U.S. patent application number 14/152597 was filed with the patent office on 2014-05-15 for recombinant vaccines and use thereof.
This patent application is currently assigned to BioNTech AG. The applicant listed for this patent is BioNTech AG. Invention is credited to Sebastian Kreiter, Ugur Sahin, Ozlem Tureci.
Application Number | 20140134201 14/152597 |
Document ID | / |
Family ID | 34441935 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134201 |
Kind Code |
A1 |
Tureci; Ozlem ; et
al. |
May 15, 2014 |
RECOMBINANT VACCINES AND USE THEREOF
Abstract
The present invention relates to fusion molecules of antigens,
the nucleic acids coding therefor and the use of such fusion
molecules and nucleic acids. In particular, said invention relates
to fusion molecules, comprising an antigen and the trans-membrane
region and cytoplasmic region of a MHC molecule and/or the
cytoplasmic region of a MHC or a SNARE molecule.
Inventors: |
Tureci; Ozlem; (Mainz,
DE) ; Sahin; Ugur; (Mainz, DE) ; Kreiter;
Sebastian; (Mainz, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioNTech AG |
Mainz |
|
DE |
|
|
Assignee: |
BioNTech AG
Mainz
DE
|
Family ID: |
34441935 |
Appl. No.: |
14/152597 |
Filed: |
January 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13471606 |
May 15, 2012 |
8637006 |
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14152597 |
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10575640 |
Feb 9, 2007 |
8178653 |
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PCT/EP2004/011512 |
Oct 13, 2004 |
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13471606 |
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Current U.S.
Class: |
424/185.1 ;
435/325; 530/350; 530/386 |
Current CPC
Class: |
C07K 14/4748 20130101;
C07K 2319/02 20130101; A61P 31/22 20180101; C07K 2319/03 20130101;
A61P 37/04 20180101; A61P 31/12 20180101; A61P 31/14 20180101; C07K
14/70539 20130101; A61K 48/00 20130101; A61P 31/20 20180101; A61K
47/64 20170801; A61P 35/00 20180101 |
Class at
Publication: |
424/185.1 ;
530/350; 530/386; 435/325 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 14, 2003 |
DE |
103 47 710.1 |
Claims
1. A fusion protein comprising an antigen, and a transmembrane
region and a cytoplasmic region of .alpha. chain of a MHC molecule
or CD1a, CD1b or CD1c but free from (i) .alpha.1, .alpha.2 and
.alpha.3 domains of the .alpha. chain of a MHC Class I molecule or
CD1a, CD1b or CD1c, (ii) .beta.2-microglobulin of a MHC Class I
molecule or CD1a, CD1b or CD1c, (iii) .alpha.1 and .alpha.2 domains
of the .alpha. chain of a MHC Class II molecule, and (iv) .beta.1
and .beta.2 domains of the .beta. chain of a MHC Class II
molecule.
2. An isolated host cell which comprises the fusion protein as
claimed in claim 1.
3. A composition which comprises the fusion protein of claim 1 in a
pharmaceutically acceptable carrier.
4. The fusion protein of claim 1, which includes an amino acid
residue sequence selected from the group consisting of SEQ ID NO:
2, 4, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
5. The fusion protein of claim 1, which is encoded by a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO: 11
or SEQ ID NO: 13.
6. The fusion protein of claim 1 which includes the amino acid
residue sequence of SEQ ID NO: 12.
7. The fusion protein of claim 1 which is encoded by a nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO: 11.
8. The fusion protein of claim 1 which is encoded by a nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO: 13.
9. The fusion protein of claim 1 comprising the amino acid residue
sequence of SEQ ID NO: 14.
10. A composition which comprises the isolated host cell of claim
2.
11. The fusion protein of claim 1 wherein the antigen is a tumor
antigen or a viral antigen.
12. The fusion protein of claim 11 wherein the tumor antigen is
selected from the group consisting of carcinoembryonic antigen,
.alpha.1-fetoprotein, isoferritin, fetal sulfoglycoprotein,
.alpha.2-H-ferroprotein, and .gamma.-fetoprotein and the viral
antigen is selected from the group consisting of a viral
ribonucleoprotein and a viral envelope protein.
13. The fusion protein of claim 1 wherein the transmembrane region
and the cytoplasmic region together comprise an amino acid residue
sequence selected from the group consisting of SEQ ID NO: 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41.
14. The fusion protein of claim 1 wherein the cytoplasmic region
comprises an amino acid residue sequence selected from the group
consisting of SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, and 42.
15. The fusion protein of claim 1 wherein the fusion protein has
the amino acid residue sequence of SEQ ID NO: 14.
16. The fusion protein of claim 1 wherein the antigen is selected
from the group consisting of ART-4, BAGE, ss catenin/m, Bcr-abL
CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, claudin-12, c-MYC, CT,
Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE,
HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT, hTRT, LAGE, LDLR/FUT,
MAGE-A, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-B,
MAGE-C, MART-1/melan-A, MC1R, myosin/m, MUC1, MUM-1, MUM-2, MUM-3,
NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor bcr-abL Pml/RARa, PRAME,
proteinase-3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3,
SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL/AML1, TPI/m, TRP-1,
TRP-2, TRP-2/INT2, TPTE, and WT.
17. The fusion protein of claim 1 comprising an amino acid residue
sequence selected from the group consisting of SEQ ID NO: 2, 4, 12,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
18. The fusion protein of claim 1, wherein the fusion protein
additionally comprises a leader sequence.
19. The fusion protein of claim 18, wherein the leader sequence is
derived from an MHC molecule.
20. The fusion protein of claim 18, wherein the fusion protein has
the following arrangement of segments: N terminus--leader
sequence/antigen/transmembrane region/cytoplasmic region--C
terminus, and the individual segments optionally are separated from
one another by linker sequences.
21. The fusion protein of claim 18, wherein the leader sequence
comprises the amino acid sequence of SEQ ID NO: 2.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/471,606, filed on May 15, 2012, which, in turn, is a
divisional of U.S. patent application Ser. No. 10/575,640, filed on
Feb. 9, 2007, now U.S. Pat. No. 8,178,653, which is the National
Stage of International Application No. PCT/EP2004/011512, filed on
Oct. 13, 2004, each of which is incorporated herein by reference in
its entirety.
[0002] This application includes biological sequence information,
which is set forth in an ASCII text file having the file name
"VOS-120-DIV1-SEQ.txt", created on Aug. 29, 2012, and having a file
size of 80,864 bytes, which is incorporated herein by
reference.
[0003] The present invention relates to fusion molecules of
antigens, to the nucleic acids coding therefor and to the use of
such fusion molecules and nucleic acids. The invention relates in
particular to fusion molecules which comprise an antigen and the
transmembrane region and cytoplasmic region of an MHC molecule or
the cytoplasmic region of an MHC molecule or of a SNARE
molecule.
[0004] Fusion molecules of the invention can be used for a large
number of applications, including in methods for inducing an immune
response in a mammal.
[0005] Antigen-specific T cell reactions are elicited by antigenic
peptides which are bound to the binding groove of glycoproteins of
the major histocompatibility complex (MHC), as part of the
mechanism of the immune system in which foreign antigens are
identified and a response to them is induced. The bound antigenic
peptides interact with T cell receptors and thus modulate an immune
response. The antigenic peptides are non-covalently bound to
certain "binding pockets" formed by polymorphic residues of the
binding groove of the MHC protein.
[0006] MHC class II molecules are heterodimeric glycoproteins
consisting of .alpha. and .beta. chains. The .alpha.1 and .beta.1
domains of these molecules fold together and form a peptide-binding
groove. Antigenic peptides bind to the MHC molecule through
interaction between anchor amino acids on the peptide and .alpha.1
and .beta.1 domains. The crystal structure of the human class II
HLA DR1 complex with an influenza virus peptide shows that the N
and C terminal ends of the bound peptide extend out of the binding
groove, so that the C terminus of the peptide lies near to the N
terminus of the .beta. chain [Brown, J. H. et al., 1993, Nature
364:33-39; Stern, L. J. et al., 1994, Nature 368:215-221]. MHC
class I molecules have different domain organizations than MHC
class II molecules but generally a similar structure with a
peptide-binding site or groove which is remote from the membrane
domains [cf. for example Rudensky, A. Y. et al., 1991, Nature
353:622-627].
[0007] The initial step in the presentation of a foreign protein
antigen is binding of the native antigen to an antigen-presenting
cell (APC). After binding to APCs, antigens penetrate into the
cells, either by phagocytosis, receptor-mediated endocytosis or
pinocytosis. Such internalized antigens are located in
intracellular membrane-bound vesicles called endosomes. Following
endosome-lysosome fusion, the antigens are processed to small
peptides by cellular proteases present in the lysosomes. The
peptides associate with the .alpha. and .beta. chains of MHC class
II molecules within these lysosomes. These MHC class II molecules,
which had previously been synthesized in the rough endoplasmic
reticulum, are transported sequentially to the Golgi complexes and
then to the lysosomal compartment. The peptide-MHC complex is
presented on the surface of APCs for T- and B-cell activation.
Therefore, the accessibility of proteolytic processing sites in the
antigen, the stability of the resulting peptides in the lysosomes
and the affinities of the peptides for MHC molecules are
determining factors for the immunogenicity of a specific
epitope.
[0008] Recombinant vaccines have particular importance in human and
veterinary medicine as agents and medicaments for the prophylaxis
and therapy of infectious diseases and cancers. The aim of
vaccination with a recombinant vaccine is to induce a specific
immune response to a defined antigen, which response has preventive
or therapeutic activity against defined diseases.
[0009] A factor which is essential for the efficacy of a
recombinant vaccine is optimal stimulation of T lymphocytes of the
immunized organism. Thus, a number of animal-experimental
investigations demonstrates that both optimal stimulation of
CD8.sup.+ and CD4.sup.+ lymphocytes is necessary for effective
immunotherapy of tumors. The known major types of recombinant
vaccines are based on recombinant proteins, synthetic peptide
fragments, recombinant viruses and nucleic acid vaccines based on
DNA or RNA. In recent years, vaccines based on DNA and RNA nucleic
acids have become increasingly important. However, only very poor
or even no stimulation of CD4.sup.+ lymphocytes can be achieved
with recombinant vaccines based on nucleic acids for very many
aims, inter alia tumour antigens. For this reason, a number of
genetic modifications has been developed with the intention of
increasing the immunogenicity of recombinant vaccines. Various
methods have been tested in this connection to date, inter alia
heterogenization of immunogens by altering the primary sequence or
by fusion to foreign epitopes, e.g. from bacteria or viruses
[Lowenadler, B. et al., 1990, Eur. J. Immunol. 20: 1541-45; Clarke,
B. E. et al., 1987, Nature 330: 381-84] and preparation of chimeric
products consisting of the actual antigen and immunomodulatory
proteins such as cytokines [Ruckert, R. et al., 1998, Eur. J.
Immunol. 28: 3312-20; Harvill, E. T., J. M. Fleming, and S. L.
Morrison, 1996, J. Immunol. 157: 3165-70]. Although vaccines based
on heterogenization induce enhanced immune responses, they have the
great disadvantage that immunostimulation against the foreign
epitope predominates and that immune responses against the actual
vaccine target remain only moderate in some cases.
[0010] A further attractive possibility is fusion to sequences of
proteins intended to permit translocation of the protein into
degrading cell compartments. However, it is now known that these
modifications lead to only a moderate improvement in stimulation of
CD4.sup.+ lymphocytes and to scarcely any enhancement of CD8.sup.+
immune responses [Wu, T. C. et al., 1995, Proc. Natl. Acad. Sci.
U.S.A. 92: 11671-11675; Bonini, C. et al., 2001, J. Immunol. 166:
5250-57, Su, Z. et al., 2002, Cancer Res. 62: 5041-5048].
[0011] It would thus be desirable for vaccines which distinctly
increase antigen presentation and thus immunogenicity in relation
to a particular antigen to be available. It would further be
desirable for it to be possible to modify vaccines systematically
in such a way that a maximum immune response by CD4.sup.+ and
CD8.sup.+ lymphocytes results, without the need to introduce
foreign epitopes.
[0012] This object is achieved according to the invention by the
subject matter of the claims.
[0013] It has been possible to establish according to the invention
that fusion molecules comprising antigen molecules and parts of
histocompatibility antigens show, when used as vaccines, an
immunogenicity which is increased >100-fold compared with the
unmodified antigens, and that surprisingly both immune responses of
CD4.sup.+ and CD8.sup.+ T lymphocytes are increased in a manner not
previously described.
[0014] The present invention relates in general to fusion molecules
of antigen molecules and to the use of such fusion molecules.
[0015] In one aspect, the invention relates to a fusion molecule
which comprises an antigen and the cytoplasmic region of a chain of
an MHC molecule, or an antigen, a transmembrane region and the
cytoplasmic region of a chain of an MHC molecule. It is preferred
for both the transmembrane region and the cytoplasmic region to be
derived from a MHC molecule. In addition, the fusion molecule
preferably comprises no MHC binding domain.
[0016] The invention further relates to a fusion molecule which
comprises an antigen and a chain of an MHC molecule or a part
thereof, where the part comprises at least the transmembrane region
and the cytoplasmic region of the chain of the MHC molecule. The
part of the chain of an MHC molecule preferably does not comprise
the MHC binding domain or parts thereof. There is thus provided in
particular a fusion molecule which comprises an antigen and a part
of a chain of an MHC molecule, which part corresponds essentially
to the sequence of the transmembrane region connected to the
cytoplasmic region of an MHC molecule, where the expression
"transmembrane region connected to the cytoplasmic region" relates
to the segment of a chain of an MHC molecule which starts with the
N-terminal end of the transmembrane region and terminates with the
C-terminal end of the cytoplasmic region, in particular the
C-terminal end of the complete chain of the MHC molecule. In this
embodiment, the connection of the transmembrane region to the
cytoplasmic region corresponds to the naturally occurring
connection between these regions.
[0017] The invention further provides a fusion molecule which
comprises an antigen and a chain of an MHC molecule or a part
thereof, where the part essentially lacks the complete N-terminal
extracellular domains of the MHC molecule.
[0018] In a particularly preferred embodiment, the fusion molecules
of the invention consist of a fusion of an antigen, where
appropriate with a leader sequence at its N-terminal end, to a
transmembrane region, preferably a transmembrane region of a chain
of an MHC molecule, at the C-terminal end of the antigen and of a
cytoplasmic region of a chain of an MHC molecule at the C-terminal
end of the transmembrane region.
[0019] In a particularly preferred embodiment, the fusion molecules
of the invention comprise a leader sequence, preferably a peptide
sequence having the properties of a secretion signal which is able
in particular to control translocation of a protein or peptide
through a membrane. It is possible to use as leader sequence the
secretion signal of any type I transmembrane protein, where the
expression "type I transmembrane protein" relates to those
transmembrane proteins whose C terminus is located in the
cytoplasm. In a particular embodiment, the leader sequence is
derived from a chain of an MHC molecule. The leader sequence is
preferably located at the N-terminal end of the fusion molecules of
the invention.
[0020] In a further aspect, the invention relates to a fusion
molecule where essentially the complete N-terminal extracellular
domains of an MHC molecule are replaced by an antigen having a
leader sequence at its N-terminal end.
[0021] It is preferred in a fusion molecule of the invention for
the antigen to be covalently connected at its N terminus to the C
terminus of a leader sequence, and the C terminus of the antigen
molecule is connected to the N terminus of the transmembrane region
which in turn is connected at the C terminus to the N terminus of
the cytoplasmic region of an MHC molecule.
[0022] Thus, the fusion molecule of the invention preferably has
the following arrangement: N terminus leader
sequence/antigen/transmembrane region/cytoplasmic region C
terminus.
[0023] In a particularly preferred embodiment, the fusion molecule
of the invention consists essentially of the leader sequence, the
antigen, the transmembrane region and the cytoplasmic region.
[0024] In a particularly preferred embodiment, the antigen is a
peptide, polypeptide or protein, and the fusion molecule of the
invention is a protein or polypeptide.
[0025] In one embodiment, a plurality of antigens which may be
identical or different are present in the fusion molecule of the
invention, i.e. at least 2, preferably 2 to 10, more preferably 2
to 5, even more preferably 2 to 3, in particular 2, antigens. These
multiply coupled antigens may be present separate from one another
or in series one after the other, where appropriate separated by a
linker, as tandem constructs. It is preferred for an immune
response to various antigens to be induced thereby on
administration.
[0026] The antigen may be complete or truncated, i.e. it contains
only a part of the natural protein or polypeptide which serves as
antigen.
[0027] The leader sequence and/or the transmembrane region of the
fusion molecules of the invention are preferably derived from MHC
molecules, in particular of class I or II. It is more preferred for
the leader sequence and/or the transmembrane region and/or the
cytoplasmic region of the fusion molecules of the invention to be
derived from MHC molecules, in particular of class I or II.
[0028] It is also possible according to the invention for one or
more, preferably flexible, linker sequences (connecting sequences)
to be present in the fusion molecule, possibly being located
between the leader sequence and the antigen, between the antigen
and the transmembrane region and/or between the transmembrane
region and the cytoplasmic region. It is preferred according to the
invention for a linker sequence to comprise about 7 to 20 amino
acids, more preferably about 8 to 16 amino acids, and in particular
about 8 to 12 amino acids.
[0029] The linker sequence in fusion molecules of the invention is
preferably flexible and thus does not hold the peptide connected
therewith in a single, unwanted conformation. The linker preferably
comprises in particular amino acids having small side chains, such
as glycine, alanine and serine, in order to make flexibility
possible. The linker sequence preferably comprises no proline
residue, which might inhibit the flexibility.
[0030] In a further embodiment, the leader sequence, the antigen,
the transmembrane region and/or the cytoplasmic region are
connected together directly without a linker.
[0031] The leader sequence preferably has the sequence shown in SEQ
ID NO: 2 or a sequence derived therefrom, or is encoded by the
sequence shown in SEQ ID NO: 1 or a sequence derived therefrom. The
transmembrane-cytoplasmic region preferably has the sequence shown
in SEQ ID NO: 4 or 6 or a sequence derived therefrom, or is encoded
by the sequence shown in SEQ ID NO: 3 or 5 or a sequence derived
therefrom.
[0032] In further preferred embodiments, the
transmembrane-cytoplasmic or the exclusively cytoplasmic region is
derived from sequence-related MHC molecules (inter alia HLA-A,
HLA-B, HLA-C, HLA-E, HLA-F, HLA-DRa, HLA-DRb, HLA-DQa, HLA-DQb,
HLA-DPa, HLA-DPb, CD1a, CD1b, CD1c). Preferred
transmembrane-cytoplasmic regions have a sequence selected from the
group consisting of the sequences depicted in SEQ ID NO: 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and sequences
derived therefrom. In further embodiments, the exclusively
cytoplasmic regions have a sequence selected from the group
consisting of the sequences depicted in SEQ ID NO: 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and sequences derived
therefrom. Further embodiments also provide for the use of varied
sequences, e.g. modified or orthologous sequences from different
organisms. Sequences particularly preferred in this connection are
those having at the C-terminal end a homology of more than 60% with
the sequences shown in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42.
[0033] In a particularly preferred embodiment, the fusion molecule
of the invention comprises the amino acid sequence shown in SEQ ID
NO: 12 or 14, or a sequence derived therefrom.
[0034] The invention further relates to a fusion molecule
comprising an antigen and a SNARE protein (in particular Cis-golgi
SNARE p28, VTI1b, membrin, pallidin, syntaxin-5, syntaxin-6,
syntaxin-7, syntaxin-8, syntaxin-10, syntaxin-10a, syntaxin-11,
syntaxin-12, syntaxin-17, VAMP-2, VAMP-3, VAMP-4, VAMP-7, VAMP8,
VTI1-a-beta, XP350893, LIPS (SEQ ID NO: 43-63)) or a sequence which
comprises one or more SNARE motifs. Targeted transport of the
antigen into a defined compartment (e.g. lysosomes and endosomes)
is possible by fusing an antigen to a SNARE protein or a SNARE
motif (preferably at the C terminus of the SNARE protein or motif).
