Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
Wo gs/26980 2 1 8 6 8 7 3 PCTtUS9SI04121
HAPTENATED PEPTIDES AND USES THEREOF
5 B~rk~round
Delayed-type hypersensitivity (DTH) is an infl~mm~ory immune reaction
mP~ ted by T Iymphocytes. DTH reactions were first observed in persons inoculated
for a second time with cowpox, who developed a reddish, painful swelling on the skin
at the site of inoculation. Similar observations were made by Robert Koch in 1890 in
10 persons with tuberculosis: Whereas normal persons showed no reaction after
subcutaneous injection of tuberculin, those previously exposed to tuberculin
developed a red, weeping skin lesion at the injection site, which reaction increased for
24 to 72 hours, then disapp~ed. This tuberculin DTH reaction became the standarddiagnostic test for tuberculosis.
A DTH reaction can occur only in an individual previously exposed
(sensitized) to a given antigen. The manifestation of the reaction can be localized (the
skin reaction at the site of injection) or systemic (whole body). Localized DTH
reactions can be elicited by subcutaneous injection of an antigen to which the
individual has been previously exposed. Within 6 to 8 hours, the skin at the site of
20 the injection becomes red, warm and swollen. The swelling and characteristic
hardness of the flesh are due to a massive infiltration of monocytic cells, particularly
macrophages, and Iymphocytes. Lysosomal hydrolases, Iymphotoxin, and other
enzymes and factors released by the infiltrating cells damage surrounding tissues and
account for the redness and necrosis of the tissue surrounding the injection site.
25 Systemic DTH reactions occur when large amounts of the antigen are introduced into
the blood stream. Typical symptoms include fever, painful joints, and Iymphopenia,
but severe cases may result in shock or even death several hours after antigen
injection.
Contact sensitivity (contact de.,nalitis) is a form of delayed-type
30 hypersensitivity caused by contact of a semi~i7ing substance with the skin, to which
the skin develops an infl~mm~tory reaction. The substances capable of inducing
contact sensitivity typically are compounds of low molecular weight, which permits
them to diffuse through the skin, and are compounds which are not antigenic by
themselves but form conjugates with proteins inside the body, which conjugates, in
35 turn, are antigenic, i.e., capable of stimulating Iymphocytes. Such compounds are
termed haptens.
Woss/26980 2 1 ~ 3 Pcr/uss5/04l2
Haptens responsible for contact sensitivities have been detected in a wide
range of industrial and natural products, including mercuric compounds in ointments,
chromate, nickel, turpentine, varnishes, resins, cosmetics and dyes. Experimentally,
contact sensitivity has been induced using substituted benzenes, e.g., picryl chloride,
5 3,4-dinitrochloro-benzene, azobenzenearsonate, dinitrophenol (DNP), trinitrophenol
(TNP), etc. (See, generally, Klein Immunology: The Science of Self and Non-self
Discrimination, 1982 (John Wiley & Sons, Inc., New York), section 10.1.6 (Haptens)
pp. 357-360.)
A prime example of naturally occurring haptens is urushiol, found in poison
10 ivy, poison oak, primrose, uncured Japanese lacquer and poison sumac. Exposure to
urushiol-cont~ining oils exuded by these plants sçn~iti7es a susceptible individual,
and re-exposure to the sensitizing agent can induce irritation, infl~mm~tion andblistering of the skin. Severe reactions can require hospitalization or can even be
fatal. (See, Kligm~n (1958) AMA Arch. of Dermatology 77:149-180).
Urushiol is a family of catechol derivatives, made up of compounds having a
catechol nucleus with a C 15 or C 17 hydrocarbon side chain attached at the 3 position
of the catechol ring. The side chains can be saturated (e.g., 3-n-pent~AIocyl catechol
and 3-n-heptadecyl catechol) or unsaturated. (See, ElSohly et al. (1982) J. Nat. Prod.
45: 532-538). Immunologically, urushiols function as haptens, in that they are not
recognized by the immllne system unless they are bound to a carrier molecule. Upon
exposure of the epidermis to urushiol, the compound forms a covalent linkage with
(or "haptenates") endogenous proteins. The haptenated proteins are most likely
recognized and processed by macrophages, then presented as antigens in a complexwith major histoco,llpat~bility complex (MHC) molecules, which complexes, in turn,
are recognized by Iymphocytes, leading to a classic DTH ,esponse.
Currently the most effective means to prevent contact sensitivities and other
hapten-m~Ai~t-od h~ ensili~rities is to avoid contact with the hapten. In many cases,
however, such as with common industrial products (e.g., lu,~enti~le, cosmetics) or
natural haptens (e.g., urushiol), avoiding the hapten may be impossible for certain
individuals, particularly if their profession involves contact with environments where
haptens to which they are sensitive are prevalent. (See, e.g., with respect to poison
ivy/oak, Oltman and Hensler (1986) Clinics in Dermatology 4:213-216.)
Many lrGatlllents have been proposed for urushiol sensitivity, but most rely on
barrier methods or amelioration of symptoms. See, e.g., U.S. Patent Nos. 4,259,318
(Duhe et al.), 4,451,453 (Lay et al.), 4,663,151 (Waali), 5,011,689 (Misenko), and
5,017,361 (Beall et al.), incorporated herein by reference.
Wo 95/26980 2 1 8 ~ 8 7 3 PCrlUss5/04121
._
Tolerization against urushiol sensitivity has been studied by several
investigators. See, e.g., Watson et al. (1981) J. Pharm. Sci. 70(7): 785-9; Stampf et
al. (1986) J. Invest. Dermatol. 86(5): 535-8; Dunn et al. (1987) J. Invest. Dermatol.
89(3):296-8. Because urushiol itself is quite toxic, tolerization has typically been
5 attempted with better tolerated analogs, such as 3-n-pentadecylcatechol acetate (see,
e.g., Watson et al. (1980) J. Invest. Dermatol. 76(3): 164-70). Such compounds have
several disadvantages, however, including rapid clearance, toxicity, low potency or
poor induction of hyposensitivity to the natural hapten, and short-lived tolerizing
effects.
Compositions and methods for tolerizing individuals against urushiol-induced
contact dermatitis have been proposed by ElSohly et al., U.S. Patent No. 4,428,965
(incorporated herein by reference) which involve the coupling of catechol derivatives
to cell n,~llll"dlle residues (i.e., cell membrane residues from autologous red blood
cells); however such compositions present difficulties in providing uniform
15 pharmaceutical formulations, and the methods are impractical in that separation of
autologous cells and coupling the catechol derivatives to the cell membranes arenecess~ry prior steps to obtaining a useful composition. In addition, the reliance on
autologous cell melllbldnes as a carrier for the catechol compound means provision of
a unique medicine for each individual treated, and no compositions for general use by
20 the urushiol-sensitive population are provided. Also, as with any medicine
inc~ ,olatillg a blood product, stringent sterile processing is required e.g., to elimin~te
components that may carry blood-borne viruses and infections, such as hepatitis B.
Accordingly, there is a need for more effective, highly specific and generally
a~ltnini~trable compositions and therapies for treating contact sensitivity to urushiol
25 and DTH-inducing haptens in general.
Summary of the Invention
The present invention provides a family of compounds for the treatment or
prevention of contact sensitivity to haptens, especially plant-derived haptens such as
30 urushiol. The present invention also provides methods for desen~i~i7ing m~mm~lc
(including hum~n~) to contact sensitivity-inducing haptens, especially plant-derived
haptens such as urushiol.
- Compounds according to the invention generally have two components: (A) a
carrier capable of binding to Class I or Class II major histocompatability complex
35 (MHC) antigens, and (B) one or more hapten molecules, covalently bound to the
peptide carrier. They may be represented by the general formula:
WO 95/26980 2 ~ $~ ~' 7 3 PCT/US9S/04121
(I) A--Bn
wherein A preferably represents a peptide of from 7 to 30 amino acid residues capable
of binding to a Class I or Class II MHC molecule. or a peptidomimetic of similarS function, B leplcsenls a hapten molecule, n Icpr~se~ts 1, 2 or 3, and--represents a
covalent bond linking each hapten molecule (B) to a functional moiety of the carrier
(A) (e.g. an amino acid residue of the carrier peptide.
Preferred compounds according to the invention have the formula:
(II) OH
HO R
A
a~C
b _ n
wherein R is a saturated or uns~u,~ted hydrocarbon radical of &om 0 to 20 carbonatoms (0 carbon atoms signifying that there is no R substituent at all), preferably 4 to
15 carbon atoms, most preferably 6 or 10 carbons atoms; A preferably represents a
20 peptide of from 7 to 30 amino acid residues capable of binding to a class I or Class II
MHC molecule; n represents 1, 2 or 3, preferably 1; and--replcsents a covalent bond
linking each of the catechol moieties (br~c~ted in the forrnula) to an amino acid
residue of the carrier peptide (A) at one of the unsub~liLuled positions a, b or c of the
catechol ring. The R substituent may be branched, unbranched or cyclic, but is
25 preferably unbranched.
The haptenated peptide compounds above can be admini~tered (usually in a
pharmaceutically acceptable carrier or diluent) to m~mm~l~ with a sensitivity (or at
risk of developing a sensitivity) to contact allergens cont~ining haptens to desensitize
them to the hapten. Particularly, the compounds of formula (II) can be used to
30 desensitize m~mm~lc, i.e., elimin~te or at least ~upple3s (reduce the severity of) the
hypersensitivity reaction, to urushiol-induced contact del.llatilis.
Brief Description of the Drawirlgs
Fig. 1 shows selected human MHC Class II binding peptides, designed in
35 accordance with the criteria for a suitable carrier peptide in accordance with the
invention.
wogs/26g80 2 1 8 68 7 ~ PCT/IJSg5/04121
Fig. 2 shows selected murine MHC Class II binding peptides suitable as
carriers in accordance with the invention, as well as the solubility of each peptide in
PBS and the affinities of each peptide for the two H-2kclass II proteins l-Ak and I-Ek
Fig. 3a-b is a graphic representation showing measurements of PDC skin
5 painted induction of DTH (as indicated by ear swelling) alone, and when pretreated
with peptide :PDC conjugates: Fig. 3a depicts the response in C3HtHeN mice and
Fig 3b depicts the response in B IO.A(4R) mice.
Fig. 4a-d is a graphic representation showing the proliferative response of T
cell (measured in CPM) from Iymph nodes (LN) of mice either treated so as to induce
10 T cell down regulation or untreated mice. 4a-b depict responses to various carriers
and carrier:PDC conjugates of A5:7:PDC imml-ni7ed mice (untreated), in C3H/HeN
mice and B lO.A(4R) mice respectively. Panel C and D present the effect of
p,et~eatl,ænt with A5:7:PDC prior to immunization with this conjugate, on the T cell
response of C3H/HeN and B 1 O.A(4R) mice, lesl,eclively (treated).
Fig. 5a-c is a graphic representation of hapten specific T cell responses in
primary cultures from three PBL individuals with recent exposure to poison oak. T
cell proliferation is measured in counts per minute (CPM).
Fig. 6a-e is a graphic representation specific T cell response in a
desen~iti7~tion experiment. Fig. 6a shows specific T cell response of AS:7:PDC
20 primed mice treated with A5:7:PDC and challenged with A5:7:PDC in the presence of
adjuvant. Fig. 6b is the negative control for the experiment showing specific T cell
response of A5:7:PDC primed mice, treated with PBS and challenged with A5:7:PDC
in the presence of adjuvant. Fig 6c is the positive control for the experiment showing
specific T cell response of A5:7:PDC primed mice treated with A5:7 peptide only and
25 challenged with A5:7:PDC in the presence of adjuvant. Fig. 6d is a toleri_ation
experiment performed at the same time showing specific T cell response of unprimed
mice tolerized with A5:7:PDC and c~ .nged with A5:7:PDC in the presence of
adjuvant. Fig. 6e shows specific T cell response of unprimed and untolerized mice
challenged with A5:7:PDC in the presence of adjuvant.
Detailed Description of Invention
The compounds (I) of the present invention are prepared by covalently
- coupling one or more hapten molecules to a carrier such as an MHC Class I or Class
lI-binding peptide, to obtain a peptide/hapten conjugate capable of forming an antigen
- 35 complex with MHC Class I or Class II molecules, which complex, in turn, is
recognized by a specific population of hapten-specific T cells. That is, the
hapten/carrier compounds of the present invention, complexed with MHC Class I or
wo 95,26980 2 1 ~ 6 ~ 7 ~ PCT/US95/04121
Class II molecules, bind to the T cell receptor (TCR) of T cells of an individual
sen~iti7.od to the particular hapten. Such recognition ordinarily leads to T cell
proliferation and the characteristic release of mediators, such as IL-2, however it is
contemplated that compounds according to the invention will be ~dmini~tered in
5 absence of adjuvants, aggregation or other cell stimulatory signals, so as to lead to
non-stim~ tory recognition by the hapten-specific T cells. In this way, the
compounds of the present invention can be used to disrupt the normal proliferation of
hapten-specific T cells or alter the T cell-m~ ed DTH response to the hapten,
resulting effectively in desensitization to the hapten. Whether the specific mechanism
10 of action of these compounds in vivo involves TCR blockade, induction of anergy,
sign~ling of apoptosis, augmentation of T cell reactivity, induction of T cell non-
responsill~ness, or some other theoretical mechanism is not critical to this invention.
~lu~lly selected compounds of formula (I) can be ~-imini~tered to a hapten-sensitive
individual to desen~iti7P it, i.e., reduce or elimin~te the DTH reaction, to the particular
15 hapten the individual sen~iti7~--d to.
The col,lpo~ ds and methods of this invention will now be described in detail
with reference to a preferred class of compounds (formula (II)) and specific
embodiments. It should be recognized, however, that the principles and methods used
to describe the preferred embodiments may be extended from this disclosure to a wide
20 range of hapten/carrier conjugates that will find uses in alleviating sensitivity to a
variety of haptens.
Compounds of the formula (II), above, may be pre~ ed by reacting a carrier
preferably a peptide or peptidomimetic with a hapten-cont~ining reagent to form a
covalent linkage between the two. When the carrier is a peptide or applupfiate
25 peptidomimetic, it is possible to react the hapten-cont~ining reagent with the amino-
terminal end of the peptide carrier, however this is not preferred, as the resulting
hapten substituent may be hindered sterically from proper presentation to T cells.
Care is ordinarily taken to block the terminal groups of the carrier peptide, e.g., by
acetylation of the amino-terminal group and amidation of the carboxyl terminus.
