U.S. patent application number 14/468479 was filed with the patent office on 2014-12-11 for antibodies and their uses for diagnosis and treatment of cytomegalovirus infection and associated diseases.
The applicant listed for this patent is Technion Research & Development Foundation Limited. Invention is credited to Oryan MAKLER, Yoram Reiter.
Application Number | 20140363440 14/468479 |
Document ID | / |
Family ID | 39735511 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140363440 |
Kind Code |
A1 |
MAKLER; Oryan ; et
al. |
December 11, 2014 |
ANTIBODIES AND THEIR USES FOR DIAGNOSIS AND TREATMENT OF
CYTOMEGALOVIRUS INFECTION AND ASSOCIATED DISEASES
Abstract
Anti CMV antibodies are provided. Thus an antibody of the
present invention comprises an antigen recognition domain capable
of binding an MHC molecule being complexed with a cytomegalovirus
(CMV) pp65 or pp64 peptide, wherein the antibody does not bind said
MHC molecule in an absence of said complexed peptide, and wherein
the antibody does not bind said peptide in an absence of said MHC
molecule. Also provided are methods of using the antibodies.
Inventors: |
MAKLER; Oryan; (Haifa,
IL) ; Reiter; Yoram; (Haifa, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Technion Research & Development Foundation Limited |
Haifa |
|
IL |
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|
Family ID: |
39735511 |
Appl. No.: |
14/468479 |
Filed: |
August 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13727643 |
Dec 27, 2012 |
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14468479 |
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12450476 |
Jan 6, 2010 |
8361473 |
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PCT/IL2008/000437 |
Mar 27, 2008 |
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13727643 |
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60929207 |
Jun 18, 2007 |
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60907343 |
Mar 29, 2007 |
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Current U.S.
Class: |
424/139.1 ;
424/178.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/088 20130101; C07K 16/2833 20130101; C07K 2317/32 20130101;
C07K 2317/21 20130101; C07K 2317/55 20130101; A61K 2039/505
20130101; C07K 2317/92 20130101 |
Class at
Publication: |
424/139.1 ;
424/178.1 |
International
Class: |
C07K 16/08 20060101
C07K016/08 |
Claims
1. A method of treating an immuno-compromised organ transplant
recipient subject having a cytomegalovirus (CMV) infection,
comprising administering to the subject a therapeutically effective
amount of an antibody comprising an antigen recognition domain
capable of binding a class I MHC molecule being complexed with a
cytomegalovirus (CMV) pp65 or pp64 peptide as set forth by SEQ ID
NO:3, wherein the antibody does not bind said MHC molecule in an
absence of said complexed peptide, and wherein the antibody does
not bind said peptide in an absence of said MHC molecule, thereby
treating the immuno-compromised organ transplant recipient subject
with CMV infection.
2. The method of claim 1, wherein said class I MHC molecule is an
HLA-A2.
3. The method of claim 1, wherein said antibody is conjugated to a
therapeutic moiety.
4. The method of claim 1, wherein the antibody an antibody
fragment.
5. The method of claim 1, wherein the antibody is an IgG subtype.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 13/727,643 filed on Dec. 27, 2012, which is a division of
U.S. patent application Ser. No. 12/450,476 filed on Jan. 6, 2010,
now U.S. Pat. No. 8,361,473, which is a National Phase of PCT
Patent Application No. PCT/IL2008/000437 having International
filing date of Mar. 27, 2008, which claims the benefit of priority
under 35 USC .sctn.119(e) of U.S. Provisional Patent Application
Nos. 60/929,207 filed on Jun. 18, 2007 and 60/907,343 filed on Mar.
29, 2007. The contents of the above applications are all
incorporated by reference as if fully set forth herein in their
entirety.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 60137SequenceListing.txt, created
on Aug. 26, 2014, comprising 220,126 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to methods of diagnosing and treating cytomegalovirus diseases and,
more particularly, but not exclusively, to antibodies capable of
same.
[0004] Of all the human herpesviruses described to date, infection
with cytomegalovirus (CMV) is considered to be the main cause of
morbidity and mortality. Approximately 70% of the world population
are carriers of the virus. Primary infection with the virus results
in a life long persistence in a latent form and is therefore
generally asymptomatic in healthy adults. However, some
individuals, such as immuno-compromised organ transplant
recipients, or individuals infected with human immunodeficiency
virus (HIV), are at high risk of developing life threatening CMV
disease due to CMV reactivation. In addition, CMV has emerged in
recent years as the most important cause of congenital infection in
the developed world, commonly leading to mental retardation and
developmental disability.
[0005] Immunity to CMV is complex and involves humoral and
cell-mediated responses. Studies showed that both natural killer
(NK) cells and cytotoxic T-lymphocytes (CTLs) are of primary
importance in prevention of recurrence. Many gene products
participate in generating the CTL response to CMV infection,
however, the high level expression frequencies of the viral protein
pp65 (e.g., Genbank Accession No. M15120; SEQ ID NO:48) suggests
pp65 as the main target of the CTL-mediated immune response. Among
all pp65 peptides, CMV specific-CTL activity in HLA-A2 positive
individuals was found to be mainly directed to the peptide
pp65.sub.495-503 (NLVPMVATV; SEQ ID NO:3) (Chee M S et al.,
1990).
[0006] Cytosolic proteins, usually synthesized in the cells, such
as CMV viral proteins, enter the class I MHC pathway of antigen
presentation. In the first step, ubiquitinated cytoplasmic proteins
are degraded by the proteasome, a cytoplasmic multiprotein complex
which generates a large portion of peptides destined for display by
class I MHC molecules. Peptides are then delivered from the
cytoplasm to the endoplasmic reticulum (ER) by the transporter
associated with antigen presentation (TAP) molecules. Newly formed
class I MHC dimers in the ER associate with and bind peptides
delivered by the TAP. Peptide binding stabilizes class I MHC
molecules and permits their movement out of the ER, through the
Golgi apparatus, to the cell surface. This pathway ensures that any
cell synthesizing viral proteins can be marked for recognition and
killing by CD8+ CTL.
[0007] Characterization of class I MHC-peptide presentation is
essential for understanding the acquired arm of the immune
response. The conventional strategy for detecting and studying rare
populations of antigen (Ag)-specific CD8+ T cells is the
application of tetrameric arrays of class I peptide-MHC complexes
(Altman J D., et al., 1996; Lee P P et al., 1999).
[0008] The diagnosis of diseases associated with CMV infection such
as retinitis, pneumonia, gastrointestinal disorders, and
encephalitis is based on clinical, histological, virological and
DNA tests.
[0009] Current methods of treating CMV in immuno-compromised (e.g.,
immuno-suppressed) subjects (e.g., HIV patients, bone marrow
transplanted subjects), especially CMV retinitis, include anti
viral drugs such as Foscarnet (FOSCAVIR.RTM.), Cidofovir
(VISTIDE.RTM.) Valganciclovir (VALCYTE.RTM.) Ganciclovir implants
(VITRASERT.RTM.) Fomivirsen (VITRAVENE.RTM.). However, the use of
these drugs may be associated with serious side effects such as
kidney damage, neutropenia and hypocalcemia. One strategy of
directly targeting CMV associated pathologies includes the use of
HLA-A2-restricted CD8(+) CTLs directed against pp65. However,
attempts to use CMV-specific CD8+ T cell clones for killing
CMV-infected retinal pigment epithelial cells have failed (Allart
S, et al., 2003; Invest Ophthalmol V is Sci. 44: 665-71).
[0010] Additional background art includes U.S. patent application
Ser. Nos. 11/203,137; 11/074,803; 10/510,229; and 11/582,416 to
Reiter Y, et al.
SUMMARY OF THE INVENTION
[0011] According to an aspect of some embodiments of the present
invention there is provided an antibody comprising an antigen
recognition domain capable of binding an MHC molecule being
complexed with a cytomegalovirus (CMV) pp65 or pp64 peptide,
wherein the antibody does not bind the MHC molecule in an absence
of the complexed peptide, and wherein the antibody does not bind
the peptide in an absence of the MHC molecule.
[0012] According to an aspect of some embodiments of the present
invention there is provided an antibody comprising a multivalent
form of the antibody of the present invention.
[0013] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient the antibody of the antibody of the present
invention.
[0014] According to an aspect of some embodiments of the present
invention there is provided a method of detecting a cell expressing
a cytomegalovirus (CMV) antigen, comprising contacting the cell
with the antibody of the present invention under conditions which
allow immunocomplex formation, wherein a presence or a level above
a predetermined threshold of the immunocomplex is indicative of CMV
expression in the cell.
[0015] According to an aspect of some embodiments of the present
invention there is provided a method of diagnosing a
cytomegalovirus (CMV) infection in a subject in need thereof,
comprising contacting a cell of the subject with the antibody of
the present invention under conditions which allow immunocomplex
formation, wherein a presence or a level above a pre-determined
threshold of the immunocomplex in the cell is indicative of the CMV
infection in the subject.
[0016] According to an aspect of some embodiments of the present
invention there is provided a method of treating a disease
associated with cytomegalovirus (CMV) infection, comprising
administering to a subject in need thereof a therapeutically
effective amount of the antibody of the present invention, thereby
treating the disease associated with CMV infection.
[0017] According to some embodiments of the invention, the
cytomegalovirus (CMV) pp65 or pp64 peptide is set forth by SEQ ID
NO:3.
[0018] According to some embodiments of the invention, the antigen
recognition domain comprises complementarity determining region
(CDR) amino acid sequences as set forth in SEQ ID NOs:24-26 and
30-32.
[0019] According to some embodiments of the invention, the antigen
recognition domain comprises complementarity determining region
(CDR) amino acid sequences as set forth in SEQ ID NOs: 36-38 and
42-44.
[0020] According to some embodiments of the invention, the antibody
being conjugated to a therapeutic moiety.
[0021] According to some embodiments of the invention, the antibody
is attached to a detectable moiety.
[0022] According to some embodiments of the invention, the antibody
being an antibody fragment.
[0023] According to some embodiments of the invention, the
multivalent form is an IgG antibody.
[0024] According to some embodiments of the invention, the subject
has a suppressed or a compromised immune system.
[0025] According to some embodiments of the invention, the CMV
infection is associated with a disease selected from the group
consisting of mononucleosis, retinitis, pneumonia, gastrointestinal
disorders, and encephalitis.
[0026] According to some embodiments of the invention, the cell is
a retina cell, lung epithelial cell, a gastrointestinal epithelial
cell or a brain cell.
[0027] According to some embodiments of the invention, the subject
is an immuno-compromised organ transplant recipient.
[0028] According to some embodiments of the invention, the subject
is infected with human immunodeficiency virus (HIV).
[0029] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0030] As used herein, the terms "comprising" and "including" or
grammatical variants thereof are to be taken as specifying the
stated features, integers, steps or components but do not preclude
the addition of one or more additional features, integers, steps,
components or groups thereof. This term encompasses the terms
"consisting of" and "consisting essentially of".
[0031] The phrase "consisting essentially of" or grammatical
variants thereof when used herein are to be taken as specifying the
stated features, integers, steps or components but do not preclude
the addition of one or more additional features, integers, steps,
components or groups thereof but only if the additional features,
integers, steps, components or groups thereof do not materially
alter the basic and novel characteristics of the claimed
composition, device or method.
[0032] The term "method" refers to manners, means, techniques and
procedures for accomplishing a given task including, but not
limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the biotechnology and
medical art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0034] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0035] In the drawings:
[0036] FIGS. 1a-d depict the specificity of recombinant Fab Abs to
the MHC class I (HLA-A2)-CMV pp65-derived peptide (NLVPMVATV; SEQ
ID NO:3) complex. FIG. 1a--A histogram depicting an ELISA assay in
which phage Fab clones were reacted with HLA-A2/pp65 complexes. Fab
clones were reacted against a specific MHC class I-peptide complex
(HLA-A2/pp65.sub.495-503, marked as "CMV") and control non-specific
complexes containing gp100.sub.280-288 peptide (SEQ ID NO:4;
YLEPGPVTA; marked as "280"). FIG. 1b--An SDS-PAGE analysis
depicting the expression and purification of HLA-A2/pp65 TCR-like
Fabs. SDS-PAGE analysis of the purified Fab proteins was performed
after metal affinity chromatography. Note the intense protein bands
purified from phage clone F5 or H9 with a molecular weight of 45
kDa. FIGS. 1c and d--Bar graphs depicting an ELISA assay of the
binding of soluble purified Fabs to the HLA-A2-peptide complexes.
The soluble clones H9 (FIG. 1c) and F5 (FIG. 1d) were reacted
against a specific complex (HLA-A2/pp65; "CMV") or control non
specific complexes containing the following peptides: EBV (280-288;
SEQ ID NO:5; GLCTLVAML), gp100 (280-288; SEQ ID NO:4), hTERT
(540-548; SEQ ID NO:6; ILAKFLHWL), gp100 (209-217; SEQ ID NO:7;
IMDQVPFSV), hTERT (865-873; SEQ ID NO:8; RLVDDFLLV), Gag (77-85;
SEQ ID NO:9; SLYNTVATL), Pol (476-484; SEQ ID NO:10; ILEPVHGV),
MART (26-35; SEQ ID NO:11; ELAGIGILTV), XAGE (SEQ ID NO:12;
GVFPSAPSPV), TARP (29-37; SEQ ID NO:13; FLRNFSLML), TAX (11-19; SEQ
ID NO:14; LLFGYPVYV). Specificity towards HLA-A2/pp65 complex can
be observed in each of the two clones.
[0037] FIGS. 2a-d are flow cytometry analyses depicting the
detection of MHC-peptide complexes on the surface of APCs using the
H9 and F5 soluble Fabs. JY or RMAS-HHD cell lines were pulsed with
various specific and nonspecific peptides. JY cells (FIGS. 2a and
c) or RMAS-HHD cells (FIGS. 2b and d) loaded with the CMV
pp65.sub.495-503 peptide (SEQ ID NO:3) or control peptides ("280",
"540"), incubated with the H9 (FIGS. 2a, b) or F5 (FIGS. 2c, d) Fab
respectively. Specific staining of the pp65 loaded cells, but not
the control cells, is shown. The same type of assay was performed
with 10 different control HLA-A2-restricted peptides (data not
shown).
[0038] FIGS. 3a-c are flow cytometry analyses depicting the
detection of MHC-peptide complexes on the surface of JY cells using
H9 Fab in its monomeric or tetrameric forms. The JY cell line was
pulsed with different peptides. FIG. 3a--JY cells loaded with
pp65.sub.495-503 peptide (SEQ ID NO:3). Incubations were with H9
Fab monomer and PE-labeled anti human Fab, or with H9 Fab tetramer
connected to PE labeled streptavidin. FIG. 3b-JY cells loaded with
pp65.sub.495-503 peptide (SEQ ID NO:3). Incubations were with H9
Fab monomer and FITC-labeled anti human Fab, or with H9 whole IgG
Ab and FITC-labeled anti human Fab. FIG. 3c-JY cells loaded with
gp100.sub.280-288 280-288 (SEQ ID NO:4) as a control. Incubations
were with H9 Fab monomer and PE-labeled anti human Fab, H9 Fab
tetramer connected to PE labeled streptavidin or with H9 IgG Ab and
PE-labeled anti human Fab. Note the specific binding of the H9 Fab
in its monomeric or tetrameric form, as well as the whole IgG H9 Ab
to JY cells pulsed with the HLA-A2-CMV peptide (pp65 495-503) but
not with JY cells when pulsed with the control peptide (gp100
280-288). Also note the increased avidity of the IgG Ab as compared
to the monomeric Fab, or the increased avidity of the tetrameric
Fab form as compared to the monomeric Fab form.
[0039] FIGS. 4a-c are graphs depicting the affinity determination
of the H9 Ab in its monomeric (FIG. 4a) or IgG (FIG. 4b) forms, as
detected by surface plasmon resonance (SPR) analysis. Each of the
forms was flowed over the relevant wells at three different
concentrations (0.05 .mu.M, 0.1 .mu.M, 0.2 .mu.M) of biotinylated
HLA-A2-pp65 495-503 complexes. As a control, H9 Ab were flowed over
wells which were coated with control biotinylated HLA-A2/pEBV
complexes (FIG. 4c). Note the absence of binding signal of the H9
Ab over the HLA-A2/pEBV complex (the concentration of HLA-A2/pEBV
complex was 0.2 .mu.M) as compared to the HLA-A2/pp65 complex (the
concentration of HLA-A2/pp65 complex was 0.2 .mu.M).
[0040] FIGS. 5a-c are flow cytometry analyses depicting the
detection of Fab sensitivity threshold (FIGS. 5a-b) and of rare
cells bearing the specific peptide-MHC complex in a heterogenous
cell population (FIG. 5c). In order to detect Fab sensitivity
threshold, JY cells were pulsed with various concentrations of
pp65.sub.495-503 peptide (0.65 nM, 0.1 .mu.M, 012 .mu.M, 0.25
.mu.M, 0.5 .mu.M, 1 .mu.M or 100 .mu.M), and incubated with H9 Fab
monomer (at a concentration of 10 .mu.g/ml) and PE-labeled anti
human Fab (FIG. 5a), or H9 Fab tetramer (at a concentration of 10
.mu.g/ml) connected to PE labeled streptavidin (FIG. 5b). Note the
significantly low concentration of the pp65.sub.495-503 peptide
needed to pulse JY cells in order to obtain a significant binding
with the H9 tetramer [e.g., a threshold of 65 nM) of the pp65
495-503 peptide] or the H9 monomer [e.g., a threshold of 0.1 .mu.M
of the pp65 495-503 peptide]. Detection of rare population of cells
bearing the specific MHC-peptide complex was by pulsing JY APCs
with the pp65 495-503 peptide and mixing them with APD cells
(HLA-A2- B cell line) at different ratios (FIG. 5c) so as to obtain
pre-determined concentrations of cells expressing the specific
MHC-peptide complex. The mixed population was stained with H9 Fab
(at a concentration of 10 .mu.g/ml), and detection sensitivity was
monitored by flow cytometry. Note the specific detection of as low
as 5% cells bearing the specific MHC-pp65 495-503 complex.
[0041] FIGS. 6a-m are flow cytometry analyses (FIGS. 6a-l) and a
bar graph (FIG. 6m) depicting the detection of the specific
HLA-A2/pp65 complex by H9 tetramer (FIG. 6e-h) or H9 IgG Ab (Data
not shown), after naturally occurring active intracellular
processing. HLA-A2 positive fibroblasts were infected with the CMV
laboratory strain AD169 (FIGS. 6a, e, i). HLA-A2 positive
uninfected fibroblasts were used as a control (FIGS. 6c, g, k) as
well as HLA-A2 negative infected fibroblasts (FIGS. 6b, f, j) or
HLA-A2 negative uninfected fibroblasts (FIGS. 6d, h, l). Incubation
were with PE labeled BB7.2 (FIGS. 6a-d), PE labeled H9 tetramer
(FIGS. 6e-h) or anti pp65 FITC mAB (FIGS. 6i-l) followed by the
secondary antibody FITC-labeled anti mouse IgG, 72 hours after
infection. Note the specific binding of the H9 tetramer to HLA-A2
positive cells following infection with CMV (FIG. 6e) as compared
to the absence of binding to HLA-A2 negative cells (FIG. 60 or to
uninfected cells (FIGS. 6g and h), demonstrating the specific
HLA-A2-CMV (pp65 495-503) complex-dependent binding of the H9
antibody to cells ex vivo. In contrast, note the non-CMV-dependent
binding of the BB7.2 Ab to HLA-A2 positive cells [same binding
efficacy in the presence (FIG. 6a) or absence (FIG. 6c) of CMV
peptide], and the non-HLA-A2-dependent binding of the Anti pp65 Ab
in CMV-infected cells [same binding efficacy in HLA-A2 positive
(FIG. 6i) or HLA-A2 negative (FIG. 6j) cells]. FIG. 6m--A
cytotoxicity assay by which H9 IgG Ab is shown to block virus
infected cells killing mediated by specific CTL line. Fibroblast
cells were radioactively labeled with S.sup.35 methionine before
infection with the CMV virus and 72 hours later the cells were
incubated with the H9 IgG Ab. CTLs were added at a target
(fibroblast cells infected with CMV):effector (CTL) ratio of 1:10
and incubated for five hours. Cells incubated with W6 Ab (an
antibody directed against HLA-A,B,C) were used as positive control,
while cells without any Ab incubation served as a reference for the
maximum killing rate. These results demonstrate the TCR-like
specificity of the H9 IgG Ab to specific CMV-infected cells.
[0042] FIGS. 7a-t are flow cytometry analyses depicting kinetic
assays which follow the dynamics between the HLA-A2 extracellular
presentation, the HLA-A2/pp65 peptide extracellular and
intracellular complex presentation and the pp65 expression, in
HLA-A2+ (positive) cells infected with the CMV wild-type (WT) AD169
strain. 36 (FIGS. 7a-e), 72 (FIGS. 7f-j), 96 (FIGS. 7k-o), and 120
(FIGS. 7p-t) hours after infection the cells were harvested and
incubated with the BB7.2 PE labeled Ab (FIGS. 7c, d, h, i, m, n, r,
s), anti pp65 Ab (FIGS. 7e, j, o, t; intracellular) and H9 IgG Ab
(FIGS. 7a, b, f, g, k, l, p, q) antibodies and analyzed by flow
cytometry. FITC-labeled anti mouse antibody and Alexa
fluor.sup.488-labeled anti human antibody were used as secondary
antibodies for the anti pp65 mAb and the H9 IgG Ab respectively.
Intracellular staining was feasible by cells permeabilization.
[0043] FIGS. 8a-t are flow cytometry analyses depicting kinetic
assays which follow the dynamics between the HLA-A2 extracellular
presentation, the HLA-A2/pp65 peptide extracellular and
intracellular complex presentation and the pp65 expression, in
HLA-A2+ (positive) cells infected with the RV798 mutant strain. 36
(FIGS. 8a-e), 72 (FIGS. 8f-j), 96 (FIGS. 8k-o), and 120 (FIGS.
8p-t) hours after infection cells were harvested and incubated with
BB7.2 PE labeled Ab (FIGS. 8c, d, h, i, m, n, r, s), anti pp65 Ab
(FIGS. 8e, j, o, t; intracellular) and H9 IgG Ab (FIGS. 8a, b, f,
g, k, l, p, q) antibodies and analyzed by flow cytometry.
FITC-labeled anti mouse antibody and Alexa fluor.sup.488-labeled
anti human antibody were used as secondary antibodies for the anti
pp65 mAb and the H9 IgG Ab respectively. Intracellular staining was
feasible by cells permeabilization.
[0044] FIGS. 9a-y are flow cytometry analyses depicting kinetic
assays which follow the dynamics between the HLA-A2 extracellular
presentation, the HLA-A2/pp65 peptide extracellular and
intracellular complex presentation and the pp65 expression, in
HLA-A2+ (positive) uninfected cells (FIGS. 9a-t) or in HLA-A2-
(negative) cells infected with the AD169 Wild Type strain of CMV.
Staining with the H9 IgG antibody, BB7.2 antibody or the anti pp65
antibodies was effected in the uninfected cells harvested at
parallel times [i.e., 36 (FIGS. 9a-e), 72 (FIGS. 9f-j), 96 (FIGS.
9k-o), and 120 (FIGS. 9p-t) hours] to the cells infected with the
viruses as described in FIGS. 7a-t and 8a-t, hereinabove. Infected
HLA-A2- (negative) cells were harvested and stained with the H9 IgG
antibody, BB7.2 antibody or the anti pp65 antibody at 120 hours
after infection with the AD169 CMV virus. Extracellular staining
with the H9 IgG antibody is shown in FIGS. 9a, f, k, p and u.
Intracellular staining with the H9 IgG antibody is shown in FIGS.
9b, g, l, q and v. Extracellular staining with the BB7.2 antibody
is shown in FIGS. 9c, h, m, r and w. Intracellular staining with
the BB7.2 antibody is shown in FIGS. 9d, i, n, s and x. Staining
with the anti pp65 antibody is shown in FIGS. 9e, j, o, t, and y.
FITC-labeled anti mouse antibody and Alexa fluor.sup.488-labeled
anti human antibody were used as secondary antibodies for the anti
pp65 mAb and the H9 IgG Ab respectively. Intracellular staining was
feasible by cells permeabilization.
[0045] FIGS. 10a-d are bar graphs depicting quantization of the
number of HLA-A2/pp65 complexes inside and on the surface of virus
infected cells. The level of fluorescence intensity on stained
cells was compared with the fluorescence intensities of calibration
beads with known numbers of PE molecules per bead, thus providing a
mean of quantifying PE-stained cells using a flow cytometer.
Incubations were with BB7.2 PE labeled Ab (FIGS. 10c and d), and H9
Ab (FIGS. 10a and b). PE-labeled anti kappa antibody was used as a
secondary antibody for the H9 IgG Ab. The calculated number of
HLA-A2/pp65 complexes inside cells (FIG. 10a) and on the surface
(FIG. 10b) as well as the number of general HLA-A2 complexes inside
the cells (FIG. 10c) and on the surface (FIG. 10d) in each time
scale, is shown for cells infected with AD169 (WT), RV798 (mutant),
and uninfected cells.
[0046] FIGS. 11a-o are confocal microscopy images of
immuno-fluorescence analyses depicting direct visualization of
HLA-A2/pp65 complexes in CMV infected fibroblasts. Infected cells
were harvested at five time scales post infection [24 (FIGS.
11a-c), 48 (FIGS. 11d-f), 72 (FIGS. 11g-i), 96 (FIGS. 11j-l) and
120 (FIGS. 11m-o) hours]. Intracellular double staining were with
the H9 Ab and Golgi marker. Secondary Ab for the H9 Ab was anti
human alexa fluor.sup.488. Secondary antibody for the Golgi marker
was anti mouse alexa fluor.sup.594. Shown are images of H9 Ab alone
(FIGS. 11a, d, g, j, m), Golgi marker alone (FIGS. 11b, e, h, k, n)
or merged images of H9 and Golgi marker (FIGS. 11c, f, I, l,
o).
[0047] FIGS. 12a-o are confocal microscopy images of
immuno-fluorescence analyses depicting direct visualization of
HLA-A2/pp65 complexes in CMV infected fibroblasts. Infected cells
were harvested at five time scales post infection [24 (FIGS.
12a-c), 48 (FIGS. 12d-f), 72 (FIGS. 12g-i), 96 (FIGS. 12j-l) and
120 (FIGS. 12m-o) hours]. Intracellular double staining were with
the H9 Ab and the ER marker. Secondary Ab for the H9 Ab was anti
human alexa fluor.sup.488. Secondary antibody for the ER marker was
anti mouse alexa fluor.sup.594. Shown are images of H9 Ab alone
(FIGS. 12a, d, g, j, m), ER marker alone (FIGS. 12b, e, h, k, n) or
merged images of H9 and ER marker (FIGS. 12c, f, I, l, o).
[0048] FIGS. 13a-j are confocal microscopy images of
immuno-fluorescence analyses depicting direct visualization of
HLA-A2/pp65 complexes of the surface (extracellular) of CMV
infected fibroblasts. The cells were extracellularly stained with
the H9 Ab, and anti human alexa fluor.sup.488 as a secondary Ab
(FIGS. 13a-e). Noninfected fibroblast cells were used as a control
(FIGS. 13f-h). Verification of the virus infection was with anti
pp65 Ab and anti mouse alexa fluor.sup.594 as a secondary Ab (FIGS.
13i-j).
[0049] FIGS. 14a-d depict the amino acid sequences (FIGS. 14a and
c; SEQ ID NOs:16 and 18) and the nucleic acid sequences (FIGS. 14b
and d; SEQ ID NOs:17 and 19) of the heavy chain (FIGS. 14a and b)
and the light chain (FIGS. 14c and d) of Fab H9. The CDRs are shown
in red; the constant regions are shown in green.
[0050] FIGS. 15a-d depict the amino acid sequences (FIGS. 15a and
c; SEQ ID NOs:20 and 22) and the nucleic acid sequences (FIGS. 15b
and d; SEQ ID NOs:21 and 23) of the heavy chain (FIGS. 15a and b)
and the light chain (FIGS. 15c and d) of Fab F5. The CDRs are shown
in red; the constant regions are shown in green.
[0051] FIGS. 16a-d are flow cytometry (FACS) analyses depicting the
detection of HLA-A2/pp65 complexes on the surface of virus-infected
cells taken from patients. PBMCs isolated from BMT recipients and
healthy donors were stained extracellular and intracellular with
the H9 Ab and the secondary anti human alexa fluor.sup.488 Ab. FIG.
16a-Confirmation of the cells' typing by staining with anti HLA-A2
(BB7.2) Ab. FIG. 16b-Extracellular staining of the BMT recipient
cells with the H9 Ab. No detection of HLA-A2/pp65 complexes is seen
in the infected cells using the H9 Ab. FIGS. 16c-d--Intracellular
staining of both BMT recipients (FIG. 16c) and health donor cells
(FIG. 16d) with the H9 Ab. A significant specific staining with the
H9 Ab of the permeabilized infected cells is seen in the BMT
recipients (FIG. 16c). In contrast, no staining of the H9 Ab is
seen in cells of the healthy control.
[0052] FIGS. 17a-i are flow cytometry (FACS) analyses depicting
examination of the proteasome inhibitor effect on the complexes
presentation. Infected (FIGS. 17a-f) fibroblasts were harvested at
three time scales post infection [48 (FIGS. 17a, d), 72 (FIGS. 17b,
e), 96 (FIGS. 17c, f) hours], and treated overnight with 10
.mu.g/ml ALLN (acetyl-leucyl-leucyl-norleucinal) (FIGS. 17a-c) or
remained untreated (FIGS. 17d-f). The cells were stained with H9 Ab
followed by anti human alexa fluor.sup.488 as a secondary Ab. FACS
analysis shows increased intensity of the signals after treatment
with the proteasome inhibitor (FIGS. 17a-c), compared to untreated
cells (FIGS. 17d-f). Control, uninfected cells (FIGS. g-i) showed
no signal while stained with the H9 Ab.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0053] The present invention, in some embodiments thereof, relates
to antibodies capable of binding MHC molecules being complexed with
cytomegalovirus (CMV) pp65 or pp64 peptides which can be used to
detect CMV infection and presentation on the cell surface and, more
particularly, but not exclusively, to methods of diagnosing and
treating diseases associated with CMV infection.
[0054] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0055] While reducing the invention to practice, the present
inventors have generated human T cell receptor (TCR)-like
antibodies directed against complexes of MHC and CMV pp65 or pp64
antigenic peptides which can recognize cells infected with CMV and
thus can be used to diagnose and treat diseases associated with CMV
infection.
[0056] As shown in the Examples section which follows, recombinant
antibodies [e.g., clones H9 (the amino acid sequence of the heavy
chain is set forth by SEQ ID NO:16; the amino acid sequence of the
light chain is set forth by SEQ ID NO:18) and F5 (the amino acid
sequence of the heavy chain is set forth by SEQ ID NO:20; the amino
acid sequence of the light chain is set forth by SEQ ID NO:22]
which can specifically recognize MHC molecules when complexed with
CMV pp65-derived peptides such as the pp65.sub.495-503 (SEQ ID
NO:3) were isolated and were found to exhibit fine specificity to
soluble or membrane-presented CMV pp65-MHC class I complex
(Examples 1 and 2 of the Examples section which follows). In
addition, multivalent forms of these antibodies (e.g., tetrameric
Fabs or bivalent IgG) which exhibit increased avidity while
preserving the specificity to the CMV pp65-MHC complex (Example 3
of the Examples section which follows) were capable of detecting as
low as 5% of subpopulations of cells bearing CMV pp65 peptide-MHC
complexes (Example 4 of the Examples section which follows).
Cytotoxicity assays using pp65-specific CD8+ T lymphocytes further
demonstrated the specificity of the TCR-like antibodies of the
invention for CMV pp65-MHC complexes by their ability to block
killing by the CTLs (Example 6 of the Examples section which
follows). Moreover, the TCR-like antibodies of the invention
enabled one, for the first time, to follow CMV pp65-MHC class I
complexes both inside and on the surface of cells infected with CMV
(Example 5 of the Examples section which follows). In addition, as
shown in FIGS. 7-9 and described in Example 7 of the Examples
section which follows, the TCR-like antibodies of the invention
demonstrated that there is no correlation between class I MHC down
regulation induced by wild-type virus and the
generation/presentation of the virus-specific
HLA-A2/pp65.sub.495-503 complex. Further quantitative data revealed
that specific HLA-A2/pp65 complexes are being generated in large
amounts and accumulated inside the infected cell in a mechanism
that is independent to the overall down regulation of HLA-A2
molecules in these cells (Example 8 of the Examples section which
follows). In addition, confocal microscopy analysis demonstrated
that immediately after CMV infection specific HLA-A2/pp65 complexes
are being generated and accumulated in the Golgi compartment and
only about 72 hours after infection are the HLA-A2/pp65 complexes
displayed on the cell surface (Example 9 of the Examples section
which follows). Moreover, as shown in FIGS. 16a-d and described in
Example 12 of the Examples section which follows, the antibodies of
the invention were shown to be capable of detecting HLA-A2/pp65
complexes in blood cells of subjects with CMV reactivation due to
immune suppression (e.g., bone marrow transplanted subjects). In
addition, as shown in FIGS. 17a-j and described in Example 13 of
the Examples section which follows, incubation of cells with a
proteasome inhibitor resulted in increased presentation of the
MHC/pp65 complexes on the cell surface.
[0057] Thus, according to an aspect of some embodiments of the
present invention there is provided an antibody comprising an
antigen recognition domain capable of binding a Major
histocompatibility complex (MHC) molecule being complexed with a
cytomegalovirus (CMV) pp65 or pp64 peptide, wherein the antibody
does not bind the MHC molecule in an absence of the complexed
peptide, and wherein the antibody does not bind the peptide in an
absence of the MHC molecule.
[0058] As used herein, the phrase "major histocompatibility complex
(MHC)" refers to a complex of antigens encoded by a group of linked
loci, which are collectively termed H-2 in the mouse and HLA in
humans. The two principal classes of the MHC antigens, class I and
class II, each comprise a set of cell surface glycoproteins which
play a role in determining tissue type and transplant
compatibility. In transplantation reactions, cytotoxic T-cells
(CTLs) respond mainly against foreign class I glycoproteins, while
helper T-cells respond mainly against foreign class II
glycoproteins.
[0059] Major histocompatibility complex (MHC) class I molecules are
expressed on the surface of nearly all cells. These molecules
function in presenting peptides which are mainly derived from
endogenously synthesized proteins to CD8+ T cells via an
interaction with the .alpha..beta. T-cell receptor. The class I MHC
molecule is a heterodimer composed of a 46-kDa heavy chain which is
non-covalently associated with the 12-kDa light chain .beta.-2
microglobulin. In humans, there are several MHC haplotypes, such
as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31,
HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, their sequences can
be found at the kabbat data base, at
htexttransferprotocol://immuno.bme.nwu.edu. Further information
concerning MHC haplotypes can be found in Paul, B. Fundamental
Immunology Lippincott-Rven Press.
[0060] Cytomegalovirus (CMV) belongs to the human herpesviruses.
There are several known strains of CMV, including strains 1042,
119, 2387. 4654, 5035, 5040, 5160, 5508, AD169, Eisenhardt, Merlin,
PT, Toledo and Towne. During viral infection, the expressed viral
proteins, e.g., pp65 of the CMV AD169 strain [GenBank Accession No.
M15120 for nucleic acid coding sequence (SEQ ID NO:48) and GenBank
Accession No. AAA45996.1 for amino acids (SEQ ID NO:50); or GenBank
Accession No. P06725 (SEQ ID NO:53)] pp64 of the CMV Towne strain
[GenBank Accession No. M67443 for nucleic acid coding sequence (SEQ
ID NO:49) and GenBank Accession No. AAA45994.1 for amino acids (SEQ
ID NO:51); or GenBank Accession No. P18139 (SEQ ID NO:52)] are
subject to proteasomal degradation and the MHC-restricted peptides
bind to the MHC molecules [e.g., MHC class I or MHC class II] and
are further presented therewith on the cell surface. The pp65 (561
amino acids in length) and pp64 (551 amino acids in length)
proteins of the CMV AD169 and Towne strains, respectively, are 99%
identical proteins and share the same amino acid sequence from
position 3-551 of pp64 and 13-561 of pp65.
[0061] As used herein the term "peptide" refers to native peptides
(either proteolysis products or synthetically synthesized peptides)
and further to peptidomimetics, such as peptoids and semipeptoids
which are peptide analogs, which may have, for example,
modifications rendering the peptides more stable while in a body,
or more immunogenic. Such modifications include, but are not
limited to, cyclization, N terminus modification, C terminus
modification, peptide bond modification, including, but not limited
to, CH.sub.2--NH, CH.sub.2--S, CH.sub.2--S.dbd.O, O.dbd.C--NH,
CH.sub.2--O, CH.sub.2--CH.sub.2, S.dbd.C--NH, CH.dbd.CH or
CF.dbd.CH, backbone modification and residue modification. Methods
for preparing peptidomimetic compounds are well known in the art
and are specified in Quantitative Drug Design, C. A. Ramsden Gd.,
Chapter 17.2, F. Choplin Pergamon Press (1992), which is
incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinunder.
[0062] As used herein in the specification and in the claims
section below the term "amino acid" is understood to include the 20
naturally occurring amino acids; those amino acids often modified
post-translationally in vivo, including for example hydroxyproline,
phosphoserine and phosphothreonine; and other unusual amino acids
including, but not limited to, 2-aminoadipic acid, hydroxylysine,
isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore,
the term "amino acid" includes both D- and L-amino acids. Further
elaboration of the possible amino acids usable according to the
invention and examples of non-natural amino acids useful in MHC-I
HLA-A2 recognizable peptide antigens are given herein under.
[0063] Based on accumulated experimental data, it is nowadays
possible to predict which of the peptides of a protein will bind to
MHC, class I. The HLA-A2 MHC class I has been so far characterized
better than other HLA haplotypes, yet predictive and/or sporadic
data is available for all other haplotypes.
[0064] With respect to HLA-A2 binding peptides, assume the
following positions (P1-P9) in a 9-mer peptide:
P1-P2-P3-P4-P5-P6-P7-P8-P9
[0065] The P2 and P2 positions include the anchor residues which
are the main residues participating in binding to MHC molecules.
Amino acid resides engaging positions P2 and P9 are hydrophilic
aliphatic non-charged natural amino (examples being Ala, Val, Leu,
Ile, Gln, Thr, Ser, Cys, preferably Val and Leu) or of a
non-natural hydrophilic aliphatic non-charged amino acid [examples
being norleucine (Nle), norvaline (Nva), .alpha.-aminobutyric
acid]. Positions P1 and P3 are also known to include amino acid
residues which participate or assist in binding to MHC molecules,
however, these positions can include any amino acids, natural or
non-natural. The other positions are engaged by amino acid residues
which typically do not participate in binding, rather these amino
acids are presented to the immune cells. Further details relating
to the binding of peptides to MHC molecules can be found in Parker,
K. C., Bednarek, M. A., Coligan, J. E., Scheme for ranking
potential HLA-A2 binding peptides based on independent binding of
individual peptide side-chains. J Immunol. 152, 163-175, 1994., see
Table V, in particular. Hence, scoring of HLA-A2.1 binding peptides
can be performed using the HLA Peptide Binding Predictions software
approachable through a worldwide web interface at
hypertexttransferprotocol://worldwideweb (dot) bimas (dot) dcrt
(dot) nih (dot) gov/molbio/hla_bind/index. This software is based
on accumulated data and scores every possible peptide in an
analyzed protein for possible binding to MHC HLA-A2.1 according to
the contribution of every amino acid in the peptide. Theoretical
binding scores represent calculated half-life of the
HLA-A2.1-peptide complex.
