Note: Descriptions are shown in the official language in which they were submitted.
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21 1~4~
PEPTIDES TOLERIZING T-CELLS AND COMPOSITIONS THEREOF
BACKGROUN~ OF THE INVENTION
.~
Several apparently disparate pathological states, such as allergy,
transplant rejection, and autoimmune diseases share the common characteristic
of being caused by deleterious immune reactions, which at least in part
10 involve activation of T cells. The cascade of molecular events leading to this
T cell activation is initiated by the formation of complexes between antigenic
peptides (a processed peptide derived from disease-causing antigenic protein
comprising at least one T cell epitope) and major histocompatibility complex
molecules (MHC). These antigen-MHC complexes are ligands that, in turn,
15 are recognized by T cell receptor (TCR) on the surface of T cells which are
specific for the disease-causing antigen. Engagement of the TCR in the
trimolecular antigen-MHC-TCR complex together with a costimulatory signal
results in activation of the T cell which is followed by a series of molecular
events characteristic of T cell activation, such as increase in tyrosine
20 phosphorylation, Ca 2+ influx, inositol phosphate turnover, the synthesis of
cytokines (i.e. IL-2. IL-3, gamma interferon) and cytokine receptors, and T
cell proliferation.
Several investigators have sought to develop treatments which
interfere with this recognition process by using high affinity MHC-binding
25 peptides that cause inhibition of T cell activation by blocking the antigen-
binding site of MHC molecules. Other investigators have attempted to devise
,treatments which result in tolerization of T cells by various mech~nisms
which include eng~ging the TCR with an antigenic peptide derived from the
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target antigen which is bound to the appropriate MHC complex but without
the appropriate costimulatory signal, and which therefore causes the target T
cells to become tolerized. Tolerized T cells will not proliferate when
stimulated with low antigen concentrations and will not produce IL-2 under
5 any conditions. It would be advantageous to provide treatment for allergy,
transplantation rejections or autoimmune disease which causes tolerization of
the targeted antigen T cells ( i.e. the T cells do not proliferate or proliferate
only minim~lly and further do not perform any of the other functions
associated with T cell activation discussed above) and moreover, it would be
10 advantageous if T cell tolerization can occur in the presence or absence of the
applopliate costimulatory signal.
SUMMARY OF THE INVENTION
The present invention provides a tolerizing peptide and a therapeutic
15 composition cont~ining at least one such tolerizing peptide which when
~lmini~tered to an individual in a therapeutically effective amount is capable
of tolerizing at least a portion of the T cells of said individual which are
specific for an antigen (e.g. an allergen. an autoantigen or a transplantation
antigen), said tolerizing peptide having the ability to combine with MHC on
20 antigen presenting cells (APC) and cause binding of T cell receptors on at
least a portion of the T cells of an individual to said peptide-MHC complex
and having at least one and preferably all of the following properties: 1) the
ability to cause no detectable or minim~l proliferation of at least a portion ofthe T cells of an individual which are specific for said antigen in an in vitro
25 assay utili7ing said peptide and histocompatible antigen-presenting cells; 2)the ability to cause no detectable or minim:~l production of IL-2 in an in vitro
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assay utili7.ing a portion of the T cells of an individual which are specific for
said antigen, said peptide~ and histocompatible antigen-presenting cells; 3) the
ability to cause no detectable or minim~l production of IL-3 in an in vitro
assay utili7ing a portion of the T cells of an individual which are specific for
5 said antigen, said peptide. and histocompatible antigen presenting cells; and 4)
the ability to cause no detectable or minim~l production of gamma IFN in an
in vitro assay utili7.ing a portion of the T cells of an individual which are
specific for said antigen~ said peptide, and histocompatible antigen presenting
cells. Said tolerizing peptide preferably has the ability to cause no detectable
10 or minim~l proliferation of a least a portion of the T cells of an individual
which are specific for said antigen when said peptide is ~rlmini.~tered in vivo
in immunogenic form. A therapeutic composition of the present invention
preferably comprises a sufficient number of tolerizing peptides to render
substantially all of the T cells of such individual which are specific for said
15 antigen unresponsive to said antigen.
The invention also provides a method for treating sensitivity to a
particular antigen in an individual by a-lmini.~tering to the individual,
preferably in nonimmunogenic form, at least one therapeutic composition of
the present invention in an amount effective to render at least a portion of, and
20 preferably all of the T cells of the individual which are specific for said
antigen unresponsive to said antigen. At least two different therapeutic
compositions of the invention can be ~-lmini.~tered simultaneously or
sequentially to the individual. Another embodiment of the present invention
provides a method for inhibiting at least a portion of an antigen specific
25 antibody response by the immune system of an individual by ~dmini.ctering to
the individual, preferably in nonimmunogenic form, at least one therapeutic
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composition of the present invention in an amount effect to render at least a
portion of the T cells of the individual which are specific for said antigen
unresponsive to said antigen.
In yet another embodiment of the invention, there is provided a
5 method of designing a tolerizing peptide of the invention and a method for
tolerizing at least a portion of a population of T cells in an individual which
are specific for an antigen ~ltili7ing said tolerizing peptide.
