Note: Descriptions are shown in the official language in which they were submitted.
CA 0220~680 1997-0~-16
CLIP INMUNONODULATORY ~ lV:
Field of the Invention
This invention relates to a peptide, CLIP, which is
demonstrated to be an immunodulatory peptide and can be
used as a composition for the treatment of immune disease.
In particular, the CLIP peptide down regulates the activity
of T-cells and also influences expression and function of
class II MHC molecules.
Background of the Invention
The class II major histocompatibility complex (MHC)
expressed by B-cells, macrophages, dendritic cells, and
thymic epithelial cells is a heterodimeric molecule with
two closely related chains. These two chains, the a and ~,
are involved in binding peptides by virtue of highly
polymorphic residues found at the N-terminal domains (1).
The class II MHC functions by binding self-peptides in the
major groove and guiding the selection of CD4+ cells in
order to initiate immune responses.
After synthesis, the class II MHC molecule associates
with the invariant chain (li) which serves to prevent
premature binding (2). The li undergoes degradation which
leaves only the peptide portion (aa residues 85-101), also
called CLIP, in contact with the a/b complex (3,4). The
CLIP fragment has also been demonstrated to prevent peptide
binding (5,6) and is, therefore, assumed to be tightly
bound to the binding groove of the MHC. It is necessary to
remove CLIP from the binding groove in order for peptide
antigens to bind to the MHC. The removal of CLIP from the
binding groove is a task facilitated by HLA-DM.
HLA-DM plays a vital role in the removal of CLIP and
allowing antigens to bind to the core binding groove of the
class II MHC. In the absence of HLA-DM, CLIP prevents the
binding of antigens in the binding groove and their
subsequent presentation on the cell surface (7). We now
show that increasing the amount of CLIP saturates the
endosome and inhibits the ability of antigen to bind. We
CA 0220~680 1997-0~-16
also now report that altering the expression of CLIP inside
the cell will clearly affect the ability of peptides to
bind the MHC. We have now more clearly defined the
function of CLIP as a regulator of the density of peptides
being presented at the surface of the APC (antigen
presenting cells). We also demonstrate that CLIP levels
modulate the activity of CD4+ T-cells and the subsets of T-
cells generated. We therefore propose that CLIP can be
used as an immunomodulatory protein in general and more
specifically it can be used to treat immune disorders
involving increased T-cell activation and inappropriate
sensitization of the immunological defences of the body
resulting in self-destruction and typically known as
autoimmune disorders. The peptide can also be used for
transplantation and for combatting infection.
Summary of the Invention
The present inventors have now characterized the
function of the CLIP peptide and its effects on
immunological function. The identification of the role of
the CLIP peptide permits the development of therapeutic
strategies in order to combat autoimmune disorders as well
as for use in tissue transplantation and for treating
infection.
The present invention relates to an isolated CLIP
peptide which can be used and administered as a therapeutic
composition for the treatment of disorders involving the
immune system.
In accordance with one embodiment the present
invention is a therapeutic composition containing the CLIP
peptide, or an active analogue or fragment thereof in a
composition suitable for oral or parenteral delivery for
treatment of immune disorders. The composition may be
delivered in a suitable vehicle, microencapsulated or
provided in a liposome and targeted to a specific site with
or without a carrier. The composition may also be
CA 0220~680 1998-08-17
administered in order to minimize or prevent tissue graft
rejection after transplantation as well as for treating
those infections where the immune response is
overcompensating and thus creating more tissue damage.
Other features and advantages of the present invention
will become apparent from the following detailed
description. It should be understood, however, that the
detailed description and the specific examples while
indicating preferred embodiments of the invention are given
by way of illustration only, since various changes and
modifications within the spirit and scope of the invention
will become apparent to those skilled in the art from this
detailed description.
Brief Description of the Drawings
The invention will now be described in relation to the
drawings in which:
Figure 1 shows the amino acid sequence of the CLIP
peptide from humans (SEQ ID NO:2) and mice (SEQ ID NO:1).
Figure 2 shows the effect of mouse CLIP peptide on the
expression of cell surface class II MHC in TA3 cells. A)
Staining of TA3 cells with mAb MK-D6 (anti-I-Ad) or 10-2.16
(anti-I-Alc) after 24 h of incubation with various
concentrations of CLIP. A dose-dependent decrease in
surface class II MHC is apparent in both instances. B)
Staining as described above for I-Ad after incubating TA3
cells with HEL or OVA for 24 h. No effect on l-Ad surface
expression is seen with HEL, which is predominantly l-Ak
restricted, while OVA significantly up-regulated l-Ad
expression. In each case, background staining was
determined using only secondary Ab (FITC-goat anti-mouse
IgG).
Figure 3 shows the effect of CLIP peptide incubation
with TA3 cells on the intracellular formation of stable,
SDS-resistant, compact MHC class II a/b chain heterodimers.
Cells were incubated for 24 h in the presence of various
CA 0220S680 1997-0~-16
concentrations of CLIP. Cell lysates were prepared and
loaded onto a 10~ SDS-PAGE gel with or without heating
(95OC, 5 min) and analyzed by Western blotting using the l-
Ad binding mAb, MK-D6. The gels were analyzed by
densitometry, and the data are presented as the percent
decrease in the compact state of l-Ad (non-boiled samples)
with respect to total intracellular l-Ad, shown as a
function of the dose of CLIP. Note that at higher CLIP
concentrations (200 ~g/ml), saturation of the system is
reached.
Figure 4 shows the binding of immunogenic peptides and
CLIP to surface class II MHC molecules. A) CLIP-incubated
cells (left) fluoresce weakly, whereas OVA-incubated cells
(right) fluoresce strongly over time indicating binding.
B) Time course of the binding of FITC-CLIP and FITC-OVA
from A), demonstrating a leveling off or plateauing of
peptide surface presentation over time in the CLIP group,
but not in the OVA peptide group. TA3 cells were incubated
in the presence of FITC-OVA-(323-339) or FITC-CLIP at 100
~g/ml. After various times, cells were washed and analyzed
by cells surface FACS. FITC-peptides were made as
described in the examples such that only one FITC group was
attached at the N-terminal of the peptide molecule.
Figure 5 shows the effect of immunization with CLIP
peptide (85-101) on the T cell response. A) Proliferation
of BALB/c-derived lymph node cells from mice immunized as
described and challenged according to the abscissa. B)
Proliferation of C3H-derived lymph node cells as described
above without the KlA2 group. C) Proliferation of BALB/c-
derived lymph node cells after enriching for APCs andmixing with nylon wool purified T cells as described. D)
Dose-response curves of the down-regulation of PPD-specific
T cell responses by CLIP. Shown is the proliferation of
BALB/c-derived cells from mice immunized as described and
challenged by using different concentrations of PPD
(micrograms per millileter) according to the abscissa. E)
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Down-regulation of OVA-specific T cell responses by CLIP.
For the above experiments, mice were immunized with Ag in
CFA or IFA using 100 ~g/footpad of CLIP and 50 ~g/footpad
of other Ags. On day 10, T cell proliferation was measured
to determine the recall response to specific Ags. Assays
were performed in the presence of priming Ag in culture
medium using 100 ~g/ml of CLIP, 50 ~g/ml of other Ags, and
40 ~g/ml of PPD unless otherwise indicated.
Figure 6 shows the effect of immunization with CLIP
peptide (85-101) on MHC class II on lymph node APCs. A)
Staining with RA3.3A1 demonstrates that immunized CLIP has
no significant effect on B220 (CD45) surface expression,
whereas in B) staining with MK-D6 shows substantial down-
regulation of surface l-Ad. In A) and B), BALB/c mice were
immunized in the hind footpads with CFA and CLIP (100
~g/footpad) using CFA and saline as a control. On day 10,
lymph node cells were harvested, stained, and analyzed by
FACS as described in the examples.
Figure 7 shows the effect of immunization with CLIP on
the proliferative response to peptide antigens.
Figure 8 shows the effect of immunization with CLIP on
the generation of TH1 and TH2 cells. A) IL-4 is assayed as
a result of CLIP immunization. B) IFN-~ is assayed as a
result of CLIP immunization.
Figure 9 shows the effect of CLIP on the antibody
response to K3, K4 and KlA2. BALB/c mice were immunized
with 50 ~g of CLIP along with either K3, K4 or KlA2 in IFA
and compared to BALB/c mice immunized without CLIP. After
two weeks a similar injection was given i.p. in IFA serum
was collected and levels of IgG1 and IgG2a antibodies were
determined as described in the examples.
Figure 10 shows the effect of CLIP on the
proliferative response of whole ovalbumin. Mice were
immunized with either ovalalbumin or ovalalbumin-CLIP with
ovalalbumin-KlA2 as the control. Lymph nodes were
CA 0220~680 1997-0~-16
harvested after 10 days, cells were cultured with 50 ~g/ml
of peptide and proliferation was assayed as described in
the examples.
