U.S. patent application number 15/187174 was filed with the patent office on 2016-11-17 for compositions and methods of identifying tumor specific neoantigens.
The applicant listed for this patent is DANA-FARBER CANCER INSTITUTE INC., THE GENERAL HOSPITAL CORPORATION. Invention is credited to Nir Hacohen, Catherine Ju-Ying Wu.
Application Number | 20160331822 15/187174 |
Document ID | / |
Family ID | 44915022 |
Filed Date | 2016-11-17 |
United States Patent
Application |
20160331822 |
Kind Code |
A1 |
Hacohen; Nir ; et
al. |
November 17, 2016 |
COMPOSITIONS AND METHODS OF IDENTIFYING TUMOR SPECIFIC
NEOANTIGENS
Abstract
The present invention related to immunotherapeutic peptides and
their use in immunotherapy, in particular the immunotherapy of
cancer. Specifically, the invention provides a method of
identifying tumor specific neoantigens that alone or in combination
with other tumor-associated peptides serve as active pharmaceutical
ingredients of vaccine compositions which stimulate anti-tumor
responses.
Inventors: |
Hacohen; Nir; (Brookline,
MA) ; Wu; Catherine Ju-Ying; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANA-FARBER CANCER INSTITUTE INC.
THE GENERAL HOSPITAL CORPORATION |
Boston
Boston |
MA
MA |
US
US |
|
|
Family ID: |
44915022 |
Appl. No.: |
15/187174 |
Filed: |
June 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14794449 |
Jul 8, 2015 |
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15187174 |
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13108610 |
May 16, 2011 |
9115402 |
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14794449 |
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61334866 |
May 14, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
G01N 2333/70539 20130101; A61K 45/06 20130101; A61K 2039/53
20130101; A61K 2039/572 20130101; A61K 39/0011 20130101; A61P 43/00
20180101; C12Q 1/6886 20130101; A61P 35/00 20180101; A61K 39/39558
20130101; G16B 15/00 20190201; A61K 2039/57 20130101; G01N 33/57492
20130101; A61P 35/02 20180101; G01N 33/6878 20130101; C12Q 2600/136
20130101; A61P 37/04 20180101; G01N 33/574 20130101; C12Q 2600/156
20130101; A61K 2039/55511 20130101; G01N 33/5011 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of inducing a tumor specific immune response in a
subject in need thereof comprising administering to the subject:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
subject-specific peptides; or (b) one or more polynucleotide
encoding the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 subject-specific peptides; wherein the subject has a tumor
and said subject-specific peptides are specific to the subject's
tumor, wherein each of said subject-specific peptides has a
different tumor neo-epitope that is an epitope specific to the
tumor of the subject, wherein each neo-epitope binds to a HLA
protein of the subject with an IC50 less than 500 nM; and wherein
each neo-epitope represents a tumor-specific non-silent mutation
selected from the group comprising (i) non-synonymous mutations
leading to different amino acids in the protein; (ii) read-through
mutations in which a stop codon is modified or deleted, leading to
translation of a longer protein with a novel tumor-specific
sequence at the C-terminus; (iii) splice site mutations that lead
to the inclusion of an intron in the mature mRNA and thus a unique
tumor-specific protein sequence; (iv) chromosomal rearrangements
that give rise to a chimeric protein with tumor-specific sequences
at the junction of two proteins (i.e., gene fusion); (v) frameshift
mutations or deletions that lead to a new open reading frame with a
novel tumor-specific protein sequence.
2. The method of claim 1 wherein the tumor specific response
comprises the induction of anti-tumor cytotoxic T cells.
3. The method of claim 1 wherein at least one subject-specific
peptide is about 8 to 50 amino acids in length.
4. The method of claim 1 wherein at least one subject-specific
peptide is greater than 15 amino acids in length.
5. The method of claim 1 wherein at least one subject-specific
peptide is about 20 to 40 amino acids in length.
6. The method of claim 1 wherein at least one subject-specific
peptide binds to the HLA protein of the subject with an 1050 less
than 250 nM.
7. The method of claim 1 wherein at least one subject-specific
peptide binds to the HLA protein of the subject with an 1050 less
than 100 nM.
8. The method of claim 1 wherein at least one subject-specific
peptide binds to the HLA protein of the subject with an 1050 less
than 50 nM.
9. The method of claim 1 comprising administering 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subject-specific
peptides.
10. The method of claim 9 further comprising administering a
peptide epitope that is capable of inducing a T helper cell
response.
11. The method of claim 10 wherein at least one subject specific
peptide is linked to the peptide epitope that is capable of
inducing a T helper cell response.
12. The method of claim 1 comprising administering one or more
polynucleotide encoding the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 subject-specific peptides.
13. The method of claim 12 further comprising administering a
polynucleotide encoding an epitope that is capable of inducing a T
helper cell response.
14. The method of claim 12, wherein the one or more polynucleotide
encoding the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 subject-specific peptides comprises a minigene.
15. The method of claim 14, wherein the minigene encodes at least
one peptide epitope that is capable of inducing a T helper cell
response.
16. The method of claim 12, wherein the one or more polynucleotide
encoding the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 subject-specific peptides comprises a viral vector.
17. The method of claim 1 wherein the tumor is a solid tumor.
18. The method of claim 1 wherein the tumor is a hematological
tumor.
19. The method of claim 1 wherein the tumor a breast tumor, an
ovarian tumor, a prostate tumor, a lung tumor, a kidney tumor, a
gastric tumor, a colon tumor, a testicular tumor, a head and neck
tumor, a pancreatic tumor, a brain tumor, a melanoma, a lymphoma or
a leukemia.
20. The method of claim 1 further comprising administering an
adjuvant.
21. The method of claim 1 further comprising administering a
carrier.
22. The method of claim 1 further comprising administering one or
more additional cancer therapeutic agent.
23. The method of claim 22 wherein the additional cancer
therapeutic agent comprises a chemotherapeutic agent, radiation, or
immunotherapy.
24. The method of claim 1 further comprising administering an
anti-immunosuppressive/immunostimulatory agent.
25. The method of claim 24 wherein the
anti-immunosuppressive/immunostimulatory agent provides a CTLA4, a
PD-1, or a PD-L1 blockade.
26. The method of claim 24 wherein the
anti-immunosuppressive/immunostimulatory agent comprises an
anti-CTLA4 antibody, an anti-PD 1 antibody, or an anti-PD-L1
antibody.
27. The method of claim 1, wherein the tumor is surgically removed
and the subject specific peptide or one or more polynucleotide is
administered at the time of the surgery.
28. The method of claim 28, wherein the subject specific peptide or
one or more polynucleotide is administered at the site of surgical
excision.
29. A method of vaccinating a subject in need thereof against a
tumor comprising: administering to the subject: (a) 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subject-specific
peptides; or (b) one or more polynucleotide encoding the 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
subject-specific peptides; or wherein the subject has a tumor and
said subject-specific peptides are specific to the subject's tumor,
wherein each of said subject-specific peptides has a different
tumor neo-epitope that is an epitope specific to the tumor of the
subject, wherein each neo-epitope binds to a HLA protein of the
subject with an IC50 less than 500 nM; and wherein each neo-epitope
represents a tumor-specific non-silent mutation selected from the
group comprising (i) non-synonymous mutations leading to different
amino acids in the protein; (ii) read-through mutations in which a
stop codon is modified or deleted, leading to translation of a
longer protein with a novel tumor-specific sequence at the
C-terminus; (iii) splice site mutations that lead to the inclusion
of an intron in the mature mRNA and thus a unique tumor-specific
protein sequence; (iv) chromosomal rearrangements that give rise to
a chimeric protein with tumor-specific sequences at the junction of
two proteins (i.e., gene fusion); (v) frameshift mutations or
deletions that lead to a new open reading frame with a novel
tumor-specific protein sequence.
30. A method of treating cancer a subject in need thereof
comprising: administering to the subject (a) 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subject-specific peptides;
or (b) one or more polynucleotide encoding the 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subject-specific
peptides; wherein the subject has a cancer and said
subject-specific peptides are specific to the subject's cancer,
wherein each of said subject-specific peptides has a different
cancer neo-epitope that is an epitope specific to the cancer of the
subject, wherein each neo-epitope binds to a HLA protein of the
subject with an IC50 less than 500 nM; and wherein each neo-epitope
represents a cancer-specific non-silent mutation selected from the
group comprising (i) non-synonymous mutations leading to different
amino acids in the protein; (ii) read-through mutations in which a
stop codon is modified or deleted, leading to translation of a
longer protein with a novel cancer-specific sequence at the
C-terminus; (iii) splice site mutations that lead to the inclusion
of an intron in the mature mRNA and thus a unique cancer-specific
protein sequence; (iv) chromosomal rearrangements that give rise to
a chimeric protein with cancer-specific sequences at the junction
of two proteins (i.e., gene fusion); (v) frameshift mutations or
deletions that lead to a new open reading frame with a novel
cancer-specific protein sequence.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/794,449 filed on Jul. 8, 2015, which is a divisional of U.S.
application Ser. No. 13/108,610 filed May 16, 2011, and which
claims benefit of and priority to U.S. provisional application No.
61/334,866, filed May 14, 2010, which is incorporated herein by
reference in its entirety.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
[0002] The contents of the text file name
"39564-502001US_ST25.txt", which was created on Jul. 19, 2011 and
is 73 KB in size, are hereby incorporated by reference it their
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the
identification of tumor specific neoantigens and the uses of these
neoantigens to produce cancer vaccines.
BACKGROUND OF THE INVENTION
[0004] Tumor vaccines are typically composed of tumor antigens and
immunostimulatory molecules (e.g. cytokines or TLR ligands) that
work together to induce antigen-specific cytotoxic T cells (CTLs)
that recognize and lyse tumor cells. At this time, almost all
vaccines contain either shared tumor antigens or whole tumor cell
preparations (Gilboa, 1999). The shared tumor antigens are
immunogenic proteins with selective expression in tumors across
many individuals and are commonly delivered to patients as
synthetic peptides or recombinant proteins (Boon et al., 2006). In
contrast, whole tumor cell preparations are delivered to patients
as autologous irradiated cells, cell lysates, cell fusions,
heat-shock protein preparations or total mRNA (Parmiani et al.,
2007). Since whole tumor cells are isolated from the autologous
patient, the cells express patient-specific tumor antigens as well
as shared tumor antigens. Finally, there is a third class of tumor
antigens that has rarely been used in vaccines due to technical
difficulties in identifying them (Sensi et al. 2006). This class
consists of proteins with tumor-specific mutations that result in
altered amino acid sequences. Such mutated proteins have the
potential to: (a) uniquely mark a tumor (relative to non-tumor
cells) for recognition and destruction by the immune system
(Lennerz et al., 2005); (b) avoid central and sometimes peripheral
T cell tolerance, and thus be recognized by more effective, high
avidity T cells receptors (Gotter et al., 2004).
[0005] Thus a need exists for a method of identifying neoepitopes
that are useful as tumor vaccines.
SUMMARY OF THE INVENTION
[0006] The present invention relates in part to the discovery of a
method of identifying peptides that are capable of elicting a tumor
specific T-cell response.
[0007] In one aspect the invention provides methods of identifying
a neoantigen by identifying a tumor specific mutation in an
expressed gene of a subject having cancer. In some aspects when the
mutation is a point mutation the method further comprises
identifying the mutant peptide having the mutation. Preferably the
mutant peptide binds to a class I HLA protein with a greater
affinity than a wild-type peptide and has an IC50 less than 500 nm;
In other aspects when the mutation is a splice-site, frameshift,
read-through or gene-fusion mutation the method further comprise
identifying the mutant polypeptide encoded by the mutation.
Preferably, the mutant polypeptide binds to a class I HLA
protein.
[0008] Optionally, the method further includes selecting peptides
or polypeptides that activate anti-tumor CD8 T cells.
[0009] The mutant peptide or polypeptide preferably binds to a
class I HLA protein with a greater affinity than a wild-type
peptide and has an IC50 less than 500 nM. Preferably, the peptide
or polypeptide has an IC50 less than 250 nM. More preferably, the
peptide or polypeptide has an IC50 less than 100 nM. Most
preferably, the peptide or polypeptide has an IC50 less than 50
nM.
[0010] The mutant peptide is about 8-10 amino acids in length. In
another aspect is about 8-50 amino acids in length. For example,
mutant peptide is greater than 10 amino acids in length, greater
than 15 amino acids in length, greater than 20 amino acids in
length, greater than 30 amino acids in length. Preferably the the
mutant peptides is about 24-40 amino acids in length.
[0011] In a further aspect the invention provides methods of
inducing a tumor specific immune response in a subject by
administering one or more peptides or polypeptides identified
according to the methods of the invention and an adjuvant. The
adjuvant is for example, a TLR-based adjuvant or a mineral oil
based adjuvant. In some aspects the peptide or polypeptide and
TLR-based adjuvant is emulsified with a mineral oil based adjuvant.
Optionally, the method further includes administering an
anti-immunosuppressive agent such as an anti-CTLA-4 antibody, an
anti-PD1 antibody an anti-PD-L1 antibody an anti-CD25 antibody or
an inhibitor of IDO.
[0012] In yet another aspect the invention provides methods of
inducing a tumor specific immune response in a subject by
administering to the subject autologous dendritic cells or antigen
presenting cells that have been pulsed with one or more of the
peptides or polypeptides identified according to the methods of the
inventions. Optionally, the method further includes administering
an adjuvant such as for example, a TLR-based adjuvant or a mineral
oil based adjuvant. In some aspects the peptide or polypeptide and
TLR-based adjuvant is emulsified with a mineral oil based adjuvant.
In some embodiments the method further includes administering an
anti-immunosuppressive agent. Anti-immunosuppressive agents include
for example an anti-CTLA-4 antibody, an anti-PD1 antibody an
anti-PD-L1 antibody an anti-CD25 antibody or an inhibitor of
IDO.
[0013] In another aspect the invention provides a method of
vaccinating or treating a subject for cancer by identifying a
plurality of tumor specific mutations in an expressed gene of the
subject, identifying mutant peptides or polypeptides having the
identified tumor specific mutations, selecting one or more of the
identified mutant peptide or polypeptides that binds to a class I
HLA protein preferably with a greater affinity than a wild-type
peptide and is capable of activating anti-tumor CD8 T-cells, and
administering to the subject the one or more selected peptides,
polypeptides or autologous dendritic cells or antigen presenting
cells pulsed with the one or more identified peptides or
polypeptides. The mutant peptide is about 8-10 amino acids in
length. In another aspect is about 8-50 amino acids in length. For
example, mutant peptide is greater than 10 amino acids in length,
greater than 15 amino acids in length, greater than 20 amino acids
in length, greater than 30 amino acids in length. Preferably, the
mutant peptides is about 24-40 amino acids in length.
[0014] Optionally, the method further includes administering an
adjuvant such as for example, a TLR-based adjuvant or a mineral oil
based adjuvant. In some aspects the peptide or polypeptide and
TLR-based adjuvant is emulsified with a mineral oil based adjuvant.
In some embodiments the method further includes administering an
anti-immunosuppressive agent. Anti-immunosuppressive agents include
for example an anti-CTLA-4 antibody, an anti-PD1 antibody an
anti-PD-L1 antibody an anti-CD25 antibody or an inhibitor of
IDO.
[0015] The method of claim 22, wherein said subject has received a
hematopoietic stem cell transplant.
[0016] The subject is a human, dog, cat, or horse. The cancer is
breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney
cancer, gastric cancer, colon cancer, testicular cancer, head and
neck cancer, pancreatic cancer, brain cancer, melanoma lymphoma,
such as B-cell lumphoma or leukemia, such as cute myelogenous
leukemia, chronic myelogenous leukemia, chronic lymphocytic
leukemia, or T cell lymphocytic leukemia.
[0017] Also included in the invention are pharmaceutical
compositions containing the peptide or polypeptide identified
according the methods of the invention and a pharmaceutically
acceptable carrier.
[0018] For example, the invention provides a composition containing
least two distinct SF3B1 peptides wherein each peptide is equal to
or less than 50 amino acids in length and contains [0019] a leucine
at amino acid position 625; [0020] a histidine at amino acid
position 626; [0021] a glutamic acid at amino acid position 700;
[0022] an aspartic acid at amino acid position 742; or [0023] an
arginine at amino acid position 903, when numbered in accordance
with wild-type SF3B1.
[0024] The invention also provides a composition containing at
least two distinct MYD88 peptides where each peptide is equal to or
less than 50 amino acids in length and contains a threonine at
amino acid position 232; a leucine at amino acid position 258; or a
proline at amino acid position 265, when numbered in accordance
with wild-type MYD88
[0025] The invention further provides composition containing at
least two distinct TP53 peptides where each peptide is equal to or
less than 50 amino acids in length and contains an arginine at
amino acid position 111; an arginine at amino acid position 215; a
serine at amino acid position 238; a glutamine at amino acid
position 248; a phenylalanine at amino acid position 255; a
cysteine at amino acid position 273 or an asparagine at amino acid
position 281, when numbered in accordance with wild-type TP53.
[0026] The invention further provides composition containing at
least two distinct ATM peptides wherein each peptide is equal to or
less than 50 amino acids in length and contain a phenylalanine at
amino acid position 1252; an arginine at amino acid position 2038;
a histidine at amino acid position 2522; or a cysteine at amino
acid position 2954, when numbered in accordance with wild-type
ATM.
[0027] A composition comprising at least two distinct Abl peptides
wherein each peptide is equal to or less than 50 amino acids in
length and contains a valine at amino acid position 244;
a valine at amino acid position 248; a glutamic acid at amino acid
position 250; an alanine at amino acid position 250; a histidine at
amino acid position 252; an arginine at amino acid position 252; a
phenylalanine at amino acid position 253; a histidine at amino acid
position 253; a lysine at amino acid position 255; a valine at
amino acid position 255; a glycine at amino acid position 276; an
isoleucine at amino acid position 315; an asparagine at amino acid
position 315; a leucine at amino acid position 317; a threonine at
amino acid position 343; a threonine at amino acid position 351; a
glycine at amino acid position 355; a valine at amino acid position
359; an alanine at amino acid position 359; an isoleucine at amino
acid position 379; a leucine at amino acid position 382; a
methionine at amino acid position 387; a proline at amino acid
position 396; an arginine at amino acid position 396; a tyrosine at
amino acid position 417; or a serine at amino acid position 486,
when numbered in accordance with wild-type ABL.
[0028] Further included in the invention is a composition
containing at least two distinct FBXW7 peptides where each peptide
is equal to or less than 50 amino acids in length and contains a
leucine at amino acid position 280; a histidine at amino acid
position 465; a cysteine at amino acid position 505; or a glutamic
acid at amino acid position 597, when numbered in accordance with
wild-type FBXW7.
[0029] In a further a aspect the invention provides a composition
containing at least two distinct MAPK1 peptides where each peptide
is equal to or less than 50 amino acids in length and contains an
asparagine at amino acid position 162; a glycine at amino acid
position 291; or a phenylalanine at amino acid position 316, when
numbered in accordance with wild-type MAPK1.
[0030] The invention also provides a composition containing at
least two distinct GNB1 peptides wherein each peptide is equal to
or less than 50 amino acids in length and contains a threonine at
amino acid position 180, when numbered in accordance with wild-type
GNB1.
[0031] Also provided by the invention is a method of treating a
subject with an imatinib resistant tumor to a HLA-A3 positive
subject a composition of Bcr-abl peptide equal to or less than 50
amino acid in length that contains a lysine at position 255 when
numbered in accordance with wild-type bcr-abl.
[0032] Further provided by the invention, is method of treating a
subject with an imatinib resistant tumor comprising administering
to the subject one or more peptides containing a bcr-abl mutation
where the peptide is equal to or less than 50 amino acid and binds
to a class I HLA protein with an IC50 less than 500 nm.
[0033] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are expressly incorporated by reference in their
entirety. In cases of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples described herein are illustrative only and
are not intended to be limiting.
[0034] Other features and advantages of the invention will be
apparent from and encompassed by the following detailed description
and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows the balance of specificity and autoimmune
toxicity using 3 classes of antigens for tumor vaccines. Whole
tumor cells may be the the least specific antigen formulation for
tumor vaccines since the full set of protein antigens expressed in
tumor cells include thousands of proteins that are also present in
other cells of the body. Overexpressed tumor antigens are slightly
more specific because they have been selected for much higher and
more selective expression in tumors compared to other cells in the
body. Nevertheless, it is impossible to test every cell in the body
for the expression of these antigens and there is a substantial
risk that other cells express them. Finally, mutated proteins
generate neoepitopes that are present only in tumor cells and
provide the greatest level of specificity.
[0036] FIG. 2 is a schema for a personalized neoantigen vaccination
strategy that can be applied to the treatment of any cancer. We
also highlight the possibility of applying this strategy in two
unique scenarios. In the first case, a patient is vaccinated in the
early period following hematopoietic stem cell transplantation
(HSCT) (e.g. as is done for CLL, CML and other leukemias). The
early post-HSCT period is a unique therapeutic setting as the
immune system is competent due to reconstitution with HSCT, thus
overcoming tumor- or treatment-induced host immune defects.
Moreover, the abundance of homeostatic cytokines in a lymphopenia
milieu, such as in the early post-HSCT setting, can contribute to
rapid expansion of T cells. In the second case, a patient is
vaccinated early in the disease course when immune competence may
be more intact in the early stages of disease, before impairment by
exposure to chemotherapy (e.g. for solid or hematopoeitic tumors).
Since the immune system is likely to be most active in these two
specific situations, we suggest that these are the ideal situations
for applying tumor vaccination strategies.
[0037] FIG. 3 shows a strategy for identifying tumor neoepitopes is
described in 3 steps: (1) using sequencing technologies, detect
gene mutations that are present in tumor but not germline DNA of a
single patient; (2) using prediction algorithms, predict whether
mutated peptides have the potential to bind personal HLA allele;
these predicted peptides may optionally be tested experimentally
for binding to appropriate HLA proteins. In addition, these genes
must also be expressed in tumor cells. (3) generate T cells ex vivo
and test whether they are able to recognize cells expressing the
mutated protein; alternatively, mass spectrometry can be used to
detect peptides eluted from tumor cell surface HLA proteins. For
chronic lymphocytic leukemia, our studies to date demonstrate that
there are an average of 23 protein-altering mutations per patient,
46 predicted binding mutant peptides and 15-25 validated binding
mutant peptides. Of these, we anticipate that .about.7-12 peptides
are expressed and processed in tumor cells (though this may differ
across tumors and patients).
[0038] FIG. 4 shows five classes of mutations generate potential
tumor neoepitopes. New tumor-specific epitopes can arise as a
result of missense, splice-site, frameshift or read-through point
mutations (red asterisk), or from the fusion of two genes (or
within the same gene). In particular, splice-site, frameshift,
read-through mutations and gene fusions can each generate novel
stretches of amino acids (in magenta) that are normally not
translated, but now are expressed and translated as a result of
mutation. Missense mutations lead to peptides with single amino
acid changes.
[0039] FIG. 5 shows the frequency of mutations per class in CLL
patients. Our studies applying next-generation sequencing to a
series of 7 CLL tumors reveal that CLL cells harbor many mutations
that provide a rich source of possible mutated peptides. We observe
that the total number of nonsilent gene alterations in CLL ranged
from 17-155 per individual, the majority of which were somatically
altered point mutations (missense). The tumors of 4 patients also
harbored splice-site mutations; for 3 patients, novel gene fusions
were identified by RNA sequencing.
[0040] FIG. 6 shows data from automated predictions (Step 2A of the
strategy in FIG. 3) of peptide binding (for peptides that harbor a
specific missense mutation) against each of a patient's 6 HLA (MHC
Class I) alleles. Magenta=strong binders; green=intermediate
binders.
[0041] FIG. 7 shows methods for confirming RNA expression of
mutated genes (Step 2B of the strategy in FIG. 3). A. For CLL
patient 7, we found that more than half of the mutated genes with
predicted HLA-binding peptides were expressed at the RNA level. B.
We have also used RNA pyrosequencing to detect expressed RNAs
harboring specific mutations found in DNA. C. We can validate novel
gene fusions that were seen by DNA sequencing using PCR-TOPO
cloning of the breakpoint region (depicted is a fusion discovered
for patient 2).
[0042] FIG. 8A-C shows a method and data for experimental
validation of HLA-peptide binding (Step 2C of the strategy in FIG.