A further possibility with such a targeted transport is for
immunogenic epitopes of the antigen to be generated and presented
in a compartment, as can be established experimentally.
[0035] SNARE proteins are membrane-associated proteins whose common
feature is the SNARE motif which comprises 60-70 amino acids. SNARE
proteins are functionally involved in the transport and fusion of
vesicles in the cell. Eukaryotic organisms have a large number of
different SNARE proteins which are associated with different
vesicle membranes in the cell (inter alia endosomal, lysosomal,
Golgi, plasma membranes). The cytoplasmic regions of the SNARE
proteins have a dual function. Firstly, they serve as trafficking
signals (address labels) which specify the destination of the
protein and of the associated membrane. Secondly, the domains may
contribute through hetero- and homoassociation (joining together)
to fusion of different vesicles (e.g. endosomes with
lysosomes).
[0036] It is also possible according to the invention for the
SNARE-antigen fusion molecules to comprise linker sequences between
the SNARE portion and the antigen portion. Also included in
relation to the antigen and the linker sequence of the
SNARE-antigen fusion molecules are all the embodiments described
above. A linker in relation to the SNARE-antigen fusion molecules
preferably comprises 80-120 amino acids. In a particular
embodiment, the linker comprises a transmembrane region. The
invention thus relates to fusion molecules which comprise a SNARE
protein or a SNARE motif fused to an antigen or a transmembrane
region and an antigen. Such fusion molecules are shown for example
in FIG. 7.
[0037] In a further aspect, the invention relates to nucleic acids
and derivatives thereof which code for the fusion molecules
described above and are preferably able to express these fusion
molecules. The term "nucleic acid" hereinafter also includes
derivatives thereof.
[0038] In a particularly preferred embodiment, the nucleic acid
which codes for a fusion molecule of the invention comprises the
nucleic acid sequence shown in SEQ ID NO: 11 or 13, or a sequence
derived therefrom.
[0039] The invention also relates to host cells which comprise a
nucleic acid of the invention.
[0040] The host cell may moreover comprise a nucleic acid which
codes for an HLA molecule. In one embodiment, the host cell
expresses the HLA molecule endogenously. In a further embodiment,
the host cell expresses the HLA molecule recombinantly. The host
cell is preferably non-proliferative. In a preferred embodiment,
the host cell is an antigen-presenting cell, in particular a
dendritic cell, a monocyte or a macrophage.
[0041] In a further aspect, the invention relates to a
pharmaceutical composition, in particular a vaccine, which
comprises one or more of the fusion molecules of the invention
and/or one or more of the nucleic acids coding therefor and/or one
or more of the host cells of the invention.
[0042] In a further aspect, the invention provides a method for
increasing the amount of MHC/peptide complexes in a cell, where the
method comprises the provision of a fusion molecule of the
invention or of a nucleic acid coding therefor for the cell. The
cell is preferably present in a living creature, and the method
comprises administering a fusion molecule of the invention or a
nucleic acid coding therefor to the living creature. In a preferred
embodiment, the cell is an antigen-presenting cell, in particular a
dendritic cell, a monocyte or a macrophage.
[0043] In a further aspect, the invention provides a method for
increasing the presentation of cell surface molecules on cells
which are able to present antigens (such as B cells and
macrophages, generally called "APC"). The antigen-presenting
activity of such cells is enhanced by providing a fusion molecule
of the invention or a nucleic acid coding therefor for the cells.
Such an enhancement of the antigen-presenting activity in turn
preferably enhances the primary activation of T cells, in
particular of CD4.sup.+ and CD8.sup.+ lymphocytes, which respond to
the antigen. The cell is preferably present in a living creature,
and the method comprises administering a fusion molecule of the
invention or a nucleic acid coding therefor to the living
creature.
[0044] In a further aspect, the invention provides a method for
inducing an immune response in a living creature, where the method
comprises the administration of a fusion molecule of the invention
and/or a nucleic acid coding therefor and/or a host cell of the
invention to the living creature.
[0045] In a further aspect, the invention provides a method for
stimulating or activating T cells, especially CD4.sup.+ and
CD8.sup.+ lymphocytes, in vitro or in a living creature, in
particular a patient, where the method comprises the provision for
the T cells or administration to the living creature of a fusion
molecule of the invention and/or a nucleic acid coding therefor
and/or a host cell of the invention. Such a stimulation or
activation is preferably expressed in an expansion, cytotoxic
reactivity and/or cytokine release by the T cells.
[0046] A further aspect provides a method for the treatment,
vaccination or immunization of a living creature, where the method
comprises the administration a fusion molecule of the invention
and/or a nucleic acid coding therefor and/or a host cell of the
invention to the living creature. In this connection, the antigens
employed in the fusion molecule of the invention or the nucleic
acid coding therefor are in particular those which are known to be
effective without the alteration according to the invention for the
intended treatment, vaccination or immunization.
[0047] The methods described above are particularly suitable for a
treatment or prophylaxis of infectious diseases caused for example
by bacteria or viruses. In particular embodiments, the antigen used
according to the invention is derived from an infectious agent such
as hepatitis A, B, C, HIV, mycobacteria, malaria pathogens, SARS
pathogens, herpesvirus, influenzavirus, poliovirus or from
bacterial pathogens such as chlamydia and mycobacteria. A
particularly beneficial application of the present invention is in
cancer immunotherapy or vaccination, where there is in particular
enhancement of activation of tumor antigen-reactive T cells, thus
improving the prospects for T-cell immunotherapy or vaccination
against tumor cells.
[0048] In specific embodiments, the antigen used according to the
invention is selected from the group consisting of the following
antigens: p53, preferably encoded by the sequence shown in SEQ ID
NO: 66, ART-4, BAGE, ss-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8,
CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M,
ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7,
HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably
MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7,
MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C,
MART-1/melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1,
NY-ESO-1, NY-BR-1, p190 minor bcr-abL Pml/RARa, PRAME,
proteinase-3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3,
SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1,
TRP-2, TRP-2/INT2, TPTE and WT, preferably WT-1, in particular
encoded by the sequence shown in SEQ ID NO: 65.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The terms "domain" or "region" relate to a particular part
of an amino acid sequence which can preferably be connected to a
specific function or structure. For example, the .alpha. and .beta.
polypeptides of an MHC class II molecule have two domains,
.alpha.1, .alpha.2, and .beta.1, .beta.2, respectively, a
transmembrane region and a cytoplasmic region. In a similar manner,
the .alpha. chain of MHC class I molecules has three domains,
.alpha.1, .alpha.2 and .alpha.3, a transmembrane region and a
cytoplasmic region.
[0050] In one embodiment, the complete domain or region is included
in a selection of the sequence of a particular domain or region for
deletion or incorporation into a fusion molecule of the invention.
In order to ensure this, the sequence of the relevant domain or
region can be extended in order to comprise parts of a linker or
even parts of the adjacent domain or region. The term "essentially"
in relation to a domain or region is to be understood in this
sense.
[0051] The term "transmembrane region" relates to the part of a
protein which essentially accounts for the portion present in a
cellular membrane and preferably serves to anchor the protein in
the membrane. A transmembrane region is preferably according to the
invention an amino acid sequence which spans the membrane once.
However, it is also possible in certain embodiments to use a
transmembrane region which spans the membrane more than once. The
transmembrane region will generally have 15-25 preferably
hydrophobic uncharged amino acids which assume for example an
.alpha.-helical conformation. The transmembrane region is
preferably derived from a protein selected from the group
consisting of MHC molecules, immunoglobulins, CD4, CD8, the CD3
.zeta. chain, the CD3 .gamma. chain, the CD3 .delta. chain and the
CD3 .epsilon. chain.
[0052] The transmembrane region typically consists in the case of
the .alpha. and .beta. chains of the MHC class II molecule of about
20 hydrophobic amino acids which are connected to the
carboxy-terminal end of the antigen. These residues allow the
protein to span the membrane. The transmembrane region terminates
with about 6-32 residues which comprise the cytoplasmic tail at the
carboxy-terminal end of each of these chains. It has been shown
that these transmembrane and cytoplasmic regions can be replaced by
sequences which signal a GPI binding, and that the chimeric
GPI-anchored class II molecules are membrane-bound (Wettstein, D.
A., J. J. Boniface, P. A. Reay, H. Schild and M. M. Davis, 1991, J.
Exp. Med. 174: 219-228). Such embodiments are encompassed by the
term "transmembrane region" according to the invention. GPI-bound
membrane anchor domains have been defined in a number of proteins,
including decay-accelerating factor (DAF), CD59 and human placental
alkaline phosphatase (HPAP) (Wettstein, D. A., J. J. et al., 1991,
J. Exp. Med. 174:219-228). For example, the 38 carboxy-terminal
amino acids of HPAP are sufficient for functioning as signal
sequence for GPI binding. If the DNA sequence coding for this
domain is connected to a secreted molecule, such as the soluble
part of the MHC class II .alpha. or .beta. chain, there is
formation of a membrane-bound chimeric molecule (Wettstein, D. A.
et al., 1991, J. Exp. Med. 174: 219-228), and a method of this type
can be employed to anchor fusion molecules of the invention to a
cell membrane.
[0053] The term "major histocompatibility complex" and the
abbreviation "MHC" relate to a complex of genes which occurs in all
vertebrates. The function of MHC proteins or molecules in signaling
between lymphocytes and antigen-presenting cells in normal immune
responses involves them binding peptides and presenting them for
possible recognition by T-cell receptors (TCR). MHC molecules bind
peptides in an intracellular processing compartment and present
these peptides on the surface of antigen-presenting cells to T
cells. The human MHC region, also referred to as HLA, is located on
chromosome 6 and comprises the class I region and the class II
region.
[0054] The term "MHC class I" or "class I" relates to the major
histocompatibility complex class I proteins or genes. Within the
human MHC class I region there are the HLA-A, HLA-B, HLA-C, HLA-E,
HLA-F, CD1a, CD1b and CD1c subregions.
[0055] The class I .alpha. chains are glycoproteins having a
molecular weight of about 44 kDa. The polypeptide chain has a
length of somewhat more than 350 amino acid residues. It can be
divided into three functional regions: an external, a transmembrane
and a cytoplasmic region. The external region has a length of 283
amino acid residues and is divided into three domains, .alpha.1,
.alpha.2 and .alpha.3. The domains and regions are usually encoded
by separate exons of the class I gene. The transmembrane region
spans the lipid bilayer of the plasma membrane. It consists of 23
usually hydrophobic amino acid residues which are arranged in an
.alpha. helix. The cytoplasmic region, i.e. the part which faces
the cytoplasm and which is connected to the transmembrane region,
typically has a length of 32 amino acid residues and is able to
interact with the elements of the cytoskeleton. The .alpha. chain
interacts with .beta.2-microglobulin and thus forms .alpha.-.beta.2
dimers on the cell surface.
[0056] The term "MHC class II" or "class II" relates to the major
histocompatibility complex class II proteins or genes. Within the
human MHC class II region there are the DP, DQ and DR subregions
for class II .alpha. chain genes and .beta. chain genes (i.e.
DP.alpha., DP.beta., DQ.alpha., DQ.beta., DR.alpha. and
DR.beta.).
[0057] Class II molecules are heterodimers each consisting of an
.alpha. chain and a .beta.chain. Both chains are glycoproteins
having a molecular weight of 31-34 kDa (a) or 26-29 kDA (.beta.).
The total length of the .alpha. chains varies from 229 to 233 amino
acid residues, and that of the .beta. chains from 225 to 238
residues. Both .alpha. and .beta. chains consist of an external
region, a connecting peptide, a transmembrane region and a
cytoplasmic tail. The external region consists of two domains,
.alpha.1 and .alpha.2 or .beta.1 and .beta.2. The connecting
peptide is respectively 13 and 9 residues long in .alpha. and
.beta. chains. It connects the two domains to the transmembrane
region which consists of 23 amino acid residues both in .alpha.
chains and in .beta. chains. The length of the cytoplasmic region,
i.e. the part which faces the cytoplasm and which is connected to
the transmembrane region, varies from 3 to 16 residues in .alpha.
chains and from 8 to 20 residues in .beta. chains.
[0058] The term "chain of an MHC molecule" relates according to the
invention to the .alpha. chain of an MHC class I molecule or to the
.alpha. and .beta. chains of an MHC class II molecule. The .alpha.
chains of an MHC class I molecule, from which the fusion molecules
of the invention can be derived, comprise the HLA-A, -B and -C
.alpha. chains. The .alpha. chains of an MHC class II molecule,
from which the fusion molecules of the invention may be derived,
comprise HLA-DR, -DP and -DQ .alpha. chains, in particular HLA-DR1,
HLA-DR2, HLA-DR4, HLA-DQ1, HLA-DQ2 and HLA-DQ8 .alpha. chains and,
in particular, .alpha. chains encoded by DRA*0101, DRA*0102,
DQA1*0301 or DQA1*0501 alleles. The .beta. chains of an MHC class
II molecule, from which the fusion molecules of the invention may
be derived, comprise HLA-DR, -DP and -DQ .beta. chains, in
particular HLA-DR1, HLA-DR2, HLA-DR4, HLA-DQ1, HLA-DQ2 and HLA-DQ8
.beta. chains and, in particular, .beta. chains encoded by DRB1*01,
DRB1*15, DRB1*16, DRB5*01, DQB1*03 and DQB1*02 alleles.
[0059] The term "MHC binding domain" relates to the "MHC class I
binding domain" and "MHC class II binding domain".
[0060] The term "MHC class I binding domain" relates to the region
of an MHC class I molecule or of an MHC class I chain which is
necessary for binding to an antigenic peptide. An MHC class I
binding domain is formed mainly by the .alpha.1 and .alpha.2
domains of the MHC class I .alpha. chain. Although the .alpha.3
domain of the .alpha. chain and .beta.2-microglobulin do not
represent essential parts of the binding domain, they are
presumably important for stabilizing the overall structure of the
MHC class I molecule and therefore the term "MHC class I binding
domain" preferably includes these regions. An MHC class I binding
domain can also be essentially defined as the extracellular domain
of an MHC class I molecule, distinguishing it from the
transmembrane and cytoplasmic regions.
[0061] The term "MHC class II binding domain" relates to the region
of an MHC class II molecule or of an MHC class II chain which is
necessary for binding to an antigenic peptide. An MHC class II
binding domain is mainly formed by the .alpha.1 and .beta.1 domains
of the MHC class II .alpha. and .beta. chains. The .alpha.2 and
.beta.2 domains of these proteins are, however, presumably also
important for stabilizing the overall structure of the MHC binding
groove, and therefore the term "MHC class II binding domain"
according to the invention preferably includes these regions. An
MHC class II binding domain can also be defined essentially as the
extracellular domain of an MHC class II molecule, distinguishing it
from the transmembrane and cytoplasmic domains.
[0062] The exact number of amino acids in the various MHC molecule
domains or regions varies depending on the mammalian species and
between gene classes within a species. When selecting the amino
acid sequence of a particular domain or region, maintenance of the
function of the domain or region is much more important than the
exact structural definition, which is based on the number of amino
acids. The skilled worker is also aware that the function can also
be maintained if rather less than the complete amino acid sequence
of the selected domain or region is used.
[0063] The term "antigen" relates to an agent against which an
immune response is to be generated. The term "antigen" includes in
particular proteins, peptides, polysaccharides, nucleic acids,
especially RNA and DNA, and nucleotides. The term "antigen" also
includes derivatized antigens as secondary substance which becomes
antigenic--and sensitizing--only through transformation (e.g.
intermediately in the molecule, by completion with body protein),
and conjugated antigens which, through artificial incorporation of
atomic groups (e.g. isocyanates, diazonium salts), display a new
constitutive specificity. In a preferred embodiment, the antigen is
a tumor antigen, i.e. a constituent of cancer cells which may be
derived from the cytoplasm, the cell surface and the cell nucleus,
in particular those antigens which are produced, preferably in
large quantity, intracellularly or as surface antigens on tumor
cells. Examples are carcinoembryonic antigen, .alpha.1-fetoprotein,
isoferritin and fetal sulfoglycoprotein, .alpha.2-H-ferroprotein
and .gamma.-fetoprotein and various viral tumor antigens. In a
further embodiment, the antigen is a viral antigen such as viral
ribonucleoproteins or envelope proteins. In particular, the antigen
or peptides thereof should be presented by MHC molecules and thus
be able to modulate, in particular, activate, cells of the immune
system, preferably CD4.sup.+ and CD8.sup.+ lymphocytes, in
particular by modulating the activity of a T-cell receptor, and
thus preferably induce T cell proliferation.
[0064] The term "MHC/peptide complex" relates to a non-covalent
complex of the binding domain of an MHC class I or MHC class II
molecule and of an MHC class I or MHC class II binding peptide.
[0065] The term "MHC binding peptide" or "binding peptide" relates
to a peptide which binds to an MHC class I and/or an MHC class II
molecule. In the case of class I MHC/peptide complexes, the binding
peptides typically have a length of 8-10 amino acids, although
longer or shorter peptides may be active. In the case of class II
MHC/peptide complexes, the binding peptides typically have a length
of 10-25 amino acids and in particular of 13-18 amino acids,
although longer and shorter peptides may be active.
[0066] Fusion molecules of the invention and the nucleic acids
coding therefor can generally be prepared by recombinant DNA
techniques such as preparation of plasmid DNA, cleavage of DNA with
restriction enzymes, ligation of DNA, transformation or
transfection of a host, cultivation of the host and isolation and
purification of the expressed fusion molecule. Such methods are
known and described for example in Sambrook et al., Molecular
Cloning (2nd edition, 1989).
[0067] DNA coding for the antigen can be obtained by isolating DNA
from natural sources or by known synthetic methods such as the
phosphate triester method; cf., for example, Oligonucleotide
Synthesis, IRL Press (M. J. Gait, editor, 1984). Synthetic
oligonucleotides can also be prepared with the aid of commercially
available automatic oligonucleotide synthesizers.
[0068] The proportions of MHC molecules in the fusion molecules of
the invention suitably correspond, in relation to the amino acid
sequence, to naturally occurring MHC molecules from humans, mice or
other rodents or other mammals or are derivatives thereof.
[0069] DNA sources coding for MHC proteins are known, such as human
lymphoblastoid cells. After isolation, the gene coding for the MHC
molecule, or an interesting part thereof, can be amplified by
polymerase chain reaction (PCR) or other known methods. Suitable
PCR primers for amplifying the gene for the MHC peptide can attach
restriction sites to the PCR product.
[0070] It is preferred according to the invention to prepare DNA
constructs which comprise nucleic acid sequences coding for the
leader sequence, the transmembrane region and the cytoplasmic
region, and which comprise a restriction cleavage site between the
leader sequence and the transmembrane region, so that essentially
any nucleotide sequence coding for an interesting antigen can be
incorporated into the construct.
[0071] In a preferred method for preparing fusion molecules of the
invention, DNA sequences are disposed in such a way that the
C-terminal end of the leader sequence is linked to the N-terminal
end of the antigen, the C-terminal end of the antigen is linked to
the N-terminal end of the transmembrane region, and the C-terminal
end of the transmembrane region is linked to the N-terminal end of
the cytoplasmic region. As discussed above, restriction cleavage
sites are preferably incorporated between the end of the leader
sequence and the start of the transmembrane region, so that
essentially any nucleic acid which codes for an interesting antigen
can be linked to the nucleic acid sequence for the transmembrane
region.
[0072] An expressed fusion molecule of the invention may be
isolated and purified in a manner known per se. Typically, the
culture medium will be centrifuged and the supernatant will then be
purified by affinity or immunoaffinity methods comprising the use
of monoclonal antibodies which bind to the expressed fusion
molecule. The fusion molecule may also comprise a sequence which
assists purification, e.g. a 6.times.His tag.
[0073] The ability of a fusion molecule of the invention to
modulate the activity of a T-cell receptor (including inactivation
of T-cell responses) can easily be determined by an in vitro assay.
Typically, T cells are provided for the assays by transformed
T-cell lines, such as T-cell hybridomas or T cells which are
isolated from a mammal such as a human or a rodent such as a mouse.
