Note: Descriptions are shown in the official language in which they were submitted.
W095130435 218 ~ 9 9 ~ F~,l/ow~. '
COMPOSITIONS AND TREATMENT FOR MULTIPLE SCLEROSIS
R~ of th.~ ~nvenrit)n
disedses are a significant human health problem and are
relatively poorly l ' ' As there is no microbial or viral culprit apparently
directly l~-r ' '~, prevention, treatment and diagnosis of such disedses must bebased on the etiology of the disease. This invariably involves a complex series of
reactions of l. ~n~ metdbolic ' ' ~ , structural r ', cells and so
forth. Implicit however in the nature of an condition is the notion that at
least one: 'i_ must be involved in creating t~he sequence of events that resultsin the symptoms. ~ ~ d~ 1;l.d~o diseases such as multiple sclerosis are
no exception.
A commonly used animal model for human multiple sclerosis is c~.
allergic ~ ,Lti~ (EAE), a '' ~. ' _ disease of the central nervous
system which can be induced in susceptible strains of mice by with
myelin basic protein (Mi3P) or its - ' T cell 1- (i.e. T cell
epitopes). MBP is one of the presumed 'i in multiple sclerosis (MS) and
hasbeency;~y~ ~r 'inbothhuman(Otaetal.,Nature,346:183-187(1990))and
rodent (Zamvil et al., Nature, 324:258-260 (1986)) systems. Ota d al., supra have
identified a major T cdl epitope recognized by MS patients, MBP amino acid residues
84-102. Minor epitopes (MBP amino acid residues 143-168, 61-82, 124-142 and 31-
50 recognized by T cells from MS patients were also described. Zamvil et al., s~pra
have shown that MBP amino acid residues 1-11 cont~in the major T cell epitope(s)causing EAE, in susceptible rodent strains.
Over the past decade, it has become apparent that peptides Cu~ to
the major T cell epitopes of native protem antigens can be used to mduce T cell non
~C~IJUl~ i!. to themselves or to the native protein antigen. Studies by Gaur et al.,
Science, 2~8:1491-1494 have shown that T cell epitopes of MBP induce reduccd T
cell ~ ,ul~ to MBP m adult mice. In these studies, tolerance to synthetic
peptides CUII. -r ' ,, to the major ~ J ' ' ~' ' ' ' of MBP (Ac 1-11
and 35-47) or the entire MBP by using an emulsion of the peptide or protein in non-
O - form (incomplete Freund's adjuvant) as described previously for
tolerdnce induction to other proteins or peptides (F. Ria et al., Nature, 343:381
(1990)). This reduced T cell l~ u.. ~ which was induced by the ~ inn
I
wo 95/30435 ~ 9 ~ Y~
of MBP T cell epitopes was able to prevent MBP-induced EAE. However, these
studies showed that Ac 1-11, the more dominant of the two epitopes, induced onlyG~ I a 50% decrease in subsequent T cell response to MBP, and the less
peptide aa 35-47 induced only .~ 'y a 20% decrease in
5 subsequent response to MBP. The mixture of the two peptides together provided a
higher decrease in subsequent T cell response to MBP.
In a completely different approach to the treatment of MS, clinical studies
have shown that interferons (IFNs) have shown some promise for treating MS. It has
been shown that ' of IFN-~ promotes ~ ... of MS, whereas
0 I~V ' ' ' IFN-~ has been shown in controlled clinical trials to suppress them
(Hillel and Bever, J. N~ fy, 46:155-164 (1993)). However, despite
evidence that _ ' of IFN-,B to patients afflicted with MS declines the
frequency and severity of attacks of MS, IFN-,B is not the long awaited cure for MS
that the medical community had hoped for (Arnason, Neurology, 43:641-643 (1993)).