The haptenic component of the compounds of formula (II) are preferably
- catechol derivatives having the general formula:
- - OH
(III) HO~R
WO 9~/26980 2 1 8 6 8 7 3 PCTIUS95/04121
In the above formula (III), R is a C0 20 hydrocarbon substituent which may be
branched, unbranched, or cyclic, and may be saturated or unsaturated. For example,
S R may be n-alkyl or n-alkenyl, branched alkyl or branched alkenyl, cycloalkyl or
cycloalkyl, phenyl, naphthyl, phenylaL~ylene, cyclohexylalkylene, etc. Preferredsubstituents will be unbranched hydrocarbon side chains of 8 to 15 carbons.
Particularly preferred substituents will be limited to fewer than about 10 carbon
atoms, most preferably at least 8 carbon atoms, where decreasing the hydrophobicity
of the side chain is desired: Natural urushiol haptens have a long unbranched side
chain of 15 or 17 carbons, which is extremely hydrophobic; and it is believed that this
hydrophobicity may play a role in the reactivity of the molecule, e.g., by embecl.ling
itself by this side chain in the hydrophobic lipid bilayer of cell ll,ell,bldnes. On the
other hand, where the hydrophobic nature of the substituent is less of a concern,
particularly preferred compounds will employ substituents that are the same as natural
urushiol, i.e., n-alkyl or n-alkenyl substituents of fifteen or seventeen carbons. Of
particular interest is n-pentadecyl catechol (PDC), i.e., the catechol derivative of
formula (III) wherein R is n-pentadecyl.
Catechol compounds can be synthesized using techniques known in the art
For example, PDC can be synth~si7~od according to the procedure of Dawson and Ng,
Org. Prep. Proc. Int. (1978) 10:167-172, or by that procedure with the modifications
as described in the working examples, below. Heptadecyl catechol (HDC) can be
synth~si7~d in an analogous manner. Alternatively, urushiols can be isolated from
plant tissue (see, e.g., ElSohly, M.A. et al. (1982) J. Natural Products 45:532-538).
The catechol compounds are then reacted with a peptide carrier to form a covalent
linkage, preferably at a side chain of one or more of the peptide amino acid residues.
For this purpose, amino acid residues having a nucleophilic side chain and having a
reactive group such as amino (-NH2), hydroxyl (-OH), or sulfhydryl (-SH) in its side
chain will typically be used. Lysine and cysteine are especially useful for this type of
conjugation, and therefore peptide carrier molecules including Iysine or cysteine
amino acid residues will be most preferred.
Generally, the compounds (II) of the invention may be prepared by reacting R-
substituted catechol derivatives (formula (III)) with a peptide carrier under conditions
that will lead to the formation of a covalent bond between the catechol molecule and
- 35 one amino acid residue of the peptide. If the peptide includes more than one residue
capable of forming a covalent linkage to the catechol compound, multiple
substitutions on the peptide can be accomplished. As will be discussed in greater
wo g5/26g80 2 1 ~ 6 8 7 3 PCT/USg5/04121
detail below, the positioning of the substituent on the peptide and the number of
substituents may have a significant effect on the potency of the conjugate. Preferred
compounds according to the present invention will have one and at most two hapten-
substituents.
S The catechol compounds may be reacted at the unsubstituted ring carbons(positions a, b and c in formula (II)) by first oxidizing the compound, e.g., with silver
oxide (Ag20), to produce a benzoquinone interm~ te. Reaction of the interm~ te
with a cysteine-cont~ining peptide generally leads to formation of a covalent bond at
the ring position ~ ent the R group (position c in formula (II)); reaction with a
lysine-cont~ining peptide generally leads to substitution at the 5-carbon (position b)
of the catechol ring. (See, e.g., Liberato et al. (1984) J. Med. Chem. 24:28-33).
The carrier peptides h~pten~tPd according to the invention must bind to an
MHC Class I or Class II molecule on antigen presenting cells. MHC Class II
molecules are normally expressed only on B Iymphocytes, macrophages, dendritic
cells, endothelial cells, and a few other cell types. An antigenic peptide binds to a
MHC molecule within a cleft on the surface of the MHC molecule. The cleft on thesurface of a MHC Class II molecule can accommodate peptides of various differentlengths. Peptides eluted from mouse and human MHC Class II molecules average 15-18 amino acid residues in length. (Hunt et al.(l992) Science 256:1817; Chicz et al.
(1993) J. Exp. Med. 178:27). MHC Class I molecules are generally present on all
nuc!~o~t~d cells and present processed protein antigen to CD8+ cells (cytotoxic T
cells). Class I molecules bind peptides of 8 to 9 amino acid residues in length and
som~timt-s 10 to 11 amino acid residues in length (Hukzo et al, J. Immunol.,
151:2572) for pres~nt~tion to antigen specific cytotoxic T cells.
Without wishing to be limited to any theory, it is believed that CD8+ as well
as CD4+ cells are effectors contributing to DTH response. Thus MHC class I
conjugate carriers which are capable of being recognized by CD8+ T cells as well as
MHC class II conjugate carriers which are capable of being recognized by CD4+ T
cells are useful in the methods of the present invention for targeting the various T
cells which are involved in the DTH response to urushiol compounds. However, it is
believed that it is possible to regulate and modulate CD4+ and CD8+ concerted T cell
response by precise targeting of the pertinent CD4+ T cell population alone, using
only an MHC class II carrier conjugate in accordance with the invention (see,
Examples 3, 4, and 5).
Peptides selected to serve as carriers for the haptenated peptides of the
invention will comprise about 7 to 30 amino acid residues, preferably about 9 to 20
amino acid residues, more preferably 9 to 15 and most preferably 9 to 13 amino acid
Wo 95/26980 2 1 8 6 8 7 3 PcrluS9S/04121
.
residues. The carrier peptide must also contain at least one amino acid residue
capable of reacting to form a covalent bond with a hapten (or reactive hapten
interm.odi~te). Lysine and cysteine are most preferred.
The present invention also includes the use of peptide-like molecules as
5 carriers such as peptidomimetics which function in a similar manner to those peptides
just described. Such peptidomimetics may incorporate unnatural amino acids, or may
include modified linkages between consecutive residues. Replacement of peptide
bonds with amide isosteres and transformation of the secondary structure of peptides
into non-peptide molecules is also contemplated (see, Goodman and Seonggu
10 Peptidomimetics for Drug Design: Burgers Medicinal Chemistry and Drug
Discovery, 5th ed.: Volume 1: Principles of Drug Design, Mandfred E. Wolff (ed.), pp
803-861 (January 1995) J.Wiley and Sons, New York). Peptidomimetics may be
particularly suitable as a carrier in that they have more lasting effects in biological
~y~ s. For example peptides are subject to attack by enzymes in biological systems.
15 A peptidomimetic which includes amide bond isosteres resemble the amide bonds of
conventional peptides but are more resistant to enzymatic cleavage in vivo.
Peptidomimetics capable of binding class II MHC are described in the literature (Hill
et al., J.lmmunoL, 152:2890-2898 (March 1994). Specific peptidomimetics suitable
as carriers in the instant invention are those which include non-peptide bonds and
20 peptide bond analogs e.g. N-methyl amide bond (NH-C2[-CO NCH3-]Cal) and
reduced bond analogs (NH-Ca2[-CH2-NH-]C1).
The individual amino acids of the carrier peptide may be characterized in one
of three categories, depending on their relative positions within the peptide and their
interaction with MHC molecules and TCRs: (1) several amino acids, usually 2 or 325 but usually not more than 7, will form an "agretope", which is a single unit of
recognition that binds to one MHC molecule or one family of MHC molecules related
by a consensus sequence; (2) several amino acids, usually 2 or 3 but usually not more
than 7, will form, after haptenation, an "epitope", which is the basic element of
recognition by a receptor, e.g., a T cell receptor; and (3) the remaining amino acids,
30 usually 4-10 amino acids, in addition to providing spacing for the agretopic and
epitopic residues, also contribute to interaction of the peptide backbone with the
MHC molecule are neither involved in binding to the MHC molecule nor to the T cell
receptor, other than to provide spacing bet~ewl the agretopic and epitopic aminoacids. The amino acids making up the agretope may be separated or contiguous in the
35 sequence of the peptide. Likewise, the amino acids which together form the epitopic
recognition sequence may be located together or separated by one to several, usually
1 to 4, amino acids. So long as binding to a MHC molecule is accomplished and at
wo gsn6980 ~ 1 ~ 6 8 7 3 PCr/USss/04121
least one site suitable for haptenation is provided, the identity and exact sequence of
the amino acids of the carrier peptide are not important.
In any particular MHC-binding peptide (e.g., a peptide of a known amino acid
sequence that binds to a known MHC molecule), the amino acid positions within the
peptide can be mapped as (1) MHC contact residues, (2) TCR contact residues or (3)
"neutral" residues not directly involved in the MHC-binding or TCR recognition
using standard techniques known to those skilled in the art. (See, e.g., Rothbard and
Gefter (1991) Ann. Rev. lmmunol. 9:527-565; and Jorgensen et al. (1992) Ann. Rev.
Immunol. 10:835-873, incorporated herein by reference). For example, the amino
acid residues of an antigenic peptide can be systematically substituted with other
residues and the substitllt~d peptides can then be tested in two different assays. In one
assay, the ability of the substituted peptides (in excess) to compete with the wild-type
peptide for binding to an MHC molecule on the surface of an antigen p,t;senlil-g cell
is ~Csesse-~ Alternatively, the ability of the substituted peptides to directly stim~ t~
antigen-specific T cells is ~csesse-l A peptide having an amino acid sub~ ulion at a
position involved in binding of the peptide with the MHC molecule will exhibit
altered binding capacity relative to the wild-type peptide. In contrast, a peptide
having a substitution in an amino acid residue at a position involved in contact of the
peptide with the T cell receptor will still be able to compete with the wild-type
peptide for binding to the MHC molecule but will have altered ability to stim~ te
antigen-specific T cells (since the substituted peptide can still bind to the MHC
molecule but is recognized differently by the T cell receptor). Finally, a neutral
amino acid position can be substituted with many amino acids without significantly
affecting the ability of the peptide to compete with the wild-type peptide for MHC
binding and to directly stimlll~e antigen-specific T cells.
The ability of wild-type and sul ~ti~uled peptides to bind to MHC molecules
can also be directly ~csessed using labeled peptides and purified MHC molecules in
well known binding assays, such as equilibrium dialysis, column binding assays, and
the like. (See, e.g., Sette (1987) Nature 328:395-399).
Several MHC-binding peptides suitable as carriers for haptens are known.
They may be entirely synthetic or derived from the sequence of natural proteins, such
as hen egg Iysozyme (HEL), bovine or human serum albumin (BSA, HSA),
ovalbumin (OVA), influenza hemagglutinin, and many others.
Although there are differences between and MHC class I and Class II binding
molecules, there are also similarities (Rothbard, JB, Curr. Biol. 4:653-655 (Jul. 1994).
Thus, with these similarities and differences in mind it is possible to choose or design
a Class I carrier using a similar analysis as described below for the design or choice of
wo 95,26980 2 1 a 6 8 7 3 PCTIUS95/04121
a Class II MHC binding carrier. For example Martin et al., J. Immunol.,151:678-687
(Jul, 1993) describe a murine MHC class I H-2b binding peptide which when
conjugated to the hapten, TNP, are recognized by TNP hapten-specific T cells.
Examples of peptide carriers specific for Class II MHC includes a peptide
of hen egg Iysozyme (HEL) sp~nning amino acid residues 52-61 can be used. It
is known that this peptide can bind to I-Ak and that the amino acid side chains at
positions S3, 56 and 59 are T cell receptor contact residues. (See, Evavold et al.
(1992) J. Immunol. 148:347-353). In the native HEL peptide, position 53 is a
tyrosine residue, position 56 is a leucine residue and position 59 is an asparagine
residue. Catechol compounds, such as PDC, or another hapten can be directly
coupled to a Iysine or other reactive amino acid substituted for leucine at position
56. The tyrosine at position 53 and the asparagine at position 59 can also be
reacted to covalently bind to a hapten molecule or can be substituted with Iysine
or cysteine, and then reaction can take place more readily at the substituted amino
acid.
An example of a peptide that binds to murine MHC Class II molecules
and is suitable as a carrier peptide is a peptide in a murine system derived from
the murine Class II MHC E chain, spanning from residues 54-66. This peptide
can bind to I-Ab or I-Ak (see binding data in Fig. 2) A residue at position 60 is a
T cell receptor contact residue. Catechol compounds (III) or other haptens can be
coupled to a lysine or other reactive amino acid substituted at position 60 for the
native amino acid (leucine).
An example of a peptide for binding to a human MHC Class II molecule
is a well-characterized peptide derived from influenza H3 hemagglutinin. The
peptide enColnr~cces amino acid residues 307-319 in the natural protein and has
the following amino acid sequence:
Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr (SEQ. ID. NO. 53)
2 3 4 5 6 7 8 9 10 11 12 13
- This peptide is known to bind to the human MHC Class II molecules HLA-DRl
and HLA-DR4 with an affinity in the low nanomolar range. T cell receptor
contact residues have been mapped to positions 308 ~position 2 above), 310
(position 4 above), 311 (position 5 above), 313 (position 7 above) and 316
- 35 (position 10 above) by substitution analysis (Krieger et al. (1991) J. Immunol.
146:2331-2340) and from cystallographic studies (Brown et al. (1993) Nature
364:33-39). The amino acid residues at positions 308, 311 and 316 are Iysines.
Wo 9s/26980 2 1 8 6 8 7 3 Pcrluss5lo4l2l
Therefore, catechol compounds (or other haptens) can be advantageously coupled
to the side chain of one or more of the Iysines at the these positions by standard
techniques. Additionally, the valine residue at position 310 can be replaced with
an amino acid residue having a reactive side chain, preferably Iysine or cysteine,
- 5 and a catechol compound can be coupled to the substituted amino acid residue.
Similarly, the asparagine residue at position 313 can be reacted with a catecholcompound to haptenate the peptide, or it can be replaced with another reactive
amino acid, preferably Iysine or cysteine, and a catechol compound can be
coupled to the substituted amino acid residue.
Based on prior studies of the structures of antigenic peptides and investigationon peptide requirements for MHC class II binding (Rothbard et al., lnt. Arch. Allergy
Immunol., 105:1-7 (Sep. 1994)), a general structure for preferred carrier peptides can
be discussed. The most preferred carriers for Class II MHC binding will be 9 to 13
amino acids in length, and the amino acid residues that are the targets for linking to a
15 hapten molecule will be spaced at specific relative positions within the molecule. lt is
believed that these particular positions within a peptide are preferentially recognized
(i.e., contacted by) the T cell receptor on antigen-specific T Iymphocytes regardless of
which protein the antigenic peptide is derived from. It is at these positions that hapten
molecules, e.g., catechol compounds, may be most advantageously bound. The
20 pattern of the amino acid residues in such preferred MHC Class II-binding carrier
peptides can be represented as follows:
(IV) (Xaa)n-Zaa-Xaa-Xaa-Zaa-Xaa-Xaa-Zaa-(Xaa)m
4 7
In the above motif, Xaa and Zaa are amino acid residues, and Zaa ,c~ sel,~ a target
residue for hapten linking. One or more of the Zaa amino acids, therefore, will have a
reactive side chain and will most preferably be a Iysine or cysteine residue. The Xaa
amino acids are selected independently and can each be different from the other. It is
30 most preferred that the Xaa amino acid at position 3 be a phenylAl~nin~, tyrosine or
isoleucine. The designations n and m rep,esent integers from 1 to 3, with n + m
equaling 2 to 6.