Suitable T-cell hybridomas are freely available or can be prepared
in a manner known per se. T cells can be isolated in a manner known
per se from a mammal; cf., for example, Shimonkevitz, R. et al.,
1983, J. Exp. Med. 158: 303.
[0074] A suitable assay for determining whether a fusion molecule
of the invention is able to modulate the activity of T cells takes
place as follows by steps 1-4 hereinafter. T cells suitably express
a marker which can be assayed and indicates the T-cell activation
or modulation of T-cell activity after activation. Thus, the mouse
T-cell hybridoma DO11.10, which expresses interleukin-2 (IL-2) on
activation, can be used. IL-2 concentrations can be measured in
order to determine whether a specific presenting peptide is able to
modulate the activity of this T-cell hybridoma. A suitable assay of
this type is carried out by the following steps:
1. T cells are obtained for example from an interesting T-cell
hybridoma or by isolation from a mammal. 2. The T cells are
cultivated under conditions which permit proliferation. 3. The
growing T cells are brought into contact with antigen-presenting
cells which in turn have been brought into contact with a fusion
molecule of the invention or with a nucleic acid coding therefor.
4. The T cells are assayed for a marker, e.g. IL-2 production is
measured.
[0075] The T cells used in the assays are incubated under
conditions suitable for proliferation. For example, a DO11.10
T-cell hybridoma is suitably incubated in complete medium (RPMI
1640, supplemented with 10% FBS, penicillin/streptomycin,
L-glutamine and 5.times.10.sup.-5 M 2-mercaptoethanol) at about
37.degree. C. with 5% CO.sub.2. Serial dilutions of the fusion
molecule of the invention can be assayed. T-cell activation signals
are provided by antigen-presenting cells which have been loaded
with the suitable antigenic peptide.
[0076] As an alternative to measuring an expressed protein such as
IL-2, it is possible to determine the modulation of T-cell
activation suitably by changes in the proliferation of
antigen-dependent T cells, as measured by known radiolabeling
methods. For example, a labeled (such as tritiated) nucleotide can
be introduced into an assay culture medium. The introduction of
such a labeled nucleotide into the DNA serves as measurand for
T-cell proliferation. This assay is unsuitable for T cells not
requiring antigen presentation for growth, such as T-cell
hybridomas. The assay is suitable for measuring the modulation of
T-cell activation by fusion molecules in the case of untransformed
T cells isolated from mammals.
[0077] The ability of a fusion molecule of the invention to induce
an immune response, including making it possible to vaccinate
against a target disease, can be determined simply by an in vivo
assay. For example, a fusion molecule of the invention or a nucleic
acid coding therefor can be administered to a mammal such as a
mouse, and blood samples be taken from the mammal at the time of
the first administration and several times at periodic intervals
thereafter (such as 1, 2, 5 and 8 weeks after administration of the
fusion molecule or of the nucleic acid coding therefor). Serum is
obtained from the blood samples and assayed for the appearance of
antibodies resulting from the immunization. Antibody concentrations
can be determined. In addition, T lymphocytes can be isolated from
the blood or from lymphatic organs and be functionally assayed for
reactivity to the antigen or epitopes derived from the antigen. All
the readout systems known to the skilled worker, inter alia
proliferation assay, cytokine secretion, cytotoxic activity,
tetramer analysis, can be used in this connection.
[0078] Methods of the invention for inducing an immune response,
including vaccination of a living creature against a target
disease, can be used in combination with known methods for inducing
an immune response. For example, a fusion molecule of the invention
or a nucleic acid coding therefor can be administered to a living
creature in an arrangement or combination with administration of a
vaccine composition in order to enhance or prolong the desired
effect of such a vaccine composition.
[0079] The term "derived" means according to the invention that a
particular entity, in particular a particular sequence, is present
in the object from which it is derived, in particular an organism
or molecule. In the case of nucleic acid and amino acid sequences,
especially particular sequence regions, "derived" additionally
means that the relevant nucleic acid or amino acid sequence is
derived, consistent with the definitions hereinafter, from a
nucleic acid or amino acid sequence which is present in the object.
Thus, the expression "sequence or region derived from an MHC
molecule" means that the sequence or region is present in an MHC
molecule or is derived, consistent with the definitions
hereinafter, from a sequence or region which is present in an MHC
molecule.
[0080] A nucleic acid is according to the invention preferably
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic
acids include according to the invention genomic DNA, cDNA, mRNA,
recombinantly prepared and chemically synthesized molecules. A
nucleic acid may according to the invention be in the form of a
molecule which is single stranded or double stranded and linear or
closed covalently to form a circle.
[0081] A sequence derived from a nucleic acid sequence or the
expression "sequence derived from a nucleic acid sequence" relates
according to the invention to homologous sequences and derivatives
of the former sequence.
[0082] Homologous nucleic acid sequences display according to the
invention at least 40%, in particular at least 50%, at least 60%,
at least 70%, at least 80%, at least 90% and preferably at least
95%, at least 98 or at least 99% identity of the nucleotides.
[0083] A nucleic acid is "homologous" to another nucleic acid in
particular when the two sequences of the complementary strands are
able to hybridize with one another and enter into a stable duplex,
the hybridization preferably taking place under conditions which
permit specific hybridization between polynucleotides (stringent
conditions). Stringent conditions are described for example in
Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,
editors, 2nd edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989 or Current Protocols in Molecular
Biology, F. M. Ausubel et al., editors, John Wiley & Sons,
Inc., New York, and relate for example to hybridization at
65.degree. C. in hybridization buffer (3.5.times.SSC, 0.02% Ficoll,
0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM
NaH.sub.2PO.sub.4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M
sodium chloride/0.15 M sodium citrate, pH 7. After the
hybridization, the membrane onto which the DNA has been transferred
is for example washed in 2.times.SSC at room temperature and then
in 0.1-0.5.times.SSC/0.1.times.SDS at temperatures of up to
68.degree. C.
[0084] "Derivative" of a nucleic acid means according to the
invention that single or multiple nucleotide substitutions,
deletions and/or additions are present in the nucleic acid. The
term "derivative" also includes in addition chemical derivatization
of a nucleic acid on a base, a sugar or phosphate of a nucleotide.
The term "derivative" also includes nucleic acids which comprise
non-naturally occurring nucleotides and nucleotide analogs.
[0085] The nucleic acids described by the invention are preferably
isolated. The term "isolated nucleic acid" means according to the
invention that the nucleic acid (i) has been amplified in vitro,
for example by polymerase chain reaction (PCR), (ii) has been
produced recombinantly by cloning, (iii) has been purified, for
example by cleavage and fractionation by gel electrophoresis, or
(iv) has been synthesized, for example by chemical synthesis. An
isolated nucleic acid is a nucleic acid which is available for
manipulation by recombinant DNA techniques.
[0086] Nucleic acids which code for fusion molecules can according
to the invention be alone or in combination with other nucleic
acids, especially heterologous nucleic acids. In preferred
embodiments, a nucleic acid is functionally connected to expression
control sequences or regulatory sequences which may be homologous
or heterologous in relation to the nucleic acid. A coding sequence
and a regulatory sequence are "functionally" connected together if
they are linked together covalently in such a way that expression
or transcription of the coding sequence is under the control or
under the influence of the regulatory sequence. If the coding
sequence is to be translated into a functional protein and where
there is a functional connection of a regulatory sequence to the
coding sequence, induction of the regulatory sequence leads to
transcription of the coding sequence without the occurrence of a
shift in reading frame in the coding sequence or of an inability of
the coding sequence to be translated into the desired protein or
peptide.
[0087] The term "expression control sequence" or "regulatory
sequence" includes according to the invention promoters, enhancers
and other control elements which control the expression of a gene.
In particular embodiments of the invention, the expression control
sequences can be regulated. The exact structure of regulatory
sequences may vary species-dependently or cell type-dependently,
but generally includes 5'-non-transcribed and 5'-non-translated
sequences which are involved in initiating transcription and
translation, respectively, such as TATA box, capping sequence, CAAT
sequence and the like. In particular, 5'-non-transcribed regulatory
sequences include a promoter region which includes a promoter
sequence for transcriptional control of the functionally connected
gene. Regulatory sequences may also include enhancer sequences or
activator sequences located upstream.
[0088] In a preferred embodiment, the nucleic acid is according to
the invention a vector, where appropriate having a promoter which
controls the expression of a nucleic acid, e.g. of a nucleic acid
which codes for a fusion molecule of the invention. In a preferred
embodiment, the promoter is a T7, T3 or SP6 promoter.
[0089] The term "vector" is used in this connection in its most
general meaning and includes any of the intermediate vehicles for a
nucleic acid which make it possible, for example, for the nucleic
acid to be introduced into prokaryotic and/or into eukaryotic cells
and, where appropriate, be integrated into a genome. Such vectors
are preferably replicated and/or expressed in the cell. An
intermediate vehicle may be adapted for example for use in
electroporation, in microprojectile bombardment, in liposomal
administration, in transfer with the aid of agrobacteria or in
insertion via DNA or RNA viruses. Vectors include plasmids,
phagemids, bacteriophages or viral genomes.
[0090] The nucleic acids which code for a fusion molecule of the
invention can be employed for transfection of host cells. Nucleic
acids mean in this connection both recombinant DNA and RNA.
Recombinant RNA can be prepared by in vitro transcription from a
DNA template. It can moreover be modified before application by
stabilizing sequences, capping and polyadenylation.
[0091] The term "host cell" relates according to the invention to
any cell which can be transformed or transfected with an exogenous
nucleic acid. The term "host cells" includes according to the
invention prokaryotic (e.g. E. coli) or eukaryotic (e.g. dendritic
cells, B cells, CHO cells, COS cells, K562 cells, yeast cells and
insect cells). Mammalian cells are particularly preferred, such as
cells from humans, mice, hamsters, pigs, goats and primates. The
cells may be derived from a large number of tissue types and
include primary cells and cell lines. Specific examples include
keratinocytes, peripheral blood leukocytes, bone marrow stem cells
and embryonic stem cells. In further embodiments, the host cell is
an antigen-presenting cell, in particular a dendritic cell, a
monocyte or macrophage. A nucleic acid may be present in the host
cell in a single or in multiple copies and is, in one embodiment,
expressed in the host cell.
[0092] The term "expression" is used according to the invention in
its most general meaning and includes the production of RNA or of
RNA and protein. It also includes partial expression of nucleic
acids. In addition, the expression may be transient or stable.
Preferred expression systems in mammalian cells include pcDNA3.1
and pRc/CMV (Invitrogen, Carlsbad, Calif.), which comprise a
selectable marker such as a gene which confers resistance to G418
(and thus makes selection of stably transfected cell lines
possible), and the enhancer-promoter sequences of cytomegalovirus
(CMV).
[0093] A nucleic acid coding for a fusion molecule of the invention
may also include a nucleic acid sequence which codes for an MHC
molecule, preferably for an HLA molecule. The nucleic acid sequence
which codes for an MHC molecule may be present on the same
expression vector as the nucleic acid which codes for the fusion
molecule, or the two nucleic acids may be present on different
expression vectors. In the latter case, the two expression vectors
can be cotransfected into a cell.
[0094] A sequence derived from an amino acid sequence or the
expression "sequence derived from an amino acid sequence" relates
according to the invention to homologous sequences and derivatives
of the former sequence.
[0095] Homologous amino acid sequences exhibit according to the
invention at least 40%, in particular at least 50%, at least 60%,
at least 70%, at least 80%, at least 90% and preferably at least
95%, at least 98 or at least 99% identity of the amino acid
residues.
[0096] "Derivatives" of a protein or polypeptide or of an amino
acid sequence in the sense of this invention include amino acid
insertion variants, amino acid deletion variants and/or amino acid
substitution variants.
[0097] Amino acid insertion variants include amino- and/or
carboxy-terminal fusions, and insertions of single or multiple
amino acids in a particular amino acid sequence. In amino acid
sequence variants with an insertion, one or more amino acid
residues are introduced into a predetermined site in an amino acid
sequence, although random insertion with suitable screening of the
resulting product is also possible. Amino acid deletion variants
are characterized by deletion of one or more amino acids from the
sequence. Amino acid substitution variants are distinguished by at
least one residue in the sequence being deleted and another residue
being inserted in its stead. The modifications are preferably
present at positions in the amino acid sequence which are not
conserved between homologous proteins or polypeptides. Amino acids
are preferably replaced by others having similar properties, such
as hydrophobicity, hydrophilicity, electronegativity, volume of the
side chain and the like (conservative substitution). Conservative
substitutions relate for example to replacement of one amino acid
by another, with both amino acids being listed in the same group
hereinafter:
1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser,
Thr (Pro, Gly) 2. negatively charged residues and their amides:
Asn, Asp, Glu, Gln 3. positively charged residues: His, Arg, Lys 4.
large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys) 5.
large aromatic residues: Phe, Tyr, Trp.
[0098] Three residues are put in parentheses because of their
particular role in protein architecture. Gly is the only residue
without a side chain and thus confers flexibility on the chain. Pro
has an unusual geometry which greatly restricts the chain. Cys can
form a disulfide bridge.
[0099] The amino acid variants described above can easily be
prepared with the aid of known peptide synthesis techniques such
as, for example, by solid phase synthesis (Merrifield, 1964) and
similar methods or by recombinant DNA manipulation. Techniques for
introducing substitution mutations at predetermined sites in DNA
which has a known or partially known sequence are well known and
include, for example, M13 mutagenesis. Manipulation of DNA
sequences to prepare proteins having substitutions, insertions or
deletions and the general recombinant methods for expression of
proteins for example in a biological system (such as mammalian,
insect, plant and viral systems) are described in detail for
example in Sambrook et al. (1989).
[0100] "Derivatives" of proteins or polypeptides also include
according to the invention single or multiple substitutions,
deletions and/or additions of any molecules which are associated
with the protein or polypeptide, such as carbohydrates, lipids
and/or proteins or polypeptides.
[0101] In one embodiment, "derivatives" of proteins or polypeptides
include those modified analogs resulting from glycosylation,
acetylation, phosphorylation, amidation, palmitoylation,
myristoylation, isoprenylation, lipidation, alkylation,
derivatization, introduction of protective/blocking groups,
proteolytic cleavage or binding to an antibody or to another
cellular ligand. Derivatives of proteins or polypeptides may also
be prepared by other methods such as, for example, by chemical
cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.2, acetylation, formylation, oxidation,
reduction or by metabolic synthesis in the presence of
tunicamycin.
[0102] The term "derivative" also extends to all functional
chemical equivalents of proteins or polypeptides.
[0103] The derivatives, described above, of proteins and
polypeptides are encompassed according to the invention by the term
"fusion molecule", even if no express reference is made
thereto.
[0104] The pharmaceutical compositions described according to the
invention can be employed therapeutically for the treatment of a
pre-existing disease or prophylactically as vaccines for
immunization.
[0105] The term "vaccine" relates according to the invention to an
antigenic preparation which comprises for example a protein, a
peptide, a nucleic acid or a polysaccharide, and which is
administered to a recipient in order to stimulate its humoral
and/or cellular immune system against one or more antigens which
are present in the vaccine preparation. The terms "vaccination" or
"immunization" relate to the process of administering a vaccine and
of stimulating an immune response against an antigen. The term
"immune response" relates to the activities of the immune system,
including activation and proliferation of specific cytotoxic T
cells after contact with an antigen.
[0106] Animal models can be employed for testing an immunizing
effect, e.g. against cancer on use of a tumor-associated antigen as
antigen. It is moreover possible for example, for human cancer
cells to be introduced into a mouse to create a tumor, and for a
nucleic acid of the invention, which codes for a fusion molecule of
the invention comprising the tumor-associated antigen, to be
administered. The effect on the cancer cells (for example reduction
in tumor size) can be measured as criterion for the efficacy of an
immunization by the nucleic acid.
[0107] As part of the composition for immunization, one or more
fusion molecules are administered with one or more adjuvants to
induce an immune response or increase an immune response. An
adjuvant is a substance which is incorporated into an antigen or is
administered together therewith and enhances the immune response.
Adjuvants are able to enhance the immune response by providing an
antigen reservoir (extracellularly or in macrophages), activating
macrophages and stimulating certain lymphocytes. Adjuvants are
known and include in a nonrestrictive manner monophosphoryl-lipid A
(MPL, SmithKline Beecham), saponins such as QS21 (SmithKline
Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18
and QS-L1 (So et al., Mol. Cells 7:178-186, 1997), incomplete
Freund's adjuvant, complete Freund's adjuvant, vitamin E,
Montanide, alum, CpG oligonucleotides (cf. Krieg et al., Nature
374:546-9, 1995) and various water-in-oil emulsions which are
prepared from biodegradable oils such as squalene and/or
tocopherol. The fusion molecules are preferably administered in a
mixture with DQS21/MPL. The ratio of DQS21 to MPL is typically
about 1:10 to 10:1, preferably about 1:5 to 5:1 and in particular
about 1:1. In a vaccine formulation for administration to humans,
DQS21 and MPL are typically present in a range from about 1 .mu.g
to about 100 .mu.g.
[0108] Other substances which stimulate an immune response in the
patient may also be administered. For example, cytokines can be
used for a vaccination because of their regulatory properties on
lymphocytes. Such cytokines include for example interleukin-12
(IL-12) which has been shown to enhance the protective effects of
vaccines (cf. Science 268:1432-1434, 1995), GM-CSF and IL-18.
[0109] The method of the invention for inducing an immune response
in a mammal generally comprises the administration of an effective
amount of a fusion molecule of the invention and/or of a nucleic
acid coding therefor, in particular in the form of a vector. DNA or
RNA which codes for a fusion molecule of the invention is
preferably administered to a mammal together with a DNA sequence
which codes for a T cell-costimulating factor, such as a gene
coding for B7-1 or B7-2.
[0110] The expression "T cell-costimulating factor" relates herein
to a molecule, in particular a peptide, which is able to provide a
costimulating signal and thus enhances an immune response, in
particular activates the proliferation of T cells in the presence
of one or more fusion molecules of the invention. Such an
activation of T-cell proliferation can be determined by generally
known assays.
[0111] These factors include costimulating molecules which are
provided in the form of proteins or nucleic acids. Examples of such
costimulating molecules are B7-1 and B7-2 (CD80 and CD86,
respectively) which are expressed on dendritic cells (DC) and
interact with the CD28 molecule expressed on T cells. This
interaction provides a costimulation (signal 2) for an
antigen/MHC/TCR-stimulated (signal 1) T cell, thus enhancing the
proliferation of the T cell and the effector function. B7 also
interacts with CTLA4 (CD152) on T cells and investigations
including CTLA4 ligands and B7 ligands show that the B7-CTLA4
interaction can enhance an antitumor immunity and CTL proliferation
(Zheng, P. et al., Proc. Natl. Acad. Sci. USA 95(11):6284-6289
(1998)).
[0112] B7 is typically not expressed on tumor cells, so that they
are not effective antigen-presenting cells (APCs) for T cells.
Induction of B7 expression would make it possible for tumor cells
more effectively to stimulate proliferation of cytotoxic T
lymphocytes and an effector function. Costimulation by a
B7/IL-6/IL-12 combination showed an induction of the IFN-gamma and
Th1 cytokine profile in a T cell population, leading to a further
enhancement of T-cell activity (Gajewski et al., J. Immunol.
154:5637-5648 (1995)).
[0113] Complete activation of cytotoxic T lymphocytes and a
complete effector function requires cooperation of T-helper cells
through the interaction between the CD40 ligand on the T-helper
cells and the CD40 molecule which is expressed by dendritic cells
(Ridge et al., Nature 393:474 (1998), Bennett et al., Nature
393:478 (1998), Schonberger et al., Nature 393:480 (1998)). The
mechanism of this costimulating signal probably relates to
increasing the B7 and associated IL-6/IL-12 production by the
dendritic cells (antigen-presenting cells). The CD40-CD40L
interaction thus complements the interactions of signal 1
(antigen/MHC-TCR) and signal 2 (B7-CD28).
[0114] The invention provides for administration of nucleic acids,
polypeptides or proteins and/or cells. Administration of DNA and
RNA is preferred.
