U.S. patent application number 11/580358 was filed with the patent office on 2007-05-03 for ex-vivo priming for generating cytotoxic t lymphocytes specific for non-tumor antigens to treat autoimmune and allergic disease.
This patent application is currently assigned to Ortho-McNeil Pharmaceutical Corp.. Invention is credited to Zeling Cai, Juli DeGraw, Michael R. Jackson, Yan Kong, Per A. Peterson, Wei-Xing Shi.
Application Number | 20070098734 11/580358 |
Document ID | / |
Family ID | 23119750 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070098734 |
Kind Code |
A1 |
Cai; Zeling ; et
al. |
May 3, 2007 |
Ex-vivo priming for generating cytotoxic T lymphocytes specific for
non-tumor antigens to treat autoimmune and allergic disease
Abstract
Cytotoxic T lymphocytes (CTLs) specific for antigenic peptides
derived from IgE molecule can be generated in vitro by stimulating
resting naive CD8 T cells with IgE peptides presented by artificial
antigen presenting cells. The IgE specific CTLs lyse the target
cells loaded with IgE peptides in vitro and inhibit antigen
specific IgE response in vivo. In addition, adoptive transfer of
the IgE specific CTL to an asthmatic mouse model can inhibit the
development of lung inflammation and airway hypersensitivity. IgE
specific CTL provides a treatment for allergic asthma and other
IgE-mediated allergic diseases. Antigenic peptides identified from
non-tumor self-antigens induce specific cytotoxic T lymphocyte
(CTL) in vitro. The CTL induced by peptides identified from CD40L
can kill activated CD4 T cells. In vitro generated CTL specific for
CD40L inhibit CD4-dependent antibody responses of all isotypes in
vivo. In contrast, CTL induced by antigenic peptides derived from
IgE specifically inhibit IgE responses, and adoptive transfer of
CD40L-specific CTL to NOD mice at early age delay the development
of diabetes in NOD mice. In vitro generated CTL specific for
non-tumor self-antigens expressed on activated CD4 T cells regulate
immune responses in vivo.
Inventors: |
Cai; Zeling; (San Diego,
CA) ; Jackson; Michael R.; (Del Mar, CA) ;
Peterson; Per A.; (Basking Ridge, NJ) ; Shi;
Wei-Xing; (San Diego, CA) ; Kong; Yan; (Belle
Mead, NJ) ; DeGraw; Juli; (San Diego, CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR
2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
Ortho-McNeil Pharmaceutical
Corp.
Raritan
NJ
|
Family ID: |
23119750 |
Appl. No.: |
11/580358 |
Filed: |
October 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10144188 |
May 13, 2002 |
|
|
|
11580358 |
Oct 13, 2006 |
|
|
|
60291300 |
May 15, 2001 |
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Current U.S.
Class: |
424/184.1 ;
435/372 |
Current CPC
Class: |
C12N 2502/50 20130101;
A61K 2035/122 20130101; C12N 2501/51 20130101; A61P 29/00 20180101;
A61K 39/0008 20130101; A61P 17/06 20180101; A61P 25/00 20180101;
A61P 37/02 20180101; A61P 37/06 20180101; A61P 19/02 20180101; A61P
11/02 20180101; C12N 2502/99 20130101; C12N 5/0636 20130101; A61P
37/08 20180101; A61P 3/10 20180101; C12N 2501/23 20130101; C12N
2501/58 20130101; A61P 11/06 20180101 |
Class at
Publication: |
424/184.1 ;
435/372 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method for producing CTL specific for one or more non-tumor
self antigen T cell epitopes, comprising: a) isolating CD8+ T cells
from a subject; b) loading antigen presenting cells (APCs) having
Class I MHC molecules with the non-tumor self antigen T cell
epitopes, c) culturing the CD8+ T cells with the APCs for a period
of time sufficient for activation of precursor CD8+ T cells
specific for the T cell epitopes; d) expanding in culture the
activated CD8+ T cells in the presence of components required for
proliferation of the activated CD8+ T cells; and e) collecting CD8+
T cells from the culture.
2. The method of claim 1 wherein the T cell epitopes are present on
IgE protein.
3. The method of claim 1 wherein the T cell epitopes are present on
CD40 ligand protein.
4-6. (canceled)
7. A method for treating a disease mediated by a disease causing
target cell, wherein the target cell has on its surface one or more
non-tumor self antigen T cell epitopes associated with Class I MHC
molecules, comprising administering to a patient in need of such
treatment, activated CD8+ T cells wherein the CD8+ T cells have
been selectively activated by culturing said CD8+ T cells in the
presence of antigen presenting cells (APC) having Class I MHC
molecules and wherein said APC are loaded with one of said
non-tumor self antigen T cell epitopes.
8. The method of claim 7 wherein the T cell epitopes are present on
IgE protein.
9. The method of claim 7 wherein the T cell epitopes are present on
CD40 ligand protein.
10. The method of claim 2 wherein said non tumor self-antigen T
cell epitopes are epitopes present on IgE protein and are selected
from the group consisting of TQSPSVFPL (SEQ ID NO:64), SLNGTTMTL
(SEQ ID NO:50), TMTLPATTL (SEQ ID NO:69), TLPATTLTL (SEQ ID NO:67),
TLSGHYATI (SEQ ID NO:68), WVDNKTFSV (SEQ ID NO:51), WLSDRTYTC (SEQ
ID NO:52), ALMRSTTKT (SEQ ID NO:65), NFMPEDISV (SEQ ID NO:79),
YATISLLTV (SEQ ID NO:58), TLTVTSTLPV (SEQ ID NO:56), SVQWLHNEV (SEQ
ID NO:76), QWLHNEVQL (SEQ ID NO:80), TLACLIQNFM (SEQ ID NO:59), or
QVMDVDLSTA (SEQ ID NO:60).
11. The method of claim 8 wherein said non tumor self-antigen T
cell epitopes are epitopes present on IgE protein and are selected
from the group consisting of TQSPSVFPL (SEQ ID NO:64), SLNGTTMTL
(SEQ ID NO:50), TMTLPATTL (SEQ ID NO:69), TLPATTLTL (SEQ ID NO:67),
TLSGHYATI (SEQ ID NO:68), WVDNKTFSV (SEQ ID NO:51), WLSDRTYTC (SEQ
ID NO:52), ALMRSTTKT (SEQ ID NO:65), NFMPEDISV (SEQ ID NO:79),
YATISLLTV (SEQ ID NO:58), TLTVTSTLPV (SEQ ID NO:56), SVQWLHNEV (SEQ
ID NO:76), QWLHNEVQL (SEQ ID NO:80), TLACLIQNFM (SEQ ID NO:59), or
QVMDVDLSTA (SEQ ID NO:60).
12. The method of claim 8 wherein the disease is an allergic
disease, allergic systemic anaphylaxis, allergic rhinitis, hay
fever, food allergy, or allergic asthma.
13. The method of claim 9 wherein the disease is autoimmune
disease.
14. The method of claim 13 wherein the disease is insulin dependent
diabetes, rheumatoid arthritis, SLE, multiple sclerosis, psoriasis,
autoimmune nephritis, multiple sclerosis, autoimmune thyroiditis,
Crohn's disease, inflammatory bowel disease, graft versus host
disease, or transplant rejection.
15. The method of claim 9 wherein the disease is an inflammatory
disease.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 10/144,188, filed May 13, 2002, which claims
priority from U.S. provisional application Ser. No. 60/291,300,
filed May 15, 2001. The disclosures of each of these patent
applications are hereby incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Immune responses to foreign antigens such as those found in
bacteria and virus protect from and eliminate infections. However,
aberrant immune responses can cause allergic diseases and
autoimmune diseases. Immune responses to foreign, sometimes
innocuous, substances such as pollen, dust mites, food antigens and
bee sting can result in allergic diseases such as hay fever, asthma
and systemic anaphylaxis. Immune responses to self-antigens such as
pancreatic islet antigens and cartilage antigens can lead to
diabetes and arthritis, respectively. The hallmark of the allergic
diseases is activation of CD4 T cells and high production of IgE by
B cells, whereas the salient feature of autoimmune diseases are
activation of CD4 T cells and over production of inflammation
cytokines. The current therapies have been focused on the treatment
of symptoms of allergy and autoimmune diseases and do not prevent
the development and progression of the diseases.
[0003] CTLs are derived from resting naive CD8 T cells and
recognize antigenic peptides presented by Major Histocompatibility
Complex (MHC) class I molecules. When resting CD8 T cells encounter
antigenic peptides/MHC complex presented by professional antigen
presenting cells, CD8 T cells will be activated and differentiated
into armed CTL. Upon recognition of peptide/MHC complexes on the
target cells, the antigen specific CTL will deliver a lethal hit
and lysis the antigen-expressing target cells, such as virus
infected target cells or tumor cells.
[0004] Activation of naive T cells in vivo is controlled by
multiple receptor-ligand interactions between T cells and
professional APC such as dendritic cells (R. M. Steinman, Annu.
Rev. Immunol. (1991) 9:271-296). It is generally accepted that two
signals are required for activation of naive T cells (C. A. Janeway
and K. Bottomly, i Cell (1994) 76:275-285). Signal 1 is induced by
the interaction between TCR and MHC/peptide complexes (R. N.
Germain, Cell (1994) 76:287-299) and is aided by binding of CD4/CD8
co-receptors to non-polymorphic regions of MHC class II/I
molecules, respectively (M. C. Miceli and J. R. Parnes, Adv.
Immunol. (1993) 53:59-122). Signal 2 is qualitatively different
from Signal 1 and is delivered via T cell costimulatory molecules
interacting with complementary ligands on APC, e.g. through CD28
interaction with B7 (P. S. Linsley and J. A. Ledbetter, Annu. Rev.
Immunol. (1993) 11:191-212; Lenschow et al., Annu. Rev. Immunol.
(1996) 14:233-258). Signals 1 and 2 function synergistically and
trigger a series of signaling events which ultimately induce T
cells to proliferate, produce cytokines and differentiate into
effector cells (Mueller et al., Annu. Rev. Immunol. (1989)
7:445-480; A. Weiss and D. R. Littman, Cell (1994) 76:263-274). The
relationship between Signals 1 and 2, however, is unclear.
[0005] Although a variety of molecules have been reported to have
costimulatory function, particular attention has been focused on
costimulation delivered via CD28-B7 interaction (R. H. Schwartz,
Cell (1992) 71:1065-1068). CD28 is a molecule with a single Ig like
domain and is constitutively expressed as a homodimer on T cells
(P. S. Linsley and J. A. Ledbetter, (1993) supra). Through its
interaction with either B7-1 or B7-2 molecules on APCs, CD28
molecules are thought to transduce unique signals that stimulate T
cell to produce growth-promoting cytokines such as IL-2 (June et
al., Immunol. Today (1994) 15:321-331), to upregulate expression of
survival factors such as Bcl-X.sub.L (Boise et al., Immunity (1995)
3:87-98) and to prevent anergy induced by Signal 1 alone (R. H.
Schwartz, Curr. Opin. Immunol. (1997) 9:351-357).
[0006] Another pair of molecules that has an important role in T
cell activation is LFA-1/ICAM-1 (Van Seventer et al., J. Immunol,
(1990) 144:4579-4586). ICAM-1 belongs to the Ig gene superfamily
and has five Ig C like domains in the extracellular regions; it is
expressed on both hemapoietic and nonhemapoietic cells. The
receptor for ICAM-1 on T cells is LFA-1 (CD11/CD18), which belongs
to the b2 integrin family (T. A. Springer, Cell (1994) 76:301-314).
The interaction of LFA-1 with ICAM-1 has potent costimulatory
function on T cells (Shimizu et al., Immunol. Rev. (1990)
114:109-143), although opinions vary on whether this function
reflects a separate signaling pathways or increased adhesion
between T cells and APC (Damle et al., J. Immunol. (1993)
151:2368-2379; Bachmann et al., Immunity (1997) 7:549-557).
[0007] In addition to B7 and ICAM-1 molecules, several other
molecules on APCs, including CD70 (Hintzen et al., J. Immunol.
(1995) 154:2612-2623) and heat-stable antigen (HSA) (Liu et al., J.
Exp. Med. (1992) 175:437-445), can exert quite potent costimulatory
function through their interaction with their respective ligands on
T cells. The implication is that T-APC interaction is highly
complex and involves multiple interactions between complementary
sets of molecules on T cells and APCs. The interaction of each set
of molecules could trigger specific signals which induce different
cellular events. The combination of the different signals may act
synergistically for optimal T cell activation and determine the
final fate of T cells. Alternatively, the function of costimulation
molecules may be redundant and the signals induced by each set of
costimulation molecules are additive. The requirement for each set
of costimulation molecules will be influenced by the strength and
characteristics of Signal 1.
[0008] In considering these two possibilities, it is important to
understand the minimal requirements for stimulating naive T cells.
Studies with CD28.sup.-/- mice indicated that CD28-B7 interaction
is highly important in some situations, but not in others
(Shahinian et al., Science (1993) 261:609-612). Likewise, the
requirement for LFA-1/ICAM interaction in primary responses is not
an invariable finding (Shier et al., J. Immunol. (1996)
157:5375-5386).
[0009] CD8 T cells recognize antigenic peptides derived mainly from
virus proteins and proteins expressed on tumor cells. However, it
has recently been reported that newly synthesized proteins are
preferentially processed by antigen-processing machinery (Schubert
et al., Nature, (2000) 404:770-774). Upon activation, immune cells
have acquired the ability to synthesize a number of new proteins,
it is possible that IgE producing B cells and activated CD4 T cells
would present a different sets of peptide/MHC complexes than the
non-IgE producing cells and resting CD4 T cells. These peptides/MHC
complexes presented on IgE producing B cells and activated CD4 T
cells would be able to be recognized by CD8 T cells. Thus, CTL
specific for these peptides/MHC complexes would be able to treat
allergy and autoimmune diseases. However, a number of tolerance
mechanisms have been able to prevent the activation the CD8 T cells
towards self-antigens in vivo.
[0010] CD8 lymphocytes (CTLs) are the arm of adaptive immunity
responsible for the recognition and elimination of infected cells,
tumor cells, and allogeneic cells. Once primed, CTL can recognize
their target antigen on a wide variety of cells and accomplish
their function by lysing the target cell and/or secreting cytokines
like TNF-alpha, or IFN-gamma.
[0011] Presentation of antigen to CD8.sup.+ CTL (cytotoxic T
lymphocytes) occurs in the context of MHC class I molecules
(MHC-I), while presentation of antigen to CD4.sup.+ HTL (helper T
lymphocytes) occurs in the context of MHC class II molecules.
[0012] Efficient induction of CD4.sup.+ T cell requires that the T
cells interact with antigen presenting cells (APC) i.e. cells that
express MHC class II and co-stimulatory molecules. APC are
dendritic cells, macrophages and activated B cells. Although nearly
all nucleated cells express MHC-I, naive CTL also require
presentation of antigen (Ag) by bone marrow-derived APC for
efficient priming (Dalyot-Herman et al., J. Immunol.,
165(12):6731-6737). Dendritic cells are highly potent inducers of
CTL responses (J. Bancherean and R. M. Steinman, Nature, (1998)
392:245-252) and are thought to be the principal APC involved in
priming CTL. Once primed, CTL can recognize their cognate Ags on a
wide variety of cells and respond by lysing the target cell and/or
secreting cytokines.
[0013] Although bone marrow-derived APC are required to efficiently
prime CTL responses (P. J. Fink and M. J. Bevan, Exp. Med. (1978)
148:755-766) activated CTL are readily able to recognize and
respond to Ag presented by a wide variety of cells. Induction of
tumor- or viral-specific CTL immune responses in vivo have been
shown to be dependent on bone marrow derived antigen-presenting
cells (Paglia et al., J. Exp. Med. (1996) 183(1):3 17-322; Labeur
et al., J. Immunol. (1999) 162(1):168-175). It is generally
accepted that bone marrow derived APC, through mechanisms unique to
these cells, take up cellular antigens either in the form of
soluble antigen, associated with chaperone molecules or by
phagocytosis.
[0014] It has long been demonstrated that responses to cellular
antigens are dependent on help delivered by CD4.sup.+ T cells. It
has also been shown that the cellular antigen had to be presented
on the same APC for recognition by the CTL and the HTL. The nature
of this help has been interpreted as a need of IL-2 necessary for
CTL expansion. Recent studies have shown that this help results
from the activation of dendritic cells by HTL and is mediated via
CD40-CD40L interaction (S. R. Clarke, J. Leukocyte Bio. (2000)
67(5):607-614).
[0015] A likely scenario for the induction of a CD8 mediated immune
response to a cellular antigen (derived from a tumor cell or an
infected cell) is therefore the following: dendritic cells acquire
antigens derived from tumor or infected cells. Interaction of
DC-antigen with CD4 cells enable the DC to activate the CD8
cells.