While the above structure and the values for n and m provide an optimal
peptide for MHC binding, much larger peptides contAining this motif (i.e., n and/or m
35 is a larger integer, e.g., up to 30 or more) could also readily bind to MHC molecules.
It should be recognized, therefore, that in synthesizing or selecting the carrier peptide
including the above structure (IV) that many additional residues can be added to the
WO 9S/26980 2 1 8 ~ 8 7 3 PCT/Ub5S~ 121
amino terminal and/or carboxy terminal ends without detrimentally affec~ing MHC
binding. Indeed, addition of more amino acid, such as polyalanine tails, may be
desirable to secure advantages unrelated to MHC binding, for example increasing the
serum half-life of the compound.
As indicated above, the Zaa amino acids are selected to provide reaction sites
for the hapten molecules while having a position within the MHC-binding carrier
peptide which places them in the optimal position for presentation to the receptor
complexes of T Iymphocytes. Any position except for position 3 are potentially
suitable for reaction sites for the hapten molecule. However prefellt;d positions
include positions 4, 7, 9, and 11. The most preferred Zaa position along the MHCbinding carrier peptide is position 7 (see Examples). The Xaa amino acids are
selected to provide suitable residues for binding to the MHC Class II antigen (i.e.,
agretopic residues) and residues for m~int~ining the spatial relationship of the other
residues involved in either MHC binding or T cell receptor p.esel.~;ll;on. The precise
combination of Xaa residues employed depends in part on the specific MHC allele to
which the MHC-binding peptide is desired to bind. For many antigenic peptides ofknown sequence, the identity of amino acid residues that are necess~ry for MHC
binding has been determined. (See, e.g., Rothbard and Gefter (1991) Ann. Rev.
lmmunol. 9:527; Evavold et al. (1992) J. Immunol. 148:347; Krieger et al. (1991) J.
Immunol. 146:2331; and Sette et al. (1993) J. Immunol. 151:3163). In addition, for
any peptide of a known amino acid sequence which binds to an MHC molecule, one
skilled in the art can map which amino acid residues at which amino acid positions
are necess~. y for binding of the peptide to an MHC molecule and which amino acid
positions can accommodate substitution with other amino acids (see, e.g. Alexander et
al., Immuni~y, 1:751-761 (1994)).
F.~peci~lly preferred peptides according to the above formula (IV) will have n
= 3 and m = 1, 2 or 3. Lysine is most preferred for one or more of the Zaa positions,
and tyrosine, isoleucine or phenyl~l~nine will preferably be at position 3. As will be
cussec~ below, all the other residues may advantageously be alanine residues.
For example, further studies on the influenza H3 hemagglutinin peptide
described above have been pelro~ ed to determine which amino acid residues are
illl~ol~lt for binding to HLA-DRl. It was found that a synthetic peptide cont~ining
only two residues from the native hemagglutinin peptide embedded within a chain of
poly~l~ninPs retained the ability to bind to HLA-DRl (Jardetzky et al. (1990) EMBO
J. 9:1797-1803). Thus, most of the amino acids within the peptide can be subs~ e~
e.g., with alanine residues. The synthetic HLA-DR l-binding peptide comprises an- amino acid sequence:
wo gs/26g80 2 1 ~ 6 8 7 3 PCT/US9S/04121
Ala-Ala-Tyr-Ala-Ala-Ala-Ala-Ala-Ala-Lys-Ala-Ala-Ala (SEQ. ID. NO. 54)
2 3 4 5 6 7 8 9 10 11 12 13
5 This synthetic peptide can also be used as a carrier for catechol compounds or other
haptens to produce haptenated peptides according to this invention. For example, the
Iysine at position 10 above can be haptenated. Furthermore, the alanines at T cell
contact positions, e.g., positions 4 and 7 in the above formula, can be substituted with-
amino acids having reactive side chains, preferably Iysine or cysteine, to provide
10 additional haptenation sites along the peptide. Particularly preferred carrier peptides,
therefore, which can be readily haptenated with catechol compounds and which bind
to a human MHC Class II molecule (e.g., HLA-DRl) have the following amino acid
sequence:
Ala-Ala-Tyr-Zaa-Ala-Ala-Zaa-Ala-Ala-Lys-Ala-Ala-Ala (SEQ. ID. NO. 55)
2 3 4 5 6 7 8 9 10 11 12 13
wherein 7~ is alanine or an amino acid residue having a nucleophilic side chain but
most preferably Iysine or cysteine.
Another synthetic polyalanine-based peptide which retains the ability to bind
to human MHC Class II molecules has similarly been defined and can be used as a
hapten carrier. This peptide has the amino acid sequence:
Ala-Ala-Ile-Zaa-Ala-Ala-7~-Ser-Ala-Zaa-Ala-Ala-Ala (SEQ. ID. NO. 56)
1 2 3 4 5 6 7 8 9 10 11 12 13
wherein Zaa is alanine or an amino acid residue having a nucleophilic side chain,
preferably Iysine or cysteine. Catechol compounds or other haptens can be coupled to
the peptide at T cell receptor contact residues, e.g., at positions 4, 7 and/or 10 of the
peptide (most preferably position 7), to produce haptenated peptide compounds
accordlng to thls mventlon.
It is further predicted that peptides can be designed which will bind to
- many different haplotypes of a MHC molecule. For example, previous studies
have shown that the same "promiscuous" peptide sequence can be eluted from
DR2, DR3, DR4, DR7, and DR8 molecules. (See, Chicz et al. (1993) J. Exp.
Med. 78:27-47). These "promiscuous" peptide sequences can be used as the
basis for designing haptenated peptides which can bind to many different MHC
14
W095,26g80 2 1 86873 PCr/US9S/04121
haplotypes (i.e., "universal binders"), using techniques discussed above. Peptides
- identified as "universal" or "nearly universal" binders are discussed in Marhall et
al. J. Immunol. 152:4946-4947 (May 1994) and in Alexander et al., supra. A
single haptenated peptide based on the polyalanine backbone discussed above
S will bind to almost all DR alleles at an affinity below 200 nM. Use of a single
carrier peptide, particularly a universal binder, as a hapten carrier would
elimin~ major disadvantages of prior methods for treating urushiol contact
sensitivity that employ autologous cell membranes as carriers: Use of a single
peptide as a substrate for the hapten would permit formulation of uniform
10 p~cpalations and would avoid the use of blood products, which are susceptible to
co~ ..in~tion, llnmit~le for general ~flmini.ctration and complicated to
formulate due to the need for m~intaining sterility at all manufacturing stages.The haptenated peptides of the invention have a further advantage in that they are
addressed directly to the cells of the immllnç system, i.e., they are intended to
bind directly to Class II-bearing APCs, and thus are believed to more efficiently
bring about their effects on the immllne system, e.g., in that more of the
~mini~tered dose of the compound will be targeted to the specific cell
populations involved in the hapten-specific hypersensitivity.
Preferred carrier peptides which have been modified or designed in
accordance with the invention to bind many different MHC haplotypes using
formulas and techniques discussed above include, but are not limited to, the following
peptides TTK (SEQ. ID. NO.I), TTK:7 (SEQ. ID. NO. 2), C03 (SEQ. ID. NO. 3),
C03:7 (SEQ. ID. NO. 4), DR002:0 (SEQ. ID. NO.S), DR002:7 (SEQ. ID. NO. 6),
DR003:0 (SEQ. ID. NO. 7), DR003:7 (SEQ. ID. NO. 8), DR004:0 (SEQ. ID. NO. 9),
DR004:7 (SEQ. ID. NO. 10), DR005:0 (SEQ. ID. NO. 11), DR005: 10 (SEQ. ID. NO.
19), DR005: 11 (SEQ. ID. NO. 20), DR005: 12 (SEQ. ID. NO. 21), DR005:2 (SEQ.
ID. NO. 12), DR005:4 (SEQ. ID. NO. 13), DR005:5 (SEQ. ID. NO. 14), DR005:6
(SEQ. ID. NO. 15), DR 005:7 (SEQ. ID. NO. 16), DR005:8 (SEQ. ID. NO. 17),
DR005:9 (SEQ. ID. NO. 18), DR006:0 (SEQ. ~D. NO. 22), 006:7 (SEQ. ID. NO. 23),
007:0 (SEQ. ID. NO. 24), 007:7 (SEQ. ID. NO. 25), 008:0 (SEQ. ID. NO. 26),
DR009:0 ~SEQ. ID. NO. 27), DR010:0 (SEQ. ID. NO. 28), DR011:0 (SEQ. ID. NO.
29), DR011.7 (SEQ. ID. NO. 30), DR012:0 (SEQ.ID. NO. 31), DR013:0 (SEQ. ID.
NO. 32), DR014:0 (SEQ. ID. NO. 33), EILAOOl:O(SEQ. ID. NO. 34), HLA001:4
(SEQ. ID. NO. 35), HLA001:5 (SEQ. ID. NO. 36), HLA001:6 (SEQ. ID. NO. 37) all
- 35 as shown in Fig. 1. Preferred peptides include DR005:7 (SEQ. ID. NO. 16), DR011 :7
(SEQ. ID. NO. 30), TTK:7 (SEQ. lD. NO. 2), and C03:7 (SEQ. ID. NO. 4) all as
shown in Fig. 1. Of the above peptides, those peptides indicated by peptide #:0 (e.g
W095/26980 2 1 8 6 8 7 3 Pcr/uss5lo4l2l
DR008:0, DR009:0), are considered "parent" peptides designed in accordance with
the invention. Such parent peptides may be substituted with lysine or cysteine at
preferred T cell contact positions (e.g. positions 4, 7, 10) in accordance with the
invention for ease of conjugation to a hapten molecule.
Additionally, peptidomimetics useful in accordance with the present invention
may be based on any of the above peptides or "parent" peptides.. For example, a
peptidomimetic based on DR005:0 (SEQ. ID. N0. I 1) may have the following
structure:
D-A-I-A-S*A-Q$A-A$A-N$E
wl.c~hl [-] is a normal peptide bond, [*] is an N-Methyl amide bond or a normal
peptide bond and [$] is a reduced bond analog or a normal peptide bond. It is
con~....p!~t~ that a peptidomimetic may include one or more [*] or[$] bonds or
15 various combinations thereof.
The MHC-binding carrier peptides can be produced by any convenient means
of peptide synthesis, but are preferably produced by chemical synthesis. See, e.g.,
Stewart, J.M. and Young, J.D. (1984) Solid Phase Peptide Synthesis 2nd ed. (Pierce
Chemical Co., Rockford L). Advantageously, the peptides may be synthesized on an20 automated peptide synthesizer using solid phase chemistry. It is contemplated that for
ease of synthesis various chemical groups may be included at the amino or carboxy
terminus. For example the amino terminus of a carrier peptide may be acylated.
Chemical groups which may be included at the carboxy terminus of a carrier peptide
include but are not limited to free acid, amide, and methoxy-ester. Peptidomimetics
25 may be produced as described in Goodman and Seonggu, supra.
In p,~pa,ing the catechol derivative-~ul,~ uled peptides of formula (II), it
may be desirable to decrease the hydrophobicity of the hydrocarbon side chain (R).
Data suggests that the hydrocarbon side chain at position 3 of the catechol nucleus of
urushiol is important in adherence and entry into the skin, while the catechol head
30 group is responsible for haptenization of endogenous proteins. (See, Baer et al.
- (1967) J. Immunol. 99:370-375). This lipid side chain can be truncated so as to be
much less hydrophobic and more highly soluble in aqueous environments. Such
compounds may also have decreased toxicity as compared to the natural urushiol,
since the natural toxicity of urushiol is associated with the ability of the long,
35 lipophilic side chain to interact with cell membrane lipids. Catechol compounds with
altered hydrocarbon side chains can still be recognized by urushiol-specific T
Iymphocytes. Thus, the use of substituted catechol compounds (III) having R
16
wo gs/26g80 2 1 8 ~ 8 7 3 PCT/USs5/04121
.
substituents of, e.g., ten carbons or fewer will yield haptenated peptides exhibiting
- enhanced p~ope~lies (e.g., increased solubility, decreased toxicity) without
elimin~ting the ability of the hapten to be recognized by hapten-specific T cells.
Other modifications which may be made to increase solubility include modification
S with polyethylene glycol (PEG) using the method of Wie et al., Int. Arch. Allergy
and Appl. Immunol., 64:84-99 (1981), to produce a peptide conjugated with PEG. In
addition, PEG can be added during chemical synthesis of a peptide of the invention.
Solubility may also be enhanced by including various chemical groups at the amino
terminus of the peptide during peptide synthesis or by chemical reaction after
synthesis. Such chemical groups include but are not limited to: sulfoacetyl,
phosphonoacetyl, lactobionyl, and carboxymethyl-polyethyleneglycol-methoxy
ethers of varying lengths.
As liccll$sed earlier, the present invention also contemplates the use of a non-
peptide carrier which may be bound to a non-MHC encoded molecule capable of
presenting antigen to T cells. Specifically, the carrier may be a lipid which can bind
to certain classes of CDl molecules which specialize in presenting certain lipidantigens to cytotoxic T cells. Recently, an antigen present~tion function has been
proposed for CD1 molecules, a family of non-MHC-encoded, non-polymorphic, ~-2
microglobin-associated glycoproteins found on most professional antigen plesehling
cells (reviewed by Parham, Peter, Nature, 372:615-616 (December 15, 1994)),.
Bec~m~n et al, Nature, 372:691-694 (1994) recently report that the CDlb moleculepresented a lipid antigen of Mycobacterium tuberculosis to human a~ T cells.
Bec~m~n et al, demonstrated that, unlike MHC Class I and Class II molecules, theantigen presented by CDlb was not a peptide, but rather a long-chain fatty acid. This
demonstrated that human T cells can recognize antigen of a totally non-protein
nature, and secondly, that lipid antigens can be presented by a specific molecule
found on antigen presenting cells.
In view of this, it is possible that CDlb molecules participate in the T cell
recognition of urushiol for the following reasons. First urushiol is a family ofcompounds that possess 15-17 carbon-long sidechains that impart lipophilic
plo~,lies to the molecules (reviewed in Tyman, Chem. Soc. Rev. 8:499-537, (1979).