Figure 11 shows the effect of CLIP on the generation
of TH1/TH2 cells in response to ovalalbumin. T-cells were
incubated with ovalalbumin and the supernatants assayed for
IL-4 and IFN-~.
Detailed Description of the Preferred Embodiments
The present invention relates to the use of a CLIP
peptide as an immunomodulatory protein which can be
isolated and used to treat immune disorders involving
increased T-cell activation, such as autoimmune disorders
as well as for tissue transplantation and for combatting
infection.
"CLIP peptide" means the portion of the MHC class II
invariant chain protein which occupies the MHC II groove
and includes human invariant chain amino acids 81 to 104
and homologous stretches of the amino acid sequence of the
MHC class II invariant chain protein of other species.
The CLIP peptide can be administered to decrease
antigen presentation to the class II MHC. Furthermore,
CLIP also is shown to increase the production of IL-4 and
decrease the production of IFN-~. The CLIP peptide is also
useful for shifting the immune T cell response from a TH1
inflammatory response to a TH2 protective response which
further supports its use as a therapeutic agent for the
treatment of immune disorders.
Also included within the scope of the invention is the
use of active fragments or analogues of a CLIP protein and
of polypeptides which include the amino acid sequence of a
CLIP peptide or fragments thereof.
By "activity" is meant any function regulated or
modulated by CLIP peptide.
In preferred embodiments, the activity is down-
regulation of the surface expression of MHC class II
CA 0220~680 1997-0~-16
molecules on the surface of antigen presenting cells and/or
the resulting reduction or prevention of a T cell response
to an antigen.
An "active fragment" or "active analogue" of MHC class
II-associated in variant chain protein or of CLIP peptide
is a fragment or analogue which retains a function
regulated or modulated by CLIP peptide.
Active analogues of human CLIP peptide include CLIP
peptides from other species. For example, murine CLIP
(Figure 1) is active in human cell systems.
One of ordinary skill in the art can readily screen
fragments or analogues of CLIP for activity by the assays
described herein. Such assays may include adding fragments
or derived analogues of CLIP to an appropriate cell culture
such as BALB/c cells which express surface I-Ad and I-Ak.
Cells can then be appropriately stained to quantitate
surface expression of I-Ad and I-Ak by FACS analysis in
order to determine any down-regulation of I-Ad or I-Ak. The
ability of CLIP fragments and/or analogues to affect
cytokine production such as IL-2 or IFN-~ can also be
assayed using lymph node cells cultured from mice immunized
with the desired fragments and/or analogues. Supernatants
from such cultures are then collected and assayed for
different cytokine concentrations.
It is also possible to assay for the ability of the
CLIP fragments and analogues to bind to cell surface class
II MHC using FACS analysis. Finally, T-cell responses in
lymph node cells from immunized BALB/c mice can be assayed
as a result of using a CLIP fragment or analogue for
immunization.
All of the assay systems are described herein in the
examples and provide a means for identifying those
fragments and analogues of CLIP which can be used as a
pharmaceutical therapeutic and immunogenic composition.
CA 0220~680 1997-0~-16
The Role of CLIP on Modulating Antigen Presentation and
Class II Expression
Considerable evidence has recently been obtained
supporting the idea that CLIP (4,5') binds in the Ag
binding groove of class II molecules (7, 14, 16, 17'). No
functional evidence exists for the in vivo role of CLIP
despite the speculation that CLIP could accumulate on class
II molecules in cells that lack HLA-DM proteins and, in
turn, block the binding of antigenic peptide to class II
MHC molecules (18'). It is now discovered that the
saturation of the endosomal compartment with exogenously
added CLIP would result in reduced surface expression of
class II molecules and would inhibit Ag presentation to
CD4+ T cells. This provides direct evidence for its
functional role and suggests novel ways to modify Ag
presentation by class II MHC molecules. More broadly
stated, the present invention now shows the immunologic
role of the CLIP peptide.
The CLIP peptide of the MHC class II pathway is able
to impede efficient Ag presentation by APCs in vivo. In
vitro, TA3-B cell hybridomas incubated with exogenous CLIP
internalize the peptide where it has a dramatic effect on
achieving compact, stable, class II heterodimers and this
is reflected as a decrease in surface class II MHC. In
vivo, administration of CLIP indirectly impedes T cell
priming, presumably by reducing the quantity of other
exogenous peptides available for presentation by APCs.
For the in vitro studies, a B cell hybridoma, TA3 was
used which is a good APC (12'). Using the I-Ad-restricted
Ag, OVA we have found that the addition of CLIP peptide
decreased the surface expression of I-Ad and I-Ak. This is
consistent with the observation that CLIP has >1000-fold
higher affinity for I-Ad than I-Ak (14'). The observed
down-regulation of surface class II MHC could be the result
of altered conformation-dependent class II epitopes, as
have been previously reported in HLA-DM mutant cell lines
CA 0220~680 1997-0~-16
(18,19') rather than of CLIP per se. These mutant cell
lines also have a large number of their class II molecules
occupied by CLIP (18'). Significantly, however, this
phenomenon is only seen in cells with defective or deleted
DM proteins. We have shown that H-2M is probably not
defective in the TA3 cell line in two significant ways.
TA3 subclones, although differing in constitutive levels of
surface I-Ad, retain relatively constant levels of H-2 M.
Also, defects in the HLA-DM protein in cell lines have been
shown to be correlated with an inability to efficiently
present native protein (19,20'). When comparing a high I-
Ad-expressing subclone of TA3 with a low I-Ad-expressing
subclone, we found no significant difference in the ability
to present either native protein or peptides (data not
shown).
It is unlikely that the CLIP-class II complexes are
recognized inefficiently by the Abs used because the MHC
down-regulation is found using Abs that target two
different MHC haplotypes (i.e. I-Ad and I-Ak) and the
Western blot analysis revealed that MK-D6 Ab binds to
stable as well as unstable heterodimers which substantiates
that MK-DK does not bind in a conformation-specific manner.
Also, this data is consistent with data showing that both
10-2.16 (anti-I-Abk) and MK-DK (anti-I-Abd). Abs
efficiently immunoprecipitate compact and SDS-unstable
dimers (15') and FACS analysis performed by others (21')
has shown that while other Abs are sensitive to
conformational changes induced by the presence or the
absence of Ii, MK-D6 remains insensitive, suggesting that
MK-D6 binds independently of conformations induced by the
peptides in the class II binding site.
The in vivo CLIP data illustrated a similar effect.
Mice immunized with CLIP had a reduction in the expression
of class II molecules on the APCs. These results support
the idea that exogenously added CLIP is taken up by the
APCs and this results in the inhibition of antigenic
CA 0220~680 1997-0~-16
peptides loading onto the MHC class II molecules in the
endosomal compartment. It is possible that the observed
reduction in class II MHC on BALB/c and C3H cells was the
result of a lack of T cell activation in the presence of
CLIP rather than a direct effect of CLIP blocking.
Decreased T cell activation has been observed in animals
immunized with CLIP and this may in turn, result in
decreased levels of MHC-up-regulating cytokines (22').
Although CLIP could apparently inhibit Ag presentation in
vivo, this effect was not apparent when cells were
subsequently challenged in vitro. It is possible that T
cell priming is more sensitive to adequate ligand density
via MHC on APCs. Should this not occur, T cells may be
tolerized rather than primed or activated. Considering
this, if APCs are presenting fewer ligands of peptide Ag
due to the presence of CLIP, a decrease in vivo priming
events should be seen. This would be consistent with
studies in which mice expressing low levels of surface
class II MHC show drastically impeded T cell responses
(23').
The observation that immunogens are capable of up-
regulating class II MHC is further evidence that
exogenously added Ags influence class II surface expression
(12). It is likely that immunogenic peptides stabilize
class II heterodimers, which leads to long lived surface
complexes.
Because of the apparent correlation between dose and
time of exposure of cells to CLIP, it is presumable that
CLIP is exerting a role in down-regulating the surface
expression of class II MHC. Further support for this comes
from Western blot analysis that demonstrates an increase in
the intracellular floppy state of MHC class II molecules
compared with that of non-CLIP incubated cells. This is
significant because it has been reported that MHC molecules
bound strictly to CLIP peptide do not achieve the compact
SDS-stable state (7').
