U.S. patent application number 17/121823 was filed with the patent office on 2021-04-08 for cellular immunity inducing vaccine.
This patent application is currently assigned to JAPANESE FOUNDATION FOR CANCER RESEARCH. The applicant listed for this patent is JAPANESE FOUNDATION FOR CANCER RESEARCH, THE JIKEI UNIVERSITY. Invention is credited to Masaki ITO, Kiyotaka SHIBA.
Application Number | 20210100885 17/121823 |
Document ID | / |
Family ID | 1000005287596 |
Filed Date | 2021-04-08 |
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United States Patent
Application |
20210100885 |
Kind Code |
A1 |
ITO; Masaki ; et
al. |
April 8, 2021 |
CELLULAR IMMUNITY INDUCING VACCINE
Abstract
A novel vaccine that can induce sufficiently high cell-mediated
immunity is disclosed. The vaccine of the present invention
contains, as an effective component, a polypeptide comprising a
tandem repeat structure in which an MHC class I epitope region
derived from an antigen protein and a spacer sequence are linked to
each other alternately and repeatedly at least three times, or a
recombinant vector which comprises a polynucleotide encoding said
polypeptide and is capable of expressing said polypeptide in vivo.
The spacer sequence is, for example, a sequence generated as an
amino acid sequence inevitably encoded by a single base sequence
which is designed such that the MHC class I epitope region derived
from the antigen protein, an MHC class II epitope region derived
from the antigen protein, and at least one
higher-order-structure-stabilizing region are encoded by different
reading frames in said single base sequence.
Inventors: |
ITO; Masaki; (Tokyo, JP)
; SHIBA; Kiyotaka; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JAPANESE FOUNDATION FOR CANCER RESEARCH
THE JIKEI UNIVERSITY |
Tokyo
Tokyo |
|
JP
JP |
|
|
Assignee: |
JAPANESE FOUNDATION FOR CANCER
RESEARCH
Tokyo
JP
THE JIKEI UNIVERSITY
Tokyo
JP
|
Family ID: |
1000005287596 |
Appl. No.: |
17/121823 |
Filed: |
December 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14901384 |
Feb 29, 2016 |
10898555 |
|
|
PCT/JP2014/067355 |
Jun 30, 2014 |
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17121823 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001192 20180801;
A61K 39/001188 20180801; A61K 2039/62 20130101; A61K 39/00117
20180801; A61K 39/001153 20180801; A61K 39/001186 20180801; Y02A
50/30 20180101; A61K 2039/64 20130101; A61K 39/0011 20130101; A61K
39/001156 20180801; A61K 39/001106 20180801; A61K 39/001162
20180801; A61K 39/001124 20180801; A61K 39/001191 20180801; C07K
19/00 20130101; C07K 14/4748 20130101; A61K 39/001151 20180801;
A61K 39/00115 20180801; C07K 14/70539 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/47 20060101 C07K014/47; C07K 14/74 20060101
C07K014/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 2, 2013 |
JP |
2013-138688 |
Claims
1. A composition comprising a pharmaceutically acceptable
additive(s) and, as an effective component, a recombinant vector
comprising a polynucleotide encoding a polypeptide having an amino
acid sequence selected from the amino acid sequences shown in SEQ
ID NOs:54-63 and is capable of expressing said polypeptide in
vivo.
2. The composition of claim 1, further comprising an adjuvant that
activates the Toll-like receptor pathway.
3. The composition of claim 1, wherein said recombinant vector
comprises a polynucleotide encoding a polypeptide having an amino
acid sequence shown in SEQ ID NO: 54 and is capable of expressing
said polypeptide in vivo.
4. The composition of claim 1, wherein said recombinant vector
comprises a polynucleotide encoding a polypeptide having an amino
acid sequence shown in SEQ ID NO: 55 and is capable of expressing
said polypeptide in vivo.
5. The composition of claim 1, wherein said recombinant vector
comprises a polynucleotide encoding a polypeptide having an amino
acid sequence shown in SEQ ID NO: 56 and is capable of expressing
said polypeptide in vivo.
6. The composition of claim 1, wherein said recombinant vector
comprises a polynucleotide encoding a polypeptide having an amino
acid sequence shown in SEQ ID NO: 57 and is capable of expressing
said polypeptide in vivo.
7. The composition of claim 1, wherein said recombinant vector
comprises a polynucleotide encoding a polypeptide having an amino
acid sequence shown in SEQ ID NO: 58 and is capable of expressing
said polypeptide in vivo.
8. The composition of claim 1, wherein said recombinant vector
comprises a polynucleotide encoding a polypeptide having an amino
acid sequence shown in SEQ ID NO: 63 and is capable of expressing
said polypeptide in vivo.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/901,384 filed Feb. 29, 2016, which is the 371 National Phase
of PCT International Application No. PCT/JP2014/067355, filed on
Jun. 30, 2014, which claims priority under 35 U.S.C. 119(a) to
Patent Application No. 2013-138688, filed in Japan on Jul. 2, 2013,
all of which are hereby expressly incorporated by reference into
the present application.
TECHNICAL FIELD
[0002] The present invention relates to a vaccine that can
effectively induce cell-mediated immunity.
BACKGROUND ART
[0003] In recent years, tumor immunotherapy using an epitope
peptide (minimal peptide sequence presented by an MHC class I or
class II molecule) is attracting attention. Epitope peptide
vaccines are administered in the form of a suspension of an epitope
peptide in an oil emulsion such as Montanide. It is thought that,
in such immunization, large amounts of the epitope peptide bind to
empty MHC molecules of antigen-presenting cells, or peptide
replacement occurs due to competition with peptides that are
already bound to MHC molecules, leading to exertion of the function
(Non-patent Documents 1 to 4). However, since this method does not
include the inherent process of antigen processing by dendritic
cells or the like, the efficiency of antigen presentation may be
low. Moreover, it has recently been reported that oil adjuvants,
which are indispensable for epitope peptide vaccines, cannot
efficiently produce an antitumor effect since inflammation at the
vaccination site causes localization of cytotoxic T cells (CTLs),
which are responsible for attacking the tumor, in the vaccination
site rather than the tumor site (Non-patent Document 5). Thus,
development of peptide vaccines that can stably produce
immunogenicity without using oil adjuvants has been hoped.
[0004] On the other hand, it is thought that, in cases where a
full-length protein is used as an immunogen, the protein is
processed by antigen-presenting cells such as dendritic cells, but
the number of epitopes per protein molecule decreases, so that the
amount of peptide presented by MHC molecules may be smaller than
that in cases of epitope peptide immunization.
[0005] Antigens administered as vaccines are recognized in the body
as foreign antigens, and incorporated into antigen-presenting
cells. The antigens are then presented by MHC class II molecules,
and tend to induce humoral immunity. For diseases such as AIDS,
malaria, and malignant tumors, induction of cell-mediated immunity
is important. In these diseases, humoral immunity is not capable of
responding to the diseases since specific antigens of the diseases
are not expressed on the cell surface, where recognition by
antibodies occur. Since the antigens specific to these diseases
undergo processing in cells as endogenous antigens, and are
presented by MHC class I molecules, only cell-mediated immunity can
produce an effect. Thus, for immunotherapies against such diseases
using a protein as an antigen, development of a system that allows
induction of cell-mediated immunity rather than humoral immunity
has been hoped.
[0006] It is known that professional antigen-presenting cells such
as dendritic cells have a mechanism that allows induction of
cell-mediated immunity against foreign antigens. This system is
called cross-presentation. In this phenomenon, antigens
incorporated as foreign antigens into antigen-presenting cells
undergo degradation by proteasome, and are presented by MHC class I
molecules. However, the types of antigens that are likely to
undergo cross-presentation and details of the mechanism of this
phenomenon are still unclear.
PRIOR ART DOCUMENTS
Non-Patent Documents
[0007] Non-patent Document 1: Yamada A, et al. Cancer Sci. 2013
January; 104(1): 15-21. doi: 10.1111/cas.12050. Epub 2012 Dec 4.
Next-generation peptide vaccines for advanced cancer.
[0008] Non-patent Document 2: Khazaie K, et al. Curr Opin Oncol.
2009 November; 21(6): 524-30. doi: 10.1097/CC0.0b013e328331a78e.
Current developments with peptide-based human tumor vaccines.
[0009] Non-patent Document 3: Perez S A, et al. Cancer. 2010 May 1;
116(9): 2071-80. doi: 10.1002/cncr.24988. A new era in anticancer
peptide vaccines.
[0010] Non-patent Document 4: Slingluff C L Jr. Cancer J. 2011
September-October; 17(5): 343-50. doi:
10.1097/PPO.0b013e318233e5b2. The present and future of peptide
vaccines for cancer: single or multiple, long or short, alone or in
combination?
[0011] Non-patent Document 5: Hailemichael Y, et al. Nat Med. 2013
April; 19(4): 465-72. doi: 10.1038/nm.3105. Epub 2013 Mar 3.
Persistent antigen at vaccination sites induces tumor-specific
CD8.sup.+ T cell sequestration, dysfunction and deletion.
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0012] Accordingly, an object of the present invention is to
provide a novel vaccine that allows induction of sufficiently high
cell-mediated immunity.
Means for Solving the Problems
[0013] From the viewpoint of obtaining a high therapeutic effect by
a vaccine, a peptide vaccine is thought to be more preferred since
it can induce cross-presentation. The present inventors thought
that the defects of conventional immunization using a short
polypeptide such as an epitope peptide or a polypeptide having a
large size such as a full-length protein can be overcome by use of
an artificial protein obtained by improving the higher-order
structure of peptide vaccines. In view of this, the present
inventors intensively studied using an artificial protein creation
technology based on the MolCraft method, which was developed by
Kiyotaka Shiba et al., and, as a result, succeeded in identifying a
structure important for artificial protein antigens which allow
induction of strong cell-mediated immunity, thereby completing the
present invention.
[0014] That is, the present invention provides a vaccine containing
as an effective component a polypeptide comprising a tandem repeat
structure in which an MHC class I epitope region and a spacer
sequence are linked to each other alternately and repeatedly at
least three times, wherein each of MHC class I epitope regions is
derived from an antigen protein and each of spacer sequences is
either (1) or (2) described below, or a recombinant vector which
comprises a polynucleotide encoding said polypeptide and is capable
of expressing said polypeptide in vivo:
[0015] (1) a sequence generated as an amino acid sequence
inevitably encoded by a single base sequence which is designed such
that said MHC class I epitope region, an MHC class II epitope
region derived from the same or a different antigen protein
mentioned above, and at least one
higher-order-structure-stabilizing region are encoded by different
reading frames in said single base sequence;
[0016] (2) a sequence which is the same amino acid sequence as (1)
except that several amino acids are substituted.
[0017] The present invention also provides a vaccine containing as
an effective component a polypeptide comprising a tandem repeat
structure in which an MHC class I epitope region and a spacer
sequence are linked to each other alternately and repeatedly at
least three times, wherein each of MHC class I epitope regions is
derived from an antigen protein and each of spacer sequences is
either (1) or (2) described below, or a recombinant vector which
comprises a polynucleotide encoding said polypeptide and is capable
of expressing said polypeptide in vivo:
[0018] (1) a sequence generated as an amino acid sequence
inevitably encoded by one reading frame in a single base sequence
which is designed such that an MHC class II epitope derived from
the same or a different antigen protein mentioned above and a
higher-order-structure-stabilizing region(s) are encoded by
different reading frames in said single base sequence, and such
that no stop codon is generated in the remaining reading frame,
said remaining reading frame being the above-mentioned one reading
frame;
[0019] (2) a sequence which is the same amino acid sequence as (1)
except that several amino acids are substituted.
Effect of the Invention
[0020] By the present invention, a peptide vaccine having an
excellent capacity to induce cell-mediated immunity is provided.
The peptide vaccine of the present invention has a
cross-presentation capacity for MHC class I and MHC class II, and
has a sufficiently high immune-inducing capacity. Even in cases
where a smaller amount of oil adjuvant is used, or where no oil
adjuvant is used, the peptide vaccine of the present invention can
induce a much higher level of, for example, not less than 100 times
higher level of, cell-mediated immunity than the original antigen
protein. By the technique of the present invention, a vaccine
having a high immune-inducing capacity can be provided even when a
peptide epitope having only weak immunogenicity is used. For
treatment and prevention of diseases such as malaria, AIDS, and
tumors, induction of cell-mediated immunity is required. By the
present invention, a vaccine effective for treatment and prevention
of such diseases can be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a diagram illustrating construction of microgenes
and artificial proteins. Panel (a) is the amino acid sequence of
native antigen ovalbumin (OVA). Panel (b) shows microgenes designed
in Examples. Panel (c) is a schematic diagram illustrating a scheme
of construction of an artificial protein and showing various motifs
and artificial proteins. Panel (d) shows the results of comparison
of the in vitro antigen-presenting capacity among various
artificial proteins and OVA. This panel also shows an SDS-PAGE
image of artificial proteins.
[0022] FIG. 2 is a diagram illustrating the flow from designing of
microgene #2101 to construction of an artificial protein gene.
[0023] FIG. 3 shows the amino acid sequences of artificial
proteins.
[0024] FIG. 4 shows the amino acid sequences of artificial
proteins.
[0025] FIG. 5 shows the results of analysis of the
antigen-presenting capacity of the artificial protein F37A. Panel
(a) is a diagram schematically showing the amino acid sequence of
F37A and the sequences of other artificial proteins. Panel (b)
shows the results of evaluation of the antigen-presenting capacity
based on the level of in vitro IL-2 production. Panel (c) shows a
diagram illustrating the structures of mutants prepared by
replacing an MHC class I epitope motif of OVA contained in F37A
with an MHC class I epitope motif of WT1 (left), and a graph
showing the results of evaluation of the antigen-presenting
capacity of each mutant based on the level of IL-2 production
(right). Panel (d) shows the results of a cross-presentation
assay.
[0026] FIG. 6 shows the amino acid sequences of artificial proteins
and mutants prepared by replacement with an MHC class I epitope
motif of WT1.
[0027] FIG. 7 shows the results of investigation of the secondary
structures of artificial proteins by circular dichroism (CD)
analysis.
[0028] FIG. 8 shows the results of Hydropathy (Kyte-Doolittle)
hydrophobicity analysis of artificial proteins using Strider 1.4f7
software.
[0029] FIG. 9 shows the results of Amphipathicity analysis using
Strider 1.4f7 software.
[0030] FIG. 10 shows the results of flow cytometric measurement of
the expression levels of CD80 and CD86 on the surface of living
CD11c.sup.+ cells stimulated with F37A for 20 hours.
[0031] FIG. 11 shows the results of investigation of induction of
antigen-specific CTLs in vivo in mice immunized with F37A. (a)
Results of a cytotoxicity assay using spleen cells collected from
the immunized mice. (b) Results of observation of tumor growth in
mice which received the immunization and subsequent tumor cell
transplantation. (c) Results of investigation of whether anti-OVA
antibodies were produced in vivo in the immunized mice or not.
[0032] FIG. 12 shows the results of a study on involvement of class
I epitopes and class II epitopes in the antigenicity of F37A. (a)
The amino acid sequences of F37A, F36C, and MT825. (b) Results of
investigation of the CTL activity by a .sup.51Cr release assay in
mice immunized together with the MPL adjuvant. (c) Results of a
tetramer assay. (d) Results of observation of tumor growth in mice
which received the immunization and subsequent tumor cell
transplantation. (e) Graphs showing tumor growth in each individual
of the immunized mice to which tumor cells were transplanted.
[0033] FIG. 13 shows a schematic diagram showing the structures of
F37A and C131B. (A) DC2.4 cells, which are antigen-presenting
cells, were treated with no inhibitor, or with cytochalasin B
(phagocytosis inhibitor), DMA (pinocytosis inhibitor), or Poly-I
(scavenger receptor A inhibitor). Thereafter, an antigen (F37A,
C131B, or OVA) was added to the cells, and the cells were then
cultured. The cultured cells were provided as samples for Western
blotting using an anti-His-tag antibody and an anti-OVA antibody to
measure the level of antigen incorporation into the
antigen-presenting cells (lane 1, untreated control; lane 2,
treatment with cytochalasin B; lane 3, treatment with DMA; lane 4,
treatment with Poly-I). The band intensity for each antigen
incorporated into the cells was measured by densitometry, and
quantified using .beta.-actin as a reference. The quantified values
were represented as graphs. (B) DC2.4 cells, which are
antigen-presenting cells, were treated with no inhibitor, or with
cytochalasin B, DMA, or Poly-I. Thereafter, an antigen (F37A,
C131B, or OVA) was added to the cells, and the cells were then
cultured. Subsequently, OVA-specific T cell hybridoma cells
(RF33.70 cells) were added to the culture to perform co-culture. By
measuring the amount of IL-2 produced into the culture supernatant,
the OVA-specific antigen-presenting capacity was evaluated.
[0034] FIG. 14 (A) Results obtained by subjecting known amounts
(100 ng, 50 ng, 10 ng, and 5 ng) of each of the F37A antigen and
the C131B antigen to electrophoresis, carrying out WB (Western
blotting) using an anti-His-tag antibody, and then detecting the
antigens by autoradiography. The band intensities were measured by
densitometry, and calibration curves were prepared based on the
antigen concentrations and the band intensities (diagrams showing
the calibration curves are not shown). (B) An antigen (F37A or
C131B) was added to DC2.4 cells, which are antigen-presenting
cells, and the cells were then cultured. By Western blotting using
an anti-His-tag antibody and an anti-OVA antibody, the level of
antigen incorporation into the antigen-presenting cells was
measured. Based on the calibration curves obtained in A, the levels
of F37A and C131B antigen incorporations into the
antigen-presenting cells were semi-quantified. Two samples were
subjected to the incorporation experiment for each of F37A and
C131B. The amounts of F37A incorporated were 6 ng and 5 ng. The
amounts of C131B incorporated were 29 ng and 22 ng. (C) In the
cases of the F37AE2 antigen (SEQ ID NO:45), which had the same
amino acid sequence as F37A except for the 5 amino acids in the
C-terminus, antigen presentation was suppressed by an SRA inhibitor
fucoidan.
MODE FOR CARRYING OUT THE INVENTION
[0035] The polypeptide used as an effective component in the
present invention is an artificial protein which is not naturally
present. The effective component polypeptide is characterized in
that it comprises a tandem repeat structure in which one MHC class
I epitope region and one spacer sequence are linked to each other
alternately and repeatedly at least three times, wherein each of
the MHC class I epitope region is derived from an antigen protein
and each of the spacer sequence is as defined in the present
description. Thanks to the structure comprising at least three
repeats each of which comprises a class I epitope region and a
spacer sequence, the polypeptide can strongly induce cell-mediated
immunity against the subject antigen protein. Usually, in the case
of peptide vaccines containing a polypeptide as an effective
component, use of a certain amount of aluminum adjuvant or oil
adjuvant is indispensable for induction of sufficient immunity in
vivo. However, the peptide vaccine of the present invention can
exhibit a sufficiently high immune-inducing capacity while reducing
the amount of the aluminum adjuvant or the oil adjuvant and hence
reducing side effects, or while using none of such adjuvants.