[0115] It was possible to show in the experiments that, compared
with the unmodified antigen, according to the invention a 100-fold
lower dose of the vaccine is sufficient to induce equivalent or
stronger immune responses. One problem on direct injection of
nucleic acid vaccines is that the dose necessary to induce immune
responses is very high. In the case of DNA vaccines, the reason is
presumably mainly based on the fact that only a fraction of the
cells take up injected DNA into the nucleus. In the case of RNA
vaccines, the problem is presumably that in particular injected RNA
is very rapidly degraded by RNAses.
[0116] It is to be expected on use of the vaccines modified
according to the invention that greatly increased immune responses
will be obtained on direct injection of nucleic acids, in
particular RNA, compared with unmodified nucleic acids.
[0117] In a preferred embodiment, a viral vector for administering
a nucleic acid which codes for a fusion molecule of the invention
is selected from the group consisting of adenoviruses,
adeno-associated viruses, poxviruses, including vacciniavirus and
attenuated poxviruses, Semliki forest virus, retroviruses, Sindbis
virus and Ty virus-like particles. Adenoviruses and retroviruses
are particularly preferred. The retroviruses are normally
replication-deficient (i.e. they are unable to produce infectious
particles).
[0118] Various methods can be employed according to the invention
to introduce nucleic acids into cells in vitro or in vivo. Such
methods include transfection of nucleic acid-calcium phosphate
precipitates, transfection of nucleic acids associated with DEAE,
transfection or infection with the above viruses carrying the
nucleic acids of interest, liposome-mediated transfection and the
like. In particular embodiments, guiding of the nucleic acid to
particular cells is preferred. In such embodiments, a carrier
employed for administering a nucleic acid to a cell (e.g. a
retrovirus or a liposome) may have a bound targeting molecule. For
example, a molecule such as an antibody which is specific for a
surface membrane protein on the target cell, or a ligand for a
receptor on the target cell, can be incorporated into the nucleic
acid carrier or bound thereto. If administration of a nucleic acid
by liposomes is desired, it is possible to incorporate proteins
which bind to a surface membrane protein which is associated with
endocytosis into the liposome formulation in order to make
targeting and/or uptake possible. Such proteins include capsid
proteins or fragments thereof, which are specific for a particular
cell type, antibodies against proteins which are internalized,
proteins which target for an intracellular site, and the like.
[0119] The nucleic acids are preferably administered together with
stabilizing substances such as RNA-stabilizing substances.
[0120] In one embodiment, the nucleic acids are administered by ex
vivo methods, i.e. by removing cells from a patient, genetically
modifying the cells, and reintroducing the modified cells into the
patient. This generally includes the introduction of a functional
copy of a gene into the cells of a patient in vitro and returning
the genetically modified cells to the patient. The functional copy
of the gene is under the functional control of regulatory elements
which permit expression of the gene in the genetically modified
cells. Transfection and transduction methods are known to the
skilled worker. The invention also provides for administration of
nucleic acids in vivo through the use of vectors such as viruses
and targeted liposomes.
[0121] Administration of polypeptides and peptides can take place
in a manner known per se.
[0122] The term "patient", "individual" or "living creature" means
according to the invention a human, non-human primate or another
animal, in particular mammal such as cow, horse, pig, sheep, goat,
dog, cat, birds such as chicken or rodent such as mouse and rat. In
a particularly preferred embodiment, the patient, the individual or
the living creature is a human.
[0123] The therapeutic compositions of the invention can be
administered in pharmaceutically acceptable preparations. Such
preparations can comprise usually pharmaceutically acceptable
concentrations of salts, buffering substances, preservatives,
carriers, supplementary immunity-increasing substances such as
adjuvants (e.g. CpG oligonucleotides) and cytokines and, where
appropriate, other therapeutic agents.
[0124] The therapeutic agents of the invention can be administered
in any conventional way, including by injection or by infusion. The
administration can take place, for example, orally, intravenously,
intraperitoneally, intramuscularly, subcutaneously,
intracutaneously, transdermally, intralymphatically, preferably by
injection into lymph nodes, especially inguinal lymph nodes,
lymphatic vessels and/or into the spleen.
[0125] The compositions of the invention are administered in
effective amounts. An "effective amount" relates to the amount
which, alone or together with further doses, achieves a desired
response or a desired effect. In the case of treatment of a
particular disease or of a particular condition, the desired
response relates to inhibition of the progress of the disease. This
includes slowing down the progression of the disease and in
particular stopping the progression of the disease. The desired
response on treatment of a disease or of a condition may also be
delaying the onset or preventing the onset of the disease or of the
condition.
[0126] An effective amount of a composition of the invention
depends on the condition to be treated, the severity of the
disease, the individual patient's parameters, including age,
physiological condition, height and weight, the duration of the
treatment, the nature of a concomitant therapy (if present), the
specific administration route and similar factors.
[0127] The pharmaceutical compositions of the invention are
preferably sterile and comprise an effective amount of the
therapeutically active substance to generate the desired response
or the desired effect.
[0128] The doses of the compositions of the invention which are
administered may depend on various parameters such as the mode of
administration, the patient's condition, the desired administration
period etc. In the case where a patient's response is inadequate
with an initial dose, it is possible to employ higher doses (or
effectively higher doses which are achieved by a different, more
localized administration route).
[0129] In general, doses of from 1 ng to 1 mg, preferably from 10
ng to 100 .mu.g, of the tumor-associated antigen are formulated and
administered for a treatment or for generating or enhancing an
immune response. If it is desired to administer nucleic acids (DNA
and RNA), doses of from 1 ng to 0.1 mg are formulated and
administered.
[0130] The pharmaceutical compositions of the invention are
generally administered in pharmaceutically acceptable amounts and
in pharmaceutically acceptable compositions. The term
"pharmaceutically acceptable" relates to a non-toxic material which
does not interact with the effect of the active ingredient of the
pharmaceutical composition. Such preparations may usually comprise
salts, buffering substances, preservatives, carriers and, where
appropriate, other therapeutic agents. When used in medicine, the
salts should be pharmaceutically acceptable. Non-pharmaceutically
acceptable salts can, however, be used to prepare pharmaceutically
acceptable salts thereof and are encompassed by the invention. Such
pharmacologically and pharmaceutically acceptable salts include in
a non-limiting manner those prepared from the following acids:
hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic,
acetic, salicylic, citric, formic, malonic, succinic acids and the
like. Pharmaceutically acceptable salts can also be prepared as
alkali metal or alkaline earth metal salts such as sodium,
potassium or calcium salts.
[0131] A pharmaceutical composition of the invention may comprise a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" relates according to the invention to one or
more compatible solid or liquid fillers, diluents or capsule
substances which are suitable for administration to a human. The
term "carrier" relates to an organic or inorganic ingredient,
natural or synthetic in nature, in which the active ingredient is
combined in order to facilitate use. The ingredients of the
pharmaceutical composition of the invention are usually such that
no interaction which substantially impairs the desired
pharmaceutical activity occurs.
[0132] The carriers are preferably sterile liquids such as water or
oils, including those derived from petroleum, animals or plants, or
being of synthetic origin, such as, for example, peanut oil,
soybean oil, mineral oil, sesame oil, sunflower oil and the like.
Saline solutions and aqueous dextrose and glycerol solutions can
also be used as aqueous carriers.
[0133] Examples of excipients and carriers are acrylic and
methacrylic derivatives, alginic acid, sorbic acid derivatives such
as .alpha.-octadecyl-.omega.-hydroxypoly(oxyethylene)-5-sorbic
acid, amino acids and their derivatives, especially amine compounds
such as choline, lecithin and phosphatidylcholine, gum arabic,
aromas, ascorbic acid, carbonates such as, for example, sodium,
potassium, magnesium and calcium carbonates and bicarbonates,
hydrogen phosphates and phosphates of sodium, potassium, calcium
and magnesium, carmellose sodium, dimethicone, colors, flavorings,
buffering substances, preservatives, thickeners, plasticizers,
gelatin, glucose syrups, malt, colloidal silicon dioxide,
hydromellose, benzoates, especially sodium and potassium benzoates,
macrogol, skim milk powder, magnesium oxide, fatty acids and their
derivatives and salts such as stearic acid and stearates,
especially magnesium and calcium stearates, fatty acid esters and
mono- and diglycerides of edible fatty acids, natural and synthetic
waxes such as beeswax, yellow wax and montan glycol wax, chlorides,
especially sodium chloride, polyvidone, polyethylene glycols,
polyvinylpyrrolidone, povidone, oils such as castor oil, soybean
oil, coconut oil, palm kernel oil, sugars and sugar derivatives,
especially mono- and disaccharides such as glucose, fructose,
mannose, galactose, lactose, maltose, xylose, sucrose, dextrose and
cellulose and their derivatives, shellac, starch and starch
derivatives, especially corn starch, tallow, talc, titanium
dioxide, tartaric acid, sugar alcohols such as glycerol, mannitol,
sorbitol and xylitol and their derivatives, glycol, ethanol and
mixtures thereof.
[0134] The pharmaceutical compositions may preferably also comprise
in addition wetting agents, emulsifiers and/or pH-buffering
agents.
[0135] In a further embodiment, the pharmaceutical compositions may
comprise an absorption enhancer. These absorption enhancers may if
desired replace an equimolar amount of the carrier in the
composition. Examples of such absorption enhancers include in a
non-limiting manner eucalyptol, N,N-diethyl-m-toluamide,
polyoxyalkylene alcohols (such as propylene glycol and polyethylene
glycol), N-methyl-2-pyrrolidone, isopropyl myristate,
dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
dimethylacetamide (DMA), urea, diethanolamine, triethanolamine and
the like (see, for example, Percutaneous Penetration Enhancers,
edited by Smith et al. (CRC Press, 1995)). The amount of absorption
enhancer in the composition may depend on the desired effects to be
achieved.
[0136] A protease inhibitor can be incorporated into the
composition of the invention in order to prevent degradation of a
peptide or protein agent and thus to increase the bioavailability.
Examples of protease inhibitors include in a non-limiting manner
aprotinin, leupepsin, pepstatin, .alpha.2-macroglobulin and trypsin
inhibitor. These inhibitors can be used alone or in
combination.
[0137] The pharmaceutical compositions of the invention can be
provided with one or more coatings. The solid oral dosage forms are
preferably provided with a coating resistant to gastric juice or
are in the form of a hardened soft gelatin capsule resistant to
gastric juice.
[0138] The pharmaceutical compositions of the invention may
comprise suitable buffering substances such as acetic acid in a
salt, citric acid in a salt, boric acid in a salt and phosphoric
acid in a salt.
[0139] The pharmaceutical compositions may also comprise where
appropriate suitable preservatives such as benzalkonium chloride,
chlorobutanol, parabens and thimerosal.
[0140] The pharmaceutical compositions are usually presented in a
unit dose form and can be produced in a manner known per se.
Pharmaceutical compositions of the invention may be for example in
the form of capsules, tablets, lozenges, solutions, suspensions,
syrups, elixirs or as emulsion.
[0141] Compositions suitable for parenteral administration usually
comprise a sterile aqueous or nonaqueous preparation of the active
agent, which is preferably isotonic with the recipient's blood.
Examples of suitable carriers and solvents are Ringer's solution
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are usually employed as dissolving or suspending medium.
[0142] The present invention is described in detail by the
following examples and figures which serve exclusively for
illustration and are not to be understood as limiting. Further
embodiments which do not go beyond the bounds of the invention and
the scope of the annexed claims are accessible to the skilled
worker on the basis of the description and the examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0143] FIG. 1: Diagrammatic representation of a fusion protein of
the invention. The fusion protein consists of an N-terminally
placed secretion signal, of a C-terminally located transmembrane
and cytoplasmic domain of a histocompatibility antigen, and of an
integrated complete or partial sequence of an antigen.
[0144] FIG. 2: Diagrammatic representation of the cassettes for
expression of fusion proteins. SP: signal peptide; MCS: multiple
cloning site; TM: transmembrane domain; MHC tail: cytoplasmic tail
of an MHC molecule; antigen: sequence coding for an antigen against
which immune responses are to be induced.
[0145] FIG. 3: Testing of the effect of various RNA doses on the
frequency of antigen-specific CD4+ T lymphocytes.
[0146] 1.times.10.sup.6 purified CD4+ lymphocytes were cocultivated
for 1 week with 2.times.10.sup.5 DC which had been transfected with
RNA in the stated amounts (0.1-10 .mu.g RNA) by electroporation. On
day 7 after stimulation, an ELISPOT was carried out under standard
conditions to detect interferon-.gamma.-secreting T lymphocytes.
The antigen-presenting cells used were DC from the same donor which
had been loaded with overlapping pp65 peptides (1.75 .mu.g/ml) or
an irrelevant control peptide. For the test, 3.times.10.sup.4
effectors were coincubated with 2.times.10.sup.4 DC for 16 h. After
standard development, the number of IFN-gamma-secreting T
lymphocytes was determined by means of a software-based video
analysis. Compared with the CMVpp65 standard RNA, there is seen to
be a massive expansion of CD4+ lymphocytes both by the CMVpp65-TM1
construct and by the CMVpp65-TM2 construct.
[0147] FIG. 4: Testing of the effect of various RNA doses on the
frequency of interferon-gamma-secreting CD8+ T lymphocytes.
1.times.10.sup.6 purified CD8+ lymphocytes were cocultivated for 1
week with 2.times.10.sup.5 DC which had been transfected with RNA
in the stated amounts (0.1-10 .mu.g RNA) by electroporation. On day
7, a standard ELISPOT was carried out to detect IFN-gamma-secreting
T lymphocytes against DC of the same donor which had been loaded
with overlapping pp65 peptides (1.75 .mu.g/ml) or an irrelevant
control peptide. 3.times.10.sup.4 effectors were coincubated with
2.times.10.sup.4 DC for 16 h. After standard development, the
number of IFN-gamma-secreting T lymphocytes was determined by means
of a software-based video analysis. There was seen to be a massive
expansion of CD8+ lymphocytes by the CMVpp65-TM1 construct and the
CMVpp65-TM2 construct. Even on use of 100.times. lower doses (0.1
.mu.g RNA), the frequency of the pp65-specific CD8+ lymphocytes was
still above the background after stimulation by DC transfected with
NYESO-RNA (data not shown). Stimulation by the CMVpp65standard
construct showed an expansion of pp65-specific lymphocytes above
the background level only with 2.5 .mu.g and above.
[0148] FIG. 5: Dose/effect profile for the expansion capacity of
various immunogens on antigen-specific lymphocytes. The immunogens
modified according to the invention exhibit a distinctly increased
potency (>100.times.) and a higher maximum effect.
[0149] FIG. 6: Comparative test of the effect of immunogens
modified according to the invention and standard immunogens on the
generation of cytotoxic immune responses. 1.times.10.sup.6 purified
CD8+ lymphocytes were cocultivated for 1 week with 2.times.10.sup.5
DC which had been transfected with 10 .mu.g of RNA by
electroporation. On day 7, a standard cytochrome cytotoxicity assay
against DC of the same donor which had been loaded with various
concentrations of overlapping pp65 peptides or an irrelevant
control peptide was carried out. 15.times.10.sup.4 effectors were
coincubated with 0.5.times.10.sup.4 DC for 4 h. After measurement
of the supernatant in a counter, the specific lysis was calculated
according to the formula: There was seen to be extensive lysis by
CD8+ lymphocytes which had been stimulated with CMVpp65-TM1 and
CMVpp65-TM2 constructs, which was above the value for the control
peptide as far as a concentration of 10 nM of the pp65 peptide
mixture (data not shown). CD8+ lymphocytes were likewise expanded
by the pp65 peptide mixture and showed a marked specific lysis, but
did not reach the level of CMVpp65-TM1 and -TM2. Only a weak
stimulation of pp65-specific cytotoxic T cells was achievable by
the CMVpp65standard construct.
[0150] FIG. 7: Diagrammatic representation of the cassettes for
expressing fusion proteins. CS: cloning site; TM: transmembrane
domain; SNARE: SNARE protein or motif; antigen: sequence coding for
an antigen against which immune responses are to be induced.
[0151] FIGS. 8A-8E: Sequences used in the examples HLA class I
TM-CM: transmembrane-cytoplasmic region of an HLA class I molecule;
HLA class II TM-CM: transmembrane-cytoplasmic region of an HLA
class II molecule.
[0152] FIG. 9: Sequences of transmembrane-cytoplasmic regions and
cytoplasmic regions of MHC molecules. The sequences show the
transmembrane-cytoplasmic region or only the cytoplasmic region of
various HLA molecules. The transmembrane region is underlined and
bold.
[0153] FIG. 10A-10C: Sequences of SNARE proteins. These sequences
are suitable for constructing the SNARE-antigen fusion molecules
(N-SNARE-antigen) of the invention.
[0154] FIG. 11: Stimulation of naive CD8+ T lymphocytes by fusion
constructs of the invention. In microtiter plates, 1.times.10.sup.5
CD8+ lymphocytes per well were stimulated against 2.times.10.sup.4
DC which were transfected with 20 .mu.g of CMVpp65-TM1 or control
RNA. The medium was supplemented with IL-6 (1000 U/ml) and IL-12
(10 ng/ml). On day +7 and +14, thawed transfected DC
(2.times.10.sup.4/well) were used for restimulation, the medium
containing IL-2 (10 U/ml) and IL-7 (5 ng/ml). On day +21, all the
populations were assayed in an ELISPOT against control peptides
(1.75 .mu.g/ml) and against pp65-overlapping peptides (1.75
.mu.g/ml). Two of the populations stimulated against CMVpp65-TM1
(Pop.1, Pop.2) showed a marked pp65 reactivity.
EXAMPLES
Example 1
Preparation of the Modified Vaccines
[0155] To prepare the modified vaccines, firstly a cassette which
permits expression of fusion genes was prepared in an expression
vector which permits transcription of RNA. For this purpose,
initially the nucleic acid which codes for a signal peptide of an
HLA molecule was amplified from human lymphocytes, and the fragment
was cloned as cDNA into a vector (SEQ ID NO: 1 and 2). The cloning
was carried out in such a way that various restriction enzyme
cleavage sites were located behind the cDNA of the signal peptide,
and further fragments can be cloned in-frame in the expression
cassette. The selected vectors were plasmids which permit in vitro
expression of RNA via a 5'-located RNA polymerase promoter T3, T7
or SP6. The next fragment cloned into this vector was a cDNA which
encodes a transmembrane domain and the cytoplasmic domain of an HLA
class I (SEQ ID NO: 3 and 4) or class II (SEQ ID NO: 5 and 6)
molecule, including stop codon. The cloning was carried out in such
a way that the resulting plasmid still has restriction enzyme
cleavage sites for cloning antigens between the two fragments (SEQ
ID NO: 7 and 8 and FIG. 1). The sequence (SEQ ID NO: 9 and 10)
coding for the human cytomegalovirus phosphoprotein 65 (pp65) was
cloned into these expression cassettes as model antigen in such a
way that a continuous ORF composed of HLA signal sequence, pp65 and
HLA transmembrane and cytoplasmic domain (SEQ ID NO: 11 and 12)
resulted. A vector which comprised the pp65 sequence with a stop
codon in the same initial vector without said fragments was
prepared for control experiments. The following nucleic acids were
used for further experiments:
CMVpp65standard: unmodified CMVpp65 sequence, standard immunogen
CMVpp65-TM1: fusion nucleic acid composed of the following
fragments: HLA class I secretion signal, pp65 ORF and HLA class I
transmembrane and cytoplasmic domain (modified immunogen).
CMVpp65-TM2: fusion nucleic acid composed of the following
fragments: HLA class I secretion signal, pp65 ORF and HLA class II
transmembrane and cytoplasmic domain (modified immunogen).
Example 2
Testing of the Modified Vaccines
[0156] The three nucleic acids (CMVpp65standard, CMVpp65TM1,
CMVpp65TM2) were employed as immunogen in stimulation tests with
autologous DCs from antigen-positive donors. In order to test CD4
and CD8 immune responses separately, purified CD4+ and CD8+
lymphocytes were used. The readout employed was the enzyme-linked
immunospot assay (ELISPOT), which is acknowledged to be the
standard assay for quantifying IFN-.lamda.-secreting T cells. A
standard chromium release assay was used to assay the effector
function of CD8+T lymphocytes. Autologous monocytes or DCs were
transfected with pp65 RNA, CMVpp65-TM1 and CMVpp65-TM2 immunogens.