15 In the clinical study described by Amason, almost all patients treated with IFN-~ had
attacks eventually~ and one in four
S m ~Iy of ~hr T
The present invention provides compositions and methods for treating multiple
20 sclerosis. t`~ of the invention comprise at least one peptide or modified
peptide of myelin basic protein (MBP) which comprise at least one T cell epitope, and
~7N-,~ in an appropriate ~ ly acceptable medium for either oral,
or ill(l~ llVU;~ ' ' ' ' " Methods of the invention include
treatment of individuals who either have MS or are suspected of being susceptible to
25 MSby ' ~ai' l 'lyeffectiveamountofac.~...l..- ~;....ofthe
invention or by _ ~ in a therapeutic regimen, a . comprising at
least one peptide or modified peptide of MBP which comprises at least one T cellepitope and further ' E. IFN-~.
30 RriPf ~Pttrtri,rti~n of ~hP Drawin,~c
Flg. la is a graphic depiction of an I-YrPtrimPnt showing the effects of ~N-
~on EAE in two groups of 10 (SJL x TLP) Fl adult female mice who were induced
EAE with guinea pig MBP in complete adjuvant plus pertussis toxin on day 0 and
35 were adll.~ d either incomplete Freund's Adjuvant only (control) or were treated
with 2000 units of IFN-,B ~ -'Iy (i.p.) on days 9, 12, 16, and 20 (indicated
by arrows on the x axis), the Y axis represents the mean clinical score (MCS) for each
~ W095130435 21~ 9 9 ~ G = ~ "
group, 0=no clinical signs of EAE, I=limp, L~llc:~ull~ , tail, 2=partial hindlimb
paralysis, ~-~,vlll~lct~ hindlimb paralysis, 4=partial to complete forelimb paralysis
and .- '
S Fig. lb is a graphic depiction of an CA~ showing the effects of MBP
peptide Ac 1-11 in two groups of 10 (SlL x TLP) Fl adult female mice who were
induced EAE with guinea pig MBP in complete adjuvant plus pertussis toxin on day 0
and were a ' ~ either PBS (control) or were treated with 250 nmol Ac 1-11
V~.'y on days lû, 13, 17, and 21 (indicated by arrows on the x aYis), the Y
aAis represents the average mean clinical score for each group as described for Fig. Ia.
Fig. Ic is a graphic depiction of an l~ showing the effects of MBP
peptide Ac 1~ ' ' ' c~ in ~ n with IFN-,~ on EAE in two groups of 10
(SJL x TLP) Fl adult female mice who were induced EAE with guinea pig MBP in
complete adjuvant plus pertussis toxin on day 0 and were: ' ' cd either PBS
(control) or were treated with 250 nmol Ac 1-11 ~I~L~ ,llv~ on days 10, 13, 17, and
21 (indicated by open arrows on the x axis), and were treated with 2000 units of IFN-
,~ , "y (i.p.) on days 9, 12, 16, and 20 (indicated by closed arrows on the x
aAis), the Y-axis indicates the mean clinical scorc as discussed for Fig. Ia.
Fig. 2 is a graphic depiction of an PYr~lin~rnt showing the effects of various
doses of IFN-,I~ (10,000 units and 2,000 units lc~J~L;~,ly) on induced EAE in two
groups of 10 (SJL x TLP) Fl adult female mice who were induced EAE with guinea
pig MBP plus pertussis toxin on day 0 and were r ' ' ' ' cd either PBS (control) or
25 were treated with 10,000 units and 2000 units ~c~ .,l) of IFN-~ t~ , " 'ly
(i.p.) on days 9,13,16, (indicated by closed arrows on the x axis), the Y-axis
indicates the mean clinical score as discussed for Fig. Ia.
Fig. 3 shows the full length amino acid sequence of human M~3P.
Fig. 4 shows the arnino acids sequences for selected MBP peptides useful in
the practice of the instant invention.