DESCRIPTION OF THE DRAWINGS
Fig la. is a graphic representation of a T cell proliferation assay of T
cell clone PL-17 to a wild type peptide of the murine hemoglobin Bd minor
chain (HbB(64-76) designated in the graph as wt Hb) or the substituted
HbB(64-76) peptide, Ser70, of the invention and using I-E ~transfected L cell
fibroblasts as antigen presenting cells (APC).
Fig lb is simila; to Fig la except that two substituted HbB(64-76)
peptides, Ser70 and Gln72, are used in the T cell proliferation assay.
Fig 2a is a graphic representation of a bioassay for IL-2 production by
Th 1 clone PL-17 stimulated with Wt Hb or substituted peptide, Ser70 and
q~l~ntit~t~d as proliferation of the IL-2 dependent cell line CTLL; and CH27 B
20 cell lymphoma were used as APC.
Fig 2b is similar to Fig. 2a except that two substituted HbB(64-76)
peptides, Ser70 and Gln72 are used in the assay.
Fig. 3a is a graphic representation in bar graph form of a bioassay for
gamma IFN production by Th 1 clone PL17 stimulated with Wt Hb and
25 substituted peptide, Ser70; L cell fibroblasts were used as antigen presenting
cells and gamma IFN was measured using an ELISA .
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Fig 3b is a graphic representation in line graph form of a bioassay for
garnma IFN production similar to that described in Fig. 3a except that
substituted peptide Gln72 was also tested.
Fig 4 is a graphic representation of a bioassay for IL-3 production by
Th 1 clone PL17 stimulated with Wt Hb or substituted peptide, Ser70; IL-3
production was quantitated as proliferation of the IL-3 dependent cell line,
GGl.12, and BlO.BR/sgSnj spleen cells were used as APC.
Fig. 5a is a graphic representation of the results of a tolerance assay
using Th 1 clone PL-17 which had been incubated overnight with either no
antigen (No Ag oln), 50 uM Ser 70, or 50 uM Gln72, and rested 1 day before
being challenged with wild type Hb peptide(64-76), DCEK-Hi7 L cells or
BlO.BR/SgSnj spleen cells were used as APC.
Fig Sb is a graphic representation of the results of a tolerance assay
using Th 1 clone PL-17 which had been incubated overnight with either no
antigen (No Ag o/n), or 50 uM Ser 70 and rested 3 days before being
challenged with wild type Hb peptide(64-76); DCEK-Hi7 L cells or
BlO.BRlSgSnj spleen cells were used as APC.
Fig. 5c is a graphic representation of the results of a tolerance assay
using Th 1 clone PL- 17 which had been incubated overnight with either no
antigen (No Ag oln)~ or 50 uM Ser 70 and rested 5 days before being
challenged with wild type Hb peptide(64-76); DCEK-Hi7 L cells or
BlO.BRlSgSnj spleen cells were used as APC.
Fig. 5d is a graphic representation of the results of a tolerance assay
using Th 1 clone PL-17 which had been incubated overnight with either no
25 antigen (No Ag oln), or 50 uM Ser 70 and rested 7 days before being
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challenged in a proliferation assay with Hb peptide(64-76); DCEK-Hi7 L cells
or B lO.BR/SgSnj spleen cells were used as APC.
Fig. 6 is a graphic representation of the results of an assay for inositol
phosphate generation by Th 1 clone PL17 cells which had been incubated with
no peptide, Hb(64-76), Ser70 or Gln72 and in which either DCEK-Hi7 L cells
or BlO.BR/SgSnj spleen cells were used as APC; total free inositol phosphate
was qu~ntit~t~d by scintillation counting.
Fig. 7a is a graphic representation of the results of a tolerance assay
using Th 1 clone Pl-17 cells which had been incubated over night with either
no antigen(No Ag o/n), 50 uM Ser70 (50uM s70 o/n), 50 uM Gln72 (50 uM
Q71 o/n) or 100 uM Gln72 (lOOuM Q72 o/n) and DCEK-Hi7 L cell
tranfectants as APC and subsequently challenged with Hb(64-76) as antigen.
Fig. 7b is a graphic representation of the results of a tolerance assay
using Th 1 clone Pl- 17 cells which had been incubated overnight with various
concentrations of substituted peptide. Ser70, and DCEK-Hi7 L cell
tranfectants or as APC and subsequently challenged with Hb(64-76) as
antigen.
Fig. 8 is a graphic representation of the results of a tolerance assay
using PL-17 cells incubated overnight with no ~ntigen, or substituted peptide,
Ser70, or with no antigen and Cyclosporin A ,or substituted peptide, Ser70,
and Cyclosporin A and with DCEK-Hi7 L cells as APC and subsequently
challenged with Hb~64-76) as antigen.
Fig. 9 is a graph depicting a FACScan analysis testing for the levels of
IL-2 receptor on the surface of PL-17 cells which had been incubated with
DCEK/Hi7 L cells alone or with substituted peptides Ser70 or Gln 72 at 50
uM.