Second, CDlb molecules are found on Langerhans cells, the major antigen-p,rse~ g cells of the human skin (Knapp et al. (eds.), Leucocyte Typing fV. White Cell
differentiation Antigens, Oxford University Press, (1989)). Finally, although T cell
35 responses to urushiol include conventionally-restricted CD4+ and CD8+ T cells
(Kalish, J. fnvest. Derrnatol. 90: 108s-l l ls (1990)), additional T cell responses to
CDlb-presented lipids cannot be ruled out. CDlb molecules may either present
WO 9S/26980 2 1 8 6 8 7 3 PCT/Ub9S~ 121
urushiol directly by the lipid portion of the molecule, or alternatively, the urushiol
may couple to other lipids that would then be presented by CDlb. To test this, the
CDlb molecule can be transfected into cells that lack MHC class I or class II
antigens, as reported in Fig. 4 of Beckman et al, supra. These cells can then be5 exposed to either purified urushiol, or to lipids or lipid extracts from cells that had
previously been reacted with urushiol. Populations of T cells from urushiol-sensitive
individuals could then be tested for the ability to recognize the urushiol-exposed
versus unexposed CDlb-transfected antigen-presenting cells. Verification of thisspecificity would require selection of a CDlb-restricted T cell line or clone as done
10 by Beckman et al., supra. Such T cells may have a significant role in hypersensitivity
to urushiol.
Thus, a CDlb binding lipid carrier may be used as a carrier molecule for
conjugation to a catechol (formula III) in accordance with the invention. Such alipid-carrier h~pten~ted molecule, when ~mini~tered to a hypersensitive individual
15 as described below, may be capable of particir~ing in the desensitization of
individuals suffering from hypersensitivity to the contact allergen.
The haptenated compounds of the invention can be ~dminictered alone or
incoll~olaled into a composition comprising a haptenated compound and a
pharm~eu~ically acceptable carrier or diluent. The term "pharm~relltically
20 acceptable carrier or diluent" is intended to include substances capable of being co-
~minictered with the active ingredient (haptenated MHC-binding peptide) and which
will not hinder its activity. Examples of such carriers and diluents include sterile
buffered saline solutions, solvents, dispersion media, delay agents, and the like. The
use of such media and agents for pharm~ceu~ic~lly active substances is well known in
25 the art. Any conventional agent compatible with the haptenated peptide can be used
with this invention.
The haptenated compounds of the invention are useful in the treatment of
contact sensitivity, e.g., to dese-n~iti7e a hypersensitive individual against exposure to
a contact allergen. The haptenated peptide compounds of the invention can also be
30 used to desensitize hapten-ser~ i7~d m~mm~l~, including hum~n~ The term
"hapten-sensitized" is used to refer to an individual susceptible to a hapten-specific
sensitivity and who has been previously exposed to the hapten. Desensitization of a
- hapten-sen~i~i7~d individual means that the hapten-sensitized individual will not
produce a hapten-specific DTH response upon exposure to the hapten or will produce
35 a response of ~limini~hPd intensity to untreated subjects. Such hapten-specific contact
sensitivity reactions are well understood and can be visually evaluated, e.g., using the
Draize system. (See, U.S. Patent No. 4,428,965). Furthermore, Example 2G
18
Wo 95/26980 2 1 8 6 8 7 3 PCT/US95/04121
describes murine desen.ci~i7~ion experiments using haptenated compounds of the
invention.
The haptenated peptides described above may be used to treat contact
sensitivity to environmental haptens. Specifically, the catechol compound-substituted
5 peptides described above can be used to treat contact sensitivity to urushiol. A
urushiol-sensitized m~mm~l can be desensitized to the urushiol contact allergen by
a~lmini~tration of a haptenated peptide in non-immunogenic form. It is believed that
the haptenated peptides, when ~minictered in non-immunogenic form induce hapten-specific non-responsiveness by causing hapten-specific T cells to become
10 nonresponsive to the hapten. The ability to induce antigen-specific T cell
nom~sl,onsiveness in a subject by ~llmini~ering to the subject a peptide derived from
the antigen has been documented both with antigens involved in allergic responses
(e.g., Fel d 1, see Briner et al. (1993) Proc. NatL Acad. Sci. USA 90:7608-7612) and
in autoimmnne ~ e~ces (e.g., experimental autoi.. ~i-.o encephalomyelitis; see
Smilek (1991) Proc. Natl. Acad. Sci. USA 88:9633-9637 and Wraith (1989) Cell
59:247-255). The invention further encompasses a composition for inducing hapten-
specific T cell nonresponsiveness in a subject comprising at least one haptenated
peptide as described previously. In addition, compositions of the instant invention
may include one or more haptenated peptides of the invention. Such compositions
20 may be ~mini~tered simultaneously or sequentially. In order to maximize the down-
regulating effect on hapten-specific T cells, it may be advantageous to ~lmini~er
several hapte~llated molecules specific for each class of hapten-specific T cell in a
single composition or multiple compositions which can be ~dmini~tPred
siml.l~neously or sequentially. For example, one haptenated molecule may comprise
25 an MHC Class I binding peptide, another haptenated molecule may comprise an
MHC Class II binding peptide and yet another haptenated molecule may comprise a
CDlb-binding lipid. In this manner a range of T cells which may participate in the
hypersensitive response to urushiol (e.g. cytotoxic T cells which recognize a peptide
bound to a Class I MHC, helper T cells which recognize peptide bound to Class II30 MHC, and cytotoxic T cells which recognize lipid bound to CDlb) may be targeted
for down regulation thereby enhancing the desensitizing effect of the compounds of
the invention.
The haptenated molecules of the present invention can be ~mini~tered in a
convenient manner such as by injection (subcutaneous, intradermal, intravenous,
35 intldpeliloneally, etc.), oral, nasal sublingual or rectal ~lrnini~tration~ or by inhalation.
Preferred routes of ~-lmini.ctration are intravenous injection and subcutaneous
injection. Depending on the route of ~mini.ctration, the peptide can be incorporated
19
wo gsl26g80 2 1 ~ -~ 8 :7 3 PCT/US95/04121
into a material to protect it from the natural conditions which may detrimentally affect
its ability to perform its intended function, such as action by enzymes, acids, and
other natural conditions which detriment~lly affect the peptides. Examples of suitable
carriers include but are not limited to physiological saline for injectable forms; starch,
S sucrose, lactose, gelatin, m~gn~ .cium stearate, acacia for oral dosage forms (solid);
water and edible oils such as peanut oil for oral dosage forms (liquid). The
haptenated peptide compound is ~dmini~tered in a non-toxic dosage concentration
sufficient to induce T cell non-responsiveness or to desensitize the subject to the
allergenic effects of the contact allergen, e.g., urushiol. The actual dosage will be
10 determined by recognized factors such as body weight, age, severity of the allergic
reaction, potency or activity of the particular active compound to be ~ ninictçred,
factors peculiar to the individual patient such as sensitivity to other medications, and
other such factors f~mili~r to those who practice in this area. An effective dosage and
the desenciti7.ing effect can be readily determined by the medical practitioner in
15 accordance with conventional techniques in the medical art.
The present invention is further illustrated by the following examples which
should in no way be construed as being further limitin~ The contents of all cited
publications not previously inco,yor~lt~d are hereby expressly incorporated by
reference. All amino acid sequences referred to herein are written in N-terminal to C-
20 terminal order.
EXAMPLE 1: T cells spe~ifi~ for PDC respond to PDC-substituted carrier
peptides
3-n-pentadecyl catechol (PDC) is a major component of poison ivy urushiol
and is capable of inducin~ and eliciting delayed-type h~el~ensilivity rashes.
(ElSohly et al. (1982) J. Nat. Prod. 45: 532-538; Baer et al. (1967) J. Immunol. 99:
370-375)
Synthesis of ~ent~dçcyl catechol
Synthesis of 3-n-pçnt~decyl catechol (PDC) was carried out according to the
procedure described by Dawson and Ng in which veratrole (1,2-dimethoxybenzene)
was first m~ ted using n-butyl lithium and reacted with l-bromopent~de~ne to
form 3-n-~en~ecyl veratrole (3-PDV). (See, Dawson and Ng (1978) Org. Prep.
Proc. Int. 10:167-172). Boron tribromide was then used to cleave the methyl ether
groups of PDV, forrning 3-PDC. A noteworthy modification in the present protocolwas the use of flash chromatography in purification of the 3-PDV and 3-PDC instead
wo gs/26980 ~ 3 PCT/US95/04121
of vacuum distillation. Structures of both 3-PDV and 3-PDC were confirrned by
- melting point, I H NMR and FAB-MS. The newly prepared 3-PDC had an Rf in TLC
analyses identical to an authentic sample obtained from the Bureau of Biologics,
FDA.
Coupling PDC to peptides and proteins
The regiospecificity and reactivity of PDC coupling to protein carriers has
been studied previously. (Liberato et al. (1984) J. Med. Chem. 24: 28-33). In general,
full-length proteins and a peptide were coupled to PDC covalently through either the
-amino group of Iysine or the sulfhydryl group of cysteine by first converting the PDC
to the o-benzoquinone using Ag20 as an oxidizing agent in THF. The freshly
p~ ,d o-benzoquinone solution was filtered directly into the peptide solution indimethylsulfoxide (DMSO) with stirring. After the removal of the reaction solvent,
the residue was taken up in water and extracted with ethyl ether to remove the excess
PDC. The PDC-~ubsliluled products were further purified by silica gel
chromatography using chloroform:ethanol:water as the solvent system. The structure
of the purified products were confirmed by amino acid analysis and mass
spectroscopy.
The carrier proteins, mouse IgG (ATCC accession no. HB 183), hen egg
Iysozyme (HEL, Sigma Chemical, St. Louis MO), or ovalbumin (OVA, Sigma
Chemical, St. Louis MO) (in range of 7-140 nanomoles) were reacted with excess
PDC (16 llmol) in pure DMSO at room te-l-pe~dture using air oxidation of the
catechol. The reaction was monitored spectrophotometrically; a characteristic
absorbance peak at about 480 nm developed as expected. (Liberato et al. (1984) J.
Med Chem. 24: 28-33). PDC was bound to an MHC-binding peptide from murine E
a protein, residues 54-66 (FEAQGALANIAVD (SEQ. ID. NO. 43) in single-letter
code), following the same procedure, after substitution of a Iysine for the leucine at
position 60 (FEAQGAKANL~VD) (SEQ. ID. NO. 45).
A monoclonal antibody was prepared against PDC by standard procedures:
Balb/c mice were immllni7~d with PDC-coupled IgG in Ribi adjuvant. Splenocytes
were fused with the SP2/O myeloma and hybridomas selected in HAT medium. A
monoclonal IgM antibody was identified which recognized PDC-substituted but not
un~ubsLiLuted IgG.
The anti-PDC monoclonal antibody was used in immunoassays of the PDC-
carrier preparations. The results showed that the anti-PDC antibody showed binding
to the PDC-substituted mouse IgG, but not the unsubstituted (control) mouse IgG
(data not shown). PDC-substituted proteins were precipitated and extracted with
Wosst26980 2 1 8 6 8 7 3 Pcrlussslo4l2l
methanol, Iyophilized and digested with trypsin before use in T-cell assays. These
treatments were found to remove toxicity in cellular assays, most likely due to
residual PDC, and to increase the solubility of the preparations.
Murine T cell responses to PDC-peptides
The ability of Class II MHC-restricted, PDC-specific T cell clones to
recognize an arbitrary peptide coupled with PDC was examined. T-cell hybridoma
clones were produced by fusing T cells derived from C3H mice that had been
imml-ni7ed with either HEL-PDC or murine IgG-PDC, and restimul~ted three days inculture with the ~ntigen~ HEL-PDC or HEL-catechol before fusion. Resulting T
hybridomas were screened for their ability to secrete interleukin-2 (IL-2) in res~,onse
to syngeneic APCs (CH27, see, e.g., Pennell et al. (1985) Proc. Natl. Acad. Sci. USA
82:3799-3803) and HEL-PDC or HEL-catechol. Two PDC-specific T hybridomas
were cloned: 9/29 F6 and 3.15.
The two independently-derived I-Ak-restricted hybridoma clones were then
tested for their ability to recognize an "~bil,~y" peptide coupled with PDC (i.e., a
peptide carrier to which the T cell hybridomas had not been previously exposed). The
peptide used for restimlll~tion was derived from the MHC Class II Ea chain, amino
acid residues 54-66. This peptide is known to bind to I-Ab (Rudensky et al. (1992)
Nature 359:429-31) and has now been discovered to bind to I-Ak.
T hybridoma 9/29 F6 was assayed for L-2 production in the presence of
APCs and PDC-~ubsli~ul~d or unsubstituted Ea peptide. Peptides in two-fold serial
dilutions were added to cultures cont~ining CH27 cells as APCs. T cells were then
added and 24-hour ~u~el~atallts tested for IL-2 using HT-2 indicator cells (murine
helper T cell line, ATCC accession no. CRL1841). Quantitation was pe.rolllRd using
the Alarnar~ Blue dye assay kit (Alamar, Sacramento CA). The data, illustrated in
Figure 3, show that the PDC-substinlt~l. Iysine-modified peptide Eas4 66
(K60:PDC-Ea, having the amino acid sequence: FEAQGAK(PDC)ANIAVD (SEQ.
ID. NO. 45), with PDC bound to the Iysine residue substituted at position 60) was
recognized (in a dose-dependent manner) by the 9/29 F6, PDC-specific T hybridomacultured with various concentrations of the K60:PDC-Ea peptide. The PDC-specificT hybridomas did not recognize the unhaptenated control Ea peptide
(FEAQGAKANIAVD) (SEQ. ID. NO. 45).
T hybridomas that were specific for PDC (3.15) and for HEL (11.6) were
assayed for L-2 production in the presence of CH27 APCs pulsed with PDC-
substituted Ea peptide, control (unsubstituted) Ea peptide, or full length HEL protein.
CH27 cells were incl-b~ted with 4 ~g/rnl of either control Ea peptide or K60:PDC-Ea
woss/26980 2 ~ 8~ 8 ~ PCT/US95/04121
peptide, or HEL protein at 20 ~g/ml, for 16-20 hours. The antigen-exposed CH27
cells were washed and diluted in two-fold steps from 1.2 x 105 cells/culture. T
hybridoma cells (5 x 104 cells/culture) specific for PDC (3.15) or specific for HEL
( I 1.6) were added to the cultures and after 24 hours supernatants were collected and
S analyzed for IL-2 release using HT-2 cells and Alamar(~) Blue dye.
The results (data not shown) indicated that APCs pulsed with the K60:PDC-E
- peptide were recognized by the PDC-specific 3.15 hybridoma, but not by the HEL-
specific, I-Ak restricted 11.6 hybridoma. Neither hybridoma recognized the
unhaptenated control Ea peptide; and APCs pulsed with HEL protein were
10 recognized only by the 11.6 hybridoma. These results establish that PDC as a hapten
can be recognized by T cells when coupled to an arbitrary peptide of appropriateMHC binding capability.