3). A. Schema for experimental validation of peptide binding to
specific HLA alleles. B. Summary of candidate mutated peptides
identified in patients 1 and 2. Shaded cells indicate that analysis
is in progress. C. Data for predicted vs experimentally verified
binding affinity of peptides generated from gene alterations
(missense mutation or gene fusion) for patient 2. A prediction
cutoff of IC.sub.50<120 nM (solid vertical line on left) results
in all peptides showing experimental binding to class I HLA.
[0043] FIG. 9 shows predicted differential binding of mutated vs
germline (i.e also called parental, wild type or normal) peptides
to HLA alleles. 12 of 25 predicted HLA binding mutated peptides of
Pt 2 have >2 fold greater binding (cutoff=red dotted line) than
parental peptides. This further increases the specificity of
mutated peptides. Mutated peptides are specific for two reasons:
first, many of the T cell receptors that recognize a mutated
peptide are not likely to detect the wild type parental peptide;
second, some of the mutated peptides can bind HLA with higher
affinity than the parental peptide. Since the first property cannot
be computationally predicted, we will focus on predicting the
second property and selecting for inclusion in vaccines only those
peptides that show higher binding to HLA for mutated relative to
wild type peptides.
[0044] FIG. 10 shows T cell reactivity against a candidate personal
CLL neoepitope (Step 3 of the strategy in FIG. 3). We observed that
T cells isolated from patient 1 post-therapy can detect a specific
mutated TLK2 peptide (peptide #7) (using the Elispot assay).
[0045] FIG. 11 shows that BCR-ABL mutations generate many peptides
predicted to bind HLA-A and HLA-B alleles. By applying the NetMHC
prediction algorithm (Nielsen et al. PLoS One. 2007, 2(8):e796), we
predicted peptides generated from the BCR-ABL mutations with
potential to bind to 8 common HLA-A and -B alleles. The most common
BCR-ABL mutations are ordered in decreasing frequency (from left to
right), and predicted IC50 of various class I MHC binding peptides
are depicted. In total, we predicted 84 peptides to bind with good
affinity, defined as an IC50 of less than 1000, across a wide range
of HLA alleles. Of all the predicted peptides, 24 of 84 (29%) were
predicted to be strong binders with an IC50<50. 42 peptides
(50%) were intermediate binders, defined as IC50 between 50 and
500. 18 peptides (21%) were weak binders defined as IC50 between
500 and 1000.
[0046] FIG. 12A-D shows BCR-ABL peptide harboring the E255K
mutation binds HLA proteins and is associated with specific,
polyfunctional T cells present in CML patients. A.
Experimentally-derived binding scores of E255K-B (and parental
peptide) to HLA A3 and supertype members. B. In CD8+ T cells
expanded from a HLAA3+E255K+patient following HSCT, we detected
IFNgamma secretion against the E255K-B (MUT) peptide and A3+
expressing APCs expressing the E255K minigene (MG). This response
was abrogated in the presence of the class I blocking antibody
(w6/32). C. IFNgamma-secreting cells were also tetramer+ for the
mutated peptide and were (D) polyfunctional, secreting IP10,
TNFalpha and GM-CSF (based on the Luminex assay).
[0047] FIG. 13A-C shows that patient-derived T cell clones can
recognize tumor-specific epitopes and kill cells presenting these
epitopes. A. Reactivity to the CD8+ T cell epitope of CML66
(peptide 66-72C) is restricted by HLA B-4403. B. CML66 mRNA can be
efficiently nucleofected into CD40L-expanded B cells. C.
CML66-specific CD8+ T cells are cytotoxic to CD40L B cells
expressing CML66 by RNA nucleofection or by peptide pulse, but not
control targets.
[0048] FIG. 14A-C shows significantly mutated genes in CLL. A. The
9 most significantly mutated genes among 64 CLL samples. N--total
covered territory in base pairs across 64 sequenced samples. p- and
q-values were calculated by comparing the probability of seeing the
observed constellation of mutations to the background mutation
rates calculated across the dataset. Red bars--genes not previously
known to be mutated in CLL; grey bars--genes in which mutation in
CLL has been previously reported. B-C. Type (missense, splice-site,
nonsense) and location of mutations in ATM, SF3B1, TP53, MYD88,
FBXW7, DDX3X, MAPK1, and GNB1 discovered among the 64 CLLs
(position and mutation in CLL samples shown above the gene)
compared to previously reported mutations in literature or in the
COSMIC database (lines show position of mutations below the
gene).
[0049] FIG. 15 shows that SF3B1 is expressed in CLL samples (7th
column in graph) and has higher expression than many control cells,
including: PBMC, M: monocyte, CC: cancer cell lines (includes K562,
Jurkat, IM9, MCF-7, Hela, Ovcar, RPMI, OTM, MCF-CAR, KM12BM and
MM1S).
[0050] FIG. 16 shows that SF3B1 mutations generate peptides that
are predicted to bind to patient-specific HLA alleles. For example,
one peptide that includes the common SF3B1 K700E mutation is
predicted to bind HLA strongly.
DETAILED DESCRIPTION OF THE INVENTION
[0051] One of the critical barriers to developing curative and
tumor-specific immunotherapy is the identification and selection of
highly restricted tumor antigens to avoid autoimmunity. Tumor
neoantigens, which arise as a result of genetic change within
malignant cells, represent the most tumor-specific class of
antigens. Neoantigens have rarely been used in vaccines due to
technical difficulties in identifying them. Our approach to
identify tumor-specific neoepitopes involves three steps. (1)
identification of DNA mutations using whole genome or whole exome
(i.e. only captured exons) or RNA sequencing of tumor versus
matched germline samples from each patient; (2) application of
validated peptide-MHC binding prediction algorithms to generate a
set of candidate T cell epitopes that may bind patient HLA alleles
and are based on non-silent mutations present in tumors; and (3)
optional demonstration of antigen-specific T cells against mutated
peptides or demonstration that a candidate peptide is bound to HLA
proteins on the tumor surface.
[0052] Accordingly, the present invention relates to methods for
identifying and/or detecting T-cell epitopes of an antigen.
Specifically, the invention provides method of identifying and/or
detecting tumor specific neoantigens that are useful in inducing a
tumor specific immune response in a subject.
[0053] In particular, the invention provides a method of
vaccinating or treating a subject by identifying a plurality of
tumor specific mutations in the genome of a subject. Mutant
peptides and polypeptides having the identified mutations and that
binds to a class I HLA protein are selected. Optionally, these
peptide and polypeptides binds to a class I HLA proteins with a
greater affinity than the wild-type peptide and/or are capable of
activating anti-tumor CD8 T-cells These peptides are administered
to the subject. Alternatively, autologous antigen-presenting cells
that have been pulsed with the peptides are administered.
[0054] The importance of mutated antigens, or neoepitopes, in the
immune control of tumors has been appreciated in seminal studies
showing that: (a) mice and humans often mount T cell responses to
mutated antigens (Parmiani et al., 2007; Sensi and Anichini, 2006);
(b) mice can be protected from a tumor by immunization with a
single mutated peptide that is present in the tumor (Mandelboim et
al., 1995); (c) spontaneous or vaccine-mediated long-term melanoma
survivors mount strong memory cytotoxic T cell (CTL) responses to
mutated antigens (Huang et al., 2004; Lennerz et al., 2005; Zhou et
al., 2005a); (d) finally, lymphoma patients show molecular
remission when immunized with patient-specific mutated
immunoglobulin proteins that are present in autologous tumor cells.
(Baskar et al., 2004). Furthermore, the CTL responses in these
patients are directed toward the mutated rather than shared regions
of the immunoglobulin protein. Additionally, such mutated peptides
have the potential to: (a) uniquely mark a tumor for recognition
and destruction by the immune system, thus reducing the risk for
autoimmunity; and (b) avoid central and peripheral T cell
tolerance, allowing the antigen to be recognized by more effective,
high avidity T cells receptors. (FIG. 1)
[0055] Identical mutations in any particular gene are rarely found
across tumors (and are even at low frequency for the most common
driver mutations). Thus, the methods of the present invention will
comprehensively identify patient-specific tumor mutations. Using
highly parallel sequencing technologies, HLA-peptide binding
prediction tools and biochemical assays the methods of the
invention will allow: (1) comprehensive identification of mutated
peptides that are expressed and bind HLA proteins present in a
patient's tumor; (2) monitoring of the natural immune response of
cancer patients to these identified neoepitopes; (3) determining
whether cytotoxic T cells that recognize these peptides in the
context of patient HLA proteins can selectively lyse autologous
tumor cells ex vivo. This strategy addresses several fundamental
questions related to how the immune system of cancer patients
interacts with tumor neoepitopes. These include: which and what
fraction of tumor neoepitopes are detected by T cells, how many T
cell precursors are able to respond to neoepitopes, how frequent
are neoepitope-specific memory and effector T cells in circulation
and in the tumor, how much avidity do T cells have for these
epitopes, are neoepitope-specific T cells functional? The answers
to these questions provide both the justification and strategy for
using tumor neoepitopes in human vaccines.
[0056] The immune system of a human can be classified into two
functional subsystems, i.e., the innate and the acquired immune
system. The innate immune system is the first line of defense
against infections, and most potential pathogens are rapidly
neutralized before they can cause, for example, a noticeable
infection. The acquired immune system reacts to molecular
structures, referred to as antigens, of the intruding organism.
There are two types of acquired immune reactions, i.e. the humoral
immune reaction and the cell-mediated immune reaction. In the
humoral immune reaction, the antibodies secreted by B cells into
bodily fluids bind to pathogen-derived antigens, leading to the
elimination of the pathogen through a variety of mechanisms, e.g.
complement-mediated lysis. In the cell-mediated immune reaction,
T-cells capable of destroying other cells are activated. If, for
example, proteins associated with a disease are present in a cell,
they are, within the cell, fragmented proteolytically to peptides.
Specific cell proteins then attach themselves to the antigen or
peptide formed in this manner and transport them to the surface of
the cell, where they are presented to the molecular defense
mechanisms, in particular T-cells, of the body. Cytotoxic T cells
recognize these antigens and kill the cells that harbor the
antigens.
[0057] The molecules which transport and present peptides on the
cell surface are referred to as proteins of the major
histocompatibility complex (MHC). The MHC proteins are classified
into MHC proteins of class I and of class II. The structures of the
proteins of the two MHC classes are very similar; however, they
differ quite considerably in their function. Proteins of MHC class
I are present on the surface of almost all cells of the body,
including most tumor cells. The proteins of MHC class I are loaded
with antigens that usually originate from endogenous proteins or
from pathogens present inside cells, and are then presented to
cytotoxic T-lymphocytes (CTLs). The MHC proteins of class II are
only present on dendritic cells, B-lymphocytes, macrophages and
other antigen-presenting cells. They present mainly peptides, which
are processed from external antigen sources, i.e. outside of the
cells, to T-helper (Th) cells. Most of the peptides bound by the
MHC proteins of class I originate from cytoplasmic proteins
produced in the healthy host organism itself and don't normally
stimulate an immune reaction. Accordingly, cytotoxic T-lymphocytes
which recognize such self-peptide-presenting MHC molecules of class
I are deleted in the thymus or, after their release from the
thymus, are deleted or inactivated, i.e. tolerized. MHC molecules
are only capable of stimulating an immune reaction when they
present peptides to non-tolerized cytotoxic T-lymphocytes.
Cytotoxic T-lymphocytes have, on their surface, both T-cell
receptors (TCR) and CD8 molecules. T-Cell receptors are capable of
recognizing and binding peptides complexed with the molecules of
MHC class I. Each cytotoxic T-lymphocyte expresses a unique T-cell
receptor which is capable of binding specific MHC/peptide
complexes.
[0058] The peptides attach themselves to the molecules of MHC class
I by competitive affinity binding within the endoplasmic reticulum,
before they are presented on the cell surface. Here, the affinity
of an individual peptide is directly linked to its amino acid
sequence and the presence of specific binding motifs in defined
positions within the amino acid sequence. If the sequence of such a
peptide is known, it is possible, for example, to manipulate the
immune system against diseased cells using, for example, peptide
vaccines.
[0059] Using computer algorithms, it is possible to predict
potential T-cell epitopes, i.e. peptide sequences, which are bound
by the MHC molecules of class I or class II in the form of a
peptide-presenting complex and then, in this form, recognized by
the T-cell receptors of T-lymphocytes. Currently, use is made, in
particular, of two programs, namely SYFPEITHI (Rammensee et al.,
Immunogenetics, 50 (1999), 213-219) and HLA_BIND (Parker et al., J.
Immunol., 152 (1994), 163-175). The peptide sequences determined in
this manner, which potentially may bind to MHC molecules of class
I, then have to be examined in vitro for their actual binding
capacity.
[0060] The technical object of the present invention is to provide
an improved method for identifying and screening potential T-cell
epitopes present in tumor cells, which method allows for
simultaneous and rapid examination of a large number of peptide
sequences, for their capability of binding to specific MHC
molecules.
[0061] In the present invention, the technical object on which it
is based is achieved by providing a method for identifying and/or
detecting mutated antigens that are present in tumors but not in
normal tissue. The method uses massively parallel genomic
sequencing of the entire coding portion of a cancer patient genome
to identify the specific mutated genes in a tumor. In order to
narrow down the mutant peptides to those with potential to bind
more strongly to HLA than the wild type peptides and thus confer
tumor specificity, well-established algorithms will be used to
predict peptides that bind any of the 6 unique class I HLA alleles
of each patient and a predicted IC50 for all 9- or 10-mer peptides
with tumor-specific mutant residues vs. those with the germline
residue will be calculated. Typically, peptides with predicted
IC50<50 nM, are generally considered medium to high affinity
binding peptides and will be selected for testing their affinity
empirically using biochemical assays of HLA-binding. Finally, it
will be determined whether the human immune system can mount
effective immune responses against these mutated tumor antigens and
thus effectively kill tumor but not normal cells.
DEFINITIONS
[0062] A "T-cell epitope" is to be understood as meaning a peptide
sequence which can be bound by the MHC molecules of class I or II
in the form of a peptide-presenting MHC molecule or MHC complex and
then, in this form, be recognized and bound by cytotoxic
T-lymphocytes or T-helper cells, respectively
[0063] A "receptor" is to be understood as meaning a biological
molecule or a molecule grouping capable of binding a ligand. A
receptor may serve, to transmit information in a cell, a cell
formation or an organism. The receptor comprises at least one
receptor unit and preferably two receptor units, where each
receptor unit may consist of a protein molecule, in particular a
glycoprotein molecule. The receptor has a structure which
complements that of a ligand and may complex the ligand as a
binding partner. The information is transmitted in particular by
conformational changes of the receptor following complexation of
the ligand on the surface of a cell. According to the invention, a
receptor is to be understood as meaning in particular proteins of
MHC classes I and II capable of forming a receptor/ligand complex
with a ligand, in particular a peptide or peptide fragment of
suitable length.
[0064] A "ligand" is to be understood as meaning a molecule which
has a structure complementary to that of a receptor and is capable
of forming a complex with this receptor. According to the
invention, a ligand is to be understood as meaning in particular a
peptide or peptide fragment which has a suitable length and
suitable binding motives in its amino acid sequence, so that the
peptide or peptide fragment is capable of forming a complex with
proteins of MHC class I or MHC class II.
[0065] A "receptor/ligand complex" is also to be understood as
meaning a "receptor/peptide complex" or "receptor/peptide fragment
complex", in particular a peptide- or peptide fragment-presenting
MHC molecule of class I or of class II.
[0066] "Proteins or molecules of the major histocompatibility
complex (MHC)", "MHC molecules", "MHC proteins" or "HLA proteins"
are to be understood as meaning, in particular, proteins capable of
binding peptides resulting from the proteolytic cleavage of protein
antigens and representing potential T-cell epitopes, transporting
them to the cell surface and presenting them there to specific
cells, in particular cytotoxic T-lymphocytes or T-helper cells. The
major histocompatibility complex in the genome comprises the
genetic region whose gene products expressed on the cell surface
are important for binding and presenting endogenous and/or foreign
antigens and thus for regulating immunological processes. The major
histocompatibility complex is classified into two gene groups
coding for different proteins, namely molecules of MHC class I and
molecules of MHC class II. The molecules of the two MHC classes are
specialized for different antigen sources. The molecules of MHC
class I present endogenously synthesized antigens, for example
viral proteins and tumor antigens. The molecules of MHC class II
present protein antigens originating from exogenous sources, for
example bacterial products. The cellular biology and the expression
patterns of the two MEW classes are adapted to these different
roles.
[0067] MEW molecules of class I consist of a heavy chain and a
light chain and are capable of binding a peptide of about 8 to 11
amino acids, but usually 9 or 10 amino acids, if this peptide has
suitable binding motifs, and presenting it to cytotoxic
T-lymphocytes. The peptide bound by the MEW molecules of class I
originates from an endogenous protein antigen. The heavy chain of
the MEW molecules of class I is preferably an HLA-A, HLA-B or HLA-C
monomer, and the light chain is .beta.-2-microglobulin.
[0068] MEW molecules of class II consist of an .alpha.-chain and a
.beta.-chain and are capable of binding a peptide of about 15 to 24
amino acids if this peptide has suitable binding motifs, and
presenting it to T-helper cells. The peptide bound by the MHC
molecules of class II usually originates from an extracellular of
exogenous protein antigen. The .alpha.-chain and the .beta.-chain
are in particular HLA-DR, HLA-DQ and HLA-DP monomers.
[0069] A "vaccine" is to be understood as meaning a composition for
generating immunity for the prophylaxis and/or treatment of
diseases. Accordingly, vaccines are medicaments which comprise
antigens and are intended to be used in humans or animals for
generating specific defense and protective substance by
vaccination.
[0070] "Isolated" means that the polynucleotide or polypeptide or
fragment, variant, or derivative thereof has been essentially
removed from other biological materials with which it is naturally
associated, or essentially free from other biological materials
derived, e.g., from a recombinant host cell that has been
genetically engineered to express the polypeptide of the
invention.
[0071] "Neoantigen" means a class of tumor antigens which arises
from tumor-specific mutations in expressed protein.
[0072] "Purified" means that the polynucleotide or polypeptide or
fragment, variant, or derivative thereof is substantially free of
other biological material with which it is naturally associated, or
free from other biological materials derived, e.g., from a
recombinant host cell that has been genetically engineered to
express the polypeptide of the invention. That is, e.g., a purified
polypeptide of the present invention is a polypeptide that is at
least about 70-100% pure, i.e., the polypeptide is present in a
composition wherein the polypeptide constitutes about 70-100% by
weight of the total composition. In some embodiments, the purified
polypeptide of the present invention is about 75%-99% by weight
pure, about 80%-99% by weight pure, about 90-99% by weight pure, or
about 95% to 99% by weight pure.
Identification of Tumor Specific Mutations
[0073] The present invention is based, on the identification of
certain mutations (e.g., the variants or alleles that are present
in cancer cells). In particular, these mutations are present in the
genome of cancer cells of a subject having cancer but not in normal
tissue from the subject.
[0074] Genetic mutations in tumors would be considered useful for
the immunological targeting of tumors if they lead to changes in
the amino acid sequence of a protein exclusively in the tumor.
Useful mutations include: (1) non-synonymous mutations leading to
different amino acids in the protein; (2) read-through mutations in
which a stop codon is modified or deleted, leading to translation
of a longer protein with a novel tumor-specific sequence at the
C-terminus; (3) splice site mutations that lead to the inclusion of
an intron in the mature mRNA and thus a unique tumor-specific
protein sequence; (4) chromosomal rearrangements that give rise to
a chimeric protein with tumor-specific sequences at the junction of
2 proteins (i.e., gene fusion); (5) frameshift mutations or
deletions that lead to a new open reading frame with a novel
tumor-specific protein sequence.
[0075] Peptides with mutations or mutated polypeptides arising from
for example, splice-site, frameshift, readthrough, or gene fusion
mutations in tumor cells may be identified by sequencing DNA, RNA
or protein in tumor versus normal cells.
[0076] Also within the scope of the inventions are peptides
including previous identified tumor specific mutations. Know tumor
mutation can be found at the Catalogue of Somatic Mutations in
Cancer (COSMIC).
[0077] A variety of methods are available for detecting the
presence of a particular mutation or allele in an individual's DNA
or RNA. Advancements in this field have provided accurate, easy,
and inexpensive large-scale SNP genotyping. Most recently, for
example, several new techniques have been described including
dynamic allele-specific hybridization (DASH), microplate array
diagonal gel electrophoresis (MADGE), pyrosequencing,
oligonucleotide-specific ligation, the TaqMan system as well as
various DNA "chip" technologies such as the Affymetrix SNP chips.
These methods require amplification of the target genetic region,
typically by PCR. Still other newly developed methods, based on the
generation of small signal molecules by invasive cleavage followed
by mass spectrometry or immobilized padlock probes and
rolling-circle amplification, might eventually eliminate the need
for PCR. Several of the methods known in the art for detecting
specific single nucleotide polymorphisms are summarized below. The
method of the present invention is understood to include all
available methods.
[0078] PCR based detection means can include multiplex
amplification of a plurality of markers simultaneously. For
example, it is well known in the art to select PCR primers to
generate PCR products that do not overlap in size and can be
analyzed simultaneously. Alternatively, it is possible to amplify
different markers with primers that are differentially labeled and
thus can each be differentially detected. Of course, hybridization
based detection means allow the differential detection of multiple
PCR products in a sample. Other techniques are known in the art to
allow multiplex analyses of a plurality of markers.
[0079] Several methods have been developed to facilitate analysis
of single nucleotide polymorphisms in genomic DNA or cellular RNA.
In one embodiment, the single base polymorphism can be detected by
using a specialized exonuclease-resistant nucleotide, as disclosed,
e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127). According to the
method, a primer complementary to the allelic sequence immediately
3' to the polymorphic site is permitted to hybridize to a target
molecule obtained from a particular animal or human. If the
polymorphic site on the target molecule contains a nucleotide that
is complementary to the particular exonuclease-resistant nucleotide
derivative present, then that derivative will be incorporated onto
the end of the hybridized primer. Such incorporation renders the
primer resistant to exonuclease, and thereby permits its detection.
Since the identity of the exonuclease-resistant derivative of the
sample is known, a finding that the primer has become resistant to
exonucleases reveals that the nucleotide present in the polymorphic
site of the target molecule was complementary to that of the
nucleotide derivative used in the reaction. This method has the
advantage that it does not require the determination of large
amounts of extraneous sequence data.
[0080] In another embodiment of the invention, a solution-based
method is used for determining the identity of the nucleotide of a
polymorphic site. Cohen, D. et al. (French Patent 2,650,840; PCT
Appln. No. WO91/02087). As in the Mundy method of U.S. Pat. No.
4,656,127, a primer is employed that is complementary to allelic
sequences immediately 3' to a polymorphic site. The method
determines the identity of the nucleotide of that site using
labeled dideoxynucleotide derivatives, which, if complementary to
the nucleotide of the polymorphic site will become incorporated
onto the terminus of the primer.
[0081] An alternative method, known as Genetic Bit Analysis or
GBA.RTM. is described by Goelet, P. et al. (PCT Appln. No.
92/15712). The method of Goelet, P. et al. uses mixtures of labeled
terminators and a primer that is complementary to the sequence 3'
to a polymorphic site. The labeled terminator that is incorporated
is thus determined by, and complementary to, the nucleotide present
in the polymorphic site of the target molecule being evaluated. In
contrast to the method of Cohen et al. (French Patent 2,650,840;
PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is
preferably a heterogeneous phase assay, in which the primer or the
target molecule is immobilized to a solid phase.
[0082] Recently, several primer-guided nucleotide incorporation
procedures for assaying polymorphic sites in DNA have been
described (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784
(1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen,
A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R.
et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA
9:107-112 (1992); Nyren, P. et al., Anal. Biochem. 208:171-175
(1993)). These methods differ from GBA.RTM. in that they all rely
on the incorporation of labeled deoxynucleotides to discriminate
between bases at a polymorphic site. In such a format, since the
signal is proportional to the number of deoxynucleotides
incorporated, polymorphisms that occur in runs of the same
nucleotide can result in signals that are proportional to the
length of the run (Syvanen, A.-C., et al., Amer. J. Hum. Genet.
52:46-59 (1993)).