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Fig. 10 is a graph depicting a FACScan analysis testing for the levels
of LFA-1 adhesion molecule on the surface of PL-17 cells which had been
incubated with DCEK/Hi7 L cells alone or in conjunction with substituted
peptides Ser70 or Gln 7~ at a concentration of 50 uM.
Fig. 1 la is a graphic representation of the results of a tolerance assay
using Th 1 clone PL-17 which had been incubated overnight with either no
antigen (No Ag o/n), or 50 uM Ser 70 and rested 3 days before being
challenged in a proliferation assay with wild type Hb peptide(64-76).
Fig. 1 lb is a graphic representation of the results of a tolerance assay
using Th 1 clone PL-17 which had been incubated overnight with either no
antigen (No Ag o/n), or 50 uM Ser 70 and rested 3 days before being
challenged in a proliferation assay with wild type Hb peptide(64-76).
DETAILED DE~CRIPTION OF THE INVENTION
The present invention provides a tolerizing peptide (also referred to herein as
a tolerizing substituted peptide) and a therapeutic composition cont~ining at
least one such tolerizing peptide which when a~lmini.ctered to an individual in
a therapeutically effective amount is capable of tolerizing at least a portion of
the T cells of said individual which are specific for an antigen (e.g. an
allergen, an ~uto~ntigen or a transplantation antigen)? said tolerizing peptide
causing no detectable or minim~l proliferation of at least a portion of the T
cells of said individual which are specific for said antigen in an in vitro assay
using histocompatible antigen-presenting cells. The terms "tolerization",
"tolerizing", "anergy" and "anergizing" etc. are used herein means that the T
cell is rendered unable to respond to stimulation with an antigen complexed to
MHC. Here tolerization includes the binding of the T cell receptor to
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complexes of the tolerizing peptide and the MHC. The term minim~l, as used
herein, when discussing minim~l T cell proliferation, minim~l lymphokine
production, etc. refers to an amount that is not significant. Histocompatible
antigen-presenting cells are antigen-presenting cells (APCs) that are
5 histocompatible with the T cell population. This is automatic if the T cells and
the APCs æ obtained from the same source. However, they may also be
prepared separately. For human-related assays, immortalized B-cell lines from
the same subject, MHC Class II compatible cell lines, or compatible
peripheral blood mononuclear cells can be used as APCs. Said tolerizing
10 peptide preferably has the ability to cause no detectable or minim~l
proliferation of a least a portion of the T cells of an individual which are
specific for said antigen when said peptide is ~tlminictered in vivo in
immunogenic form. By immunogenic form is meant a form which tends to
induce an immllne response, i.e., cause the activation of T cells or production
15 of antibodies specifically immunoreactive with the peptide. It is well known
that peptides ~lmini~tered in adjuvants tend to be immunogenic while
peptides ~clmini~tered in the absence of adjuvant and in soluble form
(nonimmunogenic form) tend to be tolerogenic. Other characteristics of
tolerizing peptides of the invention include the ability to cause no detectable
20 or minim:3l production of lymphokines, including but not limited to IL-2, IL-3, IL-4, gamma interferon, etc. in an in vitro assay ~ltili7ing a portion of the T
cells of an individual which are specific for said antigen and histocompatible
antigen-presenting cells.
A therapeutic composition of the present invention preferably
25 comprises at least one tolerizing peptide of the invention and a
pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable
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diluents include saline and aqueous buffer solutions. Pharmaceutically
acceptable carriers include polyethylene glycol (Wie et al., International
Archives of Allergy and Applied Immunology 64: 84-99 (1981)) and liposomes
(Strejan et al., Journal of Neuroimmunology 7: 27 (1984)). Such compositions
S will generally be ~(lminictered by injection (subcutaneous. intravenous, etc.),
oral ~rlministration (e.g.~ as in the form of a capsule). inhalation, transdermal
application or rectal application. The therapeutic composition of the present
invention preferably comprises a sufficient number of tolerizing peptides to
render substantially all of the T cells of such individual which are specific for
10 said antigen unresponsive to said antigen. This may turn out to be one
tolerizing peptide, two tolerizing peptides, three tolerizing peptides, etc. The
therapeutic compositions of the invention are ~flmini~tered to individuals at
dosages and for lengths of time effective to reduce sensitivity of the individual
to the antigen. As used herein, reduction in sensitivity can be defined as non-
15 responsiveness or dimunition in the symptoms to the antigen (i.e. theindividual would be less able to respond immunologically to the antigen as
determined by clinical and/or scientific procedures). This dimunition may be
subjective (i.e. the individual feels more comfortable despite the presence of
the antigen). Effective amounts of the therapeutic compositions will vary
20 according to factors such as degree of sensitivity of the individual to the
antigen. the age, sex, and weight of the individual~ etc.
The invention also provides a method for treating sensitivity to a
particular antigen in an individual by ~3dmini~t~ring to the individual,
preferably in nonimmunogenic form. at least one therapeutic composition of
25 the present invention in an amount effective to render at least a portion of, and
preferably all of the T cells of the individual which are specific for said
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antigen unresponsive to said antigen. At least two different therapeutic
compositions of the invention can be ~ minictered simultaneously or
sequentially to the individual. By rendering at least a portion of the T cells
unresponsive to the antigen, at least a portion of the antigen specific antibody
5 response is inhibited. T cell epitopes are believed to be involved in initiation
and perpetuation of the immune response to a protein or protein complex
antigen which is in turn responsible for the clinical symptoms of disease.