[0066] Hydrophilic aliphatic natural amino acids at P2 and P9 can
be substituted by synthetic amino acids, preferably Nleu, Nval
and/or .alpha.-aminobutyric acid. P9 can be also substituted by
aliphatic amino acids of the general formula
--HN(CH.sub.2).sub.nCOOH, wherein n=3-5, as well as by branched
derivatives thereof, such as, but not limited to,
##STR00001##
wherein R is, for example, methyl, ethyl or propyl, located at any
one or more of the n carbons.
[0067] The amino terminal residue (position P1) can be substituted
by positively charged aliphatic carboxylic acids, such as, but not
limited to, H.sub.2N(CH.sub.2).sub.nCOOH, wherein n=2-4 and
H.sub.2N--C(NH)--NH(CH.sub.2).sub.nCOOH, wherein n=2-3, as well as
by hydroxy Lysine, N-methyl Lysine or ornithine (Orn).
Additionally, the amino terminal residue can be substituted by
enlarged aromatic residues, such as, but not limited to,
H.sub.2N--(C.sub.6H.sub.6)--CH.sub.2--COOH, p-aminophenyl alanine,
H.sub.2N--F(NH)--NH--(C.sub.6H.sub.6)--CH.sub.2--COOH,
p-guanidinophenyl alanine or pyridinoalanine (Pal). These latter
residues may form hydrogen bonding with the OH.sup.- moieties of
the CMV pp65 residues at the MHC-1 N-terminal binding pocket, as
well as to create, at the same time aromatic-aromatic
interactions.
[0068] Derivatization of amino acid residues at positions P4-P8,
should these residues have a side-chain, such as, OH, SH or
NH.sub.2, like Ser, Tyr, Lys, Cys or Orn, can be by alkyl, aryl,
alkanoyl or aroyl. In addition, OH groups at these positions may
also be derivatized by phosphorylation and/or glycosylation. These
derivatizations have been shown in some cases to enhance the
binding to the T cell receptor.
[0069] Longer derivatives in which the second anchor amino acid is
at position P10 may include at P9 most L amino acids. In some cases
shorter derivatives are also applicable, in which the C terminal
acid serves as the second anchor residue.
[0070] Cyclic amino acid derivatives can engage position P4-P8,
preferably positions P6 and P7. Cyclization can be obtained through
amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn,
di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at
various positions in the chain (--CO--NH or --NH--CO bonds).
Backbone to backbone cyclization can also be obtained through
incorporation of modified amino acids of the formulas
H--N((CH.sub.2).sub.n--COOH)--C(R)H--COOH or
H--N((CH.sub.2).sub.n--COOH)--C(R)H--NH.sub.2, wherein n=1-4, and
further wherein R is any natural or non-natural side chain of an
amino acid.
[0071] Cyclization via formation of S--S bonds through
incorporation of two Cys residues is also possible. Additional
side-chain to side chain cyclization can be obtained via formation
of an interaction bond of the formula
--(--CH.sub.2--).sub.n--S--CH.sub.2--C--, wherein n=1 or 2, which
is possible, for example, through incorporation of Cys or homoCys
and reaction of its free SH group with, e.g., bromoacetylated Lys,
Orn, Dab or Dap.
[0072] Peptide bonds (--CO--NH--) within the peptide may be
substituted by N-methylated bonds (--N(CH.sub.3)--CO--), ester
bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds
(--CO--CH.sub.2--), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R
is any alkyl, e.g., methyl, carba bonds (--CH.sub.2--NH--),
hydroxyethylene bonds (--CH(OH)--CH.sub.2--), thioamide bonds
(--CS--NH--), olefinic double bonds (--CH.dbd.CH--), retro amide
bonds (--NH--CO--), peptide derivatives (--N(R)--CH.sub.2--CO--),
wherein R is the "normal" side chain, naturally presented on the
carbon atom.
[0073] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time. According
to some embodiments of the invention, but not in all cases
necessary, these modifications should exclude anchor amino
acids.
[0074] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as TIC,
naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0075] Various pp65 or pp64 MHC restricted peptides can be used to
form the MHC-CMV pp65 peptide complex. See for example, the
peptides described in Examples 10 and 11 of the Examples section
which follows (Tables 5-137).
[0076] According to some embodiments of the invention, the
antibodies recognize a complex formed between the MHC class I
molecule (HLA-A2) and the CMV pp65 peptide set forth by SEQ ID
NO:3.
[0077] The term "antibody" as used herein includes intact molecules
as well as functional fragments thereof, such as Fab, F(ab').sub.2,
Fv and scFv that are capable of specific binding to a human major
histocompatibility complex (MHC) class I-restricted CMV pp65 or
pp64 epitope. These functional antibody fragments are defined as
follows: (i) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule, can be produced
by digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain; (ii) Fab', the
fragment of an antibody molecule that can be obtained by treating
whole antibody with pepsin, followed by reduction, to yield an
intact light chain and a portion of the heavy chain; two Fab'
fragments are obtained per antibody molecule; (iii) F(ab').sub.2,
the fragment of the antibody that can be obtained by treating whole
antibody with the enzyme pepsin without subsequent reduction;
F(ab').sub.2 is a dimer of two Fab' fragments held together by two
disulfide bonds; (iv) Fv, defined as a genetically engineered
fragment containing the variable region of the light chain and the
variable region of the heavy chain expressed as two chains; and (v)
scFv or "single chain antibody" ("SCA"), a genetically engineered
molecule containing the variable region of the light chain and the
variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain
molecule.
[0078] Methods of making these fragments are known in the art (See
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference) and are further described herein below.
[0079] An exemplary method for generating antibodies capable of
specifically binding a CMV pp65 peptide restricted to an MHC-I
complex is described in the Examples section herein below.
[0080] In addition, such antibodies may be generated by (i)
immunizing a genetically engineered non-human mammal having cells
expressing the human major histocompatibility complex (MHC) class
I, with a soluble form of an MHC class I molecule being complexed
with the HLA-restricted epitope; (ii) isolating mRNA molecules from
antibody producing cells, such as splenocytes, of the non-human
mammal; (iii) producing a phage display library displaying protein
molecules encoded by the mRNA molecules; and (iv) isolating at
least one phage clone from the phage display library, the at least
one phage displaying the antibody specifically bindable (with an
affinity below 200 nanomolar, e.g., below 100 nanomolar, e.g.,
below 50 nanomolar, e.g., below 30 nanomolar, e.g., below 20
nanomolar, e.g., below 10 nanomolar) to the human major
histocompatibility complex (MHC) class I being complexed with the
HLA-restricted epitope. The genetic material of the phage isolate
is then used to prepare a single chain antibody or other forms of
antibodies as is further described herein below. For example, the
genetic material of the phage isolate can be used to prepare a
single chain antibody which is conjugated to an identifiable or a
therapeutic moiety. According to some embodiments of the invention,
the non-human mammal is devoid of self MHC class I molecules.
According to some embodiments of the invention, the soluble form of
the MHC class I molecule is a single chain MHC class I polypeptide
including a functional human .beta.-2 microglobulin amino acid
sequence directly or indirectly covalently linked to a functional
human MHC class I heavy chain amino acid sequence.
[0081] Recombinant MHC class I and class II complexes which are
soluble and which can be produced in large quantities are described
in, for example, Denkberg, G. et al. 2002, and further in U.S.
patent application Ser. No. 09/534,966 and PCT/IL01/00260
(published as WO 01/72768), all of which are incorporated herein by
reference. Soluble MHC class I molecules are available or can be
produced for any of the MHC haplotypes, such as, for example,
HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33,
HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, following, for example the
teachings of PCT/IL01/00260, as their sequences are known and can
be found at the kabbat data base hypertexttransferprotocol://immuno
(dot) bme (dot) nwu (dot) edu/, the contents of the site is
incorporated herein by reference. Such soluble MHC class I
molecules can be loaded with suitable HLA-restricted epitopes and
used for vaccination of non-human mammal having cells expressing
the human major histocompatibility complex (MHC) class I as is
further detailed hereinbelow.
[0082] Non-human mammal having cells expressing a human major
histocompatibility complex (MHC) class I and devoid of self major
histocompatibility complex (MHC) class I can be produced using (i)
the sequence information provided in the kabbat data base, at
hypertexttransferprotocol://immuno (dot) bme (dot) nwu (dot) edu/,
which is incorporated herein by reference and pertaining to human
MHC haplotypes, such as, for example, HLA-A2, HLA-A1, HLA-A3,
HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and
HLA-Cw8, (ii) conventional constructs preparation techniques, as
described in, for example, "Molecular Cloning: A laboratory Manual"
Sambrook et al., (1989); "Current Protocols in Molecular Biology"
Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current
Protocols in Molecular Biology", John Wiley and Sons, Baltimore,
Md. (1989); Perbal, "A Practical Guide to Molecular Cloning", John
Wiley & Sons, New York (1988); Watson et al., "Recombinant
DNA", Scientific American Books, New York; Birren et al. (eds)
"Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold
Spring Harbor Laboratory Press, New York (1998); and (iii)
conventional gene knock-in/knock-out techniques as set forth, for
example, in U.S. Pat. Nos. 5,487,992, 5,464,764, 5,387,742,
5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385,
5,175,384, 5,175,383, 4,736,866; in International Publications WO
94/23049, WO93/14200, WO 94/06908 and WO 94/28123; as well as in
Burke and Olson, Methods in Enzymology, 194:251-270, 1991;
Capecchi, Science 244:1288-1292, 1989; Davies et al., Nucleic Acids
Research, 20 (11) 2693-2698, 1992; Dickinson et al., Human
Molecular Genetics, 2(8): 1299-1302, 1993; Duff and Lincoln,
"Insertion of a pathogenic mutation into a yeast artificial
chromosome containing the human APP gene and expression in ES
cells", Research Advances in Alzheimer's Disease and Related
Disorders, 1995; Huxley et al., Genomics, 9:742-750 1991;
Jakobovits et al., Nature, 362:255-261, 1993; Lamb et al., Nature
Genetics, 5: 22-29, 1993; Pearson and Choi, Proc. Natl. Acad. Sci.
USA, 1993. 90:10578-82; Rothstein, Methods in Enzymology,
194:281-301, 1991; Schedl et al., Nature, 362: 258-261, 1993;
Strauss et al., Science, 259:1904-1907, 1993, all of which are
incorporated herein by reference.
[0083] Of particular interest is the paper by Pascolo et al.,
published in J. Exp. Med. 185: 2043-2051, 1997, which describe the
preparation of mice expressing the human HLA-A2.1, H-2Db and HHD
MHC class I molecules and devoid of mice MHC class I
altogether.
[0084] An exemplary antibody, referred to as the H9 antibody,
capable of binding to an MHC class I complexed with a CMV pp65
epitope comprises complementarity determining region (CDR) amino
acid sequences as set forth in SEQ ID NOs:24-26 (for the heavy
chain) and 30-32 (for the light chain).
[0085] Another exemplary antibody, referred to as the F5 antibody,
capable of binding to an MHC class I complexed with a CMV pp65
epitope comprises complementarity determining region (CDR) amino
acid sequences as set forth in SEQ ID NOs:36-38 (for the heavy
chain) and 42-44 (for the light chain).
[0086] The invention provides a nucleic acid construct comprising a
nucleic acid sequence encoding the CDR sequences of the heavy chain
and the light chain of the antibody of the invention. The nucleic
acid construct may further comprise a promoter for directing
expression of the nucleic acid sequence in a host cell.
[0087] According to some embodiments of the invention, the nucleic
acid construct comprising the nucleic acid sequences set forth by
SEQ ID NOs:27-29 (for the heavy chain CDRs) and SEQ ID NOs:33-35
(for the light chain CDRs).
[0088] According to some embodiments of the invention, the nucleic
acid construct comprising the nucleic acid sequences set forth by
SEQ ID NOs:39-41 (for the heavy chain CDRs) and SEQ ID NOs:45-47
(for the light chain CDRs).
[0089] According to some embodiments of the invention, the nucleic
acid construct comprising the nucleic acid sequence set forth by
SEQ ID NO:17 (for the heavy chain) and SEQ ID NO:19 (for the light
chain).
[0090] According to some embodiments of the invention, the nucleic
acid construct comprising the nucleic acid sequence set forth by
SEQ ID NO:21 (for the heavy chain) and SEQ ID NO:23 (for the light
chain).
[0091] As mentioned herein above, the antibodies of the invention
may be antibody fragments. Antibody fragments according to the
invention can be prepared by proteolytic hydrolysis of the antibody
or by expression in E. coli or mammalian cells (e.g. Chinese
hamster ovary cell culture or other protein expression systems) of
a DNA sequence encoding the fragment.
[0092] Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references
contained therein, which patents are hereby incorporated by
reference in their entirety. See also Porter, R. R., Biochem. J.,
73: 119-126, 1959. Other methods of cleaving antibodies, such as
separation of heavy chains to form monovalent light-heavy chain
fragments, further cleavage of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0093] Fv fragments comprise an association of V.sub.H and V.sub.L
chains. This association may be noncovalent, as described in Inbar
et al., Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively,
the variable chains can be linked by an intermolecular disulfide
bond or cross-linked by chemicals such as glutaraldehyde. According
to some embodiments of the invention, the Fv fragments comprise
V.sub.H and V.sub.L chains connected by a peptide linker. These
single-chain antigen binding proteins (scFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the V.sub.H and V.sub.L domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
scFvs are described, for example, by Whitlow and Filpula, Methods,
2: 97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et
al., Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat.
No. 4,946,778, which is hereby incorporated by reference in its
entirety.
[0094] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry, Methods, 2: 106-10,
1991.
[0095] According to some embodiments of the invention, the
antibodies are multivalent forms such as tetrameric Fabs or IgG1
antibodies. The advantages of the multivalent forms of the antibody
of the invention include increased avidity, yet without
compromising the antibody specificity to its target (i.e., the
MHC-CMV pp65 peptide complex). Exemplary methods for generating
tetrameric Fabs or IgG1 antibodies are described in the general
materials and experimental methods of the Examples section herein
below.
[0096] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence.
[0097] The humanized antibody optimally also will comprise at least
a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin [Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta,
Curr. Op. Struct. Biol., 2:593-596 (1992)].
[0098] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0099] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0100] It will be appreciated that once the CDRs of an antibody are
identified, using conventional genetic engineering techniques,
expressible polynucleotides encoding any of the forms or fragments
of antibodies described herein can be devised and modified in one
of many ways in order to produce a spectrum of related-products as
further described herein below.
[0101] The antibody of the invention can be used in vitro, ex vivo
and in vivo in various therapeutic or diagnostic applications.
[0102] In case the antibody of the invention is to be used for
administration into an individual (e.g., human), the human or
humanized antibody or antibody fragment will generally tend to be
better tolerated immunologically than one of non human origin since
non variable portions of non human antibodies will tend to trigger
xenogeneic immune responses more potent than the allogeneic immune
responses triggered by human antibodies which will typically be
allogeneic with the individual. It will be preferable to minimize
such immune responses since these will tend to shorten the
half-life, and hence the effectiveness, of the antibody of the
invention in the individual. Furthermore, such immune responses may
be pathogenic to the individual, for example by triggering harmful
inflammatory reactions.
[0103] Alternately, an antibody or antibody fragment of human
origin, or a humanized antibody, will also be advantageous for
applications in which a functional physiological effect, for
example an immune response against a target cell, activated by a
constant region of the antibody or antibody fragment in the
individual is desired. For example, for applications including
targeted cell killing a specific immune response is advantageous.
Such applications particularly include those in which the
functional interaction between a functional portion of the antibody
or antibody fragment, such as an Fc region, with a molecule such as
an Fc receptor or an Fc-binding complement component, is optimal
when such a functional portion is, similarly to the Fc region, of
human origin.
[0104] Depending on the application and purpose, the antibody of
the invention which includes a constant region, or a portion
thereof, of any of various isotypes may be employed. According to
some embodiments of the invention, the isotype is selected so as to
enable or inhibit a desired physiological effect, or to inhibit an
undesired specific binding of the antibody of the invention via the
constant region or portion thereof. For example, for inducing
antibody-dependent cell mediated cytotoxicity (ADCC) by a natural
killer (NK) cell, the isotype can be IgG; for inducing ADCC by a
mast cell/basophil, the isotype can be IgE; and for inducing ADCC
by an eosinophil, the isotype can be IgE or IgA. For inducing a
complement cascade the composition-of-matter may comprise an
antibody or antibody fragment comprising a constant region or
portion thereof capable of initiating the cascade. For example, the
antibody or antibody fragment may advantageously comprise a Cgamma2
domain of IgG or Cmu3 domain of IgM to trigger a C1q-mediated
complement cascade.
[0105] Conversely, for avoiding an immune response, such as the
aforementioned one, or for avoiding a specific binding via the
constant region or portion thereof, the antibody of the invention
may not comprise a constant region (be devoid of a constant
region), or a portion thereof, of the relevant isotype.
[0106] Additionally or alternatively, depending on the application
and purpose, the antibody or antibody fragment may be attached to
any of various functional moieties. An antibody or antibody
fragment, such as that of the invention, attached to a functional
moiety may be referred to in the art as an "immunoconjugate".
[0107] According to some embodiments of the invention, the
functional moiety is a detectable moiety or a toxin. An antibody or
antibody fragment attached to a toxin may be referred to in the art
as an "immunotoxin".
[0108] As is described and demonstrated in further detail
hereinbelow, a detectable moiety or a toxin may be particularly
advantageously employed in applications of the invention involving
use of the antibody of the invention to detect the complex or cells
expressing the complex of the MHC molecule and the cytomegalovirus
(CMV) pp65 peptide and/or to kill cells expressing or presenting
such a complex.
[0109] For applications involving using the antibody of the
invention to detect the antigen-presenting portion of the complex,
the detectable moiety attached to the antibody or antibody fragment
can be a reporter moiety enabling specific detection of the MHC-CMV
pp65 peptide complex bound by the antibody or antibody fragment of
the invention.
[0110] While various types of reporter moieties may be utilized to
detect the MHC-CMV pp65 peptide complex, depending on the
application and purpose, the reporter moiety can be a fluorophore
or an enzyme. Alternately, the reporter moiety may be a
radioisotope, such as [125]iodine. Further examples of reporter
moieties, including those detectable by Positron Emission
Tomagraphy (PET) and Magnetic Resonance Imaging (MRI), are well
known to those of skill in the art.
[0111] A fluorophore may be advantageously employed as a detection
moiety enabling detection of the MHC-CMV pp65 peptide complex via
any of numerous fluorescence detection methods. Depending on the
application and purpose, such fluorescence detection methods
include, but are not limited to, fluorescence activated flow
cytometry (FACS), immunofluorescence confocal microscopy,
fluorescence in-situ hybridization (FISH), fluorescence resonance
energy transfer (FRET), and the like.
[0112] Various types of fluorophores, depending on the application
and purpose, may be employed to detect the MHC-CMV pp65 peptide
complex.
[0113] Examples of suitable fluorophores include, but are not
limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC),
Cy-chrome, rhodamine, green fluorescent protein (GFP), blue
fluorescent protein (BFP), Texas red, PE-Cy5, and the like.
[0114] Preferably, the fluorophore is phycoerythrin.
[0115] As is described and illustrated in the Examples section
below, the antibody of the invention attached to a fluorophore,
such as phycoerythrin, can be used to optimally detect the MHC-CMV
pp65 peptide complex using various immunofluorescence-based
detection methods.
[0116] Ample guidance regarding fluorophore selection, methods of
linking fluorophores to various types of molecules, such as an
antibody or antibody fragment of the invention, and methods of
using such conjugates to detect molecules which are capable of
being specifically bound by antibodies or antibody fragments
comprised in such immunoconjugates is available in the literature
of the art [for example, refer to: Richard P. Haugland, "Molecular
Probes: Handbook of Fluorescent Probes and Research Chemicals
1992-1994", 5th ed., Molecular Probes, Inc. (1994); U.S. Pat. No.
6,037,137 to Oncoimmunin Inc.; Hermanson, "Bioconjugate
Techniques", Academic Press New York, N.Y. (1995); Kay M. et al.,
1995. Biochemistry 34:293; Stubbs et al., 1996. Biochemistry
35:937; Gakamsky D. et al., "Evaluating Receptor Stoichiometry by
Fluorescence Resonance Energy Transfer," in "Receptors: A Practical
Approach," 2nd ed., Stanford C. and Horton R. (eds.), Oxford
University Press, UK. (2001); U.S. Pat. No. 6,350,466 to Targesome,
Inc.]. While various methodologies may be employed to detect the
MHC-CMV pp65 peptide complex using a fluorophore, such detection is
preferably effected as described and demonstrated in the Examples
section below.
[0117] Alternately, an enzyme may be advantageously utilized as the
detectable moiety to enable detection of the antigen-presenting
portion of the complex via any of various enzyme-based detection
methods. Examples of such methods include, but are not limited to,
enzyme linked immunosorbent assay (ELISA; for example, to detect
the antigen-presenting portion of the complex in a solution),
enzyme-linked chemiluminescence assay (for example, to detect the
complex in an electrophoretically separated protein mixture), and
enzyme-linked immunohistochemical assay (for example, to detect the
complex in a fixed tissue).
[0118] Numerous types of enzymes may be employed to detect the
antigen-presenting portion of the complex, depending on the
application and purpose. For example, an antibody or antibody
fragment attached to an enzyme such as horseradish peroxidase can
be used to effectively detect the MHC-CMV pp65 peptide complex,
such as via ELISA, or enzyme-linked immunohistochemical assay.
[0119] Examples of suitable enzymes include, but are not limited
to, horseradish peroxidase (HPR), beta-galactosidase, and alkaline
phosphatase (AP).
[0120] Ample guidance for practicing such enzyme-based detection
methods is provided in the literature of the art (for example,
refer to: Khatkhatay M I. and Desai M., 1999. J Immunoassay
20:151-83; Wisdom G B., 1994. Methods Mol. Biol. 32:433-40;
Ishikawa E. et al., 1983. J Immunoassay 4:209-327; Oellerich M.,
1980. J Clin Chem Clin Biochem. 18:197-208; Schuurs A H. and van
Weemen B K., 1980. J Immunoassay 1:229-49). While various
methodologies may be employed to detect the antigen-presenting
portion of the complex using an enzyme, such detection is
preferably effected as described in the Examples section below.
[0121] The functional moiety may be attached to the antibody or
antibody fragment in various ways, depending on the context,
application and purpose.
[0122] A polypeptidic functional moiety, in particular a
polypeptidic toxin, may be advantageously attached to the antibody
or antibody fragment via standard recombinant techniques broadly
practiced in the art (for Example, refer to Sambrook et al., infra,
and associated references, listed in the Examples section which
follows).
[0123] A functional moiety may also be attached to the antibody or
antibody fragment using standard chemical synthesis techniques
widely practiced in the art [for example, refer to the extensive
guidelines provided by The American Chemical Society (for example
at: hypertexttransferprotocol://worldwideweb (dot) chemistry (dot)
org/portal/Chemistry)]. One of ordinary skill in the art, such as a
chemist, will possess the required expertise for suitably
practicing such chemical synthesis techniques.
[0124] Alternatively, a functional moiety may be attached to the
antibody or antibody fragment by attaching an affinity tag-coupled
antibody or antibody fragment of the invention to the functional
moiety conjugated to a specific ligand of the affinity tag.
[0125] Various types of affinity tags may be employed to attach the
antibody or antibody fragment to the functional moiety.
[0126] Examples of detectable moieties that can be used in the
invention include but are not limited to radioactive isotopes,
phosphorescent chemicals, chemiluminescent chemicals, fluorescent
chemicals, enzymes, fluorescent polypeptides and epitope tags. The
detectable moiety can be a member of a binding pair, which is
identifiable via its interaction with an additional member of the
binding pair, and a label which is directly visualized. In one
example, the member of the binding pair is an antigen which is
identified by a corresponding labeled antibody. In one example, the
label is a fluorescent protein or an enzyme producing a
colorimetric reaction.
[0127] When the detectable moiety is a polypeptide, the immunolabel
(i.e. the antibody conjugated to the detectable moiety) may be
produced by recombinant means or may be chemically synthesized by,
for example, the stepwise addition of one or more amino acid
residues in defined order using solid phase peptide synthetic
techniques. Examples of polypeptide detectable moieties that can be
linked to the antibodies of the invention using recombinant DNA
technology include fluorescent polypeptides, phosphorescent
polypeptides, enzymes and epitope tags.
[0128] Expression vectors can be designed to fuse proteins encoded
by the heterologous nucleic acid insert to fluorescent
polypeptides. For example, antibodies can be expressed from an
expression vector fused with a green fluorescent protein (GFP)-like
polypeptide. A wide variety of vectors are commercially available
that fuse proteins encoded by heterologous nucleic acids to the
green fluorescent protein from Aequorea victoria ("GFP"), the
yellow fluorescent protein and the red fluorescent protein and
their variants (e.g., Evrogen). In these systems, the fluorescent
polypeptide is entirely encoded by its amino acid sequence and can
fluoresce without requirement for cofactor or substrate. Expression
vectors that can be employed to fuse proteins encoded by the
heterologous nucleic acid insert to epitope tags are commercially
available (e.g., BD Biosciences, Clontech).
[0129] Alternatively, chemical attachment of a detectable moiety to
the antibodies of the invention can be effected using any suitable
chemical linkage, direct or indirect, as via a peptide bond (when
the detectable moiety is a polypeptide), or via covalent bonding to
an intervening linker element, such as a linker peptide or other
chemical moiety, such as an organic polymer. Such chimeric peptides
may be linked via bonding at the carboxy (C) or amino (N) termini
of the peptides, or via bonding to internal chemical groups such as
straight, branched or cyclic side chains, internal carbon or
nitrogen atoms, and the like. Such modified peptides can be easily
identified and prepared by one of ordinary skill in the art, using
well known methods of peptide synthesis and/or covalent linkage of
peptides. Description of fluorescent labeling of antibodies is
provided in details in U.S. Pat. Nos. 3,940,475, 4,289,747, and
4,376,110.
[0130] Exemplary methods for conjugating two peptide moieties are
described herein below:
[0131] SPDP Conjugation:
[0132] Any SPDP conjugation method known to those skilled in the
art can be used. For example, in one illustrative embodiment, a
modification of the method of Cumber et al. (1985, Methods of
Enzymology 112: 207-224) as described below, is used.
[0133] A peptide, such as an identifiable or therapeutic moiety,
(1.7 mg/ml) is mixed with a 10-fold excess of SPDP (50 mM in
ethanol) and the antibody is mixed with a 25-fold excess of SPDP in
20 mM sodium phosphate, 0.10 M NaCl pH 7.2 and each of the
reactions incubated, e.g., for 3 hours at room temperature. The
reactions are then dialyzed against PBS.
[0134] The peptide is reduced, e.g., with 50 mM DTT for 1 hour at
room temperature. The reduced peptide is desalted by equilibration
on G-25 column (up to 5% sample/column volume) with 50 mM
KH.sub.2PO.sub.4 pH 6.5. The reduced peptide is combined with the
SPDP-antibody in a molar ratio of 1:10 antibody:peptide and
incubated at 4.degree. C. overnight to form a peptide-antibody
conjugate.
[0135] Glutaraldehyde Conjugation:
[0136] Conjugation of a peptide (e.g., an identifiable or
therapeutic moiety) with an antibody can be accomplished by methods
known to those skilled in the art using glutaraldehyde. For
example, in one illustrative embodiment, the method of conjugation
by G. T. Hermanson (1996, "Antibody Modification and Conjugation,
in Bioconjugate Techniques, Academic Press, San Diego) described
below, is used.
[0137] The antibody and the peptide (1.1 mg/ml) are mixed at a
10-fold excess with 0.05% glutaraldehyde in 0.1 M phosphate, 0.15 M
NaCl pH 6.8, and allowed to react for 2 hours at room temperature.
0.01 M lysine can be added to block excess sites. After-the
reaction, the excess glutaraldehyde is removed using a G-25 column
equilibrated with PBS (10% v/v sample/column volumes)
[0138] Carbodiimide Conjugation:
[0139] Conjugation of a peptide with an antibody can be
accomplished by methods known to those skilled in the art using a
dehydrating agent such as a carbodiimide. Most preferably the
carbodiimide is used in the presence of 4-dimethyl aminopyridine.
As is well known to those skilled in the art, carbodiimide
conjugation can be used to form a covalent bond between a carboxyl
group of peptide and an hydroxyl group of an antibody (resulting in
the formation of an ester bond), or an amino group of an antibody
(resulting in the formation of an amide bond) or a sulfhydryl group
of an antibody (resulting in the formation of a thioester
bond).
[0140] Likewise, carbodiimide coupling can be used to form
analogous covalent bonds between a carbon group of an antibody and
an hydroxyl, amino or sulfhydryl group of the peptide. See,
generally, J. March, Advanced Organic Chemistry: Reaction's,
Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985.
By means of illustration, and not limitation, the peptide is
conjugated to an antibody via a covalent bond using a carbodiimide,
such as dicyclohexylcarbodiimide. See generally, the methods of
conjugation by B. Neises et al. (1978, Angew Chem., Int. Ed. Engl.
17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); E. P.
Boden et al. (1986, J. Org. Chem. 50:2394) and L. J. Mathias (1979,
Synthesis 561). The level of immunocomplex may be compared to a
control sample from a non-diseased subject, wherein an
up-regulation of immunocomplex formation is indicative of disease
associated with CMV infection. Preferably, the subject is of the
same species e.g. human, preferably matched with the same age,
weight, sex etc. It will be appreciated that the control sample may
also be of the same subject from a healthy tissue, prior to disease
progression or following disease remission.
[0141] Preferably, the affinity tag is a biotin molecule, more
preferably a streptavidin molecule.
[0142] A biotin or streptavidin affinity tag can be used to
optimally enable attachment of a streptavidin-conjugated or a
biotin-conjugated functional moiety, respectively, to the antibody
or antibody fragment due to the capability of streptavidin and
biotin to bind to each other with the highest non covalent binding
affinity (i.e., with a Kd of about 10.sup.-14 to 10.sup.-15). A
biotin affinity tag may be highly advantageous for applications
benefiting from. Thus, the antibody of invention can be a
multimeric form of the antibody or antibody fragment, which may be
optimally formed by conjugating multiple biotin-attached antibodies
or antibody fragments of the invention to a streptavidin molecule,
as described in further detail below.
[0143] As used herein the term "about" refers to plus or minus 10
percent.
[0144] Various methods, widely practiced in the art, may be
employed to attach a streptavidin or biotin molecule to a molecule
such as the antibody or antibody fragment to a functional
moiety.
[0145] For example, a biotin molecule may be advantageously
attached to an antibody or antibody fragment of the invention
attached to a recognition sequence of a biotin protein ligase. Such
a recognition sequence is a specific polypeptide sequence serving
as a specific biotinylation substrate for the biotin protein ligase
enzyme. Ample guidance for biotinylating a target polypeptide such
as an antibody fragment using a recognition sequence of a biotin
protein ligase, such as the recognition sequence of the biotin
protein ligase BirA, is provided in the literature of the art (for
example, refer to: Denkberg, G. et al., 2000. Eur. J. Immunol.
30:3522-3532). Preferably, such biotinylation of the antibody or
antibody fragment is effected as described and illustrated in the
Examples section below.
[0146] Alternately, various widely practiced methods may be
employed to attach a streptavidin molecule to an antibody fragment,
such as a single chain Fv (for example refer to Cloutier S M. et
al., 2000. Molecular Immunology 37:1067-1077; Dubel S. et al.,
1995. J Immunol Methods 178:201; Huston J S. et al., 1991. Methods
in Enzymology 203:46; Kipriyanov S M. et al., 1995. Hum Antibodies
Hybridomas 6:93; Kipriyanov S M. et al., 1996. Protein Engineering
9:203; Pearce L A. et al., 1997. Biochem Molec Biol Intl
42:1179-1188).
[0147] Functional moieties, such as fluorophores, conjugated to
streptavidin are commercially available from essentially all major
suppliers of immunofluorescence flow cytometry reagents (for
example, Pharmingen or Becton-Dickinson). Standard recombinant DNA
chemical techniques are preferably employed to produce a fusion
protein comprising streptavidin fused to a polypeptidic functional
moiety. Standard chemical synthesis techniques may also be employed
to form the streptavidin-functional moiety conjugate. Extensive
literature is available providing guidance for the expression,
purification and uses of streptavidin or streptavidin-derived
molecules (Wu S C. et al., 2002. Protein Expression and
Purification 24:348-356; Gallizia A. et al., 1998. Protein
Expression and Purification 14:192-196), fusion proteins comprising
streptavidin or streptavidin-derived molecules (Sano T. and Cantor
C R., 2000. Methods Enzymol. 326:305-11), and modified streptavidin
or streptavidin-derived molecules (see, for example: Sano T. et
al., 1993. Journal of Biological Chemistry 270:28204-28209),
including for streptavidin or streptavidin-derived molecules whose
gene sequence has been optimized for expression in E. coli
(Thompson L D. and Weber P C., 1993. Gene 136:243-6).
[0148] As mentioned, the antibody may be conjugated to a
therapeutic moiety. The therapeutic moiety can be, for example, a
cytotoxic moiety, a toxic moiety, a cytokine moiety and a second
antibody moiety comprising a different specificity to the
antibodies of the invention.
[0149] In a similar fashion to an immunolabel, an immunotoxin (i.e.
a therapeutic moiety attached to an antibody of the invention) may
be generated by recombinant or non-recombinant means. Thus, the
invention envisages a first and second polynucleotide encoding the
antibody of the invention and the therapeutic moiety, respectively,
ligated in frame, so as to encode an immunotoxin. The following
Table 1 provides examples of sequences of therapeutic moieties.
TABLE-US-00001 TABLE 1 Amino Acid sequence Nucleic Acid sequence
(Genbank (Genbank Therapeutic Moiety Accession No.) Accession No.)
Pseudomonas exotoxin AAB25018 S53109 Diphtheria toxin E00489 E00489
interleukin 2 CAA00227 A02159 CD3 P07766 X03884 CD16 AAK54251
AF372455 interleukin 4 P20096 ICRT4 HLA-A2 P01892 K02883
interleukin 10 P22301 M57627 Ricin A toxin 225988 A23903
[0150] According to some embodiments of the invention, the toxic
moiety is PE38 KDEL.
[0151] Exemplary methods of conjugating the antibodies of the
invention to peptide therapeutic agents are described herein
above.
[0152] As mentioned, the antibody of the invention, which is
capable of specifically recognizing and binding an MHC-CMV pp65
peptide complex as described above, can be used to detecting cell
expressing a cytomegalovirus (CMV) antigen.
[0153] Thus, according to an aspect of some embodiments of the
invention there is provided a method of detecting a cell expressing
a cytomegalovirus (CMV) antigen. The method is effected by
contacting the cell with the antibody under conditions which allow
immunocomplex formation, wherein a presence or a level above a
predetermined threshold of the immunocomplex is indicative of CMV
expression in the cell.
[0154] The contacting may be effected in vitro (e.g., in a cell
line), ex vivo or in vivo.
[0155] As mentioned, the method of the invention is effected under
conditions sufficient to form an immunocomplex (e.g. a complex
between the antibodies of the invention and the MHC-CMV pp65
peptide); such conditions (e.g., appropriate concentrations,
buffers, temperatures, reaction times) as well as methods to
optimize such conditions are known to those skilled in the art, and
examples are disclosed herein.
[0156] As described in the Examples section which follows, the
immunocomplex can be formed and detected within the cell or on the
cell surface. For detection in the cell, the conditions include a
permeabilization agent (e.g., a solution including saponin), to
enable penetration of the antibody inside the cell. According to
some embodiments of the invention, the immunocomplex is formed on
the surface of the cell.
[0157] Determining a presence or level of the immunocomplex of the
invention is dependent on the detectable moiety to which the
antibody is attached, essentially as described hereinabove.
[0158] A non-limiting example of the immunocomplex of the invention
is the complex formed between the antibody of the invention (e.g.,
H9 or F5) and a protein complex comprising MHC class I heavy chain
(HLA-A2) and pp65 peptide as set forth by SEQ ID NO:3.
[0159] As mentioned, the antibody of the invention, which is
capable of specifically recognizing and binding an MHC-CMV pp65
peptide complex, can be used to diagnose CMV infection in a subject
in need thereof.
[0160] Thus, according to another aspect of the invention, there is
provided a method of diagnosing a cytomegalovirus (CMV) infection
in a subject in need thereof. The method is effected by contacting
a cell of the subject with the antibody under conditions which
allow immunocomplex formation, wherein a presence or a level above
a pre-determined threshold of the immunocomplex in the cell is
indicative of the CMV infection in the subject.
[0161] As used herein the phrase "subject in need thereof" refers
to a mammal, preferably, a human subject which is suspected of
being infected with CMV.
[0162] According to some embodiments of the invention, the subject
has a suppressed or a compromised immune system, such as an
immuno-compromised organ transplant recipient or a subject infected
with human immunodeficiency virus (HIV).
[0163] According to some embodiments of the invention, the CMV
infection is associated with a disease selected from the group
consisting of mononucleosis, retinitis, pneumonia, gastrointestinal
disorders, and encephalitis.
[0164] According to some embodiments of the invention, the cell is
a retina cell, lung epithelial cell, a gastrointestinal epithelial
cell and/or a brain cell.
[0165] The antibody described herein can be used to treat a disease
associated with CMV infection.
[0166] According to an additional aspect of the invention there is
provided a method of treating a disease associated with
cytomegalovirus (CMV) infection, the method is effected by
administering to a subject in need thereof a therapeutically
effective amount of the antibody thereby treating the disease
associated with CMV infection.
[0167] The term "treating" refers to inhibiting or arresting the
development of a disease, disorder or condition and/or causing the
reduction, remission, or regression of a disease, disorder or
condition. Those of skill in the art will understand that various
methodologies and assays can be used to assess the development of a
disease, disorder or condition, and similarly, various
methodologies and assays may be used to assess the reduction,
remission or regression of a disease, disorder or condition.
[0168] The antibodies of the invention may be provided per se or
may be administered as a pharmaceutical composition.
[0169] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0170] Herein the term "active ingredient" refers to the antibodies
of the invention accountable for the biological effect.
[0171] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0172] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0173] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0174] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0175] Alternately, one may administer the pharmaceutical
composition in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
tissue region of a patient.
[0176] Pharmaceutical compositions of the invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0177] Pharmaceutical compositions for use in accordance with the
invention thus may be formulated in conventional manner using one
or more physiologically acceptable carriers comprising excipients
and auxiliaries, which facilitate processing of the active
ingredients into preparations which, can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen.