' Descr~ption of th, l
The present invention provides methods for treating multiple sclerosis
35 comprising ' ' ' ~ a Ih~,l I lly effective amount of at least one antigenic
reptide of MBP in a treatment regimen which includes a ~ y effective
amount of IFN-,B. As used herein the term "peptide" refers to an amino acid sequence
wo 9513043s 2 1 8 9 q 9 0 ~ ' --
having fewer amino acid residues than the entire amino acid sequence of the protein
from which the peptide was derived. The term "antigenic peptide" as used herein
refers to any peptide comprising at least one T cell epitope or any portion of such
peptide comprising at least one T cell epitope.
S In referring to an epitope, the epitope will be the basic element, or smallest
unit of recoglution by a receptor~ ~alL; 'y ~=' 'L . ' , h ~ y
antigens and T cell receptors, where the epitope comprises amino acids of the native
protein such as the ,, which are essential to receptor O T cell
epitopes are believed to be mvolved in initiation and ~ ,tUdliU.. of the
10 response. These T cell epitopes are thought to trigger early events at the level of the T
helper cell by being presented by an dl~ ul HLA molecule on the surface of an
antigen presenting cell, thereby . ' ~ the T cell b~ r ' with the relevant
T cell receptor for the epitope. These events lead to T cell ~lulif~ iu.., 1~ . ' '
secretion, local rl y reaction, IC~,I Ui~ of additional immune cells to the
15 site of antigen/T cell interaction and activation of the B cell cascade leading to the
production of antibodies.
Exposure of a subject to a peptide or protein which comprises at least one T
cell epitope of the ~ may modify T cell, ~l .~,.,1...l~ ;. . - such that they
become, . ~, to the ,, and do not participate m stimulatmg an
2û immune response. In addition,: ' of a protein or peptide which complises
at least one T cell epitope may modify the 1~ JhJI~ilA, secretion profile as compared
with exposure to the naturally occurring ,, (e.g. result m a decrease or IL-4
and/or increase in IL-2 causmg a mn~1ifir~tinn of THI and TH2 I r ' )-
r, ~, exposure to such a peptide may influence T cell ' r r ' which
25 normally participate in the response to the -- ~ ,. such that these T cells are
drawn away from the sites of normal exposure to the ~i~ (e.g. tissues of the
central nervous system (CNS)) to the sites of therapeutic a.l ' . -I . ,.1 ;nn of the peptide
derived therefrom. This 1l ' ' of T cell ~ ,u. - ~ , - - rnay ameliorate or
reduce the ability of the illd;V ' ' 1.~ immune system to stimulate the usual immune
30 response at the site of normal exposure to the _ ~i,, resulting in diminution of
symptoms.
~ .ny peptides derived from MBP which moderate response of a subject to
MBP ~ may be included in the methods and ~,<I..IIJU:~;LiU~ of the invention.
Such peptides may be identified, for example, by examining the structure and
35 selec~ing appropriate regions to be produced as peptides (via ~, ' expressionsystems, ~ ,dlly or otherwise) to be examined for ability to influence T cell
~ W0 95130435 2 1 8 9 9 9 ~
responses to MBP, and selecting peptides containing epitopes recognized by thesecells. Many human MBP peptides comprising T cell epitopes are described in the art.
Modified antigenic peptides are useful within the scope of the invention. For
example, a peptide can be modified so that it maintains the ability to induce T cell
S anergy and bind MHC protein without the ability to induce a strong proliferative
response or possibly, any ,ulul;f~ Live response when ~ 1 "; ~ d in ~, -
form. In this instance, critical binding residues for the T cell receptor can bedetermined using known techniques (e.g. ~ ' of each residue and
1' of the presence or absence of T cell reactivity). Those residues shown
10 to be essential to interact with the T cell receptor can be modified by replacing the
essential amino acid with another, preferably similar amino acid residue (a
Cu..~ v~i~ .) whose presence is shown to enhance, diminish but not
eliminate or not effect T ceU reactivity. In addition, those amino acid residues which
are not essential for T cell receptor interaction can be modified by being replaced by
another amino acid whose i lCul~)ul~Liull may enhance, diminish but not eliminate, or
not effect T cell reactivity. In addition, those amino acid residues which are not
essential for T cell receptor interaction can be modified by being replaced by another
amino acid whose 1 may enhance diminish or not effect T cell reactivity
but does not eliminate binding to relevant MHC.