[0036] The "MHC class I epitope derived from an antigen protein"
and the "MHC class II epitope derived from an antigen protein"
include not only epitopes whose amino acid sequences are the same
as those of the corresponding epitopes found in native antigen
proteins, but also epitopes whose amino acid sequences are the same
as the native epitope sequences except that small numbers of
residues are modified. It is known that modification of the
sequence of a native MHC class I epitope or class II epitope
increases its epitope functions such as the binding capacity to MHC
class I or class II molecules. For example, it is known that, in
the MHC class I epitope CMTWNQMNL in the tumor antigen WT1,
substitution of the second M to Y increases the binding capacity of
the epitope to MHC class I molecules (Cancer Immunol Immunother
(2002) 51: 614-620). Such a modified MHC class I or class II
epitope is also included in the "MHC class I epitope derived from
an antigen protein" or the "MHC class II epitope derived from an
antigen protein".
[0037] The tandem repeat structure is a structure comprising at
least three units linked to each other, wherein each unit is
composed of an MHC class I epitope region and a spacer sequence.
Although the upper limit of the number of the repeats is not
limited, the size of the effective component polypeptide is
preferably not more than about 500 residues from the viewpoint of
the vaccine production cost and the like. In cases where the
polynucleotide encoding the effective component polypeptide is
obtained by polymerization of microgenes by the later-mentioned MPR
method, the size of the polypeptide encoded usually becomes not
more than about 300 residues due to the general upper limit of the
size of the microgene polymer in the MPR method. Thus, the number
of the repeats in the tandem repeat structure is usually not more
than about 10.
[0038] In the tandem repeat structure, not more than several,
preferably not more than 5, more preferably not more than 3
residues may be inserted in a part of the motif-linking portions,
and/or not more than several, preferably not more than 5, more
preferably not more than 3 residues may be deleted in a part of the
motif-linking portions. Such modification of residues in the
motif-linking portions inevitably occurs due to the nature of the
MPR method. The MHC class I epitope region may contain, as
described below, a small number of the adjacent residues derived
from the original antigen protein at the end(s) of the minimal
epitope sequence, and such residues that may be contained in the
class I epitope region besides the minimal epitope sequence may be
deleted in a part of the repeat units, as long as the minimal
epitope sequences are maintained in the tandem repeat
structure.
[0039] It is not necessary that all the spacer sequence motifs in
the tandem repeat structure should be completely identical, and the
tandem repeat structure may also comprise a spacer sequence
motif(s) having not more than several, preferably not more than 6
nonidentical residues. In cases where the effective component
polypeptide is obtained from an artificial protein library prepared
by polymerization of microgenes by the MPR method, a motif sequence
not identical to the motif sequence originally defined by the
microgene is often generated due to a random reading frame shift
during the polymerization reaction process. The spacer sequence in
the present invention may be composed of a sequence generated in
such a manner, in which sequence a part of the residues (preferably
not more than 6 residues) are different from those in the spacer
sequence(s) found in other repeat unit(s).
[0040] The spacer sequence used in the present invention is a
sequence generated as an amino acid sequence inevitably encoded by
a single base sequence which is designed such that an MHC class I
epitope region derived from an antigen protein, an MHC class II
epitope region derived from the same or a different antigen protein
mentioned above, and at least one
higher-order-structure-stabilizing region are encoded by different
reading frames of the single base sequence. Preferably, the amino
acid sequence of the spacer sequence is a sequence generated by the
same reading frame as the reading frame for the class I epitope
region, which sequence occurs adjacent to the class I epitope
region. Such a base sequence designed such that a plurality of
motifs are encoded by different reading frames and such that no
stop codon occurs in any of the reading frames is sometimes called
a multifunctional base sequence or a microgene. When the amino acid
sequences encoded by two out of the three reading frames are
determined, the amino acid sequence in the remaining frame is
automatically determined.
[0041] Or, the spacer sequence used in the present invention has
the same sequence as the amino acid sequence automatically
determined as described above except that a region composed of
several amino acid residues is replaced. More specifically, the
spacer sequence may be a sequence in which a region of several
amino acid residues is replaced with an amino acid sequence derived
from a part of the MHC class II epitope region or the
higher-order-structure-stabilizing region encoded by another
reading frame. In cases where a polypeptide is prepared from a
microgene polymer prepared by the MPR method, insertion and/or
deletion of a base(s) often randomly occur(s) in a linking
portion(s) of the microgenes due to the exonuclease activity of the
polymerase, resulting in generation of a motif sequence which is
partially replaced with an amino acid sequence derived from a motif
sequence in another reading frame. In the following Examples, the
tandem repeat structure of the artificial protein F182A contains
such a partially replaced spacer sequence. It should be noted,
however, that in some cases a tandem repeat structure in which none
of the spacer sequences has such a replaced sequence may give the
polypeptide a higher capacity to induce cell-mediated immunity.
[0042] The MHC class I epitope and the MHC class II epitope may be
derived from the same antigen protein, or may be derived from
different antigen proteins. Typically, the MHC class I epitope and
the MHC class II epitope may be derived from the same antigen.
Epitope sequences capable of binding to a plurality of MHC class II
molecules are known (e.g., the pan HLA-DR-binding epitope, which is
called the PADRE epitope; see, for example, Hum Immunol. 2012
January 73(1): 1-10. and Molecular Therapy vol. 15 no. 6, 1211-1219
June 2007). In cases where a class I epitope and a class II epitope
derived from different antigen proteins are used, such an epitope
which can bind to a plurality of MHC class II molecules may be
used. Specific examples of the effective component polypeptide
using a PADRE epitope include the sequences of SEQ ID NOs: 61 and
62 in SEQUENCE LISTING (see Tables 1 and 2 below).
[0043] In the present invention, the multifunctional base sequence
may be designed such that a total of six motifs are encoded in the
three reading frames and such that no stop codon is contained in
any of the three reading frames (see FIG. 2). Among the six motifs,
one motif corresponds to an MHC class I epitope region; one motif
corresponds to an MHC class II epitope region; and two motifs
correspond to higher-order-structure-stabilizing regions. Usually,
in such cases, a multifunctional base sequence (I) encoding an MHC
class I epitope region and a multifunctional base sequence (II)
encoding an MHC class II epitope region are separately designed,
and these two multifunctional base sequences are linked to each
other while adjusting the reading frames. By this, a single
multifunctional base sequence (microgene) in which the class I
epitope, the class II epitope, and at least one
higher-order-structure-stabilizing region are encoded by different
reading frames is designed. When the amino acid sequences of two
reading frames in a multifunctional base sequence are determined
and the remaining reading frame is designed such that no stop codon
is generated therein, the amino acid sequence of this remaining
reading frame is automatically determined. Accordingly, one
automatically determined sequence motif for the class I epitope in
the multifunctional gene (I) and one automatically determined
sequence motif for the class II epitope in the multifunctional gene
(II) are obtained. In the microgene sequence after the linking, the
two higher-order-structure-stabilizing region motifs may be placed
in the same reading frame, or in different reading frames. However,
since the microgene sequence is designed such that the class I
epitope and the class II epitope are not placed in the same reading
frame, the automatically determined sequence motifs are not encoded
in the same reading frame. Among the thus obtained automatically
determined or inevitably generated sequence motifs, the sequence
motif generated for the class II epitope, which occurs adjacent to
the class I epitope region in the same reading frame as the reading
frame for the class I epitope in the microgene, is used as the
spacer sequence in the tandem repeat structure.
[0044] The "higher-order-structure-stabilizing region" is a region
having a sequence that allows a polypeptide to have a stable
higher-order structure when the polypeptide is expressed from a
nucleic acid polymer obtained by polymerization of multifunctional
base sequences. The higher-order structure of a polypeptide is
stabilized by formation of an .alpha.-helix structure(s),
.beta.-sheet structure(s), intramolecular hydrophobic bond(s),
and/or the like. Specific examples of the
higher-order-structure-stabilizing region include an
.alpha.-helix-forming region (amino acid sequence region which
tends to form an .alpha.-helix structure(s)), .beta.-sheet-forming
region (amino acid sequence region which tends to form a
.beta.-sheet structure(s)), and hydrophobic-bond-forming region
(region which is rich in amino acid residues having a hydrophobic
side chain and tends to form an intramolecular hydrophobic
bond(s)). It is known that a protein can have a stable higher-order
structure by having such structures. The
higher-order-structure-stabilizing region is preferably at least
one selected from an .alpha.-helix-forming region and a
.beta.-sheet-forming region, more preferably an
.alpha.-helix-forming region. It is known that, among amino acid
residues, there are residues that tend to form an .alpha.-helix and
residues that tend to form a .beta.-sheet. The
.alpha.-helix-forming region and the .beta.-sheet-forming region
may be constituted using such residues
[0045] The multifunctional base sequence (microgene) is preferably
designed such that, among the three reading frames, the MHC class I
epitope region is encoded in one reading frame; the MHC class II
epitope region is encoded in another reading frame; and the at
least one .alpha.-helix-forming region is encoded in the other
reading frame. As described above, the spacer sequence is an amino
acid sequence motif which occurs adjacent to the MHC class I
epitope region in the reading frame encoding the MHC class I
epitope region. For example, if the MHC class I epitope region is
encoded in the first frame, the MHC class II epitope region is
encoded in the second frame (the reading frame which occurs by
one-base shift from the first frame in the 3'-direction), and one
or two .alpha.-helix-forming regions are encoded in the third frame
(the reading frame which occurs by two-base shift from the first
frame in the 3'-direction), then the amino acid sequence generated
in the first frame may be used as the spacer sequence.
[0046] In general, MHC class I epitopes retained by antigen
proteins have a size of about 5 to 12 residues, typically about 8
to 10 residues. Although it is known that the lengths of MHC class
II epitopes are not strictly limited, MHC class II epitopes mostly
have a size of 13 to 30 residues, and a class II epitope having a
size of about 13 to 23 residues may be preferably used as the class
II epitope region motif in the present invention. Therefore, for
example, if a multifunctional base sequence is designed such that
the class I epitope is encoded in the first reading frame, the
class II epitope is encoded in the second reading frame, and the at
least one higher-order-structure-stabilizing region is encoded in
the third reading frame, then the multifunctional base sequence
usually has a size of about 30 bp to 90 bp, and the spacer sequence
obtained has a size of about 10 to 30 residues.
[0047] The "MHC class I epitope region" may contain not only the
minimal unit of the MHC class I epitope derived from an antigen
protein, but also several (for example, one to three) residues
adjacent to each side of the epitope in the amino acid sequence of
the original antigen protein. Usually, it is preferred to add at
least two amino acid residues derived from the amino acid sequence
of the original antigen protein to the N-terminus of the minimal
sequence of the MHC class I epitope, and at least one such amino
acid residue to the C-terminus, and to use the resulting amino acid
sequence as the MHC class I epitope region motif. More
specifically, for example, when the antigen protein sequence is . .
. abcdXXXXXXXXefgh . . . wherein XXXXXXXX represents a class I
epitope, cdXXXXXXXXe obtained by adding the N-terminal side "cd"
and the C-terminal side "e" to the epitope may be used as the MHC
class I epitope region motif. It is known that the C-terminal side
of the epitope is cleaved by proteasome with relatively low
sequence specificity, while the N-terminal side is cleaved by
sequence-specific aminopeptidase. By also maintaining, in the
effective component polypeptide molecule, the structures in both
sides of the epitope in the original antigen protein as described
above, antigen presentation can be allowed to occur more
efficiently, and the capacity to induce cell-mediated immunity can
be further increased. The same applies to the MHC class II epitope
region.
[0048] The sequences of MHC class I epitopes and MHC class II
epitopes in various antigen proteins have been identified, and are
known. Further, since algorithms for prediction of epitopes from
amino acid sequence information are known (for example, SYFPEITHI
algorithm software), such algorithms may be applied to an arbitrary
antigen protein for predicting epitopes that bind to MHC molecules,
and the predicted epitopes may be used as the MHC class I and class
II epitopes. Further, as described above, it is known that partial
modification of native MHC class I and class II epitopes allows
enhancement of their epitope functions (for example, the binding
capacity to MHC class I molecules or class II molecules). In the
present invention, such a modified epitope sequence may also be
used.
[0049] The spacer sequence obtained for an MHC class I epitope or
class II epitope as described above may contain a large number of
hydrophilic amino acids (e.g. R, N, D, E, Q, G, H, K, P, S, T, Y)
and have a hydrophilic property. Whether the spacer sequence is
hydrophilic or not can be investigated by Hydropathy
(Kyte-Doolittle) analysis using Strider 1.4f7 software.
[0050] The amphipathicity of the tandem repeat structure portion
may be 0.0 to 0.4. An amphipathicity analysis can be carried out
using Strider 1.4f7 software.
[0051] The polypeptide containing the tandem repeat structure
described above, which is used as an effective component, may
contain an MHC class II epitope region derived from the same
antigen protein in at least one of the N-terminal region and the
C-terminal region. By this, the capacity of the vaccine to induce
cell-mediated immunity can be further increased.
[0052] The effective component polypeptide may contain the
higher-order-structure-stabilizing region as defined above. By the
inclusion of the region which stabilizes a higher-order structure
such as an .alpha.-helix structure or a .beta.-sheet structure, the
stability of the polypeptide increases, and the production
efficiency in host cells such as E. coli can be increased.
[0053] The effective component polypeptide may also contain a tag
sequence such as a histidine tag for, e.g., convenience in
production of the polypeptide.
[0054] The effective component polypeptide may have an isoelectric
point (pI) of 6.0 to 8.6. For adjusting the isoelectric point of
the polypeptide to such a nearly neutral value, a sequence such as
DYKDHDGDYKDHDIDYKDDDDKL (SEQ ID NO: 69, triple FLAG tag sequence)
or DEDEDED (SEQ ID NO:70) may be introduced to the effective
component polypeptide, if necessary. Specific examples of effective
component polypeptides in which such sequences are introduced are
shown in SEQ ID NOs:57 to 60 in SEQUENCE LISTING (see Table 1-1
below).
[0055] Methods for designing a multifunctional base sequence is
known. For example, CyberGene software described in K. Shiba,
Journal of Molecular Catalysis B: Enzymatic 28 (2004) 145-153 may
be employed therefor. More specifically, multifunctional base
sequences can be designed by the designing methods described in,
for example, JP 4007477 B, JP 4911857 B, and JP 4989600 B. Any of
these methods may be used in the present invention.
[0056] JP 4007477 B is a designing method that is carried out by a
process in which a peptide sequence given a first function (the MHC
class I epitope peptide sequence and class II epitope peptide
sequence, in the case of the present application) is set as an
initial value, and reverse translation is performed based on the
genetic codon table in a base-by-base manner to generate all
possible base sequences encoding the peptide sequence in a
computer, followed by writing the peptide sequence population
encoded by all generated base sequences in the reading frames other
than the reading frame of the first peptide sequence in the
computer, and then selecting peptides having second and third
functions from the peptide sequence population. In this method, the
protein to be encoded by the multifunctional base sequence is
analyzed as a linked product of 20 kinds of amino acids.
[0057] The designing methods described in JP 4911857 B and JP
4989600 B are improved methods of the designing method described in
JP 4007477 B. In these methods, the protein to be encoded by the
multifunctional base sequence is analyzed as an overlapping/linked
product of very short peptide sequences of about two to eight
residues rather than a linked product of 20 kinds of amino acids.
The base sequence encoding a dipeptide is composed of six bases,
and the six bases already have the information on the translation
products in the second and the third reading frames. Therefore, by
performing analysis and calculation for the protein as an
overlapping/linked product of very short peptide sequences of about
two to eight residues, base sequences containing a stop codon(s) in
the middle of the second frame and/or the third frame can be
eliminated before the computation. By this, the computation time
and the memory size required can be largely reduced relative to the
method in which the protein is analyzed as a linked product of 20
kinds of amino acids.
[0058] In the Examples below, multifunctional base sequences
(microgenes) were designed using CyberGene software, and a
population of microgene polymers (artificial protein genes) was
constructed by a known microgene polymerization method (microgene
polymerization reaction, MPR; Kiyotaka Shiba et al., PNAS vol. 94,
pp. 3805-3810, 1997). Proteins were then expressed from these
polymers to obtain proteins having a high antigen-presenting
capacity (FIGS. 1 and 2). Such a method is known as the
MOLCRAFT.RTM. method (K. Shiba, Journal of Molecular Catalysis B:
Enzymatic 28 (2004) 145-153), and various artificial proteins have
been synthesized by this method (for example, Saito et al.,
Chemistry & Biology, Vol. 11, 765-773, 2004; Saito et al.,
Nucleic Acids Research, 2007, Vol. 35, No. 6, e38; and Kokubun et
al., Biomacromolecules 2008, 9, 3098-3105). By using the
MOLCRAFT.RTM. method, the polypeptide sequence to be used for the
vaccine of the present invention can be obtained for various
antigen proteins.
[0059] A method for producing an anti-cancer vaccine according to
the present invention using MHC class I and class II epitope
sequences derived from the WT1 protein is described below.
[0060] In the WT1 protein (SEQ ID NO:23), as MHC class I epitopes,
CMTWNQMNL (SEQ ID NO: 71, residues at positions 303 to 311 in SEQ
ID NO:23) and RMFPNAPYL (SEQ ID NO: 72, residues at positions 194
to 202 in SEQ ID NO:23), and their modified sequences (for example,
the sequence obtained by replacing the second M of CMTWNQMNL (SEQ
ID NO: 71) with Y; Cancer Immunol Immunother (2002) 51: 614-620)
may be used. In the present case, RMFPNAPYL (SEQ ID NO: 72) is
employed. RMFPNAPYL (SEQ ID NO: 72) is used for designing
multifunctional base sequences with addition of several amino acid
residues which are adjacent thereto in both sides in the original
WT1 protein (for example, addition of QA, which is adjacent in the
N-terminal side, and P, which is adjacent in the C-terminal side,
to the corresponding termini). As the MHC class II epitope,
PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 73, residues at positions 396 to
417 in SEQ ID NO:23) may be used. Briefly, the procedure is as
follows: a multifunctional base sequence encoding the class I
epitope and a multifunctional base sequence encoding the class II
epitope are separately designed, and the resulting two
multifunctional base sequences are fused with each other for
designing a microgene, followed by designing MPR primers based on
the sequence of this microgene and performing microgene
polymerization reaction by the MPR method.