These T cell epitopes are thought to trigger early events at the level of the T
helper cell by binding to an appropriate HLA molecule on the surface of an
10 APC and stimulating the relevant T cell subpopulation. If the relevant T cell
subpopulation has been rendered unresponsive to the antigen, then various
events including the activation of the B cell cascade leading to production of
antibodies will not occur. If the individual is experiencing clinical symptoms
indicative of sensitivity to a disease such as an allergic reaction or allergic
lS asthma, rheumatoid arthritis, or symptoms of graft rejection, those symptoms
can be arrested or dimini~hçd upon ~dmini~tration of the therapeutic
composition of the invention.
In yet another embodiment of the invention, there is provided a
method of dç~igning a tolerizing peptide of the invention. In one method, a
20 tolerizing peptide may be designed by first isolating an immunogenic peptide
from an antigen known to be responsible for allergy, transplantation rejection
or ~utoimmune disease. This can be done for example by using a synthetic or
recombinant peptide derived from an antigen which is previously known to be
an immunogenic peptide (i.e. contains at least one T cell epitope of the
25 antigen). This can also be done by e~mining the structure of an antigen of
interest and producing peptides (via an expression system, synthetically or
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otherwise) to be examined for their ability to influence T cell responses in a
population of T cells known to be sensitive to the antigen of interest, and
selecting appropriate peptides which contain at least one epitope recognized
by the cells. In referring to an epitope, the epitope will be the basic element
5 or smallest unit of recognition by a receptor particularly immunoglobulins,
histocompatibility antigens and T cell receptors where the epitope comprises
amino acids essential to receptor recognition.
Upon isolating or determining at least one amino acid sequence which
contains at least one epitope of the antigen of interest (an immunogenic
10 peptide), substituted peptides are generated (synthetically or otherwise) by
replacing each amino acid of the immunogenic peptide ( also referred to
herein as the wild type peptide or native peptide) with a different amino acid
(which may be a conservative arnino acid. an amino acid not found in nature
or alanine). Therefore, each substituted peptide contains one or more
15 substituted amino acid which is different from the amino acid at the same site
of the immunogenic peptide (or wild type peptide) of interest. Examples of
conservative amino acid substitutions include but are not limited to those
shown in Table I.
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Table I
Examples of Conservative Amino Acid Substitutes
s
Arnino Acid Substitutions
Ala Ser
Arg Lys
Asn Gln
Asp Glu
Cys Ala Met
Gln Asn
Glu Asp
Gly Ala
His Gln Asn Lys
Ile Leu
Leu Ile
Lys Arg
Met Leu Cys
Phe Tyr
Pro Ala
Ser Thr Ala
Thr Ser
Trp Phe Leu Ile
Tyr Phe
Val Ile
The subsituted peptides are then tested to determine whether they are
10 capable of tolerizing a portion of the T cells specific for an antigen of interest.
A substituted peptide which is determined to be a tolerizing substituted
peptide suitable for the invention has the ability to combine with MHC on
antigenic presenting cells and cause binding of T cell receptors on at least a
portion of the T cells of an individual to said peptide-MHC protein complex
15 and has at least one and preferably all of the following properties: 1) the
ability to cause no detectable or minim~l proliferation of at least a portion of
the T cells of an individual which are specific for said antigen in an in vitro
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assay utili7ing said peptide and histocompatible antigen-presenting cells; 2)
the ability to cause no detectable or minim~l production of IL-2 in an in vitro
assay utili7ing a portion of the T cells of an individual which are specific for
said antigen, said peptide, and histocompatible antigen-presenting cells; 3) the
5 ability to cause no detectable or minim~l production of IL-3 in an in vitro
assay utili7ing a portion of the T cells of an individual which are specific for
said antigen, said peptide, and histocompatible antigen presenting cells; and 4)
the ability to cause no detectable or minim~l production of gamma IFN in an
in vitro assay utili7ing a portion of the T cells of an individual which are
10 specific for said antigen, said peptide, and histocompatible antigen presenting
cells.
The invention may also include any compound that mimics a tolerizing
substituted peptide of the invention as described above such as a compound
not composed entirely of subunits joined by peptide bonds. but joined by other
15 linkages (e.g. thiolester bonds), providing that the non-peptide compound
mimics a tolerizing peptide capable of tolerizing T cells through the binding
of T cell receptors to the complex of non-peptide and MHC protein in the
absence of proliferation and/or cytokine production.
Most preferably~ a tolerizing substituted peptide (or compound which
20 mimics said peptide) of the invention causes no detectable proliferation or
minim~l proliferation of at least a portion of a population of T cells specific
for the antigen of interest and no detectable or minim~l proliferation of
lymphokines such as IL-2~ IL-3 etc.~ however~ a preferred tolerizing peptide
of the invention is capable of stimulating upregulation of IL-2 receptor (IL-
25 2R) and/or LFA- 1 adhesion molecule production thus indicating that the
substituted peptide is binding and engaging the T cell receptor in conjunction
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with the appropriate class II MHC complex and causing partial activation of
the T cell.
