mole fraction of about 0.005 to about 0.800. The
average molecular weight is between 2,000 to about 40,000 daltons
and preferably between about 3,000 to about 35,000 daltons. In a
more preferred embodiment, the average molecular weight is about
5,000 to about 25,000 daltons.
[0070] In a more preferred embodiment, the mole fraction of amino
acids of the Terpolymers is about what is preferred for Copolymer
1. The mole fraction of amino acids in Copolymer 1 is glutamic acid
(about 0.14), alanine (about 0.43), tyrosine (about 0.10) and
lysine (about 0.34). The most preferred average molecular weight
for Copolymer 1 is between about 5,000 and about 9,000 daltons.
[0071] The molar ratios of the monomers of the more preferred
terpolymer of glutamic acid, alanine and tyrosine is about 0.21 to
about 0.65 to about 0.14.
[0072] The molar ratios of the monomers of the more preferred
terpolymer of glutamic acid, alanine and lysine is about 0.15 to
about 0.48 to about 0.36.
[0073] The molar ratios of the monomers of the more preferred
terpolymer of glutamic acid, tyrosine, and lysine is about 0.26 to
about 0.16 to about 0.58.
[0074] The molar ratios of the monomers of the more preferred
terpolymer of tyrosine, alanine and lysine is about 0.10 to about
0.54 to about 0.35.
[0075] In one embodiment, the Terpolymers of the present invention
are capable of binding to an MHC class II protein which,
preferably, is associated with an autoimmune disease. Any available
method can be used to ascertain whether the Terpolymer binds to one
or more MHC class II proteins. For example, the polypeptide can be
labeled with a reporter molecule (such as a radionuclide or
biotin), mixed with a crude or pure preparation of MHC class II
protein and binding is detected if the reporter molecule adheres to
the MHC class II protein after removal of the unbound
polypeptide.
[0076] In another embodiment, the Terpolymers of the invention are
capable of binding to an MHC class II protein associated with
multiple sclerosis. A polypeptide of this embodiment can have
similar or greater affinity for the antigen binding groove of an
MHC class II protein associated with multiple sclerosis than does
Copolymer 1. Hence, the contemplated polypeptide can inhibit
binding of or displace the binding of myelin autoantigens from the
MHC class II protein. One MHC class II protein associated with
multiple sclerosis is HLA-DR4 (DRB1*1501).
[0077] In another embodiment, Copolymer 1 and the Terpolymers of
the invention are capable of binding to an MHC class II protein
associated with an arthritic condition, for example, rheumatoid
arthritis or osteoarthritis. Copolymer 1 or a Terpolymer of this
embodiment can have a greater affinity for the antigen binding
groove of an MHC class II protein associated with the autoimmune
disease than does a type II collagen 261-273 (SEQ ID NO:3) peptide.
Hence, the contemplated Copolymer 1 or Terpolymers can inhibit
binding of or displace the type II collagen 261-273 peptide from
the antigen binding groove of an MHC class II protein. The Class II
MHC protein consists of approximately equal-sized .alpha. and
.beta. subunits, both of which are transmembrane proteins. A
peptide-binding cleft is formed by parts of the amino termini of
both .alpha. and .beta. subunits. This peptide-binding cleft is the
site of presentation of the antigen to T cells. There are at least
three types of Class II MHC molecules: HLA-DR, HLA-DQ, and HLA-DP
molecules. There are also numerous alleles encoding each type of
these HLA molecules. The Class II MHC molecules are expressed
predominantly on the surfaces of B lymphocytes and antigen
presenting cells such as macrophages.
[0078] The present Terpolymers can be formulated into
pharmaceutical compositions containing a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, sweeteners and the like. The pharmaceutically
acceptable carriers may be prepared from a wide range of materials
including, but not limited to, flavoring agents, sweetening agents
and miscellaneous materials such as buffers and absorbents that may
be needed in order to prepare a particular therapeutic composition.
The use of such media and agents with pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. The present compositions may be
formulated as an injectable solution or suspension, a spray
solution or a suspension.
Therapeutic Methods Contemplated by the Invention
[0079] The present invention further provides methods for treating
and preventing autoimmune diseases in a mammal which include
administering a therapeutically effective amount of a composition
having a polypeptide containing at least three different amino
acids selected from the group consisting of the amino acids which
comprise Copolymer 1, namely glutamic acid, tyrosine, lysine, and
alanine, wherein the selected amino acids are randomly polymerized
in a linear configuration. In one embodiment the polypeptide is
Copolymer 1 or a Terpolymer.
[0080] Autoimmune diseases contemplated by the present invention
include either cell-mediated disease (e.g. T-cell) or
antibody-mediated (e.g. B cell) disorders. Such disorders can be
inter alia arthritic conditions, demyelinating diseases and
inflammatory diseases. For example, autoimmune diseases which can
be treated by the present polypeptides include multiple sclerosis,
autoimmune hemolytic anemia, autoimmune oophoritis, autoimmune
thyroiditis, autoimmune uveoretinitis, Crohn's disease, chronic
immune thrombocytopenic purpura, colitis, contact sensitivity
disease, diabetes mellitus, Graves disease, Guillain-Barre's
syndrome, Hashimoto's disease, idiopathic myxedema, myasthenia
gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis, or
systemic lupus erythematosus, The present compositions can be used
to treat one or more of these diseases.
[0081] The term "arthritic condition" as used herein is a condition
wherein at least one symptom of rheumatoid arthritis is observed in
at least one joint of a mammal, for example in a shoulder, knee,
hip, backbone or a digit of the mammal. Examples of arthritic
conditions include "Polyarthritis", which is an arthritic condition
that affects more than a single joint; "juvenile arthritis", an
arthritic condition of humans under the age of 21; and Felty's
syndrome, which can include the symptoms of neutropenia,
splenomegaly, weight loss, anemia, lymphadenopathy, and pigment
spots on the skin.
[0082] In one embodiment, any autoimmune disease can be treated by
the present polypeptides so long as the contemplated polypeptide
binds to an MHC class II protein that has been associated with the
autoimmune disease. One aspect of this embodiment provides a method
which includes selecting a polypeptide that inhibits binding of an
antigenic peptide to an MHC class II protein, for example, a method
wherein step (a) further comprises selecting the heteropolymer that
inhibits class II-specific T cell responses to an MHC class II
protein-peptide complex, and a method wherein the antigenic peptide
is associated with an autoimmune disease; in another embodiment of
the invention, a method is provided wherein the MHC class II
protein is associated with an autoimmune disease.
[0083] In another embodiment, the method for treating an autoimmune
disease in a mammal further involves inhibiting the proliferation
or function of T cells which are responsive to an autoantigen. RA
is a T cell-mediated autoimmune disease which can be treated with
the present polypeptides. The pathological process of autoimmune
diseases and immune rejection is mediated by T cells. Upon binding
to and recognition of an antigen, T cells proliferate, secrete
cytokines and recruit additional inflammatory and cytotoxic cells
to the site. The present polypeptides prevent T cell proliferation
and T cell functions such as cytokine secretion and recruitment of
inflammatory and cytotoxic cells to the site. When the autoimmune
disease is an arthritic condition the autoantigen can be collagen,
and the present polypeptides can inhibit the proliferation and
function of collagen-responsive T cells.
[0084] In another embodiment, the method for treating an autoimmune
disease in a mammal involves binding the polypeptide to an antigen
presenting cell such as a macrophage, a dendritic cell of the
lymphoid tissue or an epidermal cell. The proliferation and
functions of a T cell are activated when an appropriate antigen is
presented to it. By binding to antigen presenting cells, the
present polypeptides may block or otherwise interfere with T cell
activation.
[0085] In yet another embodiment, the method for treating an
autoimmune disease in a mammal involves binding the polypeptide to
a major histocompatibility complex class II protein which is
associated with an autoimmune disease. The Class II MHC proteins
are expressed predominantly on the surfaces of B lymphocytes and
antigen presenting cells such as macrophages. These Class II MHC
proteins have a peptide-binding cleft which is the site at which
antigenic peptides are presented to T cells. When the present
polypeptides bind to a major histocompatibility complex class II
protein, those polypeptides can block or otherwise interfere with
antigen presentation and/or T cell activation.
[0086] In another embodiment, the method for treating an autoimmune
disease in a mammal involves binding the polypeptide to Copolymer
1-reactive B cell antibodies, and/or Copolymer 1-reactive T cells.
Copolymer 1-reactive T.sub.H2/3 T cells facilitate the therapeutic
effects of Copolymer 1. When binding to Copolymer 1-reactive T
cells, the present polypeptides stimulate those T cells to
proliferate, secrete antiinflammatory cytokines and enhance the
therapeutic benefits of treatment by the present methods. According
to the present invention, the present polypeptides also bind to
autoantigen-reactive antibodies which may block the antibody from
attacking the target tissue, thereby helping to prevent the
autoimmune disease from progressing. For example, when the present
polypeptides are bound to MBP-specific antibodies, those antibodies
may not bind to MBP and thereby lead to the destruction of MBP in
the myelin sheath.
[0087] The present polypeptides may be administered by any
convenient route. In one embodiment the present polypeptides can be
administered by injection to facilitate delivery to the tissues
affected by the autoimmune disease. Thus, the present polypeptides
may, for example, be injected, ingested, inhaled, or topically
applied. The subject polypeptides may be incorporated into a cream,
solution or suspension for topical administration. The present
polypeptides are preferably administered orally, topically or by
injection without addition of an adjuvant.
Useful Kits of the Invention
[0088] Another embodiment of the invention, provides a kit for
assaying the binding of an analyte to an MHC protein, which
includes a water-soluble MHC protein which has been recombinantly
produced in a non-mammalian cell, and a means for detection of the
bound analyte on the MHC protein, and instructions for use. The MHC
protein used in the kit is an MHC class II protein selected from
the group consisting of an MHC class II HLA-DR1 protein, an MHC
class II HLA-DR2 protein and an MHC class II HLA-DR4 protein. The
kit can further comprise an autoantigenic peptide.
[0089] In a preferred embodiment, the MHC class II protein is
produced in an invertebrate or a microbial cell, such as an insect
cell or a yeast cell and is therefore devoid of bound peptide in
the antigen cleft. The means for detecting binding of the analyte
to the MHC protein can be any radioactive, fluorimetric,
chemiluminescent, enzymatic or colorimetric means known to one of
ordinary skill in the art. In a preferred embodiment, the MHC
protein is a class II HLA-DR1 or HLA-DR4 protein. Further, the kit
can include also an autoantigenic peptide, such as a collagen II
peptide, or a peptide derived from myelin basic protein, myelin
oligodendrite protein, or a peptide from some other protein
implicated in an autoimmune disease.