CA 0220~680 1997-0~-16
Defective Ag presentation has been shown to be linked
to the inability of HLA-DM molecules (the human counterpart
to murine H-2M molecules) to remove invariant peptides and
that once functional DM molecules are transfected to these
cells, normal Ag presentation resumes (18'). The
observations may be linked to the function of the H-2M
accessory protein, which is believed to play a vital role
in the endocytic pathway (6, 24') similar to that of HLA-
DM. By incubating APCs with CLIP, we have in effect
artificially enhanced its presence in the endocytic
pathway. Here, we believe that the compartment of peptide
loading (CPL or CIIV) is saturated with CLIP and H-2M is
unable to perform adequately in removing the peptide
occurring as a result of self-processing activity. Because
of this, CLIP remains bound with MHC molecules and prevents
them from having their binding sites occupied by exogenous
peptides. For this to be true, it must be demonstrated
that exogenous CLIP is in fact being internalized. The
preliminary confocal microscopy results (data not shown)
revealed that CLI-P is in fact internalized over time and
colocalizes intracellularly with a vesicle that is rich in
class II MHC and is proximal to the cell surface. Also,
incubating TA3 cells with FITC-CLIP or FITC-OVA-(323-339)
demonstrated that OVA peptide binds strongly and
immediately to surface class II molecules, whereas CLIP
does not. Moreover, these observations do not appear to be
brought about by the ability of CLIP to down-regulate class
II MHC, since down-regulation is not apparent until after
4h of culture. These observations lead to a strong
indication that exogenous CLIP is being internalized along
the endocytic pathway. It has also been shown that class
II peptide complexes once formed are virtually irreversible
(25'). Given this, it is difficult to rationalize
exogenous CLIP exerting its effect in any other way than by
interfering at the stage where APC is endogenously loading
the processed Ag onto class II molecules, since the
CA 0220~680 1997-0~-16
mycobacterial components ln CFA require processing before
presentation. Therefore, other than associating with class
II MHC, as is the case with OVA-(323-339) and other
immunogenic peptides, CLIP is most likely interfering
endogenously, perhaps with H-2M, which prevents the export
of functional class II-immunogenic peptide complexes to the
cell surface.
The functional effect of CLIP on T cell responses is
further supported by its role in Ag presentation. We found
that CLIP inhibited the T cell response when injected
simultaneously with Ag. This suggests that either CLIP
prevents presentation of Ag to T cells or blocks T cell
function. Cell mixing experiments supported the functional
role of CLIP in blocking Ag presentation by the APCs. When
T cells and APCs from mice immunized with CLIP plus CFA or
with CFA alone were mixed, we found that APCs from the
CLIP/CFRA-immunized mice inhibited the presentation of PPD
to T cells from the CFA alone group. Mixing T cells from
CLIP-immunized mice with APCs from saline-immunized mice
revealed a similarly reduced in vitro T cell response,
suggesting that T cells were inefficiently primed in vivo
due to inefficient presentation and low class II MHC on the
APCs (23').
The data in Figure 5A, where a different peptide of
similar size to CLIP is used in its place for immunization,
shows that in vivo T cell priming is impaired not as the
result of direct competition between CLIP and other
immunized Ag but rather as a result of the interaction
between CLIP and the endocytic pathway.
These results support the idea that the level of
intracellular CLIP is normally in an equilibrium that
affords the cell a balance between efficient peptide
presentation and maintaining the pool of class II MHC to
present antigenic peptide. When this equilibrium is
altered by exogenous CLIP, efficient peptide presentation
by MHC class II molecules is drastically impeded, which in
CA 0220~680 1997-0~-16
turn, down-regulates Ag-speclfic T cell responses.
Saturating quantities of CLIP peptide prevents adequate
peptide loading of exogenous antigenic peptide, which in
turn impedes the formation of the compact state of class II
heterodimers. The ultimate effect of reducing the number
of cell surface-stable class II heterodimers is a reduction
in the efficiency of APC Ag presentation. In support of
these results, studies in HLA-DMB mutants have demonstrated
that the inability to remove Ii peptides prevents the
formation of stable HLA-DR molecules (18'). These data
demonstrate the ability of CLIP, administered exogenously,
to down-regulate the immune response by blocking efficient
Ag presentation to T cells. Because of its ability to bind
to MHC class II a/~ heterodimers in the peptide loading
compartment, exogenously added CLIP inhibits the principal
function of the H-2M molecule in situ. These studies
confirm the intracellular role of CLIP in Ag presentation.
The Role of Clip On the Immune Response Induced on TH1 and
TH2 by Different Peptide Antigens
Also investigated was the effect of CLIP on the immune
response induced by different peptide antigens with
different affinities for MHC. The peptide K3 has a low
affinity and has been shown to induce a TH2 type of
response while KlA2 with a high affinity has been shown to
induce a TH1 type of response. The peptide K4 has an
intermediate affinity and induces both TH1 and TH2 types of
responses (8'). We now show that saturating the endosomal
compartment with exogenously added CLIP would down regulate
the antigen presentation which in turn may affect the
cytokine profile and perhaps completely alter the immune
response to a specific antigen.
When immunizing CLIP with the three peptide antigens a
variation in response to the peptides K3, K4 and KlA2 was
noted. The peptide KlA2 induced a strong proliferative
response whereas the peptide K3 generated only a weak
CA 0220~680 l997-0~-l6
14
proliferative response. The peptide K4, on the other hand,
produced a moderate response. This confirms earlier work
done with these peptides (8'). This variation in response
is a consequence of the fact that the peptides, though
cross reactive, have minor differences in their amino acid
structure at key residues resulting in different affinities
for I-Ad.
The inventor has shown that when K3, K4 and KlA2 were
injected along with CLIP, a significant decrease in the
proliferative response towards each of the peptides was
observed. Also observed was that K3 and K4 consistently
demonstrated a significant drop in response when immunized
with CLIP whereas KlA2 experienced a reduction ranging from
minimal to significant (data not shown). It is reasonable
to assume that CLIP, like the peptide antigens K3, K4 and
KlA2, becomes internalized and localized in the endosome.
As both CLIP and the peptides are able to bind to class II
MHC, they compete with each other for the privilege. As a
result, antigens experience a decreased rate of binding to
the MHC and a lower level of presentation. This results in
a lower ligand density and is, therefore, responsible for
the observed weaker immune response. The peptide KlA2, due
to its high affinity, was able to compete more effectively
with CLIP than the other peptides and therefore did not
experience as great a decline in the level of peptide
binding or presentation and in turn, did not show as
noticeable a decrease in response.
The degree to which CLIP is able to inhibit antigen
binding and subsequent antigen presentation is related to
the affinity of the antigen for I-Ad. High affinity
antigens appear to be effective at competing with CLIP and
therefore are still able to mount a significant response.
Lower affinity antigens appear to be less capable of doing
this and a greater drop in proliferative response is
observed.
CA 0220~680 1997-0~-16
It has been previously demonstrated that ligand
density, along with the type of APC and particular epitope
presented, is an important factor in determining whether a
Thl-like or a Th2-like response is generated (8', 17').
Important for our study, it has been shown that a high
ligand density is responsible for generating Thl cell
responses, where as Th2 cell responses result from a low
ligand density. If, as evidence indicates, CLIP is truly
altering the expression of peptides at the surface of APC's
and as a result is affecting T-cell activation and
proliferation then it would be expected that CLIP would
also affect cytokine profiles. This is, indeed, what we
found.
The notion that CLIP down regulates MHC expression is
supported by its ability to alter cytokine secretion. We
observed that CLIP causes greater amounts of IL-4 to be
released in mice immunized with either K3, K4, or KlA2.
This increase in IL-4 would correspond to an increase in
the relative amount of Th2 cells generated by the immune
response. Also observed, was the fact that CLIP induced a
decrease in the amount of IFN~ secreted, indicating there
was a drop proportion of Thl cells. All in all, CLIP
demonstrated an ability to shift the T-helper cell response
away from a Thl-like and towards a Th2-like.
The results from the antibody studies also reveal a
shift from a Thl towards a Th2 response. Immunization with
CLIP was observed to change the proportions of IgGl and
IgG2a antibodies. As a result of CLIP, antibody responses
to each peptide showed a significant increase in the
proportions of IgGl antibodies, as well as a decrease in
the proportion of IgGa antibodies. As IgGl antibodies are
associated with a Th2 response and IgG2a antibodies with a
Thl response (13'), this shows again that there is a switch
from Thl to Th2 cells.
The change in T-helper cell subsets is an important
aspect of a treatment with a CLIP-containing immunogenic
CA 0220~680 l997-0~-l6
16
composition. It alters the very nature of the immune
response as it does not stimulate antibody production
because the peptide is non-antigenic. Rather than being
inclined towards an inflammatory response (Thl), an immune
reaction can be directed towards a more protective response
(Th2).
Under normal situations, immune systems rarely see
peptide antigens as described above but rather encounter
protein antigens. Protein antigens require an important
additional step, in that it is necessary for them to
undergo processing before binding to class II MHC. The
inventor has shown that CLIP had a similar effect on the
ovalbumin as it did on the peptides K3, K4, and KlA2. It
caused a decrease in proliferative response. It also
induced an increase in the levels of Il-4 secretion and a
correlating decrease in IFN-~ levels. Apparently, there is
no difference in the effect of CLIP on peptides or protein.