[0061] First, in a computer, each of QARMFPNAPYLP (SEQ ID NO: 74)
and PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 73) is set as an initial
value (first sequence). Reverse translation into base sequences is
then performed based on the genetic codon table in a base-by-base
manner to generate all possible base sequences encoding the peptide
sequence in the computer. Subsequently, from the base sequences
encoding QARMFPNAPYLP (SEQ ID NO: 74) and the base sequences
encoding PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 73), sequences encoding
a higher-order-structure-stabilizing region in another reading
frame are selected (second sequences) for each first sequence. In
cases where CyberGene software is used, when QARMFPNAPYLP (SEQ ID
NO: 74) and PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 73) are fed as first
sequences, a number of sequence candidates that tend to form an
.alpha.-helix structure and/or .beta.-sheet structure are
described. Hence, sequences which tend to have a stable structure
may be selected therefrom as the second sequences. Simple reverse
translation of the first-sequence motifs produces a vast number of
combinations of DNA sequences. However, since the CyberGene program
is designed such that cases where identical motif sequences are
generated in a plurality of reading frames and cases where a stop
codon(s) is/are generated in any of the reading frames are
eliminated, the number of candidates for the multifunctional base
sequences obtained from the motif sequences fed is much smaller
than the theoretical number of the combinations. By assigning a
first sequence and a second sequence, the third sequence can be
automatically determined. Hundreds or more of multifunctional base
sequences each of which encodes an epitope motif in one reading
frame and a higher-order-structure-stabilizing motif in one of the
other reading frames are output. These sequences are ranked based
on the tendency to form a structure. About 10 top-ranked sequences
are selected from those sequences.
[0062] By linking a multifunctional sequence (I) encoding
QARMFPNAPYLP (SEQ ID NO: 74) in the first frame and a
multifunctional sequence (II) encoding PGCNKRYFKLSHLQMHSRKHTG (SEQ
ID NO: 73) in the first frame obtained as described above to each
other, a microgene sequence encoding the MHC class I epitope and
class II epitope of WT1, and at least one
higher-order-structure-stabilizing region, in different reading
frames is obtained. Among the candidate sequences, sequences in
which no stop codon is generated even in cases where the frame is
shifted are selected, and adjustment of the sequences is carried
out. In this process, the linking site is appropriately adjusted
such that the MHC class I motif QARMFPNAPYLP (SEQ ID NO: 74) and
the MHC class II motif PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 73) are
not placed in the same reading frame. A third sequences, which are
automatically determined and have no given function, occur for the
class I motif QARMFPNAPYLP (SEQ ID NO: 74) and the class II motif
PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 73), respectively. As a result
of the linking of the multifunctional base sequences (I) and (II)
to each other such that the MHC class I motif and the MHC class II
motif are not placed in the same reading frame, the third sequence
generated for the class II motif is placed in the same reading
frame as that of the MHC class I motif. Accordingly, the spacer
sequence in the present invention can be understood as follows.
That is, when a multifunctional base sequence is designed such that
an MHC class II epitope region (epitope derived from the same
antigen as the antigen from which the class I epitope employed for
the polypeptide is derived) and a
higher-order-structure-stabilizing region(s) are encoded by
different reading frames, a certain amino acid sequence is encoded
by the remaining reading frame, which sequence is the spacer
sequence in the present invention.
[0063] By the process described above, a spacer sequence for the
polypeptide to be used as an effective component in the present
invention can be obtained. This spacer sequence may be linked to
the MHC class I motif QARMFPNAPYLP (SEQ ID NO: 74), and a tandem
repeat structure of the resulting sequence may be constructed for
designing the effective-component polypeptide. Or, microgenes may
be polymerized while allowing random frame shifts to occur by the
MPR method, and proteins may be expressed from the resulting
polymers (artificial protein genes). Thereafter, proteins
containing a structure in which the class I epitope and the spacer
sequence are linked tandemly and repeatedly three times or more may
be selected, and their capacities to induce cell-mediated immunity
may be investigated.
[0064] The MPR primers used in the MPR method are designed such
that the sense primer and the antisense primer form complementary
base pairs of several bases (usually about eight bases) in the
3'-end region of each of the primers. However, a mismatch is
provided for the 3'-end base. In the polymerization reaction using
the MPR primers, the primers anneal to each other in a part of the
3'-side region of each primer, and, as a result, complementary
strands are synthesized for the single-stranded portions by the
polymerization reaction. In the MPR method, the primers themselves
also act as templates. Each MPR primer may be used at a
concentration of about 40 nM to 2000 nM. By performing two-step
reaction cycles using a thermal cycler, the microgenes are tandemly
linked to each other and thus a microgene polymer is synthesized.
As the polymerase, a DNA polymerase having 3'.fwdarw.5' exonuclease
activity is used. During the polymerization reaction process,
fluctuation occurs in the linking portions of the microgenes,
randomly causing deletion and/or insertion of a base(s). This
causes shifts of the reading frame, leading to creation of a
library of artificial genes which produces the encoded polypeptide
sequences in various numbers and various combinations.
[0065] The thus obtained genes are incorporated into an appropriate
protein expression vector by a well-known conventional method. By
allowing expression of the proteins, an artificial protein library
can be obtained. A His-tag fusion protein expression vector may be
used for introduction of the genes into appropriate host cells such
as E. coli or insect cells, and the expressed proteins may be
purified by a conventional method using the His-tag. Since the
expression efficiency in the host cells varies among the artificial
genes, the amount of protein purified also needs to be evaluated
for preparation of the artificial protein library.
[0066] The artificial proteins expressed from the artificial genes
may be evaluated by conventional methods for their
antigen-presenting capacities and capacities to induce
cell-mediated immunity. Artificial genes encoding artificial
proteins having a structure in which the class I epitope motif and
the spacer sequence obtained by the design of the multifunctional
base sequence are linked to each other tandemly and repeatedly
three times or more may be selected, and the selected artificial
genes may be evaluated for the antigen-presenting capacities and
capacities to induce cell-mediated immunity.
[0067] For example, in the evaluation of the antigen-presenting
capacity, each obtained artificial protein may be added to
antigen-presenting cells, and presentation of the epitope of
interest on MHC class I or class II molecules may be measured in
vitro using epitope-specific CD8.sup.+ T cells or the like. In a
method in which the presentation capacity of the class I epitope is
evaluated using epitope-specific CD8.sup.+ T cells, CD8.sup.+ T
cells having T cell receptors (TCRs) specific to the class I
epitope are co-cultured with antigen-presenting cells to which the
artificial protein is added. After incorporation and processing of
the artificial protein followed by presentation of the epitope on
MHC class I molecules, CD8.sup.+ T cells recognize the epitope via
TCRs, and produce IL-2 specifically to the antigen. By selecting
artificial proteins which cause high production of the IL-2,
artificial proteins which cause antigen presentation via
cross-presentation can be screened.
[0068] Taking into account the in vitro antigen-presenting
capacity, the amount of protein purified, and the like, artificial
proteins having a high capacity to induce CD8.sup.+ cytotoxic T
cells (capacity to induce cell-mediated immunity) in vivo can be
selected by a conventional method. For example, animals such as
mice (excluding human) are immunized by intracutaneous,
subcutaneous, intraperitoneal, or another mode of administration of
about 100 .mu.g of each candidate artificial protein together with
20 .mu.g of adjuvant MPL (monophosphoryl lipid A) at least once,
preferably about three times at two-week intervals. Thereafter,
spleen cells may be removed and subjected to a tetramer assay by
flow cytometry using a tetramer reagent for detection of CD8.sup.+
T cells having epitope-specific TCRs, to evaluate the capacity of
each protein to induce CD8.sup.+ cytotoxic T cells. Or, selection
of artificial proteins having high cytotoxicity may also be carried
out by performing a functional cytotoxicity assay using recombinant
E.G7 tumor cells which express each antigen protein, and their
parent cells, EL-4. Furthermore, E.G7 cells expressing each antigen
protein may be inoculated to immunized animals, and then artificial
proteins that suppress tumor growth may be selected.
[0069] By carrying out the MolCraft method according to the
procedure described above, preferred examples of the polypeptide to
be used as an effective component of the vaccine, comprising a
tandem repeat structure in which an MHC class I epitope derived
from an arbitrary antigen protein and a spacer sequence according
to the definition in the present invention are linked to each other
repeatedly three times or more can be obtained. Once the amino acid
sequence of the polypeptide and the base sequence of the
polynucleotide (artificial gene) encoding the amino acid sequence
are specified, the polypeptide can be produced by a method well
known in the art. For example, the polynucleotide may be
incorporated into an appropriate expression vector, and the
polypeptide may then be expressed in host cells such as E. coli or
insect cells, followed by recovering and purifying the polypeptide.
The polynucleotide itself can be obtained by PCR amplification
using, as a template, the artificial gene obtained in the MPR
process in the MolCraft method, or can be prepared by chemical
synthesis in cases where the sequence of the polynucleotide has
been determined. The polypeptide expressed in E. coli cells can be
used as an effective component for pharmaceuticals after removing
endotoxin by a method such as the Triton X-114 method. The
polypeptide whose sequence has been determined can also be
chemically synthesized by a conventional method such as the Fmoc
method or the tBoc method. The polypeptide obtained by chemical
synthesis, as it is or as a long-chain polypeptide prepared by
enzymatic linking, may be refolded to form a higher-order structure
required for use in the present invention.
[0070] The vaccine of the present invention can be produced for
various antigen proteins. Vaccines for tumor antigens and cancer
stem cell antigens may be provided as anti-cancer vaccines
(therapeutic or prophylactic agents for cancer), and vaccines for
antigens of pathogens and parasites may be provided as vaccines for
prevention and treatment of infections. The present invention can
be favorably applied to diseases whose prevention and treatment are
significantly dependent on cell-mediated immunity. Specific
examples of the tumor antigens include WT1, survivin, survivin-B2,
MAGE-A3, MEGE-A4, tyrosinase, gp100, Melan-A, TRP-2, SNRPD1, CDK4,
NY-ESO-1, HER2, MUC-1, CD20, and p53. Examples of the cancer stem
cell antigens include CD44, CD133, LGR5, and Dclkl. Examples of the
viral antigens include constituent proteins of viruses such as
hepatitis viruses (HBV, HCV, and the like), human papillomavirus,
and human immunodeficiency virus. Examples of the parasite antigens
include Plasmodium proteins. Using MHC class I epitopes and class
II epitopes of these antigens as motifs, vaccines of the present
invention can be designed and produced.
[0071] As specific examples of the vaccine peptide of the present
invention, examples of the amino acid sequences of polypeptides
designed for tumor antigen proteins are shown in Tables 1-1 to 1-3
below.
[Table 1-1]
TABLE-US-00001 [0072] Tumor antigen WT1 (Wilms tumor 1) WT1-derived
MHC class I epitope CYTWNWNL (SEQ ID NO: 75) WT1-derived MHC class
I epitope RMFNAPYL (SEQ ID NO: 72) WT1-derived MHC class II epitope
KRYFKLSHLQMEISRKH (SEQ ID NO: 76) .alpha.-Helix structure sequence
##STR00001## (SEQ. ID NO: 14) Examples of vaccine peptides: AkiKaze
A24 (SEQ ID NO: 54)
MRGSHHHHHHGSVDWGTRLPKRYFKLSHLQMHSRKHGSLECYTWNQMNLGATDFSGSS
CSSCRDQRGWPLECYTWNQMNLGATDFSGSSCSSCRDQRGWPLECYTWNQMNLGATDF
##STR00002## RYFKLSHLQMHSRKHGD AkiKaze A2 (SEQ ID NO: 55)
MRGSHHHHHHGSVDWGTRLPKRYFKLSHLQMHSRKHGSVDQARMFPNAPYLPSTDFSG
SSCSSCRDQRGWPQARMFPNAPYLPSTDFSGSSCSSCRDQRGWPQARMFPNAPYLPSTDF
##STR00003## FKLSHLQMHSRKHGD AkiKaze A242 (SEQ ID NO: 56)
MRGSHHHHHHGSVDWGTRLPKRYFKLSHLQMHSRKHGSLECYTWNQMNLGATDFSGSS
CSSCRDQRGWPLECYTWNQMNLGATDFSGSSCSSCRDQRGWPLECYTWNQMNLGATDF
SGSSCSSCRDQRGWPVDQARMFPNAPYLPSTDFSGSSCSSCRDQRGWPQARMFPNAPYLP
##STR00004## ##STR00005## WT1 A2 8110 (SEQ ID NO: 57)
MRGSHHHHHHGSVDWGTGSYVQCSLSSFLRNKRYFKLSHLQMHSRKHGSVDQARMFPN
APYLPSTDFSGSSCSSCRDQRGWPQARMFPNAPYLPSTDFSGSSCSSCRDQRGWPQARMF
PNAPYLPSTDFSGSSCSSCRDQRGWPLERLAVCSIVILLIFLLAEQALLQALALADALAEAGS
YVQCSLSSFLRNKRYFKLSHLQMHSRKHVDDYKDHDGDYKDHDIDYKDDDDKLVDKLL
ESIINFEKLTDKLGD WT1 A24 8112 (SEQ ID NO: 58)
MRGSHHHHHHGSVDWGTGSYVQCSLSSFLRNKRYFKLSHLQMHSRKHGSLECYTWNQM
NLGAGTSVTSSSRTCRCTRGSTLECYTWNQMNLGAGTSVTSSSRTCRCTRGSTLECYTWN
QMNLGAGTSVTSSSRTCRCTRGSTVDLERLAVCSMLLIFLLAEQALLQALALADALAEAG
SYVQCSLSSFLRNKRYFKLSHLQMHSRKHVDDYKDHDGDYKDHDIDYKDDDDKLVDKL
LESIINFEKLTDKLGD WT1 A24 839 (SEQ ID NO: 59)
MRGSHHHHHHGSVDWGTGSYVQCSLSSFLRNKRYFKLSHLQMHSRKHGSLECYTWNQM
NLGATDFSGSSCSSCRDQRGWPLECYTWNQMNLGATDFSGSSCSSCRDQRGWPLECYTW
NQMNLGATDFSGSSCSSCRDQRGWPVDLERLAVCSIVILLIFLLAEQALLQALALADALAE
AGSYVQCSLSSFLRNKRYFKLSHLQMHSRKHVDDEDEDEDVDKLLESIINFEKLTDKLGD WT1 A2
8310 (SEQ ID NO: 60)
MRGSHHHHHHGSVDWGTGSYVQCSLSSFLRNKRYFKLSHLQMHSRKHGSVDQARMFPN
APYLPSTDFSGSSCSSCRDQRGWPQARMFPNAPYLPSTDFSGSSCSSCRDQRGWPQARMF
PNAPYLPSTDFSGSSCSSCRDQRGWPLERLAVCSMLLIFLLAEQALLQALALADALAEAGS
YVQCSLSSFLRNKRYFKLSHLQMHSRKHVDDEDEDEDVDKLLESIINFEKLTDKLGD
TABLE-US-00002 TABLE 1-2 Tumor antigen WT1 (Wilms tumor 1)
WT1-derived MHC class I epitope CYTWNQMNL (SEQ ID NO: 75)
WT1-derived MHC class I epitope RUFPNAPYL (SEQ ID NO: 72) Pan
HLA-DR-binding epitope (PADRE) AKFVAAWTLKAAA (SEQ ID NO: 77)
.alpha.-Helix structure sequence ##STR00006## (SEQ ID NO: 14)
Examples of vaccine peptides: His PADRE WT1 A2 (SEQ ID NO: 61)
MRGSHHHHHHGSVDGTRLPAKFVAAWTLKAAAGSVDQARMFPNAPYLPSTDFSGSSCS
SCRDQRGWPQARMFPNAPYLPSTDFSGSSCSSCRDQRGWPQARMFPNAPYLPSTDFSGS
##STR00007## AWTLKAAAGD His PADRE WT1 A24 (SEQ ID NO: 62)
MRGSHHHHHHGSVDGTRLPAKFVAAWTLKAAAGSLECYTWNQMNLGATDFSGSSCSS
CRDQRGWPLECYTWNQMNLGATDFSGSSCSSCRDQRGWPLECYTWNQMNLGATDFSG
##STR00008## FVAAWTLKAAAGD
TABLE-US-00003 TABLE 1-3 Tumor antigen gp100 gp100-derived MHC
class I epitope KVPRNQDWL (SEQ ID NO: 78) gp100-derived MHC class
II epitope WNRQLYPEWTEAQRLD (SEQ ID NO: 79) .alpha.-Helix structure
sequence ##STR00009## (SEQ ID NO: 14) Examples of vaccine peptides:
gp100 7172 (SEQ ID NO:63)
MRGSHHHHHHGSVDWGTRLPKAWNRQLYPEWTEAQRLDCWGSATKVPRNQDWLGV
TDFSGSSCSSCRDQRGWPATKVPRNQDWLGVTDFSGSSCSSCRDQRGWPATKVPRNQD
##STR00010## RSLPKAWNRQLYPEWTEAQRLDCWVDKLGDLG
[0073] The administration route of the vaccine of the present
invention to the body may be oral administration or parenteral
administration. Parenteral administration such as intramuscular
administration, subcutaneous administration, intravenous
administration, or intraarterial administration is preferred. The
dose is appropriately selected depending on the conditions and
symptoms of the disease to be prevented/treated, the age and the
body weight of the animal to which the vaccine is to be
administered, and the like. The effective dose per subject animal
per day may be usually 0.1 .mu.g to 500 mg, for example, 1 .mu.g to
100 mg. The vaccine may be administered at one time, or dividedly
in several times. For example, the vaccine may be administered
dividedly in several times at intervals of several days to several
months.
[0074] The formulation of the vaccine is not limited. The vaccine
may be composed of only the polypeptide, or may be formulated by
mixing the polypeptide with a pharmaceutically acceptable
additive(s) suitable for each administration route, such as
carriers, diluents, and vehicles. Methods of formulation and
additives which may be used are well known in the field of
formulation of pharmaceuticals. Specific examples of the
formulation include oral preparations such as tablets, capsules,
granules, powders, and syrups; and parenteral preparations such as
inhalants, injection solutions, suppositories, and solutions.