[0083] A number of initiatives are currently underway to obtain
sequence information directly from millions of individual molecules
of DNA or RNA in parallel. Real-time single molecule
sequencing-by-synthesis technologies rely on the detection of
fluorescent nucleotides as they are incorporated into a nascent
strand of DNA that is complementary to the template being
sequenced. In one method, oligonucleotides 30-50 bases in length
are covalently anchored at the 5' end to glass cover slips. These
anchored strands perform two functions. First, they act as capture
sites for the target template strands if the templates are
configured with capture tails complementary to the surface-bound
oligonucleotides. They also act as primers for the template
directed primer extension that forms the basis of the sequence
reading. The capture primers function as a fixed position site for
sequence determination using multiple cycles of synthesis,
detection, and chemical cleavage of the dye-linker to remove the
dye. Each cycle consists of adding the polymerase/labeled
nucleotide mixture, rinsing, imaging and cleavage of dye. In an
alternative method, polymerase is modified with a fluorescent donor
molecule and immobilized on a glass slide, while each nucleotide is
color-coded with an acceptor fluorescent moiety attached to a
gamma-phosphate. The system detects the interaction between a
fluorescently-tagged polymerase and a fluorescently modified
nucleotide as the nucleotide becomes incorporated into the de novo
chain. Other sequencing-by-synthesis technologies also exist.
[0084] Preferably, any suitable sequencing-by-synthesis platform
can be used to identify mutations. As described above, four major
sequencing-by-synthesis platforms are currently available: the
Genome Sequencers from Roche/454 Life Sciences, the 1G Analyzer
from Illumina/Solexa, the SOLiD system from Applied BioSystems, and
the Heliscope system from Helicos Biosciences.
Sequencing-by-synthesis platforms have also been described by
Pacific BioSciences and VisiGen Biotechnologies. Each of these
platforms can be used in the methods of the invention. In some
embodiments, a plurality of nucleic acid molecules being sequenced
is bound to a support (e.g., solid support). To immobilize the
nucleic acid on a support, a capture sequence/universal priming
site can be added at the 3' and/or 5' end of the template. The
nucleic acids may be bound to the support by hybridizing the
capture sequence to a complementary sequence covalently attached to
the support. The capture sequence (also referred to as a universal
capture sequence) is a nucleic acid sequence complementary to a
sequence attached to a support that may dually serve as a universal
primer.
[0085] As an alternative to a capture sequence, a member of a
coupling pair (such as, e.g., antibody/antigen, receptor/ligand, or
the avidin-biotin pair as described in, e.g., US Patent Application
No. 2006/0252077) may be linked to each fragment to be captured on
a surface coated with a respective second member of that coupling
pair.
[0086] Subsequent to the capture, the sequence may be analyzed, for
example, by single molecule detection/sequencing, e.g., as
described in the Examples and in U.S. Pat. No. 7,283,337, including
template-dependent sequencing-by-synthesis. In
sequencing-by-synthesis, the surface-bound molecule is exposed to a
plurality of labeled nucleotide triphosphates in the presence of
polymerase. The sequence of the template is determined by the order
of labeled nucleotides incorporated into the 3' end of the growing
chain. This can be done in real time or can be done in a
step-and-repeat mode. For real-time analysis, different optical
labels to each nucleotide may be incorporated and multiple lasers
may be utilized for stimulation of incorporated nucleotides.
[0087] Any cell type or tissue may be utilized to obtain nucleic
acid samples for use in the diagnostics described herein. In a
preferred embodiment, the DNA or RNA sample is obtained from a
tumor or a bodily fluid, e.g., blood, obtained by known techniques
(e.g. venipuncture) or saliva. Alternatively, nucleic acid tests
can be performed on dry samples (e.g. hair or skin).
[0088] Alternatively, protein mass spectrometry may be used to
identify or validate the presence of mutated peptides bound to MHC
proteins on tumor cells. Peptides can be acid-eluted from tumor
cells or from HLA molecules that are immunoprecipitated from tumor,
and then identified using mass spectrometry.
Neoantigenic Peptides
[0089] The invention further includes isolated peptides that
comprise the tumor specific mutations identified by the methods of
the invention, peptides that comprise know tumor specific
mutations, and mutant polypeptides or fragments thereof identified
by the method of the invention. These peptides and polypeptides are
referred to herein as "neoantigenic peptides" or "neoantigenic
polypeptides". The term "peptide" is used interchangeably with
"mutant peptide" and "neoantigenic peptide" in the present
specification to designate a series of residues, typically L-amino
acids, connected one to the other, typically by peptide bonds
between the .alpha.-amino and carboxyl groups of adjacent amino
acids. Similarly, the term "polypeptide" is used interchangeably
with "mutant polypeptide" and "neoantigenic polypeptide" in the
present specification to designate a series of residues, typically
L-amino acids, connected one to the other, typically by peptide
bonds between the .alpha.-amino and carboxyl groups of adjacent
amino acids. The polypeptides or peptides can be a variety of
lengths, either in their neutral (uncharged) forms or in forms
which are salts, and either free of modifications such as
glycosylation, side chain oxidation, or phosphorylation or
containing these modifications, subject to the condition that the
modification not destroy the biological activity of the
polypeptides as herein described.
[0090] In certain embodiments the size of the at least one
neoantigenic peptide molecule may comprise, but is not limited to,
about 5, about 6, about 7, about 8, about 9, about 10, about 11,
about 12, about 13, about 14, about 15, about 16, about 17, about
18, about 19, about 20, about 21, about 22, about 23, about 24,
about 25, about 26, about 27, about 28, about 29, about 30, about
31, about 32, about 33, about 34, about 35, about 36, about 37,
about 38, about 39, about 40, about 41, about 42, about 43, about
44, about 45, about 46, about 47, about 48, about 49, about 50,
about 60, about 70, about 80, about 90, about 100, about 110, about
120 or greater amino molecule residues, and any range derivable
therein. In specific embodiments the neoantigenic peptide molecules
are equal to or less than 50 amino acids.
[0091] In some embodiments the particular neoantigenic peptides and
polypeptides of the invention are: for MHC Class I 13 residues or
less in length and usually consist of between about 8 and about 11
residues, particularly 9 or 10 residues; for MHC Class II, 15-24
residues.
[0092] A longer peptide may be designed in several ways. In one
case, when HLA-binding peptides are predicted or known, a longer
peptide could consist of either: (1) individual binding peptides
with an extensions of 2-5 amino acids toward the N- and C-terminus
of each corresponding gene product; (2) a concatenatation of some
or all of the binding peptides with extended sequences for each. In
another case, when sequencing reveals a long (>10 residues)
neoepitope sequence present in the tumor (e.g. due to a frameshift,
read-through or intron inclusion that leads to a novel peptide
sequence), a longer peptide would consist of: (3) the entire
stretch of novel tumor-specific amino acids--thus bypassing the
need for computational prediction or in vitro testing of peptide
binding to HLA proteins. In both cases, use of a longer peptide
allows endogenous processing by patient cells and may lead to more
effective antigen presentation and induction of T cell
responses.
[0093] The neoantigenic peptides and polypeptides bind an HLA
protein. In some aspect the neoantigenic peptides and polypeptides
binds an HLA protein. with greater affinity than a wild-type
peptide. The neoantigenic peptide or polypeptide has an IC50 of at
least less than 5000 nM, at least less than 500 nM, at least less
then 250 nM, at least less than 200 nM, at least less than 150 nM,
at least less than 100 nM, at least less than 50 nM or less.
[0094] The neoantigenic peptides and polypeptides does not induce
an autoimmune response and/or invoke immunological tolerance when
administered to a subject.
[0095] The invention also provides compositions comprising at least
two or more neoantigenic peptides. In some embodiments the
composition contains at least two distint peptides. Preferably, the
at least two distint peptides are derived from the same
polypeptide. By distint polypeptides is meant that the peptide vary
by length, amino acid sequence or both. The peptides are derived
from any polypeptide know to or have been found to by the methods
of the invention to contain a tumor specific mutation. Suitable
polypeptides from which the neoantigenic peptides may be derived
can be found for example at the COSMIC database. COSMIC curates
comprehensive information on somatic mutations in human cancer. The
peptide contains the tumor specific mutation. In some aspects the
tumor specific mutation is a driver mutation for a particular
cancer type. In some aspects, the peptides are derived from a SF3B1
polypeptide, a MYD88 polypeptide, a TP53 polypeptide, an ATM
polypeptide, an Abl polypeptide, A FBXW7 polypeptide, a DDX3X
polypeptide, a MAPK1 polypeptide of a GNB1 polypeptide.
[0096] By a SF3B1 peptide is meant that the peptide contains a
portion of a SF3B1 polypeptide. Preferably, a SF3B1 peptide
includes either leucine at amino acid position 625; a histidine at
amino acid position 626; a glutamic acid at amino acid position
700; an aspartic acid at amino acid position 742; or an arginine at
amino acid position 903, when numbered in accordance with wild-type
SF3B1. A wild type SF3B1 is shown in Table A (SEQ ID NO:1).
TABLE-US-00001 TABLE A Wild Type SF3B1 (SEQ ID NO: 1)
makiakthedieaqireiqgkkaaldeaqgvgldstgyydqeiyggsdsr
fagyvtsiaateledddddyssstsllgqkkpgyhapvallndipqsteq
ydpfaehrppkiadredeykkhrrtmiisperldpfadggktpdpkmnar
tymdvmreqhltkeereirqqlaekakagelkvvngaaasqppskrkrrw
dqtadqtpgatpkklsswdqaetpghtpslrwdetpgrakgsetpgatpg
skiwdptpshtpagaatpgrgdtpghatpghggatssarknrwdetpkte
rdtpghgsgwaetprtdrggdsigetptpgaskrksrwdetpasqmggst
pvltpgktpigtpamnmatptpghimsmtpeqlqawrwereidernrpls
deeldamfpegykvlpppagyvpirtparkltatptplggmtgfhmqted
rtmksvndqpsgnlpflkpddiqyfdkllvdvdestlspeeqkerkimkl
llkikngtppmrkaalrqitdkarefgagplfnqilpllmsptledqerh
llvkvidrilyklddlvrpyvhkilvviepllidedyyarvegreiisnl
akaaglatmistmrpdidnmdeyvrnttarafavvasalgipsllpflka
vckskkswqarhtgikivqqiailmgcailphlrslveiiehglvdeqqk
vrtisalaiaalaeaatpygiesfdsvlkplwkgirqhrgkglaaflkai
gyliplmdaeyanyytrevmlilirefqspdeemkkivlkvvkqccgtdg
veanyikteilppffkhfwqhrmaldrrnyrqlvdttvelankvgaaeii
srivddlkdeaeqyrkmvmetiekimgnlgaadidhkleeqlidgilyaf
qeqttedsvmlngfgtvvnalgkrvkpylpqicgtvlwrinnksakvrqq
aadlisrtavvmktcqeeklmghlgvvlyeylgeeypevlgsilgalkai
vnvigmhkmtppikdllprltpilknrhekvqencidlvgriadrgaeyv
sarewmricfellellkahkkairratvntfgyiakaigphdvlatllnn
lkvqerqnrvcttvaiaivaetcspftvlpalmneyrvpelnvqngvlks
lsflfeyigemgkdyiyavtplledalmdrdlvhrqtasavvqhmslgvy
gfgcedslnhllnyvwpnvfetsphviqavmgaleglrvaigpermlqyc
lqglfhparkvrdvywkiynsiyigsqdaliahypriynddkntyiryel dyil
[0097] By a MYD88 peptide is meant that the peptide contains a
portion of a MYD88 polypeptide. Preferably, a MYD88 peptide
includes either a threonine at amino acid position 232; a leucine
at amino acid position 258; or a proline at amino acid position
265, when numbered in accordance with wild-type MYD88 when numbered
in accordance with wild-type MYD88. A wild type MYD88 is shown in
Table B (SEQ ID NO:2).
TABLE-US-00002 TABLE B Wild Type MYD88 (SEQ ID NO: 2)
mrpdraeapgppamaaggpgagsaapvsstsslplaalnmrvrrrlslfl
nvrtqvaadwtalaeemdfeyleirqletqadptgrlldawqgrpgasvg
rllelltklgrddvllelgpsieedcqkyilkqqqeeaekplqvaavdss
vprtaelagittlddplghmperfdaficycpsdiqfvqemirqleqtny
rlklcvsdrdvlpgtcvwsiaseliekrcrrmvvvvsddylqskecdfqt
kfalslspgahqkrlipikykamkkefpsilrfitvcdytnpctkswfwt rlakalslp
[0098] By a TP53 peptide is meant that the peptide contains a
portion of a TP53 polypeptide. Preferably, a TP53 peptide includes
either an arginine at amino acid position 111; an arginine at amino
acid position 215; a serine at amino acid position 238; a glutamine
at amino acid position 248; a phenylalanine at amino acid position
255; a cysteine at amino acid position 273 or an asparagine at
amino acid position 281, when numbered in accordance with wild-type
TP53. A wild type TP53 is shown in Table C (SEQ ID NO:3).
TABLE-US-00003 TABLE C Wild Type TP53 (SEQ ID NO: 3)
meepqsdpsvepplsgetfsdlwkllpennvlsplpsqamddlmlspddi
eqwftedpgpdeaprmpeaappvapapaaptpaapapapswplsssvpsq
ktyqgsygfrlgflhsgtaksvtctyspalnkmfcqlaktcpvqlwvdst
pppgtrvramaiykqsqhmtevvrrcphhercsdsdglappqhlirvegn
lrveylddrntfrhsvvvpyeppevgsdcttihynymcnsscmggmnrrp
iltiitledssgnllgrnsfevrvcacpgrdrrteeenlrkkgephhelp
pgstkralpnntssspqpkkkpldgeyftlqirgrerfemfrelnealel
kdaqagkepggsrahsshlkskkgqstsrhkklmfktegpdsd
[0099] By an ATM peptide is meant that the peptide contains a
portion of a SF3B1 polypeptide. Preferably, a ATM peptide includes
either a phenylalanine at amino acid position 1252; an arginine at
amino acid position 2038; a histidine at amino acid position 2522;
or a cysteine at amino acid position 2954, when numbered in
accordance with wild-type ATM. A wild type ATM is shown in Table D
(SEQ ID NO:4).
TABLE-US-00004 [0099] TABLE D Wild Type ATM (SEQ ID NO: 4)
mslvlndlliccrqlehdraterkkevekfkrlirdpetikhldrhsdsk
qgkylnwdavfrflqkyiqketeclriakpnvsastqasrqkkmqeissl
vkyfikcanrraprlkcqellnyimdtvkdssngaiygadcsnillkdil
syrkywceisqqqwlelfsvyfrlylkpsqdvhrvlvariihavtkgccs
qtdglnskfldffskaiqcarqeksssglnhilaaltiflktlavnfrir
vcelgdeilptllyiwtqhrindslkeviielfqlqiyihhpkgaktqek
gayestkwrsilynlydllvneishigsrgkyssgfrniavkenlielma
dichqvfnedtrsleisqsytttqressdysvpckrkkielgwevikdhl
qksqndfdlvpwlqiatqliskypaslpncelspllmilsqllpqqrhge
rtpyvlrcltevalcqdkrsnlessqksdllklwnkiwcitfrgisseqi
qaenfgllgaiiqgslvevdrefwklftgsacrpscpavccltlalttsi
vpgtvkmgieqnmcevnrsfslkesimkwllfyqlegdlenstevppilh
snfphlvlekilvsltmknckaamnffqsvpecehhqkdkeelsfsevee
lflqttfdkmdfltivrecgiekhqssigfsvhqnlkesldrcllglseq
llnnysseitnsetivrcsrllvgvlgcycymgviaeeeaykselfqkak
slmqcagesitlfknktneefrigslrnmmqlctrclsnctkkspnkias
gfflrlltsklmndiadickslasfikkpfdrgevesmeddtngnlmeve
dqssmnlfndypdssvsdanepgesqstigainplaeeylskqdllfldm
lkflclcvttaqtntvsfraadirrkllmlidsstleptkslhlhmylml
lkelpgeeyplpmedvlellkplsnvcslyrrdqdvcktilnhvlhvvkn
lgqsnmdsentrdaqgqfltvigafwhltkerkyifsvrmalvnclktll
eadpyskwailnvmgkdfpvnevftqfladnhhqvrmlaaesinrlfqdt
kgdssrllkalplklqqtafenaylkagegmremshsaenpetldeiynr
ksvlltliavvlscspicekqalfalcksvkenglephlvkkvlekvset
fgyrrledfmashldylvlewlnlqdteynlssfpfillnytniedfyrs
cykvliphlvirshfdevksianqiqedwkslltdcfpkilvnilpyfay
egtrdsgmaqqretatkvydmlksenllgkqidhlfisnlpeivvellmt
lhepanssasqstdlcdfsgdldpapnpphfpshvikatfayisnchktk
lksileilskspdsyqkillaiceqaaetnnvykkhrilkiyhlfvslll
kdiksglggawafvlrdviytlihyinqrpscimdvslrsfslccdllsq
vcqtavtyckdalenhlhvivgtliplvyegvevqkqvldllkylvidnk
dnenlyitiklldpfpdhvvfkdlritqqkikysrgpfslleeinhflsv
svydalpltrleglkdlrrqlelhkdqmvdimrasqdnpqdgimvklvvn
llqlskmainhtgekevleavgsclgevgpidfstiaiqhskdasytkal
klfedkelqwtfimltylnntlvedcvkvrsaavtclknilatktghsfw
eiykmttdpmlaylqpfrtsrkkflevprfdkenpfeglddinlwiplse
nhdiwiktltcafldsggtkceilqllkpmcevktdfcqtvlpylihdil
lqdtneswrnllsthvqgfftsclrhfsqtsrsttpanldsesehffrcc
ldkksqrtmlavvdymrrqkrpssgtifndafwldlnylevakvaqscaa
hftallyaeiyadkksmddqekrslafeegsqsttisslsekskeetgis
lqdllleiyrsigepdslygcgggkmlqpitrlrtyeheamwgkalvtyd
letaipsstrqagiiqalqnlglchilsvylkgldyenkdwcpeleelhy
qaawrnmqwdhctsvskevegtsyheslynalqslrdrefstfyeslkya
rvkeveemckrslesvyslyptlsrlqaigelesigelfsrsvthrqlse
vyikwqkhsqllkdsdfsfqepimalrtvileilmekemdnsqrecikdi
ltkhlvelsilartfkntqlperaifqikqynsyscgvsewqleeaqvfw
akkeqslalsilkqmikkldascaannpslkltyteclrvcgnwlaetcl
enpavimqtylekavevagnydgessdelrngkmkaflslarfsdtqyqr
ienymkssefenkqallkrakeevgllrehkiqtnrytvkvqreleldel
alralkedrkrflckavenyincllsgeehdmwvfrlcslwlensgvsev
ngmmkrdgmkiptykflplmyqlaarmgtkmmgglgfhevinnlisrism
dhphhtlfiilalananrdefltkpevarrsritknvpkgssqldedrte
aanriictirsrrpqmvrsvealcdayiilanldatqwktqrkginipad
qpitklknledvvvptmeikvdhtgeygnlvtiqsfkaefrlaggvnlpk
iidcvgsdgkerrqlvkgrddlrqdavmqqvfqmcntllqrntetrkrkl
tictykvvplsqrsgvlewctgtvpigeflvnnedgahkryrpndfsafq
cqkkmmevqkksfeekyevfmdvcqnfqpvfryfcmekfldpaiwfekrl
aytrsvatssivgyilglgdrhvgnilineqsaelvhidlgvafeqgkil
ptpetvpfrltrdivdgmgitgvegvfrrccektmevmrnsqetlltive
vllydplfdwtmnplkalylqqrpedetelhptlnaddqeckrnlsdidq
sfnkvaervlmrlqeklkgveegtvlsvggqvnlliqqaidpknlsrlfp gwkawv
[0100] By an Abl peptide is meant that the peptide contains a
portion of an Abl polypeptide. Preferably, a Bcr-abl peptide
includes a valine at amino acid position 244; a valine at amino
acid position 248; a glutamic acid at amino acid position 250; an
alanine at amino acid position 250; a histidine at amino acid
position 252; an arginine at amino acid position 252; a
phenylalanine at amino acid position 253; a histidine at amino acid
position 253; a lysine at amino acid position 255; a valine at
amino acid position 255; a glycine at amino acid position 276; an
isoleucine at amino acid position 315; an asparagine at amino acid
position 315; a leucine at amino acid position 317; a threonine at
amino acid position 343; a threonine at amino acid position 351; a
glycine at amino acid position 355; a valine at amino acid position
359; an alanine at amino acid position 359; an isoleucine at amino
acid position 379; a leucine at amino acid position 382; a
methionine at amino acid position 387; a proline at amino acid
position 396; an arginine at amino acid position 396; a tyrosine at
amino acid position 417; or a serine at amino acid position 486,
when numbered in accordance with wild-type Abl. A wild type Abl is
shown in Table E (SEQ ID NO:5).
TABLE-US-00005 TABLE E Wild Type Ab1 (SEQ ID NO: 5)
MLEICLKLVGCKSKKGLSSSSSCYLEEALQRPVASDEEPQGLSEAARWN
SKENLLAGPSENDPNLEVALYDFVASGDNTLSITKGEKLRVLGYNHNGE
WCEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYLLSSGING
SFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRENTLA
ELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMK
HKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIK
HPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYM
ATQISSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDT
YTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYP
GIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIH
QAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPELPTKTRTSRRAA
EHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDE
RLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEE
GRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHL
WKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVT
LPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPP
RLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHK
EEAGKGSALGTPAAAEPVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSS
ESPGRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAK
TKATSLVDAVNSDAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAP
VPSTLPSASSALAGDQPSSTAFTPLISTRVSLRKTRQPPERIASGAITK
GVVLDSTEALCLAISRNSEQMASHSAVLEAGKNLYTFCVSYVDSIQQMR
NKFAFREAINKLENNLRELQICPATAGSGPAATQDFSKLLSSVKEISDI VQR
[0101] By a FBXW7 peptide is meant that the peptide contains a
portion of a FBXW7 polypeptide. Preferably, a FBXW7peptide includes
either a leucine at amino acid position 280; a histidine at amino
acid position 465; a cysteine at amino acid position 505; or a
glutamic acid at amino acid position 597, when numbered in
accordance with wild-type FBXW7. A wild type FBXW7 is shown in
Table F (SEQ ID N06).
TABLE-US-00006 TABLE F Wild Type FBXW7 (SEQ ID NO: 6)
mnqellsvgskrrrtggslrgnpsssqvdeeqmnrvveeeqqqqlrqqee
ehtarngevvgveprpggqndsqqgqleennnrfisvdedssgnqeeqee
deehageqdeedeeeeemdqesddfdqsddssredehthtnsvtnsssiv
dlpvhqlsspfytkttkmkrkldhgsevrsfslgkkpckvseytsttglv
pcsatpttfgdlraangqgqqrrritsvqpptglqewlkmfqswsgpekl
laldelidsceptqvkhmmqviepqfqrdfisllpkelalyvlsflepkd
llqaaqtcrywrilaednllwrekckeegideplhikrrkvikpgfihsp
wksayirqhridtnwrrgelkspkvlkghddhvitclqfcgnrivsgsdd
ntlkvwsavtgkclrtlvghtggvwssqmrdniiisgstdrtlkvwnaet
gecihtlyghtstvrcmhlhekrvvsgsrdatlrvwdietgqclhvlmgh
vaavrcvqydgrrvvsgaydfmvkvwdpetetclhtlqghtnrvyslqfd
gihvvsgsldtsirvwdvetgncihtltghqsltsgmelkdnilvsgnad
stvkiwdiktgqclqtlqgpnkhqsavtclqfnknfvitssddgtvklwd
lktgefirnlvtlesggsggvvwrirasntklvcavgsrngteetkllvl dfdvdmk
[0102] By a DDX3X peptide is meant that the peptide contains a
portion of a DDX3X polypeptide. A DDX3X peptide is a peptide that
is the result of a missence mutation at amino acid position 24; a
splice site at amino acid position 342 or a frame shift at amino
acid position 410 when numbered in accordance with wild-type DDX3X.
A wild type DDX3X is shown in Table G (SEQ ID NO:7).