The CLIP peptide has been demonstrated to be effective
at transforming the immune response. It is able to down
regulate the strength of the proliferative response without
eliminating it. CLIP is also able to alter the nature of
the T-helper subset populations and as a result change the
way in which the immune system responds to antigens. The
Th2 subset is important for the generation of humoral
immune responses whereas the Thl subset plays a critical
role in generating delayed-type hypersensitivity immune
responses. The Thl subsets are often found to also be
involved with autoimmune conditions. They play an
important role in causing inflammatory disorders as well.
A technique such as using CLIP, provides a means by which
to manipulate Thl subsets could prove useful in controlling
autoimmune disorders. CLIP may be an effective tool to
modulate responses and shift them away from a harmful,
disease causing Thl immune response towards a Th2 response
which often associated with protection.
Peptides
CA 0220~680 1997-0~-16
CLIP peptides or fragments thereof may be prepared by
any suitable peptide synthetic method.
Chemical synthesis may be employed, for example
standard solid phase peptide synthetic techniques may be
used. In standard solid phase peptide synthesis, peptides
of varying length can be prepared using commercially
available equipment. This equipment can be obtained from
Applied Biosystems (Foster City, CA.). The reaction
conditions in peptide synthesis are optimized to prevent
isomerization of stereochemical centres, to prevent side
reactions and to obtain high yields. The peptides are
synthesized using standard automated protocols, using t-
butoxycarbonyl-alpha-amino acids, and following the
manufacturer's instructions for blocking interfering
groups, protecting the amino acid to be reacted, coupling,
deprotecting and capping of unreacted residues. The solid
support is generally based on a polystyrene resin, the
resin acting both as a support for the growing peptide
chain, and as a protective group for the carboxy terminus.
Cleavage from the resin yields the free carboxylic acid.
Peptides are purified by HPLC techniques, for example on a
preparative C18 reverse phase column, using acetonitrile
gradients in 0.1% trifluoroacetic acid, followed by vacuum
drying.
CLIP peptides may also be produced by recombinant
synthesis. A DNA sequence encoding the desired peptide is
prepared, for example by cloning the required fragment from
the DNA sequence encoding the complete invariant chain
protein, obtainable from genomic DNA or from commercially
available genomic or cDNA libraries, and subcloning into an
expression plasmid DNA. Suitable mammalian expression
plasmids include pRC/CMV from Invitrogen Inc. The gene
construct is expressed in a suitable cell line, such as a
Cos or CHO cell line and the expressed peptide is extracted
and purified by conventional methods. Suitable methods for
recombinant synthesis of peptides are described in
CA 0220~680 1997-0~-16
"Molecular Cloning" (Sambrook, Fritsch and Maniatis, Cold
Spring Harbor Laboratory Press, 1989).
Analogues of CLIP peptides may be prepared by similar
synthetic methods. The term "analogue" extends to any
functional and/or chemical equivalent of a CLIP peptide and
includes peptides having one or more conservative amino
acid substitutions, peptides incorporating unnatural amino
acids and peptides having modified side chains.
Examples of side chain modifications contemplated by
the present invention include modification of amino groups
such as by reductive alkylation by reaction with an
aldehyde followed by reduction with NaBH4; amidation with
methylacetimidate; acetylation with acetic anhydride;
carbamylation of amino groups with cyanate;
trinitrobenzylation of amino groups with 2, 4, 6,
trinitrobenzene sulfonic acid (TNBS); alkylation of amino
groups with succinic anhydride and tetrahydrophthalic
anhydride; and pyridoxylation of lysine with pyridoxal-5'-
phosphate followed by reduction with NaBH4.
The guanidino group of arginine residues may be
modified by the formation of heterocyclic condensation
products with reagents such as 2, 3-butanedione,
phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide
activation via -acylisourea formation followed by
subsequent derivatisation, for example, to a corresponding
amide.
Sulfhydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide;
performic acid oxidation to cysteic acid; formation of
mixed disulphides with other thiol compounds; reaction with
maleimide, maleic anhydride or other substituted maleimide;
formation of mercurial derivatives using 4-
chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid,
phenylmercury chloride, 2-chloromercuric-4-nitrophenol and
CA 0220~680 1997-0~-16
19
other mercurials; carbamylation with cyanate at alkaline
pH.
Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the
indole ring with 2-hydroxy-5-nitrobenzyl bromide or
sulphonyl halides. Tyrosine residues may be altered by
nitration with tetranitromethane to form a 3-nitrotyrosine
derivative.
Modification of the imidazole ring of a histidine
residue may be accomplished by alkylation with iodacetic
acid derivatives of N-carbethoxylation with
diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not
limited to, use of norleucine, 4-amino butyric acid, 4-
amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic
acid-, t-butylglycine, norvaline, phenylglycine, ornithine,
sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-
thienyl alanine and/or D-isomers or amino acids.
Examples of conservative amino acid substitutions are
substitutions within the following five groups of amino
acids (amino acids are identified by the conventional
single letter code): Group 1: F Y W; Group 2: V L I; Group
3: H K R; Group 4: M S T P A G; Group 5: D E.
Also included in the present invention are CLIP
analogues such as those described in Gautam et al., (1995)
and Jensen (1996), the contents of which are incorporated
herein by reference. These studies examined the effect of
amino acid substitution on CLIP binding to the MHC groove
and described analogues retaining CLIP binding activity.
It is predicted that these analogues will be effective in
the methods of the present invention.
The discovery that the CLIP peptide is internalized
and decreases surface expression of class II MHC molecules
as well as inhibits antigen presentation to T cells and
decreases T cell function, clearly points to a therapeutic
CA 0220~680 l997-0~-l6
role for this peptide. In addition, the finding that CLIP
alters cytokine production and alters TH1 and TH2 responses
clearly indicates its role as a therapeutic composition for
those conditions involving inappropriate immune responses
5 and in particular autoimmune disorders such as lupus,
diabetes, rheumatoid arthritis just to name a few.
Autoimmune disorders result from a failure of the immune
system to discriminate between foreign antigen and self
antigens and can result in death.
In accordance with one embodiment of the invention,
CLIP peptide or an active fragment or analogue thereof is
administered to a mammal in need of treatment as a
pharmaceutical composition comprising a solution of the
peptide, and optionally including a pharmaceutically
15 acceptable carrier.
A pharmaceutical composition comprising a solution of
a CLIP peptide acts as an anti-inflammatory and leads to
down-regulation of the expression of MHC class II
molecules.
The composition may be administered in a safe and
effective amount to any living organism including humans
and animals. By safe and effective as used herein is meant
providing sufficient potency in order to decrease, prevent,
ameliorate or treat the immune condition affecting a
25 subject while avoiding serious side effects. Such a safe
and effective amount will vary depending on the age of the
subject, the physical condition of the subject being
treated, the severity of the immune condition, the duration
of treatment and the nature of any concurrent therapy.
Administration of a therapeutically active amount of the
pharmaceutical composition of the present invention is
defined as an amount effective, at dosages and for periods
of time necessary to achieve the desired result. This may
also vary according to factors such as the disease state,
35 age, sex, and weight of the subject, and the ability of the
CLIP peptide to elicit a desired response in the subject.
CA 0220~680 1997-0~-16
Dosage regimes may be adjusted to provide the optimum
therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally
reduced as indicated by the exigencies of the therapeutic
situation.
By pharmaceutical carrier as used herein is meant one
or more biologically compatible solid or liquid delivery
systems. By biologically compatible as used herein is
meant that the components of the composition are capable of
being commingled, without interacting in a manner that
would substantially decrease the pharmaceutical efficacy of
the total CLIP composition which includes the liposome
delivery system under ordinary use. Some examples of
compatible materials useful as pharmaceutical carriers are
sugars, starches, cellulose and its derivatives, powdered
tragacanth, malt, gelatin, collagen, talc, stearic acids,
magnesium stearate, calcium sulfate, vegetable oils,
polyols, agar, alginic acids, pyrogen-free water, isotonic
saline, phosphate buffer, and other suitable non-toxic
substances used in pharmaceutical formulations. Other
excipients such as wetting agents and lubricants, tableting
agents, stabilizers, anti-oxidants and preservatives are
also contemplated.
The peptide solution may be administered
therapeutically by injection or by oral, nasal, buccal,
rectal, vaginal, transdermal or ocular routes in a variety
of formulations, as is known to those in the art.
For oral administration, various techniques can be
used to improve peptide stability, based for example on
chemical modification, formulation and use of protease
inhibitors. Stability can be improved if synthetic amino
acids are used, such as peptoids or betidamino acids, or if
metabolically stable analogues are prepared.