DCs were loaded with overlapping peptides for pp65 and with control
peptide as maximum stimulation control. The DCs treated in this way
were coincubated with CD4+ or CD8+ lymphocytes overnight or for 7
days. The readout took place against autologous monocytes or DCs
which had been pulsed with pp65 overlapping peptides or with a CMV
fibroblast lysate. The investigation of CD4+ immune responses
surprisingly revealed that both modified immunogens (CMVpp65-TM1
and CMVpp65-TM2) not only induced an enhanced immune response to
the CMVpp65standard immunogen, but also induced a maximum level of
antigen-specified IFN-gamma secretion in CD4+ lymphocytes (FIG. 3).
The percentage of antigen-specific CD4+ cells after stimulation by
the modified pp65 constructs was moreover equal to or even higher
than after stimulation with pp65 overlapping peptides. As expected,
the CMVpp65standard immunogen showed no relevant stimulation of
CD4+ lymphocytes.
[0157] An even more surprising result emerged on investigation of
CD8 immune responses after stimulation with the immunogens. It was
possible to show that the use of the modified expression cassettes
for stimulating CD8+ lymphocytes likewise led to a proportion of
specifically IFN-.lamda.-secreting cells which is comparable to
that after stimulation with pp65 overlapping peptides.
Surprisingly, the modified RNA constructs were far superior to the
unmodified CMVpp65standard immunogens in this case too (FIGS. 4 and
5). The results in the cytotoxicity assay showed that both
modifications led to a not previously described drastic increase in
cytotoxicity compared with CMVpp65standard RNA (FIG. 6). In this
case too there was surprisingly seen to be a superiority of the
modified immunogens over the overlapping pp65 peptides.
Example 3
Stimulation of Naive CD8+ T Lymphocytes by HLA Fusion Antigens
[0158] In order to attest the possibility of priming and subsequent
expansion of naive CD8+ lymphocytes by the fusion constructs of the
invention, dendritic cells of a CMV-negative donor were transfected
with RNA of the unmodified CMVpp65 or with CMVpp65-TM1 RNA or with
a control RNA (NY-Eso-1). The transfected dendritic cells were
employed to stimulate autologous CD8+ lymphocytes. 2 restimulations
were carried out with frozen transfected dendritic cells at weekly
intervals. For the readout, on day +21 after the first stimulation,
all cell populations were assayed in an IFN.gamma. ELISpot assay
against autologous dendritic cells which were loaded either with
pp65 overlapping peptides or, as control, with irrelevant
overlapping peptides. It was found in this case that pp65-reactive
CD8+ T lymphocyte populations were generated by stimulation with
CMVpp65-TM1 RNA in two cases (FIG. 11). Stimulations with the
dendritic cells transfected with the unmodified CMVpp65 RNA or with
control RNA by contrast showed no significant pp65 reactivity.
Example 4
Use of HLA Fusion Antigens for Stimulating Tumor Cell-Reactive T
Lymphocytes
[0159] In order to be able to expand CD8+ and CD4+ T lymphocytes
against defined tumor antigens, the following antigen sequences
were cloned as inserts into fusion constructs of the invention: the
tumor antigen TPTE (Koslowski et al., 2004, PMID 15342378), the
tumor antigen PRAME (Ikeda et al., 1997, PMID 9047241) in variant 1
(SEQ ID NO: 64), the tumor antigen WT1 as variant C (SEQ ID NO: 65)
and the tumor antigen p53 (SEQ ID NO: 66). For the functional
validation, human dendritic cells of an HLA* A 0201-positive donor
were transfected either with WT1-HLA-TM1-RNA, with unmodified
WT1-RNA or irrelevant control RNA and used as target cells. After
coincubation with WT1-reactive CD8+ T-cell clones for 8 or 16
hours, IFN.gamma. was quantified in the supernatant. It was seen
that secretion was a factor of 6-9 higher after coincubation with
WT1-HLA-TM1 transfected dendritic cells by comparison with
coincubation after transfection with unmodified WT1.
[0160] In a series of experiments, the following results were
achieved in summary and confirmed several times: [0161] The
modified immunogens lead to a distinctly enhanced stimulation and
expansion of antigen-specific CD4+ lymphocytes (increased
proliferation of CD4+ lymphocytes) [0162] The modified immunogens
lead to a distinctly enhanced stimulation and expansion of
antigen-specific CD8+ lymphocytes (increased proliferation of CD8+
lymphocytes) [0163] The modified immunogens lead to a distinctly
enhanced cytokine release from antigen-specific CD4+ lymphocytes
and CD8+ lymphocytes (increased cytokine release=increased
activation) [0164] The modified immunogens lead to a distinctly
enhanced cytotoxic reactivity of antigen-specific CD8+ lymphocytes
(increased cytotoxic effect) [0165] The modified immunogens are
100.times. more potent in relation to the expansion of
antigen-specific CD8+ lymphocytes [0166] The modified immunogens
have, even at a 100.times. lower dose, a stronger effect on the
expansion of antigen-specific CD4+ lymphocytes than standard
immunogens
[0167] In summary, therefore, it can be said that the modifications
according to the invention of an antigen result in a more than
100-fold increased potency (leftward shift in the dose-effect
curve) and a drastically increased biological activity. Compared
with the unmodified antigen sequences customary to date, it is
possible to generate an immunogen which has a quantitatively and
qualitatively greater efficacy as vaccine.
[0168] An important result of the invention is that
antigen-specific CD4+ and CD8+ lymphocytes are optimally stimulated
and expanded simultaneously. Stimulation of CD8+ and CD4+
lymphocytes is crucially important for the efficacy in particular
of therapeutic vaccines.
Sequence CWU 1
1
66178DNAHomo sapiens 1atgcgggtca cggcgccccg aaccctcatc ctgctgctct
cgggagccct ggccctgacc 60gagacctggg ccggctcc 78226PRTHomo sapiens
2Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala 1
5 10 15 Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser 20 25 3168DNAHomo
sapiens 3atcgtgggca ttgttgctgg cctggctgtc ctagcagttg tggtcatcgg
agctgtggtc 60gctactgtga tgtgtaggag gaagagctca ggtggaaaag gagggagcta
ctctcaggct 120gcgtccagcg acagtgccca gggctctgat gtgtctctca cagcttga
168455PRTHomo sapiens 4Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu
Ala Val Val Val Ile 1 5 10 15 Gly Ala Val Val Ala Thr Val Met Cys
Arg Arg Lys Ser Ser Gly Gly 20 25 30 Lys Gly Gly Ser Tyr Ser Gln
Ala Ala Ser Ser Asp Ser Ala Gln Gly 35 40 45 Ser Asp Val Ser Leu
Thr Ala 50 55 5129DNAHomo sapiens 5cagagcaaga tgctgagtgg agtcgggggc
tttgtgctgg gcctgctctt ccttggggcc 60gggctgttca tctacttcag gaatcagaaa
ggacactctg gacttcagcc aagaggattc 120ctgagctga 129642PRTHomo sapiens
6Gln Ser Lys Met Leu Ser Gly Val Gly Gly Phe Val Leu Gly Leu Leu 1
5 10 15 Phe Leu Gly Ala Gly Leu Phe Ile Tyr Phe Arg Asn Gln Lys Gly
His 20 25 30 Ser Gly Leu Gln Pro Arg Gly Phe Leu Ser 35 40
724DNAArtificial SequenceDNA of restriction site in human HLA class
I domains 7ctgcaggtcg actctagagg atcc 2488PRTArtificial
SequenceRestriction site in human HLA class I domains 8Leu Gln Val
Asp Ser Arg Gly Ser 1 5 91683DNAHuman cytomegalovirus 9atggagtcgc
gcggtcgccg ttgtcccgaa atgatatccg tactgggtcc catttcgggg 60cacgtgctga
aagccgtgtt tagtcgcggc gatacgccgg tgctgccgca cgagacgcga
120ctcctgcaga cgggtatcca cgtacgcgtg agccagccct cgctgatctt
ggtatcgcag 180tacacgcccg actcgacgcc atgccaccgc ggcgacaatc
agctgcaggt gcagcacacg 240tactttacgg gcagcgaggt ggagaacgtg
tcggtcaacg tgcacaaccc cacgggccga 300agcatctgcc ccagccagga
gcccatgtcg atctatgtgt acgcgctgcc gctcaagatg 360ctgaacatcc
ccagcatcaa cgtgcaccac tacccgtcgg cggccgagcg caaacaccga
420cacctgcccg tagctgacgc tgtgattcac gcgtcgggca agcagatgtg
gcaggcgcgt 480ctcacggtct cgggactggc ctggacgcgt cagcagaacc
agtggaaaga gcccgacgtc 540tactacacgt cagcgttcgt gtttcccacc
aaggacgtgg cactgcggca cgtggtgtgc 600gcgcacgagc tggtttgctc
catggagaac acgcgcgcaa ccaagatgca ggtgataggt 660gaccagtacg
tcaaggtgta cctggagtcc ttctgcgagg acgtgccctc cggcaagctc
720tttatgcacg tcacgctggg ctctgacgtg gaagaggacc tgacgatgac
ccgcaacccg 780caacccttca tgcgccccca cgagcgcaac ggctttacgg
tgttgtgtcc caaaaatatg 840ataatcaaac cgggcaagat ctcgcacatc
atgctggatg tggcttttac ctcacacgag 900cattttgggc tgctgtgtcc
caagagcatc ccgggcctga gcatctcagg taacctgttg 960atgaacgggc
agcagatctt cctggaggta caagccatac gcgagaccgt ggaactgcgt
1020cagtacgatc ccgtggctgc gctcttcttt ttcgatatcg acttgctgct
gcagcgcggg 1080cctcagtaca gcgagcaccc caccttcacc agccagtatc
gcatccaggg caagcttgag 1140taccgacaca cctgggaccg gcacgacgag
ggtgccgccc agggcgacga cgacgtctgg 1200accagcggat cggactccga
cgaagaactc gtaaccaccg agcgcaagac gccccgcgtc 1260accggcggcg
gcgccatggc gggcgcctcc acttccgcgg gccgcaaacg caaatcagca
1320tcctcggcga cggcgtgcac gtcgggcgtt atgacacgcg gccgccttaa
ggccgagtcc 1380accgtcgcgc ccgaagagga caccgacgag gattccgaca
acgaaatcca caatccggcc 1440gtgttcacct ggccgccctg gcaggccggc
atcctggccc gcaacctggt gcccatggtg 1500gctacggttc agggtcagaa
tctgaagtac caggaattct tctgggacgc caacgacatc 1560taccgcatct
tcgccgaatt ggaaggcgta tggcagcccg ctgcgcaacc caaacgtcgc
1620cgccaccggc aagacgcctt gcccgggcca tgcatcgcct cgacgcccaa
aaagcaccga 1680ggt 168310561PRTHuman cytomegalovirus 10Met Glu Ser
Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly 1 5 10 15 Pro
Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr 20 25
30 Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val
35 40 45 Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr
Pro Asp 50 55 60 Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln
Val Gln His Thr 65 70 75 80 Tyr Phe Thr Gly Ser Glu Val Glu Asn Val
Ser Val Asn Val His Asn 85 90 95 Pro Thr Gly Arg Ser Ile Cys Pro
Ser Gln Glu Pro Met Ser Ile Tyr 100 105 110 Val Tyr Ala Leu Pro Leu
Lys Met Leu Asn Ile Pro Ser Ile Asn Val 115 120 125 His His Tyr Pro
Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val 130 135 140 Ala Asp
Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg 145 150 155
160 Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys
165 170 175 Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr
Lys Asp 180 185 190 Val Ala Leu Arg His Val Val Cys Ala His Glu Leu
Val Cys Ser Met 195 200 205 Glu Asn Thr Arg Ala Thr Lys Met Gln Val
Ile Gly Asp Gln Tyr Val 210 215 220 Lys Val Tyr Leu Glu Ser Phe Cys
Glu Asp Val Pro Ser Gly Lys Leu 225 230 235 240 Phe Met His Val Thr
Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met 245 250 255 Thr Arg Asn
Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe 260 265 270 Thr
Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser 275 280
285 His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu
290 295 300 Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn
Leu Leu 305 310 315 320 Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln
Ala Ile Arg Glu Thr 325 330 335 Val Glu Leu Arg Gln Tyr Asp Pro Val
Ala Ala Leu Phe Phe Phe Asp 340 345 350 Ile Asp Leu Leu Leu Gln Arg
Gly Pro Gln Tyr Ser Glu His Pro Thr 355 360 365 Phe Thr Ser Gln Tyr
Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr 370 375 380 Trp Asp Arg
His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp 385 390 395 400
Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys 405
410 415 Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr
Ser 420 425 430 Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala
Cys Thr Ser 435 440 445 Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu
Ser Thr Val Ala Pro 450 455 460 Glu Glu Asp Thr Asp Glu Asp Ser Asp
Asn Glu Ile His Asn Pro Ala 465 470 475 480 Val Phe Thr Trp Pro Pro
Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu 485 490 495 Val Pro Met Val
Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu 500 505 510 Phe Phe
Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu 515 520 525
Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln 530
535 540 Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His
Arg 545 550 555 560 Gly 111962DNAArtificial SequenceDNA encoding
human HLA class I domains and CMV pp65 11atgcgggtca cggcgccccg
aaccctcatc ctgctgctct cgggagccct ggccctgacc 60gagacctggg ccggctccct
gcaggtcgac tctagaggat ccaccatgga gtcgcgcggt 120cgccgttgtc
ccgaaatgat atccgtactg ggtcccattt cggggcacgt gctgaaagcc
180gtgtttagtc gcggcgatac gccggtgctg ccgcacgaga cgcgactcct
gcagacgggt 240atccacgtac gcgtgagcca gccctcgctg atcttggtat
cgcagtacac gcccgactcg 300acgccatgcc accgcggcga caatcagctg
caggtgcagc acacgtactt tacgggcagc 360gaggtggaga acgtgtcggt
caacgtgcac aaccccacgg gccgaagcat ctgccccagc 420caggagccca
tgtcgatcta tgtgtacgcg ctgccgctca agatgctgaa catccccagc
480atcaacgtgc accactaccc gtcggcggcc gagcgcaaac accgacacct
gcccgtagct 540gacgctgtga ttcacgcgtc gggcaagcag atgtggcagg
cgcgtctcac ggtctcggga 600ctggcctgga cgcgtcagca gaaccagtgg
aaagagcccg acgtctacta cacgtcagcg 660ttcgtgtttc ccaccaagga
cgtggcactg cggcacgtgg tgtgcgcgca cgagctggtt 720tgctccatgg
agaacacgcg cgcaaccaag atgcaggtga taggtgacca gtacgtcaag
780gtgtacctgg agtccttctg cgaggacgtg ccctccggca agctctttat
gcacgtcacg 840ctgggctctg acgtggaaga ggacctgacg atgacccgca
acccgcaacc cttcatgcgc 900ccccacgagc gcaacggctt tacggtgttg
tgtcccaaaa atatgataat caaaccgggc 960aagatctcgc acatcatgct
ggatgtggct tttacctcac acgagcattt tgggctgctg 1020tgtcccaaga
gcatcccggg cctgagcatc tcaggtaacc tgttgatgaa cgggcagcag
1080atcttcctgg aggtacaagc catacgcgag accgtggaac tgcgtcagta
cgatcccgtg 1140gctgcgctct tctttttcga tatcgacttg ctgctgcagc
gcgggcctca gtacagcgag 1200caccccacct tcaccagcca gtatcgcatc
cagggcaagc ttgagtaccg acacacctgg 1260gaccggcacg acgagggtgc
cgcccagggc gacgacgacg tctggaccag cggatcggac 1320tccgacgaag
aactcgtaac caccgagcgc aagacgcccc gcgtcaccgg cggcggcgcc