SUMMARY OF THE INVENTION
[0016] Immune cells, such as IgE producing B cells and activated
CD4 T cells play a central role in the pathogenesis of allergic
diseases and autoimmune diseases. The present invention utilizes
cytotoxic T lymphocytes (CTLs) to eliminate or inhibit the immune
cells that cause the allergy and/or autoimmune diseases. Thus, the
development and progression of diseases can be prevented or
interrupted by the methods of the present invention.
[0017] The present invention provides a method for producing CTL
specific for one or more non-tumor self antigen T cell epitopes,
comprising: [0018] a. isolating CD8.sup.+ T cells from a subject;
[0019] b. loading antigen presenting cells (APC's) having Class I
MHC molecules with the T cell epitopes; [0020] c. culturing the
CD8.sup.+ T cells with the antigen-loaded APC's for a period of
time sufficient for activation of precursor CD8.sup.+ T cells
specific for the T cell epitopes; [0021] d. expanding in culture
the activated CD8.sup.+ T cells in the presence of components
required for proliferation of the activated CD8.sup.+ T cells; and,
[0022] e. collecting CD8.sup.+ T cells from the culture.
[0023] The present invention also provides CD8.sup.+ T cells that
are specifically cytotoxic for a disease causing target cell,
wherein the target cell has on its surface one or more non-tumor
self antigen T cell epitopes associated with Class I MHC molecules,
and wherein the CD8.sup.+ T cells have been selectively activated
by interaction with Class I MHC molecules associated with the
non-tumor self antigen T cell epitopes.
[0024] The present invention also provides a method for treating a
disease mediated by a disease causing target cell, wherein the
target cell has on its surface one or more non-tumor self antigen T
cell epitopes associated with Class I MHC molecules, comprising
administering to a patient in need of such treatment, activated
CD8.sup.+ T cells wherein the CD8.sup.+ T cells have been
selectively activated by interaction with Class I MHC molecules
associated with the non-tumor self antigen T cell epitopes.
[0025] The present invention demonstrates that by making and using
artificial antigen presenting cells, tolerance of CD8 T cells to
self antigens was broken and CTLs specific for antigenic peptides
identified from IgE or CD40L proteins were generated. Adoptive
transfer of the in vitro generated CTLs specific for CD40L to NOD
mice dramatically delayed the development of diabetes, and CTLs
specific for IgE peptides inhibited the production of IgE and
reduced lung inflammation in an asthmatic mouse model. The above
system is potentially applicable to human diseases that are caused
by CD4 T cells and by IgE producing B cells. Autoimmune diseases
that caused by CD4 T cells are diabetes, rheumatoid arthritis, SLE,
multiple sclerosis and psoriasis. Whereas allergic diseases
mediated by IgE are systemic anaphylaxis caused by drugs, venoms
and peanuts, allergic rhinitis, food allergy, and allergic asthma.
In addition other self-antigens that expressed on immune cells can
also be used for generation of CTLs in vitro as well in vivo for
treatment of autoimmune diseases and allergic diseases. Antigenic
peptides, proteins or RNA and DNA encoding the non tumor antigens
expressed in non tumor cells can also be used to develop vaccines
for treatment or prevention of allergy and autoimmune diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1:
[0027] The amino acid sequences of IgE.sup.a SEQ ID NO: 102 and
IgE.sup.b SEQ ID NO: 103 constant regions were aligned with vector
NTI software. The sequence differences between the two alleles are
bold and underlined.
[0028] FIG. 2, Panels A, B, C and D:
[0029] CD8.sup.+ T cells were purified from lymph nodes of CBF1/J
mice (A, B and D) or from B6, interferon .gamma. knock out mice
(IFN.gamma..sup.-/-) or perforin (PF.sup.-/-) knock out mice (C).
The purified CD8 T cells were cultured with indicated IgE peptides
presented by SC2 cells transfected with D.sup.b MHC class I, B7-1
(CD80) and ICAM-1 (CD54) molecules. Low dose of recombinant IL-2
(20 units/ml) was added to the culture at Day 3 and every other day
thereafter. On Day 9, CTL activity was measured against .sup.51Cr
labeled RMAS cells loaded with or without indicated IgE peptides.
In FIG. 2, Panel D, anti-D.sup.b mAb (20 .mu.g/ml) was added at the
beginning of CTL assay.
[0030] FIG. 3:
[0031] Adult CBF1/J mice (8 to 12 weeks) were immunized
intraperitoneally with 50 .mu.g ovalbumin (OVA) precipitated with
Alum Hydroxide on Day 1 and Day 14 respectively. Serum IgE, IgG1
and IgG2a were measured by ELISA on Day 28. Two weeks after the
second immunization, the mice were challenged with OVA intranasally
every other day for three treatments. IgE-specific CTLs or control
CTLs (5.times.10.sup.6) were give intravenously one day after each
challenge. Serum IgG and IgE were measured again two weeks after
the last CTL therapy.
[0032] FIG. 4, Panels A, B, C and D:
[0033] CBF1/J mice were immunized as in FIG. 3. Two weeks after the
second immunization, two different doses (5.times.10.sup.6 and
10.times.10.sup.6) of anti-IgE CTLs were given intravenously three
times every other day. Three weeks after the CTL treatment, serum
IgE and OVA-specific IgE was measured and challenged with OVA
intranasally every other day for three treatments. After the last
challenge, bronchial alveolar lavage (BAL) was collected and the
total cells in BAL were counted. Eotaxin in the BAL was measured by
ELISA and Eosinophils cells in the BAL were differentiated by HE
staining.
[0034] FIG. 5, Panels A and B:
[0035] CBF1/J mice were immunized with OVA/Alum at Day 1 and Day
14. Two weeks after the second immunization, mice were injected
every other day for three treatments with PBS, anti-IgE CTL or a
control CTL (anti-influenza CTL) as indicated. Three weeks after
the last treatment, mice were challenged with OVA intranasally
every other day for three treatments. One day after the last
challenge with OVA, airway responsiveness to methacholine for each
mouse was measured by whole body plethrography. Two independent
experiments were shown in Panels A and B respectively.
[0036] FIG. 6, Panels A and B:
[0037] Adult CBF1/J mice (8 to 12 weeks) were immunized
intraperitoneally with 50 .mu.g ovalbumin (OVA) precipitated with
Alum Hydroxide on Day 1 and Day 14 respectively. Two weeks after
the second immunization, the mice were given IgE-specific CTLs
(5.times.10.sup.6) or PBS intravenously. Three weeks after the last
treatment, mice were challenged with OVA intranasally every other
day for two to three treatments. One day after the last challenge
with OVA, the BAL was prepared from each mouse and the lung from
each mouse was fixed and stained with HE. A representative HE
staining of lung tissue from mice received PBS (Panel A) or from
mice received anti-IgE CTL (Panel B) was shown.
[0038] FIG. 7:
[0039] The amino acid sequence deduced from cDNA encoding the human
IgE constant region (SEQ ID NO: 104). Total RNA was prepared from
U266 cell line, which produces human IgE. The total RNA was reverse
transcribed and amplified by PCR with two oligoes encoding the 5'
and 3' human IgE constant region respectively. The cDNA was cloned
into pcDNA3 vector and sequenced.
[0040] FIG. 8:
[0041] Drosophila cells transfected with human HLA-A2 class I cDNA
were cultured with a titrated concentration of indicated IgE
peptides or control peptide (H690) overnight at room temperature
and further cultured at 37.degree. C. for an additional two hours.
The cells were washed and stained with anti-HLA-A2 mAb and analyzed
by flow cytometry. The mean fluorescence intensity was indicated at
Y axis and the peptide concentration was indicated at X axis.
[0042] FIG. 9, Panels A, B, C and D:
[0043] CD8 T cells were purified from individual donors and
cultured with Drosophila cells transfected with HLA-A2, hB7-1,
hB7-2, hICAM-1 and hLFA-3 molecules in the presence of indicated
peptides. After being cultured for six days, low doses of hIL-2 was
added to the culture and re-stimulated with peptides loaded
autologous adherent cells for an additional seven days. The CTLs
were then harvested and the specific killing activities were tested
with .sup.51Cr labeled T2 cells that loaded with indicated peptides
by a standard chromium release assay.
[0044] FIG. 10:
[0045] The amino acid sequence of human IgE was derived as
described as in FIG. 6. The antigenic peptides that contain nine
amino acids were underlines and the antigenic peptides that contain
ten amino acids were shown in bold (SEQ ID NO:105).
[0046] FIG. 11, Panels A and B:
[0047] TAP 2 deficient RMA.S cells (right panel) or L.sup.d
transfected RMA.S cells (left panel) were incubated with indicated
concentration of peptides at 28.degree. C. overnight and then
incubated at 37.degree. C. for two to four hours. The cells were
harvested and stained with mAb specific for L.sup.d (right panel)
or for D.sup.b (left panel) and analyzed with FACScan.
[0048] FIG. 12, Panels A and B:
[0049] CD8.sup.+ T cells were purified from LN of B10.D2 mice and
cultured with Drosophila cells transfected with L.sup.d, B7-1 and
ICAM-1 in the presence of CD40L.186 peptide (left panel) or QL9
peptide (right panel). IL-2 (20 U/ml) was added to the culture at
Days 3 and 5. On Day 7, CTL activity was measured against .sup.51Cr
labeled RMAS.L.sup.d target cells in the presence of indicated
peptides.
[0050] FIG. 13, Panels A and B:
[0051] Purified CD8.sup.+ T cells from B6 mice were cultured with
Drosophila cells transfected with D.sup.b, B7-1 and ICAM-1 in the
presence of Ig E.44 peptide (left panel) or Ig E.366 peptide (right
panel). IL-2 (20 U/ml) was added to the culture on Days 3 and 5.
CTL was harvested on Day 7 and their specific activity was measured
against .sup.51Cr labeled RMA.S target cells in the presence of
indicated peptides.
[0052] FIG. 14, Panels A and B:
[0053] Purified CD4 or CD8 T cells were activated with plate-bound
anti-CD3 and anti-CD28 for forty hours (top panel) or for indicated
time (bottom panel) and were stained with indicated mAb.1
[0054] FIG. 15, Panels A, B, C and D:
[0055] CD40L specific CTL were generated as described in FIG. 2.
CD4 cells used as targets were purified from wild type,
CD40L.sup.-/- or .mu.2m.sup.-/- mice and activated with anti-CD3
and anti-CD28 for forty hours.
[0056] FIG. 16, Panels A and B:
[0057] B10.D2 (top panel) or B6 (bottom panel) were immunized with
OVA+CFA and treated with Ab or CTL as indicated. The spleen cells
were measured for OVA-producing B cells by ELISA spot at Day 21
after immunization.
[0058] FIG. 17, Panels A, B, C, D and E:
[0059] B10.D2 mice were immunized with OVA+CFA on Day 1. Anti-CD40L
CTL or anti-CD40L Ab were given at Days 1, 3, 5. Serum was
collected on Day 14 and OVA-specific immunoglobulins were measured
by ELISA.
[0060] FIG. 18, Panels A, B and C:
[0061] CD8 T cells were purified from C57BL/6 mice and cultured
with Drosophila cells transfected with D.sup.b, B7-1 and ICAM-1 in
the presence of IgE.44 peptide (A), IgE.366 peptide (B) and IgE.125
(C). IL-2 (20 units/ml) was added to the culture on Day 3 and 5.
CTLs were harvested on Day 7 and their specific killing activity
was measured against .sup.51Cr labeled RMA.S target cells in the
presence or absence of indicated peptides.
[0062] FIG. 19, Panels A and B:
[0063] CD8 T cells were purified from C57BL/6 (B6), perforin knock
out mice (pf.sup.-/-) and IFN.gamma. knock out mice
(IFN.gamma..sup.-/-) were cultured with Drosophila cells
transfected with D b, B7-1 and ICAM-1 in the presence of IgE.44
peptide. IL-2 (20 units/ml) was added to the culture on Day 3 and
5. CTLs were tasted on Day 7 and their specific killing activity
was measured against 51Cr labeled RMA.S target cells in the
presence or absence of IgE.44 peptide. In Panel A, CTL activity was
measured in the presence or absence of 10 .mu.g/ml of anti-D.sup.b
monoclonal antibody.
[0064] FIG. 20:
[0065] CD19.sup.+ B cells were purified from human PBMC and
cultured with IL-4 (10 ng/ml) and anti-CD40 mAb (5 mg/ml). Anti-IgE
CTLs were generated as described on FIG. 9 in the presence of
indicated IgE peptides (B) IgE47 and 96, (C) IgE 884 and 890. CTLs
were added at Day 4 to the culture B and C. On Day 6, the culture
supernatant was collected and human IgE was measured by ELISA. In
culture A, no CTLs were added and no B cells in culture D.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0066] The present invention provides in one embodiment, a method
for treating a subject with non-tumor self-antigen T cell epitopes
comprising: [0067] a. preparing a naturally occurring antigen
presenting cell (APC) or a non-naturally occurring
antigen-presenting cell line (nnAPC), wherein said APC or said
nnAPC is capable of presenting up to about fifteen different
peptide molecules that is associated with allergic and/or
autoimmune disease, preferably about ten different peptide-epitope
molecules, simultaneously where each peptide is about six to twelve
amino acids in length, preferably about eight to ten amino acids in
length and in a concentration range of about 10 nM to 100 .mu.M;
[0068] b. harvesting CD8.sup.+ cells from said subject or a
suitable donor; [0069] c. stimulating said CD8.sup.+ cells with
said APC or said nnAPC cell line; [0070] d. adding said CD8.sup.+
cells to media that contains a cytokine, such as, IL-2, IL-7 or
CGM, preferably, IL-2, or IL-2 and IL-7 in combination; [0071] e.
mixing unsuspended peripheral blood monocytes, or alternatively,
CD8-depleted peripheral blood monocytes collected from said subject
or a suitable donor with about 10 to 50 .mu.g/ml of a peptide;
[0072] f. irradiating said peripheral blood monocyte suspension
with a sufficient dose of .gamma.-radiation necessary to sterilize
all components in the suspension, except the desired peripheral
blood monocytes, such as a dose in the range of about 3,000 to
7,000 rads, preferably about 5,000 rads; [0073] g. isolating
adherent peripheral blood monocytes; [0074] h. loading said
adherent peripheral blood monocytes with about 10 ng/ml to 10 g/ml
of said each peptide; [0075] i. combining said CD8.sup.+ cells with
said adherent peripheral blood monocytes at a ratio of about ten
CD8.sup.+ cells to one peripheral blood monocyte; [0076] j.
optionally stimulating said combined suspension of CD8.sup.+ cells
and peripheral blood monocytes for about six to seven days; [0077]
k. optionally stimulating said suspension of CD8.sup.+ cells and
peripheral blood monocytes with IL-2 and IL-7 in media; [0078] l.
optionally assaying CD8.sup.+ suspension for suitable CTL activity,
and optionally assaying for CTL purity, sterility and endotoxin
content; and [0079] m. inoculating said subject with CD8.sup.+
suspension.
[0080] Another embodiment of the present invention provides a
method for treating a subject comprising, [0081] a. preparing a
naturally occurring antigen presenting cell (APC) or a
non-naturally occurring antigen-presenting cell line (nnAPC),
wherein said APC or said nnAPC is capable of presenting up to about
fifteen different peptide-epitope molecules that is associated with
allergic and/or autoimmune disease, preferably about ten peptides,
simultaneously where each peptide is eight to ten amino acids in
length; [0082] b. harvesting CD8.sup.+ cells from said subject;
[0083] c. stimulating said CD8.sup.+ cells with said APC or said
nnAPC cell line for about six to seven days; [0084] d. stimulating
said CD8.sup.+ cells with IL-2 and IL-7 in media; [0085] e. mixing
peripheral blood monocytes collected from said subject with about
20 .mu.g/ml of each peptide; [0086] f. irradiating said
CD8-depleted peripheral blood monocyte suspension with about 5,000
rads of .gamma.-radiation; [0087] g. isolating adherent peripheral
blood monocytes; [0088] h. loading said adherent peripheral blood
monocytes with about 100 ng/ml of said epitope; [0089] i. combining
said CD8.sup.+ cells with said adherent peripheral blood monocytes
at a ratio of about ten CD8.sup.+ cells to one peripheral blood
monocyte; [0090] j. stimulating said combined suspension of
CD8.sup.+ cells and peripheral blood monocytes for about six to
seven days; [0091] k. stimulating said suspension of CD8.sup.+
cells and peripheral blood monocytes with IL-2 and IL-7 in media;
[0092] l. assaying CD8.sup.+ suspension for suitable CTL activity,
purity, sterility and endotoxin content; and [0093] m. inoculating
said subject with CD8.sup.+ suspension.
