Note: Descriptions are shown in the official language in which they were submitted.
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WO 94/27634 216 4 3 2 ~ PCT/AU94/00292
CRYPTIC PEPTIDES FOR USE IN INDIJCING
IMMUNOLOGIC TOLERANCE
Background of the Invention
S Feeding antigens has been a classical method for inducing imml~nnlogical
unresponsiveness or oral tolerance (Asherson, G. L., et al., (1977), Cell Immunol., 33:145;
Asherson, G. L., et al. (1979), Immunology, 36:449; Challacombe, S. J., and Tomasi, T.J.,
(1980), J. Exp. Med., 152:1459, Bruce, M. G., and Ferguson, A. (1986), Immunology, 57:627;
Mowat, A. M., et al., (1982), Immunology, 45:105; and Strobel, S., et al., (1983),
Immunology, 49:451). Although regarded as being an important physiological response to
dietary antigens (Mowat, A. M., (1987), Immunology Today, 8:93), it has been suggested that
oral tolerance could be used to control aberrant immunological responses such as those found
in autoimmune ~ e~e, Thompson, H. S. G., and Staines, A. (1990), Immunol. Today,11 :396, and allergy.
The most extensively studied model system for autoimmune disease has been that of
experimental allergic encephalomyelitis (EAE). It has been shown that rats fed a tolerizing
dose of myelin basic protein (MBP) prior to sen~iti7~tion can be protected from an
encephalitogenic challenge with MBP (Miller, A. et al., (1991), ~ Exp. Med., 174:791;
Whitacre, C. C., et al., (1991), J. Immunol., 147:2155; and Miller, A., et al., (1992),
Proc. Natl. Acad. Sci. USA, 89:421). However, there have been conflicting views as to the
mech~ni.~m~ involved in inducing oral tolerance. For exarnple, Whitacre et al., (1991),
J. Immunol., 147:2155, found they were unable to transfer ~u~re~ion using T cells from
tolerized ~nim~l~, but showed that clonal anergy may be an important mech~ni~m for down-
regulating the effector function of CD4+ MBP-reactive T cells. Alternatively, Miller, A. et
al., (1991), J. Exp. Med., 174:791, have shown that suppression can be transferred to naive
recipients who receive CD8+ T cells from tolerized ~nim~l~ These s~ cssel (Ts) cells
through the release of a soluble cytokine were reported to be able to inhibit the in vitro
response of a MBP-specific CD4+ T cell line and could also bring about a by-stander
~u~l~;ssion of unrelated T cells (Miller, A., (1991), cifed supra). The immllnoregulatory
cytokine released by Ts cells was later defined as TGF-131 (Miller, A., et al. (1992)
Proc. Na~l. Acad. Sci. USA 89:421).
Peptides derived from a variety of protein antigens, including bacterial and viral
pathogens, ~to~ntigens~ allergens and other ~xl,e.illlental antigens such as hen egg Iysozyme
(HEL), ovalburnin (OVA) and lambda lc~lessol (cl) have been e~mined for the ability to
stimulate antigen-specific T cells. A wide size spectrum of peptides has been reported to
serve as 1[` cell epitopes. For e~nnple, a peptide derived from Hepatitis B surface antigen
(HBsAg amino acid residues 19-33) has recently been shown to stim--l~te T cell responses in
a majority of human subjects who had been immlmi~rl with a recombinant hepatitis B
vaccine (Schad, V.C. et al., (1991) Seminars in Immunol., 3:217-224). A major
WO 94/27634 PCT/AU94/00292
2~ ~32~ -2-
mycobacterial antigen 65-kD protein has also been epitope-mapped (Lamb, J.R. et al., (1987)
EMBO J., 6(5):1245-1249). T cell epilcl,es have been identified in the peptides comprised of
amino acid residues 112-132 and 437-459 of the 65-kD protein. MBP has also been epitope-
mapped in both human (Ota, K. et al., (1990) Nature, 346: 183- 187 ) and rodent (Zamvil et
al., (1986) Nature, 324:258-260) systems.
T cell epitopes present in allergenic proteins have very recently been described(O'Hehir, R. et al., (1991) Ann. Rev. Immunol., 9:67-95 ). Several peptides derived from the
house dust mite allergen Der p I have been shown to be T cell-reactive (Thomas, W.R., et al.
In Epitopes of Atopic Allergens Procee~1ings of Workshop from XIV Congress of the
European Academy of Allergy and Clinical ~mmlm~logy, Berlin (Sept. 1989) pp. 77-82;
O'Hehir, R.E. (1991) Annual Review Immunology 9:67-95; Stewart, G.A. et al. In: Epitopes
of Atopic Allergens Procee-lin~ of Workshop from XIV Congress of the European Academy
of Allergy and Clinical Immunology, Berlin (Sept. 1989) pp. 41-47; and Yessel, H. et al. In:
T Cell Activation in Health and Disease: Discrimination Between Immunity and Tolerance,
Conference 22-26 (Sept. 1990) Trinity College, Oxford U.K.). A T cell-stimulatory peptide
derived from the short ragweed allergen ~m~ I (amino acid residues 54-65) has also been
reported (Rothbard, J.B. et al., (1988) Cell, 52:515-523). Using a panel of T cell clones
derived from a rye grass-allergic individual, Perez ~ ~L demons~ ed that T cell epitopes are
contained within amino acid residues 191-210 of the protein allergen I~ I (Perez, M. et al.,
(1990) J. Biol. Chem. 265(27): 16210-16215 .
Srmm~-y of the Invention
This invention pertains to methods of inducing immllnologic tolerance to a protein
antigen in a subject, such as human, by ~imini~tt~ring a tolerizing amount of a composition
25 comprising at least one cryptic peptide derived from the antigen and a ph~rm~ceutically
acceptable carrier. Compositions which include a cryptic peptide derived from a protein
antigen, such as an allergen or ~llto~nti~en, can be ~tlmini~tered to induce tolerance in a naive
or pre-sen~iti7tod individual. Preferably, the composition is ~tlminist~red orally to treat
sensitivity in an individual to an allergen or allto~ntigen.
Brief D&Y~ ;ItionoftheD...~ gs
Figure I is a graphic representation of the responses of T cells isolated from mice
in....~ i7~d with ~ I and analyzed for response to selected peptides derived from I~er p I
by tritiated thymidine incorporation.
Figure 2a and 2b are graphic representations of the responses of T cells isolated from
mice immunized with a selected peptide derived from ~ I and analyzed for response to
either Der p I protein (panel a) or the a~n)p-iate peptide (panel b).
WO 94l27634 ~ 1 ~ 4 3 ~ ~ PCT/AU94/00292
-3 -
Figure 3 is a schematic representation of the location of T cell epitopes recognized by
mice in the 1~ I protein sequence where immlln~dominant epitopes are represented with
h~tchPd squares, cryptic epitopes are represented by dotted squares and the absence of
epitopes is r~lesenled by black squares.