Formulation may be, for example, in liposomes for
improved stability. Oral administration of peptides may be
accompanied by protease inhibitors such as aprotinin,
CA 0220~680 1997-0~-16
soybean trypsin inhibitor or FK-448, to provide protection
for the peptide. Suitable methods for preparation of oral
formulations of peptide drugs have been described, for
example, by Saffran et al., (1979) (use of trasylol
protease inhibitor); Lundin et al. (1986) and Vilhardt et
al., (1986).
The composition containing the CLIP peptide of the
present invention can also be administered in an solution
or emulsion contained within phospholipid vesicles called
liposomes. The liposomes may be unilamellar or
multilamellar and are formed of constituents selected from
phosphatidylcholine, dipalmitoylphosphatidylcholine,
cholesterol, phsphatidylethanolamine, phsophatidylserine,
demyristoylphosphatidylcholine and combinations thereof.
The multilamellar liposomes comprise multilamellar vesicles
of similar composition to unilamellar vesicles, but are
prepared so as to result in a plurality of compartments in
which the CLIP containing solution or emulsion is
entrapped. Additionally, other adjuvants and modifiers may
be included in the liposomal formulation such as
polyethyleneglycol, other peptides or other materials.
The liposomes containing the CLIP composition may also
have modifications such as having antibodies immobilized
onto the surface of the liposome in order to target their
delivery.
Due to the high surface area and extensive vascular
network, the nasal cavity provides a good site for
absorption of both lipophilic and hydrophilic drugs,
especially when coadministered with absorption enhancers.
The nasal absorption of peptide-based drugs can be improved
by using aminoboronic acid derivatives, amastatin, and
other enzyme inhibitors as absorption enhancers and by
using surfactants such as sodium glycolate, as described in
Amidon et al., (1994).
The transdermal route provides good control of
delivery and maintenance of the therapeutic level of drug
CA 0220~680 l997-0~-l6
over a prolonged period of time. A means of increasing
skin permeability is desirable, to provide for systemic
access of peptides. For example, iontophoresis can be used
as an active driving force for charged peptides or chemical
enhancers such as the nonionic surfactant n-decylmethyl
sulfoxide (NDMS) can be used.
Transdermal delivery of peptides is described in
Amidon et al. (1994) and Choi et al. (1990).
Peptides may also be conjugated with water soluble
polymers such as polyethylene glycol, dextran or albumin or
incorporated into drug delivery systems such as polymeric
matrices to increase plasma half-life.
More generally, formulations suitable for particular
modes of administration of peptides are described, for
example, in Remington's Pharmaceutical Sciences, latest
edition, Mack Publishing Company (Easton, PA.)
Immunogenic Compositions
In accordance with a further embodiment of the invention,
CLIP peptide or an active fragment or analogue thereof is
administered as an immunogenic composition to a mammal in need
of treatment.
Immunogenic compositions of CLIP peptide do not stimulate
any antibody response (B.J. Rider et al. (1996), Molec.
Immunol., v. 33, p. 625) but do modulate the response of the T
cell arm of the immune system to an antigen. In addition to
down-regulating surface expression of MHC class II molecules,
immunogenic compositions of CLIP cause a shift in the ratio of
THl:TH2 helper cells.
Immunogenic compositions of CLIP or an active fragment or
analogue thereof may be prepared by combining the selected
peptide with a suitable adjuvant.
Adjuvants may be employed which not only enhance but
selectively modulate the type of immune response to the
administered peptide; for example monophosphoryl lipid A (MPL)
favours a TH1 type response, while QS21 (Cambridge Biotech)
favours a cytotoxic T cell response.
CA 0220~680 l997-0~-l6
24
Aluminum hydroxide and aluminum phosphate (collectively
commonly referred to as alum) are adjuvants commonly used in
human and veterinary vaccines. Olive oil emulsions or other
human-approved emulsifying agents may also be used.
An adjuvant should be non-toxic, capable of stimulating a
sustained immune response and compatible with the peptide.
Immunogenic compositions containing proteins or peptides
are generally well known in the art, as exemplified by U.S.
Patents 4,601,903; 4,599,231; 4,599,230; and 4,596,792; all of
which references are incorporated herein by reference.
Immunogenic compositions may be prepared as injectables, as
liquid solutions or as emulsions. CLIP peptides or analogues
or fragments thereof may be mixed with pharmaceutically
acceptable excipients which are compatible with the peptides,
fragments or analogues. Such excipients may include water,
saline, dextrose, glycerol, ethanol, and combinations thereof.
The immunogenic compositions of the invention may further
contain auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, or adjuvants to enhance the
effectiveness of the vaccines.
Immunogenic compositions may be administered
parenterally, or by injection subcutaneously or
intramuscularly. Alternatively, the immunogenic compositions
formed according to the present invention may be formulated
and delivered in a manner to evoke an immune response at
mucosal surfaces. The oral, nasal, vaginal, gastrointestinal,
respiratory or other mucosal route of vaccine administration
may be preferred to combat infections which take place at
mucosal surfaces, for example in the respiratory, digestive or
urogenital tracts. Nasal immunization has been shown to be
efficacious in generating both respiratory tract mucosal
immunity and systemic immunity. Inhalation of an aerosol
formulation may also be used to combat lung or respiratory
tract infections.
Delivery systems for mucosal immunization include lipid
vesicles, biodegradable microcapsules, attenuated bacteria,
live viral vectors and bacterial toxins or subunits thereof.
CA 0220~680 l997-0~-l6
For examples, cholera toxin B subunit may be conjugated to an
antigen for improved mucosal immunization.
Alternatively, other modes of administration including
suppositories and oral formulations may be desirable. For
suppositories, binders and carriers may include, for example,
polyalkalene glycols or triglycerides. Oral formulations may
include normally employed excipients such as, for example,
pharmaceutical grades of saccharine, cellulose and magnesium
carbonate. Immunogenic compositions may take the form of
solutions, aerosols, suspensions, tablets, pills, capsules,
sustained release formulations or powders and may comprise 10-
95% of peptide 15 or peptide 42 or an analogue or fragment of
one of these peptides.
The immunogenic compositions are administered in a manner
compatible with the dosage formulation, and in such amount as
will be therapeutically effective and immunogenic. The
quantity of immunogenic composition to be administered depends
on the subject to be treated, including, for example, the
weight of the subject and the capacity of the subject's immune
system to produce a cell-mediated immune response. The dosage
may also depend on the route of administration. However,
suitable dosage ranges are readily determinable by one skilled
in the art and may be of the order of micrograms of the
peptides, analogues or fragments thereof.
Varying concentrations of CLIP may be used depending on
the affinity of the antigen involved.
Nucleic acid molecules encoding CLIP peptide or a
fragment or an analogue thereof may also be used for
immunization. For example, DNA in a plasmid vector may be
administered directly, in saline, by injection, preferably by
intramuscular injection, for genetic immunization. It is
believed that the DNA is expressed in vivo to give the encoded
peptide antigen which stimulates an immune response. DNA may
also be administered by constructing a live vector such as
Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus
including the DNA. Some live vectors that have been used to
carry heterologous antigens to the immune system are discussed
CA 0220~680 l998-08-l7
26
in, for example, O'Hagan (31). Processes for the direct
injection of DNA into subjects for genetic immunization are
described in, for example, Ulmer et al., (32).
The examples are described for the purposes of
illustration and are not intended to limit the scope of the
invention.
Methods of molecular genetics, protein and peptide
biochemistry and immunology referred to but not explicitly
described in this disclosure and examples are reported in
the scientific literature and are well known to those
skilled in the art.
EXAMPLES
Example 1
Materials and Methods
Mice
Female BALB/c (I-Ad) and C3H/HEI (I-Ak) mice, 7 to 12
wk of age, were purchased from The Jackson Laboratory (Bar
Harbor, ME).
Antigens
CFA, IFA, OVA, hen egg lyzozyme (HEL), and purified
protein derivative of Mycobacterium tuberculosis (PPD) used
for immunizations and in vitro were purchased from sigma
Chemical Co. (St. Louis, MO). Mouse CLIP-(85-101), KlA2,
and OVA-(323-339) peptides were synthesized in this
laboratory using an Applied Biosystem 431A peptide
synthesizer (Foster City, CA), as previously described (10,
11), according to the sequence KPVSQMRMATPLLMRPM (SEQ ID
NO:1) (CLIP), EYKEYAAYAEYAEYA (SEQ ID NO:3) (KlA2), or
ISQAVHAAHAEINEAGR (SEQ ID NO:4) (OVA). KlA2 has been
described previously (10) and shows an IC50 value of 0.27 ~M
for binding to purified class II I-Ad molecules (11).
