Leukemia associated genes

The invention describes leukemia associated genes, including fragments and biologically functional variants thereof. Also included are polypeptides and fragments thereof encoded by such genes, and antibodies relating thereto. Methods and products also are provided for diagnosing and treating conditions characterized by expression of a preproTRH, tryptase-L and/or Oct-T1 gene product.

FIELD OF THE INVENTION 
This invention relates to nucleic acid molecules and encoded polypeptides 
which are expressed preferentially in leukemia. The nucleic acid molecules 
and encoded polypeptides are useful in, inter alia, diagnostic and 
therapeutic contexts. 
BACKGROUND OF THE INVENTION 
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 specific" genes. These tumor specific 
genes are markers for the tumor phenotype. The expression of tumor 
specific genes can also be an essential event in the process of 
tumorigenesis. 
Typically, the host recognizes as foreign the tumor specific genes which 
are not expressed in normal non-tumorigenic cells. Thus, the expression of 
tumor specific genes can provoke an immune response against the tumor 
cells by the host. Tumor specific 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 
The discovery of tumor specific expression of a gene provides a means of 
identifying a cell as a tumor cell. Diagnostic compounds can be based on 
the tumor specific gene, and used to determine the presence and location 
of tumor cells. Further, when the tumor specific gene contributes to an 
aspect of the tumor phenotype (e.g., unregulated growth or metastasis), 
the tumor specific gene can be used to provide therapeutics such as 
antisense nucleic acids which can reduce or substantially eliminate 
expression of that gene, thereby reducing or substantially eliminating the 
phenotypic aspect which depends on the expression of the particular tumor 
specific gene. 
As previously noted, the polypeptide products of tumor specific 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 specific 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 specific 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. 
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 
autoimnmune 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. 
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 for further information on this family of genes. Also, 
see U.S. patent application Ser. No. 807,043, filed Dec. 12, 1991, now 
U.S. Pat. No. 5,342,774. 
In U.S. patent application Ser. No. 938,334, now 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. 
In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 and 
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-Cw16 molecules, also known as HLA-C*1601. The disclosure shows that a 
given TRAP can yield a plurality of TRAs. 
In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, and 
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. 
In U.S. patent application Ser. No. 08/032,978, now U.S. Pat. No. 
5,620,886, and 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. 
In U.S. patent application Ser. No. 079,110, now U.S. Pat. No. 5,571,711 
and entitled "Isolated Nucleic Acid Molecules Coding For BAGE Tumor 
Rejection Antigen Precursors" and Ser. No. 196,630, filed Feb. 15, 1994, 
and entitled "Isolated Peptides Which form Complexes with MHC Molecule 
HLA-C-Clone 10 and Uses Thereof" the entire disclosures of which are 
incorporated herein by reference, an unrelated tumor rejection antigen 
precursor, the so-called "BAGE" precursor, is described. TRAs are derived 
from the TRAP and also are described. They form complexes with MHC 
molecule HLA-C-Clone 10. 
In U.S. patent application Ser. No. 096,039, filed Jul. 22, 1993 and 
entitled "Isolated Nucleic Acid Molecules Coding for GAGE Tumor Rejection 
Antigen Precursors" and Ser. No. 250,162, now U.S. Pat. No. 5,610,013 and 
entitled "Method for Diagnosing a Disorder by Determining Expression of 
GAGE Tumor Rejection Antigen Precursors", the entire disclosures of which 
are incorporated herein by reference, another unrelated tumor rejection 
antigen precursor, the so-called "GAGE" precursor, is described. The GAGE 
precursor is not related to the BAGE or the MAGE family. 
In U.S. patent application Ser. No. 08/408,015, filed Mar. 21, 1995, and 
entitled "RAGE Tumor Rejection Antigen Precursors", incorporated herein by 
reference in its entirety, another TRAP is taught which is not derived 
from any of the foregoing genes. The TRAP is referred to as RAGE. In U.S. 
patent application Ser. No. 08/530,015, filed Sep. 20, 1995, and entitled 
"Isolated RAGE-1 Derived Peptides Which Complex with HLA-B7 Molecules and 
Uses Thereof", also incorporated by reference, the TRA derived form one 
member of the RAGE family of genes is taught to be presented by HLA-B7 
molecules. This disclosure shows that additional TRAPs and TRAs can be 
derived from different sources. 
In U.S. patent application Ser. no. 08/253,503, now U.S. Pat. No.5,589,334, 
and entitled "Isolated Nucleic Acid Molecule Which Codes for a Tumor 
Rejection Antigen Precursor Which is Processed to an Antigen Presented by 
HLA-B44", incorporated herein by reference in its entirety, another TRAP 
is taught which is not derived from any of the foregoing genes. The gene 
encoding the TRAP is referred to as MUM-1. A tumor rejection antigen, 
LB-33B, is described in the application. 
In U.S. patent application Ser. No. 08/373,636, filed Jan. 17, 1995, and 
entitled "Isolated Nucleic Acid Molecule Which Codes for a Tumor Rejection 
Antigen Precursor Which is Processed to Antigens Presented by HLA 
Molecules and Uses Thereof", incorporated herein by reference in its 
entirety, other TRAPs are taught which are derived from LB33 and presented 
by HLA-B13, HLA-Cw6, HLA-A28 and HLA-A24. 
In PCT publication WO96/10577, published Apr. 11, 1996, and entitled 
"Isolated Nucleic Acid Molecule Coding for a Tumor Rejection Antigen 
Precursor DAGE and Uses Thereof", incorporated herein by reference in its 
entirety, another TRAP is taught which is not derived from any of the 
foregoing genes. The TRAP was referred to as DAGE, but is now referred to 
as PRAME. A tumor rejection antigen is described in the application which 
is presented by HLA-A24. 
In U.S. patent application Ser. No. 08/487,135, filed Jun. 7, 1995, and 
entitled "Isolated Nucleic Acid Molecule, Peptides Which Form Complexes 
with MHC Molecule HLA-A2 and Uses Thereof", incorporated herein by 
reference in its entirety, another TRAP is taught which is not derived 
from any of the foregoing genes. The TRAP is referred to as NAG. Various 
TRAs derived from NAG and presented by HLA-A2 are taught in this 
application. 
In U.S. patent application Ser. No. 08/403,388, now U.S. Pat. No. 
5,587,289, and entitled "Isolated Nucleic Acid Molecules Which Are Members 
of the MAGE-Xp Family and Uses Thereof", incorporated herein by reference 
in its entirety, three TRAPs are taught which are not derived from any of 
the foregoing genes. These TRAPs are referred to as MAGE-Xp2, MAGE-Xp3 and 
MAGE-Xp4. 
The work which is presented by the papers, patents and patent applications 
described above deal, for the most part, with the MAGE family of genes, 
the BAGE gene, the GAGE gene and the RAGE family of genes. 
In U.S. patent application Ser. No. 08/672,351, filed Jun. 25, 1996, and 
entitled "Brain Glycogen Phosphorylase Cancer Antigen", incorporated 
herein by reference in its entirety, another TRAP is taught which is not 
derived from any of the foregoing genes. This TRAP is a gene which is 
expressed normally in the brain and retinal pigmented epithelium. It was 
discovered that the brain glycogen phosphorylase gene is expressed in 
melanoma cells, and encodes tumor rejection antigens and precursors 
thereof. It now has been discovered that additional genes similarly are 
expressed in a tumor associated pattern in leukemia cells. 
These three genes which are believed to encode tumor rejection antigen 
precursors are referred to generally as leukemia associated genes. These 
genes do not show homology to the MAGE family of genes, to the BAGE gene, 
the GAGE gene, the RAGE family of genes, the LB33/MUM-1 gene, the NAG 
gene, the MAGE-Xp family of genes or the brain glycogen phosphorylase 
gene. Two of the genes are known genes which were not previously known to 
be expressed in a leukemia associated manner. One of the genes is an 
unknown gene. Thus the invention relates to the genes expressed 
specifically in certain leukemia cells, tumor rejection antigen precursors 
encoded by such genes, as well as related molecules and applications of 
these various entities. 
The invention is elaborated upon further in the disclosure which follows. 
SUMMARY OF THE INVENTION 
The invention provides isolated nucleic acid molecules encoding leukemia 
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 leukemia 
associated nucleic acid molecules 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 leukemia associated gene. 