Synthesis of the Terpolymers of the Invention
[0090] The Terpolymers can be made by any procedure available to
one of skill in the art. For example, the Terpolymers can be made
under condensation conditions using the desired molar fraction of
amino acids in solution or by solid phase synthetic procedures.
Condensation conditions include the proper temperature, pH and
solvent conditions for condensing the carboxyl group of one amino
acid with the amino group of another amino acid to form a peptide
bond. Condensing agents, for example, dicyclohexyl-carbodiimide,
can be used to facilitate the formation of the peptide bond.
Blocking groups can be used to protect functional groups, such as
the side chain moieties and some of the amino or carboxyl groups,
against undesired side reactions.
[0091] For example, the process disclosed in U.S. Pat. No.
3,849,550 can be used where the N-carboxyanhydrides of tyrosine,
alanine, .gamma.-benzyl glutamate and N-trifluoroacetyl-lysine are
polymerized at ambient temperatures in anhydrous dioxane with
diethylamine as an initiator. The .gamma.-carboxyl group of the
glutamic acid can be deblocked by hydrogen bromide in glacial
acetic acid. The trifluoroacetyl groups are removed from lysine by
1 molar piperidine. One of skill in the art readily understands
that the process can be adjusted to make polypeptides containing
the desired amino acids, for example, only three of the four amino
acids in Copolymer 1 by selectively eliminating the reactions that
relate to any one of glutamic acid, alanine, tyrosine or lysine.
For purposes of the application, the terms "ambient temperature"
and "room temperature" mean a temperature ranging from about 20 to
about 26 degrees Centigrade (.degree. C.).
[0092] The average molecular weight of the Terpolymers can be
adjusted during polypeptide synthesis or after the Terpolymers have
been made. To adjust the average molecular weight during
polypeptide synthesis, the synthetic conditions or the amounts of
amino acids are adjusted so that synthesis stops when the
polypeptide reaches the approximate length which is desired. After
synthesis, polypeptides with the desired average molecular weight
can be obtained by any available size selection procedure, for
example, chromatography of the polypeptides on a molecular weight
sizing column or gel, and collection of the average molecular
weight ranges desired. The present polypeptides can also be
partially hydrolyzed to remove high molecular weight species, for
example, by acid or enzymatic hydrolysis, and then purified to
remove the acid or enzymes.
[0093] In one embodiment, the present invention provides a process
for preparing Terpolymers with a desired average molecular weight
which includes reacting a protected polypeptide with hydrobromic
acid to form a polypeptide having the desired average molecular
weight profile. The reaction is performed for a time and at a
temperature which is predetermined by one or more test reactions.
During the test reactions, the time and temperature are varied and
the average molecular weight range of a given batch of test
polypeptides is determined. The test conditions which provide the
optimal average molecular weight range for that batch of
polypeptides are used for the batch. Thus, polypeptides having the
desired average molecular weight profile, can be produced by a
process which includes reacting the protected polypeptide with
hydrobromic acid for a time and at a temperature predetermined by
test reaction.
[0094] In a preferred embodiment, a test sample of protected
polypeptide from a given batch is reacted with hydrobromic acid for
about 10-50 hours at a temperature of about 20-28.degree. C. The
best conditions for that batch are determined by running several
test reactions. For example, in one embodiment, the protected
polypeptide is reacted with hydrobromic acid for about 17 hours at
a temperature of about 26.degree. C.
[0095] The examples which follow describe the invention in detail
with respect to showing how certain specific representative
embodiments thereof can be made, the materials, apparatus and
process steps being understood as examples that are intended to be
illustrative only. In particular, the invention is not intended to
be limited to the methods, materials, conditions, process
parameters, apparatus and the like specifically recited herein.
[0096] Throughout this application, various publications, patents,
and patent applications have been referred to. The teachings and
disclosures of these publications, patents, and patent applications
in their entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
the present invention pertains.
[0097] It is to be understood and expected that variations in the
principles of invention herein disclosed may be made by one skilled
in the art and it is intended that such modifications are to be
included within the scope of the present invention.
Example I
Preparation of Polypeptides
Preparation of Protected Polypeptides
[0098] Protected polypeptides are prepared as described by
Teitelbaum et al., using the N-carboxyanhydride (NCA) blocked amino
acids tyrosine, alanine, glutamic acid and trifluoroacetyllysine
dissolved in dioxane. 1 EUR. J. IMMUN. 242 (1971). The carboxylate
group on glutamic acid is blocked with a benzyl (BZ) group.
[0099] The polymerization process is initiated by the addition of
0.01-0.02% diethylamine. The reaction mixture is stirred at room
temperature for 20 hours and then poured into water. The protected
polypeptide product is filtered, washed with water and dried. The
removal of the .gamma.-benzyl blocking groups from the glutamate
residue is carried out by treating the protected polypeptide with
33% hydrobromic acid in glacial acetic acid at room temperature for
6-12 hours with stirring. The product is poured into excess water,
filtered, washed and dried, yielding the protected polypeptide.
Preparing Polypeptides with Molecular Weight 7,000.+-.2,000 Da
[0100] Treatment of .gamma.-benzyl protected polypeptides 33% HBr
in acetic acid not only insures removal of the benzyl protecting
group from the .gamma.-carboxylate of the glutamate residue but
also cleaves the polypeptides into smaller polypeptides. The time
needed for obtaining a polypeptide of molecular weight
7,000.+-.2,000 daltons depends on the reaction temperature and the
size of protected polypeptide. At temperatures of between
20.degree. C. to 28.degree. C. a test reaction is performed on
every batch for different time periods, for example, from 10-50
hours. A curve of average molecular weight obtained over time is
drawn. The time needed for obtaining molecular weight
7,000.+-.2,000 Da is calculated from the curve and the reaction is
performed on a larger scale. On average, at 26.degree. C., the time
period for obtaining a mixture of polypeptides with a molecular
weight of 7,000.+-.2,000 Da is 17 hours. The product is poured into
excess water, filtered, washed and dried, yielding a polypeptide
with a desired range of average molecular weights.
Preparation of Low-Toxicity Lysine-Containing Polypeptides
[0101] Protected trifluoroacetyl-polypeptide (20 g) is dispersed in
1 liter of water and 100 g piperidine is added. The mixture is
stirred for 24 hours at room temperature and filtered. The solution
of crude polypeptide is distributed into dialysis bags and dialyzed
at 10-20.degree. C. in water until pH 8 is attained. The
polypeptide solution is dialyzed in 0.3% acetic acid and then again
in water until pH 5.5 to 6.0 is obtained. This solution is then
concentrated and lyophilized to dryness.
Synthesis of: Molecular weight 7,600 TGA, poly[L-Tyr.sup.0.136,
L-Glu.sup.0.21, L-Ala.sup.0.648]
1) Raw Materials:
TABLE-US-00001 [0102] L-Ala-NCA 18.74 g L-Tyr-NCA 6.5 g
5-BZ-L-Glu-NCA 13 g Diethylamine 0.165 g Dioxane 790 ml
HBr/AcOH(33%) 480 ml Phenol 4.8 g Piperidine 13.2 ml Deionized
water Acetic acid
2) Procedure:
[0103] L-Tyr-NCA (6.5 g) is placed in dioxane (211 ml) and heated
to 60.degree. C. for 10 min, then filtered. 5-BZ-L-Glu-NCA (13 g)
is placed in dioxane (226 ml) and stirred at 20-25.degree. C. for
10 min, then filtered. L-Ala-NCA (18.74 g) is placed in dioxane
(350 ml) and stirred at 20-25.degree. C. for 10 min, then
filtered.
[0104] A 2 L Erlenmeyer equipped with a magnetic stirrer, is
charged with the solution of L-Tyr-NCA, the solution of
5-BZ-L-Glu-NCA and the solution of L-Ala-NCA. Diethylamine (0.165
g) in dioxane (2.5 ml) is introduced to the reaction mixture and
stirred at 20-25.degree. C. for 20 hours. The reaction mixture is
added to deionized water (1 L), filtered and dried at 60.degree. C.
under vacuum. Yield -25.8 g.
[0105] A solution of phenol (4.8 g) in HBr/AcOH (33%; 480 ml) is
stirred for 20 hours. A 2 L Erlenmeyer equipped with a magnetic
stirrer, is charged with a solution of HBr/AcOH,
poly[5-BZ-L-Glu,L-Ala,L-LTyr] and stirred at 26.+-.1.degree. C. for
16 hours. The reaction mixture is added to deionized water (2 L)
and stirred for 1 hour. The precipitate is filtered and washed with
deionized water until the pH is 6. The product is dried in a
circulating air oven at 40.degree. C., for about 40 hours. Yield
-24 g.
[0106] A 2 L Erlenmeyer equipped with a magnetic stirrer, is
charged with a piperidine (13.2 ml), deionized water (1200 ml), and
poly[L-Glu,L-Ala,L-LTyr], then stirred at 20-25.degree. C. for 24
hours. The resulting solution is ultrafiltered through
polyethersulphone membranes with a cutoff of 5000 daltons until two
thirds of the original volume is removed. The original volume is
restored by addition of fresh deionized water. The process is
repeated 5 times, until the impurity content is less than 1% by
HPLC. The resulting solution (at full volume) is acidified to pH
4.4 with acetic acid. The solution is ultrafiltered to pH 5.5-6 and
the volume is reduced to one third. The resulting solution is
lyophilized to dryness.
Synthesis of Molecular Weight 8850 GAL,
poly[L-Glu.sup.0.153,L-Ala.sup.0.479,L-Lys.sup.0.365]
1) Raw Materials
TABLE-US-00002 [0107] L-Ala-NCA 14 g N6-TFA-L-Lys-NCA 22.9 g
5-BZ-L-Glu-NCA 9.8 g Diethylamine 0.12 g Dioxane 850 ml HBr/AcOH
(33%) 480 ml Phenol 4.8 g Piperidine 13.2 ml Deionized water Acetic
acid
2) Procedure
[0108] N6-TFA-L-Lys-NCA (22.9 g) in dioxane (420 ml) is stirred at
20-25.degree. C. for 10 min. and filtered. 5-BZ-L-Glu-NCA (9.8 g)
in dioxane (170 ml) is stirred at 20-25.degree. C. for 10 min. and
filtered. L-Ala-NCA (14 g) in dioxane (260 ml) is stirred at
20-25.degree. C. for 10 min. and filtered. A 2 L Erlenmeyer
equipped with a magnetic stirrer, is charged with the solution of
N6-TFA-L-Lys-NCA, the solution of 5-BZ-L-Glu-NCA and the solution
of L-Ala-NCA. Diethylamine (0.12 g) in dioxane (2.5 ml) is added,
and the reaction mixture is stirred at 20-25.degree. C. for 20
hours. The reaction mixture is added to deionized water (1 L),
filtered and dried at 60.degree. C. under vacuum.