One hypothesis for why a tolerizing substituted peptide of the
invention functions as described herein may be that the contact of a peptide-
S MHC complex with the T cell receptor triggers multiple intracellularpathways to be activated. It is possible, therefore~ that the substituted peptide
of the invention which must bind the T cell receptor differently than the nativeor wild type immunogenic peptide. stimulates fewer of these pathways than
the native peptide, and the intracellular pathway triggered is that which causes10 the T cells to become tolerized.
The above-described tolerizing peptide may be used in a method for
tolerizing at least a portion of a population of T cells in an individual which
are specific for an antigen by first generating a tolerizing peptide by the
methods described above and ~(lminictering said peptide in a therapeutic
15 composition in an amount effective to cause tolerization of at least a portion
of said population of T cells.
The invention is further illustrated in the following non-limiting
examples.
20 F.x~nlple 1
The Thl clone PL-17 ( also clecign~tPd herein as PL-17 cells) is
specific for the known immunogenic peptide of the murine hemoglobin ,Bd
minor chain, Hb,B(64-76) (also designated herein as native peptide. native
immunogenic peptide or wild type peptide (Wt Hb)) in the context of I-Ek.
25 Substituted Hb(64-76) peptides were generated by introducing conservative,
14
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2i~4409
single amino acid substitutions at any one of amino acid residues 67-76 of the
native peptide as is shown in Table II.
Table II
Peptide Sequence
Peptide D~cign~tion
64 65 66 67 68 69 70 71 72 73 74 75 76
Hb(64-76) G K K V I T A F N E G L K
Ala67-Hb(64-76) A
Leu68-Hb(64-76) L
Ser69-Hb(64-76) S
Ser70-Hb(64-76) S
Tyr71-Hb(64-76) Y
Gln72-Hb(64-76) Q
AsF73-Hb(64-76) D
Ala74-Hb(64-76) A
Ile75-Hb(64-76)
Arg76-Hb(64-76) R
The Hb(64-76) peptide and substituted peptides were synthesized
using a DuPont RaMPS apparatus, purified by reverse phase HPLC on a
Beckman 6300 amino acid analyzer.
1~
Using an I-Ek-transfected L cell fibroblast, DCEK-Hi7, as APC, it was
found that the peptide generated by substituting Ser for Ala at position 70
(also referred to herein as S70, Ser70 or Ser70-Hb(64-76)) ablated the
proliferative response completely, even at a 10-fold greater concentration than
20 that which gives maximal proliferation (Example 2) with the native
immunogenic peptide (Figs. la and lb). In addition, substituted peptide
Gln72 (also referred to herein as G72, Gly72-Hb(64-76)) could not stimulate
PL-17 to produce cytokines. Figs. 2a and 2b show that. while the T cells
1~
W O 94/06828 PC~/US93~084 ~
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make significant amount of IL-2 when presented with native Hb(64-76), no
detectable IL-2 was seen on stimulation with either of the substituted peptides.Reports by others have suggested that the production of IL-3 and IFN-~ by
Thl clones can occur by TCR engagement alone while an additional
5 costimulus is required for IL-2 production . Therefore, supernatants were
analyzed (Example 2) after stimulation with the peptide analogs for IFN-
~(Figs. 3a and 3b) or IL-3 (Fig. 4) production (Example 2). However, as for
the IL-2 profile, only the native immunogenic peptide Hb(64-76) could
provide applopliate activation to allow PL-17 to synthesize IFN-~ (Figs. 3a
10 and 3b) and IL-3 (Fig. 4). When fresh BlOBR/SgSnj spleen cells were used
as APC in analogous experiments similar results were found (data not shown).
One of the earliest intracellular events to occur after TCR engagement
is activation of phospholipase C which catalyses the breakdown of membrane
phospholipids and generation of inositol phosphates. As discussed in the
15 above hypothesis, it is possible that an earlier activation event than cytokine
production might be stimulated by the substituted Hb peptides. Thus, after
stimulating the T cells with native Hb(64-76) or one of the substituted
peptides (Ser70 or Gly72) and DCEK-Hi7 cells PL-17 cells were assayed for
total inositol phosphate production (Example 2). Although the native Hb964-
20 76) peptide allowed production of .~ignific~nt levels of inositol phosphates, thesubstituted peptides Ser70 and Gln72 did not (Fig. 6). Thus, for the Th 1
clone PL-17, introducing conservative amino acid substitutions at various
residues of the native immunogenic peptide, resulted in loss of the activation
signals that stimulate detectable proliferation, cytokine production, and
25 inositol phosphate generation.
16
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Further, it is known that upon activation. T cells upregulate various
cell surface molecules, including LFA- 1 and IL-2R. Therefore a comparison
of the levels of these molecules on PL-17 after stimulation with Hb(64-76).