[0178] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0179] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0180] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0181] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0182] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0183] For administration by nasal inhalation, the active
ingredients for use according to the invention are conveniently
delivered in the form of an aerosol spray presentation from a
pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0184] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0185] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0186] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0187] The pharmaceutical composition of the invention may also be
formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0188] Pharmaceutical compositions suitable for use in context of
the invention include compositions wherein the active ingredients
are contained in an amount effective to achieve the intended
purpose. More specifically, a therapeutically effective amount
means an amount of active ingredients (the antibody of the
invention or the nucleic acid construct encoding same) effective to
prevent, alleviate or ameliorate symptoms of a pathology, (e.g., a
disease associated with cytomegalovirus infection) or prolong the
survival of the subject being treated.
[0189] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0190] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0191] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0192] Dosage amount and interval may be adjusted individually to
provide plasma or brain levels of the active ingredient are
sufficient to induce or suppress the biological effect (minimal
effective concentration, MEC). The MEC will vary for each
preparation, but can be estimated from in vitro data. Dosages
necessary to achieve the MEC will depend on individual
characteristics and route of administration. Detection assays can
be used to determine plasma concentrations.
[0193] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0194] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0195] Compositions of the invention may, if desired, be presented
in a pack or dispenser device, such as an FDA approved kit, which
may contain one or more unit dosage forms containing the active
ingredient. The pack may, for example, comprise metal or plastic
foil, such as a blister pack. The pack or dispenser device may be
accompanied by instructions for administration and use. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition, as if further detailed
above.
[0196] As used herein the term "about" refers to .+-.10%.
[0197] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0198] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0199] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0200] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
GENERAL MATERIALS AND EXPERIMENTAL METHODS
Generation of Biotinylated Single-Chain MHC/Peptide Complexes
[0201] Single-chain MHC (scMHC)/peptide complexes were produced by
in vitro refolding of inclusion bodies produced in Escherichia
coli, as described (Denkberg, G. et al., 2000). Briefly, a
single-chain .beta..sub.2-microglobulin (.beta..sub.2m)-HLA/A2
(scMHC) construct, in which the .beta..sub.2m and HLA-A2 genes are
connected to each other by a flexible peptide linker (wherein the
.beta.2m gene is translationally fused upstream of the gene
encoding the MHC heavy chain) (HLA-A2), was engineered to contain
the BirA recognition sequence for site-specific biotinylation at
the C terminus (scMHC-BirA). In vitro refolding was performed in
the presence of a 5-10 molar excess of the antigenic peptides, as
described (Denkberg, G. et al., 2000). Correctly folded MHC/peptide
complexes were isolated and purified by anion exchange Q-Sepharose
chromatography (Pharmacia, Peapack, N.J.), followed by
site-specific biotinylation using the BirA enzyme (Avidity, Denver,
Colo.), as previously described (Altman, J. D. et al., 1996). The
homogeneity and purity of the scMHC-peptide complexes were analyzed
by various biochemical means, including SDS-PAGE, size exclusion
chromatography, and ELISA, as previously described (Denkberg, G. et
al., 2000).
[0202] Selection of Phage Antibodies on Biotinylated Complexes
[0203] Selection of phage Abs on biotinylated complexes was
preformed, as described (Denkberg, G., et al., 2002; Lev A., et
al., 2002). Briefly, a large human Fab library containing
3.7.times.10.sup.10 different Fab clones (De Haard, H J., et al.,
1999) was used for the selection. Phages (10.sup.13) were first
preincubated with streptavidin-coated paramagnetic beads (200
.mu.l; Dynal, Oslo, Norway) to deplete the streptavidin binders.
The remaining phages were subsequently used for panning with
decreasing amounts of biotinylated scMHC-peptide complexes. The
streptavidin-depleted library was incubated in solution with
soluble biotinylated scHLA-A2/pp65 complexes (500 nM for the first
round, and 100 nM for the following rounds) for 30 minutes at room
temperature (RT).
[0204] Streptavidin-coated magnetic beads (200 .mu.l for the first
round of selection, and 100 .mu.l for the second and third rounds)
were added to the mixture and incubated for 10-15 minutes at RT.
The beads were washed extensively 12 times with PBS/Tween 0.1%, and
additional two washes were with PBS. Bound phages were eluted with
triethylamine (100 mM, 5 minutes at RT), followed by neutralization
with Tris-HCl (1 M, pH 7.4), and used to infect E. coli TG1 cells
(OD=0.5) for 30 minutes at 37.degree. C.
[0205] The diversity of the selected Abs was determined by DNA
fingerprinting using a restriction endonuclease (BstNI), which is a
frequent cutter of Ab V gene sequences. The Fab DNA of different
clones was PCR amplified using the primers pUC-reverse
[5'-AGCGGATAACAATTTCACACAGG-3' (SEQ ID NO:1)] and fd-tet-seq24
[5'-TTTGTCGTCTTTCCAGACGTTAGT-3' (SEQ ID NO:2)], followed by
digestion with BstNI (NEB, Beverly, Mass.) (2 hours, 60.degree. C.)
and analysis on agarose gel electrophoresis.
[0206] Expression and Purification of Soluble Recombinant Fab
Abs
[0207] Fab Abs were expressed and purified, as described recently
(Denkberg, G., et al., 2000). BL21 bacterial cells were grown to
OD.sub.600=0.8-1.0 and induced to express the recombinant Fab Ab by
the addition of 1 mM isopropyl 13-D-thiogalactoside (IPTG) for 3-4
hours at 30.degree. C. Periplasmic content was released using the
B-PER solution (Pierce, Rockford, Ill.), which was applied onto a
prewashed TALON column (Clontech, Palo Alto, Calif.). Bound Fabs
were eluted using 0.5 ml of 100 mM imidazole in PBS. The eluted
Fabs were dialyzed twice against PBS (overnight, 4.degree. C.) to
remove residual imidazole.
[0208] ELISA with Phage Clones and Purified Fab Abs
[0209] The binding specificities of individual phage clones and
soluble Fab were determined by ELISA using biotinylated
scMHC-peptide complexes. ELISA plates (Falcon) were coated
overnight with BSA-biotin (1 .mu.g/well). After having been washed,
the plates were incubated (1 hour, RT) with streptavidin (1
.mu.g/well), washed extensively, and further incubated (1 hour, RT)
with 0.5 .mu.g of MHC/peptide complexes. The plates were blocked
for 30 minutes at RT with PBS/2% skim milk and subsequently were
incubated for 1 hour at RT with phage clones (.about.10.sup.9
phages/well) or various concentrations of soluble purified Fab.
After having been washed, the plates were incubated with
HRP-conjugated/anti-human Fab Ab (for soluble Fabs) or
HRP-conjugated anti-M13 phage (for phage-displayed Fabs). Detection
was performed using tetramethylbenzidine reagent (Sigma-Aldrich,
St. Louis, Mo.). The HLA-A2-restricted peptides used for
specificity studies of the Fab phage clones or purified Fab Abs are
as described in Examples 1 and 2 below.
[0210] Generation of Fluorescently-Labeled Tetrameric Fab
[0211] The genes encoding the L and H chain of Fab H9 were cloned
separately into a T7-promotor pET-based expression vector. The L
chain gene was engineered to contain the BirA recognition sequence
for site-specific biotinylation at the C terminus. These constructs
were expressed separately in E. coli BL21 cells and upon induction
with IPTG, intracellular inclusion bodies that contain large
amounts of the recombinant protein accumulated. Inclusion bodies of
both chains were purified, solubilized, reduced with 10 mg/ml DTE
(Dithioerithrol), and subsequently refolded at a 1:1 ratio in a
redox-shuffling buffer system containing 0.1 M Tris, 0.5 M
arginine, and 0.09 mM oxidized glutathione, pH 8.0. Correctly
folded Fab was then isolated and purified by anion exchange MonoQ
chromatography (Pharmacia). The Fab peak fractions were
concentrated using Centricon-30 (Amicon, Beverly, Mass.) to 1
mg/ml, and the buffer was exchanged to Tris-HCl (10 mM, pH 8.0).
Biotinylation was performed using the BirA enzyme (Avidity), as
previously described. Excess biotin was removed from biotinylated
Fabs using a G-25 desalting column. PE-labeled streptavidin
(Jackson ImmunoResearch, West Grove, Pa.) was added at a molar
ratio of 1:4 to produce fluorescent tetramers of the biotinylated
Fab.
[0212] Generation of Whole IgG from Recombinant Fab
[0213] To transform the recombinant fragments into whole IgG
molecules, the eukaryotic expression vector pCMV/myc/ER
(Invitrogen) was used. The heavy and the light chains of the Fab
were cloned separately. Each shuttle expression vector carries a
different antibiotics resistance gene and thus expression was
facilitated by co-transfection of the two constructs into human
embryonic kidney HEK293 cells. Cotransfections of HEK293 cells were
performed using the nonliposomal transfection reagent FuGene 6
(Roche, Brussels, Belgium) according to the manufacturer's
instructions. The transfection was performed with serum free medium
containing 0.8 mg/ml of G418, and 100 .mu.g/ml of hygromycin.
Forty-eight hours after transfection limiting dilutions were
performed into medium containing 0.8 mg/ml of G418, and 100
.mu.g/ml of hygromycin. Cells were plated in 96-well plates at 1000
cells per well. Medium was exchanged after 5 and 10 days. Wells in
which a single colony grew up to 50% of the well were further
trypsinized with 20 .mu.l and 20 .mu.l medium and splitted into two
wells: 10 .mu.l into a 24 well plate (backup) and 30 .mu.l into a
24 well plate (experiment). When the plate reached 80% confluency,
serum starvation was initiated by reducing each day serum
percentile to 0.5%. After 48 hours of incubation with 0.5% fetal
calf serum (FCS), screening of cell culture supernatants was
performed by ELISA and FACS assays. The IgG secreting clones that
exhibited the best binding reactivity as detected by ELISA, FACS
and the highest amount of protein, were selected for antibody
production and purification.
[0214] Protein A-SEPHAROSE.TM. 4 Fast Flow beads (Amersham) were
prepared according to the manufacturer's instructions. Briefly,
supernatant was loaded on the Protein A-Sepharose beads at 15-50
ml/h. Unbound immunoglobulins were washed with 0.001 M
NaH.sub.2PO.sub.4 and 0.019 M Na.sub.2HPO.sub.4. Bound
immunoglobulins were then eluted with 0.1 M citric acid at pH 3.
Five fractions were collected with 250 .mu.l of elusion buffer and
immediately neutralized with 80 .mu.l of Tris-HCL pH 9. IgG
concentration was measured using the Pierce protein assay. The
eluted protein was dialyzed against PBS pH 7.4 over night. 10 mgs
of IgG were produced from 1 L of culture supernatant.
[0215] Flow Cytometry
[0216] The B cell line RMAS-HHD, which is transfected with a
single-chain .beta..sub.2m-HLA-A2 gene, the EBV-transformed
HLA-A2.sup.+ JY cells, and the HLA-A2- B cell line APD-70 were used
to determine the reactivity of the recombinant Fab Abs with cell
surface-expressed HLA-A2/peptide complexes. Peptide pulsing was
performed as indicated: 10.sup.6 cells were washed twice with
serum-free RPMI and incubated overnight at 26.degree. C. or
37.degree. C., respectively, in medium containing 1-50 .mu.M of the
peptide. The RMAS-HHD cells were subsequently incubated at
37.degree. C. for 2-3 hours to stabilize cell surface expression of
MHC-peptide complexes.
[0217] Cells were incubated for 60 minutes at 4.degree. C. with
recombinant Fab Abs (10 .mu.g/ml) in 100 .mu.l PBS. After one wash,
the cells were incubated with 1 .mu.g anti-human Fab (Jackson
ImmunoResearch) for another 60 minutes at 4.degree. C. After three
washes, the cells were resuspended in ice-cold PBS. The cells were
analyzed by a FACStar flow cytometer (BD Biosciences, San Jose,
Calif.).
[0218] Surface Plasmon Resonance
[0219] 0.0025 mg/ml of biotinylated HLA-A2/pp65 or control
HLA-A2/EBV complexes were bound to a streptavidin (SA) sensor chip
(Biacore, Uppsala, Sweden) per well. Measurements of 780-800 RU
were detected for each well after complexes binding. Soluble
isolated antibodies in their monomeric/IgG form were diluted in PBS
at three concentration (0.05 .mu.M, 0.1 .mu.M, 0.2 .mu.M) and were
flowed over the relevant wells at a rate of 10 .mu.l/min at room
temperature. Responses were recorded using Biacore 2000 and
analyzed using BlAevaluation software 3.2 (Biacore, Uppsala,
Sweden).
[0220] Cell Infection
[0221] Human fibroblasts which express the HLA-A2 allele were
obtained from primary cultures of foreskins and grown in Dulbecco's
modified Eagle's medium (DMEM), containing 2 mM Glutamine, 100 IU
of penicillin/ml, 10% fetal calf serum (FCS), non essential amino
acid (1:100), sodium Pyruvate (1:100) and 10 mM hepes. The cells
were infected at an MOI of 0.5-1 with the laboratory strain
AD169.sup.41 and harvested at five time scales for FACS analysis.
MHC expression on virus infected or uninfected cells was determined
using PE conjugated anti HLA-A2 (BB7.2) monoclonal antibody.
Detection of infection was with anti pp65 monoclonal antibody
(clone IL11, Virusys, Sykesville, Md. USA) and anti mouse PE as
secondary antibody. For intracellular staining cells were fixed
with 0.3% formaldehyde and then permeabilized with PBS containing
0.05% Saponin and 1% goat serum used for blocking.
[0222] Cytotoxicity Assay
[0223] Target cells were cultured in 48-well plates in DMEM medium
plus 10% FCS and were grown up until confluent. Cells were washed
and incubated overnight with 15 .mu.Ci/ml (1 Ci 37 GBq) [.sup.35S]
methionine (NEN). After 1 hour of incubation with the IgG H9 (10-20
.mu.g/ml or the indicated concentration at 37.degree. C.), effector
CTL cells were added at a target:effector ratio of 1:3 respectively
and incubated for 5 hours at 37.degree. C. After incubation,
[.sup.35S] methionine release from target cells was measured in a
50-.mu.l sample of the culture supernatant. All assays were
performed in triplicate.
[0224] Confocal Microscopy
[0225] Infected and noninfected fibroblast cells were fixed for 10
minutes with 0.5% paraformaldehyde, and washed twice with PBS
containing 0.1% bovine serum albumin (BSA). The cells were
permeabilized and incubated with anti pp65 mAb, H9 IgG, anti
calnexin (Chemicon, cat. No. MAB3126), and/or cis-golgi matrix
protein (GM130) (BD transduction laboratories, cat No. 610822) in
the presence of a PBS medium containing 0.05% saponin, 1% fetal
bovine serum, and 0.1% BSA, for 40 minutes at 4.degree. C. Cells
were subsequently washed and further incubated with goat anti mouse
secondary Ab conjugated to Alexa-flour.sup.594 (Molecular Probes,
cat. No. A21216), and goat anti human secondary Ab conjugated to
Alexa-flour.sup.488 (Molecular Probes, cat. No. A11013),
respectively. DRAQ5 (Alexis Biochemicals) was added to the stained
cells before they were washed again. Images were collected on a LSM
510 META laser scanning microscope (Carl Zeiss Microimaging Inc)
using a .times.63 oil immersion objective numerical aperture 1.32,
at different zoom factors. Alexa Fluor.sup.488 was excited using an
argon laser at 488 nm. Alexa Fluor.sup.594 was excited using a
krypton laser at 568 mm Differential interference contrast images
were collected simultaneous with the fluorescence images using the
transmitted light detector. Z stacks of images were collected using
a step increment of 0.3 .mu.m between planes. All pictures were
taken with identical settings.
[0226] Isolation of PBMCs
[0227] Samples of 20-30 ml blood obtained from healthy donors or
BMT patients, containing 500 units (U) of heparin was added to 50
ml sterile tubes containing 15 ml LYMPHOPREP.TM. (Axis shield PoC
AS, Oslo Norway). The blood was added gently without mixing between
the Ficoll and the blood. The tubes were centrifuged for 30 minutes
at 1000 g without brakes. The upper layer that contains the serum
was removed and the Buffy coat that contains the peripheral blood
mononuclear cells (PBMCs) was transferred to new tubes. The PBMCs
were washed twice with 40 ml of phosphate buffer saline (PBS) and 2
mM EDTA (centrifuged at 700 g for 8 minutes). The PBMCs were
resuspended in 20 ml PBS, counted, centrifuged at 500 g for 8
minutes and resuspended in PBMCs medium at 1-5.times.10.sup.6
cells/ml. About 70.times.10.sup.6 cells are isolated from a total
of 50 ml blood sample.
Example I
Selection and Cloning of Recombinant Antibodies Specific for
HLA-A2-PP65 Complex
Experimental Results
[0228] Selection of Recombinant Antibodies Specific for HLA-A2/pp65
Complexes
[0229] Recombinant peptide-HLA-A2 complexes that present the
pp65.sub.495-503 (SEQ ID NO:3) CMV-derived peptide were generated
using a single-chain MHC (scMHC) construct according to the method
previously described previously (Denkberg G., et al., 2000). In
this construct, the extracellular domains of HLA-A2 are connected
into a single-chain molecule with .beta..sub.2m using a 15-aa
flexible linker (the .beta.2m is translationally fused upstream of
the MHC heavy chain). The scMHC-peptide complexes were produced by
in vitro refolding of inclusion bodies in the presence of the pp65
495-503 peptide (SEQ ID NO:3). The refolded scHLA-A2/pp65 complexes
were found to be pure, homogenous, and monomeric by SDS-PAGE and
size exclusion chromatography analyses (data not shown).
Recombinant scMHC-peptide complexes generated by this strategy were
previously characterized in detail for their biochemical,
biophysical, and biological properties, and were found to be
correctly folded and functional (Denkberg G., et al., 2000;
Denkberg G., et al., 2001).
[0230] A large human Fab library containing 3.7.times.10.sup.10
different Fab clones was used for the selection on biotinylatd
HLA-A2/pp65 complexes (De Haard H J., et al., 1999). Phage
displayed antibodies which were capable of binding to the specific
biotinylated HLA-A2/peptide complex were selected as previously
described (Denkberg G., et al., 2002; Lev A., et al., 2002).
Enrichment in phage titer was observed after three rounds of
panning (Table 2, hereinbelow). Specificity of the selected phage
antibodies against the complex was analyzed by a differential ELISA
assay in which binding was tested against specific (pp65 495-503
peptide; SEQ ID NO:3) and non specific (gp100 280-288 peptide; SEQ
ID NO:4) biotinylated HLA-A2/peptide complexes. These were
immobilized to wells through BSA-biotin-streptavidin. As shown in
FIG. 1a, a high percentage of specific clones was observed; 54
clones of the 96 screened (56%), were peptide specific and bound
the specific peptide/MHC used in the selection (i.e., the
scHLA-A2/pp65 complex).
TABLE-US-00002 TABLE 2 Table 2: Results of the amounts of phages
counted before and after each panning (inputs and outputs).
Enrichment of the outputs can be seen in each panning round. Round
of Panning Phage input Phage output Enrichment 1.sup.st 10.sup.12 4
.times. 10.sup.5 2.sup.nd 1.5 .times. 10.sup.12 5 .times. 10.sup.6
75 3.sup.rd 5 .times. 10.sup.12 1.5 .times. 10.sup.9 750
[0231] Cloning of Two Fab Clones with Specificity to the
HLA-A2-pp65.sub.495-503 Complex
[0232] The diversity within the selected TCR-like Fabs was assessed
by DNA fingerprint analysis using the BstNI restriction enzyme. The
analysis revealed two different clones, termed H9 and F5 with
HLA-A2/pp65 specificity (data not shown). DNA sequencing analysis
confirmed these observations. The nucleic acid and amino acid
sequences of the heavy and light chains of H9 Fab clone are
provided in FIGS. 14a-d (SEQ ID NOs:16-19). The nucleic acid and
amino acid sequences of the heavy and light chains of F5 Fab clone
are provided in FIGS. 15a-d (SEQ ID NOs:20-23). The amino acid
sequences of the CDRs of the H9 and F5 Fab Abs are provided in
Table 3, hereinbelow. The nucleic acid sequences of the CDRs of the
H9 and F5 Fab Abs are provided in Table 4, hereinbelow.
TABLE-US-00003 TABLE 3 Amino acid sequences of the CDRs of the Fab
antibodies Fab clone CDRs heavy chain CDRs light chain H9 SYAISW
RASQSVSSSYLA (SEQ ID NO: 24; CDR1) (SEQ ID NO: 30; CDR1)
GIIPIFGTANYAQKFQG GASSRAT (SEQ ID NO: 25; CDR2) (SEQ ID NO: 31;
CDR2) GDLYYYDSSGYPRYYFDY QHYSTSPGFT (SEQ ID NO: 26; CDR3) (SEQ ID
NO: 32; CDR3) F5 SSNYYWG TRSTGSITSNYVH (SEQ ID NO: 36; CDR1) (SEQ
ID NO: 42; CDR1) AIYYSGSTYYNPSLKS EDNERPS (SEQ ID NO: 37; CDR2)
(SEQ ID NO: 43; CDR2) RIGVAGQWYFDLWGRGTLVTVSS QSYDDSNHISV (SEQ ID
NO: 38; CDR3) (SEQ ID NO: 44; CDR3) Table 3: CDRs (amino acid
sequences) of the heavy and light chains of Fabs H9 and F5.
TABLE-US-00004 TABLE 4 Nucleic acid sequences of the CDRs of the
Fab antibodies Fab clone CDRs heavy chain CDRs light chain H9
GCTATGCTATCAGCTG AGGGCCAGTCAGAGTGTTAGCAGCA (SEQ ID NO: 27; CDR1)
GCTACTTAGC GGGATCATCCCTATCTTTGGTACAGCAAAC (SEQ ID NO: 33; CDR1)
TACGCACAGAAGTTCCAGGG GGTGCATCCAGCAGGGCCACT (SEQ ID NO: 28; CDR2)
(SEQ ID NO: 34; CDR2) GGGGATCTGTATTACTATGATAGTAGTG
AGCACTATAGCACCTCACCTGG GTTATCCGCGATACTACTTTGACTA GTTCACT (SEQ ID
NO: 29; CDR3) (SEQ ID NO:35; CDR3) F5 AGCAGTAATTACTACTGGGGC
ACCCGCAGCACTGGCAGCATTAC (SEQ ID NO: 39; CDR1) CAGCAACTATGTGCAC
GCTATCTATTATAGTGGGAGCACCTACTAC (SEQ ID NO: 45; CDR1)
AACCCGTCCCTCAAGAGT GAGGATAACGAAAGACCCTCT (SEQ ID NO: 40; CDR2) (SEQ
ID NO: 46; CDR2) CGTATAGGAGTGGCTGGCCAATGGTATTTC
CAGTCTTATGATGACAGCAATC GATCTCTGGGGCCGTGGCACCCTGGTCAC ATATTTCTGTC
CGTCTCAAGC (SEQ ID NO: 47; CDR3) (SEQ ID NO: 41; CDR3) Table 4:
CDRs (nucleic acid sequences) of the heavy and light chains of Fabs
H9 and F5.
[0233] Production of the Recombinant, Soluble Fab Clones
[0234] The isolated Fab clones with specificity toward the
HLA-A2/pp65 complex (H9, F5) were produced in a soluble form in E.
coli BL21 cells. These Fabs which are tagged at the CH1 domain with
a hexahistidine sequence, were purified from the periplasmic
fraction by metal affinity chromatography. SDS-PAGE analysis
revealed the level of purification and the expected molecular size
of the Fab antibodies (FIG. 1b).
[0235] These data demonstrate the isolation of recombinant
antibodies with peptide-specific, MHC restricted binding to the
CMV-derived T cell epitope pp65.sub.495-503 (SEQ ID NO:3).
Example 2
Characterization of HLA-A21PP65-Specific TCR-Like Recombinant
Antibodies
Experimental Results
[0236] HLA-A2/pp65-Specific TCR-Like Recombinant Antibodies Exhibit
Binding Characteristics and Fine Specificity of a TCR-Like
Molecule
[0237] The specificity of the two recombinant monoclonal Fab
antibodies to the MHC-CMV peptide complex was tested by ELISA
(FIGS. 1c and d). To determine the correct folding of the bound
complexes and their stability during the binding assays, the
ability of the complexes to react with the conformation-specific
mAb, w6/32, that recognizes HLA complexes only when folded
correctly and when containing peptide was monitored. As shown in
FIGS. 1c and d, the soluble Fab Abs reacted only with the specific
HLA-A2/pp65 complex but not with other control HLA-A2/peptide
complexes containing viral epitopes derived from the TAX protein
(e.g., TAX 11-19; SEQ ID NO:14), Gag (e.g., Gag 77-85; SEQ ID NO:9)
or Pol (e.g., Pol 476-484; SEQ ID NO:10), or a variety of
tumor-associated epitopes such as telomerase epitopes [e.g., hTERT
540 (SEQ ID NO:6) or hTERT 865 (SEQ ID NO:8)], melanoma gp100
epitopes [e.g., 209 (SEQ ID NO:7) or 280 (SEQ ID NO:4)], XAGE (SEQ
ID NO:12), TARP (SEQ ID NO:13) and MART-1-derived epitopes (e.g.,
MART 26-35; SEQ ID NO:11) (Pascolo S., et al., 1997). Thus, these
peptide-specific and MHC-restricted Fab antibodies exhibit the
binding characteristics and fine specificity of a TCR-like
molecule.
[0238] HLA-A2/pp65-Specific TCR-Like Recombinant Antibodies
Specifically Bind MHC-Peptide Complexes Presented on Cells
[0239] To demonstrate that the isolated Fab antibodies can bind the
specific MHC-peptide complex not only in the recombinant soluble
form, but also in the native form, as expressed on the cell
surface, the present inventors used murine TAP2 (transporter
associated with antigen presentation)-deficient RMA-S cells
transfected with the human HLA-A2 gene in a single-chain format
(Pascolo S., et al., 1997) (HLA-A2.1/Db-.beta..sub.2m single chain,
RMA-S--HHD cells). The pp65.sub.495-503 peptide and control
peptides were loaded on RMA-S--HHD cells and the ability of the
selected Fab Abs to bind to peptide-loaded cells was monitored by
flow cytometry. Peptide-induced MHC stabilization of the TAP2
mutant RMA-S-HHD cells was demonstrated by the reactivity of mAbs
w6/32 (HLA conformation dependent) and BB7.2 (HLA-A2 specific) with
peptide-loaded, but not unloaded cells (data not shown). As shown
in FIGS. 2b and d, Fabs H9 and F5 reacted only with pp65-loaded
RMA-S-HHD cells, but not with cells loaded with the EBV derived
peptide. Similar results were observed in FACS analysis using 10
other HLA-A2-restricted peptides (data not shown).
[0240] In addition, the present inventors used the TAP.sup.+
EBV-transformed B-lymphoblast HLA-A2.sup.+ JY cells as APCs. These
cells have normal TAP; consequently, peptide loading is facilitated
by the exchange of endogenously derived peptides with
HLA-A2-restricted peptides supplied externally by incubation of the
cells with the desired peptides. As shown in FIGS. 2a and c, the
Fab antibodies recognize only JY cells loaded with the specific
pp65 peptide to which they were selected, but not with control
HLA-A2-restricted peptides derived from melanoma gp100 [G9-154 (SEQ
ID NO:15) and G9-280 (SEQ ID NO:4) epitopes] and MART1 peptides
(SEQ ID NO:11), or a telomerase human telomerase reverse
transcriptase (hTERT)-derived peptide (T540 epitope; SEQ ID NO:6).
As a control, peptide-loaded HLA-A2.sup.-/HLA-A1.sup.+ APD B cells
were used. No binding of the Fab Abs to these cells was observed
(data not shown). These results demonstrate the ability of the
selected Fabs to detect specifically complexes of HLA-A2 in
association with the pp65.sub.495-503 peptide (SEQ ID NO:3), on the
surface of cells.
[0241] These results demonstrate the fine specificity of the
recombinant Fab clones H9 and F5 to soluble or membrane-presented
CMV-MHC class I complex.
Example 3
Generation of Multivalent Antibody Forms and their Binding to
Peptide-Pulsed APCs
Experimental Results
[0242] Increased Avidity of Fab Tetramers to Peptide Pulsed
APCs
[0243] Fab fragments isolated from the phage library are
monovalent. To increase the avidity of these fragments, Fab
tetramers were generated. This approach was previously used to
increase the binding avidity of peptide-MHC complexes to the TCR or
to increase the sensitivity of recombinant Ab molecules (Cloutier S
M., et al., 2000). To form a Fab tetramer with H9, a BirA tag
sequence for site-specific biotinylation was introduced at the
C-terminus of the light chain. The Fab domains were expressed
separately in E. coli and were refolded in vitro followed by
purification and in vitro biotinylation using the E. coli-derived
BirA enzyme (Cohen C J., et al., 2002). H9 Fab tetramers were
generated with a fluorescently labeled streptavidin and their
reactivity was examined by flow cytometry with JY pulsed cells. As
shown in FIG. 3a the fluorescence intensity measured on
peptide-pulsed JY cells with the H9 Fab tetramer was significantly
higher compared to the reactivity of the H9 Fab monomer. The
specificity, however, was not altered (FIG. 3c).
[0244] Increased Avidity of Whole IgG Antibodies to Peptide Pulsed
APCs
[0245] Another strategy for increasing the avidity was by creating
a whole IgG antibody molecule which is bivalent. To transform the
recombinant Fab fragment into a whole IgG molecule, eukaryotic
shuttle expression vectors containing the constant regions of IgG1
for the heavy chain and a vector containing the constant domain of
a kappa light chain were used. Recombinant H9 Fab-derived IgG was
produced from these expression vectors by co-transfection of the
two constructs into human embryonic kidney HEK293 cells. After
proper selection and generation of stable secreting clones,
purified TCR-like whole IgG molecules were produced and tested for
binding specifically towards APCs pulsed with the pp65495-503
peptide. As shown in FIG. 3b, the binding specificity of the whole
IgG molecule was maintained. As expected, the fluorescence
intensity observed with the IgG was significantly higher compared
to that of the Fab monomer. JY cells pulsed with control peptide
(derived from gp100) were incubated with the three H9 constructs
(monomer, tetramer, whole IgG Ab) to confirm specificity (FIG.
3c).
[0246] These results demonstrate the generation of bivalent (IgG)
or tetrameric Fab antibodies and the increased avidity, yet without
compromising specificity of the recombinant antibodies to the
CMV-MHC class I complex.
Example 4
The TCR-Like Antibodies of the Invention are Highly Specific and
Sensitive to MHC-CMV Peptide Complexes
Experimental Results
[0247] Determination of Binding Affinity of the Recombinant
TCR-Like Antibodies
[0248] Binding affinity determination of the H9 Ab was performed by
surface plasmon resonance (SPR) analysis using streptavidin sensor
chips coated with biotinylated HLA-A2/pp65 or control HLA-A2/EBV
complexes. The apparent affinity of the monomeric/IgG forms of the
H9 Ab indicated K.sub.D values of 8 nM and 5 nM, respectively. The
time necessary for binding of the H9 Fab/IgG Ab to the specific
complexes (K.sub.on) was 1.05.times.10.sup.5 l/Ms and
5.99.times.10.sup.5 l/Ms, respectively. The dissociation rate (Kd
or K.sub.off) of the H9 Fab was 8.79.times.10.sup.4 l/s compared to
the H9 IgG Ab, which was 3.52.times.10.sup.-3 l/s (FIGS. 4a, b). No
significant binding of the antibodies was detected when control
HLA-A2/EBV complexes were immobilized to the sensor chip (FIG.
4c).
[0249] The Recombinant TCR-Like Antibodies are Highly Specific to
the MHC-pp65 Complex
[0250] To study the sensitivity of ligand recognition by the Fab
and its derivatives the reactivity threshold was examined by
peptide titration on JY cells which were pulsed with different
concentrations of the pp65 495-503 peptide. As shown in FIGS. 5a
and b, peptide titration of pulsed JY demonstrated that the
staining intensity was dependent on the concentration of the
peptide used for pulsing, and that peptide concentrations at the
low nM range were sufficient for Fab tetramer (FIG. 5b) but not for
the monomer (FIG. 5a). Thus, the tetrameric form of H9 Fab was able
to detect much lower numbers of peptide/HLA-A2 complexes on the
surface of peptide-pulsed JY cells than the monomer. Similar
results were observed with the whole IgG molecule (data not shown).
Overall, these and additional studies revealed that the H9 tetramer
and IgG molecules are capable of detecting HLA-A2/pp65 complexes on
cells pulsed with as low as .about.100 nM pp65.sub.495-503
peptide.
[0251] The Recombinant TCR-Like Antibodies can Detect Low Amounts
of MHC-pp65 Complexes Presented on Cells in a Mixed Population of
Cells
[0252] The TCR-like Fab were further used to detect APCs bearing
the specific peptide-MHC complexes in a heterogeneous cell
population. This can verify the ability of the TCR-like Fab
molecules to detect complexes on individual cell samples in a mixed
cell population. To simulate the situation of a heterogeneous
population of cells in which only a small fraction might express
the specific peptide-MHC complex, pp65 peptide pulsed JY cells were
mixed with HLA-A2.sup.-/HLA-A1.sup.+ APD B cells at various ratios
and the reactivity of H9 Fab was analyzed by flow cytometry. As
shown in FIG. 5c, staining with H9 Fab tetramer allows accurate
identification of the admixed pp65 JY pulsed cells that express on
their surface HLA-A2/pp65 complexes, using a simple one-color flow
cytometry analysis. Using various ratios of mixtures between pulsed
and nonpulsed cells, the H9 Fab was shown capable of detecting as
low as 5% pp65 JY pulsed cells within a background population of
95% nonpulsed cells (FIG. 5c).
[0253] Altogether, these results demonstrate detection of cell
subpopulation bearing CMV peptide-MHC complexes.
Example 5
The TCR-Like Antibodies of the Invention can Detect HLA-A2/PP65
Complexes on Surface of Viral-Infected Cells
Experimental Results
[0254] Detection of HLA-A2/pp65 Complexes on the Surface of
Virus-Infected Cells
[0255] To test the ability of the isolated Fab to bind specifically
HLA-A2/pp65 complexes produced under naturally occurring
physiological Antigen (Ag) processing, HLA-A2 positive fibroblasts
were infected with the CMV laboratory strain AD169 at multiplicity
of infection (MOI) of 0.5 (FIGS. 6a-l). HLA-A2 negative fibroblasts
infected with the virus, were used as control in addition to
uninfected HLA-A2 negative and positive cells. 72 hours after
infection, infected and control cells were incubated with the
tetrameric form of H9. To verify the expression of HLA-A2 molecules
on the surface of infected, versus uninfected cells, the human
fibroblasts were also stained with PE-labeled BB7.2. Confirmation
for efficiency of virus infection was monitored with anti pp65 mAb
and the secondary antibody FITC-labeled anti mouse IgG. As shown in
FIGS. 6a and c, there was a somewhat decrease in the expression of
HLA-A2 complexes on the surface of the virus infected cells, due to
the virus well known down regulation mechanism of the MHC
expression. However, despite the relatively low amount of HLA-A2
expressed on the cell surface, there was still specific staining of
infected cells with the H9 tetramer (FIGS. 6e and g), suggesting
that the isolated antibody was able to detect not only complexes
presented on peptide pulsed APCs but also specific MHC-peptide
complexes expressed after active and naturally occurring endogenous
intracellular processing. The H9 Ab showed no binding at all in the
control uninfected cells (FIGS. 6g and h) as well as in the HLA-A2
negative cells (FIG. 6f), indicating its fine specificity towards
HLA-A2/pp65 complexes presented on the cell surface. Staining with
the anti pp65 mAb revealed the expression of the pp65 protein after
successful infection of the fibroblasts (FIGS. 6i and j).
[0256] The specificity of the H9 Ab was verified using a control
TCR-like Ab (2F1) which recognizes specifically class I MHC
complexes in association with the gp100 280-288 peptide. No
staining was visible in this assay, confirming again the H9
tetramer's specificity (data not shown).
[0257] These results demonstrate, for the first time, the ability
to follow the CMV-MHC class I complexes on the cells surface of APC
as well as inside infected cells.
Example 6
The TCR-Like Antibodies of the Invention can Compete with CTLs on
Specific HLA-A2/PP65 Sites and Thereby Prevent CTL-Mediated
Cytotoxicity
Experimental Results
[0258] The H9 Ab can Prevent CTL-Mediated Cytotoxicity Directed
Against the HLA-A2-pp65 Complex
[0259] The specificity of the H9 Ab to the MHC-pp65 495-503 complex
presented on cells was further demonstrated by the specific
inhibition of CTL-mediated cell killing by the H9 antibody.
Briefly, fibroblast cells were radioactively labeled with
S.sup.35-methionine before infection with the CMV virus and 72
hours later the cells were incubated with H9 Ab. CTLs from a line
targeted to the pp65 (495-503) epitope were added at a target
(i.e., fibroblast cells)-effector (i.e., CTL) ratio of 1:10 and
incubated for five hours. Cells incubated with anti-HLA-A2 W6/32
MAb were used as positive control, while cells without any Ab
incubation served as a reference for maximal killing. As shown in
FIG. 6m, maximal percentage of killing was observed in the virus
infected cells which were not incubated with Abs (CMV CTL alone).
However, incubation with the H9 IgG Ab exhibited .about.60%
blockage of killing by the CTLs (CMV CTL+H9).
[0260] The cytotoxicity assay demonstrated the capability of the
isolated antibody to recognize specifically complexes presented on
virus infected cells and its potential to compete with the same
sites recognized by CTLs, leading to the blockage of killing by
these effector cells.
Example 7
The TCR-Like Antibodies of the Invention are Valuable Tools for
Following the Dynamics of HLA-A2/PP65 Expression in Cells Infected
with the CMV Virus
Experimental Results
[0261] The Dynamics of HLA-A2/pp65 Complex Expression in Cells
Infected with Wild-Type and Mutant Virus
[0262] The fact that the H9 Ab was able to detect specific
complexes on virus infected cells enabled to follow the expression
levels of the complexes throughout the virus infection cycle. Based
on precedent results which showed down regulation of MHC class I
expression after viral infection (Ahn, K. et al. 1996), the present
inventors investigated whether the generation and presentation of
HLA-A2/pp65 complexes throughout various time points after
infection is influenced by the down regulation mechanism. To this
end two strategies were employed; (i) the intracellular versus
extracellular staining with H9 or anti-HLA-A2 BB7.2 Abs which
enabled to determine if the level of the complexes
generation/expression is correlated with their uptake to the cell
surface; (ii) the usage of a mutant strain of CMV which does not
induce down regulation of MHC class I. The level of expression of
HLA-A2/pp65 complexes in cells infected with the wild type AD169
strain was compared to that in cells infected with the mutant
strain. For this purpose, the genetically modified CMV strain RV798
(Jones T R and Sun L., 1997), which lacks most of the genes
responsible for the down regulation mechanism of MHC class I (US2
to US11 genes), was employed.
