U.S. patent application number 09/923831 was filed with the patent office on 2002-08-22 for tumor associated nucleic acids and uses therefor.
Invention is credited to Boon-Falleur, Thierry, Martelange, Valerie, Smet, Charles De.
Application Number | 20020115142 09/923831 |
Document ID | / |
Family ID | 26821090 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115142 |
Kind Code |
A1 |
Martelange, Valerie ; et
al. |
August 22, 2002 |
Tumor associated nucleic acids and uses therefor
Abstract
The invention describes sdp3.8 tumor associated nucleic acids,
including fragments and biologically functional variants thereof.
Also included are polypeptides and fragments thereof encoded by
such nucleic acids, and antibodies relating thereto. Methods and
products also are provided for diagnosing and treating conditions
characterized by expression of a sdp3.8 gene product.
Inventors: |
Martelange, Valerie;
(Brussels, BE) ; Smet, Charles De; (Brussels,
BE) ; Boon-Falleur, Thierry; (Brussels, BE) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Family ID: |
26821090 |
Appl. No.: |
09/923831 |
Filed: |
August 7, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09923831 |
Aug 7, 2001 |
|
|
|
09567995 |
May 10, 2000 |
|
|
|
6303756 |
|
|
|
|
09567995 |
May 10, 2000 |
|
|
|
09183706 |
Oct 30, 1998 |
|
|
|
6245525 |
|
|
|
|
09183706 |
Oct 30, 1998 |
|
|
|
09122989 |
Jul 27, 1998 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/183; 435/320.1; 435/325; 536/23.1 |
Current CPC
Class: |
C07K 14/4748
20130101 |
Class at
Publication: |
435/69.1 ;
435/183; 435/320.1; 435/325; 536/23.1 |
International
Class: |
C12P 021/02; C12N
005/06; C07H 021/04; C12N 009/00 |
Claims
1. An isolated nucleic acid molecule selected from the group
consisting of: (a) nucleic acid molecules which hybridize under
stringent conditions to a nucleic acid molecule having a nucleotide
sequence set forth as SEQ ID NO:42, and which code for a sarcoma
associated gene product, (b) deletions, additions and substitutions
of the nucleic acid molecules of (a), which code for a sarcoma
associated gene product, (c) nucleic acid molecules that differ
from the nucleic acid molecules of (a) or (b) in codon sequence due
to the degeneracy of the genetic code, and (d) complements of (a),
(b) or (c).
2. The isolated nucleic acid molecule of claim 1, wherein the
isolated nucleic acid molecule comprises the nucleic acid sequence
set forth as SEQ ID NO: 1.
3. The isolated nucleic acid molecule of claim 1, wherein the
isolated nucleic acid molecule comprises the nucleic acid sequence
set forth as nucleotides 208-2151 of SEQ ID NO:42.
4. The isolated nucleic acid molecule of claim 1, wherein the
isolated nucleic acid molecule comprises the nucleic acid sequence
set forth as SEQ ID NO:42.
5. An isolated nucleic acid molecule comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO:1 and
nucleotides 208-2151 of SEQ ID NO:42.
6. An isolated nucleic acid molecule selected from the group
consisting of: (a) an unique fragment of the nucleotide sequence
set forth as nucleotides 1-2340 of SEQ ID NO:42 between 12 and 2339
nucleotides in length, and (b) complements of (a).
7. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is at least 14 contiguous
nucleotides.
8. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is at least 16 contiguous
nucleotides.
9. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is at least 18 contiguous
nucleotides.
10. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is at least 20 contiguous
nucleotides.
11. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is at least 22 contiguous
nucleotides.
12. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is at least 25 contiguous
nucleotides.
13. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is at least 30 contiguous
nucleotides.
14. The isolated nucleic acid molecule of claim 6, wherein the
isolated nucleic acid molecule is between 12 and 32 contiguous
nucleotides.
15. An expression vector comprising the isolated nucleic acid
molecule of any of claims 1-14 operably linked to a promoter.
16. A host cell transformed or transfected with the expression
vector of claim 15.
17. An isolated polypeptide encoded by the isolated nucleic acid
molecule of any of claims 1-5.
18. An isolated polypeptide comprising a fragment of the
polypeptide of claim 17 at least 9 amino acids in length.
19. The isolated polypeptide of claim 18, wherein the fragment
binds to a polypeptide-binding agent.
20. The isolated polypeptide of claim 19, wherein the fragment
binds to an antibody or a cytotoxic T lymphocyte.
21. An isolated polypeptide which selectively binds a protein
encoded by the isolated nucleic acid molecule of any of claims
1-5.
22. The isolated polypeptide of claim 21, wherein the isolated
polypeptide is an Fab or F(ab).sub.2 fragment of an antibody.
23. The isolated polypeptide of claim 21, wherein the isolated
polypeptide is a fragment of an antibody, the fragment including a
CDR3 region selective for the protein.
24. The isolated polypeptide of claim 21, wherein the isolated
polypeptide is an antibody selected from the group consisting of
monoclonal antibodies, humanized antibodies and chimeric
antibodies.
25. A method for diagnosing a disorder characterized by expression
of a tumor associated nucleic acid molecule, comprising: contacting
a biological sample isolated from a subject with an agent that is
specific for the tumor associated nucleic acid molecule, wherein
the tumor associated nucleic acid molecule hybridizes under
stringent conditions to a molecule having a nucleotide sequence set
forth as SEQ ID NO:42, and determining the interaction between the
agent and the tumor associated nucleic acid molecule as a
determination of the disorder.
26. The method of claim 25 wherein the agent is a nucleic acid
molecule comprising a molecule having a nucleotide sequence set
forth as SEQ ID NO:42, fragments thereof, and complements
thereof.
27. The method of claim 25, wherein the agent comprises a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO:42 or a
fragment thereof.
28. The method of claim 25, wherein the biological sample is
isolated from a non-testis tissue.
29. The method of claim 25 wherein the interaction is determined by
amplifying at least a portion of the nucleic acid molecule.
30. A method for diagnosing a disorder characterized by expression
of a tumor associated polypeptide, comprising: contacting a
biological sample isolated from a subject with an agent that binds
the tumor associated polypeptide as claimed in claim 17 or claim
18, and determining binding between the tumor associated
polypeptide and the agent as a determinant of the disorder.
31. The method of claim 30, wherein the tumor associated
polypeptide is a polypeptide encoded by the nucleotide sequence set
forth as SEQ ID NO:42 and fragments thereof.
32. The method of claim 30, wherein the agent is an antibody or
fragment thereof.
33. A method for treating a subject with a disorder characterized
by expression of a tumor associated nucleic acid as claimed in
claim 1, comprising administering to the subject an effective
amount of an agent which reduces the expression of the tumor
associated nucleic acid, sufficient to ameliorate the disorder.
34. The method of claim 33 wherein the agent which reduces the
expression of the tumor associated nucleic acid is an antisense
nucleic acid which hybridizes to the tumor associated nucleic
acid.
35. A composition comprising: an antisense nucleic acid which binds
to a tumor associated nucleic acid which hybridizes under stringent
conditions to a nucleic acid having a nucleotide sequence set forth
as SEQ ID NO:42, and reduces the expression of the tumor associated
nucleic acid, and a pharmaceutically acceptable carrier.
36. A kit for detecting the presence of the expression of a tumor
associated polypeptide precursor comprising a first isolated
nucleic acid molecule consisting of a 20-32 nucleotide contiguous
segment of SEQ ID NO:42, and a second isolated nucleic acid
molecule consisting of a 20-32 nucleotide contiguous segment of the
complement of SEQ ID NO:42, wherein the contiguous segments are
nonoverlapping.
37. The kit of claim 36, wherein the first and the second isolated
nucleic acid molecules are constructed and arranged to selectively
amplify at least a portion of an isolated nucleic acid molecule
comprising SEQ ID NO:42.
38. A method for treating a subject with a disorder characterized
by expression of a tumor associated nucleic acid as claimed in
claim 1, comprising administering to the subject an amount of an
agent, which enriches selectively in the subject the presence of
complexes of an HLA molecule and a polypeptide encoded by the tumor
associated nucleic acid as claimed in claim 1, effective to
ameliorate the disorder.
39. The method of claim 38, wherein the disorder is cancer.
40. A method for treating a subject having a condition
characterized by expression of a tumor associated antigen encoded
by a tumor associated nucleic acid as claimed in claim 1 in cells
of the subject, comprising: (i) removing an immunoreactive cell
containing sample from the subject, (ii) contacting the
immunoreactive cell containing sample to a host cell under
conditions favoring production of cytolytic T cells against the
tumor associated antigen which is a fragment of the precursor,
(iii) introducing the cytolytic T cells to the subject in an amount
effective to lyse cells which express the tumor associated antigen,
wherein the host cell is transformed or transfected with an
expression vector comprising the isolated nucleic acid molecule of
claim 1 operably linked to a promoter.
41. The method of claim 40, wherein the host cell recombinantly
expresses an HLA molecule which binds the tumor associated
antigen.
42. The method of claim 40, wherein the host cell endogenously
expresses an HLA molecule which binds the tumor associated
antigen.
43. A method for producing a tumor associated polypeptide
comprising providing a nucleic acid molecule comprising a tumor
associated nucleic acid molecule operably linked to a promoter,
wherein the tumor associated nucleic acid molecule encodes the
tumor associated polypeptide or a fragment thereof, wherein the
tumor associated polypeptide comprises SEQ ID NO:42, or a fragment
thereof expressing the nucleic acid molecule in an expression
system, and isolating the tumor associated polypeptide or a
fragment thereof from the expression system.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of copending
U.S. application Ser. No. 09/567,995, filed May 10, 2000, which is
a divisional of U.S. application Ser. No. 09/183,706, filed Oct.
30, 1998, now issued as U.S. Pat. No. 6,245,525, which was a
continuation-in-part of U.S. application Ser. No. 09/122,989 filed
Jul. 27, 1998.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid molecules and encoded
polypeptides which are expressed preferentially in tumors,
particularly in sarcomas. The nucleic acid molecules and encoded
polypeptides are useful in, inter alia, diagnostic and therapeutic
contexts.
BACKGROUND OF THE INVENTION
[0003] The phenotypic changes which distinguish a tumor cell from
its normal counterpart are often the result of one or more changes
to the genome of the cell. The genes which are expressed in tumor
cells, but not in normal counterparts, can be termed "tumor
associated" genes. These tumor associated genes are markers for the
tumor phenotype. The expression of tumor associated genes can also
be an essential event in the process of tumorigenesis.
[0004] Typically, the host recognizes as foreign the tumor
associated genes which are not expressed in normal non-tumorigenic
cells. Thus, the expression of tumor associated genes can provoke
an immune response against the tumor cells by the host. Tumor
associated genes can also be expressed in normal cells within
certain tissues without provoking an immune response. In such
tissues, expression of the gene and/or presentation of an
ordinarily immunologically recognizable fragment of the protein
product on the cell surface may not provoke an immune response
because the immune system does not "see" the cells inside these
immunologically privileged tissues. Examples of immunologically
privileged tissues include brain and testis.
[0005] The discovery of tumor associated expression of a gene
provides a means of identifying a cell as a tumor cell. Diagnostic
compounds can be based on the tumor associated gene, and used to
determine the presence and location of tumor cells. Further, when
the tumor associated gene contributes to an aspect of the tumor
phenotype (e.g., unregulated growth or metastasis), the tumor
associated gene can be used to provide therapeutics such as
antisense nucleic acids which can reduce Qr substantially eliminate
expression of that gene, thereby reducing or substantially
eliminating the phenotypic aspect which depends on the expression
of the particular tumor associated gene.
[0006] As previously noted, the polypeptide products of tumor
associated genes can be the targets for host immune surveillance
and provoke selection and expansion of one or more clones of
cytotoxic T lymphocytes specific for the tumor associated gene
product. Examples of this phenomenon include proteins and fragments
thereof encoded by the MAGE family of genes, the tyrosinase gene,
the Melan-A gene, the BAGE gene, the GAGE gene, the RAGE family of
genes, the PRAME gene and the brain glycogen phosphorylase gene, as
are detailed below. Thus, tumor associated expression of genes
suggests that such genes can encode proteins which will be
recognized by the immune system as foreign and thus provide a
target for tumor rejection. Such genes encode "tumor rejection
antigen precursors", or TRAPs, which may be used to generate
therapeutics for enhancement of the immune system response to
tumors expressing such genes and proteins.
[0007] The process by which the mammalian immune system recognizes
and reacts to foreign or alien materials is a complex one. An
important facet of the system is the T cell response. This response
requires that T cells recognize and interact with complexes of cell
surface molecules, referred to as human leukocyte antigens ("HLA"),
or major histocompatibility complexes ("MHCs"), and peptides. The
peptides are derived from larger molecules which are processed by
the cells which also present the HLA/MHC molecule. See in this
regard Male et al., Advanced Immunology (J. P. Lipincott Company,
1987), especially chapters 6-10. The interaction of T cells and
complexes of HLA/peptide is restricted, requiring a T cell specific
for a particular combination of an HLA molecule and a peptide. If a
specific T cell is not present, there is no T cell response even if
its partner complex is present. Similarly, there is no response if
the specific complex is absent, but the T cell is present. The
mechanism is involved in the immune system's response to foreign
materials, in autoimmune pathologies, and in responses to cellular
abnormalities. Much work has focused on the mechanisms by which
proteins are processed into the HLA binding peptides. See, in this
regard, Barinaga, Science 257: 880, 1992; Fremont et al., Science
257: 919, 1992; Matsumura et al., Science 257: 927, 1992; Latron et
al., Science 257: 964, 1992.
[0008] The mechanism by which T cells recognize cellular
abnormalities has also been implicated in cancer. For example, in
PCT application PCT/US92/04354, filed May 22, 1992, published on
Nov. 26, 1992, and incorporated by reference, a family of genes is
disclosed, which are processed into peptides which, in turn, are
expressed on cell surfaces, which can lead to lysis of the tumor
cells by specific CTLs. The genes are said to code for "tumor
rejection antigen precursors" or "TRAP" molecules, and the peptides
derived therefrom are referred to as "tumor rejection antigens" or
"TRAs". See Traversari et al., J. Exp. Med. 176:1453-1457, 1992;
van der Bruggen et al., Science 254: 1643,1991; De Plaen et al.,
Immunogenetics 40:360-369, 1994 and U.S. Pat. No. 5,342,774 for
further information on this family of genes.
[0009] In U.S. Pat. No. 5,405,940, the disclosure of which is
incorporated by reference, nonapeptides are taught which are
presented by the HLA-A1 molecule. The reference teaches that given
the known specificity of particular peptides for particular HLA
molecules, one should expect a particular peptide to bind one HLA
molecule, but not others. This is important, because different
individuals possess different HLA phenotypes. As a result, while
identification of a particular peptide as being a partner for a
specific HLA molecule has diagnostic and therapeutic ramifications,
these are only relevant for individuals with that particular HLA
phenotype. There is a need for further work in the area, because
cellular abnormalities are not restricted to one particular HLA
phenotype, and targeted therapy requires some knowledge of the
phenotype of the abnormal cells at issue.
[0010] In U.S. Pat. No. 5,629,166, incorporated by reference, the
fact that the MAGE-1 expression product is processed to a second
TRA is disclosed. This second TRA is presented by HLA-Cwl6
molecules, also known as HLA-C*1601. The disclosure shows that a
given TRAP can yield a plurality of TRAs.
[0011] In U.S. Pat. No. 5,487,974, incorporated by reference
herein, tyrosinase is described as a tumor rejection antigen
precursor. This reference discloses that a molecule which is
produced by some normal cells (e.g., melanocytes), is processed in
tumor cells to yield a tumor rejection antigen that is presented by
HLA-A2 molecules.
[0012] In U.S. Pat. No. 5,620,886, incorporated herein by reference
in its entirety, a second TRA, not derived from tyrosinase is
taught to be presented by HLA-A2 molecules. The TRA is derived from
a TRAP, but is coded for by a known MAGE gene. This disclosure
shows that a particular HLA molecule may present TRAs derived from
different sources.
[0013] Additional TRAPs are disclosed in U.S. Pat. Nos. 5,571,711,
5,610,013, 5,587,289 and 5,589,334, as well as PCT publication
WO96/10577. The TRAPs are processed to tumor rejection antigens,
which are presented by a variety of HLA molecules.
[0014] Presently there is a need for additional cancer antigens for
development of therapeutics and diagnosis applicable to a greater
number of cancer patients having various cancers.
SUMMARY OF THE INVENTION
[0015] It now has been discovered that an additional gene, sdp3.8
(HAGE), unrelated to any of the foregoing TRAPs, is expressed in a
tumor associated pattern in sarcoma cells. The invention provides
isolated sdp3.8 nucleic acid molecules encoding tumor associated
polypeptides. The invention also provides expression vectors
containing those molecules and host cells transfected with those
molecules, as well as isolated polypeptides encoded by the tumor
associated nucleic acid molecules (including tumor rejection
antigen precursors and fragments of the isolated polypeptides). The
foregoing isolated nucleic acid molecules and polypeptides can be
used in the diagnosis or treatment of conditions characterized by
the expression of a tumor associated gene.
[0016] According to one aspect of the invention, an isolated
nucleic acid molecule is provided. The molecule hybridizes under
stringent conditions to a nucleic acid having a nucleotide sequence
as set forth in SEQ ID NO:42. The isolated nucleic acid molecule is
a tumor associated polypeptide precursor and codes for a sdp3.8
tumor associated polypeptide, preferably a polypeptide which
comprises a polypeptide which binds an HLA molecule. The invention
also embraces deletions, additions and substitutions of the
foregoing nucleic acids which code for a sdp3.8 tumor associated
polypeptide. The invention further embraces nucleic acid molecules
that differ from the foregoing isolated nucleic acid molecules in
codon sequence due to the degeneracy of the genetic code. The
invention also embraces complements of the foregoing nucleic acids.
In certain embodiments, the isolated nucleic acid molecule
comprises the nucleotide of SEQ ID NO:1, nucleotides 208-2151 of
SEQ ID NO:42, or SEQ I) NO:42. In preferred embodiments, the
isolated nucleic acid molecule comprises the coding region of the
foregoing nucleic acids.
[0017] In another aspect of the invention, an isolated nucleic acid
molecule comprising the nucleic acid sequence set forth as SEQ ID
NO:1 or nucleotides 208-2151 of SEQ ID NO:42 is provided.