According to one aspect of the invention, methods for diagnosing a disorder 
characterized by the expression of a leukemia associated nucleic acid 
molecule or a leukemia associated polypeptide are provided. The methods 
involve contacting a biological sample isolated from a subject with an 
agent that is specific for the leukemia associated nucleic acid molecule 
or an expression product thereof. In certain embodiments, the leukemia 
associated nucleic acid molecule hybridizes under stringent conditions to 
a molecule having a nucleotide sequence selected from the group consisting 
of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. In these 
certain embodiments, the leukemia associated nucleic acid optionally codes 
for a leukemia associated polypeptide. In other embodiments, the agent is 
a binding agent which selectively binds to a leukemia 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 selected from the group consisting of SEQ ID 
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, 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 other preferred embodiments, the 
leukemia associated polypeptide comprises a polypeptide having an amino 
acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID 
NO:4, SEQ ID NO:6, SEQ ID NO:8 and fragments thereof. In particularly 
preferred embodiments, the agent which binds the leukemia associated 
polypeptide is an antibody. In the foregoing embodiments, the biological 
sample preferably is isolated from a non-fetal-brain tissue, a 
non-mastocyte tissue, or a non-fetal-testis tissue. In certain of the 
foregoing embodiments, the leukemia associated nucleic acids and 
polypeptides are fragments of the foregoing sequences. 
The recognition that peptides derived from leukemia 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 leukemia 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 leukemia associated polypeptide. The method then provides 
for determining the interaction between the agent and the tumor rejection 
antigen derived from a leukemia associated polypeptide as a determination 
of the disorder. In certain embodiments, the tumor rejection antigen 
derived from a leukemia associated polypeptide comprises the amino acid 
sequence of a fragment of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID 
NO:8. 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-fetal-brain, 
non-mastocyte or non-fetal testis tissue. In certain embodiments, the 
agent is an antibody. 
The above-described method provides diagnosis of a disorder based on the 
presence of leukemia 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 leukemia 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 leukemia associated polypeptide is a 
peptide comprising the amino acids of a fragment of SEQ ID NO:2, SEQ ID 
NO:4, SEQ ID NO:6 or SEQ ID NO:8. 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-fetal-brain, non-mastocyte or non-fetal testis tissue. In certain 
embodiments, the agent is an antibody. 
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 
leukemia associated nucleic acid or polypeptide is provided. The method 
involves administering to the subject an agent which reduces the 
expression of the leukemia associated nucleic acid or polypeptide to 
ameliorate the disorder. The agent is administered in an effective amount. 
In certain embodiments, the leukemia 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 
rejection antigen derived from a leukemia associated polypeptide encoded 
by a nucleic acid molecule selected from the group consisting of SEQ ID 
NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, sufficient to ameliorate 
the disorder. Preferably, the tumor rejection antigen derived from a 
leukemia associated polypeptide is a peptide derived from the polypeptide 
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. Another method 
involves 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 leukemia 
associated polypeptide. Preferably the complexes are formed of HLA and the 
certain leukemia associated peptides as described above. In other 
embodiments, the leukemia 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 leukemia associated nucleic acid 
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID 
NO:5, SEQ ID NO:7 and fragments thereof. 
According to another aspect of the invention, a composition is provided. 
The composition comprises an antisense nucleic acid which binds to a 
leukemia associated nucleic acid selected from the group consisting of SEQ 
ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and fragments thereof. The 
antisense nucleic acid reduces the expression of the leukemia associated 
nucleic acid. The composition also includes a pharmaceutically acceptable 
carrier. 
The invention in another aspect involves a kit for detecting the presence 
of the expression of a leukemia 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:1 and 
complements thereof, a 12-32 nucleotide contiguous segment of SEQ ID NO:3 
and complements thereof, a 12-32 nucleotide contiguous segment of SEQ ID 
NO:5, a 12-32 nucleotide contiguous segment of SEQ ID NO:7 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:1, the nucleic acid sequence of SEQ ID NO:3, the nucleic acid sequence 
of SEQ ID NO:5, the nucleic acid sequence of SEQ ID NO:7, 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:1 and 
another of the primers is a complement of another contiguous segment of 
SEQ ID NO:1. In other preferred embodiments, one of the primers is a 
contiguous segment of SEQ ID NO:3 and another of the primers is the 
complement of another contiguous segment of SEQ ID NO:3. In still other 
preferred embodiments, one of the primers is a contiguous segment of SEQ 
ID NO:5 and another of the primers is the complement of another contiguous 
segment of SEQ ID NO:5. In yet other preferred embodiments, one of the 
primers is a contiguous segment of SEQ ID NO:7 and another of the primers 
is the complement of another contiguous segment of SEQ ID NO:7. 
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 preproTRH, tryptase-L, Oct-T1 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 preproTRH, tryptase-L or Oct-T1. 
According to another aspect of the invention, the use of isolated 
preproTRH, tryptase-L, Oct-T1 or fragments thereof in the manufacture of a 
medicament is provided. Preferred fragments of the preproTRH, tryptase-L 
and Oct-T1 molecules are described above. The use of antisense nucleic 
acids which bind to a leukemia 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. 
According to another aspect of the invention, the use of isolated 
preproTRH, tryptase-L, Oct-T1 or fragments thereof, including antisense 
nucleic acids, in the manufacture of a medicament for the treatment of 
cancer is provided. 
According to still another 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 selected from 
the group consisting of SEQ ID NO:3 or SEQ ID NO:5. The isolated nucleic 
acid molecule is a leukemia associated polypeptide precursor and codes for 
a tryptase-L leukemia associated polypeptide. The invention further 
embraces nucleic acid molecules that differ from the foregoing isolated 
nucleic acid molecules in codon sequence to the degeneracy of the genetic 
code. The invention also embraces complements of the foregoing nucleic 
acids. In preferred embodiments, the isolated nucleic acid molecule 
comprises the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:5. In 
particularly preferred embodiments, the isolated nucleic acid molecule 
comprises the coding region of the foregoing nucleic acids. 
According to another aspect of the invention, an isolated nucleic acid 
molecule is provided which comprises a molecule selected from the group 
consisting of a unique fragment of nucleotides 487-1499 of SEQ ID NO:3 or 
SEQ ID NO:5 between 12 and 1012 nucleotides in length, a unique fragment 
of nucleotides 1665-1774 of SEQ ID NO:5 between 12 and 109 nucleotides in 
length and complements thereof. In preferred embodiments, the unique 
fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguous nucleotides 
of the foregoing. In another embodiment, the isolated nucleic acid 
molecule consists of between 12 and 32 contiguous nucleotides of the 
foregoing. 
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. 
According to another aspect of the invention, an isolated tryptase-L 
polypeptide is provided which is encoded by a nucleic acid molecule which 
hybridizes under stringent conditions to a molecule having the nucleic 
acid sequence of SEQ ID NO:3, the nucleic acid sequence of SEQ ID NO:5, 
nucleic acid molecules which vary from the foregoing according to the 
degeneracy of the genetic code, and complements of any of the foregoing 
nucleic acid molecules. 
According to yet another aspect of the invention, an isolated polypeptide 
is provided which comprises a unique fragment of SEQ ID NO:4 or SEQ ID 
NO:6 between 9 and 189 amino acids in length. Preferably, the unique 
fragment of the isolated polypeptide binds to a polypeptide-binding agent. 
In other preferred embodiments, the unique fragment of the isolated 
polypeptide binds to an antibody or a cytotoxic T lymphocyte. 
The invention also provides isolated polypeptides which selectively bind a 
tryptase-L 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 tryptase-L proteins of the invention). The isolated binding 
polypeptides include monoclonal antibodies. 
In connection with any of the isolated nucleic acids encoding a leukemia 
associated polypeptide as described above, especially a tumor rejection 
antigen derived from a leukemia 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. 
The invention also embraces functional variants and equivalents of all of 
the molecules described above. 
These and other objects of the invention will be described in fuirther 
detail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES 
SEQ ID NO:1 is the nucleotide sequence of the preproTRH gene. 
SEQ ID NO:2 is the amino acid sequence of the polypeptide encoded by the 
preproTRH gene. 
SEQ ID NO:3 is the nucleotide sequence of the tryptase-L gene clone 
NVB352/1. 
SEQ ID NO:4 is the amino acid sequence of the polypeptide encoded by the 
tryptase-L gene clone NVB352/1. 
SEQ ID NO:5 is the nucleotide sequence of the tryptase-L gene clone 
NVB352/3. 
SEQ ID NO:6 is the amino acid sequence of the polypeptide encoded by the 
tryptase-L gene clone NVB352/3. 
SEQ ID NO:7 is the nucleotide sequence of the SIAX DP2-64 (Oct-T1) gene. 
SEQ ID NO:8 is the amino acid sequence of the polypeptide encoded by the 
SIAX DP2-64 (Oct-T1) gene. 
SEQ ID NO:9 is a sense primer for specific PCR amplification of preproTRH. 
SEQ ID NO:10 is an antisense primer for specific PCR amplification of 
preproTRH. 
SEQ ID NO:11 is a sense primer for specific PCR amplification of 
tryptase-L. 
SEQ ID NO:12 is an antisense primer for specific PCR amplification of 
tryptase-L. 
SEQ ID NO:13 is a sense primer for specific PCR amplification of SIAX 
DP2-64 (Oct-T1). 
SEQ ID NO:14 is an antisense primer for specific PCR amplification of SIAX 
DP2-64 (Oct-T1). 