[0075] Conventional peptide vaccines need to be administered in
combination with a certain amount of oil adjuvant or aluminum
adjuvant in order to induce sufficient immunity in vivo. Examples
of such adjuvants that are clinically used at present include Alum
(aluminum salt), MF59 (oil emulsion), and Montanide (e.g.,
Montanide ISA 51VG; oil emulsion). It is thought that oil adjuvants
and aluminum adjuvants support immunity through, for example,
suppression of antigen degradation, induction of inflammatory cells
by tissue destruction, and/or maturation of antigen-presenting
cells. However, occurrence of inflammatory reaction (stiffness) in
the skin due to such adjuvants has been a problem, and it has also
been pointed out that induced CTLs accumulate in the site of
inoculation of the adjuvant, preventing effective suppression of
tumor growth, which is problematic. The vaccine of the present
invention can strongly induce cell-mediated immunity while reducing
side effects by reducing the usage of such a problematic oil
adjuvant or aluminum adjuvant, or without using such adjuvants. The
artificial proteins prepared in the Examples below using OVA do not
enhance expression of costimulatory molecules (CD80, CD86, and the
like) by TLR (Toll-like Receptor) pathway stimulation. In such
cases, an adjuvant that stimulates the TLR pathway such as a TLR
ligand may be used in combination. The term "used in combination"
means that the vaccine and the adjuvant are administered to the
subject either at the same time or sequentially. In cases where the
vaccine and the adjuvant are administered at the same time, the
vaccine may be formulated such that the vaccine further contains
the adjuvant. Some peptide sequences that mimic the TLR ligand
function have been identified, and known examples of peptide
sequences that mimic the TLR-4 ligand function include APPHALS and
QEINSSY (PLoS ONE, February 2012, Volume 7, Issue 2, e30839). By
introduction of such a peptide sequence into the sequence of the
effective component polypeptide, an adjuvant function may also be
given to the polypeptide. Thus, modes in which a peptide sequence
having an adjuvant function is introduced into the effective
component polypeptide are also included in the modes in which the
adjuvant is "used in combination". Examples of adjuvants which
stimulate the TLR pathway and are clinically used include MPL.
[0076] The vaccine of the present invention may be a vaccine
containing as an effective component a recombinant vector which
comprises a polynucleotide encoding the artificial polypeptide
described above and is capable of expressing the polypeptide in
vivo. Vaccines in such a form are also called gene vaccines. The
polynucleotide may be either DNA or RNA, and is preferably DNA. The
vector to be used for production of the gene vaccine is not limited
as long as the vector allows expression in cells of the subject
animal (preferably in mammalian cells), and may be either a plasmid
vector or a viral vector. Any known vector in the field of gene
vaccines may be used. The polynucleotide such as DNA or RNA
encoding the artificial polypeptide described above can be easily
prepared by a conventional method as mentioned above. The
incorporation of the polynucleotide into the vector can be carried
out using a method well known in the art.
[0077] The administration route of the gene vaccine is preferably a
parenteral administration route such as intramuscular
administration, subcutaneous administration, intravenous
administration, or intraarterial administration. The dose may be
appropriately selected depending on the type of the antigen and the
like, and is usually about 0.1 .mu.g to 100 mg, for example, about
1 .mu.g to 10 mg, in terms of the weight of the gene vaccine per
1-kg body weight.
[0078] As methods of using a gene vaccine, in vivo methods, in
which the gene vaccine is directly introduced into the body; and ex
vivo methods, in which a certain kind of cells are collected from
the subject animal, and the gene is then introduced into the cells
ex vivo, followed by returning the resulting cells into the body;
are known (for example, Nikkei Science, April 1994, p. 20-45; The
Pharmaceutical Monthly, 1994, Vol. 36, No. 1, p. 23-48;
Experimental Medicine, Extra Edition, 1994, Vol. 12, No. 15). In
vivo methods are more preferred.
[0079] In cases where the vaccine is administered by an in vivo
method, the vaccine may be administered through an appropriate
administration route depending on the disease to be treated,
symptoms, and the like. The vaccine may be administered by, for
example, intravenous, intraarterial, subcutaneous, or intramuscular
administration. In cases where the vaccine is administered by an in
vivo method, the vaccine may be formulated into a preparation such
as a solution. In general, the vaccine is in the form of, for
example, an injection solution containing as an effective component
DNA encoding the polypeptide of the present invention. If
necessary, a conventionally used carrier may be added thereto. In
case of a liposome or membrane fusion liposome (Sendai virus
(HVJ)-liposome or the like) containing the DNA, the liposome may be
formulated into a liposome preparation such as a suspension, frozen
preparation, or centrifugally concentrated frozen preparation.
[0080] The vaccine of the present invention may be used in
combination with other pharmaceutical(s). For example, a vaccine of
the present invention designed for a tumor antigen may be used in
combination with other anticancer drug(s).
[0081] At present, in tumor immunotherapy, immune checkpoint
inhibitors are attracting attention (Nature Reviews Cancer 12,
252-264 (April 2012)). In the living body, a system for inhibitory
control of excessive immune reaction is present. Molecules
expressed in antigen-presenting cells (APCs) and molecules
expressed in T cells, for example, PD-L1 and PD-1; CD80 and CTLA4;
MHC class I or MHC class II and KIR or LAW; and GLA9 and TIM3 have
been identified so far. By their interaction, a negative signal is
transmitted to T cells to cause inhibition of the T-cell reaction.
This mechanism is called immune checkpoint.
[0082] Administration of a humanized anti-CTAL-4 antibody,
anti-PD-L1 antibody, or anti-PD-1 antibody (immune checkpoint
inhibitor), which have functions to inhibit the immune checkpoint,
shows a drastic therapeutic effect in melanoma and lung cancer
(Clin Cancer Res. 2013 Oct. 1; 19(19): 5300-9). It is also reported
that a severe autoimmune disease occurs at the same time since
breakdown of immune tolerance to self occurs. This fact indicates
that tumor immunity that attacks cancer cells is originally
established in cancer patients, and suggests that a system in which
expression of PD-L1 and production of various cytokines by cancer
cells cause negative control of immune checkpoint, resulting in
suppression of the tumor immunity, is functioning, and that, as a
result, suppression of the growth of tumor cells becomes
impossible, leading to progression of the cancer. That is,
tumor-bearing patients are in a braked state where tumor immunity
is suppressed. It is thought that administration of the immune
checkpoint inhibitor leads to releasing of such a brake against
tumor immunity, and allows functioning of the tumor immunity that
attacks cancer cells, which is originally retained by the patient,
leading to production of the antitumor effect. Although no
practical evidence has been obtained for the fact that the tumor
immunity originally retained by tumor-bearing patients functions to
protect against development of cancer, tumor immunotherapy may
largely change in the future due to the progress in scientific
understanding of, and drastic therapeutic effects of specific
inhibitors of, immune checkpoint.
[0083] Immune checkpoint inhibitors are assumed to produce immunity
against a mutant protein having a mutation called passenger
mutation, which mutant protein does not necessarily accumulate in
cancer cells since the mutation does not affect the protein
function although it causes amino acid substitution, rather than
immunity against an antigen which is overexpressed in cancer, or
the so-called cancer antigen, which has a driver mutation (mutation
which contributes to the growth of cancer cells, such as an amino
acid substitution, gene fusion, deletion, or insertion that causes
accumulation in cancer cells). That is, antigens targeted by tumor
immunity induced by immune checkpoint inhibitors may largely vary
among individuals. Although immune checkpoint inhibitors induce
strong antitumor immunity, they are not necessarily effective for
all patients, and reported to show different effectiveness on
different types of cancer.
[0084] Taking these facts into account, it is suggested that
induction of strong tumor immunity may be achieved, and a stronger
antitumor effect may therefore be obtained, by inducing immunity
against a tumor antigen using an artificial protein vaccine
according to the present invention while controlling the
immunosuppressed state of the tumor-bearing patient using an immune
checkpoint inhibitor, that is, by using an artificial protein
vaccine according to the present invention and an immune checkpoint
inhibitor in combination.
Examples
[0085] The present invention is described below more concretely
based on Examples. However, the present invention is not limited to
the Examples.
1. Design of Microgenes for Creating Artificial Proteins
[0086] From native antigen OVA (SEQ ID NO:24), OVA-I:
.circle-solid. (OVA MHC class I epitope, OVA258-265, SIINFEKL; SEQ
ID NO: 67) and OVA-II: .box-solid. (OVA MHC class II epitope,
OVA324-340, ISQAVHAAHAEINEAGR; SEQ ID NO: 2) were selected (FIG.
1a).
[0087] Microgenes #2101 and #6101, in which the MHC class I epitope
OVA-I is encoded in the first sequence, and the MHC class II
epitope OVA-II is encoded in the second sequence, were designed
using CyberGene software, developed by Kiyotaka Shiba et al. (K.
Shiba, Journal of Molecular Catalysis B: Enzymatic 28 (2004)
145-153) (FIG. 1b). The designing process is shown in 1) to 5) in
FIG. 2. Since the two amino acids adjacent to the N-terminus of the
MHC class I epitope (SIINFEKL) in native antigen OVA are known to
influence degradation by aminopeptidase in the cell, two amino
acids LE derived from the OVA full-length antigen were added to the
N-terminus of OVA-I. The one amino acid in the C-terminus was also
selected such that T derived from the OVA full-length antigen is
conserved (LESIINFEKLT; SEQ ID NO: 1), and used in a motif for
designing microgenes.
[0088] First, the multifunctional base sequence (I) encoding the
OVA-I motif LESIINFEKLT (SEQ ID NO: 1) and the multifunctional base
sequence (II) encoding the OVA-II motif ISQAVHAAHAEINEAGR (SEQ ID
NO: 2) were separately designed using CyberGene. If possible codons
were written out by reverse translation from the OVA-I motif and
the OVA-II motif, the combinations of DNA sequences amounted to
248,832 and 169,869,312, respectively, but DNA sequences such as
those having a stop codon in any of the reading frames were
eliminated by CyberGene. By assigning the OVA-I motif and the
OVA-II motif to the first sequences, respectively, and assigning of
an amino acid sequence which tends to form an .alpha.-helix
structure or a .beta.-sheet structure to the second sequence,
hundreds or more of gene sequences were assigned to each of the
motifs. For each case, sequences having structures with higher
stabilities were selected. Examples of multifunctional base
sequences (I) and (II) obtained as a result are shown in 4) in FIG.
2.
[0089] The resulting multifunctional base sequences (I) and (II)
were linked to each other to design microgenes #2101 (SEQ ID NO:11)
and #6101 (SEQ ID NO:15). The amino acid sequences encoded by the
three reading frames of #2101 are shown in SEQ ID NOs:12 to 14. The
first frame (SEQ ID NO:12) encodes the MHC class I epitope; the
second frame (SEQ ID NO:13) encodes the MHC class II epitope; and
the third frame (SEQ ID NO:14) encodes two .alpha.-helix motifs.
The amino acid sequences encoded by the three reading frames of
#6101 are shown in SEQ ID NOs:16 to 18. The first frame (SEQ ID
NO:16) encodes the MHC class I epitope; the second frame (SEQ ID
NO:17) encodes the MHC class II epitope and a .beta.-sheet motif;
and the third frame (SEQ ID NO:18) encodes an .alpha.-helix
motif.
2. Creation of Artificial Protein Library Using MolCraft Method
[0090] Using the MolCraft method developed by Kiyotaka Shiba et al.
(K. Shiba, Journal of Molecular Catalysis B: Enzymatic 28 (2004)
145-153), peptide motif sequences (Table 2) such as the OVA MHC
class I and class II epitopes, protein-stabilizing sequences
including .alpha.-helix, and sequences automatically defined by
CyberGene were combinatorially linked to each other to synthesize
artificial protein genes. A summary of the process of synthesis of
the artificial protein gene using #2101 by the MPR method (Kiyotaka
Shiba et al., PNAS vol. 94, pp. 3805-3810, 1997) is shown in 6) to
9) in FIG. 2.
[0091] In the polymerization reaction of #2101, 2101-S primer
(CTCGAGAGTATCATCAACTTCGAGAAGCTTACCGATTTCTCAGGCT; SEQ ID NO:19) and
2101-AS primer (GCGGCCAGCCTCGTTGATCTCTGCATGAGCTGCATGAACTGCCTGAGAT;
SEQ ID NO:20) were used. In the polymerization reaction of #6101,
6101-S primer (CTCGAAAGTATTATCAATTTCGAAAAACTCACCGATTTCTCAGGCT; SEQ
ID NO:21) and 6101-AS primer (having the same sequence as 2101-AS)
were used. A total of 50 .mu.L of polymerization reaction solution
was prepared such that the reaction solution had the following
composition: 2.6 .mu.L of Vent DNA polymerases having 3'.fwdarw.5'
exonuclease activity (2 units/.mu.L, NEW ENGLAND BioLabs), 5 .mu.L
of 10.times.ThermoPol Reaction Buffer (NEW ENGLAND BioLabs,
1.times.ThermoPol Reaction Buffer: Tris-HCl pH 8.8, 10 mM potassium
chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, and 0.1%
Triton X-100), 350 .mu.M dNTP, 400 nM each of MPR primers S and AS
(20 pmol each of the primers was used). The polymerization reaction
was carried out using a thermal cycler under the following
conditions: 94.degree. C. for 10 minutes.fwdarw.60.degree. C. for
10 minutes.fwdarw.30 cycles of (94.degree. C. for 10
seconds.fwdarw.60.degree. C. for 1 minute).fwdarw.60.degree. C. for
7 minutes.fwdarw.4.degree. C..infin..
[0092] As described above, 134 kinds of artificial protein genes
were synthesized, and each gene was cloned into an expression
vector. As a result of checking expression of a protein from each
of 62 kinds of genes in E. coli, 40 kinds of genes were found to
show expression of the protein.
TABLE-US-00004 TABLE 2 .circle-solid. LESIINFEKLT MHC class I SEQ
ID NO: 1 .box-solid. ISQAVHAAHAEINEAGR MHC class II SEQ ID NO: 2
.tangle-solidup. REYHQLREAYR .alpha.-helix SEQ ID NO: 3
FLRQFMQLMQRSTRLA .alpha.-helix SEQ ID NO: 4 .diamond-solid.
SKVLSISKNSP .beta.-sheet SEQ ID NO: 5 .quadrature. SRVSSTSRSLP SEQ
ID NO: 6 .DELTA. RKYYQFRKTHR SEQ ID NO: 7 .gradient.
DFSGSSCSSCRDQRGWP SEQ ID NO: 8 (.gradient./) DLRQFTCRDQRGWP SEQ ID
NO: 9 .largecircle. MRGSHHHHHH His-tag SEQ ID NO: 10 .diamond.
Other sequences (.gradient./): Sequence generated by a frame shift
which occurred in a middle part of .gradient..
3. In Vitro Antigen-Presenting Function Assay
[0093] From the library of these artificial proteins, 8 kinds of
artificial proteins (F138A, G142A, G142C, F182A, F58B, F58C, F112A,
and F112C shown in FIG. 1c) were first selected, and subjected to
an in vitro antigen-presenting capacity assay. The amino acid
sequences of the artificial proteins are shown in FIGS. 3 and 4,
and SEQ ID NOs:26 to 43.
[0094] Each artificial protein was added to antigen-presenting
cells (DC2.4 dendritic cell line), and co-cultured with T cells
(RF33.70) that recognized an OVA-specific epitope, followed by
measuring the IL-2 productivity to evaluate the antigen-presenting
capacity.
[0095] As a result, only clone F182A (SEQ ID NO:26) caused
production of IL-2 to show the antigen-presenting capacity at a
concentration of 10 .mu.g/ml (FIG. 1d). The size and the purity of
the protein were checked by SDS-PAGE (FIG. 1d). Native OVA did not
show the antigen-presenting capacity at a concentration of 10
.mu.g/ml. Similar results were obtained in an experiment using
bone-marrow-derived dendritic cells, wherein only F182A. among, the
8 kinds of artificial proteins, caused production of IL-2 to show
the antigen-presenting capacity (data not shown). From these
results, F182A artificial protein was found to have a capacity to
induce cell-mediated immunity.
4. Artificial Antigen which Shows Antigen-Presenting Capacity, and
its Characteristic Amino Acid Sequence 4-1. F37A Artificial Protein
Shows 100-Fold Stronger Antigen Presentation than Native OVA
[0096] Subsequently, from the library, additional 8 kinds of
artificial proteins including artificial proteins having a
structure similar to that of F182A were selected, and subjected to
evaluation of their antigen-presenting capacities in vitro. At an
antigen concentration of 10 .mu.g/ml, not only F182A, but also F37A
(SEQ ID NO:44) and F36C (SEQ ID NO:31) showed the
antigen-presenting capacity. All of F 182A, F37A, and F36C, which
showed the antigen-presenting capacity. were found to have a common
sequence pattern. That is, they had the sequence of
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
(a part or all of .gradient. may be (.gradient./)). This is a
structure in which LESIINFEKLTDFSGSSCSSCRDQRGWP
((.circle-solid..gradient., SEQ ID NO:22) or
LESIINFEKLTDLRQFTCRDQRGWP (.circle-solid.(.gradient./), SEQ ID
NO:53) is tandernly repeated three times. Such sequences are
hereinafter represented as
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
including those in which a part or all of .gradient. is
(.gradient./).
[0097] Native OVA did not show the antigen-presenting capacity
until the concentration increased to 1000 .mu.g/ml. Thus, F182A,
F37A, and F36C were shown to have a 100-fold higher
antigen-presenting capacity than OVA.
4-2. Characteristic Sequence Pattern of F37A Acts on
Antigen-presenting Capacity
[0098] In order to clarify the fact that the sequence
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.,
which is common to the artificial proteins that showed antigen
presentation, is important for the antigen presentation, the OVA-I
sequences (.circle-solid.) in the F37A sequence were replaced one
by one with an MHC class I epitope sequence (RMFPNAPYL, residues at
positions 194 to 202 in SEQ ID NO:23) of WT1 (Wilms tumor 1) to
prepare mutants (FIG. 5c). The sequence of W.gradient. is shown in
SEQ ID NO:25. The amino acid sequences of the artificial proteins
are shown in FIG. 6 and SEQ ID NOs:44 to 52.
[0099] The antigen-presenting capacities of these proteins were
investigated to find that, when even as few as one OVA-I sequence
was replaced with the WT1 sequence, artificial proteins lost their
antigen-presenting capacity in antigen-presenting cells which was
co-cultured with T-cells (RF33.70) recognizing an OVA-specific
epitope. F37AE2 is an artificial protein having the same amino acid
sequence as the, amino acid sequence of F37A except that the three
amino acids in the C-terminus of F37A arc replaced with 5 amino
acids different therefrom, and contains
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
as it is. F37AE2 showed an increase in the IL-2 productivity
dependently on the antigen concentration. From these results, it
was revealed that the artificial proteins having stronger
antigen-presenting capacities than native OVA protein function
through the characteristic
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
sequence, and that these artificial proteins allow highly efficient
presentation of the epitope peptide on MHC class I molecules
irrespective of the fact that they are foreign antigens.
[0100] Since F37A stably showed high protein productivity in E.
coli, and had the highest antigen-presenting capacity of the three
artificial proteins, F37A was used to carry out the following
experiments.