[0018] According to another aspect of the invention, an isolated
nucleic acid molecule is provided which comprises a unique fragment
of nucleotides 1-2340 of SEQ ID NO:42 that is 12 or more
nucleotides in length and complements thereof. In preferred
embodiments, the fragment is at least 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 27, 30, 40, 50 or more contiguous
nucleotides of the foregoing, and every integer therebetween. In
another embodiment, the isolated nucleic acid molecule consists of
between 12 and 32 contiguous nucleotides of the foregoing. In still
another embodiment, the sequence of the unique fragment includes 1,
2, 3, 4, 5, 6, or 7 contiguous nucleotides nonidentical to the
sequence claimed in claim 1. Preferred fragments encode immunogenic
fragments of the polypeptide encoded by the sdp3.8 nucleic acid
described herein.
[0019] Methods for identifying sdp3.8 related nucleic acids,
including full-length sdp3.8 cDNAs and sdp3.8 genomic DNAs, are
also included in the invention. The methods include contacting a
nucleic acid sample (such as a cDNA library, genomic library,
genomic DNA isolate, etc.) with a nucleic acid probe or primer
derived from a sdp3.8 nucleic acid such as SEQ ID NO: 1 or 42. The
nucleic acid sample and the probe or primer hybridizes to
complementary nucleotide sequences of nucleic acids in the sample,
if any are present, allowing detection of sdp3.8 related nucleic
acids. Preferably the probe or primer is detectably labeled. The
specific conditions, reagents, and the like can be selected by one
of ordinary skill in the art to selectively identify sdp3.8 related
nucleic acids.
[0020] According to yet another aspect of the invention, the
invention involves expression vectors, and host cells transformed
or transfected with such expression vectors, comprising the nucleic
acid molecules described above. The expression vectors optionally
include a nucleic acid molecule which codes for an HLA molecule. Of
course, an HLA-encoding nucleic acid molecule can also be contained
in a separate expression vector. Host cells transformed or
transfected with the foregoing expression vectors are also
provided.
[0021] According to another aspect of the invention, an isolated
sdp3.8 polypeptide is provided which is encoded by the foregoing
nucleic acid molecules. Fragments of such polypeptides also are
provided. Preferably, the fragment of the isolated polypeptide
binds to a polypeptide-binding agent. In other preferred
embodiments, the fragment of the isolated polypeptide is an
immunogenic peptide, such as a fragment which binds to an antibody
or a cytotoxic T lymphocyte.
[0022] The invention also provides isolated polypeptides which
selectively bind a sdp3.8 protein or fragments thereof. Isolated
binding polypeptides include antibodies and fragments of antibodies
(e.g., Fab, F(ab).sub.2, Fd and antibody fragments which include a
CDR3 region which binds selectively to the sdp3.8 proteins of the
invention). The isolated binding polypeptides include monoclonal
antibodies, humanized antibodies and chimeric antibodies.
[0023] In connection with any of the isolated nucleic acids
encoding a tumor associated polypeptide as described above,
especially a tumor rejection antigen derived from a tumor
associated polypeptide, the invention also embraces degenerate
nucleic acids that differ from the isolated nucleic acid in codon
sequence only due to the degeneracy of the genetic code or
complements of any of the foregoing nucleic acids.
[0024] According to still another aspect of the invention, methods
for diagnosing a disorder characterized by the expression of a
tumor associated nucleic acid molecule or a tumor associated
polypeptide are provided. The methods involve contacting a
biological sample isolated from a subject with an agent that is
specific for the tumor associated nucleic acid molecule or an
expression product thereof. In certain embodiments, the tumor
associated nucleic acid molecule hybridizes under stringent
conditions to a molecule having a nucleotide sequence set forth as
SEQ ID NO:42. In these certain embodiments, the tumor associated
nucleic acid optionally codes for a tumor associated polypeptide.
In other embodiments, the agent is a binding agent which
selectively binds to a sdp3.8 tumor associated polypeptide, such as
an antibody, cytotoxic T lymphocyte, polypeptide, and the like. The
methods further involve determining the interaction or binding
between the agent and the nucleic acid molecule or expression
product thereof as a determination of the disorder. In preferred
embodiments, the agent is a nucleic acid molecule comprising a
molecule having a nucleotide sequence set forth as SEQ ID NO:42,
fragments thereof, and complements thereof. In certain embodiments,
the interaction between the agent and the nucleic acid molecule is
determined by amplifying at least a portion of the nucleic acid
molecule. In preferred embodiments, the agent which binds the tumor
associated polypeptide is an antibody. In the foregoing
embodiments, the biological sample preferably is isolated from a
non-testis tissue. In certain of the foregoing embodiments, the
tumor associated nucleic acids and polypeptides are fragments of
the foregoing sequences.
[0025] The recognition that peptides derived from tumor associated
polypeptides may be presented by HLA molecules and recognized by
CTLs permits diagnosis of certain disorders. Thus, according to
another aspect of the invention, a method for diagnosis of a
disorder characterized by expression of a tumor rejection antigen
derived from a tumor associated polypeptide is provided. The method
involves contacting a biological sample isolated from a subject
with an agent that is specific for the tumor rejection antigen
derived from a tumor associated polypeptide. The method then
provides for determining the interaction between the agent and the
tumor rejection antigen derived from a tumor associated polypeptide
as a determination of the disorder. In certain embodiments, the
tumor rejection antigen derived from a tumor associated polypeptide
comprises the amino acid sequence of a polypeptide encoded by SEQ
ID NO:42 or nucleic acid molecules which hybridize thereto under
stringent conditions. In preferred embodiments, the tumor rejection
antigen comprises between 7 and 100 consecutive amino acids (7, 8,
9, 10, 22, 12, 13, 14, 15, and so on including every integer
therebetween up to 100) of the foregoing sequences. Preferably, the
biological sample is isolated from non-testis tissue. In certain
embodiments, the agent is an antibody.
[0026] The above-described method provides diagnosis of a disorder
based on the presence of tumor associated TRAs. Another aspect of
the invention provides methods for diagnosing a disorder
characterized by the expression of a tumor rejection antigen
derived from a tumor associated polypeptide which forms a complex
with HLA molecules. The method involves contacting a biological
sample isolated from a subject with an agent that binds the complex
and then determining binding between the complex and the agent as a
determination of the disorder. In one embodiment, the tumor
rejection antigen derived from a sdp3.8 tumor associated
polypeptide is a peptide comprising the amino acids of a fragment
of a polypeptide encoded by SEQ ID NO:42 or nucleic acid molecules
which hybridize thereto under stringent conditions. In preferred
embodiments, the tumor rejection antigen comprises between 7 and
100 consecutive amino acids of the foregoing sequences. Preferably,
the biological sample is isolated from non-testis tissue. In
certain embodiments, the agent is an antibody. Any of the foregoing
diagnostic methods can be applied sequentially over time to permit
determination of the prognosis or progression of the disorder.
[0027] In addition to diagnosis of disorders, treatment of certain
disorders is also desirable. According to another aspect of the
invention, methods for treating a subject with a disorder
characterized by expression of a tumor associated nucleic acid or
polypeptide is provided. The method involves administering to the
subject an agent which reduces the expression of the sdp3.8 tumor
associated nucleic acid or polypeptide to ameliorate the disorder.
The agent is administered in an effective amount. In other
embodiments, the tumor associated nucleic acid or polypeptide is a
nucleic acid and the agent is an antisense nucleic acid. The
antisense nucleic acid preferably hybridizes to a tumor associated
nucleic acid set forth as SEQ ID NO: 1 or nucleic acid molecules
which hybridize thereto under stringent conditions and fragments
thereof.
[0028] In another aspect of the invention, the tumor associated
nucleic acid or polypeptide is a tumor rejection antigen and the
method involves administering to the subject an amount of an agent
which enriches selectively in the subject the presence of complexes
of HLA and a tumor associated polypeptide or fragment thereof
encoded by SEQ ID NO:42 or nucleic acid molecules which hybridize
thereto under stringent conditions, sufficient to ameliorate the
disorder. In certain embodiments, the disorder is cancer.
[0029] Still other treatment methods provided by the invention
involve administering to a subject in need of such treatment an
amount of autologous cytolytic T cells sufficient to ameliorate the
disorder, wherein the autologous cytolytic T cells are specific for
complexes of an HLA molecule and a tumor rejection antigen derived
from a sdp3.8 tumor associated polypeptide. Preferably the
complexes are formed of HLA and the certain tumor associated
peptides as described above. The methods in certain embodiments
include removing an immunoreactive cell containing sample from the
subject, contacting the immunoreactive cell containing sample to a
host cell under conditions favoring production of cytolytic T cells
against the tumor associated antigen. The cytolytic T cells are
introduced to the subject in an amount effective to lyse cells
which express the tumor associated antigen. Preferably the host
cell is transformed or transfected with an expression vector
comprising the isolated nucleic acid molecule of claim 1 operably
linked to a promoter. In certain embodiments the host cell
recombinantly expresses an HLA molecule which binds the tumor
associated antigen. In other embodiments the host cell endogenously
expresses an HLA molecule which binds the tumor associated
antigen.
[0030] According to another aspect of the invention, a composition
is provided. The composition comprises an antisense nucleic acid
which binds to a tumor associated nucleic acid set forth as SEQ ID
NO:42, and fragments thereof. The antisense nucleic acid reduces
the expression of the tumor associated nucleic acid. The
composition also includes a pharmaceutically acceptable
carrier.
[0031] The invention in another aspect involves a kit for detecting
the presence of the expression of a tumor associated polypeptide
precursor. Such kits employ two or more of the above-described
nucleic acid molecules isolated in separate containers and packaged
in a single package. In one such kit, a pair of isolated nucleic
acid molecules is provided, each of the pair consisting essentially
of a molecule selected from the group consisting of a 12-32
nucleotide contiguous segment of SEQ ID NO:42 and complements
thereof, and wherein the contiguous segments are nonoverlapping.
Preferably, the pair of isolated nucleic acid molecules is
constructed and arranged to selectively amplify at least a portion
of an isolated nucleic acid molecule which hybridizes under
stringent conditions to a molecule selected from the group
consisting of the nucleic acid sequence of SEQ ID NO:42, nucleic
acid molecules which differ from the above in codon sequence due to
the degeneracy of the genetic code and complements thereof. In
certain embodiments, the pair of isolated nucleic acid molecules is
PCR primers. Preferably one of the primers is a contiguous segment
of SEQ ID NO:42 and another of the primers is a complement of
another contiguous segment of SEQ ID NO:42.
[0032] According to yet another aspect of the invention, methods
for producing a tumor associated polypeptide are provided. The
methods include providing a nucleic acid molecule comprising a
sdp3.8 tumor associated nucleic acid molecule operably linked to a
promoter. The tumor associated nucleic acid molecule encodes the
tumor associated polypeptide or a fragment thereof, wherein the
tumor associated polypeptide is SEQ ID NO:4 or a fragment thereof.
The methods also include expressing the nucleic acid molecule in an
expression system, and isolating the tumor associated polypeptide
or a fragment thereof from the expression system.
[0033] The invention in another aspect also provides pharmaceutical
preparations containing the agents and/or cells of the preceding
paragraphs. In one embodiment, the preparation contains a
pharmaceutically effective amount of sdp3.8 polypeptides encoded by
the foregoing nucleic acids, or a fragment thereof, that binds an
HLA molecule along with pharmaceutically acceptable diluents,
carriers or excipients. In another embodiment, the preparation
contains a pharmaceutically effective amount of isolated autologous
cytolytic T cells specific for complexes of an HLA molecule and a
tumor rejection antigen derived from such sdp3.8 polypeptides.
[0034] According to another aspect of the invention, the use of
isolated sdp3.8 polypeptides or nucleic acids, or fragments
thereof, in the manufacture of a medicament is provided. Preferred
fragments of the sdp3.8 molecules are described above. The use of
antisense nucleic acids which bind to a tumor associated nucleic
acid in the manufacture of a medicament is also provided. In
certain embodiments, the medicament is an injectable medicament, an
oral medicament, or an inhalable medicament.
[0035] According to another aspect of the invention, the use of
isolated sdp3.8 polypeptides or nucleic acids, or fragments
thereof, including antisense nucleic acids, in the manufacture of a
medicament for the treatment of cancer is provided.
[0036] The invention also embraces functional variants and
equivalents of all of the molecules described above.
[0037] These and other objects of the invention will be described
in further detail in connection with the detailed description of
the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The examples which follow show the isolation of nucleic acid
molecules which code for polypeptides and are expressed
preferentially in tumor cells, i.e. which are tumor associated
genes. It is believed that the isolated nucleic acid molecules
encode sdp3.8 polypeptides because the nucleic acid molecules were
initially isolated from expressed mRNA via RT-PCR amplification.
Hence, one aspect of the invention is an isolated nucleic acid
molecule which includes all or a fragment of the nucleotide
sequence set forth in SEQ ID NO:42. This sequence does not encode a
previously recognized tumor rejection antigen precursor, such as a
MAGE, BAGE, GAGE, RAGE, LB33/MUM-1, PRAME, NAG, MAGE-Xp or brain
glycogen phosphorylase sequence, as will be seen by comparing them
to the sequence of any of the genes described in the
references.
[0039] The invention thus involves in one aspect sdp3.8 (HAGE)
nucleic acids, encoded polypeptides, functional modifications and
variants of the foregoing, useful fragments of the foregoing, as
well as therapeutics and diagnostics related thereto.
[0040] The invention provides nucleic acid molecules which can code
for a sdp3.8 polypeptide and which hybridize under stringent
conditions to a nucleic acid molecule consisting of the nucleotide
sequence set forth in SEQ ID NO:42. Such nucleic acids are termed
tumor associated polypeptide precursors, and may be DNA, RNA, or
composed of mixed deoxyribonucleotides and ribonucleotides. The
tumor associated polypeptide precursors can also incorporate
synthetic non-natural nucleotides.
[0041] The invention thus encompasses other tumor associated
nucleic acids, some of which may be expressed in normal tissues. A
tumor associated nucleic acid or polypeptide is a nucleic acid or
polypeptide expressed preferentially in cancer cells, such as
tumors including sarcomas, carcinomas, etc. Various methods for
determining the expression of a nucleic acid and/or a polypeptide
in normal and tumor cells are known to those of skill in the art
and are described further below. As used herein, tumor associated
polypeptides include proteins, protein fragments, and peptides. In
particular, tumor associated polypeptides include TRAPs and
TRAs.
[0042] The term "stringent conditions" as used herein refers to
parameters with which the art is familiar. More specifically,
stringent conditions, as used herein, refers to hybridization at
65.degree. C. in hybridization buffer (3.5.times. SSC, 0.02%
Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 25
mM NaH.sub.2PO.sub.4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M
sodium chloride/0.015M sodium citrate, pH 7; SDS is sodium dodecyl
sulphate; and EDTA is ethylenediaminetetracetic acid. After
hybridization, the membrane upon which the nucleic acid is
transferred is washed at 2.times. SSC at room temperature and then
at 0.1.times. SSC/0.1.times. SDS at 65.degree. C. SSC is 0.15M
sodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecyl
sulphate; and EDTA is ethylenediamine tetraacetic acid. After
hybridization, the support upon which the nucleic acid is
transferred is washed, for example, in 2.times. SSC at room
temperature and then at 0.1-0.5.times. SSC/0.1.times. SDS at
temperatures up to 68.degree. C. The foregoing set of hybridization
conditions is but one example of stringent hybridization conditions
known to one of ordinary skill in the art.
[0043] There are other conditions, reagents, and so forth which can
be used, which result in stringent hybridization (see, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York, 1989, or Current Protocols in Molecular Biology,
F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New
York). The skilled artisan will be familiar with such conditions,
and thus they are not given here. It will be understood, however,
that the skilled artisan will be able to manipulate the conditions
in a manner to permit the clear identification of homologs and
alleles of sdp3.8 nucleic acid molecules of the invention. The
skilled artisan also is familiar with the methodology for screening
cells, preferably cancer cells, and libraries for expression of
such molecules which then are routinely isolated, followed by
isolation of the pertinent nucleic acid and sequencing. Thus sdp3.8
nucleic acids including full-length cDNAs and genomic DNAs are
provided by the invention.
[0044] In general homologs and alleles typically will share at
least 40% nucleotide identity and/or at least 50% amino acid
identity to the coding region of tumor associated nucleic acids, in
some instances will share at least 50% nucleotide identity and/or
at least 65% amino acid identity and in still other instances will
share at least 60% nucleotide identity and/or at least 75% amino
acid identity. Preferred homologs and alleles share nucleotide and
amino acid identities with SEQ ID NO:42 and encode polypeptides of
greater than 80%, more preferably greater than 90%, still more
preferably greater than 95% and most preferably greater than 99%
identity. The percent identity can be calculated using various,
publicly available software tools developed by NCBI (Bethesda, Md.)
that can be obtained through the internet
(ftp:/ncbi.nlm.nih.gov/pub/). Exemplary tools include the BLAST
system available at http://www.ncbi.nlm.nih.gov, which uses
algorithms developed by Altschul et al. (Nucleic Acids Res.
25:3389-3402, 1997). Pairwise and ClustalW alignments (BLOSUM30
matrix setting) as well as Kyte-Doolittle hydropathic analysis can
be obtained using the MacVector sequence analysis software (Oxford
Molecular Group). Complements of the foregoing nucleic acids also
are embraced by the invention.
[0045] Also provided are nucleic acid molecules which include the
nucleotide sequence of SEQ ID NO:1 or nucleotides 208-2151 of SEQ
ID NO:42, and fragments thereof.
[0046] The nucleic acids disclosed herein are useful as probes and
amplification primers for determining the expression of sdp3.8
genes according to standard hybridization procedures. The nucleic
acids also can be used to express tumor associated polypeptides in
vitro or in vivo, by, e.g., operably linking the nucleic acid to a
promoter and transcribing and translating the nucleic acid in an
expression system. The nucleic acids also can be used to prepare
fragments of such polypeptides useful for e.g., preparation of
antibodies. Many other uses will be apparent to the skilled
artisan.
[0047] In screening for related nucleic acids, such as nucleic acid
molecules related in nucleotide sequence to sdp3.8, a Southern blot
may be performed using the foregoing conditions, together with a
radioactive probe (e.g. SEQ ID NO: 1). After washing the membrane
to which the nucleic acid is finally transferred, the membrane can
be placed against x-ray film to detect the radioactive signal. In
screening for the expression of tumor associated nucleic acids,
Northern blot hybridizations using the foregoing conditions (see
also the Examples) can be performed on samples taken from cancer
patients or subjects suspected of having a condition characterized
by expression of tumor associated nucleic acids. Amplification
protocols such as polymerase chain reaction using primers which
hybridize to the sequences presented also can be used for detection
of the tumor associated nucleic acids or expression products
thereof.