SEQ ID NO:15 is a sense primer for specific PCR amplification of 
.beta.-actin. 
SEQ ID NO:16 is an antisense primer for specific PCR amplification of 
.beta.-actin. 
DETAILED DESCRIPTION OF THE INVENTION 
The examples which follow show the isolation of nucleic acid molecules 
which code for polypeptides and are expressed preferentially in malignant 
hemopathies, i.e. which are leukemia associated genes. These isolated 
nucleic acid molecules include nucleic acid molecules which encode 
preproTRH, tryptase-L and Oct-T1. In particular, the tryptase-L nucleic 
acids are different from previously disclosed tryptase coding sequences 
described supra. Hence, one aspect of the invention is an isolated nucleic 
acid molecule which includes all or a unique portion of the nucleotide 
sequence set forth in SEQ ID NO:3 or SEQ ID NO:5. This sequence, and the 
other leukemia associated gene sequences do 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. 
The invention thus involves in one aspect preproTRH, tryptase-L and Oct-T1 
nucleic acids, encoded polypeptides, functional modifications and variants 
of the foregoing, useful fragments of the foregoing, as well as 
therapeutics and diagnostics related thereto. 
According to one aspect of the invention, methods for diagnosing a disorder 
that is characterized by expression of a leukemia associated nucleic acid 
or polypeptide are provided. The methods involve contacting a biological 
sample isolated from a subject with an agent specific for the leukemia 
associated nucleic acid or polypeptide to detect the presence of the 
leukemia associated nucleic acid or polypeptide in the biological sample. 
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 leukemia 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. 
The biological sample can be located in vivo or in vitro. For example, the 
biological sample can be a hematopoietic tissue in vivo and the agent 
specific for the leukemia 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 hematopoietic tissue that express the 
leukemia associated gene products). Alternatively, the biological sample 
can be located in vitro (e.g., a blood sample, bone marrow biopsy, tissue 
extract). In a particularly preferred embodiment, the biological sample 
can be a cell-containing sample, more preferably a sample containing 
hematopoietic cells. 
Also a part of the invention are those nucleic acid sequences which also 
code for a preproTRH, tryptase-L or Oct-T1 polypeptide and which hybridize 
under stringent conditions to a nucleic acid molecule consisting of the 
nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or 
SEQ ID NO:7, respectively. Such nucleic acids are termed leukemia 
associated polypeptide precursors, and may be DNA, RNA, or composed of 
mixed deoxyribonucleotides and ribonucleotides. The leukemia associated 
polypeptide precursors can also incorporate synthetic non-natural 
nucleotides. 
The invention thus encompasses other leukemia associated nucleic acids, 
some of which previously were identified in normal tissues. A leukemia 
associated nucleic acid or polypeptide is a nucleic acid or polypeptide 
expressed preferentially in leukemias and solid forms of leukemia cell 
malignancies, such as lymphomas. Various methods for determining the 
expression of a nucleic acid and/or a polypeptide in normal and leukemia 
cells are known to those of skill in the art and are described further 
below. As used herein, leukemia associated polypeptides include proteins, 
protein fragments, and peptides. In particular, leukemia associated 
polypeptides include TRAPs and TRAs. 
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.2 PO.sub.4 (pH 7), 0.5% SDS, 2 mM 
EDTA). SSC is 0.15M sodium chloride/0.15M 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. 
There are other conditions, reagents, and so forth which can be used, which 
result in the same degree of stringency (see, e.g. Molecular Cloning: A 
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring 
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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 tryptase-L 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. 
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 leukemia 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. Watson-Crick complements of the 
foregoing nucleic acids also are embraced by the invention. 
The nucleic acids disclosed herein are useful for determining the 
expression of preproTRH, tryptase-L and Oct-T1 genes according to standard 
hybridization procedures. The nucleic acids also can be used to express 
leukemia associated polypeptides in vitro or in vivo. 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. 
In screening for related genes, such as tryptase-L family members, a 
Southern blot may be performed using the foregoing conditions, together 
with a radioactive probe. 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. 
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. 
The invention also provides isolated unique fragments of SEQ ID NO:1, SEQ 
ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or complements thereof. A unique 
fragment is one that is a `signature` for the larger nucleic acid. It is, 
for example, long enough to assure that its precise sequence is not found 
in molecules outside of the tryptase-L family as defined herein. Unique 
fragments can be used as probes in Southern blot assays to identify family 
members 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 
nucleotides or more are preferred for certain uses such as Southern 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 for generating immunoassay components. Unique fragments 
further can be used as antisense molecules to inhibit the expression of 
the leukemia associated nucleic acids and encoded proteins of the 
invention, particularly for therapeutic purposes as described in greater 
detail below. 
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 SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and 
complements thereof will require longer segments to be unique while others 
will require only short segments, typically between 12 and 32 nucleotides 
(e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 
29, 30, 31 and 32 nucleotides long). 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 non-family members. A comparison of the sequence of the 
fragment to those on known data bases typically is all that is necessary, 
although in vitro confirmatory hybridization and sequencing analysis may 
be performed. 
For any pair of PCR primers constructed and arranged to selectively 
amplify, for example, a tryptase-L nucleic acid, a tryptase-L specific 
primer may be used. Such a primer is a contiguous stretch of tryptase-L 
which hybridizes selectively to tryptase-L and not other tryptase nucleic 
acids. Such a specific primer would fully hybridize to a contiguous 
stretch of nucleotides only in tryptase-L, but would hybridize at most 
only in part to tryptase genes that do not share the nucleotides to which 
the tryptase-L specific primer binds. For efficient PCR priming and 
tryptase-L identification, the tryptase-L specific primer should be 
constructed and arranged so it does not hybridize efficiently at its 3' 
end to tryptase genes other than tryptase-L. Preferably the area of 
non-identity is at least one to four nucleotides in length and forms the 
3' end of the tryptase-L 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. Primers for selective amplification of 
tryptase-L preferably are selected from portions of SEQ ID NO:3 which 
share lesser homology with tryptases other than tryptase-L. In such cases, 
selective amplification of tryptase-L can be achieved with one tryptase-L 
specific primer and one primer which hybridizes to tryptases generically. 
Preferably, however, both primers are tryptase-L specific primers are 
described hereinabove. Exemplary primers include SEQ ID NO:11 and SEQ ID 
NO:12, which are derived from SEQ ID NO:3, respectively. 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. 
Similarly, one of ordinary skill in the art can select primers from the 
nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:7 for selective 
amplification of preproTRH or Oct-T1 mRNA sequences, respectively. For 
example, exemplary primers specific for preproTRH include SEQ ID NO:9 and 
SEQ ID NO:10, which are derived from SEQ ID NO:1. Exemplary primers 
specific for Oct-T1 include SEQ ID NO:13 and SEQ ID NO:14, which are 
derived from SEQ ID NO:7. For amplification of tryptase-L, primers can be 
designed which are specific for each clone (i.e., which amplify a portion 
of either SEQ ID NO:3 or SEQ ID NO:5), or which amplify both clones (i.e., 
SEQ ID NO:3 and SEQ ID NO:5). As demonstrated in the Examples below, 
primer pairs specific to preproTRH, tryptase-L or Oct-T1 can be used to 
distinguish the expression of the genes in cells and tissues. 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 primers above. One 
of ordinary skill in the art can determine with no more than routine 
experimentation the preferred primers for selective amplification of 
particular leukemia associated 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. 
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. 
The invention also provides isolated polypeptides which include unique 
fragments of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. Such 
polypeptides are useful, for example, alone or as fusion proteins to 
generate antibodies, as a components of an immunoassay, or for determining 
the binding specificity of HLA molecules and/or CTL clones for preproTRH, 
tryptase-L and Oct-T1 proteins. 
A unique fragment of a tryptase-L protein, for example, generally has the 
features and characteristics of unique fragments as discussed above in 
connection with nucleic acids. As will be recognized by those skilled in 
the art, the size of the unique fragment will depend upon factors such as 
whether the fragment constitutes a portion of a conserved protein domain. 
Thus, some regions of SEQ ID NO:4 (or SEQ ID NO:6), 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). 
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 unique fragment of a polypeptide 
include interaction with antibodies, interaction with other polypeptides 
or fragments thereof, selective binding of nucleic acids, and enzymatic 
activity. A tumor rejection antigen is an example of a unique fragment of 
a tumor specific polypeptide which retains the functional capability of 
HLA binding and interaction with cytotoxic 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 cytotoxic T lymphocyte 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). 
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 unique 
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. 
The skilled artisan will also realize that conservative amino acid 
substitutions may be made in tryptase-L polypeptides to provide 
functionally active homologs of the foregoing polypeptides, i.e, the 
homologs retain the functional capabilities of the preproTRH, tryptase-L 
or Oct-T1 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. 