1380atggcgggcg cctccacttc cgcgggccgc aaacgcaaat cagcatcctc
ggcgacggcg 1440tgcacgtcgg gcgttatgac acgcggccgc cttaaggccg
agtccaccgt cgcgcccgaa 1500gaggacaccg acgaggattc cgacaacgaa
atccacaatc cggccgtgtt cacctggccg 1560ccctggcagg ccggcatcct
ggcccgcaac ctggtgccca tggtggctac ggttcagggt 1620cagaatctga
agtaccagga attcttctgg gacgccaacg acatctaccg catcttcgcc
1680gaattggaag gcgtatggca gcccgctgcg caacccaaac gtcgccgcca
ccggcaagac 1740gccttgcccg ggccatgcat cgcctcgacg cccaaaaagc
accgaggtgg atccatcgtg 1800ggcattgttg ctggcctggc tgtcctagca
gttgtggtca tcggagctgt ggtcgctact 1860gtgatgtgta ggaggaagag
ctcaggtgga aaaggaggga gctactctca ggctgcgtcc 1920agcgacagtg
cccagggctc tgatgtgtct ctcacagctt ga 196212653PRTArtificial
SequenceFusion protein of human HLA class I domains and CMV pp65
12Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala 1
5 10 15 Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Leu Gln Val Asp Ser
Arg 20 25 30 Gly Ser Thr Met Glu Ser Arg Gly Arg Arg Cys Pro Glu
Met Ile Ser 35 40 45 Val Leu Gly Pro Ile Ser Gly His Val Leu Lys
Ala Val Phe Ser Arg 50 55 60 Gly Asp Thr Pro Val Leu Pro His Glu
Thr Arg Leu Leu Gln Thr Gly 65 70 75 80 Ile His Val Arg Val Ser Gln
Pro Ser Leu Ile Leu Val Ser Gln Tyr 85 90 95 Thr Pro Asp Ser Thr
Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val 100 105 110 Gln His Thr
Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn 115 120 125 Val
His Asn Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met 130 135
140 Ser Ile Tyr Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser
145 150 155 160 Ile Asn Val His His Tyr Pro Ser Ala Ala Glu Arg Lys
His Arg His 165 170 175 Leu Pro Val Ala Asp Ala Val Ile His Ala Ser
Gly Lys Gln Met Trp 180 185 190 Gln Ala Arg Leu Thr Val Ser Gly Leu
Ala Trp Thr Arg Gln Gln Asn 195 200 205 Gln Trp Lys Glu Pro Asp Val
Tyr Tyr Thr Ser Ala Phe Val Phe Pro 210 215 220 Thr Lys Asp Val Ala
Leu Arg His Val Val Cys Ala His Glu Leu Val 225 230 235 240 Cys Ser
Met Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp 245 250 255
Gln Tyr Val Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser 260
265 270 Gly Lys Leu Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu
Asp 275 280 285 Leu Thr Met Thr Arg Asn Pro Gln Pro Phe Met Arg Pro
His Glu Arg 290 295 300 Asn Gly Phe Thr Val Leu Cys Pro Lys Asn Met
Ile Ile Lys Pro Gly 305 310 315 320 Lys Ile Ser His Ile Met Leu Asp
Val Ala Phe Thr Ser His Glu His 325 330 335 Phe Gly Leu Leu Cys Pro
Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly 340 345 350 Asn Leu Leu Met
Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile 355 360 365 Arg Glu
Thr Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe 370 375 380
Phe Phe Asp Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu 385
390 395 400 His Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu
Glu Tyr 405 410 415 Arg His Thr Trp Asp Arg His Asp Glu Gly Ala Ala
Gln Gly Asp Asp 420 425 430 Asp Val Trp Thr Ser Gly Ser Asp Ser Asp
Glu Glu Leu Val Thr Thr 435 440 445 Glu Arg Lys Thr Pro Arg Val Thr
Gly Gly Gly Ala Met Ala Gly Ala 450 455 460 Ser Thr Ser Ala Gly Arg
Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala 465 470 475 480 Cys Thr Ser
Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr 485 490 495 Val
Ala Pro Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His 500 505
510 Asn Pro Ala Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala
515 520 525 Arg Asn Leu Val Pro Met Val Ala Thr Val Gln Gly Gln Asn
Leu Lys 530 535 540 Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr
Arg Ile Phe Ala 545 550 555 560 Glu Leu Glu Gly Val Trp Gln Pro Ala
Ala Gln Pro Lys Arg Arg Arg 565 570 575 His Arg Gln Asp Ala Leu Pro
Gly Pro Cys Ile Ala Ser Thr Pro Lys 580 585 590 Lys His Arg Gly Gly
Ser Ile Val Gly Ile Val Ala Gly Leu Ala Val 595 600 605 Leu Ala Val
Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys Arg 610 615 620 Arg
Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser 625 630
635 640 Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala 645 650
131923DNAArtificial SequenceDNA encoding human HLA class I/II
domains and CMV pp65 13atgcgggtca cggcgccccg aaccctcatc ctgctgctct
cgggagccct ggccctgacc 60gagacctggg ccggctccct gcaggtcgac tctagaggat
ccaccatgga gtcgcgcggt 120cgccgttgtc ccgaaatgat atccgtactg
ggtcccattt cggggcacgt gctgaaagcc 180gtgtttagtc gcggcgatac
gccggtgctg ccgcacgaga cgcgactcct gcagacgggt 240atccacgtac
gcgtgagcca gccctcgctg atcttggtat cgcagtacac gcccgactcg
300acgccatgcc accgcggcga caatcagctg caggtgcagc acacgtactt
tacgggcagc 360gaggtggaga acgtgtcggt caacgtgcac aaccccacgg
gccgaagcat ctgccccagc 420caggagccca tgtcgatcta tgtgtacgcg
ctgccgctca agatgctgaa catccccagc 480atcaacgtgc accactaccc
gtcggcggcc gagcgcaaac accgacacct gcccgtagct 540gacgctgtga
ttcacgcgtc gggcaagcag atgtggcagg cgcgtctcac ggtctcggga
600ctggcctgga cgcgtcagca gaaccagtgg aaagagcccg acgtctacta
cacgtcagcg 660ttcgtgtttc ccaccaagga cgtggcactg cggcacgtgg
tgtgcgcgca cgagctggtt 720tgctccatgg agaacacgcg cgcaaccaag
atgcaggtga taggtgacca gtacgtcaag 780gtgtacctgg agtccttctg
cgaggacgtg ccctccggca agctctttat gcacgtcacg 840ctgggctctg
acgtggaaga ggacctgacg atgacccgca acccgcaacc cttcatgcgc
900ccccacgagc gcaacggctt tacggtgttg tgtcccaaaa atatgataat
caaaccgggc 960aagatctcgc acatcatgct ggatgtggct tttacctcac
acgagcattt tgggctgctg 1020tgtcccaaga gcatcccggg cctgagcatc
tcaggtaacc tgttgatgaa cgggcagcag 1080atcttcctgg aggtacaagc
catacgcgag accgtggaac tgcgtcagta cgatcccgtg 1140gctgcgctct
tctttttcga tatcgacttg ctgctgcagc gcgggcctca gtacagcgag
1200caccccacct tcaccagcca gtatcgcatc cagggcaagc ttgagtaccg
acacacctgg 1260gaccggcacg acgagggtgc cgcccagggc gacgacgacg
tctggaccag cggatcggac 1320tccgacgaag aactcgtaac caccgagcgc
aagacgcccc gcgtcaccgg cggcggcgcc 1380atggcgggcg cctccacttc
cgcgggccgc aaacgcaaat cagcatcctc ggcgacggcg 1440tgcacgtcgg
gcgttatgac acgcggccgc cttaaggccg agtccaccgt
cgcgcccgaa 1500gaggacaccg acgaggattc cgacaacgaa atccacaatc
cggccgtgtt cacctggccg 1560ccctggcagg ccggcatcct ggcccgcaac
ctggtgccca tggtggctac ggttcagggt 1620cagaatctga agtaccagga
attcttctgg gacgccaacg acatctaccg catcttcgcc 1680gaattggaag
gcgtatggca gcccgctgcg caacccaaac gtcgccgcca ccggcaagac
1740gccttgcccg ggccatgcat cgcctcgacg cccaaaaagc accgaggtgg
atcccagagc 1800aagatgctga gtggagtcgg gggctttgtg ctgggcctgc
tcttccttgg ggccgggctg 1860ttcatctact tcaggaatca gaaaggacac
tctggacttc agccaagagg attcctgagc 1920tga 192314640PRTArtificial
SequenceHuman HLA class I/II domains and CMV pp65 fusion protein
14Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala 1
5 10 15 Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Leu Gln Val Asp Ser
Arg 20 25 30 Gly Ser Thr Met Glu Ser Arg Gly Arg Arg Cys Pro Glu
Met Ile Ser 35 40 45 Val Leu Gly Pro Ile Ser Gly His Val Leu Lys
Ala Val Phe Ser Arg 50 55 60 Gly Asp Thr Pro Val Leu Pro His Glu
Thr Arg Leu Leu Gln Thr Gly 65 70 75 80 Ile His Val Arg Val Ser Gln
Pro Ser Leu Ile Leu Val Ser Gln Tyr 85 90 95 Thr Pro Asp Ser Thr
Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val 100 105 110 Gln His Thr
Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn 115 120 125 Val
His Asn Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met 130 135
140 Ser Ile Tyr Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser
145 150 155 160 Ile Asn Val His His Tyr Pro Ser Ala Ala Glu Arg Lys
His Arg His 165 170 175 Leu Pro Val Ala Asp Ala Val Ile His Ala Ser
Gly Lys Gln Met Trp 180 185 190 Gln Ala Arg Leu Thr Val Ser Gly Leu
Ala Trp Thr Arg Gln Gln Asn 195 200 205 Gln Trp Lys Glu Pro Asp Val
Tyr Tyr Thr Ser Ala Phe Val Phe Pro 210 215 220 Thr Lys Asp Val Ala
Leu Arg His Val Val Cys Ala His Glu Leu Val 225 230 235 240 Cys Ser
Met Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp 245 250 255
Gln Tyr Val Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser 260
265 270 Gly Lys Leu Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu
Asp 275 280 285 Leu Thr Met Thr Arg Asn Pro Gln Pro Phe Met Arg Pro
His Glu Arg 290 295 300 Asn Gly Phe Thr Val Leu Cys Pro Lys Asn Met
Ile Ile Lys Pro Gly 305 310 315 320 Lys Ile Ser His Ile Met Leu Asp
Val Ala Phe Thr Ser His Glu His 325 330 335 Phe Gly Leu Leu Cys Pro
Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly 340 345 350 Asn Leu Leu Met
Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile 355 360 365 Arg Glu
Thr Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe 370 375 380
Phe Phe Asp Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu 385
390 395 400 His Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu
Glu Tyr 405 410 415 Arg His Thr Trp Asp Arg His Asp Glu Gly Ala Ala
Gln Gly Asp Asp 420 425 430 Asp Val Trp Thr Ser Gly Ser Asp Ser Asp
Glu Glu Leu Val Thr Thr 435 440 445 Glu Arg Lys Thr Pro Arg Val Thr
Gly Gly Gly Ala Met Ala Gly Ala 450 455 460 Ser Thr Ser Ala Gly Arg
Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala 465 470 475 480 Cys Thr Ser
Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr 485 490 495 Val
Ala Pro Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His 500 505
510 Asn Pro Ala Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala
515 520 525 Arg Asn Leu Val Pro Met Val Ala Thr Val Gln Gly Gln Asn
Leu Lys 530 535 540 Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr
Arg Ile Phe Ala 545 550 555 560 Glu Leu Glu Gly Val Trp Gln Pro Ala
Ala Gln Pro Lys Arg Arg Arg 565 570 575 His Arg Gln Asp Ala Leu Pro
Gly Pro Cys Ile Ala Ser Thr Pro Lys 580 585 590 Lys His Arg Gly Gly
Ser Gln Ser Lys Met Leu Ser Gly Val Gly Gly 595 600 605 Phe Val Leu
Gly Leu Leu Phe Leu Gly Ala Gly Leu Phe Ile Tyr Phe 610 615 620 Arg
Asn Gln Lys Gly His Ser Gly Leu Gln Pro Arg Gly Phe Leu Ser 625 630
635 640 1566PRTHomo sapiens 15Pro Ser Ser Gln Pro Thr Ile Pro Ile
Val Gly Ile Ile Ala Gly Leu 1 5 10 15 Val Leu Phe Gly Ala Val Ile
Thr Gly Ala Val Val Ala Ala Val Met 20 25 30 Trp Arg Arg Lys Ser
Ser Asp Arg Lys Gly Gly Ser Tyr Ser Gln Ala 35 40 45 Ala Ser Ser
Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Cys 50 55 60 Lys
Val 65 1624PRTHomo sapiens 16Gly Ser Tyr Ser Gln Ala Ala Ser Ser
Asp Ser Ala Gln Gly Ser Asp 1 5 10 15 Val Ser Leu Thr Ala Cys Lys
Val 20 1763PRTHomo sapiens 17Pro Ser Ser Gln Ser Thr Val Pro Ile
Val Gly Ile Val Ala Gly Leu 1 5 10 15 Ala Val Leu Ala Val Val Val
Ile Gly Ala Val Val Ala Ala Val Met 20 25 30 Cys Arg Arg Lys Ser
Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala 35 40 45 Ala Cys Ser
Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala 50 55 60
1821PRTHomo sapiens 18Gly Ser Tyr Ser Gln Ala Ala Cys Ser Asp Ser
Ala Gln Gly Ser Asp 1 5 10 15 Val Ser Leu Thr Ala 20 1967PRTHomo
sapiens 19Pro Ser Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Val Ala
Gly Leu 1 5 10 15 Ala Val Leu Ala Val Leu Ala Val Leu Gly Ala Met
Val Ala Val Val 20 25 30 Met Cys Arg Arg Lys Ser Ser Gly Gly Lys
Gly Gly Ser Cys Ser Gln 35 40 45 Ala Ala Ser Ser Asn Ser Ala Gln
Gly Ser Asp Glu Ser Leu Ile Ala 50 55 60 Cys Lys Ala 65 2014PRTHomo
sapiens 20Ser Ala Gln Gly Ser Asp Glu Ser Leu Ile Ala Cys Lys Ala 1
5 10 2162PRTHomo sapiens 21Pro Ala Ser Gln Pro Thr Ile Pro Ile Val
Gly Ile Ile Ala Gly Leu 1 5 10 15 Val Leu Leu Gly Ser Val Val Ser
Gly Ala Val Val Ala Ala Val Ile 20 25 30 Trp Arg Lys Lys Ser Ser
Gly Gly Lys Gly Gly Ser Tyr Ser Lys Ala 35 40 45 Glu Trp Ser Asp
Ser Ala Gln Gly Ser Glu Ser His Ser Leu 50 55 60 2220PRTHomo
sapiens 22Gly Ser Tyr Ser Lys Ala Glu Trp Ser Asp Ser Ala Gln Gly
Ser Glu 1 5 10 15 Ser His Ser Leu 20 2366PRTHomo sapiens 23Gln Ser
Pro Gln Pro Thr Ile Pro Ile Val Gly Ile Val Ala Gly Leu 1 5 10 15
Val Val Leu Gly Ala Val Val Thr Gly Ala Val Val Ala Ala Val Met 20
25 30 Trp Arg Lys Lys Ser Ser Asp Arg Asn Arg Gly Ser Tyr Ser Gln
Ala 35 40 45 Ala Val Thr Asp Ser Ala Gln Gly Ser Gly Val Ser Leu
Thr Ala Asn 50 55 60 Lys Val 65 2427PRTHomo sapiens 24Arg Asn Arg
Gly Ser Tyr Ser Gln Ala Ala Val Thr Asp Ser Ala Gln 1 5 10 15 Gly
Ser Gly Val Ser Leu Thr Ala Asn Lys Val 20 25 2537PRTHomo sapiens
25Val Val Cys Ala Leu Gly Leu Thr Val Gly Leu Val Gly Ile Ile Ile 1
5 10 15 Gly Thr Ile Phe Ile Ile Lys Gly Leu Arg Lys Ser Asn Ala Ala
Glu 20 25 30 Arg Arg Gly Pro Leu 35 2612PRTHomo sapiens 26Arg Lys
Ser Asn Ala Ala Glu Arg Arg Gly Pro Leu 1 5 10 2738PRTHomo sapiens
27Met Leu Ser Gly Val Gly Gly Phe Val Leu Gly Leu Leu Phe Leu Ala 1
5 10 15 Gly Leu Phe Ile Tyr Phe Arg Asn Gln Lys Gly His Ser Gly Leu
Gln 20 25 30 Pro Arg Gly Phe Leu Ser 35 2812PRTHomo sapiens 28Gly
His Ser Gly Leu Gln Pro Arg Gly Phe Leu Ser 1 5 10 2937PRTHomo
sapiens 29Val Val Cys Ala Leu Gly Leu Ser Val Gly Leu Met Gly Ile
Val Val 1 5 10 15 Gly Thr Val Phe Ile Ile Gln Gly Leu Arg Ser Val
Gly Ala Ser Arg 20 25 30 His Gln Gly Pro Leu 35 3010PRTHomo sapiens
30Val Gly Ala Ser Arg His Gln Gly Pro Leu 1 5 10 3131PRTHomo
sapiens 31Met Leu Ser Gly Ile Gly Gly Phe Val Leu Gly Leu Ile Phe
Leu Gly 1 5 10 15 Leu Gly Leu Ile Ile His His Arg Ser Gln Lys Gly
Leu Leu His 20 25 30 328PRTHomo sapiens 32Arg Ser Gln Lys Gly Leu
Leu His 1 5 3337PRTHomo sapiens 33Val Leu Cys Ala Leu Gly Leu Val
Leu Gly Leu Val Gly Ile Ile Val 1 5 10 15 Gly Thr Val Leu Ile Ile
Lys Ser Leu Arg Ser Gly His Asp Pro Arg 20 25 30 Ala Gln Gly Thr
Leu 35 3412PRTHomo sapiens 34Arg Ser Gly His Asp Pro Arg Ala Gln
Gly Thr Leu 1 5 10 3533PRTHomo sapiens 35Thr Leu Thr Gly Ala Gly
Gly Phe Val Leu Gly Leu Ile Ile Cys Gly 1 5 10 15 Val Gly Ile Phe
Met His Arg Arg Ser Lys Lys Val Gln Arg Gly Ser 20 25 30 Ala
369PRTHomo sapiens 36Ser Lys Lys Val Gln Arg Gly Ser Ala 1 5
3726PRTHomo sapiens 37Phe Ile Ile Leu Ala Val Ile Val Pro Leu Leu
Leu Leu Ile Gly Leu 1 5 10 15 Ala Leu Trp Phe Arg Lys Arg Cys Phe