[0109] A solution of phenol (4.8 g) in HBr/AcOH (33%; 480 ml) is
stirred for 20 hours. A 2 L Erlenmeyer equipped with a magnetic
stirrer, is charged with a solution of HBr/AcOH,
poly[5-BZ-L-Glu,L-Ala,N6-TFA-L-Lys] (24 g) and is stirred at
26.+-.1.degree. C. for 16 hours. The reaction mixture is added to
deionized water (2 L) and stirred for 1 hour. The precipitate is
filtered and washed with deionized water until pH=6. The product is
dried in a circulating air oven at 40.degree. C., about 40
hours.
[0110] A 2 L Erlenmeyer equipped with a magnetic stirrer, is
charged with piperidine (13.2 ml), deionized water (1200 ml) and
poly[L-Glu,L-Ala,N6-TFA-L-Lys] (24 g) is stirred at 20-25.degree.
C. for 24 hours. The solution is ultrafiltered through
polyethersulphone membranes with a cut off of 5000 daltons until
two thirds of the original volume are removed. The original volume
is restored by addition of fresh deionized water. The process is
repeated 5 times, until the impurity content is less than 1% (by
HPLC). The resulting solution (at full volume) is acidified to pH
4.4 with acetic acid. The solution is ultrafiltered until the pH is
5.5-6 and the volume is reduced to one third. The resulting
solution is lyophilized to dryness.
Synthesis of Molecular Weight 11,050 TGL,
poly[L-Tyr.sup.0.162,L-Glu.sup.0.259,L-Lys.sup.0.579]
1) Raw Materials
TABLE-US-00003 [0111] 5-BZ-L-Glu-NCA 10.34 g N6-TFA-L-Lys-NCA 24.16
g L-Tyr-NCA 5.2 g Diethylamine 0.095 g Dioxane 810 ml HBr/AcOH
(33%) 460 ml Phenol 4.6 g Piperidine 150 ml Deionized water Acetic
acid
2) Procedure
[0112] L-Tyr-NCA (5.2 g) in dioxane (180 ml) is heated to
60.degree. C. for 10 min, and filtered. N6-TFA-L-Lys-NCA (24.16 g)
in dioxane (450 ml) is stirred at 20-25.degree. C. for 10 min. and
filtered. 5-BZ-L-Glu-NCA (10.35 g) in dioxane (180 ml) is stirred
at 20-25.degree. C. for 10 min. and filtered.
[0113] A 2 L Erlenmeyer equipped with a magnetic stirrer, is
charged with the solution of N6-TFA-L-Lys-NCA, the solution of
L-Tyr-NCA and the solution of 5-BZ-L-Glu-NCA. Diethylamine (0.095
g) in dioxane (2.5 ml) is introduced, and the reaction mixture is
stirred at 20-25.degree. C. for 20 hours. The reaction mixture is
added to deionized water (0.7 L), filtered and dried at 60.degree.
C. under vacuum.
[0114] A solution of phenol (4.6 g) in HBr/AcOH (33%; 460 ml) is
stirred for 20 hours. A 2 L Erlenmeyer equipped with a magnetic
stirrer, is charged with the solution of HBr/AcOH and the
poly[5-BZ-L-Glu,L-Tyr,N6-TFA-L-Lys]. This mixture is stirred at
26.+-.1.degree. C. for 16 hours and then the mixture is added to
deionized water (1.5 L) and stirred for 1/2 hour. The precipitate
is filtered and washed with deionized water until the pH is
5.5.
[0115] A 2 L Erlenmeyer equipped with a magnetic stirrer, is
charged with a piperidine (150 ml), deionized water (1400 ml), and
poly[L-Glu,L-Tyr,N6-TFA-L-Lys] (50 g) and is stirred at
20-25.degree. C. for 24 hours. The resulting solution of
poly[L-Glu, L-Tyr, L-Lys] is ultrafiltered through
polyethersulphone membranes with a cut off of 5000 daltons until
two thirds of the original volume were removed. The original volume
is restored by addition of fresh deionized water. The process is
repeated 5 times, until the impurity content is less than 1% (by
HPLC). The resulting solution (at full volume) is acidified to pH
4.2 with acetic acid. The solution is ultrafiltered to a pH of
5.5-6 and the volume is reduced to one third. The resulting
solution is lyophilized to dryness.
Synthesis of Molecular Weight 20,000 TAL, poly[L-Tyr.sup.0.102,
L-Ala.sup.0.542, L-Lys.sup.0.353]
1) Raw Materials
TABLE-US-00004 [0116] L-Ala-NCA 14 g N6-TFA-L-Lys-NCA 22.9 g
L-Tyr-NCA 4.9 g Diethylamine 0.1 g Dioxane 850 ml HBr/AcOH (33%)
500 ml Phenol 5 g Piperidine 162 ml Deionized water Acetic acid
2) Procedure
[0117] L-Tyr-NCA (4.9 g) in dioxane (170 ml) is heated to
60.degree. C. for 10 min, and filtered. N6-TFA-L-Lys-NCA (22.9 g)
in dioxane (417 ml) is stirred at 20-25.degree. C. for 10 min. and
filtered. L-Ala-NCA (14 g) in dioxane (260 ml) is stirred at
20-25.degree. C. for 10 min. and filtered.
[0118] A 2 L Erlenmeyer equipped with a magnetic stirrer, is
charged with the solution of N6-TFA-L-Lys-NCA, the solution of
Tyr-NCA in dioxane and the solution of L-Ala-NCA. Diethylamine (0.1
g) in dioxane (2 ml) is introduced and the mixture is stirred at
20-25.degree. C. for 20 hours. The reaction mixture is then added
to deionized water (1 L), filtered and dried at 60.degree. C. under
vacuum.
[0119] A solution of phenol (5 g) in HBr/AcOH (33%; 500 ml) is
stirred for 20 hours. A 2 L Erlenmeyer equipped with a magnetic
stirrer, is charged with the solution of HBr/AcOH and the
poly[L-Ala,L-Tyr,N6-TFA-L-Lys] and is stirred at 26.+-.1.degree. C.
for 16 hours. The reaction mixture is then added to deionized water
(2 L) and stirred for 1/2 hour. The precipitate is filtered and
washed with deionized water until the pH is 5.5.
[0120] A 2 L Erlenmeyer equipped with a magnetic stirrer, is
charged with a piperidine (162 ml), water (1500 ml) and the
poly[L-Ala,L-Tyr,N6-TFA-L-Lys] (30 g), and is stirred at
20-25.degree. C. for 24 hours. The resulting solution of
poly[L-Ala,L-Tyr,L-Lys] is ultrafiltered through polyethersulphone
membranes with a cut off of 5000 daltons until two thirds of the
original volume were removed. The original volume is restored by
addition of fresh deionized water. The process is repeated 5 times,
until the impurity content is less than 1% (by HPLC). The resulting
solution (at full volume) is acidified to pH 4.4 with acetic acid.
The solution is ultrafiltered to pH 5.5-6 and the volume is reduced
to one third. The resulting solution is lyophilized to dryness.
Example 2
Low Molecular Weight Polypeptides
In Vivo Tests
[0121] Three batches of copolymer-1 having an average molecular
weight of 7.3 and 8.4 KDa (less than 2.5% copolymer-1 species over
40 KDa) and 22 KDa (more than 5% copolymer-1 species over 40 KDa)
were subjected to the toxicity test described below. Five mice were
used in each experimental group.
Method
[0122] Copolymer-1 is dissolved in distilled water to yield a
solution of 2 mg/ml of the active ingredient. Each mouse is
injected with 0.5 ml of the test solution into the lateral tail
vein. Mice were observed for mortality and relevant clinical signs
over a 48 hour period. Observations were recorded 10 minutes, 24
hours and 48 hours post-injection. If, at the end of 48 hours, all
the animals were alive and no adverse signs had been observed, then
the batch is designated "non-toxic". If, however, one or more of
the mice had died or had shown adverse signs, then the batch is
designated "toxic".
Results
[0123] Three of five mice died after 48 hours when treated with the
22 kDa average molecular weight Copolymer 1 polypeptides.
Accordingly, this high average molecular weight batch is designated
"toxic". The Copolymer 1 batches having average molecular weights
of 7.3 and 8.4 KDa were both designated "non-toxic."
In Vitro Rat Basophilic Leukemia Cell Degranulation Tests
[0124] Histamine (or serotonin) release from basophile is an in
vitro model for immediate hypersensitivity. The Rat Basophilic
Leukemia cell line, RBL-2H.sub.o, is uniform and easy to maintain
in culture but is a highly sensitive and reproducible system for
testing for degranulation. E. L. Basumian, et al., 11 EUR. J.
IMMUNOL. 317 (1981). The physiological stimulus for histamine
release involves binding of the antigen to membrane-bound IgE
molecules, which triggers an intricate biochemical cascade.
Degranulation is induced by non-IgE-mediated stimuli, including
various peptides and synthetic polymers, e.g. polylysine. R. P.
Siraganian, TRENDS IN PHARMACOLOGICAL SCIENCES 432 (October 1983).
The RBL degranulation test is, therefore, used to screen out those
batches of COP-1 which evoke substantial degranulation and thus
might elicit undesirable local and/or systemic side effects.
Method
[0125] Rat Basophilic Leukemia cells (RBL-2H.sub.o), are loaded
with [H.sup.3]-serotonin, followed by incubation with 100 .mu.g of
the COP-1 to be tested. Batches of COP-1 which induce non-specific
degranulation, release [H.sup.3]-serotonin into the medium. The
radioactivity in the medium is counted by a scintillation counter
and the total radiolabeled serotonin incorporated into the cells is
determined in the pelleted cells. Percent degranulation is
calculated as the percentage of serotonin released out of the total
incorporated.