Peptides were purified using reverse phase HPLC on a C18
analytical column (YMC, Kuse-gun, Kyoto, Japan), using a
linear gradient of water-acetonitrile (1.37%
acetonitrile/min). FITC-CLIP-(85-101) and FITC-OVA-(323-
339) were prepared using an N-hydroxysuccinamide-FITC (NHS,
CA 0220~680 1997-0~-16
Pierce Immunotechnology, Rockford, IL) ester llnkage
according to the following protocol. Five parts of NHS-
FITC were dissolved in N-methylpyrrolidone with one part of
peptide bound to solid support that still had side chain
groups protected, leaving only the N terminus free, thus
ensuring that only one FITC group can react per peptide
molecule. Two parts of DIEA (diisopropylethylamine) were
subsequently added. After 4 to 8 h, the reaction mixture
was filtered through a medium fritted glass funnel; cleaved
from supporting resin and protecting groups using a
cleavage mixture (Applied Biosystems) of crystalline
phenol, 1,2-ethanedithiol, thioanisole, deionized H2O, and
trifluoroacetic acid; and purified on a Sephadex G-25
column (Pharmacia Biotech, Quebec, Canada), and the FITC-
conjugated peptides were purified by HPLC as previously
described (10). Peptides used in this study were dissolved
at a concentration of 1 mg/ml in either physiologic saline
or medium and filtered through a 0.22-~m pore size filter
for sterilization.
K3 and K4, were prepared by the Merrifield solid-phase
technique on a Beckman 990C Peptide Synthesizer (Palo Alto,
CA) as previously described (14'). The crude preparations
were purified by HPLC on a C-18 reverse phase semi-
preparative SynChropak RP-P column (synchrom, Linden, IN),
using a linear gradient from water to acetonitrile (1.37~
acetronitrile/min). For functional assays, peptides were
dissolved in saline by adjusting pH to 7.2 with 0.1 N NaOH
and were sterilized by filtation through a 0.22 ~m filter.
Abbreviations used for amino acids: K, lysine; E, glutamic
acid; Y tyrosine; A, alanine. Purfied protein derivative
(PPD, Statens Seruminstifut Tuberculin Department
Copenhagen, Denmark) was used at a concentration of 40~g/ml
for a positive control.
An tibodies
CA 0220~680 1997-0~-16
28
mAb-producing hybridomas 10-2.15 (anti-I-A~ ), MK-D6
(Anti-I-A~d), and RA3.3A1 (anti-B220), used for Western
blotting and FACS analysis, were purchased from the
American Type Culture Collection (Rockville, MD).
Horseradish peroxidase-conjugated rabbit and anti-mouse IgG
Abs, used for Western blotting, were purchased from
Amersham Canada Ltd. (Oakville, Canada).
Cells and hybridomas
TA3 cells were obtained from Dr. L. Glimcher, Harvard
Medical School (Boston, MA). As previously described (12).
TA3 cell lines exhibit a reduction in surface I-Ad
expression when passaged in culture over time. In the high
expressing clones, I-Ad surface expression exceeds I-Ak,
whereas low expressing clones have greatly reduced I-Ad
compared to I-Ak. For these studies, an intermediate
expressing subclone of the TA3 cell line (TA3.11) that has
similar levels of I-Ad and I-Ak expression was used (12).
Immunization and T cell proliferation assay
Mice were immunized in the bind footpads with CLIP
peptide (100 ~g), KlA2 peptide (100 ~g), or OVA (50 ~g)
emulsified with CFA or IFA (Sigma Chemical Co., St. Louis,
MO) using a saline group as a control. After 10 days,
popliteal lymph nodes were excised, and a single cell
suspension was prepared. Cells from CFA/CLIP-immunized
mice and those from control mice were separated as follows.
Cell suspensions were incubated for 1 h at 37~C, and the
resulting nonadherent supernatant fraction was enriched for
T cells on a nylon wool column (13). Adherent macrophages
were pooled with column-bound B cells from CFA/CLIP-
immunized mice and in turn pooled with T cells enrichedfrom control mice. In other experiments, cell suspensions
were used without enrichment. Cells were cultured in 96-
well flat-bottom plates (Becton Dickinson Co., Rutherford,
NJ) at 2 x 105 cells/well in the presence or the absence of
either of both challenge peptides (100 ~g/ml for CLIP
CA 0220~680 l997-0~-l6
29
peptide, 40 ~g/ml of PPD unless otherwise indicated, and 50
~g/ml for OVA) in 200 ml of culture medium (RPMI 1640 (Life
Technologies, Grand Island, NY) supplemented with 10% FCS
(Bockneck, Canada), 10 mM HEPES, 2 mM L-glutamine, 5 x 10-5
5 M 2-ME, and 1 U/ml penicillin-streptomycin). After 3 days,
cultures were pulsed wlth 1 ~Ci/well of [3H]TdR (New England
Nuclear-DuPont, Boston, MA) for 16 to 20 h. Incorporation
of [3H]TdR was measured using a liquid scintillation counter
(LKB Instruments, Gaithersburg, MD). None of the peptides,
including CLIP, was toxic to the cells in culture over a
wide range of doses, as determined by trypan blue dye
exclusion.
In another experiment, BALB/c mice were immunlzed with
50~g of either K3, K4 or KlA2 peptide along with either
15 saline, CLIP or ovalbumin (323 - 339) peptide emulsified in
IFA ( Sigma Chemical Co.) in both the hind foot pads.
Another group of mice was immunized with 50 ~g of ovalbumin
protein (Sigma Chemical Co.) along with either saline,
CLIP, or KlA2. After ten days, popliteal lymph nodes were
20 removed and single cell suspension was prepared. The cells
were then cultured in 96-well flat bottom plates (Becton
Dickinson and CO., NJ) at 2x105 cells/well in the presence
or absence of the peptide (50~g/ml) in 200 ~l of culture
medium [RPMI 1640, (Gibco, Grand Island, NY) supplemented
25 with 10% FCS (Bockneck, Rexdale, ON, Canada), 10mM HEPES,
2mM L-glutamine, 5x105 M 2-mercaptoethanol and 10/ml
penicillin/streptomycin]. After 3 days, cultures were
pulsed with l~Ci/well of [3H]-thymidine (NEN Du Pont,
Boston, MA) for 16-20 h. Incorporation of [3H] thymidine
30 was measured using a liquid scintillation counter (LKB
Instruments, Gaithersburg, MD).
FACS analysis
FACS analysis was performed on both TA3 cells and
murine lymphocytes. For TA3 cells, 1 x 106 cells were
35 incubated with or without CLIP-(85-101) or OVA-(323-339)
- CA 0220~680 1997-0~-16
peptides at different times and concentrations. Cells were
also incubated with OVA or HEL proteins. Cells were then
stained for I-Ad and I-Ak surface expression. In other
experiment, cells were incubated with FITC-CLIP (100 ~g/ml)
or FITC-OVA-(323-339) peptide (100 ~g/ml) for various times
and then analyzed by flow cytometry (12) (Becton Dickinson,
CA). Live cells were gated based on propidium iodide
exclusion and side/forward laser scatter. Gated cells
(20,000e vents/sample) were subsequently analyzed using
LYSYS software (Becton Dickinson, Mountain View, CA).
For murine lymphocytes, lymph nodes from CLIP- or non-
CLIP-immunized mice (described below) were harvested, and a
single cell suspension was prepared. Cells were stained
for I-Ad (BALB/c) or I-Ak (C3H) and B220 surface expression
and subjected to flow cytometric FACScan analysis as
described above.
Western blot analysis
TA3.11 cells (2 x 106) were incubated with or without
varying concentrations of CLIP peptide (100, 200, and 500
~g/ml) for 24 h at 37~C. Cells were resuspended in lysis
buffer (1% Triton X-100, 1% BSA, 1 mM iodoacetamide, 0.2
U/ml aprotinin, lmM PMSF, 0.01 M Tris, 0.14 M NaCl, and
0.025% NaN3, pH 8.0) and incubated for 1 h at 4~C. Lysate
was cleared by microcentrifugation (10,000 x g. 30 min) and
added to an equal volume of 2 x SDS-sample buffer (25% 4 x
Tris-SDS (pH 6.8; 6% Tris and 0.4 g of SDS), 20% glycerol,
4% SDS, and 2% 2-ME). Samples were either boiled for 5 min
or not boiled and analyzed by electrophoresis by 10% SDS-
PAGE. Gels were blotted onto Immobilon paper (Millipore
30 Corp., Bedford, MA) and incubated with primary Ab MK-D6
(anti-I-A~d). Secondary horseradish peroxidase-conjugated
goat anti-mouse IgG (Amersham Canada) was subsequently
added to the washed blot and incubated. Washed blots were
developed using the enhanced chemiluminescence method
(Amersham Canada) and exposed to x-ray film (Dupont De
Nemours Co., Wilmington, DE).