TABLE-US-00007 TABLE F Wild Type DDX3X (SEQ ID NO: 7)
mshvavenalgldqqfagldlnssdnqsggstaskgryipphlrnreatk
gfydkdssgwssskdkdayssfgsrsdsrgkssffsdrgsgsrgrfddrg
rsdydgigsrgdrsgfgkferggnsrwcdksdeddwskplppserleqel
fsggntginfekyddipveatgnncpphiesfsdvemgeiimgnieltry
trptpvqkhaipiikekrdlmacaqtgsgktaafllpilsqiysdgpgea
lramkengrygrrkqypislvlaptrelavqiyeearkfsyrsrvrpcvv
yggadigqqirdlergchllvatpgrlvdmmergkigldfckylvldead
rmldmgfepqirriveqdtmppkgvrhtmmfsatfpkeiqmlardfldey
iflavgrvgstsenitqkvvwveesdkrsflldllnatgkdsltlvfvet
kkgadsledflyhegyactsihgdrsqrdreealhqfrsgkspilvatav
aargldisnvkhvinfdlpsdieeyvhrigrtgrvgnlglatsffnerni
nitkdlldllveakqevpswlenmayehhykgssrgrskssrfsggfgar
dyrqssgassssfsssrasssrsgggghgssrgfggggyggfynsdgygg nynsqgvdwwgn
[0103] By a MAPK1 peptide is meant that the peptide contains a
portion of a MAPK1 polypeptide. Preferably, a MAPK1 peptide
includes either an asparagine at amino acid position 162; a glycine
at amino acid position 291; or a phenylalanine at amino acid
position 316, when numbered in accordance with wild-type MAPK1. A
wild type MAPK1 is shown in Table H (SEQ ID NO:8).
TABLE-US-00008 TABLE F Wild Type MAPK1 (SEQ ID NO: 8)
maaaaaagagpemvrgqvfdvgprytnlsyigegaygmvcsaydnvnkvr
vaikkispfehqtycqrtlreikillrfrheniigindiiraptieqmkd
vyivqdlmetdlykllktqhlsndhicyflyqilrglkyihsanvlhrdl
kpsnlllnttcdlkicdfglarvadpdhdhtgflteyvatrwyrapeiml
nskgytksidiwsvgcilaemlsnrpifpgkhyldqlnhilgilgspsqe
dlnciinlkarnyllslphknkvpwnrlfpnadskaldlldkmltfnphk
rieveqalahpyleqyydpsdepiaeapfkfdmelddlpkeklkelifee tarfqpgyrs
[0104] By a GNB1 peptide is meant that the peptide contains a
portion of a GNB1 polypeptide. Preferably, a GNB1 peptide includes
a threonine at amino acid position 180, when numbered in accordance
with wild-type GNB1. A wild type GNB1 is shown in Table I (SEQ ID
NO9).
TABLE-US-00009 TABLE I Wild Type GNB1 (SEQ ID NO: 9)
mseldqlrqeaeqlknqirdarkacadatlsqitnnidpvgriqmrtrrt
lrghlakiyamhwgtdsrllvsasqdgkliiwdsyttnkvhaiplrsswv
mtcayapsgnyvacggldnicsiynlktregnvrvsrelaghtgylsccr
flddnqivtssgdttcalwdietgqqtttftghtgdvmslslapdtrlfv
sgacdasaklwdvregmcrqtftghesdinaicffpngnafatgsddatc
rlfdlradqelmtyshdniicgitsysfsksgrlllagyddfncnvwdal
kadragvlaghdnrvsclgvtddgmavatgswdsflkiwn
[0105] Neoantigenic peptides and polypeptides having the desired
activity may be modified as necessary to provide certain desired
attributes, e.g. improved pharmacological characteristics, while
increasing or at least retaining substantially all of the
biological activity of the unmodified peptide to bind the desired
MHC molecule and activate the appropriate T cell. For instance, the
neoantigenic peptide and polypeptides may be subject to various
changes, such as substitutions, either conservative or
non-conservative, where such changes might provide for certain
advantages in their use, such as improved MHC binding. By
conservative substitutions is meant replacing an amino acid residue
with another which is biologically and/or chemically similar, e.g.,
one hydrophobic residue for another, or one polar residue for
another. The substitutions include combinations such as Gly, Ala;
Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. The effect of single amino acid substitutions may also be
probed using D-amino acids. Such modifications may be made using
well known peptide synthesis procedures, as described in e.g.,
Merrifield, Science 232:341-347 (1986), Barany & Merrifield,
The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press),
pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide
Synthesis, (Rockford, III., Pierce), 2d Ed. (1984).
[0106] The neoantigenic peptide and polypeptides can also be
modified by extending or decreasing the compound's amino acid
sequence, e.g., by the addition or deletion of amino acids. The
peptides, polypeptides or analogs can also be modified by altering
the order or composition of certain residues, it being readily
appreciated that certain amino acid residues essential for
biological activity, e.g., those at critical contact sites or
conserved residues, may generally not be altered without an adverse
effect on biological activity. The non-critical amino acids need
not be limited to those naturally occurring in proteins, such as
L-.alpha.-amino acids, or their D-isomers, but may include
non-natural amino acids as well, such as
(.beta.-.gamma.-.delta.-amino acids, as well as many derivatives of
L-.alpha.-amino acids.
[0107] Typically, a series of peptides with single amino acid
substitutions are employed to determine the effect of electrostatic
charge, hydrophobicity, etc. on binding. For instance, a series of
positively charged (e.g., Lys or Arg) or negatively charged (e.g.,
Glu) amino acid substitutions are made along the length of the
peptide revealing different patterns of sensitivity towards various
MHC molecules and T cell receptors. In addition, multiple
substitutions using small, relatively neutral moieties such as Ala,
Gly, Pro, or similar residues may be employed. The substitutions
may be homo-oligomers or hetero-oligomers. The number and types of
residues which are substituted or added depend on the spacing
necessary between essential contact points and certain functional
attributes which are sought (e.g., hydrophobicity versus
hydrophilicity). Increased binding affinity for an MHC molecule or
T cell receptor may also be achieved by such substitutions,
compared to the affinity of the parent peptide. In any event, such
substitutions should employ amino acid residues or other molecular
fragments chosen to avoid, for example, steric and charge
interference which might disrupt binding.
[0108] Amino acid substitutions are typically of single residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final peptide. Substitutional variants
are those in which at least one residue of a peptide has been
removed and a different residue inserted in its place. Such
substitutions generally are made in accordance with the following
Table when it is desired to finely modulate the characteristics of
the peptide.
TABLE-US-00010 Original Residue Exemplary Substitution Ala Ser Arg
Lys, His Asn Gln Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Lys;
Arg Ile Leu; Val Leu Ile; Val Lys Arg; His Met Leu; Ile Phe Tyr;
Trp Ser Thr Thr Ser Trp Tyr; Phe Tyr Trp; Phe Val Ile; Leu Pro
Gly
[0109] Substantial changes in function (e.g., affinity for MHC
molecules or T cell receptors) are made by selecting substitutions
that are less conservative than those in above Table, i.e.,
selecting residues that differ more significantly in their effect
on maintaining (a) the structure of the peptide backbone in the
area of the substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in peptide properties will be those in which (a)
hydrophilic residue, e.g. seryl, is substituted for (or by) a
hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or
alanyl; (b) a residue having an electropositive side chain, e.g.,
lysl, arginyl, or histidyl, is substituted for (or by) an
electronegative residue, e.g. glutamyl or aspartyl; or (c) a
residue having a bulky side chain, e.g. phenylalanine, is
substituted for (or by) one not having a side chain, e.g.,
glycine.
[0110] The peptides and polypeptides may also comprise isosteres of
two or more residues in the neoantigenic peptide or polypeptides.
An isostere as defined here is a sequence of two or more residues
that can be substituted for a second sequence because the steric
conformation of the first sequence fits a binding site specific for
the second sequence. The term specifically includes peptide
backbone modifications well known to those skilled in the art. Such
modifications include modifications of the amide nitrogen, the
.alpha.-carbon, amide carbonyl, complete replacement of the amide
bond, extensions, deletions or backbone crosslinks. See, generally,
Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, Vol. VII (Weinstein ed., 1983).
[0111] Modifications of peptides and polypeptides with various
amino acid mimetics or unnatural amino acids are particularly
useful in increasing the stability of the peptide and polypeptide
in vivo. Stability can be assayed in a number of ways. For
instance, peptidases and various biological media, such as human
plasma and serum, have been used to test stability. See, e.g.,
Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986).
Half life of the peptides of the present invention is conveniently
determined using a 25% human serum (v/v) assay. The protocol is
generally as follows. Pooled human serum (Type AB, non-heat
inactivated) is delipidated by centrifugation before use. The serum
is then diluted to 25% with RPMI tissue culture media and used to
test peptide stability. At predetermined time intervals a small
amount of reaction solution is removed and added to either 6%
aqueous trichloracetic acid or ethanol. The cloudy reaction sample
is cooled (4.degree. C.) for 15 minutes and then spun to pellet the
precipitated serum proteins. The presence of the peptides is then
determined by reversed-phase HPLC using stability-specific
chromatography conditions.
[0112] The peptides and polypeptides may be modified to provide
desired attributes other than improved serum half life. For
instance, the ability of the peptides to induce CTL activity can be
enhanced by linkage to a sequence which contains at least one
epitope that is capable of inducing a T helper cell response.
Particularly preferred immunogenic peptides/T helper conjugates are
linked by a spacer molecule. The spacer is typically comprised of
relatively small, neutral molecules, such as amino acids or amino
acid mimetics, which are substantially uncharged under
physiological conditions. The spacers are typically selected from,
e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or
neutral polar amino acids. It will be understood that the
optionally present spacer need not be comprised of the same
residues and thus may be a hetero- or homo-oligomer. When present,
the spacer will usually be at least one or two residues, more
usually three to six residues. Alternatively, the peptide may be
linked to the T helper peptide without a spacer.
[0113] The neoantigenic peptide may be linked to the T helper
peptide either directly or via a spacer either at the amino or
carboxy terminus of the peptide. The amino terminus of either the
neoantigenic peptide or the T helper peptide may be acylated.
Exemplary T helper peptides include tetanus toxoid 830-843,
influenza 307-319, malaria circumsporozoite 382-398 and
378-389.
[0114] Proteins or peptides may be made by any technique known to
those of skill in the art, including the expression of proteins,
polypeptides or peptides through standard molecular biological
techniques, the isolation of proteins or peptides from natural
sources, or the chemical synthesis of proteins or peptides. The
nucleotide and protein, polypeptide and peptide sequences
corresponding to various genes have been previously disclosed, and
may be found at computerized databases known to those of ordinary
skill in the art. One such database is the National Center for
Biotechnology Information's Genbank and GenPept databases located
at the National Institutes of Health website. The coding regions
for known genes may be amplified and/or expressed using the
techniques disclosed herein or as would be known to those of
ordinary skill in the art. Alternatively, various commercial
preparations of proteins, polypeptides and peptides are known to
those of skill in the art.
[0115] In a further aspect of the invention provides a nucleic acid
(e.g. polynucleotide) encoding a neoantigenic peptide of the
invention. The polynucleotide may be e.g. DNA, cDNA, PNA, CNA, RNA,
either single- and/or double-stranded, or native or stabilized
forms of polynucleotides, such as e.g. polynucleotides with a
phosphorothiate backbone, or combinations thereof and it may or may
not contain introns so long as it codes for the peptide. Of course,
only peptides that contain naturally occurring amino acid residues
joined by naturally occurring peptide bonds are encodable by a
polynucleotide. A still further aspect of the invention provides an
expression vector capable of expressing a polypeptide according to
the invention. Expression vectors for different cell types are well
known in the art and can be selected without undue experimentation.
Generally, the DNA is inserted into an expression vector, such as a
plasmid, in proper orientation and correct reading frame for
expression. If necessary, the DNA may be linked to the appropriate
transcriptional and translational regulatory control nucleotide
sequences recognized by the desired host, although such controls
are generally available in the expression vector. The vector is
then introduced into the host through standard techniques. Guidance
can be found e.g. in Sambrook et al. (1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.
Vaccine Compositions
[0116] The present invention is directed to an immunogenic
composition, e.g., a vaccine composition capable of raising a
specific T-cell response. The vaccine composition comprises mutant
peptides and mutant polypeptides corresponding to tumor specific
neoantigens identified by the methods described herein.
[0117] A person skilled in the art will be able to select preferred
peptides, polypeptide or combination of thereof by testing, for
example, the generation of T-cells in vitro as well as their
efficiency and overall presence, the proliferation, affinity and
expansion of certain T-cells for certain peptides, and the
functionality of the T-cells, e.g. by analyzing the IFN-.gamma.
production or tumor killing by T-cells. Usually, the most efficient
peptides are then combined as a vaccine.
[0118] A suitable vaccine will preferably contain between 1 and 20
peptides, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 different peptides, further preferred
6, 7, 8, 9, 10 11, 12, 13, or 14 different peptides, and most
preferably 12, 13 or 14 different peptides.
[0119] In one embodiment of the present invention the different
peptides and/or polypeptides are selected so that one vaccine
composition comprises peptides and/or polypeptides capable of
associating with different MHC molecules, such as different MHC
class I molecule. Preferably, one vaccine composition comprises
peptides and/or polypeptides capable of associating with the most
frequently occurring MHC class I molecules. Hence vaccine
compositions according to the invention comprises different
fragments capable of associating with at least 2 preferred, more
preferably at least 3 preferred, even more preferably at least 4
preferred MHC class I molecules.
[0120] The vaccine composition is capable of raising a specific
cytotoxic T-cells response and/or a specific helper T-cell
response.
[0121] The vaccine composition can further comprise an adjuvant
and/or a carrier. Examples of useful adjuvants and carriers are
given herein below. The peptides and/or polypeptides in the
composition can be associated with a carrier such as e.g. a protein
or an antigen-presenting cell such as e.g. a dendritic cell (DC)
capable of presenting the peptide to a T-cell.
[0122] Adjuvants are any substance whose admixture into the vaccine
composition increases or otherwise modifies the immune response to
the mutant peptide. Carriers are scaffold structures, for example a
polypeptide or a polysaccharide, to which the neoantigenic
peptides, is capable of being associated. Optionally, adjuvants are
conjugated covalently or non-covalently to the peptides or
polypeptides of the invention.
[0123] The ability of an adjuvant to increase the immune response
to an antigen is typically manifested by a significant increase in
immune-mediated reaction, or reduction in disease symptoms. For
example, an increase in humoral immunity is typically manifested by
a significant increase in the titer of antibodies raised to the
antigen, and an increase in T-cell activity is typically manifested
in increased cell proliferation, or cellular cytotoxicity, or
cytokine secretion. An adjuvant may also alter an immune response,
for example, by changing a primarily humoral or Th response into a
primarily cellular, or Th response.
[0124] Suitable adjuvants include, but are not limited to 1018 ISS,
aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA,
dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch,
ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A,
Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide
ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM. vector
system, PLG microparticles, resiquimod, SRL172, Virosomes and other
Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,
Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass.,
USA) which is derived from saponin, mycobacterial extracts and
synthetic bacterial cell wall mimics, and other proprietary
adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as
incomplete Freund's or GM-CSF are preferred. Several immunological
adjuvants (e.g., MF59) specific for dendritic cells and their
preparation have been described previously (Dupuis M, et al., Cell
Immunol. 1998; 186(1):18-27; Allison A C; Dev Biol Stand. 1998;
92:3-11). Also cytokines may be used. Several cytokines have been
directly linked to influencing dendritic cell migration to lymphoid
tissues (e.g., TNF-alpha), accelerating the maturation of dendritic
cells into efficient antigen-presenting cells for T-lymphocytes
(e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589,
specifically incorporated herein by reference in its entirety) and
acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J
Immunother Emphasis Tumor Immunol. 1996 (6):414-418).
[0125] CpG immunostimulatory oligonucleotides have also been
reported to enhance the effects of adjuvants in a vaccine setting.
Without being bound by theory, CpG oligonucleotides act by
activating the innate (non-adaptive) immune system via Toll-like
receptors (TLR), mainly TLR9. CpG triggered TLR9 activation
enhances antigen-specific humoral and cellular responses to a wide
variety of antigens, including peptide or protein antigens, live or
killed viruses, dendritic cell vaccines, autologous cellular
vaccines and polysaccharide conjugates in both prophylactic and
therapeutic vaccines. More importantly, it enhances dendritic cell
maturation and differentiation, resulting in enhanced activation of
TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, even
in the absence of CD4 T-cell help. The TH1 bias induced by TLR9
stimulation is maintained even in the presence of vaccine adjuvants
such as alum or incomplete Freund's adjuvant (IFA) that normally
promote a TH2 bias. CpG oligonucleotides show even greater adjuvant
activity when formulated or co-administered with other adjuvants or
in formulations such as microparticles, nano particles, lipid
emulsions or similar formulations, which are especially necessary
for inducing a strong response when the antigen is relatively weak.
They also accelerate the immune response and enabled the antigen
doses to be reduced by approximately two orders of magnitude, with
comparable antibody responses to the full-dose vaccine without CpG
in some experiments (Arthur M. Krieg, Nature Reviews, Drug
Discovery, 5, June 2006, 471-484). U.S. Pat. No. 6,406,705 B1
describes the combined use of CpG oligonucleotides, non-nucleic
acid adjuvants and an antigen to induce an antigen-specific immune
response. A commercially available CpG TLR9 antagonist is dSLIM
(double Stem Loop Immunomodulator) by Mologen (Berlin, GERMANY),
which is a preferred component of the pharmaceutical composition of
the present invention. Other TLR binding molecules such as RNA
binding TLR 7, TLR 8 and/or TLR 9 may also be used.
[0126] Other examples of useful adjuvants include, but are not
limited to, chemically modified CpGs (e.g. CpR, Idera),
Poly(I:C)(e.g. polyi:CI2U), non-CpG bacterial DNA or RNA as well as
immunoactive small molecules and antibodies such as
cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016,
sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632,
pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175,
which may act therapeutically and/or as an adjuvant. The amounts
and concentrations of adjuvants and additives useful in the context
of the present invention can readily be determined by the skilled
artisan without undue experimentation. Additional adjuvants include
colony-stimulating factors, such as Granulocyte Macrophage Colony
Stimulating Factor (GM-CSF, sargramostim).
[0127] A vaccine composition according to the present invention may
comprise more than one different adjuvants. Furthermore, the
invention encompasses a therapeutic composition comprising any
adjuvant substance including any of the above or combinations
thereof. It is also contemplated that the peptide or polypeptide,
and the adjuvant can be administered separately in any appropriate
sequence.
[0128] A carrier may be present independently of an adjuvant. The
function of a carrier can for example be to increase the molecular
weight of in particular mutant in order to increase their activity
or immunogenicity, to confer stability, to increase the biological
activity, or to increase serum half-life. Furthermore, a carrier
may aid presenting peptides to T-cells. The carrier may be any
suitable carrier known to the person skilled in the art, for
example a protein or an antigen presenting cell. A carrier protein
could be but is not limited to keyhole limpet hemocyanin, serum
proteins such as transferrin, bovine serum albumin, human serum
albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones,
such as insulin or palmitic acid. For immunization of humans, the
carrier must be a physiologically acceptable carrier acceptable to
humans and safe. However, tetanus toxoid and/or diptheria toxoid
are suitable carriers in one embodiment of the invention.
Alternatively, the carrier may be dextrans for example
sepharose.
[0129] Cytotoxic T-cells (CTLs) recognize an antigen in the form of
a peptide bound to an MHC molecule rather than the intact foreign
antigen itself. The MHC molecule itself is located at the cell
surface of an antigen presenting cell. Thus, an activation of CTLs
is only possible if a trimeric complex of peptide antigen, MHC
molecule, and APC is present. Correspondingly, it may enhance the
immune response if not only the peptide is used for activation of
CTLs, but if additionally APCs with the respective MHC molecule are
added. Therefore, in some embodiments the vaccine composition
according to the present invention additionally contains at least
one antigen presenting cell.
[0130] The antigen-presenting cell (or stimulator cell) typically
has an MHC class I or II molecule on its surface, and in one
embodiment is substantially incapable of itself loading the MHC
class I or II molecule with the selected antigen. As is described
in more detail below, the MHC class I or II molecule may readily be
loaded with the selected antigen in vitro.
[0131] Preferably, the antigen presenting cells are dendritic
cells. Suitably, the dendritic cells are autologous dendritic cells
that are pulsed with the neoantigenic peptide. The peptide may be
any suitable peptide that gives rise to an appropriate T-cell
response. T-cell therapy using autologous dendritic cells pulsed
with peptides from a tumor associated antigen is disclosed in
Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al.
(1997) The Prostate 32, 272-278.
[0132] Thus, in one embodiment of the present invention the vaccine
composition containing at least one antigen presenting cell is
pulsed or loaded with one or more peptides of the present
invention. Alternatively, peripheral blood mononuclear cells
(PBMCs) isolated from a patient may be loaded with peptides ex vivo
and injected back into the patient.
[0133] As an alternative the antigen presenting cell comprises an
expression construct encoding a peptide of the present invention.
The polynucleotide may be any suitable polynucleotide and it is
preferred that it is capable of transducing the dendritic cell,
thus resulting in the presentation of a peptide and induction of
immunity.
Therapeutic Methods
[0134] The invention further provides a method of inducing a tumor
specific immune response in a subject, vaccinating against a tumor,
treating and or alleviating a symptom of cancer in a subject by
administering the subject a neoantigenic peptide or vaccine
composition of the invention.
[0135] The subject has been diagnosed with cancer or is at risk of
developing cancer. The subject has a imatinib resistant tumor. The
subject is a human, dog, cat, horse or any animal in which a tumor
specific immune response is desired. The tumor is any solid tumor
such as breast, ovarian, prostate, lung, kidney, gastric, colon,
testicular, head and neck, pancreas, brain, melanoma, and other
tumors of tissue organs and hematological tumors, such as lymphomas
and leukemias, including acute myelogenous leukemia, chronic
myelogenous leukemia, chronic lymphocytic leukemia, T cell
lymphocytic leukemia, and B cell lymphomas.
[0136] The peptide or composition of the invention is administered
in an amount sufficient to induce a CTL response.
[0137] In specific embodiments, the invention provides methods of
treating an imatinib resistant tumor by administering to a subject
one or more neoantigenic peptides that contain a bcr-abl mutation.
In some embodiments the subject is HLA-A3. Bcr-abl mutations
include for example T315I, E255K, M351T, Y253H, Q252H, F317L,
F359V, G250E, Y253F, E355G, E255V, M244V, L248V, G250A, Q252R,
D276G, T315N, M343T, F359A, V379I, F382L, L387M, H396P, H396R,
S417Y, F486S.
[0138] The neoantigenic peptide, polypeptide or vaccine composition
of the invention can be administered alone or in combination with
other therapeutic agents. The therapeutic agent is for example, a
chemotherapeutic agent, radiation, or immunotherapy. Any suitable
therapeutic treatment for a particular cancer may be administered.
Examples of chemotherapeutic agents include, but are not limited
to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin,
capecitabine, carboplatin, carmustine, cladribine, cisapride,
cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC),
dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alpha,
etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine,
granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha,
irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna,
methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone,
omeprazole, ondansetron, paclitaxel (Taxol.RTM.), pilocarpine,
prochloroperazine, rituximab, tamoxifen, taxol, topotecan
hydrochloride, trastuzumab, vinblastine, vincristine and
vinorelbine tartrate. For prostate cancer treatment, a preferred
chemotherapeutic agent with which anti-CTLA-4 can be combined is
paclitaxel (Taxol.RTM.).
[0139] In addition, the subject may be further administered an
anti-immunosuppressive/immunostimulatory agent. For example, the
subject is further administered an anti-CTLA antibody or anti-PD-1
or anti-PD-L1. Blockade of CTLA-4 or PD-L1 by antibodies can
enhance the immune response to cancerous cells in the patient. In
particular, CTLA-4 blockade has been shown effective when following
a vaccination protocol.
[0140] The optimum amount of each peptide to be included in the
vaccine composition and the optimum dosing regimen can be
determined by one skilled in the art without undue experimentation.
For example, the peptide or its variant may be prepared for
intravenous (i.v.) injection, sub-cutaneous (s.c.) injection,
intradermal (i.d.) injection, intraperitoneal (i.p.) injection,
intramuscular (i.m.) injection. Preferred methods of peptide
injection include s.c., i.d., i.p., i.m., and i.v. Preferred
methods of DNA injection include i.d., i.m., s.c., i.p. and i.v.
For example, doses of between 1 and 500 mg 50 .mu.g and 1.5 mg,
preferably 125 .mu.g to 500 .mu.g, of peptide or DNA may be given
and will depend from the respective peptide or DNA. Doses of this
range were successfully used in previous trials (Brunsvig P F, et
al., Cancer Immunol Immunother. 2006; 55(12):1553-1564; M.
Staehler, et al., ASCO meeting 2007; Abstract No 3017). Other
methods of administion of the vaccine composition are known to
thoses skilled in the art.