Results
[0126] Four batches of Copolymer 1, with average molecular weight
between 8,250-14,500 were analyzed for both percentage of the
species with molecular weight over 40 kDa and for degranulation of
RBL's. Results are summarized in Table 1.
TABLE-US-00005 TABLE 1 % of species with M.W. Average M.W. over 40
KDa % Serotonin Release 6,250 <2.5 12.4 7,300 <2.5 21.0
13,000 >5 66.9 14,500 >5 67.8
[0127] As can be seen, when the percentage of high average
molecular weight species is low (less than 2.5%), the percent
release of serotonin is also low, and vice versa. These data
indicate that lower average molecular weight Copolymer 1
polypeptides are preferable to higher average molecular weight
Copolymer 1 polypeptides.
Example 3
Suppression of EAE by the Polypeptides
[0128] Injection of Copolymer 1 in incomplete Freund's adjuvant
before disease induction can suppress experimental allergic
encephalomyelitis (EAE). This suppression appears to be mediated by
Copolymer 1-specific suppressor T cells of the T.sub.H2 type which
cross react with myelin basic protein. Lando et al., 123 J.
IMMUNOL. 2156 (1979); Aharoni et al., 17 EUR. J. IMMUNOL. 23
(1993); Aharoni et al., 94 PROC. NATL. ACAD. SCI. USA 10821 (1997).
Other researchers have observed that the therapeutic effect of
Copolymer 1 in multiple sclerosis patients is also associated with
the induction of T.sub.H2 cells. Lahat et al., 244 J. Neurol. 129
(1997). In this example, EAE is suppressed to by different
polypeptides of the present invention.
Methods
Copolymer 1
[0129] Copolymer 1 batches #02095 and 55495, with average molecular
weights of 6000 Da and 5800 Da, respectively, were obtained from
Teva Pharmaceutical Industries (Petach Tikva, Israel).
Terpolymers
[0130] Four terpolymers were obtained from Teva Pharmaceutical
Industries (Petach Tikva, Israel). The properties of these
terpolymers are provided below: [0131] 1) The GAL terpolymer
(SD-1689) is a mixture of polypeptides containing the following
mole fraction of amino acids glutamic acid (0.153), alanine (0.479)
and lysine (0.365). The range of GAL average molecular weights is
about 4650 daltons to about 20,050 daltons. The average molecular
weight of this GAL preparation is 8850 daltons. [0132] 2) The TGA
terpolymer (SD-1690) is a mixture of polypeptides containing the
following mole fraction of amino acids tyrosine (0.136), glutamic
acid (0.210) and alanine (0.648). The range of TGA average
molecular weights is about 1000 daltons to about 40,000 daltons.
The average molecular weight of this TGA preparation is 7600
daltons. [0133] 3) The TAL terpolymer (SD-1691) is a mixture of
polypeptides containing the following mole fraction of amino acids
tyrosine (0.102), alanine (0.542) and lysine (0.353). The range of
TAL average molecular weights is about 5700 daltons to about 34,400
daltOns. The average molecular weight of this TAL preparation is
about 20,000 daltons. [0134] 4) The GTL terpolymer (SD-1697) is a
mixture of polypeptides containing the following mole fraction of
amino acids glutamic acid (0.259), tyrosine (0.162) and lysine
(0.579). The range of GTL average molecular weights is about 4,000
daltons to about 23,500 daltons. The average molecular weight of
this GTL preparation is about 11050 daltons.
[0135] A control polypeptide is used, consisting of a mixture of
polypeptides containing a 1:1:1 mixture of amino acids alanine,
glutamic acid and tyrosine, with an average molecular weight 26,700
Da. This polypeptide is obtained from Sigma Chemical Company (St.
Louis, Mo.).
Induction of EAE
[0136] Two to three month old female (SJL/J.times.BALB/c)FI mice
are injected in all four footpads with mouse spinal cord homogenate
(3.5 mg/mouse) emulsified in a 1:1 ratio in complete Freund's
adjuvant (CFA) supplemented with 4 mg/ml H37Ra. Pertussis toxin
(0.25 ml, 250 ng, Sigma) is injected intravenously, immediately
after and 48 hr later. Mice are examined daily from day 10 post
induction for clinical signs of EAE which were scored on a 0-5
scale as described in Lando et al., 123 J. IMMUNOL. 2156
(1979).
EAE Suppression by Injection with Incomplete Adjuvant
[0137] The tested polypeptides (10 mg/mouse) are injected in
incomplete Freund's adjuvant (ICFA) subcutaneously in one nuchal
area in 2-3 spots. EAE is induced 3 weeks later as described
above.
EAE Suppression by Oral Administration
[0138] In a second test, female Lewis rats are fed 5 mg/kg guinea
pig BP or Copolymer 1 dissolved in phosphate buffered saline (PBS)
at 2-3 day intervals before EAE induction. EAE is induced two days
after the last feeding by injection of 25 .mu.g guinea pig MBP
emulsified in 1:1 CFA containing 4 mg/ml mycobacterium tuberculosis
(H37Ra) (Difco Lab, Detroit, Mich.). A total volume of 0.1 ml is
injected into each of two hindfoot pads. Control rats are mock fed
with phosphate buffered saline.
[0139] The efficacy of orally administered Copolymer 1 for
preventing EAE in rats is compared to that of rats fed guinea pig
MBP by the method of Higgins et al. 140 J. IMMUNOL. 440 (1988).
[0140] Animals were examined daily from day 10 post induction for
signs of disease. EAE is scored as follows: 0=no disease; 1=limp
tail; 2=hind limb paralysis; 3=paralysis of all four limbs;
4=moribund condition; and 5=death.
RESULTS
[0141] EAE Suppression by Injection with Incomplete Adjuvant
[0142] Table 2 illustrates the effects of administering the present
polypeptides by injection in ICFA prior to induction of EAE. Of the
four polypeptides tested, Copolymer 1 and TAL caused the greatest
EAE suppression. GTL also exhibited good suppressive activity. GAL
and TGA, were somewhat less effective.
TABLE-US-00006 TABLE 2 Suppression of EAE in mice by Injection of
the Present Polypeptides Suppression % Polypeptide Incidence MMS*
Incidence MMS* None 11/11 4.0 (Negative Control) ICFA 11/11 3.4 0.0
15.0 (Negative Control) Copolymer 1 2/11 0.45 82.0 89.0 (Positive
Control) SD-1689 - GAL 7/8 3.5 12.5 12.5 SD-1690 - TGA 6/8 2.5 25.0
37.5 SD-1691 - TAL 3/8 1.1 62.5 72.5 SD-1697 - GTL 5/8 1.5 37.5
62.5 D-Copolymer 1 11/11 4.1 0.0 0.0 *MMS = Mean Maximal Score
D-Copolymer 1 is a copolymer of d-lysine, d-tyrosine, d-glutamic
acid and d-alanine in the molar ratios of Copolymer 1.
[0143] Table 3 illustrates the efficacy of orally administered
Copolymer 1 in preventing the clinical manifestations of EAE in
Lewis rats compared to rats receiving only phosphate buffered
saline (PBS) or guinea pig basic protein (GPBP).
TABLE-US-00007 TABLE 3 Suppression of EAE in Rats by Oral
Administration of the Present Polypeptides Fed Antigen Incidence
MMS .+-. SD Mean Onset (days) PBS (Control) 27/28 (96%) 1.8 .+-.
0.5 11.9 GPBP 10/17 (59%) 0.9 .+-. 0.5 11.4 p = 0.0026 Copolymer 1
13/28 (46%) 0.78 .+-. 0.45 12.6 p = 0.00005 Each numerical value
represents the cumulative results of 3-5 independent experiments.
The p values represent the statistical significance of the
difference from the control (PBS) group. Mean maximal score is
calculated for the entire group.
[0144] These data indicate that the present polypeptides are
therapeutically effective for preventing the onset and severity of
EAE when administered either orally or by injection.
Example 4
Binding to Antigen Presenting Cells
[0145] Several of the present polypeptides bind efficiently to
living antigen presenting cells.
Methods
Copolymer 1
[0146] Copolymer 1 batches #02095 and 55495, with average molecular
weights of 6000 Da and 5800 Da, respectively, were obtained from
Teva Pharmaceutical Industries (Petach Tikva, Israel).
Terpolymers
[0147] GAL, TGA, TAL, GTL and control polypeptides are as described
above under Example 3.
Biotinylation of Antigens
[0148] Biotinylation of Copolymer 1 and Terpolymers is performed at
0.degree. C. with biotin-N-hydroxysuccinimide (Sigma) according to
Fridkis-Hareli et al. 91 PROC. NATL. ACAD. SCI. USA 4872
(1994).
Binding of Biotinylated Antigens to Antigen Presenting Cells
[0149] The biotinylated polypeptides were examined for binding to
living antigen presenting cells of mouse and human origin, using
fluorescently labeled streptavidin and FACS analysis. Adherent
spleen cells from (SJL/J.times.BALB/c)FI mice, or EBV transformed
human B cells of DR7,w ll haplotype (1.times.10.sup.6/100 .mu.l),
were incubated at 37.degree. C. for 20 hr. with 50 .mu.g
biotinylated Copolymer 1 or Terpolymers dissolved in 100 .mu.l PBS
containing 0.1% BSA. The cells were then incubated at 4.degree. C.
for 30 min. with phycoerythrin-conjugated streptavidin (Jackson
Immuno Research, West Grove, Pa.) at a concentration of 0.5
.mu.g/100 .mu.l cell suspension. After each incubation the cells
were washed three times with PBS containing 0.1% BSA. Thereafter,
cells were analyzed by flow cytometry using FACScan
(Becton-Dickinson, Mountain View, Calif.). For each analysis, 5000
cells were examined. Dead cells were excluded on the basis of
forward and side-angle light scatter.
Epstein-Barr Virus (EBV) Transformed B-Cell Lines
[0150] EBV-transformed B-cell lines were initiated according to
Teitelbaum et al., 89 PROC. NATL. ACAD. SCI. USA 137 (1992).
Approximately 20.times.10.sup.6 peripheral blood mononuclear cells
were cultured with 895.8 cell line supernatant, for 1 hr at
37.degree. C. The cells were then washed and cultured in RPMI
medium with 10% FCS and cyclosporin A (10 .mu.g/ml) to deplete T
cells.