S Figure 4 is a graphic representation of the responses of T cells isolated from mice fed
with buffer (panel a), peptide GEX p57-130 (panel b), peptide GEX plO1-154 (panel c), or
recombinant protein, GEX ~2 I (1-222) (panel d) followed by immunization with ~ÇLI2 I
and analyzed for response to ~ I in vitro by IL-3/GM-(: SF (panels a-d) or IL-2
production (panels e-h).
Figure S is a graphic le~lese~ ion of the responses of T cells isolated from mice fed
recombinant protein GEX ~ II (1-129), peptide GEX plO1-154, or peptide GEX pl88-222 followed by immllni7~tion with Der p I and analyzed for response to 1~ I (panel a and
d), peptide pl 10-131 (panel b and e), or peptide p78-100 (panel c and f) by IL-3/GM-CSF
(panels a-c) or IL-2 production (panels d-f).
Figure 6 is a graphic representation of the responses of T cells isolated from mice fed
with either buffer or recombinant fusion peptide (GEX pl31-187) followed by immnni7~tion
with ~2 I and analyzed for response to I~Y2 I by IL-2 production.
Detailed lDescription of the Invention
This invention pertains to methods for inducing immlmologic tolerance to a protein
antigen in a subject by ~-lmini~tçring at least one cryptic peptide derived from the antigen.
Protein antigens are known to contain certain ~letermin~ntx or epitopes which, upon
presentation with a particular class II major histocompatibility (MHC) molecule will activate
T cells of a subject upon exposure to the native protein antigen. Rather than the T cell
response being limited by the presence of one or two letermin~nt~ on an antigen, it appears
that the T cell response prer~ ially utilizes a selected nurnber of cletermin~ntx. Thus, a
hierarchy of T cell d~ ..t usage exists for a mlllticlel~. Il~ill~ll~ protein antigen.
Accordingly, the T cell cl~l~....i ..~..lx or epitopes for a particular protein antigen can be
divided into categories based on in vitro T cell proliferation assays in which protein antigen-
30 primed T cells are cultured with a selected concentration of a peptide derived from the protein
antigen and the amount of proliferation by the T cells in response to the peptide is determined
by, for example, triti~te-l thymidine incorporation.
By this assay, a peptide is categorized as comprising an immlln~clomin~nt T cellepitope if the peptide consistently in~ es one of the highest T cell proliferative responses in
35 antigen-primed T cells in the subject tested. Relative to an immunodominant epitope, a
peptide which comprises a minor T cell epitope recalls in vitro T cell proliferation to a more
variable and lesser extent. Those peptides which recall T cell proliferation of less than 2 fold
WO 94/27634 PCT/AU94/00292
~ 1~4~ 4
the background level of media alone are categorized as either not comprising a T cell epitope
or comprising a cryptic T cell epitope. Cryptic epitopes are those determin~nt~ in a protein
antigen which, due to processing and presentation of the native protein antigen to the
a~lopliate MHC molecule, are not normally revealed to the imml-ne system. However, a
5 peptide comprising a cryptic epitope is capable oftolerizing T cells, 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 cryptic epi~ol,es in a peptide categorized by the
10 above-described assay, antigen-primed T cells are cultured in vitro in the presence of each
peptide separately to establish peptide-reactive T cell 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 proliferation upon ch~llenge with the peptide and the protein
antigen from which the peptide is derived.
The presence of cryptic epitopes in a protein antigen is due to a lack of exposure of
certain epitopes to the immllne system which may result from normal processing of the
protein antigen which fails to reveal the epitope to the a~ropliate class II MHC molecule.
~lt~rn~tively, the end product of antigen processing may be a large fragment which hides the
cryptic epitope and hinders access to the MHC molecule or the T cell receptor on T cells
20 specific for the epitope. Additionally, other epitopes on the same protein antigen may
compete with the cryptic epitope for binding to the same restriction element or may have a
higher affinity and availability for a different restriction element, thus preventing cryptic
epitope interaction with MHC.
Cryptic peptides of the invention comprise at least one cryptic epitope derived from a
25 protein antigen (i.e., the peptide comprises at least apprnximAtely 7 amino acid residues).
Such peptides can comprise as many amino acid residues as desired and preferably comprise
at least about 7, more preferably at least about 15, even more preferably at least about 20 and
most preferably at least about 25 amino acid residues of a protein antigen. A peptide length
of about 20-40 amino acid residues is preferred as increases in length of a peptide may result
30 in difficulty in peptide synthesis as well as retention of an undesirable property (e.g.,
immllnoglobulin binding or enzymatic activity) due to m~int~n~nce of conformational
similarity between the peptide and the protein antigen, such as an allergen from which it is
derived. If desired, the amino acid sequences of one or more peptides can be produced and
joined by a linker to increase sensitivity to processing by antigen-presenting cells. Such
35 linker can be any non-epitope amino acid sequence or other a~propl;ate linking or joining
agent. For example, two cryptic peptides can be joined or a cryptic peptide and a peptide
WO 94/27634 216 ~ 3 ~ ~ PCT/AU94/00292
-5 -
compri~ an imml~nodominant or minor epitope derived from the protein antigen can be
linked.
Cryptic peptides can be produced by recombinant DNA techniques in a host cell
transformed with a nucleic acid vector directing t;x~res~ion of a nucleotide sequence coding
S for such peptide, or by chemical synthto~i~, or in certain limited situations by chemical
cleavage of protein antigen such as an allergen. When produced by recombinant techniques,
host cells transformed with nucleic acid vectors directing expression of a nucleotide sequence
coding for a peptide are cultured in a medium suitable for the cells. The peptides may be
secreted and harvested from a mixture of cells and cell culture medium. Alternatively, the
peptide may be retained cytoplasmically and the cells harvested, Iysed and the peptide
isolated a~d purified. Peptides can be isolated using techniques known in the art for
purifying peptides or proteins including ion-exchange chromatography, gel filtration
chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with
antibodies specific for the peptide or the protein antigen from which the peptide is derived, or
a portion lhereof. The cryptic peptides described herein are isolated such that the peptide is
snbst~nti~lly free of cellular material or culture medium when produced by recombinant
DNA techniques, or substantially free of chemical precursors or other chemicals when
synth~ci7.-d chemically, or obtained by chemical cleavage of a protein allergen or other
protein antigen.
To obtain cryptic peptides of the invention where the T cell ~ilo~es of a protein
antigen are urlknown or ill-defined, the protein structure of the antigen can be reviewed and
the sequence divided into at least t~,vo peptide fragments of desired lengths. For example, the
protein sequence of a protein antigen can be systematically divided into at least two non-
overlapping fr~gment~ of desired length or overlapping fr~gment~ of desired length. As an
illustrative example, the known amino acid sequence of Der p I, a major allergen of
nermato~hz~oides ~teror~ys~inus having an amino acid sequence of 229 residues (shown in
SEQ ID NO: 1), can be divided into peptide fragments of about 20-35 amino acid residues in
length, with each fragment overlapping with another by about 10 amino acids. To maximize
the potential of including T cell epitopes in the peptide fr~gment~, areas of overlap and length
of each fragment can be designed to m~int~in the presence of T cell epitopes predicted using
algo~ s (Rothbard, J. and Taylor, W.R. (1988) EMBO J. 7:93-100; and Berzofsky, J.A.