[0048] The invention also includes degenerate nucleic acids which
include alternative codons to those present in the native
materials. For example, serine residues are encoded by the codons
TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is
equivalent for the purposes of encoding a serine residue. Thus, it
will be apparent to one of ordinary skill in the art that any of
the serine-encoding nucleotide triplets may be employed to direct
the protein synthesis apparatus, in vitro or in vivo, to
incorporate a serine residue. Similarly, nucleotide sequence
triplets which encode other amino acid residues include, but are
not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC,
CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT
(threonine codons); AAC and AAT (asparagine codons); and ATA, ATC
and ATT (isoleucine codons). Other amino acid residues may be
encoded similarly by multiple nucleotide sequences. Thus, the
invention embraces degenerate nucleic acids that differ from the
biologically isolated nucleic acids in codon sequence due to the
degeneracy of the genetic code.
[0049] The invention also provides isolated unique fragments of SEQ
ID NO:42 or complements thereof, particularly "unique" fragments. A
unique fragment is one that is a `signature` for the larger nucleic
acid. It, for example, is long enough to assure that its precise
sequence is not found in molecules within the human genome outside
of the HAGE (sdp3.8) nucleic acids defined herein (and human
alleles). Those of ordinary skill in the art may apply no more than
routine procedures to determine if a fragment is unique within the
human genome. Unique fragments, however, exclude fragments
completely composed of the nucleotide sequences of any of the
GenBank accession numbers listed in Table V or other previously
published sequences as of the filing date of the priority documents
for sequences listed in a respective priority document or the
filing date of this application for sequences listed for the first
time in this application which overlap the sequences of the
invention.
[0050] A fragment which is completely composed of the sequence
described in the foregoing GenBank deposits is one which does not
include any of the nucleotides unique to the sequences of the
invention. Thus, a unique fragment must contain a nucleotide
sequence other than the exact sequence of those in GenBank or
fragments thereof. The difference may be an addition, deletion or
substitution with respect to the GenBank sequence or it may be a
sequence wholly separate from the GenBank sequence.
[0051] Unique fragments can be used as probes in Southern and
Northern blot assays to identify such nucleic acids, or can be used
in amplification assays such as those employing PCR. As known to
those skilled in the art, large probes such as 200, 250, 300 or
more nucleotides are preferred for certain uses such as Southern
and Northern blots, while smaller fragments will be preferred for
uses such as PCR. Unique fragments also can be used to produce
fusion proteins for generating antibodies or determining binding of
the polypeptide fragments, or for generating immunoassay
components. Likewise, unique fragments can be employed to produce
nonfused fragments of sdp3.8 polypeptides, useful, for example, in
the preparation of antibodies, and in immunoassays. Unique
fragments further can be used as antisense molecules to inhibit the
expression of sdp3.8 nucleic acids and polypeptides, particularly
for therapeutic purposes as described in greater detail below.
[0052] As will be recognized by those skilled in the art, the size
of the unique fragment will depend upon its conservancy in the
genetic code. Thus, some regions of sdp3.8 sequences, such as SEQ
ID NO:42, and complements thereof will require longer segments to
be unique while others will require only short segments, typically
between 12 and 32 nucleotides or more in length (e.g. 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31
and 32 or more), up to the entire length of the disclosed sequence.
The invention embraces each and every fragment of the sdp3.8
sequence, beginning at the first nucleotide, the second nucleotide
and so on, up to 12 nucleotides short of the end, and ending
anywhere from nucleotide number 12, 13, 14 and so on, up to the
very last nucleotide (provided the sequence is unique as described
above).
[0053] Many segments of the polypeptide coding region of novel
tumor associated nucleic acids, or complements thereof, that is 20
or more nucleotides in length will be unique. Those skilled in the
art are well versed in methods for selecting such sequences,
typically on the basis of the ability of the unique fragment to
selectively distinguish the sequence of interest from other
sequences in the human genome. A comparison of the sequence of the
fragment to those on known databases typically is all that is
necessary, although in vitro confirmatory hybridization and
sequencing analysis may be performed.
[0054] A unique fragment can be a functional fragment. A functional
fragment of a nucleic acid molecule of the invention is a fragment
which retains some functional property of the larger nucleic acid
molecule, such as coding for a functional polypeptide, binding to
proteins, regulating transcription of operably linked nucleic
acids, and the like. One of ordinary skill in the art can readily
determine using the assays described herein and those well known in
the art to determine whether a fragment is a functional fragment of
a nucleic acid molecule using no more than routine
experimentation.
[0055] For any pair of PCR primers constructed and arranged to
selectively amplify, for example, a sdp3.8 nucleic acid, a sdp3.8
specific primer may be used. Such a primer is a contiguous stretch
of sdp3.8 which hybridizes selectively to sdp3.8 nucleic acids.
Such a specific primer would fully hybridize to a contiguous
stretch of nucleotides only in sdp3.8 nucleic acids, but would
hybridize at most only in part to genes that do not share the
nucleotides to which the sdp3.8 specific primer binds. For
efficient PCR priming and sdp3.8 nucleic acid identification, the
sdp3.8 specific primer should be constructed and arranged so it
does not hybridize efficiently at its 3' end to genes other than
sdp3.8. Preferably the area of non-identity is at least one to four
nucleotides in length and forms the 3' end of the sdp3.8 specific
primer. The kinetics of hybridization then will strongly favor
hybridization at the 5' end. In this instance, 3' initiated PCR
extension will occur only when both the 5' and 3' ends hybridize to
the nucleic acid. Exemplary primers include SEQ ID NO:2 and SEQ ID
NO:3, which are derived from SEQ ID NO: 1. Other exemplary primers
can differ from the above by addition or deletion of 1, 2, 3, 4, 5,
or more nucleotides from the 5' end of the primer. One of ordinary
skill in the art can determine with no more than routine
experimentation the preferred primers for selective amplification
of sdp3.8 and related genes. Additional methods which can
distinguish nucleotide sequences of substantial homology, such as
ligase chain reaction ("LCR") and other methods, will be apparent
to skilled artisans.
[0056] As used herein with respect to nucleic acids, the term
"isolated" means: (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which polymerase chain reaction (PCR) primer sequences
have been disclosed is considered isolated but a nucleic acid
sequence existing in its native state in its natural host is not.
An isolated nucleic acid may be substantially purified, but need
not be. For example, a nucleic acid that is isolated within a
cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein because it is readily manipulable by standard
techniques known to those of ordinary skill in the art. An isolated
nucleic acid molecule as used herein is not a naturally occurring
chromosome.
[0057] The invention also provides isolated polypeptides which
include translation products of SEQ ID NO:42, related SAGE (sdp3.8)
nucleic acids (such as cDNAs including the full-length coding
region of sdp3.8, e.g. SEQ ID NO:42) and fragments thereof. Such
polypeptides are useful, for example, alone or as fusion proteins
to generate antibodies, as a component of an immunoassay, or for
determining the binding specificity of HLA molecules and/or CTL
clones for sdp3.8 proteins. Tumor associated polypeptides can be
isolated from biological samples including tissue or cell
homogenates, and can also be expressed recombinantly in a variety
of prokaryotic and eukaryotic expression systems by constructing an
expression vector appropriate to the expression system, introducing
the expression vector into the expression system, and isolating the
recombinantly expressed protein. Short polypeptides, including
antigenic peptides (such as are presented by MHC molecules on the
surface of a cell for immune recognition) also can be synthesized
chemically using well-established methods of peptide synthesis.
[0058] Thus, as used herein with respect to polypeptides,
"isolated" means separated from its native environment and present
in sufficient quantity to permit its identification or use.
Isolated, when referring to a protein or polypeptide, means, for
example: (i) selectively produced by expression of a recombinant
nucleic acid or (ii) purified as by chromatography or
electrophoresis. Isolated proteins or polypeptides may, but need
not be, substantially pure. The term "substantially pure" means
that the proteins or polypeptides are essentially free of other
substances with which they may be found in nature or in vivo
systems to an extent practical and appropriate for their intended
use. Substantially pure polypeptides may be produced by techniques
well known in the art. Because an isolated protein may be admixed
with a pharmaceutically acceptable carrier in a pharmaceutical
preparation, the protein may comprise only a small percentage by
weight of the preparation. The protein is nonetheless isolated in
that it has been separated from the substances with which it may be
associated in living systems, i.e. isolated from other
proteins.
[0059] A fragment of a sdp3.8 protein, for example, generally has
the features and characteristics of fragments including unique
fragments as discussed above in connection with nucleic acids. As
will be recognized by those skilled in the art, the size of a
fragment which is unique will depend upon factors such as whether
the fragment constitutes a portion of a conserved protein domain.
Thus, some regions of sdp3.8 polypeptides will require longer
segments to be unique while others will require only short
segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8,
9, 10, 11 and 12 amino acids long).
[0060] Unique fragments of a polypeptide preferably are those
fragments which retain a distinct functional capability of the
polypeptide. Functional capabilities which can be retained in a
fragment of a polypeptide include interaction with antibodies,
interaction with other polypeptides or fragments thereof, selective
binding of nucleic acids, and enzymatic activity. One important
activity is the ability to act as a signature for identifying the
polypeptide. Another is the ability to complex with HLA and to
provoke in a human an immune response. A tumor rejection antigen is
an example of a fragment of a tumor associated polypeptide which
retains the functional capability of HLA binding and interaction
with T lymphocytes. Tumor rejection antigens presented by HLA class
I molecules typically are 9 amino acids in length, although
peptides of 8, 9 and 10 and more amino acids also retain the
capability to interact with HLA and T lymphocytes to an extent
effective to provoke a cytotoxic T lymphocyte response (see, e.g.,
Van den Eynde & Brichard, Curr. Opin. Immunol. 7:674-681, 1995;
Coulie et al., Stem Cells 13:393-403, 1995). Similarly, tumor
rejection antigens (e.g., 10-20 amino acids in length) can interact
with HLA class II molecules and T helper lymphocytes, provoking
proliferation and response of the T helper lymphocytes (see, e.g.,
Van den Eynde & van der Bruggen, Curr. Opin. Immunol.
9:684-693, 1997; Topalian et al., J. Exp. Med. 183:1965-1971,
1996).
[0061] Those skilled in the art are well versed in methods for
selecting unique amino acid sequences, typically on the basis of
the ability of the fragment to selectively distinguish the sequence
of interest from non-family members. A comparison of the sequence
of the fragment to those on known data bases typically is all that
is necessary.
[0062] The invention embraces variants of the tumor associated
polypeptides described above. As used herein, a "variant" of a
tumor associated polypeptide is a polypeptide which contains one or
more modifications to the primary amino acid sequence of a tumor
associated polypeptide. Modifications which create a tumor
associated polypeptide variant can be made to a tumor associated
polypeptide 1) to reduce or eliminate an activity of the tumor
associated polypeptide; 2) to enhance a property of the tumor
associated polypeptide, such as protein stability in an expression
system or the stability of protein-protein binding; 3) to provide a
novel activity or property to a tumor associated polypeptide, such
as addition of an antigenic epitope or addition of a detectable
moiety; or 4) to provide equivalent or better binding to an HLA
molecule. Modifications to a tumor associated polypeptide are
typically made to the nucleic acid which encodes the tumor
associated polypeptide, and can include deletions, point mutations,
truncations, amino acid substitutions and additions of amino acids
or non-amino acid moieties. Alternatively, modifications can be
made directly to the polypeptide, such as by cleavage, addition of
a linker molecule, addition of a detectable moiety, such as biotin,
addition of a fatty acid, and the like. Modifications also embrace
fusion proteins comprising all or part of the tumor associated
amino acid sequences, e.g., SEQ ID NOs:43. One of skill in the art
will be familiar with methods for predicting the effect on protein
conformation of a change in protein sequence, and can thus "design"
a variant tumor associated polypeptide according to known methods.
One example of such a method is described by Dahiyat and Mayo in
Science 278:82-87, 1997, whereby proteins can be designed de novo.
The method can be applied to a known protein to vary a only a
portion of the polypeptide sequence. By applying the computational
methods of Dahiyat and Mayo, specific variants of a tumor
associated polypeptide can be proposed and tested to determine
whether the variant retains a desired conformation. general,
variants include tumor associated polypeptides which are modified
specifically to alter a feature of the polypeptide unrelated to its
desired physiological activity. For example, cysteine residues can
be substituted or deleted to prevent unwanted disulfide linkages.
Similarly, certain amino acids can be changed to enhance expression
of a tumor associated polypeptide by eliminating proteolysis by
proteases in an expression system (e.g., dibasic amino acid
residues in yeast expression systems in which KEX2 protease
activity is present).
[0063] Mutations of a nucleic acid which encode a tumor associated
polypeptide preferably preserve the amino acid reading frame of the
coding sequence, and preferably do not create regions in the
nucleic acid which are likely to hybridize to form secondary
structures, such a hairpins or loops, which can be deleterious to
expression of the variant polypeptide.
[0064] Mutations can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid which encodes the polypeptide. Variant polypeptides
are then expressed and tested for one or more activities to
determine which mutation provides a variant polypeptide with the
desired properties. Further mutations can be made to variants (or
to non-variant tumor associated polypeptides) which are silent as
to the amino acid sequence of the polypeptide, but which provide
preferred codons for translation in a particular host. The
preferred codons for translation of a nucleic acid in, e.g., E.
coli, are well known to those of ordinary skill in the art. Still
other mutations can be made to the noncoding sequences of a tumor
associated gene or cDNA clone to enhance expression of the
polypeptide. The activity of variants of tumor associated
polypeptides can be tested by cloning the gene encoding the variant
tumor associated polypeptide into a bacterial or mammalian
expression vector, introducing the vector into an appropriate host
cell, expressing the variant tumor associated polypeptide, and
testing for a functional capability of the tumor associated
polypeptides as disclosed herein. For example, the variant tumor
associated polypeptide can be tested for reaction with autologous
or allogeneic sera as disclosed in the Examples. Preparation of
other variant polypeptides may favor testing of other activities,
as will be known to one of ordinary skill in the art.
[0065] The skilled artisan will also realize that conservative
amino acid substitutions may be made in sdp3.8 polypeptides to
provide functional variants of the foregoing polypeptides, i.e, the
variants which the functional capabilities of the sdp3.8
polypeptides. As used herein, a "conservative amino acid
substitution" refers to an amino acid substitution which does not
alter the relative charge or size characteristics of the protein in
which the amino acid substitution is made. Conservative
substitutions of amino acids include substitutions made amongst
amino acids within the following groups: (a) M, I, L, V; (b) F, Y,
W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
[0066] For example, upon determining that a peptide derived from a
sdp3.8 polypeptide is presented by a MHC molecule and recognized by
CTLs (e.g., as described in the Examples), one can make
conservative amino acid substitutions to the amino acid sequence of
the peptide, particularly at residues which are thought not to be
direct contact points with the MHC molecule. For example, methods
for identifying functional variants of HLA class II binding
peptides are provided in a published PCT application of Strominger
and Wucherpfennig (PCT/US96/03182). Peptides bearing one or more
amino acid substitutions also can be tested for concordance with
known HLA/MHC motifs prior to synthesis using, e.g. the computer
program described by D'Amaro and Drijfhout (D'Amaro et al., Human
Immunol. 43:13-18, 1995; Drijfhout et al., Human Immunol. 43:1-12,
1995) or as described below in the Examples. The substituted
peptides can then be tested for binding to the MHC molecule and
recognition by CTLs when bound to MHC. These variants can be tested
for improved stability and are useful, inter alia, in vaccine
compositions.
[0067] Functional variants of sdp3.8 polypeptides, i.e., variants
of polypeptides which retain the function of the natural
polypeptides, can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such
as are found in references which compile such methods, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York, 1989, or Current Protocols in Molecular Biology,
F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
For example, exemplary functional variants of the sdp3.8
polypeptides include conservative amino acid substitutions of
polypeptides encoded by SEQ ID NO:42. Conservative amino-acid
substitutions in the amino acid sequence of sdp3.8 polypeptides to
produce functional variants of sdp3.8 polypeptides typically are
made by alteration of the nucleic acid encoding a sdp3.8
polypeptide (e.g. SEQ ID NO:42). Such substitutions can be made by
a variety of methods known to one of ordinary skill in the art. For
example, amino acid substitutions may be made by PCR-directed
mutation, site-directed mutagenesis according to the method of
Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or
by chemical synthesis of a gene encoding a sdp3.8 polypeptide.
Where amino acid substitutions are made to a small unique fragment
of a sdp3.8 polypeptide, such as a 9 amino acid peptide, the
substitutions can be made by directly synthesizing the peptide. The
activity of functional variants or fragments of sdp3.8 polypeptides
can be tested by cloning the gene encoding the altered sdp3.8
polypeptide into a bacterial or mammalian expression vector,
introducing the vector into an appropriate host cell, expressing
the altered sdp3.8 polypeptide, and testing for a functional
capability of the sdp3.8 polypeptides as disclosed herein.
[0068] As mentioned above, the invention embraces antisense
oligonucleotides that selectively bind to a tumor associated gene
nucleic acid molecule, including those encoding a sdp3.8 protein,
to decrease transcription and/or translation of tumor associated
genes. This is desirable in virtually any medical condition wherein
a reduction in tumor associated gene product expression is
desirable, including to reduce any aspect of a malignant cell
phenotype attributable to tumor associated gene expression, such as
expression of sdp3.8. Antisense molecules, in this manner, can be
used to slow down or arrest such aspects of a malignant cell
phenotype as found in, inter alia, sarcomas and carcinomas.
[0069] As used herein, the term "antisense oligonucleotide" or
"antisense" describes an oligonucleotide that is an
oligoribonucleotide, oligodeoxyribonucleotide, modified
oligoribonucleotide, or modified oligodeoxyribonucleotide which
hybridizes under physiological conditions to DNA comprising a
particular gene or to an MRNA transcript of that gene and, thereby,
inhibits the transcription of that gene and/or the translation of
that mRNA. The antisense molecules are designed so as to interfere
with transcription or translation of a target gene upon
hybridization with the target gene. Those skilled in the art will
recognize that the exact length of the antisense oligonucleotide
and its degree of complementarity with its target will depend upon
the specific target selected, including the sequence of the target
and the particular bases which comprise that sequence. It is
preferred that the antisense oligonucleotide be constructed and
arranged so as to bind selectively with the target under
physiological conditions, i.e., to hybridize substantially more to
the target sequence than to any other sequence in the target cell
under physiological conditions. Based upon SEQ ID NO:42 or upon
allelic or homologous genomic and/or cDNA sequences, one of skill
in the art can easily choose and synthesize any of a number of
appropriate antisense molecules for use in accordance with the
present invention. In order to be sufficiently selective and potent
for inhibition, such antisense oligonucleotides should comprise at
least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and,
more preferably, at least 15 consecutive bases which are
complementary to the target. Most preferably, the antisense
oligonucleotides comprise a complementary sequence of 20-30 bases.