Results
[0151] Table 4 illustrates the binding of terpolymers and Copolymer
1 polypeptides to living antigen presenting cells, including mouse
spleen macrophages and human EBV transformed B-cell lines. Data
illustrating both the percent of binding and the intensity of cell
staining are provided (Table 4 I+II). TAL bound the most
efficiently--better even than Copolymer 1. GAL and GTL also bound
very well--the same or even somewhat better than Copolymer 1. TGA
bound well but somewhat less than Copolymer 1.
[0152] TAL bound most efficiently both to spleen macrophages of
(SJL/J.times.BALB/c) F, mice and to EBV-transformed B cells from a
normal DR7.w11 donor, as expressed by the intensity of the binding
(Table 4).
TABLE-US-00008 TABLE 4 Binding of Terpolymers to antigen presenting
cells Polypeptide % Binding MFI* I. Antigen presenting cells from
mouse spleen macrophages Copolymer 1 85 493 (positive control)
SD-1689 - GAL 88 600 SD-1690 - TGA 74 39 SD-1691 - TAL 100 1929
SD-1697 - GTL 90 973 II. Antigen presenting cells from human EBV
transformed B-cell line Copolymer 1 96 727 (positive control)
SD-1689 - GAL 95 661 SD-1690 - TGA 72 49 SD-1691 - TAL 100 1438
SD-1697 - GTL 97 1057 *MFI = Mean Fluorescence Intensity
Example 5
The Present Polypeptides Bind to Purified Human Leukocyte Antigens
(HLA)
[0153] This example illustrates that polypeptides of the present
invention bind to human B cells and to purified human lymphocyte
antigens with high affinity, including the HLA-DR1, HLA-DR2 and
HLA-DR4 molecules.
Methods
Cell Lines and Antibodies
[0154] Homozygous EBV-transformed human B lymphocyte lines used for
immunoaffinity purification of HLA-DR1, -DR2 and -DR4 molecules
were LG-2 (DRB1-), MGAR (DRB1-1501) and Preiss
(DRB-0401/DRB4-0101), respectively.
[0155] Cells were grown in RPMI 1640 supplemented with 10% FCS, 2
mM glutamine, 50 U/ml penicillin G and 50 .mu.g/ml streptomycin in
roller bottles and stored as pellets at -80.degree. C. The anti-DR
hybridoma LB3.1 (IgG2b) is grown in serum-free medium
(Macrophage-SFM, Gibco BRL). See Gorga et al., 103 CELL. IMMUNOL.
160 (1986).
Protein Purification
[0156] Immunoaffinity purification of HLA-DR1, -DR2 and -DR4
molecules is performed, with minor modifications, as previously
reported by Gorga et al., 262 J. BIOL. CHEM. 16087 (1987). Briefly,
detergent soluble membrane preparations from LG-2, MGAR and Priess
cells were passed at a flow rate of approximately 11 ml/hr through
a series of columns in the following sequence: Sepharose CL-6B (30
ml), normal mouse serum-Aff-gel 10 (10 ml), protein A-Sepharose
CL-4B (5 ml) and LB3.1-protein A-Sepharose CL-4B (5 ml). DR2a
(DRB5*0101) and Drw53 (DRB4*0101), the products of DR genes linked
to the DRB1 alleles were not removed from the MGAR and Priess
lysates before passage through the LB3.1 immunoaffinity column, and
contaminate the DR2 and DR4 preparations in the amount of 5-10%.
All the subsequent steps were as previously described by Gorga et
al., 262 J. BIOL. CHEM. 16087 (1987). The eluate is dialyzed
against 0.1% deoxycholate, 10 mM Tris-HCl, pH 8.0 and concentrated
on a Centriprep 30 membrane (Amicon). Protein concentrations were
determined by bicinchonitric acid assay (Pierce Chemical Co.).
Polypeptides and Control Antigens
[0157] Copolymer 1 [0158] Copolymer 1 batches #55495 and 52596,
with average molecular weight of 5800 daltons and 8,150 daltons,
respectively, were obtained from Teva Pharmaceutical Industries
(Petach Tikva, Israel). Copolymer 1 batch 52596 had a molar ratio
of 1 tyrosine:1.5 glutamic acid:4.3 alanine:3.1 lysine. [0159]
Myelin Basic Protein and Hemagglutinin Control Peptides [0160] MBP
peptides were synthesized on an Applied Biosystems Peptide
Synthesizer using solid phase techniques. Barany et al., THE
PEPTIDES 1 (1979). Peptides purified by reversed-phase HPLC. The
peptides used were HA 306-318, having the sequence PKYVKQNTLKLAT
(MW 1718) (SEQ ID NO: 2), and MBP 84 102, having the sequence
DENPVVHFFKNIVTPRTPP (MW 2529) (SEQ ID NO: 1). [0161] Terpolymers
[0162] GAL, TGA, TAL, GTL and control polypeptides are as described
above under Example 3.
Polypeptide Labeling
[0163] Biotinylation of the various polypeptides is performed as in
Example 4. Unreacted biotin is removed by dialysis
(Spectra/Por.RTM. membrane MWCO 500, Spectrum Medical
Industries).
Assays for Polypeptide Binding to Class II MHC Proteins
[0164] Solutions: The solutions used in this assay were the
following. Binding buffer is 20 mM 2-[N-morpholino]ethanesulfonic
acid (MES), 1% n-octyl .beta.-D-glycopyranoside, 140 mM NaCl, 0.05%
NaN.sub.3, pH 5.0, unless otherwise specified. PBS is 150 mM sodium
chloride, 7.5 mM sodium phosphate, dibasic, 2.5 mM sodium
phosphate, monobasic, pH 7.2. TBS is 137 mM sodium chloride, 25 mM
Tris pH 8.0, 2.7 mM potassium chloride. TTBS is TBS plus 0.05%
Tween-20. [0165] Microtiter Assay Plate Preparation: Ninety-six
well microtiter immunoassay plates (PRO-BIND.TM., Falcon) were
coated with 1 .mu.g/well affinity-purified LB3.1 monoclonal
antibodies in PBS (100 .mu.l total) for 18 hrs at 4.degree. C. The
wells were then blocked with TBS containing 3% BSA for 1 hr at
37.degree. C. and washed three times with TTBS. Before sample
addition, 50 .mu.l of TBS/1% BSA is added to each well. [0166]
Binding reactions: Detergent-solubilized HLA-DR1, -DR2 and -DR4
molecules (0.5 .mu.g/sample) were incubated with biotinylated
control peptides at various concentrations for 40 hours at
37.degree. C. in 50 .mu.l of the binding buffer and transferred to
prepared microtiter assay plates and incubated for 1 hr at
37.degree. C. for capture of polypeptide-class II complexes. [0167]
Inhibition reactions: Biotinylated polypeptides at a final
concentration of 1.5 .mu.M in 50 .mu.l of binding buffer were
coincubated with unlabeled polypeptides as well as the peptides HA
306-318 (SEQ ID NO: 2) or MBP 84-102 (SEQ ID NO: 1), used as
inhibitors, and HLA-DR molecules for 40 hr at 37.degree. C. [0168]
Detection of class II/polypeptide complexes: Bound
polypeptide-biotin is detected using streptavidin-conjugated
alkaline phosphatase as follows. Plates were washed three times
with TTBS and incubated with 100 .mu.l of streptavidin-conjugated
alkaline phosphatase (1:3000, BioRad) for 1 hr at 37.degree. C.,
followed by addition of p-nitrophenyl phosphate in triethanolamine
buffer (BioRad). The absorbency at 410 nm is monitored by a
microplate reader (Dynatech MR4000).
Results
Terpolymers Binding to Class II MHC Proteins
[0169] Detergent-soluble HLA-DR1, HLA-DR2 and HLA-DR4 proteins were
purified from homozygous EBV-transformed B cell lines LG-2
(DRB1*0101), MGAR (DRB1*1501) and Priess (DRB1*0401), respectively,
as described previously by Fridkis-Hareli et al., 160 J. IMMUNOL.
4386 (1998). Three different preparations of Copolymer 1 had bound
to these molecules with high affinity. Id. To determine the
affinity of the terpolymers for HLA-DR proteins, binding assays
were carried out with biotinylated Terpolymers, and compared to
Copolymer 1. The polypeptides were incubated at a range of
concentrations with purified HLA-DR1, HLA-DR2 and HLA-DR4 molecules
at pH 5.0 followed by capture with class II-specific mAb and
detection with alkaline phosphatase-streptavidin.
[0170] Binding by TAL and Copolymer 1 to detergent-soluble HLA-DR1
and HLA-DR4 molecules is better than that of GAL, TGA or GTL.
However, GTL and Copolymer 1 bound better than the other
polypeptides to HLA-DR2, based on the saturation binding curves
(FIG. 2A), and on K.sub.d values, calculated from the
double-reciprocal plots of the binding data (FIG. 2B, Table 5).
[0171] Competitive binding assays were carried out with
biotinylated Copolymer 1 and the following unlabeled inhibitors:
Copolymer 1, TAL, TGA, GAL, TGL, MBP 84-102 and HA 306-318
polypeptides (FIG. 3). The MBP 84-102 (SEQ ID NO: 1) peptide is a
poor inhibitor of the binding of Copolymer 1 to HLA-DR2. The
binding of biotinylated Copolymer 1 to detergent-soluble HLA-DR1
and -DR4 molecules is efficiently inhibited by unlabeled TGA, TAL,
HA 306-318 (SEQ ID NO: 2) and Copolymer 1. However, binding of
biotinylated Copolymer 1 to detergent-soluble HLA-DR1 and -DR4
molecules is inhibited more than 10-fold less by TGL, as indicated
by 50% inhibitory dosages (IC.sub.50) (FIG. 3, Table 5). Similarly,
GAL is also a poor inhibitor of Copolymer 1 binding to HLA-DR1 and
-DR4 molecules. In general, the binding pattern to HLA-DR2 is
similar to that observed for HLA-DR1 (Table 5). These results show
that the polypeptides of three amino acids, in particular TAL and
TGA bind to class II MHC molecules with an affinity range similar
to that of antigenic peptides and of Copolymer 1. Hence, TAL and
TGA are effective competitors for the class II MHC molecules to
which Copolymer 1 binds. Based on their binding capacity, the
polypeptides could be arranged in the following order: [0172] (A)
binding to HLA-DR1: Copolymer 1>TAL>GTL>TGA>>GAL;
[0173] (B) binding to HLA-DR2: Copolymer
1>GTL>TAL>GAL>TGA; [0174] (C) binding to HLA-DR4:
TAL>Copolymer 1>>TGA>GTL>GAL.