[0263] As shown in FIGS. 7a-t, 8a-t and 9a-y, the general
expression of HLA-A2 class I MHC was followed throughout four time
points (36, 72, 96 and 120 hours) after cell infection with AD169
WT CMV strain (FIGS. 7a-t) and RV798 mutant CMV strain (FIGS.
8a-t), as well as the expression of specific HLA-A2 complexes in
association with the pp65 495-503 peptide using the H9 IgG Ab. The
infection efficiency was monitored by following the expression of
the pp65 protein in infected cells through the use of an anti-pp65
MAb. Detection with H9 or BB7.2 Abs was performed in each time
point by intracellular and extracellular staining. To verify the
specificity of the reagents used for detection, especially the
reactivity of the anti-HLA-A2/pp65 495-503 TCR-like antibody,
controls which were uninfected HLA-A2 positive fibroblasts (FIGS.
9a-t) or CMV infected human fibroblasts that are HLA-A2 negative
(FIGS. 9u-y) were used. The results show progressive expression of
pp65 in cells infected with wild-type (FIGS. 7e, j, o, t) and
mutant (FIGS. 8e, j, o, t) CMV strains while in non-infected cells
(FIGS. 9e, j, o, t) no expression was observed. The expression of
pp65 in cells that were infected with the mutant stain RV798 was
somewhat higher. Staining with the anti pp65 Ab also indicated that
the cells begin to express the pp65 protein less than 36 hours
after infection (data not shown). These data are in agreement with
previous studies (Soderberg-Naucler C., et al., 1998). Expression
of HLA-A2 on the surface of cells infected with wild-type virus
clearly showed a phenotype involving significant down regulation of
HLA-A2 expression (FIGS. 7c, h, m and r) compared to the uninfected
fibroblasts (FIGS. 9c, h, m, r). This down regulation is increased
over time through the progression of the time points. Also, the
intracellular expression of HLA-A2 in infected cells seemed to be
higher than the amount in the uninfected cells (Compare FIGS. 7d,
i, n and s to FIGS. 9d, i, n and s, respectively). These data are
in agreement with previous studies (Ahn K., et al., 1996).
[0264] When cells were infected with wild-type virus, a specific
and gradual increase in staining with the H9 IgG TCR-like antibody
was observed indicating the generation of HLA-A2/pp65 495-503
complexes inside infected cells (FIGS. 7b, g, l and q) as well as
their presentation on the cell surface (FIGS. 7a, f, k and p).
However, although the amount of complexes which bear the pp65
495-503 peptide seemed to be quite low at the cell surface (e.g.,
compare FIG. 7f with 7g), intracellular staining of these specific
complexes revealed a very significant large pool of complexes
inside the cell. This might indicate that although the pp65 is well
processed inside the cell and its peptides are deposited on the
class I MHC, it is avoided from being displayed on the cell surface
as part of the virus evasion mechanisms. Interestingly, there was
no correlation between HLA-A2 down regulation as clearly observed
through the progression of time and the significant increase in the
intracellular pools of HLA-A2/pp65 495-503 complexes or their
expression on the cell surface. Most striking is that after 120
hours the expression of HLA-A2 is very low however both
intracellular pools are very high and surface expression is
significant.
[0265] The Reactivity of the H9 IgG Molecule to the MHC-CMV pp65
Peptide Complex Both Inside and on the Surface of Cells is Highly
Specific
[0266] Non-infected HLA-A2 positive cells were stained with
anti-HLA-A2 antibody BB7.2 both inside (FIGS. 9d, i, n, s) and on
the surface (FIGS. 9c, h, m, r). As shown, there were no observed
alterations in HLA-A2 expression inside the cells as well as its
presentation on the cell surface throughout the time points tested
after infection. In contrary to the infected fibroblasts, the
amount of complexes as determined using the BB7.2 antibody on the
cell surface of non-infected cells seemed to be higher than their
amount inside the cells (compare FIGS. 9c, h, m, r with FIGS. 9d,
i, n, s, respectively). No pools of complexes were observed inside
the cells (FIGS. 9d, i, n, s) as seen in the infected fibroblasts
(FIGS. 7d, i, n, s). This implies that the HLA-A2 complexes
expressed inside the uninfected cells are freely presented on the
cell surface, in contrast to the infected cells (see FIGS. 7d, i,
n, s). In contrast, the H9 TCR-like antibody was not reactive with
uninfected cells both inside (FIGS. 9b, g, l, q) and on the cell
surface (FIGS. 9a, f, k, p), indicating its fine specificity
towards its antigen.
[0267] When HLA-A2 negative human fibroblasts were infected with
wild-type CMV, pp65 expression was clearly observed (FIG. 9y),
however, no reactivity with the anti-HLA-A2 antibody (FIGS. 9w, x)
or the H9 TCR-like antibody (FIGS. 9u, v) was observed inside or on
the surface of the infected cells indicating the highly specific
reactivity of the molecules.
[0268] The presentation of HLA-A2/pp65 complexes was further
examined both inside the cells and on their surface less than 24
hours after infection. These studies demonstrated that although
pp65 is expressed, there is no presentation of its peptides on
HLA-A2 molecules (Data not shown).
[0269] FIGS. 8a-t follow the dynamics of antigen presentation in
the mutant strain RV798. The infected cells were efficiently
infected with the virus as observed from the staining with
anti-pp65 (FIGS. 8e, j, o, t). It was clearly observed that the
effect of the mutant virus on HLA-A2 expression inside and on the
surface of infected cells was diminished, thus the mutant virus no
longer significantly down regulates HLA-A2 expression, as expected.
When using the H9 IgG TCR-like antibody, similar to the results
observed with wild-type CMV, a gradual increase over time of
intracellular pools of HLA-A2/pp65 495-503 complexes inside
infected cells was observed (FIGS. 8b, g, l, q) as well as their
gradual appearance on the cell surface (FIGS. 8a, f, k, p). Also,
it was quite evident that the number of HLA-A2/pp65 495-503
complexes inside the infected cells was higher than those on the
cell surface (Compare FIGS. 8b, g, l, q to FIGS. 8a, f, k, p,
respectively). This may indicate that although the mutant virus
does not activate the down regulation mechanism, there are still
HLA-A2 pools as well as specific HLA-A2/pp65 pools inside the
cells, which are avoided from being presented on the cell
surface.
[0270] In general, these results present the usage of the H9 Ab to
follow the dynamic expression and kinetics of HLA-A2/pp65 495-503
presentation intracellularly and on the surface of infected cells
as a function of time after viral infection. Most striking is the
observation that there is no correlation between class I MHC down
regulation induced by wild-type virus and the
generation/presentation of the viral specific HLA-A2/pp65 495-503
complex. On the contrary, the down regulation did not affect the
generation of a significant and large intracellular pool of viral
complexes and their appearance over time on the cell surface.
Similar studies using the H9 antibody and a mutant virus that
abolishes class I MHC down regulation showed a similar pattern of
expression inside the cell and on its surface with somewhat
increased number of complexes on both compared to wild-type virus
especially between 24-72 hours after infection.
Example 8
The TCR-Like Antibodies of the Invention can be Used to Quantify
the Number of HLA-A2/PP65 Complexes on Viral Infected CELLS
[0271] The knowledge of the number of complexes presented on the
cell surface can be used to understand how the immune system
identifies viral infection. Related to the studies presented
herein, the present inventors attempted to quantify and compare the
number of complexes generated inside the infected cells to those
presented on the cell surface, as follows.
Experimental Results
[0272] Quantization of the Number of HLA-A2/pp65 Complexes on the
Surface of Infected Cells
[0273] The unique H9 IgG TCR-like antibody enables the present
inventors to directly quantify the number and percentage of
specific HLA-A2/pp65 complexes among HLA-A2-derived complexes which
are displayed on the cell surface. Staining of virus infected cells
with the H9 IgG TCR-like antibody enabled the present inventors to
directly count the number of complexes on the surface of the
infected cells using a PE-labeled anti kappa secondary monoclonal
antibody that generates a 1:1 binding stoichiometry with the H9 IgG
molecule. The level of fluorescence intensity resulting from
specific reactivity of the H9 IgG antibody on infected cells can be
directly correlated with the fluorescence intensities of
calibration beads with known numbers PE molecules per bead
(QuantiBRITE PE beads; BD Biosciences), using simple flow cytometry
calibrations. This strategy enabled the present inventors to
determine the number of PE molecules bound to the cells and thereby
the number of sites which are bound by the H9 antibody.
[0274] In agreement to the results presented on FIGS. 7-9 (Example
7, hereinabove), there was an immediate and massive down regulation
of HLA-A2 complexes (using the BB7 Ab) from the cell surface after
infection with the CMV wild-type strain (FIG. 10d). In all time
points there were about 5,000 complexes observed on the cell
surface compared to .about.25,000 complexes in the uninfected
cells, implying that there was over 85% decrease in the amount of
HLA-A2 complexes presented on the cell surface (FIG. 10d). The
number of HLA-A2 complexes inside the cells in infected vs
uninfected cells remained almost the same (FIG. 10c). The number of
HLA-A2/pp65 complexes presented on the cell surface was gradually
increased over time (FIG. 10b). Specific complexes were observed
using the H9 antibody starting at 36 hours after infection and the
number reached to approximately 400 sites/cell 120 hours after
infection (FIG. 10c). This implies that 120 hours after infection
with the virus, about 10%-15% of the HLA-A2 complexes presented on
the cell surfaces bear the pp65 495-503 peptide. Interestingly, the
number of these specific complexes inside the cells reaches to
.about.2000/cell after 120 hours (FIG. 10A). This number is close
to the total number of HLA-A2 complexes inside the cell, suggesting
that most of the HLA-A2 complexes which accumulate inside infected
cells are
[0275] HLA-A2/pp65. This might also suggest that most of these
specific complexes which are generated inside the cells are avoided
from being presented on the surface.
[0276] Using the mutant virus, the same number of HLA-A2 molecules
on the cell surface was observed as in the uninfected cells (FIG.
10d). The number of sites reached to approximately 20,000 (FIG.
10d). However, the number of complexes quantified inside the cells
was significantly higher than the number observed in the uninfected
cells, and approached to .about.10,000 (FIG. 10c) compared to
.about.1,000 in the uninfected cells (FIG. 10c). As for HLA-A2/pp65
complexes, there were .about.400 sites detected on the cell surface
(FIG. 10b), implying that similar to cells infected with wild-type
virus most of viral HLA-A2/pp65 complexes are avoided from being
transported to the cell surface. The percentage of these complexes
amongst HLA-A2 complexes on the cell surface is very low. However,
the number of HLA-A2/pp65 complexes inside the infected cells
reached to approximately 3,000 (FIG. 10a) in each time point after
72 hours thus until 120 hours after infection there is an
accumulation of the specific complexes inside the cell. This
accumulation might lead to the observation that after this time
point, most of the complexes inside the cell are composed of
HLA-A2/pp65.
[0277] These data provide a quantitative measure to the observation
that specific HLA-A2/pp65 complexes are being generated in large
amounts and accumulated inside the infected cell in a mechanism
that is independent to the overall down regulation of HLA-A2
molecules in these cells. The accumulation was observed with
wild-type and mutant virus strains and for both the accumulated
HLA-A2/pp65 complexes were avoided from being presented in large
amounts on the cell surface.
[0278] These results visualize large intracellular pools of the
viral complexes after infection, follow and quantify their
expression on the surface. These results demonstrate that despite
significant down regulation of MHC expression by wild-type virus
large pools of specific viral complexes are generated
intracellularly, and their export to the cell surface occurs in a
limited quantity. These studies describe the first attempt to
directly visualize and analyze the dynamics of a naturally
occurring viral-derived human MHC-peptide complex after viral
infection.
[0279] The data also demonstrate the ability of the TCR-like
antibody of the instant application to detect and accurately
quantify the number of HLA-A2/peptide complexes on the surface of
infected cells under naturally occurring intracellular processing.
These results can be used to follow the effectiveness of viral
strategies for immunization.
Example 9
Visualization Through Confocal Microscopy Imaging of HLA-A2/PP65
Expression in Virus-Infected Cells
Experimental Results
[0280] Visualization Through Confocal Microscopy Imaging of
HLA-A2/PP65 Expression in Virus-Infected Cells
[0281] Confocal microscopy of CMV infected cells stained with the
H9 IgG TCR-like antibody enabled the present inventors to visualize
and image the specific HLA-A2/pp65 complexes generated inside the
cells, as well as their display on the cell surface. Moreover, it
enabled the present inventors to localize the complexes inside the
cell during the virus infection cycle.
[0282] CMV infected cells were harvested every 24 hours for 5 days.
At each time point cells were stained with the H9 Ab, and anti
human alexa fluor.sup.488 as a secondary Ab. The cells were also
stained intracellularly with the H9 Ab, anti calnexin, cis Golgi
matrix protein (GM130), and anti pp65 Ab, after fixation and
permeabilization. Secondary antibody for the ER marker, Golgi
marker and anti pp65 was anti mouse alexa fluor.sup.594.
Noninfected fibroblast cells were used as a control.
[0283] The results of these assays further demonstrate and image
the significant pool of specific HLA-A2/pp65 complexes generated
inside infected cells (FIGS. 11a-o, 12a-o). The data also show that
the specific complexes are densely colocalized with the cis-golgi
apparatus (FIGS. 11a-o). This co-localization is observed clearly
after 24 hours in comparison with the later time points, in which
the complexes are more widely distributed and co-localized to the
ER/cytosol as indicated by co-staining with the various
localization markers (FIGS. 12a-o). Additionally, as time
progresses, a significant enlargement of the Golgi apparatus is
observed, as part of the morphological changes of the infected
cells. Extracellular staining of the HLA-A2/pp65 complexes showed
their display on the cell surface only after 72 hours post
infection (FIGS. 13a-e). These results are with complete agreement
with the flow cytometry analysis of the kinetic of HLA-A2/pp65
epitope presentation as shown in FIGS. 7a-t, 8a-t and 9a-y.
Confocal microscopy analysis of control noninfected cells showed no
staining with the H9 Ab (FIGS. 13f-h), indicating its fine
specificity towards the HLA-A2/pp65 complexes. Staining with anti
pp65 Ab confirmed the effectiveness of the viral infection in the
experiments (FIGS. 13i-j).
[0284] These results visualize the present inventors' finding that
specific HLA-A2/pp65 complexes are being generated and accumulate
in infected cells and are localized in the Golgi compartment. They
are prevented from being displayed on the cell surface at early
time points and only 72 hours after infection they can be imaged on
the cell surface. The fact that the specific complexes are
prevented from being displayed on the cell surface is only
temporary. Progressed time scales showed that the complexes are
being significantly displayed on the cell surface. The intermediate
time points clearly show that the complexes are less co-localized
with the Golgi due to their movement to the cell membrane. The
phenomena of Golgi enlargement is usually attributed to an
extensive synthesis of proteins after viral infection. These
results can imply that this enlargement is also due to the specific
accumulation of complexes in the Golgi.
Example 10
CMV PP64 MHC Restricted Peptides
[0285] Tables 5-70 hereinbelow provide the user parameters and
scoring information used to select CMV PP64 restricted peptides
(each of 9 or 10 amino acids in length) of various HLA molecules.
The analysis was performed using the Bimas software
[hypertexttransferprotocol://worldwideweb-bimas (dot) cit (dot) nih
(dot) gov/molbio/hla_bind/]. The scoring results and the sequences
of the selected peptides (according to each user parameters and
scoring information) are provided in Table 137 in Example 11,
hereinbelow. The CMV PP64 kDa protein used for analysis is provided
by SEQ ID NO:52 [(GenBank Accession No. P18139; PP65_HCMVT 64 kDa
lower matrix phosphoprotein--Human cytomegalovirus (strain Towne)
(HHV-5) (Human herpesvirus 5)].
TABLE-US-00005 TABLE 5 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A1
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported 15 back
in scoring output table
TABLE-US-00006 TABLE 6 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A1
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported 20 back
in scoring output table
TABLE-US-00007 TABLE 7 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0201 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
48 scoring output table
TABLE-US-00008 TABLE 8 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0201 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 53 scoring output table
TABLE-US-00009 TABLE 9 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0205 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
50 scoring output table
TABLE-US-00010 TABLE 10 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0205 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 47 scoring output table
TABLE-US-00011 TABLE 11 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A24
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
61 scoring output table
TABLE-US-00012 TABLE 12 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A24
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
76 scoring output table
TABLE-US-00013 TABLE 13 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A3
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
23 scoring output table
TABLE-US-00014 TABLE 14 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A3
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
21 scoring output table
TABLE-US-00015 TABLE 15 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A68.1 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
79 scoring output table
TABLE-US-00016 TABLE 16 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A68.1 length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
77 scoring output table
TABLE-US-00017 TABLE 17 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_1101 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
5 scoring output table
TABLE-US-00018 TABLE 18 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_1101 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 4 scoring output table
TABLE-US-00019 TABLE 19 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3101 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
10 scoring output table
TABLE-US-00020 TABLE 20 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3101 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 17 scoring output table
TABLE-US-00021 TABLE 21 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3302 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
39 scoring output table
TABLE-US-00022 TABLE 22 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3302 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 34 scoring output table
TABLE-US-00023 TABLE 23 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B14
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
61 scoring output table
TABLE-US-00024 TABLE 24 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B14
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
60 scoring output table
TABLE-US-00025 TABLE 25 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B40
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
36 scoring output table
TABLE-US-00026 TABLE 26 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B40
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
42 scoring output table
TABLE-US-00027 TABLE 27 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B60
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
41 scoring output table
TABLE-US-00028 TABLE 28 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B60
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
49 scoring output table
TABLE-US-00029 TABLE 29 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B61
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
44 scoring output table
TABLE-US-00030 TABLE 30 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B61
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
45 scoring output table
TABLE-US-00031 TABLE 31 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B62
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
57 scoring output table
TABLE-US-00032 TABLE 32 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B62
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
54 scoring output table
TABLE-US-00033 TABLE 33 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B7
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
55 scoring output table
TABLE-US-00034 TABLE 34 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B7
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
62 scoring output table
TABLE-US-00035 TABLE 35 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B8
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
12 scoring output table
TABLE-US-00036 TABLE 36 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B8
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 542 number of top-scoring subsequences reported back in
14 scoring output table
TABLE-US-00037 TABLE 37 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2702 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
71 scoring output table
TABLE-US-00038 TABLE 38 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2702 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 72 scoring output table
TABLE-US-00039 TABLE 39 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2705 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
276 scoring output table
TABLE-US-00040 TABLE 40 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2705 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 283 scoring output table
TABLE-US-00041 TABLE 41 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3501 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
72 scoring output table
TABLE-US-00042 TABLE 42 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3501 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 81 scoring output table
TABLE-US-00043 TABLE 43 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3701 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
90 scoring output table
TABLE-US-00044 TABLE 44 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3701 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 100 scoring output table
TABLE-US-00045 TABLE 45 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3801 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
50 scoring output table
TABLE-US-00046 TABLE 46 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3801 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 59 scoring output table
TABLE-US-00047 TABLE 47 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3901 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
100 scoring output table
TABLE-US-00048 TABLE 48 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3901 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 102 scoring output table
TABLE-US-00049 TABLE 49 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3902 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
61 scoring output table
TABLE-US-00050 TABLE 50 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3902 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 69 scoring output table
TABLE-US-00051 TABLE 51 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_4403 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
47 scoring output table
TABLE-US-00052 TABLE 52 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_4403 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 56 scoring output table
TABLE-US-00053 TABLE 53 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5101 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
139 scoring output table
TABLE-US-00054 TABLE 54 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5101 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 127 scoring output table
TABLE-US-00055 TABLE 55 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5102 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
149 scoring output table
TABLE-US-00056 TABLE 56 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5102 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 140 scoring output table
TABLE-US-00057 TABLE 57 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5103 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
scoring output table 89
TABLE-US-00058 TABLE 58 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5103 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 86 scoring output table
TABLE-US-00059 TABLE 59 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5201 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
111 scoring output table
TABLE-US-00060 TABLE 60 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5201 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
back in 120 scoring output table
TABLE-US-00061 TABLE 61 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5801 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 551 number of subsequence scores
calculated 543 number of top-scoring subsequences reported back in
55 scoring output table
TABLE-US-00062 TABLE 62 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5801 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
50 back in scoring output table
TABLE-US-00063 TABLE 63 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0301 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 543 number of top-scoring subsequences reported
back 99 in scoring output table
TABLE-US-00064 TABLE 64 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0301 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
91 back in scoring output table
TABLE-US-00065 TABLE 65 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0401 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 543 number of top-scoring subsequences reported
88 back in scoring output table
TABLE-US-00066 TABLE 66 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0401 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
96 back in scoring output table
TABLE-US-00067 TABLE 67 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0602 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 543 number of top-scoring subsequences reported
115 back in scoring output table
TABLE-US-00068 TABLE 68 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0602 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
117 back in scoring output table
TABLE-US-00069 TABLE 69 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0702 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 543 number of top-scoring subsequences reported
61 back in scoring output table
TABLE-US-00070 TABLE 70 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0702 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 551 number of subsequence
scores calculated 542 number of top-scoring subsequences reported
73 back in scoring output table
Example 11
CMV PP65 MHC Restricted Peptides
[0286] Tables 71-136 hereinbelow provide the user parameters and
scoring information used to select CMV PP65 restricted peptides
(each of 9 or 10 amino acids in length) of various HLA molecules.
The analysis was performed using the Bimas software
[hypertexttransferprotocol://worldwideweb-bimas (dot) cit (dot) nih
(dot) gov/molbio/hla_bind/]. The scoring results and the sequences
of the selected peptides (according to each user parameters and
scoring information) are provided in Table 137, hereinbelow. The
CMV PP65 kDa protein used for analysis is provided by SEQ ID NO:53
[GenBank Accession No. P06725; PP65_HCMVA 65 kDa lower matrix
phosphoprotein--Human cytomegalovirus (strain AD169) (HHV-5) (Human
herpesvirus 5)].
TABLE-US-00071 TABLE 71 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A1
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 15 back
in scoring output table
TABLE-US-00072 TABLE 72 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A1
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences reported 20 back
in scoring output table
TABLE-US-00073 TABLE 73 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0201 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 48 back
in scoring output table
TABLE-US-00074 TABLE 74 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0201 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
53 back in scoring output table
TABLE-US-00075 TABLE 75 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0205 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 51 back
in scoring output table
TABLE-US-00076 TABLE 76 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_0205 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
47 back in scoring output table
TABLE-US-00077 TABLE 77 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A24
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
64 scoring output table
TABLE-US-00078 TABLE 78 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A24
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences reported 76 back
in scoring output table
TABLE-US-00079 TABLE 79 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A3
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 23 back
in scoring output table
TABLE-US-00080 TABLE 80 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected A3
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences reported 21 back
in scoring output table
TABLE-US-00081 TABLE 81 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A68.1 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 79 back
in scoring output table
TABLE-US-00082 TABLE 82 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A68.1 length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences reported 77 back
in scoring output table
TABLE-US-00083 TABLE 83 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_1101 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 5 back
in scoring output table
TABLE-US-00084 TABLE 84 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_1101 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported 4
back in scoring output table
TABLE-US-00085 TABLE 85 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3101 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 10 back
in scoring output table
TABLE-US-00086 TABLE 86 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3101 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
17 back in scoring output table
TABLE-US-00087 TABLE 87 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3302 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 40 back
in scoring output table
TABLE-US-00088 TABLE 88 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
A_3302 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
36 back in scoring output table
TABLE-US-00089 TABLE 89 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B14
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 65 back
in scoring output table
TABLE-US-00090 TABLE 90 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B14
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences reported 63 back
in scoring output table
TABLE-US-00091 TABLE 91 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B40
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported 35 back
in scoring output table
TABLE-US-00092 TABLE 92 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B40
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences 42 reported back
in scoring output table
TABLE-US-00093 TABLE 93 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B60
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences 42 reported back
in scoring output table
TABLE-US-00094 TABLE 94 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B60
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences 50 reported back
in scoring output table
TABLE-US-00095 TABLE 95 User Parameters and Scoring Information
User Parameters and Scoring Information method selected to limit
number of results cutoff score cutoff score selected 1 HLA molecule
type selected B61 length selected for subsequences to be scored 9
echoing mode selected for input sequence Y echoing format numbered
lines length of user's input peptide sequence 561 number of
subsequence scores calculated 553 number of top-scoring
subsequences 44 reported back in scoring output table
TABLE-US-00096 TABLE 96 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B61
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences 46 reported back
in scoring output table
TABLE-US-00097 TABLE 97 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B62
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences 57 reported back
in scoring output table
TABLE-US-00098 TABLE 98 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B62
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences 54 reported back
in scoring output table
TABLE-US-00099 TABLE 99 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B7
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences 57 reported back
in scoring output table
TABLE-US-00100 TABLE 100 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B7
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences 63 reported back
in scoring output table
TABLE-US-00101 TABLE 101 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B8
length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences 12 reported back
in scoring output table
TABLE-US-00102 TABLE 102 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected B8
length selected for subsequences to be scored 10 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 552 number of top-scoring subsequences 15 reported back
in scoring output table
TABLE-US-00103 TABLE 103 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2702 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences 74 reported back
in scoring output table
TABLE-US-00104 TABLE 104 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2702 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences 74 reported back
in scoring output table
TABLE-US-00105 TABLE 105 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2705 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences 282 reported back
in scoring output table
TABLE-US-00106 TABLE 106 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_2705 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences 287
reported back in scoring output table
TABLE-US-00107 TABLE 107 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3501 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
74 scoring output table
TABLE-US-00108 TABLE 108 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3501 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back 82 in scoring output table
TABLE-US-00109 TABLE 109 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3701 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back 93
in scoring output table
TABLE-US-00110 TABLE 110 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3701 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back 103 in scoring output table
TABLE-US-00111 TABLE 111 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3801 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
51 scoring output table
TABLE-US-00112 TABLE 112 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3801 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 59 scoring output table
TABLE-US-00113 TABLE 113 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3901 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
104 scoring output table
TABLE-US-00114 TABLE 114 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3901 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 105 scoring output table
TABLE-US-00115 TABLE 115 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3902 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
63 scoring output table
TABLE-US-00116 TABLE 116 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_3902 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 70 scoring output table
TABLE-US-00117 TABLE 117 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_4403 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
47 scoring output table
TABLE-US-00118 TABLE 118 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_4403 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 57 scoring output table
TABLE-US-00119 TABLE 119 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5101 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
141 scoring output table
TABLE-US-00120 TABLE 120 method selected to limit number of results
cutoff score 1 HLA molecule type selected B_5101 length selected
for subsequences to be scored 10 echoing mode selected for input
sequence Y echoing format numbered lines length of user's input
peptide sequence 561 number of subsequence scores calculated 552
number of top-scoring subsequences reported back in 128 scoring
output table
TABLE-US-00121 TABLE 121 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5102 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
151 scoring output table
TABLE-US-00122 TABLE 122 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5102 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 140 scoring output table
TABLE-US-00123 TABLE 123 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5103 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
90 scoring output table
TABLE-US-00124 TABLE 124 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5103 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 86 scoring output table
TABLE-US-00125 TABLE 125 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5201 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
114 scoring output table
TABLE-US-00126 TABLE 126 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5201 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 120 scoring output table
TABLE-US-00127 TABLE 127 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5801 length selected for subsequences to be scored 9 echoing mode
selected for input sequence Y echoing format numbered lines length
of user's input peptide sequence 561 number of subsequence scores
calculated 553 number of top-scoring subsequences reported back in
55 scoring output table
TABLE-US-00128 TABLE 128 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
B_5801 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 49 scoring output table
TABLE-US-00129 TABLE 129 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0301 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 553 number of top-scoring subsequences reported
back in 103 scoring output table
TABLE-US-00130 TABLE 130 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0301 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 93 scoring output table
TABLE-US-00131 TABLE 131 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0401 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 553 number of top-scoring subsequences reported
back in 90 scoring output table
TABLE-US-00132 TABLE 132 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0401 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 98 scoring output table
TABLE-US-00133 TABLE 133 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0602 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 553 number of top-scoring subsequences reported
back in 119 scoring output table
TABLE-US-00134 TABLE 134 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0602 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 120 scoring output table
TABLE-US-00135 TABLE 135 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0702 length selected for subsequences to be scored 9 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 553 number of top-scoring subsequences reported
back in 62 scoring output table
TABLE-US-00136 TABLE 136 method selected to limit number of results
cutoff score cutoff score selected 1 HLA molecule type selected
Cw_0702 length selected for subsequences to be scored 10 echoing
mode selected for input sequence Y echoing format numbered lines
length of user's input peptide sequence 561 number of subsequence
scores calculated 552 number of top-scoring subsequences reported
back in 74 scoring output table
[0287] Table 137 hereinbelow, depicts subsequence residue listing
(Sequence), SEQ ID NO: and scoring results [Rank and Score (the
estimate of half time of disassociation of a molecule containing
this subsequence)] obtained according to the user parameters and
scoring information summarized in Tables 5-136, hereinabove, for
HLA restricted peptides derived from the CMV pp65 (SEQ ID NO:53) or
pp64 (SEQ ID NO:52) polypeptides. For each row, a reference to the
relevant "user parameters and scoring information Table" is made by
indicating the "Table No." on the last column
TABLE-US-00137 Lengthy table referenced here
US20140363440A1-20141211-T00001 Please refer to the end of the
specification for access instructions.
Example 12
Detection of HLA-A2/PP65 Complexes on the Surface of Virus-Infected
Cells of Patients
[0288] The ability of the H9 Ab to detect HLA-A2/pp65 complexes was
further evaluated in heterogeneous population of cells taken from
CMV infected individuals. Briefly, samples were taken from bone
marrow transplanted (BMT) patients whom are under reactivation of
CMV infection due to immuno-suppression. Healthy donors were used
as a control to verify the H9 Fab specificity.
Experimental Results
[0289] Peripheral blood mononuclear cells (PBMCs) were isolated
from samples taken from BMT patients and healthy donors. The
isolated cells were stained with the H9 Ab and the secondary anti
human alexa fluor.sup.488 Ab. For intracellular staining with the
H9 Ab, the cells were permeabilized as described under "General
Materials and Experimental Methods".
[0290] Both healthy donors and BMT patients were HLA-A2+ (i.e.,
express the HLA-A2 allele) as detected by the anti HLA-A2 Ab
(BB7.2) and anti mouse alexa fluor.sup.488 Abs (FIG. 16a and data
not shown). Extracellular staining with the H9 Ab did not detect
complexes of the HLA-A2/pp65 on the surface of infected cells taken
from BMT patients or healthy controls (FIG. 16b and data not
shown). However, as shown in FIGS. 16c and d, intracellular
staining with the H9 Ab demonstrated a significant binding of the
antibody to the infected cells from BMT patients (FIG. 16c) as
compared to the control cells taken from healthy donors (FIG. 16d).
These results confirm the ability of the isolated H9 Ab to detect
specific HLA-A2/pp65 complexes not only after directed infection
with laboratory strain of the CMV, but also complexes derived from
cells undergoing reactivation of the virus e.g., due to
immuno-suppression.
Example 13
Proteasome Inhibitor Effect on HLA-A2/PP65 Complexes in Virus
Infected Cells
Experimental Results
[0291] The Release of Complexes Accumulation from their
Intracellular Location to the Cell Surface by Proteasome
Inhibitor
[0292] The proteasome inhibitor acetyl-leucyl-leucyl-norleucinal
(ALLN; available from CALBIOCHEM Cat. No. 208750) was used in order
to understand the mechanism by which complexes are prevented from
reaching the cell membrane. The effect of the proteasome inhibitor
was examined by FACS analysis, while treating the infected cells
with ALLN at three time scales after infection. At each time scale,
the cells were extracellularly stained with the H9 Ab and anti
human alexa-flour.sup.488 as a secondary Ab. As shown in FIGS.
17a-i there was a significant effect of the inhibitor on the
presentation of the complexes on the cell surface. Presence of the
inhibitor at each time scale caused an increased presentation of
the complexes on the cell surface compared to untreated cells. The
effect of the increased presentation was more significant at the
lower time scales, and seamed to reach a steady state at 96 hours
post infection. Control uninfected cells showed no staining with
the H9 Ab. Thus, incubation with the proteasome inhibitor ALLN
increased presentation of the MHC/pp65 complexes on the cell
surface.
SUMMARY
[0293] In this study, the present inventors have demonstrated the
selection of recombinant Fab Abs directed against a human viral T
cell epitope derived from CMV, from a large nonimmune human Ab
phage library. These Abs exhibit an exquisite, very specific, and
special binding pattern: they can bind in a peptide-specific manner
only to HLA-A2/pp65 complexes; hence, these are recombinant Abs
with T cell Ag receptor-like specificity. In contrast to the
inherent low affinity of TCRs, these molecules display the high
affinity binding characteristics of Abs, in the nM range, while
retaining TCR specificity. The present inventors have demonstrated
here the ability of these Abs to bind specifically to recombinant
class I peptide-MHC complexes, as well as to complexes presented on
the surface of peptide pulsed APCs.
[0294] An important feature of the TCR-like Fab Abs isolated in
this study is their capability to detect TCR ligands at cell
surface densities close to the threshold limit for T cell
recognition. The H9 HLA-A2/pp65-specific TCR-like Fab Ab was able
to detect in a reproducible manner as low as 100 sites/cell. Using
flow cytometry, it was possible to use the H9 Fab Ab to detect the
specific ligand on cells pulsed with peptide concentrations similar
to those required to activate T cell hybridoma or CTL lines to
cytokine secretion and within a few fold of the minimal
concentration able to sensitize target cells for lysis in a
short-term assay (Porgador A., et al., 1997).
[0295] These data indicate that when applied to dissociated cell
populations using flow cytometry, the detection of ligand with H9
and other TCR-like Fabs with similar affinity approaches the
sensitivity of T cells, and hence that these molecules are suitable
reagents for evaluating antigenic complex expression at low, but
physiologically relevant levels. In this study, the detection
sensitivity of specific ligand was observed with as low as 100
complexes per cell. Thus, this principle has been applied in this
study to mixtures of peptide pulsed HLA-A2+ JY cells, and the
HLA-A2- B cell line APD. By using the H9 tetramer in a single-step
staining for flow cytometry, it was possible to readily identify
pp65 495-503 peptide pulsed JY cells admixed with APD cells in as
low proportion as 5%.
[0296] The avidity of the TCR-like Ab molecules was improved by
making the recombinant monovalent molecules into multivalent
molecules. This was feasible by altering the basic Fab form to a
tetrameric molecule or to a whole bivalent IgG Ab.
[0297] Detection of class I MHC complexes in association with the
pp65 495-503 peptide on virus infected cells, showed the ability of
the H9 Ab to recognize complexes not only on the surface of peptide
pulsed APCs, but also complexes which were produced by naturally
occurring active antigen processing. Cytotoxicity assays directed
to virus infected cells confirmed these findings. The blockage of
killing by the CTLs after incubation with the H9 Ab showed a
competition between the cytotoxic T-cell receptor and the H9
TCR-like Ab on the same site presented on the virus infected
cell.
[0298] Using the H9 Ab at various time points following infection
the present inventors could track the presentation level of
HLA-A2/pp65 complexes during the course of virus infection cycle.
Specific staining with the H9 Ab lead to the observation that the
expression level of the specific HLA-A2/pp65 complexes on the cell
surface does not represent the overall quantity of these specific
complexes, because as shown most of them are located inside the
cell. The results presented herein demonstrate the existence of a
significant large pool of specific HLA-A2/pp65 complexes inside
virus infected cells, which increased as a function of time after
viral infection. The use of a CMV mutant strain which lacks the
genes responsible for MHC class I down regulation revealed similar
findings. Large pools of specific complexes, bearing the pp65
495-503 peptide, were found inside the cells. In contrast to the
uninfected cells, there is a large amount of MHC class I complexes
inside the cells which are infected with the wild-type/mutant
strain.
[0299] The results of the kinetic assays also clearly show that
there is a great correlation between the pp65 expression level and
its presentation level. Both increase as time goes by. Moreover,
the timing of the pp65 expression might precede the processing and
presentation of this protein, as presented in the results.
[0300] This work provides also quantitative data about the number
of specific HLA-A2/pp65 complexes generated inside infected cells
as well as presented on the cell surface after active intracellular
processing by virus infected cells. The results revealed for the
first time the number of sites which are presented on the cell
surface and recognized by the immune system. Moreover, quantization
of general HLA-A2 complexes enabled the present inventors to
determine the percentage of complexes down regulated after viral
infection. It also enabled the present inventors to compare between
the number of general complexes and the number of specific
HLA-A2/pp65 complexes inside the cells and on their surface. This
analysis enables the determination of the percentage of the
specific complexes among the general complexes. The results
indicated quantitatively that most of the complexes inside the
virus infected cells are bearing the pp65 495-503 peptide. Large
numbers of specific complexes were also found in the cells infected
with the mutant strain, strengthening the previous data, regarding
the pools which are prevented from being translocated to the
membrane.
[0301] Confocal immunofluorescence microscopy enabled for the first
time direct visualization of the intracellular and extracellular
sites of peptide-MHC molecules throughout virus infection cycle, as
well as determination of their localization inside the cell. This
visualization revealed the colocalization of the HLA-A2/pp65
complexes with the cis-golgi apparatus. It also showed the exact
movement of the complexes from this location to the cell surface,
in correlation to the virus infection kinetics. At the progressed
time scales there was a significant display of the complexes on the
cell surface.
[0302] The study presented here shows the usage of an isolated
human recombinant Ab towards a specific viral peptide-MHC class I
in the following: (i) tracking the level of specific complexes
throughout time scale which represents a viral infection cycle;
(ii) tracking the number of complexes throughout time scale inside
the cell and on its surface and analysis of this data; (iii)
visualization of complexes in a viral infection system which
demonstrate the intracellular localization of the complexes
throughout time scale, and; (iv) detection of the correlation
between protein expression and its derived peptide presentation on
HLA-A2 complexes after processing.
[0303] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0304] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
REFERENCES
Additional References are Cited in Text
[0305] 1. Ahn, K. et al. Human cytomegalovirus inhibits antigen
presentation by a sequential multistep process Proc. Natl. Acad.
Sci. U.S.A 93, 10990-10995 (1996). [0306] 2. Allart S, et al.,
2003; Invest Ophthalmol V is Sci. 44: 665-71 [0307] 3. Altman, J.
D. et al. Phenotypic analysis of antigen-specific T lymphocytes.
Science 274, 94-96 (1996). [0308] 4. Chee, M. S. et al. Analysis of
the protein-coding content of the sequence of human cytomegalovirus
strain AD169. Curr. Top. Microbiol. Immunol. 154, 125-169 (1990).
[0309] 5. Cohen, C. J. et al. Direct detection and quantitation of
a distinct T-cell epitope derived from tumor-specific epithelial
cell-associated mucin using human recombinant antibodies endowed
with the antigen-specific, major histocompatibility
complex-restricted specificity of T cells Cancer Res. 62, 5835-5844
(2002). [0310] 6. Cloutier, S. M. et al. Streptabody, a high
avidity molecule made by tetramerization of in vivo biotinylated,
phage display-selected scFv fragments on streptavidin. Mol.
Immunol. 37, 1067-1077 (2000). [0311] 7. De Haard, H. J. et al. A
large non-immunized human Fab fragment phage library that permits
rapid isolation and kinetic analysis of high affinity antibodies.