[0094] Another embodiment of the present invention provides a
method for treating a subject with autoimmune disease, including,
but not limited to, rheumatoid arthritis, lupus, psoriasis,
autoimmune nephritis, multiple sclerosis, insulin dependent
diabetes, autoimmune thyroiditis, Crohn's disease, inflammatory
bowel disease, graft versus host disease and transplant rejection,
and/or allergic diseases, including, but not limited to, food
allergy, hay fever, allergic rhinitis, allergic asthma and venom
allergy, comprising: [0095] a. preparing a naturally occurring
antigen-presenting cell (APC) or a non-naturally occurring
antigen-presenting cell line (nnAPC), wherein said APC or said
nnAPC is capable of presenting up to about fifteen different
peptide-epitope molecules that is associated with such diseases,
preferably about ten peptides, simultaneously where each peptide is
eight to ten amino acids in length; [0096] b. harvesting CD8.sup.+
cells from said subject; [0097] c. stimulating said CD8.sup.+ cells
with said APC or said nnAPC cell line for about six to seven days;
[0098] d. stimulating said CD8.sup.+ cells with IL-2 and IL-7 in
media; [0099] e. mixing peripheral blood monocytes collected from
said subject with about 20 .mu.g/ml of each peptide said APC or
said nnAPC can present; [0100] f. irradiating said CD8-depleted
peripheral blood monocyte suspension with about 5,000 rads of
.gamma.-radiation; [0101] g. isolating adherent peripheral blood
monocytes; [0102] h. loading said adherent peripheral blood
monocytes with about 100 ng/ml of said epitope; [0103] i. combining
said CD8.sup.+ cells with said adherent peripheral blood monocytes
at a ratio of about ten CD8.sup.+ cells to one peripheral blood
monocyte; [0104] j. stimulating said combined suspension of
CD8.sup.+ cells and peripheral blood monocytes for about six to
seven days; [0105] k. stimulating said suspension of CD8.sup.+
cells and peripheral blood monocytes with IL-2 and IL-7 in media;
[0106] l. assaying CD8.sup.+ suspension for suitable CTL activity,
purity, sterility and endotoxin content; and [0107] m. inoculating
said subject with CD8.sup.+ suspension.
[0108] Another embodiment of the present invention is a method of
treating allergic and/or autoimmune diseases wherein the nnAPC
presents the following peptides, SEQ ID NO:15 to SEQ ID NO: 49.
[0109] Another embodiment of the present invention is a method of
treating a non-cancer disease or disease condition that results in
an insufficient or inadequate immune response that is normally
associated with Class I HLA molecules, wherein the treatment
eliminates infected or transformed cells wherein said elimination
has been demonstrated to be mediated by CTLs.
[0110] Another embodiment of the present invention is a method of
treating a non-cancer disease or disease condition that results in
an insufficient or inadequate immune response that is normally
associated with Class I HLA molecules, wherein infected or
transformed cells that have been shown to be susceptible to
elimination by CTL are treated by the method comprising: [0111] a.
preparing a naturally occurring antigen presenting cell (APC) or a
non-naturally occurring antigen-presenting cell line (nnAPC),
wherein said APC or said nnAPC is capable of presenting up to about
fifteen different peptide molecules that is associated with said
disease or disease condition, preferably about ten different
peptide epitope molecules, simultaneously where each peptide is
about six to twelve amino acids in length, preferably about eight
to ten amino acids in length and in a concentration range of about
10 nM to 100 .mu.M; [0112] b. harvesting CD8.sup.+ cells from said
subject or a suitable donor; [0113] c. stimulating said CD8.sup.+
cells with said APC or said nnAPC cell line; [0114] d. adding said
CD8.sup.+ cells to media that contains a cytokine, such as, IL-2,
IL-7 or CGM, preferably, IL-2, or IL-2 and IL-7 in combination;
[0115] e. mixing unsuspended peripheral blood monocytes, or
alternatively, CD8-depleted peripheral blood monocytes collected
from said subject or a suitable donor with about 10 to 50 g/ml of a
peptide; [0116] f. irradiating said peripheral blood monocyte
suspension with a sufficient dose of y-radiation necessary to
sterilize all components in the suspension, except the desired
peripheral blood monocytes, such as a dose in the range of about
3,000 to 7,000 rads, preferably about 5,000 rads; [0117] g.
isolating adherent peripheral blood monocytes; [0118] h. loading
said adherent peripheral blood monocytes with about 10 ng/ml to 10
g/ml of said each peptide; [0119] i. combining said CD8.sup.+ cells
with said adherent peripheral blood monocytes at a ratio of about
ten CD8.sup.+ cells to one peripheral blood monocyte; [0120] j.
optionally stimulating said combined suspension of CD8.sup.+ cells
and peripheral blood monocytes for about six to seven days; [0121]
k. optionally stimulating said suspension of CD8.sup.+ cells and
peripheral blood monocytes with IL-2 and IL-7 in media; [0122] l.
optionally assaying CD8.sup.+ suspension for suitable CTL activity,
and optionally assaying for CTL purity, sterility and endotoxin
content; and [0123] m. inoculating said subject with CD8.sup.+
suspension.
[0124] The present invention provides a non-naturally occurring
antigen-presenting cell (nnAPC) derived from Drosophila
melanogaster cells transfected with DNA for expression, wherein the
nnAPC is capable of simultaneously presenting up to fifteen
different peptide molecules associated with allergic and/or
autoimmune disease, preferably ten peptide molecules that are
encoded by the DNA.
[0125] The present invention provides a non-naturally occurring
antigen-presenting cell (nnAPC) derived from Drosophila
melanogaster cells transfected with human class I HLA, binding, and
co-stimulatory molecule's DNA for expression, wherein the nnAPC is
capable of presenting up to fifteen different peptide molecules
associated with allergic and/or autoimmune disease, preferably ten
peptide molecules that are encoded by the DNA simultaneously.
[0126] Another embodiment of the present invention provides a nnAPC
that presents peptides that are associated with various desired
functions that enhance the treatment of the subject. For example,
in addition to peptides associated with the disease or disease
condition being treated, the nnAPC can present peptides associated
with accessory molecules such as, lymphocyte function antigens
(LFA-1, LFA-2 and LFA-3), intercellular adhesion molecule 1
(ICAM-1), T-cell co-stimulatory factors (CD2, CD28, B7) enhance
cell-cell adhesion or transduce additional cell activation
signals.
[0127] Another embodiment of the present invention provides a nnAPC
that presents peptides that are associated with allergic and/or
autoimmune diseases. For example, the peptides associated or
derived from IgE may be presented with peptides associated or
derived from an allergen, or further in combination with CD40L
peptides.
[0128] Another embodiment of the present invention provides a
method for manufacturing non-naturally occurring antigen-presenting
cell (nnAPC) capable of presenting up to ten different peptide
molecules associated with allergic and/or autoimmune disease
simultaneously, said method comprising of the step: [0129] a.
preparing a insect cell line from Drosophila melanogaster eggs;
alternatively preparing an insect cell line, where cells are grown
for twelve days, selected with peptides, preferably tetramers, that
are capable of identifying the desired cells, and then expanding
said desired cells with OKT3 and IL-2. [0130] b. growing said
insect cells a media that is suitable for growing insect cells,
preferably Schneider.TM.'s Drosophila Medium; [0131] c. making a
pRmHa-3 plasmid from a pRmHa-1 expression vector, where said
pRmHa-3 plasmid includes a metallothionein promoter, metal response
consensus sequences and an alcohol dehydrogenase gene bearing a
polyadenylation signal isolated from Drosophila melanogaster;
[0132] d. inserting into said pRmHa-3 plasmid complementary DNA for
human class I HLA A2.1, B7.1, B7.2, ICAM-1, .beta.-2 microglobulin
and LFA-3, wherein A2.1 can be substituted with any human class I
DNA sequence; [0133] e. transfecting said insect cells with a
phshneo plasmid and said pRmHa-3 plasmid containing complementary
DNA; [0134] f. creating nnAPC by contacting said insect cells with
CuSO.sub.4 to induce expression of the transfected genes in said
insect cells.
[0135] Professional antigen presenting cells, such as dendritic
cells and macrophages, can be loaded with IgE peptides
(Dalyot-Herman et al. (2000) supra) or IgE recombinant proteins
(Paglia et al. (1996) supra) or transduced with virus encoding IgE
or fragments of IgE (Yang et al., Cellular Immunology (2000)
204:29-37). These modified professional antigen-presenting cells
can then be used to activate IgE specific CD8 T cells and generate
IgE specific CTLs in vitro. Alternatively, non-professional antigen
presenting cells can also be transfected or transduced with a
number of genes that encode costimulation molecules plus the genes
that encode IgE and a fragment of IgE. The modified
non-professional antigen presenting cells thus can be used to
stimulate IgE specific CD8 T cells for generation of IgE specific
CTLs.
[0136] The insect cells of the present invention are grown in a
media suitable for growing insects, hereinafter referenced to as
"insect growth media". Insect growth media are commercially
available from a number of vendors, such as, Schneider.TM.'s
Drosophila Medium, Grace's Insect Media, and TC-100 Insect Media.
Alternatively, insect growth media can be prepared by one of
ordinary skill in the art. Typically, the media will include
components necessary to promote and sustain the growth of insects
cells, such as, inorganic salts (for example, calcium chloride,
magnesium sulfate, potassium chloride, potassium phosphate, sodium
bicarbonate, sodium chloride, and sodium phosphate), amino acids
various carbohydrate and chemical species (Imogene Schneider, Exp.
Zool. (1964) 156(1):91-104). Alternatively, the media can also
include vitamins, minerals, and other components that aid in the
growth of insect cells.
[0137] Following is a list of abbreviations and definitions used in
the present specification.
ABBREVIATIONS
[0138] TABLE-US-00001 ABBREVIATIONS APC Antigen-presenting cells
CD8.sup.+ CD8.sup.+ T cells CTL Cytotoxic T lymphocyte FAS Also
known as CD95, epitope on T cells ICAM Intercellular adhesion
molecule IL Interleukin LFA Lymphocyte function antigens MHC Major
histocompatibility complex nnAPC Non-naturally occurring
antigen-presenting cell PBMC Peripheral blood mononuclear cell PBS
Phosphate-buffered saline PCR Polymerase chain reaction RPMI
Roswell Park Memorial Institute RWJPRI The R. W. Johnson
Pharmaceutical Research Institute T Target TCR T cell antigen
receptor
[0139] Following is a list of abbreviations used in the present
specification for various peptide epitopes. The individual amino
acid residues are identified according to a single letter code that
is readily known and used by those of ordinary skill in the
art.
AMINO ACID ABBREVIATIONS
[0140] TABLE-US-00002 ABBREVIATIONS AMINO ACID 3-Letter 1-Letter
alanine ala A valine val V leucine leu L isoleucine ile I proline
pro P phenylalanine phe F trytophan tyr W methionine met M glycine
gly G serine ser S threonine thr T cysteine cys C tyrosine tyr Y
asparagine asn N glutamine gln Q aspartic acid asp D glutamic acid
glu E lysine lys K arginine arg R histidine his H
PEPTIDE EPITOPE ABBREVIATIONS
[0141] As used herein the term IgE 11 refers to the amino acid
sequence KPCKGTASM (SEQ ID NO: 1).
[0142] As used herein the term IgE 209 refers to the amino acid
sequence IPPSPLDLY (SEQ ID NO: 2).
[0143] As used herein the term IgE 366 refers to the amino acid
sequence GSNQGFFIF(SEQ ID NO: 3).
[0144] As used herein the term IgE 29 refers to the amino acid
sequence FPNPVTVTW (SEQ ID NO: 4).
[0145] As used herein the term IgE 105 refers to the amino acid
sequence HSSCDPNAF (SEQ ID NO: 5).
[0146] As used herein the term IgE 114 refers to the amino acid
sequence HSTIQLYCF (SEQ ID NO: 6).
[0147] As used herein the term IgE 363 refers to the amino acid
sequence KSNGSNQGF (SEQ ID NO: 7).
[0148] As used herein the term IgE 307 refers to the amino acid
sequence RSAPEVYVF (SEQ ID NO: 8).
[0149] As used herein the term IgE 44 refers to the amino acid
sequence MSTVNFPAL (SEQ ID NO: 9).
[0150] As used herein the term IgE 411 refers to the amino acid
sequence TSLGNTSLR (SEQ ID NO: 10).
[0151] As used herein the terrn IgE 16 refers to the amino acid
sequence TASMTLGCL (SEQ ID NO: 11).
[0152] As used herein, the terrn IgE 159 refers to the amino acid
sequence of ASTCSKLNI (SEQ ID NO: 12).
[0153] As used herein, the term IgE 125 refers to the amino acid
sequence of GHILNDVSV (SEQ ID NO: 13).
[0154] As used herein the term CD40L 17 refers to the amino acid
sequence LPASMKIFM (SEQ ID NO: 15).
[0155] As used herein the term CD40L 186 refers to the amino acid
sequence RPFIVGLWL (SEQ ID NO: 16).
[0156] As used herein the term CD40L 118 refers to the amino acid
sequence DPQIAAHVV (SEQ ID NO: 17).
[0157] As used herein the term CD40L 220 refers to the amino acid
sequence QSVHLGGVF (SEQ ID NO: 18).
[0158] As used herein the term CD40L 9 refers to the amino acid
sequence SPRSVATGL (SEQ ID NO: 19).
[0159] As used herein the term CD40L 195 refers to the amino acid
sequence KPSIGSERI (SEQ ID NO: 20).
[0160] As used herein the term CD40L 252 refers to the amino acid
sequence FSSFGLLKL (SEQ ID NO: 21).
[0161] As used herein the term CD40L 7 refers to the amino acid
sequence QPSPRSVAT (SEQ ID NO: 22).
[0162] As used herein the term CD40L 181 refers to the amino acid
sequence EPSSQRPFI (SEQ ID NO: 23).
[0163] As used herein the term CD40L 79 refers to the amino acid
sequence LSLLNCEEM (SEQ ID NO: 24).
[0164] As used herein, the term CD40L 152 refers to the amino acid
sequence of VMLENGKQL (SEQ ID NO: 25).
[0165] As used herein, the term CD40L 146 refers to the amino acid
sequence of TMKSNLVML (SEQ ID NO: 26).
[0166] As used herein, the term CD40L 235 refers to the amino acid
sequence of SVFVNVTEA (SEQ ID NO: 27).
[0167] As used herein, the term CD40L 38 refers to the amino acid
sequence of GSVLFAVYL (SEQ ID NO: 28).
[0168] As used herein, the term CD40L 19 refers to the amino acid
sequence of ASMKIFMYL (SEQ ID NO: 29).
[0169] As used herein the term CD40L 24 refers to the amino acid
sequence FMYLLTVFL (SEQ ID NO: 30).
[0170] As used herein the term CD40L 167 refers to the amino acid
sequence GLYYIYAQV (SEQ ID NO: 31).
[0171] As used herein the term CD40L 22 refers to the amino acid
sequence KIFMYLLTV (SEQ ID NO: 32).
[0172] As used herein the term CD40L 36 refers to the amino acid
sequence MIGSALFAV (SEQ ID NO: 33).
[0173] As used herein the term CD40L 58 refers to the amino acid
sequence NLHEDFVFM (SEQ ID NO: 34).
[0174] As used herein the term CD40L 170 refers to the amino acid
sequence YIYAQVTFC (SEQ ID NO: 35).
[0175] As used herein the term CD40L 26 refers to the amino acid
sequence YLLTVFLIT (SEQ ID NO: 36).
[0176] As used herein the term CD40L 231 refers to the amino acid
sequence LQPGASVFV (SEQ ID NO: 37).
[0177] As used herein the term CD40L 45 refers to the amino acid
sequence YLHRRLDKI (SEQ ID NO: 38).
[0178] As used herein the term CD40L 147 refers to the amino acid
sequence TMSNNLVTL (SEQ ID NO: 39).
[0179] As used herein, the term CD40L 229 refers to the amino acid
sequence of FELQPGASV (SEQ ID NO: 40).
[0180] As used herein, the term CD40L 160 refers to the amino acid
sequence of QLTVKRQGL (SEQ ID NO: 41).
[0181] As used herein, the term CD40L 35 refers to the amino acid
sequence of QMIGSALFA (SEQ ID NO: 42).
[0182] As used herein, the term CD40L 185 refers to the amino acid
sequence of SQAPFIASL (SEQ ID NO: 43).
[0183] As used herein, the term CD40L 19 refers to the amino acid
sequence of ISMKIFMYL (SEQ ID NO: 44).
[0184] As used herein, the term CD40L 153 refers to the amino acid
sequence of VTLENGKQL (SEQ ID NO: 45).