Gln72 or Ser 70. FACScan (FACS) analysis (Example 3) revealed that,
5 while stimulation with Gln72 generally did not increase either receptor above
control levels, Ser70 stimulation upregulated both LFA-l (Fig. 10) and IL-2R
(Fig. 9) levels significantly. These results suggested that Ser70 was deliveringa partial signal to PL-17 upon TCR engagement that was independent of
inositol phosphate generation. These results further suggest that Gln72 was
10 not binding to the T cell receptor which is a requisite of a tolerizing
substituted peptide of the invention as described above.
With the hypothesis that partial T cell stimulation might lead to
tolerance induction instead of T cell activation and proliferation, the ability of
the T cells to respond to the immunogenic peptide after being presented with
15 the substituted peptides Ser70 and Gln72 was shown. A challenge
proliferation assay was conducted with PL-17 cells that had been previously
stimulated with DCEK-Hi7 (Fig. 1 la) or B lO.BRJSgSnj spleen cells (Fig.
1 lb) as a source of live, functional APC alone, or with Ser70 or Gln72 peptide
analogs. Although T cells which had previously seen Gln72 analog responded
20 norrnally in the challenge assay, PL-17 which had been previously presented
with Ser70 analog were now completely unresponsive to the immunogenic
peptide (Fig. 1 la and Fig. 1 lb), although they could respond well to
exogenous IL-2 (data not shown) or PMA and Ionomycin (data not shown).
These results suggest that a substituted peptide which differs from the native
25 immunogenic peptide only by a single conservative amino acid substitution is
capable of transducing a partial signal to the T cell (partial activation of the T
WO 94/06828 PC~/US9~/084~
214~09
cell) that causes it to become unresponsive to subsequent challenge with the
native immunogenic peptide and is therefore deemed unresponsive and
tolerized. The observation was extended further by studying the tolerizing
ability of another substituted peptide, Asp73, which had given similar results
5 as Ser70 in proliferation, cytokine production and FACS assays (data not
shown). PL-17 cells which had previously been presented Asp73 were
completely unable to proliferate when challenged with Hb(64-76) peptide.
However, it appeared that more of Asp73 substituted peptide than Ser70
substituted peptide was needed to induce this anergic state (at least 30~LM
10 Asp73 compared to lO~M Ser70) suggesting that perhaps changes to these
two positions of the native peptide have a different effect on the manner in
which the TCR is engaged by the substituted peptide-MHC complex.
Next, studies investigating how long-term this state of
unresponsiveness could last were conducted. When T cells were rested for 1,
3, 5, and 7 days (Example 5) before challenging with Hb(64-76) they still
could not mount a proliferative response (Figs. 5a-5d), leading to the
conclusion that the substituted peptide Ser70 had induced PL- 17 into a state ofprofound anergy. In similar experiments (Example 6), it was shown that
varying concentrations including relatively low concentrations of Ser70 were
20 able to induce tolerance (Figs. 7a and 7b). However, the results in Fig 7a
inflic~te that Gln72 was unable to induce tolerance indicating that it is not
binding to the TCR and partially activating the T cells as is required of a
tolerizing substituted peptide of the invention.
In previous studies. using chemically fixed APC (i.e. APC treated with
25 ECDI) and immunogenic peptide to induce anergy, it was shown that addition
of cyclosporin A or allogenic spleen to the culture prevented the T cells from
18
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becoming unresponsive to subsequent challenge with live fixed APC .
Similarly in the present system it was found that addition of cyclosporin A
with Ser70 prevented this induction of anergy (Fig. 8), although adding T-
depleted allogenic spleen cells as a source of costimulation had no effect (data
5 not shown). These results suggested that the mechanism of anergy induced by
substituted peptide Ser70 on a professional APC is similar to, but also
distinctly different from, that caused by presentation of peptide by a fixed
APC.
In other studies it was shown that non-immunogenic peptide analogs
10 could function in an antagonistic manner by competing for TCR sites without
transducing any detectable signal to the T cell. However, in this system Ser70
causes the T cells to become anergized and therefore must be sending some
signal to the T cell whereas Gln 72 is not anergizing T cells and is therefore
not sending a signal to the T cell. The underlying mechanism of how this is
15 occurring is as yet unknown. However, since costimulation from APC is
provided in this system, this tolerization occurs even in the presence of
costimulatory signals.
One might envisage that contact of a peptide - MHC complex with a
TCR triggers multiple intracellular pathways to be activated, each perhaps
20 connected to different chains of the CD3 complex. In support of this idea.
many recent studies have shown that several of the molecules that make up the
CD3 complex contain functional domains and are capable of .sign~ling
independent of the rest of the complex . It is possible~ therefore, that the
substituted peptides, which must bind the TCR differently than the
25 immunogenic peptide, stimulate fewer of these pathways than the native
peptide and, for Ser70 and Asp73, the intracellular pathway triggered is that
19
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which causes anergy. If this is true, then the signal for anergy induction is a
normal part of the T cell response to all antigenic stimuli but is normally over-
ridden by the stimulatory pathways which allow a proliferative response. By
selecting substituted peptides that are recognized only slightly differently by
the T cell than the native peptide one is able to separate these activation
pathways.