J. Biol. Chem. 274, 18218-18230 (1999). [0312] 8. Denkberg, G.,
Cohen, C. J., Segal, D., Kirkin, A. F. & Reiter, Y. Recombinant
human single-chain MHC-peptide complexes made from E. coli By in
vitro refolding: functional single-chain MHC-peptide complexes and
tetramers with tumor associated antigens. Eur. J. Immunol. 30,
3522-3532 (2000). [0313] 9. Denkberg, G., Cohen, C. J. &
Reiter, Y. Critical role for CD8 in binding of MHC tetramers to
TCR: CD8 antibodies block specific binding of human tumor-specific
MHC-peptide tetramers to TCR. J. Immunol. 167, 270-276 (2001).
[0314] 10. Denkberg, G. et al. Direct visualization of distinct T
cell epitopes derived from a melanoma tumor-associated antigen by
using human recombinant antibodies with MHC-restricted T cell
receptor-like specificity. Proc. Natl. Acad. Sci. U.S.A 99,
9421-9426 (2002). [0315] 11. Jones, T. R. & Sun, L. Human
cytomegalovirus US2 destabilizes major histocompatibility complex
class I heavy chains J. Virol. 71, 2970-2979 (1997). [0316] 12.
Lee, P. P. et al. Characterization of circulating T cells specific
for tumor-associated antigens in melanoma patients. Nat Med 5,
677-685 (1999). [0317] 13. Lev, A. et al. Isolation and
characterization of human recombinant antibodies endowed with the
antigen-specific, major histocompatibility complex-restricted
specificity of T cells directed toward the widely expressed tumor
T-cell epitopes of the telomerase catalytic subunit. Cancer Res.
62, 3184-3194 (2002). [0318] 14. Pascolo, S. et al.
HLA-A2.1-restricted education and cytolytic activity of CD8(+) T
lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain
transgenic H-2Db beta2m double knockout mice. J. Exp. Med. 185,
2043-2051 (1997). [0319] 15. Porgador, A., Yewdell, J. W., Deng, Y.
P., Bennink, J. R. & Germain, R. N. Localization, quantitation,
and in situ detection of specific peptide MHC class I complexes
using a monoclonal antibody. Immunity 6, 715-726 (1997). [0320] 16.
Soderberg-Naucler, C., Fish, K. N. & Nelson, J. A. Growth of
human cytomegalovirus in primary macrophages. Methods 16,
126-+(1998).
TABLE-US-LTS-00001 [0320] LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140363440A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 1
1
904123DNAArtificial sequenceSingle strand DNA oligonucleotide
1agcggataac aatttcacac agg 23224DNAArtificial sequenceSingle strand
DNA oligonucleotide 2tttgtcgtct ttccagacgt tagt 2439PRTArtificial
sequenceCMV pp65 (495-503) HLA-A2 restricted peptide 3Asn Leu Val
Pro Met Val Ala Thr Val 1 5 49PRTArtificial sequencegp100 (280-288)
derived HLA control peptide 4Tyr Leu Glu Pro Gly Pro Val Thr Ala 1
5 59PRTArtificial sequenceEBV (280-288) derived HLA control peptide
5Gly Leu Cys Thr Leu Val Ala Met Leu 1 5 69PRTArtificial
sequencehTERT (540-548) derived HLA control peptide 6Ile Leu Ala
Lys Phe Leu His Trp Leu 1 5 79PRTArtificial sequencemelanoma gp100
(209-217) derived HLA control peptide 7Ile Met Asp Gln Val Pro Phe
Ser Val 1 5 89PRTArtificial sequencehTERT (865-873) derived HLA
control peptide 8Arg Leu Val Asp Asp Phe Leu Leu Val 1 5
99PRTArtificial sequenceGag (77-85) derived HLA control peptide
9Ser Leu Tyr Asn Thr Val Ala Thr Leu 1 5 108PRTArtificial
sequencePol (476-484) derived HLA control peptide 10Ile Leu Glu Pro
Val His Gly Val 1 5 1110PRTArtificial sequenceMART-1 (26-35)
derived HLA control peptide 11Glu Leu Ala Gly Ile Gly Ile Leu Thr
Val 1 5 10 1210PRTArtificial sequenceXAGE derived HLA control
peptide 12Gly Val Phe Pro Ser Ala Pro Ser Pro Val 1 5 10
139PRTArtificial sequenceTARP (29-37) derived HLA control peptide
13Phe Leu Arg Asn Phe Ser Leu Met Leu 1 5 149PRTArtificial
sequenceTAX (11-19) derived HLA control peptide 14Leu Leu Phe Gly
Tyr Pro Val Tyr Val 1 5 159PRTArtificial sequenceMelanoma gp100
(154-162) derived HLA control peptide 15Lys Thr Trp Gly Gln Tyr Trp
Gln Val 1 5 16230PRTArtificial sequenceH9 Fab heavy chain
polypeptide sequence 16Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Leu Tyr Tyr Tyr Asp Ser Ser Gly Tyr Pro Arg Tyr
100 105 110 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 210 215
220 Val Glu Pro Lys Ser Cys 225 230 17690DNAArtificial sequenceH9
Fab heavy chain coding sequence 17gaagtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc
agctatgcta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggaggg atcatcccta tctttggtac agcaaactac 180gcacagaagt
tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac
240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc
gagaggggat 300ctgtattact atgatagtag tggttatccg cgatactact
ttgactactg gggccagggc 360accctggtca ccgtctcaag cgcctccacc
aagggcccat cggtcttccc cctggcaccc 420tcctccaaga gcacctctgg
gggcacagcg gccctgggct gcctggtcaa ggactacttc 480cccgaaccgg
tgacggtgtc gtggaactca ggcgccctga ccagcggcgt ccacaccttc
540ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtagtgac
cgtgccctcc 600agcagcttgg gcacccagac ctacatctgc aacgtgaatc
acaagcccag caacaccaag 660gtggacaaga aagttgagcc caaatcttgt
69018197PRTArtificial sequenceH9 Fab light chain polypeptide
sequence 18Leu Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro 1 5 10 15 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser 20 25 30 Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu 35 40 45 Val Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 65 70 75 80 Glu Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln His Tyr Ser Thr Ser 85 90 95 Pro Gly
Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Arg Arg Thr 100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115
120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe 180 185 190 Asn Arg Gly Glu Cys 195
19597DNAArtificial sequenceH9 Fab light chain coding sequence
19cttgaaacga cactcacgca gtctccaggc accctgtctt tgtctccagg ggaaagagcc
60accctctcct gcagggccag tcagagtgtt agcagcagct acttagcctg gtaccagcag
120aaacctggcc aggctcccag gctcgtcatc tatggtgcat ccagcagggc
cactggcatc 180ccagacaggt tcagtggcag tgggtctggg acagacttca
ctctcaccat cagcagactg 240gagcctgaag attttgcagt ttattactgt
cagcactata gcacctcacc tgggttcact 300tttggccagg ggaccaagct
ggagatcaga cgaactgtgg ctgcaccatc tgtcttcatc 360ttcccgccat
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat
420aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct
ccaatcgggt 480aactcccagg agagtgtcac agagcaggac agcaaggaca
gcacctacag cctcagcagc 540catcagggcc tgagctcgcc cgtcacaaag
agcttcaaca ggggagagtg ttaataa 59720225PRTArtificial sequenceF5 Fab
heavy chain polypeptide sequence 20Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Asn Tyr Tyr Trp
Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile
Gly Ala Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60
Leu Lys Ser Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65
70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr 85 90 95 Cys Ala Arg Arg Ile Gly Val Ala Gly Gln Trp Tyr
Phe Asp Leu Trp 100 105 110 Gly Arg Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220 Cys 225 21675DNAArtificial sequenceF5 Fab heavy
chain coding sequence 21caggtgcagc tgcaggagtc cggcccagga ctggtgaagc
cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agcagtaatt
actactgggg ctggatccgc 120cagcccccag ggaaggggct ggagtggatt
ggtgctatct attatagtgg gagcacctac 180tacaacccgt ccctcaagag
tcgagtcgcc atatccgtag acacgtccaa gaaccagttc 240tcgctgaagt
tgagttctgt gaccgccgca gacacggctg tctattactg tgcgagacgt
300ataggagtgg ctggccaatg gtatttcgat ctctggggcc gtggcaccct
ggtcaccgtc 360tcaagcgcct ccaccaaggg cccatcggtc ttccccctgg
caccctcctc caagagcacc 420tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga accggtgacg 480gtgtcgtgga actcaggcgc
cctgaccagc ggcgtccaca ccttcccggc tgtcctacag 540tcctcaggac
tctactccct cagcagcgta gtgaccgtgc cctccagcag cttgggcacc
600cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga
caagaaagtt 660gagcccaaat cttgt 67522199PRTArtificial sequenceF5 Fab
light chain polypeptide sequence 22Leu Asn Phe Met Leu Thr Gln Pro
His Ser Val Ser Gly Ser Pro Gly 1 5 10 15 Lys Thr Val Thr Ile Ser
Cys Thr Arg Ser Thr Gly Ser Ile Thr Ser 20 25 30 Asn Tyr Val His
Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr 35 40 45 Val Ile
Cys Glu Asp Asn Glu Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60
Ser Gly Ser Ile Asp Ile Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser 65
70 75 80 Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Tyr Asp 85 90 95 Asp Ser Asn His Ile Ser Val Phe Gly Thr Gly Thr
Lys Val Thr Val 100 105 110 Leu Gly Gln Pro Lys Ala Asn Pro Cys Leu
Ile Ser Asp Phe Tyr Pro 115 120 125 Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Gly Ser Pro Val Lys Ala 130 135 140 Gly Val Glu Thr Thr Lys
Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 145 150 155 160 Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 165 170 175 Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 180 185
190 Val Ala Pro Ala Glu Cys Ser 195 23660DNAArtificial sequenceF5
Fab light chain coding sequence 23cttaatttta tgctgactca gccccactct
gtgtcggggt ctccggggaa gacggttacc 60atctcctgca cccgcagcac tggcagcatt
accagcaact atgtgcactg gtaccagcag 120cgcccgggca gttcccccac
cactgtgatc tgtgaggata acgaaagacc ctctggggtc 180cctgatcgat
tctctggctc catcgacatc tcctccaact ctgcctccct caccatctct
240ggactgaaga ctgaggacga ggctgactac tactgtcagt cttatgatga
cagcaatcat 300atttctgtct tcggtactgg gaccaaggtc accgtcctag
gtcagcccaa ggccaacccc 360tgtctgatca gtgacttcta cccgggagct
gtgacagtgg cctggaaggc agatggcagc 420cccgtcaagg cgggagtgga
gaccaccaaa ccctccaaac agagcaacaa caagtacgcg 480gccagcagct
acctgagcct gacgcccgag cagtggaagt cccacagaag ctacagctgc
540caggtcacgc atgaggggag caccgtggag aagacagtgg cccctgcaga
atgttcataa 600actgtcactc tgttcccgcc ctcctctgag gagctccaag
ccaacaaggc cacactagtg 660246PRTArtificial sequenceH9 Fab VH CDR1
24Ser Tyr Ala Ile Ser Trp 1 5 2517PRTArtificial sequenceH9 Fab VH
CDR2 25Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
Gln 1 5 10 15 Gly 2618PRTArtificial sequenceH9 Fab VH CDR3 26Gly
Asp Leu Tyr Tyr Tyr Asp Ser Ser Gly Tyr Pro Arg Tyr Tyr Phe 1 5 10
15 Asp Tyr 2716DNAArtificial sequenceH9 Fab VH CDR1 coding sequence
27gctatgctat cagctg 162850DNAArtificial sequenceH9 Fab VH CDR2
coding sequence 28gggatcatcc ctatctttgg tacagcaaac tacgcacaga
agttccaggg 502953DNAArtificial sequenceH9 Fab VH CDR3 coding
sequence 29ggggatctgt attactatga tagtagtggt tatccgcgat actactttga
cta 533012PRTArtificial sequenceH9 Fab VL CDR1 30Arg Ala Ser Gln
Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 317PRTArtificial sequenceH9
Fab VL CDR2 31Gly Ala Ser Ser Arg Ala Thr 1 5 3210PRTArtificial
sequenceH9 Fab VL CDR3 32Gln His Tyr Ser Thr Ser Pro Gly Phe Thr 1
5 10 3335DNAArtificial sequenceH9 Fab VL CDR1 coding sequence
33agggccagtc agagtgttag cagcagctac ttagc 353421DNAArtificial
sequenceH9 Fab VL CDR2 coding sequence 34ggtgcatcca gcagggccac t
213529DNAArtificial sequenceH9 Fab VL CDR3 coding sequence
35agcactatag cacctcacct gggttcact 29367PRTArtificial sequenceF5 Fab
VH CDR1 36Ser Ser Asn Tyr Tyr Trp Gly 1 5 3716PRTArtificial
sequenceF5 Fab VH CDR2 37Ala Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr
Asn Pro Ser Leu Lys Ser 1 5 10 15 3823PRTArtificial sequenceF5 Fab
VH CDR3 38Arg Ile Gly Val Ala Gly Gln Trp Tyr Phe Asp Leu Trp Gly
Arg Gly 1 5 10 15 Thr Leu Val Thr Val Ser Ser 20 3921DNAArtificial
sequenceF5 Fab VH CDR1 coding sequence 39agcagtaatt actactgggg c
214048DNAArtificial sequenceF5 Fab VH CDR2 coding sequence
40gctatctatt atagtgggag cacctactac aacccgtccc tcaagagt
484169DNAArtificial sequenceF5 Fab VH CDR3 coding sequence
41cgtataggag tggctggcca atggtatttc gatctctggg gccgtggcac cctggtcacc
60gtctcaagc 694213PRTArtificial sequenceF5 Fab VL CDR1 42Thr Arg
Ser Thr Gly Ser Ile Thr Ser Asn Tyr Val His 1 5 10 437PRTArtificial
sequenceF5 Fab VL CDR2 43Glu Asp Asn Glu Arg Pro Ser 1 5
4411PRTArtificial sequenceF5 Fab VL CDR3 44Gln Ser Tyr Asp Asp Ser
Asn His Ile Ser Val 1 5 10 4539DNAArtificial sequenceF5 Fab VL CDR1
coding sequence 45acccgcagca ctggcagcat taccagcaac tatgtgcac
394621DNAArtificial sequenceF5 Fab VL CDR2 coding sequence
46gaggataacg aaagaccctc t 214733DNAArtificial sequenceF5 Fab VL
CDR3 coding sequence 47cagtcttatg atgacagcaa tcatatttct gtc
33484102DNAHuman cytomegalovirus 48aagcttacgg aaaatacgac agagaagacg
agtcctgtca ctttagccat ggtctgcggc 60gatctctaaa cagaggaccc tgataatggg
aaacggacac taggcgtccg cgccatacgg 120gattaaaaca aaaaaaaatc
ggtggtggtg tgtgatgggg tgtggtgacg gtggggcttc 180gcctcttttt
tttgtaataa aaaaagacac tgaataatcc gcggttgtct ctgtgtagaa
240cgtttttatt tcgggttccg cgtttggtcg cctgcctatg taaggcggcg
gccgcagagg 300gcgcgccgct cagtcgccta cacccgtacg cgcaggcagc
atggagtcgc gcggtcgccg 360ttgtcccgaa atgatatccg tactgggtcc
catttcgggg cacgtgctga aagccgtgtt 420tagtcgcggc gatacgccgg
tgctgccgca cgagacgcga ctcctgcaga cgggtatcca 480cgtacgcgtg
agccagccct cgctgatctt ggtatcgcag tacacgcccg actcgacgcc
540atgccaccgc ggcgacaatc agctgcaggt gcagcacacg tactttacgg
gcagcgaggt 600ggagaacgtg tcggtcaacg tgcacaaccc cacgggccga
agcatctgcc ccagccagga 660gcccatgtcg atctatgtgt acgcgctgcc
gctcaagatg ctgaacatcc ccagcatcaa 720cgtgcaccac tacccgtcgg
cggccgagcg caaacaccga cacctgcccg tagctgacgc 780tgtgattcac
gcgtcgggca agcagatgtg gcaggcgcgt ctcacggtct cgggactggc
840ctggacgcgt cagcagaacc agtggaaaga gcccgacgtc tactacacgt
cagcgttcgt 900gtttcccacc aaggacgtgg cactgcggca cgtggtgtgc
gcgcacgagc tggtttgctc 960catggagaac acgcgcgcaa ccaagatgca
ggtgataggt gaccagtacg tcaaggtgta 1020cctggagtcc ttctgcgagg
acgtgccctc cggcaagctc tttatgcacg tcacgctggg 1080ctctgacgtg
gaagaggacc tgacgatgac ccgcaacccg caacccttca tgcgccccca
1140cgagcgcaac ggctttacgg tgttgtgtcc caaaaatatg ataatcaaac
cgggcaagat 1200ctcgcacatc atgctggatg tggcttttac ctcacacgag
cattttgggc tgctgtgtcc 1260caagagcatc ccgggcctga gcatctcagg
taacctgttg atgaacgggc agcagatctt 1320cctggaggta caagccatac
gcgagaccgt ggaactgcgt cagtacgatc ccgtggctgc 1380gctcttcttt
ttcgatatcg acttgctgct gcagcgcggg cctcagtaca gcgagcaccc
1440caccttcacc agccagtatc gcatccaggg caagcttgag taccgacaca
cctgggaccg 1500gcacgacgag ggtgccgccc agggcgacga cgacgtctgg
accagcggat cggactccga 1560cgaagaactc gtaaccaccg agcgcaagac
gccccgcgtc accggcggcg gcgccatggc 1620gggcgcctcc acttccgcgg
gccgcaaacg caaatcagca tcctcggcga cggcgtgcac 1680gtcgggcgtt
atgacacgcg gccgccttaa ggccgagtcc accgtcgcgc ccgaagagga
1740caccgacgag gattccgaca acgaaatcca caatccggcc gtgttcacct
ggccgccctg 1800gcaggccggc atcctggccc gcaacctggt gcccatggtg
gctacggttc agggtcagaa 1860tctgaagtac caggaattct tctgggacgc
caacgacatc taccgcatct tcgccgaatt 1920ggaaggcgta tggcagcccg
ctgcgcaacc caaacgtcgc cgccaccggc aagacgcctt 1980gcccgggcca
tgcatcgcct cgacgcccaa aaagcaccga ggttgagcca cccgccgcac
2040gcgcttagga cgactctata aaaacccacg tccactcaga cacgcaactt
ttggccgcca 2100cacctgtcac cgctgctata tttgcgacag ttgccggaac
ccttcccgac ctcccacgaa 2160gacccgttca cctttgcgca tcccctgacc
ctccccccat cccgccttcg caatgtctca 2220ggcatcgtcc tcgcccggtg
agggaccctc gtcggaagcg gccgcgatca gcgaggccga 2280agccgccagc
ggaagctttg gtcgcctgca ctgccaggtg cttcggctca tcaccaacgt
2340ggaaggcggc tcgctggaag ccggtcgtct gcgactcctg gacctgcgta
ccaacataga 2400ggtgagccgg ccctcggttc tctgctgttt tcaggagaac
aaatctccgc acgacaccgt 2460agacctgacc gacttaaaca tcaagggccg
ctgcgtggtg ggcgaacagg accgactgct 2520ggtggacctc aacaactttg
gcccacgacg cctgacgcca ggctcagaaa acaacacggt 2580ctcggtactg
gcctttgcgc tgccgctgga ccgcgtgccc gttagcggac tgcacctctt
2640tcagagccag cggcgcggcg gcgaagaaaa tcggccgcga atggaagcgc
gcgccatcat 2700ccgccgcacg gctcaccact gggccgtgcg actgaccgta
acgccgaact ggcgccgcag 2760aaccgacagc agtttggagg cagggcagat
ctttgtcagc cagttcgcct ttcgcgccgg 2820cgccatcccg ctgacgctgg
tagacgccct ggagcagctg gcctgttcgg accctaacac 2880gtacatccac
aaaacggaga cggacgaacg aggccaatgg atcatgctgt ttctgcatca
2940cgactcaccg cacccgccga ccagcgtgtt tctgcacttt tcggtttaca
cgcatcgcgc 3000cgaggtggtg gcgcgacaca atccgtaccc gcacctacga
cgcttgccgg acaacggctt 3060ccagctgttg attcccaaaa gttttacgct
gacgcgcata catcccgagt acatcgtgca 3120gatccagaat gctttcgaga
ccaatcagac tcacgacacc atctttttcc cggaaaacat 3180cccgggcgtc
tccatagaag ccggcccgct acccgatcgt gtgcgaatca ccctccgcgt
3240cacgctgacc ggcgatcagg ccgttcattt ggaacaccga cagccgctag
gccgcatcca 3300ctttttccgc cgtgggtttt ggactctcac acccggtaaa
ccggacaaaa tcaagcgtcc 3360ccaggtgcag ctgcgcgccg gtctctttcc
acggagcaac gtcatgcgcg gcgccgtctc 3420cgagtttctc ccgcagtccc
ccggattacc acccaccgag gaagaggagg aagaagagga 3480agaggacgac
gaagatgacc tctcctccac accgacgccg acccccctgt ccgaagccat
3540gtttgccggc ttcgaggaag ccagcggcga cgaggactcg gacacccaag
ccggactgtc 3600cccggcactg atcctgaccg gacaaagacg tcgaagcggt
aacaacgggg ctctcacgct 3660cgtcatcccc tcgtggcacg tctttgcgag
ccttgacgac ttggtaccat taacggtgag 3720cgtgcagcac gccgcactac
gacctacctc ttatctgcgc agcgacatgg acggcgacgt 3780gcgtaccgcg
gcagacatca gcagcacgtt gcggtccgtg cccgcgccac gaccctcacc
3840catcagcacc gcttccactt ccagcacccc acgcagtcga ccccgcatct
agagagagac 3900ttctttgttt ttcccccgcg tgtttttccc attccctgta
tttatttcta aataataaaa 3960acacagagac gttgataata accgcggtgt
gctttattag ggtatcacgg tgtagaaaaa 4020aaagagaggg aagccctaaa
tatagcgtct ctcttactcg agcttattga gcgcagccac 4080aaaaatccgc
cgattcagat ct 4102491932DNAHuman cytomegalovirus 49gccatggcat
ccgtactggg tcccatttcg gggcacgtgc tgaaagccgt gtttagtcgc 60ggcgacacgc
cggtgctgcc gcacgagacg cgactcctgc agacgggtat ccacgtgcgc
120gtgagccagc cctcgctgat cctggtgtcg cagtacacgc ccgactcgac
gccatgccac 180cgcggcgaca atcagctgca ggtgcagcac acgtacttta
cgggcagcga ggtggagaac 240gtgtcggtca acgtgcacaa ccccacgggc
cggagcatct gccccagcca agagcccatg 300tcgatctatg tgtacgcgct
gccgctcaag atgctgaaca tccccagcat caacgtgcac 360cactacccgt
cggcggccga gcgcaaacac cgacacctgc ccgtagctga cgctgtgatt
420cacgcgtcgg gcaagcagat gtggcaggcg cgtctcacgg tctcgggact
ggcctggacg 480cgtcagcaga accagtggaa agagcccgac gtctactaca
cgtcagcgtt cgtgtttccc 540accaaggacg tggcactgcg gcacgtggtg
tgcgcgcacg agctggtttg ctccatggag 600aacacgcgcg caaccaagat
gcaggtgata ggtgaccagt acgtcaaggt gtacctggag 660tccttctgcg
aggacgtgcc ctccggcaag ctctttatgc acgtcacgct gggctctgac
720gtggaagagg acctgacgat gacccgcaac ccgcaaccct tcatgcgccc
ccacgagcgc 780aacggcttta cggtgttgtg tcccaaaaat atgataatca
aaccgggcaa gatctcgcac 840atcatgctgg atgtggcttt tacctcacac
gagcattttg ggctgctgtg tcccaagagc 900atcccgggcc tgagcatctc
aggtaaccta ttgatgaacg ggcagcagat cttcctggag 960gtgcaagcga
tacgcgagac cgtggaactg cgtcagtacg atcccgtggc tgcgctcttc
1020tttttcgata tcgacttgct gctgcagcgc gggcctcagt acagcgaaca
ccccaccttc 1080accagccagt atcgcatcca gggcaagctt gagtaccgac
acacctggga ccggcacgac 1140gagggtgccg cccagggcga cgacgacgtc
tggaccagcg gatcggactc cgacgaggaa 1200ctcgtaacca ccgagcgcaa
gacgccccgc gttaccggcg gcggcgccat ggcgggcgcc 1260tccacttccg
cgggccgcaa acgcaaatca gcatcctcgg cgacggcgtg cacggcgggc
1320gttatgacac gcggccgcct taaggccgag tccaccgtcg cgcccgaaga
ggacaccgac 1380gaggattccg acaacgaaat ccacaatccg gccgtgttca
cctggccgcc ctggcaggcc 1440ggcatcctgg cccgcaacct ggtgcccatg
gttgctacgg ttcagggtca gaatctgaag 1500taccaggagt tcttctggga
cgccaacgac atctaccgca tcttcgccga attggaaggc 1560gtatggcagc
ccgctgcgca acccaaacgt cgccgccacc ggcaagacgc cttgcccggg
1620ccatgcatcg cctcgacgcc caaaaagcac cgaggttgag ccacccgccg
cgcacgctta 1680ggacgactct ataaaaaccc acgtccactc agacacgcga
cttttggccg ccacacctgt 1740cgccgctgct atatttgcga cagttgccgg
aacccttccc gacctcccac gaagacccgt 1800tcacctttgc gcatcccctg
accccccccc tcatcccgcc ttcgcgatgt ctcaggcatc 1860gtcctcgccc
ggtgagggac cctcgtcgga agcggccgcg atcagcgagg ccgaagccgc
1920cagcggaagc tt 193250548PRTHuman cytomegalovirus 50Met Glu Ser
Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly 1 5 10 15 Pro
Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr 20 25
30 Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val
35 40 45 Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr
Pro Asp 50 55 60 Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln
Val Gln His Thr 65 70 75 80 Tyr Phe Thr Gly Ser Glu Val Glu Asn Val
Ser Val Asn Glu Pro Met 85 90 95 Ser Ile Tyr Val Tyr Ala Leu Pro
Leu Lys Met Leu Asn Ile Pro Ser 100 105 110 Ile Asn Val His His Tyr
Pro Ser Ala Ala Glu Arg Lys His Arg His 115 120 125 Leu Pro Val Ala
Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp 130 135 140 Gln Ala
Arg Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn 145 150 155
160 Gln Trp Lys Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro
165 170 175 Thr Lys Asp Val Ala Leu Arg His Val Val Cys Ala His Glu
Leu Val 180 185 190 Cys Ser Met Glu Asn Thr Arg Ala Thr Lys Met Gln
Val Ile Gly Asp 195 200 205 Gln Tyr Val Lys Val Tyr Leu Glu Ser Phe
Cys Glu Asp Val Pro Ser 210 215 220 Gly Lys Leu Phe Met His Val Thr
Leu Gly Ser Asp Val Glu Glu Asp 225 230 235 240 Leu Thr Met Thr Arg
Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg 245 250 255 Asn Gly Phe
Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly 260 265 270 Lys
Ile Ser His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His 275 280
285 Phe Gly Leu Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly
290 295 300 Asn Leu Leu Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln
Ala Ile 305 310 315 320 Arg Glu Thr Val Glu Leu Arg Gln Tyr Asp Pro
Val Ala Ala Leu Phe 325 330 335 Phe Phe Asp Ile Asp Leu Leu Leu Gln
Arg Gly Pro Gln Tyr Ser Glu 340 345 350 His Pro Thr Phe Thr Ser Gln
Tyr Arg Ile Gln Gly Lys Leu Glu Tyr 355 360 365 Arg His Thr Trp Asp
Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp 370 375 380 Asp Val Trp
Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr 385 390 395 400
Glu Arg Lys Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala 405
410 415 Ser Thr Ser Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr
Ala 420 425 430 Cys Thr Ser Gly Val Met Thr Arg Gly Arg Leu Lys Ala
Glu Ser Thr 435 440 445 Val Ala Pro Glu Glu Asp Thr Asp Glu Asp Ser
Asp Asn Glu Ile His 450 455 460 Asn Pro Ala Val Phe Thr Trp Pro Pro
Trp Gln Ala Gly Ile Leu Ala 465 470 475 480 Arg Asn Leu Val Pro Met
Val Ala Thr Val Gln Gly Gln Asn Leu Lys 485 490 495 Tyr Gln Glu Phe
Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala 500 505 510 Glu Leu
Glu Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg 515 520 525
His Arg Gln Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys 530
535 540 Lys His Arg Gly 545 51551PRTHuman cytomegalovirus 51Met Ala
Ser Val Leu Gly Pro Ile Ser Gly His Val Leu Lys Ala Val 1 5 10 15
Phe Ser Arg Gly Asp Thr Pro Val Leu Pro His Glu Thr Arg Leu Leu 20
25 30 Gln Thr Gly Ile His Val Arg Val Ser Gln Pro Ser Leu Ile Leu
Val 35 40 45 Ser Gln Tyr Thr Pro Asp Ser Thr Pro Cys His Arg Gly
Asp Asn Gln 50 55 60 Leu Gln Val Gln His Thr Tyr Phe Thr Gly Ser
Glu Val Glu Asn Val 65 70 75 80 Ser Val Asn Val His Asn Pro Thr Gly
Arg Ser Ile Cys Pro Ser Gln 85 90 95 Glu Pro Met Ser Ile Tyr Val
Tyr Ala Leu Pro Leu Lys Met Leu Asn 100 105 110 Ile Pro Ser Ile Asn
Val His His Tyr Pro Ser Ala Ala Glu Arg Lys 115 120 125 His Arg His
Leu Pro Val Ala Asp Ala Val Ile His Ala Ser Gly Lys 130 135 140 Gln
Met Trp Gln Ala Arg Leu Thr Val Ser Gly Leu Ala Trp Thr Arg 145 150
155 160 Gln Gln Asn Gln Trp Lys Glu Pro Asp Val Tyr Tyr Thr Ser Ala
Phe 165 170 175 Val Phe Pro Thr Lys Asp Val Ala Leu Arg His Val Val
Cys Ala His 180 185 190 Glu Leu Val Cys Ser Met Glu Asn Thr Arg Ala
Thr Lys Met Gln Val 195 200 205 Ile Gly Asp Gln Tyr Val Lys Val Tyr
Leu Glu Ser Phe Cys Glu Asp 210 215 220 Val Pro Ser Gly Lys Leu Phe
Met His Val Thr Leu Gly Ser Asp Val 225 230 235 240 Glu Glu Asp Leu
Thr Met Thr Arg Asn Pro Gln Pro Phe Met Arg Pro 245 250 255 His Glu
Arg Asn Gly Phe Thr Val Leu Cys Pro Lys Asn Met Ile Ile 260 265 270
Lys Pro Gly Lys Ile Ser His Ile Met Leu Asp Val Ala Phe Thr Ser 275
280 285 His Glu His Phe Gly Leu Leu Cys Pro Lys Ser Ile Pro Gly Leu
Ser 290 295 300 Ile Ser Gly Asn Leu Leu Met Asn Gly Gln Gln Ile Phe
Leu Glu Val 305 310 315 320 Gln Ala Ile Arg Glu Thr Val Glu Leu Arg
Gln Tyr Asp Pro Val Ala 325 330 335 Ala Leu Phe Phe Phe Asp Ile Asp
Leu Leu Leu Gln Arg Gly Pro Gln 340 345 350 Tyr Ser Glu His Pro Thr
Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys 355 360 365 Leu Glu Tyr Arg
His Thr Trp Asp Arg His Asp Glu Gly Ala Ala Gln 370 375 380 Gly Asp
Asp Asp Val Trp Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu 385 390 395
400 Val Thr Thr Glu Arg Lys Thr Pro Arg Val Thr Gly Gly Gly Ala Met
405 410 415 Ala Gly Ala Ser Thr Ser Ala Gly Arg Lys Arg Lys Ser Ala
Ser Ser 420 425 430 Ala Thr Ala Cys Thr Ala Gly Val Met Thr Arg Gly
Arg Leu Lys Ala 435 440 445 Glu Ser Thr Val Ala Pro Glu Glu Asp Thr
Asp Glu Asp Ser Asp Asn 450 455 460 Glu Ile His Asn Pro Ala Val Phe
Thr Trp Pro Pro Trp Gln Ala Gly 465 470 475 480 Ile Leu Ala Arg Asn
Leu Val Pro Met Val Ala Thr Val Gln Gly Gln 485 490 495 Asn Leu Lys
Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg 500 505 510 Ile
Phe Ala Glu Leu Glu Gly Val Trp Gln Pro Ala Ala Gln Pro Lys 515 520
525 Arg Arg Arg His Arg Gln Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser
530 535 540 Thr Pro Lys Lys His Arg Gly 545 550 52551PRTHuman
cytomegalovirus 52Met Ala Ser Val Leu Gly Pro Ile Ser Gly His Val
Leu Lys Ala Val 1 5 10 15 Phe Ser Arg Gly Asp Thr Pro Val Leu Pro
His Glu Thr Arg Leu Leu 20 25 30 Gln Thr Gly Ile His Val Arg Val
Ser Gln Pro Ser Leu Ile Leu Val 35 40 45 Ser Gln Tyr Thr Pro Asp
Ser Thr Pro Cys His Arg Gly Asp Asn Gln 50 55 60 Leu Gln Val Gln
His Thr Tyr Phe Thr Gly Ser Glu Val Glu Asn Val 65 70 75 80 Ser Val
Asn Val His Asn Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln 85 90 95
Glu Pro Met Ser Ile Tyr Val Tyr Ala Leu Pro Leu Lys Met Leu Asn 100
105 110 Ile Pro Ser Ile Asn Val His His Tyr Pro Ser Ala Ala Glu Arg
Lys 115 120 125 His Arg His Leu Pro Val Ala Asp Ala Val Ile His Ala
Ser Gly Lys 130 135 140 Gln Met Trp Gln Ala Arg Leu Thr Val Ser Gly
Leu Ala Trp Thr Arg 145 150 155 160 Gln Gln Asn Gln Trp Lys Glu Pro
Asp Val Tyr Tyr Thr Ser Ala Phe 165 170 175 Val Phe Pro Thr Lys Asp
Val Ala Leu Arg His Val Val Cys Ala His 180 185 190 Glu Leu Val Cys
Ser Met Glu Asn Thr Arg Ala Thr Lys Met Gln Val 195 200 205 Ile Gly
Asp Gln Tyr Val Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp 210 215 220
Val Pro Ser Gly Lys Leu Phe Met His Val Thr Leu Gly Ser Asp Val 225
230 235 240 Glu Glu Asp Leu Thr Met Thr Arg Asn Pro Gln Pro Phe Met
Arg Pro 245 250 255 His Glu Arg Asn Gly Phe Thr Val Leu Cys Pro Lys
Asn Met Ile Ile 260 265 270 Lys Pro Gly Lys Ile Ser His Ile Met Leu
Asp Val Ala Phe Thr Ser 275 280 285 His Glu His Phe Gly Leu Leu Cys
Pro Lys Ser Ile Pro Gly Leu Ser 290 295 300 Ile Ser Gly Asn Leu Leu
Met Asn Gly Gln Gln Ile Phe Leu Glu Val 305 310 315 320 Gln Ala Ile
Arg Glu Thr Val Glu Leu Arg Gln Tyr Asp Pro Val Ala 325 330 335 Ala
Leu Phe Phe Phe Asp Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln 340 345
350 Tyr Ser Glu His Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys
355 360 365 Leu Glu Tyr Arg His Thr Trp Asp Arg His Asp Glu Gly Ala
Ala Gln 370 375 380 Gly Asp Asp Asp Val Trp Thr Ser Gly Ser Asp Ser
Asp Glu Glu Leu 385 390 395 400 Val Thr Thr Glu Arg Lys Thr Pro Arg
Val Thr Gly Gly Gly Ala Met 405 410 415 Ala Gly Ala Ser Thr Ser Ala
Gly Arg Lys Arg Lys Ser Ala Ser Ser 420 425 430 Ala Thr Ala Cys Thr
Ala Gly Val Met Thr Arg Gly Arg Leu Lys Ala 435 440 445 Glu Ser Thr
Val Ala Pro Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn 450 455 460 Glu
Ile His Asn Pro Ala Val Phe Thr Trp Pro Pro Trp Gln Ala Gly 465 470
475 480 Ile Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val Gln Gly
Gln 485 490 495 Asn Leu
Lys Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg 500 505 510
Ile Phe Ala Glu Leu Glu Gly Val Trp Gln Pro Ala Ala Gln Pro Lys 515
520 525 Arg Arg Arg His Arg Gln Asp Ala Leu Pro Gly Pro Cys Ile Ala
Ser 530 535 540 Thr Pro Lys Lys His Arg Gly 545 550 53561PRTHuman
cytomegalovirus 53Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile
Ser Val Leu Gly 1 5 10 15 Pro Ile Ser Gly His Val Leu Lys Ala Val
Phe Ser Arg Gly Asp Thr 20 25 30 Pro Val Leu Pro His Glu Thr Arg
Leu Leu Gln Thr Gly Ile His Val 35 40 45 Arg Val Ser Gln Pro Ser
Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp 50 55 60 Ser Thr Pro Cys
His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr 65 70 75 80 Tyr Phe
Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn 85 90 95
Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr 100
105 110 Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn
Val 115 120 125 His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His