[0185] As used herein, the term CD40L 126 refers to the amino acid
sequence of VISEASSKT (SEQ ID NO: 46).
[0186] As used herein, the term CD40L 227 refers to the amino acid
sequence of GVFELQPGA (SEQ ID NO: 47).
[0187] As used herein, the term CD40L 20 refers to the amino acid
sequence of SMKIFMYLL (SEQ ID NO: 48).
[0188] As used herein, the term CD40L 165 refers to the amino acid
sequence of RQGLYYIYA (SEQ ID NO: 49).
[0189] As used herein, the term IgE 47 refers to the amino acid
sequence of SLNGTTMTL (SEQ ID NO: 50).
[0190] As used herein, the term IgE 96 refers to the amino acid
sequence of WVDNKTFSV (SEQ ID NO: 51).
[0191] As used herein, the term IgE 185 refers to the amino acid
sequence of WLSDRTYTC (SEQ ID NO: 52).
[0192] As used herein, the term IgE 309 refers to the amino acid
sequence of ALSDRTYTC (SEQ ID NO: 53).
[0193] As used herein, the term IgE 876 refers to the amino acid
sequence of SLLTVSGAWA (SEQ ID NO: 54).
[0194] As used herein, the term IgE 883 refers to the amino acid
sequence of WLEDGQVMDV (SEQ ID NO: 55).
[0195] As used herein, the term IgE 884 refers to the amino acid
sequence of TLTVTSTLPV (SEQ ID NO: 56).
[0196] As used herein, the term IgE 887 refers to the amino acid
sequence of QMFTCRVAHT (SEQ ID NO: 57).
[0197] As used herein, the term IgE 890 refers to the amino acid
sequence of YATISLLTV (SEQ ID NO: 58).
[0198] As used herein, the term IgE 895 refers to the amino acid
sequence of TLACLIQNFM (SEQ ID NO: 59).
[0199] As used herein, the term IgE 898 refers to the amino acid
sequence of QVMDVDLSTA (SEQ ID NO: 60).
TERMS AND DEFINITIONS
[0200] As used herein, the term "adoptive immunotherapy" refers the
administration of donor or autologous T lymphocytes for the
treatment of a disease or disease condition, wherein the disease or
disease condition results in an insufficient or inadequate immune
response that is normally associated with Class I HLA molecules.
Adoptive immunotherapy is an appropriate treatment for any disease
or disease condition where the elimination of infected or
transformed cells has been demonstrated to be achieved by CTLs. For
example, disease or disease conditions include but are not limited
to cancer and/or tumors, such as, melanoma, prostate, breast,
colo-rectal, stomach, throat and neck, pancreatic, cervical,
ovarian, bone, leukemia and lung cancer; viral infections, such as,
hepatitis B, hepatitis C, human immunodeficiency virus; and
bacterial infections, such as, malaria; tuberculosis, and lysteria
monocytogenesis.
[0201] As used herein, the term "B7.1" refers to a co-stimulatory
molecule associated with antigen-presenting cells.
[0202] As used herein, the term "BCNU" refers to carmustine, also
known as, 1,3-bis (2chloroethyl)-1-nitrosourea.
[0203] As used herein, the term "BSE" refers to bovine spongiform
encephalitis.
[0204] As used herein, the term "CD" refers to clusters of
differentiation, T lymphocytes (originally), B lymphocytes,
monocytes, macrophages, and granulocytes grouped by antigen
epitopes and function.
[0205] As used herein, the term "DTIC" refers to dacarbazine,
5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide.
[0206] As used herein, the term "ex vivo" or "ex vivo therapy"
refers to a therapy where biological materials, typically cells,
are obtained from a patient or a suitable alternate source, such
as, a suitable donor, and are modified, such that the modified
cells can be used to treat a pathological condition which will be
improved by the long-term or constant delivery of the therapeutic
benefit produced by the modified cells. Treatment includes the
re-introduction of the modified biological materials, obtained from
either the patient or from the alternate source, into the patient.
A benefit of ex vivo therapy is the ability to provide the patient
the benefit of the treatment, without exposing the patient to
undesired collateral effects from the treatment. For example,
cytokines are often administered to patients with cancer or viral
infections to stimulate expansion of the patient's CTLs. However,
cytokines often cause the onset of flu like symptoms in the
patients. In an ex vivo procedure, cytokines are used to stimulate
expansion of the CTLs outside of the patient's body, and the
patient is spared the exposure and the consequent side effects of
the cytokines. Alternatively under suitable situations, or
conditions, where appropriate and where the subject can derive
benefit, the subject can be treated concurrently with low level
dosages of a interferon.
[0207] As used herein, the term "HEPES" refers to
N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid buffer.
[0208] As used herein, the term "HLA-A2.1" refers to a HLA Class I
molecule found in approximately 45% of Caucasians.
[0209] As used herein, the term "MPC-10" refers to a magnetic
particle concentrator.
[0210] As used herein, the term "NK cells" refers to natural killer
cells.
[0211] As used herein, the term "OKT3" refers to ORTHOCLONE OKT3,
muromonab-CD3, anti-CD3 monoclonal antibody.
[0212] As used herein, the term "TAP-1, 2" refers to Transporter
Associated with Antigen Processing-1, 2.
[0213] As used herein, the term "Th cells" refers to Helper T
cells, CD4.sup.+.
[0214] As used herein, the term "C-lectin" refers to a peptide of
the sequence that has been found to be associated with ovarian
cancer.
[0215] As used herein, the term "major histocompatibility complex"
or "MHC" is a generic designation meant to encompass the
histocompatibility antigen systems described in different species
including the human leucocyte antigens (HLA).
[0216] As used herein, the terms "epitope," "peptide epitope,"
"antigenic peptide" and "immunogenic peptide" refers to a peptide
derived from an antigen capable of causing a cellular immune
response in a mammal. Such peptides may also be reactive with
antibodies from an animal immunized with the peptides. Such
peptides may be about five to twenty amino acid in length
preferably about eight to fifteen amino acids in length, and most
preferably about nine to ten amino acids in length.
[0217] As used herein, the term "analog" includes any polypeptide
having an amino acid residue sequence substantially identical to
the polypeptide sequence of the present invention in which one or
more residues have been conservatively substituted with a
functionally similar residue and which displays the functional
aspects of the present invention as described herein. Examples of
conservative substitutions include the substitution of one
non-polar (hydrophobic) residue such as isoleucine, valine, leucine
or methionine for another, the substitution of one polar
(hydrophilic) residue for another such as between arginine and
lysine, between glutamine and asparagine, between glycine and
serine, the substitution of one basic residue such as lysine,
arginine or histidine for another, or the substitution of one
acidic residue, such as aspartic acid or glutamic acid or
another.
[0218] As used herein, the term "conservative substitution" also
includes the use of a chemically derivatized residue in place of a
non-derivatized residue.
[0219] As used herein, the term "chemical derivative" refers to a
subject polypeptide having one or more residues chemically
derivatized by reaction of a functional side group. Examples of
such derivatized molecules include for example, those molecules in
which free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups may be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups may be derivatized to form O-acyl or O-alkyl derivatives.
The imidazole nitrogen of histidine may be derivatized to form
N-im-benzylhistidine. Also included as chemical derivatives are
those proteins or peptides which contain one or more
naturally-occurring amino acid derivatives of the twenty standard
amino acids. For example, 4-hydroxyproline may be substituted for
proline; 5-hydroxylysine may be substituted for lysine;
3-methylhistidine may be substituted for histidine; homoserine may
be substituted for serine; and ornithine may be substituted for
lysine. Proteins or polypeptides of the present invention also
include any polypeptide having one or more additions and/or
deletions or residues relative to the sequence of a polypeptide
whose sequence is encoded is the corresponding nucleic sequence of
the present invention, so long as the requisite activity is
maintained.
[0220] Cytolytic T cells (CD8.sup.+) are the main line of defense
against viral infections. CD8.sup.+ lymphocytes specifically
recognize and kill host cells that are infected by a virus.
Theoretically, it should be possible to harness the immune system
to combat other types of diseases including cancer. However, few in
vitro/ex vivo procedures have been available for specifically
activating CTLs. The identification of key allergic and/or
autoimmune antigens noted herein and a method for specific in vitro
activation CTLs described below now allow testing of the concept of
adoptive immunotherapy of allergic and/or autoimmune diseases.
[0221] All naive T cells require two signals for activation to
elicit an immune response. For CD8.sup.+ lymphocytes (CTLs), the
first signal, which imparts specificity, consists of presentation
to the CD8.sup.+ cell of an immunogenic peptide fragment (epitope)
of the antigen bound to the Class I MHC (HLA) complex present on
the surface of antigen-presenting cells (APCs). This complex is
recognized specifically by a T cell antigen receptor (TCR), which
communicates the signal intracellularly.
[0222] Binding to the T cell receptor is necessary but not
sufficient to induce T cell activation, and usually will not lead
to cell proliferation or cytokine secretion. Complete activation
requires a second co-stimulatory signal(s), these signals serve to
further enhance the activation cascade. Among the co-stimulatory
molecules on antigen-presenting cells, B7 and cell adhesion
molecules (integrins) such as ICAM-1 assist in this process by
binding to CD28 and LFA-1, respectively, on the T cell. When a
CD8.sup.+ cell interacts with an antigen-presenting cell bearing an
immunogenic peptide (epitope) bound by a Class I MHC molecule in
the presence of appropriate co-stimulatory molecule interactions,
the CD8.sup.+ cell becomes a fully activated cytolytic T cell.
[0223] Lymphocyte-mediated cell killing involves a sequence of
biological events beginning with the binding of the CD8.sup.+ CTL
to an antigen-bearing target (tumor) cell by means of the
recognition process described above for T cell activation. The
interaction begins with the binding of antigen in association with
an MHC Class I molecule on the APC or target cell to the T cell
antigen receptor (TCR). Accessory molecules such as lymphocyte
function antigens (LFA-1, LFA-2 and LFA-3), intercellular adhesion
molecule 1 (ICAM-1), T cell co-stimulatory factors (CD2, CD28, B7)
enhance cell-cell adhesion or transduce additional cell activation
signals.
[0224] After cell-cell interaction, the CTL kills the target cell
through the action of soluble cytolytic mediators (perforin and
granzymes stored in cytoplasmic granules in the T cell) and a CTL
surface molecule (FAS ligand). After the cytolytic attack, target
cells die by necrosis (membrane perforation and organelle
destruction) or apoptosis (chromatin condensation, DNA
fragmentation and membrane blebbing).
[0225] The mechanisms of lymphocyte-mediated cytolysis is
graphically depicted in FIG. 2. In Panel A of FIG. 2, after binding
to the target cell, cytoplasmic granules in the CTL are rapidly
reoriented toward the target cell for release of granules
containing perforin and granzymes into the intercellular space.
These proteolytic enzymes form pores in the plasma membrane of the
target cell eventually leading to cell necrosis. In Panel B, after
binding to the target cell, the level of FAS expression on the CTL
increases. The interaction of FAS and the FAS receptor on the
target cell leads to apoptosis. Proteases such as CPP32 and others
related to IL-1b-converting enzyme (ICE) have been implicated in
the induction of apoptosis.
[0226] It is possible to use naturally-occurring antigen-presenting
cells, for example, dendritic cells, macrophages, autologous tumor
cells for in vitro CD8.sup.+ activation. However, the efficiency of
activation following this approach is low. This is because the
Class I molecules of native APCs contain many other types of
peptide epitopes besides tumor epitopes. Most of the peptides are
derived from normal innocuous cell proteins, resulting in a
dilution of the number of active native APCs that would actually be
effective against a tumor (Allison et al., Curr. Op. Immunol.
(1995) 7:682-686).
[0227] A more direct and efficient approach to this problem is to
specifically activate CD8.sup.+ cells only with those epitopes
relevant to combating a specific disease, (such as allergic and/or
autoimmune disease). To this end, an artificial antigen presenting
cell is created by expressing MHC Class I molecules in Drosophila
melanogaster (fruit fly) cells. Since Drosophila does not have an
immune system, the TAP-1,2 peptide transporters involved in loading
peptide epitopes onto class I molecules are absent. As a result,
the class I molecules appear on the Drosophila cell surface as
empty vessels. By incubating these transfected Drosophila cells
with exogenous peptides that bind to the class I molecules, such
as, cancer or tumor specific epitopes, including but limited to,
melanoma specific epitopes, it is possible to occupy every class I
molecule with the same peptide. High density expression of class I
molecules containing a single peptide in these Drosophila APCs
permit generation of cytotoxic CD8.sup.+ T cells in vitro which are
completely specific for the antigen peptide. Methods and procedures
for preparing Drosophila cells are taught in U.S. Pat. No.
5,529,921 issued Jun. 25, 1996 entitled "In Vitro Activation of
Cytotoxic T-Cells Using Insect Cells Expressing Human Class I MHC
and .beta.2-Microglobulin", and U.S. Pat. No. 5,314,813 issued May
24, 1994 entitled "Drosophila Cell Lines Expressing Genes Encoding
MHC Class I Antigens And .beta.2-Microglobulin and Capable of
Assembling Empty Complexes and Methods of Making Said Cell Lines".
In particular, U.S. Pat. No. 5,529,921 discloses at column 26, line
56 to column 28, line 22 various methods of separating out and/or
enriching cultures of precursor cells.
[0228] Additionally, this feature eliminates the need for in vivo
stimulation of the immune system with various cytokines. Thereby
resulting in a treatment that fore goes the side effects caused by
cytokines. Alternatively under suitable situations, or conditions,
where appropriate and where the subject can derive benefit, the
subject can be treated concurrently with low level dosages of
.alpha. interferon.
[0229] Eliminating the need for in vivo stimulation with cytokines
provides an improvement to the quality of patient care. Treatment
regimes that include the administration of cytokines to patients
often result in the patient developing flu-like symptoms, such as
nausea, vomiting, and fever. These side reactions are generally not
life threatening, although a particularly severe reaction occurring
in a patient who is already in a weaken condition could result in a
life endangering situation. Another consideration is the adverse
impact such side reactions have on patient acceptance and
compliance of an otherwise beneficial treatment regime. Removing
the need for in vivo stimulation with cytokines results in a
treatment regime that improves the comfort of the patient, and
provides the clinician with an effective method of treatment that
his or her patient is more likely to comply with.
[0230] The utility of this method for adoptive immunotherapy has
been demonstrated in mice using transfected Drosophila cells as
APCs and CD8.sup.+ cells from the 2C line of T cell receptor (TCR)
transgenic mice. In this system, purified CD8.sup.+ 2C cells are
highly responsive to in vitro peptides presented by MHC Class I
(L.sup.d)-transfected Drosophila cells also bearing the
co-stimulatory molecules B7-1 and ICAM-1. Transfected Drosophila
cells as a probe for defining the minimal requirements for
stimulating unprimed CD8.sup.+ T cells (Cai et al., P.N.A.S. USA
(1996) 93:14736-14741). Alternatively, when un-separated mouse
spleen cells are used as responders in place of purified 2C cells,
the need for co-stimulatory molecules does not apply. In this
instance, the CD8.sup.+ cells in the spleen population receive
"bystander" co-stimulation from activated B cells. Utilizing this
finding, it has been possible to show that MHC Class I
(L.sup.d)-transfected Drosophila cells are able to induce normal
DBA/2 mouse spleen cells to respond to syngeneic P815 mastocytoma
tumor-specific peptides in vitro in the absence of added
lymphokines. Injection of these CTLs into DBA/2 mice bearing P815
mastocytoma led to rapid tumor regression (Sun et al., Immunity
(1996) 4:555-564).
[0231] The use of any natural, or artificial, antigen presenting
cell (APC) system to generate cytotoxic T lymphocytes in vitro is
limited by the antigen specificities these systems are capable of
generating.
[0232] The following APC systems have been utilized to generate
antigen-specific CTL's to single epitopes: [0233] 1. Human
dendritic cells (DC) pulsed with defined peptides; [0234] 2.
Peripheral blood mononuclear cells (PBMCs) which have been driven
to lymphoblasts and pulsed with peptides; [0235] 3. Lymphoblastoid
cell lines (LCL) where the natural peptides are acid-stripped and
loaded with the peptides of interest; [0236] 4. Drosophila cells
engineered to express empty class I molecules; and Mouse 3T3 cells
transfected with human class I and co-stimulatory molecules. (J-B.
Latouche and M. Sadelain, Nature Biotech (2000) 18:405-409).
[0237] Dendritic cells (DCs) are considered the primary antigen
presenting cell system in humans because of their wide application
in presenting primary antigen cells. Self or foreign proteins are
processed within a DC. The resultant peptide epitopes are presented
by HLA molecules, and are transported to the surface of the DC.