E~mI~le 2
Studies showing PL-17 Th 1 clone (PL-17 cells) proliferative,
lymphokine and inositol generation responses to substituted peptides of Hb
(64-76) of the invention were concll~cted
The proliferation assay (results shown in Figs la and lb) was
performed in 96 well flat bottomed plates in 200~11 RPMI-1640 media
co~ ;"i~-g lO~o fetal calf serum (Hyclone, Logan, UT), 2 mM glut~mine,
50~1g/ml gentamicin, lOmM Hepes buffer, and 2-ME (2xlO-5M). PL-17 clone
at 2.104 cells/well, mitomycin-C treated DCEK-Hi7 L cell fibroblasts
(77,ug/ml in HBSS for 90 minutes at 37C, Sigma Chemical Co., St. Louis,
MO) transfected with the I-Ek construct at 5x104 cells/well, and Hb peptides
(0-lOO,uM) were added to the appropriate wells. The assay was incubated at
37C for 72 hours with the addition of 3H-thymidine (0.4111Ci/well) during the
last 20 hours. The results shown in Figs la and lb in(1ic~t~. that the substituted
peptide Ser70 ablated the proliferative response of the T cells even at a 10 fold
greater concentration than that which gives maximal proliferation with the
immunogenic peptide Hb(64-76). In addition, substituted peptide Gln72
could not stimulate proliferation.
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IL-2 (Figs. 2a and 2b) and gamma IFN (Figs 3a and 3b) lymphokine
responses were assessed using 24-hour (IL-2) or 48-hour (gamma IFN)
supernatants from the above cultures. For IL-2 detection, the CH-27 B cell
lymphoma (mitomycin-C treated as above, 3-5x104 cells/well) was used as
S APC instead of L cell fibroblasts tO increase assay sensitivity. IL-2 was
qu~ntit~ted in a bioassay as proliferation of the IL-2 dependent cell line
CTLL. Briefly, CTLL cells (5x103 cells/well) were incubated with test
supern~t~ntc for 48 hours. Tritated-thymidine was included during the final
20 hours. The results in Figs 2a and 2b indicate that while the cells make a
cignific~nt amount of IL-2 when presented with Hb(64-72), no detectable IL-2
was seen on stimulation with either of the substituted peptides.
Gamma IF~T was measured using an ELISA as described with reagents
provided by R. Schreiber (Washington University, St. Louis, MO).
Monoclonal antibody specific for gamma IFN (H22) was adsorbed onto
Immulon 2 (Dynatech) 96-well plates overnight at 4C in carbonate buffer
pH9.6. Bound gamma IFN was identified with a polyvalent rabbit anti-
murine gamma IFN followed by peroxidase conjugated goat anti-rabbit IgG
(TAGO). The substrate was developed with the ABTS reagent and read at
414 nm. As shown in Figs 3a and 3b, the results indicate that only the
immunogenic peptide Hb(64-76) could provide the appropriate activation to
allow PL-17 cells to synthesize gamma IFN.
IL-3 was qll~ntit~ted in a bioassay as proliferation of the IL-3
dependent cell line GGl.12 (J. McKearn, Monsanto. Chesterfield, MO) ( ).
Briefly, GG1.12 cells (lx104 cells/well) were incubated with test supernatants
discussed above (collected at 48 hours) for 48 hours~ with 3H-thymidine
included during the last 20 hours. As shown in Fig. 4, the results inrlicate that
WO 94/06828 PC~r/US93/08~
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only the immunogenic peptide Hb(64-76) could provide appropriate activation
to allow PL-17 to synthesize gamma IFN.
For inositol phosphate generation detection PL-17 cells were
incubated overnight at 1-2x107 cells/ml in Inositol-free RPMI containing 20-
50~LCi/ml myo [2-3H] inositol (Amersham) and 10% FCS (dialised in PBS to
remove inositol). The cells were then washed in HBSS and resuspended in
RPMI 1640 complete media and lOmM LiCl (an inositol-1-phosphatase
inhibitor). The T cells at 7-lOx105 cells were incubated in 96 well flat
bottomed plates with the indicated doses of Hb(64-76) or peptide analogs and
DCEK-Hi7 cL cells (5.104/well) or irradiated B lO.BR/SgSnj spleen
cells(5.105/well) for 90-120 minutc.s The samples were assayed for
accumulation of free inositol phosphates. Briefly, the cultures were extracted
with lml of a 1/2 mixture of chloroform and methonol followed by 0.25ml of
chloroform and H20. The phases were separated by centrifugation and the
H2O-soluble fraction placed on a 0.25ml AG1-X8 formate ion-exchange
column (Bio-Rad), then washed extensively with SmM myo-inositol. Total
free inositol phosphate was eluted from the column with 0. lM formic acid,
lM sodium formate in a volume of 1.5ml, and the ratio label was qu~ntit~ted
by scintiliation counting. As shown in Fig 6, the results indicate that althoughthe immunogenic peptide Hb(64-76) allowed production of signific:~nt levels
of inositol phosphates. the substituted peptides Ser70 and Gln 72 did not.
For some proliferation and lymphokine assays the L cell transfectants
were substituted by irradiated BlO.BR/S~Snj spleen cells (2000 rads, 5xlO~
cells/well).