Leu Pro Val 130 135 140 Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln
Met Trp Gln Ala Arg 145 150 155 160 Leu Thr Val Ser Gly Leu Ala Trp
Thr Arg Gln Gln Asn Gln Trp Lys 165 170 175 Glu Pro Asp Val Tyr Tyr
Thr Ser Ala Phe Val Phe Pro Thr Lys Asp 180 185 190 Val Ala Leu Arg
His Val Val Cys Ala His Glu Leu Val Cys Ser Met 195 200 205 Glu Asn
Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val 210 215 220
Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu 225
230 235 240 Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu
Thr Met 245 250 255 Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu
Arg Asn Gly Phe 260 265 270 Thr Val Leu Cys Pro Lys Asn Met Ile Ile
Lys Pro Gly Lys Ile Ser 275 280 285 His Ile Met Leu Asp Val Ala Phe
Thr Ser His Glu His Phe Gly Leu 290 295 300 Leu Cys Pro Lys Ser Ile
Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu 305 310 315 320 Met Asn Gly
Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr 325 330 335 Val
Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp 340 345
350 Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr
355 360 365 Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg
His Thr 370 375 380 Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp
Asp Asp Val Trp 385 390 395 400 Thr Ser Gly Ser Asp Ser Asp Glu Glu
Leu Val Thr Thr Glu Arg Lys 405 410 415 Thr Pro Arg Val Thr Gly Gly
Gly Ala Met Ala Gly Ala Ser Thr Ser 420 425 430 Ala Gly Arg Lys Arg
Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ser 435 440 445 Gly Val Met
Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro 450 455 460 Glu
Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala 465 470
475 480 Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn
Leu 485 490 495 Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys
Tyr Gln Glu 500 505 510 Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile
Phe Ala Glu Leu Glu 515 520 525 Gly Val Trp Gln Pro Ala Ala Gln Pro
Lys Arg Arg Arg His Arg Gln 530 535 540 Asp Ala Leu Pro Gly Pro Cys
Ile Ala Ser Thr Pro Lys Lys His Arg 545 550 555 560 Gly
549PRTArtificial sequenceHLA restricted peptide 54Ala Ala Glu Arg
Lys His Arg His Leu 1 5 559PRTArtificial sequenceHLA restricted
peptide 55Ala Ala Leu Phe Phe Phe Asp Ile Asp 1 5 5610PRTArtificial
sequenceHLA restricted peptide 56Ala Ala Leu Phe Phe Phe Asp Ile
Asp Leu 1 5 10 579PRTArtificial sequenceHLA restricted peptide
57Ala Ala Gln Gly Asp Asp Asp Val Trp 1 5 5810PRTArtificial
sequenceHLA restricted peptide 58Ala Ala Gln Gly Asp Asp Asp Val
Trp Thr 1 5 10 599PRTArtificial sequenceHLA restricted peptide
59Ala Ala Gln Pro Lys Arg Arg Arg His 1 5 6010PRTArtificial
sequenceHLA restricted peptide 60Ala Ala Gln Pro Lys Arg Arg Arg
His Arg 1 5 10 619PRTArtificial sequenceHLA restricted peptide
61Ala Cys Thr Ala Gly Val Met Thr Arg 1 5 629PRTArtificial
sequenceHLA restricted peptide 62Ala Cys Thr Ser Gly Val Met Thr
Arg 1 5 6310PRTArtificial sequenceHLA restricted peptide 63Ala Asp
Ala Val Ile His Ala Ser Gly Lys 1 5 10 649PRTArtificial sequenceHLA
restricted peptide 64Ala Glu Leu Glu Gly Val Trp Gln Pro 1 5
6510PRTArtificial sequenceHLA restricted peptide 65Ala Glu Leu Glu
Gly Val Trp Gln Pro Ala 1 5 10 6610PRTArtificial sequenceHLA
restricted peptide 66Ala Glu Arg Lys His Arg His Leu Pro Val 1 5 10
679PRTArtificial sequenceHLA restricted peptide 67Ala Glu Ser Thr
Val Ala Pro Glu Glu 1 5 6810PRTArtificial sequenceHLA restricted
peptide 68Ala Glu Ser Thr Val Ala Pro Glu Glu Asp 1 5 10
6910PRTArtificial sequenceHLA restricted peptide 69Ala Phe Thr Ser
His Glu His Phe Gly Leu 1 5 10 709PRTArtificial sequenceHLA
restricted peptide 70Ala Phe Val Phe Pro Thr Lys Asp Val 1 5
7110PRTArtificial sequenceHLA restricted peptide 71Ala Phe Val Phe
Pro Thr Lys Asp Val Ala 1 5 10 729PRTArtificial sequenceHLA
restricted peptide 72Ala Gly Ala Ser Thr Ser Ala Gly Arg 1 5
7310PRTArtificial sequenceHLA restricted peptide 73Ala Gly Ala Ser
Thr Ser Ala Gly Arg Lys 1 5 10 749PRTArtificial sequenceHLA
restricted peptide 74Ala Gly Ile Leu Ala Arg Asn Leu Val 1 5
7510PRTArtificial sequenceHLA restricted peptide 75Ala Gly Ile Leu
Ala Arg Asn Leu Val Pro 1 5 10 769PRTArtificial sequenceHLA
restricted peptide 76Ala Gly Arg Lys Arg Lys Ser Ala Ser 1 5
7710PRTArtificial sequenceHLA restricted peptide 77Ala Gly Arg Lys
Arg Lys Ser Ala Ser Ser 1 5 10 789PRTArtificial sequenceHLA
restricted peptide 78Ala Gly Val Met Thr Arg Gly Arg Leu 1 5
7910PRTArtificial sequenceHLA restricted peptide 79Ala Gly Val Met
Thr Arg Gly Arg Leu Lys 1 5 10 809PRTArtificial sequenceHLA
restricted peptide 80Ala His Glu Leu Val Cys Ser Met Glu 1 5
8110PRTArtificial sequenceHLA restricted peptide 81Ala His Glu Leu
Val Cys Ser Met Glu Asn 1 5 10 829PRTArtificial sequenceHLA
restricted peptide 82Ala Ile Arg Glu Thr Val Glu Leu Arg 1 5
839PRTArtificial sequenceHLA restricted peptide 83Ala Leu Phe Phe
Phe Asp Ile Asp Leu 1 5 8410PRTArtificial sequenceHLA restricted
peptide 84Ala Leu Phe Phe Phe Asp Ile Asp Leu Leu 1 5 10
859PRTArtificial sequenceHLA restricted peptide 85Ala Leu Pro Gly
Pro Cys Ile Ala Ser 1 5 8610PRTArtificial sequenceHLA restricted
peptide 86Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr 1 5 10
879PRTArtificial sequenceHLA restricted peptide 87Ala Leu Pro Leu
Lys Met Leu Asn Ile 1 5 889PRTArtificial sequenceHLA restricted
peptide 88Ala Leu Arg His Val Val Cys Ala His 1 5 8910PRTArtificial
sequenceHLA restricted peptide 89Ala Leu Arg His Val Val Cys Ala
His Glu 1 5 10 909PRTArtificial sequenceHLA restricted peptide
90Ala Met Ala Gly Ala Ser Thr Ser Ala 1 5 919PRTArtificial
sequenceHLA restricted peptide 91Ala Asn Asp Ile Tyr Arg Ile Phe
Ala 1 5 929PRTArtificial sequenceHLA restricted peptide 92Ala Pro
Glu Glu Asp Thr Asp Glu Asp 1 5 9310PRTArtificial sequenceHLA
restricted peptide 93Ala Pro Glu Glu Asp Thr Asp Glu Asp Ser 1 5 10
949PRTArtificial sequenceHLA restricted peptide 94Ala Gln Gly Asp
Asp Asp Val Trp Thr 1 5 9510PRTArtificial sequenceHLA restricted
peptide 95Ala Gln Gly Asp Asp Asp Val Trp Thr Ser 1 5 10
969PRTArtificial sequenceHLA restricted peptide 96Ala Gln Pro Lys
Arg Arg Arg His Arg 1 5 9710PRTArtificial sequenceHLA restricted
peptide 97Ala Gln Pro Lys Arg Arg Arg His Arg Gln 1 5 10
989PRTArtificial sequenceHLA restricted peptide 98Ala Arg Leu Thr
Val Ser Gly Leu Ala 1 5 9910PRTArtificial sequenceHLA restricted
peptide 99Ala Arg Leu Thr Val Ser Gly Leu Ala Trp 1 5 10
1009PRTArtificial sequenceHLA restricted peptide 100Ala Arg Asn Leu
Val Pro Met Val Ala 1 5 10110PRTArtificial sequenceHLA restricted
peptide 101Ala Arg Asn Leu Val Pro Met Val Ala Thr 1 5 10
1029PRTArtificial sequenceHLA restricted peptide 102Ala Ser Gly Lys
Gln Met Trp Gln Ala 1 5 10310PRTArtificial sequenceHLA restricted
peptide 103Ala Ser Gly Lys Gln Met Trp Gln Ala Arg 1 5 10
1049PRTArtificial sequenceHLA restricted peptide 104Ala Ser Ser Ala
Thr Ala Cys Thr Ala 1 5 1059PRTArtificial sequenceHLA restricted
peptide 105Ala Ser Ser Ala Thr Ala Cys Thr Ser 1 5
1069PRTArtificial sequenceHLA restricted peptide 106Ala Ser Thr Ser
Ala Gly Arg Lys Arg 1 5 10710PRTArtificial sequenceHLA restricted
peptide 107Ala Ser Thr Ser Ala Gly Arg Lys Arg Lys 1 5 10
10810PRTArtificial sequenceHLA restricted peptide 108Ala Ser Val
Leu Gly Pro Ile Ser Gly His 1 5 10 1099PRTArtificial sequenceHLA
restricted peptide 109Ala Thr Ala Cys Thr Ala Gly Val Met 1 5
11010PRTArtificial sequenceHLA restricted peptide 110Ala Thr Ala
Cys Thr Ala Gly Val Met Thr 1 5 10 1119PRTArtificial sequenceHLA
restricted peptide 111Ala Thr Ala Cys Thr Ser Gly Val Met 1 5
11210PRTArtificial sequenceHLA restricted peptide 112Ala Thr Ala
Cys Thr Ser Gly Val Met Thr 1 5 10 1139PRTArtificial sequenceHLA
restricted peptide 113Ala Thr Val Gln Gly Gln Asn Leu Lys 1 5
11410PRTArtificial sequenceHLA restricted peptide 114Ala Thr Val
Gln Gly Gln Asn Leu Lys Tyr 1 5 10 11510PRTArtificial sequenceHLA
restricted peptide 115Ala Val Phe Ser Arg Gly Asp Thr Pro Val 1 5
10 11610PRTArtificial sequenceHLA restricted peptide 116Ala Val Phe
Thr Trp Pro Pro Trp Gln Ala 1 5 10 11710PRTArtificial sequenceHLA
restricted peptide 117Ala Val Ile His Ala Ser Gly Lys Gln Met 1 5
10 1189PRTArtificial sequenceHLA restricted peptide 118Ala Trp Thr
Arg Gln Gln Asn Gln Trp 1 5 11910PRTArtificial sequenceHLA
restricted peptide 119Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys 1 5
10 1209PRTArtificial sequenceHLA restricted peptide 120Cys Ala His
Glu Leu Val Cys Ser Met 1 5 1219PRTArtificial sequenceHLA
restricted peptide 121Cys Glu Asp Val Pro Ser Gly Lys Leu 1 5
12210PRTArtificial sequenceHLA restricted peptide 122Cys Glu Asp
Val Pro Ser Gly Lys Leu Phe 1 5 10 12310PRTArtificial sequenceHLA
restricted peptide 123Cys His Arg Gly Asp Asn Gln Leu Gln Val 1 5
10 12410PRTArtificial sequenceHLA restricted peptide 124Cys Ile Ala
Ser Thr Pro Lys Lys His Arg 1 5 10 1259PRTArtificial sequenceHLA
restricted peptide 125Cys Pro Glu Met Ile Ser Val Leu Gly 1 5
12610PRTArtificial sequenceHLA restricted peptide 126Cys Pro Glu
Met Ile Ser Val Leu Gly Pro 1 5 10 1279PRTArtificial sequenceHLA
restricted peptide 127Cys Pro Lys Asn Met Ile Ile Lys Pro 1 5
12810PRTArtificial sequenceHLA restricted peptide 128Cys Pro Lys
Asn Met Ile Ile Lys Pro Gly 1 5 10 1299PRTArtificial sequenceHLA
restricted peptide 129Cys Pro Lys Ser Ile Pro Gly Leu Ser 1 5
13010PRTArtificial sequenceHLA restricted peptide 130Cys Pro Lys
Ser Ile Pro Gly Leu Ser Ile 1 5 10 1319PRTArtificial sequenceHLA
restricted peptide 131Cys Pro Ser Gln Glu Pro Met Ser Ile 1 5
13210PRTArtificial sequenceHLA restricted peptide 132Cys Pro Ser
Gln Glu Pro Met Ser Ile Tyr 1 5 10 1339PRTArtificial sequenceHLA
restricted peptide 133Cys Ser Met Glu Asn Thr Arg Ala Thr 1 5
13410PRTArtificial sequenceHLA restricted peptide 134Cys Ser Met
Glu Asn Thr Arg Ala Thr Lys 1 5 10 13510PRTArtificial sequenceHLA
restricted peptide 135Cys Thr Ala Gly Val Met Thr Arg Gly Arg 1 5
10 13610PRTArtificial sequenceHLA restricted peptide 136Cys Thr Ser
Gly Val Met Thr Arg Gly Arg 1 5 10 1379PRTArtificial sequenceHLA
restricted peptide 137Asp Ala Leu Pro Gly Pro Cys Ile Ala 1 5
13810PRTArtificial sequenceHLA restricted peptide 138Asp Ala Leu
Pro Gly Pro Cys Ile Ala Ser 1 5 10 1399PRTArtificial sequenceHLA
restricted peptide 139Asp Ala Asn Asp Ile Tyr Arg Ile Phe 1 5
14010PRTArtificial sequenceHLA restricted peptide 140Asp Ala Asn
Asp Ile Tyr Arg Ile Phe Ala 1 5 10 1419PRTArtificial sequenceHLA
restricted peptide 141Asp Ala Val Ile His Ala Ser Gly Lys 1 5
14210PRTArtificial sequenceHLA restricted peptide 142Asp Ala Val
Ile His Ala Ser Gly Lys Gln 1 5 10 1439PRTArtificial sequenceHLA
restricted peptide 143Asp Asp Asp Val Trp Thr Ser Gly Ser 1 5
14410PRTArtificial sequenceHLA restricted peptide 144Asp Asp Val
Trp Thr Ser Gly Ser Asp Ser 1 5 10 1459PRTArtificial sequenceHLA
restricted peptide 145Asp Glu Asp Ser Asp Asn Glu Ile His 1 5
14610PRTArtificial sequenceHLA restricted peptide 146Asp Glu Asp
Ser Asp Asn Glu Ile His Asn 1 5 10 1479PRTArtificial sequenceHLA
restricted peptide 147Asp Glu Glu Leu Val Thr Thr Glu Arg 1 5
14810PRTArtificial sequenceHLA restricted peptide 148Asp Glu Glu
Leu Val Thr Thr Glu Arg Lys 1 5 10 1499PRTArtificial sequenceHLA
restricted peptide 149Asp Glu Gly Ala Ala Gln Gly Asp Asp 1 5
15010PRTArtificial sequenceHLA restricted peptide 150Asp Glu Gly
Ala Ala Gln Gly Asp Asp Asp 1 5 10 1519PRTArtificial sequenceHLA
restricted peptide 151Asp Ile Tyr Arg Ile Phe Ala Glu Leu 1 5
15210PRTArtificial sequenceHLA restricted peptide 152Asp Leu Leu
Leu Gln Arg Gly Pro Gln Tyr 1 5 10 1539PRTArtificial sequenceHLA
restricted peptide 153Asp Asn Glu Ile His Asn Pro Ala Val 1 5
15410PRTArtificial sequenceHLA restricted peptide 154Asp Asn Glu
Ile His Asn Pro Ala Val Phe 1 5 10 15510PRTArtificial sequenceHLA
restricted peptide 155Asp Asn Gln Leu Gln
Val Gln His Thr Tyr 1 5 10 1569PRTArtificial sequenceHLA restricted
peptide 156Asp Pro Val Ala Ala Leu Phe Phe Phe 1 5
15710PRTArtificial sequenceHLA restricted peptide 157Asp Pro Val
Ala Ala Leu Phe Phe Phe Asp 1 5 10 1589PRTArtificial sequenceHLA
restricted peptide 158Asp Gln Tyr Val Lys Val Tyr Leu Glu 1 5
15910PRTArtificial sequenceHLA restricted peptide 159Asp Gln Tyr
Val Lys Val Tyr Leu Glu Ser 1 5 10 1609PRTArtificial sequenceHLA
restricted peptide 160Asp Arg His Asp Glu Gly Ala Ala Gln 1 5
16110PRTArtificial sequenceHLA restricted peptide 161Asp Arg His
Asp Glu Gly Ala Ala Gln Gly 1 5 10 1629PRTArtificial sequenceHLA
restricted peptide 162Asp Ser Asp Glu Glu Leu Val Thr Thr 1 5
16310PRTArtificial sequenceHLA restricted peptide 163Asp Ser Asp
Asn Glu Ile His Asn Pro Ala 1 5 10 16410PRTArtificial sequenceHLA
restricted peptide 164Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile 1 5
10 1659PRTArtificial sequenceHLA restricted peptide 165Asp Thr Pro
Val Leu Pro His Glu Thr 1 5 16610PRTArtificial sequenceHLA
restricted peptide 166Asp Thr Pro Val Leu Pro His Glu Thr Arg 1 5
10 1679PRTArtificial sequenceHLA restricted peptide 167Asp Val Ala
Phe Thr Ser His Glu His 1 5 16810PRTArtificial sequenceHLA
restricted peptide 168Asp Val Ala Phe Thr Ser His Glu His Phe 1 5
10 1699PRTArtificial sequenceHLA restricted peptide 169Asp Val Ala
Leu Arg His Val Val Cys 1 5 17010PRTArtificial sequenceHLA
restricted peptide 170Asp Val Ala Leu Arg His Val Val Cys Ala 1 5
10 1719PRTArtificial sequenceHLA restricted peptide 171Asp Val Glu
Glu Asp Leu Thr Met Thr 1 5 17210PRTArtificial sequenceHLA
restricted peptide 172Asp Val Glu Glu Asp Leu Thr Met Thr Arg 1 5
10 1739PRTArtificial sequenceHLA restricted peptide 173Asp Val Pro
Ser Gly Lys Leu Phe Met 1 5 17410PRTArtificial sequenceHLA
restricted peptide 174Asp Val Pro Ser Gly Lys Leu Phe Met His 1 5
10 1759PRTArtificial sequenceHLA restricted peptide 175Asp Val Trp
Thr Ser Gly Ser Asp Ser 1 5 17610PRTArtificial sequenceHLA
restricted peptide 176Asp Val Trp Thr Ser Gly Ser Asp Ser Asp 1 5
10 1779PRTArtificial sequenceHLA restricted peptide 177Asp Val Tyr
Tyr Thr Ser Ala Phe Val 1 5 17810PRTArtificial sequenceHLA
restricted peptide 178Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe 1 5
10 1799PRTArtificial sequenceHLA restricted peptide 179Glu Asp Ser
Asp Asn Glu Ile His Asn 1 5 1809PRTArtificial sequenceHLA
restricted peptide 180Glu Asp Thr Asp Glu Asp Ser Asp Asn 1 5
1819PRTArtificial sequenceHLA restricted peptide 181Glu Asp Val Pro
Ser Gly Lys Leu Phe 1 5 18210PRTArtificial sequenceHLA restricted
peptide 182Glu Asp Val Pro Ser Gly Lys Leu Phe Met 1 5 10
1839PRTArtificial sequenceHLA restricted peptide 183Glu Glu Asp Leu
Thr Met Thr Arg Asn 1 5 18410PRTArtificial sequenceHLA restricted
peptide 184Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn 1 5 10
1859PRTArtificial sequenceHLA restricted peptide 185Glu Glu Leu Val
Thr Thr Glu Arg Lys 1 5 18610PRTArtificial sequenceHLA restricted
peptide 186Glu Glu Leu Val Thr Thr Glu Arg Lys Thr 1 5 10
1879PRTArtificial sequenceHLA restricted peptide 187Glu Phe Phe Trp
Asp Ala Asn Asp Ile 1 5 18810PRTArtificial sequenceHLA restricted
peptide 188Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr 1 5 10
18910PRTArtificial sequenceHLA restricted peptide 189Glu Gly Ala
Ala Gln Gly Asp Asp Asp Val 1 5 10 1909PRTArtificial sequenceHLA
restricted peptide 190Glu His Phe Gly Leu Leu Cys Pro Lys 1 5
19110PRTArtificial sequenceHLA restricted peptide 191Glu His Phe
Gly Leu Leu Cys Pro Lys Ser 1 5 10 1929PRTArtificial sequenceHLA
restricted peptide 192Glu His Pro Thr Phe Thr Ser Gln Tyr 1 5
19310PRTArtificial sequenceHLA restricted peptide 193Glu His Pro
Thr Phe Thr Ser Gln Tyr Arg 1 5 10 1949PRTArtificial sequenceHLA
restricted peptide 194Glu Ile His Asn Pro Ala Val Phe Thr 1 5
19510PRTArtificial sequenceHLA restricted peptide 195Glu Ile His
Asn Pro Ala Val Phe Thr Trp 1 5 10 1969PRTArtificial sequenceHLA
restricted peptide 196Glu Leu Glu Gly Val Trp Gln Pro Ala 1 5
19710PRTArtificial sequenceHLA restricted peptide 197Glu Leu Glu
Gly Val Trp Gln Pro Ala Ala 1 5 10 1989PRTArtificial sequenceHLA
restricted peptide 198Glu Leu Arg Gln Tyr Asp Pro Val Ala 1 5
19910PRTArtificial sequenceHLA restricted peptide 199Glu Leu Arg
Gln Tyr Asp Pro Val Ala Ala 1 5 10 2009PRTArtificial sequenceHLA
restricted peptide 200Glu Leu Val Cys Ser Met Glu Asn Thr 1 5
20110PRTArtificial sequenceHLA restricted peptide 201Glu Leu Val
Cys Ser Met Glu Asn Thr Arg 1 5 10 2029PRTArtificial sequenceHLA
restricted peptide 202Glu Leu Val Thr Thr Glu Arg Lys Thr 1 5
2039PRTArtificial sequenceHLA restricted peptide 203Glu Met Ile Ser
Val Leu Gly Pro Ile 1 5 20410PRTArtificial sequenceHLA restricted
peptide 204Glu Met Ile Ser Val Leu Gly Pro Ile Ser 1 5 10
20510PRTArtificial sequenceHLA restricted peptide 205Glu Asn Thr
Arg Ala Thr Lys Met Gln Val 1 5 10 2069PRTArtificial sequenceHLA
restricted peptide 206Glu Pro Asp Val Tyr Tyr Thr Ser Ala 1 5
20710PRTArtificial sequenceHLA restricted peptide 207Glu Pro Asp
Val Tyr Tyr Thr Ser Ala Phe 1 5 10 2089PRTArtificial sequenceHLA
restricted peptide 208Glu Pro Met Ser Ile Tyr Val Tyr Ala 1 5
20910PRTArtificial sequenceHLA restricted peptide 209Glu Pro Met
Ser Ile Tyr Val Tyr Ala Leu 1 5 10 2109PRTArtificial sequenceHLA
restricted peptide 210Glu Arg Lys His Arg His Leu Pro Val 1 5
21110PRTArtificial sequenceHLA restricted peptide 211Glu Arg Lys
His Arg His Leu Pro Val Ala 1 5 10 2129PRTArtificial sequenceHLA
restricted peptide 212Glu Arg Lys Thr Pro Arg Val Thr Gly 1 5
21310PRTArtificial sequenceHLA restricted peptide 213Glu Arg Lys
Thr Pro Arg Val Thr Gly Gly 1 5 10 2149PRTArtificial sequenceHLA
restricted peptide 214Glu Arg Asn Gly Phe Thr Val Leu Cys 1 5
21510PRTArtificial sequenceHLA restricted peptide 215Glu Arg Asn
Gly Phe Thr Val Leu Cys Pro 1 5 10 2169PRTArtificial sequenceHLA
restricted peptide 216Glu Ser Phe Cys Glu Asp Val Pro Ser 1 5
21710PRTArtificial sequenceHLA restricted peptide 217Glu Ser Arg
Gly Arg Arg Cys Pro Glu Met 1 5 10 21810PRTArtificial sequenceHLA
restricted peptide 218Glu Ser Thr Val Ala Pro Glu Glu Asp Thr 1 5
10 2199PRTArtificial sequenceHLA restricted peptide 219Glu Thr Arg
Leu Leu Gln Thr Gly Ile 1 5 22010PRTArtificial sequenceHLA
restricted peptide 220Glu Thr Arg Leu Leu Gln Thr Gly Ile His 1 5
10 2219PRTArtificial sequenceHLA restricted peptide 221Glu Val Glu
Asn Val Ser Val Asn Val 1 5 22210PRTArtificial sequenceHLA
restricted peptide 222Glu Val Glu Asn Val Ser Val Asn Val His 1 5
10 2239PRTArtificial sequenceHLA restricted peptide 223Glu Val Gln
Ala Ile Arg Glu Thr Val 1 5 22410PRTArtificial sequenceHLA
restricted peptide 224Glu Val Gln Ala Ile Arg Glu Thr Val Glu 1 5
10 2259PRTArtificial sequenceHLA restricted peptide 225Glu Tyr Arg
His Thr Trp Asp Arg His 1 5 2269PRTArtificial sequenceHLA
restricted peptide 226Phe Ala Glu Leu Glu Gly Val Trp Gln 1 5
2279PRTArtificial sequenceHLA restricted peptide 227Phe Cys Glu Asp
Val Pro Ser Gly Lys 1 5 22810PRTArtificial sequenceHLA restricted
peptide 228Phe Cys Glu Asp Val Pro Ser Gly Lys Leu 1 5 10
2299PRTArtificial sequenceHLA restricted peptide 229Phe Asp Ile Asp
Leu Leu Leu Gln Arg 1 5 2309PRTArtificial sequenceHLA restricted
peptide 230Phe Phe Asp Ile Asp Leu Leu Leu Gln 1 5
23110PRTArtificial sequenceHLA restricted peptide 231Phe Phe Asp
Ile Asp Leu Leu Leu Gln Arg 1 5 10 2329PRTArtificial sequenceHLA
restricted peptide 232Phe Phe Phe Asp Ile Asp Leu Leu Leu 1 5
23310PRTArtificial sequenceHLA restricted peptide 233Phe Phe Phe
Asp Ile Asp Leu Leu Leu Gln 1 5 10 2349PRTArtificial sequenceHLA
restricted peptide 234Phe Phe Trp Asp Ala Asn Asp Ile Tyr 1 5
23510PRTArtificial sequenceHLA restricted peptide 235Phe Phe Trp
Asp Ala Asn Asp Ile Tyr Arg 1 5 10 2369PRTArtificial sequenceHLA
restricted peptide 236Phe Gly Leu Leu Cys Pro Lys Ser Ile 1 5
23710PRTArtificial sequenceHLA restricted peptide 237Phe Gly Leu
Leu Cys Pro Lys Ser Ile Pro 1 5 10 23810PRTArtificial sequenceHLA
restricted peptide 238Phe Leu Glu Val Gln Ala Ile Arg Glu Thr 1 5
10 23910PRTArtificial sequenceHLA restricted peptide 239Phe Met His
Val Thr Leu Gly Ser Asp Val 1 5 10 24010PRTArtificial sequenceHLA
restricted peptide 240Phe Met Arg Pro His Glu Arg Asn Gly Phe 1 5
10 2419PRTArtificial sequenceHLA restricted peptide 241Phe Pro Thr
Lys Asp Val Ala Leu Arg 1 5 24210PRTArtificial sequenceHLA
restricted peptide 242Phe Pro Thr Lys Asp Val Ala Leu Arg His 1 5
10 2439PRTArtificial sequenceHLA restricted peptide 243Phe Ser Arg
Gly Asp Thr Pro Val Leu 1 5 2449PRTArtificial sequenceHLA
restricted peptide 244Phe Thr Gly Ser Glu Val Glu Asn Val 1 5
24510PRTArtificial sequenceHLA restricted peptide 245Phe Thr Gly
Ser Glu Val Glu Asn Val Ser 1 5 10 2469PRTArtificial sequenceHLA
restricted peptide 246Phe Thr Ser His Glu His Phe Gly Leu 1 5
24710PRTArtificial sequenceHLA restricted peptide 247Phe Thr Ser
His Glu His Phe Gly Leu Leu 1 5 10 24810PRTArtificial sequenceHLA
restricted peptide 248Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys 1 5
10 2499PRTArtificial sequenceHLA restricted peptide 249Phe Thr Val
Leu Cys Pro Lys Asn Met 1 5 25010PRTArtificial sequenceHLA
restricted peptide 250Phe Thr Val Leu Cys Pro Lys Asn Met Ile 1 5
10 25110PRTArtificial sequenceHLA restricted peptide 251Phe Thr Trp
Pro Pro Trp Gln Ala Gly Ile 1 5 10 2529PRTArtificial sequenceHLA
restricted peptide 252Phe Val Phe Pro Thr Lys Asp Val Ala 1 5
25310PRTArtificial sequenceHLA restricted peptide 253Phe Val Phe
Pro Thr Lys Asp Val Ala Leu 1 5 10 2549PRTArtificial sequenceHLA
restricted peptide 254Phe Trp Asp Ala Asn Asp Ile Tyr Arg 1 5
25510PRTArtificial sequenceHLA restricted peptide 255Phe Trp Asp
Ala Asn Asp Ile Tyr Arg Ile 1 5 10 2569PRTArtificial sequenceHLA
restricted peptide 256Gly Ala Ala Gln Gly Asp Asp Asp Val 1 5
25710PRTArtificial sequenceHLA restricted peptide 257Gly Ala Ala
Gln Gly Asp Asp Asp Val Trp 1 5 10 2589PRTArtificial sequenceHLA
restricted peptide 258Gly Ala Met Ala Gly Ala Ser Thr Ser 1 5
25910PRTArtificial sequenceHLA restricted peptide 259Gly Ala Met
Ala Gly Ala Ser Thr Ser Ala 1 5 10 2609PRTArtificial sequenceHLA
restricted peptide 260Gly Ala Ser Thr Ser Ala Gly Arg Lys 1 5
26110PRTArtificial sequenceHLA restricted peptide 261Gly Ala Ser
Thr Ser Ala Gly Arg Lys Arg 1 5 10 26210PRTArtificial sequenceHLA
restricted peptide 262Gly Asp Asp Asp Val Trp Thr Ser Gly Ser 1 5
10 26310PRTArtificial sequenceHLA restricted peptide 263Gly Asp Asn
Gln Leu Gln Val Gln His Thr 1 5 10 2649PRTArtificial sequenceHLA
restricted peptide 264Gly Asp Gln Tyr Val Lys Val Tyr Leu 1 5
26510PRTArtificial sequenceHLA restricted peptide 265Gly Asp Gln
Tyr Val Lys Val Tyr Leu Glu 1 5 10 26610PRTArtificial sequenceHLA
restricted peptide 266Gly Asp Thr Pro Val Leu Pro His Glu Thr 1 5
10 2679PRTArtificial sequenceHLA restricted peptide 267Gly Phe Thr
Val Leu Cys Pro Lys Asn 1 5 26810PRTArtificial sequenceHLA
restricted peptide 268Gly Phe Thr Val Leu Cys Pro Lys Asn Met 1 5
10 2699PRTArtificial sequenceHLA restricted peptide 269Gly Gly Ala
Met Ala Gly Ala Ser Thr 1 5 27010PRTArtificial sequenceHLA
restricted peptide 270Gly Gly Ala Met Ala Gly Ala Ser Thr Ser 1 5
10 2719PRTArtificial sequenceHLA restricted peptide 271Gly Gly Gly
Ala Met Ala Gly Ala Ser 1 5 27210PRTArtificial sequenceHLA
restricted peptide 272Gly Gly Gly Ala Met Ala Gly Ala Ser Thr 1 5
10 2739PRTArtificial sequenceHLA restricted peptide 273Gly His Val
Leu Lys Ala Val Phe Ser 1 5 27410PRTArtificial sequenceHLA
restricted peptide 274Gly His Val Leu Lys Ala Val Phe Ser Arg 1 5
10 27510PRTArtificial sequenceHLA restricted peptide 275Gly Ile His
Val Arg Val Ser Gln Pro Ser 1 5 10 27610PRTArtificial sequenceHLA
restricted peptide 276Gly Ile Leu Ala Arg Asn Leu Val Pro Met 1 5
10 27710PRTArtificial sequenceHLA restricted peptide 277Gly Lys Ile
Ser His Ile Met Leu Asp Val 1 5 10 2789PRTArtificial sequenceHLA
restricted peptide 278Gly Lys Leu Glu Tyr Arg His Thr Trp 1 5
27910PRTArtificial sequenceHLA restricted peptide 279Gly Lys Leu
Glu Tyr Arg His Thr Trp Asp 1 5 10 2809PRTArtificial sequenceHLA
restricted peptide 280Gly Lys Leu Phe Met His Val Thr Leu 1 5
2819PRTArtificial sequenceHLA restricted peptide 281Gly Lys Gln Met
Trp Gln Ala Arg Leu 1 5 28210PRTArtificial sequenceHLA restricted
peptide 282Gly Lys Gln Met Trp Gln Ala Arg Leu Thr 1 5 10
2839PRTArtificial sequenceHLA restricted peptide 283Gly Leu Ala Trp
Thr Arg Gln Gln Asn 1 5 2849PRTArtificial sequenceHLA restricted
peptide 284Gly Leu Ser Ile Ser Gly Asn Leu Leu 1 5
28510PRTArtificial sequenceHLA restricted peptide 285Gly Leu Ser
Ile Ser Gly Asn Leu Leu Met 1 5 10 28610PRTArtificial sequenceHLA
restricted peptide 286Gly Asn Leu Leu Met Asn Gly Gln Gln Ile 1 5
10 2879PRTArtificial sequenceHLA restricted peptide 287Gly Pro Cys
Ile Ala Ser Thr Pro Lys 1 5 28810PRTArtificial sequenceHLA
restricted peptide 288Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys 1 5
10 2899PRTArtificial sequenceHLA restricted peptide 289Gly Pro Ile
Ser Gly His Val Leu Lys 1 5 29010PRTArtificial sequenceHLA
restricted peptide 290Gly Pro Ile Ser Gly His Val Leu Lys Ala 1 5
10 2919PRTArtificial sequenceHLA restricted peptide 291Gly Pro Gln
Tyr Ser Glu His Pro Thr 1 5 29210PRTArtificial sequenceHLA
restricted peptide 292Gly Pro Gln Tyr Ser Glu His Pro Thr Phe 1
5
10 2939PRTArtificial sequenceHLA restricted peptide 293Gly Gln Asn
Leu Lys Tyr Gln Glu Phe 1 5 29410PRTArtificial sequenceHLA
restricted peptide 294Gly Gln Asn Leu Lys Tyr Gln Glu Phe Phe 1 5
10 2959PRTArtificial sequenceHLA restricted peptide 295Gly Gln Gln
Ile Phe Leu Glu Val Gln 1 5 29610PRTArtificial sequenceHLA
restricted peptide 296Gly Gln Gln Ile Phe Leu Glu Val Gln Ala 1 5
10 2979PRTArtificial sequenceHLA restricted peptide 297Gly Arg Lys
Arg Lys Ser Ala Ser Ser 1 5 29810PRTArtificial sequenceHLA
restricted peptide 298Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala 1 5
10 2999PRTArtificial sequenceHLA restricted peptide 299Gly Arg Leu
Lys Ala Glu Ser Thr Val 1 5 30010PRTArtificial sequenceHLA
restricted peptide 300Gly Arg Leu Lys Ala Glu Ser Thr Val Ala 1 5
10 3019PRTArtificial sequenceHLA restricted peptide 301Gly Arg Arg
Cys Pro Glu Met Ile Ser 1 5 30210PRTArtificial sequenceHLA
restricted peptide 302Gly Arg Arg Cys Pro Glu Met Ile Ser Val 1 5
10 3039PRTArtificial sequenceHLA restricted peptide 303Gly Arg Ser
Ile Cys Pro Ser Gln Glu 1 5 30410PRTArtificial sequenceHLA
restricted peptide 304Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro 1 5
10 3059PRTArtificial sequenceHLA restricted peptide 305Gly Ser Asp
Ser Asp Glu Glu Leu Val 1 5 30610PRTArtificial sequenceHLA
restricted peptide 306Gly Ser Asp Ser Asp Glu Glu Leu Val Thr 1 5
10 3079PRTArtificial sequenceHLA restricted peptide 307Gly Ser Asp
Val Glu Glu Asp Leu Thr 1 5 30810PRTArtificial sequenceHLA
restricted peptide 308Gly Ser Asp Val Glu Glu Asp Leu Thr Met 1 5
10 3099PRTArtificial sequenceHLA restricted peptide 309Gly Ser Glu
Val Glu Asn Val Ser Val 1 5 31010PRTArtificial sequenceHLA
restricted peptide 310Gly Ser Glu Val Glu Asn Val Ser Val Asn 1 5
10 3119PRTArtificial sequenceHLA restricted peptide 311Gly Val Met
Thr Arg Gly Arg Leu Lys 1 5 31210PRTArtificial sequenceHLA
restricted peptide 312Gly Val Met Thr Arg Gly Arg Leu Lys Ala 1 5
10 31310PRTArtificial sequenceHLA restricted peptide 313Gly Val Trp
Gln Pro Ala Ala Gln Pro Lys 1 5 10 31410PRTArtificial sequenceHLA
restricted peptide 314His Ala Ser Gly Lys Gln Met Trp Gln Ala 1 5
10 31510PRTArtificial sequenceHLA restricted peptide 315His Glu His
Phe Gly Leu Leu Cys Pro Lys 1 5 10 3169PRTArtificial sequenceHLA
restricted peptide 316His Glu Leu Val Cys Ser Met Glu Asn 1 5
31710PRTArtificial sequenceHLA restricted peptide 317His Glu Leu
Val Cys Ser Met Glu Asn Thr 1 5 10 3189PRTArtificial sequenceHLA
restricted peptide 318His Glu Arg Asn Gly Phe Thr Val Leu 1 5
31910PRTArtificial sequenceHLA restricted peptide 319His Glu Arg
Asn Gly Phe Thr Val Leu Cys 1 5 10 32010PRTArtificial sequenceHLA
restricted peptide 320His Glu Thr Arg Leu Leu Gln Thr Gly Ile 1 5
10 3219PRTArtificial sequenceHLA restricted peptide 321His Phe Gly
Leu Leu Cys Pro Lys Ser 1 5 32210PRTArtificial sequenceHLA
restricted peptide 322His Phe Gly Leu Leu Cys Pro Lys Ser Ile 1 5
10 3239PRTArtificial sequenceHLA restricted peptide 323His His Tyr