[0141] The inventive pharmaceutical composition may be compiled so
that the selection, number and/or amount of peptides present in the
composition is/are tissue, cancer, and/or patient-specific. For
instance, the exact selection of peptides can be guided by
expression patterns of the parent proteins in a given tissue to
avoid side effects. The selection may be dependent on the specific
type of cancer, the status of the disease, earlier treatment
regimens, the immune status of the patient, and, of course, the
HLA-haplotype of the patient. Furthermore, the vaccine according to
the invention can contain individualized components, according to
personal needs of the particular patient. Examples include varying
the amounts of peptides according to the expression of the related
neoantigen in the particular patient, unwanted side-effects due to
personal allergies or other treatments, and adjustments for
secondary treatments following a first round or scheme of
treatment.
[0142] For a composition to be used as a vaccine for cancer,
peptides whose endogenous parent proteins are expressed in high
amounts in normal tissues will be avoided or be present in low
amounts in the composition of the invention. On the other hand, if
it is known that the tumor of a patient expresses high amounts of a
certain protein, the respective pharmaceutical composition for
treatment of this cancer may be present in high amounts and/or more
than one peptide specific for this particularly protein or pathway
of this protein may be included.
[0143] Pharmaceutical compositions comprising the peptide of the
invention may be administered to an individual already suffering
from cancer. In therapeutic applications, compositions are
administered to a patient in an amount sufficient to elicit an
effective CTL response to the tumor antigen and to cure or at least
partially arrest symptoms and/or complications. An amount adequate
to accomplish this is defined as "therapeutically effective dose."
Amounts effective for this use will depend on, e.g., the peptide
composition, the manner of administration, the stage and severity
of the disease being treated, the weight and general state of
health of the patient, and the judgment of the prescribing
physician, but generally range for the initial immunization (that
is for therapeutic or prophylactic administration) from about 1.0
.mu.g to about 50,000 .mu.g of peptide for a 70 kg patient,
followed by boosting dosages or from about 1.0 .mu.g to about
10,000 .mu.g of peptide pursuant to a boosting regimen over weeks
to months depending upon the patient's response and condition by
measuring specific CTL activity in the patient's blood. It must be
kept in mind that the peptide and compositions of the present
invention may generally be employed in serious disease states, that
is, life-threatening or potentially life threatening situations,
especially when the cancer has metastasized. In such cases, in view
of the minimization of extraneous substances and the relative
nontoxic nature of the peptide, it is possible and may be felt
desirable by the treating physician to administer substantial
excesses of these peptide compositions.
[0144] For therapeutic use, administration should begin at the
detection or surgical removal of tumors. This is followed by
boosting doses until at least symptoms are substantially abated and
for a period thereafter.
[0145] The pharmaceutical compositions (e.g., vaccine compositions)
for therapeutic treatment are intended for parenteral, topical,
nasal, oral or local administration. Preferably, the pharmaceutical
compositions are administered parenterally, e.g., intravenously,
subcutaneously, intradermally, or intramuscularly. The compositions
may be administered at the site of surgical excision to induce a
local immune response to the tumor. The invention provides
compositions for parenteral administration which comprise a
solution of the peptides and vaccine compositions are dissolved or
suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered
water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.
These compositions may be sterilized by conventional, well known
sterilization techniques, or may be sterile filtered. The resulting
aqueous solutions may be packaged for use as is, or lyophilized,
the lyophilized preparation being combined with a sterile solution
prior to administration. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc.
[0146] The concentration of peptides of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0147] The peptide of the invention may also be administered via
liposomes, which target the peptides to a particular cells tissue,
such as lymphoid tissue. Liposomes are also useful in increasing
the half-life of the peptides. Liposomes include emulsions, foams,
micelles, insoluble monolayers, liquid crystals, phospholipid
dispersions, lamellar layers and the like. In these preparations
the peptide to be delivered is incorporated as part of a liposome,
alone or in conjunction with a molecule which binds to, e.g., a
receptor prevalent among lymphoid cells, such as monoclonal
antibodies which bind to the CD45 antigen, or with other
therapeutic or immunogenic compositions. Thus, liposomes filled
with a desired peptide of the invention can be directed to the site
of lymphoid cells, where the liposomes then deliver the selected
therapeutic/immunogenic peptide compositions. Liposomes for use in
the invention are formed from standard vesicle-forming lipids,
which generally include neutral and negatively charged
phospholipids and a sterol, such as cholesterol. The selection of
lipids is generally guided by consideration of, e.g., liposome
size, acid lability and stability of the liposomes in the blood
stream. A variety of methods are available for preparing liposomes,
as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9;
467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and
5,019,369.
[0148] For targeting to the immune cells, a ligand to be
incorporated into the liposome can include, e.g., antibodies or
fragments thereof specific for cell surface determinants of the
desired immune system cells. A liposome suspension containing a
peptide may be administered intravenously, locally, topically, etc.
in a dose which varies according to, inter alia, the manner of
administration, the peptide being delivered, and the stage of the
disease being treated.
[0149] For solid compositions, conventional or nanoparticle
nontoxic solid carriers may be used which include, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talcum, cellulose, glucose, sucrose,
magnesium carbonate, and the like. For oral administration, a
pharmaceutically acceptable nontoxic composition is formed by
incorporating any of the normally employed excipients, such as
those carriers previously listed, and generally 10-95% of active
ingredient, that is, one or more peptides of the invention, and
more preferably at a concentration of 25%-75%.
[0150] For aerosol administration, the immunogenic peptides are
preferably supplied in finely divided form along with a surfactant
and propellant. Typical percentages of peptides are 0.01%-20% by
weight, preferably 1%-10%. The surfactant must, of course, be
nontoxic, and preferably soluble in the propellant. Representative
of such agents are the esters or partial esters of fatty acids
containing from 6 to 22 carbon atoms, such as caproic, octanoic,
lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic
acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.1%-20% by weight of the
composition, preferably 0.25-5%. The balance of the composition is
ordinarily propellant. A carrier can also be included as desired,
as with, e.g., lecithin for intranasal delivery.
[0151] For therapeutic or immunization purposes, nucleic acids
encoding the peptide of the invention and optionally one or more of
the peptides described herein can also be administered to the
patient. A number of methods are conveniently used to deliver the
nucleic acids to the patient. For instance, the nucleic acid can be
delivered directly, as "naked DNA". This approach is described, for
instance, in Wolff et al., Science 247: 1465-1468 (1990) as well as
U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acids can also
be administered using ballistic delivery as described, for
instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of
DNA can be administered. Alternatively, DNA can be adhered to
particles, such as gold particles.
[0152] The nucleic acids can also be delivered complexed to
cationic compounds, such as cationic lipids. Lipid-mediated gene
delivery methods are described, for instance, in U.S. Pat. No.
9,618,372 WOAWO 96/18372; U.S. Pat. No. 9,324,640 WOAWO 93/24640;
Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988);
U.S. Pat. No. 5,279,833 USA Rose U.S. Pat. Nos. 5,279,833;
9,106,309 WOAWO 91/06309; and Felgner et al., Proc. Natl. Acad.
Sci. USA 84: 7413-7414 (1987).
[0153] The peptides and polypeptides of the invention can also be
expressed by attenuated viral hosts, such as vaccinia or fowlpox.
This approach involves the use of vaccinia virus as a vector to
express nucleotide sequences that encode the peptide of the
invention. Upon introduction into an acutely or chronically
infected host or into a noninfected host, the recombinant vaccinia
virus expresses the immunogenic peptide, and thereby elicits a host
CTL response. Vaccinia vectors and methods useful in immunization
protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another
vector is BCG (Bacille Calmette Guerin). BCG vectors are described
in Stover et al. (Nature 351:456-460 (1991)). A wide variety of
other vectors useful for therapeutic administration or immunization
of the peptides of the invention, e.g., Salmonella typhi vectors
and the like, will be apparent to those skilled in the art from the
description herein.
[0154] A preferred means of administering nucleic acids encoding
the peptide of the invention uses minigene constructs encoding
multiple epitopes. To create a DNA sequence encoding the selected
CTL epitopes (minigene) for expression in human cells, the amino
acid sequences of the epitopes are reverse translated. A human
codon usage table is used to guide the codon choice for each amino
acid. These epitope-encoding DNA sequences are directly adjoined,
creating a continuous polypeptide sequence. To optimize expression
and/or immunogenicity, additional elements can be incorporated into
the minigene design. Examples of amino acid sequence that could be
reverse translated and included in the minigene sequence include:
helper T lymphocyte, epitopes, a leader (signal) sequence, and an
endoplasmic reticulum retention signal. In addition, MHC
presentation of CTL epitopes may be improved by including synthetic
(e.g. poly-alanine) or naturally-occurring flanking sequences
adjacent to the CTL epitopes.
[0155] The minigene sequence is converted to DNA by assembling
oligonucleotides that encode the plus and minus strands of the
minigene. Overlapping oligonucleotides (30-100 bases long) are
synthesized, phosphorylated, purified and annealed under
appropriate conditions using well known techniques. The ends of the
oligonucleotides are joined using T4 DNA ligase. This synthetic
minigene, encoding the CTL epitope polypeptide, can then cloned
into a desired expression vector.
[0156] Standard regulatory sequences well known to those of skill
in the art are included in the vector to ensure expression in the
target cells. Several vector elements are required: a promoter with
a down-stream cloning site for minigene insertion; a
polyadenylation signal for efficient transcription termination; an
E. coli origin of replication; and an E. coli selectable marker
(e.g. ampicillin or kanamycin resistance). Numerous promoters can
be used for this purpose, e.g., the human cytomegalovirus (hCMV)
promoter. See, U.S. Pat. Nos. 5,580,859 and 5,589,466 for other
suitable promoter sequences.
[0157] Additional vector modifications may be desired to optimize
minigene expression and immunogenicity. In some cases, introns are
required for efficient gene expression, and one or more synthetic
or naturally-occurring introns could be incorporated into the
transcribed region of the minigene. The inclusion of mRNA
stabilization sequences can also be considered for increasing
minigene expression. It has recently been proposed that
immunostimulatory sequences (ISSs or CpGs) play a role in the
immunogenicity of DNA' vaccines. These sequences could be included
in the vector, outside the minigene coding sequence, if found to
enhance immunogenicity.
[0158] In some embodiments, a bicistronic expression vector, to
allow production of the minigene-encoded epitopes and a second
protein included to enhance or decrease immunogenicity can be used.
Examples of proteins or polypeptides that could beneficially
enhance the immune response if co-expressed include cytokines
(e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g. LeIF)
or costimulatory molecules. Helper (HTL) epitopes could be joined
to intracellular targeting signals and expressed separately from
the CTL epitopes. This would allow direction of the HTL epitopes to
a cell compartment different than the CTL epitopes. If required,
this could facilitate more efficient entry of HTL epitopes into the
MHC class II pathway, thereby improving CTL induction. In contrast
to CTL induction, specifically decreasing the immune response by
co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may
be beneficial in certain diseases.
[0159] Once an expression vector is selected, the minigene is
cloned into the polylinker region downstream of the promoter. This
plasmid is transformed into an appropriate E. coli strain, and DNA
is prepared using standard techniques. The orientation and DNA
sequence of the minigene, as well as all other elements included in
the vector, are confirmed using restriction mapping and DNA
sequence analysis. Bacterial cells harboring the correct plasmid
can be stored as a master cell bank and a working cell bank.
[0160] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). A variety
of methods have been described, and new techniques may become
available. As noted above, nucleic acids are conveniently
formulated with cationic lipids. In addition, glycolipids,
fusogenic liposomes, peptides and compounds referred to
collectively as protective, interactive, non-condensing (PINC)
could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0161] Target cell sensitization can be used as a functional assay
for expression and MHC class I presentation of minigene-encoded CTL
epitopes. The plasmid DNA is introduced into a mammalian cell line
that is suitable as a target for standard CTL chromium release
assays. The transfection method used will be dependent on the final
formulation. Electroporation can be used for "naked" DNA, whereas
cationic lipids allow direct in vitro transfection. A plasmid
expressing green fluorescent protein (GFP) can be co-transfected to
allow enrichment of transfected cells using fluorescence activated
cell sorting (FACS). These cells are then chromium-51 labeled and
used as target cells for epitope-specific CTL lines. Cytolysis,
detected by 51 Cr release, indicates production of MHC presentation
of mini gene-encoded CTL epitopes.
[0162] In vivo immunogenicity is a second approach for functional
testing of minigene DNA formulations. Transgenic mice expressing
appropriate human MHC molecules are immunized with the DNA product.
The dose and route of administration are formulation dependent
(e.g. IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one
days after immunization, splenocytes are harvested and restimulated
for 1 week in the presence of peptides encoding each epitope being
tested. These effector cells (CTLs) are assayed for cytolysis of
peptide-loaded, chromium-51 labeled target cells using standard
techniques. Lysis of target cells sensitized by MHC loading of
peptides corresponding to minigene-encoded epitopes demonstrates
DNA vaccine function for in vivo induction of CTLs.
[0163] Peptides may be used to elicit CTL ex vivo, as well. The
resulting CTL, can be used to treat chronic tumors in patients that
do not respond to other conventional forms of therapy, or will not
respond to a peptide vaccine approach of therapy. Ex vivo CTL
responses to a particular tumor antigen are induced by incubating
in tissue culture the patient's CTL precursor cells (CTLp) together
with a source of antigen-presenting cells (APC) and the appropriate
peptide. After an appropriate incubation time (typically 1-4
weeks), in which the CTLp are activated and mature and expand into
effector CTL, the cells are infused back into the patient, where
they will destroy their specific target cell (i.e., a tumor cell).
In order to optimize the in vitro conditions for the generation of
specific cytotoxic T cells, the culture of stimulator cells is
maintained in an appropriate serum-free medium.
[0164] Prior to incubation of the stimulator cells with the cells
to be activated, e.g., precursor CD8+ cells, an amount of antigenic
peptide is added to the stimulator cell culture, of sufficient
quantity to become loaded onto the human Class I molecules to be
expressed on the surface of the stimulator cells. In the present
invention, a sufficient amount of peptide is an amount that will
allow about 200, and preferably 200 or more, human Class I MHC
molecules loaded with peptide to be expressed on the surface of
each stimulator cell. Preferably, the stimulator cells are
incubated with >2 .mu.g/ml peptide. For example, the stimular
cells are incubates with >3, 4, 5, 10, 15, or more .mu.g/ml
peptide.
[0165] Resting or precursor CD8+ cells are then incubated in
culture with the appropriate stimulator cells for a time period
sufficient to activate the CD8+ cells. Preferably, the CD8+ cells
are activated in an antigen-specific manner. The ratio of resting
or precursor CD8+ (effector) cells to stimulator cells may vary
from individual to individual and may further depend upon variables
such as the amenability of an individual's lymphocytes to culturing
conditions and the nature and severity of the disease condition or
other condition for which the within-described treatment modality
is used. Preferably, however, the lymphocyte:stimulator cell ratio
is in the range of about 30:1 to 300:1. The effector/stimulator
culture may be maintained for as long a time as is necessary to
stimulate a therapeutically useable or effective number of CD8+
cells.
[0166] The induction of CTL in vitro requires the specific
recognition of peptides that are bound to allele specific MHC class
I molecules on APC. The number of specific MHC/peptide complexes
per APC is crucial for the stimulation of CTL, particularly in
primary immune responses. While small amounts of peptide/MHC
complexes per cell are sufficient to render a cell susceptible to
lysis by CTL, or to stimulate a secondary CTL response, the
successful activation of a CTL precursor (pCTL) during primary
response requires a significantly higher number of MHC/peptide
complexes. Peptide loading of empty major histocompatability
complex molecules on cells allows the induction of primary
cytotoxic T lymphocyte responses. Peptide loading of empty major
histocompatability complex molecules on cells enables the induction
of primary cytotoxic T lymphocyte responses.
[0167] Since mutant cell lines do not exist for every human MHC
allele, it is advantageous to use a technique to remove endogenous
MHC-associated peptides from the surface of APC, followed by
loading the resulting empty MEW molecules with the immunogenic
peptides of interest. The use of non-transformed (non-tumorigenic),
noninfected cells, and preferably, autologous cells of patients as
APC is desirable for the design of CTL induction protocols directed
towards development of ex vivo CTL therapies. This application
discloses methods for stripping the endogenous MHC-associated
peptides from the surface of APC followed by the loading of desired
peptides.
[0168] A stable MEW class I molecule is a trimeric complex formed
of the following elements: 1) a peptide usually of 8-10 residues,
2) a transmembrane heavy polymorphic protein chain which bears the
peptide-binding site in its .alpha.1 and .alpha.2 domains, and 3) a
non-covalently associated non-polymorphic light chain,
.beta.2microglobulin. Removing the bound peptides and/or
dissociating the .beta.2microglobulin from the complex renders the
MEW class I molecules nonfunctional and unstable, resulting in
rapid degradation. All MHC class I molecules isolated from PBMCs
have endogenous peptides bound to them. Therefore, the first step
is to remove all endogenous peptides bound to MHC class I molecules
on the APC without causing their degradation before exogenous
peptides can be added to them.
[0169] Two possible ways to free up MHC class I molecules of bound
peptides include lowering the culture temperature from 37.degree.
C. to 26.degree. C. overnight to destabilize .beta.2microglobulin
and stripping the endogenous peptides from the cell using a mild
acid treatment. The methods release previously bound peptides into
the extracellular environment allowing new exogenous peptides to
bind to the empty class I molecules. The cold-temperature
incubation method enables exogenous peptides to bind efficiently to
the MEW complex, but requires an overnight incubation at 26.degree.
C. which may slow the cell's metabolic rate. It is also likely that
cells not actively synthesizing MEW molecules (e.g., resting PBMC)
would not produce high amounts of empty surface MHC molecules by
the cold temperature procedure.
[0170] Harsh acid stripping involves extraction of the peptides
with trifluoroacetic acid, pH 2, or acid denaturation of the
immunoaffinity purified class I-peptide complexes. These methods
are not feasible for CTL induction, since it is important to remove
the endogenous peptides while preserving APC viability and an
optimal metabolic state which is critical for antigen presentation.
Mild acid solutions of pH 3 such as glycine or citrate-phosphate
buffers have been used to identify endogenous peptides and to
identify tumor associated T cell epitopes. The treatment is
especially effective, in that only the MHC class I molecules are
destabilized (and associated peptides released), while other
surface antigens remain intact, including MHC class II molecules.
Most importantly, treatment of cells with the mild acid solutions
do not affect the cell's viability or metabolic state. The mild
acid treatment is rapid since the stripping of the endogenous
peptides occurs in two minutes at 4.degree. C. and the APC is ready
to perform its function after the appropriate peptides are loaded.
The technique is utilized herein to make peptide-specific APCs for
the generation of primary antigen-specific CTL. The resulting APC
are efficient in inducing peptide-specific CD8+ CTL.
[0171] Activated CD8+ cells may be effectively separated from the
stimulator cells using one of a variety of known methods. For
example, monoclonal antibodies specific for the stimulator cells,
for the peptides loaded onto the stimulator cells, or for the CD8+
cells (or a segment thereof) may be utilized to bind their
appropriate complementary ligand. Antibody-tagged molecules may
then be extracted from the stimulator-effector cell admixture via
appropriate means, e.g., via well-known immunoprecipitation or
immunoassay methods.
[0172] Effective, cytotoxic amounts of the activated CD8+ cells can
vary between in vitro and in vivo uses, as well as with the amount
and type of cells that are the ultimate target of these killer
cells. The amount will also vary depending on the condition of the
patient and should be determined via consideration of all
appropriate factors by the practitioner. Preferably, however, about
1.times.10.sup.6 to about 1.times.10.sup.12, more preferably about
1.times.10.sup.8 to about 1.times.10.sup.11, and even more
preferably, about 1.times.10.sup.9 to about 1.times.10.sup.10
activated CD8+ cells are utilized for adult humans, compared to
about 5.times.10.sup.6-5.times.10.sup.7 cells used in mice.
[0173] Preferably, as discussed above, the activated CD8+ cells are
harvested from the cell culture prior to administration of the CD8+
cells to the individual being treated. It is important to note,
however, that unlike other present and proposed treatment
modalities, the present method uses a cell culture system that is
not tumorigenic. Therefore, if complete separation of stimulator
cells and activated CD8+ cells is not achieved, there is no
inherent danger known to be associated with the administration of a
small number of stimulator cells, whereas administration of
mammalian tumor-promoting cells may be extremely hazardous.
[0174] Methods of re-introducing cellular components are known in
the art and include procedures such as those exemplified in U.S.
Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to
Rosenberg. For example, administration of activated CD8+ cells via
intravenous infusion is appropriate.
[0175] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
A Strategy to Identify Neoepitopes for Vaccination
[0176] Our approach to identify tumor-specific neoepitopes involves
3 steps. (1) Identification of DNA mutations using whole genome or
whole exome (i.e. only captured exons) sequencing of tumor versus
matched germline samples from each patient. Our preliminary studies
demonstrate that CLL cells contain many distinct genetic changes
that alter amino acid sequence and could generate potential novel T
cell epitopes. (2) Application of highly validated peptide-MHC
binding prediction algorithms to generate a set of candidate T cell
epitopes based on non-silent mutations present in tumors. We will
confirm expression of mutated genes as RNA in CLL samples, and then
confirm the peptide-HLA binding predictions using an experimental
approach to quantify binding of candidate peptides to HLA alleles.
(3) Generation of antigen-specific T cells against mutated
peptides.
Example 2
Tumor and Normal Genome Sequencing for the Identification of
Mutated Genes in Tumors of Patients with Chronic Lymphocytic
Leukemia (Step 1)
[0177] To detect tumor-specific mutations (that are not present in
normal tissues), samples were collected from tumors and from normal
tissues of each patient. For leukemias, tumors were purified using
magnetic bead isolation or fluorescence-activated cell sorting
using antibodies specific to tumor cells, e.g., the tumor cells of
patients with chronic lymphocytic leukemia (CLL) express the CD5
and CD19 surface markers. Skin fibroblasts were used as a normal
tissue control. DNA or RNA for sequencing was purified from
isolated tumor or normal tissue cells. For melanoma, ovarian and
other solid tumors (in which there is contamination with non-tumor
cells), DNA and RNA were isolated from relatively homogeneous
short-term cultures of tumor cells or from laser-captured tumor.
PBMCs were used as normal control cells. For all samples, PBMCs
were cryopreserved until needed for expansion of mutated
peptide-specific T cells. Finally, short-term cultures of tumor
cells were also cryopreserved for later use as targets of expanded
T cells. Isolated genomic DNA or RNA was tested for nucleic acid
integrity and purity prior to sequencing.
[0178] For each sample of DNA, whole genomic DNA was sheared and
sequenced, or coding exons were captured by complementary
oligonucleotides using hybrid selection and then sequenced (Gnirke
et al., Nat Biotechnol. 2009, 27(2):182-9). DNA and RNA libraries
were generated and sequenced using Illumina next-generation
sequencing instruments.
[0179] Sequencing of 64 patients with chronic lymphocytic leukemia
(CLL) yielded an average of 23 non-silent mutations that alter
protein amino acid sequences (FIG. 3) in the tumor relative to the
germline DNA sequence. These non-silent mutations fall into 5
distinct classes with the potential to generate neoepitopes:
missense, splice-site, frame-shift (indel, insertions and
deletions), read-through and gene fusions (FIG. 4). The frequencies
of these mutations vary across individual patients (FIG. 5). All
these mutations provide potential neoepitopes for immunization,
with frame-shift, read-through and splice-site (e.g. with retained
introns) mutations generating longer stretches of novel peptides,
missense mutations leading to short peptides with single amino acid
changes and finally, fusion genes generating hybrid peptides with
novel junction sequences.
Example 3
Identification of HLA-Binding Peptides Derived From Expressed
Proteins Harboring Tumor-Specific Mutations (Step 2)
[0180] The next question is whether mutated genes may generate
peptides that can be presented by patient MHC/HLA proteins. First,
several algorithms were used to predict 30 and 137 HLA-binding
peptides with IC50 scores <500 nM from 10 missense mutations of
Patient 1, and from 53 missense 1 indel and 2 gene fusions of
Patient 2. An example for one missense mutation in a patient with 6
specific HLA alleles is shown with 2 predicted binding peptides out
of 54 combinations of 9-mers peptides and HLA alleles (FIG. 6). To
confirm that these genes are expressed in tumors, we measure RNA
levels for the mutated genes (using several approaches that depend
on the mutation class, FIG. 7), and found that 98% of mutated genes
with HLA binding peptides were expressed.