Functionally equivalent variants of preproTRH, tryptase-L and/or Oct-T1 
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, N.Y., 1989, or 
Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John 
Wiley & Sons, Inc., New York. For example, exemplary functionally 
equivalent variants of the tryptase-L polypeptides include conservative 
amino acid substitutions of SEQ ID NO:4 or and SEQ ID NO:6. Conservative 
amino-acid substitutions in the amino acid sequence of tryptase-L 
polypeptides to produce functionally equivalent variants of tryptase-L 
polypeptides typically are made by alteration of the nucleic acid encoding 
tryptase-L (SEQ ID NO:3, SEQ ID NO:5). 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 tryptase-L polypeptide. Where amino acid substitutions are 
made to a small unique fragment of a tryptase-L polypeptide, such as a 9 
amino acid peptide, the substitutions can be made by directly synthesizing 
the peptide. The activity of functionally equivalent fragments of 
tryptase-L polypeptides can be tested by cloning the gene encoding the 
altered tryptase-L polypeptide into a bacterial or mammalian expression 
vector, introducing the vector into an appropriate host cell, expressing 
the altered tryptase-L polypeptide, and testing for a functional 
capability of the tryptase-L polypeptides as disclosed herein. 
Functionally equivalent variants of the preproTRH and Oct-T1 polypeptides 
can be prepared in a like manner. 
As mentioned above, the invention embraces antisense oligonucleotides that 
selectively bind to a leukemia associated gene nucleic acid molecule, 
including those encoding a preproTRH protein, a tryptase-L protein or a 
Oct-T1 protein, to decrease transcription and/or translation of leukemia 
associated genes. This is desirable in virtually any medical condition 
wherein a reduction in leukemia associated gene product expression is 
desirable, including to reduce any aspect of a malignant hemopathy cell 
phenotype attributable to leukemia associated gene expression, such as 
expression of preproTRH, tryptase-L and/or Oct-T1. Antisense molecules, in 
this manner, can be used to slow down or arrest such aspects of a 
malignant leukemia cell phenotype as found in, inter alia, leukemia and 
solid forms such as lymphoma. 
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:1, 
SEQ ID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7, or upon allelic or homologous 
genomic and/or DNA 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 
NOs:1, 3, 5 and 7 disclose cDNA sequences, one of ordinary skill in the 
art may easily derive the genomic DNA corresponding to the cDNAs of SEQ ID 
NOs:1, 3, 5 and 7. Thus, the present invention also provides for antisense 
oligonucleotides which are complementary to the genomic DNA corresponding 
to SEQ ID NOs:1, 3, 5 and 7. Similarly, antisense to allelic or homologous 
DNAs and genomic DNAs are enabled without undue experimentation. 
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. 
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. 
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 intemucleoside linkages are 
phosphorothioates, alkylphosphonates, phosphorodithioates, is phosphate 
esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, 
phosphate triesters, acetamidates, peptides, and carboxymethyl esters. 
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 leukemia associated 
proteins, together with pharmaceutically acceptable carriers. 
It will also be recognized from the examples that the invention embraces 
the use of the preproTRH, tryptase-L and Oct-T1 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. In instances in which a human HLA class I molecule presents 
tumor rejection antigens derived from the preproTRH, tryptase-L and Oct-T1 
genes, 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 genes 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 
preproTRH, tryptase-L and/or Oct-T1 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 preproTRH, 
tryptase-L and/or Oct-T1 TRA. Further, cell-free transcription systems may 
be used in lieu of cells. 
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 or alkaline phosphatase), and genes 
which visibly affect the phenotype of transformed or transfected cells, 
hosts, colonies or plaques. 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. 
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. 
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 TAT 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. 
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, N.Y., 
1989. Cells are genetically engineered by the introduction into the cells 
of heterologous DNA (RNA) encoding the preproTRH, tryptase-L or Oct-T1 
tumor specific polypeptide or fragment or variant thereof. That 
heterologous DNA (RNA) is placed under operable control of transcriptional 
elements to permit the expression of the heterologous DNA in the host 
cell. 
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.alpha., 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 Warnier et al., in 
intradermal injection in mice for immunization against P1A (Int. J. 
Cancer. 67:303-310, 1996). 
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. 
The invention also involves agents which bind to leukemia associated 
polypeptides including preproTRH, tryptase-L and Oct-T1, and in certain 
embodiments preferably to unique fragments of the preproTRH, tryptase-L 
and Oct-T1 polypeptides. Such binding partners can be used in screening 
assays to detect the presence or absence of a preproTRH, tryptase-L or 
Oct-T1 polypeptide and in purification protocols to isolate preproTRH, 
tryptase-L or Oct-T1 polypeptides. Likewise, such binding partners can be 
used to selectively target drugs, toxins or other molecules to leukemia 
cells which present preproTRH, tryptase-L or Oct-T1 leukemia associated 
polypeptides. In this manner, cells present in solid or non-solid tumors 
which express preproTRH, tryptase-L or Oct-T1 leukemia associated 
polypeptides can be treated with cytotoxic compounds. 
The invention, therefore, involves antibodies or fragments of antibodies 
having the ability to selectively bind to preproTRH, tryptase-L or Oct-T1 
leukemia associated polypeptides, and preferably to unique fragments 
thereof. Antibodies include polyclonal and monoclonal antibodies, prepared 
according to conventional methodology. 
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. 
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 Modern 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. 
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 (FR1 
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. 
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. 
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 leukemia associated polypeptides including preproTRH, 
tryptase-L or Oct-T1. 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. 
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 preproTRH, tryptase-L or Oct-T1 
leukemia associated polypeptide. This process can be repeated through 
several cycles of reselection of phage that bind to a preproTRH, 
tryptase-L or Oct-T1 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 preproTRH, tryptase-L or 
Oct-T1 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 leukemia 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 leukemia 
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 
leukemia associated genes such as those leukemia cells which present 
preproTRH, tryptase-L or Oct-T1 polypeptides on the cell surface. Such 
binding agent molecules can also be prepared to bind complexes of an 
preproTRH, tryptase-L or Oct-T1 polypeptide and an HLA molecule by 
selecting the binding agent using such complexes. Drug molecules that 
would disable or destroy leukemia cells which express such complexes or 
preproTRH, tryptase-L or Oct-T1 polypeptides are known to those skilled in 
the art and are commercially available. For example, the immunotoxin art 
provides examples of toxins which are effective when delivered to a cell 
by an antibody or fragment thereof. Examples of toxins include 
ribosome-damaging toxins derived from plants or bacterial such as ricin, 
abrin, saporin, Pseudomonas endotoxin, diphtheria toxin, A chain toxins, 
blocked ricin, etc. 
The skilled artisan can determine which HLA molecule binds to tumor 
rejection antigens derived from preproTRH, tryptase-L and/or Oct-T1 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 leukemia associated genes such as preproTRH, tryptase-L 
and/or Oct-T1 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 U.S. 
patent application Ser. No. 08/530,569. 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 II 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. 
The invention as described herein has a number of uses, some of which are 
described herein. First, the invention permits the artisan to diagnose a 
disorder characterized by expression of the TRAP. These methods involve 
determining expression of the TRAP gene, and/or TRAs derived therefrom, 
such as a TRA presented by HLA-A2, HLA-A26, HLA-B7, etc. 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, 
assaying with binding partners for complexes of TRA and HLA, such as 
antibodies, is especially preferred. An alternate method for determination 
is a TNF release assay, of the type described supra. 
The isolation of the TRAP gene also makes it possible to isolate the TRAP 
molecule tself, especially TRAP molecules containing the acmcnino acid 
sequences coded for by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID 
NO:8. Other TRAPs or TRAs encoded by leukemia associated genes and 
recognized by other CTL clones and/or presented by other HLA molecules may 
be isolated by the procedures detailed herein. (There are numerous HLA 
molecules known to those skilled in the art, including but not limited to, 
those encoded by HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G genes.) A 
variety of methodologies well-known to the skilled practitioner can be 
utilized to obtain isolated TRAP molecules. The protein may be purified 
from cells which naturally produce the protein. Alternatively, an 
expression vector may be introduced into cells to cause production of the 
protein. In another method, mRNA transcripts may be microinjected or 
otherwise introduced into cells to cause production of the encoded 
protein. Translation of mRNA in cell-free extracts such as the 
reticulocyte lysate system also may be used to produce protein. Peptides 
comprising TRAs of the invention may also be synthesized in vitro. Those 
skilled in the art also can readily follow known methods for isolating 
proteins in order to obtain isolated TRAPs and/or TRAs derived therefrom. 
These include, but are not limited to, immunochromotography, HPLC, 
size-exclusion chromatography, ion-exchange chromatography and 
immune-affinity chromatography. 
These isolated 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 
leukemia 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). 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, leukemias 
and lymphomas in particular. 
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. 