However, it was found that DCs would not consistently generate in
vitro, CTLs directed against four different peptides. This would
have provided CTLs having activity corresponding to each of the
four peptides. In addition, it was also found that the phenotype of
the DC at the time of peptide pulsing, mature or immature, did not
effect the outcome.
[0238] Alternatively, Drosophila cell stimulation usually resulted
in CTLs directed against up to ten different types of peptides.
This provides CTLs that are active to each of the ten peptides.
[0239] The ability of Drosophila cells and DC to elicit CTL
responses were evaluated, initially to a single peptide epitope,
following the standard stimulation protocols for each, in order to
compare DCs and transfected Drosophila cells. Immature DCs were
generated by culturing for one week autologous monocytes in the
presence of IL-4 and GM-CSF. Mature DCs were obtained from immature
DCs by addition of TNF .alpha. to the culture medium twenty-four
hours prior to harvesting. DCs (immature and mature) were
harvested, pulsed with peptides and mixed with purified CD8 cells
following the procedure used for the stimulation of CD8 cells and
peptide-pulsed Drosophila cells. Drosophila cells were found to be
generally better stimulators than DC. Further, DCs displaying
either the immature or mature phenotype were not as efficient as
Drosophila cells in eliciting specific CTL responses when defined
peptides were used to pulse the APCs. This is particularly
surprising, because of the dominant role played by DCs in the
immune system.
Preparation of Cytotoxic Lymphocytes
[0240] CD8.sup.+ cells isolated from leukapheresis samples by
positive selection with anti-CD8 antibody are stimulated against
IgE and/or CD40L associated peptides presented by Drosophila cells
expressing Human Class I molecules (HLA-A2.1), B7.1, ICAM-1, LFA-3
and B7.2. CD8.sup.+ cells are re-stimulated for two rounds with
autologous monocytes loaded with the peptide epitope in the
presence of IL-2 and IL-7. CTLs are non-specifically expanded with
OKT3 and IL-2. CTL activity is measured against cells and purity of
CD8.sup.+ T cells is assessed by flow cytometry.
[0241] The manufacturing processes and protocols are done according
to Good Laboratory Practices and Good Manufacturing Practices.
"Good Laboratory Practices" and "Good Manufacturing Practices" are
standards of laboratory and manufacturing practices, which are set
by United States Food and Drug Administration, and are readily
known to those of skill in the art. The CTLs are monitored for
identity, viability, CTL activity, sterility, and endotoxin
content.
[0242] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE 1
Manufacture of Drosophila Antigen-Presenting Cells
[0243] The Schneider S2 cell line was prepared from Drosophila
melanogaster (Oregon-R) eggs according to published procedures and
has been deposited with the American Type Culture Collection (CRL
10974). S2 cells are grown in commercial Schneider's Drosophila
medium supplemented with 10% fetal bovine serum.
[0244] The pRmHa-3 plasmid vector for expressing MHC Class I and
co-stimulatory proteins in S2 cells was derived from the pRmHa-1
expression vector constructed as described in the literature. It
contains a metallothionein promoter, metal response consensus
sequences and an alcohol dehydrogenase gene bearing a
polyadenylation signal isolated from Drosophila melanogaster.
Complementary DNAs for Fransfection were Prepared as Follows:
[0245] HLA-A2.1 and .beta.-2 microglobulin: Reverse
transcription-PCR from K562 cells using primers derived from the
published sequence. [0246] B7.1: Reverse transcription-PCR from
K562 cells using primers derived from the published sequence.
[0247] ICAM-1: Reverse transcription-PCR from K562 cells using
primers derived from the published sequence. [0248] B7.2: Reverse
transcription-PCR from HL-60 cells (ATCC CCL-240) using primers
derived from the published sequence. [0249] LFA-3: Reverse
transcription-PCR from HL-60 cells (ATCC CCL-240) using primers
derived from the published sequence.
[0250] Complementary DNAs were individually inserted into the
pRmHa-3 vector. S2 cells were transfected with a mixture of
HLA-A2.1, B7.1 and ICAM-1 plasmid DNAs and the phshneo plasmid
using the calcium phosphate precipitation method. Stably
transfected cells were selected by culturing in Schneider's medium
containing geneticin. Twenty-four hours before use, expression of
the transfected genes was induced by addition of CuSO.sub.4. The
level of expression was assessed by flow cytometry using
anti-HLA-A2.1, anti-B7.1 and anti-ICAM-1 antibodies. HLA expression
by greater than 30% of the cells is necessary for efficient in
vitro activation of CD8.sup.+ lymphocytes.
Isolation of Human CD8.sup.+ Cells
[0251] CD8.sup.+ cells are isolated from leukapheresis samples by
positive selection using the Dynabeads.TM. isolation procedure
(Dynal). An anti-human CD8 mouse monoclonal antibody (50 .mu.g/ml
in human gamma globulin [Gammagard.RTM.]) is added to washed cells
in Dulbecco's PBS supplemented with 1% human serum albumin
(Baxter-Hyland) and 0.2% Na citrate. After incubation at 4.degree.
C. for forty-five minutes with gentle mixing, the cells are washed
and re-suspended in the same buffer containing Dynal magnetic beads
(Dynabeads.TM.) coated with sheep anti-mouse IgG at a bead to cell
ratio of 1:1. The cells and beads are placed into a sterile tube
and gently mixed at 4.degree. C. for forty-five minutes. At the end
of this time, the antibody-bound cells are removed magnetically
using the MPC-1.RTM. separator according to the manufacturer's
instructions (Dynal). Dissociation of the CD8 cell-bead complex is
achieved by incubation at 37.degree. C. for forty-five minutes in
the presence of CD8 peptide59-70 (AAEGLDTQRFSG, SEQ.ID.NO.:61).
Free beads are removed magnetically and the CD8 cells are counted
and analyzed by flow cytometry to evaluate purity. Recovery of
CD8.sup.+ cells is typically greater than 80%. Table 1 summarizes
the cell composition of fourteen separate CD8.sup.+ preparations
from normal human PBMC preparations by positive selection with
anti-CD8 antibody. TABLE-US-00003 TABLE 1 Purification of CD8.sup.+
Cells by Positive Selection Analyzed by Flow Cytometry PBMC POST
SELECTION CELL TYPE Mean % (Range) Mean % (Range) CD8 T cells 15%
(7-24) 82% (56-95) CD4 T cells 36% (14-52) 2% (0.1-10) CD 14
Monocytes 15% (7-26) 0.8% (0.2-2) CD15 Neutrophils 12% (8-21) 0.6%
(0.1-3) CD19 B cells 2% (0.4-7) 3% (0.5-9) CD56 NK cells 6% (2-17)
6% (0.1-20)
In Vitro Immunization of Purified Human CD8.sup.+ Cells
[0252] Primary Stimulation: Transfected Drosophila S2 cells are
incubated in Schneider's medium (10.sup.6 cells/ml) supplemented
with 10% fetal calf serum and CuSO.sub.4 at 27.degree. C. for
twenty-four hours. Cells are harvested, washed and re-suspended in
Insect X-press medium (BioWhittaker) containing 100 .mu.g/ml human
tyrosinase.sub.369-377 (RWJPRI). Following incubation at 27.degree.
C. for three hours, the S2 cells are mixed with CD8.sup.+ cells at
a ratio of 1:10 in RPMI medium (Gibco) supplemented with 10%
autologous serum. The cell mixture is incubated for four days at
37.degree. C. during which the Drosophila cells die off. On Day 5,
IL-2 (20 U/ml) and IL-7 (30 U/ml) are added with a media change to
selectively expand the tyrosinase-specific CTL population.
[0253] Re-stimulation: Frozen, autologous, CD8-depleted PBMCs,
obtained at the time of leukapheresis, are thawed, washed and
re-suspended at 10.sup.6 cells/ml in RPMI medium containing 10%
autologous serum (as a source of .beta.2 microglobulin) and 20
.mu.g/ml of peptide epitope. Following .gamma.-irradiation (5,000
rads), the cells are incubated at 37.degree. C. for two hours.
Non-adherent cells are removed by washing with Dulbecco's PBS.
Adherent monocytes are loaded with the tyrosinase epitope by
incubation for 90 minutes in Hepes-buffered RPMI medium containing
10% autologous serum and 10 .mu.g/ml of peptide epitope. The
supernatant is removed and the Drosophila-activated CD8.sup.+ cell
suspension (3.times.10.sup.6 cells/ml in RPMI medium with 10%
autologous serum) is added at a ratio of ten CD8.sup.+ cells to one
adherent monocyte. After three to four days of culture at
37.degree. C., IL-2 (20 U/ml) and IL-7 (30 U/ml) are added with a
medium change to selectively expand the epitope-specific CTL
population.
[0254] Non-specific Expansion: CD8's non-specifically expanded and
culturing them in RPMI medium supplemented with autologous serum,
anti-CD3 monoclonal antibody (OKT.RTM.3), IL-2 and .gamma.
irradiated autologous PBMCs.
Assays for Activity and Purity
[0255] CTL Assay: Epitope-bearing (target) cells are used as target
cells in a .sup.51Cr release assay. 5.times.10.sup.6 target cells
in RPMI medium containing 4% fetal calf serum, 1% HEPES buffer and
0.25% gentamycin are labeled at 37.degree. C. for one hour with 0.1
mCi .sup.51Cr. Cells are washed four times and diluted to 10.sup.5
cells/ml in RPMI with 10% fetal bovine serum (HyClone). In a
96-well microtiter plate, 100 .mu.l effector CTLs and 100 .mu.l
peptide-loaded, .sup.51Cr-labeled target cells are combined at
ratios of 100:1, 20:1 and 4:1 (effector: target). K562 cells are
added at a ratio of 20:1 (K562) to reduce natural killer cell
background lysis. Non-specific lysis is assessed using cells
labeled with .sup.51Cr as described above, but not bearing the
epitope cell line. Controls to measure spontaneous release and
maximum release of .sup.51Cr are included in duplicate. After
incubation at 37.degree. C. for six hours, the plates are
centrifuged and the supernatants counted to measure .sup.51Cr
release. Percent specific lysis is calculated using the following
equation: cpm sample-cpm spontaneous release/cpm maximum
release-cpm spontaneous release.times.100
[0256] Flow Cytometry: CD8.sup.+ cells, before and after in vitro
activation, were analyzed for a number of cell surface markers
using fluorescent monoclonal antibodies and FACS analysis. Results
from a typical activation protocol using cells from a healthy donor
is shown in Table 2. TABLE-US-00004 TABLE 2 Flow Cytometry Analysis
of In Vitro Activated CD8.sup.+ Cells PRE- POST- MARKER/ ACTIVATION
ACTIVATION CELL TYPE Mean % Mean % CD8 T cell 98 99
TCR.alpha..beta. T cell receptor 98 92 CD 44 lymph node homing
receptor 91 99 CD45RO memory T cell 58 88 CD45RA 41 31 CD62L HEV
homing receptor 24 38 CD56 NK cell 1 11 CD25 activated T cell 0.1
29
[0257] In addition to activity and purity, CTL preparations will be
assayed for sterility and endotoxin content. TABLE-US-00005
REAGENTS REAGENT SUPPLIER GRADE NOTES Rh IL-2 Chiron USP sterile
solution Rh IL-7 Genzyme Research lyophilized, sterile solution
Peptide RWJPRI Research Dynabeads .RTM. Dynal GMP sheep anti-mouse
IgG M-450 magnetic beads Human serum Baxter USP sterile,
non-pyrogenic albumin hepatitis virus-free, 25% solution Fetal
bovine Gemini Research sterile, BSE-, serum endotoxin-
mycoplasma-free Gammagard .RTM. Baxter USP sterile, human immune
globulin solution for injection Anti-CD8 RWJPRI Research mouse
anti-human CD8 antibody monoclonal antibody CD8 RWJPRI Research
release of CD8.sup.+ cells peptide.sub.59-70 from magnetic beads
W6/32 ATCC Research mouse anti-human HLA-A, B, C monoclonal
antibody
[0258] TABLE-US-00006 CELL LINES CELL LINE SUPPLIER NOTES
Drosophila S2 ATCC CRL 10974 M14 UCSD HLA-A2.1 human melanoma K562
ATCC Human erythroleukemic cell line; target for NK cells JY cells
ATCC EBV-transformed, human B cell line expressing HLA-A2.1 and B7
P815 and P1024 ATCC DBA/2 mouse mastocytoma cell lines Jurkat A2.1
ATCC acute T cell leukemia transfected with human HLA-A2.1 ATCC:
American Type Culture Collection
EXAMPLE 2
Trial of Cytotoxic T Cell Infusions Against IgE Producing Cells
Purpose of Trial
[0259] This example teaches the effectiveness of cytotoxic T Cell
infusions in the treatment of allergic diseases as assessed
according to the following factors: [0260] 1. Safety and toleration
of re-infused autologous CTLs after in vitro immunization; [0261]
2. Kinetics of infused CTLs in the systemic circulation factoring
in limiting dilution analysis; [0262] 3. Whole body disposition of
CTLs by radioscintigraphy; [0263] 4. Cell composition of biopsied
nodules by immunohistology (CTLs, TH, NK, B cells); and [0264] 5.
Regression of measurable lesions and duration of response over two
months. Treatment with Ex Vivo Generated Autologous CTLs
[0265] All patients will receive, at least, a single infusion of
autologous CTLs. The number of cycles and the dose of cells
administered to each patient are summarized in Table 1. The number
of cells generated in vitro is dependent on patient-related factors
such as the numbers of PBMCs isolated from the aphaeresis procedure
and the number of CD8.sup.+ T cells present in each PBMC
preparation. Since all of the cells generated in vitro are
re-infused into the donor, doses administered to each patient are
necessarily varied. In an attempt to normalize the doses between
patients, a calculated "potency" score is recorded for each dose.
The value is obtained by multiplying the total number of cells by
the lytic activity obtained with peptide-loaded target cells.
Patients are entered into a second, third or fourth cycle of
treatment based on their clinical status at the end of each cycle.
The total number of naive CD8.sup.+ T cells isolated is dependent
on its percentage in each of the PBMC preparations. The percent of
CD8.sup.+ T cells varies among the patients. The procedure for
generating CTLs ex vivo is taught in the Specification and Example
1, above.
Up-Regulation of Class I and Melanoma-Associated Antigens in
Response to IFN.alpha.-2b
[0266] In an attempt to enhance the ability of the antigen-specific
CTLs to lyse IgE producing cells in vivo, low dose IFN.alpha.-2b is
administered for five consecutive days prior to the CTL infusion,
and thrice weekly for an additional four weeks. One way to measure
an in vivo response to the cytokine is to evaluate biopsies
obtained at serial time points by immunohistochemical analysis for
positive staining with specific antibodies.
Antigenic Specificity of Ex Vivo-Generated CTLs
[0267] CTLs generated from all patients are evaluated on the day of
release against peptide-loaded T2 targets, an HLA-A2 IgE producing
M-14 clone 4 cell line and an autologous M-14 cell line, if biopsy
material was available to establish a line. Each prepared dose of
cells is evaluated for its cytolytic activity. Peptide-loaded T2
cells, presenting either each peptide alone, or all peptides
simultaneously, are used to determine the specificity of the CTL
response generated for each patient. The ability to lyse
endogenously-expressed, HLA-A2-associated, antigen-bearing cells is
assessed with an HLA-A2 matched line or an autologous cell line. In
addition to cytolytic activity, antigen-specificity is evaluated
with an established method for detecting intracellular gamma
interferon production, made in response to a specific peptide
stimulus. The CTLs generated at the end of the ex vivo protocol are
evaluated by this method. The percent of cells specific for each of
the peptides is recorded individually. The total number of specific
cells in each bulk CD8 culture from a patient is calculated by
adding each of the peptide specificities detected in that
population of T cells. An increase in the total number of specific
cells is detected with each successive treatment cycle.
Presence of Anergic State Did Not Preclude Ability to Generate CTLs
or Prevent a Clinical Response
[0268] Most of the patients treated under this protocol receive
previous medical intervention. A pretreatment skin test is
performed to determine if an anergic response to a panel of seven
common antigens correlates with either an inability to generate
CTLs ex vivo, or prevent a documented clinical response. The
ability to generate CTLs ex vivo does not correlate with the
patient's pretreatment skin test results.
EXAMPLE 3
[0269] IgE plays an essential role in the pathogenesis of allergic
asthma. Here, we show that cytotoxic T lymphocytes (CTLs) specific
for antigenic peptides derived from IgE molecule can be generated
in vitro by stimulating resting naive CD8 T cells with IgE peptides
presented by artificial antigen presenting cells. The IgE specific
CTLs lyse the target cells loaded with IgE peptides in vitro and
inhibit antigen specific IgE response in vivo. In addition,
adoptive transfer of the IgE specific CTL to an asthmatic mouse
model can inhibit the development of lung inflammation and airway
hypersensitivity. Thus, IgE specific CTL may provide a treatment
for allergic asthma and other IgE-mediated allergic diseases.