TABLE-US-00009 [0174] TABLE 5 Affinity of the present polypeptides
for purified human HLA-DR1, -DR2 and -DR4 molecules DR1.sup.2 DR2
DR4 Polypeptide.sup.1 K.sub.d.sup.3 IC.sub.50.sup.4 K.sub.d
IC.sub.50 K.sub.d IC.sub.50 Copolymer 1 7.4 8.8 8.2 10.1 1.5 10.8
TAL 2.0 3.3 1.7 2.7 2.0 6.5 GAL 0.5 .sup.5 1.7 .sup.5 0.3 .sup.5
TGA 1.0 1.3 0.8 9.5 0.8 1.0 TGL 0.4 43.0 5.0 25.4 0.8 43.0 Effect
of superantigens on the binding of polypeptides to HLA-DR molecules
Bacterial superantigens SEA, SEB and TSST-1 have been shown to
inhibit Copolymer 1 binding to purified HLA-DR molecules only at
very high concentrations. Fridkis-Hareli et al., 160 J. IMMUNOL.
4386 (1998). To examine the effect of these superantigens on the
binding of Terpolymers to purified HLA-DR1, HLA-DR2 and HLA-DR4
molecules, competitive binding assays were carried out with
unlabeled SEA, SEB and TSST-1. .sup.1Copolymer 1 polypeptides with
average MW of 5,800; TAL, MW 20,000; GAL, MW 8,850; TGA, MW 7,600;
and TGL, MW 11,050, were incubated at a range of concentrations
with purified HLA-DR1, -DR2 and -DR4 molecules at pH 5.0 followed
by capture with class II-specific mAb and peptide detection with
alkaline phosphatase-streptavidin. .sup.2Detergent-soluble HLA-DR1,
-DR2 and -DR4 molecules were purified as described in Materials and
Methods. .sup.3K.sub.d, the dissociation constant at equilibrium,
is calculated from the slope of the double reciprocal plot (FIG.
2B), and is expressed as .times.10.sup.-8 M. .sup.4IC.sub.50,
inhibitory concentration giving 50% inhibition, is calculated based
on the competitive binding assays (FIG. 3), and is expressed as
.times.10.sup.-6 M. .sup.5 IC.sub.50 values for GAL could not be
determined exactly, but were less than 1000 .mu.M (see FIG. 3).
[0175] Binding of TAL to HLA-DR1, HLA-DR2 and HLA-DR4 is only
inhibited by the superantigens at high molar ratios of
superantigen: for example, fifty times the amount of superantigen
is needed to inhibit TAL binding (FIG. 4A). However, binding of TGA
and GAL is inhibited more significantly by the superantigens (FIGS.
4B and C), indicating that these polypeptides bound the HLA
antigens with lower affinity.
Example 6
Inhibition of MBP-Induced T Cell Proliferation
[0176] This example illustrates that the proliferation of T cells
which are normally activated by myelin basic protein (MBP) can be
inhibited by simultaneous exposure to the present polypeptides.
Methods
Copolymer 1
[0177] Copolymer 1 batches #02095 and 55495, with average molecular
weights of 6000 Da and 5800 Da, respectively, were obtained from
Teva Pharmaceutical Industries (Petach Tikva, Israel).
Myelin Basic Protein Peptides were synthesized on an Applied
Biosystems Peptide Synthesizer using solid phase techniques. Barany
et al., THE PEPTIDES 1 (1979). Peptides purified by reversed-phase
HPLC. The peptides used were HA 306-318, having the sequence
PKYVKQNTLKLAT (MW 1718) (SEQ ID NO: 2), and MBP 84 102, having the
sequence DENPVVHFFKNIVTPRTPP (MW 2529) (SEQ ID NO: 1).
Terpolymers
[0178] GAL, TGA, TAL, GTL and control polypeptides are as described
above under Example 3.
T Cell Lines and Clones
[0179] Myelin Basic Protein (MBP) specific T-cell lines originated
from spleens of mice which, ten days earlier, were immunized with
20 .mu.g of the 84-102 peptide of MBP emulsified in complete
Freund's adjuvant supplemented with 4 mg/ml of Mycobacterium
tuberculosis H37Ra. Cells were cultured and selected in vitro using
the immunizing antigen (0.2-1 mg/plate), in culture medium (RPMI, 2
mM glutamine, 1 mM sodium pyruvate, non essential amino acids,
5.times.10.sup.-5 M 2-mercaptoethanol, 100 .mu.g/ml penicillin, 100
.mu.g/ml streptomycin), supplemented with 1% autologous serum.
After 4 days, cells were transferred to culture medium containing
10% FCS and supplemented with 10% supernatant of Con A activated
normal spleen cells as T cell growth factor (TCGF). Every fourteen
to twenty one days, cells were stimulated by exposure to the
immunizing antigen presented on syngeneic irradiated (3000 rad)
spleen cells (50.times.10.sup.6/plate) for 3 days, followed by
propagation in TCGF medium. Cloning of T cell lines is performed by
limiting dilution at 0.3 cells/well in microliter plate in the
presence of the antigen (2-10 .mu.g/well) and irradiated syngeneic
spleen cells (5.times.10.sup.6/well). [0180] Human T cell lines
were derived from peripheral blood mononuclear cells according to
Teitelbaum et al., 89 PROC. NATL. ACAD. SCI. USA 137 (1992).
Approximately 5.times.10.sup.6 cells were incubated in each well of
a 24-well culture plate with Copolymer 1 or MBP (50 .mu.g/ml) in
culture medium supplemented with 10% heat-inactivated autologous
serum. After 7 days of culture, the cells were transferred to
culture medium containing 10% fetal calf serum and recombinant
human interleukin 2 (20 units/mil). The cells were grown
continuously in this medium with periodic exposure to antigen
presented on irradiated (3000 rad), autologous mononuclear cells,
every 14-18 days.
Proliferation Assay
[0181] Cells of T lines or clones were tested for their specific
proliferative response 10-21 days after antigenic stimulation. T
Cells (1.5.times.10.sup.4) were cultured in triplicates with
5.times.10.sup.5 irradiated spleen cells and with the indicated
antigens in a final volume of 0.2 ml in 10% FCS culture medium. At
the end of 48 hr incubation, cultures were pulsed with 1
.mu.Ci[.sup.3H]-thymidine (standard deviation <20% of the mean
cpm), and harvested 6-12 h later.
Inhibition Studies
[0182] Inhibition of the T-cell proliferative response is studied
by adding various concentrations of Copolymer 1 and Terpolymers
plus the stimulating MBP antigen to the proliferation assay system.
Inhibition is calculated as percent inhibition using equation
2:
% Inhibition=[1-(cpm with inhibitor/cpm without
inhibitor)].times.100. 2
Results
[0183] FIG. 1 illustrates how copolymer 1 and terpolymers effect
the proliferation of T cells which are specific for certain myelin
basic protein (MBP) peptides. In general, T cells proliferate when
exposed to the antigen to which they were sensitized. Thus, MBP
and, in particular, certain antigenic peptides from MBP stimulate
the proliferation of MBP-specific T cells.
[0184] Copolymer 1 and Terpolymers did not stimulate proliferation
of T cells which were specific for the 84-102 peptide of MBP.
Instead, they significantly inhibited the proliferation of T cells
exposed to this MBP antigen.
[0185] TAL exhibited the most efficient inhibition of proliferation
in T cells specific for the MBP 84-102 antigen. Inhibition by TAL
is even greater than that provided by Copolymer 1. TGA induced
lower inhibition of T cell proliferation. GAL and GTL inhibited T
cell proliferation in a dose responsive manner similar to Copolymer
1.
Example 7
Terpolymers are Recognized by Some Copolymer 1-Specific T Cells
[0186] Terpolymers can stimulate some Copolymer 1-specific T cell
lines to proliferate and secrete the cytokine, IL-4.
Methods
Copolymer 1
[0187] Copolymer 1 batches #02095 and 55495, with average molecular
weight of 6000 Da and 5800 Da, respectively, were obtained from
Teva Pharmaceutical Industries (Petach Tikva, Israel).
[0188] Myelin Basic Protein Peptides were synthesized on an Applied
Biosystems Peptide Synthesizer using solid phase techniques. Barany
et al., THE PEPTIDES 1 (1979). Peptides purified by reversed-phase
HPLC. The peptides used were HA 306-318, having the sequence
PKYVKQNTLKLAT (MW 1718) (SEQ ID NO: 2), and MBP 84 102, having the
sequence DENPVVHFFKNIVTPRTPP (MW 2529) (SEQ ID NO: 1).
Terpolymers
[0189] GAL, TGA, TAL, GTL and control polypeptides are as described
above under Example 3.
T Cell Lines and Clones
[0190] Mouse T cell lines and clones were established according to
Aharoni et al., 23 Eur. J. Immunol. 17 (1993); Aharoni et al., 94
PROC. NATL. ACAD. SCI. USA 10821 (1997). Copolymer 1 specific lines
were originated from spleens of mice which, 15 to 35 days earlier,
had been rendered unresponsive to EAE by subcutaneously injecting
each mouse with 5-10 mg Copolymer 1, emulsified in incomplete
Freund's adjuvant (Difco). Alternatively, Copolymer 1 specific
lines were obtained from the lymph nodes of mice which had been
immunized ten days earlier with 200 .mu.g Copolymer 1 emulsified in
complete Freund's adjuvant (Difco) supplemented with 4 mgiml of
Mycobacterium tuberculosis H37Ra (Difco). [0191] Copolymer
1-specific T cell lines were used and T cell clones LN-1, LN-2,
S-3, 5-22-5, C-14, and C-52. The LN-2 antibodies were derived from
lymph nodes of (SJL/J.times.BALB/c) F.sub.1 mice injected with
Copolymer 1 in CFA. The 5-22-5 antibodies were obtained from
spleens of (SJL/J.times.BALB/C) F.sub.1 mice injected with
Copolymer 1 in CFA, according to Aharoni at al., 23 EUR. J.
IMMUNOL. 17 (1993); Aharoni et al., PROC. NATL. ACAD. SCI. USA
10821 (1997). [0192] Human T cell clones were derived from
peripheral blood mononuclear cells according Teitelbaum at al., 89
PROC. NATL. ACAD. SCI. USA 137 (1992). Approximately
5.times.10.sup.6 cells were incubated in each well of a 24-well
culture plate with Copolymer 1 or MBP (50 .mu.g/ml) in culture
medium supplemented with 10% heat-inactivated autologous serum.