Therapeutic approaches based upon the disclosure are premised on a response 
by a subject's immune system, leading to lysis of TRA presenting cells, 
such as HLA-B7 cells. One such approach is the administration of 
autologous CTLs specific to the 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. 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. Other CTLs specific to preproTRH, tryptase-L and/or Oct-T1 
may be isolated and administered by similar methods. 
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. 
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 leukemia 
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 leukemia associated gene derived TRA is being presented, 
and the subject is an appropriate candidate for the therapeutic approaches 
set forth supra. 
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 preproTRH, tryptase-L or Oct-T1 TRA may be 
operably linked to promoter and enhancer sequences which direct expression 
of the preproTRH, tryptase-L or Oct-T1 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 preproTRH, tryptase-L or 
Oct-T1 TRAs. Nucleic acids encoding a preproTRH, tryptase-L or Oct-T1 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. 
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 preproTRH, tryptase-L and/or Oct-T1 
encoded TRAP, and/or TRAs derived therefrom. Initial doses can be followed 
by booster doses, following immunization protocols standard in the art. 
As part of the immunization protocols, substances which potentiate the 
immune response may be administered with nucleic acid or peptide 
components of a cancer vaccine. Such immune response potentiating compound 
may be classified as either adjuvants or cytokines. 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 include MPL (SmithKline Beecham), a 
congener obtained after purification and acid hydrolysis of Salmonella 
Minnesota Re 595 lipopolysaccharide, QS21 (SmithKline Beecham), a pure 
QA-21 saponin purified from Quillja saponaria extract, and various 
water-in-oil emulsions prepared from biodegradable oils such as squalene 
and/or tocopherol. Cytokines are also useful in vaccination protocols as a 
result of lymphocyte stimulatory properties. Many 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 (Science 268: 1432-1434, 1995). 
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. 
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. 
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. 
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. 
The invention also contemplates gene therapy. The procedure for performing 
ex vivo gene therapy 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 which 
contains a defective copy of the gene, 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 gene therapy using vectors such as 
adenovirus also is contemplated according to the invention. 
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. This can be monitored by 
routine methods or can be monitored according to diagnostic methods of the 
invention discussed herein. 
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. 
EXAMPLES 
Example 1 
Representational Difference Analysis--Subtractive cDNA hybridizations 
between leukemia and normal leukoccytes. 
1. Procedure 
Tester population: PBL from a female patient (SIAX, LB-1079) with newly 
diagnosed acute myelogenous leukemia (FAB-M2 subtype), with a chromosomal 
rearrangement t(8;21)(q22;q22) detected by cytogenetic analysis. The PBL 
were collected by leukapheresis before starting chemotherapy 
(approximately 70% of the PBL were leukemia blast cells), purified on a 
Lymphoprep (Ficol1) gradient, washed and frozen in culture medium and DMSO 
at -80.degree. C. For RNA preparation, 6.times.10.sup.8 cells were thawed, 
washed and centrifuged as a dry cell pellet, containing 4.6.times.10.sup.8 
cells. 
Driver population: PBL from a normal person were collected by 
leukapheresis, they were purified on a Lymphoprep (Ficol1) gradient, 
washed and frozen at -80.degree. C. as a dry cell pellet after 
centrifugation, containing 5.times.10.sup.8 cells. 
Total RNA preparation: The guanidinium isothiocyanate/cesium chloride 
procedure was used L. G. Davis, M. D. Dibner, J. F. Battey, Basic Methods 
in Molecular Biology, Elsevier, N.Y., 1986, p. 130-135!. 390 .mu.g and 50 
.mu.g were obtained from tester and driver populations respectively. 
mRNA purification: Oligo-dT columns (Pharmacia mRNA Purification Kit, 
Pharmacia, Uppsala, Sweden) were used according to the manufacturer's 
protocol. 
cDNA preparation: cDNA was prepared according to the manufacturer's 
protocol (Amersham, cDNA Synthesis Module). 6 .mu.g and 2 .mu.g were 
obtained from tester and driver populations respectively. 
Difference analysis: The cDNA Difference Analysis Protocol of Hubank and 
Schatz (Nucleic Acids Res. 22: 5640-5648, 1994) was followed for the 
preparation of tester representation (TR), driver representation (DR), 
difference product 1 and difference product 2. Some particularities 
concerning the preparation of tester representation must be mentioned: 1) 
only 600 ng (and not 1.2 .mu.g have been used in the initial ligation of 
adaptors; 2) the PCR reaction has been divided in 40 aliquots. Some 
particularities concerning the preparation of difference product 1 (DP1) 
must be mentioned: only 0.2 .mu.g TR and 20 .mu.g DR (and not 0.4 and 40 
.mu.g) were used in 5 .mu.l hybridization buffer. Some particularities 
concerning the preparation of difference product 2 (DP2) must be 
mentioned: only 2.5 ng TR and 20 .mu.g DR (and not 50 ng and 40 .mu.g) 
were used in 5 .mu.l hybridization buffer. Finally, no third difference 
product could be obtained. 
Cloning of the tester representation and difference products 1 and 2: The 
cDNAs of the tester representation and difference products 1 and 2 
obtained after digestion with DpnII were cloned by common ligation into 
the BamHI cloning site of vector pTZ18R, digested with BamHI (DpnII and 
BamHI cohesive ends are mutually compatible) and dephosphorylated with 
Calf Intestinal Phosphatase. The ligation products were used to transform 
competent Top10F' bacteria by electroporation. Transformed bacteria were 
selected on agar plates with ampicillin, their plasmid DNA was purified by 
miniprep DNA extraction (Qiaprep, Qiagen), and analyzed by BamHI digestion 
and DNA sequencing. 
2. Results 
Sequencing of 16 clones with DP2 products: Some clones contained more than 
1 insert. Note also that 1 single mRNA can provide more than one amplified 
DpnII restriction fragment. Because two thirds of the individual inserts 
were derived from the myeloperoxidase (MPO) mRNA, more bacterial colonies 
were isolated after hybridization with a myeloperoxydase-specific 
oligonucleotide probe. 48 MPO-negative colonies were selected and grown 
with ampicillin, their plasmid DNA was purified by miniprep DNA extraction 
(Qiaprep, Qiagen), and analysed by DNA sequencing. 
Conclusions: The majority of cDNA fragments amplified by the RDA method, 
using leukemia cells as tester population, and normal PBL as driver 
population, derived from the myeloperoxidase mRNA. The MPO gene is highly 
expressed in normal and leukemic myeloid progenitor cells, but not in 
differentiated white blood cells. This is the reason why derived cDNA 
fragments are amplified by RDA. This also explains the presence of CD34 
which was also amplified by RDA when normal PBL are used as the driver 
population. The presence of cDNA fragments derived from highly expressed 
ubiquitous RNAs (28S and 40S ribosomal RNA genes) probably results from a 
residual background after two cycles of differential hybridization. Some 
cDNA fragments are derived from known genes. 
For the last two categories, we have designed specific PCR primer pairs, 
derived from the sequence obtained from the cloned fragments. RT-PCR was 
performed on a few leukemia, normal PBL and normal bone marrow samples. 
The genes expressed only in leukemia samples were further tested for their 
expression on a larger panel of normal and leukemic tissues. Only three 
genes appear to show a leukemia-specific expression pattern: 
The prepro TRH gene. 
The gene encoding an mRNA with strong (but not complete) homology with the 
five known tryptase genes. This gene will be further referred to as 
tryptase-L (L for leukemia). 
An apparently new gene, further referred to as SIAX DP2-64. 
The cDNAs of these three genes have been cloned from a cDNA library 
obtained from the same leukemia (SIAX, LB-1079), and sequenced. We have 
tested their expression by RT-PCR on normal tissue, leukemia and solid 
tumor samples. These data are further detailed in the following pages. 
Example 2 
Pre-pro-thyrotropin-releasing hormone gene (preproTRH) 
Cloning the cDNA: The cDNA library NVB32 was prepared as follows: Total RNA 
was extracted from the thawed leukemic PBL obtained from patient SIAX, 
LB-1079 (see tester population, RDA protocol, Example 1). Poly-A RNA was 
purified on oligo-dT columns (Pharmacia mRNA Purification Kit), according 
to the manufacturer's protocol. cDNA was prepared with Superscript kit 
(Gibco-BRL), according to the manufacturer's protocol, with random 
primers. The cDNA fragments were ligated to the EcoRI adaptors, 
fractionated on a chromatography column (4 fractions: A, B, C, and D were 
obtained) and ligated into the EcoRI digested and dephosphorylated 
pcDSRalpha vector. The ligation products were used to transform Top10F' 
competent bacteria by electroporation. Transformed bacteria colonies were 
obtained after selective growth with ampicillin, pooled with respect to 
fraction, and kept frozen at -80.degree. C. Fraction B appeared to have 
the largest inserts. 