CA 0220~680 1997-0~-16
Cyto~; n~ assays: To determine the cytokine production,
lymph node cells (2x106) form immunized mice were cultured
as described above in 24-well flat bottom plates (Corning
Glass Works, Coming, NY) in 2 ml medium in presence or
absence of the peptides K3, K4 or KlA2 50~g/ml).
Supernatents were collected at different time periods and
assayed for different cytokine contents. IL-2 contents
were assayed by culturing supernatent wisth CTLL cells for
20 h. Cultures were then pulsed with [3H]-thymidine
(l~Ci/well) and incorporation of [3H]-thymidine was measured
as described above. Recombinant IL-2 (20U/ml)
(Collaborative Biomedical Products, Bedford, MA) was used
as positive control. IL-4 was assayed using IL-4 dependent
CT4.S cells (kindly supplied by Dr. B. Chan, Robarts
Research Institute, London, Ont.) CT4.S cells were
cultured with supermatant for 30 h. [3H]-thymidine
(l~Ci/well) was added and cultured for additional 18 h.
Incorporation of [3H]-thymidine was assayed as described
above. IL-4 containing suplernatant from murine rlL-4 cDNA
transfected X63Ag8-653 myeloma cells (X63, kindly suppliced
by Dr. B.Chan, Robarts Research Institute, London, Ont.)
was used as positive control at 20U/ml (19). Supermatants
were also assayed for IFN-y was used as positive control at
4Ong/ml.
Antibody response: Mice were immunized with 30~M K3, K4 or
KlA2 in 50~1 saline emulsified in 50~1 of CFA in one hind
footpad. After two weeks second shot of the respective
peptide (30~M) in IFA was given intraperitoneally. Blood
was collected at different time intervals after second shot
and sera collected. The isotype of antibodies generated
against K3,K4 and KlA2 were detected by ELISA assay using
isotype specific Goat anti-mouse lg antibody.
Flow Cytometric Analysis: A20 hybridoma cells were stained
for the FACS analysis cells were incubated with or without
various concentrations of CLIP or ovalbumin peptide for 24
- CA 0220~680 1997-0~-16
hours at 37~C in 24 well lplates at a concentration of 2x105
cells/well. Cells were then washed and incubated with
MKD.6 (anti -Ad) antibodies. These cells were then washed
and stained with flourescein isothiocynate (FlTC)-
conjugated goat anti-mouse lgGFc (Jackson Laboratories, Bar
Harbor, ME). For negative control, cells were stained with
only FlTC-conjugated secondary antibody.
Example 2
Effect of incubation of TA3 cells with CLIP peptide or
10 immunogenic peptides and proteins
TA3 cells, were incubated with various concentrations
of CLIP peptide-(85-101), OVA peptide-(323-339), HEL, or
OVA for various lengths of times (15 min to 24 h) at 37~C.
Cells were then stained for the surface expression of I-Ad
or I-Ak and analyzed by flow cytometry (Fig. 2, A and B).
Surface I-Ad was significantly decreased in a dose-dependent
fashion, while the decrease in I-Ak was less pronounced
(Fig. lA). Maximal down-regulation was observed after 24 h
using 100 ~g/ml of peptide. Previous work has demonstrated
that the AakA~d heterodimer has increased affinity for CLIP
over AakA~k heterodimers (7, 14) based on competition
assays, and this is a likely explanation for the observed
difference in MHC class II surface down-regulation between
phenotypes.
Figure 2B demonstrates the effect of incubating the
same cells used above with immunogenic Ags. The I-Ad-
restricted Ag OVA resulted in an increase in surface I-Ad,
whereas the I-Ak-restricted Ag HEL did not. Using a peptide
fragment (323-339) of OVA also resulted in a similar
increase in surface I-Ad (data not shown). These results
are significant because they suggest that 1) CLIP peptide
decreases the surface expression of class II expansion in a
promiscuous fashion and 2) immunogenic Ags up-regulate
class II expression in a class II MHC-restricted fashion,
thus confirming previous studies with TA3 cells (12).
CA 0220~680 1997-0~-16
Example 3
Incubation of TA3 cells with CLIP peptide increases the
intracellular floppy state of I-Ad complexes
It has prevlously been shown (15) that the unstable
state of class II molecules is associated with a/~/Li
complexes that migrate slower on SDS-PAGE than molecules in
the stable state, which is a reflection of stable binding
of the a/~ heterodimer with peptide. It has also been
reported that binding of CLIP peptides to MHC class II
molecules results in the inability of a/~ complexes to
achieve the compact state (7). To determine whether
exogenously added CLIP peptide has an effect on the
stability of a/~ hetereodimers, TA3.11 cells were cultured
with varying amounts of the CLIP peptide (Fig. 3). A dose-
dependent increase in the ratio of unstable state of I-Ad
molecules (nonboiled) was observed compared with total I-Ad
(boiled) of cell lysates, and a point of saturation occurs
at a CLIP concentration of 200 ~g/ml.
Example 4
Incubation of TA3 cells with CLIP demonstrates the
exogenous CLIP does not bind to class II molecules on the
cell surface
To determine whether exogenous CLIP was binding
directly to cell surface class II MHC or was associating
internally, TA3 cells were incubated in the presence of
FITC-CLIP or FITC-OVA-(323-339), as a control, for various
time periods. Cells were subsequently analyzed by FACS
analysis. Figure 4 (A and B) shows that the immunogenic
peptide OVA-(323-339) bound to TA3 cell surface Ags to a
much greater extent than CLIP, despite the promiscuity of
CLIP for binding to class II molecules. This demonstrates
that immunogenic peptides are more capable than CLIP of
binding to surface class II MHC. Further, time course
studies show that CLIP binding appears to plateau at 3 h,
CA 0220~680 1997-0~-16
34
whereas binding of OVA peptide continues to increase with
time over a 24-h period.
.Yr le 5
Immunization of Ags with CLIP peptide inhibits in vitro T
cell recall response
The inventor investigated what effect(s) CLIP may have
in vivo by immunizing BALB/c mice and C3H/HeJ mice with
CLIP peptide (100 mg/footpad) in CFA or IFA and, in another
experiment, with OVA (50 ~g/footpad). Harvested popliteal
lymph node cells were challenged in vitro with the priming
Ag, PPD, CLIP, or OVA, and the T cell response was
measured. Figure 4(A and B) shows the effect of immunizing
with CLIP peptide in concert with CFA. BALB/c mice
immunized (Fig. 5a) with CLIP and CFA exhibited a
significant decrease in T cell response to PPD over that of
mice immunized with CFA alone. Similarly, in C3H/HeJ mice,
(Fig. 5B), immunization with CFA and CLIP produced a
decrease in the in vitro T cell proliferative response to
PPD over that of mice immunized with CFA alone.
To determine whether the decreased response was due
solely to competition between CFA and CLIP peptide, BALB/c
mice were also immunized with CAFA and an unrelated I-Ad-
restricted synthetic peptide, KlA2 (EYKEYAAYA(EYA)2) (10,
11). Mice immunized with CFA/saline were used as controls.
The T cell response was measured as described above (Fig.
5A), and no decrease in the PPD or KlA2 response was
observed, suggesting that CLIP interfered with the PPD
while KlA2 has no such effect.
To determine whether CLIP exerts its effects on APCs
or T cells, cell mixing experiments were performed using T
cells from CFA/saline-immunized or CLIP/CFA-immunized mice.
The results of this experiment are shown in Figure 5C.
When T cells from control CFA/saline-immunized mice were
mixed with APCs from CLIP/CFA-immunized mice, the T cell
response was reduced to a similar level as that observed
when both APCs and T cells were derived from CLIP/CFA-
CA 0220~680 1997-0~-16
immunized mice. When APCs from the CFA/saline-immunized
group were mixed with T cells from CLIP/CFA-immunized mice,
a T cell response similar to that of the CFA/CLIP group was
observed. We believe that this is the result of a lack of
priming of T cells in the presence of CLIP, and this lack
of priming subsequently yields fewer activated T cells in
vitro.
To evaluate the magnitude of the down-regulatory
effect of CLIP to PPD, dose-response studies were
performed. PPD was used at concentrations of 100, 10, 1,
and 0.1 ~g/ml. Figure 5D shows that there was a
consistently impeded recall response to PPD in the
CFA/CLIP-immunized group compared with that in the group
immunized with CFA alone.
Finally, to exclude the possibility of the down-
regulatory effect being specific to PPD, OVA was used as a
priming Ag with CFA and CLIP. As with PPD, the OVA recall
response was also reduced when CLIP was co-immunized with
OVA (Fig. 4E).