[0270] Cytotoxic T lymphocytes are derived from resting naive CD8 T
cells. In the present of antigens and co-stimulations, resting
naive CD8 T cells can be activated and differentiated into armed
cytotoxic T cells, which can destroy the target cells that express
the antigens. CTLs play an essential role in immunity against virus
and intracellular pathogens by lysis the infected cells and/or
through the effect of cytokines CTL produced.
Identification of Antigenic Peptides from IgE Protein Sequence:
[0271] Two alleles of mouse IgE (IgEa and IgEb) have been described
previously (P06336). The alignment of the amino acid sequences of
the IgEa and IgEb shown that 95% of the amino acid sequences are
identical. A fourteen amino acids differences are located at the
junction region between CH1 and CH2 region and another five amino
acid differences are located at the junction region between the CH3
and CH4 region. The amino acid sequence of IgEb was analyzed for 9
mer peptide sequences that contain binding motifs for Ld and Db MHC
class I molecules by using the software of the Bioinformatics &
Molecular Analysis Section available at
http://bimas.dcrt.nih.gov/molbio/hla_bind/. This program ranks
potential nonapeptides based on a predicted half-time of
dissociation to MHC class I molecules. Based on the ranking
analysis, eight peptides with Ld binding motifs and five peptides
with Db binding motifs were selected for synthesis (Table 1).
[0272] The binding capacity of these synthetic peptides to L.sup.d
and D.sup.b class I molecules were tested in an MHC class I
stabilization assay (Cai et al. (1996) supra). Antigen-transporting
deficient (TAP.sup.-) RMAS cells (H-2.sup.b) or L.sup.d transfected
RMAS (RMAS-L.sup.d) cells were cultured in the presence of a
titrated concentration of peptides at 27.degree. C. After overnight
culturing at 27.degree. C., these cells were further cultured for
two hours at 37.degree. C. and the surface expression of L.sup.d or
D.sup.b on the cells were analyzed by flow cytometry. As shown in
Table 1, two IgE peptides, IgE 11 and IgE366 bind to L.sup.d
strongly, whereas IgE 114 binds L.sup.d weakly. Of the five
peptides predicted bind to D.sup.b, only IgE44 binds D.sup.b
strongly and two peptides, IgE16 and IgE125, bind D.sup.b weakly.
Interestingly, IgE366 originally predicted binding L.sup.d binds
both L.sup.d and D.sup.b. Thus, a total of six peptides were
identified that bind to either L.sup.d or D.sup.b MHC class I
molecules.
TABLE 1
Mouse IgE amino acid sequence: SEQ ID NO: 14
[0273] 1 sirnpqlypl kpckgtasmt Igclvkdyep npvtvtwysd slnmstvnfp
[0274] 51 algselkvtt sqvtswgksa knftchvthp psfnesrtil vrpvnitept
[0275] 101 lellhsscdp naffistiqly cfiyghilnd vsvswlmddr eitdtlaqtv
[0276] 151 likeegklas tcsklniteq qwmsestftc kvtsqgvdyl ahtrrcpdhe
[0277] 201 prgvitylip pspldlyqng apkltclvvd leseknvnvt wnqekktsvs
[0278] 251 asqwytkhhn nattsitsil pvvakdwieg ygyqcivdhp dfpkpivrsi
[0279] 301 tktpgqrsap evyvfpppee esedkrtltc liqnffpedi
svqwlgdgkl
[0280] 351 isnsqhstm plksngsnqg ffifsrleva ktlwtqrkqf tcqvihealq
401 kprklektis tslgntslpr s TABLE-US-00007 TABLE 1 Identification
of Antigenic Peptides of Mouse IgE MHC Sequence Stabili- Peptide
Sel- Peptide Identification zation name ected sequence Number
Score.sup.a of MHC.sup.b IgE 11 L.sup.d KPCKGTASM SEQ ID NO: 1 195
++ IgE 209 L.sup.d IPPSPLDLY SEQ ID NO: 2 90 - IgE 366 L.sup.d
GSNQGFFIF SEQ ID NO: 3 65 ++.sup.c IgE 29 L.sup.d FPNPVTVTW SEQ ID
NO: 4 60 - IgE 105 L.sup.d HSSCDPNAF SEQ ID NO: 5 50 - IgE 114
L.sup.d HSTIQLYCF SEQ ID NO: 6 50 + IgE 363 L.sup.d KSNGSNQGF SEQ
ID NO: 7 50 - IgE 307 L.sup.d RSAPEVYVF SEQ ID NO: 8 50 - IgE 44
D.sup.b MSTVNFPAL SEQ ID NO: 9 937 ++ IgE 411 D.sup.b TSLGNTSLR SEQ
ID NO: 10 44 - IgE 16 D.sup.b TASMTLGCL SEQ ID NO: 11 22 + IgE 159
D.sup.b ASTCSKLNI SEQ ID NO: 12 19 - IgE 125 D.sup.b GHILNDVSV SEQ
ID NO: 13 30 + .sup.aCalculated score in arbitrary units. .sup.bThe
ratio of fluorescence intensity with peptides - without
peptide/without peptides less than two-fold is scored as "+` and
more than two fold is calculate as "++". .sup.cIgE 366 also
stabilizes D.sup.b class I molecules.
Generation of IgE Peptide Specific CTLs in Vitro
[0281] The ability of these IgE peptides in eliciting CTL responses
was evaluated in vitro. As previously described, Drosophila cells
transfected with MHC class I plus B7-1 and ICAM-1 are potent
antigen presenting cells (APC) in activation of resting naive CD8 T
cells in vitro. Resting naive CD8 T cells were purified from mouse
lymph nodes and cultured with peptide loaded Drosophila cells
transfected with L.sup.d or D.sup.b plus B7-1 and ICAM-1 in the
absence of cytokines. IL-2 (20 units/ml) was added at Day 3 and
every other day thereafter. The CTL activity towards peptides
loaded RMAS (K.sup.b, D.sup.b) cells or RMAS-L.sup.d cells were
measured on Day 9. As shown in FIG. 1, CTLs induced by IgE 44
peptide specifically lysed the RMAS cells loaded with IgE 44
peptides, neither the target cells alone nor the target cells
loaded with other IgE peptides were recognized by the IgE44
specific CTLs.
[0282] No specific CTL activity was induced by IgE16 or IgE 125
peptides, which have been show to bind D.sup.b. IgE366 was
originally identified as L.sup.d binding peptide, interestingly, in
addition to inducing L.sup.d restricted CTLs by IgE366, IgE366 also
induce D.sup.b restricted CTLs (FIG. 2, Panel B). Of the three
L.sup.d binding peptides, in addition to IgE366, IgE11 also induces
antigen specific CTLs. The killing of IgE specific CTL is poreforin
dependent and is independent of the expression of IFN.gamma. (FIG.
2, Panel C). Moreover, the CTL induced by IgE peptides are MHC
restricted because the killing of IgE44 loaded RMAS targets by
IgE44 specific CTL was completely blocked by anti-D.sup.b mAb (FIG.
2, Panel D). FACS analysis of these CTL revealed that they are
.alpha..beta. TCR positive CD8.sup.+ T cells and no expression of
NK cell marker (DX5 or NK1.1) were detected on these cells (data
not shown).
Inhibition of IgE Responses by Anti-IgE Specific CTLs.
[0283] Because CTLs induced by IgE peptides kills the target cell
specifically in vitro, we were interested in seeing if these CTLs
could inhibit the IgE responses in vivo. Mice have very low serum
IgE and do not develop allergic response spontaneously. Ovalbumin
precipitated with Alum Hydroxyde has been used to induce antigen
specific IgE responses in mice. As shown in FIG. 3, after two
immunizations with OVA plus alum hydroxyde, both total serum IgE
and ova-specific IgE in the immunized mice were high and the IgE
level was further increased after intranasal challenge of these
mice with OVA. TABLE-US-00008 TABLE 2 The Effect of Anti-IgE CTL on
Airway Inflammation.sup.a Eosinophilic Hyperplasia Treatment
Inflammation.sup.b infiltration of BALT.sup.c PBS (5) 3, 1, 2, 2, 0
2, 0, 2, 3, 0 2, 0, 2, 3, 0 Anti-IgE CTL(5) 0, 0, 0, 1, 0 0, 0, 0,
0, 0 0, T, 0, 1, 0 Control CTL(4) 3, 3, 2, 3 2, 3, 3, 1 3, 2, 2, 2
Normal mice(4) 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 .sup.aAdult CBF1/J
mice were immunized with 50 .mu.g ovalbumin (OVA) plus Alum
hydroxide intraperitoneally on Day 1 and Day 14. Two weeks after
the second immunization, 5 .times. 106 anti-IgE CTL or a control
CTL or PBS were given every other day for three times. Three weeks
after the last CTL treatment, the mice were # challenged with OVA
intranasally every other day for three times. One day after the
last challenge, bronchial alveolar lavage was collected and lung
tissue was collected from each mice and stained with HE staining.
The lung inflammation of each mouse was independently evaluated by
a pathologist. .sup.bScore: O = Normal; T = trace; 1 = mild; 2 =
mild to moderated; 3 = moderate; 4 = severe .sup.cBALT = Bronchial
Associated Lymphoid Hyperplasia.
[0284] TABLE-US-00009 TABLE 3 HLA-A2 Peptide Motif Search for Human
IgE Score (Estimate Half Start Subsequence Sequence Time of
Disassociation Posi- Residue Identification of HLA-2 Containing
Rank tion Listing No. this Subsequence) 1 185 WLSDRTYTC (SEQ ID NO:
52) 93.696 2 96 WVDNKTFSV (SEQ ID NO: 51) 64.948 3 71 LLTVSGAWA
(SEQ ID NO: 62) 46.451 4 365 QLPDARHST (SEQ ID NO: 63) 30.553 5 3
TQSPSVFPL (SEQ ID NO: 64) 28.893 6 309 ALMRSTTKT (SEQ ID NO: 65)
27.572 7 59 TLTLSGHYA (SEQ ID NO: 66) 27.324 8 54 TLPATTLTL (SEQ ID
NO: 67) 21.362 9 47 SLNGTTMTL (SEQ ID NO: 50) 21.362 10 61
TLSGHYATI (SEQ ID NO: 68) 15.649 11 52 TMTLPATTL (SEQ ID NO: 69)
15.428 12 178 LTLSQKHWL (SEQ ID NO: 70) 10.264 13 66 YATISLLTV (SEQ
ID NO: 58) 10.220 14 154 QVMDVDLST (SEQ ID NO: 71) 9.892 15 17
NIPSNATSV (SEQ ID NO: 72) 9.563 16 133 LLCLVSGYT (SEQ ID NO: 73)
9.058 17 403 FICRAVHEA (SEQ ID NO: 74) 7.227 18 236 TITCLVVDL (SEQ
ID NO: 75) 6.756 19 356 SVQWLHNEV (SEQ ID NO: 76) 6.086 20 155
VMDVDLSTA (SEQ ID NO: 77) 5.612
[0285] TABLE-US-00010 TABLE 4 HLA-A2 Peptide Motif Search for Human
IgE by Neuro-Network Sequence Net Output C150 Start End Sequence
Identification No. 0.747555 5.71921 223 231 RPSPFDLFI (SEQ ID NO:
78) 0.695169 8.21283 349 357 NFMPEDISV (SEQ ID NO: 79) 0.628452
13.021 358 366 QWLHNEVQL (SEQ ID NO: 80) 0.60628 15.1782 33 41
GYFPEPVMV (SEQ ID NO: 81) 0.53619 24.6281 54 62 TLPATTLT (SEQ ID
NO: 82) 0.45981 41.7417 108 116 DFTPPTVKI (SEQ ID NO: 83) 0.406526
60.3147 229 237 LFIRKSPTI (SEQ ID NO: 84) 0.382602 71.153 96 104
WVDNKTFSV (SEQ ID NO: 51) 0.373791 75.6184 148 156 TWLEDGQVM (SEQ
ID NO: 85) 0.34985 89.2174 61 69 TLSGHYATI (SEQ ID NO: 68) 0.348214
90.2317 396 404 EWEQKDEFI (SEQ ID NO: 86) 0.344683 92.4594 278 286
LTVTSTLPV (SEQ ID NO: 87) 0.317372 111.656 128 136 PPTIQLLCL (SEQ
ID NO: 88) 0.29653 128.947 170 178 ELASTQSEL (SEQ ID NO: 89)
0.292132 132.924 236 244 TITCLVVDL (SEQ ID NO: 75) 0.272911 151.798
106 114 SRDFTPPTV (SEQ ID NO: 90) 0.26747 157.612 213 221 NPRGVSAYL
(SEQ ID NO: 91) 0.252711 174.529 10 18 PLTRCCKNI (SEQ ID NO: 92)
0.227935 207.107 147 155 ITWLEDGQV (SEQ ID NO: 93) 0.220931 217.374
234 242 SPTITCLVV (SEQ ID NO: 94) 0.219179 220.02 47 55 SLNGTTMTL
(SEQ ID NO: 50) 0.218951 220.368 384 392 FFVFSRLEV (SEQ ID NO: 95)
0.199355 252.309 139 147 GYTPGTINI (SEQ ID NO: 96) 0.188573 271.82
123 131 GGGHFPPTI (SEQ ID NO: 97) 0.170795 307.296 245 253
APSKGTVNL (SEQ ID NO: 98) 0.136633 389.134 302 310 THPHLPRAL (SEQ
ID NO: 99) 0.124225 423.96 284 292 LPVGTRDWI (SEQ ID NO: 100)
0.115665 449.785 378 386 KTKGSGFFV (SEQ ID NO: 101)
EXAMPLE 4
[0286] In the presence of specific antigen and constimulation,
resting CD8 T cells can be activated and differentiated into CTL,
which plays an essential role in anti-virus immune response.
Recently, it has also been shown that tumor associated antigens
specific CTL generated in vitro can be used in treating cancer
patients. Here we show that antigenic peptides identified from
non-tumor self-antigens can induce specific cytotoxic T lymphocyte
(CTL) in vitro. The CTL induced by peptides identified from CD40L,
a self antigen transiently expressed on activated CD4 T cells, can
kill activated CD4 T cells and the killing can be blocked either by
the antibody (Ab) specific for the restricting class I molecule or
by the Ab recognizing CD8 molecule. In addition, neither activated
CD4 T cells generated from CD40L.sup.-/- mice nor from 2m.sup.-/-
mice are killed by the CD40L specific CTL, demonstrating that the
killing of activated CD4 T cells by CD40L specific CTL is
antigen-dependent and MHC restricted. Importantly, in vitro
generated CTL specific for CD40L inhibit CD4-dependent antibody
responses of all isotypes in vivo. In contrast, CTL induced by
antigenic peptides derived from IgE specifically inhibit IgE
responses and adoptive transfer of CD40L-specific CTL to NOD mice
at early age delay the development of diabetes in NOD mice. Thus,
in vitro generated CTL specific for non-tumor self-antigens
expressed on activated CD4 T cells can regulate immune responses in
vivo.
[0287] Allergic diseases, such as hay fever, asthma and systemic
anaphylaxis, are immune responses to innocuous substances. The
hallmark of the diseases is activation of CD4 cells and over
production of IgE by B cells. The current therapies have been
focused on the treatment of symptoms and do not prevent the
development and progression of the diseases. Because
allergen-activated CD4 cells and IgE producing B cells play a
central role in the pathogenesis of allergy, our strategy is to use
autologous CTL to eliminate activated CD4 T cells and IgE producing
B cells, thus preventing the development and progression of the
diseases. Two molecules, CD40 ligand (CD40L) and IgE, were selected
as target antigens for CTL therapy. Three antigenic peptides from
CD40L and two antigenic peptides from IgE were identified. CTLs
specific for these peptides have been generated and the function of
these CTLs has been evaluated both in vitro and in vivo.
[0288] Three antigenic epitopes from CD40L and two epitopes from
IgE molecules were identified. Synthetic peptides of the antigenic
epitopes were able to bind to class I molecules and to activate
resting naive CD8 T cells in vitro.
[0289] CTLs were generated by stimulation of CD8 T cells with CD40L
or IgE peptides presented by Drosophila cells expressing MHC class
I, B7-1 and ICAM-1 molecules. The CTLs thus generated in vitro
killed peptide-loaded target cells specifically. CD40L-peptide
specific CTL killed activated CD4 T cells and the recognition was
dependent on the expression of CD40L and MHC class I molecules.