Pro Ser Ala Ala Glu Arg 1 5 32410PRTArtificial sequenceHLA
restricted peptide 324His His Tyr Pro Ser Ala Ala Glu Arg Lys 1 5
10 3259PRTArtificial sequenceHLA restricted peptide 325His Ile Met
Leu Asp Val Ala Phe Thr 1 5 32610PRTArtificial sequenceHLA
restricted peptide 326His Ile Met Leu Asp Val Ala Phe Thr Ser 1 5
10 3279PRTArtificial sequenceHLA restricted peptide 327His Leu Pro
Val Ala Asp Ala Val Ile 1 5 32810PRTArtificial sequenceHLA
restricted peptide 328His Leu Pro Val Ala Asp Ala Val Ile His 1 5
10 3299PRTArtificial sequenceHLA restricted peptide 329His Asn Pro
Thr Gly Arg Ser Ile Cys 1 5 3309PRTArtificial sequenceHLA
restricted peptide 330His Pro Thr Phe Thr Ser Gln Tyr Arg 1 5
33110PRTArtificial sequenceHLA restricted peptide 331His Pro Thr
Phe Thr Ser Gln Tyr Arg Ile 1 5 10 3329PRTArtificial sequenceHLA
restricted peptide 332His Arg Gly Asp Asn Gln Leu Gln Val 1 5
33310PRTArtificial sequenceHLA restricted peptide 333His Arg Gly
Asp Asn Gln Leu Gln Val Gln 1 5 10 3349PRTArtificial sequenceHLA
restricted peptide 334His Arg His Leu Pro Val Ala Asp Ala 1 5
33510PRTArtificial sequenceHLA restricted peptide 335His Arg His
Leu Pro Val Ala Asp Ala Val 1 5 10 3369PRTArtificial sequenceHLA
restricted peptide 336His Arg Gln Asp Ala Leu Pro Gly Pro 1 5
33710PRTArtificial sequenceHLA restricted peptide 337His Arg Gln
Asp Ala Leu Pro Gly Pro Cys 1 5 10 33810PRTArtificial sequenceHLA
restricted peptide 338His Thr Trp Asp Arg His Asp Glu Gly Ala 1 5
10 3399PRTArtificial sequenceHLA restricted peptide 339His Thr Tyr
Phe Thr Gly Ser Glu Val 1 5 3409PRTArtificial sequenceHLA
restricted peptide 340His Val Leu Lys Ala Val Phe Ser Arg 1 5
3419PRTArtificial sequenceHLA restricted peptide 341His Val Arg Val
Ser Gln Pro Ser Leu 1 5 34210PRTArtificial sequenceHLA restricted
peptide 342His Val Arg Val Ser Gln Pro Ser Leu Ile 1 5 10
3439PRTArtificial sequenceHLA restricted peptide 343His Val Val Cys
Ala His Glu Leu Val 1 5 34410PRTArtificial sequenceHLA restricted
peptide 344His Val Val Cys Ala His Glu Leu Val Cys 1 5 10
3459PRTArtificial sequenceHLA restricted peptide 345His Tyr Pro Ser
Ala Ala Glu Arg Lys 1 5 3469PRTArtificial sequenceHLA restricted
peptide 346Ile Ala Ser Thr Pro Lys Lys His Arg 1 5
34710PRTArtificial sequenceHLA restricted peptide 347Ile Ala Ser
Thr Pro Lys Lys His Arg Gly 1 5 10 3489PRTArtificial sequenceHLA
restricted peptide 348Ile Cys Pro Ser Gln Glu Pro Met Ser 1 5
34910PRTArtificial sequenceHLA restricted peptide 349Ile Cys Pro
Ser Gln Glu Pro Met Ser Ile 1 5 10 3509PRTArtificial sequenceHLA
restricted peptide 350Ile Phe Ala Glu Leu Glu Gly Val Trp 1 5
3519PRTArtificial sequenceHLA restricted peptide 351Ile Phe Leu Glu
Val Gln Ala Ile Arg 1 5 3529PRTArtificial sequenceHLA restricted
peptide 352Ile Gly Asp Gln Tyr Val Lys Val Tyr 1 5
35310PRTArtificial sequenceHLA restricted peptide 353Ile Gly Asp
Gln Tyr Val Lys Val Tyr Leu 1 5 10 3549PRTArtificial sequenceHLA
restricted peptide 354Ile His Ala Ser Gly Lys Gln Met Trp 1 5
3559PRTArtificial sequenceHLA restricted peptide 355Ile His Asn Pro
Ala Val Phe Thr Trp 1 5 3569PRTArtificial sequenceHLA restricted
peptide 356Ile His Val Arg Val Ser Gln Pro Ser 1 5
35710PRTArtificial sequenceHLA restricted peptide 357Ile His Val
Arg Val Ser Gln Pro Ser Leu 1 5 10 3589PRTArtificial sequenceHLA
restricted peptide 358Ile Ile Lys Pro Gly Lys Ile Ser His 1 5
35910PRTArtificial sequenceHLA restricted peptide 359Ile Ile Lys
Pro Gly Lys Ile Ser His Ile 1 5 10 3609PRTArtificial sequenceHLA
restricted peptide 360Ile Lys Pro Gly Lys Ile Ser His Ile 1 5
36110PRTArtificial sequenceHLA restricted peptide 361Ile Lys Pro
Gly Lys Ile Ser His Ile Met 1 5 10 3629PRTArtificial sequenceHLA
restricted peptide 362Ile Leu Ala Arg Asn Leu Val Pro Met 1 5
36310PRTArtificial sequenceHLA restricted peptide 363Ile Leu Ala
Arg Asn Leu Val Pro Met Val 1 5 10 36410PRTArtificial sequenceHLA
restricted peptide 364Ile Leu Val Ser Gln Tyr Thr Pro Asp Ser 1 5
10 3659PRTArtificial sequenceHLA restricted peptide 365Ile Met Leu
Asp Val Ala Phe Thr Ser 1 5 36610PRTArtificial sequenceHLA
restricted peptide 366Ile Met Leu Asp Val Ala Phe Thr Ser His 1 5
10 3679PRTArtificial sequenceHLA restricted peptide 367Ile Asn Val
His His Tyr Pro Ser Ala 1 5 36810PRTArtificial sequenceHLA
restricted peptide 368Ile Asn Val His His Tyr Pro Ser Ala Ala 1 5
10 3699PRTArtificial sequenceHLA restricted peptide 369Ile Pro Gly
Leu Ser Ile Ser Gly Asn 1 5 37010PRTArtificial sequenceHLA
restricted peptide 370Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu 1 5
10 3719PRTArtificial sequenceHLA restricted peptide 371Ile Pro Ser
Ile Asn Val His His Tyr 1 5 37210PRTArtificial sequenceHLA
restricted peptide 372Ile Pro Ser Ile Asn Val His His Tyr Pro 1 5
10 3739PRTArtificial sequenceHLA restricted peptide 373Ile Gln Gly
Lys Leu Glu Tyr Arg His 1 5 37410PRTArtificial sequenceHLA
restricted peptide 374Ile Gln Gly Lys Leu Glu Tyr Arg His Thr 1 5
10 3759PRTArtificial sequenceHLA restricted peptide 375Ile Arg Glu
Thr Val Glu Leu Arg Gln 1 5 37610PRTArtificial sequenceHLA
restricted peptide 376Ile Arg Glu Thr Val Glu Leu Arg Gln Tyr 1 5
10 3779PRTArtificial sequenceHLA restricted peptide 377Ile Ser Gly
His Val Leu Lys Ala Val 1 5 37810PRTArtificial sequenceHLA
restricted peptide 378Ile Ser Gly His Val Leu Lys Ala Val Phe 1 5
10 3799PRTArtificial sequenceHLA restricted peptide 379Ile Ser His
Ile Met Leu Asp Val Ala 1 5 38010PRTArtificial sequenceHLA
restricted peptide 380Ile Ser His Ile Met Leu Asp Val Ala Phe 1 5
10 38110PRTArtificial sequenceHLA restricted peptide 381Ile Ser Val
Leu Gly Pro Ile Ser Gly His 1 5 10 3829PRTArtificial sequenceHLA
restricted peptide 382Ile Tyr Val Tyr Ala Leu Pro Leu Lys 1 5
38310PRTArtificial sequenceHLA restricted peptide 383Ile Tyr Val
Tyr Ala Leu Pro Leu Lys Met 1 5 10 3849PRTArtificial sequenceHLA
restricted peptide 384Lys Ala Val Phe Ser Arg Gly Asp Thr 1 5
38510PRTArtificial sequenceHLA restricted peptide 385Lys Ala Val
Phe Ser Arg Gly Asp Thr Pro 1 5 10 3869PRTArtificial sequenceHLA
restricted peptide 386Lys Asp Val Ala Leu Arg His Val Val 1 5
38710PRTArtificial sequenceHLA restricted peptide 387Lys Asp Val
Ala Leu Arg His Val Val Cys 1 5 10 3889PRTArtificial sequenceHLA
restricted peptide 388Lys Glu Pro Asp Val Tyr Tyr Thr Ser 1 5
38910PRTArtificial sequenceHLA restricted peptide 389Lys Glu Pro
Asp Val Tyr Tyr Thr Ser Ala 1 5 10 39010PRTArtificial sequenceHLA
restricted peptide 390Lys His Arg His Leu Pro Val Ala Asp Ala 1 5
10 3919PRTArtificial sequenceHLA restricted peptide 391Lys Ile Ser
His Ile Met Leu Asp Val 1 5 39210PRTArtificial sequenceHLA
restricted peptide 392Lys Ile Ser His Ile Met Leu Asp Val Ala 1 5
10 39310PRTArtificial sequenceHLA restricted peptide 393Lys Leu Glu
Tyr Arg His Thr Trp Asp Arg 1 5 10 3949PRTArtificial sequenceHLA
restricted peptide 394Lys Leu Phe Met His Val Thr Leu Gly 1 5
39510PRTArtificial sequenceHLA restricted peptide 395Lys Leu Phe
Met His Val Thr Leu Gly Ser 1 5 10 3969PRTArtificial sequenceHLA
restricted peptide 396Lys Met Leu Asn Ile Pro Ser Ile Asn 1 5
39710PRTArtificial sequenceHLA restricted peptide 397Lys Met Leu
Asn Ile Pro Ser Ile Asn Val 1 5 10 3989PRTArtificial sequenceHLA
restricted peptide 398Lys Met Gln Val Ile Gly Asp Gln Tyr 1 5
39910PRTArtificial sequenceHLA restricted peptide 399Lys Met Gln
Val Ile Gly Asp Gln Tyr Val 1 5 10 4009PRTArtificial sequenceHLA
restricted peptide 400Lys Asn Met Ile Ile Lys Pro Gly Lys 1 5
40110PRTArtificial sequenceHLA restricted peptide 401Lys Asn Met
Ile Ile Lys Pro Gly Lys Ile 1 5 10 4029PRTArtificial sequenceHLA
restricted peptide 402Lys Pro Gly Lys Ile Ser His Ile Met 1 5
40310PRTArtificial sequenceHLA restricted peptide 403Lys Pro Gly
Lys Ile Ser His Ile Met Leu 1 5 10 4049PRTArtificial sequenceHLA
restricted peptide 404Lys Gln Met Trp Gln Ala Arg Leu Thr 1 5
40510PRTArtificial sequenceHLA restricted peptide 405Lys Gln Met
Trp Gln Ala Arg Leu Thr Val 1 5 10 4069PRTArtificial sequenceHLA
restricted peptide 406Lys Arg Lys Ser Ala Ser Ser Ala Thr 1 5
40710PRTArtificial sequenceHLA restricted peptide 407Lys Arg Lys
Ser Ala Ser Ser Ala Thr Ala 1 5 10 4089PRTArtificial sequenceHLA
restricted peptide 408Lys Arg Arg Arg His Arg Gln Asp Ala 1 5
40910PRTArtificial sequenceHLA restricted peptide 409Lys Arg Arg
Arg His Arg Gln Asp Ala Leu 1 5 10 4109PRTArtificial sequenceHLA
restricted peptide 410Lys Ser Ala Ser Ser Ala Thr Ala Cys 1 5
41110PRTArtificial sequenceHLA restricted peptide 411Lys Ser Ala
Ser Ser Ala Thr Ala Cys Thr 1 5 10 4129PRTArtificial sequenceHLA
restricted peptide 412Lys Ser Ile Pro Gly Leu Ser Ile Ser 1 5
41310PRTArtificial sequenceHLA restricted peptide 413Lys Ser Ile
Pro Gly Leu Ser Ile Ser Gly 1 5 10 41410PRTArtificial sequenceHLA
restricted peptide 414Lys Thr Pro Arg Val Thr Gly Gly Gly Ala 1 5
10 4159PRTArtificial sequenceHLA restricted peptide 415Lys Val Tyr
Leu Glu Ser Phe Cys Glu 1 5 41610PRTArtificial sequenceHLA
restricted peptide 416Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp 1 5
10 4179PRTArtificial sequenceHLA restricted peptide 417Lys Tyr Gln
Glu Phe Phe Trp Asp Ala 1 5 41810PRTArtificial sequenceHLA
restricted peptide 418Lys Tyr Gln Glu Phe Phe Trp Asp Ala Asn 1 5
10 4199PRTArtificial sequenceHLA restricted peptide 419Leu Ala Arg
Asn Leu Val Pro Met Val 1 5 42010PRTArtificial sequenceHLA
restricted peptide 420Leu Ala Arg Asn Leu Val Pro Met Val Ala 1 5
10 4219PRTArtificial sequenceHLA restricted peptide 421Leu Ala Trp
Thr Arg Gln Gln Asn Gln 1 5 42210PRTArtificial sequenceHLA
restricted peptide 422Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp 1 5
10 4239PRTArtificial sequenceHLA restricted peptide 423Leu Cys Pro
Lys Asn Met Ile Ile Lys 1 5 4249PRTArtificial sequenceHLA
restricted peptide 424Leu Cys Pro Lys Ser Ile Pro Gly Leu 1 5
42510PRTArtificial sequenceHLA restricted peptide 425Leu Cys Pro
Lys Ser Ile Pro Gly Leu Ser 1 5 10 4269PRTArtificial sequenceHLA
restricted peptide 426Leu Glu Gly Val Trp Gln Pro Ala Ala 1 5
42710PRTArtificial sequenceHLA restricted peptide 427Leu Glu Ser
Phe Cys Glu Asp Val Pro Ser 1 5 10 4289PRTArtificial sequenceHLA
restricted peptide 428Leu Glu Val Gln Ala Ile Arg Glu Thr 1 5
42910PRTArtificial sequenceHLA restricted peptide 429Leu Glu Val
Gln Ala Ile Arg Glu Thr Val 1 5 10 4309PRTArtificial sequenceHLA
restricted peptide 430Leu Glu Tyr
Arg His Thr Trp Asp Arg 1 5 43110PRTArtificial sequenceHLA
restricted peptide 431Leu Glu Tyr Arg His Thr Trp Asp Arg His 1 5
10 4329PRTArtificial sequenceHLA restricted peptide 432Leu Phe Phe
Phe Asp Ile Asp Leu Leu 1 5 43310PRTArtificial sequenceHLA
restricted peptide 433Leu Phe Phe Phe Asp Ile Asp Leu Leu Leu 1 5
10 4349PRTArtificial sequenceHLA restricted peptide 434Leu Phe Met
His Val Thr Leu Gly Ser 1 5 43510PRTArtificial sequenceHLA
restricted peptide 435Leu Phe Met His Val Thr Leu Gly Ser Asp 1 5
10 4369PRTArtificial sequenceHLA restricted peptide 436Leu Gly Pro
Ile Ser Gly His Val Leu 1 5 43710PRTArtificial sequenceHLA
restricted peptide 437Leu Gly Pro Ile Ser Gly His Val Leu Lys 1 5
10 4389PRTArtificial sequenceHLA restricted peptide 438Leu Gly Ser
Asp Val Glu Glu Asp Leu 1 5 43910PRTArtificial sequenceHLA
restricted peptide 439Leu Gly Ser Asp Val Glu Glu Asp Leu Thr 1 5
10 44010PRTArtificial sequenceHLA restricted peptide 440Leu Lys Ala
Val Phe Ser Arg Gly Asp Thr 1 5 10 4419PRTArtificial sequenceHLA
restricted peptide 441Leu Lys Met Leu Asn Ile Pro Ser Ile 1 5
44210PRTArtificial sequenceHLA restricted peptide 442Leu Lys Met
Leu Asn Ile Pro Ser Ile Asn 1 5 10 44310PRTArtificial sequenceHLA
restricted peptide 443Leu Lys Tyr Gln Glu Phe Phe Trp Asp Ala 1 5
10 44410PRTArtificial sequenceHLA restricted peptide 444Leu Leu Cys
Pro Lys Ser Ile Pro Gly Leu 1 5 10 4459PRTArtificial sequenceHLA
restricted peptide 445Leu Leu Leu Gln Arg Gly Pro Gln Tyr 1 5
44610PRTArtificial sequenceHLA restricted peptide 446Leu Leu Leu
Gln Arg Gly Pro Gln Tyr Ser 1 5 10 4479PRTArtificial sequenceHLA
restricted peptide 447Leu Leu Met Asn Gly Gln Gln Ile Phe 1 5
44810PRTArtificial sequenceHLA restricted peptide 448Leu Leu Met
Asn Gly Gln Gln Ile Phe Leu 1 5 10 4499PRTArtificial sequenceHLA
restricted peptide 449Leu Leu Gln Arg Gly Pro Gln Tyr Ser 1 5
4509PRTArtificial sequenceHLA restricted peptide 450Leu Leu Gln Thr
Gly Ile His Val Arg 1 5 45110PRTArtificial sequenceHLA restricted
peptide 451Leu Leu Gln Thr Gly Ile His Val Arg Val 1 5 10
4529PRTArtificial sequenceHLA restricted peptide 452Leu Met Asn Gly
Gln Gln Ile Phe Leu 1 5 4539PRTArtificial sequenceHLA restricted
peptide 453Leu Asn Ile Pro Ser Ile Asn Val His 1 5
45410PRTArtificial sequenceHLA restricted peptide 454Leu Asn Ile
Pro Ser Ile Asn Val His His 1 5 10 4559PRTArtificial sequenceHLA
restricted peptide 455Leu Pro Gly Pro Cys Ile Ala Ser Thr 1 5
45610PRTArtificial sequenceHLA restricted peptide 456Leu Pro Gly
Pro Cys Ile Ala Ser Thr Pro 1 5 10 4579PRTArtificial sequenceHLA
restricted peptide 457Leu Pro His Glu Thr Arg Leu Leu Gln 1 5
45810PRTArtificial sequenceHLA restricted peptide 458Leu Pro His
Glu Thr Arg Leu Leu Gln Thr 1 5 10 4599PRTArtificial sequenceHLA
restricted peptide 459Leu Pro Leu Lys Met Leu Asn Ile Pro 1 5
46010PRTArtificial sequenceHLA restricted peptide 460Leu Pro Leu
Lys Met Leu Asn Ile Pro Ser 1 5 10 4619PRTArtificial sequenceHLA
restricted peptide 461Leu Pro Val Ala Asp Ala Val Ile His 1 5
46210PRTArtificial sequenceHLA restricted peptide 462Leu Pro Val
Ala Asp Ala Val Ile His Ala 1 5 10 4639PRTArtificial sequenceHLA
restricted peptide 463Leu Gln Arg Gly Pro Gln Tyr Ser Glu 1 5
46410PRTArtificial sequenceHLA restricted peptide 464Leu Gln Arg
Gly Pro Gln Tyr Ser Glu His 1 5 10 4659PRTArtificial sequenceHLA
restricted peptide 465Leu Gln Thr Gly Ile His Val Arg Val 1 5
46610PRTArtificial sequenceHLA restricted peptide 466Leu Gln Thr
Gly Ile His Val Arg Val Ser 1 5 10 4679PRTArtificial sequenceHLA
restricted peptide 467Leu Gln Val Gln His Thr Tyr Phe Thr 1 5
46810PRTArtificial sequenceHLA restricted peptide 468Leu Gln Val
Gln His Thr Tyr Phe Thr Gly 1 5 10 4699PRTArtificial sequenceHLA
restricted peptide 469Leu Arg His Val Val Cys Ala His Glu 1 5
47010PRTArtificial sequenceHLA restricted peptide 470Leu Arg His
Val Val Cys Ala His Glu Leu 1 5 10 4719PRTArtificial sequenceHLA
restricted peptide 471Leu Arg Gln Tyr Asp Pro Val Ala Ala 1 5
47210PRTArtificial sequenceHLA restricted peptide 472Leu Arg Gln
Tyr Asp Pro Val Ala Ala Leu 1 5 10 4739PRTArtificial sequenceHLA
restricted peptide 473Leu Ser Ile Ser Gly Asn Leu Leu Met 1 5
47410PRTArtificial sequenceHLA restricted peptide 474Leu Ser Ile
Ser Gly Asn Leu Leu Met Asn 1 5 10 47510PRTArtificial sequenceHLA
restricted peptide 475Leu Thr Met Thr Arg Asn Pro Gln Pro Phe 1 5
10 4769PRTArtificial sequenceHLA restricted peptide 476Leu Thr Val
Ser Gly Leu Ala Trp Thr 1 5 47710PRTArtificial sequenceHLA
restricted peptide 477Leu Thr Val Ser Gly Leu Ala Trp Thr Arg 1 5
10 4789PRTArtificial sequenceHLA restricted peptide 478Leu Val Cys
Ser Met Glu Asn Thr Arg 1 5 47910PRTArtificial sequenceHLA
restricted peptide 479Leu Val Cys Ser Met Glu Asn Thr Arg Ala 1 5
10 4809PRTArtificial sequenceHLA restricted peptide 480Leu Val Ser
Gln Tyr Thr Pro Asp Ser 1 5 48110PRTArtificial sequenceHLA
restricted peptide 481Leu Val Ser Gln Tyr Thr Pro Asp Ser Thr 1 5
10 48210PRTArtificial sequenceHLA restricted peptide 482Leu Val Thr
Thr Glu Arg Lys Thr Pro Arg 1 5 10 48310PRTArtificial sequenceHLA
restricted peptide 483Met Ala Gly Ala Ser Thr Ser Ala Gly Arg 1 5
10 4849PRTArtificial sequenceHLA restricted peptide 484Met Ala Ser
Val Leu Gly Pro Ile Ser 1 5 4859PRTArtificial sequenceHLA
restricted peptide 485Met Glu Asn Thr Arg Ala Thr Lys Met 1 5
48610PRTArtificial sequenceHLA restricted peptide 486Met Glu Asn
Thr Arg Ala Thr Lys Met Gln 1 5 10 4879PRTArtificial sequenceHLA
restricted peptide 487Met His Val Thr Leu Gly Ser Asp Val 1 5
48810PRTArtificial sequenceHLA restricted peptide 488Met Ile Ile
Lys Pro Gly Lys Ile Ser His 1 5 10 4899PRTArtificial sequenceHLA
restricted peptide 489Met Ile Ser Val Leu Gly Pro Ile Ser 1 5
4909PRTArtificial sequenceHLA restricted peptide 490Met Leu Asp Val
Ala Phe Thr Ser His 1 5 4919PRTArtificial sequenceHLA restricted
peptide 491Met Leu Asn Ile Pro Ser Ile Asn Val 1 5
49210PRTArtificial sequenceHLA restricted peptide 492Met Leu Asn
Ile Pro Ser Ile Asn Val His 1 5 10 49310PRTArtificial sequenceHLA
restricted peptide 493Met Asn Gly Gln Gln Ile Phe Leu Glu Val 1 5
10 4949PRTArtificial sequenceHLA restricted peptide 494Met Gln Val
Ile Gly Asp Gln Tyr Val 1 5 49510PRTArtificial sequenceHLA
restricted peptide 495Met Gln Val Ile Gly Asp Gln Tyr Val Lys 1 5
10 4969PRTArtificial sequenceHLA restricted peptide 496Met Arg Pro
His Glu Arg Asn Gly Phe 1 5 49710PRTArtificial sequenceHLA
restricted peptide 497Met Arg Pro His Glu Arg Asn Gly Phe Thr 1 5
10 49810PRTArtificial sequenceHLA restricted peptide 498Met Ser Ile
Tyr Val Tyr Ala Leu Pro Leu 1 5 10 49910PRTArtificial sequenceHLA
restricted peptide 499Met Thr Arg Gly Arg Leu Lys Ala Glu Ser 1 5
10 5009PRTArtificial sequenceHLA restricted peptide 500Met Thr Arg
Asn Pro Gln Pro Phe Met 1 5 50110PRTArtificial sequenceHLA
restricted peptide 501Met Thr Arg Asn Pro Gln Pro Phe Met Arg 1 5
10 5029PRTArtificial sequenceHLA restricted peptide 502Met Val Ala
Thr Val Gln Gly Gln Asn 1 5 50310PRTArtificial sequenceHLA
restricted peptide 503Met Val Ala Thr Val Gln Gly Gln Asn Leu 1 5
10 50410PRTArtificial sequenceHLA restricted peptide 504Asn Asp Ile
Tyr Arg Ile Phe Ala Glu Leu 1 5 10 5059PRTArtificial sequenceHLA
restricted peptide 505Asn Glu Ile His Asn Pro Ala Val Phe 1 5
50610PRTArtificial sequenceHLA restricted peptide 506Asn Glu Ile
His Asn Pro Ala Val Phe Thr 1 5 10 5079PRTArtificial sequenceHLA
restricted peptide 507Asn Gly Phe Thr Val Leu Cys Pro Lys 1 5
50810PRTArtificial sequenceHLA restricted peptide 508Asn Gly Phe
Thr Val Leu Cys Pro Lys Asn 1 5 10 5099PRTArtificial sequenceHLA
restricted peptide 509Asn Gly Gln Gln Ile Phe Leu Glu Val 1 5
5109PRTArtificial sequenceHLA restricted peptide 510Asn Ile Pro Ser
Ile Asn Val His His 1 5 51110PRTArtificial sequenceHLA restricted
peptide 511Asn Ile Pro Ser Ile Asn Val His His Tyr 1 5 10
5129PRTArtificial sequenceHLA restricted peptide 512Asn Leu Lys Tyr
Gln Glu Phe Phe Trp 1 5 5139PRTArtificial sequenceHLA restricted
peptide 513Asn Leu Leu Met Asn Gly Gln Gln Ile 1 5
51410PRTArtificial sequenceHLA restricted peptide 514Asn Leu Leu
Met Asn Gly Gln Gln Ile Phe 1 5 10 51510PRTArtificial sequenceHLA
restricted peptide 515Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr 1 5
10 5169PRTArtificial sequenceHLA restricted peptide 516Asn Met Ile
Ile Lys Pro Gly Lys Ile 1 5 51710PRTArtificial sequenceHLA
restricted peptide 517Asn Met Ile Ile Lys Pro Gly Lys Ile Ser 1 5
10 5189PRTArtificial sequenceHLA restricted peptide 518Asn Pro Ala
Val Phe Thr Trp Pro Pro 1 5 51910PRTArtificial sequenceHLA
restricted peptide 519Asn Pro Ala Val Phe Thr Trp Pro Pro Trp 1 5
10 5209PRTArtificial sequenceHLA restricted peptide 520Asn Pro Gln
Pro Phe Met Arg Pro His 1 5 52110PRTArtificial sequenceHLA
restricted peptide 521Asn Pro Gln Pro Phe Met Arg Pro His Glu 1 5
10 5229PRTArtificial sequenceHLA restricted peptide 522Asn Pro Thr
Gly Arg Ser Ile Cys Pro 1 5 52310PRTArtificial sequenceHLA
restricted peptide 523Asn Pro Thr Gly Arg Ser Ile Cys Pro Ser 1 5
10 5249PRTArtificial sequenceHLA restricted peptide 524Asn Gln Leu
Gln Val Gln His Thr Tyr 1 5 52510PRTArtificial sequenceHLA
restricted peptide 525Asn Gln Leu Gln Val Gln His Thr Tyr Phe 1 5
10 5269PRTArtificial sequenceHLA restricted peptide 526Asn Gln Trp
Lys Glu Pro Asp Val Tyr 1 5 52710PRTArtificial sequenceHLA
restricted peptide 527Asn Gln Trp Lys Glu Pro Asp Val Tyr Tyr 1 5
10 5289PRTArtificial sequenceHLA restricted peptide 528Asn Thr Arg
Ala Thr Lys Met Gln Val 1 5 52910PRTArtificial sequenceHLA
restricted peptide 529Asn Thr Arg Ala Thr Lys Met Gln Val Ile 1 5
10 5309PRTArtificial sequenceHLA restricted peptide 530Asn Val His
His Tyr Pro Ser Ala Ala 1 5 5319PRTArtificial sequenceHLA
restricted peptide 531Asn Val His Asn Pro Thr Gly Arg Ser 1 5
53210PRTArtificial sequenceHLA restricted peptide 532Asn Val His
Asn Pro Thr Gly Arg Ser Ile 1 5 10 53310PRTArtificial sequenceHLA
restricted peptide 533Asn Val Ser Val Asn Val His Asn Pro Thr 1 5
10 5349PRTArtificial sequenceHLA restricted peptide 534Pro Ala Ala
Gln Pro Lys Arg Arg Arg 1 5 5359PRTArtificial sequenceHLA
restricted peptide 535Pro Ala Val Phe Thr Trp Pro Pro Trp 1 5
5369PRTArtificial sequenceHLA restricted peptide 536Pro Cys His Arg
Gly Asp Asn Gln Leu 1 5 5379PRTArtificial sequenceHLA restricted
peptide 537Pro Cys Ile Ala Ser Thr Pro Lys Lys 1 5
5389PRTArtificial sequenceHLA restricted peptide 538Pro Asp Val Tyr
Tyr Thr Ser Ala Phe 1 5 53910PRTArtificial sequenceHLA restricted
peptide 539Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val 1 5 10
54010PRTArtificial sequenceHLA restricted peptide 540Pro Glu Met
Ile Ser Val Leu Gly Pro Ile 1 5 10 5419PRTArtificial sequenceHLA
restricted peptide 541Pro Gly Lys Ile Ser His Ile Met Leu 1 5
5429PRTArtificial sequenceHLA restricted peptide 542Pro Gly Leu Ser
Ile Ser Gly Asn Leu 1 5 54310PRTArtificial sequenceHLA restricted
peptide 543Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu 1 5 10
5449PRTArtificial sequenceHLA restricted peptide 544Pro His Glu Arg
Asn Gly Phe Thr Val 1 5 54510PRTArtificial sequenceHLA restricted
peptide 545Pro His Glu Arg Asn Gly Phe Thr Val Leu 1 5 10
5469PRTArtificial sequenceHLA restricted peptide 546Pro His Glu Thr
Arg Leu Leu Gln Thr 1 5 5479PRTArtificial sequenceHLA restricted
peptide 547Pro Met Ser Ile Tyr Val Tyr Ala Leu 1 5
5489PRTArtificial sequenceHLA restricted peptide 548Pro Pro Trp Gln
Ala Gly Ile Leu Ala 1 5 54910PRTArtificial sequenceHLA restricted
peptide 549Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg 1 5 10
55010PRTArtificial sequenceHLA restricted peptide 550Pro Gln Pro
Phe Met Arg Pro His Glu Arg 1 5 10 5519PRTArtificial sequenceHLA
restricted peptide 551Pro Gln Tyr Ser Glu His Pro Thr Phe 1 5
55210PRTArtificial sequenceHLA restricted peptide 552Pro Gln Tyr
Ser Glu His Pro Thr Phe Thr 1 5 10 5539PRTArtificial sequenceHLA
restricted peptide 553Pro Arg Val Thr Gly Gly Gly Ala Met 1 5
55410PRTArtificial sequenceHLA restricted peptide 554Pro Arg Val
Thr Gly Gly Gly Ala Met Ala 1 5 10 5559PRTArtificial sequenceHLA
restricted peptide 555Pro Ser Ala Ala Glu Arg Lys His Arg 1 5
5569PRTArtificial sequenceHLA restricted peptide 556Pro Ser Leu Ile
Leu Val Ser Gln Tyr 1 5 5579PRTArtificial sequenceHLA restricted
peptide 557Pro Ser Gln Glu Pro Met Ser Ile Tyr 1 5
5589PRTArtificial sequenceHLA restricted peptide 558Pro Thr Phe Thr
Ser Gln Tyr Arg Ile 1 5 5599PRTArtificial sequenceHLA restricted
peptide 559Pro Val Ala Asp Ala Val Ile His Ala 1 5
5609PRTArtificial sequenceHLA restricted peptide 560Pro Val Leu Pro
His Glu Thr Arg Leu 1 5 56110PRTArtificial sequenceHLA restricted
peptide 561Pro Val Leu Pro His Glu Thr Arg Leu Leu 1 5 10
5629PRTArtificial sequenceHLA restricted peptide 562Gln Ala Gly Ile
Leu Ala Arg Asn Leu 1 5 56310PRTArtificial sequenceHLA restricted
peptide 563Gln Ala Gly Ile Leu Ala Arg Asn Leu Val 1 5 10
5649PRTArtificial sequenceHLA restricted peptide 564Gln Ala Ile Arg
Glu Thr Val Glu Leu 1 5 56510PRTArtificial sequenceHLA restricted
peptide 565Gln Ala Ile Arg Glu Thr Val Glu Leu Arg 1 5 10
5669PRTArtificial sequenceHLA restricted peptide 566Gln Ala Arg Leu
Thr Val Ser Gly Leu 1 5 56710PRTArtificial sequenceHLA restricted
peptide 567Gln Ala Arg Leu Thr Val Ser Gly Leu Ala 1 5 10
5689PRTArtificial sequenceHLA restricted peptide 568Gln Asp Ala Leu
Pro
Gly Pro Cys Ile 1 5 56910PRTArtificial sequenceHLA restricted
peptide 569Gln Asp Ala Leu Pro Gly Pro Cys Ile Ala 1 5 10
5709PRTArtificial sequenceHLA restricted peptide 570Gln Glu Phe Phe
Trp Asp Ala Asn Asp 1 5 57110PRTArtificial sequenceHLA restricted
peptide 571Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile 1 5 10
5729PRTArtificial sequenceHLA restricted peptide 572Gln Glu Pro Met
Ser Ile Tyr Val Tyr 1 5 57310PRTArtificial sequenceHLA restricted
peptide 573Gln Glu Pro Met Ser Ile Tyr Val Tyr Ala 1 5 10
5749PRTArtificial sequenceHLA restricted peptide 574Gln Gly Asp Asp
Asp Val Trp Thr Ser 1 5 5759PRTArtificial sequenceHLA restricted
peptide 575Gln Gly Lys Leu Glu Tyr Arg His Thr 1 5
57610PRTArtificial sequenceHLA restricted peptide 576Gln Gly Lys
Leu Glu Tyr Arg His Thr Trp 1 5 10 57710PRTArtificial sequenceHLA
restricted peptide 577Gln Gly Gln Asn Leu Lys Tyr Gln Glu Phe 1 5
10 57810PRTArtificial sequenceHLA restricted peptide 578Gln His Thr
Tyr Phe Thr Gly Ser Glu Val 1 5 10 5799PRTArtificial sequenceHLA
restricted peptide 579Gln Ile Phe Leu Glu Val Gln Ala Ile 1 5
58010PRTArtificial sequenceHLA restricted peptide 580Gln Ile Phe
Leu Glu Val Gln Ala Ile Arg 1 5 10 5819PRTArtificial sequenceHLA
restricted peptide 581Gln Leu Gln Val Gln His Thr Tyr Phe 1 5
58210PRTArtificial sequenceHLA restricted peptide 582Gln Leu Gln
Val Gln His Thr Tyr Phe Thr 1 5 10 5839PRTArtificial sequenceHLA
restricted peptide 583Gln Met Trp Gln Ala Arg Leu Thr Val 1 5
58410PRTArtificial sequenceHLA restricted peptide 584Gln Met Trp
Gln Ala Arg Leu Thr Val Ser 1 5 10 5859PRTArtificial sequenceHLA
restricted peptide 585Gln Asn Leu Lys Tyr Gln Glu Phe Phe 1 5
58610PRTArtificial sequenceHLA restricted peptide 586Gln Asn Leu
Lys Tyr Gln Glu Phe Phe Trp 1 5 10 5879PRTArtificial sequenceHLA
restricted peptide 587Gln Asn Gln Trp Lys Glu Pro Asp Val 1 5
58810PRTArtificial sequenceHLA restricted peptide 588Gln Asn Gln
Trp Lys Glu Pro Asp Val Tyr 1 5 10 5899PRTArtificial sequenceHLA
restricted peptide 589Gln Pro Ala Ala Gln Pro Lys Arg Arg 1 5
59010PRTArtificial sequenceHLA restricted peptide 590Gln Pro Ala
Ala Gln Pro Lys Arg Arg Arg 1 5 10 5919PRTArtificial sequenceHLA
restricted peptide 591Gln Pro Phe Met Arg Pro His Glu Arg 1 5
59210PRTArtificial sequenceHLA restricted peptide 592Gln Pro Phe
Met Arg Pro His Glu Arg Asn 1 5 10 5939PRTArtificial sequenceHLA
restricted peptide 593Gln Pro Lys Arg Arg Arg His Arg Gln 1 5
59410PRTArtificial sequenceHLA restricted peptide 594Gln Pro Lys
Arg Arg Arg His Arg Gln Asp 1 5 10 5959PRTArtificial sequenceHLA
restricted peptide 595Gln Pro Ser Leu Ile Leu Val Ser Gln 1 5
59610PRTArtificial sequenceHLA restricted peptide 596Gln Pro Ser
Leu Ile Leu Val Ser Gln Tyr 1 5 10 5979PRTArtificial sequenceHLA
restricted peptide 597Gln Gln Ile Phe Leu Glu Val Gln Ala 1 5
59810PRTArtificial sequenceHLA restricted peptide 598Gln Gln Ile
Phe Leu Glu Val Gln Ala Ile 1 5 10 5999PRTArtificial sequenceHLA
restricted peptide 599Gln Gln Asn Gln Trp Lys Glu Pro Asp 1 5
60010PRTArtificial sequenceHLA restricted peptide 600Gln Gln Asn
Gln Trp Lys Glu Pro Asp Val 1 5 10 6019PRTArtificial sequenceHLA
restricted peptide 601Gln Arg Gly Pro Gln Tyr Ser Glu His 1 5
60210PRTArtificial sequenceHLA restricted peptide 602Gln Arg Gly
Pro Gln Tyr Ser Glu His Pro 1 5 10 6039PRTArtificial sequenceHLA
restricted peptide 603Gln Val Ile Gly Asp Gln Tyr Val Lys 1 5
60410PRTArtificial sequenceHLA restricted peptide 604Gln Val Ile
Gly Asp Gln Tyr Val Lys Val 1 5 10 60510PRTArtificial sequenceHLA
restricted peptide 605Gln Val Gln His Thr Tyr Phe Thr Gly Ser 1 5
10 6069PRTArtificial sequenceHLA restricted peptide 606Gln Trp Lys
Glu Pro Asp Val Tyr Tyr 1 5 60710PRTArtificial sequenceHLA
restricted peptide 607Gln Trp Lys Glu Pro Asp Val Tyr Tyr Thr 1 5
10 6089PRTArtificial sequenceHLA restricted peptide 608Gln Tyr Asp
Pro Val Ala Ala Leu Phe 1 5 60910PRTArtificial sequenceHLA
restricted peptide 609Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe 1 5
10 61010PRTArtificial sequenceHLA restricted peptide 610Gln Tyr Arg
Ile Gln Gly Lys Leu Glu Tyr 1 5 10 6119PRTArtificial sequenceHLA
restricted peptide 611Gln Tyr Ser Glu His Pro Thr Phe Thr 1 5
61210PRTArtificial sequenceHLA restricted peptide 612Gln Tyr Ser
Glu His Pro Thr Phe Thr Ser 1 5 10 6139PRTArtificial sequenceHLA
restricted peptide 613Gln Tyr Thr Pro Asp Ser Thr Pro Cys 1 5