Although oligonucleotides may be chosen which are antisense to any
region of the gene or mRNA transcripts, in preferred embodiments
the antisense oligonucleotides correspond to N-terminal or 5'
upstream sites such as translation initiation, transcription
initiation or promoter sites. In addition, 3'-untranslated regions
may be targeted. Targeting to mRNA splicing sites has also been
used in the art but may be less preferred if alternative MRNA
splicing occurs. In addition, the antisense is targeted,
preferably, to sites in which MRNA secondary structure is not
expected (see, e.g., Sainio et al., Cell Mol. Neurobiol.
14(5):439-457, 1994) and at which proteins are not expected to
bind. Finally, although, SEQ ID NO:42 discloses a cDNA sequence,
one of ordinary skill in the art may easily derive the
corresponding genomic DNA. Thus, the present invention also
provides for antisense oligonucleotides which are complementary to
the full length cDNA and/or genomic DNA corresponding to SEQ ID
NO:42. Similarly, antisense to allelic or homologous DNAs and
genomic DNAs are enabled without undue experimentation.
[0070] In one set of embodiments, the antisense oligonucleotides of
the invention may be composed of "natural" deoxyribonucleotides,
ribonucleotides, or any combination thereof. That is, the 5' end of
one native nucleotide and the 3' end of another native nucleotide
may be covalently linked, as in natural systems, via a
phosphodiester internucleoside linkage. These oligonucleotides may
be prepared by art recognized methods which may be carried out
manually or by an automated synthesizer. They also may be produced
recombinantly by vectors.
[0071] In preferred embodiments, however, the antisense
oligonucleotides of the invention also may include "modified"
oligonucleotides. That is, the oligonucleotides may be modified in
a number of ways which do not prevent them from hybridizing to
their target but which enhance their stability or targeting or
which otherwise enhance their therapeutic effectiveness.
[0072] The term "modified oligonucleotide" as used herein describes
an oligonucleotide in which (1) at least two of its nucleotides are
covalently linked via a synthetic internucleoside linkage (i.e., a
linkage other than a phosphodiester linkage between the 5' end of
one nucleotide and the 3' end of another nucleotide) and/or (2) a
chemical group not normally associated with nucleic acids has been
covalently attached to the oligonucleotide. Preferred synthetic
internucleoside linkages are phosphorothioates, alkylphosphonates,
phosphorodithioates, phosphate esters, alkylphosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, peptides, and carboxymethyl esters.
[0073] The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose. Modified oligonucleotides also
can include base analogs such as C-5 propyne modified bases (Wagner
et al., Nature Biotechnology 14:840-844, 1996). The present
invention, thus, contemplates pharmaceutical preparations
containing modified antisense molecules that are complementary to
and hybridizable with, under physiological conditions, nucleic
acids encoding tumor associated proteins, together with
pharmaceutically acceptable carriers.
[0074] It will also be recognized from the examples that the
invention embraces the use of the sdp3.8 sequences in expression
vectors, as well as to transfect host cells and cell lines, be
these prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells,
COS cells, yeast expression systems and recombinant baculovirus
expression in insect cells). Especially useful are mammalian cells
such as mouse, hamster, pig, goat, primate, etc. They can be of a
wide variety of tissue types, including mast cells, fibroblasts,
oocytes and lymphocytes, and they may be primary cells or cell
lines. Specific examples include dendritic cells, U293 cells,
peripheral blood leukocytes, bone marrow stem cells and embryonic
stem cells. The expression vectors require that the pertinent
sequence, i.e., those nucleic acids described supra, be operably
linked to a promoter.
[0075] Especially preferred are nucleic acids encoding a series of
epitopes, known as "polytopes". The epitopes can be arranged in
sequential or overlapping fashion (see, e.g., Thomson et al., Proc.
Natl. Acad. Sci. USA 92:5845-5849, 1995; Gilbert et al., Nature
Biotechnol. 15:1280-1284, 1997), with or without the natural
flanking sequences, and can be separated by unrelated linker
sequences if desired. The polytope is processed to generated
individual epitopes which are recognized by the immune system for
generation of immune responses.
[0076] Thus, for example, peptides derived from the polypeptide
having an amino acid sequence encoded by the nucleic acid of SEQ ID
NO:42, and which are presented by MHC molecules and recognized by
CTL or T helper lymphocytes can be combined with peptides from
other tumor rejection antigens (e.g. by preparation of hybrid
nucleic acids or polypeptides) to form "polytopes". Exemplary tumor
associated peptide antigens that can be administered to induce or
enhance an immune response are derived from tumor associated genes
and encoded proteins including MAGE-1, MAGE-2, MAGE-3, MAGE-4,
MAGE-5, MAGE-6, MAGE-7, MAGE-8, MAGE-9,MAGE-10, MAGE-11, MAGE-12,
MAGE-13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7,
GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2),
MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brain glycogen
phosphorylase, Melan-A, MAGE-5 C1, MAGE-C2, NY-ESO-1, LAGE-1,
SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7. For
example, antigenic peptides characteristic of tumors include those
listed in Table I below.
1TABLE I Exemplary Antigens SEQ ID Gene MHC Peptide Position NO:
MAGE-1 HLA-A1 EADPTGHSY 161-169 4 HLA-Cw16 SAYGEPRKL 230-238 5
MAGE-3 HLA-A1 EVDPIGHLY 168-176 6 HLA-A2 FLWGPRALV 271-279 7
HLA-B44 MEVDPIGHLY 167-176 8 BAGE HLA-Cw16 AARAVFLAL 2-10 9 GAGE-1,
2 HLA-Cw16 YRPRPRRY 9-16 10 RAGE HLA-B7 SPSSNRIRNT 11-20 11 GnT-V
HLA-A2 VLPDVFIRC(V) 2-10/11 12, 13 MUM-1 HLA-B44 EEKLIVVLF exon 14
EEKLSVVLF 2/intron 15 (wild type) CDK4 HLA-A2 ACDPHSGHFV 23-32 16
ARDPHSGHFV 17 (wild type) .beta.-catenin HLA-A24 SYLDSGIHF 29-37 18
SYLDSGIHS (wild type) 19 Tyrosinase HLA-A2 MLLAVLYCL 1-9 20 HLA-A2
YMNGTMSQV 369-377 21 HLA-A2 YMDGTMSQV 369-377 37 HLA-A24 AFLPWHRLF
206-214 22 HLA-B44 SEIWRDIDF 192-200 23 HLA-B44 YEIWRDIDF 192-200
24 HLA-DR4 QNILLSNAPLGPQFP 56-70 25 HLA-DR4 DYSYLQDSDPDSFQD 448-462
26 Melan- HLA-A2 (E)AAGIGILTV 26/27-35 27, A.sup.MART-1 28 HLA-A2
ILTVILGVL 32-40 29 gp HLA-A2 KTWGQYWQV 154-162 30 100.sup.Pmell17
HLA-A2 ITDQVPFSV 209-217 31 HLA-A2 YLEPGPVTA 280-288 32 HLA-A2
LLDGTATLRL 457-466 33 HLA-A2 VLYRYGSFSV 476-485 34 PRAME HLA-A24
LYVDSLFFL 301-309 35 MAGE-6 HLA-Cw16 KISGGPRISYPL 292-303 36
[0077] See, for example, PCT application publication no.
WO96/10577. Other examples will be known to one of ordinary skill
in the art (for example, see Coulie, Stem Cells 13:393-403, 1995),
and can be used in the invention in a like manner as those
disclosed herein. One of ordinary skill in the art can prepare
polypeptides comprising one or more sdp3.8 peptides and one or more
of the foregoing tumor rejection peptides, or nucleic acids
encoding such polypeptides, according to standard procedures of
molecular biology.
[0078] Thus polytopes are groups of two or more potentially
immunogenic or immune response stimulating peptides which can be
joined together in various arrangements (e.g. concatenated,
overlapping). The polytope (or nucleic acid encoding the polytope)
can be administered in a standard immunization protocol, e.g. to
animals, to test the effectiveness of the polytope in stimulating,
enhancing and/or provoking an immune response.
[0079] The peptides can be joined together directly or via the use
of flanking sequences to form polytopes, and the use of polytopes
as vaccines is well known in the art (see, e.g., Thomson et al.,
Proc. Acad. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et
al., Nature Biotechnol. 15(12):1280-1284, 1997; Thomson et al., J.
Immunol. 157(2):822-826, 1996; Tam et al., J. Exp. Med.
171(1):299-306, 1990). For example, Tam showed that polytopes
consisting of both MHC class I and class II binding epitopes
successfully generated antibody and protective immunity in a mouse
model. Tam also demonstrated that polytopes comprising "strings" of
epitopes are processed to yield individual epitopes which are
presented by MHC molecules and recognized by CTLs. Thus polytopes
containing various numbers and combinations of epitopes can be
prepared and tested for recognition by CTLs and for efficacy in
increasing an immune response.
[0080] It is known that tumors express a set of tumor antigens, of
which only certain subsets may be expressed in the tumor of any
given patient. Polytopes can be prepared which correspond to the
different combination of epitopes representing the subset of tumor
rejection antigens expressed in a particular patient. Polytopes
also can be prepared to reflect a broader spectrum of tumor
rejection antigens known to be expressed by a tumor type. Polytopes
can be introduced to a patient in need of such treatment as
polypeptide structures, or via the use of nucleic acid delivery
systems known in the art (see, e.g., Allsopp et al., Eur. J
Immunol. 26(8):1951-1959, 1996). Adenovirus, pox virus, Ty-virus
like particles, adeno-associated virus, plasmids, bacteria, etc.
can be used in such delivery. One can test the polytope delivery
systems in mouse models to determine efficacy of the delivery
system. The systems also can be tested in human clinical
trials.
[0081] In instances in which a human HLA class I molecule presents
tumor rejection antigens derived from sdp3.8 nucleic acids, the
expression vector may also include a nucleic acid sequence coding
for the HLA molecule that presents any particular tumor rejection
antigen derived from these nucleic acids and polypeptides.
Alternatively, the nucleic acid sequence coding for such a HLA
molecule can be contained within a separate expression vector. In a
situation where the vector contains both coding sequences, the
single vector can be used to transfect a cell which does not
normally express either one. Where the coding sequences for the
tumor rejection antigen precursor and the HLA molecule which
presents it are contained on separate expression vectors, the
expression vectors can be cotransfected. The tumor rejection
antigen precursor coding sequence may be used alone, when, e.g. the
host cell already expresses a HLA molecule which presents a TRA
derived from sdp3.8 TRAPs. Of course, there is no limit on the
particular host cell which can be used. As the vectors which
contain the two coding sequences may be used in any
antigen-presenting cells if desired, and the gene for tumor
rejection antigen precursor can be used in host cells which do not
express a HLA molecule which presents a sdp3.8 TRA. Further,
cell-free transcription systems may be used in lieu of cells.
[0082] As used herein, a "vector" may be any of a number of nucleic
acids into which a desired sequence may be inserted by restriction
and ligation for transport between different genetic environments
or for expression in a host cell. Vectors are typically composed of
DNA although RNA vectors are also available. Vectors include, but
are not limited to, plasmids and phagemids. A cloning vector is one
which is able to replicate in a host cell, and which is further
characterized by one or more endonuclease restriction sites at
which the vector may be cut in a determinable fashion and into
which a desired DNA sequence may be ligated such that the new
recombinant vector retains its ability to replicate in the host
cell. In the case of plasmids, replication of the desired sequence
may occur many times as the plasmid increases in copy number within
the host bacterium or just a single time per host before the host
reproduces by mitosis. In the case of phage, replication may occur
actively during a lytic phase or passively during a lysogenic
phase. An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. Vectors may further contain one or more marker
sequences suitable for use in the identification of cells which
have or have not been transformed or transfected with the vector.
Markers include, for example, genes encoding proteins which
increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art (e.g.
.beta.-galactosidase, luciferase or alkaline phosphatase), and
genes which visibly affect the phenotype of transformed or
transfected cells, hosts, colonies or plaques (e.g., green
fluorescent protein). Preferred vectors are those capable of
autonomous replication and expression of the structural gene
products present in the DNA segments to which they are operably
joined. Preferred vectors are those capable of autonomous
replication and expression of the structural gene products present
in the DNA segments to which they are operably joined.
[0083] As used herein, a coding sequence and regulatory sequences
are said to be "operably" joined when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
sequences. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operably joined if induction of a promoter in the 5' regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide.
[0084] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribing and 5'
non-translating sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CAAT sequence, and the like. Especially, such 5'
non-transcribing regulatory sequences will include a promoter
region which includes a promoter sequence for transcriptional
control of the operably joined gene. Regulatory sequences may also
include enhancer sequences or upstream activator sequences as
desired. The vectors of the invention may optionally include 5'
leader or signal sequences, 5' or 3'. The choice and design of an
appropriate vector is within the ability and discretion of one of
ordinary skill in the art.
[0085] Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See Molecular Cloning: A Laboratory Manual, J. Sambrook,
et al., eds., Second Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York, 1989. Cells are genetically
engineered by the introduction into the cells of heterologous DNA
(RNA) encoding the sdp3.8 tumor associated polypeptide or fragment
or variant thereof. The heterologous DNA (RNA) is placed under
operable control of transcriptional elements to permit the
expression of the heterologous DNA in the host cell.
[0086] Preferred systems for MRNA expression in mammalian cells are
those such as pRc/CMV (available from Invitrogen, Carlsbad, Calif.)
that contain a selectable marker such as a gene that confers G418
resistance (which facilitates the selection of stably transfected
cell lines) and the human cytomegalovirus (CMV) enhancer-promoter
sequences. Additionally, suitable for expression in primate or
canine cell lines is the pCEP4 vector (Invitrogen), which contains
an Epstein Barr virus (EBV) origin of replication, facilitating the
maintenance of plasmid as a multicopy extrachromosomal element.
Another expression vector is the pEF-BOS plasmid containing the
promoter of polypeptide Elongation Factor 1 a, which stimulates
efficiently transcription in vitro. The plasmid is described by
Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use
in transfection experiments is disclosed by, for example, Demoulin
(Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferred
expression vector is an adenovirus, described by
Stratford-Perricaudet, which is defective for E1 and E3 proteins
(J. Clin. Invest. 90:626-630, 1992). The use of the adenovirus as
an Adeno.P1A recombinant is disclosed by Wamier et al., in
intradermal injection in mice for immunization against P1A (Int. J
Cancer, 67:303-310, 1996). Also included are bacterial systems for
delivery of antigens to eukaryotic cells, such as those which
utilize Yersinia (e.g. Stambach and Bevan, J. Immunol. 153:1603,
1994) and Listeria (Dietrich et al., Nature Biotechnol. 16:181,
1998). Still other delivery and expression systems will be known to
one of ordinary skill in the art.
[0087] The invention also embraces so-called expression kits, which
allow the artisan to prepare a desired expression vector or
vectors. Such expression kits include at least separate portions of
each of the previously discussed coding sequences. Other components
may be added, as desired, as long as the previously mentioned
sequences, which are required, are included.
[0088] The invention also permits the construction of tumor
associated gene "knock-outs" in cells and in animals, providing
materials for studying certain aspects of cancer and immune system
responses to cancer.
[0089] The invention as described herein has a number of uses, some
of which are described elsewhere herein. First, the invention
permits isolation of the tumor associated protein molecules. A
variety of methodologies well-known to the skilled practitioner can
be utilized to obtain isolated tumor associated molecules. The
polypeptide may be purified from cells which naturally produce the
polypeptide by chromatographic means or immunological recognition.
Alternatively, an expression vector may be introduced into cells to
cause production of the polypeptide. In another method, mRNA
transcripts may be microinjected or otherwise introduced into cells
to cause production of the encoded polypeptide. Translation of mRNA
in cell-free extracts such as the reticulocyte lysate system also
may be used to produce polypeptide. Those skilled in the art also
can readily follow known methods for isolating tumor associated
polypeptides. These include, but are not limited to,
immunochromatography, HPLC, size-exclusion chromatography,
ion-exchange chromatography and immune-affinity chromatography.
[0090] The isolation and identification of tumor associated nucleic
acids also makes it possible for the artisan to diagnose a disorder
characterized by expression of tumor associated nucleic acids or
polypeptides. These methods involve determining expression of one
or more tumor associated nucleic acids, and/or encoded tumor
associated polypeptides and/or peptides derived therefrom. In the
former situation, such determinations can be carried out via any
standard nucleic acid determination assay, including the polymerase
chain reaction, or assaying with labeled hybridization probes. In
the latter situation, such determinations can be carried out by
screening patient antisera for recognition of the polypeptide or by
assaying biological samples with binding partners (e.g.,
antibodies) for tumor associated polypeptides or complexes of
antigens derived therefrom and HLA molecules.
[0091] The invention also makes it possible isolate proteins which
bind to tumor associated polypeptides as disclosed herein,
including antibodies and cellular binding partners of the tumor
associateds. Additional uses are described further herein.
[0092] The invention also provides, in certain embodiments,
"dominant negative" polypeptides derived from tumor associated
polypeptides. A dominant negative polypeptide is an inactive
variant of a protein, which, by interacting with the cellular
machinery, displaces an active protein from its interaction with
the cellular machinery or competes with the active protein, thereby
reducing the effect of the active protein. For example, a dominant
negative receptor which binds a ligand but does not transmit a
signal in response to binding of the ligand can reduce the
biological effect of expression of the ligand. Likewise, a dominant
negative catalytically-inactive kinase which interacts normally
with target proteins but does not phosphorylate the target proteins
can reduce phosphorylation of the target proteins in response to a
cellular signal. Similarly, a dominant negative transcription
factor which binds to a promoter site in the control region of a
gene but does not increase gene transcription can reduce the effect
of a normal transcription factor by occupying promoter binding
sites without increasing transcription.
[0093] The end result of the expression of a dominant negative
polypeptide in a cell is a reduction in function of active
proteins. One of ordinary skill in the art can assess the potential
for a dominant negative variant of a protein, and using standard
mutagenesis techniques to create one or more dominant negative
variant polypeptides. For example, one of ordinary skill in the art
can modify the sequence of tumor associated polypeptides by
site-specific mutagenesis, scanning mutagenesis, partial gene
deletion or truncation, and the like. See, e.g., U.S. Pat. No.
5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
The skilled artisan then can test the population of mutagenized
polypeptides for diminution in a selected and/or for retention of
such an activity. Other similar methods for creating and testing
dominant negative variants of a protein will be apparent to one of
ordinary skill in the art.
[0094] The invention also involves agents which bind to tumor
associated polypeptides encoded by sdp3.8 nucleic acid molecules
("sdp3.8 polypeptides"), and in certain embodiments preferably to
unique fragments of the sdp3.8 polypeptides. Such binding partners
can be used in screening assays to detect the presence or absence
of a sdp3.8 polypeptide and in purification protocols to isolate
sdp3.8 polypeptides. Likewise, such binding partners can be used to
selectively target drugs, toxins or other molecules to leukemia
cells which present sdp3.8 tumor associated polypeptides. In this
manner, cells present in solid or non-solid tumors which express
sdp3.8 tumor associated polypeptides can be treated with cytotoxic
compounds. Such agents also can be used to inhibit the native
activity of the tumor associated polypeptides, for example, by
binding to such polypeptides.