(1989) Philos. Tran* R. Soc. Lond. 323:535-544). Preferably, human T cell epitopes within
a protein antigen can be predicted using known HLA class II binding specific amino acid
residues. 1he res~llting peptide fragments can be produced by recombinant DNA techniques
35 or chemical synthP~
The peptide fragments derived from a protein antigen are tested to d~l~ , . li, ,e those
fr~gment~ having T cell stimulating activity (i.e., proliferation, lymphokine secretion and/or
W094/27634 21~432(~ PCT/AU9410~292
induction of T cell anergy/tolerization) and thus comprise at least one T cell epitope. For
example, human T cell stim~ ting activity can be tested by culturing T cells obtained from a
subject, such as a human, sensitive to a protein antigen (i.e., a subject which has an imml-ne
response to the protein antigen) with a peptide fragment derived from the protein antigen and
dett~rminin~ the presence of proliferation by T cells in response to the peptide. The presence
of proliferation by T cells can be ~let~rmin~cl by, for example, uptake of tritiated thymidine.
Tmmlln~dominant T cell epitopes, minor T cell epitopes and cryptic epitopes can be
identified as described in Example 3. To confirm the presence of a cryptic epitope in a
selected peptide, T cells are obtained from an individual sensitive to the protein antigen and
cultured with each of the cryptic peptides separately to establish peptide-reactive T cell lines.
The presence of T cell proliferation or induction of T cell tolerance in response to the peptide
and the protein antigen from which the peptide is derived confirms the presence of at least
one cryptic epitope in the peptide.
Cryptic peptides of the invention can be derived from a protein antigen such as an
allergen or ~lto~ntigen. When derived from an allergen, the cryptic peptide can be derived
from any known protein allergen, such as an allergen of the following genus: the genus
Derm~topha~oides; the genus ~li~; the genus ~mbrosia; the genus T olillm; the genus
Cryptomeria; the genus Alter~ria; the genus ~ ; the genus Betula; the genus Quercus: the
genus ~21~; the genus ~rtemi~ia the genus Planta~o; the genus p~rietaria; the genus C~nine;
the genus Rlattella; the genus ~; the genus Peripl~neta; and the genus Sor~hl-m Cryptic
peptides recognized by mice in ~ I, a major allergen of the species Derm~topha~oides
ptero~ us, have been ~letermint?cl in mice and comprise amino acid residues 120-143 of
I (SEQ ID NO:1), amino acid residues 144-169 of ~ I (SEQ ID NO:l) and amino
acid residues 131-187 of r2çL~ I (SEQ ID NO:l).
Cryptic peptides can also be derived from protein antigens other than allergens where
immunologic tolerance to an ~ to~ntigen is desired. ~l~to~ntigens from which cryptic
peptides can be derived include insulin, glutamic acid decarboxylase (64K), PM- 1 and
carboxypeptidase for use in treating diabetes; myelin basic protein for use in treating multiple
sclerosis; rh factor for use in treating erythroblastosis fetalis; acetylcholine receptors for use
in keating my~th~ni~ gravis; thyroid rece;~Lol~ for use in treating Graves Disease; basement
membrane protein for use in treating Good Pasture's syndrome; and thyroid proteins for use
in treating thyroiditis.
According to one aspect of this invention, cryptic peptides derived from a protein
antigen are ~tlminiett?red to a subject to induce immllnnlogic tolerance in the subject to the
protein antigen. ~e term subject includes living or~ni~m~ capable of mounting an immune
response to a protein antigen, e.g., m~mm~ Examples of subjects include hllm~n~, rats,
mice, dogs, cats, horses, cows and transgenic species thereof. Tmmlm~logic tolerance refers
WO 94/27634 216 4 3 2 6 PCT/AU94/00292
to a condition in a subject where a block in the development, growth or differentiation of
specific Iymphocytes in the subject results upon ~lminictration of a tolerizing amount of a
cryptic peptide of the invention. Tolerance results from the interaction of antigen with
antigen receptors on Iymphocytes under conditions in which the Iymphocytes, instead of
5 becoming activated, are deleted or rendered unresponsive. Tolerance may also be due to the
action of specific T or B Iymphocytes or other regulatory meçh~ni~m~ that prevent
Iymphocyte activation. One mech~ni~m for inhibiting an immune response is the stimulation
of a class of Iymphocytes, called s~p,~;sser T cells, whose principal function is to suppress
the activation of specific T and B Iymphocytes. In this situation, inhibition is mediated not
10 by the antigen itself but by regulatory cells that are in~llce~l by the antigen. Another
proposed meçh~ni~m for tolerance is a response by the immune system to antigen in which
unique or idiotypic determin~nte of lymphocytes or antibodies specific for the antigen are
targeted. This response results in a network of complementary idiotypes and antiidiotypes
which block the stim~ tion of antigen-specific cells. Finally, the products of activation of B
15 and T Iyrnphocytes, namely antibodies and cytokines, respectively, are themselves capable of
regulating specific immunity to result in tolerance in addition to functioning as the principle
effector rnolecules of lymphocytes.
In order to induce immunologic tolerance in a subject, a tolerizing amount of a cryptic
peptide derived from a protein antigen is ~timinictered to the subject. A tolerizing amount is
20 defined as a dosage of cryptic peptide necessary to induce immunologic tolerance in a
subject, such as a human to the antigen from which the cryptic peptide is derived.
Tmmllnologic tolerance in a subject is indicated by non-responsiveness or ~liminution in
symptoms to the protein antigen, such an an allergen or ~lto~ntigen, as determin~d by
standard clinical procedures (see e.g., Varney et al,, (1990) British Medical Journal 302:265-
25 269). When tolerance to an allergen is sought, such non-responsiveness includes ~liminntion
in allergen in~ ecl allergic symptoms. As referred to herein, a ~liminntion in symptoms to an
allergen includes any reduction in the allergic response of a subject, such as a human, to the
allergen following a tre~tm~nt regimen with a cryptic peptide as described herein. This
fliminntion in symptoms may be ~letermin~d subjectively in a human (e.g., the patient feels
30 more comfortable upon exposure to the allergen), or clinically, such as with a standard skin
test.
Cryptic peptides derived from a protein antigen are typically ~(imini~tered to a subject
in the form of a composition which includes a ph~rm~ceutically acceptable carrier or diluent.
A-lminictration of a composition of the present invention to induce immlln~logic tolerance in
35 a subject to a protein antigen can be carried out using known procedures, at dosages and for
periods of time effective to tolerize the subject to the protein antigen. Effective amounts of
the composition will vary according to factors such as the degree of sensiliviLy of the subject
-
WO 94/27634 ~ 1 ~; 4 ~ ~ ~ PCT/AU94/00292
to the antigen, the age, sex, and weight of the subject, and the ability of the cryptic peptide(s)
to induce tolerance in the subject. Dosage regima may be adjusted to provide the optimum
therapeutic response. For example, several divided doses may be ~tlmini.~tered daily or the
dose may be proportionally reduced as in-lic~tçcl by the exigencies of the therapeutic
5 situation.