EXAMPLE 2: ~rt~n-spe~ c down regulation of DTH re~ se induced by
15 PDC in a murine model
The purpose of the following expelil"el,t~ is to study MHC class II restricted
T cell responses to urushiol haptens in a mouse model with an H-2k MHC
background. Synthetically prepared 3-pent~lecyl-catechol (PDC) which contains a
20 saturated 15 carbon sidechain and is a major component of urushiol, was used as the
hapten in these studies. Liberato et al. J. Med. Chem. 24:PDC is known to couple to
peptides and proteins specifically through cysteine and Iysine residues. The following
experiments specifically address the position of the hapten on the carrier peptide, the
cross-reactivity of synthetic hapten conjugates with t cells induced from skin painting
25 with the hapten alone, and whether or not such synthetic compounds can down-
regulate in vivo DTH responses induced by cutaneous exposure to the free hapten.
PROCEDURES:
30 Molecular Modelin~
A model of the binding of peptides to the I-Ak molecule was developed based
on the crystal structure of the HLA-DRI molecule con~ining the hemaglutinin
peptide HA 307-319 (Wiley, Stominger et al.,) using the QUANTA modeling package
from Molecular Simulations, Inc
- Peptide Synthesis:
wo gs/26980 2 1 8 6 ~ 7 3 PCr/USs5/04121
Peptides were synthesized employing solid phase techniques, either using an
Applied Biosystems Peptide synthesizer or an Advanced Chemtech robotics system
utili7.ing FaxtMOCTM chernistry with commercially available Wang resins, and Fmoc
protected amino acids, as previously described. Fig. 2 lists the names and sequences
of the peptides used in this study. For synthetic ease, all of the peptides used were
acetylated at the amino terminus and were amidated at the carboxy terminus. All
peptides were purified using reverse phase HPLC and their structures were confirmed
by amino acid analysis and fast atom bombardment mass spectrometry.
Synthesis of TNP-Coupled Peptides:
The synthesis of these conjugates was done while the peptides were still
coupled to the solid phase support using standard protocols Good et al., Selected
Methods in Cellular Immunology, B Mishell and S.M. Shiigi (eds), p 343 (New York,
1980).
Synthesis of PDC-Coupled Peptides
A quantity of 0.03 mmol of the desired carrier peptide was dissolved in dry
DMSO:DMF (1:5 v:v, approximately 10ml.). While the peptide was dissolving, a
stock solution of PDC converted to its quinone form was prepared by dissolving 0.05
mmol of PDC in 2.5 ml of MeCN, followed by the addition of 1.35 mg of powdered
AgO. This mixture was swirled for 25 minutes, then filtered through a 0.1 ~lm PTGE
filte directly into the peptide solution. The resulting mixture was ~git~ted (rocked) for
4 hours at room te.l.pe.~lt~re.
After the agitation was stopped, the excess PDC/quinone was removed by the
slow addition of 20 ml of dry diethyl ether (added in 1.0 ml aliquots with swirling)
over the course of one hour. The resulting p-ccipa~e was separated from the ether
solution, washed twice with ether, and dissolved in 50C tli~tilled water. After the
aqueous solution had cooled, it was extracted twice with dichlorometh~ne. Then the
aqueous solution was dried (rotary evaporation) and was purified by reverse phase
chromatography (C-18 HEMA cartridges, Alltech) using either acidic (TFA-water-
acetonitrile) or basic (diisopropylethylamine-water-acetonitrile) solvent systems, and
the purity of the material was ~csesse~ by mass spectroscopy. Final yields ranged
from 28% to 79% based on starting peptide.
Purification of I-Ak Proteins
The procedures used for detergent solubilized and affinity purification of H-2-
I-Ak molecules were sirnilar to those described by Babbit et al., Nature, 317:359
.
24
wo gst26g80 2 1 8 6 8 7 3 PCT/USg5/04121
,
(1985). Briefly, the B Iymphoblastoid cell line CH27, which expresses I-Aka and
- chains, was grown in RPMI 1640 medium supplemented with 10% heat inactivated
FCS, 2 mM glut~min-~" and antibiotics. Cells were harvested, washed in PBS, Iysed
with 1 % NP-40 and the supernatant separated from nuclear debris by centrifugation.
The solubilized MHC class II proteins were affinity purified using the monoclonal
antibody, 10.3.6.2 (28), coupled to Sepharose, after being passed through a Sepharose
CL-4B precolumn. MHC class II proteins were eluted with 1 % octyl-~-D-
glucopyranoside (octyl glucoside), S0 mM phosphate at pH 11.5, and imm~ tely
neutrali_ed using 1 M phosl)hate at pH 6.0, then concentrated to approximately 1mg/ml using an Amicon stirred cell concentrator. Finally, the concentrated protein
solution was fractionated by gel filtration in PBS + 1.0 % octyl glucoside + 0.2%
NaN3 using a 75 x 2.5 column of BioGel A0.5 (BioRad), and collecting the fractions
that corresponded to the molecular weight of the a~-heterodimer (nominal 60 kD).The purity of the final material was assayed by SDS PAGE, HPLC gel filtration, and
Edman sequencing.
MHC Class II Peptide Binding and Inhibition Assays:
For direct binding assays, an optimal concentration of affinity purified I-Ak(35nM) was incubated with serial dilutions of a peptide HADP7.47,
AAYKAAKAAAAAA (SEQ. ID. NO. 38), modified with long chain biotin at the
amino terminus (NLCB:HADP7.47) in PBS cont~ining 0.02% dodecyl maltoside at
pH 5.5, in 96 well polypropylene plates (Costar) for 16-20 hours at 37C. In studies
optimi7ing the assay, between 5 to 10% of the I-Ak molecules were capable of
binding added peptide. Therefore the effective concentration of I-Ak was
approximately 3.5 nM. The conditions of the assays were shown to be in ligand
excess, because a two-fold reduction (of these class II molecules) did not change the
measured ED50 values. The I-Ak -peptide complexes (50 111) were transferred, in
duplicate, to wells of a 96-well microtiter plate precoated eith the monoclonal
antibody- 10.3.6.2 and blocked with heat inactivated fetal calf serum. An additional
50 ,ul of 50 mM TRIS pH 7.0 cont~ining 0.02% Tween 20 and 0.05% NaN3.
Europium labeled streptavidin (Pharmacia) was added and incubated overnight. After
washing, complexes were measllred by the addition of 0.1 M acetate/phth~l~te buffer,
pH 3.2, cont~ining 0.1% Triton X-100, 15 llM 2-naphthoyltrifluoroacetone and 50 ~1
M tri-N-octylphospine oxide, which released the ch~l~ted eul~ph~ from steptavidin
35 and formed a highly fluorescent micellar solution. The resultant fluorescence was
measured using a fluorescent plate reader (DELPHIA, LKB/Pharmacia). The data
wo gs/26g80 2 1 ~ 6 8 7 3 PcrluS9S/04121
was analyzed using a fitting algorithm to a theoretical binding equation that calculated
the concentration of peptide giving a half-maximal signal (ED50).
The inhibition assay format was identical to the procedure described above
with the exception that the unlabeled peptide antagonist was serially diluted and
5 incubated with constant concentrations of NLCB:HADP7.47 and the I-Ak protein.
The concentration of unlabeled peptide that prevented 50% of the labeled peptidefrom binding was the IC50 value. The concentration of the NLCH:HADP7.47 used in
each assay was experim~n~lly determined to be at least one sixth of its measuredED50 in order to assure the inhibition as primarily measuring the binding
10 characteristics of the competitor peptide. This was confirmed by comparing the IC50
of unlabeled HADP7.47 (SEQ. ID. NO. 38) to the measured ED50 of the labeled
peptide, demonstrating that the IC50 solely ll-ea~uf~d the affinity of the antogonist for
the receptor and was independent of the presence of the agonist. However, the IC50
and ED50 values are referred to being equivalent to the KDaPparent and not a true KD,
15 because of the known decomposision of MHC class II molecules during the course of
the assay (Sette et al., J. Immunol., 148:844 (1992); Barany and Merrifield, ThePeptides, E.Gross and J. Meienhofer, eds. Academic Press, New York). Therefore,
the incubation time was chosed to permit sufficient amount of complex to be formed
to allow ease of detection, but was minimi7Pd to limit the amount of decomposition of
20 the receptor. The presence of endogenous peptides makes the assay an exchangereaction, rather than a simple binding event. Consequently, the inherent
characteristics of the MHC molecules prevent the assay from ever ~ ining true
equilibrium. However, by balancing the time of incubation, the KDaPparent can
approximate the true KD.
Animals:
C3H/HeN/HeN [Kk-IAk I-Ek Dk] mice were purchased from Simonsen Labs
(Gilroy, CA.). BlO.A(5R) [Kk-Ak Db], AT.L [Ks I-As/k I-EkDd], C57Bl/6J [Kb I-
AbDb], B10.A(5R) [Kb l-Ab I-Eb/dDd], PLJ/SJL [KU/s I-AU~s I-EU/s DU/S] mice were30 purchased from Jackson Labs (Bar Harbor, Maine).
Cells were obtained from draining inguinal or popliteal lymph nodes (LN) of
variously treated mice. Syngeneic spleen cells [irradiated 3000 rads and lysed with
Red Blood Cell Lysing Solution ~SIGMA)] were used as antigen presenting cells
(APCs). Syngeneic spleen cells (prepared as above) were hapten conjugated at room
35 temperature for 30 minutes by exposure to 20 rnM purified PDC, purified urushiol, or
TNP chloride. Following extensive washing with PBS, the hapten conjugated
preparations were used as a source of antigen.
26
W095126980 21 86873 PCT~ 35,~121
T cell cultures were supplemented with recombinant mouse IL-2 (40 units/ml
- from Genzyme, Cambridge, MA) after 3 to 4 days and every 2 to 3 days thereafter.
Cultures were re-stimulated with fresh APCs and antigen every 10 to 14 days.
T cell Activiation Assays:
T cells (2x104 per well) and APCs (5.105 per well) were incubated in 96 well
microtiter plates with a penal of antigens and applol,liate controls for 72 hours. 3H
thymidine (0.5 mCi/well) ~!vas added for an additional 6 to 8 hours of incubation
before harvesting the plates and assessing the incorporated 3H thymidine ( 1205
Betaplate liquid scintillation counter, LKB Wallac) indicated in counts per minute
(CPM).
Alternatively, supern~t~n~ from the cultures were harvested after 24 hours of
incubation with antigen. L-2 dependent HT-2 cells (5000 cells/well) were added to
the culture supernatants and incub~ttod for 16 hours, and the amount of 3H thymidine
incorporated by the cells was determined as in the previous paragraph.
PDC Induced DTH Response in the H-2k Mouse Model:
Mice (age-m~tched for each separate experiment) were sensitized by the
application of PDC dissolved in DMS0 (500 mg/ml) and diluted in acetone (2 mg in100 ~11) to the shaved lower abdomen. Mice were routinely challenged 4 days after
sensitization by the application of the catechol dissolved as above (30 ~lg in 10 ~1) to
both the dorsal and ventral sides of the right ear. The left ear of each mouse was
challenged with the solvent (acetone:DMS0) alone as an irritation control. Ear
thickness was measured using a spring-loaded micrometer. Results were expressed as
the difference in swelling between the right ear and the left ear in units of 10-2 mm.
In experiments where other variables were tested, the following controls were
included: "primary response" (negative control group) mice were skin-painted with
acetone:DMS0 and challenged as above; and "secondary response" (positive controlgroup) mice were painted with PDC and ch~llenged as above.
Anti~en-Specific T Cell Tolerization:
According to the protocol published by G. Shearer and M. Gefter (31 ) mice
were treated with soluble antigen (25-100 ~g per mouse) ~lmini~tered wither
intravenously or subcutaneously 10 and 5 days prior to the challenge. The challenge
is given subcutaneously (25-100 ~lg/mouse) in RIBI adjuvant (RIBI Immunochemical,
Hamilton, MT).
W09st26980 21 86a73 PCT/US95/04121
ANALYSIS:
A. Carrier Modeling and Synthesis:
Carrier Modeling was based on the logic that by knowing the prop~.~ies of a
5 peptide that are required for binding to a specific MHC class II protein, as well as
which residues seem to be generally more important for T cell recognition, it should
be possible to design a peptide that will specifically bind the desired MHC class II
allele? have little or no immunogenic plopellies, and provide suitable reactivity for
conjugation of a desired hapten at a position that would optimally interact with C cell
10 receptors. Additional constraints of high solubility and aqueous stability were
imposed because of the biological necessity of having a soluble compound (carrier:
hapten conjugate) for injection into ~nim~l~ or addition to cell culture.
The choice of the I-Ak binding parent peptides HEL 52-61 and IEa 54-66
were made because of their known immunological behavior (31). The requirements
15 for I-Ak binding were det~ led by ..,ea~ult;hlg the binding of peptides (based on
these two parents) that contained single amino acid substitutions at each position of
the parent peptide (nominally 19 peptides for each position of the parent sequence),
and then elucidating the most probable ~lignm~nt of the peptide sets (unpublished
data), similar to the SAR (structure activity relation) study of the I-AU system20 (described in 32). Using the ~lignm~nts obtained in this fashion, along with the
published crystal structure of the human MHC class II allele DRB 1*0101 (33), a
semi-qu~ntit~ive model of I-Ak:peptide complexes was form~ te~l
Using several computer generted models (not shown) of the I-Ak molecule
including 2 different peptides (HEL 52-61 (SEQ. ID. N0. 41) and I-Ea 54-66 (SEQ.25 ID. N0. 43)) complexed with the molecule at various angles, it appeared likely that
residues in the peptide not closely associated with the I-Ak molecule were necessary
requilelllents for the ability of the peptide to bind the MHC Class II molecule. The
binding data for the single amino acid variants of HEL 52-61 (SEQ. ID. N0. 41) and
I-Ea 54-66 (SEQ. ID. N0. 43) support the latter assumption. Given this information
30 and assumptions, the carrier peptide series A5 (Fig. 5) was design~ted and
syn~hesi7~-d with Iysine at the fourth, seventh, or tenth position (peptides A5:4 (SEQ.
ID. N0. 47), A5:7 (SEQ. ID. N0. 48), or A5:10 (SEQ. ID. N0. 49) respectively)
since these positions are, hypothetically, the most exposed to the TCR (being the least
involved with the I-Ak molecule). The rem~ining residues were determined to be
35 necessary for peptide binding, increasing the solubility of the carrier, and minimi7ing
the exposure of the peptide side chains to putative TCRs.