[0290] The function of CD40L-specific CTL were also evaluated in
vivo. Antigen-specific antibody response was inhibited by
anti-CD40L CTL. The effect of anti-CD40L CTL and anti-IgE CTL on
allergy and autoimmune diseases will be investigated in animal
models. TABLE-US-00011 TABLE 5 MHC Class I Binding Motif Search for
Mouse CD40L Sequence Start Identification Score Rank Position AA
Sequence Number Number 1 17 LPASMKIFM SEQ ID NO: 15 150.00
(L.sup.d) 2 186 RPFIVGLWL SEQ ID NO: 16 150.00 (L.sup.d) 3 118
DPQIAAHVV SEQ ID NO: 17 90.00 (L.sup.d) 4 220 QSVHLGGVF SEQ ID NO:
18 50.00 (L.sup.d) 5 9 SPRSVATGL SEQ ID NO: 19 45.00 (L.sup.d) 6
195 KPSIGSERI SEQ ID NO: 20 39.00 (L.sup.d) 7 252 FSSFGLLKL SEQ ID
NO: 21 32.50 (L.sup.d) 8 7 QPSPRSVAT SEQ ID NO: 22 30.00 (L.sup.d)
9 181 EPSSQRPFI SEQ ID NO: 23 30.00 (L.sup.d) 10 79 LSLLNCEEM SEQ
ID NO: 24 25.00 (L.sup.d) 1 79 LSLLNCEEM SEQ ID NO: 24 5713.03
(D.sup.b) 2 152 VMLENGKQL SEQ ID NO: 25 5160.15 (D.sup.b) 3 146
TMKSNLVML SEQ ID NO: 26 2648.88 (D.sup.b) 4 235 SVFVNVTEA SEQ ID
NO: 27 95.12 (D.sup.b) 5 38 GSVLFAVYL SEQ ID NO: 28 46.87 (D.sup.b)
6 19 ASMKIFMYL SEQ ID NO: 29 46.87 (D.sup.b) # Estimate of half
time of disassociation of a molecule containing this
subsequence.
[0291] TABLE-US-00012 TABLE 6 HLA-A2 Peptide Motif Search for Human
CD40L Score (Estimate of Half-Time of Start Subsequence Sequence
Disassociation of a Posi- Residue Identification Molecule
Containing Rank tion Listing Number this Subsequence) 1 24
FMYLLTVFL SEQ ID NO: 30 1249.083 2 167 GLYYIYAQV SEQ ID NO: 31
333.850 3 22 KIFMYLLTV SEQ ID NO: 32 284.846 4 36 MIGSALFAV SEQ ID
NO: 33 216.879 5 58 NLHEDFVFM SEQ ID NO: 34 212.854 6 170 YIYAQVTFC
SEQ ID NO: 35 127.199 7 26 YLLTVFLIT SEQ ID NO: 36 98.803 8 231
LQPGASVFV SEQ ID NO: 37 65.934 9 45 YLHRRLDKI SEQ ID NO: 38 54.086
10 147 TMSNNLVTL SEQ ID NO: 39 35.485 11 229 FELQPGASV SEQ ID NO:
40 23.018 12 160 QLTVKRQGL SEQ ID NO: 41 21.362 13 35 QMIGSALFA SEQ
ID NO: 42 19.734 14 185 SQAPFIASL SEQ ID NO: 43 18.930 15 19
ISMKIFMYL SEQ ID NO: 44 9.166 16 153 VTLENGKQL SEQ ID NO: 45 7.652
17 126 VISEASSKT SEQ ID NO: 46 7.142 18 227 GVFELQPGA SEQ ID NO: 47
6.594 19 20 SMKIFMYLL SEQ ID NO: 48 4.720 20 165 RQGLYYIYA SEQ ID
NO: 49 4.156
[0292] TABLE-US-00013 TABLE 7 Summary of CTL Activity Generated
From PBMC in Different Donors Sequence Identification Specific IgE
Peptide AA Sequence Number Killing * 47 SLNGTTMTL .sup.1 SEQ ID NO:
50 7/8 96 WVDNKTFSV .sup.1 SEQ ID NO: 51 3/8 185 WLSDRTYTC SEQ ID
NO: 52 0/8 308 ALSDRTYTC SEQ ID NO: 53 0/3 876 SLLTVSGAWA SEQ ID
NO: 54 0/5 883 WLEDGQVMDV SEQ ID NO: 55 1/5 884 TLTVTSTLPV .sup.2
SEQ ID NO: 56 8/8 887 QMFTCRVAHT SEQ ID NO: 57 1/4 890 YATISLLTV
.sup.1 SEQ ID NO: 58 4/5 895 TLACLIQNFM .sup.2 SEQ ID NO: 59 3/4
898 QVMDVDLSTA .sup.2 SEQ ID NO: 60 3/4 x/N x: number of donor from
whom anti-IgE CTL was generated; N: number of donor tested CD8+ T
cells were purified from PBMC and cultured with Drosophila cells
transfected with A2.1, B7.1 and ICAM-1 in the presence of IgE
peptides. Statistics indicated the capability of IgE peptide to
generate specific CTL response from different donors. .sup.1 and
.sup.2 indicate anti-IgE CTL was generated from 9-mer and 10-mer
respectively.
[0293]
Sequence CWU 1
1
105 1 9 PRT Artificial Sequence Peptide antigen 1 Lys Pro Cys Lys
Gly Thr Ala Ser Met 1 5 2 9 PRT Artificial Sequence Peptide antigen
2 Ile Pro Pro Ser Pro Leu Asp Leu Tyr 1 5 3 9 PRT Artificial
Sequence Peptide antigen 3 Gly Ser Asn Gln Gly Phe Phe Ile Phe 1 5
4 9 PRT Artificial Sequence Peptide antigen 4 Phe Pro Asn Pro Val
Thr Val Thr Trp 1 5 5 9 PRT Artificial Sequence Peptide antigen 5
His Ser Ser Cys Asp Pro Asn Ala Phe 1 5 6 9 PRT Artificial Sequence
Peptide antigen 6 His Ser Thr Ile Gln Leu Tyr Cys Phe 1 5 7 9 PRT
Artificial Sequence Peptide antigen 7 Lys Ser Asn Gly Ser Asn Gln
Gly Phe 1 5 8 9 PRT Artificial Sequence Peptide antigen 8 Arg Ser
Ala Pro Glu Val Tyr Val Phe 1 5 9 9 PRT Artificial Sequence Peptide
antigen 9 Met Ser Thr Val Asn Phe Pro Ala Leu 1 5 10 9 PRT
Artificial Sequence Peptide antigen 10 Thr Ser Leu Gly Asn Thr Ser
Leu Arg 1 5 11 9 PRT Artificial Sequence Peptide antigen 11 Thr Ala
Ser Met Thr Leu Gly Cys Leu 1 5 12 9 PRT Artificial Sequence
Peptide antigen 12 Ala Ser Thr Cys Ser Lys Leu Asn Ile 1 5 13 9 PRT
Artificial Sequence Peptide antigen 13 Gly His Ile Leu Asn Asp Val
Ser Val 1 5 14 421 PRT Mus musculus 14 Ser Ile Arg Asn Pro Gln Leu
Tyr Pro Leu Lys Pro Cys Lys Gly Thr 1 5 10 15 Ala Ser Met Thr Leu
Gly Cys Leu Val Lys Asp Tyr Glu Pro Asn Pro 20 25 30 Val Thr Val
Thr Trp Tyr Ser Asp Ser Leu Asn Met Ser Thr Val Asn 35 40 45 Phe
Pro Ala Leu Gly Ser Glu Leu Lys Val Thr Thr Ser Gln Val Thr 50 55
60 Ser Trp Gly Lys Ser Ala Lys Asn Phe Thr Cys His Val Thr His Pro
65 70 75 80 Pro Ser Phe Asn Glu Ser Arg Thr Ile Leu Val Arg Pro Val
Asn Ile 85 90 95 Thr Glu Pro Thr Leu Glu Leu Leu His Ser Ser Cys
Asp Pro Asn Ala 100 105 110 Phe His Ser Thr Ile Gln Leu Tyr Cys Phe
Ile Tyr Gly His Ile Leu 115 120 125 Asn Asp Val Ser Val Ser Trp Leu
Met Asp Asp Arg Glu Ile Thr Asp 130 135 140 Thr Leu Ala Gln Thr Val
Leu Ile Lys Glu Glu Gly Lys Leu Ala Ser 145 150 155 160 Thr Cys Ser
Lys Leu Asn Ile Thr Glu Gln Gln Trp Met Ser Glu Ser 165 170 175 Thr
Phe Thr Cys Lys Val Thr Ser Gln Gly Val Asp Tyr Leu Ala His 180 185
190 Thr Arg Arg Cys Pro Asp His Glu Pro Arg Gly Val Ile Thr Tyr Leu
195 200 205 Ile Pro Pro Ser Pro Leu Asp Leu Tyr Gln Asn Gly Ala Pro
Lys Leu 210 215 220 Thr Cys Leu Val Val Asp Leu Glu Ser Glu Lys Asn
Val Asn Val Thr 225 230 235 240 Trp Asn Gln Glu Lys Lys Thr Ser Val
Ser Ala Ser Gln Trp Tyr Thr 245 250 255 Lys His His Asn Asn Ala Thr
Thr Ser Ile Thr Ser Ile Leu Pro Val 260 265 270 Val Ala Lys Asp Trp
Ile Glu Gly Tyr Gly Tyr Gln Cys Ile Val Asp 275 280 285 His Pro Asp
Phe Pro Lys Pro Ile Val Arg Ser Ile Thr Lys Thr Pro 290 295 300 Gly
Gln Arg Ser Ala Pro Glu Val Tyr Val Phe Pro Pro Pro Glu Glu 305 310
315 320 Glu Ser Glu Asp Lys Arg Thr Leu Thr Cys Leu Ile Gln Asn Phe
Phe 325 330 335 Pro Glu Asp Ile Ser Val Gln Trp Leu Gly Asp Gly Lys
Leu Ile Ser 340 345 350 Asn Ser Gln His Ser Thr Thr Thr Pro Leu Lys
Ser Asn Gly Ser Asn 355 360 365 Gln Gly Phe Phe Ile Phe Ser Arg Leu
Glu Val Ala Lys Thr Leu Trp 370 375 380 Thr Gln Arg Lys Gln Phe Thr
Cys Gln Val Ile His Glu Ala Leu Gln 385 390 395 400 Lys Pro Arg Lys
Leu Glu Lys Thr Ile Ser Thr Ser Leu Gly Asn Thr 405 410 415 Ser Leu
Pro Arg Ser 420 15 9 PRT Artificial Sequence Peptide antigen 15 Leu
Pro Ala Ser Met Lys Ile Phe Met 1 5 16 9 PRT Artificial Sequence
Peptide antigen 16 Arg Pro Phe Ile Val Gly Leu Trp Leu 1 5 17 9 PRT
Artificial Sequence Peptide antigen 17 Asp Pro Gln Ile Ala Ala His
Val Val 1 5 18 9 PRT Artificial Sequence Peptide antigen 18 Gln Ser
Val His Leu Gly Gly Val Phe 1 5 19 9 PRT Artificial Sequence
Peptide antigen 19 Ser Pro Arg Ser Val Ala Thr Gly Leu 1 5 20 9 PRT
Artificial Sequence Peptide antigen 20 Lys Pro Ser Ile Gly Ser Glu
Arg Ile 1 5 21 9 PRT Artificial Sequence Peptide antigen 21 Phe Ser
Ser Phe Gly Leu Leu Lys Leu 1 5 22 9 PRT Artificial Sequence
Peptide antigen 22 Gln Pro Ser Pro Arg Ser Val Ala Thr 1 5 23 9 PRT
Artificial Sequence Peptide antigen 23 Glu Pro Ser Ser Gln Arg Pro
Phe Ile 1 5 24 9 PRT Artificial Sequence Peptide antigen 24 Leu Ser
Leu Leu Asn Cys Glu Glu Met 1 5 25 9 PRT Artificial Sequence
Peptide antigen 25 Val Met Leu Glu Asn Gly Lys Gln Leu 1 5 26 9 PRT
Artificial Sequence Peptide antigen 26 Thr Met Lys Ser Asn Leu Val
Met Leu 1 5 27 9 PRT Artificial Sequence Peptide antigen 27 Ser Val
Phe Val Asn Val Thr Glu Ala 1 5 28 9 PRT Artificial Sequence
Peptide antigen 28 Gly Ser Val Leu Phe Ala Val Tyr Leu 1 5 29 9 PRT
Artificial Sequence Peptide antigen 29 Ala Ser Met Lys Ile Phe Met
Tyr Leu 1 5 30 9 PRT Artificial Sequence Peptide antigen 30 Phe Met
Tyr Leu Leu Thr Val Phe Leu 1 5 31 9 PRT Artificial Sequence
Peptide antigen 31 Gly Leu Tyr Tyr Ile Tyr Ala Gln Val 1 5 32 9 PRT
Artificial Sequence Peptide antigen 32 Lys Ile Phe Met Tyr Leu Leu
Thr Val 1 5 33 9 PRT Artificial Sequence Peptide antigen 33 Met Ile
Gly Ser Ala Leu Phe Ala Val 1 5 34 9 PRT Artificial Sequence
Peptide antigen 34 Asn Leu His Glu Asp Phe Val Phe Met 1 5 35 9 PRT
Artificial Sequence Peptide antigen 35 Tyr Ile Tyr Ala Gln Val Thr
Phe Cys 1 5 36 9 PRT Artificial Sequence Peptide antigen 36 Tyr Leu
Leu Thr Val Phe Leu Ile Thr 1 5 37 9 PRT Artificial Sequence
Peptide antigen 37 Leu Gln Pro Gly Ala Ser Val Phe Val 1 5 38 9 PRT
Artificial Sequence Peptide antigen 38 Tyr Leu His Arg Arg Leu Asp
Lys Ile 1 5 39 9 PRT Artificial Sequence Peptide antigen 39 Thr Met
Ser Asn Asn Leu Val Thr Leu 1 5 40 9 PRT Artificial Sequence
Peptide antigen 40 Phe Glu Leu Gln Pro Gly Ala Ser Val 1 5 41 9 PRT
Artificial Sequence Peptide antigen 41 Gln Leu Thr Val Lys Arg Gln
Gly Leu 1 5 42 9 PRT Artificial Sequence Peptide antigen 42 Gln Met
Ile Gly Ser Ala Leu Phe Ala 1 5 43 9 PRT Artificial Sequence
Peptide antigen 43 Ser Gln Ala Pro Phe Ile Ala Ser Leu 1 5 44 9 PRT
Artificial Sequence Peptide antigen 44 Ile Ser Met Lys Ile Phe Met
Tyr Leu 1 5 45 9 PRT Artificial Sequence Peptide antigen 45 Val Thr
Leu Glu Asn Gly Lys Gln Leu 1 5 46 9 PRT Artificial Sequence
Peptide antigen 46 Val Ile Ser Glu Ala Ser Ser Lys Thr 1 5 47 9 PRT
Artificial Sequence Peptide antigen 47 Gly Val Phe Glu Leu Gln Pro
Gly Ala 1 5 48 9 PRT Artificial Sequence Peptide antigen 48 Ser Met
Lys Ile Phe Met Tyr Leu Leu 1 5 49 9 PRT Artificial Sequence
Peptide antigen 49 Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala 1 5 50 9 PRT
Artificial Sequence Peptide antigen 50 Ser Leu Asn Gly Thr Thr Met
Thr Leu 1 5 51 9 PRT Artificial Sequence Peptide antigen 51 Trp Val
Asp Asn Lys Thr Phe Ser Val 1 5 52 9 PRT Artificial Sequence
Peptide antigen 52 Trp Leu Ser Asp Arg Thr Tyr Thr Cys 1 5 53 9 PRT
Artificial Sequence Peptide antigen 53 Ala Leu Ser Asp Arg Thr Tyr
Thr Cys 1 5 54 10 PRT Artificial Sequence Peptide antigen 54 Ser
Leu Leu Thr Val Ser Gly Ala Trp Ala 1 5 10 55 10 PRT Artificial
Sequence Peptide antigen 55 Trp Leu Glu Asp Gly Gln Val Met Asp Val
1 5 10 56 10 PRT Artificial Sequence Peptide antigen 56 Thr Leu Thr
Val Thr Ser Thr Leu Pro Val 1 5 10 57 10 PRT Artificial Sequence
Peptide antigen 57 Gln Met Phe Thr Cys Arg Val Ala His Thr 1 5 10
58 9 PRT Artificial Sequence Peptide antigen 58 Tyr Ala Thr Ile Ser
Leu Leu Thr Val 1 5 59 10 PRT Artificial Sequence Peptide antigen
59 Thr Leu Ala Cys Leu Ile Gln Asn Phe Met 1 5 10 60 10 PRT
Artificial Sequence Peptide antigen 60 Gln Val Met Asp Val Asp Leu
Ser Thr Ala 1 5 10 61 12 PRT Artificial Sequence Peptide antigen 61
Ala Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly 1 5 10 62 9 PRT
Artificial Sequence Peptide antigen 62 Leu Leu Thr Val Ser Gly Ala
Trp Ala 1 5 63 9 PRT Artificial Sequence Peptide antigen 63 Gln Leu
Pro Asp Ala Arg His Ser Thr 1 5 64 9 PRT Artificial Sequence
Peptide antigen 64 Thr Gln Ser Pro Ser Val Phe Pro Leu 1 5 65 9 PRT
Artificial Sequence Peptide antigen 65 Ala Leu Met Arg Ser Thr Thr
Lys Thr 1 5 66 9 PRT Artificial Sequence Peptide antigen 66 Thr Leu
Thr Leu Ser Gly His Tyr Ala 1 5 67 9 PRT Artificial Sequence
Peptide antigen 67 Thr Leu Pro Ala Thr Thr Leu Thr Leu 1 5 68 9 PRT
Artificial Sequence Peptide antigen 68 Thr Leu Ser Gly His Tyr Ala