6149PRTArtificial sequenceHLA restricted peptide 614Gln Tyr Val Lys
Val Tyr Leu Glu Ser 1 5 61510PRTArtificial sequenceHLA restricted
peptide 615Gln Tyr Val Lys Val Tyr Leu Glu Ser Phe 1 5 10
6169PRTArtificial sequenceHLA restricted peptide 616Arg Cys Pro Glu
Met Ile Ser Val Leu 1 5 6179PRTArtificial sequenceHLA restricted
peptide 617Arg Glu Thr Val Glu Leu Arg Gln Tyr 1 5
6189PRTArtificial sequenceHLA restricted peptide 618Arg Gly Asp Asn
Gln Leu Gln Val Gln 1 5 61910PRTArtificial sequenceHLA restricted
peptide 619Arg Gly Asp Asn Gln Leu Gln Val Gln His 1 5 10
6209PRTArtificial sequenceHLA restricted peptide 620Arg Gly Asp Thr
Pro Val Leu Pro His 1 5 62110PRTArtificial sequenceHLA restricted
peptide 621Arg Gly Pro Gln Tyr Ser Glu His Pro Thr 1 5 10
6229PRTArtificial sequenceHLA restricted peptide 622Arg Gly Arg Leu
Lys Ala Glu Ser Thr 1 5 62310PRTArtificial sequenceHLA restricted
peptide 623Arg Gly Arg Leu Lys Ala Glu Ser Thr Val 1 5 10
6249PRTArtificial sequenceHLA restricted peptide 624Arg Gly Arg Arg
Cys Pro Glu Met Ile 1 5 62510PRTArtificial sequenceHLA restricted
peptide 625Arg Gly Arg Arg Cys Pro Glu Met Ile Ser 1 5 10
6269PRTArtificial sequenceHLA restricted peptide 626Arg His Asp Glu
Gly Ala Ala Gln Gly 1 5 62710PRTArtificial sequenceHLA restricted
peptide 627Arg His Asp Glu Gly Ala Ala Gln Gly Asp 1 5 10
6289PRTArtificial sequenceHLA restricted peptide 628Arg His Leu Pro
Val Ala Asp Ala Val 1 5 62910PRTArtificial sequenceHLA restricted
peptide 629Arg His Leu Pro Val Ala Asp Ala Val Ile 1 5 10
6309PRTArtificial sequenceHLA restricted peptide 630Arg His Val Val
Cys Ala His Glu Leu 1 5 63110PRTArtificial sequenceHLA restricted
peptide 631Arg His Val Val Cys Ala His Glu Leu Val 1 5 10
6329PRTArtificial sequenceHLA restricted peptide 632Arg Ile Phe Ala
Glu Leu Glu Gly Val 1 5 63310PRTArtificial sequenceHLA restricted
peptide 633Arg Ile Phe Ala Glu Leu Glu Gly Val Trp 1 5 10
6349PRTArtificial sequenceHLA restricted peptide 634Arg Ile Gln Gly
Lys Leu Glu Tyr Arg 1 5 63510PRTArtificial sequenceHLA restricted
peptide 635Arg Ile Gln Gly Lys Leu Glu Tyr Arg His 1 5 10
6369PRTArtificial sequenceHLA restricted peptide 636Arg Lys His Arg
His Leu Pro Val Ala 1 5 6379PRTArtificial sequenceHLA restricted
peptide 637Arg Lys Ser Ala Ser Ser Ala Thr Ala 1 5
63810PRTArtificial sequenceHLA restricted peptide 638Arg Lys Ser
Ala Ser Ser Ala Thr Ala Cys 1 5 10 6399PRTArtificial sequenceHLA
restricted peptide 639Arg Leu Lys Ala Glu Ser Thr Val Ala 1 5
6409PRTArtificial sequenceHLA restricted peptide 640Arg Leu Leu Gln
Thr Gly Ile His Val 1 5 64110PRTArtificial sequenceHLA restricted
peptide 641Arg Leu Leu Gln Thr Gly Ile His Val Arg 1 5 10
6429PRTArtificial sequenceHLA restricted peptide 642Arg Leu Thr Val
Ser Gly Leu Ala Trp 1 5 64310PRTArtificial sequenceHLA restricted
peptide 643Arg Leu Thr Val Ser Gly Leu Ala Trp Thr 1 5 10
64410PRTArtificial sequenceHLA restricted peptide 644Arg Asn Gly
Phe Thr Val Leu Cys Pro Lys 1 5 10 6459PRTArtificial sequenceHLA
restricted peptide 645Arg Asn Leu Val Pro Met Val Ala Thr 1 5
64610PRTArtificial sequenceHLA restricted peptide 646Arg Asn Leu
Val Pro Met Val Ala Thr Val 1 5 10 64710PRTArtificial sequenceHLA
restricted peptide 647Arg Asn Pro Gln Pro Phe Met Arg Pro His 1 5
10 6489PRTArtificial sequenceHLA restricted peptide 648Arg Pro His
Glu Arg Asn Gly Phe Thr 1 5 64910PRTArtificial sequenceHLA
restricted peptide 649Arg Pro His Glu Arg Asn Gly Phe Thr Val 1 5
10 6509PRTArtificial sequenceHLA restricted peptide 650Arg Gln Asp
Ala Leu Pro Gly Pro Cys 1 5 65110PRTArtificial sequenceHLA
restricted peptide 651Arg Gln Asp Ala Leu Pro Gly Pro Cys Ile 1 5
10 6529PRTArtificial sequenceHLA restricted peptide 652Arg Gln Gln
Asn Gln Trp Lys Glu Pro 1 5 65310PRTArtificial sequenceHLA
restricted peptide 653Arg Gln Gln Asn Gln Trp Lys Glu Pro Asp 1 5
10 6549PRTArtificial sequenceHLA restricted peptide 654Arg Gln Tyr
Asp Pro Val Ala Ala Leu 1 5 65510PRTArtificial sequenceHLA
restricted peptide 655Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe 1 5
10 6569PRTArtificial sequenceHLA restricted peptide 656Arg Arg Cys
Pro Glu Met Ile Ser Val 1 5 65710PRTArtificial sequenceHLA
restricted peptide 657Arg Arg Cys Pro Glu Met Ile Ser Val Leu 1 5
10 6589PRTArtificial sequenceHLA restricted peptide 658Arg Arg His
Arg Gln Asp Ala Leu Pro 1 5 65910PRTArtificial sequenceHLA
restricted peptide 659Arg Arg His Arg Gln Asp Ala Leu Pro Gly 1 5
10 6609PRTArtificial sequenceHLA restricted peptide 660Arg Arg Arg
His Arg Gln Asp Ala Leu 1 5 66110PRTArtificial sequenceHLA
restricted peptide 661Arg Arg Arg His Arg Gln Asp Ala Leu Pro 1 5
10 66210PRTArtificial sequenceHLA restricted peptide 662Arg Ser Ile
Cys Pro Ser Gln Glu Pro Met 1 5 10 6639PRTArtificial sequenceHLA
restricted peptide 663Arg Val Ser Gln Pro Ser Leu Ile Leu 1 5
66410PRTArtificial sequenceHLA restricted peptide 664Arg Val Ser
Gln Pro Ser Leu Ile Leu Val 1 5 10 6659PRTArtificial sequenceHLA
restricted peptide 665Arg Val Thr Gly Gly Gly Ala Met Ala 1 5
6669PRTArtificial sequenceHLA restricted peptide 666Ser Ala Ala Glu
Arg Lys His Arg His 1 5 66710PRTArtificial sequenceHLA restricted
peptide 667Ser Ala Ala Glu Arg Lys His Arg His Leu 1 5 10
6689PRTArtificial sequenceHLA restricted peptide 668Ser Ala Phe Val
Phe Pro Thr Lys Asp 1 5 66910PRTArtificial sequenceHLA restricted
peptide 669Ser Ala Phe Val Phe Pro Thr Lys Asp Val 1 5 10
6709PRTArtificial sequenceHLA restricted peptide 670Ser Ala Gly Arg
Lys Arg Lys Ser Ala 1 5 67110PRTArtificial sequenceHLA restricted
peptide 671Ser Ala Gly Arg Lys Arg Lys Ser Ala Ser 1 5 10
6729PRTArtificial sequenceHLA restricted peptide 672Ser Ala Ser Ser
Ala Thr Ala Cys Thr 1 5 67310PRTArtificial sequenceHLA restricted
peptide 673Ser Ala Ser Ser Ala Thr Ala Cys Thr Ala 1 5 10
67410PRTArtificial sequenceHLA restricted peptide 674Ser Ala Ser
Ser Ala Thr Ala Cys Thr Ser 1 5 10 6759PRTArtificial sequenceHLA
restricted peptide 675Ser Ala Thr Ala Cys Thr Ala Gly Val 1 5
67610PRTArtificial sequenceHLA restricted peptide 676Ser Ala Thr
Ala Cys Thr Ala Gly Val Met 1 5 10 6779PRTArtificial sequenceHLA
restricted peptide 677Ser Ala Thr Ala Cys Thr Ser Gly Val 1 5
67810PRTArtificial sequenceHLA restricted peptide 678Ser Ala Thr
Ala Cys Thr Ser Gly Val Met 1 5 10 6799PRTArtificial sequenceHLA
restricted peptide 679Ser Asp Asn Glu Ile His Asn Pro Ala 1 5
68010PRTArtificial sequenceHLA restricted peptide 680Ser Asp Asn
Glu Ile His Asn Pro Ala Val 1 5 10 6819PRTArtificial sequenceHLA
restricted peptide 681Ser Asp Ser Asp Glu Glu Leu Val Thr 1 5
68210PRTArtificial sequenceHLA restricted peptide 682Ser Asp Ser
Asp Glu Glu Leu Val Thr Thr 1 5 10 6839PRTArtificial sequenceHLA
restricted peptide 683Ser Asp Val Glu Glu Asp Leu Thr Met 1 5
68410PRTArtificial sequenceHLA restricted peptide 684Ser Asp Val
Glu Glu Asp Leu Thr Met Thr 1 5 10 6859PRTArtificial sequenceHLA
restricted peptide 685Ser Glu His Pro Thr Phe Thr Ser Gln 1 5
68610PRTArtificial sequenceHLA restricted peptide 686Ser Glu His
Pro Thr Phe Thr Ser Gln Tyr 1 5 10 6879PRTArtificial sequenceHLA
restricted peptide 687Ser Glu Val Glu Asn Val Ser Val Asn 1 5
68810PRTArtificial sequenceHLA restricted peptide 688Ser Glu Val
Glu Asn Val Ser Val Asn Val 1 5 10 6899PRTArtificial sequenceHLA
restricted peptide 689Ser Phe Cys Glu Asp Val Pro Ser Gly 1 5
69010PRTArtificial sequenceHLA restricted peptide 690Ser Phe Cys
Glu Asp Val Pro Ser Gly Lys 1 5 10 6919PRTArtificial sequenceHLA
restricted peptide 691Ser Gly His Val Leu Lys Ala Val Phe 1 5
69210PRTArtificial sequenceHLA restricted peptide 692Ser Gly His
Val Leu Lys Ala Val Phe Ser 1 5 10 6939PRTArtificial sequenceHLA
restricted peptide 693Ser Gly Lys Leu Phe Met His Val Thr 1 5
69410PRTArtificial sequenceHLA restricted peptide 694Ser Gly Lys
Leu Phe Met His Val Thr Leu 1 5 10 6959PRTArtificial sequenceHLA
restricted peptide 695Ser Gly Lys Gln Met Trp Gln Ala Arg 1 5
69610PRTArtificial sequenceHLA restricted peptide 696Ser Gly Lys
Gln Met Trp Gln Ala Arg Leu 1 5 10 69710PRTArtificial sequenceHLA
restricted peptide 697Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn 1 5
10 6989PRTArtificial sequenceHLA restricted peptide 698Ser Gly Ser
Asp Ser Asp Glu Glu Leu 1 5 69910PRTArtificial sequenceHLA
restricted peptide 699Ser Gly Ser Asp Ser Asp Glu Glu Leu Val 1 5
10 7009PRTArtificial sequenceHLA restricted peptide 700Ser Gly Val
Met Thr Arg Gly Arg Leu 1 5 70110PRTArtificial sequenceHLA
restricted peptide 701Ser Gly Val Met Thr Arg Gly Arg Leu Lys 1 5
10 7029PRTArtificial sequenceHLA restricted peptide 702Ser His Glu
His Phe Gly Leu Leu Cys 1 5 70310PRTArtificial sequenceHLA
restricted peptide 703Ser His Glu His Phe Gly Leu Leu Cys Pro 1 5
10 7049PRTArtificial sequenceHLA restricted peptide 704Ser His Ile
Met Leu Asp Val Ala Phe 1 5 70510PRTArtificial sequenceHLA
restricted peptide 705Ser His Ile Met Leu Asp Val Ala Phe Thr 1 5
10 7069PRTArtificial sequenceHLA
restricted peptide 706Ser Ile Cys Pro Ser Gln Glu Pro Met 1 5
70710PRTArtificial sequenceHLA restricted peptide 707Ser Ile Cys
Pro Ser Gln Glu Pro Met Ser 1 5 10 7089PRTArtificial sequenceHLA
restricted peptide 708Ser Ile Asn Val His His Tyr Pro Ser 1 5
70910PRTArtificial sequenceHLA restricted peptide 709Ser Ile Asn
Val His His Tyr Pro Ser Ala 1 5 10 71010PRTArtificial sequenceHLA
restricted peptide 710Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn 1 5
10 7119PRTArtificial sequenceHLA restricted peptide 711Ser Ile Ser
Gly Asn Leu Leu Met Asn 1 5 7129PRTArtificial sequenceHLA
restricted peptide 712Ser Ile Tyr Val Tyr Ala Leu Pro Leu 1 5
71310PRTArtificial sequenceHLA restricted peptide 713Ser Ile Tyr
Val Tyr Ala Leu Pro Leu Lys 1 5 10 7149PRTArtificial sequenceHLA
restricted peptide 714Ser Leu Ile Leu Val Ser Gln Tyr Thr 1 5
7159PRTArtificial sequenceHLA restricted peptide 715Ser Met Glu Asn
Thr Arg Ala Thr Lys 1 5 71610PRTArtificial sequenceHLA restricted
peptide 716Ser Met Glu Asn Thr Arg Ala Thr Lys Met 1 5 10
7179PRTArtificial sequenceHLA restricted peptide 717Ser Gln Glu Pro
Met Ser Ile Tyr Val 1 5 71810PRTArtificial sequenceHLA restricted
peptide 718Ser Gln Glu Pro Met Ser Ile Tyr Val Tyr 1 5 10
7199PRTArtificial sequenceHLA restricted peptide 719Ser Gln Pro Ser
Leu Ile Leu Val Ser 1 5 72010PRTArtificial sequenceHLA restricted
peptide 720Ser Gln Pro Ser Leu Ile Leu Val Ser Gln 1 5 10
7219PRTArtificial sequenceHLA restricted peptide 721Ser Gln Tyr Arg
Ile Gln Gly Lys Leu 1 5 72210PRTArtificial sequenceHLA restricted
peptide 722Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu 1 5 10
7239PRTArtificial sequenceHLA restricted peptide 723Ser Gln Tyr Thr
Pro Asp Ser Thr Pro 1 5 72410PRTArtificial sequenceHLA restricted
peptide 724Ser Gln Tyr Thr Pro Asp Ser Thr Pro Cys 1 5 10
7259PRTArtificial sequenceHLA restricted peptide 725Ser Arg Gly Asp
Thr Pro Val Leu Pro 1 5 72610PRTArtificial sequenceHLA restricted
peptide 726Ser Arg Gly Asp Thr Pro Val Leu Pro His 1 5 10
7279PRTArtificial sequenceHLA restricted peptide 727Ser Arg Gly Arg
Arg Cys Pro Glu Met 1 5 72810PRTArtificial sequenceHLA restricted
peptide 728Ser Arg Gly Arg Arg Cys Pro Glu Met Ile 1 5 10
72910PRTArtificial sequenceHLA restricted peptide 729Ser Ser Ala
Thr Ala Cys Thr Ala Gly Val 1 5 10 73010PRTArtificial sequenceHLA
restricted peptide 730Ser Ser Ala Thr Ala Cys Thr Ser Gly Val 1 5
10 7319PRTArtificial sequenceHLA restricted peptide 731Ser Thr Pro
Cys His Arg Gly Asp Asn 1 5 7329PRTArtificial sequenceHLA
restricted peptide 732Ser Thr Ser Ala Gly Arg Lys Arg Lys 1 5
7339PRTArtificial sequenceHLA restricted peptide 733Ser Thr Val Ala
Pro Glu Glu Asp Thr 1 5 7349PRTArtificial sequenceHLA restricted
peptide 734Ser Val Leu Gly Pro Ile Ser Gly His 1 5
73510PRTArtificial sequenceHLA restricted peptide 735Ser Val Leu
Gly Pro Ile Ser Gly His Val 1 5 10 73610PRTArtificial sequenceHLA
restricted peptide 736Ser Val Asn Val His Asn Pro Thr Gly Arg 1 5
10 7379PRTArtificial sequenceHLA restricted peptide 737Thr Ala Cys
Thr Ala Gly Val Met Thr 1 5 73810PRTArtificial sequenceHLA
restricted peptide 738Thr Ala Cys Thr Ala Gly Val Met Thr Arg 1 5
10 7399PRTArtificial sequenceHLA restricted peptide 739Thr Ala Cys
Thr Ser Gly Val Met Thr 1 5 74010PRTArtificial sequenceHLA
restricted peptide 740Thr Ala Cys Thr Ser Gly Val Met Thr Arg 1 5
10 7419PRTArtificial sequenceHLA restricted peptide 741Thr Ala Gly
Val Met Thr Arg Gly Arg 1 5 74210PRTArtificial sequenceHLA
restricted peptide 742Thr Ala Gly Val Met Thr Arg Gly Arg Leu 1 5
10 7439PRTArtificial sequenceHLA restricted peptide 743Thr Asp Glu
Asp Ser Asp Asn Glu Ile 1 5 7449PRTArtificial sequenceHLA
restricted peptide 744Thr Glu Arg Lys Thr Pro Arg Val Thr 1 5
7459PRTArtificial sequenceHLA restricted peptide 745Thr Gly Gly Gly
Ala Met Ala Gly Ala 1 5 74610PRTArtificial sequenceHLA restricted
peptide 746Thr Gly Gly Gly Ala Met Ala Gly Ala Ser 1 5 10
7479PRTArtificial sequenceHLA restricted peptide 747Thr Gly Ser Glu
Val Glu Asn Val Ser 1 5 74810PRTArtificial sequenceHLA restricted
peptide 748Thr Gly Ser Glu Val Glu Asn Val Ser Val 1 5 10
7499PRTArtificial sequenceHLA restricted peptide 749Thr Lys Asp Val
Ala Leu Arg His Val 1 5 75010PRTArtificial sequenceHLA restricted
peptide 750Thr Lys Asp Val Ala Leu Arg His Val Val 1 5 10
75110PRTArtificial sequenceHLA restricted peptide 751Thr Lys Met
Gln Val Ile Gly Asp Gln Tyr 1 5 10 75210PRTArtificial sequenceHLA
restricted peptide 752Thr Leu Gly Ser Asp Val Glu Glu Asp Leu 1 5
10 7539PRTArtificial sequenceHLA restricted peptide 753Thr Met Thr
Arg Asn Pro Gln Pro Phe 1 5 75410PRTArtificial sequenceHLA
restricted peptide 754Thr Met Thr Arg Asn Pro Gln Pro Phe Met 1 5
10 7559PRTArtificial sequenceHLA restricted peptide 755Thr Pro Cys
His Arg Gly Asp Asn Gln 1 5 75610PRTArtificial sequenceHLA
restricted peptide 756Thr Pro Cys His Arg Gly Asp Asn Gln Leu 1 5
10 7579PRTArtificial sequenceHLA restricted peptide 757Thr Pro Asp
Ser Thr Pro Cys His Arg 1 5 75810PRTArtificial sequenceHLA
restricted peptide 758Thr Pro Asp Ser Thr Pro Cys His Arg Gly 1 5
10 7599PRTArtificial sequenceHLA restricted peptide 759Thr Pro Arg
Val Thr Gly Gly Gly Ala 1 5 76010PRTArtificial sequenceHLA
restricted peptide 760Thr Pro Arg Val Thr Gly Gly Gly Ala Met 1 5
10 7619PRTArtificial sequenceHLA restricted peptide 761Thr Pro Val
Leu Pro His Glu Thr Arg 1 5 76210PRTArtificial sequenceHLA
restricted peptide 762Thr Pro Val Leu Pro His Glu Thr Arg Leu 1 5
10 7639PRTArtificial sequenceHLA restricted peptide 763Thr Arg Ala
Thr Lys Met Gln Val Ile 1 5 76410PRTArtificial sequenceHLA
restricted peptide 764Thr Arg Ala Thr Lys Met Gln Val Ile Gly 1 5
10 7659PRTArtificial sequenceHLA restricted peptide 765Thr Arg Gly
Arg Leu Lys Ala Glu Ser 1 5 76610PRTArtificial sequenceHLA
restricted peptide 766Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr 1 5
10 7679PRTArtificial sequenceHLA restricted peptide 767Thr Arg Leu
Leu Gln Thr Gly Ile His 1 5 76810PRTArtificial sequenceHLA
restricted peptide 768Thr Arg Leu Leu Gln Thr Gly Ile His Val 1 5
10 7699PRTArtificial sequenceHLA restricted peptide 769Thr Arg Asn
Pro Gln Pro Phe Met Arg 1 5 77010PRTArtificial sequenceHLA
restricted peptide 770Thr Arg Asn Pro Gln Pro Phe Met Arg Pro 1 5
10 7719PRTArtificial sequenceHLA restricted peptide 771Thr Arg Gln
Gln Asn Gln Trp Lys Glu 1 5 77210PRTArtificial sequenceHLA
restricted peptide 772Thr Arg Gln Gln Asn Gln Trp Lys Glu Pro 1 5
10 7739PRTArtificial sequenceHLA restricted peptide 773Thr Ser Ala
Phe Val Phe Pro Thr Lys 1 5 77410PRTArtificial sequenceHLA
restricted peptide 774Thr Ser Ala Gly Arg Lys Arg Lys Ser Ala 1 5
10 77510PRTArtificial sequenceHLA restricted peptide 775Thr Ser Gly
Ser Asp Ser Asp Glu Glu Leu 1 5 10 7769PRTArtificial sequenceHLA
restricted peptide 776Thr Ser Gly Val Met Thr Arg Gly Arg 1 5
77710PRTArtificial sequenceHLA restricted peptide 777Thr Ser Gly
Val Met Thr Arg Gly Arg Leu 1 5 10 7789PRTArtificial sequenceHLA
restricted peptide 778Thr Ser His Glu His Phe Gly Leu Leu 1 5
77910PRTArtificial sequenceHLA restricted peptide 779Thr Ser His
Glu His Phe Gly Leu Leu Cys 1 5 10 7809PRTArtificial sequenceHLA
restricted peptide 780Thr Ser Gln Tyr Arg Ile Gln Gly Lys 1 5
78110PRTArtificial sequenceHLA restricted peptide 781Thr Ser Gln
Tyr Arg Ile Gln Gly Lys Leu 1 5 10 7829PRTArtificial sequenceHLA
restricted peptide 782Thr Thr Glu Arg Lys Thr Pro Arg Val 1 5
78310PRTArtificial sequenceHLA restricted peptide 783Thr Thr Glu
Arg Lys Thr Pro Arg Val Thr 1 5 10 7849PRTArtificial sequenceHLA
restricted peptide 784Thr Val Ala Pro Glu Glu Asp Thr Asp 1 5
78510PRTArtificial sequenceHLA restricted peptide 785Thr Val Ala
Pro Glu Glu Asp Thr Asp Glu 1 5 10 78610PRTArtificial sequenceHLA
restricted peptide 786Thr Val Glu Leu Arg Gln Tyr Asp Pro Val 1 5
10 7879PRTArtificial sequenceHLA restricted peptide 787Thr Val Leu
Cys Pro Lys Asn Met Ile 1 5 78810PRTArtificial sequenceHLA
restricted peptide 788Thr Val Leu Cys Pro Lys Asn Met Ile Ile 1 5
10 7899PRTArtificial sequenceHLA restricted peptide 789Thr Val Gln
Gly Gln Asn Leu Lys Tyr 1 5 7909PRTArtificial sequenceHLA
restricted peptide 790Thr Val Ser Gly Leu Ala Trp Thr Arg 1 5
7919PRTArtificial sequenceHLA restricted peptide 791Thr Trp Asp Arg
His Asp Glu Gly Ala 1 5 79210PRTArtificial sequenceHLA restricted
peptide 792Thr Trp Asp Arg His Asp Glu Gly Ala Ala 1 5 10
7939PRTArtificial sequenceHLA restricted peptide 793Thr Trp Pro Pro
Trp Gln Ala Gly Ile 1 5 79410PRTArtificial sequenceHLA restricted
peptide 794Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu 1 5 10
79510PRTArtificial sequenceHLA restricted peptide 795Thr Tyr Phe
Thr Gly Ser Glu Val Glu Asn 1 5 10 7969PRTArtificial sequenceHLA
restricted peptide 796Val Ala Ala Leu Phe Phe Phe Asp Ile 1 5
7979PRTArtificial sequenceHLA restricted peptide 797Val Ala Asp Ala
Val Ile His Ala Ser 1 5 79810PRTArtificial sequenceHLA restricted
peptide 798Val Ala Asp Ala Val Ile His Ala Ser Gly 1 5 10
7999PRTArtificial sequenceHLA restricted peptide 799Val Ala Phe Thr
Ser His Glu His Phe 1 5 80010PRTArtificial sequenceHLA restricted
peptide 800Val Ala Phe Thr Ser His Glu His Phe Gly 1 5 10
8019PRTArtificial sequenceHLA restricted peptide 801Val Ala Leu Arg
His Val Val Cys Ala 1 5 80210PRTArtificial sequenceHLA restricted
peptide 802Val Ala Leu Arg His Val Val Cys Ala His 1 5 10
8039PRTArtificial sequenceHLA restricted peptide 803Val Ala Thr Val
Gln Gly Gln Asn Leu 1 5 80410PRTArtificial sequenceHLA restricted
peptide 804Val Ala Thr Val Gln Gly Gln Asn Leu Lys 1 5 10
8059PRTArtificial sequenceHLA restricted peptide 805Val Cys Ala His
Glu Leu Val Cys Ser 1 5 80610PRTArtificial sequenceHLA restricted
peptide 806Val Cys Ala His Glu Leu Val Cys Ser Met 1 5 10
8079PRTArtificial sequenceHLA restricted peptide 807Val Cys Ser Met
Glu Asn Thr Arg Ala 1 5 8089PRTArtificial sequenceHLA restricted
peptide 808Val Glu Glu Asp Leu Thr Met Thr Arg 1 5
80910PRTArtificial sequenceHLA restricted peptide 809Val Glu Glu
Asp Leu Thr Met Thr Arg Asn 1 5 10 8109PRTArtificial sequenceHLA
restricted peptide 810Val Glu Leu Arg Gln Tyr Asp Pro Val 1 5
81110PRTArtificial sequenceHLA restricted peptide 811Val Glu Leu
Arg Gln Tyr Asp Pro Val Ala 1 5 10 8129PRTArtificial sequenceHLA
restricted peptide 812Val Glu Asn Val Ser Val Asn Val His 1 5
81310PRTArtificial sequenceHLA restricted peptide 813Val Glu Asn
Val Ser Val Asn Val His Asn 1 5 10 8149PRTArtificial sequenceHLA
restricted peptide 814Val Phe Pro Thr Lys Asp Val Ala Leu 1 5
81510PRTArtificial sequenceHLA restricted peptide 815Val Phe Pro
Thr Lys Asp Val Ala Leu Arg 1 5 10 8169PRTArtificial sequenceHLA
restricted peptide 816Val Phe Ser Arg Gly Asp Thr Pro Val 1 5
81710PRTArtificial sequenceHLA restricted peptide 817Val Phe Ser
Arg Gly Asp Thr Pro Val Leu 1 5 10 8189PRTArtificial sequenceHLA
restricted peptide 818Val Phe Thr Trp Pro Pro Trp Gln Ala 1 5
81910PRTArtificial sequenceHLA restricted peptide 819Val His His
Tyr Pro Ser Ala Ala Glu Arg 1 5 10 8209PRTArtificial sequenceHLA
restricted peptide 820Val His Asn Pro Thr Gly Arg Ser Ile 1 5
82110PRTArtificial sequenceHLA restricted peptide 821Val His Asn
Pro Thr Gly Arg Ser Ile Cys 1 5 10 8229PRTArtificial sequenceHLA
restricted peptide 822Val Ile Gly Asp Gln Tyr Val Lys Val 1 5
82310PRTArtificial sequenceHLA restricted peptide 823Val Ile Gly
Asp Gln Tyr Val Lys Val Tyr 1 5 10 8249PRTArtificial sequenceHLA
restricted peptide 824Val Ile His Ala Ser Gly Lys Gln Met 1 5
82510PRTArtificial sequenceHLA restricted peptide 825Val Ile His
Ala Ser Gly Lys Gln Met Trp 1 5 10 8269PRTArtificial sequenceHLA
restricted peptide 826Val Lys Val Tyr Leu Glu Ser Phe Cys 1 5
8279PRTArtificial sequenceHLA restricted peptide 827Val Leu Cys Pro
Lys Asn Met Ile Ile 1 5 82810PRTArtificial sequenceHLA restricted
peptide 828Val Leu Cys Pro Lys Asn Met Ile Ile Lys 1 5 10
8299PRTArtificial sequenceHLA restricted peptide 829Val Leu Gly Pro
Ile Ser Gly His Val 1 5 83010PRTArtificial sequenceHLA restricted
peptide 830Val Leu Gly Pro Ile Ser Gly His Val Leu 1 5 10
8319PRTArtificial sequenceHLA restricted peptide 831Val Leu Lys Ala
Val Phe Ser Arg Gly 1 5 83210PRTArtificial sequenceHLA restricted
peptide 832Val Leu Lys Ala Val Phe Ser Arg Gly Asp 1 5 10
8339PRTArtificial sequenceHLA restricted peptide 833Val Leu Pro His
Glu Thr Arg Leu Leu 1 5 8349PRTArtificial sequenceHLA restricted
peptide 834Val Met Thr Arg Gly Arg Leu Lys Ala 1 5
8359PRTArtificial sequenceHLA restricted peptide 835Val Asn Val His
Asn Pro Thr Gly Arg 1 5 83610PRTArtificial sequenceHLA restricted
peptide 836Val Asn Val His Asn Pro Thr Gly Arg Ser 1 5 10
8379PRTArtificial sequenceHLA restricted peptide 837Val Pro Met Val
Ala Thr Val Gln Gly 1 5 83810PRTArtificial sequenceHLA restricted
peptide 838Val Pro Met Val Ala Thr Val Gln Gly Gln 1 5 10
8399PRTArtificial sequenceHLA restricted peptide 839Val Pro Ser Gly
Lys Leu Phe Met His 1 5 84010PRTArtificial sequenceHLA restricted
peptide 840Val Pro Ser Gly Lys Leu Phe Met His Val 1 5 10
8419PRTArtificial sequenceHLA restricted peptide 841Val Gln Ala Ile
Arg Glu Thr Val Glu 1 5 84210PRTArtificial sequenceHLA restricted
peptide 842Val Gln Ala Ile Arg Glu Thr Val Glu Leu 1 5 10
84310PRTArtificial sequenceHLA restricted peptide 843Val Gln Gly
Gln Asn Leu Lys Tyr Gln Glu 1 5 10 8449PRTArtificial sequenceHLA
restricted peptide 844Val Gln His Thr Tyr
Phe Thr Gly Ser 1 5 84510PRTArtificial sequenceHLA restricted
peptide 845Val Gln His Thr Tyr Phe Thr Gly Ser Glu 1 5 10
8469PRTArtificial sequenceHLA restricted peptide 846Val Arg Val Ser
Gln Pro Ser Leu Ile 1 5 84710PRTArtificial sequenceHLA restricted
peptide 847Val Arg Val Ser Gln Pro Ser Leu Ile Leu 1 5 10
8489PRTArtificial sequenceHLA restricted peptide 848Val Ser Gln Pro
Ser Leu Ile Leu Val 1 5 84910PRTArtificial sequenceHLA restricted
peptide 849Val Ser Gln Pro Ser Leu Ile Leu Val Ser 1 5 10
8509PRTArtificial sequenceHLA restricted peptide 850Val Ser Gln Tyr
Thr Pro Asp Ser Thr 1 5 8519PRTArtificial sequenceHLA restricted
peptide 851Val Ser Val Asn Val His Asn Pro Thr 1 5
85210PRTArtificial sequenceHLA restricted peptide 852Val Thr Gly
Gly Gly Ala Met Ala Gly Ala 1 5 10 8539PRTArtificial sequenceHLA
restricted peptide 853Val Thr Thr Glu Arg Lys Thr Pro Arg 1 5
85410PRTArtificial sequenceHLA restricted peptide 854Val Thr Thr
Glu Arg Lys Thr Pro Arg Val 1 5 10 8559PRTArtificial sequenceHLA
restricted peptide 855Val Val Cys Ala His Glu Leu Val Cys 1 5
85610PRTArtificial sequenceHLA restricted peptide 856Val Val Cys
Ala His Glu Leu Val Cys Ser 1 5 10 8579PRTArtificial sequenceHLA
restricted peptide 857Val Trp Gln Pro Ala Ala Gln Pro Lys 1 5
85810PRTArtificial sequenceHLA restricted peptide 858Val Trp Gln
Pro Ala Ala Gln Pro Lys Arg 1 5 10 8599PRTArtificial sequenceHLA
restricted peptide 859Val Tyr Ala Leu Pro Leu Lys Met Leu 1 5
86010PRTArtificial sequenceHLA restricted peptide 860Val Tyr Ala
Leu Pro Leu Lys Met Leu Asn 1 5 10 8619PRTArtificial sequenceHLA
restricted peptide 861Val Tyr Leu Glu Ser Phe Cys Glu Asp 1 5
86210PRTArtificial sequenceHLA restricted peptide 862Val Tyr Leu
Glu Ser Phe Cys Glu Asp Val 1 5 10 8639PRTArtificial sequenceHLA
restricted peptide 863Val Tyr Tyr Thr Ser Ala Phe Val Phe 1 5
8649PRTArtificial sequenceHLA restricted peptide 864Trp Asp Ala Asn
Asp Ile Tyr Arg Ile 1 5 86510PRTArtificial sequenceHLA restricted
peptide 865Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe 1 5 10
8669PRTArtificial sequenceHLA restricted peptide 866Trp Asp Arg His
Asp Glu Gly Ala Ala 1 5 8679PRTArtificial sequenceHLA restricted
peptide 867Trp Lys Glu Pro Asp Val Tyr Tyr Thr 1 5
86810PRTArtificial sequenceHLA restricted peptide 868Trp Lys Glu
Pro Asp Val Tyr Tyr Thr Ser 1 5 10 8699PRTArtificial sequenceHLA
restricted peptide 869Trp Pro Pro Trp Gln Ala Gly Ile Leu 1 5
87010PRTArtificial sequenceHLA restricted peptide 870Trp Pro Pro
Trp Gln Ala Gly Ile Leu Ala 1 5 10 8719PRTArtificial sequenceHLA
restricted peptide 871Trp Gln Ala Gly Ile Leu Ala Arg Asn 1 5
87210PRTArtificial sequenceHLA restricted peptide 872Trp Gln Ala
Gly Ile Leu Ala Arg Asn Leu 1 5 10 8739PRTArtificial sequenceHLA
restricted peptide 873Trp Gln Ala Arg Leu Thr Val Ser Gly 1 5
87410PRTArtificial sequenceHLA restricted peptide 874Trp Gln Ala
Arg Leu Thr Val Ser Gly Leu 1 5 10 8759PRTArtificial sequenceHLA
restricted peptide 875Trp Gln Pro Ala Ala Gln Pro Lys Arg 1 5
87610PRTArtificial sequenceHLA restricted peptide 876Trp Gln Pro
Ala Ala Gln Pro Lys Arg Arg 1 5 10 8779PRTArtificial sequenceHLA
restricted peptide 877Trp Thr Arg Gln Gln Asn Gln Trp Lys 1 5
8789PRTArtificial sequenceHLA restricted peptide 878Tyr Ala Leu Pro
Leu Lys Met Leu Asn 1 5 87910PRTArtificial sequenceHLA restricted
peptide 879Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile 1 5 10
8809PRTArtificial sequenceHLA restricted peptide 880Tyr Asp Pro Val
Ala Ala Leu Phe Phe 1 5 88110PRTArtificial sequenceHLA restricted
peptide 881Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe 1 5 10
8829PRTArtificial sequenceHLA restricted peptide 882Tyr Phe Thr Gly
Ser Glu Val Glu Asn 1 5 88310PRTArtificial sequenceHLA restricted
peptide 883Tyr Phe Thr Gly Ser Glu Val Glu Asn Val 1 5 10
8849PRTArtificial sequenceHLA restricted peptide 884Tyr Leu Glu Ser
Phe Cys Glu Asp Val 1 5 8859PRTArtificial sequenceHLA restricted
peptide 885Tyr Pro Ser Ala Ala Glu Arg Lys His 1 5
88610PRTArtificial sequenceHLA restricted peptide 886Tyr Pro Ser
Ala Ala Glu Arg Lys His Arg 1 5 10 8879PRTArtificial sequenceHLA
restricted peptide 887Tyr Gln Glu Phe Phe Trp Asp Ala Asn 1 5
88810PRTArtificial sequenceHLA restricted peptide 888Tyr Gln Glu
Phe Phe Trp Asp Ala Asn Asp 1 5 10 8899PRTArtificial sequenceHLA
restricted peptide 889Tyr Arg His Thr Trp Asp Arg His Asp 1 5
89010PRTArtificial sequenceHLA restricted peptide 890Tyr Arg His
Thr Trp Asp Arg His Asp Glu 1 5 10 8919PRTArtificial sequenceHLA
restricted peptide 891Tyr Arg Ile Phe Ala Glu Leu Glu Gly 1 5
89210PRTArtificial sequenceHLA restricted peptide 892Tyr Arg Ile
Phe Ala Glu Leu Glu Gly Val 1 5 10 8939PRTArtificial sequenceHLA
restricted peptide 893Tyr Arg Ile Gln Gly Lys Leu Glu Tyr 1 5
89410PRTArtificial sequenceHLA restricted peptide 894Tyr Arg Ile
Gln Gly Lys Leu Glu Tyr Arg 1 5 10 8959PRTArtificial sequenceHLA
restricted peptide 895Tyr Ser Glu His Pro Thr Phe Thr Ser 1 5
89610PRTArtificial sequenceHLA restricted peptide 896Tyr Ser Glu
His Pro Thr Phe Thr Ser Gln 1 5 10 8979PRTArtificial sequenceHLA
restricted peptide 897Tyr Thr Pro Asp Ser Thr Pro Cys His 1 5
89810PRTArtificial sequenceHLA restricted peptide 898Tyr Thr Pro
Asp Ser Thr Pro Cys His Arg 1 5 10 8999PRTArtificial sequenceHLA
restricted peptide 899Tyr Thr Ser Ala Phe Val Phe Pro Thr 1 5
90010PRTArtificial sequenceHLA restricted peptide 900Tyr Thr Ser
Ala Phe Val Phe Pro Thr Lys 1 5 10 9019PRTArtificial sequenceHLA
restricted peptide 901Tyr Val Lys Val Tyr Leu Glu Ser Phe 1 5
90210PRTArtificial sequenceHLA restricted peptide 902Tyr Val Lys
Val Tyr Leu Glu Ser Phe Cys 1 5 10 9039PRTArtificial sequenceHLA
restricted peptide 903Tyr Val Tyr Ala Leu Pro Leu Lys Met 1 5
90410PRTArtificial sequenceHLA restricted peptide 904Tyr Val Tyr
Ala Leu Pro Leu Lys Met Leu 1 5 10
* * * * *
References