Example 6
Immunization with CLIP peptide decreases surface expression
of class II MHC
To determine whether the class II MHC down-regulation
observed with CLIP in vitro could be occurring in vivo,
BALB/c mice were immunized as described above, and the
cells from lymph nodes were stained either for I-Ad surface
expression or for B220 (CD45) as a control. Figure 6 (A
and B) shows the results of FACS analysis on B lymphocytes
and macrophages obtained from the lymph nodes of BALB/c
mice. I-Ad surface expression was significantly reduced in
the CLIP-immunized group compared with that in the saline
control group (Fig. 6B), whereas B220 expression was not
(Fig. 6A). Similar results were obtained in C3H/HeJ-
derived (I-Ak) lymph node cells (data not shown). These
results suggest that the functional results obtained above
CA 0220~680 1997-0~-16
36
may be explained by the lower level of class II expression
in CLIP-immunized mice.
Example 7
Effect of CLIP on the cell surface expression of MHC
class 11 : A20 hybridoma cells were incubated with various
concentrations of CLIP(85-101) or ovalbumin peptide (323 -
339) for 24 hours. Cells were stained for l-Ad expression
and analyzed by flow cytometry. The data presented in
Figure 7 show that CLIP down regulated the surface
expression of MHC class ll on A20 cells in a dose dependent
manner. The immunogenic peptide, ovalbumin, on the other
hand, up regulated the level of class ll expression. These
results suggest that addition of exogenous CLIP interferes
with its efficlent removal from the MHC molecules by H-2M
and thus, affects the transport of MHC to the cell surface,
where as, the immunogenic peptides bind to the MHC and thus
up regulate class ll expression.
Example 8
T i zation with CLIP inhibits the In Vivo response to
peptide: As the previous results suggested CLIP down
regulates the Cell surface expression of mHC, further
experiments were done to study its effect on the antigen
presentation in vivo. Mice were immunized with the CLIP
(50 ~g/footpad) along with peptide antigens K3, K4, or KlA2
(50 ~g/footpad). Peptide K3 has low affinity for MHC while
KlA2 has high affinity. K4 has an intermediate affinity
for MHC (8'). Ovalbumin peptide (323-339) was used as a
control in place of CLIP. Ten days later, lymph nodes were
harvested and T-cell proliferation was assayed. Mice
immunized with the peptides along with CLIP showed a
significant reduction in the proliferative response over
mice immunized with peptides alone (Figure 8). However,
the magnitude of the down regulation of the response
depended on the affinity of the peptide for the MHC. The
inhibition of response to the high affinity peptide KlA2
CA 0220~680 1997-0~-16
was to a lesser extent than K4 (intermediate affinity) and
K3 (low affinity). This suggests that exogenously added
CLIP competes with the peptide antigens for binding to MHC
and thus, inhibits the presentation of the antigens to T
cells resulting in down regulation of the response.
Example 9
Immunization with CLIP shifts the response toward TH2: We
have reported earlier that high affinity peptide KlA2
induces the generation of TH1 type response while low
affinity peptide K3 induces TH2 type of response. The
peptide K4 with intermediate affinity induces both TH1 and
TH2 type of responses (8'). To analyze whether down
regulation of antigen presentation by CLIP alters the
differentiation of TH1 and TH2 cells in response to peptide
antigens, mice were immunized with peptides in presence of
CLIP and draining lymph nodes were harvested after ten
days. Cells were then cultured in presence of peptides,
supernatants were collected and assayed for the presence of
IL-4 and IFN-r. The data presented in Figure 9 shows that
immunization with peptide in presence of CLIP resulted in
a decrease in IFN-r production by K4 and KlA2 primed cells.
On the other hand, IL-4 production by K3 and K4 was up
regulated and a little amount of IL-4 was detected in KlA2
primed cell culture. These results suggest that
immunization with CLIP along with peptide antigens shifts
the response toward TH2 type.
Example 10
T ; zation with CLIP shifts the isotype of peptide
specific an~;hoA;es from IgG2a to IgG1: Mice were immunized
with either K3, K4 or KlA2 peptides emulsified in CFA. Two
weeks later mice were given a second injection of peptides
emulsified in IFA. Mice were bled after two weeks of
second injection and serum was separated. The serum was
then assayed for the presence of IgG1, IgG2a antibodies.
The peptide K3 induces antibodies of IgG1 isotype with very
low levels of IgG2a isotype antibodies. Immunization with
CA 0220~680 1997-0~-16
CLIP along with the peptide increased the levels of IgG1
antibodies indicating an increase in the Th2 response. On
the other hand, immunization with K4 and CLIP did not seem
to have an effect on IgG1 response but down regulated the
IgG2a response suggesting down regulation of TH1 type of
response while TH2 response is maintained. The third
peptide, KlA2, displayed the biggest shift in antibody
response upon addition of CLIP. The IgG1 antibody response
increased significantly when CLIP was added, where as, the
IgG2a response was significantly down regulated suggesting
a shift from a strong TH1 to a TH2 response (Figure 10).
These results confirm the results obtained from cytokine
studies.
Example 11
Immunization with CLIP down regulates the Proliferative
Response to Ovalbumin Protein: In order to extend the
results observed for peptide antigens, CLIP peptide was
also used in conjunction with protein antigen. As protein
antigens require processing before they are able to bind to
MHC it was of interest to determine whether there would be
an observable difference between them and peptide antigens.
Mice were immunized with ovalbumin protein with or without
CLIP in both the hind footpads. After 10 days, lymph nodes
were harvested and a T-cell prolifeation assay was set up.
The results presented in Figure 11 show that addition of
CLIP down regulated the prlilferative response of mice to
ovalbumin protein. Possibly, exogenously added CLIP
interferes with the binding of peptide fragments of
ovalbumin to MHC molecules thus, decreasing the
presentation to T cells resulting in down regulation of
proliferative response.
Further, effect of immunization with CLIP on the
generation of TH1 and TH2 response by ovalbumin was tested.
The results presented in figure 6 show a higher level of
Il-4 secretion upon immunization with CLIP and a decreased
level of IFN-~ suggesting a shift from TH1 to TH2 type of
CA 0220~680 1997-0~-16
39
response. These results suggest that CLIP can inhibit the
response to both peptide and protein antigens.
Example 12
Effect of increasing doses of CLIP on the antigen
presentation: To find out the effect of increasing
immunization doses of CLIP on the antigen presentation in
vivo mice were immunized with various concentrations of
CLIP in CFA. After 10 days, lymph nodes were removed and
proliferation was assayed in response to recall antigen
PPD. Our results show that CLIP down regulated the
proliferated response in a dose dependent manner. The
effect of CLIP reached its maximum at a concentration of 10
~g/ml. Inhibitory effect of CLIP at 100 ~g/ml was not much
different than at 10 ~g/ml.
~Y: l~ 13
A group of NOD mice, an accepted animal model of
autoimmune or Type I diabetes, are treated with an
immunogenic composition of CLIP peptide (10 to 50 ~g/mouse)
and incomplete Freund's adjuvant at around 4 to 8 weeks of
age, one treatment per week for 4 weeks.
A control group of NOD mice are treated with adjuvant
alone. Development of diabetes is followed by testing for
urine glucose and, when urine glucose is detected, by
determining blood glucose. Urine and blood glucose are
measured by conventional methods.
Immunization with CLIP peptide prevents or delays the
development of diabetes in the test group of NOD mice,
compared to the control group.
- CA 0220~680 l997-0~-l6
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CA 0220~680 l998-08-l7
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: THE UNIVERSITY OF WESTERN ONTARIO
(B) STREET: Office of Research Services, Stevenson-Lawon Building
(C) CITY: London, Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): N6A 5B9
(ii) TITLE OF INVENTION: CLIP IMMUNOMODULATORY PEPTIDE
(iii) NUMBER OF SEQUENCES:4
(iv) CORRESPONDENCE ADDRESS:
Patricia A. Rae (Dr.)
Sim & McBurney
330 University Avenue, 6th Floor
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(vi) CURRENT APPLICATION DATA:
(A)APPLICATION NUMBER: 2,205,680
(B) FILING DATE: 16-MAY-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) PATENT AGENT INFORMATION
(A) NAME: Patricia A. Rae (Dr.)
(B) REFERENCE NUMBER: 6310-12/PAR
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Lys Pro Val Ser Gln Met Arg Met Met Ala Thr Pro Leu Leu Met Arg
1 5 10 15
Pro Met
CA 0220~680 l998-08-l7
46
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Pro Lys Pro Pro Lys Pro Val Ser Lys Met Arg Met Ala Thr Pro
1 5 10 15
Leu Leu Met Gln Ala Leu Pro Met
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Glu Lys Tyr Lys Glu Tyr Ala Ala Tyr Ala Glu Tyr Ala Glu Tyr Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly
1 5 10 15
Arg