Cryptic peptides may be ~lmini~tered to a subject in a convenient manner such as by
injection (subcutaneous, intravenous, etc.), oral ~lmini~tration, inhalation, intranasal,
transdermal application, or rectal ~imini~tration. Preferred routes of ~lmini~tration to induce
immlmologic tolerance in a subject are oral and intranasal ~mini~tration. See O'Hehir, R.E.
et al. (1993) Eur. J. Clin. Invest. 23(12): 763-772). Depending on the route of ~timini.~tration,
the active compound (i.e., the cryptic peptide) may be coated with in a material to protect the
compound from the action of enzymes, acids and other natural conditions which may
inactivate the compound.
To atlmini~t~r a cryptic peptide or peptides by enteral ~(lmini~tration, it may be
15 necessary to coat the peptide with, or co-~-lmini~ter the peptide with, a material to prevent its
inactivation. For example, the cryptic peptide may be ~lmini~tered to a subject in an
a~ropl;ate diluent, co-~lmini~tered with enzyme inhibitors or in an al,L,rop.iate carrier such
as liposomes. Ph~rm~eutically acceptable diluents include saline and aqueous buffer
solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate
20 (DEP) and trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as
conventional liposomes (Strejan et al., (1984) J. Neuroimmunol. 7:27). For purposes of
inducing tolerance, the composition is preferably ~-lmini~tered in non-immlln-~genic form,
e.g., one that does not contain adjuvant.
The active compound may also be ~rlmini~tered pale~lleldlly. Dispersions can also be
25 p~ d in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under
ordinary conditions of storage and use, these pl~lions may contain a preservative to
prevent the growth of microorg~ni~m~
Ph~rm~e~ltical compositions suitable for injectable use include sterile aqueous
solutions (where water soluble) or dispersions and sterile powders for the extemporaneous
30 ~ ,paL~lion of sterile injectable solutions or dispersion. In all cases, the composition must be
sterile and must be fluid to the extent that easy syringability exists. It must be stable under
the conditions of m~nllf~ctllre and storage and must be preserved against the cont~min~ting
action of microorg~ni.~m.~ such as bacteria and fungi. The carrier can be a solvent or
dispersion medium co~ g, for example, water, ethanol, polyol (for example, glycerol,
35 propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof,
and vegetable oils. The proper fluidity can be m~int~inPrl for example, by the use of a
coating such as licithin, by the m~ t~ ce of the required particle size in the case of
WO94/27634 ~ 64326 PCl IAU941~0292
dispersion and by the use of surfactants. Prevention of the action of microorg~ni~m~ can be
achieved by various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, ~or example, sugars, polyalcohols such as manitol,
sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition an agent which delays
absorption, for example, al.~ u.~l monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating active compound in the
required amount in an a~plopliate solvent with one or a combination of ingredients
enumera~ed above, as required, followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those enumerated above. In the
case of slerile powders for the plc~dlion of sterile injectable solutions, the preferred
methods of prcpa~dlion are vacuum drying and freeze-drying which yields a powder of the
active ingredient (i.e., a peptide of the invention) plus any additional desired ingredient from
a previously sterile-filtered solution thereof.
When a cryptic peptide or peptides as herein described is suitably protected, asdescribed above, the peptide may be orally ~lmini~tered, for example, with an inert diluent or
an ~c~imil~hle edible carrier. The peptide and other ingredients may also be enclosed in a
hard or soft shell gelatin capsule, colllp-cs~ed into tablets, or incorporated directly into the
individual's diet. For oral ~ nninictration~ the active compound may be incorporated with
excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. Such compositions and plc~ Lions should contain
at least 1% by weight of active compound. The percentage of the compositions andp~ lions may, of course, be varied and may conveniently be between about 5 to about
80% of the weight of the unit. The amount of active compound in such compositions is such
that a suitable dosage will be obtained. Preferred compositions or pl~dlions according to
the present invention are prepared so that an oral dosage unit contains between from about 10
mg to about 200 mg of active compound.
As used herein "ph~rm~e~1tically acceptable carrier" includes any and all solvents,
dispersion media, coatings, antih~.teri~l and antifungal agents, isotonic and absorption
delaying agents, and the like. The use of such media and agents for ph~n~ceutically active
substances is well known in the art. Except insofar as any conventional media or agent is
incnmp~tible with the active compound, use thereof in the composition is contemplated.
Supplementary active compounds can also be incorporated into the compositions.
It may be advantageous to form~ te compositions in dosage unit form for ease of
lmini~tration and uniro,~ y of dosage. Dosage unit ffirm as used herein refers to
WO 94/27634 PCT/AU94100292
21 ~43~
-10-
physically discrete units suited as unitary dosages for the m~mm~ n subjects to be treated;
each unit cont~ining a predete-minP~l quantity of active compound calculated to produce the
desired effect in association with the required ph~rm~ceutical carrier. The specification for
the dosage unit forms of the invention are dictated by and directly dependent on (a) the
5 unique characteristics of the active compound and the particular effect to be achieved, and (b)
the limitations inherent in the art of compounding such an active compound for the tre~tnnent
of the subject.
Compositions of the invention can include one or more cryptic peptides or a cryptic
peptide and a peptide comprising an immlln~dominant or minor epitope which are
10 ~(lmini~tpred to subjects, such as hllm~n~, who are naive or pre-sen~iti7çcl to the protein
antigen from which the peptide is derived, at dosages and for lengths of time effective to
induce tolerance in the subject to the antigen. For example, an amount of one or more of the
same or of di~eLenl compositions effective to induce tolerance in a subject can be
~llmini~tered simultaneously or sequentially. A composition comprising at least two peptides
15 (e.g., a physical mixture of at least two peptides), can also be used in methods of tolerization.
For example, a cryptic peptide and a peptide comprising an immunodominant epitope can be
co-~-lmini~tered.
The fact that tolerance can be incluce~l by ~tlminictering a cryptic peptide of the
invention (i.e., a peptide which does not contain an epitope recognized during imml-ni7~tion
20 when the entire protein antigen is presented to a subject) is significant. Peptides effective in
immlmotherapy may therefore not simply be limited to those identified by T-cell clones or
polyclonal responses of sP-n~iti7Pd individuals. Adminiskation of a cryptic peptide may avoid
the potential limitations inherent in ~q~lmini~tering a peptide cont~ining immlln~dominant
epitopes to sçn~iti7~cl individuals. The use of cryptic peptides also offers the potential for
25 modifying immllne responses without having to redirect the development of T-cell clones
which have already progressed along Thl and Th2 or equivalent ~lhw~ys.
It is also possible to modify the skucture of cryptic peptides useful in methods of the
invention for such purposes as increasing solubility, enhancing therapeutic or preventive
efficacy, or stability (e.g., shelf life ex vivo, and resi~t~nce to proteolytic degradation in vivo).
30 A modified peptide can be produced in which the amino acid sequence has been altered, such
as by amino acid substitution, deletion, or addition, to modify the ability of the peptide to
induce tolerance, or to which a component has been added for the same purpose.