The B fraction of the NVB329 cDNA library was screened for preproTRH clones 
by hybridizing 12,000 colonies with a .sup.32 P-labelled PCR probe 
amplified from SIAX cDNA. Sixteen colonies were found to be positive, with 
two of them containing a plasmid with a .about.1.6 kb insert, 
corresponding to the predicted mRNA size. Only one of both recombinant 
plasmids (clone CHM327-3A/8) had its insert oriented in the right 
direction. 
2. Sequencing: Clone CHM327-3A/8 has been fully sequenced (SEQ ID NO:1; see 
FIG. 1), using the Delta-Taq Sequencing Kit (Amersham). Its sequence is 
completely identical to the published sequence of preproTRH, except for 
the presence of an additional guanosine in the leukemia cDNA, in the 3' 
untranslated region (the protein sequence is not affected by this 
difference). 
3. Expression of the gene: Expression of the preproTRH gene in normal, 
leukemia and solid tumor tissues was tested by RT-PCR, as detailed in the 
protocol herein. 
a. expression in normal tissues: A positive signal was found in a fetal 
brain sample. Absence of PCR amplification was found in the following 
samples: adult brain, colon, liver, ovary, skin, placenta, lung, kidney, 
testis, endometrium, bladder, normal peripheral blood leukocytes, normal 
bone marrow. These results are similar to the expression pattern found in 
the literature, where the preproTRH gene is found expressed in 
hypothalamus and other parts of the central nervous system. 
b. expression in solid tumors: The following tumor tissues were tested, and 
were found to be negative for the expression of the preproTRH gene: 
malignant melanoma, breast cancer, laryngeal carcinoma, lung NSCLC, 
bladder carcinoma, stomach cancer, lung SCLC, testicle tumor, uterine 
carcinoma, renal carcinoma, colon carcinoma, tongue cancer, esophageal 
cancer, ovarian cancer, sarcoma, skin carcinoma. Note that immunoreactive 
TRH has been detected in human tumors, derived from the neural crest 
(Wilber, J. F., Clin. Endocr. 59: 3, 1984). 
c. Expression in malignant hemopathies: the summary 
acute myeloid leukemias: 11 positive samples (49 tested) 
chronic myeloid leukemias: no positive sample (5 tested) 
acute lymphoid leukemias: 4 positive samples (15 tested) 
chronic lymphoid leukemias: no positive sample (2 tested) 
multiple myeloma: no positive sample (1 tested) 
Thus, the expression of the preproTRH gene is found in 23% of acute 
leukemia samples. 
The results can also be presented in relation with the most frequent 
chromosomal abnormalities found in the acute leukemias: 
t(9;22)(q34;q11): 1 positive sample (3 tested) 
t(8;21)(q22;q22): 11 positive samples (12 tested) 
t(3;21)(q26;q22): 1 positive sample (2 tested) 
t(12;21)(p13;q22): 2 positive samples (2 tested) 
Inv(16)(p13;q22): 2 positive samples (8 tested) 
t(15;17)(q22;q21): no positive samples (4 tested) 
11q23 rearrangement: no positive samples (3 tested) 
trisomy 8: no positive samples (4 tested) 
del 5/5q or del 7/7q: 3 positive samples (8 tested) 
There is a clear correlation between the preproTRH gene expression and the 
acute leukemias with rearrangement of the AML1 gene, located on 21q22, and 
encoding the AMLcl transcription factor. 
4. Conclusion: The preproTRH gene is expressed in human acute leukemia 
cells. This is a new concept. The expression of the gene is related to 
chromosomal rearrangements involving the AML1 gene, which are involved in 
leukemogenesis. 
This gene is expressed in normal tissues located in the central nervous 
system. However, the preproTRH protein is processed through specialized 
enzymatic pathways in neuron hypothalamic cells to produce the modified 
tripeptide neurohormone TRH. PreproTRH may be processed differently in 
leukemic cells. It could accumulate in cell compartments where peptides 
are efficiently processed and presented to HLA class I molecules. In this 
case, leukemic cells would carry strong antigenic peptides, while neurons 
would not. Moreover, cells from the CNS are protected against the 
cell-mediated immunune system. 
The preproTRH gene is expressed in leukemic cells, but not in normal bone 
marrow, nor in normal PBL. Therefore, its specific and sensitive detection 
by RT-PCR or other methods is potentially useful as a leukemia-specific 
tumor marker, for the detection of minimal residual disease, or for the 
quantitative evaluation of response to treatment after induction 
chemotherapy. 
Example 3 
Tryptase-L 
1. Cloning the cDNA: The cDNA library NVB329 was prepared as detailed in 
Example 2 above. The B fraction of the NVB329 cDNA library was screened 
for tryptase-L clones by hybridizing 40,000 colonies with a .sup.33 
P-labelled oligonucleotide probe derived from the sequence of the 
amplified fragment SIAX DP2-04. Three colonies were found to be positive, 
and each contained a plasmid (NVB352/1, 2, 3) with a .+-.2.2-2.5 kb insert 
oriented in the right direction. 
2. Sequencing: Two of the three independent inserts NVB352/1 and NVB352/3 
have been sequenced and the partial sequence of clone NVB352/2 has been 
determined (NVB352/1=SEQ ID NO:3; NVB352/3=SEQ ID NO:5; see FIGS. 2 and 
3), using the Delta-Taq Sequencing Kit (Amersham). The three cDNAs come 
from the same primary RNA transcript, whose sequence has a strong homology 
with the sequence of the five published tryptase mRNAs (tryptases alpha, 
beta, I, II, III). The three cDNA clones contain one or two segments that 
are not present in the tryptase mRNA sequence, and that contain consensus 
nucleotides similar to those found in the beginning and at the end of 
introns. The genomic DNAs of the five known tryptase genes contain introns 
at the same locations. Therefore, we consider that these additional 
sequences in the 3 cDNA clones are unspliced introns. The deduced protein 
sequence, if we exclude the unspliced intron sequences, is very similar to 
the tryptase proteins, and the consensus amino acids for the serine 
protease activity are conserved. Note that the protein sequence is 
modified by the unspliced introns in the mRNA. 
3. Expression of the gene: Expression of the tryptase-L gene in normal, 
leukemia and solid tumor tissues was tested by RT-PCR, as detailed in the 
protocol herewith. 
a. Expression in normal tissues: No positive signal was found in the 
following normal tissue samples: fetal and adult brain, colon, liver, 
ovary, skin, placenta, lung, kidney, testis, endometrium, bladder, normal 
peripheral blood leukocytes, normal bone marrow. Note that tryptases are 
genes specifically expressed in mastocytes. We have not yet tested 
tryptase-L expression in normal mastocytes by RT-PCR. 
b. Expression in solid tumors: The following tumor tissues were tested, and 
were found to be negative for the expression of the tryptase-L gene: 
malignant melanoma, breast cancer, laryngeal carcinoma, lung NSCLC, 
bladder carcinoma, stomach cancer, lung SCLC, testicle tumor, uterine 
carcinoma, renal carcinoma, colon carcinoma, tongue cancer, esophageal 
cancer, varian cancer, sarcoma, skin carcinoma. 
c. Expression in malignant hemopathies: Thirteen samples have been tested 
to date (detailed data are enclosed herewith). Six are positive for the 
expression of tryptase-L, all of them are AMLs. Due to the small sample 
size, we cannot draw any statistical conclusions, but it is noteworthy 
that three of the positive samples are AMLs with a t(8;21) translocation. 
4. Conclusion: The tryptase-L gene is expressed in human acute myeloid 
leukemia cells. Its expression has not been found in the normal tissues 
tested, but it is possible that normal mastocytes express the gene. The 
expression of the gene is possibly related to the t(8;21) chromosomal 
rearrangements. 
The tryptase-L gene is expressed in leukemic cells, but not in normal bone 
marrow, nor in normal PBL. Therefore, its specific and sensitive detection 
by RT-PCR and other methods is useful as a leukemia-specific tumor marker, 
for the detection of minimal residual disease, or for the quantitative 
evaluation of response to treatment after induction chemotherapy. 
Example 4 
SIAX DP2-64/Oct-T1 
1. Cloning the eDNA: The cDNA library NVB329 was prepared as detailed in 
Example 2. The B fraction of the NVB329 cDNA library was screened for SIAX 
DP2-64 clones by hybridizing 40,000 colonies with a .sup.32 P-labelled PCR 
probe amplified from SIAX cDNA. One colony was found to be positive (clone 
CHM329/2-15). The same library was rescreened for SIAX DP2-64 clones by 
hybridizing 40,000 colonies with a .sup.32 P-labelled PCR probe amplified 
from SIAX cDNA. 11 colonies were found to be positive (clones CHM363/3, 5, 
8). 
2. Sequencing: Clone CHM329/2-15 has been fully sequenced (SEQ ID NO:7; see 
FIGS. 4-6), using the Delta-Taq Sequencing Kit (Amersham). Its 3' half has 
no homology with known genes, while its 5' is completely identical to the 
3' end of the Oct-T1 gene, a member of the POU family of transcription 
factors. (Bhargava A. K. et al., Differential expression of 4 members of 
the POU family of proteins in activated and PMA-treated Jurkat T cells. 