28
Wos5/26980 ~l;B~8~ PCr/USs5/04121
Fig. 2 lists the peptides made in this study, as well as their solubility in PBS- and their affinities for the two H-2k class II proteins I-Ak and I-Ek. Studies showed
that substitution of Iysine in the carrier sequence has little effect on the affinity of the
peptide for I-Ak (data not shown). Studies also indicated a relative insensitivity of
peptide binding to I-Ak when PDC was conjugated to the A5 carrier series (data not
shown). The data in Fig. 2 shows that it is possible to m~int~in or even improveMHC class II binding while dramatically increasing the solubility of the peptide.
These results support the deterrnination of which residues are important for MHCclass II binding. Fig. 2 illustrates that the I-Ek molecule, in general, binds peptides
with a much higher affinity (lower IC50 value) as compared to I-Ak. This
phenomenon has been well documtontecl in the literature and somewhat complicatescomparison of peptide binding to the two MHC class II proteins. However, the
important observation is that the peptides designed to specifically bind I-Ak do not
bind I-Ek significantly, and vice versa.
B. The Role of the peptide carrier in Class II binding and T cell recognition
In order to dissect the molecular specifity of T cell recognition of PDC
murine T cell lines specific for PDC were established.
Restriction analysis, utili7ing spleen cells from different mouse strains as
APC's, revealed that the recognition of A5:7:PDC (SEQ. ID. NO. 48) conjugate is
restricted by I-Ak (data not shown). In addition, the T cell line was shown, by FACS
analysis, to be CD4+ and CD8- (data not shown).
A T cell line established from lymph nodes of C3H/HeN rnice immllni7ed
with A5:7:PDC (SEQ. ID. NO. 48) show specificity (data not shown). Two discrete
peptide calriers with PDC conjugated at the 7th position-A5:7:PDC (SEQ. ID. NO.
48) and I-Ea 54-66, K60:PDC (SEQ. ID. NO. 45) activated the T cell line as
indicated by 3H-thyrnidine uptake. In contrast, PDC conjugation at different positions
on the carrier-A5:4:PDC (SEQ. ID. NO. 47), A5:10:PDC (SEQ. ID. NO. 49), or the
carriers alone gave virtually no response (data not shown). Possible toxicity ofAS:4:PDC (SEQ. ID. NO. 47) and AS:lO:PDC (SEQ. ID. NO. 49) conjugates or
- rnitrogenicity of AS:5:PDC (FEDQKSLENIARD (SEQ. ID. NO. 61)) were ruled out
by testing viability of T cells and using naive lymph nodes or an irrelevant, I-Ak
restricted T cell line, respectively. These results indicate that conjugation position is
crucial for T cell recognition of the hapten. Control peptide:hapten conjugates, 35 consisting of HLA-DR (DR2:7 (SEQ. ID. NO. 6)) of I-Ek (ES:7 (SEQ. ID. NO. 51))
binding peptides with PDC anchored to the 7th position, did not elicit a proliferative
29
wo gs/26980 2 1 8 6 8 7 3 PcrluS9S/04121
response. These results confirm the importance of MHC binding property of the
carrier that is a imperative for PDC-specific, I-Ak restricted T cell recognition.
Syngeneic spleen cells, conjugated with PDC or urushiol, but not TNP, also
stim~ ted this T cell line (data not shown), demonstrating that T cells from
A5:7:PDC (SEQ. ID. NO. 48) immllni7~d mice respond to PDC, but not to TNP,
when it is conjugated to a variety of carriers. This suggests that these T cellsrecognize a common determinant in the multiple carrier:hapten conjugates. The
results of these experiments indicate that the hapten-specific T cell line, generated
using a single carrier coupled to PDC, recognize the hapten in a carrier independent
manner.
C. T cells induced by PDC skin-painting can be activated in a hapten-specif~lc
fashion by a rationally designed peptide:PDC conjugate
The hapten urushiol binds to epidermal cells, both keratinocytes and
Langerhans cells. Langerhans cells conjugated with urushiol then migrate throughIymphatics to Iymph nodes, where they present antigen (ha~t~,na~d peptides) to Tcells and thereby induce allergic contact sensitivity (twenty-five) to determinewhether a hapten specific T cell response, to the carrier:PCD conjugate (A5:7:PCD
(SEQ. ID. NO. 48)) can be recalled from draining LN of mice sensitized with PDC
following the skin painting. The following studies were conducted.
T cells from draining LN of PDC skin-painted mice were stimulated by
AS:7:PDC (SEQ. ID. NO. 48) (data not shown). An unrelated hapten conjugated ontoan I-Ak binding carrier (HEL 52-61, K56:TNP (SEQ. ID. NO. 41)), as well as carrier
peptides alone, failed to activate these T cells, indicating that PDC sensitization elicits
a hapten-specific T cell response. Recognition of A5:7:PDC (SEQ. ID. NO. 48)
conjugate by LN T cells from PDC skin painted mice supports the hypothesis that T
cell recognition of the hapten can be independent of carrier sequence.
Data showed that T cells primed by PDC skin painting respond to A5:7:PDC
but not A5:4:PDC (SEQ. ID. NO. 47) nor A5:10:PDC (SEQ. ID. NO. 49) (data not
shown), suggesting that T cell recognition of the hapten is restricted to a specific (7th)
position on the carrier.
The lack of response to PDC linked at positions other than the seventh could
be due to inferior immunogenicity, low precursor frequence or both. Attempts to
generate PDC specific T cell lines by immllni7~tion with the A5:4:PDC (SEQ. ID.
NO. 47) or A5: lO:PDC (SEQ. ID. NO. 49) were nn~ccessful, while imrnunization
with the compound A5:7:PDC (SEQ. ID. NO. 48) generated potent, PDC-specific T
wo g5/26980 2 1 S ~ ~7 3 Pcrlussslo4l2l
cells. Therefore, the seventh position of the carrier peptide appears to be the most
favored site for hapten recognition by T cells
D. T cells in PDC s~ ed mice fail to recognize PDC in the context of an I-
5 Ek binding peptide
C3H/HeN mice express both I-Ak and I-Ek class II MHC molecules. In order
to determine whether a hapten-specific, I-Ek restricted T cell response in mice
sensitized with PDC can be obtained, a similar experiment as described in section C
was conducted, employing I-Ek binding peptide conjugated to PDC at positions 4th,
10 7th or 10th.
T cells from draining LN of C3H/HeN mice sensitized with PDC responded to
A5:7:PDC (SEQ. ID. NO. 48) but not E5:4:PDC (SEQ. ID. NO. 50), E5:7:PDC (SEQ.
ID. NO. 51) or E5:10:PDC (SEQ. ID. NO. 52) (data not shown). ~mml-ni7~tion with
the above conjugates did not yield a PDC specific T cell. These results suggest that
15 the PDC specific T cell repertoire, triggered by hapten conjugation of cutaneous
proteins from PDC skin-p~inting, is predominantly composted of I-Ak restricted Tcells.
E. Antigen-~pe~ifir~ down-regulation of DTH induced by PDC can be
20 achieved by treatment with a single rationally designed peptide-conjugate.
To determine whether a class II binding peptide conjugated to PDC can be
used to down regulate a PDC-induced DTH response, C3H/HeN and BlO.A(4R) mice
were injected with PDC conjugated to different carrier peptides prior to PDC were
challenged. Figures 3a and 3b ,~ sent two separate experiments. Tlc~n~ t with
25 A5:7:PDC (SEQ. ID. NO. 48) reduced the DTH response down to background level,indicating that pre-Ll~allnellt with A5:7:PDC (SEQ. ID. NO. 48) results in
downregulation of the effector T cells m.~ ting the DTH lespol-se. T,eatlllent with
the same carrier peptide coupled to PDC at a different TCR contact residue failed to
do so. Additionally, the relatively insoluble carrier:PDC conjugate (I-Ea 54-66,30 K60:PDC (SEQ. ID. NO. 45)) with the hapten conjugated at the preferred TCR
contact position, failed to decrease the DTH response in BlO.A(4R) mice. This
failure to induce downregulation of the DTH response may have been due to the
insolubility of the compound.
35 F. Treatment with peptide:PDC conjugate abrogates the hapten specific T
cell proliferative response, and correlates with ~imini.ched IL-2 secretion.
W095/26980 2~ a6873 PCT/US95/04121
To further dissect the downregulation of DTH by peptide:PDC conjugate, the
invitro response of cells from LN of pre-injected mice were tested. C3H/HeN and
B IO.A(4R) mice were treated with either A5:7:PDC (SEQ. ID. N0. 48) ("tolerized")
or PBS ("control"), followed by immuni7~tion with the above conjugate in the
presence of adjuvant (RIBI). T cells from draining LN were tested with a panel of
peptide:hapten compounds. Tmmnni7ed mice demonstrated a vigorous hapten-
specific T cell response to A5:7:PDC (SEQ. ID. N0. 48) (Fig. 4a and b). Again,
hapten-specific T cells exhibited a narrowly defined specificity that did not cross react
with the other conjugates containing PDC at a different TCR contact residue. A
carrier specific response was not ~e~ected even following deliberate immllni7~tion,
indicating that we used an nonimm~mogenic peptide carrier, ot achieve predominantly
a hapten-specific T cell response. T cells from the "tolerized" group failed to
proliferate following a challenge with the A5:7:PDC (SEQ. ID. N0. 48)
peptide:hapten conjugate (Fig. 4c and d). T cell responses to allogeneic cells or Con-
A were similar in the tested groups (data not shown). The above result suggests that
treatment with a class II binding peptide coupled to PDC at a discrete position can
induce T cell unresponsiveness to the hapten-peptide conjugate.
Failure to proliferate was correlated with ~liminich~cl IL-2 secretion (data notshown). Exogenous IL-2 added to LN T cells from mice tolerized with A5:7:PDC
(SEQ. ID. N0. 48) restored anti-PDC reactivity in a dose dependent fashion (2 and 5
stim~ tion index at a concentration of 2.5 and 5 U/ml recombinant mouse IL-2,
respectively) (data not shown). In spite of the increase of T cell proliferative response
in the untreated group, an incignific~n~ change of the stimnl~tion index was observed
(10, 10 and 11 stimlll~tion index at a concentration of 0, 2.5 and 5 U/ml recol..binant
25 mouse L-2, respectively). The above result suggests that the unresponsiveness to
PDC, induced by treatment with A5:7:PDC (SEQ. ID. N0. 48) conjugate, can be
reversed by the additin of exogeneous IL-2, a m~.ch~nicm consistent with T cell non-
responsiveness .
Mitogenic as well as allogeneic responses (mixed lymphocyte reaction - MLR)
30 in the tolerized group were similar to the control group (data not shown) indicating
that the state of in~uce~ u.~leslJonsiveness is confined to the peptide:PDC conjugate
alone and is not ~csoci~ted with general immuno~u~ c~sion.
Similar results were obtained when mice received a challenge of PDC-skin
painting (data not shown); T cell responses to A5:7:PDC (SEQ. ID. N0. 48)
35 following PDC skin painting (generating diverse species of hapten conjugated
proteins) were down-regulated by pre-treatment (tolerization) with A5:7:PDC (SEQ.
wo 95/26980 2 1 8 6 8 7 3 Pcr/uss5/04l2l
._
ID. NO. 48), indicating that the tolerance persist even after challenge with varied
hapten conjugates.
G. D~s~n i~ Complete Abrogation of PDC-specific T cell response in
5 A5:7:PDC primed C3H mice
Treatment of PDC-sensitized (primed) mice with a single class II binding
peptide:PDC conjugate (A5:7:PDC (SEQ. ID. NO. 48)) was shown to attenuate
completely the hapten specific T cell response.
A desensitization experiment was conducted according to the following
protocol. C3H mice were primed by injection with 25 ~g of A5:7:PDC (SEQ. ID.
NO. 48) conjugate in the presence of Ribi (an adjuvant). One month lager the mice
received 4 weekly injections of A5:7:PDC (SEQ. ID. NO. 48) conjugate (25 ~
g/injection in PBS, subc~lt~nloously ). Two weeks post ~ nt, the mice were
challenged with 25 llg of A5:7:PDC (SEQ. ID. NO. 48) conjugate in the presence of
Ribi. Lymph node T cells from the A5:7:PDC (SEQ. ID. NO. 48) desensitized group
failed to respond to the haptenic compounds (Fig 6a). Lymph node T cells from
primed mice treated with PBS or with the carrier peptide (A5:7 (SEQ. ID. NO. 48))
alone exhibited a vigorous PDC specific response (Figs 6b and Fig. 6c, respectively).
Responses to allogenic antigens (MLR) or to Con-A mitogen were intact for all
groups.
The efficacy of the tre~tmtont in primed mice (desensitization experiment, Fig.
6a) was similar to, if not better than the tre~tm~.nt in naive mice (tolerization
experiment, Fig. 6d). Similar tolerization experiments are described above in
Example 2F. For comparison, Fig. 6e shows the vigorous response of T cells from
unprimed and untolerized mice challenged with A5:7:PDC (SEQ. ID. NO. 48) in the
presence of adjuvant.
H. Cross tolerization of PDC specific r~cponce u~sing a distinct carrier
c~ to PDC at position 7.
In order to demonstrate that the down regulation of the hapten specific T cell
response is-indeed carrier independent and hapten-specific, a distinct, I-Ak binding
carrier, A6:7 (having the amino acid sequence YDDNGAKQNAAER (SEQ. ID. NO.
62)) was coupled to PDC at the 7th position and was used for cross tolerization. C3H
mice were treated with A5:7:PDC (SEQ. ID. NO. 48) or A6:7:PDC (SEQ. ID. NO.
62) peptide using the same protocol (2 s.q. injections) and challenged with A5:7:PDC
(SEQ. ID. NO. 48). Treatment with A6:7:PDC (SEQ. ID. NO. 62) conjugated was
shown to induce unresponsiveness to A5:7:PDC (SEQ. ID. NO. 48) challenge (data
WO95/26980 21 86873 PCr/US9S/04121
not shown), indicating that the tolerizdtion is specific to the hapten and is independent
of the carrier peptide.
F.Y~rle 3: Hapten~e~ peptides which bind human MHC molecules and
5 s~im~ e hapten specific T cell responses
PROCEDURES.
Design /Selection of Carrier Peptides for peptide:PDC conju~ation
A set of peptides was designed to bind a broad range of ~A DR alleles. The
peptides were tested for binding to HLA class II (using represent~tive DR alleles) and
their solubility was ~csessed (see, earlier Examples). In addition, two natural
peptides, complement C3 precursor 753-756 and Protein kinase TTK818-830 were
selected based on a ~ b~ce criteria using multiple criteria including the following:
15 protein must be a humdn protein, must be a unique se.~uence, must have a solubility
coefficient that is less than 0 etc. All of the peptides de-sign~d or chosen for this study
are listed in Fig. 1. One peptide, DR005 (SEQ. ID. NO. 11) was selected and
conjugated with PDC at the 7th position as described in Examples 1 and 2. This
peptide:PDC conjugate triggered hapted-specific T cell response in DR4Dw4
20 transgenic ~nim~l.c (data not shown) and therefore was put to use in the human T cell
proliferation assay system described below.