After 7 days of culture, the cells were transferred to culture
medium containing 10% fetal calf serum and recombinant human
interleukin 2 (20 units/ml). The cells were grown continuously in
this medium with periodic exposure to antigen presented on
irradiated (3000 rad), autologous mononuclear cells, every 14-18
days. The C-52 T-cell clone was derived from a DR7, w11 donor, also
according to Teitelbaum at al., 89 PROC. NATL. ACAD. SCI. USA 137
(1992).
Proliferation Assay
[0193] Cells of T lines or clones were tested for their specific
proliferative response 10-21 days after antigenic stimulation. T
Cells (1.5.times.10.sup.4) were cultured in triplicates with
5.times.10.sup.6 irradiated spleen cells or with human
EBV-transformed B cells (5.times.10.sup.4), and with the indicated
antigens in a final volume of 0.2 ml in 10% FCS culture medium. At
the end of 48 hr incubation, cultures were pulsed with 1
.mu.Ci[.sup.3H]-thymidine and then harvested 6-12 hr later. The
variations of triplicates from their mean were under 20%.
cell line cross-reacted only with TAL. No cell lines cross reacted
with GTL or with AGT (1:1:1) which has the same amino acids as TGA
but in a different mole fraction than TGA or Copolymer 1.
TABLE-US-00010 TABLE 6 Cross reactivity of Terpolymers with
Copolymer 1 for Copolymer 1 specific T-cell lines and clones I.
Murine T-cell lines and clones Cross reactivity with Copolymer 1
(%) T-cells LN-3 S-3 LN-1 S-22-5 Polypeptide prol*
IL-4.sup..dagger-dbl. prol* IL-4.sup..dagger-dbl. prol*
IL-4.sup..dagger-dbl. prol* IL-4.sup..dagger-dbl. SD-1689 - 130 139
49 11 0 1 0 0 GAL SD-1690 - 3 6 102 107 0 1 0 0 TGA SD-1691 - 3 7 2
3 64 120 0 0 TAL SD-1697 - 2 4 12 6 0 1 0 0 GTL 1:1:1 AGT 2 0 2 2 1
0 2 0T II. Human T-cell Clones Cross reactivity with Copolymer 1
(%) T-cells C-14 C-52 Polypeptide proliferation proliferation
SD-1689 - GAL 4 75 SD-1690 - TGA 1 58 SD-1691 - TAL 0 0 SD-1697 -
GTL 0 5 *prol = as measured by proliferation. .sup..dagger-dbl.IL-4
= as measured by Interleukin-4.
Example 8
Terpolymers Cross-React with Anti-Copolymer 1 Antibodies
[0194] Antibodies directed against Copolymer 1 cross-react with
Terpolymers which lack either tyrosine, glutamic acid or alanine.
However, Terpolymers lacking lysine are not recognized efficiently
by anti-Copolymer 1 antibodies.
Antibodies
[0195] Mouse anti-MBP and anti-Copolymer 1 monoclonal antibodies
were obtained by fusion of MBP- or Copolymer 1-immunized spleen
cells from SJL/J mice, with the NSW murine plasmacytoma cell line.
Teitelbaum of al., Proc. Natl. Acad. Sci. USA 9528 (1991).
Radioimmunoassay
[0196] Flexible plastic microtiter plates were coated with
Copolymer 1 or Terpolymers (2 .mu.g/ml). After 16 hr incubation at
room temperature, plates were washed three times and saturated for
2 hr with PBS containing 2% bovine serum albumin, 0.05% Tween 20,
0.1% sodium azide, 10 mM EDTA, and heparin at 5 units/mil ("PBS
buffer"). Monoclonal antibody supernatants (50 .mu.l), were added
to the wells for a 2 hr incubation, and the wells were washed again
with PBS buffer. .sup.125I-labeled goat anti-mouse Fab antibodies
(1.times.10.sup.5 cpm/well) were added for overnight incubation at
4.degree. C. After extensive washing, radioactivity is measured in
a gamma counter.
Methods
ELISA Assay for Antibody Cross-Reactivity
[0197] A standard ELISA assay is employed using anti-Copolymer 1
polyclonal antibodies and microtiter plates coated with 2 .mu.g/ml
of terpolymer preparation.
Copolymer 1
[0198] Copolymer 1 with the following amino acid composition is
obtained from Teva Pharmaceutical Industries (Petach Tikva,
Israel).
TABLE-US-00011 Amino Acid Molar Fraction L-glutamic acid 0.141
L-alanine 0.427 L-tyrosine 0.095 L-lysine 0.338
Terpolymers
[0199] The four Terpolymers of Example 1 were used.
Results
[0200] Table 7 indicated that anti-Copolymer 1 polyclonal
antibodies cross react with Terpolymers which lack either tyrosine,
glutamic acid or alanine. The relatively high percentage binding of
the terpolymers lacking glutamic acid (TAL) might be explained by
its high average molecular weight. Terpolymers which lack lysine
are not efficiently recognized by anti-Copolymer 1 antibodies.
These data suggest that charged amino acids like lysine may play a
role in the recognition and binding of Copolymer 1 and
Terpolymers.
TABLE-US-00012 TABLE 7 Percent Binding in separate experiments
Terpolymers MW* 1 2 3 3 Mean S.D. TAL 20,000 111.1 130.1 115.8
114.0 117.8 8.48 GAL 8,850 7.5 10.8 9.3 9.2 9.2 1.35 GTL 11,050
98.7 87.0 n.d..sup..dagger-dbl. n.d..sup..dagger-dbl. 92.9 8.30 TGA
7,600 87.9 79.3 n.d..sup..dagger-dbl. n.d..sup..dagger-dbl. 83.6
6.12 *daltons. .sup..dagger-dbl.n.d.--not determined.
Cross Reactivity of Terpolymers with Copolymer 1-Reactive
Monoclonal Antibodies
[0201] The cross reactivity of the Terpolymers with Copolymer 1 at
the level of B cell response is tested using monoclonal antibodies
(mAbs), that are either reactive with both Copolymer 1 and MBP
(mAbs 2-2-18 and 3-1-45), or are reactive with only Copolymer I
(mAbs 3-3-9 and 5-7-2). See Teitelbaum et al., 88 Proc. Natl. Acad.
Sci. USA 9528 (1991).
[0202] Table 8 illustrates that the Terpolymers differed in their
ability to bind these mAbs. TGA and GTL were not recognized by any
of the Copolymer 1 specific mAbs. On the other hand, TAL and GAL
bound to Copolymer 1 and MBP specific mAbs with an affinity which
is similar to that of Copolymer 1. GAL and TAL differed only in the
binding to one mAb i.e. 5-7-2 which bound to TAL and not to
GAL.
TABLE-US-00013 TABLE 8 Cross reactivity of Terpolymers with MBP-
and Copolymer 1-reactive B cell antibodies Antibody Cross
reactivity with Copolymer 1 (%) Polypeptide 2-2-18 3-1-45 3-3-9
5-7-2 SD-1689 - GAL 96 98 107 10 SD-1690 - TGA 3 2 1 10 SD-1691 -
TAL 96 98 103 106 SD-1697 - GTL 1 2 1 1 2-2-18 is anti mouse MBP
monoclonal antibody cross reactive with Copolymer 1 3-1-45 is anti
Copolymer 1 monoclonal antibody cross reactive with MBP 3-3-9 is
anti Copolymer 1 monoclonal antibody non cross reactive with MBP
5-7-2 is anti Copolymer 1 monoclonal antibody non cross reactive
with MBP
Example 9
Copolymer 1 and Terpolymers Compete with Collagen for Binding to
Human Leukocyte Antigens and Inhibit Collagen-Specific T-Cell
Response
[0203] This example illustrates that Copolymer 1, TAL, GAL, GTL,
and TGA compete for binding to MHC proteins with the rheumatoid
arthritis-associated immunodominant collagen antigen, CII 261-273
(SEQ ID NO: 3).
Methods
Protein Expression and Purification
[0204] Recombinant HLA-DR1 and HLA-DR4 molecules (encoded by
DRA/DRB I *0101 and *0401, respectively) were expressed in
Drosophila S2 cells as described in Stern, L. et al. 68 CELL 465
(1992) and Dessen, A. et al. 7 IMMUNITY 473 (1997). Cells were
grown in roller bottles at 26.degree. C. in Excell 401 medium
(Sigma, St. Louis, Mo.) supplemented with 0-5% fetal bovine serum
(Sigma). Cells were induced by addition of CuSO.sub.4 to 1 mM final
concentration, then incubated an additional 4-5 days.
Immunoaffinity purification of recombinant HLA-DR1 and HLA-DR4 is
performed as previously reported by Stern, L. et al. 68 CELL 465
(1992) and Dessen, A. et al. 7 IMMUNITY 473 (1997). Supernatant
from harvested cells is sequentially passed through Protein A,
Protein G and Protein A-LB3.1 columns, followed by elution of the
bound HLA-DR with 50 mM 3cyclohexylamino-1-propane sulfonic acid
(CAPS), pH 11.5, and neutralized with 200 mm phosphate (pH 6.0).
The eluate is concentrated on a Centriprep 10 membrane (Amicon).
Protein concentrations were determined by bicinchoninic acid assay
(Pierce Chemical Co.).
Polypeptide Labeling
[0205] The present polypeptides and the HA 306-318 peptide were
biotinylated as in Examples 4 and 7.
Class II MHC Protein Binding Assay
[0206] In this assay, water-soluble recombinantly-produced proteins
were incubated with biotinylated polypeptides of the present
invention and varying quantities of unlabeled competitor
polypeptides, collagen CII peptides or influenza virus HA peptides.
Assays were performed in 96-well microliter immunoassay plates
(PRO-BOND.TM., Falcon) which were coated with affinity-purified
LB3.1 monoclonal antibodies. Antibody coating is performed by
placing 100 .mu.l of 10.0 .mu.g/ml LB3.1 monoclonal antibodies in
each well and incubating at 4.degree. C. for 18 hrs. Microtiter
wells were then blocked with Tris buffered saline (TBS) containing
3% bovine serum albumin (BSA) for 1 hr at 37.degree. C. and washed
three times with TTBS. Before sample addition, 50 .mu.l of TBS
containing 1% BSA is added to each well. Phosphate buffered saline
(PBS) is 150 mM sodium chloride, 7.5 mM sodium phosphate dibasic,
2.5 mM sodium phosphate monobasic, pH 7.2. Tris buffered saline
(TBS) is 137 mM sodium chloride, 25 mM IRIS pH 8.0, 2.7 mM
potassium chloride. TTBS is TBS with 0.05% Tween-2G. Other
solutions used in this assay are described in Fridkis-Hareli, M. et
al., 160 J. IMMUNOL. 4386 (1998).