For example, a peptide can be modified so that it m~int~in~ the ability to induce T cell
anergy or tolerance and bind MHC proteins. In this instance, critical binding residues for the
35 T cell receptor (i.e., the amino acid residues which comprise the cryptic epitope) can be
let~rmined using known techniques (e.g., substitution of each residue, such as, for example,
with alanine and determination of presence or absence of T cell reactivity). Those residues
WO 94/27634 216 ~ ~ 2 ~ PCT/AU94/00292
-1 1-
shown to be essential can be modified by replacing the essçnti~l amino acid with another,
preferably similar amino acid residue (a conservative substitution) whose presence is shown
to çnh~nce, dimini~h, but not elimin~te or affect T cell reactivity. In addition, those amino
acid residues which are not essential for T cell interaction can be modified by being replaced
5 by another amino acid whose incorporation may enhance, tlimini~h or not affect T cell
reactivity, but not elimin~te binding to relevant MHC. Preferred amino acid substitutions for
non-essential amino acids include, but are not limited to substitutions with ~l~nine, glutamic
acid or a methyl amino acid.
~nother example of a modification of peptides is sllbstitlltion of cysteine residues
10 preferably with alanine, or glutamic acid, or alternatively with serine or threonine to
minimi7P dimerization via disulfide linkages.
In order to enhance stability and/or reactivity, peptides can also be modified to
incorporate one or more polymorphisms in the amino acid sequence of a protein antigen
reslllting from natural allelic variation. Additionally, D-amino acids, non-natural amino acids
15 or non-arnino acid analogues can be substituted or added to produce a modified peptide
within the scope of this invention. Furthermore, peptides can be modified using the
polyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al. supra) to produce
a peptide conjugated with PEG. Modifications of peptides can also include
reduction/alkylation (Tarr in: Methods of Protein Microcharacterization, J.E. Silver ed.
20 Humana Press, Clifton, NJ, pp 155-194 (1986)); acylation (Tarr, supra); esterification (Tarr,
supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected
Methods in ~ellular lmmunology, WH Freeman, San Francisco, CA (1980); U.S. Patent
4,939,239); or mild formalin treatment (Marsh, (1971) International Archives of Allergy and
Applied Immunology 41: 199-215).
To f~cilit~te purification and potentially increase solubility of peptides, it is possible
to add reporter group(s) to the peptide backbone. For example, poly-hi~ti~line can be added
to a peptide to purify the peptide on immobilized metal ion affinity chromatography
(Hochuli, E. et al., (1988) Bio/Technology, 6:1321-1235). In addition, specific endoprotease
cleavage sites can be introduced, if desired, between a reporter group and amino acid
30 sequences of a peptide to f~rilit~te isolation of peptides free of irrelevant sequences. In order
to sllcce,s~fully tolerize a subject to a protein antigen, it may be necessary to increase the
solubility of a peptide by adding functional groups to the peptide or by not including
hydrophobic regions in the peptide.
To potentially aid proper antigen processing of T cell ~; ilo~es within a peptide,
35 canonical protease sensitive sites can be recombinantly or synthetically engint?ered within the
peptide. For example, charged amino acid pairs, such as KK or RR, can be introduced within
a peptide during recombinant construction of the peptide. The resulting peptide can be
,
wO 94/Z7634 '~ ~ 6 432 6 i'CT/AU94100292
rendered sensitive to cathepsin and/or other trypsin-like enzymes cleavage to generate
portions of the peptide CO~ Iirlg one or more T cell epitopes. In addition, such charged
amino acid residues can result in an increase in solubility of a peptide.
Site-directed mutagenesis of DNA encoding a peptide can be used to modify the
structure ofthe peptide. Such methods may involve PCR (Ho et al, (1989) Gene 77:51-59)
or total synthesis of mllt~te~l genes (Hostomsky, Z., et al., (1989)
Biochem. Biophys. Res. Comm. 161:1056-1063). To enhance bacterial expression, the
aforementioned methods can be used in conjunction with other procedures to change the
eucaryotic codons in DNA constructs encoding peptides to ones preferentially used in E. ~QIi,
yeast, m~mm~ n cells or other eucaryotic cells.
This invention is further illustrated by the following non-limiting examples. The
co~ of all references and published patent applications cited throughout this application
are hereby incorporated by reference. The following methodology described in the Materials
and Methods section was used throughout the examples set forth below.
MATERIALS AND METHODS
Animals and Ant~en~
Female B10 and BALB congenic mice, and inbred C57BL/6J were purchased from
the Animal Resource Centre, Murdoch, Western Australia at 6-8 weeks of age.
The house dust mite allergen Der p I was affinity purified from spent mite medium
(SMM) using previously described techniques (Hoyne, G.F. et al. (1993) cited supra;
Lombardo et al. J. Immunol. 144:1353-1360 and Chapman (1989) Advances in Biosciences
74:281-295). Ovalbumin (OVA) crystalline Grade V was purchased from the Sigma
Chemical ColllLpally, St. Louis, MO. Overlapping synthetic peptides derived from the
published 1~ I sequence (Chua et al. (1988) J. Eicp. Med. 167:175-182) were synth~ei7ed
using standard t-BOC ch~rni~try and peptides were purified by reverse phase highperformance liquid chromatography (HPLC) and the sequence of individual peptides were
checked to verify identity. The peptides used in this study comprised the following dmino
acid residues derived from the ~ÇLp I sequence (Chua et al. (1988) cited supra): 1-20, 13-
39, 21-49, 40-60, 50-71, 61-84, 78-100, 85-109, 101-119, 110-131, 120-143, 132-157, 144-
169, 158-180, 170-191, 181-204, 197-222.