Initiation of Urushiol-Specific T Cell Cultures
Blood samples (~75ml) received from 73 individuals with poison-ivy/oak
25 hypersensitivity, following contact with to poison oak 5-90 days earlier. T cell
cultures were initi~ted from T cells (5Xl04/well in 96 well Costdr plate) separated
from the blood following centrifugation on a Ficoll gradient and stim~ ted with
urushiol haptenated autologous cells at a concentration of 25 ~LM in AIM-V medium
without serum. The urushiol used herein was obtained from the FDA. Urushiol
30 haptenated autologous cells were prepared as follows: Briefly, 2X106 cells separated
from the blood, resuspended in 200~LI of PBS and incubated with 10mM of the hapten
(urushiol) for 30 minutes at room tel,lpe.d~ule. The cells are washed three times with
PBS, resuspended in 200~1 of AIM-V and sonicated for 30 seconds using tip
sonicator. On day 7 the T cell cultures are fed with fresh AIM-V medium containing
35 l0U/ml of rH IL-2 and split or transferred to 24 well Costar plates when necessary.
Each patient was given a patient number and each T cell line is referred to with regard
to the patient that was timulated with one antigen.
WO 95/26980 2 1 ~ 3 PCI/US95/04121
T cell cloning:
On day 11 - 14 of the culture T cells are seeded in 96 well Costar plates at a
concentration of 0.3 cells/well in the presence of 2x104 JY EBV cells (4000R) and
2x105 PBLs (3000R) in AIM-V medium containing I ~g/ml o PHA and 20U/ml of rH
L-2.
Fourteen days later the growing clones from the 96 well plates are transfered
to 24 well plates and are expanded with using the following mixture suspension:
2x105 JY EBV cells (4000R) and 2x106 PBLs (3000R) in AIM-V medium cont~ining
1 ~g/ml of PHA and 20 U/ml or rH L-2.
The specificity of the clones is tested (on day 11-14 to the culture) with
urushiol-haptenated cells at concentration ranging from 100~M to 3.3 ~lM in
duplicates using 2 fold dilutions. Positive T cell clones are then further tested with
various peptide:PDC conjugates to define their fine specificity.
Proliferation Assay
Responder cells are rested without rH IL-2 for 3-4 days prior to the assay. On
day 11-14 of the T cell culture, the T cells are collected washed three times to remove
L-2. The cells are then added at 5x104 cell/well to U-shape 96 well plates along with
lx105 irradiated (3000R) autologous peripheral blood mononuclear cells. Antigensprepared from urushiol, PDC, PIXT (poison ivy extract) or TNP h~ptçn~ted
autologous cells are tested in various concentration ranging from from 100 ~lM to 3.3
IlM in duplicates using 2 fold dilutions. Preparation of haptenated cells are made
freshly for each experiment according to the above protocol. In some experimentsDR005:5 (SEQ. ID. NO. 14) (peptide designed to bind wide range of DR alleles) and
DR005:7:PDC (SEQ. ID. NO. 16) conjugate were also tested. The antigens were
tested in several concentration ranging from 100,uM to 0.1~m. After 72 hours of
culture, 100 ~11 of supematant was havested for assaying release of the Iymphokine
interferon gamma (using an ELISA kit from Endogen) while the rem~inder is pulsedwith [3H]Thymidine for the final 18 hours of culture. Cells are harvested onto
filtermats for liquid scintillation counting T cell proliferation is monitored by
thimidine incorporation using Beta plate reader. Supematants from microtiter plate
assay are incub~ d in ELISA plates coated with anti gIFN, anti L-4 and anti L- 10
antibodies (Endogene, cytokines kit). Cultures are scored as positive for proliferation
if CPM due to tritiated thymidine uptake is greater-than or equal to three-fold the
background in the llnc~im~ tçd controls, net counts are at least 1000 CPM, and the
Wo95/26980 21 8~ PCT/US95/04121
standard error of the mean is less than the net CPM. Stimulation index (SI) is defined
as the CPM divided by the CPM of the control.
ANALYSIS
Hapten specific T cell response was demonstrated in primary cultures from
PBL of individuals with recent exposure to poison oak. Fig SA-C shows the
proliferative response of T cells from representative primary T cell cultures (from
patients #486, #471, and #551) to urushiol, PDC, and PIXT (poison ivy extract)
sonicate of h~pten~ted cells and to DR005:7:PDC (SEQ. ID. NO. 16) conjugate but
not to the carrier alone (DR005:7 (SEQ. ID. NO. 16)). These results confirm that T
cells are capable of recognizing the arrier peptide with PDC conjugated to the 7th
position, but do not recognize the carrier peptide without the PDC
Other studies:
l S Using procedures similar to those described above, S poison Oak sensitive
individuals were screened for urushiol specificity. Several clones from each
individual were then assayed for recognition of PDC on 4 different carriers
DR005:7:PDC(SEQ. ID. NO. 16), DR011:7:PDC (SEQ. ID. NO. 30) and TTK:7:PDC
(SEQ. ID. NO. 2) carrier peptide from human tyrosine kinase protein) and C03:7:PDC
(SEQ. ID. NO. 4) (carrier peptide from the third domain of human complement, all as
shown in Fig. S) as well as recognition to PIXT, PDC, urushiol and TNP haptenated
autologous cells. Four out of S patients responded to all 4 carrier PDC conjugates
whereas all patients responded to 3 out of 4 carrier PDC conjugates (Data not Shown).
The T cell proliferative response to the various carrier:PDC conjugates varied from
patient to patient but not among clones of the same patients. Data showed that T cell
clones responding to various carriers also responded to PIXT, PDC, Urushiol but not
to TNP haptenated autologous cells demonc~rating that T cell specificity is confined
to haptens and hapten:conjugates from urushiol but not to unrelated haptens. The data
also suggests that T cell recognition of PDC conjugates was independent of the
carrier.
Other Studies:
The effect of the hapten anchoring position onto the class II binding carrier
peptide on T cell activation was also studied. The HLA-DR binding carrier, DR005(SEQ. ID. NO. 11) was coupled with PDC at positions 2, 4, 5, 6, 8, 9, 10, 11, and 12
as described in earlier examples. T cell clones from 2 patients responded to DR005
(SEQ. ID. NO. l l) carrier with PDC anchored to various positions in an
36
Wo 95/26980 2 1 8 6 ~ 7 3 PCr/US9S/04121
undiscrimin~ing fashion. Furthermore EBV lines expressing different HLA-DR were
capable of presenting the DR005:PDC conjugate although with different levels of
efficiency (Data not shown) This result appears to suggest that recogntion of the
hapten is completely carrier independent, which as di~cllssed earlier makes possible a
"universal type carrier".
Example 4: CD4/CD8 pheonotype of primary T cell cultures followin~ anti~enic
T cell cutlures followin.~ anti.~enic stimulation.
FACS analysis:
The method described by Vedder and Harlan (1988) J. Clin. Invest. 81:676,
was used in this analysis. Briefly, cells (0.5-lxl06) are incubated with 20~1 of anti-
CD4 antibodies conjugated with PE, together with 20~11 of anti CD8 antibodies
conjugated with FITC for 30 minutes on ice. The cells are washed and monitored for
staining using FACS STAR (Becton Dickinson).
Analysis of Results:
In the manner described above, T cells from PBLs of 25 individuals were
stained with anti-CD4 and anti CD8 antibodies prior to antigenic stimulation (Day 0)
and following primary (Pl) and secondary P2 antigenic stimulation. In 24 out of 25
cases, the CD4/CD8 ratio decreased from an average of 3.0 (Day 0) to 1.1 (Pl) and
0.8 (P2). This result suggests the induction of CD8+ cells following stimulation with
the hapten.
Example 5: Cytokine profile of Urushiol-specific T cell clone from Poison Oak-
s~ ilive patient
Cytokine Assay:
Cytokine levels in supernatants (from Example 5) were detected by two site
sandwich ELISA as previously descnbed (Cherwinski et al, J. Exp Med. 166:1229
(1987); Abrams et al, J. Immunol., 140:131 (1988)). Briefly, supernatants from
microtiter plate assays are inc~b~ted in ELISA plates coated with anti yIFN, anti IL-4
- and anti IL-10 antibodies (Endogen, cytokines kit) followed by incubation with a .
secondary matched antibody (supplied in the kit). The plates are washed and the
35 singal is visualized using calorimetric reaction. A standard curved generated by use
of known concentration of the cytokines is employed to convert cytokine levels read
as O.D to concentration (p~/ml).
37
wo 95,26g80 2 ~ 8 ~ ~ 7 3 PCT/USgS/04121
Analysis:
Cytokine profile of urushiol specific T cell clone from poison oak-sensitive
patients was shown to be that of the TH1 type, classified as the mediators of DTH
responses Analysis of the supernatants from cultures of T cell clone from patient
470.8 with various antigens revealed that high levels of yIFN, IL-8 and TNFoc were
secreted whereas no secretion of L-4 was detec~ecl (data not shown)
FY~mrle 6: Hapt~n~te~ peptides which bind to human MHC molecules
A series of haptenated peptides that bind to human MHC Class II molecules
HLA-DR 1 and/or HLA-DR4 and which have PDC ~t~ch~d to Iysines at known T cell
contact residues are synthesi
Choice of carrier peptide
PDC-substituted peptides are prepared using the sequence of influenza H3
h~m~gglutinin (HA) amino acid residues 307-319 (SEQ ID NO:53). This is a well-
characteriæd native human Class II MHC-binding peptide: Three-dimensional
crystallographic data on the binding of this peptide to DR1 have been published
(Brown et al. (1993) Nature 364:33-39); the binding affinity of the peptide to DRl
and DR4 is low nanomolar; and T cell receptor contact residues at positions 308, 310,
311, 313, and 316 have been identified from sul)slilulion analysis (Krieger et al.
(1991) J. ImmunoL 146:2331-2340) as well as from crystallography. The valine
residue at position 310 and the asparagine at position 313 are replaced by Iysine in
separate syntheses to provide carriers that can be PDC-~ub~liluled at those sites.
PDC-coupled peptides
Using the approach employed to produce PDC coupled peptides as desribed in
Example 2, a panel of peptides derived from HA307 31g (shown in Table 1 below)
are produced and characterized. Each peptide is singly substituted with a PDC.
38
Wo 95/26980 ~ 1 ~ 6 81 3 PCTIUS9S/04121
TABLE 1
Panel of DR1/DR4-binding PDC peptides on a HA307 31g backbone
5 l . HA307 31g(control - unsubstituted) = PKYVKQNTLKLAT (SEQ. ID.
NO. 53)
2. HA307 31gK308:PDC = PK(PDC)YVKQNTLKLAT (SEQ. ID. NO. 53)
3. HA307 31gK310:PDC = PKYK(PDC)KQNTLKLAT (SEQ. ID. NO. 57)
4. HA307 31gK311 :PDC = PKYVK(PDC)PNTLKLAT (SEQ. ID. NO. 58)
5- HA307 31gK313:PDC = PKYVKQK(PDC)TLKLAT (SEQ. ID. NO. 59)
6. HA307 31gK316:PDC = PKYVKQNTLK(PDC)LAT (SEQ. ID. NO. 53)
Binding inhibition assays are performed, as described in Example 2 for the
TNP-coupled peptides, to verify the binding of each haptenated HA peptide to
15 isolated DR4 molecules.
Establishing and testing T cell lines derived from DR l and DR4 donors of known
urushiol hypersensitivity to define in vitro antigenicity of each peptide
T cell clones are established from several DRl and DR4 donors who have
20 experienced rashes from natural contact with Rhus species. To assure an adequate
source of peripheral blood as a source of T cells and APC, app~op.iate tissue-typed
volunteers donate up to 1 unit of blood for these studies. The published procedure of
Kalish et al. is followed in esserlti~l detail to establish T cell lines (Kalish and
Johnson (1990) J. Immunol. 145:3703-3713). Heparinized peripheral venous blood is
25 separated into peripheral mononuclear cells on Ficoll-Hypaque and T cells areisolated by erythrocyte rosetting. The rçsulting peripheral blood T cells are plated in
U-bottom 96 well plates, using the inner 60 wells, along with 2 x 105/ well irradiated
(4000R) peripheral blood mononuclear cells and antigen in the form of the PDC-
coupled carriers used at concentrations beginning at 50 llg/ml. The antigen
30 concentration is a key factor that is systematically varied at initiation of replicate
cultures for optimal stim~ tion. Carriers are hen eggwhite Iysozyme (HEL-PDC),
ovalbumin (OVA-PDC) and keyhole-limpet hemocyanin (KLH-PDC). Several
carriers are tested since all may not be processed and presented equally well by each
individual's peripheral blood monocytes, and because T cells specific for some
35 carriers may be stiml~l~ted in certain individuals. However, after the initial expansion
and pre-screening, lines are assayed for recognition of PDC-coupled versus
uncoupled peptides not related to the initial carriers. T cells added per well are 3 x
39
WO 95/26980 ;2'~ 6 8 7 3 PCT/US95/04121
102, I x 103, 3 x 103, 9 x 103 and 3 x 104 in 60 replicates each. Culturing is
performed in Gibco AIM V medium, I % human AB serum, supplemented with
glutamine and antibiotics. After 6 days, recombinant L-2 (40 U/ml) and L-4 (19
U/ml) are added to all cultures. Growing cultures are assayed after an additional
S week for response to the initi~ting antigens. A sample of 75 ~1 from each culture is
transferred to a 96-well round-bottom plate and washed. The rem~ining culture is fed
with medium/L-2/IL-4. Each sample is washed and split to 6 wells (three duplicates)
containin~ freshly-thawed autologous, irradiated PBL and either PDC-antigen, Garrier
only or medium only. After 48 hrs, assay wells are pulsed with [3H]thymudine andcounted. Positives are selected for expansion according to the following criteria: a)
stimulation index (CPM experimentaVCPM control) 2 3Ø b) ~ CPM 2 1000, c)
standard error < /~ CPM. Cultures positive on the ini~i~ting antigen are expan~Pd to
48 well plates in the presence of IL-2/IL-4 for 6-8 days and tested in duplicate for
proliferation in response to each of the synthetic peptides.
Proliferation assay of T cell lines using PDC-peptides:
Responder cells are rested without Iymphokines for 3-4 days prior to assays.