Cys 20 25 386PRTHomo sapiens 38Arg Lys Arg Cys Phe Cys 1 5
3930PRTHomo sapiens 39Ile Val Leu Ala Ile Ile Val Pro Ser Leu Leu
Leu Leu Leu Cys Leu 1 5 10 15 Ala Leu Trp Tyr Met Arg Arg Arg Ser
Tyr Gln Asn Ile Pro 20 25 30 409PRTHomo sapiens 40Arg Arg Arg Ser
Tyr Gln Asn Ile Pro 1 5 4130PRTHomo sapiens 41Trp Ile Ala Leu Val
Val Ile Val Pro Leu Val Ile Leu Ile Val Leu 1 5 10 15 Val Leu Trp
Phe Lys Lys His Cys Ser Tyr Gln Asp Ile Leu 20 25 30 4210PRTHomo
sapiens 42Lys Lys His Cys Ser Tyr Gln Asp Ile Leu 1 5 10
43250PRTHomo sapiens 43Met Ala Ala Gly Thr Ser Ser Tyr Trp Glu Asp
Leu Arg Lys Gln Ala 1 5 10 15 Arg Gln Leu Glu Asn Glu Leu Asp Leu
Lys Leu Val Ser Phe Ser Lys 20 25 30 Leu Cys Thr Ser Tyr Ser His
Ser Ser Thr Arg Asp Gly Arg Arg Asp 35 40 45 Arg Tyr Ser Ser Asp
Thr Thr Pro Leu Leu Asn Gly Ser Ser Gln Asp 50 55 60 Arg Met Phe
Glu Thr Met Ala Ile Glu Ile Glu Gln Leu Leu Ala Arg 65 70 75 80 Leu
Thr Gly Val Asn Asp Lys Met Ala Glu Tyr Thr Asn Ser Ala Gly 85 90
95 Val Pro Ser Leu Asn Ala Ala Leu Met His Thr Leu Gln Arg His Arg
100 105 110 Asp Ile Leu Gln Asp Tyr Thr His Glu Phe His Lys Thr Lys
Ala Asn 115 120 125 Phe Met Ala Ile Arg Glu Arg Glu Asn Leu Met Gly
Ser Val Arg Lys 130 135 140 Asp Ile Glu Ser Tyr Lys Ser Gly Ser Gly
Val Asn Asn Arg Arg Thr 145 150 155 160 Glu Leu Phe Leu Lys Glu His
Asp His Leu Arg Asn Ser Asp Arg Leu 165 170 175 Ile Glu Glu Thr Ile
Ser Ile Ala Met Ala Thr Lys Glu Asn Met Thr 180 185 190 Ser Gln Arg
Gly Met Leu Lys Ser Ile His Ser Lys Met Asn Thr Leu 195 200 205 Ala
Asn Arg Phe Pro Ala Val Asn Ser Leu Ile Gln Arg Ile Asn Leu 210 215
220 Arg Lys Arg Arg Asp Ser Leu Ile Leu Gly Gly Val Ile Gly Ile Cys
225 230 235 240 Thr Ile Leu Leu Leu Leu Tyr Ala Phe His 245 250
44128PRTHomo sapiens 44Met Gly Ala Ser Leu Thr Ser Pro Gly Thr Gln
Glu Lys Leu Ile Arg 1 5 10 15 Asp Phe Asp Glu Lys Gln Gln Glu Ala
Asn Lys Met Leu Thr Gln Met 20 25 30 Glu Glu Glu Leu His Tyr Ala
Pro Val Ser Phe His Asn Pro Met Met 35 40 45 Ser Lys Leu Gln Asp
Tyr Gln Lys Asp Leu Ala Gln Phe His Leu Glu 50 55 60 Ala Arg Thr
Met Pro Gly Asp Arg Gly Asp Met Lys Tyr Gly Thr Tyr 65 70 75 80 Ala
Val Glu Asn Glu His Met Asn Arg Leu Gln Ser Gln Arg Ala Met 85 90
95 Leu Leu Gln Gly Thr Lys Ser Leu Gly Arg Ala Thr Gln Glu Thr Asp
100 105 110 Gln Ile Gly Ser Glu Ile Ser Glu Glu Leu Gly Asn Gln Arg
Asp Gln 115 120 125 45212PRTHomo sapiens 45Met Asp Pro Leu Phe Gln
Gln Thr His Lys Gln Val His Glu Ile Gln 1 5 10 15 Ser Cys Met Gly
Arg Leu Glu Thr Ala Asp Lys Gln Ser Val His Ile 20 25 30 Val Glu
Asn Glu Ile Gln Ala Ser Ile Asp Gln Ile Phe Ser Arg Leu 35 40 45
Glu Arg Leu Glu Ile Leu Ser Ser Lys Glu Pro Pro Asn Lys Arg Gln 50
55 60 Asn Ala Arg Leu Arg Val Asp Gln Leu Lys Tyr Asp Val Gln His
Leu 65 70 75 80 Gln Thr Ala Leu Arg Asn Phe Gln His Arg Arg His Ala
Arg Glu Gln 85 90 95 Gln Glu Arg Gln Arg Glu Glu Leu Leu Ser Arg
Thr Phe Thr Thr Asn 100 105 110 Asp Ser Asp Thr Thr Ile Pro Met Asp
Glu Ser Leu Gln Phe Asn Ser 115 120 125 Ser Leu Gln Lys Val His Asn
Gly Met Asp Asp Leu Ile Leu Asp Gly 130 135 140 His Asn Ile Leu Asp
Gly Leu Arg Thr Gln Arg Leu Thr Leu Lys Gly 145 150 155 160 Thr Gln
Lys Lys Ile Leu Asp Ile Ala Asn Met Leu Gly Leu Ser Asn 165 170 175
Thr Val Met Arg Leu Ile Glu Lys Arg Ala Phe Gln Asp Lys Tyr Phe 180
185 190 Met Ile Gly Gly Met Leu Leu Thr Cys Val Val Met Phe Leu Val
Val 195 200 205 Gln Tyr Leu Thr 210 46172PRTHomo sapiens 46Met Ser
Val Pro Gly Pro Ser Ser Pro Asp Gly Ala Leu Thr Arg Pro 1 5 10 15
Pro Tyr Cys Leu Glu Ala Gly Glu Pro Thr Pro Gly Leu Ser Asp Thr 20
25 30 Ser Pro Asp Glu Gly Leu Ile Glu Asp Leu Thr Ile Glu Asp Lys
Ala 35 40 45 Val Glu Gln Leu Ala Glu Gly Leu Leu Ser His Tyr Leu
Pro Asp Leu 50 55 60 Gln Arg Ser Lys Gln Ala Leu Gln Glu Leu Thr
Gln Asn Gln Val Val 65 70
75 80 Leu Leu Asp Thr Leu Glu Gln Glu Ile Ser Lys Phe Lys Glu Cys
His 85 90 95 Ser Met Leu Asp Ile Asn Ala Leu Phe Ala Glu Ala Lys
His Tyr His 100 105 110 Ala Lys Leu Val Asn Ile Arg Lys Glu Met Leu
Met Leu His Glu Lys 115 120 125 Thr Ser Lys Leu Lys Lys Arg Ala Leu
Lys Leu Gln Gln Lys Arg Gln 130 135 140 Lys Glu Glu Leu Glu Arg Glu
Gln Gln Arg Glu Lys Glu Phe Glu Arg 145 150 155 160 Glu Lys Gln Leu
Thr Ala Arg Pro Ala Lys Arg Met 165 170 47301PRTHomo sapiens 47Met
Ser Cys Arg Asp Arg Thr Gln Glu Phe Leu Ser Ala Cys Lys Ser 1 5 10
15 Leu Gln Thr Arg Gln Asn Gly Ile Gln Thr Asn Lys Pro Ala Leu Arg
20 25 30 Ala Val Arg Gln Arg Ser Glu Phe Thr Leu Met Ala Lys Arg
Ile Gly 35 40 45 Lys Asp Leu Ser Asn Thr Phe Ala Lys Leu Glu Lys
Leu Thr Ile Leu 50 55 60 Ala Lys Arg Lys Ser Leu Phe Asp Asp Lys
Ala Val Glu Ile Glu Glu 65 70 75 80 Leu Thr Tyr Ile Ile Lys Gln Asp
Ile Asn Ser Leu Asn Lys Gln Ile 85 90 95 Ala Gln Leu Gln Asp Phe
Val Arg Ala Lys Gly Ser Gln Ser Gly Arg 100 105 110 His Leu Gln Thr
His Ser Asn Thr Ile Val Val Ser Leu Gln Ser Lys 115 120 125 Leu Ala
Ser Met Ser Asn Asp Phe Lys Ser Val Leu Glu Val Arg Thr 130 135 140
Glu Asn Leu Lys Gln Gln Arg Ser Arg Arg Glu Gln Phe Ser Arg Ala 145
150 155 160 Pro Val Ser Ala Leu Pro Leu Ala Pro Asn His Leu Gly Gly
Gly Ala 165 170 175 Val Val Leu Gly Ala Glu Ser His Ala Ser Lys Asp
Val Ala Ile Asp 180 185 190 Met Met Asp Ser Arg Thr Ser Gln Gln Leu
Gln Leu Ile Asp Glu Gln 195 200 205 Asp Ser Tyr Ile Gln Ser Arg Ala
Asp Thr Met Gln Asn Ile Glu Ser 210 215 220 Thr Ile Val Glu Leu Gly
Ser Ile Phe Gln Gln Leu Ala His Met Val 225 230 235 240 Lys Glu Gln
Glu Glu Thr Ile Gln Arg Ile Asp Glu Asn Val Leu Gly 245 250 255 Ala
Gln Leu Asp Val Glu Ala Ala His Ser Glu Ile Leu Lys Tyr Phe 260 265
270 Gln Ser Val Thr Ser Asn Arg Trp Leu Met Val Lys Ile Phe Leu Ile
275 280 285 Leu Ile Val Phe Phe Ile Ile Phe Val Val Phe Leu Ala 290
295 300 48255PRTHomo sapiens 48Met Ser Met Glu Asp Pro Phe Phe Val
Val Lys Gly Glu Val Gln Lys 1 5 10 15 Ala Val Asn Thr Ala Gln Gly
Leu Phe Gln Arg Trp Thr Glu Leu Leu 20 25 30 Gln Asp Pro Ser Thr
Ala Thr Arg Glu Glu Ile Asp Trp Thr Thr Asn 35 40 45 Glu Leu Arg
Asn Asn Leu Arg Ser Ile Glu Trp Asp Leu Glu Asp Leu 50 55 60 Asp
Glu Thr Ile Ser Ile Val Glu Ala Asn Pro Arg Lys Phe Asn Leu 65 70
75 80 Asp Ala Thr Glu Leu Ser Ile Arg Lys Ala Phe Ile Thr Ser Thr
Arg 85 90 95 Gln Val Val Arg Asp Met Lys Asp Gln Met Ser Thr Ser
Ser Val Gln 100 105 110 Ala Leu Ala Glu Arg Lys Asn Arg Gln Ala Leu
Leu Gly Asp Ser Gly 115 120 125 Ser Gln Asn Trp Ser Thr Gly Thr Thr
Asp Lys Tyr Gly Arg Leu Asp 130 135 140 Arg Glu Leu Gln Arg Ala Asn
Ser His Phe Ile Glu Glu Gln Gln Ala 145 150 155 160 Gln Gln Gln Leu
Ile Val Glu Gln Gln Asp Glu Gln Leu Glu Leu Val 165 170 175 Ser Gly
Ser Ile Gly Val Leu Lys Asn Met Ser Gln Arg Ile Gly Gly 180 185 190
Glu Leu Glu Glu Gln Ala Val Met Leu Glu Asp Phe Ser His Glu Leu 195
200 205 Glu Ser Thr Gln Ser Arg Leu Asp Asn Val Met Lys Lys Leu Ala
Lys 210 215 220 Val Ser His Met Thr Ser Asp Arg Arg Gln Trp Cys Ala
Ile Ala Ile 225 230 235 240 Leu Phe Ala Val Leu Leu Val Val Leu Ile
Leu Phe Leu Val Leu 245 250 255 49261PRTHomo sapiens 49Met Ser Tyr
Thr Pro Gly Val Gly Gly Asp Pro Ala Gln Leu Ala Gln 1 5 10 15 Arg
Ile Ser Ser Asn Ile Gln Lys Ile Thr Gln Cys Ser Val Glu Ile 20 25
30 Gln Arg Thr Leu Asn Gln Leu Gly Thr Pro Gln Asp Ser Pro Glu Leu
35 40 45 Arg Gln Gln Leu Gln Gln Lys Gln Gln Tyr Thr Asn Gln Leu
Ala Lys 50 55 60 Glu Thr Asp Lys Tyr Ile Lys Glu Phe Gly Ser Leu
Pro Thr Thr Pro 65 70 75 80 Ser Glu Gln Arg Gln Arg Lys Ile Gln Lys
Asp Arg Leu Val Ala Glu 85 90 95 Phe Thr Thr Ser Leu Thr Asn Phe
Gln Lys Val Gln Arg Gln Ala Ala 100 105 110 Glu Arg Glu Lys Glu Phe
Val Ala Arg Val Arg Ala Ser Ser Arg Val 115 120 125 Ser Gly Ser Phe
Pro Glu Asp Ser Ser Lys Glu Arg Asn Leu Val Ser 130 135 140 Trp Glu
Ser Gln Thr Gln Pro Gln Val Gln Val Gln Asp Glu Glu Ile 145 150 155
160 Thr Glu Asp Asp Leu Arg Leu Ile His Glu Arg Glu Ser Ser Ile Arg
165 170 175 Gln Leu Glu Ala Asp Ile Met Asp Ile Asn Glu Ile Phe Lys
Asp Leu 180 185 190 Gly Met Met Ile His Glu Gln Gly Asp Val Ile Asp
Ser Ile Glu Ala 195 200 205 Asn Val Glu Asn Ala Glu Val His Val Gln
Gln Ala Asn Gln Gln Leu 210 215 220 Ser Arg Ala Ala Asp Tyr Gln Arg
Lys Ser Arg Lys Thr Leu Cys Ile 225 230 235 240 Ile Ile Leu Ile Leu
Val Ile Gly Val Ala Ile Ile Ser Leu Ile Ile 245 250 255 Trp Gly Leu
Asn His 260 50236PRTHomo sapiens 50Met Ala Pro Asp Pro Trp Phe Ser
Thr Tyr Asp Ser Thr Cys Gln Ile 1 5 10 15 Ala Gln Glu Ile Ala Glu
Lys Ile Gln Gln Arg Asn Gln Tyr Glu Arg 20 25 30 Lys Gly Glu Lys
Ala Pro Lys Leu Thr Val Thr Ile Arg Ala Leu Leu 35 40 45 Gln Asn
Leu Lys Glu Lys Ile Ala Leu Leu Lys Asp Leu Leu Leu Arg 50 55 60
Ala Val Ser Thr His Gln Ile Thr Gln Leu Glu Gly Asp Arg Arg Gln 65
70 75 80 Asn Leu Leu Asp Asp Leu Val Thr Arg Glu Arg Leu Leu Leu
Ala Ser 85 90 95 Phe Lys Asn Glu Gly Ala Glu Pro Asp Leu Ile Arg
Ser Ser Leu Met 100 105 110 Ser Glu Glu Ala Lys Arg Gly Ala Pro Asn
Pro Trp Leu Phe Glu Glu 115 120 125 Pro Glu Glu Thr Arg Gly Leu Gly
Phe Asp Glu Ile Arg Gln Gln Gln 130 135 140 Gln Lys Ile Ile Gln Glu
Gln Asp Ala Gly Leu Asp Ala Leu Ser Ser 145 150 155 160 Ile Ile Ser
Arg Gln Lys Gln Met Gly Gln Glu Ile Gly Asn Glu Leu 165 170 175 Asp
Glu Gln Asn Glu Ile Ile Asp Asp Leu Ala Asn Leu Val Glu Asn 180 185
190 Thr Asp Glu Lys Leu Arg Asn Glu Thr Arg Arg Val Asn Met Val Asp
195 200 205 Arg Lys Ser Ala Ser Cys Gly Met Ile Met Val Ile Leu Leu
Leu Leu 210 215 220 Val Ala Ile Val Val Val Ala Val Trp Pro Thr Asn
225 230 235 51200PRTHomo sapiens 51Met Ser Leu Glu Asp Pro Phe Phe
Val Val Arg Gly Glu Val Gln Lys 1 5 10 15 Ala Val Asn Thr Ala Arg
Gly Leu Tyr Gln Arg Trp Cys Glu Leu Leu 20 25 30 Gln Glu Ser Ala
Ala Val Gly Arg Glu Glu Leu Asp Trp Thr Thr Asn 35 40 45 Glu Leu
Arg Asn Gly Leu Arg Ser Ile Glu Trp Asp Leu Glu Asp Leu 50 55 60
Glu Glu Thr Ile Gly Ile Val Glu Ala Asn Pro Gly Lys Pro Ala Ala 65
70 75 80 Gln Lys Ser Pro Ser Asp Leu Leu Asp Ala Ser Ala Val Ser
Ala Thr 85 90 95 Ser Arg Tyr Ile Glu Glu Gln Gln Ala Thr Gln Gln
Leu Ile Met Asp 100 105 110 Glu Gln Asp Gln Gln Leu Glu Met Val Ser
Gly Ser Ile Gln Val Leu 115 120 125 Lys His Met Ser Gly Arg Val Gly
Glu Glu Leu Asp Glu Gln Gly Ile 130 135 140 Met Leu Asp Ala Phe Ala
Gln Glu Met Asp His Thr Gln Ser Arg Met 145 150 155 160 Asp Gly Val
Leu Arg Lys Leu Ala Lys Val Ser His Met Thr Ser Asp 165 170 175 Arg
Arg Gln Trp Cys Ala Ile Ala Val Leu Val Gly Val Leu Leu Leu 180 185
190 Val Leu Ile Leu Leu Phe Ser Leu 195 200 52249PRTHomo sapiens
52Met Ser Leu Glu Asp Pro Phe Phe Val Val Arg Gly Glu Val Gln Lys 1
5 10 15 Ala Val Asn Thr Ala Arg Gly Leu Tyr Gln Arg Trp Cys Glu Leu
Leu 20 25 30 Gln Glu Ser Ala Ala Val Gly Arg Glu Glu Leu Asp Trp
Thr Thr Asn 35 40 45 Glu Leu Arg Asn Gly Leu Arg Ser Ile Glu Trp
Asp Leu Glu Asp Leu 50 55 60 Glu Glu Thr Ile Gly Ile Val Glu Ala
Asn Pro Gly Lys Phe Lys Leu 65 70 75 80 Pro Ala Gly Asp Leu Gln Glu
Arg Lys Val Phe Val Glu Arg Met Arg 85 90 95 Glu Ala Val Gln Glu
Met Lys Asp His Met Val Ser Pro Thr Ala Val 100 105 110 Ala Phe Leu
Glu Arg Asn Asn Arg Glu Ile Leu Ala Gly Lys Pro Ala 115 120 125 Ala
Gln Lys Ser Pro Ser Asp Leu Leu Asp Ala Ser Ala Val Ser Ala 130 135
140 Thr Ser Arg Tyr Ile Glu Glu Gln Gln Ala Thr Gln Gln Leu Ile Met
145 150 155 160 Asp Glu Gln Asp Gln Gln Leu Glu Met Val Ser Gly Ser
Ile Gln Val 165 170 175 Leu Lys His Met Ser Gly Arg Val Gly Glu Glu
Leu Asp Glu Gln Gly 180 185 190 Ile Met Leu Asp Ala Phe Ala Gln Glu
Met Asp His Thr Gln Ser Arg 195 200 205 Met Asp Gly Val Leu Arg Lys
Leu Ala Lys Val Ser His Met Thr Ser 210 215 220 Asp Arg Arg Gln Trp
Cys Ala Ile Ala Val Leu Val Gly Val Leu Leu 225 230 235 240 Leu Val
Leu Ile Leu Leu Phe Ser Leu 245 53287PRTHomo sapiens 53Met Lys Asp
Arg Leu Ala Glu Leu Leu Asp Leu Ser Lys Gln Tyr Asp 1 5 10 15 Gln
Gln Phe Pro Asp Gly Asp Asp Glu Phe Asp Ser Pro His Glu Asp 20 25
30 Ile Val Phe Glu Thr Asp His Ile Leu Glu Ser Leu Tyr Arg Asp Ile
35 40 45 Arg Asp Ile Gln Asp Glu Asn Gln Leu Leu Val Ala Asp Val
Lys Arg 50 55 60 Leu Gly Lys Gln Asn Ala Arg Phe Leu Thr Ser Met
Arg Arg Leu Ser 65 70 75 80 Ser Ile Lys Arg Asp Thr Asn Ser Ile Ala
Lys Ala Phe Arg Ala Arg 85 90 95 Gly Glu Val Ile His Cys Lys Leu
Arg Ala Met Lys Glu Leu Ser Glu 100 105 110 Ala Ala Glu Ala Gln His
Gly Pro His Ser Ala Val Ala Arg Ile Ser 115 120 125 Arg Ala Gln Tyr
Asn Ala Leu Thr Leu Thr Phe Gln Arg Ala Met His 130 135 140 Asp Tyr
Asn Gln Ala Glu Met Lys Gln Arg Asp Asn Cys Lys Ile Arg 145 150 155
160 Ile Gln Arg Gln Leu Glu Ile Met Gly Lys Glu Val Ser Gly Asp Gln
165 170 175 Ile Glu Asp Met Phe Glu Gln Gly Lys Trp Asp Val Phe Ser
Glu Asn 180 185 190 Leu Leu Ala Asp Val Lys Gly Arg Gly Pro Pro Thr
Thr Arg Ser Arg 195 200 205 Ala Ala Thr Ala Asn Cys Cys Ala Trp Arg
Ala Ala Ile Arg Asp Val 210 215 220 His Glu Leu Phe Leu Gln Met Ala
Val Leu Val Glu Lys Gln Ala Asp 225 230 235 240 Thr Leu Asn Val Ile
Glu Leu Asn Val Gln Lys Thr Val Asp Tyr Thr 245 250 255 Gly Gln Ala
Lys Ala Gln Val Arg Lys Ala Val Gln Tyr Glu Glu Lys 260 265 270 Asn
Pro Cys Arg Thr Leu Cys Cys Phe Cys Cys Pro Cys Leu Lys 275 280 285
54276PRTHomo sapiens 54Met Ser Tyr Gly Pro Leu Asp Met Tyr Arg Asn
Pro Gly Pro Ser Gly 1 5 10 15 Pro Gln Leu Arg Asp Phe Ser Ser Ile
Ile Gln Thr Cys Ser Gly Asn 20 25 30 Ile Gln Arg Ile Ser Gln Ala
Thr Ala Gln Ile Lys Asn Leu Met Ser 35 40 45 Gln Leu Gly Thr Lys
Gln Asp Ser Ser Lys Leu Gln Glu Asn Leu Gln 50 55 60 Gln Leu Gln
His Ser Thr Asn Gln Leu Ala Lys Glu Thr Asn Glu Leu 65 70 75 80 Leu
Lys Glu Leu Gly Ser Leu Pro Leu Pro Leu Ser Thr Ser Glu Gln 85 90
95 Arg Gln Gln Arg Leu Gln Lys Glu Arg Leu Met Asn Asp Phe Ser Ala
100 105 110 Ala Leu Asn Asn Phe Gln Ala Val Gln Arg Arg Val Ser Glu
Lys Glu 115 120 125 Lys Glu Ser Ile Ala Arg Ala Arg Ala Gly Ser Arg
Leu Ser Ala Glu 130 135 140 Glu Arg Gln Arg Glu Glu Gln Leu Val Ser