Additionally, peptides of the invention can be modified by replacing an amino
acid shown to be essential to interact with MHC protein complex with another
preferably similar amino acid residue (cu..~,_. v c~liv~ ' ) whose presence is
shown to enhance, diminish but not eliminate or not effect T cell activity. In addition,
amino acid residues which are not essential for interaction with the MHC protein25 complex can be modified by being replaced by another amino acid whose
il~Cul~ul~iu~ may enhance, not effect, or diminish but not eliminate T cell reactivity.
Preferred atnino acid . ' for nu.. ~ ' arnino acids include but are not
limited to ' with alanine, glutamic acid, or a methyl amino acid.
Another example of a n f~rlifi~rion of peptides is ~ ' of cysteine
30 residues preferably with serine, threonine, leucine or glutamic acid to minimize
,1:, . ,. .;, ~ ;~ ~1I via disulfide linkages. In addition peptides may be modified to increase
the solubility of a peptide for use in buffered aqueous solutions such as
,UI~ y acceptable carriers or diluents by adding functional groups to the
peptide, terminal portions of the peptide, or by not including hydrophobic T cell
35 epitopes or regions containing l-ydlu~ ùbic epitopes in the peptides or llydlu~ ubil,
regions of the protein or peptide. For example, to increase solubility, charged amino
acids or charged amino acid pairs or triplets may be added to the carboxy or amino
WO 95130435 I ~
2189990
terminus of the peptide. Examples of charged amino acids include argil:une (R),
Iysine (K), histidine (H), glutarnic acid (E~ nd àspartic acid (D).
Additionally peptides comprising "cryptic epitopes" may be determined and
are also useful in the methods and compositions of the invention. Cryptic epitopes are
those ,1~ in a protein antigen which, due to processing and I of
the native protein antigen to the appropriate MHC molecule, are not normally
revealed to the immune system. However, a peptide comprising a cryptic epitope is
capable of causing T cells to become non-responsive, and when a subject is primed
with the peptide, T cells obtained from the subject will proliferate in vitro in response
to the peptide or the protein antigen from which the peptide is derived. Peptides
which comprise at least one cryptic epitope derived from a protein antigen are referred
to herein as "cryptic peptides". To confirm the presence of crvptic epitopes a T cell
,Ul Ulif~ liu.l assay may be used as is known in the art in which antigen primed T cells
are cultured in vitro in the presence of each peptide separately to establish peptide-
reactive T ceU lines. A peptide is considered to comprise at least one cryptic epitope
if a T cell line can be established with a given peptide and T cells are capable of
~ulif~ liu~ upon challenge with the peptide and the protein antigen from which the
peptide is derived.
Antigenic peptides useful within in the ~ and methods of the
present invention include the following peptides or portions thereof having residue
numbers which correspond the amino acid residues of the human MBP protem shown
in Fig. 3: a peptide comprising all or a portion of amino acid residues 84-106 of
human MBP, a peptide comprising all or a portion of amino acid residues 84-102 of
human MBP, a peptide comprising all or a portion of amino acid residues 89-101 of
human MBP, a peptide comprising all or a portion of amino acid residues 140-172 of
human MBP, a peptide comprising all or a portion of amino acid residues 143-168 of
human MBP, a peptide comprising all or a portion of amino acid residues 142-167 of
human MBP and a peptide comprising all or a portion of arrlino acids residues 13-25
of human MBP. Sequences of selected peptides are shown in Figure 4. Any of these30 peptides may be modified as described or may extend upstream omlufrom
their carboxy or amino termini so long as they maintain their antigenic quality of
comprising at least one T cell epitope.