Proc.Natl.Acad.Sci. 90:10260-10264, November 1993). 
Clones CHM363/3, CHM363/5 and CHM363/8 were sequenced in their 5' 
extremities, each of which were completely homologous to the Oct-T1 mRNA 
sequence (see FIG. 4). Clone CHM363/5 has been almost entirely sequenced 
(see FIG. 5), and is almost completely identical to the Oct-T1 sequence, 
with minor differences in the 3' untranslated region. Therefore, we can 
conclude that our SIAX DP2-64 is identical to the previously identified 
Oct-T1 mRNA. It appears that the published sequence lacks the 3' 
extremity. We have cloned and sequenced the missing 600 base pairs 
together with the poly-A tail. 
3. Expression of the gene: Expression of the Oct-T1 gene in normal, 
leukemia and solid tumor tissues was tested by RT-PCR, as detailed in the 
protocol herewith. 
a. Expression in normal tissue: No positive signal was found in the 
following samples: adult brain, colon, liver, ovary, skin, placenta, lung, 
kidney, endometrium, bladder, normal peripheral blood leukocytes, normal 
bone marrow. A positive signal was detected in all the testis samples 
tested, with the exception of a fetal testis sample. 
b. Expression in solid tumors: The following tumor tissues were tested, and 
were found to be negative for the expression of the Oct-T1 gene: malignant 
melanoma, breast cancer, laryngeal carcinoma, lung NSCLC, bladder 
carcinoma, stomach cancer, lung SCLC, testicle tumor, uterine carcinoma, 
renal carcinoma, colon carcinoma, tongue cancer, esophageal cancer, 
ovarian cancer, c-skin carcinoma. A positive signal was obtained with an 
undifferentiated lung sarcoma, but four other sarcoma samples were found 
to be negative. 
c. Expression in malignant hemopathies: 
summary: 
acute myeloid leukemias: 6 positive samples (49 tested) 
chronic myeloid leukemias: no positive samples (5 tested) 
acute lymphoid leukemias: 11 positive samples (15 tested) 
chronic lymphoid leukemias: no positive samples (2 tested) 
multiple myeloma: no positive sample (1 tested) 
Thus, the expression of the Oct-T1 gene is found in 11% of acute myeloid 
leukemia and 73% of acute lymphoid leukemia samples. 
The results can also be presented in relation with the most frequent 
chromosomal abnormalities found in the acute leukemias: 
t(9;22)(q34;q11): 1 positive sample (3 tested) 
t(8;21)(q22;q22): 10 positive samples (11 tested) 
t(3;21)(q26;q22): no positive samples (2 tested) 
t(12;21)(p13;q22): 2 positive samples (2 tested) 
Inv(16)(p13;q22): no positive samples (8 tested) 
t(15;17)(q22;q21): no positive samples (4 tested) 
11q23 rearrangement: no positive samples (3 tested) 
trisomy 8: no positive samples (4 tested) 
del 5/5q or del 7/7q: 3 positive samples (8 tested) 
There is a clear correlation between the Oct-T1 gene expression and the 
acute leukemia with rearrangement of the AML1 gene, located on 21q22, and 
encoding the AML1 transcription factor. 
4. Conclusion: The Oct-T1 gene is expressed in human acute leukemia cells. 
The gene is particularly frequently expressed in ALL. The expression of 
the gene is related to chromosomal rearrangements involving the AML1 gene, 
which are involved in leukemogenesis. 
Therefore, it may be possible to immunize leukemia patients against 
antigens derived from the Oct-T1 protein in the form of peptides presented 
by HLA class I molecules, and present at the surface of leukemia cells 
expressing the gene. These antigens should not be present on testis 
germinal cells, since these do not express HLA class I molecules. 
The Oct-T1 gene is expressed in leukemic cells, but not in normal bone 
marrow, nor in normal PBL. Therefore, its specific and sensitive detection 
by RT-PCR is potentially useful as a leukemia-specific tumor marker, for 
the detection of minimal residual disease, or for the quantitative 
evaluation of response to treatment after induction chemotherapy. 
Example 5 
RT-PCR assavs for the expression of the preproTRH, the tryptase-L, and the 
Oct-T1/SIAX DP2-64 genes 
Isolation of total RNA from tumor samples (quickly frozen at -80.degree. 
C.) was performed by the guanidinium isothiocyanate/cesium chloride 
procedure (Davis et al., supra). cDNA synthesis was accomplished by 
extension with oligo(dT).sub.15. cDNA was then amplified by PCR with pairs 
of oligonucleotide primers that are highly specific for each tested gene. 
To ensure that the RNA was not degraded, a PCR assay with primers specific 
for .beta.-actin was carried out. 
1. cDNA synthesis The concentration of the RNA to be tested was adjusted to 
in 1 .mu.l of total RNA/3.25 .mu.l of water. The following reagents were 
mixed in a reaction tube placed in melting ice: 
______________________________________ 
Reverse Transcriptase Buffer 5X 
4 .mu.l 
(Life Technologies Inc., Gaithersburg, MD) 
dATP 10 mM 1 .mu.l 
dCTP 10 mM 1 .mu.l 
dGTP 10 mM 1 .mu.l 
dTTP 10 mM 1 .mu.l 
Dithiothreitol 100 mM 2 .mu.l 
oligo(dT).sub.15 20 .mu.M 2 .mu.l 
Rnasin 40 units/.mu.l (Promega Corp.) 
0.5 .mu.l 
M-MLV reverse transcriptase 200 units/.mu.l 
(Life Technologies, Inc.) 1 .mu.l 
Add 2 .mu.g of template RNA 
6.5 .mu.l 
Total volume: 20 .mu.l 
______________________________________ 
The reaction components were mixed and incubated at 42.degree. C. for 60 
min. The mixture was then chilled on ice. Water was added (80 .mu.l) to 
obtain a final volume of 100 .mu.l. The mixture was store at -20.degree. 
C. until used in PCR. 
2. PCR amplification 
a. Primers 
__________________________________________________________________________ 
preproTRH: sense primer (SEQ ID NO: 7): OPC376: 5'-CCAGCGGCTGCAAGGGGACCA-3 
antisense primer (SEQ ID NO: 8): OPC377: 5'-TGCCCGCCGACCAGGGTGCT-3' 
tryptase-L: sense primer (SEQ ID NO: 9): OPC314: 5'-CCCAAGAAGCCCTGAGC-3' 
antisense primer (SEQ ID NO: 10): OPC315: 5'-CAAGAAAGGGGAGGGGG-3' 
Oct-T1: sense primer (SEQ ID NO: 11): OPC406: 5'-CTGATCTAGTCCCAAGTCACC-3' 
. 
antisense primer (SEQ ID NO: 12): OPC407: 5'-ACAGCACTTGATCCAGAGTGG-3' 
.beta.-actin sense primer (SEQ ID NO: 13): OPC236: 5'-GGCATCGTGATGGACTCCG- 
3' 
antisense primer (SEQ ID NO: 14): OPC237: 5'-GCTGGAAGGTGGACAGCGA-3' 
__________________________________________________________________________ 
b. PCR reaction: The following reagents were mixed in a reaction tube 
placed in melting ice: 
H.sub.2 O: 18.5 .mu.l 
PCR buffer 10.times. (Dynazyme): 2.5 .mu.l 
dNTP (10 mM each): 0.25 .mu.l 
sense primer (20 .mu.M): 0.5 .mu.l 
antisense primer (20 .mu.M): 0.5 .mu.l 
Dynazyme: 0.25 .mu.l 
cDNA was added (2.5 .mu.l corresponding to 50 ng of total RNA), and the 
reaction mixture mixed. One drop of mineral oil (Sigma M-3516) was layered 
on top of the PCR solution. The reaction tube was transferred to the 
thermocycler for amplification. Positive control: cDNA from SIAX/LB-1079; 
Negative control: water. 
Thermal cycles: The PCR reactions were cycled as follows: 
______________________________________ 
First denaturation: 
94.degree. for 5 min 
Denaturation: 
94.degree. for 1 min 
Annealing: 
preproTRH 72.degree. for 1 min 28 cycles for amplification of 
preproTRH gene 
tryptase-L 
59.degree. for 2 min 35 cycles for amplification of tryptase-L 
gene 
Oct-T1 63.degree. for 2 min 27 cycles for amplification of Oct-T1 
gene 
.beta.-actin 
68.degree. for 2 min 23 cycles for amplification of 
.beta.-actin 
gene 
Extension: 
72.degree. for 3 min 
Final extension: 
72.degree. for 15 min 
______________________________________ 
The reactions were stored at 4.degree. C. until used in agarose gel 
electrophoresis. 
3. Gel electrophoresis Aliquots (10 .mu.l) of the PCR reaction were 
electrophoresed on a 1% agarose gel stained with ethidium bromide. 