[0095] The invention, therefore, involves antibodies or fragments
of antibodies having the ability to selectively bind to sdp3.8
tumor associated polypeptides, and preferably to unique fragments
thereof. Antibodies include polyclonal and monoclonal antibodies,
prepared according to conventional methodology.
[0096] The antibodies of the present invention thus are prepared by
any of a variety of methods, including administering protein,
fragments of protein, cells expressing the protein or fragments
thereof and the like to an animal to induce polyclonal antibodies.
The production of monoclonal antibodies is according to techniques
well known in the art. As detailed herein, such antibodies may be
used for example to identify tissues expressing protein or to
purify protein. Antibodies also may be coupled to specific labeling
agents for imaging or to antitumor agents, including, but not
limited to, methotrexate, radioiodinated compounds, toxins such as
ricin, other cytostatic or cytolytic drugs, and so forth.
Antibodies prepared according to the invention also preferably are
specific for the TRA/HLA complexes described herein.
[0097] Significantly, as is well-known in the art, only a small
portion of an antibody molecule, the paratope, is involved in the
binding of the antibody to its epitope (see, in general, Clark, W.
R. (1986) The Experimental Foundations of Modem Immunology Wiley
& Sons, Inc., New York; Roitt, I. (1991) Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and
Fc regions, for example, are effectors of the complement cascade
but are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab').sub.2
fragment, retains both of the antigen binding sites of an intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0098] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, 1986; Roitt,
1991). In both the heavy chain Fd fragment and the light chain of
IgG immunoglobulins, there are four framework regions (FRI through
FR4) separated respectively by three complementarity determining
regions (CDR1 through CDR3). The CDRs, and in particular the CDR3
regions, and more particularly the heavy chain CDR3, are largely
responsible for antibody specificity.
[0099] It is now well-established in the art that the non-CDR
regions of a mammalian antibody may be replaced with similar
regions of conspecific or heterospecific antibodies while retaining
the epitopic specificity of the original antibody. This is most
clearly manifested in the development and use of "humanized"
antibodies in which non-human CDRs are covalently joined to human
FR and/or Fc/pFc' regions to produce a functional antibody. Thus,
for example, PCT International Publication Number WO 92/04381
teaches the production and use of humanized murine RSV antibodies
in which at least a portion of the murine FR regions have been
replaced by FR regions of human origin. Such antibodies, including
fragments of intact antibodies with antigen-binding ability, are
often referred to as "chimeric" antibodies.
[0100] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab').sub.2, Fab, Fv
and Fd fragments; chimeric antibodies in which the Fc and/or FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by homologous human or non-human sequences; chimeric
F(ab').sub.2 fragment antibodies in which the FR and/or CDR1 and/or
CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences; chimeric Fab fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light
chain CDR3 regions have been replaced by homologous human or
non-human sequences; and chimeric Fd fragment antibodies in which
the FR and/or CDR1 and/or CDR2 regions have been replaced by
homologous human or non-human sequences. The present invention also
includes so-called single chain antibodies. Thus, the invention
involves polypeptides of numerous size and type that bind
specifically to tumor associated polypeptides including sdp3.8
polypeptides. These polypeptides may be derived also from sources
other than antibody technology. For example, such polypeptide
binding agents can be provided by degenerate peptide libraries
which can be readily prepared in solution, in immobilized form or
as phage display libraries. Combinatorial libraries also can be
synthesized of peptides containing one or more amino acids.
Libraries further can be synthesized of peptoids and non-peptide
synthetic moieties.
[0101] Phage display can be particularly effective in identifying
binding peptides useful according to the invention. Briefly, one
prepares a phage library (using e.g. m13, fd, or lambda phage),
displaying inserts from 4 to about 80 amino acid residues using
conventional procedures. The inserts may represent a completely
degenerate or biased array. One then can select phage-bearing
inserts which bind to a sdp3.8 tumor associated polypeptide. This
process can be repeated through several cycles of reselection of
phage that bind to a sdp3.8 polypeptide. Repeated rounds lead to
enrichment of phage bearing particular sequences. DNA sequence
analysis can be conducted to identify the sequences of the
expressed polypeptides. The minimal linear portion of the sequence
that binds to the sdp3.8 polypeptide can be determined. One can
repeat the procedure using a biased library containing inserts
containing part or all of the minimal linear portion plus one or
more additional degenerate residues upstream or downstream thereof.
Thus, the tumor associated polypeptides of the invention can be
used to screen peptide libraries, including phage display
libraries, to identify and select peptide binding partners of the
tumor associated polypeptides of the invention. Such molecules can
be used, as described, for screening assays, for diagnostic assays,
for purification protocols or for targeting drugs, toxins and/or
labeling agents (e.g. radioisotopes, fluorescent molecules, etc.)
to cells which express tumor associated genes such as those
leukemia cells which present sdp3.8 polypeptides on the cell
surface. Such binding agent molecules can also be prepared to bind
complexes of an sdp3.8 polypeptide and an HLA molecule by selecting
the binding agent using such complexes.
[0102] A tumor associated antigen polypeptide, or a fragment
thereof, also can be used to isolate their native binding partners.
Isolation of such binding partners may be performed according to
well-known methods. For example, isolated tumor associated antigen
polypeptides can be attached to a substrate (e.g., chromatographic
media, such as polystyrene beads, or a filter), and then a solution
suspected of containing the binding partner may be applied to the
substrate. If a binding partner which can interact with tumor
associated antigen polypeptides is present in the solution, then it
will bind to the substrate-bound tumor associated antigen
polypeptide. The binding partner then may be isolated.
[0103] As detailed herein, the foregoing antibodies and other
binding molecules may be used for example to identify tissues
expressing protein or to purify protein. Antibodies also may be
coupled to specific diagnostic labeling agents for imaging of cells
and tissues that express tumor associated polypeptides or to
therapeutically useful agents according to standard coupling
procedures. Diagnostic agents include, but are not limited to,
barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium,
diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoate
sodium and radiodiagnostics including positron emitters such as
fluorine-18 and carbon-11, gamma emitters such as iodine-123,
technitium-99m, iodine-131 and indium-111, nuclides for nuclear
magnetic resonance such as fluorine and gadolinium. Other
diagnostic agents useful in the invention will be apparent to one
of ordinary skill in the art. As used herein, "therapeutically
useful agents" include any therapeutic molecule which desirably is
targeted selectively to a cell expressing one of the cancer
antigens disclosed herein, including antineoplastic agents,
radioiodinated compounds, toxins, other cytostatic or cytolytic
drugs, and so forth. Antineoplastic therapeutics are well known and
include: aminoglutethimide, azathioprine, bleomycin sulfate,
busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide,
cyclosporine, cytarabidine, dacarbazine, dactinomycin,
daunorubicin, doxorubicin, taxol, etoposide, fluorouracil,
interferon-.alpha., lomustine, mercaptopurine, methotrexate,
mitotane, procarbazine HCl, thioguanine, vinblastine sulfate and
vincristine sulfate. Additional antineoplastic agents include those
disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and
Bruce A. Chabner), and the introduction thereto, pp.1202-1263, of
Goodman and Gilman's "The Pharmacological Basis of Therapeutics",
Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions
Division). Toxins can be proteins such as, for example, pokeweed
anti-viral protein, cholera toxin, pertussis toxin, ricin, gelonin,
abrin, diphtheria exotoxin, or Pseudomonas exotoxin. Toxin moieties
can also be high energy-emitting radionuclides such as
cobalt-60.
[0104] The skilled artisan can determine which HLA molecule binds
to tumor rejection antigens derived from sdp3.8 tumor rejection
antigen precursors by, e.g., experiments utilizing antibodies to
block specifically individual HLA class I molecules. For example,
antibodies which bind selectively to HLA-A2 will prevent efficient
presentation of TRAs specifically presented by HLA-A2. Thus, if
TRAs derived from a tumor associated gene such as HAGE (sdp3.8) are
presented by HLA-A2, then the inclusion of anti-HLA-A2 antibodies
in an in vitro assay will block the presentation of these TRAs. An
assay for determining the nature of the HLA molecule is found in
International ApplicationNo. PCT/US96/04037. Briefly, in
determining the HLA molecule type, inhibition experiments were
carried out where the production of tumor necrosis factor (TNF) by
cytotoxic T lymphocyte (CTL) clone 263/17 was tested in the
presence of monoclonal antibodies directed against HLA molecules or
against CD4/CD8 accessory molecules. Four monoclonal antibodies
were found to inhibit the production of TNF by CTL 263/17:
monoclonal antibody W6/32, which is directed against all HLA class
I molecules (Parham et al., J. Immunol. 123:342, 1979), antibody
B1.23.2 which recognizes HLA-B and C molecules (Rebai et al.,
Tissue Antigens 22:107, 1983), antibody ME-1 which specifically
recognizes HLA-B7 (Ellis et al., Hum. Immunol. 5:49, 1982) and
antibody B9.4.1 against CD8. No inhibition was found with
antibodies directed against HLA Class 11 DR molecules (L243:
Lampson et al., J. Immunol. 125:293, 1980), against HLA-A3 (GAPA 3:
Berger et al., Hybridoma 1:87, 1982) or against CD4 (13B.8.82). The
conclusion was that CTL 263/17 was of the CD8 type, and recognized
an antigen presented by HLA-B7. Similar experiments using widely
available anti-HLA antibodies can be performed to determine the
nature of a HLA molecule which presents a HAGE antigen.
[0105] Thus isolated tumor associated polypeptide molecules when
processed and presented as the TRA, or as complexes of TRA and HLA,
such as HLA-A2, HLA-A26 or HLA-B7, etc. may be combined with
materials such as adjuvants to produce vaccines useful in treating
disorders characterized by expression of the TRAP molecule. In
addition, vaccines can be prepared from cells which present the
TRA/HLA complexes on their surface, such as non-proliferative
cancer cells, non-proliferative transfectants, etcetera. In all
cases where cells are used as a vaccine, these can be cells
transfected with coding sequences for one or both of the components
necessary to provoke a CTL response, or be cells which already
express both molecules without the need for transfection. Vaccines
also encompass naked DNA or RNA, encoding a tumor associated TRA or
precursor thereof, which may be produced in vitro and administered
via injection, particle bombardment, nasal aspiration and other
methods. Vaccines of the "naked nucleic acid" type have been
demonstrated to provoke an immunological response including
generation of CTLs specific for the peptide encoded by the naked
nucleic acid (Science 259:1745-1748, 1993).
[0106] When "disorder" is used herein, it refers to any
pathological condition where the tumor rejection antigen precursor
is expressed. An example of such a disorder is cancer, sarcomas and
carcinomas in particular.
[0107] As used herein, a subject is a human, non-human primate,
cow, horse, pig, sheep, goat, dog, cat or rodent. In all
embodiments human tumor antigens and human subjects are
preferred.
[0108] Samples of tissue and/or cells for use in the various
methods described herein can be obtained through standard methods
such as tissue biopsy, including punch biopsy and cell scraping,
and collection of blood or other bodily fluids by aspiration or
other methods.
[0109] In certain embodiments of the invention, an immunoreactive
cell sample is removed from a subject. By "immunoreactive cell" is
meant a cell which can mature into an immune cell (such as a B
cell, a helper T cell, or a cytolytic T cell) upon appropriate
stimulation. Thus immunoreactive cells include CD34.sup.+
hematopoietic stem cells, immature T cells and immature B cells.
When it is desired to produce cytolytic T cells which recognize a
tumor associated antigen, the immunoreactive cell is contacted with
a cell which expresses a tumor associated antigen under conditions
favoring production, differentiation and/or selection of cytolytic
T cells; the differentiation of the T cell precursor into a
cytolytic T cell upon exposure to antigen is similar to clonal
selection of the immune system.
[0110] Some therapeutic approaches based upon the disclosure are
premised on a response by a subject's immune system, leading to
lysis of antigen presenting cells, such as sarcoma or carcinoma
cells which present one or more tumor associated antigens. One such
approach is the administration of autologous CTLs specific to a
tumor associated antigen/MHC complex to a subject with abnormal
cells of the phenotype at issue. It is within the skill of the
artisan to develop such CTLs in vitro. An example of a method for T
cell differentiation is presented in International Application
number PCT/US96/05607. Generally, a sample of cells taken from a
subject, such as blood cells, are contacted with a cell presenting
the complex and capable of provoking CTLs to proliferate. The
target cell can be a transfectant, such as a COS cell of the type
described supra. These transfectants present the desired complex of
their surface and, when combined with a CTL of interest, stimulate
its proliferation. COS cells, such as those used herein are widely
available, as are other suitable host cells. Specific production of
a CTL is well known to one of ordinary skill in the art. The
clonally expanded autologous CTLs then are administered to the
subject.
[0111] Another method for selecting antigen-specific CTL clones has
recently been described (Altman et al., Science 274:94-96, 1996;
Dunbar et al., Curr. Biol. 8:413-416, 1998), in which fluorogenic
tetramers of MHC class I molecule/peptide complexes are used to
detect specific CTL clones. Briefly, soluble MHC class I molecules
are folded in vitro in the presence of .beta..sub.2-microglobulin
and a peptide antigen which binds the class I molecule. After
purification, the MHC/peptide complex is purified and labeled with
biotin. Tetramers are formed by mixing the biotinylated peptide-MHC
complex with labeled avidin (e.g. phycoerythrin) at a molar ratio
or 4:1. Tetramers are then contacted with a source of CTLs such as
peripheral blood or lymph node. The tetramers bind CTLs which
recognize the peptide antigen/MHC class I complex. Cells bound by
the tetramers can be sorted by fluorescence activated cell sorting
to isolate the reactive CTLs. The isolated CTLs then can be
expanded in vitro for use as described herein.
[0112] To detail a therapeutic methodology, referred to as adoptive
transfer (Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al.,
Science 257: 238, 1992; Lynch et al, Eur. J. Immunol. 21:
1403-1410, 1991; Kast et al., Cell 59: 603-614, 1989), cells
presenting the desired complex are combined with CTLs leading to
proliferation of the CTLs specific thereto. The proliferated CTLs
are then administered to a subject with a cellular abnormality
which is characterized by certain of the abnormal cells presenting
the particular complex. The CTLs then lyse the abnormal cells,
thereby achieving the desired therapeutic goal.
[0113] The foregoing therapy assumes that at least some of the
subject's abnormal cells present the relevant HLA/TRA complex. This
can be determined very easily, as the art is very familiar with
methods for identifying cells which present a particular HLA
molecule, as well as how to identify cells expressing DNA of the
pertinent sequences, in this case a tumor associated gene sequence.
Once cells presenting the relevant complex are identified via the
foregoing screening methodology, they can be combined with a sample
from a patient, where the sample contains CTLs. If the complex
presenting cells are lysed by the mixed CTL sample, then it can be
assumed that a tumor associated gene derived TRA is being
presented, and the subject is an appropriate candidate for the
therapeutic approaches set forth supra.
[0114] Adoptive transfer is not the only form of therapy that is
available in accordance with the invention. CTLs can also be
provoked in vivo, using a number of approaches. One approach is the
use of non-proliferative cells expressing the complex. The cells
used in this approach may be those that normally express the
complex, such as irradiated tumor cells or cells transfected with
one or both of the genes necessary for presentation of the complex.
Chen et al., Proc. Natl. Acad. Sci. USA 88: 110-114 (1991)
exemplifies this approach, showing the use of transfected cells
expressing HPV E7 peptides in a therapeutic regime. Various cell
types may be used. Similarly, vectors carrying one or both of the
genes of interest may be used. Viral or bacterial vectors are
especially preferred. For example, nucleic acids which encode a
sdp3.8 TRA may be operably linked to promoter and enhancer
sequences which direct expression of the sdp3.8 TRA in certain
tissues or cell types. The nucleic acid may be incorporated into an
expression vector. Expression vectors may be unmodified
extrachromosomal nucleic acids, plasmids or viral genomes
constructed or modified to enable insertion of exogenous nucleic
acids, such as those encoding sdp3.8 TRAs. Nucleic acids encoding a
sdp3.8 TRA also may be inserted into a retroviral genome, thereby
facilitating integration of the nucleic acid into the genome of the
target tissue or cell type. In these systems, the gene of interest
is carried by a microorganism, e.g., a Vaccinia virus, retrovirus
or the bacteria BCG, and the materials defacto "infect" host cells.
The cells which result present the complex of interest, and are
recognized by autologous CTLs, which then proliferate.
[0115] A similar effect can be achieved by combining a TRAP or a
stimulatory fragment thereof with an adjuvant to facilitate
incorporation into HLA presenting cells in vivo. The TRAP is
processed to yield the peptide partner of the HLA molecule while
the TRA is presented without the need for further processing.
Generally, subjects can receive an intradermal injection of an
effective amount of a sdp3.8 encoded TRAP, and/or TRAs derived
therefrom. TRAs from sdp3.8 also can be combined with TRAs from
other tumor associated polypeptides in a polytope arrangement as
described above. Initial doses can be followed by booster doses,
following immunization protocols standard in the art.
[0116] The invention involves the use of various materials
disclosed herein to "immunize" subjects or as "vaccines". As used
herein, "immunization" or "vaccination" means increasing or
activating an immune response against an antigen. It does not
require elimination or eradication of a condition but rather
contemplates the clinically favorable enhancement of an immune
response toward an antigen. Generally accepted animal models can be
used for testing of immunization against cancer using a tumor
associated antigen nucleic acid. For example, human cancer cells
can be introduced into a mouse to create a tumor, and one or more
tumor associated nucleic acids can be delivered by the methods
described herein. The effect on the cancer cells (e.g., reduction
of tumor size) can be assessed as a measure of the effectiveness of
the tumor associated nucleic acid immunization. Of course, testing
of the foregoing animal model using more conventional methods for
immunization include the administration of one or more tumor
associated polypeptides or peptides derived therefrom, optionally
combined with one or more adjuvants and/or cytokines to boost the
immune response. Methods for immunization, including formulation of
a vaccine composition and selection of doses, route of
administration and the schedule of administration (e.g. primary and
one or more booster doses), are well known in the art. The tests
also can be performed in humans, where the end point is to test for
the presence of enhanced levels of circulating CTLs against cells
bearing the antigen, to test for levels of circulating antibodies
against the antigen, to test for the presence of cells expressing
the antigen and so forth.