Pl~alion of reconnh;n~nt protein~
Inserts encoding either the whole er p I or ~ II protein (from spent mite
medium, the Commonwealth Serum Laboratories, Melbourne, Australia) or recombinant
constructs (formed from the restriction endonuclease fr~gm~nt~tion of the relevant cDNA;
1-- WO 94/27634 216 4 3 ~ ~ PCTtAU94/00292
see Chua et al (1990) Int. Arch. AllergyAppl. Immunol. 91:118-123), were ligated to the p-
GEX vector and transforrned into F~cherichia~Qli (Smith, D. B., and Johnson, K. S., (1988)
Gene, 67:31). The procedures for the molecular cloning of these products have been
described elsewhere (Chua, K. Y., et al., (1988) J. Exp. Med., 167:175 and Chua, K. Y., et al.,
S (1990) Int. ~rch. Allergy Appl. Immunol., 91: 124). Log phase ~ coli cells l~ rolllled with
pGEX based protein or peptide constructs were in-luced to express the recombinant protein
by adding 0.1 mM isopropylthiogalactosi(l~e (IPTG) (Promega) to the culture with ~h~kin~
for 60 minutes at 37C. Because large quantities of fusion peptides were required they were
prepared from solubilized inclusions. Bacterial pellets were resuspended in tris buffered
saline with 1 mM EDTA and transferred to a homogenizing bottle cont~ining 0.1 mm glass
beads and were homogenized using a Braun MSK Homogenizer for five mimltes The lysate
was removed after ultracentrifugation at 10,000 g for 10 minlltes at 4C. The pellet was
washed twice with 1.75 M g~l~nitline HCL cont~ining 1 M NaCl and 1% triton-X 100 (BDH
Chemicals) by thoroughly aspirating in a pipette and then centrifugation. The pellet was then
dissolved by incubating it in 8 M urea with 50 mM NaC1 and 1 mM ethylene di~minetetraacetic acid (EDTA) for 2 hours at 37C. The sarnple was dialyzed in 3-
(cyclohexylamino)-propanesulfonic acid (CAPS) buffer pH 10.7 and the pH was slowly
adjusted to pH 9.6. The recombin~nt material was then clarified by centrifugation at 10 000 g
and the concentration of the soluble m~teri~l was estim~te-l against standard quantities of
bovine serurn alburnin (BSA) using SDS-polyacrylarnide gel electrophoresis (SDS-PAGE)
and staiIling with Coomassie blue. Recombinant peptides used in this study included GEX
I (amino acid residues 1-222), GEX I~ II (amino acid residues 1-129), GEX pl-14
(amino acid residues 1-14 of E2ÇL~ I), GEX p60-111 (amino acid residues 60-111 f~ I),
GEX p98-140 (amino acid residues 98-140 of ~ I), GEX plO1-154 (amino acid residues
101-154 of I2~ I), GEX p57-130 (amino acid residues 57-130 of ~ I), GEX pl88-222(amino acid residues 188-222 of I2~ I)-
Tntlllction of Or~l Toler~nce
Mice were lightly anesthetized under ether and fed- intragastrically by a tube with 3
mg of protein or peptide on 3 consecutive days. Antigens were dissolved in CAPS buffer and
arlmini.~tered in a volume of 0.2 ml. Mice were immllni7~d subcutaneously at the base of tail
7 days af~er the last feed with 100 mg of native protein em~ ified in complete Freund's
Adjuvant (CFA) in a volume of 0.2 ml.
Clllhlre M~ inm
Lymph node cells were cultured in Dulbecco's Modified Eagles medium (DME)
supplemented with 2% fetal calf serum (FCS), 50 mM, 2-ME, 2 mM L-GI~lt~mine and 20
wO 94/27634 2 ~ ~ ~3 2 ~ PCT/AU94/00292
-14-
mg/ml ge~ lycin. FDC-P1 cells (Kelso, A., (1990), J. Immunol., 145:2167) were
m~int~ined in DME + 5% FCS while CTLL-2 cells (Krillis, S. (1978) J. Immunol. 120:20)
were m~int~inPd in Rosewall Park Memorial Institute (RPMI) medium + 10% FCS.
5 TCell A~,vs
The periaortic and inguinal lymph nodes were collected from immlmi7Pd mice and
single cell ~u~ell~ions were prepared by t;x~lessi~lg the nodes through a stainless steel wire
mesh. Cells were washed and cultured at 4 x 105 cells in a volurne of 0.2 ml in DME culture
medium in a 96 well flat bottom tissue culture plate. Protein or peptide antigens were added
10 at various concentrations and the cells were incubated at 37C for 24 hours. Supern~t~nt~
were collected and stored at -20C until required. The Der p I used for all in vitro assays was
the allergen isolated from spent mite medium (SMM).
Lymrhokine A~ys
FDC-Pl cells proliferate m~xim~lly in response to IL-3 and GM-CSF and
subm~xim~lly to IFN-y or IL-4 (Kelso, A. (1990) cited supra). 2x103 cells were added in 50
~Ll DME + 5% FCS to 50 ,ul of culture ~u~ in 96 well flat bottom tissue culture plates.
The cells were inc~lb~ted for 40 hours at 37C and then pulsed with 1 ~lCi 3H-Thymidine for
another 4-6 hours at 37C. The cells were then harvested onto glass fiber filter mats and
samples counted for 3H-Thymidine incull,old~ion using liquid scintillation ~e~ metry or
for latter ~x~c,;lllents due to its acquisition, on a Packard matrix 9600 direct beta counter
(Packard Instrllment~, Meriden, CT).
The CTLL-2 cell line will proliferate m~xim~lly with IL-2 but only poorly in thepresence of IL-4 (Kelso, A. (1990) J. Immunol. 145:2167). Supern~t~nt~ were cultured with
5000 CTLL-2 cells per well for 24 hours at 37C and pulsed with 1 ,uCi of 3H-thymidine
(3H-Tdr). Cells were harvested onto glass fiber filter mats and the amount of radioactivity
incorporated was determined as described above.
F.x~mrle 1 n~h,.llli~ ion of Immllnodominant, Minor ~n-l Cryptic
T Cell Fcpitopes Reco~ni7~ by Mice in Der p I
It has been previously shown that H2b mice are high responders to ~ I while H2k,H2d and H2q mice are low responders (Hoyne, G. (1992) Ph.D. Thesis, T cell Recognition
During Mucosal and Systemic Responses, Ulliv~ y of Western Australia). To determine
the location of T-cell epitopes on I~er p I, B 10 mice were immlmi7~d subcutaneously with
100 ~g of ~ I in CFA and after 8 days the periaortic and inguinal lymph nodes were
ex~mined for antigen specific lymphokine release (IL-3/GM-CSF) using a panel of
overlapping peptides. In three separate ~ ;lllents the greatest response was found to
~\ WO 94/27634 216 ~ 3 2 ~ PCT/AU94/00292
-15-
peptide pl l 0-131 (amino acid residues 110-131 of ~ I) while lower responses were also
seen to peptides p78-100 (amino acid residues 78-100 of ~ I) and p21 -49 (amino acid
residues 21 -49 of ~ I). No other peptides could stimulate a response. An example of the
results of one such experiment is shown in Figure 1 in which the following peptides were
5 used: peptide pl 10-131 ([1); and peptide p78-100 (~) and peptide p21-49 (-).
To test for cryptic epitopes, mice were immllni7~-1 with all the peptides and responses
to ~çLp I and the immlmi~in~ peptide were measured in the presence of spleen adherent
cells. Peptide pl20-143 (amino acid residues 120-143 of ~ÇLP I) and peptide pl44-169
(amino acid residues 144-169 of Der p I) were able to sensitize mice so they could recall
10 responses to both intact ~ I protein (Figure 2a) and the peptides (Figure 2b) respectively.
The results of Figure 2 show the mean IL-3/GM-CSF response of triplicate samples. The
following peptides are shown in the Figure: peptide pl20-143 (O); peptide pl44-169 (~);
peptide pl32-157 (O); and peptide pl58-180 (X).