4-3. Circular Dichroism Analysis (CD) (FIG. 7)
[0101] Native OVA and artificial proteins containing many
.alpha.-helix structures (F182C, F37C, and F36B) showed graph
patterns indicating typical .alpha.-helix structures. On the other
hand, F36A and F182B showed graph patterns characterized by random
coils.
[0102] F182A, F37C, and F36C, which exhibited antigenicity, showed
a common, characteristic graph pattern. They were found to have a
secondary structure which was thought to contain at least an
.alpha.-helix structure, although the graph pattern was evidently
different from that shown by native OVA protein. Influence of such
a secondary structure on the production of antigenicity was
suggested.
[0103] Table 3 summarizes biochemical characteristics of the
artificial proteins used in the experiment, the numbers of the
OVA-I: .circle-solid. and OVA-II: .box-solid. epitope sequences
contained in each protein, and the presence/absence of in vitro
antigenicity of each protein.
TABLE-US-00005 TABLE 3 No. of No. of total Isoelectric OVA-I OVA-II
Antigenecity Code Residues M.W.* Point* epitope epitope Uniqe Motif
pattern (in vitro)* native OVA 386 42.911 5.19 1 1 + OVA-I peptide
8 963 5.72 1 0 OVA-II peptide 17 1773 6.00 0 1 F182A 190 21.051
4.71 4 2 .circle-solid..gradient. .circle-solid.(.gradient./
).circle-solid..gradient..circle-solid..gradient. + F37A 181 20.313
7.17 3 2
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
+ F36C 120 13.844 8.60 3 0
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
+ F37AE2 183 20.517 7.90 3 2
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
+ MT819 184 20.657 8.30 2 2
.circle-solid..gradient..circle-solid..gradient. - MT820 184 20.627
8.30 2 2 .circle-solid..gradient. .circle-solid..gradient. - MT821
184 20.657 8.30 2 2
.circle-solid..gradient..circle-solid..gradient. - MT822 185 20.803
8.64 1 2 .circle-solid..gradient. - MT823 185 20.603 8.64 1 2
.circle-solid..gradient. - MT824 188 20.803 6.64 1 2
.circle-solid..gradient. - MT825 186 20.948 8.90 0 2 - F138A 158
17.963 8.55 2 1 .circle-solid..gradient..circle-solid..gradient. -
G142A 241 27.003 9.36 4 3 .circle-solid..gradient.
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
- G142C 243 29.034 11.12 2 1 .circle-solid..gradient.
.circle-solid..gradient. - F58B 163 1.250 9.20 2 2
.circle-solid..gradient. .circle-solid..gradient. - F58C 166 19.390
11.21 1 2 .circle-solid..gradient. F112A 207 23.513 11.16 1 2
.circle-solid..gradient. - F112C 211 24.985 10.74 2 2
.circle-solid..gradient. .circle-solid..gradient. - F182B 202
22.632 10.73 1 2 .circle-solid..gradient. - F182C 194 23.900 11.11
2 0 .circle-solid..gradient. .circle-solid..gradient. - F37B 182
20.505 11.24 1 3 .circle-solid..gradient. - F37C 182 22.060 11.24 1
1 .circle-solid..gradient. - F36A 115 12.066 11.00 0 3 - F36B 117
14.469 11.65 0 0 - MT290 113 12.158 10.09 0 0 - MT332 140 16.449
11.69 0 0 - MT297 104 12.251 11.90 0 0 -
[0104] G142A (SEQ ID NO:35) had four MHC class I epitopes and two
MHC class II epitopes, but did not show antigenicity. This fact
suggests that the presence of many MHC class I epitopes in the
protein does not necessarily contribute to the induction of
antigenicity.
[0105] Proteins in which the .circle-solid..gradient. sequence is
repeated twice such as F138A and G142A did not show antigenicity.
All of the proteins that showed antigenicity contained
characteristic
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient..
Thus, the characteristic structure formed by repeating of the
.circle-solid..gradient. sequence three or more times, found in
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.,
was suggested to be important for the induction of
antigenicity.
[0106] The .gradient. sequence (DFSGSSCSSCRDQRGWP, SEQ ID NO: 8) is
a sequence proposed as the third sequence by the algorithm of
CyberGene software developed by Kiyotaka Shiba et al., when the MHC
class II sequence is set as the first sequence, and a sequence that
forms an .alpha.-helix is set as the second sequence.
[0107] All of the proteins that showed antigenicity were found to
have isoelectric points of nearly neutral pHs ranging from 6.0 to
8.6. This fact suggests that a neutral isoelectric point is
important for antigenicity of the artificial protein.
[0108] Using Strider 1.4f7 software, Hydropathy (Kyte-Doolittle)
hydrophobicity analysis was carried out with a window setting of 9.
As a result, it was shown that the .gradient. sequence
(DFSGSSCSSCRDQRGWP, SEQ ID NO:8) located downstream of each MHC
class I sequence contained a lot of hydrophilic amino acids (e.g.,
R, N, D, E, Q, G, H, K, P, S, T, and Y) and exhibited hydrophilic
characteristics (FIG. 8), suggesting that it is important that the
sequence located downstream of each MHC class I sequence in the
three repeats of MHC class I has a hydrophilic property.
[0109] Further, using Strider 1.4f7 software, Amphipathicity
analysis of the proteins was carried out, with a window setting of
9. As a result, the region of characteristic
.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.
sequence had an amphipathicity between 0.0 and 0.4 (FIG. 9). Thus,
it was suggested that such a structure without extreme deviation of
amphipathicity is important for the exhibition of antigenicity.
5. F37A Artificial Protein Allows Antigen Presentation through
Cross-Presentation
[0110] In order to confirm that the artificial proteins are
incorporated into antigen-presenting cells and allow
antigen-presentation of the epitope through cross-presentation, the
proteins were treated with an inhibitor of proteasome involved in
cross-presentation, Epoxomicin>or MG132, and evaluated for their
capacities to induce cell-mediated immunity. As a result, the
capacities of F182A and F37A to induce cell-mediated immunity were
suppressed (FIG. 5d). Treatment with a lysosome inhibitor
(Chloroquine), which has an effect to promote cross-presentation,
enhanced the capacities of F182A and F37A to induce cell-mediated
immunity.
[0111] From these findings, it could be confirmed that F182A and
F37A are incorporated into antigen-presenting cells as foreign
antigens and undergo proteasomal degradation, followed by
presentation of the peptide epitope on MHC class I molecules, that
is, the antigen presentation of F182A and F37A occurs through the
so-called cross-presentation.
6. F37A does not Exhibit its Antigen-Presenting Capacity through
Maturation of Dendritic Cells
[0112] It is known that induction of immunity by antigen-presenting
cells requires not only antigen presentation on MHC molecules, but
also expression of costimulatory molecules (CD80 and CD86), that
is, maturation of the antigen-presenting cells. In view of this,
F37A was added to BMDCs, bone marrow-derived dendritic cells,
induced from mouse bone marrow monocytes using GM-CSF, and
expression of the maturation markers CD80 and CD86 was
investigated.
[0113] The results are shown in FIG. 10. F37A did not affect the
expression of CD80 and CD86. Thus, it was suggested that F37A does
not cause antigen presentation by affecting the maturation of
antigen-presenting cells. It should be noted that LPS derived from
E. coli or from the environment was removed from the artificial
protein and OVA using Triton X-114, and that the LPS concentration
in the sample was confirmed to be not more than 0.5 EU/mg.
7. F37A Artificial Protein Strongly Induces Cell-Mediated Immunity
In Vivo
[0114] Subsequently, an antigen was intradermally administered to
C57B1/6 mice at 100 mg/mouse three times at two-week intervals to
perform immunization. As the groups to be studied, an OVA-I peptide
group (OVA MHC class I epitope, OVA257-264, SIINFEKL was
administered), native OVA protein group, and F37A artificial
protein group were set. The immunization was carried out using, as
an adjuvant, MPL (monophosphoryl lipid A) or Freund's adjuvant CFA
(a complete adjuvant (supplemented with killed tubercle bacillus)
was used once, and an incomplete adjuvant was used twice).
[0115] Spleen cells were removed from the immunized mice, and
subjected to mixed culture in the presence of IL-2 (10 ng/ml) with
EG7-OVA cells (OVA-expressing tumor cells) inactivated by 100 Gy
X-ray radiation, thereby performing in vitro stimulation.
Thereafter, for detection of functional OVA-specific T cells, a
Cromium-51 releasing assay (cytotoxicity assay) targeting EL-4
(cells not expressing OVA, corresponding to parent cells of
E.G7-OVA) and EG7-OVA cells (cells expressing OVA) was carried
out.
[0116] The results of the cytotoxicity assay are shown in FIG. 11a.
Without use of an adjuvant, OVA-specific cytotoxic T cells, CTLs,
were not detected in any of the groups immunized with the OVA-I
peptide, native OVA protein, or F37A (FIG. 11a, top row).
[0117] In the cases where an antigen was administered together with
an adjuvant MPL, the F37A group showed significantly stronger CTL
induction compared to the OVA-I peptide group and the OVA group
(FIG. 11a, middle row). Thus, it could be confirmed that F37A is
capable of inducing cell-mediated immunity even without use of
Freund's oil adjuvant.
[0118] In the cases where CFA was used as the adjuvant, the OVA-I
peptide group and the OVA group also showed CTL induction. The CTL
induction capacity of the F37A group tended to be higher than those
of the OVA-I peptide group and the OVA group (FIG. 11a, bottom
row).
[0119] These results indicate that F37A can more strongly induce
CTLs than native OVA protein also in vivo.
8. Tumor Growth Suppression Effect of F37A
[0120] Subsequently, the tumor suppression effect on an
OVA-expressing tumor was studied. Mice were immunized in the same
manner as described above, and EG7-OVA tumor cells
(2.times.10.sup.6 cells) were subcutaneously administered to the
back of each mouse. Thereafter, the tumor diameter was measured
every week. As a result, no difference in the tumor diameter were
found among the groups without use of an adjuvant, at Week 3 after
the inoculation of the tumor cells.
[0121] However, in the cases where MPL was used as an adjuvant, the
OVA immunization group and the F37A immunization group showed a
significant tumor growth suppression effect (FIG. 11b). Thus, it
could be confirmed that F37A is capable of inducing cell-mediated
immunity even without use of Freund's oil adjuvant, and that the
induced cell-mediated immunity is functional.
[0122] Among the groups in which the CFA adjuvant was used, the
F37A immunization group showed significant suppression of the tumor
growth.
[0123] These results indicate that CTLs induced by immunization
with F37A are functional CTLs that can attack OVA-expressing tumor
cells.
9. F37A Exhibits Not Only Capacity to Induce Cell-Mediated
Immunity, but also Induction of Humoral Immunity
[0124] Serum was collected from immunized mice, and whether or not
anti-OVA antibodies were produced was investigated by the ELISA
method using OVA as an antigen (FIG. 11c).
[0125] The group immunized with the OVA-I peptide did not show
production of anti-OVA antibodies irrespective of whether an
adjuvant was used or not.
[0126] On the other hand, the F37A immunization group showed
production of anti-OVA antibodies by use of the adjuvant MPL or
CFA. However, the amount of the antibodies produced was obviously
lower than that in the OVA protein immunization group.
[0127] From the results on the CTL induction capacity and the
antibody productivity, it, was found that, although native OVA
protein has a capacity to induce both cell-mediated immunity and
humoral immunity, it is more likely to induce humoral immunity.
[0128] On the other hand, it was found that, although F37A has a
capacity to induce both cell-mediated immunity and humoral
immunity, it is more likely to induce cell-mediated immunity rather
than humoral immunity.
10. MHC Class II Epitope in F37A is not Indispensable for
OVA-Specific CTL Induction, and MHC Class I Epitope Functions for
Induction of Cell-Mediated Immunity
[0129] In order to investigate whether the MHC class II epitope
sequence OVA-II is involved in the OVA-specific CTL induction by
F37A, mice were immunized with F36C artificial protein, which had
no MHC class II epitope. In addition, in order to clarify that the
MHC class I epitope sequence OVA-I present in F37A functions for
induction of cell-mediated immunity, mice were immunized with.
MT825 artificial protein, in which all three OVA-I sequences were
replaced with WT1 MHC class I epitopes. In the immunization, 100
.mu.g of an antigen was intraperitoneally administered together
with the adjuvant MPL (20 .mu.g/mouse) three times at two-week
intervals (FIG. 12a).
11. F36C, which has same Characteristic Sequence Pattern
(.circle-solid..gradient..circle-solid..gradient..circle-solid..gradient.-
) as that of F37A but does not have MHC Class II Epitope Sequence,
can Induce CTLs
[0130] As a result of a CTL assay of the immunized mice, the F37A
immunization group showed a significantly higher level of induction
of CTLs compared to the MT825 immunization group and the OVA
immunization group (FIG. 12b). F36C, which had the same
characteristic sequence pattern
(.circle-solid..gradient..circle-solid..gradient..circle-solid..g-
radient.) as that of F37A but did not have the MHC class II epitope
sequence, also showed a tendency to induce CTLs. MT825, which had
no OVA-I, did not induce CTLs at all. From these results, it was
suggested that induction of OVA-specific CTLs does not necessarily
require the MHC class II epitope sequence.
12. OVA-I Sequence Functions for OVA-Specific CTL Induction by
F37A
[0131] Since MT825 showed no CTL induction capacity, it was found
that the OVA-I (OVA MHC class I epitope, SIINFEKL) sequence in F37A
is indispensable for the OVA-specific CTL induction.
13. F37A Strongly Induces OVA-Specific CTLs (Tetramer Assay)
[0132] The presence of OVA-I peptide (SIINFEKL)-specific
CD8-positive T cells in the immunized mice was confirmed by an
assay using a tetramer reagent specific to the OVA-I sequence. The
tetramer reagent is a tetramer containing the epitope peptide OVA-I
bound to an MHC class I molecule, and cells expressing
OVA-I-specific T cell receptors (TCRs) of T cells can be quantified
with the reagent.
[0133] The results of the tetramer assay are shown in FIG. 12c.
F37A showed a significantly higher level of induction of
tetramer-positive cells compared to the MT825 immunization group.
OVA and F36C also showed a tendency to induce the tetramer.
[0134] From these results, it was suggested that the CTLs which
attack the OVA tumor cells described above are tetramer-positive
CD8 cells specific to the OVA-I sequence of F37A.
14. F37A and F36C Have Capacity to Suppress Tumor Growth
[0135] To mice immunized in the same manner as described above,
EG7-OVA cells (2.times.10.sup.6 cells) were inoculated, and the
tumor diameter was measured. At Week 3 after the tumor inoculation,
the F37A and F36C immunization groups showed significant
suppression of the tumor growth (FIG. 12d). FIG. 12e shows the
tumor growth in each mouse. The OVA immunization group also showed
a tendency to suppress the tumor growth.
[0136] As a result of comparison of the survival curve among the
mice, prolonged survival was found in the OVA immunization group,
F37A immunization group, and F36C immunization group. Among these,
F37A showed the strongest effect of prolonging the survival (FIG.
12e, right end in the bottom row).
[0137] From these results, it was revealed that F37A can suppress
the tumor growth more strongly than native OVA protein.
[0138] Although the tumor growth suppression was also found in
F36C, which had no MHC class II sequence, a stronger tumor
suppression capacity was found in F37A, which had MHC class II
sequences. It was therefore suggested that, while an MHC class II
sequence is not necessarily required in the induction phase
(induction of CTLs), an antigen having both MHC class I and MHC
class II sequences exhibits a stronger effect in the effector phase
(when the immunity functions to attack the tumor).
[0139] F37A, which had a structure in which a sequence composed of
an MHC class I sequence and a spacer sequence defined by CyberGene
which were linked to each other was tandemly repeated three times
and an MHC class II epitope was present at both of the N-terminus
and the C-terminus, most strongly induced cell-mediated immunity
both in vitro and in vivo and suppressed the tumor growth. Thus, it
is thought that F37A provide us with a characteristic structure
that functions as an antigen for vaccines which strongly induce
cell-mediated immunity.
15. Analysis of Mechanism of Antigen Presentation caused by
F37A
[0140] At present, little is known about the intracellular pathway
of cross-presentation, in which a foreign antigen is incorporated
into antigen-presenting cells and an antigen epitope is presented
on MHC class I molecules. The mechanism of antigen presentation
caused by F37A, which is capable of inducing strong cell-mediated
immunity through cross-presentation, was investigated.
[0141] F37A (SEQ ID NO:44), which shows the antigen-presenting
capacity, comprises three MHC class I epitopes and two class II
epitopes, and comprises a tandem repeat structure in which the
class I epitope and the spacer sequence are linked to each other
alternately and repeatedly three times. On the other hand, C131B
(SEQ ID NO:64) comprises three MHC class I epitopes and three class
II epitopes, but its molecular context (e.g. combination of the
order of epitope sequences) is different from that of F37A. C131B
does not comprise the tandem repeat structure described above, and
shows no antigen-presenting capacity (FIG. 13A).
[0142] First, the uptake of F37A and C131B by antigen-presenting
cells was investigated. As a result, the uptake of F37A by
antigen-presenting cells was found to be lower than the uptake of
C131B (FIG. 14A, 14B). The uptake of C131B tended to be larger than
the uptake of F37A. However, as shown in FIG. 13, C131B did not
show antigen-presenting capacity at all. Thus, it was suggested
that the uptake of F37A by antigen-presenting cells has no
influence on the antigen-presenting function of F37A.
[0143] Subsequently, the fact that the mode of uptake of F37A by
antigen-presenting cells is associated with the enhancement of the
antigen-presenting capacity was investigated.
[0144] Examples of the mode of antigen uptake in cross-presentation
that have been reported so far include macropinocytosis,
non-specific phagocytosis, and receptor-mediated phagocytosis.
Native antigen OVA is uptaken through mannose receptors of
antigen-presenting cells (Burgdorf S, Kautz A, Bohnert V, Knolle
PA, Kurts C (2007) Distinct pathways of antigen uptake and
intracellular routing in CD4 and CD8 T cell activation. Science
316: 612-616.).
[0145] Since F37A was prepared using E. coil, it is not
glycosylated. Therefore, it is thought that, unlike native antigen
OVA, F37A is uptaken by antigen-presenting cells by a mechanism
other than the pathway through the mannose receptor.
[0146] Antigen presenting cells (DC2.4 cells) were preliminarily
treated with cytochalasin B (phagocytosis inhibitor),
5-(N,N-dimethyl)amiloride (DMA, pinocytosis inhibitor), or Poly-I
(class A scavenger receptor (SRA) inhibitor), and an antigen (F37A,
C131B, or OVA) was then added to the treated cells as well as
untreated cells. Thereafter, the cells were cultured, and evaluated
for the uptake of the antigen and for the antigen-presenting
capacities in vitro.