Cells are washed three times to remove IL-2, resuspended in fresh AIM V medium,
1% AB serum, and added at 5 x 104 cells/well to flat-bottomed 96 well plates along
with 1 x 105 irradiated autologous peripheral blood monuclear cells. Each 48-well
culture is split into 14 wells (final volume 200 microliters) for testing duplicate
cultures of each of the control and PDC-peptides at 1-10 micromolar in the panel as
well as a medium-only control. After 24 hours of culture, 100 ~Ll of supernatant is
harvested for assaying release of the lymphokine inte.relon-gamma (antibodies for
immunoassays provided by Dr. Giorgio Trinchieri, Wistar Institute) while the
remainder are pulsed with [3H]thymidine for the final 18 hours of culture. Cells are
harvested onto filtermats for liquid scintillation counting.
Analysis of results:
Cultures are scored as positive for proliferation if CPM due to tritiated
thymidine uptake is greater than or equal to three-fold the background in the
unstimulated controls, net counts are at least 1000 CPM, and the standard error of the
mean is less than the net CPM. For interferon gamma assays, backgrounds are
usually below the level of detection (<1 U/rnl) while stimulated cultures produce 80-
100 U/ml (Kalish and Johnson (1990) J. Immunol. 145:3706-3713). Since the
peptides are DR-binding, Class II MHC-restricted T cells rather than Class I-restricted
responses are measured.
W095/269~1U 2~ PCr/USsS~41~1
These studies provide information on the ability of PDC-coupled peptides to
be recognized by human T cells. Principally, data on the T cell antigenicity of each
peptide is obtained and conditions for assays are established. A determination of a
significant response to each peptide is made, and the frequency of PDC-peptide
S reactive T cells from different donors is appro~im~te~l Peptides that are widely
recognized in urushiol-responsive patients can then be tested for efficacy in a
delayed-type hypersensitivity model in transgenic mice expressing human DR4 Class
II MHC molecules in a manner described in Example 3.
In these mice, typical hapten-specific hypersensitivity reactions can be
m~ ted (e.g., as described in Éxample 3) by hapten-specific T cells which recogni_e
the hapten bound to DR4 molecules. Thus, these mice can be used to examine the
ability of PDC-coupled peptides which bind to DR4 to induce urushiol-specific T cell
nonresponsiveness and clesen~iti7~ urushiol-sensitized subjects.
Example 7: Couplin~ of peniriilqmine to a MHC-bindin~ peptide
Penicill~mine (3-1llel-;apto-D-valine) was coupled to a cysteine residue within
a synthetic MHC Class II-binding peptide having the amino acid sequence:
Ala-Ala-Tyr-Lys-Ala-Ala-Cys-Ala-Ala-Ala-Ala-Ala-Ala (SEQ. ID. N0. 60)
1 2 3 4 5 6 7 8 9 10 1 1 12 13
Iodine was used as an oxidant to permit disulfide bonding between the cysteine
sulfhydryl and the ll.elcapto group of D-penicill~mine. To the peptide in acetic acid
was added 10 equivalents of D-penicill~mine, followed by addition of iodine/acetic
acid until the solution turned slightly yellow, eventually becoming colorless. Then an
additional 10 equivalents of iodine/acetic acid was added until a stable color
developed. Mixture was stirred 1 hr, then rotary evaporated to remove acetic
acid/iodine, and the peptide purified by HPLC. Mass spectroscopy confirmed the
coupling.
The penicillamine-substituted peptide is tested for recognition by
penicill~min~-reactive T cell isolated from blood donated from arthritis patients taken
off penicillamine after exhibiting a sensitivity. Demonstration of specific T cell
recognition could lead to a desen~iti7~ion regimen in line with the principles
discussed herein.
41
W O95/26980 2 ~ ~ ~ 8 ~ 3 PCTrUS9S/04121
SEQUENCE LISTING
~1) GENERAL INFORMATION:
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: IMMULOGIC PHARMACEUTICAL CORPORATION
(B) STREET: 610 Lincoln Street
(C) CITY: Waltham
(D) STATE: MA
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02154
(G) TELEPHONE: (617) 466-6000
(H) TELEFAX: (617)466-6040
( ii ) TITLE OF INVENTION: HAPTENATED PEPTIDES AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 62
( iv ) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/222,206
~B) FILING DATE: April 1, 1994
(viii) ATTORNEY/AGENT INFORMATION:
~A) NAME: Vanstone, Darlene A.
tB) REGISTRATION NUMBER: 35,279
(C) REFERENCE/DOCKET NUMBER: 079.2PCT
( ix ) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 466-6000
(B) TELEFAX: (617) 466-6040
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRAN~N~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: in.ternal
(xi) S~:Qu N~ DESCRIPTION: SEQ ID NO: 1:
Glu His Tyr Ser Gly Gly Glu Ser His Asn Ser Ser Ser
42
WO 95/26980 2 1 8 ~ 8 / 3 PCT/US95/04121
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Glu His Tyr Ser Gly Gly Lys Ser His Asn Ser Ser Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 3:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) sTRANn~nN~cs
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Glu Asp Ile Ile Ala Glu Glu Asn Ile Val Ser Arg Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 4:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRAN~N~ss:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Glu Asp Ile Ile Ala Glu Lys Asn Ile Val Ser Arg Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 5:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
43
W O 95/26980 2 ~ 7 ~ PCTrUS95/04121
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) ~:y~NC~ DESCRIPTION: SEQ ID NO: 5:
Ala Ala Ile Ala Ser Ala Ala Ser Ala Ala Ala Gln Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
~C) STRANDEDNESS:
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Ala Ala Ile Ala Ser Ala Lys Ser Ala Ala Ala Gln Ala
1 0
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Asp Ala Ile Ala Ser Ala Ala Ser Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 8:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
44
wo g5t26980 2 1 ~ ~ ~ 7 3 PCTtUS9S/04121
-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Asp Ala Ile Ala Ser Ala Lys Ser Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
` (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) ~u~c~ DESCRIPTION: SEQ ID NO: 9:
Asp Ala Ile Ala Ser Ala Ala Asn Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Asp Ala Ile Ala Ser Ala Lys Asn Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Asp Ala Ile Ala Ser Ala Ala Gln Ala Ala Ala Asn Glu
1 5 10
W O 95/26980 2 1 8 6 8 7 3 PCTAUS9S/04121
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Asp Lys Ile Ala Ser Ala Ala Gln Ala Ala Ala Asn Glu
1 5 lo
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) ~:QU~C~: DESCRIPTION: SEQ ID NO: 13:
Asp Ala Ile Lys Ser Ala Ala Gln Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 14:
~ Uk~C~ CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) ~ u~C~ DESCRIPTION: SEQ ID NO: 14:
Asp Ala Ile Ala Lys Ala Ala Gln Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 15:
_ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
46
W 095/26980 21~B73 PCT/U~5~ 121
-
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
S (v) FRAGMENT TYPE: internal
0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Asp Ala Ile Ala Ser Lys Ala Gln Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(c) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Asp Ala Ile Ala Ser Ala Lys Gln Ala Ala Ala Asn Glu
1 5 10
35 ( 2) INFORMATION FOR SEQ ID NO: 17:
(i) ~Q~ N~: CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRAN~ N~:ss:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) ~QU~NC~ DESCRIPTION: SEQ ID NO: 17:
Asp Ala Ile Ala Ser Ala Ala Lys Ala Ala Ala Asn Glu
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(c) STRAN~N~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
47
WO95/26980 2 ~ ~3 6 8 7 3 PCT/US95/04121
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Asp Ala Ile Ala Ser Ala Ala Gln Lys Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANv~vN~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asp Ala Ile Ala Ser Ala Ala Gln Ala Lys Ala Asn Glu
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
~B) TYPE: amino acid
(C) STRANv~vN~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Asp Ala Ile Ala Ser Ala Ala Gln Ala Ala Lys Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 21:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRA~ :SS:
(D) TOPOLOGY: linear
(iil MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
- 60
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Asp Ala Ile Ala Ser Ala Ala Gln Ala Ala Ala Lys Glu
5 10
48
21 ~6873
WO 95/26g80 PCI~/US95/04121
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Asp Ala Ile Ala Ser Ala Ala Ser Ala Ala Ala Arg Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Asp Ala Ile Ala Ser Ala Lys Ser Ala Ala Ala Arg Glu
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~n~s
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DE5CRIPTION: SEQ ID NO: 24:
Asp Ala Tyr Ala Ser Ala Ala Ser Ala Ala Ala Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 25:
~ (i) S~:Q~CE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
49
WO 9S/26980 2 1 8 6 8 7 3 PCT/US9S/04121
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Asp Ala Tyr Ala Ser Ala Lys Ser Ala Ala Ala Asn Glu
(2) INFORMATION FOR SEQ ID NO: 26:
( i ) ~hQUh'N~h CHARACTERISTICS:
~A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Asp Ala Ile Ala Ser Ala Ala Ser Ala Ala Ala Asn Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS-
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Asn Ala Ile Ala Ser Ala Ala Ser Ala Ala Ala Asn Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANn~nN~S:
( D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
wo 95/26g80 ~ I ~ 6 8 ~ 3 PCT~US95/04121
_
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Ser Ala Ile Ala Ala Asn Ala Ser Ala Ala Ala Asn Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRAN~:~N~.SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Ser Ala Tyr Ala Ala Asn Ala Ser Ala Ala Ala Asn Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
( C ) STR~Nl ~ NN l~ S
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Ser Ala Tyr Ala Ala Asn Lys Ser Ala Ala Ala Asn Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
- Ala Gly Tyr Arg Ser Asn Tyr Thr Tyr Tyr Ala Tyr Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 32:
W O95/26980 2 1 8 6 8 7 3 PCTrUS9S104121
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(c) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Ala Gly Tyr Arg Ser Asn Tyr Thr Ala Gln Ala Gln Ala
l 5 l0
20 (2) INFORMATION FOR SEQ ID NO: 33:
QU~N~: CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
( c ) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Gly Gly Tyr Ala Gly Glu Ala Gly Pro Ala Ala Gly Gly
l 5 l0
40 ( 2) INFORMATION FOR SEQ ID NO: 34:
:QUkNC~: CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(c) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Thr Leu Ser Ala Ala Ala Ala Asn Leu
l 5
60 (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
wog5/26980 21 ~6873 PCT/US95/04121
._ ,
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
s
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Thr Leu Ser Lys Ala Ala Ala Asn Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANv~vN~ss:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
2S
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Thr Leu Ser Ala Lys Ala Ala Asn Leu
(2) INFORMATION FOR SEQ ID NO: 37:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Thr Leu Ser Ala Ala Ala Lys Asn Leu
(2) INFORMATION FOR SEQ ID NO: 38:
(i) ~:Qu~: CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
W O 95/26980 21 86873 PCT/U~3~ 121
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Ala Ala Tyr Lys Ala Ala Lys Ala Ala Ala Ala Ala Ala
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 39:
Qu~N~ CHARACTERISTICS:
(A) LENGTH: l0 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Asp Tyr Gly Ile Leu Gln Ile Asn Ser Arg
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l0 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
Asp Lys Gly Ile Leu Gln Ile Asn Ser Arg
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l0 amino acids
(B) TYPE: amino acid
(C) sTRANn~N~s:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
Asp Tyr Gly Ile Lys Gln Ile Asn Ser Arg
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 42:
54
wo gs/26g80 2 1 8 6 8 7 3 PCT/US95/04121
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
- (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Asp Tyr Gly Ile Leu Gln Ile Lys Ser Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
( D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Phe Glu Ala Lys Gly Ala Leu Ala Asn Ile Ala Val Asp
(2) INFORMATION FOR SEQ ID NO: 45:
- 60
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
wo gs/26g80 2 1 8 6 8 7 3 PCT/US95/04121
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Phe Glu Ala Gln Gly Ala Lys Ala Asn Ile Ala Val Asp
0 1 5 10
(2) INFORMATION FOR SEQ ID NO: 46:
(i) S~QU~C~ CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Phe Glu Ala Gln Gly Ala Leu Ala Asn Lys Ala Val Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRA~:UN~:SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Phe Glu Asp Lys Gly Ser Leu Glu Asn Ile Ala Arg Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 48:
~ :Qu~C~: CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRAN~N~SS:
(D) TOPOLOGY: linear
- ( ii ) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
56
W 095/26980 2 1 8 ~ ~ 7 3 PCTnUS95/04121
.. _
Phe Glu Asp Gln Gly Ser Lys Glu Asn Ile Ala Arg Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Phe Glu Asp Gln Gly Ser Leu Glu Asn Lys Ala Arg Asp
1 5 10
25 ( 2) lN~O~ ~TION FOR SEQ ID NO: 50:
(i) s~Quk~CE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Ala Ala Ile Lys Ala Ala Ala Ala Ala Ala Arg Ala Ala
1 5 10
45 ( 2) INFORMATION FOR SEQ ID NO: 51:
QU~N~: CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Ala Ala Ile Ala Ala Ala Lys Ala Ala Ala Arg Ala Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
W O95/26980 21 86~ PCTrUS95/04121
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
. (xi) ~Qu~C~: DESCRIPTION: SEQ ID NO: 52:
Ala Ala Ile Ala Ala Ala Ala Ala Ala Lys Arg Ala Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 53:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids-
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:-54:
Ala Ala Tyr Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 55:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
6S
(ii) MOLECULE TYPE: peptide
W O 95/26980 2 1 ~ 6 8 7 3 PCTAUS95/04121
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D~ OTHER INFORMATION:/note= "Wherein Xaa is alanine or
lysine or cysteine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Ala Ala Tyr Xaa Ala Ala Xaa Ala Ala Lys Ala Ala Ala
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 56:
QU~NCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRAN~:~N~:SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/note= ~Wherein Xaa is alanine or
lysine or cysteine~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Ala Ala Ile Xaa Ala Ala Xaa Ser Ala Xaa Ala Ala Ala
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) sTR~N~nN~ss:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Pro Lys Tyr Lys Lys Gln Asn Thr Leu Lys Leu Ala Thr
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
59
W 095/26980 2 1 ~ ~ ~ 7 3 PCTrUS95/04121
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
s
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Pro Lys Tyr Val Lys Pro Asn Thr Leu Lys Leu Ala Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
( D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Pro Lys Tyr Val Lys Gln Lys Thr Leu Lys Leu Ala Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Ala Ala Tyr Lys Ala Ala Cys Ala Ala Ala Ala Ala Ala
1 5 10
55 ( 2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
- 60 (c) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
W Og5/2080 21 8 6 8 7 3 rCT/U~gS~121
.
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Phe Glu Asp Gln Lys Ser Leu Glu Asn Ile Ala Arg Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 62:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 13 amino acids
~B) TYPE: amino acid
~C) sTRA~nF~nN~s
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: peptide
~v) FRAGMENT TYPE: internal
~xi) ~QU~NC~ DESCRIPTION: SEQ ID NO: 62:
Tyr Asp Asp Asn Gly Ala Lys Gln Asn Ala Ala Glu Arg
1 5 10
61