[0117] As part of the immunization compositions, one or more tumor
associated polypeptides or stimulatory fragments thereof are
administered with one or more adjuvants to induce an immune
response or to increase an immune response. An adjuvant is a
substance incorporated into or administered with antigen which
potentiates the immune response. Adjuvants may enhance the
immunological response by providing a reservoir of antigen
(extracellularly or within macrophages), activating macrophages and
stimulating specific sets of lymphocytes. Adjuvants of many kinds
are well known in the art. Specific examples of adjuvants include
monophosphoryl lipid A (MPL, SmithKline Beecham), a congener
obtained after purification and acid hydrolysis of Salmonella
minnesota Re 595 lipopolysaccharide; saponins including QS21
(SmithKline Beecham), a pure QA-21 saponin purified from Quillja
saponaria extract; DQS21, described in PCT application WO96/33739
(SmithKline Beecham); QS-7, QS-17, QS-18, and QS-L1 (So et al.,
Mol. Cells 7:178-186, 1997); incomplete Freund's adjuvant; complete
Freund's adjuvant; montanide; and various water-in-oil emulsions
prepared from biodegradable oils such as squalene and/or
tocopherol. Preferably, the peptides are administered mixed with a
combination of DQS21/MPL. The ratio of DQS21 to MPL typically will
be about 1:10 to 10: 1, preferably about 1:5 to 5:1 and more
preferably about 1:1. Typically for human administration, DQS21 and
MPL will be present in a vaccine formulation in the range of about
1 .mu.g to about 100 .mu.g. Other adjuvants are known in the art
and can be used in the invention (see, e.g. Goding, Monoclonal
Antibodies: Principles and Practice, 2nd Ed., 1986). Methods for
the preparation of mixtures or emulsions of peptide and adjuvant
are well known to those of skill in the art of vaccination.
[0118] Other agents which stimulate the immune response of the
subject can also be administered to the subject. For example, other
cytokines are also useful in vaccination protocols as a result of
their lymphocyte regulatory properties. Many other cytokines useful
for such purposes will be known to one of ordinary skill in the
art, including interleukin-12 (IL-12) which has been shown to
enhance the protective effects of vaccines (see, e.g., Science
268:1432-1434, 1995), GM-CSF and IL-18. Thus cytokines can be
administered in conjunction with antigens and adjuvants to increase
the immune response to the antigens.
[0119] There are a number of additional immune response
potentiating compounds that can be used in vaccination protocols.
These include costimulatory molecules provided in either protein or
nucleic acid form. Such costimulatory molecules include the B7-1
and B7-2 (CD80 and CD86 respectively) molecules which are expressed
on dendritic cells (DC) and interact with the CD28 molecule
expressed on the T cell. This interaction provides costimulation
(signal 2) to an antigen/MHC/TCR stimulated (signal 1) T cell,
increasing T cell proliferation and effector function. B7 also
interacts with CTLA4 (CD152) on T cells and studies involving CTLA4
and B7 ligands indicate that the B7-CTLA4 interaction can enhance
antitumor immunity and CTL proliferation (Zheng et al., Proc. Nat'l
Acad. Sci. USA 95:6284-6289, 1998).
[0120] B7 typically is not expressed on tumor cells so they are not
efficient antigen presenting cells (APCs) for T cells. Induction of
B7 expression would enable the tumor cells to stimulate more
efficiently CTL proliferation and effector function. A combination
of B7/IL-6/IL-12 costimulation has been shown to induce IFN-gamma
and a Th1 cytokine profile in the T cell population leading to
further enhanced T cell activity (Gajewski et al., J. Immunol.
154:5637-5648, 1995). Tumor cell transfection with B7 has been
discussed in relation to in vitro CTL expansion for adoptive
transfer immunotherapy by Wang et al. (J. Immunother. 19:1-8,
1996). Other delivery mechanisms for the B7 molecule would include
nucleic acid (naked DNA) immunization (Kim et al., Nature
Biotechnol. 15:7:641-646, 1997) and recombinant viruses such as
adeno and pox (Wendtner et al., Gene Ther. 4:726-735, 1997). These
systems are all amenable to the construction and use of expression
cassettes for the coexpression of B7 with other molecules of choice
such as the antigens or fragment(s) of antigens discussed herein
(including polytopes) or cytokines. These delivery systems can be
used for induction of the appropriate molecules in vitro and for in
vivo vaccination situations. The use of anti-CD28 antibodies to
directly stimulate T cells in vitro and in vivo could also be
considered.
[0121] Lymphocyte function associated antigen-3 (LFA-3) is
expressed on APCs and some tumor cells and interacts with CD2
expressed on T cells. This interaction induces T cell IL-2 and
IFN-gamma production and can thus complement but not substitute,
the B7/CD28 costimulatory interaction (Parra et al., J. Immunol.,
158:637-642, 1997; Fenton et al., J. Immunother., 21:95-108,
1998).
[0122] Lymphocyte function associated antigen-3 (LFA-1) is
expressed on leukocytes and interacts with ICAM-1 expressed on APCs
and some tumor cells. This interaction induces T cell IL-2 and
IFN-gamma production and can thus complement but not substitute,
the B7/CD28 costimulatory interaction (Fenton et al., 1998). LFA-1
is thus a further example of a costimulatory molecule that could be
provided in a vaccination protocol in the various ways discussed
above for B7.
[0123] Complete CTL activation and effector function requires Th
cell help through the interaction between the Th cell CD40L (CD40
ligand) molecule and the CD40 molecule expressed by DCs (Ridge et
al., Nature 393:474, 1998; Bennett et al., Nature 393:478, 1998;
Schoenberger et al., Nature 393:480, 1998). This mechanism of this
costimulatory signal is likely to involve upregulation of B7 and
associated IL-6/IL-12 production by the DC (APC). The CD40-CD40L
interaction thus complements the signal 1 (antigen/MHC-TCR) and
signal 2 (B7-CD28) interactions.
[0124] The use of anti-CD40 antibodies to stimulate DC cells
directly, would be expected to enhance a response to tumor
associated antigens which are normally encountered outside of an
inflammatory context or are presented by non-professional APCs
(tumor cells). In these situations Th help and B7 costimulation
signals are not provided. This mechanism might be used in the
context of antigen pulsed DC based therapies or in situations where
Th epitopes have not been defined within known tumor associated
antigen precursors.
[0125] The invention contemplates delivery of nucleic acids,
polypeptides or peptides for vaccination. Delivery of polypeptides
and peptides can be accomplished according to standard vaccination
protocols which are well known in the art. In another embodiment,
the delivery of nucleic acid is accomplished by ex vivo methods,
i.e. by removing a cell from a subject, genetically engineering the
cell to include a tumor associated nucleic acid, and reintroducing
the engineered cell into the subject. One example of such a
procedure is outlined in U.S. Pat. No. 5,399,346 and in exhibits
submitted in the file history of that patent, all of which are
publicly available documents. In general, it involves introduction
in vitro of a functional copy of a gene into a cell(s) of a
subject, and returning the genetically engineered cell(s) to the
subject. The functional copy of the gene is under operable control
of regulatory elements which permit expression of the gene in the
genetically engineered cell(s). Numerous transfection and
transduction techniques as well as appropriate expression vectors
are well known to those of ordinary skill in the art, some of which
are described in PCT application WO95/00654. In vivo nucleic acid
delivery using vectors such as viruses and targeted liposomes also
is contemplated according to the invention.
[0126] In preferred embodiments, a virus vector for delivering a
nucleic acid encoding a tumor associated polypeptide is selected
from the group consisting of adenoviruses, adeno-associated
viruses, poxviruses including vaccinia viruses and attenuated
poxviruses, Semliki Forest virus, Venezuelan equine encephalitis
virus, retroviruses, Sindbis virus, and Ty virus-like particle.
Examples of viruses and virus-like particles which have been used
to deliver exogenous nucleic acids include: replication-defective
adenoviruses (e.g., Xiang et al., Virology 219:220-227, 1996; Eloit
et al., J. Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine
15:335-339, 1997), amodifiedretrovirus (Townsend et al., J. Virol.
71:3365-3374, 1997), a nonreplicating retrovirus (Irwin et al., J.
Virol. 68:5036-5044, 1994), a replication defective Semliki Forest
virus (Zhao et al., Proc. Natl. Acad. Sci. USA 92:3009-3013, 1995),
canarypox virus and highly attenuated vaccinia virus derivative
(Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353, 1996),
non-replicative vaccinia virus (Moss, Proc. Nat. Acad. Sci. USA
93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol.
Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis
et al., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et
al., Virology 212:587-594, 1995), and Ty virus-like particle
(Allsopp et al., Eur. J. Immunol 26:1951-1959, 1996). In preferred
embodiments, the virus vector is an adenovirus.
[0127] Another preferred virus for certain applications is the
adeno-associated virus, a double-stranded DNA virus. The
adeno-associated virus is capable of infecting a wide range of cell
types and species and can be engineered to be
replication-deficient. It further has advantages, such as heat and
lipid solvent stability, high transduction frequencies in cells of
diverse lineages, including hematopoietic cells, and lack of
superinfection inhibition thus allowing multiple series of
transductions. The adeno-associated virus can integrate into human
cellular DNA in a site-specific manner, thereby minimizing the
possibility of insertional mutagenesis and variability of inserted
gene expression. In addition, wild-type adeno-associated virus
infections have been followed in tissue culture for greater than
100 passages in the absence of selective pressure, implying that
the adeno-associated virus genomic integration is a relatively
stable event. The adeno-associated virus can also function in an
extrachromosomal fashion.
[0128] In general, other preferred viral vectors are based on
non-cytopathic eukaryotic viruses in which non-essential genes have
been replaced with the gene of interest. Non-cytopathic viruses
include retroviruses, the life cycle of which involves reverse
transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Adenoviruses and
retroviruses have been approved for human gene therapy trials. In
general, the retroviruses are replication-deficient (i.e., capable
of directing synthesis of the desired proteins, but incapable of
manufacturing an infectious particle). Such genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of genes in vivo. Standard protocols
for producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell lined with plasmid,
production of recombinant retroviruses by the packaging cell line,
collection of viral particles from tissue culture media, and
infection of the target cells with viral particles) are provided in
Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual,"
W. H. Freeman Co., New York (1990) and Murry, E. J. Ed. "Methods in
Molecular Biology," vol. 7, Humana Press, Inc., Cliffton, N.J.
(1991).
[0129] Preferably the foregoing nucleic acid delivery vectors: (1)
contain exogenous genetic material that can be transcribed and
translated in a mammalian cell and that can induce an immune
response in a host, and (2) contain on a surface a ligand that
selectively binds to a receptor on the surface of a target cell,
such as a mammalian cell, and thereby gains entry to the target
cell.
[0130] Various techniques may be employed for introducing nucleic
acids of the invention into cells, depending on whether the nucleic
acids are introduced in vitro or in vivo in a host. Such techniques
include transfection of nucleic acid-CaPO.sub.4 precipitates,
transfection of nucleic acids associated with DEAE, transfection or
infection with the foregoing viruses including the nucleic acid of
interest, liposome mediated transfection, and the like. For certain
uses, it is preferred to target the nucleic acid to particular
cells. In such instances, a vehicle used for delivering a nucleic
acid of the invention into a cell (e.g., a retrovirus, or other
virus; a liposome) can have a targeting molecule attached thereto.
For example, a molecule such as an antibody specific for a surface
membrane protein on the target cell or a ligand for a receptor on
the target cell can be bound to or incorporated within the nucleic
acid delivery vehicle. Preferred antibodies include antibodies
which selectively bind a tumor associated polypeptide, alone or as
a complex with a MHC molecule. Especially preferred are monoclonal
antibodies. Where liposomes are employed to deliver the nucleic
acids of the invention, proteins which bind to a surface membrane
protein associated with endocytosis may be incorporated into the
liposome formulation for targeting and/or to facilitate uptake.
Such proteins include capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling, proteins that target intracellular
localization and enhance intracellular half life, and the like.
Polymeric delivery systems also have been used successfully to
deliver nucleic acids into cells, as is known by those skilled in
the art. Such systems even permit oral delivery of nucleic
acids.
[0131] When administered, the therapeutic compositions of the
present invention are administered in pharmaceutically acceptable
preparations. Such preparations may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, supplementary immune
potentiating agents such as adjuvants and cytokines and optionally
other therapeutic agents.
[0132] The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active ingredients. The term
"physiologically acceptable" refers to a non-toxic material that is
compatible with a biological system such as a cell, cell culture,
tissue, or organism. The characteristics of the carrier will depend
on the route of administration. Physiologically and
pharmaceutically acceptable carriers include diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials
which are well known in the art.
[0133] The therapeutics of the invention can be administered by any
conventional route, including injection or by gradual infusion over
time. The administration may, for example, be oral, intravenous,
intraperitoneal, intramuscular, intracavity, subcutaneous, or
transdermal. When antibodies are used therapeutically, a preferred
route of administration is by pulmonary aerosol. Techniques for
preparing aerosol delivery systems containing antibodies are well
known to those of skill in the art. Generally, such systems should
utilize components which will not significantly impair the
biological properties of the antibodies, such as the paratope
binding capacity (see, for example, Sciarra and Cutie, "Aerosols,"
in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp
1694-1712). Those of skill in the art can readily determine the
various parameters and conditions for producing antibody aerosols
without resort to undue experimentation. When using antisense
preparations of the invention, slow intravenous administration is
preferred.
[0134] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like.
[0135] The preparations of the invention are administered in
effective amounts. An effective amount is that amount of a
pharmaceutical preparation that alone, or together with further
doses, stimulates the desired response. In the case of treating
cancer, the desired response is inhibiting the progression of the
cancer. This may involve only slowing the progression of the
disease temporarily, although more preferably, it involves halting
the progression of the disease permanently. In the case of
stimulating an immune response, the desired response is an increase
in antibodies or T lymphocytes which are specific for the
immunogen(s) employed. These responses can be monitored by routine
methods or can be monitored according to diagnostic methods of the
invention discussed herein.
[0136] Where it is desired to stimulate an immune response using a
therapeutic composition of the invention, this may involve the
stimulation of a humoral antibody response resulting in an increase
in antibody titer in serum, a clonal expansion of cytotoxic
lymphocytes, or some other desirable immunologic response. It is
believed that doses of immunogens ranging from one
nanogram/kilogram to 100 milligrams/kilogram, depending upon the
mode of administration, would be effective. The preferred range is
believed to be between 500 nanograms and 500 micrograms per
kilogram. The absolute amount will depend upon a variety of
factors, including the material selected for administration,
whether the administration is in single or multiple doses, and
individual patient parameters including age, physical condition,
size, weight, and the stage of the disease. These factors are well
known to those of ordinary skill in the art and can be addressed
with no more than routine experimentation.
[0137] According to another aspect of the invention, methods for
diagnosing or determining the prognosis of a disorder that is
characterized by expression of a HAGE (sdp3.8) tumor associated
nucleic acid or polypeptide are provided. The methods involve
contacting a biological sample isolated from a subject with an
agent specific for the tumor associated nucleic acid or polypeptide
to detect the presence of the tumor associated nucleic acid or
polypeptide in the biological sample. Optionally, a series of tests
is carried out over time to determine the subject's prognosis with
respect to progression or regression of the disorder.
[0138] As used herein, "contacting" means placing the biological
sample in sufficient proximity to the agent and under the
appropriate conditions of, e.g., concentration, temperature, time,
ionic strength, to allow the specific interaction between the agent
and tumor associated nucleic acid or polypeptide that are present
in the biological sample. In general, the conditions for contacting
the agent with the biological sample are conditions known by those
of ordinary skill in the art to facilitate a specific interaction
between a molecule and its cognate (e.g., a protein and its
receptor cognate, an antibody and its protein antigen cognate, a
nucleic acid and its complementary sequence cognate) in a
biological sample. Exemplary conditions for facilitating a specific
interaction between a molecule and its cognate are described in
U.S. Pat. No. 5,108,921, issued to Low et al.
[0139] The biological sample can be located in vivo or in vitro.
For example, the biological sample can be a tissue in vivo and the
agent specific for the tumor associated nucleic acid or polypeptide
can be used to detect the presence of such molecules in the
hematopoietic tissue (e.g., for imaging portions of the tissue that
express the tumor associated gene products). Alternatively, the
biological sample can be located in vitro (e.g., a blood sample,
tumor biopsy, tissue extract). In a particularly preferred
embodiment, the biological sample can be a cell-containing sample,
more preferably a sample containing tumor cells.
EXAMPLES
Example 1
[0140] Isolation of a Nucleic Acid Specifically Expressed by
Sarcoma Cell Line LB-23
[0141] Specific cDNA fragments of sarcoma cell line LB-23 were
enriched by subtraction of cDNA fragments found in normal uterus,
breast, colon and heart, according to the representational
difference analysis method (RDA) described for cDNA by Hubank &
Schatz (Nucl. Acids Res. 22:5640, 1994).
[0142] Briefly, cellular cDNAs obtained by reverse transcription
with an oligo-dT primer on poly-A RNA of the normal and sarcoma
cell samples were digested by restriction enzyme DpnII. The DpnII
fragments of each origin were ligated with the same set of
adapters, divided in several groups and separately amplified by
PCR. The PCR products originating from the same sample were pooled
and digested again by DpnII. The DpnII fragments from the sarcoma
cell line (the tester) were ligated with a new adapter set and
hybridized with an excess of DpnII fragments derived from the
normal tissues (the driver). The hybridization mixture was then
submitted to PCR amplification using the new adapter set. Only
those DpnII fragments derived from the tester and absent in the
driver were expected to be amplified exponentially because they
carry primer-complementary sequences at both ends. Three cycles of
subtractive hybridization and amplification were performed. The
final PCR mixture products were then cloned and sequenced.
[0143] The sequencing of 106 cDNA clones yielded 42 different
sequences. Twenty-seven sequences corresponded to genes recorded in
databases: most of these genes are involved in cell proliferation,
whereas the remaining genes are ectopically expressed
differentiation genes, mitochondrial genes, oncogenes, or genes
with unknown function. Fifteen cDNA clones showed no homology to
any recorded gene. Among the 15 unknown sequences, one, named
sdp3.8, appeared to have tumor associated expression as seen by
RT-PCR. The sequence of the sdp3.8 clone was 323 bp long (SEQ ID
NO: 1). The sdp3.8 cDNA nucleotide sequence has four open reading
frames (ORFs) beginning at nucleotides 27 (12 amino acids), 103 (35
amino acids), 170 (50 amino acids) and 210 (36 amino acids). The
amino acid sequences of the ORFs are set forth in SEQ ID NOs:38-41,
respectively.
Example 2
[0144] Expression of Gene sdp3.8 in Normal Tissues and Tumor
Samples
[0145] The expression pattern of the sdp3.8 messenger was
determined by RT-PCR analysis of normal tissues and tumor samples.