[0147] As a result, the uptake of F37A by the antigen-presenting
cells tended to be suppressed by Poly-I. On the other hand, the
uptake of C131B and OVA was enhanced relative to poly-I (FIG. 13A),
although the mechanism of this phenomenon is unclear. The
antigen-presenting capacity of F37A was suppressed by cytochalasin
B, DMA, and Poly-I. In particular, Poly-I strongly suppressed the
antigen presentation (FIG. 13B). In the cases of the F37AE2 antigen
(SEQ ID NO:45), which had the same amino acid sequence as F37A
except for the 5 amino acids in the C-terminus, antigen
presentation was suppressed by an SRA inhibitor fucoidan (FIG.
14C).
[0148] SRA is a cell membrane receptor expressed in macrophages,
dendritic cells and the like, and responsible for uptake and
processing of oxidized LDL and the like. It is also known that a
protein composed of HSP (heat shock protein) bound to an antigen is
uptaken by antigen-presenting cells through SRA, and induces
cell-mediated immunity through cross-presentation (Murshid A, Gong
J, Calderwood S K (2012) The role of heat shock proteins in antigen
cross presentation. Front Immunol 3: 63.). Taking these facts into
account, it was suggested that the uptake of F37A by
antigen-presenting cells through SRA, which is due to the
difference in the molecular context between F37A and C131B, leads
to exhibition of the strong antigen-presenting capacity of F37A.
Sequence CWU 1
1
83111PRTHomo sapiens 1Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr1
5 10217PRTHomo sapiens 2Ile Ser Gln Ala Val His Ala Ala His Ala Glu
Ile Asn Glu Ala Gly1 5 10 15Arg311PRTArtificialalpha helix 3Arg Glu
Tyr His Gln Leu Arg Glu Ala Tyr Arg1 5 10416PRTArtificialalpha
helix 4Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu
Ala1 5 10 15511PRTArtificialbeta sheet 5Ser Lys Val Leu Ser Ile Ser
Lys Asn Ser Pro1 5 10611PRTArtificialpeptide sequence 6Ser Arg Val
Ser Ser Thr Ser Arg Ser Leu Pro1 5 10711PRTArtificialpeptide
sequence 7Arg Lys Tyr Tyr Gln Phe Arg Lys Thr His Arg1 5
10817PRTArtificialpeptide sequence 8Asp Phe Ser Gly Ser Ser Cys Ser
Ser Cys Arg Asp Gln Arg Gly Trp1 5 10 15Pro914PRTArtificialpeptide
sequence 9Asp Leu Arg Gln Phe Thr Cys Arg Asp Gln Arg Gly Trp Pro1
5 101010PRTArtificialHis tag 10Met Arg Gly Ser His His His His His
His1 5 101185DNAArtificialmicrogene #2101 11ctcgagagta tcatcaactt
cgagaagctt accgatttct caggcagttc atgcagctca 60tgcagagatc aacgaggctg
gccgc 851228PRTArtificialpeptide sequence encoded in frame 1 of
microgene #2101 12Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp
Phe Ser Gly Ser1 5 10 15Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp
Pro 20 251328PRTArtificialpeptide sequence encoded in frame 2 of
microgene #2101 13Ser Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Ile
Ser Gln Ala Val1 5 10 15His Ala Ala His Ala Glu Ile Asn Glu Ala Gly
Arg 20 251427PRTArtificialpeptide sequence encoded in frame 3 of
microgene #2101 14Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe
Leu Arg Gln Phe1 5 10 15Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala
20 251585DNAArtificialmicrogene #6101 15ctcgaaagta ttatcaattt
cgaaaaactc accgatttct caggcagttc atgcagctca 60tgcagagatc aacgaggctg
gccgc 851628PRTArtificialpeptide sequence encoded in frame 1 of
microgene #6101 16Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp
Phe Ser Gly Ser1 5 10 15Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp
Pro 20 251728PRTArtificialpeptide sequence encoded in frame 2 of
microgene #6101 17Ser Lys Val Leu Ser Ile Ser Lys Asn Ser Pro Ile
Ser Gln Ala Val1 5 10 15His Ala Ala His Ala Glu Ile Asn Glu Ala Gly
Arg 20 251827PRTArtificialpeptide sequence encoded in frame 3 of
microgene #6101 18Arg Lys Tyr Tyr Gln Phe Arg Lys Thr His Arg Phe
Leu Arg Gln Phe1 5 10 15Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala
20 251946DNAArtificialMPR primer 2101-S 19ctcgagagta tcatcaactt
cgagaagctt accgatttct caggct 462049DNAArtificialMPR primer 2101-AS,
6101-AS 20gcggccagcc tcgttgatct ctgcatgagc tgcatgaact gcctgagat
492146DNAArtificialMPR primer 6101-S 21ctcgaaagta ttatcaattt
cgaaaaactc accgatttct caggct 462228PRTArtificialtandem repeat unit
22Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser1
5 10 15Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro 20
2523517PRTHomo sapiens 23Met Gln Asp Pro Ala Ser Thr Cys Val Pro
Glu Pro Ala Ser Gln His1 5 10 15Thr Leu Arg Ser Gly Pro Gly Cys Leu
Gln Gln Pro Glu Gln Gln Gly 20 25 30Val Arg Asp Pro Gly Gly Ile Trp
Ala Lys Leu Gly Ala Ala Glu Ala 35 40 45Ser Ala Glu Arg Leu Gln Gly
Arg Arg Ser Arg Gly Ala Ser Gly Ser 50 55 60Glu Pro Gln Gln Met Gly
Ser Asp Val Arg Asp Leu Asn Ala Leu Leu65 70 75 80Pro Ala Val Pro
Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val 85 90 95Ser Gly Ala
Ala Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly 100 105 110Ala
Ser Ala Tyr Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro 115 120
125Pro Pro Pro Pro Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro
130 135 140Ser Trp Gly Gly Ala Glu Pro His Glu Glu Gln Cys Leu Ser
Ala Phe145 150 155 160Thr Val His Phe Ser Gly Gln Phe Thr Gly Thr
Ala Gly Ala Cys Arg 165 170 175Tyr Gly Pro Phe Gly Pro Pro Pro Pro
Ser Gln Ala Ser Ser Gly Gln 180 185 190Ala Arg Met Phe Pro Asn Ala
Pro Tyr Leu Pro Ser Cys Leu Glu Ser 195 200 205Gln Pro Ala Ile Arg
Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly 210 215 220Thr Pro Ser
Tyr Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro225 230 235
240Asn His Ser Phe Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu
245 250 255Gly Glu Gln Gln Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys
His Thr 260 265 270Pro Thr Asp Ser Cys Thr Gly Ser Gln Ala Leu Leu
Leu Arg Thr Pro 275 280 285Tyr Ser Ser Asp Asn Leu Tyr Gln Met Thr
Ser Gln Leu Glu Cys Met 290 295 300Thr Trp Asn Gln Met Asn Leu Gly
Ala Thr Leu Lys Gly Val Ala Ala305 310 315 320Gly Ser Ser Ser Ser
Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser 325 330 335Thr Gly Tyr
Glu Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala 340 345 350Gln
Tyr Arg Ile His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val 355 360
365Arg Arg Val Pro Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu
370 375 380Thr Ser Glu Lys Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys
Asn Lys385 390 395 400Arg Tyr Phe Lys Leu Ser His Leu Gln Met His
Ser Arg Lys His Thr 405 410 415Gly Glu Lys Pro Tyr Gln Cys Asp Phe
Lys Asp Cys Glu Arg Arg Phe 420 425 430Ser Arg Ser Asp Gln Leu Lys
Arg His Gln Arg Arg His Thr Gly Val 435 440 445Lys Pro Phe Gln Cys
Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp 450 455 460His Leu Lys
Thr His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys465 470 475
480Pro Phe Ser Cys Arg Trp Pro Ser Cys Gln Lys Lys Phe Ala Arg Ser
485 490 495Asp Glu Leu Val Arg His His Asn Met His Gln Arg Asn Met
Thr Lys 500 505 510Leu Gln Leu Ala Leu 51524386PRTGallus gallus
24Met Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe1
5 10 15Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys
Pro 20 25 30Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala
Lys Asp 35 40 45Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp
Lys Leu Pro 50 55 60Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr
Ser Val Asn Val65 70 75 80His Ser Ser Leu Arg Asp Ile Leu Asn Gln
Ile Thr Lys Pro Asn Asp 85 90 95Val Tyr Ser Phe Ser Leu Ala Ser Arg
Leu Tyr Ala Glu Glu Arg Tyr 100 105 110Pro Ile Leu Pro Glu Tyr Leu
Gln Cys Val Lys Glu Leu Tyr Arg Gly 115 120 125Gly Leu Glu Pro Ile
Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu 130 135 140Leu Ile Asn
Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn145 150 155
160Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val
165 170 175Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Thr Phe Lys
Asp Glu 180 185 190Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln
Glu Ser Lys Pro 195 200 205Val Gln Met Met Tyr Gln Ile Gly Leu Phe
Arg Val Ala Ser Met Ala 210 215 220Ser Glu Lys Met Lys Ile Leu Glu
Leu Pro Phe Ala Ser Gly Thr Met225 230 235 240Ser Met Leu Val Leu
Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu 245 250 255Glu Ser Ile
Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn 260 265 270Val
Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 275 280
285Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr
290 295 300Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser
Ala Glu305 310 315 320Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala
His Ala Glu Ile Asn 325 330 335Glu Ala Gly Arg Glu Val Val Gly Ser
Ala Glu Ala Gly Val Asp Ala 340 345 350Ala Ser Val Ser Glu Glu Phe
Arg Ala Asp His Pro Phe Leu Phe Cys 355 360 365Ile Lys His Ile Ala
Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val 370 375 380Ser
Pro3852529PRTArtificialtandem repeat unit with a substitution of
WT1 MHC class I epitope for OVA MHC class I epitope 25Leu Glu Arg
Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe Ser Gly1 5 10 15Ser Ser
Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro 20
2526190PRTArtificialF182A 26Met Arg Gly Ser His His His His His His
Gly Ser Val Asp Trp Gly1 5 10 15Thr Pro Glu Ile Asn Glu Ala Gly Arg
Leu Glu Ser Ile Ile Asn Phe 20 25 30Glu Lys Leu Thr Asp Phe Ser Gly
Ser Ser Cys Ser Ser Cys Arg Asp 35 40 45Gln Arg Gly Trp Pro Ser Arg
Val Ser Ser Thr Ser Arg Ser Leu Pro 50 55 60Ile Ser Gln Ala Val His
Ala Ala His Ala Glu Ile Asn Glu Ala Gly65 70 75 80Arg Ser Arg Val
Ser Ser Thr Ser Arg Ser Leu Pro Ile Ser His Ala 85 90 95Glu Ile Asn
Glu Ala Gly Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu 100 105 110Thr
Asp Leu Arg Gln Phe Thr Cys Arg Asp Gln Arg Gly Trp Pro Leu 115 120
125Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser
130 135 140Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Gln Ser Ile
Ile Asn145 150 155 160Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser
Cys Ser Ser Cys Arg 165 170 175Asp Gln Arg Trp Leu Ala Leu Glu Gly
Gly Ser Gly Val Asn 180 185 19027202PRTArtificialF182B 27Met Arg
Gly Ser His His His His His His Thr Asp Pro Ser Thr Val1 5 10 15Pro
Gln Arg Ser Thr Arg Leu Ala Ala Ser Arg Val Ser Ser Thr Ser 20 25
30Arg Ser Leu Pro Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile
35 40 45Asn Glu Ala Gly Pro Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr
Arg 50 55 60Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg
Leu Ala65 70 75 80Ala Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr Arg
Phe Leu Met Gln 85 90 95Arg Ser Thr Arg Leu Ala Ser Arg Val Ser Ser
Thr Ser Arg Ser Leu 100 105 110Pro Ile Ser Gly Ser Ser His Ala Glu
Ile Asn Glu Ala Gly Arg Ser 115 120 125Arg Val Ser Ser Thr Ser Glu
Lys Leu Thr Asp Phe Ser Ser Ser Ser 130 135 140Cys Ser Ser Cys Arg
Asp Gln Arg Gly Trp Pro Gln Ser Ile Ile Asn145 150 155 160Phe Glu
Lys Leu Thr Asp Ser Ser Gly Ser Ser Cys Ser Ser Cys Arg 165 170
175Asp Gln Arg Gly Trp Pro Ser Arg Gly Asp Leu Gly Leu Ile Asn Leu
180 185 190Thr Lys Phe Ser Lys Glu Phe Arg Pro Ala 195
20028194PRTArtificialF182C 28Met Arg Gly Ser His His His His His
His Gly Ile Arg Arg Gln Trp1 5 10 15Arg Tyr Pro Arg Asp Gln Arg Gly
Trp Pro Pro Arg Glu Tyr His Gln 20 25 30Leu Arg Glu Ala Tyr Arg Phe
Leu Arg Gln Phe Met Gln Leu Met Gln 35 40 45Arg Ser Thr Arg Leu Ala
Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu 50 55 60Thr Asp Phe Ser Gly
Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly65 70 75 80Trp Pro Leu
Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser 85 90 95Cys Arg
Asp Gln Arg Gly Trp Pro Arg Glu Tyr His Gln Leu Arg Glu 100 105
110Ala Tyr Arg Ser Gln Ala Val His Met Gln Arg Ser Thr Arg Leu Ala
115 120 125Ala Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe Leu
Arg Gln 130 135 140Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala
Ala Glu Tyr His145 150 155 160Gln Leu Arg Glu Ala Tyr Arg Phe Leu
Arg Gln Phe Met Gln Leu Met 165 170 175Gln Arg Ser Thr Arg Ala Gly
Pro Arg Arg Gly Asp Leu Gly Val Lys 180 185 190Leu
Asn29115PRTArtificialF36A 29Met Arg Gly Ser His His His His His His
Gly Ser Val Asp Gly Thr1 5 10 15Pro Thr Arg Leu Ala Ala Ser Arg Val
Ser Ser Thr Ser Arg Ser Leu 20 25 30Pro Ile Ser Gln Ala Val His Ala
Ala His Ala Glu Ile Asn Glu Ala 35 40 45Gly Arg Ser Arg Val Ser Ser
Thr Ser Arg Ser Leu Pro Ile Ser Gln 50 55 60Ala Val His Ala Ala His
Ala Glu Ile Asn Glu Ala Gly Arg Arg Val65 70 75 80Ser Ser Thr Ser
Arg Ser Leu Pro Ile Ser Gln Ala Val His Ala Ala 85 90 95His Ala Glu
Ile Asn Glu Ala Gly Arg Leu Glu Ser Ile Ile Asn Gly 100 105 110Asp
Leu Gly 11530117PRTArtificialF36B 30Met Arg Gly Ser His His His His
His His Thr Asp Pro Ser Thr Val1 5 10 15Pro Gln Arg Gly Trp Pro Pro
Arg Glu Tyr His Gln Leu Arg Glu Ala 20 25 30Tyr Arg Phe Leu Arg Gln
Phe Met Gln Leu Met Gln Arg Ser Thr Arg 35 40 45Leu Ala Ala Arg Glu
Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe Leu 50 55 60Arg Gln Phe Met
Gln Leu Met Gln Arg Ser Thr Arg Leu Ala Ala Glu65 70 75 80Tyr His
Gln Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe Met Gln 85 90 95Leu
Met Gln Arg Ser Thr Arg Leu Ala Ala Ser Arg Val Ser Ser Thr 100 105
110Gly Ile Trp Val Asn 11531120PRTArtificialF36C 31Met Arg Gly Ser
His His His His His His Gly Ile Arg Arg Gln Trp1 5 10 15Arg Tyr Pro
Asn Glu Ala Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu 20 25 30Lys Leu
Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln 35 40 45Arg
Gly Trp Pro Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp 50 55
60Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro65
70 75 80Gln Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser
Ser 85 90 95Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Glu
Tyr His 100 105 110Gln Arg Gly Ser Gly Leu Ile Asn 115
12032182PRTArtificialF37B 32Met Arg Gly Ser His His His His His His
Thr Asp Pro Ser Thr Val1 5 10 15Pro Ala Tyr Arg Phe Leu Arg Gln Phe
Met Gln Leu Met Gln Arg Ser 20 25
30Thr Arg Leu Ala Ala Ser Arg Val Ser Ser Thr Ser Arg Ser Leu Pro
35 40 45Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala
Gly 50 55 60Arg Ser Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Ile Ser
Gln Ala65 70 75 80Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly
Arg Ser Arg Val 85 90 95Ser Ser Thr Ser Arg Ser Leu Pro Ile Ser Gln
Ala Val His Ala Ala 100 105 110His Ala Glu Ile Asn Glu Ala Gly Arg
Leu Glu Ser Ile Ile Asn Phe 115 120 125Glu Lys Leu Thr Asp Phe Ser
Gly Ser Ser Cys Ser Ser Cys Arg Asp 130 135 140Gln Arg Gly Trp Pro
Pro Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr145 150 155 160Arg Phe
Leu Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu 165 170
175Ala Gly Ile Trp Val Asn 18033185PRTArtificialF37C 33Met Arg Gly
Ser His His His His His His Gly Ile Arg Arg Gln Trp1 5 10 15Arg Tyr
Pro Leu Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg 20 25 30Asp
Gln Arg Gly Trp Pro Pro Arg Glu Tyr His Gln Leu Arg Glu Ala 35 40
45Tyr Arg Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg
50 55 60Leu Ala Ala Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe
Leu65 70 75 80Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu
Ala Ala Arg 85 90 95Glu Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe Leu
Arg Gln Phe Met 100 105 110Gln Leu Met Gln Arg Ser Thr Arg Leu Ala
Ala Ser Arg Val Ser Ser 115 120 125Thr Ser Arg Ser Leu Pro Ile Ser
Gln Ala Val His Ala Ala His Ala 130 135 140Glu Ile Asn Glu Ala Gly
Arg Leu Glu Ser Ile Ile Asn Phe Glu Lys145 150 155 160Leu Thr Asp
Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg 165 170 175Gly
Trp Arg Gly Ser Gly Leu Ile Asn 180 18534158PRTArtificialF138A
34Met Arg Gly Ser His His His His His His Gly Ser Val Asp Trp Gly1
5 10 15Thr Leu Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly
Cys 20 25 30Ser Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Ile Ser Gln
Ala Val 35 40 45His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Arg
Glu Tyr His 50 55 60Gln Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe
Met Gln Leu Met65 70 75 80Gln Arg Ser Thr Arg Leu Ala Ala Glu Ser
Ile Ile Asn Phe Glu Lys 85 90 95Leu Thr Asp Phe Ser Gly Ser Ser Cys
Ser Ser Cys Arg Asp Gln Arg 100 105 110Gly Trp Pro Leu Glu Ser Ile
Ile Asn Phe Glu Lys Leu Thr Asp Phe 115 120 125Ser Gly Ser Ser Cys
Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro 130 135 140Arg Glu Tyr
His Gln Leu Arg Glu Ala Tyr Gly Asp Leu Gly145 150
15535239PRTArtificialG142A 35Met Arg Gly Ser His His His His His
His Gly Ser Val Asp Trp Gly1 5 10 15Thr Gly Thr Pro Ile Ser Gln Ala
Val His Ala Ala His Ala Glu Ile 20 25 30Asn Glu Ala Gly Leu Glu Ser
Ile Ile Asn Phe Glu Lys Leu Thr Asp 35 40 45Phe Ser Gly Ser Ser Cys
Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro 50 55 60Arg Ser Lys Val Leu
Ser Ile Ser Lys Asn Ser Pro Ile Ser Gln Ala65 70 75 80Val His Ala
Ala His Ala Glu Ile Asn Glu Ala Gly Arg Leu Glu Ser 85 90 95Ile Ile
Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser Cys Ser 100 105
110Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Lys Tyr Tyr Gln Phe
115 120 125Arg Lys Thr His Arg Phe Leu Arg Gln Phe Met Gln Leu Met
Gln Arg 130 135 140Ser Thr Arg Leu Ala Ala Ser Lys Val Leu Ser Ile
Ser Lys Asn Ser145 150 155 160Pro Ile Ser Gln Ala Val His Ala Ala
His Ala Glu Ile Asn Glu Ala 165 170 175Gly Arg Leu Glu Ser Ile Ile
Asn Phe Glu Lys Leu Thr Asp Phe Ser 180 185 190Gly Ser Ser Cys Ser
Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu 195 200 205Ser Ile Ile
Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser Cys 210 215 220Ser
Ser Cys Arg Asp Gln Arg Gly Trp Pro Gly Ile Trp Val Asn225 230
23536237PRTArtificialG142C 36Met Arg Gly Ser His His His His His
His Gly Ile Arg Arg Gln Trp1 5 10 15Arg Tyr Pro Asp Phe Ser Gly Ser
Ser Cys Ser Ser Cys Arg Asp Gln 20 25 30Arg Gly Trp Pro Arg Lys Tyr
Tyr Gln Phe Arg Lys Thr His Arg Phe 35 40 45Leu Arg Gln Phe Met Gln
Leu Met Gln Arg Ser Thr Arg Leu Ala Ala 50 55 60Leu Glu Ser Ile Ile
Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser65 70 75 80Ser Cys Ser
Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Lys Tyr 85 90 95Tyr Gln
Phe Arg Lys Thr His Arg Phe Leu Arg Gln Phe Met Gln Leu 100 105
110Met Gln Arg Ser Thr Arg Leu Ala Ala Ser Lys Val Leu Ser Ile Ser
115 120 125Lys Asn Ser Pro Ile Ser Gln Ala Val His Ala Ala His Ala
Glu Ile 130 135 140Asn Glu Ala Gly Arg Leu Glu Ser Ile Ile Asn Phe
Glu Lys Leu Thr145 150 155 160Asp Phe Ser Gly Ser Ser Cys Ser Ser
Cys Arg Asp Gln Arg Gly Trp 165 170 175Pro Pro Arg Lys Tyr Tyr Gln
Phe Arg Lys Thr His Arg Phe Leu Arg 180 185 190Gln Phe Met Gln Leu
Met Gln Arg Ser Thr Arg Leu Ala Ala Arg Lys 195 200 205Tyr Tyr Gln
Phe Arg Lys Thr His Arg Phe Leu Arg Gln Phe Met Gln 210 215 220Leu
Met Gln Arg Ser Thr Arg Leu Ala Gly Asp Leu Gly225 230
23537163PRTArtificialF58B 37Met Arg Gly Ser His His His His His His
Thr Asp Pro Ser Thr Val1 5 10 15Pro Arg Gly Trp Pro Gln Ser Ile Ile
Asn Phe Glu Lys Leu Thr Asp 20 25 30Phe Ser Gly Ser Ser Cys Ser Ser
Cys Arg Asp Gln Arg Gly Trp Pro 35 40 45Pro Arg Glu Tyr His Gln Leu
Arg Glu Ala Tyr Arg Phe Leu Arg Gln 50 55 60Phe Met Gln Leu Met Gln
Arg Ser Thr Arg Leu Ala Ala Ser Arg Val65 70 75 80Ser Ser Thr Ser
Arg Ser Leu Pro Ile Ser Gln Ala Val His Ala Ala 85 90 95His Ala Glu
Ile Asn Glu Ala Gly Arg Leu Glu Ser Ile Ile Asn Phe 100 105 110Glu
Lys Leu Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp 115 120
125Gln Arg Gly Trp Pro Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Ile
130 135 140Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala
Gly Gly145 150 155 160Asp Leu Gly38166PRTArtificialF58C 38Met Arg
Gly Ser His His His His His His Gly Ile Arg Arg Gln Trp1 5 10 15Arg
Tyr Pro Glu Ala Gly Arg Arg Val Ser Ser Thr Ser Arg Ser Leu 20 25
30Pro Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala
35 40 45Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe
Ser 50 55 60Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro
Pro Arg65 70 75 80Glu Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe Leu
Arg Gln Phe Met 85 90 95Gln Leu Met Gln Arg Ser Thr Arg Leu Ala Ala
Ser Arg Val Ser Ser 100 105 110Thr Ser Arg Ser Leu Pro Ile Ser Gln
Ala Val His Ala Ala His Ala 115 120 125Glu Ile Asn Glu Ala Gly Arg
Glu Tyr His Gln Leu Arg Glu Ala Tyr 130 135 140Arg Phe Leu Arg Gln
Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu145 150 155 160Ala Gly
Ile Trp Val Asn 16539207PRTArtificialF112A 39Met Arg Gly Ser His
His His His His His Gly Ser Val Asp Gly Thr1 5 10 15Arg Glu Ala Tyr
Arg Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg 20 25 30Ser Thr Arg
Leu Ala Ala Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr 35 40 45Arg Phe
Leu Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu 50 55 60Ala
Ala Ser Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Ile Ser Gln65 70 75
80Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Ser Arg Val
85 90 95Ser Ser Thr Ser Arg Ser Leu Pro Ile Ser Gln Ala Val His Ala
Ala 100 105 110His Ala Glu Ile Asn Glu Ala Gly Arg Ser Arg Val Ser
Ser Thr Ser 115 120 125Arg Ser Leu Pro Ile Ser Gln Ala Val His Ala
Ala His Ala Glu Ile 130 135 140Asn Glu Ala Gly Arg Arg Glu Tyr His
Gln Leu Arg Glu Ala Tyr Leu145 150 155 160Phe Leu Arg Gln Phe Met
Gln Leu Met Gln Arg Ser Thr Arg Leu Ala 165 170 175Leu Glu Ser Ile
Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser 180 185 190Ser Cys
Ser Ser Cys Arg Asp Gln Arg Gly Gly Ile Trp Val Asn 195 200
20540211PRTArtificialF112C 40Met Arg Gly Ser His His His His His
His Gly Ile Arg Arg Gln Trp1 5 10 15Arg Tyr Pro Arg Ser Leu Pro Ile
Ser Gln Gly Ser Ser Cys Ser Ser 20 25 30Cys Arg Asp Gln Arg Gly Trp
Pro Leu Glu Ser Ile Ile Asn Phe Glu 35 40 45Lys Leu Thr Asp Phe Ser
Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln 50 55 60Arg Gly Trp Pro Pro
Arg Glu Tyr His Pro Leu Arg Glu Ala Tyr Arg65 70 75 80Phe Leu Arg
Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala 85 90 95Arg Glu
Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe 100 105
110Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala Ala Arg Glu Tyr His
115 120 125Gln Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe Met Gln
Leu Met 130 135 140Gln Arg Ser Thr Arg Leu Ala Ala Glu Ser Ile Ile
Asn Phe Glu Lys145 150 155 160Leu Thr Tyr Phe Ser Gly Ser Ser Cys
Ser Ser Cys Arg Asp Gln Arg 165 170 175Gly Trp Pro Ser Arg Val Ser
Ser Thr Ser Arg Ser Leu Pro Ile Ser 180 185 190Gln Ala Val His Ala
Ala His Ala Glu Ile Asn Glu Ala Gly Ser Gly 195 200 205Leu Ile Asn
21041113PRTArtificialMT290 41Met Arg Gly Ser His His His His His
His Gly Ile Arg Arg Arg Tyr1 5 10 15Pro Glu Ser Leu Ala Arg Ala Tyr
Gly Glu Leu Ala Ser Arg Ala Glu 20 25 30Ser Leu Ala Arg Ala Tyr Gly
Glu Leu Ala Ser Arg Ala Glu Ser Leu 35 40 45Ala Arg Ala Tyr Gly Glu
Leu Ala Ser Arg Ala Glu Ser Leu Ala Arg 50 55 60Ala Tyr Gly Glu Leu
Ala Ser Arg Ala Glu Ser Leu Ala Arg Ala Tyr65 70 75 80Gly Glu Leu
Ala Ser Arg Gly Lys Ser Cys Lys Gly Val Trp Arg Thr 85 90 95Cys Lys
Pro Ser Gly Lys Ser Cys Lys Gly Gly Gly Ser Gly Leu Ile 100 105
110Asn42104PRTArtificialMT297 42Met Arg Gly Ser His His His His His
His Gly Ser Val Asp Gly Thr1 5 10 15Arg Thr Ser Lys Pro Asn Gly Lys
Ser Tyr Arg Val Val Trp Arg Thr 20 25 30Ser Lys Pro Asn Gly Lys Ser
Tyr Arg Val Val Trp Arg Thr Ser Lys 35 40 45Pro Asn Gly Lys Ser Tyr
Arg Val Val Trp Arg Thr Ser Lys Pro Asn 50 55 60Gly Lys Ser Tyr Arg
Val Val Trp Arg Thr Ser Lys Pro Asn Glu Lys65 70 75 80Ser Tyr Arg
Val Val Trp Arg Thr Ser Lys Pro Asn Arg Lys Val Leu 85 90 95Gln Gly
Arg Gly Ile Trp Val Asn 10043141PRTArtificialMT332 43Met Arg Gly
Ser His Phe His His His His His Gly Ile Arg Arg Arg1 5 10 15Tyr Pro
Leu Gln Gly Arg Met Glu Asn Leu Gln Ala Glu Arg Lys Val 20 25 30Leu
Gln Gly Arg Met Glu Asn Leu Gln Ala Glu Lys Gly Ser Ser Gly 35 40
45Pro Tyr Gly Glu Ser Ser Gly Arg Glu Arg Phe Phe Arg Ala Val Trp
50 55 60Arg Ile Phe Arg Gln Arg Lys Val Leu Gln Gly Arg Met Glu Asn
Leu65 70 75 80Gln Ala Glu Lys Gly Ser Ser Gly Pro Tyr Gly Glu Ser
Ser Gly Arg 85 90 95Glu Arg Phe Phe Arg Ala Val Trp Arg Ile Phe Arg
Gln Arg Lys Gly 100 105 110Ser Ser Gly Pro Tyr Gly Glu Ser Ser Gly
Arg Glu Arg Phe Phe Arg 115 120 125Ala Val Trp Arg Ile Phe Arg Gly
Ser Gly Leu Ile Asn 130 135 14044181PRTArtificialF37A 44Met Arg Gly
Ser His His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg
Leu Pro Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30Asn
Glu Ala Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr 35 40
45Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp
50 55 60Pro Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser
Gly65 70 75 80Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro
Leu Glu Ser 85 90 95Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly
Ser Ser Cys Ser 100 105 110Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro
Arg Glu Tyr His Gln Leu 115 120 125Arg Glu Ala Tyr Arg Phe Leu Arg
Gln Phe Met Gln Leu Met Gln Arg 130 135 140Ser Thr Arg Leu Ala Ala
Ser Arg Val Ser Ser Thr Ser Arg Ser Leu145 150 155 160Pro Ile Ser
Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala 165 170 175Gly
Gly Asp Leu Gly 18045183PRTArtificialF37AE2 45Met Arg Gly Ser His
His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30Asn Glu Ala
Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr 35 40 45Asp Phe
Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp 50 55 60Pro
Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly65 70 75
80Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu Ser
85 90 95Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser Cys
Ser 100 105 110Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Glu Tyr
His Gln Leu 115 120 125Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe Met
Gln Leu Met Gln Arg 130 135 140Ser Thr Arg Leu Ala Ala Ser Arg Val
Ser Ser Thr Ser Arg Ser Leu145 150 155 160Pro Ile Ser Gln Ala Val
His Ala Ala His Ala Glu Ile Asn Glu Ala 165 170 175Gly Gly Ser Gly
Leu Ile Asn 18046184PRTArtificialMT819 46Met Arg Gly Ser His
His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30Asn Glu Ala
Gly Arg Leu Glu Arg Met Phe Pro Asn Ala Pro Tyr Leu 35 40 45Thr Asp
Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly 50 55 60Trp
Pro Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser65 70 75
80Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu
85 90 95Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser
Cys 100 105 110Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Glu
Tyr His Gln 115 120 125Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe
Met Gln Leu Met Gln 130 135 140Arg Ser Thr Arg Leu Ala Ala Ser Arg
Val Ser Ser Thr Ser Arg Ser145 150 155 160Leu Pro Ile Ser Gln Ala
Val His Ala Ala His Ala Glu Ile Asn Glu 165 170 175Ala Gly Gly Ser
Gly Leu Ile Asn 18047184PRTArtificialMT820 47Met Arg Gly Ser His
His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30Asn Glu Ala
Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr 35 40 45Asp Phe
Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp 50 55 60Pro
Leu Glu Arg Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe Ser65 70 75
80Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu
85 90 95Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser
Cys 100 105 110Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Glu
Tyr His Gln 115 120 125Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe
Met Gln Leu Met Gln 130 135 140Arg Ser Thr Arg Leu Ala Ala Ser Arg
Val Ser Ser Thr Ser Arg Ser145 150 155 160Leu Pro Ile Ser Gln Ala
Val His Ala Ala His Ala Glu Ile Asn Glu 165 170 175Ala Gly Gly Ser
Gly Leu Ile Asn 18048184PRTArtificialMT821 48Met Arg Gly Ser His
His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30Asn Glu Ala
Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr 35 40 45Asp Phe
Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp 50 55 60Pro
Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly65 70 75
80Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu Arg
85 90 95Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe Ser Gly Ser Ser
Cys 100 105 110Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Glu
Tyr His Gln 115 120 125Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe
Met Gln Leu Met Gln 130 135 140Arg Ser Thr Arg Leu Ala Ala Ser Arg
Val Ser Ser Thr Ser Arg Ser145 150 155 160Leu Pro Ile Ser Gln Ala
Val His Ala Ala His Ala Glu Ile Asn Glu 165 170 175Ala Gly Gly Ser
Gly Leu Ile Asn 18049185PRTArtificialMT822 49Met Arg Gly Ser His
His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30Asn Glu Ala
Gly Arg Leu Glu Arg Met Phe Pro Asn Ala Pro Tyr Leu 35 40 45Thr Asp
Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly 50 55 60Trp
Pro Leu Glu Arg Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe65 70 75
80Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu
85 90 95Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser
Ser 100 105 110Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg
Glu Tyr His 115 120 125Gln Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln
Phe Met Gln Leu Met 130 135 140Gln Arg Ser Thr Arg Leu Ala Ala Ser
Arg Val Ser Ser Thr Ser Arg145 150 155 160Ser Leu Pro Ile Ser Gln
Ala Val His Ala Ala His Ala Glu Ile Asn 165 170 175Glu Ala Gly Gly
Ser Gly Leu Ile Asn 180 18550185PRTArtificialMT823 50Met Arg Gly
Ser His His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg
Leu Pro Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30Asn
Glu Ala Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr 35 40
45Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp
50 55 60Pro Leu Glu Arg Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe
Ser65 70 75 80Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp
Pro Leu Glu 85 90 95Arg Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe
Ser Gly Ser Ser 100 105 110Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp
Pro Pro Arg Glu Tyr His 115 120 125Gln Leu Arg Glu Ala Tyr Arg Phe
Leu Arg Gln Phe Met Gln Leu Met 130 135 140Gln Arg Ser Thr Arg Leu
Ala Ala Ser Arg Val Ser Ser Thr Ser Arg145 150 155 160Ser Leu Pro
Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn 165 170 175Glu
Ala Gly Gly Ser Gly Leu Ile Asn 180 18551185PRTArtificialMT824
51Met Arg Gly Ser His His His His His His Gly Ser Val Asp Trp Gly1
5 10 15Thr Arg Leu Pro Ile Ser Gln Ala Val His Ala Ala His Ala Glu
Ile 20 25 30Asn Glu Ala Gly Arg Leu Glu Arg Met Phe Pro Asn Ala Pro
Tyr Leu 35 40 45Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp
Gln Arg Gly 50 55 60Trp Pro Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu
Thr Asp Phe Ser65 70 75 80Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln
Arg Gly Trp Pro Leu Glu 85 90 95Arg Met Phe Pro Asn Ala Pro Tyr Leu
Thr Asp Phe Ser Gly Ser Ser 100 105 110Cys Ser Ser Cys Arg Asp Gln
Arg Gly Trp Pro Pro Arg Glu Tyr His 115 120 125Gln Leu Arg Glu Ala
Tyr Arg Phe Leu Arg Gln Phe Met Gln Leu Met 130 135 140Gln Arg Ser
Thr Arg Leu Ala Ala Ser Arg Val Ser Ser Thr Ser Arg145 150 155
160Ser Leu Pro Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn
165 170 175Glu Ala Gly Gly Ser Gly Leu Ile Asn 180
18552186PRTArtificialMT825 52Met Arg Gly Ser His His His His His
His Gly Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro Ile Ser Gln Ala
Val His Ala Ala His Ala Glu Ile 20 25 30Asn Glu Ala Gly Arg Leu Glu
Arg Met Phe Pro Asn Ala Pro Tyr Leu 35 40 45Thr Asp Phe Ser Gly Ser
Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly 50 55 60Trp Pro Leu Glu Arg
Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe65 70 75 80Ser Gly Ser
Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu 85 90 95Glu Arg
Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe Ser Gly Ser 100 105
110Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Pro Arg Glu Tyr
115 120 125His Gln Leu Arg Glu Ala Tyr Arg Phe Leu Arg Gln Phe Met
Gln Leu 130 135 140Met Gln Arg Ser Thr Arg Leu Ala Ala Ser Arg Val
Ser Ser Thr Ser145 150 155 160Arg Ser Leu Pro Ile Ser Gln Ala Val
His Ala Ala His Ala Glu Ile 165 170 175Asn Glu Ala Gly Gly Ser Gly
Leu Ile Asn 180 1855325PRTArtificialtandem repeat unit 53Leu Glu
Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Leu Arg Gln Phe1 5 10 15Thr
Cys Arg Asp Gln Arg Gly Trp Pro 20 2554193PRTArtificial
SequenceAkiKaze A24 54Met Arg Gly Ser His His His His His His Gly
Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro Lys Arg Tyr Phe Lys Leu
Ser His Leu Gln Met His 20 25 30Ser Arg Lys His Gly Ser Leu Glu Cys
Tyr Thr Trp Asn Gln Met Asn 35 40 45Leu Gly Ala Thr Asp Phe Ser Gly
Ser Ser Cys Ser Ser Cys Arg Asp 50 55 60Gln Arg Gly Trp Pro Leu Glu
Cys Tyr Thr Trp Asn Gln Met Asn Leu65 70 75 80Gly Ala Thr Asp Phe
Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln 85 90 95Arg Gly Trp Pro
Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly 100 105 110Ala Thr
Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg 115 120
125Gly Trp Pro Val Asp Leu Glu Pro Arg Glu Tyr His Gln Leu Arg Glu
130 135 140Ala Tyr Arg Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg
Ser Thr145 150 155 160Arg Leu Ala Ala Ser Arg Val Ser Ser Thr Ser
Arg Ser Leu Pro Lys 165 170 175Arg Tyr Phe Lys Leu Ser His Leu Gln
Met His Ser Arg Lys His Gly 180 185 190Asp55193PRTArtificial
SequenceAkiKaze A2 55Met Arg Gly Ser His His His His His His Gly
Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro Lys Arg Tyr Phe Lys Leu
Ser His Leu Gln Met His 20 25 30Ser Arg Lys His Gly Ser Val Asp Gln
Ala Arg Met Phe Pro Asn Ala 35 40 45Pro Tyr Leu Pro Ser Thr Asp Phe
Ser Gly Ser Ser Cys Ser Ser Cys 50 55 60Arg Asp Gln Arg Gly Trp Pro
Gln Ala Arg Met Phe Pro Asn Ala Pro65 70 75 80Tyr Leu Pro Ser Thr
Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg 85 90 95Asp Gln Arg Gly
Trp Pro Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr 100 105 110Leu Pro
Ser Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp 115 120
125Gln Arg Gly Trp Pro Leu Glu Pro Arg Glu Tyr His Gln Leu Arg Glu
130 135 140Ala Tyr Arg Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg
Ser Thr145 150 155 160Arg Leu Ala Ala Ser Arg Val Ser Ser Thr Ser
Arg Ser Leu Pro Lys 165 170 175Arg Tyr Phe Lys Leu Ser His Leu Gln
Met His Ser Arg Lys His Gly 180 185 190Asp56286PRTArtificial
SequenceAkiKaze A242 56Met Arg Gly Ser His His His His His His Gly
Ser Val Asp Trp Gly1 5 10 15Thr Arg Leu Pro Lys Arg Tyr Phe Lys Leu
Ser His Leu Gln Met His 20 25 30Ser Arg Lys His Gly Ser Leu Glu Cys
Tyr Thr Trp Asn Gln Met Asn 35 40 45Leu Gly Ala Thr Asp Phe Ser Gly
Ser Ser Cys Ser Ser Cys Arg Asp 50 55 60Gln Arg Gly Trp Pro Leu Glu
Cys Tyr Thr Trp Asn Gln Met Asn Leu65 70 75 80Gly Ala Thr Asp Phe
Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln 85 90 95Arg Gly Trp Pro
Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly 100 105 110Ala Thr
Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg 115 120
125Gly Trp Pro Val Asp Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr Leu
130 135 140Pro Ser Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg
Asp Gln145 150 155 160Arg Gly Trp Pro Gln Ala Arg Met Phe Pro Asn
Ala Pro Tyr Leu Pro 165 170 175Ser Thr Asp Phe Ser Gly Ser Ser Cys
Ser Ser Cys Arg Asp Gln Arg 180 185 190Gly Trp Pro Gln Ala Arg Met
Phe Pro Asn Ala Pro Tyr Leu Pro Ser 195 200 205Thr Asp Phe Ser Gly
Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly 210 215 220Trp Pro Leu
Glu Pro Arg Glu Tyr His Gln Leu Arg Glu Ala Tyr Arg225 230 235
240Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala
245 250 255Ala Ser Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Lys Arg
Tyr Phe 260 265 270Lys Leu Ser His Leu Gln Met His Ser Arg Lys His
Gly Asp 275 280 28557252PRTArtificial SequenceWT1 A2 8110 57Met Arg
Gly Ser His His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr
Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg Asn Lys 20 25
30Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His Gly
35 40 45Ser Val Asp Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr Leu Pro
Ser 50 55 60Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln
Arg Gly65 70 75 80Trp Pro Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr
Leu Pro Ser Thr 85 90 95Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg
Asp Gln Arg Gly Trp 100 105 110Pro Gln Ala Arg Met Phe Pro Asn Ala
Pro Tyr Leu Pro Ser Thr Asp 115 120 125Phe Ser Gly Ser Ser Cys Ser
Ser Cys Arg Asp Gln Arg Gly Trp Pro 130 135 140Leu Glu Arg Leu Ala
Val Cys Ser Met Leu Leu Ile Phe Leu Leu Ala145 150 155 160Glu Gln
Ala Leu Leu Gln Ala Leu Ala Leu Ala Asp Ala Leu Ala Glu 165 170
175Ala Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg Asn Lys
180 185 190Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys
His Val 195 200 205Asp Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp
His Asp Ile Asp 210 215 220Tyr Lys Asp Asp Asp Asp Lys Leu Val Asp
Lys Leu Leu Glu Ser Ile225 230 235 240Ile Asn Phe Glu Lys Leu Thr
Asp Lys Leu Gly Asp 245 25058252PRTArtificial SequenceWT1 A24 8112
58Met Arg Gly Ser His His His His His His Gly Ser Val Asp Trp Gly1
5 10 15Thr Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg Asn
Lys 20 25 30Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys
His Gly 35 40 45Ser Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly
Ala Gly Thr 50 55 60Ser Val Thr Ser Ser Ser Arg Thr Cys Arg Cys Thr
Arg Gly Ser Thr65 70 75 80Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn
Leu Gly Ala Gly Thr Ser 85 90 95Val Thr Ser Ser Ser Arg Thr Cys Arg
Cys Thr Arg Gly Ser Thr Leu 100 105 110Glu Cys Tyr Thr Trp Asn Gln
Met Asn Leu Gly Ala Gly Thr Ser Val 115 120 125Thr Ser Ser Ser Arg
Thr Cys Arg Cys Thr Arg Gly Ser Thr Val Asp 130 135 140Leu Glu Arg
Leu Ala Val Cys Ser Met Leu Leu Ile Phe Leu Leu Ala145 150 155
160Glu Gln Ala Leu Leu Gln Ala Leu Ala Leu Ala Asp Ala Leu Ala Glu
165 170 175Ala Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg
Asn Lys 180 185
190Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His Val
195 200 205Asp Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp
Ile Asp 210 215 220Tyr Lys Asp Asp Asp Asp Lys Leu Val Asp Lys Leu
Leu Glu Ser Ile225 230 235 240Ile Asn Phe Glu Lys Leu Thr Asp Lys
Leu Gly Asp 245 25059236PRTArtificial SequenceWT1 A24 839 59Met Arg
Gly Ser His His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr
Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg Asn Lys 20 25
30Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His Gly
35 40 45Ser Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly Ala Thr
Asp 50 55 60Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly
Trp Pro65 70 75 80Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly
Ala Thr Asp Phe 85 90 95Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln
Arg Gly Trp Pro Leu 100 105 110Glu Cys Tyr Thr Trp Asn Gln Met Asn
Leu Gly Ala Thr Asp Phe Ser 115 120 125Gly Ser Ser Cys Ser Ser Cys
Arg Asp Gln Arg Gly Trp Pro Val Asp 130 135 140Leu Glu Arg Leu Ala
Val Cys Ser Met Leu Leu Ile Phe Leu Leu Ala145 150 155 160Glu Gln
Ala Leu Leu Gln Ala Leu Ala Leu Ala Asp Ala Leu Ala Glu 165 170
175Ala Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg Asn Lys
180 185 190Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys
His Val 195 200 205Asp Asp Glu Asp Glu Asp Glu Asp Val Asp Lys Leu
Leu Glu Ser Ile 210 215 220Ile Asn Phe Glu Lys Leu Thr Asp Lys Leu
Gly Asp225 230 23560236PRTArtificial SequenceWT1 A2 8310 60Met Arg
Gly Ser His His His His His His Gly Ser Val Asp Trp Gly1 5 10 15Thr
Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg Asn Lys 20 25
30Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His Gly
35 40 45Ser Val Asp Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr Leu Pro
Ser 50 55 60Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln
Arg Gly65 70 75 80Trp Pro Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr
Leu Pro Ser Thr 85 90 95Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg
Asp Gln Arg Gly Trp 100 105 110Pro Gln Ala Arg Met Phe Pro Asn Ala
Pro Tyr Leu Pro Ser Thr Asp 115 120 125Phe Ser Gly Ser Ser Cys Ser
Ser Cys Arg Asp Gln Arg Gly Trp Pro 130 135 140Leu Glu Arg Leu Ala
Val Cys Ser Met Leu Leu Ile Phe Leu Leu Ala145 150 155 160Glu Gln
Ala Leu Leu Gln Ala Leu Ala Leu Ala Asp Ala Leu Ala Glu 165 170
175Ala Gly Ser Tyr Val Gln Cys Ser Leu Ser Ser Phe Leu Arg Asn Lys
180 185 190Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys
His Val 195 200 205Asp Asp Glu Asp Glu Asp Glu Asp Val Asp Lys Leu
Leu Glu Ser Ile 210 215 220Ile Asn Phe Glu Lys Leu Thr Asp Lys Leu
Gly Asp225 230 23561186PRTArtificial SequenceHis PADRE WT1 A2 61Met
Arg Gly Ser His His His His His His Gly Ser Val Asp Gly Thr1 5 10
15Arg Leu Pro Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala
20 25 30Gly Ser Val Asp Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr Leu
Pro 35 40 45Ser Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp
Gln Arg 50 55 60Gly Trp Pro Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr
Leu Pro Ser65 70 75 80Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys
Arg Asp Gln Arg Gly 85 90 95Trp Pro Gln Ala Arg Met Phe Pro Asn Ala
Pro Tyr Leu Pro Ser Thr 100 105 110Asp Phe Ser Gly Ser Ser Cys Ser
Ser Cys Arg Asp Gln Arg Gly Trp 115 120 125Pro Leu Glu Pro Arg Glu
Tyr His Gln Leu Arg Glu Ala Tyr Arg Phe 130 135 140Leu Arg Gln Phe
Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala Ala145 150 155 160Ser
Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Ala Lys Phe Val Ala 165 170
175Ala Trp Thr Leu Lys Ala Ala Ala Gly Asp 180
18562186PRTArtificial SequenceHis PADRE WT1 A24 62Met Arg Gly Ser
His His His His His His Gly Ser Val Asp Gly Thr1 5 10 15Arg Leu Pro
Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 20 25 30Gly Ser
Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly Ala Thr 35 40 45Asp
Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp 50 55
60Pro Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly Ala Thr Asp65
70 75 80Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp
Pro 85 90 95Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly Ala Thr
Asp Phe 100 105 110Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg
Gly Trp Pro Val 115 120 125Asp Leu Glu Pro Arg Glu Tyr His Gln Leu
Arg Glu Ala Tyr Arg Phe 130 135 140Leu Arg Gln Phe Met Gln Leu Met
Gln Arg Ser Thr Arg Leu Ala Ala145 150 155 160Ser Arg Val Ser Ser
Thr Ser Arg Ser Leu Pro Ala Lys Phe Val Ala 165 170 175Ala Trp Thr
Leu Lys Ala Ala Ala Gly Asp 180 18563203PRTArtificial Sequencegp100
7172 63Met Arg Gly Ser His His His His His His Gly Ser Val Asp Trp
Gly1 5 10 15Thr Arg Leu Pro Lys Ala Trp Asn Arg Gln Leu Tyr Pro Glu
Trp Thr 20 25 30Glu Ala Gln Arg Leu Asp Cys Trp Gly Ser Ala Thr Lys
Val Pro Arg 35 40 45Asn Gln Asp Trp Leu Gly Val Thr Asp Phe Ser Gly
Ser Ser Cys Ser 50 55 60Ser Cys Arg Asp Gln Arg Gly Trp Pro Ala Thr
Lys Val Pro Arg Asn65 70 75 80Gln Asp Trp Leu Gly Val Thr Asp Phe
Ser Gly Ser Ser Cys Ser Ser 85 90 95Cys Arg Asp Gln Arg Gly Trp Pro
Ala Thr Lys Val Pro Arg Asn Gln 100 105 110Asp Trp Leu Gly Val Thr
Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys 115 120 125Arg Asp Gln Arg
Gly Trp Pro Ala Arg Glu Tyr His Gln Leu Arg Glu 130 135 140Ala Tyr
Arg Phe Leu Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr145 150 155
160Arg Leu Ala Ala Ser Arg Val Ser Ser Thr Ser Arg Ser Leu Pro Lys
165 170 175Ala Trp Asn Arg Gln Leu Tyr Pro Glu Trp Thr Glu Ala Gln
Arg Leu 180 185 190Asp Cys Trp Val Asp Lys Leu Gly Asp Leu Gly 195
20064216PRTArtificial SequenceC131B 64Met Arg Gly Ser His His His
His His His Thr Asp Pro Ser Thr Val1 5 10 15Pro Leu Arg Glu Ala Tyr
Arg Ile Ser Gln Ala Val His Ala Ala His 20 25 30Ala Glu Ile Asn Glu
Ala Gly Arg Leu Glu Ser Ile Ile Asn Phe Glu 35 40 45Lys Leu Thr Glu
Phe Leu Arg Gln Phe Met Gln Leu Met Arg Arg Ser 50 55 60Thr Arg Leu
Ala Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Phe65 70 75 80Leu
Arg Gln Phe Met Gln Leu Met Gln Arg Ser Thr Arg Pro Arg Glu 85 90
95Tyr His Gln Leu Arg Glu Ala Tyr Arg Ile Ser Gln Ala Val His Ala
100 105 110Ala His Ala Glu Ile Asn Glu Ala Gly Arg Ser Arg Val Ser
Ser Thr 115 120 125Ser Arg Ser Leu Pro Asn Phe Ser Gly Ser Ser Cys
Ser Ser Cys Arg 130 135 140Asp Gln Arg Gly Trp Pro Pro Arg Glu Tyr
His Gln Leu Arg Glu Ala145 150 155 160Tyr Arg Ile Ser Gln Ala Val
His Ala Ala His Ala Glu Ile Asn Glu 165 170 175Ala Gly Arg Leu Glu
Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Phe 180 185 190Leu Arg Gln
Phe Met Gln Leu Met Gln Arg Ser Thr Arg Leu Ala Ala 195 200 205Arg
Gly Gly Ser Gly Leu Ile Asn 210 2156512PRTArtificial SequenceWT1
MHC class I epitope 65Leu Glu Arg Met Phe Pro Asn Ala Pro Tyr Leu
Thr1 5 106684PRTArtificial SequenceTandem repeat structure 66Leu
Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser1 5 10
15Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu Ser Ile
20 25 30Ile Asn Phe Glu Lys Leu Thr Asp Phe Ser Gly Ser Ser Cys Ser
Ser 35 40 45Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu Ser Ile Ile Asn
Phe Glu 50 55 60Lys Leu Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser Cys
Arg Asp Gln65 70 75 80Arg Gly Trp Pro678PRTArtificial SequenceMHC
class I epitope of OVA 67Ser Ile Ile Asn Phe Glu Lys Leu1
56887PRTArtificial SequenceTandem repeat structure 68Leu Glu Arg
Met Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe Ser Gly1 5 10 15Ser Ser
Cys Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu Arg 20 25 30Met
Phe Pro Asn Ala Pro Tyr Leu Thr Asp Phe Ser Gly Ser Ser Cys 35 40
45Ser Ser Cys Arg Asp Gln Arg Gly Trp Pro Leu Glu Arg Met Phe Pro
50 55 60Asn Ala Pro Tyr Leu Thr Asp Phe Ser Gly Ser Ser Cys Ser Ser
Cys65 70 75 80Arg Asp Gln Arg Gly Trp Pro 856923PRTArtificial
Sequencetriple FLAG tag sequence 69Asp Tyr Lys Asp His Asp Gly Asp
Tyr Lys Asp His Asp Ile Asp Tyr1 5 10 15Lys Asp Asp Asp Asp Lys Leu
20707PRTArtificial Sequencepeptide sequence 70Asp Glu Asp Glu Asp
Glu Asp1 5719PRTHomo sapiens 71Cys Met Thr Trp Asn Gln Met Asn Leu1
5729PRTHomo sapiens 72Arg Met Phe Pro Asn Ala Pro Tyr Leu1
57322PRTHomo sapiens 73Pro Gly Cys Asn Lys Arg Tyr Phe Lys Leu Ser
His Leu Gln Met His1 5 10 15Ser Arg Lys His Thr Gly 207412PRTHomo
sapiens 74Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr Leu Pro1 5
10759PRTArtificial SequenceWT1-derived MHC class I epitope 75Cys
Tyr Thr Trp Asn Gln Met Asn Leu1 57616PRTArtificial
SequenceWT1-derived MHC class II epitope 76Lys Arg Tyr Phe Lys Leu
Ser His Leu Gln Met His Ser Arg Lys His1 5 10 157713PRTArtificial
SequencePan HLA-DR-binding epitope (PADRE) 77Ala Lys Phe Val Ala
Ala Trp Thr Leu Lys Ala Ala Ala1 5 10789PRTArtificial
Sequencegp100-derived MHC class I epitope 78Lys Val Pro Arg Asn Gln
Asp Trp Leu1 57916PRTArtificial Sequencegp100-derived MHC class II
epitope 79Trp Asn Arg Gln Leu Tyr Pro Glu Trp Thr Glu Ala Gln Arg
Leu Asp1 5 10 158012PRTArtificial Sequencepeptide sequence (SEQ ID
NO1 + E) shown in Fig. 2 80Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu
Thr Glu1 5 108116PRTArtificial Sequencepeptide sequence shown in
Fig. 2 81Phe Ser Gly Ser Ser Cys Ser Ser Cys Arg Asp Gln Arg Gly
Trp Pro1 5 10 158236DNAArtificial Sequencenucleotide sequence shown
in Fig. 2 82ctcgagagta tcatcaactt cgagaagctt accgag
368351DNAArtificial Sequencenucleotide sequence shown in Fig. 2
83atttctcagg cagttcatgc agctcatgca gagatcaacg aggctggccg c 51
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