Thr Ile 1 5 69 9 PRT Artificial Sequence Peptide antigen 69 Thr Met
Thr Leu Pro Ala Thr Thr Leu 1 5 70 9 PRT Artificial Sequence
Peptide antigen 70 Leu Thr Leu Ser Gln Lys His Trp Leu 1 5 71 9 PRT
Artificial Sequence Peptide antigen 71 Gln Val Met Asp Val Asp Leu
Ser Thr 1 5 72 9 PRT Artificial Sequence Peptide antigen 72 Asn Ile
Pro Ser Asn Ala Thr Ser Val 1 5 73 9 PRT Artificial Sequence
Peptide antigen 73 Leu Leu Cys Leu Val Ser Gly Tyr Thr 1 5 74 9 PRT
Artificial Sequence Peptide antigen 74 Phe Ile Cys Arg Ala Val His
Glu Ala 1 5 75 9 PRT Artificial Sequence Peptide antigen 75 Thr Ile
Thr Cys Leu Val Val Asp Leu 1 5 76 9 PRT Artificial Sequence
Peptide antigen 76 Ser Val Gln Trp Leu His Asn Glu Val 1 5 77 9 PRT
Artificial Sequence Peptide antigen 77 Val Met Asp Val Asp Leu Ser
Thr Ala 1 5 78 9 PRT Artificial Sequence Peptide antigen 78 Arg Pro
Ser Pro Phe Asp Leu Phe Ile 1 5 79 9 PRT Artificial Sequence
Peptide antigen 79 Asn Phe Met Pro Glu Asp Ile Ser Val 1 5 80 9 PRT
Artificial Sequence Peptide antigen 80 Gln Trp Leu His Asn Glu Val
Gln Leu 1 5 81 9 PRT Artificial Sequence Peptide antigen 81 Gly Tyr
Phe Pro Glu Pro Val Met Val 1 5 82 8 PRT Artificial Sequence
Peptide antigen 82 Thr Leu Pro Ala Thr Thr Leu Thr 1 5 83 9 PRT
Artificial Sequence Peptide antigen 83 Asp Phe Thr Pro Pro Thr Val
Lys Ile 1 5 84 9 PRT Artificial Sequence Peptide antigen 84 Leu Phe
Ile Arg Lys Ser Pro Thr Ile 1 5 85 9 PRT Artificial Sequence
Peptide antigen 85 Thr Trp Leu Glu Asp Gly Gln Val Met 1 5 86 9 PRT
Artificial Sequence Peptide antigen 86 Glu Trp Glu Gln Lys Asp Glu
Phe Ile 1 5 87 9 PRT Artificial Sequence Peptide antigen 87 Leu Thr
Val Thr Ser Thr Leu Pro Val 1 5 88 9 PRT Artificial Sequence
Peptide antigen 88 Pro Pro Thr Ile Gln Leu Leu Cys Leu 1 5 89 9 PRT
Artificial Sequence Peptide antigen 89 Glu Leu Ala Ser Thr Gln Ser
Glu Leu 1 5 90 9 PRT Artificial Sequence Peptide antigen 90 Ser Arg
Asp Phe Thr Pro Pro Thr Val 1 5 91 9 PRT Artificial Sequence
Peptide antigen 91 Asn Pro Arg Gly Val Ser Ala Tyr Leu 1 5 92 9 PRT
Artificial Sequence Peptide antigen 92 Pro Leu Thr Arg Cys Cys Lys
Asn Ile 1 5 93 9 PRT Artificial Sequence Peptide antigen 93 Ile Thr
Trp Leu Glu Asp Gly Gln Val 1 5 94 9 PRT Artificial Sequence
Peptide antigen 94 Ser Pro Thr Ile Thr Cys Leu Val Val 1 5 95 9 PRT
Artificial Sequence Peptide antigen 95 Phe Phe Val Phe Ser Arg Leu
Glu Val 1 5 96 9 PRT Artificial Sequence Peptide antigen 96 Gly Tyr
Thr Pro Gly Thr Ile Asn Ile 1 5 97 9 PRT Artificial Sequence
Peptide antigen 97 Gly Gly Gly His Phe Pro Pro Thr Ile 1 5 98 9 PRT
Artificial Sequence Peptide antigen 98 Ala Pro Ser Lys Gly Thr Val
Asn Leu 1 5 99 9 PRT Artificial Sequence Peptide antigen 99 Thr His
Pro His Leu Pro Arg Ala Leu 1 5 100 9 PRT Artificial Sequence
Peptide antigen 100 Leu Pro Val Gly Thr Arg Asp Trp Ile 1 5 101 9
PRT Artificial Sequence Peptide antigen 101 Lys Thr Lys Gly Ser Gly
Phe Phe Val 1 5 102 388 PRT Artificial Sequence Peptide antigen 102
Thr Val Thr Trp Tyr Ser Asp Ser Leu Asn Met Ser Thr Val Asn Phe 1 5
10 15 Pro Ala Leu Gly Ser Glu Leu Lys Val Thr Thr Ser Gln Val Thr
Ser 20 25 30 Trp Gly Lys Ser Ala Lys Asn Phe Thr Cys His Val Thr
His Pro Pro 35 40 45 Ser Phe Asn Glu Ser Arg Thr Ile Leu Val Arg
Pro Val Asn Ile Thr 50 55 60 Glu Pro Thr Leu Glu Leu Leu His Ser
Ser Cys Asp Pro Asn Ala Phe 65 70 75 80 His Ser Thr Ile Gln Leu Tyr
Cys Phe Ile Tyr Gly His Ile Leu Asn 85 90 95 Asp Val Ser Val Ser
Trp Leu Met Asp Asp Arg Glu Ile Thr Asp Thr 100 105 110 Leu Ala Gln
Thr Val Leu Ile Lys Glu Glu Gly Lys Leu Ala Ser Thr 115 120 125 Cys
Ser Lys Leu Asn Ile Thr Glu Gln Gln Trp Met Ser Glu Ser Thr 130 135
140 Phe Thr Cys Lys Val Thr Ser Gln Gly Val Asp Tyr Leu Ala His Thr
145 150 155 160 Arg Arg Cys Pro Asp His Glu Pro Arg Gly Val Ile Thr
Tyr Leu Ile 165 170 175 Pro Pro Ser Pro Leu Asp Leu Tyr Gln Asn Gly
Ala Pro Lys Leu Thr 180 185 190 Cys Leu Val Val Asp Leu Glu Ser Glu
Lys Asn Val Asn Val Thr Trp 195 200 205 Asn Gln Glu Lys Lys Thr Ser
Val Ser Ala Ser Gln Trp Tyr Thr Lys 210 215 220 His His Asn Asn Ala
Thr Thr Ser Ile Thr Ser Ile Leu Pro Val Val 225 230 235 240 Ala Lys
Asp Trp Ile Glu Gly Tyr Gly Tyr Gln Cys Ile Val Asp His 245 250 255
Pro Asp Phe Pro Lys Pro Ile Val Arg Ser Ile Thr Lys Thr Pro Gly 260
265 270 Gln Arg Ser Ala Pro Glu Val Tyr Val Phe Pro Pro Pro Glu Glu
Glu 275 280 285 Ser Glu Asp Lys Arg Thr Leu Thr Cys Leu Ile Gln Asn
Phe Phe Pro 290 295 300 Glu Asp Ile Ser Val Gln Trp Leu Gly Asp Gly
Lys Leu Ile Ser Asn 305 310 315 320 Ser Gln His Ser Thr Thr Thr Pro
Leu Lys Ser Asn Gly Ser Asn Gln 325 330 335 Gly Phe Phe Ile Phe Ser
Arg Leu Glu Val Ala Lys Thr Leu Trp Thr 340 345 350 Gln Arg Lys Gln
Phe Thr Cys Gln Val Ile His Glu Ala Leu Gln Lys 355 360 365 Pro Arg
Lys Leu Glu Lys Thr Ile Ser Thr Ser Leu Gly Asn Thr Ser 370 375 380
Leu Arg Pro Ser 385 103 423 PRT Artificial Sequence Peptide antigen
103 Ser Ile Arg Asn Pro Gln Leu Tyr Pro Leu Lys Pro Cys Lys Gly Thr
1 5 10 15 Ala Ser Met Thr Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Asn Pro 20 25 30 Val Thr Val Thr Trp Tyr Ser Asp Ser Leu Asn Met
Ser Thr Val Asn 35 40 45 Phe Pro Ala Leu Gly Ser Glu Leu Lys Val
Thr Thr Ser Gln Val Thr 50 55 60 Ser Trp Gly Lys Ser Ala Lys Asn
Phe Thr Cys His Val Thr His Pro 65 70 75 80 Pro Ser Phe Asn Glu Ser
Arg Thr Ile Leu Val Arg Pro Val Thr His 85 90 95 Ser Leu Ser Pro
Pro Trp Ser Tyr Ser Ile His Arg Cys Asp Pro Asn 100 105 110 Ala Phe
His Ser Thr Ile Gln Leu Tyr Cys Phe Ile Tyr Gly His Ile 115 120 125
Leu Asn Asp Val Ser Val Ser Trp Leu Met Asp Asp Arg Glu Ile Thr 130
135 140 Asp Thr Leu Ala Gln Thr Val Leu Ile Lys Glu Glu Gly Lys Leu
Ala 145 150 155 160 Ser Thr Cys Ser Lys Leu Asn Ile Thr Glu Gln Gln
Trp Met Ser Glu 165 170 175 Ser Thr Phe Thr Cys Arg Val Thr Ser Gln
Gly Val Asp Tyr Leu Ala 180 185 190 His Thr Arg Arg Cys Pro Asp His
Glu Pro Arg
Gly Ala Ile Thr Tyr 195 200 205 Leu Ile Pro Pro Ser Pro Leu Asp Leu
Tyr Gln Asn Gly Ala Pro Lys 210 215 220 Leu Thr Cys Leu Val Val Asp
Leu Glu Ser Glu Lys Asn Val Asn Val 225 230 235 240 Thr Trp Asn Gln
Glu Lys Lys Thr Ser Val Ser Ala Ser Gln Trp Tyr 245 250 255 Thr Lys
His His Asn Asn Ala Thr Thr Ser Ile Thr Ser Ile Leu Pro 260 265 270
Val Val Ala Lys Asp Trp Ile Glu Gly Tyr Gly Tyr Gln Cys Val Val 275
280 285 Asp Arg Pro Asp Phe Pro Lys Pro Ile Val Arg Ser Ile Thr Leu
Pro 290 295 300 Gln Val Ser Gln Arg Ser Ala Pro Glu Val Tyr Val Phe
Pro Pro Pro 305 310 315 320 Glu Glu Glu Ser Glu Asp Lys Arg Thr Leu
Thr Cys Leu Ile Gln Asn 325 330 335 Phe Phe Pro Glu Asp Ile Ser Val
Gln Trp Leu Gly Asp Gly Lys Leu 340 345 350 Ile Ser Asn Ser Gln His
Ser Thr Thr Thr Pro Leu Lys Ser Asn Gly 355 360 365 Ser Asn Gln Gly
Phe Phe Ile Phe Ser Arg Leu Glu Val Ala Lys Thr 370 375 380 Leu Trp
Thr Gln Arg Lys Gln Phe Thr Cys Gln Val Ile His Glu Ala 385 390 395
400 Leu Gln Lys Pro Arg Lys Leu Glu Lys Thr Ile Ser Thr Ser Leu Gly
405 410 415 Asn Thr Ser Leu Arg Pro Ser 420 104 428 PRT Homo
sapiens 104 Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys
Cys Lys 1 5 10 15 Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly
Cys Leu Ala Thr 20 25 30 Gly Tyr Phe Pro Glu Pro Val Met Val Thr
Trp Asp Thr Gly Ser Leu 35 40 45 Asn Gly Thr Thr Met Thr Leu Pro
Ala Thr Thr Leu Thr Leu Ser Gly 50 55 60 His Tyr Ala Thr Ile Ser
Leu Leu Thr Val Ser Gly Ala Trp Ala Lys 65 70 75 80 Gln Met Phe Thr
Cys Arg Val Ala His Thr Pro Ser Ser Thr Asp Trp 85 90 95 Val Asp
Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe Thr Pro Pro 100 105 110
Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro 115
120 125 Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly
Thr 130 135 140 Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp
Val Asp Leu 145 150 155 160 Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu
Leu Ala Ser Thr Gln Ser 165 170 175 Glu Leu Thr Leu Ser Gln Lys His
Trp Leu Ser Asp Arg Thr Tyr Thr 180 185 190 Cys Gln Val Thr Tyr Gln
Gly His Thr Phe Glu Asp Ser Thr Lys Lys 195 200 205 Cys Ala Asp Ser
Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro 210 215 220 Ser Pro
Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu 225 230 235
240 Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser
245 250 255 Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu
Glu Lys 260 265 270 Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu
Pro Val Gly Thr 275 280 285 Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln
Cys Arg Val Thr His Pro 290 295 300 His Leu Pro Arg Ala Leu Met Arg
Ser Thr Thr Lys Thr Ser Gly Pro 305 310 315 320 Arg Ala Ala Pro Glu
Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly 325 330 335 Ser Arg Asp
Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met Pro 340 345 350 Glu
Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp 355 360
365 Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe
370 375 380 Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu
Gln Lys 385 390 395 400 Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala
Ala Ser Pro Ser Gln 405 410 415 Thr Val Gln Arg Ala Val Ser Val Asn
Pro Gly Lys 420 425 105 436 PRT Homo sapiens 105 Ala Ser Thr Gln
Ser Pro Ser Val Phe Pro Leu Thr Arg Cys Cys Lys 1 5 10 15 Asn Ile
Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr 20 25 30
Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr Gly Ser Leu 35
40 45 Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu Ser
Gly 50 55 60 His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala
Trp Ala Lys 65 70 75 80 Gln Met Phe Thr Cys Arg Val Ala His Thr Pro
Ser Ser Thr Asp Trp 85 90 95 Val Asp Asn Lys Thr Phe Ser Val Cys
Ser Arg Asp Phe Thr Pro Pro 100 105 110 Thr Val Lys Ile Leu Gln Ser
Ser Cys Asp Gly Gly Gly His Phe Pro 115 120 125 Pro Thr Ile Gln Leu
Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr 130 135 140 Ile Asn Ile
Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu 145 150 155 160
Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser 165
170 175 Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr
Thr 180 185 190 Cys Gln Val Thr Tyr Gln His Thr Phe Glu Asp Ser Thr
Lys Lys Cys 195 200 205 Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr
Leu Ser Arg Pro Ser 210 215 220 Pro Phe Asp Leu Phe Ile Arg Lys Ser
Pro Thr Ile Thr Cys Leu Val 225 230 235 240 Val Asp Leu Ala Pro Ser
Lys Gly Thr Val Asn Leu Thr Trp Ser Arg 245 250 255 Ala Ser Gly Lys
Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln 260 265 270 Arg Asn
Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr Arg 275 280 285
Asp Trp Ile Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly 290
295 300 Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala
Leu 305 310 315 320 Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala
Ala Pro Glu Val 325 330 335 Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly
Ser Arg Asp Lys Arg Thr 340 345 350 Leu Ala Cys Leu Ile Gln Asn Phe
Met Pro Glu Asp Ile Ser Val Gln 355 360 365 Trp Leu His Asn Glu Val
Gln Pro Asp Ala Arg His Ser Thr Thr Gln 370 375 380 Pro Arg Lys Thr
Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu 385 390 395 400 Val
Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala 405 410
415 Val His Glu Ala Ala Ser Pro Ser Gln Thr Gln Arg Ala Val Ser Val
420 425 430 Asn Pro Gly Lys 435
* * * * *
References