Fxample 3
22
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Studies using a FACscan analyzer were used to determine the presence
of LFA 1 adhesion molecule and IL-2 receptors (IL-2R) on PL-17 cells and
the results are shown in Figs 9 and 10. PL-17 (5x105 cells/well) were
incubated with mitomycin C treated DCEK/Hi7 L cells (5xlOs cells/well)
alone or with the analog peptides Ser70-Hb(64-76) or Gln72-Hb(64-76) at
50~1M in 24 well plates for 48 hours. The T cells were then separated from
the L cells by centrifugation over Ficoll-Paque (1.077g/ml~ Pharmacia LKB,
NJ) at 300 rpm for 15 minutes, and washed three times before preparing for
FACS analysis. For each group 1.105 cells were incubated for 30 minutes on
ice with a, FO441.9 (anit-LFA1. Rag IgG), or b, anti-IL-2R (rat IgG, E.,
Unanue, Washington University, St. Louis, MO), washed twice in PBS
cont~ining o.5% BSA and 0.1% sodium azide, incubated for 30 minutes on ice
with ~;llC-labelled goat anti-rat IgG (Southern Biotech.), then washed twice
again and resuspended in PBS as above. The cells were analyzed on a
FACScan analyzer.
As shown in Figs 9 and 10, the results indicate that Hb (64-76) and
Ser70 Hb (64-76) stimulation upregulated both LFA-1 (Fig 10) and IL-2R
(Fig. 9) levels significantly. These results suggest that Ser70 was delivering apartial signal to PL-17 upon TCR engagement that was independent of inositol
phophate generation.
F.xam~le 4
Studies in the form of tolerance assays to determine whether
substituted peptide Ser70-Hb(64-76) induces anergy of PL-17 were conducted
and the results are shown in Figs. 1 la and 1 lb.
WO g4/06828 PCr/US93/084~
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PL-17 cells (5xlOs/well) and mitomycin-C treated (Fig. 1 la), DCEK-
Hi7 L cell transfectants (5xlO5/well) or (Fig. 1 lb), BlO.BR/SgSnj spleen
cells (SxlO6/well) were incubated alone or with 50,uM of the indicated analog
peptide for 20-24 hours at 37C in 24 well tissue culture plates in a final
5 volume of 700~L1. The T cells were then separated form the APC by
centrifugation over Ficoll-Paque (1.077g/ml, Pharmacia LKB, NJ) at 3000
rpm. for 15 minutes, washed three times in HBSS and rested for three days in
48 well tissue culture plates in RPMI media. T cells were then challenged in a
proliferation assay as described in the Example 2, using Hb(64-76) as antigen
10 (Fig. 1 la). Mitomycin-C treated DCEK-Hi7 L cell transfectants or CH-27
cells (3-SxlO4/well) can replace the spleen cells in the challenge assay. (data
not shown)
As shown in Figs. 1 la and 1 lb, the results indicate that although T
cells which had previously seen Gln72 responded nor nally in the challenge
15 assay, PL-17 which had been previously presented with Ser70 were now
completely unresponsive to the immunogenic peptide Hb(64-76).
Ex~,n ?le 5
Studies showing that Ser70 -induced anergy is a) long-term and b)
20 prevented by Cyclosporin A were conducted and the results are shown in Figs.
Sa-d and in Fig 8.
PL-17 cells were used in the tolerance assay with Mitomycin C treated
DCEK-Hi7 L cells as described in Example 4 with the addition of Cyclosporin
A (l,ug/ml) (Pharmacia) to those cells used in the studies shown in Fig. 8. T
25 cells were separated from APC by ficoll as described in Example 2. The T
cells were then rested in 48 well tissue culture plates for a)1 day (Fig 5a) b) 3
24
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days (Fig. 5b), c) 5 days (Fig. Sc) and d) 7 days (Fig. 5d) before being
- ch~llenged in a proliferation assay as described in Example 2.
The results in Figs 5a-5d indicate that even though T cells had been
rested for up to seven days before challenging with Hb(64-76), they still could
5 not mount a proliferative response which indicates that the substituted peptide
Ser 70 had acted to induced PL-17 into a state of tolerization. Furthermore,
the results shown in Fig 8 indicate that the addition of cyclosporin A to the
culture prevented T cells from becoming unresponsive to subsequent
ch~ nge with live APC.
F.xample 6
Studies showing that various concentrations of the substituted peptide
Ser70 of the invention is capable of inducing tolerization were conducted and
are shown in Figs 7a and 7b.
PL-17 were used in tolerance assays as described in Example 5 except
that the T cells were incubated overnight with various concentrations of
substituted peptides Ser 70 (S70) (Fig. 7a, and 7b) and Gln 72 (Q72) (Fig 7a
only) in the presence of APC.
The results shown in Fig 7a inclirat~ that those T cells incubated with
20 Ser70 were tolerized whereas those T cells incubated with no antigen or with
Gln72 were not tolerized. The results shown in Fig. 7b indicate that those T
cells incubated with very low concentrations of Ser70 were still tolerized even
at high concentrations of immunogenic peptide Hb(64-76)