Two primers were selected for PCR analysis: a sense primer, sdp3.8S
(5'-TAGAGAGGAAGGTTTGAAAT-3'; SEQ ID NO:2), located at nucleotides
9-28 of SEQ ID NO:1, and an antisense primer, sdp3.8A
(5'-ATGTGCAGGTAGATTGGGAT-3'- ; SEQ ID NO:3), located at nucleotides
187-206 of SEQ ID NO:1. Total RNA of normal or tumor samples of the
indicated origins were converted to cDNA. The cDNA corresponding to
50 ng of total RNA was then amplified by PCR with primers sdp3.8S
and sdp3.8A with 0.625U of TaKaRa Taq polymerase for 30 cycles
(94.degree. C., 1 min; 57.degree. C., 2 min; 72.degree. C., 2 min)
with 15' at 72.degree. C. for the final extension. The PCR using
the same primers also was performed using genomic DNA as a
substrate. The primers are located in different exons, as
determined by the different sizes of PCR products obtained on cDNA
(196 bp) or genomic DNA (approximately 3 kb).
[0146] Sdp3.8 is not expressed in a panel of normal tissues tested
(Table II), with the exception of testis. Thus sdp3.8 shares an
expression pattern with other tumor associated genes in that it is
expressed only in immune privileged normal tissues such as testis.
Among tumoral samples (Table III), sdp3.8 is frequently expressed
in sarcomas (32%). Sdp 3.8 is expressed with lesser frequency in
epidermoid carcinoma (10%), non-small cell lung carcinoma (7%),
head & neck carcinoma (5%), brain tumors, neuroblastoma and
uveal melanoma tumor samples.
2TABLE II Expression of gene sdp3.8 in normal tissues (RT-PCR)
Tissue Sample code detection of sdp3.8 ovary CLO7 - kidney BA21 -
kidney BA4 - adrenal glands LB539 - adrenal glands LB538 - uterus
LB1022 - breast LB673 .+-. breast LB520 - sperm LB568 - skin LB243
- skin LB148 - brain JNO9 - testis LB882 +++ testis LB881 +++ heart
CLO3 - prostate CLO9 - stomach LB189 - lung LB264 - lung LB176 -
colon LB298 - bladder HM83 - liver LB898 - liver CLO10 - bone
marrow LB1039 - PBL LB490 - retina SH8 -
[0147]
3TABLE III Expression of gene sdp3.8 in tumors (RT-PCR) sdp3.8
Sample number positive (%) Cutaneous melanoma 47 0 Primary 25 0
Metastatic 22 0 Uveal melanoma 5 1 Neuroblastoma 2 1 Bladder
carcinoma 30 0 Breast carcinoma 14 0 Lung carcinoma NSCLC 27 2 7%
Epidermoid carcinoma 20 2 10% Bronchiolo-alveolar carcinoma 2 0
Adenocarcinoma 13 0 Sarcoma 19 6 32% Brain tumors 6 1 Prostate
adenocarcinoma 2 0 Head & neck carcinoma 20 1 5% Colorectal
carcinoma 19 0 Leukemia 25 0 Renal tumors 16 0 Uterine tumors 5 0
Esophageal carcinoma 14 0 Myeloma 5 0 Mesothelioma 4 0 Thyroid 5
0
Example 3
[0148] Isolation of a Full Length sdp3.8 cDNA Clone.
[0149] To obtain the complete sdp3.8 cDNA, a classical cDNA library
was screened with a sdp3.8 probe. The cDNA library was constructed
with LB451 testis RNA in pcDNA1/Amp as described for SK29-MEL.1 RNA
in U.S. Pat. No. 5,519,117. Approximately 250,000 bacteria were
plated on nylon membranes. Duplicates were made and treated to
denature and fix the bacterial DNA. A sdp3.8 specific probe was
generated by performing RT-PCR (reverse transcription-PCR) using
LB23-SARC RNA as template and sdp3.8 specific primers as described
above. The 196 bp sdp3.8 PCR product was purified on a sepharose
CL-6B column, then labeled using random primers, Klenow DNA
polymerase and .alpha.-.sup.32-P-dCTP according to standard
procedures. Treated duplicate nylon membranes were hybridized with
the sdp3.8 specific probe (overnight incubation at 65 C), then
washed in stringent conditions, and autoradiographed overnight.
Positive spots were obtained. A secondary screening was performed
according to standard procedures, and a bacterial clone was
obtained which contained the complete sdp3.8 cDNA. The complete
sdp3.8 cDNA clone was sequenced and found to be 2365 nucleotides
long (SEQ ID NO:42) inclusive of the poly A tail, or about 2340
nucleotides long without the poly A tail. An open reading frame
runs through the cDNA, with the first ATG at nucleotide 208 and a
stop codon at nucleotide 2152. It encodes a putative protein of 648
amino-acids (SEQ ID NO:43). The gene was found to have homology to
p68 RNA helicase, and so was called HAGE, for Helicase-related
AntiGEn.
Example 4
[0150] Identification of the Portion of Tumor Associated Genes
Encoding Tumor Rejection Antigens.
[0151] In a first method, available CTL clones directed against
antigens presented by autologous tumor cells shown to express one
or more of the tumor associated genes are screened for specificity
against COS cells transfected with sdp3.8 genes and autologous HLA
alleles as described by Brichard et al. (Eur. J. Immunol.
26:224-230, 1996). CTL recognition of sdp3.8 is determined by
measuring release of TNF from the cytolytic T lymphocyte or by
.sup.51Cr release assay (Herin et al., Int. J. Cancer 39:390-396,
1987). If a CTL clone specifically recognizes a transfected COS
cell, shorter fragments of the coding sequences are prepared and
tested by transfecting COS cells to identify the region of the gene
that encodes the peptide recognized by the CTL. Fragments of sdp3.8
are prepared by exonuclease III digestion or other standard
molecular biology methods such as PCR. Synthetic peptides are
prepared and tested to confirm the exact sequence of the
antigen.
[0152] Alternatively, CTL clones are generated by stimulating the
peripheral blood lymphocytes (PBLs) of a patient with autologous
normal cells transfected with DNA clones encoding sdp3.8
polypeptides (e.g. SEQ ID NO: 1) or with irradiated PBLs loaded
with synthetic peptides corresponding to the putative proteins and
matching the consensus for the appropriate HLA class I molecule to
localize the antigenic peptide within the sdp3.8 clones (see, e.g.,
van der Bruggen et al., Eur. J. Immunol.24:3038-3043, 1994; Herman
et al., Immunogenetics 43:377-383, 1996). Localization of one or
more antigenic peptides in a protein sequence can be aided by HLA
peptide binding predictions made according to established rules for
binding potential (e.g., Parker et al, J. Immunol. 152:163, 1994;
Rammensee et al., Immunogenetics 41:178-228, 1995). HLA binding
predictions can conveniently be made using an algorithm available
via the Internet on the National Institutes of Health World Wide
Web site at URL http://bimas.dcrt.nih.gov. For example, several
predicted HLA binding motifs for the four potential sdp3.8 ORFs
based on SEQ ID NO: 1 (SEQ ID Nos:38-41) or the long open reading
frame (SEQ ID NO:43) of the HAGE cDNA (SEQ ID NO:42) are listed in
the table below:
4TABLE IV Predicted HLA binding motifs in HAGE (sdp3.8)
polypeptides HLA Binding score SEQ ID NO/amino acids molecule
(t.sub.1/2 disassociation) SEQ43/AA297-305 A_0201 746
SEQ43/AA380-388 A3 180 SEQ40/AA19-27 A24 126 SEQ43/AA44-52 A68.1
400 SEQ43/AA489-497 A68.1 240 SEQ40/AA2-10 B7 200 SEQ41/AA2-10 B7
80 SEQ43/AA29-37 B_2705 3000 SEQ43/AA435-443 B_2705 2000
SEQ41/AA27-35 B_2705 2000 SEQ43/AA17-25 B_2705 1000 SEQ41/AA8-16
B_2705 1000 SEQ41/AA16-24 B_2705 600 SEQ41/AA15-23 B_2705 600
SEQ39/AA24-32 B44 120 SEQ40/AA24-32 B44 90 SEQ39/AA27-35 Cw_0301
125
[0153] Alternatively, CTL clones obtained by stimulation of
lymphocytes with autologous tumor cells which express sdp3.8 are
screened for specificity against COS cells transfected with sdp3.8
cDNA and autologous HLA alleles as described by Brichard et al.
(Eur. J. Immunol. 26:224-230, 1996).
[0154] Optionally, shorter fragments of HAGE/sdp3.8 cDNAs are
generated by PCR. Shorter fragments are used to provoke TNF release
or .sup.51Cr release as above.
Example 5
[0155] Identification of Tumor Associated Gene Encoded Tumor
Rejection Antigen Peptides
[0156] Synthetic peptides corresponding to portions of the shortest
fragment of sdp3.8 which provokes TNF release are prepared.
Progressively shorter peptides are synthesized to determine the
optimal sdp3.8 tumor rejection antigen peptides for a given HLA
molecule.
[0157] Synthetic peptides are tested for lysis of HLA expressing
cells according to known procedures. For example, if the HLA which
presents a peptide of interest is determined to be HLA-A2, then T2
cells can be used. T2 cells are HLA-A2+cells which have an
antigen-processing defect resulting in an increased capacity to
present exogenous peptides. T2 cells are mixed with a synthetic
peptide corresponding to the CTL-reactive portion of sdp3.8. CTL
cells are added and lysis is measured after 4 hours to determine
which peptides efficiently stimulate the lysis of T2 cells bearing
HLA-A2. Other HLA expressing cells are known in the art or can be
prepared by transfection with specific HLA clones.
[0158] To determine the optimal size of the synthetic peptide,
peptides of decreasing size are synthesized based on the sequence
of the peptide determined above, by successively removing one amino
acid from the amino terminal end or the carboxy terminal end of the
peptide. These peptides are tested for the ability to induce cell
lysis of appropriate HLA expressing cells by CTL cells in a dose
response assay. Lyophilized peptides are dissolved at 20 mg/ml in
DMSO, then diluted to 2 mg/ml in 10 mM acetic acid and stored at
-80 C. Target cells, e.g. HLA-A2.sup.+ T2 cells, are labeled with
.sup.51Cr, as described above, for 1 hour at 37 C followed by
extensive washing to remove unincorporated label. To confirm the
necessity of the interaction of the peptide with the HLA, T2 cells
optionally can be pretreated with an anti-HLA-A2 antibody, such as
MA2.1 (Wolfel et al., Eur. J. Immunol. 24: 759-764, 1994), and then
are incubated in 96-well microplates in the presence of various
concentrations of peptides for 30 minutes at 37 C. CTLs which
recognize the peptide presented by the HLA are then added in an
equal volume of medium at an effector:target ratio of 30:1.
Chromium-51 release is measured after 4 hours.
Example 6
[0159] Normal Cells are not Lysed by CTLs which Lyse Cells
Expressing Tumor Associated Genes
[0160] This example describes CTL lysis experiments with various
cell lines with or without incubation with the tumor associated
gene derived peptides determined above. Tumor cells which express
sdp3.8, normal B cells transformed with EBV (B-EBV) from the
patient who is the source of the tumor cells, and normal peripheral
blood lymphocytes from the same patient (PBL) are tested for lysis
by CTL cells in a dose response assay. These cells are incubated
with CTLs at the effector/target ratios determined to be optimal in
the dose response assays detailed above, and assayed for lysis as
described above. Lysis of only the sdp3.8-expressing tumor cells by
the CTLs, demonstrates that B-EBV and PBL cells of the patient are
not recognized by the CTLs because such cells do not normally
express the tumor rejection antigen derived from sdp3.8
proteins.
[0161] It is next determined whether these cells would be lysed by
CTL if pulsed with a peptide derived from sdp3.8. The peptides
selected on the basis of the experiments above are tested for the
ability to induce cell lysis of sdp3.8-expressing tumor cells,
B-EBV cells, and non-autologous cells which express the appropriate
HLA by CTL cells in a dose response assay as in previous examples.
B-EBV and PBL pulsed with preferred peptides are now lysed by CTLs,
as are sdp3.8-expressing tumor cells and the non-autologous cells
pulsed with preferred peptides.
5TABLE V HAGE (sdp3.8) Sequence Homologies (GenBank accession
numbers) AF018044, U60880, L22944, U85943, U89924, AF073995,
AF019886, X90639, U52949, AF033115, U93301, AF018052, AF018056,
U93295, L39119, U60876, Y10545, U65226, D44443, AJ000542, AF018066,
X74324, AJ005168, AF018036, U60877, AF018053, D88984, U93302,
U89672, AJ001134, U14118, AF018054, U93294, AC005501, AF046856,
Y17556, AJ010397, AF018071, AF033196, AF045229, AF013625, AF015523,
U93712, AF073473, AJ001044, AF019671, AF019720, AF041461, U15425,
AJ006789, D16247, D49729, L33810, U31958, AC005372, U92795,
AF011925, AF056022, AF054142, AF019721, U36623, AF028729, U67221,
U13667, AB010259, U25746, U46006, U89673, U83666, Z46845, AF030780,
U13563, AF034540, AF048848, AF054140, U90261, AF034793, AF030777,
AF075440, J04847, AF034805, AF034539, AF030779, AF021811, AF054065,
AF019734, U89574, AF054135, Z12125, U58669, U82970, U37222,
AF053372, AF030774, Y16911, L38622, AF056702, X65627, AF030773,
U00031, X57328, AB008265, AF000984, M83211, L25126, AF045381,
Z38117, AB003909, AF000985, L08427, AC004120, Z54202, U93298,
U49757, Y16912, X85122, AF018055, Z77249, AF000982, AP000983,
U50553, AF061337, U32699, AC002483, Z83822, AE001174, AA883800,
AA948168, R82515, F20206, F20451, F20430, F20197, F20205, AA514191,
F20377, F20455, F20201, F20494, P20380, P20443, AA514190, F20198,
P20182, F20192, F20472, P20456, F20460, F20484, P20187, F20189,
F20495, F20208, F20446, P20373, P20471, F20468, F20457, P20404,
P20490, P20412, F20212, P20449, F20461, F20499, F20450, F20376,
F20464, F20487, F20188, F20459, F20193, F20486, F20448, F20407,
F20194, P20497, P20502, P20181, F20508, P20453, P20374, P20209,
P20509, P20389, AA933999, P20485, F20214, F20410, F20388, F20500,
F20470, F20406, F20386, F20213, F20488, P20190, F20203, P20199,
F20431, P20180, F20191, P20408, P20489, P20196, P20387, F21475,
P21480, P20379, P20507, F20503, F20465, F20382, P20204, P20381,
P20411, F20416, F20215, P20458, F20419, P20447, F20378, F20462,
F20216, F20429, F20210, P20418, P20385, F20384, F20383, P20505,
P20202, P20498, F20207, F20218, F20445, P20454, F20463, P20390,
F20501, P22435, P20195, P20220, AA996393, P20375, H43402, H30783,
AA356553, R82572, R13011, AA643791, P20405, T85890, T82153, AA3
16798, AA248948, H65151, AA883852, A1018020, R18664, R46313,
A1050031, T89754, AA070542, H04357, AA148692, AP037645, AA453316,
AA573298, W52969, AA591070, U24210, W91597, W91767, W91680,
AA681183, AA166485, AA920867, AA120483, AA574856, AA895748,
AA144517, AA983064, AA124406, AA116852, AA199025, AA791735, W29675,
AA105648, AA645800, AA020429, AA541947, AA709485, AA174503,
AA172504, AA086670, AP027363, AP064731, AP062411, AP064739, U83004,
U19683, AA514079, A1137622, P20150, A1099575, A1043754, A1028830,
Z30733, A1010783, N82908, C91736, AA440301, T41860, AA395139,
AA849867, A1008158, AA851373, A1007729, AA042545.
[0162] The terms and expressions which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
[0163] All of the references described herein are incorporated by
reference.