[0181] The HLA binding capacity of all predicted peptides that pass
RNA expression validation are then experimentally validated by
performing competitive binding assays with test peptides versus
reference peptides known to bind to the HLA allele. (Sidney et al.
Curr Protoc Immunol. 2001, Chapter 18:Unit 18.3) (FIG. 8A). Of the
subset that we submitted for experimental confirmation of HLA
binding, 8 of 17 (47%) predicted peptides from missense mutations
in Pt 1 were confirmed to have high binding affinities for HLA
alleles (IC.sub.50<500)(FIG. 8B). For Pt 2, 25 of 49 predicted
peptides were experimentally confirmed as HLA binding (FIG. 8B).
These results suggest that all peptides with predicted
IC.sub.50<150 nM show HLA binding experimentally, while a
cut-off of <500 nM generates true binding peptides 40-50% of the
time (FIG. 8C). Of note, 12 of the 25 confirmed mutated peptides of
Pt 2 have >2-fold better binding affinity than the germline
peptide (FIG. 9). While such peptides are preferable for
incorporating in a tumor vaccine to reduce the chance of T cells
cross-reacting with the germline peptide, peptides that do not show
differential binding may still provide tumor-specific responses due
to differential recognition of mutant vs. germline peptide by the T
cell receptor.
Example 4
CD8+ T Cell Responses Against Mutated Peptides Identified by
Sequencing CLL Patient Samples (Step 3)
[0182] Based on the predicted or experimentally verified
HLA-binding mutated peptides, we can now determine whether T cells
can be generated to recognize these tumor-specific mutated
peptides. We thus synthesized peptides with binding scores of less
than 1000 nM that are derived from genes with validated expression
in tumor cells. To generate T cells of desired specificity, we
stimulated T cells of the sequenced patients with peptide-pulsed
(either using an individual peptide or a peptide pool) autologous
APCs (dendritic cells and CD40L-expanded autologous B cells) on a
weekly basis, in the presence of IL-2 and IL-7. After 3-4 rounds of
stimulation, the expanded CD8+ cells were tested on ELISpot for
evidence of reactivity against the peptide, based on IFNgamma
secretion. Of the 17 candidate peptides of Patient 1 (FIG. 10), we
have detected IFNgamma secretion in T cells against autologous DCs
pulsed with a mutated peptide from the TLK2 gene.
Example 5
Mutated Bcr-Abl Gene Binds to Patient MHC/HLA Proteins and can
Elicit Mutant-Peptide-Specific CD8+ T Cells
[0183] We performed a more complete study of T cell responses to
tumor-specific mutant peptides in patients with another type of
leukemia, chronic myeloid leukemia (CML). CML is defined by the
expression of a tumor-specific translocation, the product of the
BCR-ABL gene fusion. Mutations in BCR-ABL develop in CML patients
who develop drug resistance to front-line pharmacologic therapy
with imatinib mesylate, which targets BCR-ABL. Potentially, these
mutations may generate neoepitopes that T cells from the host, or
an engrafted normal donor, can recognize when bound to MHC
proteins; these T cells are likely to be minimally tolerized.
[0184] We considered the 20 most common mutations that evolve in
patients with resistance to imatinib, and predicted the binding of
9- and 10-mer peptides tiled around each mutation. Using either the
NetMHC (Nielsen et al. PLoS One. 2007, 2(8):e796) or IEDB (Vita R
et al. Nucleic Acids Res. 2010, 38:D854-62) predictive algorithms,
we predicted binding of 84 peptides from 20 common mutations to one
or more 8 common HLA alleles (IC.sub.50<1000), with many
peptides derived from the three most common mutations. 24 of 84
peptides were predicted to be strong binders (IC.sub.50<50)
(FIG. 14), 42 peptides intermediate binders
(50<IC.sub.50<500), and 18 peptides weak binders
(500<IC.sub.50<1000).
[0185] We focused our attention on a mutant peptide generated from
the E255K (E255K-B.sub.255-263) mutation (KVYEGVWKK)(SEQ ID NO: 10)
that is predicted to bind with high affinity to HLA-A3.
(IC.sub.50=33.1). Using a competitive MHC binding assay (FIG. 8A),
we experimentally confirmed the high binding affinity of E255K-B
for HLA-A3 (IC.sub.50=17 nM) with .about.10-fold stronger
HLA-binding of the mutant peptide compared to the parental
(wildtype) peptide (FIG. 15A). E255K-B was also experimentally
verified to bind other A3 supertype family members HLA-A*1101 and
HLA-A*68. We next generated T cell lines against E255K-B from a
normal HLA-A3+ donor and 2 E255K+/HLA-A3+ CML patients that each
demonstrated greater specificity against the mutated than the
parental peptide (FIG. 15B, C). E255K-B appears to be endogenously
processed and presented since T cells reactive for E255K-B also
responded to HLA-A3+ APCs transfected with a minigene encompassing
227 base pairs surrounding the E255K mutation. Finally, E255K
reactivity in one patient developed only following curative
allo-HSCT (FIG. 15D). These studies demonstrate that
leukemia-driven genetic alterations can provide novel immunogenic
tumor-specific antigen targets that are associated with clinical
response in vivo. Our approach to identifying immunogenic T cell
epitopes of mutated BCR-ABL thus illustrates an effective strategy
for applying bioinformatics tools to discover T cell epitopes from
mutated genes.
Example 6
Patient T Cell Clones that Recognize Tumor Epitopes can Selectively
Kill Cells Presenting Mutated Epitopes
[0186] Confirmation of target specificity of T cells is best
addressed by characterization of individual T cell clones. We
therefore typically isolate mutated peptide-specific T cell clones
by limiting dilution of reactive T cell lines and then use standard
chromium release assays to screen for T cell clones that
demonstrate differential killing of mutated vs germline
peptide-pulsed autologous APCs. Using a standard dilution series
for each peptide, we measure the concentration of peptide required
for 50% killing. If the ratio of wild type to mutant peptides
needed for 50% killing is greater than 10-fold, we conclude that
there is differential recognition of these peptides by T cells, as
seen previously for mutated tumor antigens. We have carried out
this procedure for a CML tumor antigen, CML66. To determine whether
CML66-peptide-specific T cells recognize processed and presented
epitopes, CML66-peptide-reactive T cells were incubated with
autologous APCs transduces to express the entire CML66 protein. We
expressed CML66 by nucleofection of either plasmid DNA, or in vitro
transcribed RNA (in DCs, CD40L-expanded B cells, or K562 cells with
engineered HLA molecules). As shown in FIG. 12A, stimulated T cells
were specific to HLA-B4403 bound CML66-derived peptide epitope
(peptide 66-72C). Since whole CML66 protein was efficiently
expressed when CD40L-expanded B cells were nucleofected with CML66
mRNA (FIG. 12B), we were able to use these cells (or peptide pulsed
cells) as targets in a standard chromium release assay and found
that the T cells lysed these targets cell effectively (FIG. 12C).
Comparable assays, including lysing of patient-matched tumor cells,
are being carried out for each of the mutated peptide-specific T
cell lines generated from each cancer patient (e.g. using the T
cell lines described in Examples 6 and 7).
Example 7
Mutated Tumor Drivers as Potential Tumor Antigens
[0187] Of 1188 nonsilent mutations across 64 patients, we
identified 8 recurrent mutations, including SF3B1 (16% of CLL
patients), TP53 (12.5%), MYD88 (9%), ATM (9%), FBXW7 (6%), MAPK1
(5%), GNB1 (3%) and M6PR (3%) (FIG. 11). These mutations
(especially the most frequent ones: SF3B1, TP53, MYD88 and ATM) are
predicted to be driver mutations that are essential for tumor
development or progression. These driver genes represent promising
tumor-specific antigens for inclusion in a vaccine.
[0188] SF3B1 is the most frequently mutated gene in CLL, is mutated
at conserved sites, is highly expressed in CLL patients (FIG. 12),
and has not been previously described. The most common SF3B1
mutation was K700E (40% of SF3B1 mutations); genotyping of an
additional 89 independent CLL patients uncovered 6 more patient
tumors harboring this mutation. By applying peptide-HLA binding
algorithms to the SF3B1 mutations, we predict binding of the
mutated peptides to the most common HLA-A2 allele (FIG. 13). If a
peptide that harbors the most common mutation in CLL (SF3B1 K700E)
binds the most common class I HLA allele (HLA-A2), then this
peptide is an excellent candidate for inclusion in a CLL vaccine
for many CLL patients.
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Sequence CWU 1
1
2111304PRTHomo sapiens 1Met Ala Lys Ile Ala Lys Thr His Glu Asp Ile
Glu Ala Gln Ile Arg 1 5 10 15 Glu Ile Gln Gly Lys Lys Ala Ala Leu
Asp Glu Ala Gln Gly Val Gly 20 25 30 Leu Asp Ser Thr Gly Tyr Tyr
Asp Gln Glu Ile Tyr Gly Gly Ser Asp 35 40 45 Ser Arg Phe Ala Gly
Tyr Val Thr Ser Ile Ala Ala Thr Glu Leu Glu 50 55 60 Asp Asp Asp
Asp Asp Tyr Ser Ser Ser Thr Ser Leu Leu Gly Gln Lys 65 70 75 80 Lys
Pro Gly Tyr His Ala Pro Val Ala Leu Leu Asn Asp Ile Pro Gln 85 90
95 Ser Thr Glu Gln Tyr Asp Pro Phe Ala Glu His Arg Pro Pro Lys Ile
100 105 110 Ala Asp Arg Glu Asp Glu Tyr Lys Lys His Arg Arg Thr Met
Ile Ile 115 120 125 Ser Pro Glu Arg Leu Asp Pro Phe Ala Asp Gly Gly
Lys Thr Pro Asp 130 135 140 Pro Lys Met Asn Ala Arg Thr Tyr Met Asp
Val Met Arg Glu Gln His 145 150 155 160 Leu Thr Lys Glu Glu Arg Glu
Ile Arg Gln Gln Leu Ala Glu Lys Ala 165 170 175 Lys Ala Gly Glu Leu
Lys Val Val Asn Gly Ala Ala Ala Ser Gln Pro 180 185 190 Pro Ser Lys
Arg Lys Arg Arg Trp Asp Gln Thr Ala Asp Gln Thr Pro 195 200 205 Gly
Ala Thr Pro Lys Lys Leu Ser Ser Trp Asp Gln Ala Glu Thr Pro 210 215
220 Gly His Thr Pro Ser Leu Arg Trp Asp Glu Thr Pro Gly Arg Ala Lys
225 230 235 240 Gly Ser Glu Thr Pro Gly Ala Thr Pro Gly Ser Lys Ile
Trp Asp Pro 245 250 255 Thr Pro Ser His Thr Pro Ala Gly Ala Ala Thr
Pro Gly Arg Gly Asp 260 265 270 Thr Pro Gly His Ala Thr Pro Gly His
Gly Gly Ala Thr Ser Ser Ala 275 280 285 Arg Lys Asn Arg Trp Asp Glu
Thr Pro Lys Thr Glu Arg Asp Thr Pro 290 295 300 Gly His Gly Ser Gly
Trp Ala Glu Thr Pro Arg Thr Asp Arg Gly Gly 305 310 315 320 Asp Ser
Ile Gly Glu Thr Pro Thr Pro Gly Ala Ser Lys Arg Lys Ser 325 330 335
Arg Trp Asp Glu Thr Pro Ala Ser Gln Met Gly Gly Ser Thr Pro Val 340
345 350 Leu Thr Pro Gly Lys Thr Pro Ile Gly Thr Pro Ala Met Asn Met
Ala 355 360 365 Thr Pro Thr Pro Gly His Ile Met Ser Met Thr Pro Glu
Gln Leu Gln 370 375 380 Ala Trp Arg Trp Glu Arg Glu Ile Asp Glu Arg
Asn Arg Pro Leu Ser 385 390 395 400 Asp Glu Glu Leu Asp Ala Met Phe
Pro Glu Gly Tyr Lys Val Leu Pro 405 410 415 Pro Pro Ala Gly Tyr Val
Pro Ile Arg Thr Pro Ala Arg Lys Leu Thr 420 425 430 Ala Thr Pro Thr
Pro Leu Gly Gly Met Thr Gly Phe His Met Gln Thr 435 440 445 Glu Asp
Arg Thr Met Lys Ser Val Asn Asp Gln Pro Ser Gly Asn Leu 450 455 460
Pro Phe Leu Lys Pro Asp Asp Ile Gln Tyr Phe Asp Lys Leu Leu Val 465
470 475 480 Asp Val Asp Glu Ser Thr Leu Ser Pro Glu Glu Gln Lys Glu
Arg Lys 485 490 495 Ile Met Lys Leu Leu Leu Lys Ile Lys Asn Gly Thr
Pro Pro Met Arg 500 505 510 Lys Ala Ala Leu Arg Gln Ile Thr Asp Lys
Ala Arg Glu Phe Gly Ala 515 520 525 Gly Pro Leu Phe Asn Gln Ile Leu
Pro Leu Leu Met Ser Pro Thr Leu 530 535 540 Glu Asp Gln Glu Arg His
Leu Leu Val Lys Val Ile Asp Arg Ile Leu 545 550 555 560 Tyr Lys Leu
Asp Asp Leu Val Arg Pro Tyr Val His Lys Ile Leu Val 565 570 575 Val
Ile Glu Pro Leu Leu Ile Asp Glu Asp Tyr Tyr Ala Arg Val Glu 580 585
590 Gly Arg Glu Ile Ile Ser Asn Leu Ala Lys Ala Ala Gly Leu Ala Thr
595 600 605 Met Ile Ser Thr Met Arg Pro Asp Ile Asp Asn Met Asp Glu
Tyr Val 610 615 620 Arg Asn Thr Thr Ala Arg Ala Phe Ala Val Val Ala
Ser Ala Leu Gly 625 630 635 640 Ile Pro Ser Leu Leu Pro Phe Leu Lys
Ala Val Cys Lys Ser Lys Lys 645 650 655 Ser Trp Gln Ala Arg His Thr
Gly Ile Lys Ile Val Gln Gln Ile Ala 660 665 670 Ile Leu Met Gly Cys
Ala Ile Leu Pro His Leu Arg Ser Leu Val Glu 675 680 685 Ile Ile Glu
His Gly Leu Val Asp Glu Gln Gln Lys Val Arg Thr Ile 690 695 700 Ser
Ala Leu Ala Ile Ala Ala Leu Ala Glu Ala Ala Thr Pro Tyr Gly 705 710
715 720 Ile Glu Ser Phe Asp Ser Val Leu Lys Pro Leu Trp Lys Gly Ile
Arg 725 730 735 Gln His Arg Gly Lys Gly Leu Ala Ala Phe Leu Lys Ala
Ile Gly Tyr 740 745 750 Leu Ile Pro Leu Met Asp Ala Glu Tyr Ala Asn
Tyr Tyr Thr Arg Glu 755 760 765 Val Met Leu Ile Leu Ile Arg Glu Phe
Gln Ser Pro Asp Glu Glu Met 770 775 780 Lys Lys Ile Val Leu Lys Val
Val Lys Gln Cys Cys Gly Thr Asp Gly 785 790 795 800 Val Glu Ala Asn
Tyr Ile Lys Thr Glu Ile Leu Pro Pro Phe Phe Lys 805 810 815 His Phe
Trp Gln His Arg Met Ala Leu Asp Arg Arg Asn Tyr Arg Gln 820 825 830
Leu Val Asp Thr Thr Val Glu Leu Ala Asn Lys Val Gly Ala Ala Glu 835
840 845 Ile Ile Ser Arg Ile Val Asp Asp Leu Lys Asp Glu Ala Glu Gln
Tyr 850 855 860 Arg Lys Met Val Met Glu Thr Ile Glu Lys Ile Met Gly
Asn Leu Gly 865 870 875 880 Ala Ala Asp Ile Asp His Lys Leu Glu Glu
Gln Leu Ile Asp Gly Ile 885 890 895 Leu Tyr Ala Phe Gln Glu Gln Thr
Thr Glu Asp Ser Val Met Leu Asn 900 905 910 Gly Phe Gly Thr Val Val
Asn Ala Leu Gly Lys Arg Val Lys Pro Tyr 915 920 925 Leu Pro Gln Ile
Cys Gly Thr Val Leu Trp Arg Leu Asn Asn Lys Ser 930 935 940 Ala Lys
Val Arg Gln Gln Ala Ala Asp Leu Ile Ser Arg Thr Ala Val 945 950 955
960 Val Met Lys Thr Cys Gln Glu Glu Lys Leu Met Gly His Leu Gly Val
965 970 975 Val Leu Tyr Glu Tyr Leu Gly Glu Glu Tyr Pro Glu Val Leu
Gly Ser 980 985 990 Ile Leu Gly Ala Leu Lys Ala Ile Val Asn Val Ile
Gly Met His Lys 995 1000 1005 Met Thr Pro Pro Ile Lys Asp Leu Leu
Pro Arg Leu Thr Pro Ile 1010 1015 1020 Leu Lys Asn Arg His Glu Lys
Val Gln Glu Asn Cys Ile Asp Leu 1025 1030 1035 Val Gly Arg Ile Ala
Asp Arg Gly Ala Glu Tyr Val Ser Ala Arg 1040 1045 1050 Glu Trp Met
Arg Ile Cys Phe Glu Leu Leu Glu Leu Leu Lys Ala 1055 1060 1065 His
Lys Lys Ala Ile Arg Arg Ala Thr Val Asn Thr Phe Gly Tyr 1070 1075
1080 Ile Ala Lys Ala Ile Gly Pro His Asp Val Leu Ala Thr Leu Leu
1085 1090 1095 Asn Asn Leu Lys Val Gln Glu Arg Gln Asn Arg Val Cys
Thr Thr 1100 1105 1110 Val Ala Ile Ala Ile Val Ala Glu Thr Cys Ser
Pro Phe Thr Val 1115 1120 1125 Leu Pro Ala Leu Met Asn Glu Tyr Arg
Val Pro Glu Leu Asn Val 1130 1135 1140 Gln Asn Gly Val Leu Lys Ser
Leu Ser Phe Leu Phe Glu Tyr Ile 1145 1150 1155 Gly Glu Met Gly Lys
Asp Tyr Ile Tyr Ala Val Thr Pro Leu Leu 1160 1165 1170 Glu Asp Ala
Leu Met Asp Arg Asp Leu Val His Arg Gln Thr Ala 1175 1180 1185 Ser
Ala Val Val Gln His Met Ser Leu Gly Val Tyr Gly Phe Gly 1190 1195
1200 Cys Glu Asp Ser Leu Asn His Leu Leu Asn Tyr Val Trp Pro Asn
1205 1210 1215 Val Phe Glu Thr Ser Pro His Val Ile Gln Ala Val Met
Gly Ala 1220 1225 1230 Leu Glu Gly Leu Arg Val Ala Ile Gly Pro Cys
Arg Met Leu Gln 1235 1240 1245 Tyr Cys Leu Gln Gly Leu Phe His Pro
Ala Arg Lys Val Arg Asp 1250 1255 1260 Val Tyr Trp Lys Ile Tyr Asn
Ser Ile Tyr Ile Gly Ser Gln Asp 1265 1270 1275 Ala Leu Ile Ala His
Tyr Pro Arg Ile Tyr Asn Asp Asp Lys Asn 1280 1285 1290 Thr Tyr Ile
Arg Tyr Glu Leu Asp Tyr Ile Leu 1295 1300 2309PRTHomo sapiens 2Met
Arg Pro Asp Arg Ala Glu Ala Pro Gly Pro Pro Ala Met Ala Ala 1 5 10
15 Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr Ser Ser
20 25 30 Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg Leu
Ser Leu 35 40 45 Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp
Thr Ala Leu Ala 50 55 60 Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile
Arg Gln Leu Glu Thr Gln 65 70 75 80 Ala Asp Pro Thr Gly Arg Leu Leu
Asp Ala Trp Gln Gly Arg Pro Gly 85 90 95 Ala Ser Val Gly Arg Leu
Leu Glu Leu Leu Thr Lys Leu Gly Arg Asp 100 105 110 Asp Val Leu Leu
Glu Leu Gly Pro Ser Ile Glu Glu Asp Cys Gln Lys 115 120 125 Tyr Ile
Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro Leu Gln Val 130 135 140
Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu Ala Gly Ile 145
150 155 160 Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg Phe
Asp Ala 165 170 175 Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val
Gln Glu Met Ile 180 185 190 Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu
Lys Leu Cys Val Ser Asp 195 200 205 Arg Asp Val Leu Pro Gly Thr Cys
Val Trp Ser Ile Ala Ser Glu Leu 210 215 220 Ile Glu Lys Arg Cys Arg
Arg Met Val Val Val Val Ser Asp Asp Tyr 225 230 235 240 Leu Gln Ser
Lys Glu Cys Asp Phe Gln Thr Lys Phe Ala Leu Ser Leu 245 250 255 Ser
Pro Gly Ala His Gln Lys Arg Leu Ile Pro Ile Lys Tyr Lys Ala 260 265
270 Met Lys Lys Glu Phe Pro Ser Ile Leu Arg Phe Ile Thr Val Cys Asp
275 280 285 Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe Trp Thr Arg Leu
Ala Lys 290 295 300 Ala Leu Ser Leu Pro 305 3393PRTHomo sapiens
3Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln 1
5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val
Leu 20 25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu
Ser Pro Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly
Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Pro Val
Ala Pro Ala Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro
Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser Gln Lys
Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu His
Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala
Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135
140 Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met
145 150 155 160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val
Arg Arg Cys 165 170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly
Leu Ala Pro Pro Gln 180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu
Arg Val Glu Tyr Leu Asp Asp 195 200 205 Arg Asn Thr Phe Arg His Ser
Val Val Val Pro Tyr Glu Pro Pro Glu 210 215 220 Val Gly Ser Asp Cys
Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser 225 230 235 240 Ser Cys
Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255
Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260
265 270 Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu
Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro
Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser
Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe
Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg
Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln Ala Gly
Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu Lys
Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380
Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390 43056PRTHomo sapiens
4Met Ser Leu Val Leu Asn Asp Leu Leu Ile Cys Cys Arg Gln Leu Glu 1
5 10 15 His Asp Arg Ala Thr Glu Arg Lys Lys Glu Val Glu Lys Phe Lys
Arg 20 25 30 Leu Ile Arg Asp Pro Glu Thr Ile Lys His Leu Asp Arg
His Ser Asp 35 40 45 Ser Lys Gln Gly Lys Tyr Leu Asn Trp Asp Ala
Val Phe Arg Phe Leu 50 55 60 Gln Lys Tyr Ile Gln Lys Glu Thr Glu
Cys Leu Arg Ile Ala Lys Pro 65 70 75 80 Asn Val Ser Ala Ser Thr Gln
Ala Ser Arg Gln Lys Lys Met Gln Glu 85 90 95 Ile Ser Ser Leu Val
Lys Tyr Phe Ile Lys Cys Ala Asn Arg Arg Ala 100 105 110 Pro Arg Leu
Lys Cys Gln Glu Leu Leu Asn Tyr Ile Met Asp Thr Val 115 120 125 Lys
Asp Ser Ser Asn Gly Ala Ile Tyr Gly Ala Asp Cys Ser Asn Ile 130 135
140 Leu Leu Lys Asp Ile Leu Ser Val Arg Lys Tyr Trp Cys Glu Ile Ser
145 150 155 160 Gln Gln Gln Trp Leu Glu Leu Phe Ser Val Tyr Phe Arg
Leu Tyr Leu 165 170 175 Lys Pro Ser Gln Asp Val His Arg Val Leu Val
Ala Arg Ile Ile His 180 185 190 Ala Val Thr Lys Gly Cys Cys Ser Gln
Thr Asp Gly Leu Asn Ser Lys 195 200 205 Phe Leu Asp Phe Phe Ser Lys
Ala Ile Gln Cys Ala Arg Gln Glu Lys 210 215 220 Ser Ser Ser Gly Leu
Asn His Ile Leu Ala Ala Leu Thr Ile Phe Leu 225 230 235 240 Lys Thr
Leu Ala Val Asn Phe Arg Ile Arg Val Cys Glu Leu Gly Asp 245 250 255
Glu Ile Leu Pro Thr Leu Leu Tyr Ile Trp Thr Gln His Arg Leu Asn 260
265 270 Asp Ser
Leu Lys Glu Val Ile Ile Glu Leu Phe Gln Leu Gln Ile Tyr 275 280 285
Ile His His Pro Lys Gly Ala Lys Thr Gln Glu Lys Gly Ala Tyr Glu 290
295 300 Ser Thr Lys Trp Arg Ser Ile Leu Tyr Asn Leu Tyr Asp Leu Leu
Val 305 310 315 320 Asn Glu Ile Ser His Ile Gly Ser Arg Gly Lys Tyr
Ser Ser Gly Phe 325 330 335 Arg Asn Ile Ala Val Lys Glu Asn Leu Ile
Glu Leu Met Ala Asp Ile 340 345 350 Cys His Gln Val Phe Asn Glu Asp
Thr Arg Ser Leu Glu Ile Ser Gln 355 360 365 Ser Tyr Thr Thr Thr Gln
Arg Glu Ser Ser Asp Tyr Ser Val Pro Cys 370 375 380 Lys Arg Lys Lys
Ile Glu Leu Gly Trp Glu Val Ile Lys Asp His Leu 385 390 395 400 Gln
Lys Ser Gln Asn Asp Phe Asp Leu Val Pro Trp Leu Gln Ile Ala 405 410
415 Thr Gln Leu Ile Ser Lys Tyr Pro Ala Ser Leu Pro Asn Cys Glu Leu
420 425 430 Ser Pro Leu Leu Met Ile Leu Ser Gln Leu Leu Pro Gln Gln
Arg His 435 440 445 Gly Glu Arg Thr Pro Tyr Val Leu Arg Cys Leu Thr
Glu Val Ala Leu 450 455 460 Cys Gln Asp Lys Arg Ser Asn Leu Glu Ser
Ser Gln Lys Ser Asp Leu 465 470 475 480 Leu Lys Leu Trp Asn Lys Ile
Trp Cys Ile Thr Phe Arg Gly Ile Ser 485 490 495 Ser Glu Gln Ile Gln
Ala Glu Asn Phe Gly Leu Leu Gly Ala Ile Ile 500 505 510 Gln Gly Ser
Leu Val Glu Val Asp Arg Glu Phe Trp Lys Leu Phe Thr 515 520 525 Gly
Ser Ala Cys Arg Pro Ser Cys Pro Ala Val Cys Cys Leu Thr Leu 530 535
540 Ala Leu Thr Thr Ser Ile Val Pro Gly Thr Val Lys Met Gly Ile Glu
545 550 555 560 Gln Asn Met Cys Glu Val Asn Arg Ser Phe Ser Leu Lys
Glu Ser Ile 565 570 575 Met Lys Trp Leu Leu Phe Tyr Gln Leu Glu Gly
Asp Leu Glu Asn Ser 580 585 590 Thr Glu Val Pro Pro Ile Leu His Ser
Asn Phe Pro His Leu Val Leu 595 600 605 Glu Lys Ile Leu Val Ser Leu
Thr Met Lys Asn Cys Lys Ala Ala Met 610 615 620 Asn Phe Phe Gln Ser
Val Pro Glu Cys Glu His His Gln Lys Asp Lys 625 630 635 640 Glu Glu
Leu Ser Phe Ser Glu Val Glu Glu Leu Phe Leu Gln Thr Thr 645 650 655
Phe Asp Lys Met Asp Phe Leu Thr Ile Val Arg Glu Cys Gly Ile Glu 660
665 670 Lys His Gln Ser Ser Ile Gly Phe Ser Val His Gln Asn Leu Lys
Glu 675 680 685 Ser Leu Asp Arg Cys Leu Leu Gly Leu Ser Glu Gln Leu
Leu Asn Asn 690 695 700 Tyr Ser Ser Glu Ile Thr Asn Ser Glu Thr Leu
Val Arg Cys Ser Arg 705 710 715 720 Leu Leu Val Gly Val Leu Gly Cys
Tyr Cys Tyr Met Gly Val Ile Ala 725 730 735 Glu Glu Glu Ala Tyr Lys
Ser Glu Leu Phe Gln Lys Ala Lys Ser Leu 740 745 750 Met Gln Cys Ala
Gly Glu Ser Ile Thr Leu Phe Lys Asn Lys Thr Asn 755 760 765 Glu Glu
Phe Arg Ile Gly Ser Leu Arg Asn Met Met Gln Leu Cys Thr 770 775 780
Arg Cys Leu Ser Asn Cys Thr Lys Lys Ser Pro Asn Lys Ile Ala Ser 785
790 795 800 Gly Phe Phe Leu Arg Leu Leu Thr Ser Lys Leu Met Asn Asp
Ile Ala 805 810 815 Asp Ile Cys Lys Ser Leu Ala Ser Phe Ile Lys Lys
Pro Phe Asp Arg 820 825 830 Gly Glu Val Glu Ser Met Glu Asp Asp Thr
Asn Gly Asn Leu Met Glu 835 840 845 Val Glu Asp Gln Ser Ser Met Asn
Leu Phe Asn Asp Tyr Pro Asp Ser 850 855 860 Ser Val Ser Asp Ala Asn
Glu Pro Gly Glu Ser Gln Ser Thr Ile Gly 865 870 875 880 Ala Ile Asn
Pro Leu Ala Glu Glu Tyr Leu Ser Lys Gln Asp Leu Leu 885 890 895 Phe
Leu Asp Met Leu Lys Phe Leu Cys Leu Cys Val Thr Thr Ala Gln 900 905
910 Thr Asn Thr Val Ser Phe Arg Ala Ala Asp Ile Arg Arg Lys Leu Leu
915 920 925 Met Leu Ile Asp Ser Ser Thr Leu Glu Pro Thr Lys Ser Leu
His Leu 930 935 940 His Met Tyr Leu Met Leu Leu Lys Glu Leu Pro Gly
Glu Glu Tyr Pro 945 950 955 960 Leu Pro Met Glu Asp Val Leu Glu Leu
Leu Lys Pro Leu Ser Asn Val 965 970 975 Cys Ser Leu Tyr Arg Arg Asp
Gln Asp Val Cys Lys Thr Ile Leu Asn 980 985 990 His Val Leu His Val
Val Lys Asn Leu Gly Gln Ser Asn Met Asp Ser 995 1000 1005 Glu Asn
Thr Arg Asp Ala Gln Gly Gln Phe Leu Thr Val Ile Gly 1010 1015 1020
Ala Phe Trp His Leu Thr Lys Glu Arg Lys Tyr Ile Phe Ser Val 1025
1030 1035 Arg Met Ala Leu Val Asn Cys Leu Lys Thr Leu Leu Glu Ala
Asp 1040 1045 1050 Pro Tyr Ser Lys Trp Ala Ile Leu Asn Val Met Gly
Lys Asp Phe 1055 1060 1065 Pro Val Asn Glu Val Phe Thr Gln Phe Leu
Ala Asp Asn His His 1070 1075 1080 Gln Val Arg Met Leu Ala Ala Glu
Ser Ile Asn Arg Leu Phe Gln 1085 1090 1095 Asp Thr Lys Gly Asp Ser
Ser Arg Leu Leu Lys Ala Leu Pro Leu 1100 1105 1110 Lys Leu Gln Gln
Thr Ala Phe Glu Asn Ala Tyr Leu Lys Ala Gln 1115 1120 1125 Glu Gly
Met Arg Glu Met Ser His Ser Ala Glu Asn Pro Glu Thr 1130 1135 1140
Leu Asp Glu Ile Tyr Asn Arg Lys Ser Val Leu Leu Thr Leu Ile 1145
1150 1155 Ala Val Val Leu Ser Cys Ser Pro Ile Cys Glu Lys Gln Ala
Leu 1160 1165 1170 Phe Ala Leu Cys Lys Ser Val Lys Glu Asn Gly Leu
Glu Pro His 1175 1180 1185 Leu Val Lys Lys Val Leu Glu Lys Val Ser
Glu Thr Phe Gly Tyr 1190 1195 1200 Arg Arg Leu Glu Asp Phe Met Ala
Ser His Leu Asp Tyr Leu Val 1205 1210 1215 Leu Glu Trp Leu Asn Leu
Gln Asp Thr Glu Tyr Asn Leu Ser Ser 1220 1225 1230 Phe Pro Phe Ile
Leu Leu Asn Tyr Thr Asn Ile Glu Asp Phe Tyr 1235 1240 1245 Arg Ser
Cys Tyr Lys Val Leu Ile Pro His Leu Val Ile Arg Ser 1250 1255 1260
His Phe Asp Glu Val Lys Ser Ile Ala Asn Gln Ile Gln Glu Asp 1265
1270 1275 Trp Lys Ser Leu Leu Thr Asp Cys Phe Pro Lys Ile Leu Val
Asn 1280 1285 1290 Ile Leu Pro Tyr Phe Ala Tyr Glu Gly Thr Arg Asp
Ser Gly Met 1295 1300 1305 Ala Gln Gln Arg Glu Thr Ala Thr Lys Val
Tyr Asp Met Leu Lys 1310 1315 1320 Ser Glu Asn Leu Leu Gly Lys Gln
Ile Asp His Leu Phe Ile Ser 1325 1330 1335 Asn Leu Pro Glu Ile Val
Val Glu Leu Leu Met Thr Leu His Glu 1340 1345 1350 Pro Ala Asn Ser
Ser Ala Ser Gln Ser Thr Asp Leu Cys Asp Phe 1355 1360 1365 Ser Gly
Asp Leu Asp Pro Ala Pro Asn Pro Pro His Phe Pro Ser 1370 1375 1380
His Val Ile Lys Ala Thr Phe Ala Tyr Ile Ser Asn Cys His Lys 1385
1390 1395 Thr Lys Leu Lys Ser Ile Leu Glu Ile Leu Ser Lys Ser Pro
Asp 1400 1405 1410 Ser Tyr Gln Lys Ile Leu Leu Ala Ile Cys Glu Gln
Ala Ala Glu 1415 1420 1425 Thr Asn Asn Val Tyr Lys Lys His Arg Ile
Leu Lys Ile Tyr His 1430 1435 1440 Leu Phe Val Ser Leu Leu Leu Lys
Asp Ile Lys Ser Gly Leu Gly 1445 1450 1455 Gly Ala Trp Ala Phe Val
Leu Arg Asp Val Ile Tyr Thr Leu Ile 1460 1465 1470 His Tyr Ile Asn
Gln Arg Pro Ser Cys Ile Met Asp Val Ser Leu 1475 1480 1485 Arg Ser
Phe Ser Leu Cys Cys Asp Leu Leu Ser Gln Val Cys Gln 1490 1495 1500
Thr Ala Val Thr Tyr Cys Lys Asp Ala Leu Glu Asn His Leu His 1505
1510 1515 Val Ile Val Gly Thr Leu Ile Pro Leu Val Tyr Glu Gln Val
Glu 1520 1525 1530 Val Gln Lys Gln Val Leu Asp Leu Leu Lys Tyr Leu
Val Ile Asp 1535 1540 1545 Asn Lys Asp Asn Glu Asn Leu Tyr Ile Thr
Ile Lys Leu Leu Asp 1550 1555 1560 Pro Phe Pro Asp His Val Val Phe
Lys Asp Leu Arg Ile Thr Gln 1565 1570 1575 Gln Lys Ile Lys Tyr Ser
Arg Gly Pro Phe Ser Leu Leu Glu Glu 1580 1585 1590 Ile Asn His Phe
Leu Ser Val Ser Val Tyr Asp Ala Leu Pro Leu 1595 1600 1605 Thr Arg
Leu Glu Gly Leu Lys Asp Leu Arg Arg Gln Leu Glu Leu 1610 1615 1620
His Lys Asp Gln Met Val Asp Ile Met Arg Ala Ser Gln Asp Asn 1625
1630 1635 Pro Gln Asp Gly Ile Met Val Lys Leu Val Val Asn Leu Leu
Gln 1640 1645 1650 Leu Ser Lys Met Ala Ile Asn His Thr Gly Glu Lys
Glu Val Leu 1655 1660 1665 Glu Ala Val Gly Ser Cys Leu Gly Glu Val
Gly Pro Ile Asp Phe 1670 1675 1680 Ser Thr Ile Ala Ile Gln His Ser
Lys Asp Ala Ser Tyr Thr Lys 1685 1690 1695 Ala Leu Lys Leu Phe Glu
Asp Lys Glu Leu Gln Trp Thr Phe Ile 1700 1705 1710 Met Leu Thr Tyr
Leu Asn Asn Thr Leu Val Glu Asp Cys Val Lys 1715 1720 1725 Val Arg
Ser Ala Ala Val Thr Cys Leu Lys Asn Ile Leu Ala Thr 1730 1735 1740
Lys Thr Gly His Ser Phe Trp Glu Ile Tyr Lys Met Thr Thr Asp 1745
1750 1755 Pro Met Leu Ala Tyr Leu Gln Pro Phe Arg Thr Ser Arg Lys
Lys 1760 1765 1770 Phe Leu Glu Val Pro Arg Phe Asp Lys Glu Asn Pro
Phe Glu Gly 1775 1780 1785 Leu Asp Asp Ile Asn Leu Trp Ile Pro Leu
Ser Glu Asn His Asp 1790 1795 1800 Ile Trp Ile Lys Thr Leu Thr Cys
Ala Phe Leu Asp Ser Gly Gly 1805 1810 1815 Thr Lys Cys Glu Ile Leu
Gln Leu Leu Lys Pro Met Cys Glu Val 1820 1825 1830 Lys Thr Asp Phe
Cys Gln Thr Val Leu Pro Tyr Leu Ile His Asp 1835 1840 1845 Ile Leu
Leu Gln Asp Thr Asn Glu Ser Trp Arg Asn Leu Leu Ser 1850 1855 1860
Thr His Val Gln Gly Phe Phe Thr Ser Cys Leu Arg His Phe Ser 1865
1870 1875 Gln Thr Ser Arg Ser Thr Thr Pro Ala Asn Leu Asp Ser Glu
Ser 1880 1885 1890 Glu His Phe Phe Arg Cys Cys Leu Asp Lys Lys Ser
Gln Arg Thr 1895 1900 1905 Met Leu Ala Val Val Asp Tyr Met Arg Arg
Gln Lys Arg Pro Ser 1910 1915 1920 Ser Gly Thr Ile Phe Asn Asp Ala
Phe Trp Leu Asp Leu Asn Tyr 1925 1930 1935 Leu Glu Val Ala Lys Val
Ala Gln Ser Cys Ala Ala His Phe Thr 1940 1945 1950 Ala Leu Leu Tyr
Ala Glu Ile Tyr Ala Asp Lys Lys Ser Met Asp 1955 1960 1965 Asp Gln
Glu Lys Arg Ser Leu Ala Phe Glu Glu Gly Ser Gln Ser 1970 1975 1980
Thr Thr Ile Ser Ser Leu Ser Glu Lys Ser Lys Glu Glu Thr Gly 1985
1990 1995 Ile Ser Leu Gln Asp Leu Leu Leu Glu Ile Tyr Arg Ser Ile
Gly 2000 2005 2010 Glu Pro Asp Ser Leu Tyr Gly Cys Gly Gly Gly Lys
Met Leu Gln 2015 2020 2025 Pro Ile Thr Arg Leu Arg Thr Tyr Glu His
Glu Ala Met Trp Gly 2030 2035 2040 Lys Ala Leu Val Thr Tyr Asp Leu
Glu Thr Ala Ile Pro Ser Ser 2045 2050 2055 Thr Arg Gln Ala Gly Ile
Ile Gln Ala Leu Gln Asn Leu Gly Leu 2060 2065 2070 Cys His Ile Leu
Ser Val Tyr Leu Lys Gly Leu Asp Tyr Glu Asn 2075 2080 2085 Lys Asp
Trp Cys Pro Glu Leu Glu Glu Leu His Tyr Gln Ala Ala 2090 2095 2100
Trp Arg Asn Met Gln Trp Asp His Cys Thr Ser Val Ser Lys Glu 2105
2110 2115 Val Glu Gly Thr Ser Tyr His Glu Ser Leu Tyr Asn Ala Leu
Gln 2120 2125 2130 Ser Leu Arg Asp Arg Glu Phe Ser Thr Phe Tyr Glu
Ser Leu Lys 2135 2140 2145 Tyr Ala Arg Val Lys Glu Val Glu Glu Met
Cys Lys Arg Ser Leu 2150 2155 2160 Glu Ser Val Tyr Ser Leu Tyr Pro
Thr Leu Ser Arg Leu Gln Ala 2165 2170 2175 Ile Gly Glu Leu Glu Ser
Ile Gly Glu Leu Phe Ser Arg Ser Val 2180 2185 2190 Thr His Arg Gln
Leu Ser Glu Val Tyr Ile Lys Trp Gln Lys His 2195 2200 2205 Ser Gln
Leu Leu Lys Asp Ser Asp Phe Ser Phe Gln Glu Pro Ile 2210 2215 2220
Met Ala Leu Arg Thr Val Ile Leu Glu Ile Leu Met Glu Lys Glu 2225
2230 2235 Met Asp Asn Ser Gln Arg Glu Cys Ile Lys Asp Ile Leu Thr
Lys 2240 2245 2250 His Leu Val Glu Leu Ser Ile Leu Ala Arg Thr Phe
Lys Asn Thr 2255 2260 2265 Gln Leu Pro Glu Arg Ala Ile Phe Gln Ile
Lys Gln Tyr Asn Ser 2270 2275 2280 Val Ser Cys Gly Val Ser Glu Trp
Gln Leu Glu Glu Ala Gln Val 2285 2290 2295 Phe Trp Ala Lys Lys Glu
Gln Ser Leu Ala Leu Ser Ile Leu Lys 2300 2305 2310 Gln Met Ile Lys
Lys Leu Asp Ala Ser Cys Ala Ala Asn Asn Pro 2315 2320 2325 Ser Leu
Lys Leu Thr Tyr Thr Glu Cys Leu Arg Val Cys Gly Asn 2330 2335 2340
Trp Leu Ala Glu Thr Cys Leu Glu Asn Pro Ala Val Ile Met Gln 2345
2350 2355 Thr Tyr Leu Glu Lys Ala Val Glu Val Ala Gly Asn Tyr Asp
Gly 2360 2365 2370 Glu Ser Ser Asp Glu Leu Arg Asn Gly Lys Met Lys
Ala Phe Leu 2375 2380 2385 Ser Leu Ala Arg Phe Ser Asp Thr Gln Tyr
Gln Arg Ile Glu Asn 2390 2395 2400 Tyr Met Lys Ser Ser Glu Phe Glu
Asn Lys Gln Ala Leu Leu Lys 2405 2410 2415 Arg Ala Lys Glu Glu Val
Gly Leu Leu Arg Glu His Lys Ile Gln 2420 2425 2430 Thr Asn Arg Tyr
Thr Val Lys Val Gln Arg Glu Leu Glu Leu Asp 2435 2440 2445 Glu Leu
Ala Leu Arg Ala Leu Lys Glu Asp Arg Lys Arg Phe Leu 2450 2455 2460
Cys Lys Ala Val Glu Asn Tyr Ile Asn Cys Leu Leu Ser Gly Glu 2465
2470 2475 Glu His Asp Met Trp Val Phe Arg Leu Cys Ser Leu Trp Leu
Glu 2480 2485 2490 Asn Ser Gly Val Ser Glu Val Asn Gly Met Met Lys
Arg Asp Gly 2495 2500
2505 Met Lys Ile Pro Thr Tyr Lys Phe Leu Pro Leu Met Tyr Gln Leu
2510 2515 2520 Ala Ala Arg Met Gly Thr Lys Met Met Gly Gly Leu Gly
Phe His 2525 2530 2535 Glu Val Leu Asn Asn Leu Ile Ser Arg Ile Ser
Met Asp His Pro 2540 2545 2550 His His Thr Leu Phe Ile Ile Leu Ala
Leu Ala Asn Ala Asn Arg 2555 2560 2565 Asp Glu Phe Leu Thr Lys Pro
Glu Val Ala Arg Arg Ser Arg Ile 2570 2575 2580 Thr Lys Asn Val Pro
Lys Gln Ser Ser Gln Leu Asp Glu Asp Arg 2585 2590 2595 Thr Glu Ala
Ala Asn Arg Ile Ile Cys Thr Ile Arg Ser Arg Arg 2600 2605 2610 Pro
Gln Met Val Arg Ser Val Glu Ala Leu Cys Asp Ala Tyr Ile 2615 2620
2625 Ile Leu Ala Asn Leu Asp Ala Thr Gln Trp Lys Thr Gln Arg Lys
2630 2635 2640 Gly Ile Asn Ile Pro Ala Asp Gln Pro Ile Thr Lys Leu
Lys Asn 2645 2650 2655 Leu Glu Asp Val Val Val Pro Thr Met Glu Ile
Lys Val Asp His 2660 2665 2670 Thr Gly Glu Tyr Gly Asn Leu Val Thr
Ile Gln Ser Phe Lys Ala 2675 2680 2685 Glu Phe Arg Leu Ala Gly Gly
Val Asn Leu Pro Lys Ile Ile Asp 2690 2695 2700 Cys Val Gly Ser Asp
Gly Lys Glu Arg Arg Gln Leu Val Lys Gly 2705 2710 2715 Arg Asp Asp
Leu Arg Gln Asp Ala Val Met Gln Gln Val Phe Gln 2720 2725 2730 Met
Cys Asn Thr Leu Leu Gln Arg Asn Thr Glu Thr Arg Lys Arg 2735 2740
2745 Lys Leu Thr Ile Cys Thr Tyr Lys Val Val Pro Leu Ser Gln Arg
2750 2755 2760 Ser Gly Val Leu Glu Trp Cys Thr Gly Thr Val Pro Ile
Gly Glu 2765 2770 2775 Phe Leu Val Asn Asn Glu Asp Gly Ala His Lys
Arg Tyr Arg Pro 2780 2785 2790 Asn Asp Phe Ser Ala Phe Gln Cys Gln
Lys Lys Met Met Glu Val 2795 2800 2805 Gln Lys Lys Ser Phe Glu Glu
Lys Tyr Glu Val Phe Met Asp Val 2810 2815 2820 Cys Gln Asn Phe Gln
Pro Val Phe Arg Tyr Phe Cys Met Glu Lys 2825 2830 2835 Phe Leu Asp
Pro Ala Ile Trp Phe Glu Lys Arg Leu Ala Tyr Thr 2840 2845 2850 Arg
Ser Val Ala Thr Ser Ser Ile Val Gly Tyr Ile Leu Gly Leu 2855 2860
2865 Gly Asp Arg His Val Gln Asn Ile Leu Ile Asn Glu Gln Ser Ala
2870 2875 2880 Glu Leu Val His Ile Asp Leu Gly Val Ala Phe Glu Gln
Gly Lys 2885 2890 2895 Ile Leu Pro Thr Pro Glu Thr Val Pro Phe Arg
Leu Thr Arg Asp 2900 2905 2910 Ile Val Asp Gly Met Gly Ile Thr Gly
Val Glu Gly Val Phe Arg 2915 2920 2925 Arg Cys Cys Glu Lys Thr Met
Glu Val Met Arg Asn Ser Gln Glu 2930 2935 2940 Thr Leu Leu Thr Ile
Val Glu Val Leu Leu Tyr Asp Pro Leu Phe 2945 2950 2955 Asp Trp Thr
Met Asn Pro Leu Lys Ala Leu Tyr Leu Gln Gln Arg 2960 2965 2970 Pro
Glu Asp Glu Thr Glu Leu His Pro Thr Leu Asn Ala Asp Asp 2975 2980
2985 Gln Glu Cys Lys Arg Asn Leu Ser Asp Ile Asp Gln Ser Phe Asn
2990 2995 3000 Lys Val Ala Glu Arg Val Leu Met Arg Leu Gln Glu Lys
Leu Lys 3005 3010 3015 Gly Val Glu Glu Gly Thr Val Leu Ser Val Gly
Gly Gln Val Asn 3020 3025 3030 Leu Leu Ile Gln Gln Ala Ile Asp Pro
Lys Asn Leu Ser Arg Leu 3035 3040 3045 Phe Pro Gly Trp Lys Ala Trp
Val 3050 3055 51130PRTHomo sapiens 5Met Leu Glu Ile Cys Leu Lys Leu
Val Gly Cys Lys Ser Lys Lys Gly 1 5 10 15 Leu Ser Ser Ser Ser Ser