As a result of the work described herein it has been discovered that a
cnnlhin~ion of an antigenic peptide derived from MBP and IFN-~, when
P ' I in a therapeutic regimen, has a synergistic effect (Fig. Ic) which
, ~ diminishes the clinical symptoms of EAE in mice to a far greater extent
than the effect of each on mitigating the symptoms of EAE when ~' ' alon~
WO 95130435 2
(Figs. 1 a-b), and which is greater than what one would expect for a merely additive
effect of the peptide plus IFN-~.
As EAE serves as a mouse model of human MS and is induced by MBP, it is
expected that a similar effect would also be seen in humans. Therefore, the present
5 invention provides a method of treating individuals who have multiple sclerosis or are
susceptible to developing multiple sclerosis, which comprises A~ an
effective amount of an antigenic peptide derived from MBP in non- ~,
form ~I.e. without adjuvant) in a therapeutic regimen which also includes the
- ' of IFN-I~.
~.' of a cnmrnC;tinn cr~n~rricin~ at least one antigenic peptide of
MBP in a therapeutic regimen which includes r ' ' ' ' " of lFN-~ can be carried
out using known procedures at dosages and for periods of time to effectively reduce,
eliminate or prevent the symptoms associates with multiple sclerosis. Effective
amounts of either antigenic peptide or IFN-~ when a.l." ~r .. ~I together in a
15 therapeutic regimen va~y according to factors such as the degree of sensitivity and
5llcn-r~ihili~y of the individual to MS, the age, sex, and weight of the individual, and
the ability of the antigenic MBP peptide to elicit an antigenic response in the
individual. The active C~ ' (i.e. the MBP peptide or, . thereof and
IFN-~) may be A ' ' ' ~li in a convenient manner such as by injection
20 ( ' ~,..V~,O etc.), oral A~' ' ' ' '' , inhalation, i
application or rectal: ' Depending on the route of ' the
active compound may be coated with a material to protect the compound from the
action of enzymes, acids and other natural conditions which may inactivate the
compound.
For example, preferably about 1 1lg-3mg and more preferably about 20-500 ~Lg
of antigenic peptide derived from MBP per dosage unit may be: ' ' by
injection. Preferably, a dosage unit of 100-10,000 units of IFN-~ may be
~ by injection. The dosage regimen of these two: . ' may be
adjusted to provide the optimum therapeutic response. For exAmple, IFN-~ and a
C~ q~ comprising antigenic peptide derived from MBP may be: '
' '.~/ or may preferably be r ' ' ' ' at least six hours apart, preferably
at least 12 hours apart, or more preferably at least 24 hours aparL The therapeutic
regimen of A~' ' ' ' ' g, both antigenic peptide and ~N-~ may continue over a
period of days or weeks and may be reduced or extended as indicated by the
exigencies of the therapeutic situation.
The present invention also provides a novel rC~mrnci~inn comprising a
physical mixture of an antigenic peptide derived from MBP and IFN-,~ in a
21 s~ggO - :
""~., .,.;, ~lly acceptable carrier or diluent. This ~ mr--citinn may be used as part of a
therapeutic regimen for treating or preventing multiple sclerosis in an individual.
This inveMion is further illustrated by the following non-limiting example.
EXAMPLE
Svnthesis of mouse MBP Deptide Ac l-l 1
Mouse MBP peptide Ac 1-11 was synthesized using standard Fmoc/tBoc synthesis andpurified by HPLC.