15 Fx~m~le 2 Tn~lnrtioll of Oral Toler~nce in ~ice by A~lmini.~tration of Fusion Peptides
A number of recombinant peptides were generated by restriction enzyme digestion of
I cDNA. These fragments were cloned into the pGEX t;~ression vector as describedabove and transformed into E~ ~QIi- The recombinant peptides chosen for use in this study
20 were expressed as fusions attached to the glutathione-S-transferase protein of Schi~tosom~
japol icum. The fusion proteins and peptides were solubilized from bacterial cell pellets and
dialyzed into CAPS buffer pH 9.6. The recombinant peptides listed in Figure 3 were chosen
on the basis of the known T-cell epitope data described above. Recombinant peptides were
selected for the presence of immlln~dol,lhlani (h~trh~d squares) or cryptic epitopes (dotted
25 squares) or the absence of T cell epitopes (black squares) within the sequence. Thus, control
peptides GEX pl -23 (amino acid residues 1-23 of ~ I) and GEX pl 88-222 (amino acid
residues 197-222 of ~ I) did not contain any T cell epitopes. GEX p57-130 (amino acid
residues 57-130 of Der p I) cont~ined two epitopes while GEX plO1-154 (amino acid
residues 101-154 of ~ I) and GEX p98-140 (amino acid residues 57-130 of Der p I)30 contains the single immunodomin~nt epitope (amino acid residues 110-131), while GEX
pl 31 - 187 (amino acid residues 131 -187 of ~ I) contains the cryptic epitopes.Following a previously characterized regime for inducing oral tolerance (Hoyne, G.
F., (1993), Immunology 78:534-540), mice were fed 3 mg of fusion peptide on 3 consecutive
days and after a further 7 days were immunized subcutaneously with native protein in CFA.
35 In vitro lymphokine assays were then pelrolllled 7 days later using the periaortic and inguinal
lymph nodes ~timlll~t~d with either protein or synthetic peptides. Experiments were
performed to show that feeding mice CAPS buffer or the recombinant GEX ~ I ( l -222)
WO 94/27634 2, ~ ~ 4 3 2 ~ PCT/AU94/00292
-16-
fusion protein did not effect the IL-2 or IL-3/GM-CSF responses of mice to subcutaneous
injection of OVA in CFA.
To test whether orally ~lminictered peptides could induce tolerance, control mice
were fed CAPS buffer (Figure 4, panels a and e), while test ~nim~l~ received 3 mg on three
consecutive days of either GEX ~ I (1-222) (Figure 4 panels d and h) or the fusion
peptides GEX pS7-130 (Figure 4, panels b and f) or GEX plO1-154 (Figure 4, panels c and
g). One week later the response to immuni7~tion with native Der p I in CFA was determined
Mice fed CAPS buffer showed strong responses to the ~ I protein in vitro secreting both
IL-3/GM-CSF (Figure 4, panel a) and IL-2 (Figure 4, panel e) in response to TCR triggering.
10 On the other hand, mice fed GEX Der p I (1-222) or either ofthe two peptides GEX pS7-130
or GEX 101-154 had depressed IL-2 responses (Figure 4, panels f-h). The more pronounced
inhibition of IL-2 responses was a con~ t~nt feature of all experiment~ of this nature. Mice
were also fed with GEX p98- 140 and an equal degree of tolerance induced by this peptide
was found.
To ex~mine how the development of oral tolerance effected responses to T cell
epitopes on the allergen mice were fed 3 mg on three consecutive days of either GEX plOl -
154 or GEX p 188-222 and GEX ~ II (1 - 129) as a control. One week later mice were
immuni7ecl with ~ I and the responses of draining lymph node cells were measured to the
protein and peptides in vitro. The data shows the response for individual mice in each group
20 at the following antigen concentrations: ~ I, 20 ,ug/ml; and peptide pl 10- 131 and peptide
p78-100, 10 ,uM. As seen in Figure 5 feeding mice either GEX pl88-222 or GEX 12~ II
(1-129) did not affect the capacity of their lymph node cells to secrete either IL-3/GM-CSF or
IL-2 upon in vitro challenge with either protein or with the imm~mogenic peptides pl 10-131
or p78-100. However, in contrast, the lymphokine responses of GEX plOl-154 fed mice
25 were m~rkPrlly reduced and thus appear to have become tolerant to the whole protein (Figure
5). The tolerance induced by feeding one epitope appears to affect T cells specific for other
epitopes on the allergen. Subsequent experimt?nt~ using GEX p61-100 which con~i,ls one
epitope and the fusion peptide GEX p 1 -23 as a control gave the sarne result.
To determine whether a peptide co~ in~ a cryptic epitope could influence the
30 immune response, mice were fed 3mg on three consecutive days of GEX pl 31 - 187 (Figure 6
(-)) which contains the cryptic epitope found on peptide 144-169 while control mice were fed
with CAPS buffer (Figure 6 (Cl)). One week later mice were immuni7~d with I2~ I in
CFA. Lymph node cells were cultured in vitro with I2Ç~ I and sUpern~t~ntc assayed ~or IL-
2. Each data point in Figure 6 represents the mean response of 5 ~nim~lc per group +
35 standard deviation. The responses of cryptic peptide fed mice were statistically different (p <
0.05 t-test). As shown in Figure 6 lymph node cells from control mice showed strong
WO 94/27634 ~ 1 6 4 3 ~ 6 PCT/AU9~/00292
responses to Der p I in vitro by secreting IL-2, but mice fed the cryptic epitope displayed
much weaker lymphokine response in vitro.
The results presented here show that feeding fusion peptides cont~ining dominant or
cryptic T-cell epitopes can inhibit T cell responses to subcutaneous immllni7~tion with the
S whole antigen. In the case of fusion peptides co~ g ~lomin~nt epitopes the inhibition was
profound and was measured by depressed IL-2 and GM-CSF release from draining lymph
node cells challenged in vitro with whole allergen or the immunodominant peptides. This
included responses to peptides cont~ining residues which were not present on the fusion used
for feeding. For example, feeding the fusion peptide GEX p 101 - 154 inhibited the ability of
10 Per p I immunization to induce T cells which react with the whole allergen and with
synthetic peptides pl 10-131 and p78-100. This effect may therefor be mediated by a soluble
factor. The inhibition was otherwise specific because it could not be induced by the Der p II
fusion protein. Similar data has been obtained by Miller, A., et al., (1991), J: E~cp. Med.,
174:791; Whitacre, C. C., et al., (1991), J. Immunol., 147:2155, and Miller, A., et al., (1992),
Proc. Natl. Acad. Sci. USA, 89:421, who found that oral tolerance to MBP was mediated by
TGF-~31 and could be shown to suppress bystander responses in an in vitro model.Feeding two fusion proteins that did not contain T-cell epitopes did not inhibit the
imm~lne responses. However, feeding the fusion peptide GEX pl 31-187 which contained the
cryptic epitope found in peptide pl44-169 did significantly inhibit. The degree of inhibition
was not as marked as for the fusions cont~ining dominant epitopes but presumably could be
increased by extending the feeding regime or increasing the dose. Feeding the fusion
peptides was also found to Sell!~iti7e T cells in the MLN so they release GM-CSF on
stimlll~tion in vitro with Der p I or synthetic peptides including the cryptic peptide pl44-169
after feeding peptide GEX p 131 - 187. The presence of these sen~iti7Pd cells in oral tolerance
has recently been described for OVA (Hoyne, G. F., et al., (1993), Immunology 78:534-540).