Sequence CWU 1
1
43 1 323 DNA Homo sapiens 1 gatcaaatta gagaggaagg tttgaaatgg
caaaaaacaa agtgggcaga tttaccacca 60 attaagaaaa acttttataa
agagtccact gccacaagtg ccatgtcaaa agtagaagca 120 gatagttgga
ggaaagaaaa ttttaatata acgtgggatg acttgaagga tggggagaaa 180
cgacctatcc caatctacct gcacatttga tgacgccttt caatgttatc ctgaggttat
240 ggaaaacatt aaaaaggcag gttttcaaaa gccaacacct attcagtcac
aggcatggcc 300 attgtgttg caaggaatag atc 323 2 20 DNA Homo sapiens 2
tagagaggaa ggtttgaaat 20 3 20 DNA Homo sapiens 3 atgtgcaggt
agattgggat 20 4 9 PRT Homo sapiens 4 Glu Ala Asp Pro Thr Gly His
Ser Tyr 1 5 5 9 PRT Homo sapiens 5 Ser Ala Tyr Gly Glu Pro Arg Lys
Leu 1 5 6 9 PRT Homo sapiens 6 Glu Val Asp Pro Ile Gly His Leu Tyr
1 5 7 9 PRT Homo sapiens 7 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5
8 10 PRT Homo sapiens 8 Met Glu Val Asp Pro Ile Gly His Leu Tyr 1 5
10 9 9 PRT Homo sapiens 9 Ala Ala Arg Ala Val Phe Leu Ala Leu 1 5
10 8 PRT Homo sapiens 10 Tyr Arg Pro Arg Pro Arg Arg Tyr 1 5 11 10
PRT Homo sapiens 11 Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr 1 5 10
12 9 PRT Homo sapiens 12 Val Leu Pro Asp Val Phe Ile Arg Cys 1 5 13
10 PRT Homo sapiens 13 Val Leu Pro Asp Val Phe Ile Arg Cys Val 1 5
10 14 9 PRT Homo sapiens 14 Glu Glu Lys Leu Ile Val Val Leu Phe 1 5
15 9 PRT Homo sapiens 15 Glu Glu Lys Leu Ser Val Val Leu Phe 1 5 16
10 PRT Homo sapiens 16 Ala Cys Asp Pro His Ser Gly His Phe Val 1 5
10 17 10 PRT Homo sapiens 17 Ala Arg Asp Pro His Ser Gly His Phe
Val 1 5 10 18 9 PRT Homo sapiens 18 Ser Tyr Leu Asp Ser Gly Ile His
Phe 1 5 19 9 PRT Homo sapiens 19 Ser Tyr Leu Asp Ser Gly Ile His
Ser 1 5 20 9 PRT Homo sapiens 20 Met Leu Leu Ala Val Leu Tyr Cys
Leu 1 5 21 9 PRT Homo sapiens 21 Tyr Met Asn Gly Thr Met Ser Gln
Val 1 5 22 9 PRT Homo sapiens 22 Ala Phe Leu Pro Trp His Arg Leu
Phe 1 5 23 9 PRT Homo sapiens 23 Ser Glu Ile Trp Arg Asp Ile Asp
Phe 1 5 24 9 PRT Homo sapiens 24 Tyr Glu Ile Trp Arg Asp Ile Asp
Phe 1 5 25 15 PRT Homo sapiens 25 Gln Asn Ile Leu Leu Ser Asn Ala
Pro Leu Gly Pro Gln Phe Pro 1 5 10 15 26 15 PRT Homo sapiens 26 Asp
Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp 1 5 10 15
27 10 PRT Homo sapiens 27 Glu Ala Ala Gly Ile Gly Ile Leu Thr Val 1
5 10 28 9 PRT Homo sapiens 28 Ala Ala Gly Ile Gly Ile Leu Thr Val 1
5 29 9 PRT Homo sapiens 29 Ile Leu Thr Val Ile Leu Gly Val Leu 1 5
30 9 PRT Homo sapiens 30 Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5 31
9 PRT Homo sapiens 31 Ile Thr Asp Gln Val Pro Phe Ser Val 1 5 32 9
PRT Homo sapiens 32 Tyr Leu Glu Pro Gly Pro Val Thr Ala 1 5 33 10
PRT Homo sapiens 33 Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu 1 5 10
34 10 PRT Homo sapiens 34 Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val 1
5 10 35 9 PRT Homo sapiens 35 Leu Tyr Val Asp Ser Leu Phe Phe Leu 1
5 36 12 PRT Homo sapiens 36 Lys Ile Ser Gly Gly Pro Arg Ile Ser Tyr
Pro Leu 1 5 10 37 9 PRT Homo sapiens 37 Tyr Met Asp Gly Thr Met Ser
Gln Val 1 5 38 12 PRT Homo sapiens 38 Met Ala Lys Asn Lys Val Gly
Arg Phe Thr Thr Asn 1 5 10 39 35 PRT Homo sapiens 39 Met Ser Lys
Val Glu Ala Asp Ser Trp Arg Lys Glu Asn Phe Asn Ile 1 5 10 15 Thr
Trp Asp Asp Leu Lys Asp Gly Glu Lys Arg Pro Ile Pro Ile Tyr 20 25
30 Leu His Ile 35 40 50 PRT Homo sapiens 40 Met Gly Arg Asn Asp Leu
Ser Gln Ser Thr Cys Thr Phe Asp Asp Ala 1 5 10 15 Phe Gln Cys Tyr
Pro Glu Val Met Glu Asn Ile Lys Lys Ala Gly Phe 20 25 30 Gln Lys
Pro Thr Pro Ile Gln Ser Gln Ala Trp Pro Ile Val Leu Gln 35 40 45
Gly Ile 50 41 36 PRT Homo sapiens 41 Met Thr Pro Phe Asn Val Ile
Leu Arg Leu Trp Lys Thr Leu Lys Arg 1 5 10 15 Gln Val Phe Lys Ser
Gln His Leu Phe Ser His Arg His Gly Pro Leu 20 25 30 Cys Cys Lys
Glu 35 42 2365 DNA H. sapiens CDS (208)...(2151) 42 ttggtaccga
gctcggatcc ctagtaacgg ccgccagtgt gctggaaaga gtgggcggga 60
tagagagcgt gggcgggggg gctagcctcg tgcgggctcc ttaagtagcg gctgcgtggc
120 ttccctggca cgctagtctt acgacgtcac ggtcaggtgg tgcagagctg
gacggcaacg 180 acgtcggacg cgccccttct tggaaca atg tcc cac cac gga
gga gct ccc aag 234 Met Ser His His Gly Gly Ala Pro Lys 1 5 gcc tct
acg tgg gtc gtt gct agt cgg cga agc tcg aca gtg tcc cga 282 Ala Ser
Thr Trp Val Val Ala Ser Arg Arg Ser Ser Thr Val Ser Arg 10 15 20 25
gcg cca gag agg agg ccg gcg gag gag ttg aat cga aca ggt cct gag 330
Ala Pro Glu Arg Arg Pro Ala Glu Glu Leu Asn Arg Thr Gly Pro Glu 30
35 40 gga tat agt gtc ggc aga ggt ggt cgc tgg aga ggc acc tct agg
ccc 378 Gly Tyr Ser Val Gly Arg Gly Gly Arg Trp Arg Gly Thr Ser Arg
Pro 45 50 55 ccg gag gcc gtg gcc gct ggt cac gag gaa ctg ccg ctg
tgt ttt gct 426 Pro Glu Ala Val Ala Ala Gly His Glu Glu Leu Pro Leu
Cys Phe Ala 60 65 70 ttg aag agc cac ttt gtt ggc gcg gta atc ggt
cgt ggt ggg tca aaa 474 Leu Lys Ser His Phe Val Gly Ala Val Ile Gly
Arg Gly Gly Ser Lys 75 80 85 ata aag aat ata caa agt aca aca aac
acc aca atc caa ata ata caa 522 Ile Lys Asn Ile Gln Ser Thr Thr Asn
Thr Thr Ile Gln Ile Ile Gln 90 95 100 105 gaa caa cca gaa tca tta
gtc aaa att ttt ggc agc aag gca atg caa 570 Glu Gln Pro Glu Ser Leu
Val Lys Ile Phe Gly Ser Lys Ala Met Gln 110 115 120 acg aaa gca aaa
gca gtg ata gac aat ttt gtt aaa aag cta gaa gaa 618 Thr Lys Ala Lys
Ala Val Ile Asp Asn Phe Val Lys Lys Leu Glu Glu 125 130 135 aat tac
aat tca gaa tgc gga att gat act gca ttc caa cct tct gtt 666 Asn Tyr
Asn Ser Glu Cys Gly Ile Asp Thr Ala Phe Gln Pro Ser Val 140 145 150
gga aaa gat gga agc aca gat aac aat gtt gtt gca gga gat cgg cca 714
Gly Lys Asp Gly Ser Thr Asp Asn Asn Val Val Ala Gly Asp Arg Pro 155
160 165 ttg ata gat tgg gat caa att aga gag gaa ggt ttg aaa tgg caa
aaa 762 Leu Ile Asp Trp Asp Gln Ile Arg Glu Glu Gly Leu Lys Trp Gln
Lys 170 175 180 185 aca aag tgg gca gat tta cca cca att aag aaa aac
ttt tat aaa gag 810 Thr Lys Trp Ala Asp Leu Pro Pro Ile Lys Lys Asn
Phe Tyr Lys Glu 190 195 200 tcc act gcc aca agt gcc atg tca aaa gta
gaa gca gat agt tgg agg 858 Ser Thr Ala Thr Ser Ala Met Ser Lys Val
Glu Ala Asp Ser Trp Arg 205 210 215 aaa gaa aat ttt aat ata acg tgg
gat gac ttg aag gat ggg gag aaa 906 Lys Glu Asn Phe Asn Ile Thr Trp
Asp Asp Leu Lys Asp Gly Glu Lys 220 225 230 cga cct atc ccc aat cct
acc tgc aca ttt gat gac gcc ttt caa tgt 954 Arg Pro Ile Pro Asn Pro
Thr Cys Thr Phe Asp Asp Ala Phe Gln Cys 235 240 245 tat cct gag gtt
atg gaa aac att aaa aag gca ggt ttt caa aag cca 1002 Tyr Pro Glu
Val Met Glu Asn Ile Lys Lys Ala Gly Phe Gln Lys Pro 250 255 260 265
aca cct att cag tca cag gca tgg ccc att gtg ttg caa gga ata gat
1050 Thr Pro Ile Gln Ser Gln Ala Trp Pro Ile Val Leu Gln Gly Ile
Asp 270 275 280 ctt ata gga gta gcc cag act gga aca gga aag aca ttg
tgt tat tta 1098 Leu Ile Gly Val Ala Gln Thr Gly Thr Gly Lys Thr
Leu Cys Tyr Leu 285 290 295 atg cct gga ttt att cat ctg gtc ctt caa
ccc agc ctt aaa ggt caa 1146 Met Pro Gly Phe Ile His Leu Val Leu
Gln Pro Ser Leu Lys Gly Gln 300 305 310 agg aat aga ccc ggc atg tta
gtt cta act ccc act cgg gaa tta gca 1194 Arg Asn Arg Pro Gly Met
Leu Val Leu Thr Pro Thr Arg Glu Leu Ala 315 320 325 ctt caa gta gaa
gga gaa tgt tgc aaa tat tca tat aaa ggg ctt cgg 1242 Leu Gln Val
Glu Gly Glu Cys Cys Lys Tyr Ser Tyr Lys Gly Leu Arg 330 335 340 345
agt gtt tgt gta tat ggt ggt gga aat aga gat gaa caa ata gaa gag
1290 Ser Val Cys Val Tyr Gly Gly Gly Asn Arg Asp Glu Gln Ile Glu
Glu 350 355 360 ctt aaa aaa ggt gta gat atc ata att gca act ccc gga
aga ttg aat 1338 Leu Lys Lys Gly Val Asp Ile Ile Ile Ala Thr Pro
Gly Arg Leu Asn 365 370 375 gat ctg caa atg agt aac ttc gtc aat ctg
aag aat ata acc tac ttg 1386 Asp Leu Gln Met Ser Asn Phe Val Asn
Leu Lys Asn Ile Thr Tyr Leu 380 385 390 gtt tta gat gaa gca gac aag
atg ttg gac atg gga ttt gaa ccc cag 1434 Val Leu Asp Glu Ala Asp
Lys Met Leu Asp Met Gly Phe Glu Pro Gln 395 400 405 ata atg aag att
ttg tta gat gtg cgc cca gat agg cag aca gtt atg 1482 Ile Met Lys
Ile Leu Leu Asp Val Arg Pro Asp Arg Gln Thr Val Met 410 415 420 425
acc agt gct aca tgg cct cat tca gtt cat cgc ctc gca caa tct tat
1530 Thr Ser Ala Thr Trp Pro His Ser Val His Arg Leu Ala Gln Ser
Tyr 430 435 440 ttg aaa gaa cca atg att gtc tat gtt ggt aca ttg gat
cta gtt gct 1578 Leu Lys Glu Pro Met Ile Val Tyr Val Gly Thr Leu
Asp Leu Val Ala 445 450 455 gta agt tca gtg aag caa aat ata att gta
acc acc gag gaa gag aaa 1626 Val Ser Ser Val Lys Gln Asn Ile Ile
Val Thr Thr Glu Glu Glu Lys 460 465 470 tgg agt cac atg caa act ttt
cta cag agt atg tca tcc aca gac aaa 1674 Trp Ser His Met Gln Thr
Phe Leu Gln Ser Met Ser Ser Thr Asp Lys 475 480 485 gtc att gtc ttc
gtt tct cga aaa gct gtt gcg gat cac tta tca agt 1722 Val Ile Val
Phe Val Ser Arg Lys Ala Val Ala Asp His Leu Ser Ser 490 495 500 505
gac cta ata ctt gga aat ata tca gta gag tct ctg cat gga gat aga
1770 Asp Leu Ile Leu Gly Asn Ile Ser Val Glu Ser Leu His Gly Asp
Arg 510 515 520 gaa cag aga gat cgg gag aaa gca tta gag aac ttt aaa
aca ggc aaa 1818 Glu Gln Arg Asp Arg Glu Lys Ala Leu Glu Asn Phe
Lys Thr Gly Lys 525 530 535 gtg aga ata cta att gca act gat cta gcc
tct aga gga ctt gat gtc 1866 Val Arg Ile Leu Ile Ala Thr Asp Leu
Ala Ser Arg Gly Leu Asp Val 540 545 550 cat gac gtt aca cat gtc tat
aat ttt gac ttt cca cgg aat att gaa 1914 His Asp Val Thr His Val
Tyr Asn Phe Asp Phe Pro Arg Asn Ile Glu 555 560 565 gaa tac gta cac
cga ata ggg cgc acg gga aga gca ggg agg act ggt 1962 Glu Tyr Val
His Arg Ile Gly Arg Thr Gly Arg Ala Gly Arg Thr Gly 570 575 580 585
gtt tcc att aca act ttg act aga aat gat tgg agg gtt gcc tct gaa
2010 Val Ser Ile Thr Thr Leu Thr Arg Asn Asp Trp Arg Val Ala Ser
Glu 590 595 600 ttg att aat att ctg gaa aga gca aat cag agt att cca
gag gag ctt 2058 Leu Ile Asn Ile Leu Glu Arg Ala Asn Gln Ser Ile
Pro Glu Glu Leu 605 610 615 gta tca atg gct gag agg ttt gag gca cat
caa cgg aaa agg gaa atg 2106 Val Ser Met Ala Glu Arg Phe Glu Ala
His Gln Arg Lys Arg Glu Met 620 625 630 gaa aga aaa atg gaa aga cct
caa gga agg ccc aag aag ttt cat 2151 Glu Arg Lys Met Glu Arg Pro
Gln Gly Arg Pro Lys Lys Phe His 635 640 645 taatgtcttc tgtactagtg
gggtagagaa ttcaagattt tttagaaata tagtaagaca 2211 gaagtattgg
acatgttggc agtatgaaga gaccggactg atttgactga ttcttaaaat 2271
aatagtgttt gaaaatatag aatccagtgt tttatacttt ctttaataaa aatagaagta
2331 tttaaactta aaaaaaaaaa aaaaaaaaaa aaaa 2365 43 648 PRT H.
sapiens 43 Met Ser His His Gly Gly Ala Pro Lys Ala Ser Thr Trp Val
Val Ala 1 5 10 15 Ser Arg Arg Ser Ser Thr Val Ser Arg Ala Pro Glu
Arg Arg Pro Ala 20 25 30 Glu Glu Leu Asn Arg Thr Gly Pro Glu Gly
Tyr Ser Val Gly Arg Gly 35 40 45 Gly Arg Trp Arg Gly Thr Ser Arg
Pro Pro Glu Ala Val Ala Ala Gly 50 55 60 His Glu Glu Leu Pro Leu
Cys Phe Ala Leu Lys Ser His Phe Val Gly 65 70 75 80 Ala Val Ile Gly
Arg Gly Gly Ser Lys Ile Lys Asn Ile Gln Ser Thr 85 90 95 Thr Asn
Thr Thr Ile Gln Ile Ile Gln Glu Gln Pro Glu Ser Leu Val 100 105 110
Lys Ile Phe Gly Ser Lys Ala Met Gln Thr Lys Ala Lys Ala Val Ile 115
120 125 Asp Asn Phe Val Lys Lys Leu Glu Glu Asn Tyr Asn Ser Glu Cys
Gly 130 135 140 Ile Asp Thr Ala Phe Gln Pro Ser Val Gly Lys Asp Gly
Ser Thr Asp 145 150 155 160 Asn Asn Val Val Ala Gly Asp Arg Pro Leu
Ile Asp Trp Asp Gln Ile 165 170 175 Arg Glu Glu Gly Leu Lys Trp Gln
Lys Thr Lys Trp Ala Asp Leu Pro 180 185 190 Pro Ile Lys Lys Asn Phe
Tyr Lys Glu Ser Thr Ala Thr Ser Ala Met 195 200 205 Ser Lys Val Glu
Ala Asp Ser Trp Arg Lys Glu Asn Phe Asn Ile Thr 210 215 220 Trp Asp
Asp Leu Lys Asp Gly Glu Lys Arg Pro Ile Pro Asn Pro Thr 225 230 235
240 Cys Thr Phe Asp Asp Ala Phe Gln Cys Tyr Pro Glu Val Met Glu Asn
245 250 255 Ile Lys Lys Ala Gly Phe Gln Lys Pro Thr Pro Ile Gln Ser
Gln Ala 260 265 270 Trp Pro Ile Val Leu Gln Gly Ile Asp Leu Ile Gly
Val Ala Gln Thr 275 280 285 Gly Thr Gly Lys Thr Leu Cys Tyr Leu Met
Pro Gly Phe Ile His Leu 290 295 300 Val Leu Gln Pro Ser Leu Lys Gly
Gln Arg Asn Arg Pro Gly Met Leu 305 310 315 320 Val Leu Thr Pro Thr
Arg Glu Leu Ala Leu Gln Val Glu Gly Glu Cys 325 330 335 Cys Lys Tyr
Ser Tyr Lys Gly Leu Arg Ser Val Cys Val Tyr Gly Gly 340 345 350 Gly
Asn Arg Asp Glu Gln Ile Glu Glu Leu Lys Lys Gly Val Asp Ile 355 360
365 Ile Ile Ala Thr Pro Gly Arg Leu Asn Asp Leu Gln Met Ser Asn Phe
370 375 380 Val Asn Leu Lys Asn Ile Thr Tyr Leu Val Leu Asp Glu Ala
Asp Lys 385 390 395 400 Met Leu Asp Met Gly Phe Glu Pro Gln Ile Met
Lys Ile Leu Leu Asp 405 410 415 Val Arg Pro Asp Arg Gln Thr Val Met
Thr Ser Ala Thr Trp Pro His 420 425 430 Ser Val His Arg Leu Ala Gln
Ser Tyr Leu Lys Glu Pro Met Ile Val 435 440 445 Tyr Val Gly Thr Leu
Asp Leu Val Ala Val Ser Ser Val Lys Gln Asn 450 455 460 Ile Ile Val
Thr Thr Glu Glu Glu Lys Trp Ser His Met Gln Thr Phe 465 470 475 480
Leu Gln Ser Met Ser Ser Thr Asp Lys Val Ile Val Phe Val Ser Arg 485
490 495 Lys Ala Val Ala Asp His Leu Ser Ser Asp Leu Ile Leu Gly Asn
Ile 500 505 510 Ser Val Glu Ser Leu His Gly Asp Arg Glu Gln Arg Asp
Arg Glu Lys 515 520 525 Ala Leu Glu Asn Phe Lys Thr Gly Lys Val Arg
Ile Leu Ile Ala Thr 530 535 540 Asp Leu Ala Ser Arg Gly Leu Asp Val
His Asp Val Thr His Val Tyr 545 550 555 560 Asn Phe Asp Phe Pro Arg
Asn Ile Glu Glu Tyr Val His Arg Ile Gly 565 570 575 Arg Thr Gly Arg
Ala Gly Arg Thr Gly Val Ser Ile Thr Thr Leu Thr 580 585 590 Arg Asn
Asp Trp Arg Val Ala Ser Glu Leu Ile Asn Ile Leu Glu Arg 595 600 605
Ala Asn Gln Ser Ile Pro Glu Glu Leu Val Ser Met Ala Glu Arg Phe 610
615 620 Glu Ala His Gln Arg Lys Arg Glu Met Glu Arg Lys Met Glu Arg
Pro 625 630 635 640 Gln Gly Arg Pro Lys Lys Phe His 645
* * * * *
References