~n(llu~tion of EAF
EAE was induced in 6 to 8 week old female (SJL x PL)FI mice (Jackson Labs, Bar
Harbor, ME) by immunizing mice with 100 ~g purified guinea pig MBP in CFA (GIBCOLab., Grand Island, NY) containing 400 ILg H37RA strain M. tuberculosis (DIFCO Lab.,
Detroit, MI) ,-,~ at the base of the tail. 200 ng Pertussis Toxin (JHL
BIOSCENCE, Lenexa, Kansas) was given twice illil~ .lU~I~' (i.V.) on the day of
"~ ", and also 48 hours later. Mice were monitored daily for disease symptoms and
were scored for disease severity on the following scale 0=no clinical signs of EAE, l=limp,
UIIIC~ ;VC tail, 2=partial hindlimb paralysis, 3=complete hindlimb paralysis, 4=partial to
complete forelimb paralysis and 5=moribund. Data are expressed as the mean of the disease
severity score on each day including all the animals in the group. Mice were followed for 26
days. Once a mouse died of EAE, a score of 5 was mcluded in calculations for all subsequeM
days,
Effect of IFN-,B on EAF
In a titration experimeM for the purposes of determining the effects of various dosages
of IFN-~3 on EAE (Fig. 2). One group of mice was treatea ~ ~ r ' 'ly with PBS on days
9, 13, and 16 (control) after EAE induction, one group of mice were treated with 10,000 units
of IFN-,~ on days 9, 13, and 16 (open circle) and one group of mice were treated with 2,000
units of IFN-,~ on days 9, 13, and 16 (closed circle). As shown in Fig. 2, the symptoms of
EAE are similar at each time point for both dosages of IFN-,~, thereby indicating that the lower
dosage would be suitable for experimeMs with IFN-,~ and is a preferred dosage as the chances
of toxicity due to a higher dose of IFN-~ are less likely. 2000 units of IFN-~ were then used
in the remaining I~Yr^rim.~n~ shown in Figs. la-lc as this dosage showed il~ ,llL in
clinical score.
AMEND0 SHE~T
WO 9513043~ 1 Ir~
2~ ~g~go
As shown in Fig la a control group of mice was treated with PBS ar,d another
group of mice were treated with 2000 units of lFN-~ i.p. on days 9, 12, 16, and 20.
As shown in Fig. Ia, the group treated with IFN-~ only had slightly less severe
symptoms during the time course as those of the control group.
F,ffrrt of mn~lcp ~RP rPrtirlP Ac 1-11 OD E~F
The effect of mouse MBP peptide Ac 1-11 was determined and the results are
shown in Fig. Ib. One group of mice was treated i.p. with PBS on days 10, 13, 17,
and 21 (control) after EAE mduction, and one group of mice was treated i.v. with 250
nmol of peptide Ac 1-11 on days 10, 13, 17, and 21. The mice were monitored as
described above. As shown in Fig lb, the mice treated with Ac 1-11 had less severe
symptoms than those of the control group.
F,ffP~t of trp ~fmr~nf with a ~ of pe~tide ~Ar 1~ on F~AF~
Theeffectsoftreatmentwithac~mhin~finn of peptideAc 1-11 and~N-~ are
shown in Fig. lc. One group of mice was treated i.p. with PBS (control) after EAE
induction, and one group of mice was treated i.v. with 250 nmol of peptide Ac 1-11
on days 10, 13, 17 and 21 (open arrows) and treated i.p. with 2000 units of IFN-,B on
days 9, 12, 16, and 20. As shown in Fig. lc, the group of mice treated with a
20 ~ of peptide and ~7N-,~ showed a marked decrease in the severity of
symptoms as compared with the control group as well as compared to treatment with
either IFN-~ alone or peptide alone as shown in Figs. la and Ib indicating a
synergistic effect of the, ' Therefore, a treatment regimen which includes
a .. ,.1.;. ~ ." of peptide and IFN-,B provides an enhanced effect on ~ the
25 severity of the symptoms of EAE.
Although the invention has been described with reference to its preferred
L~IIIJO~' ', other: b~ ' can achieve the same results. Variations and
to the present invention will be obvious to those skilled in the art and is
intended to cover in the appended claims all such mnr~ifir~tinnc and I ~ ' that
30 follow in the true spirit and scope of the invention.