Phe Asp Ser His Glu Glu 145 150 155 160 Trp Asn Gln Met Gln Ser Gln
Glu Asp Glu Val Ala Ile Thr Glu Gln 165 170 175 Asp Leu Glu Leu Ile
Lys Glu Arg Glu Thr Ala Ile Arg Gln Leu Glu 180 185 190 Ala Asp Ile
Leu Asp Val Asn Gln Ile Phe Lys Asp Leu Ala Met Met 195 200 205 Ile
His Asp Gln Gly Asp Leu Ile Asp Ser Ile Glu Ala Asn Val Glu 210 215
220 Ser Ser Glu Val His Val Glu Arg Ala Thr Glu Gln Leu Gln Arg Ala
225 230 235 240 Ala Tyr Tyr Gln Lys Lys Ser Arg Lys Lys Met Cys Ile
Leu Val Leu 245 250 255 Val Leu Ser Val Ile Ile Leu Ile Leu Gly Leu
Ile Ile Trp Leu Val 260 265 270 Tyr Lys Thr Lys 275 55302PRTHomo
sapiens 55Met Ser Glu Asp Glu Glu Lys Val Lys Leu Arg Arg Leu Glu
Pro Ala 1 5 10 15 Ile Gln Lys Phe Ile Lys Ile Val Ile Pro Thr Asn
Leu Glu Arg Leu 20 25 30 Arg Lys His Gln Ile Asn Ile Glu Lys Tyr
Gln Arg Cys Arg Ile Trp 35 40 45 Asp Lys Leu His Glu Glu His Ile
Asn Ala Gly Arg Thr Val Gln Gln 50 55 60 Leu Arg Ser Asn Ile Arg
Glu Ile Glu Lys Leu Cys Leu Lys Val Arg 65 70 75 80 Lys Asp Asp Leu
Val Leu Leu Lys Arg Met Ile Asp Pro Val Lys Glu 85 90 95 Glu Ala
Ser Ala Ala Thr Ala Glu Phe Leu Gln Leu His Leu Glu Ser 100 105 110
Val Glu Glu Leu Lys Lys Gln Phe Asn Asp Glu Glu Thr Leu Leu Gln
115
120 125 Pro Pro Leu Thr Arg Ser Met Thr Val Gly Gly Ala Phe His Thr
Thr 130 135 140 Glu Ala Glu Ala Ser Ser Gln Ser Leu Thr Gln Ile Tyr
Ala Leu Pro 145 150 155 160 Glu Ile Pro Gln Asp Gln Asn Ala Ala Glu
Ser Arg Glu Thr Leu Glu 165 170 175 Ala Asp Leu Ile Glu Leu Ser Gln
Leu Val Thr Asp Phe Ser Leu Leu 180 185 190 Val Asn Ser Gln Gln Glu
Lys Ile Asp Ser Ile Ala Asp His Val Asn 195 200 205 Ser Ala Ala Val
Asn Val Glu Glu Gly Thr Lys Asn Leu Gly Lys Ala 210 215 220 Ala Lys
Tyr Lys Leu Ala Ala Leu Pro Val Ala Gly Ala Leu Ile Gly 225 230 235
240 Gly Met Val Gly Gly Pro Ile Gly Leu Leu Ala Cys Phe Lys Val Ala
245 250 255 Gly Ile Ala Ala Ala Leu Gly Gly Gly Val Leu Gly Phe Thr
Gly Gly 260 265 270 Lys Leu Ile Gln Arg Lys Lys Gln Lys Met Met Glu
Lys Leu Thr Ser 275 280 285 Ser Cys Pro Asp Leu Pro Ser Gln Thr Asp
Lys Lys Cys Ser 290 295 300 56116PRTHomo sapiens 56Met Ser Ala Thr
Ala Ala Thr Ala Pro Pro Ala Ala Pro Ala Gly Glu 1 5 10 15 Gly Gly
Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Asn Arg Arg Leu 20 25 30
Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg Val 35
40 45 Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu
Asp 50 55 60 Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe
Glu Thr Ser 65 70 75 80 Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys
Asn Leu Lys Met Met 85 90 95 Ile Ile Leu Gly Val Ile Cys Ala Ile
Ile Leu Ile Ile Ile Ile Val 100 105 110 Tyr Phe Ser Ser 115
57100PRTHomo sapiens 57Met Ser Thr Gly Pro Thr Ala Ala Thr Gly Ser
Asn Arg Arg Leu Gln 1 5 10 15 Gln Thr Gln Asn Gln Val Asp Glu Val
Val Asp Ile Met Arg Val Asn 20 25 30 Val Asp Lys Val Leu Glu Arg
Asp Gln Lys Leu Ser Glu Leu Asp Asp 35 40 45 Arg Ala Asp Ala Leu
Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser Ala 50 55 60 Ala Lys Leu
Lys Arg Lys Tyr Trp Trp Lys Asn Cys Lys Met Trp Ala 65 70 75 80 Ile
Gly Ile Thr Val Leu Val Ile Phe Ile Ile Ile Ile Ile Val Trp 85 90
95 Val Val Ser Ser 100 58141PRTHomo sapiens 58Met Pro Pro Lys Phe
Lys Arg His Leu Asn Asp Asp Asp Val Thr Gly 1 5 10 15 Ser Val Lys
Ser Glu Arg Arg Asn Leu Leu Glu Asp Asp Ser Asp Glu 20 25 30 Glu
Glu Asp Phe Phe Leu Arg Gly Pro Ser Gly Pro Arg Phe Gly Pro 35 40
45 Arg Asn Asp Lys Ile Lys His Val Gln Asn Gln Val Asp Glu Val Ile
50 55 60 Asp Val Met Pro Glu Asn Ile Thr Lys Val Ile Glu Arg Gly
Glu Arg 65 70 75 80 Leu Asp Glu Leu Gln Asp Lys Ser Glu Ser Leu Ser
Asp Asn Ala Thr 85 90 95 Ala Phe Ser Asn Arg Ser Lys Gln Leu Arg
Arg Gln Met Trp Trp Arg 100 105 110 Gly Cys Lys Ile Lys Ala Ile Met
Ala Leu Val Ala Ala Ile Leu Leu 115 120 125 Leu Val Ile Ile Ile Leu
Ile Val Met Lys Tyr Arg Thr 130 135 140 59220PRTHomo sapiens 59Met
Ala Ile Leu Phe Ala Val Val Ala Arg Gly Thr Thr Ile Leu Ala 1 5 10
15 Lys His Ala Trp Cys Gly Gly Asn Phe Leu Glu Val Thr Glu Gln Ile
20 25 30 Leu Ala Lys Ile Pro Ser Glu Asn Asn Lys Leu Thr Tyr Ser
His Gly 35 40 45 Asn Tyr Leu Phe His Tyr Ile Cys Gln Asp Arg Ile
Val Tyr Leu Cys 50 55 60 Ile Thr Asp Asp Asp Phe Glu Arg Ser Arg
Ala Phe Asn Phe Leu Asn 65 70 75 80 Glu Ile Lys Lys Arg Phe Gln Thr
Thr Tyr Gly Ser Arg Ala Gln Thr 85 90 95 Ala Leu Pro Tyr Ala Met
Asn Ser Glu Phe Ser Ser Val Leu Ala Ala 100 105 110 Gln Leu Lys His
His Ser Glu Asn Lys Gly Leu Asp Lys Val Met Glu 115 120 125 Thr Gln
Ala Gln Val Asp Glu Leu Lys Gly Ile Met Val Arg Asn Ile 130 135 140
Asp Leu Val Ala Gln Arg Gly Glu Arg Leu Glu Leu Leu Ile Asp Lys 145
150 155 160 Thr Glu Asn Leu Val Asp Ser Ser Val Thr Phe Lys Thr Thr
Ser Arg 165 170 175 Asn Leu Ala Arg Ala Met Cys Met Lys Asn Leu Lys
Leu Thr Ile Ile 180 185 190 Ile Ile Ile Val Ser Ile Val Phe Ile Tyr
Ile Ile Val Ser Pro Leu 195 200 205 Cys Gly Gly Phe Thr Trp Pro Ser
Cys Val Lys Lys 210 215 220 60100PRTHomo sapiens 60Met Glu Glu Ala
Ser Glu Gly Gly Gly Asn Asp Arg Val Arg Asn Leu 1 5 10 15 Gln Ser
Glu Val Glu Gly Val Lys Asn Ile Met Thr Gln Asn Val Glu 20 25 30
Arg Ile Leu Ala Arg Gly Glu Asn Leu Glu His Leu Arg Asn Lys Thr 35
40 45 Glu Asp Leu Glu Ala Thr Ser Glu His Phe Lys Thr Thr Ser Gln
Lys 50 55 60 Val Ala Arg Lys Phe Trp Trp Lys Asn Val Lys Met Ile
Val Leu Ile 65 70 75 80 Cys Val Ile Val Phe Ile Ile Ile Leu Phe Ile
Val Leu Phe Ala Thr 85 90 95 Gly Ala Phe Ser 100 61203PRTHomo
sapiens 61Met Ser Ser Asp Phe Glu Gly Tyr Glu Gln Asp Phe Ala Val
Leu Thr 1 5 10 15 Ala Glu Ile Thr Ser Lys Ile Ala Arg Val Pro Arg
Leu Pro Pro Asp 20 25 30 Glu Lys Lys Gln Met Val Ala Asn Val Glu
Lys Gln Leu Glu Glu Ala 35 40 45 Lys Glu Leu Leu Glu Gln Met Asp
Leu Glu Val Arg Glu Ile Pro Pro 50 55 60 Gln Ser Arg Gly Met Tyr
Ser Asn Arg Met Arg Ser Tyr Lys Gln Glu 65 70 75 80 Met Gly Lys Leu
Glu Thr Asp Phe Lys Arg Ser Arg Ile Ala Tyr Ser 85 90 95 Asp Glu
Val Arg Asn Glu Leu Leu Gly Asp Asp Gly Asn Ser Ser Glu 100 105 110
Asn Gln Arg Ala His Leu Leu Asp Asn Thr Glu Arg Leu Glu Arg Ser 115
120 125 Ser Arg Arg Leu Glu Ala Gly Tyr Gln Ile Ala Val Glu Thr Glu
Gln 130 135 140 Ile Gly Gln Glu Met Leu Glu Asn Leu Ser His Asp Arg
Glu Lys Ile 145 150 155 160 Gln Arg Ala Arg Glu Arg Leu Arg Glu Thr
Asp Ala Asn Leu Gly Lys 165 170 175 Ser Ser Arg Ile Leu Thr Gly Met
Leu Arg Arg Gly Cys Ser Val Lys 180 185 190 Lys Gln Cys Asn Leu Ser
Leu Ala Pro Lys Ala 195 200 62269PRTHomo sapiens 62Met Arg Asp Arg
Leu Pro Asp Leu Thr Ala Cys Arg Lys Asn Asp Asp 1 5 10 15 Gly Asp
Thr Val Val Val Val Glu Lys Asp His Phe Met Asp Asp Phe 20 25 30
Phe His Gln Val Glu Glu Ile Arg Asn Ser Ile Asp Lys Ile Thr Gln 35
40 45 Tyr Val Glu Glu Val Lys Lys Asn His Ser Ile Ile Leu Ser Ala
Pro 50 55 60 Asn Pro Glu Gly Lys Ile Lys Glu Glu Leu Glu Asp Leu
Asn Lys Glu 65 70 75 80 Ile Lys Lys Thr Ala Asn Lys Ile Arg Ala Lys
Leu Lys Ala Ile Glu 85 90 95 Gln Ser Phe Asp Gln Asp Glu Ser Gly
Asn Arg Thr Ser Val Asp Leu 100 105 110 Arg Ile Arg Arg Thr Gln His
Ser Val Leu Ser Arg Lys Phe Val Glu 115 120 125 Ala Met Ala Glu Tyr
Asn Glu Ala Gln Thr Leu Phe Arg Glu Arg Ser 130 135 140 Lys Gly Arg
Ile Gln Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr Thr 145 150 155 160
Asp Asp Glu Leu Glu Glu Met Leu Glu Ser Gly Lys Pro Ser Ile Phe 165
170 175 Thr Ser Asp Ile Ile Ser Asp Ser Gln Ile Thr Arg Gln Ala Leu
Asn 180 185 190 Glu Ile Glu Ser Arg His Lys Asp Ile Met Lys Leu Glu
Thr Ser Ile 195 200 205 Arg Glu Leu His Glu Met Phe Met Asp Met Ala
Met Phe Val Glu Thr 210 215 220 Gln Gly Glu Met Ile Asn Asn Ile Glu
Arg Asn Val Met Asn Ala Thr 225 230 235 240 Asp Tyr Val Glu His Ala
Lys Glu Glu Thr Lys Lys Ala Ile Lys Tyr 245 250 255 Gln Ser Lys Ala
Arg Arg Val Ser Leu Ala Ser Lys Asn 260 265 63222PRTHomo sapiens
63Gln Met Ala Ala Leu Ala Pro Leu Pro Pro Leu Pro Ala Gln Phe Lys 1
5 10 15 Ser Ile Gln His His Leu Arg Thr Ala Gln Glu His Asp Lys Arg
Asp 20 25 30 Pro Val Val Ala Tyr Tyr Cys Arg Leu Tyr Ala Met Gln
Thr Gly Met 35 40 45 Lys Ile Asp Ser Lys Thr Pro Glu Cys Arg Lys
Phe Leu Ser Lys Leu 50 55 60 Met Asp Gln Leu Glu Ala Leu Lys Lys
Gln Leu Gly Asp Asn Glu Ala 65 70 75 80 Ile Thr Gln Glu Ile Val Gly
Cys Ala Leu Glu Asn Tyr Ala Leu Lys 85 90 95 Met Phe Leu Tyr Ala
Asp Asn Glu Asp Arg Ala Gly Arg Phe His Lys 100 105 110 Asn Met Ile
Lys Ser Phe Tyr Thr Ala Ser Leu Leu Ile Asp Val Ile 115 120 125 Thr
Val Phe Gly Glu Leu Thr Asp Glu Asn Val Lys His Arg Lys Tyr 130 135
140 Ala Arg Trp Lys Ala Thr Tyr Ile His Asn Cys Leu Lys Glu Trp Gly
145 150 155 160 Asp Ser Ser Ser Arg Pro Cys Trp Glu Leu Lys Lys Ile
Met Ile Leu 165 170 175 Lys Lys Met Lys Met Leu Glu Gln Pro Leu Cys
Pro Leu Ser Gln Leu 180 185 190 Ser His His His Leu Gln Leu Met Thr
Gln Gln His Ala Ile Arg Gln 195 200 205 Leu Tyr Trp Asn Thr Asp Ser
Ser Gly Cys Thr Arg Ser Ser 210 215 220 641527DNAHomo sapiens
64atggaacgaa ggcgtttgtg gggttccatt cagagccgat acatcagcat gagtgtgtgg
60acaagcccac ggagacttgt ggagctggca gggcagagcc tgctgaagga tgaggccctg
120gccattgccg ccctggagtt gctgcccagg gagctcttcc cgccactctt
catggcagcc 180tttgacggga gacacagcca gaccctgaag gcaatggtgc
aggcctggcc cttcacctgc 240ctccctctgg gagtgctgat gaagggacaa
catcttcacc tggagacctt caaagctgtg 300cttgatggac ttgatgtgct
ccttgcccag gaggttcgcc ccaggaggtg gaaacttcaa 360gtgctggatt
tacggaagaa ctctcatcag gacttctgga ctgtatggtc tggaaacagg
420gccagtctgt actcatttcc agagccagaa gcagctcagc ccatgacaaa
gaagcgaaaa 480gtagatggtt tgagcacaga ggcagagcag cccttcattc
cagtagaggt gctcgtagac 540ctgttcctca aggaaggtgc ctgtgatgaa
ttgttctcct acctcattga gaaagtgaag 600cgaaagaaaa atgtactacg
cctgtgctgt aagaagctga agatttttgc aatgcccatg 660caggatatca
agatgatcct gaaaatggtg cagctggact ctattgaaga tttggaagtg
720acttgtacct ggaagctacc caccttggcg aaattttctc cttacctggg
ccagatgatt 780aatctgcgta gactcctcct ctcccacatc catgcatctt
cctacatttc cccggagaag 840gaagagcagt atatcgccca gttcacctct
cagttcctca gtctgcagtg cctgcaggct 900ctctatgtgg actctttatt
tttccttaga ggccgcctgg atcagttgct caggcacgtg 960atgaacccct
tggaaaccct ctcaataact aactgccggc tttcggaagg ggatgtgatg
1020catctgtccc agagtcccag cgtcagtcag ctaagtgtcc tgagtctaag
tggggtcatg 1080ctgaccgatg taagtcccga gcccctccaa gctctgctgg
agagagcctc tgccaccctc 1140caggacctgg tctttgatga gtgtgggatc
acggatgatc agctccttgc cctcctgcct 1200tccctgagcc actgctccca
gcttacaacc ttaagcttct acgggaattc catctccata 1260tctgccttgc
agagtctcct gcagcacctc atcgggctga gcaatctgac ccacgtgctg
1320tatcctgtcc ccctggagag ttatgaggac atccatggta ccctccacct
ggagaggctt 1380gcctatctgc atgccaggct cagggagttg ctgtgtgagt
tggggcggcc cagcatggtc 1440tggcttagtg ccaacccctg tcctcactgt
ggggacagaa ccttctatga cccggagccc 1500atcctgtgcc cctgtttcat gcctaac
1527651296DNAHomo sapiens 65atgggctccg acgtgcggga cctgaacgcg
ctgctgcccg ccgtcccctc cctgggtggc 60ggcggcggct gtgccctgcc tgtgagcggc
gcggcgcagt gggcgccggt gctggacttt 120gcgcccccgg gcgcttcggc
ttacgggtcg ttgggcggcc ccgcgccgcc accggctccg 180ccgccacccc
cgccgccgcc gcctcactcc ttcatcaaac aggagccgag ctggggcggc
240gcggagccgc acgaggagca gtgcctgagc gccttcactg tccacttttc
cggccagttc 300actggcacag ccggagcctg tcgctacggg cccttcggtc
ctcctccgcc cagccaggcg 360tcatccggcc aggccaggat gtttcctaac
gcgccctacc tgcccagctg cctcgagagc 420cagcccgcta ttcgcaatca
gggttacagc acggtcacct tcgacgggac gcccagctac 480ggtcacacgc
cctcgcacca tgcggcgcag ttccccaacc actcattcaa gcatgaggat
540cccatgggcc agcagggctc gctgggtgag cagcagtact cggtgccgcc
cccggtctat 600ggctgccaca cccccaccga cagctgcacc ggcagccagg
ctttgctgct gaggacgccc 660tacagcagtg acaatttata ccaaatgaca
tcccagcttg aatgcatgac ctggaatcag 720atgaacttag gagccacctt
aaagggccac agcacagggt acgagagcga taaccacaca 780acgcccatcc
tctgcggagc ccaatacaga atacacacgc acggtgtctt cagaggcatt
840caggatgtgc gacgtgtgcc tggagtagcc ccgactcttg tacggtcggc
atctgagacc 900agtgagaaac gccccttcat gtgtgcttac ccaggctgca
ataagagata ttttaagctg 960tcccacttac agatgcacag caggaagcac
actggtgaga aaccatacca gtgtgacttc 1020aaggactgtg aacgaaggtt
ttctcgttca gaccagctca aaagacacca aaggagacat 1080acaggtgtga
aaccattcca gtgtaaaact tgtcagcgaa agttctcccg gtccgaccac
1140ctgaagaccc acaccaggac tcatacaggt aaaacaagtg aaaagccctt
cagctgtcgg 1200tggccaagtt gtcagaaaaa gtttgcccgg tcagatgaat
tagtccgcca tcacaacatg 1260catcagagaa acatgaccaa actccagctg gcgctt
1296661179DNAHomo sapiens 66atggaggagc cgcagtcaga tcctagcgtc
gagccccctc tgagtcagga aacattttca 60gacctatgga aactacttcc tgaaaacaac
gttctgtccc ccttgccgtc ccaagcaatg 120gatgatttga tgctgtcccc
ggacgatatt gaacaatggt tcactgaaga cccaggtcca 180gatgaagctc
ccagaatgcc agaggctgct ccccgcgtgg cccctgcacc agcagctcct
240acaccggcgg cccctgcacc agccccctcc tggcccctgt catcttctgt
cccttcccag 300aaaacctacc agggcagcta cggtttccgt ctgggcttct
tgcattctgg gacagccaag 360tctgtgactt gcacgtactc ccctgccctc
aacaagatgt tttgccaact ggccaagacc 420tgccctgtgc agctgtgggt
tgattccaca cccccgcccg gcacccgcgt ccgcgccatg 480gccatctaca
agcagtcaca gcacatgacg gaggttgtga ggcgctgccc ccaccatgag
540cgctgctcag atagcgatgg tctggcccct cctcagcatc ttatccgagt
ggaaggaaat 600ttgcgtgtgg agtatttgga tgacagaaac acttttcgac
atagtgtggt ggtgccctat 660gagccgcctg aggttggctc tgactgtacc
accatccact acaactacat gtgtaacagt 720tcctgcatgg gcggcatgaa
ccggaggccc atcctcacca tcatcacact ggaagactcc 780agtggtaatc
tactgggacg gaacagcttt gaggtgcgtg tttgtgcctg tcctgggaga
840gaccggcgca cagaggaaga gaatctccgc aagaaagggg agcctcacca
cgagctgccc 900ccagggagca ctaagcgagc actgcccaac aacaccagct
cctctcccca gccaaagaag 960aaaccactgg atggagaata tttcaccctt
cagatccgtg ggcgtgagcg cttcgagatg 1020ttccgagagc tgaatgaggc
cttggaactc aaggatgccc aggctgggaa ggagccaggg 1080gggagcaggg
ctcactccag ccacctgaag tccaaaaagg gtcagtctac ctcccgccat
1140aaaaaactca tgttcaagac agaagggcct gactcagac 1179
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