Cys Tyr Leu Glu Glu Ala Leu Gln Arg Pro 20 25 30 Val Ala Ser Asp
Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala Arg Trp 35 40 45 Asn Ser
Lys Glu Asn Leu Leu Ala Gly Pro Ser Glu Asn Asp Pro Asn 50 55 60
Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser Gly Asp Asn Thr Leu 65
70 75 80 Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu Gly Tyr Asn
His Asn 85 90 95 Gly Glu Trp Cys Glu Ala Gln Thr Lys Asn Gly Gln
Gly Trp Val Pro 100 105 110 Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu
Glu Lys His Ser Trp Tyr 115 120 125 His Gly Pro Val Ser Arg Asn Ala
Ala Glu Tyr Leu Leu Ser Ser Gly 130 135 140 Ile Asn Gly Ser Phe Leu
Val Arg Glu Ser Glu Ser Ser Pro Gly Gln 145 150 155 160 Arg Ser Ile
Ser Leu Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg Ile 165 170 175 Asn
Thr Ala Ser Asp Gly Lys Leu Tyr Val Ser Ser Glu Ser Arg Phe 180 185
190 Asn Thr Leu Ala Glu Leu Val His His His Ser Thr Val Ala Asp Gly
195 200 205 Leu Ile Thr Thr Leu His Tyr Pro Ala Pro Lys Arg Asn Lys
Pro Thr 210 215 220 Val Tyr Gly Val Ser Pro Asn Tyr Asp Lys Trp Glu
Met Glu Arg Thr 225 230 235 240 Asp Ile Thr Met Lys His Lys Leu Gly
Gly Gly Gln Tyr Gly Glu Val 245 250 255 Tyr Glu Gly Val Trp Lys Lys
Tyr Ser Leu Thr Val Ala Val Lys Thr 260 265 270 Leu Lys Glu Asp Thr
Met Glu Val Glu Glu Phe Leu Lys Glu Ala Ala 275 280 285 Val Met Lys
Glu Ile Lys His Pro Asn Leu Val Gln Leu Leu Gly Val 290 295 300 Cys
Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Phe Met Thr Tyr 305 310
315 320 Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg Gln Glu Val
Asn 325 330 335 Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile Ser Ser
Ala Met Glu 340 345 350 Tyr Leu Glu Lys Lys Asn Phe Ile His Arg Asp
Leu Ala Ala Arg Asn 355 360 365 Cys Leu Val Gly Glu Asn His Leu Val
Lys Val Ala Asp Phe Gly Leu 370 375 380 Ser Arg Leu Met Thr Gly Asp
Thr Tyr Thr Ala His Ala Gly Ala Lys 385 390 395 400 Phe Pro Ile Lys
Trp Thr Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe 405 410 415 Ser Ile
Lys Ser Asp Val Trp Ala Phe Gly Val Leu Leu Trp Glu Ile 420 425 430
Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile Asp Leu Ser Gln Val 435
440 445 Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Pro Glu Gly
Cys 450 455 460 Pro Glu Lys Val Tyr Glu Leu Met Arg Ala Cys Trp Gln
Trp Asn Pro 465 470 475 480 Ser Asp Arg Pro Ser Phe Ala Glu Ile His
Gln Ala Phe Glu Thr Met 485 490 495 Phe Gln Glu Ser Ser Ile Ser Asp
Glu Val Glu Lys Glu Leu Gly Lys 500 505 510 Gln Gly Val Arg Gly Ala
Val Ser Thr Leu Leu Gln Ala Pro Glu Leu 515 520 525 Pro Thr Lys Thr
Arg Thr Ser Arg Arg Ala Ala Glu His Arg Asp Thr 530 535 540 Thr Asp
Val Pro Glu Met Pro His Ser Lys Gly Gln Gly Glu Ser Asp 545 550 555
560 Pro Leu Asp His Glu Pro Ala Val Ser Pro Leu Leu Pro Arg Lys Glu
565 570 575 Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu Asp Glu Arg Leu
Leu Pro 580 585 590 Lys Asp Lys Lys Thr Asn Leu Phe Ser Ala Leu Ile
Lys Lys Lys Lys 595 600 605 Lys Thr Ala Pro Thr Pro Pro Lys Arg Ser
Ser Ser Phe Arg Glu Met 610 615 620 Asp Gly Gln Pro Glu Arg Arg Gly
Ala Gly Glu Glu Glu Gly Arg Asp 625 630 635 640 Ile Ser Asn Gly Ala
Leu Ala Phe Thr Pro Leu Asp Thr Ala Asp Pro 645 650 655 Ala Lys Ser
Pro Lys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly Ala 660 665 670 Leu
Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser Pro His Leu Trp Lys 675 680
685 Lys Ser Ser Thr Leu Thr Ser Ser Arg Leu Ala Thr Gly Glu Glu Glu
690 695 700 Gly Gly Gly Ser Ser Ser Lys Arg Phe Leu Arg Ser Cys Ser
Ala Ser 705 710 715 720 Cys Val Pro His Gly Ala Lys Asp Thr Glu Trp
Arg Ser Val Thr Leu 725 730 735 Pro Arg Asp Leu Gln Ser Thr Gly Arg
Gln Phe Asp Ser Ser Thr Phe 740 745 750 Gly Gly His Lys Ser Glu Lys
Pro Ala Leu Pro Arg Lys Arg Ala Gly 755 760 765 Glu Asn Arg Ser Asp
Gln Val Thr Arg Gly Thr Val Thr Pro Pro Pro 770 775 780 Arg Leu Val
Lys Lys Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp 785 790 795 800
Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro Asn Leu Thr Pro Lys 805
810 815 Pro Leu Arg Arg Gln Val Thr Val Ala Pro Ala Ser Gly Leu Pro
His 820 825 830 Lys Glu Glu Ala Gly Lys Gly Ser Ala Leu Gly Thr Pro
Ala Ala Ala 835 840 845 Glu Pro Val Thr Pro Thr Ser Lys Ala Gly Ser
Gly Ala Pro Gly Gly 850 855 860 Thr Ser Lys Gly Pro Ala Glu Glu Ser
Arg Val Arg Arg His Lys His 865 870 875 880 Ser Ser Glu Ser Pro Gly
Arg Asp Lys Gly Lys Leu Ser Arg Leu Lys 885 890 895 Pro Ala Pro Pro
Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly 900 905 910 Lys Pro
Ser Gln Ser Pro Ser Gln Glu Ala Ala Gly Glu Ala Val Leu 915 920 925
Gly Ala Lys Thr Lys Ala Thr Ser Leu Val Asp Ala Val Asn Ser Asp 930
935 940 Ala Ala Lys Pro Ser Gln Pro Gly Glu Gly Leu Lys Lys Pro Val
Leu 945 950 955 960 Pro Ala Thr Pro Lys Pro Gln Ser Ala Lys Pro Ser
Gly Thr Pro Ile 965 970 975 Ser Pro Ala Pro Val Pro Ser Thr Leu Pro
Ser Ala Ser Ser Ala Leu 980 985 990 Ala Gly Asp Gln Pro Ser Ser Thr
Ala Phe Ile Pro Leu Ile Ser Thr 995 1000 1005 Arg Val Ser Leu Arg
Lys Thr Arg Gln Pro Pro Glu Arg Ile Ala 1010 1015 1020 Ser Gly Ala
Ile Thr Lys Gly Val Val Leu Asp Ser Thr Glu Ala 1025 1030 1035 Leu
Cys Leu Ala Ile Ser Arg Asn Ser Glu Gln Met Ala Ser His 1040 1045
1050 Ser Ala Val Leu Glu Ala Gly Lys Asn Leu Tyr Thr Phe Cys Val
1055 1060 1065 Ser Tyr Val Asp Ser Ile Gln Gln Met Arg Asn Lys Phe
Ala Phe 1070 1075 1080 Arg Glu Ala Ile Asn Lys Leu Glu Asn Asn Leu
Arg Glu Leu Gln 1085 1090 1095 Ile Cys Pro Ala Thr Ala Gly Ser Gly
Pro Ala Ala Thr Gln Asp 1100 1105 1110 Phe Ser Lys Leu Leu Ser Ser
Val Lys Glu Ile Ser Asp Ile Val 1115 1120 1125 Gln Arg 1130
6707PRTHomo sapiens 6Met Asn Gln Glu Leu Leu Ser Val Gly Ser Lys
Arg Arg Arg Thr Gly 1 5 10 15 Gly Ser Leu Arg Gly Asn Pro Ser Ser
Ser Gln Val Asp Glu Glu Gln 20 25 30 Met Asn Arg Val Val Glu Glu
Glu Gln Gln Gln Gln Leu Arg Gln Gln 35 40 45 Glu Glu Glu His Thr
Ala Arg Asn Gly Glu Val Val Gly Val Glu Pro 50 55 60 Arg Pro Gly
Gly Gln Asn Asp Ser Gln Gln Gly Gln Leu Glu Glu Asn 65 70 75 80 Asn
Asn Arg Phe Ile Ser Val Asp Glu Asp Ser Ser Gly Asn Gln Glu 85 90
95 Glu Gln Glu Glu Asp Glu Glu His Ala Gly Glu Gln Asp Glu Glu Asp
100 105 110 Glu Glu Glu Glu Glu Met Asp Gln Glu Ser Asp Asp Phe Asp
Gln Ser 115 120 125 Asp Asp Ser Ser Arg Glu Asp Glu His Thr His Thr
Asn Ser Val Thr 130 135 140 Asn Ser Ser Ser Ile Val Asp Leu Pro Val
His Gln Leu Ser Ser Pro 145 150 155 160 Phe Tyr Thr Lys Thr Thr Lys
Met Lys Arg Lys Leu Asp His Gly Ser 165 170 175 Glu Val Arg Ser Phe
Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu 180 185 190 Tyr Thr Ser
Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr 195 200 205 Phe
Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 210 215
220 Ile Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met
225 230 235 240 Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu
Asp Glu Leu 245 250 255 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His
Met Met Gln Val Ile 260 265 270 Glu Pro Gln Phe Gln Arg Asp Phe Ile
Ser Leu Leu Pro Lys Glu Leu 275 280 285 Ala Leu Tyr Val Leu Ser Phe
Leu Glu Pro Lys Asp Leu Leu Gln Ala 290 295 300 Ala Gln Thr Cys Arg
Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu 305 310 315 320 Trp Arg
Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile 325 330 335
Lys Arg Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys 340
345 350 Ser Ala Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg
Gly 355 360 365 Glu Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp
His Val Ile 370 375 380 Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile Val
Ser Gly Ser Asp Asp 385 390 395 400 Asn Thr Leu Lys Val Trp Ser Ala
Val Thr Gly Lys Cys Leu Arg Thr 405 410 415 Leu Val Gly His Thr Gly
Gly Val Trp Ser Ser Gln Met Arg Asp Asn 420 425 430 Ile Ile Ile Ser
Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala 435 440 445 Glu Thr
Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val 450 455 460
Arg Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 465
470 475 480 Ala Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu
His Val 485 490 495 Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln
Tyr Asp Gly Arg 500 505 510 Arg Val Val Ser Gly Ala Tyr Asp Phe Met
Val Lys Val Trp Asp Pro 515 520 525 Glu Thr Glu Thr Cys Leu His Thr
Leu Gln Gly His Thr Asn Arg Val 530 535 540 Tyr Ser Leu Gln Phe Asp
Gly Ile His Val Val Ser Gly Ser Leu Asp 545 550 555 560 Thr Ser Ile
Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr 565 570 575 Leu
Thr Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn 580
585 590 Ile Leu Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp
Ile 595 600 605 Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn
Lys His Gln 610 615 620 Ser Ala Val Thr Cys Leu Gln Phe Asn Lys Asn
Phe Val Ile Thr Ser 625 630 635 640 Ser Asp Asp Gly Thr Val Lys Leu
Trp Asp Leu Lys Thr Gly Glu Phe 645 650 655 Ile Arg Asn Leu Val Thr
Leu Glu Ser Gly Gly Ser Gly Gly Val Val 660 665 670 Trp Arg Ile Arg
Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser 675 680 685 Arg Asn
Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val 690 695 700
Asp Met Lys 705 7662PRTHomo sapiens 7Met Ser His Val Ala Val Glu
Asn Ala Leu Gly Leu Asp Gln Gln Phe 1 5 10 15 Ala Gly Leu Asp Leu
Asn Ser Ser Asp Asn Gln Ser Gly Gly Ser Thr 20 25 30 Ala Ser Lys
Gly Arg Tyr Ile Pro Pro His Leu Arg Asn Arg Glu Ala 35 40 45 Thr
Lys Gly Phe Tyr Asp Lys Asp Ser Ser Gly Trp Ser Ser Ser Lys 50 55
60 Asp Lys Asp Ala Tyr Ser Ser Phe Gly Ser Arg Ser Asp Ser Arg Gly
65 70 75 80 Lys Ser Ser Phe Phe Ser Asp Arg Gly Ser Gly Ser Arg Gly
Arg Phe 85 90 95 Asp Asp Arg Gly Arg Ser Asp Tyr Asp Gly Ile Gly
Ser Arg Gly Asp 100 105 110 Arg Ser Gly Phe Gly Lys Phe Glu Arg Gly
Gly Asn Ser Arg Trp Cys 115 120 125 Asp Lys Ser Asp Glu Asp Asp Trp
Ser Lys Pro Leu Pro Pro Ser Glu 130 135 140 Arg Leu Glu Gln Glu Leu
Phe Ser Gly Gly Asn Thr Gly Ile Asn Phe 145 150 155 160 Glu Lys Tyr
Asp Asp Ile Pro Val Glu Ala Thr Gly Asn Asn Cys Pro 165 170 175 Pro
His Ile Glu Ser Phe Ser Asp Val Glu Met Gly Glu Ile Ile Met 180 185
190 Gly Asn Ile Glu Leu Thr Arg Tyr Thr Arg Pro Thr Pro Val Gln Lys
195 200 205 His Ala Ile Pro Ile Ile Lys Glu Lys Arg Asp Leu Met Ala
Cys Ala 210 215 220 Gln Thr Gly Ser Gly Lys Thr Ala Ala Phe Leu Leu
Pro Ile Leu Ser 225 230 235 240 Gln Ile Tyr Ser Asp Gly Pro Gly Glu
Ala Leu Arg Ala Met Lys Glu 245 250 255 Asn Gly Arg Tyr Gly Arg Arg
Lys Gln Tyr Pro Ile Ser Leu Val Leu 260 265 270 Ala Pro Thr Arg Glu
Leu Ala Val Gln Ile Tyr Glu Glu Ala Arg Lys 275 280 285 Phe Ser Tyr
Arg Ser Arg Val Arg Pro Cys Val Val Tyr Gly Gly Ala 290 295 300 Asp
Ile Gly Gln Gln Ile Arg Asp Leu Glu Arg Gly Cys His Leu Leu 305 310
315 320 Val Ala Thr Pro Gly Arg Leu Val Asp Met Met Glu Arg Gly Lys
Ile 325 330 335 Gly Leu Asp Phe Cys Lys Tyr Leu Val Leu Asp Glu Ala
Asp Arg Met 340 345 350 Leu Asp Met Gly Phe Glu Pro Gln Ile Arg Arg
Ile Val Glu Gln Asp 355 360 365 Thr Met Pro Pro Lys Gly Val Arg His
Thr Met Met Phe Ser Ala Thr 370 375 380 Phe Pro Lys Glu Ile Gln Met
Leu Ala Arg Asp Phe Leu Asp Glu Tyr 385 390 395 400 Ile Phe Leu Ala
Val Gly Arg Val Gly Ser Thr Ser Glu Asn Ile Thr 405 410 415 Gln Lys
Val Val Trp Val Glu Glu Ser Asp Lys Arg Ser Phe Leu Leu 420 425 430
Asp Leu Leu Asn Ala Thr Gly Lys Asp Ser Leu Thr Leu Val Phe Val 435
440 445 Glu Thr Lys Lys Gly Ala Asp Ser Leu Glu Asp Phe Leu Tyr His
Glu 450 455 460 Gly Tyr Ala Cys Thr Ser Ile His Gly Asp Arg Ser Gln
Arg Asp Arg 465 470 475 480 Glu Glu Ala Leu His Gln Phe Arg Ser Gly
Lys Ser Pro Ile Leu Val 485 490 495 Ala Thr Ala Val Ala Ala Arg Gly
Leu Asp Ile Ser Asn Val Lys His 500 505 510 Val Ile Asn Phe Asp Leu
Pro Ser Asp Ile Glu Glu Tyr Val His Arg 515 520 525 Ile Gly Arg Thr
Gly Arg Val Gly Asn Leu Gly Leu Ala Thr Ser Phe 530 535 540 Phe Asn
Glu Arg Asn Ile Asn Ile Thr Lys Asp Leu Leu Asp Leu Leu 545 550 555
560 Val Glu Ala Lys Gln Glu Val Pro Ser Trp Leu Glu Asn Met Ala Tyr
565 570 575 Glu His His Tyr Lys Gly Ser Ser Arg Gly Arg Ser Lys Ser
Ser Arg 580 585 590 Phe Ser Gly Gly Phe Gly Ala Arg Asp Tyr Arg Gln
Ser Ser Gly Ala 595 600 605 Ser Ser Ser Ser Phe Ser Ser Ser Arg Ala
Ser Ser Ser Arg Ser Gly 610 615 620 Gly Gly Gly His Gly Ser Ser Arg
Gly Phe Gly Gly Gly Gly Tyr Gly 625 630 635 640 Gly Phe Tyr Asn Ser
Asp Gly Tyr Gly Gly Asn Tyr Asn Ser Gln Gly 645 650 655 Val Asp Trp
Trp Gly Asn 660 8360PRTHomo sapiens 8Met Ala Ala Ala Ala Ala Ala
Gly Ala Gly Pro Glu Met Val Arg Gly 1 5 10 15 Gln Val Phe Asp Val
Gly Pro Arg Tyr Thr Asn Leu Ser Tyr Ile Gly 20 25 30 Glu Gly Ala
Tyr Gly Met Val Cys Ser Ala Tyr Asp Asn Val Asn Lys 35 40 45 Val
Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr Tyr 50 55
60 Cys Gln Arg Thr Leu Arg Glu Ile Lys Ile Leu Leu Arg Phe Arg His
65 70 75 80 Glu Asn Ile Ile Gly Ile Asn Asp Ile Ile Arg Ala Pro Thr
Ile Glu 85 90 95 Gln Met Lys Asp Val Tyr Ile Val Gln Asp Leu Met
Glu Thr Asp Leu 100 105 110 Tyr Lys Leu Leu Lys Thr Gln His Leu Ser
Asn Asp His Ile Cys Tyr 115 120 125 Phe Leu Tyr Gln Ile Leu Arg Gly
Leu Lys Tyr Ile His Ser Ala Asn 130 135 140 Val Leu His Arg Asp Leu
Lys Pro Ser Asn Leu Leu Leu Asn Thr Thr 145 150 155 160 Cys Asp Leu
Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ala Asp Pro 165 170 175 Asp
His Asp His Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg Trp 180 185
190 Tyr Arg Ala Pro Glu Ile Met Leu Asn Ser Lys Gly Tyr Thr Lys Ser
195 200 205 Ile Asp Ile Trp Ser Val Gly Cys Ile Leu Ala Glu Met Leu
Ser Asn 210 215 220 Arg Pro Ile Phe Pro Gly Lys His Tyr Leu Asp Gln
Leu Asn His Ile 225 230 235 240 Leu Gly Ile Leu Gly Ser Pro Ser Gln
Glu Asp Leu Asn Cys Ile Ile 245 250 255 Asn Leu Lys Ala Arg Asn Tyr
Leu Leu Ser Leu Pro His Lys Asn Lys 260 265 270 Val Pro Trp Asn Arg
Leu Phe Pro Asn Ala Asp Ser Lys Ala Leu Asp 275 280 285 Leu Leu Asp
Lys Met Leu Thr Phe Asn Pro His Lys Arg Ile Glu Val 290 295 300 Glu
Gln Ala Leu Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp Pro Ser 305 310
315 320 Asp Glu Pro Ile Ala Glu Ala Pro Phe Lys Phe Asp Met Glu Leu
Asp 325 330 335 Asp Leu Pro Lys Glu Lys Leu Lys Glu Leu Ile Phe Glu
Glu Thr Ala 340 345 350 Arg Phe Gln Pro Gly Tyr Arg Ser 355 360
9340PRTHomo sapiens 9Met Ser Glu Leu Asp Gln Leu Arg Gln Glu Ala
Glu Gln Leu Lys Asn 1 5 10 15 Gln Ile Arg Asp Ala Arg Lys Ala Cys
Ala Asp Ala Thr Leu Ser Gln 20 25 30 Ile Thr Asn Asn Ile Asp Pro
Val Gly Arg Ile Gln Met Arg Thr Arg 35 40 45 Arg Thr Leu Arg Gly
His Leu Ala Lys Ile Tyr Ala Met His Trp Gly 50 55 60 Thr Asp Ser
Arg Leu Leu Val Ser Ala Ser Gln Asp Gly Lys Leu Ile 65 70 75 80 Ile
Trp Asp Ser Tyr Thr Thr Asn Lys Val His Ala Ile Pro Leu Arg 85 90
95 Ser Ser Trp Val Met Thr Cys Ala Tyr Ala Pro Ser Gly Asn Tyr Val
100 105 110 Ala Cys Gly Gly Leu Asp Asn Ile Cys Ser Ile Tyr Asn Leu
Lys Thr 115 120 125 Arg Glu Gly Asn Val Arg Val Ser Arg Glu Leu Ala
Gly His Thr Gly 130 135 140 Tyr Leu Ser Cys Cys Arg Phe Leu Asp Asp
Asn Gln Ile Val Thr Ser 145 150 155 160 Ser Gly Asp Thr Thr Cys Ala
Leu Trp Asp Ile Glu Thr Gly Gln Gln 165 170 175 Thr Thr Thr Phe Thr
Gly His Thr Gly Asp Val Met Ser Leu Ser Leu 180 185 190 Ala Pro Asp
Thr Arg Leu Phe Val Ser Gly Ala Cys Asp Ala Ser Ala 195 200 205 Lys
Leu Trp Asp Val Arg Glu Gly Met Cys Arg Gln Thr Phe Thr Gly 210 215
220 His Glu Ser Asp Ile Asn Ala Ile Cys Phe Phe Pro Asn Gly Asn Ala
225 230 235 240 Phe Ala Thr Gly Ser Asp Asp Ala Thr Cys Arg Leu Phe
Asp Leu Arg 245 250 255 Ala Asp Gln Glu Leu Met Thr Tyr Ser His Asp
Asn Ile Ile Cys Gly 260 265 270 Ile Thr Ser Val Ser Phe Ser Lys Ser
Gly Arg Leu Leu Leu Ala Gly 275 280 285 Tyr Asp Asp Phe Asn Cys Asn
Val Trp Asp Ala Leu Lys Ala Asp Arg 290 295 300 Ala Gly Val Leu Ala
Gly His Asp Asn Arg Val Ser Cys Leu Gly Val 305 310 315 320 Thr Asp
Asp Gly Met Ala Val Ala Thr Gly Ser Trp Asp Ser Phe Leu 325 330 335
Lys Ile Trp Asn 340 109PRTArtificial SequenceChemically synthesized
peptide 10Lys Val Tyr Glu Gly Val Trp Lys Lys 1 5 118PRTArtificial
SequenceParental peptide 11Leu Met Pro Lys His Phe Ile Arg 1 5
128PRTArtificial SequenceMissense mutant peptide 12Leu Met Pro Lys
Leu Phe Ile Arg 1 5 139PRTArtificial SequenceChemically synthesized
peptide 13Ala Ser Ile Leu Leu Met Thr Val Thr 1 5 149PRTArtificial
SequenceChemically synthesized peptide 14Ser Ile Leu Leu Met Thr
Val Thr Ser 1 5 159PRTArtificial SequenceChemically synthesized
peptide 15Ile Leu Leu Met Thr Val Thr Ser Ile 1 5 169PRTArtificial
SequenceChemically synthesized peptide 16Leu Leu Met Thr Val Thr
Ser Ile Asp 1 5 179PRTArtificial SequenceChemically synthesized
peptide 17Leu Met Thr Val Thr Ser Ile Asp Arg 1 5 189PRTArtificial
SequenceChemically synthesized peptide 18Met Thr Val Thr Ser Ile
Asp Arg Phe 1 5 199PRTArtificial SequenceChemically synthesized
peptide 19Thr Val Thr Ser Ile Asp Arg Phe Leu 1 5 209PRTArtificial
SequenceChemically synthesized peptide 20Val Thr Ser Ile Asp Arg
Phe Leu Ala 1 5 219PRTArtificial SequenceChemically synthesized
peptide 21Thr Ser Ile Asp Arg Phe Leu Ala Val 1 5
* * * * *