Fx~mrle 3 Determin~tion of Tmmllnodomin~nt ~rinor ~ncl
Cryptic T Cell F~pitopes Reco~ni7~ by ~n
Aller~ic Tn-livi~ln~l jn ner p I
To ~letermine T cell epitopes recognized by an allergic individual in the ~
I protein sequence a T cell line can be established by cnltllrin~ mite-allergic patient peripheral
blood white cells in complete medium at 2 x 106/ml in the presence of 20 llg purified native
Der p I/ml. After 7 days of culture at 37C in a humidified CO2 incubator the viable cells
can be isolated by centrifugation with Iymphocyte separation medium (LSM, Organon
Technica, Durham, NC) and cultured in complete medium cont~ining recombinant IL-2 and
recombinant IL-4 for 2-3 additional weeks. When the T cells are "rested" and no longer
responsive to growth factors, a secondary proliferation assay can be performed by culturing 2
432~ ~
WO 94/27634 PCT/AU94/00292
-18-
x 104 T cells in 200 ~LI complete medium wlth 5 x 104 gamma-irradiated (3500 Rads)
peripheral white blood cells as antigen presçnting cells in the presence of various
concentrations of peptides derived from the intact protein. The cultures can then pulsed with
tritiated thymidine (1 ~lCi/well) on day 3 and harvested onto glass fiber filters on day 4.
5 Peptides stim~ ting tritium incorporation at least 2-fold over the medium control are defined
as cont~ininf~ T cell epitopes naturally exposed to the T cells when presented with the entire
protein (i.e., the peptides comprise at least one minor or immunodominant epitope). Those
peptides stim~ ting tritium incorporation of less than 2-fold above the medium control either
do not contain a T cell epitope or contain a cryptic epitope (i.e., an epitope not normally
10 exposed to T cells when the entire protein is presented). To confirm the presence of a cryptic
epitope in these peptides, T cell lines can be established by c~ rin~ peripheral blood white
cells from the same individual in the presence of each peptide separately to establish peptide-
reactive T cell lines. The "rested" T cells can then be challenged with each peptide and the
I2~ I protein. A peptide which comprises at least one cryptic epitope is capable of
15 stim~ ting the proliferation of the T cell line in the presence of the peptide or the entire
protein at a level at least 2-fold above the medium control or is capable of tolerizing T cells.
I~QUIV~T ,Fl~TS
Those skilled in the art will recognize, or be able to ascertain using no more than
20 routine experiment~tion~ many equivalents to the specific embo-liment~ of the invention
described herein. Such equivalents are intended to be encomp~secl by the following claims.
94/27634 ~ ~ 6 43 2 6 PCT/AU94/00292
-19-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: lN~'l'l'l'U'l'~ FOR CHILD HEALTH RESEARCH
(B) STREET: P.O. BOX 885
(C) CITY: WEST PERTH
(D) STATE: W~:~l~N AUSTRALIA
(E) COUNTRY: AUSTRALIA
(F) POSTAL CODE (ZIP): 6872
(ii) TITLE OF LNv~N-llON: CRYPTIC PEPTIDES FOR USE IN IN~U~1NG
IMMUNOLOGIC TOLERANCE
(iii) NUMBER OF S~QU~N~S: 2
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy diRk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII TEXT
(V) ~UK~NL APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/072,832
(B) FILING DATE: 2-JUN-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MANDRAGOURAS, AMY E.
(B) REGISTRATION NUMBER: 36,207
(C) REFERENCE/DOCKET NUMBER: IMI-037CPPC
W O 94/27634 ~ ~ ~ 4 3 2 ~ PCT/AU94/00292
-20-
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400
(B) TELEFAX: (617) 227-5941
5 ( 2) INFORMATION FOR SEQ ID NO:1:
( i ) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 834 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 8 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..738
(Xi) S~QU~N~ DESCRIPTION: SEQ ID NO:l:
AAA AAC CGA TTT TTG ATG AGT GCA GAA GCT TTT GAA CAC CTC AAA ACT 48
Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Ly& Thr
1 5 10 15
25 CAA TTC GAT TTG AAT GCT GAA ACT AAC GCC TGC AGT ATC AAT GGA AAT 96
Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cy8 Ser Ile Asn Gly Asn
20 25 30
GCT CCA GCT GAA ATC GAT TTG CGA CAA ATG CGA ACT GTC ACT CCC ATT144
30 Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile
35 40 45
CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT GCC192
Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala
W 0 94/27634 2 ~ PCT/AU94/00292
GCA ACT GAA TCA GCT TAT TTG GCT CAC CGT AAT CAA TCA TTG GAT CTT 240
Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser ~eu Asp Leu
65 70 75 80
5 GCT GA~ CAA GAA TTA GTC GAT TGT GCT TCC CAA CAC GGT TGT CAT GGT 288
Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly
85 90 9S
GAT ACC ATT CCA CGT GGT ATT GAA TAC ATC CAA CAT AAT GGT GTC GTC 336
0 Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val
100 105 110
CAA GAA AGC TAC TAT CGA TAC GTT GCA CGA GAA CAA TCA TGC CGA CGA 384
Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg
115 120 125
CCA AAT GCA CAA CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC CCA 432
Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro
130 135 140
CCA AAT GCA AAC A~A ATT CGT GAA GCT TTG GCT CAA ACC CAC AGC GCT 480
Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala
145 150 155 160
25 ATT GCC GTC ATT ATT GGC ATC AAA GAT TTA GAC GCA TTC CGT CAT TAT 528
Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr
165 170 175
GAT GGC CGA ACA ATC ATT CAA CGC GAT AAT GGT TAC CAA CCA AAC TAT 576
30 Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr
180 185 lgO
CAC GCT GTC AAC ATT GTT GGT TAC AGT AAC GCA CAA GGT GTC GAT TAT 624
His Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr
195 200 205
_
W O 94/27634 '~ 1 ~ 4 3 ~ ~ PCT/~U94100292
TGG ATC GTA CGA AAC AGT TGG GAT ACC AAT TGG GGT GAT A~T GGT TAC 672
Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr
210 215 220
5 GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA 720
Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro
225 230 235 240
TAT GTT GTC ATT CTC TAAACAAAAA GACAATTTCT TATATGATTG TCACTAATTT 775
10 Tyr Val Val Ile Leu
245
ATTTAAAATC AAAATTTTTT AGAAAATGAA TA~ATTCATT CACAAAAATT PAa~U~aA 834
(2) INFORMATION FOR SEQ ID NO:2:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 245 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:2:
LYB Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr
1 5 10 15
30 Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn
Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile
Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala
WO 94/27634 216 43 PCT/AIJ94/00~9
Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser Leu Asp Leu
65 70 75 80
Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly
85 90 95
Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val
100 105 110
Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg
115 120 125
Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro
130 135 140
Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala
145 150 155 160
Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Ary His Tyr
165 170 175
Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr
180 185 190
His Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr
195 200 205
Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr
210 215 220
Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro
225 230 235 240
35 Tyr Val Val Ile Leu
245