[0207] Binding analysis is performed by incubating the water
soluble DR molecules with biotinylated polypeptides of the present
invention and varying concentrations of unlabeled inhibitors
(Copolymer 1, TAL, GAL, GTL, TGA, Collagen CII 261-273 peptide or
HA 306-318 peptide). The collagen type CII peptide 261-273, has SEQ
ID NO: 3 (AGFKGEQGPKGEP) and a molecular weight of 1516. The
concentration of DR employed is 0.15 .mu.M. The final concentration
of biotinylated Copolymer 1 or terpolymers is 1.5 .mu.M. Incubation
is for 40 hr at 37.degree. C. in 50 .mu.l binding buffer at pH
5.0.
[0208] Bound label is detected using streptavidin-conjugated
alkaline phosphatase as follows. Plates were washed three times
with TTBS and incubated with 100 .mu.l of streptavidin-conjugated
alkaline phosphatase (1:3000, BioRad, Richmond, Va.) for 1 hr at
37.degree. C., followed by addition of p-nitrophenyl phosphate in
triethanolamine buffer (BioRad). The absorbency at 410 nm is
monitored by a microplate reader (model MR4000, Dynatech,
Chantilly, Va.).
T Cell Hybridoma and Antigen Presenting Cell (APC) Binding
Assays
[0209] Copolymer 1 and Terpolymers were tested to ascertain if they
could inhibit activation of T cells responsive to the collagen CII
peptide. Mouse DR1-restricted 3.19 and 19.3 T cell hybridomas and
mouse DR4-restricted 3838 and 03 T cell hybridomas were used.
Rosloniec, E. F., et al., 185 J. EXP. MED. 1113-1122 (1997);
Andersson, E. C., et al., PROC. NATL. ACAD. Sci. USA (1998).
Antigen presenting cells (APCs) were L cells transfected with DR1
(the L57.23 cell line provided by Rosloniec, E. F., et al., 185 J.
Exp. Med. 1113-1122 (1997)), L cells transfected with DR4, and
Priess cells (DRB1*0401/DR84*0101). T cell stimulation experiments
were performed in 96-well microliter plates in a total volume of
0.2 ml. Irradiated (3000-rad) APC (2.5.times.10.sup.4/well) were
coincubated with CII 261-273 (40 .mu.g/ml) and varying
concentrations of the present polypeptides for 2 hr at 37.degree.
C., then T cells (5.times.10.sup.4/well) were added and the
incubation is continued for 24 hr at 37.degree. C. Supernatants (30
.mu.l) were removed and incubated with IL-2 dependent CTL-L
(5.times.10.sup.4/well) for 12 hr, followed by labeling with
.sup.3H-thymidine (1 .mu.Ci/well) for 12 hr. Plates were harvested
and the radioactivity is monitored using a 1450 microbeta Plus
liquid scintillation counter (Wallac, Gaithersburg, Md.).
Results
Class II MHC Protein Binding Assay
[0210] The recombinant water-soluble HLA-DR1 and -DR4 proteins
produced in insect cells were largely free of bound autoantigens or
other peptides. Hence, data obtained from insect cell produced
proteins can more accurately indicate the actual binding affinities
for polypeptides. Fridkis-Hareli et al., 160 J. IMMUNOL. 4386
(1998) (the entire contents of which are hereby incorporated herein
by reference).
[0211] Competitive binding of each of the Copolymer 1 and
Terpolymers to HLA-DR1, HLA-DR2 and HLA-DR4 molecules is depicted
in FIGS. 5(A and B). Binding of each of the Copolymer 1 (YEAK, top
panel FIG. 5A) and Terpolymers is substantially greater than that
of the CII 261-273 (SEQ ID NO: 3) peptide, as judged by quantity of
CII 261-273 (SEQ ID NO: 3) peptide required for 50% inhibition. As
also observed above, TAL bound HLA-DR1 and HLA-DR4 with greater
affinity than did Copolymer 1. The kinetics of inhibition by
unlabeled TAL (YAK, bottom panel FIG. 5A) polypeptides were also
somewhat superior to that of the influenza virus peptide HA 306-318
(SEQ ID NO: 2). However, the influenza virus peptide HA 306-318
inhibited the binding of Copolymer 1 and of Terpolymers more
efficiently than the CII 261-273 peptide.
T Cell Hybridoma and Antigen Presenting Cell Binding Assay
Results
[0212] Copolymer 1 and Terpolymers also inhibited DR1-restricted T
cell activation by CII collagen peptide (FIG. 6). T cell activation
was detected by observing IL-2 production by
DR1-restricted-CII-specific T cell hybridomas. Collagen peptide CII
261-273 (SEQ ID NO: 3) at varying concentrations was coincubated
with one of the present polypeptides and then T cells (clone 3.19
or 19.3 as indicated) were added, and the mixtures were further
incubated. The supernatants from these incubated cells are removed,
and were assayed for IL-2 by observing whether the supernatant
induced proliferation of IL-2-dependent cytotoxic T lymphocytes
(CTL-L). The extent of inhibition by TAL is shown as solid circles
( ), by TGA as solid triangles (.DELTA.), by GTL as open triangles
(.DELTA.), and by Copolymer 1 as solid squares (.box-solid.).
Percent inhibition of CTL-L proliferation shown on the ordinate was
calculated using equation 1.
[0213] Again, TAL is the most potent inhibitor. However, GTL and
Copolymer 1 were also potent inhibitors of T cell activation by the
CII collagen peptide. TGA inhibited activation less efficiently.
Similar results were obtained with other batches of Copolymer 1 and
Terpolymers.
[0214] A similar inhibition of activation of DR4-restricted T cells
was observed, as shown in FIGS. 7(A and B). IL-2 production was
used to assess activation of DR4-restricted CII-specific T cell
hybridomas (3838 and D3). The presence of different polypeptides
inhibited IL-2 production, indicating that they inhibited
DR4-restricted T cell activation. FIG. 7A shows the effects of
coincubating irradiated 3838 or D3 Priess cells with collagen
peptide CII 261-273 (SEQ ID NO: 3) at the fixed concentration of 40
.mu.g/ml, and with varying concentrations of polypeptides, for 2 hr
at 37.degree. C. FIG. 7B shows the effects of incubating L cells
transfected with a gene encoding HLA-DR4 with collagen peptide CII
261-273 (SEQ ID NO: 3) at the fixed concentration of 40 .mu.g/ml,
and with varying concentrations of GAL, TAL, GTL, TGA, and
Copolymer 1, for 2 hr at 37.degree. C. T cells were then added
(clones 3838 or D3 as indicated), and samples were further
incubated for 24 hr at 37.degree. C. Supernatants (30 .mu.l) were
then removed, and were assayed for activation by IL-2-induced
proliferation of IL-2-dependent cytotoxic T lymphocytes (CTL-L).
Each polypeptide mixture was tested in duplicate. The concentration
of the present polypeptides is indicated on the abscissa. The
extent of inhibition by TAL is shown as solid circles ( ), by TGA
as solid triangles (.tangle-solidup.), by GTL as open triangles
(.DELTA.), and by Copolymer 1 as solid squares (.box-solid.).
Percent inhibition of CTL-L proliferation shown on the ordinate was
calculated using equation 1.
Example 10
Copolymer 1 Inhibits Activation of T Cells Responsive to a
Myasthenia Gravis Antigenic Peptide
Methods
Copolymer 1
[0215] Copolymer 1 was obtained from Teva Pharmaceutical Industries
(Petach Tikva, Israel).
[0216] Myasthenia Gravis-Related Peptides were synthesized on an
Applied Biosystems Peptide Synthesizer using solid phase
techniques. Barany et al., THE PEPTIDES 1 (1979). Peptides were
purified by reversed-phase HPLC. The peptides used were the p259
peptide, see Zisman et al., Hum. Immuol. 1995 November;
44(3):121-30; Brocke et al. Immunology 1990 April;
69(4):495-500.
IL-2 Secretion
[0217] Secretion of IL-2 by the cell line WCB AB in response to the
myasthenia gravis peptides and/or Copolymer 1 were evaluated. Cells
(1.5.times.10.sup.4) were incubated with the indicated antigen.
Secretion of IL-2 by the cell line WCB AB was used as a measure of
activation of that T cell line.
Results
[0218] Table 9 indicates that the p259 peptide stimulates T cell
secretion. However, when Copolymer 1 is incubated with the p259
peptide, T cell secretion of IL-2 is inhibited in a dose-related
fashion. At 100 .mu.M Copolymer 1 inhibits about 91% of IL-2
secretion (Table 10), indicating that Copolymer 1 is a potent
inhibitor of T cell activation.
TABLE-US-00014 TABLE 9 IL-2 secretion from WCB AB line in response
to p259 p 259 conc. Avg OD IL-2 (.mu.M) (450 nm) SD % CV (pg/ml) 0
0.077 0.01 0 0.25 0.135 0.00 24 0.5 0.227 0.01 3.9 72 1 0.387 0.01
2.9 159 2 0.725 0.02 2.4 347
TABLE-US-00015 TABLE 10 IL-2 secretion from WCB AB line in response
to Cop1 alone or 2 .mu.M p259 + Cop1 Cop1 Avg OD IL-2 (.mu.M) (450
nm) SD % CV (pg/ml) % Inhibition 0 0.725 0.02 2.4 347 1 0.086 0.01
0 2 0.079 0.00 0 10 0.086 0.00 0 20 0.266 0.01 8.0 93 73 60 0.166
0.01 40 88 100 0.151 0.01 11.2 32 91 Absorbance is the average of 3
samples. Confidence values were calculated relative to the
absorbance of a blank (% CV = SD/(Avg - blank)*100).
Sequence CWU 1
1
3119PRTArtificial Sequencesynthetic peptide (MBP residues 84-102)
1Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg1 5
10 15Thr Pro Pro213PRTArtificial Sequencesynthetic peptide (HA
residues 306-318) 2Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala
Thr1 5 10 313PRTArtificial Sequencesynthetic peptide (CII amino
acids 261-273) 3Ala Gly Phe Lys Gly Glu Gln Gly Pro Lys Gly Glu
Pro1 5 10
* * * * *