TABLE 1 
__________________________________________________________________________ 
GENE EXPRESSION IN MALIGNANT HEMOPATHIES 
Code 
Diagnosis 
Karyotype TRH 
OCT-T1 
Tryp-L 
__________________________________________________________________________ 
CHIL 
AML-M0 none - - 
DRIA 
AML-M0 46, XX, t(4;11)(q21;q23) 
+/- 
- - 
DUMA 
AML-M0 46, XY + + 
UMON 
AML-M0 45, XX, t(9;22), -7 + +++ 
CABU 
AML-M1 47, XY, t(9;11)(p22;q23), +21 
- - + 
DEVA 
AML-M1 46, XX - +/- 
ELCA 
AML-M1 46, XY, t(8;21)(q22;q22) 
++ +++ + 
ETIT 
AML-M1 46, XX, t(4;1;18)(q27;p35;q21), -2, -4, 
- - 
GREM 
AML-M1 46, XX - +/- 
LENN 
AML-M1 47, XX, +21 + - 
MELU 
AML-M1 46, XX - - 
KRIM 
AML-M1 46, XY - - 
BENA 
AML-M1 46, XY - - 
ALVA 
AML-M2 42, XX, +8 - - - 
AUWE 
AML-M2 46, XY, -5, der(7)t(5;7)(q21;q2), +8 
- - +++ 
BOUR 
AML-M2 45, X, -Y, t(8;17;21)(q22;q11;q22) 
++ +++ 
DUWE 
AML-M2 47, XY, t(9;22)(q34;q11),(+12), i(17q) 
- - 
EVEL 
AML-M2 46, XX - - - 
SIAX 
AML-M2 46, XX, t(8;21)(q22;q22) 
+++ 
+++ 
MARO 
AML-M2 44, XX, -4, del(5)(q14;q31), -6, -7, 
+/- 
- 
ENIS 
AML-M2 46, XY, t(8;21)(q22;q22) 
++ + 
RAMA 
AML-M2 46, XX +++ 
+++ 
LEMO 
AML-M3 46, XY, t(15;17)(q22;q12) 
+/- 
- 
KARA 
AML-M3 46, XY, t(15;17)(q22;q12) 
+/- 
+/- 
DEVI 
AML-M3 46, XY, t(15;17)(q22;q12) 
- - - 
COWE 
AML-M3 46, XX, t(15;17)(q22;q12) 
+/- 
- 
LUSE 
AML-M4 46, XX - - 
REIB 
AML-M4 46, XX +/- 
- 
RICO 
AML-M4 46, XY - - 
AERT 
AML-M4 46, XX - - 
GIHA 
AML-M4 46, XY - - 
ERTE 
AML-M4Eo 
46, XX, inv(16) + - 
TEEN 
AML-M5 46, XY, t(8;11)(p22;q23) 
- - 
DOMA 
AML-M5 46, XX, del(7)(q22) +++ 
- 
ROCH 
AML-M5a - - 
DANA 
AML-M5b 
46, XY - - 
LATT 
AML-M5b 
1) 46, XY, -5, ins(12;?)(p12;?), +M 
- - 
LIAK 
AML-M5b 
1) 46, XX, inv(16) - - + 
OBBE 
AML-M5b 
46, XX - - 
RYRO 
AML-M5b 
46,XX - - 
LINT 
AML-M5b 
46, XY, inv(16) + - 
RONI 
AML-M5b 
46, XX - - 
TERE 
AML-M5b 
47, XY, t(2;7)(p1?4;p22), +8 10! 
- - 
ERNI 
AML-M5b 
del 1q, trisomie 8 - - 
LOLI 
AML-M5b 
46, XY - - 
NIES 
AML-M6 44, XY, del(2)(q12;q14), del(7)(q32), 
+/- 
- 
REUT 
AML-M6 46, XX +/- 
- 
GALU 
AML-M6 44, XX, t(5;17)(p14;q11), -14, -20 
+/- 
- 
OLBE 
AML-M6 46, XX, t(21;21) - - 
GRAD 
CLL 46, XX - - 
JEUM 
CLL 46, XY - - 
DERU 
CML 46, XX, t(9;22)(q34;q11) 
- - 
LOUY 
CML 46, XY, t(9;22) - - 
NEIR 
CML 46, XY, t(9;22)(q34;q11) 
- - 
VIER 
CML 46, XY, t(9;22) - - - 
CAVA 
ALL 1) 47, XY, t(9;22)(q34;q11), -9, 
- ++ 
AGUI 
common ALL 
1)47,XY,t(9;22)(q34;q11),-9 
- ++ 
DRON 
common ALL 
46, XX +/- 
- 
GERN 
common ALL 
45, XY, -7, t(7;16)(q11.2;q24), -18, +mar 
+ + 
ISER 
common ALL 
46, XX, -7, del(12)(p11.?2), +mar 
- + 
LULL 
common ALL 
47, X, der(X), del(1)(q25;q42), -3, t(6;22) 
+/- 
++ 
MEUL 
common ALL 
47,XX,-2,der(2)t(2;9)(?p16;p23),del(12)(p13) 
- ++ 
SACK 
common ALL 
not known - - 
WIRA 
common ALL 
46, XY + + 
DANN 
common ALL 
46, XX ++ + - 
POEL 
common ALL 
46, XY + ++ 
FURM 
common ALL 
46, XY, -18, +mar +/- 
++ 
AMAY 
common ALL - - 
QUEL 
MM 81,XX,?add(5)(q3?2),-8,-9,-12,+15,-16,+22, 
31 - 
ORBA 
MM 
BETT 
T-ALL 46, XY, del (7)(p13) 
+/- 
+ - 
JASI 
T-ALL 46, XX - + 
OUSA 
T-ALL 47, XY, +7 - - 
__________________________________________________________________________ 
N.B. results are expressed as relative intensity on the agarose gel, as 
compared with positive control (SIAX), arbitrarily assigned +++ (+, ++, 
+++ results are considered positive; -, +/- results are considered 
negative). 
Example 6 
Identification of the portion of leukemia associated genes encoding a tumor 
rejection antigens. 
In a first method, available CTL clones directed against antigens presented 
by autologous tumor cells shown to express one or more of the leukemia 
associated genes are screened for specificity against COS cells 
transfected with preproTRH, tryptase-L and/or Oct-T1 genes and autologous 
HLA alleles as described by Brichard et al. (Eur. J. Immunol. 26:224-230, 
1996). CTL recognition of preproTRH, tryptase-L and/or Oct-T1 is 
determined by measuring release of TNF from the cytolytic T lymphocyte or 
by .sup.51 Cr 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 preproTRH, tryptase-L and/or 
Oct-T1 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. 
Alternatively, CTL clones are generated by stimulating the peripheral blood 
lymphocytes (PBLs) of a patient with autologous normal cells transfected 
with DNA clones encoding preproTRH, tryptase-L and/or Oct-T1 polypeptides 
(e.g. SEQ ID NOs:1, 3, 5 and/or 7) 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 preproTRH, tryptase-L and/or Oct-T1 clones 
(see, e.g., van der Bruggen et al., Eur. J. Immunol. 24:3038-3043, 1994; 
MAGE3 peptides presented by HLA.A2). The HLA type of the patient from 
which the leukemia cells from which preproTRH, tryptase-L and Oct-T1 were 
isolated is: A2, A26, B7, B56, Cw1, DR1, DR8, DQ4, DQ5. 
Optionally, shorter fragments of preproTRH, tryptase-L and/or Oct-T1 cDNAs 
are generated by PCR. Shorter fragments are used to provoke TNF release or 
.sup.51 Cr release as above. 
Example 7 
Identification of leukemia associated gene encoded tumor rejection antigen 
peptides 
Synthetic peptides corresponding to portions of the shortest fragment of 
preproTRH, tryptase-L and/or Oct-T1 which provokes TNF release are 
prepared. Progressively shorter peptides are synthesized to determine the 
optimal preproTRH, tryptase-L and/or Oct-T1 tumor rejection antigen 
peptides for a given HLA molecule. 
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.sup.+ 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 
preproTRH, tryptase-L or Oct-T1. 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. 
To determine the optimal size of the synthetic peptide, peptides of 
decreasing size are synthesized based on the sequence of the peptide 
deterined 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.degree. C. Target cells, e.g. 
HLA-A2.sup.+ T2 cells, are labeled with .sup.51 Cr, as described above, 
for 1 hour at 37.degree. 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.degree. 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 8 
Determination of the recognition of homologous peptides of genes related to 
leukemia associated genes by CTLs 
As noted above, Oct-T1 and tryptase-L have high amino acid homology to 
other Oct family transcription factors and other tryptases. To demonstrate 
that a tumor rejection antigen derived from Oct-T1 and/or tryptase-L is 
specific for these genes, peptides of other Oct and tryptase proteins 
which correspond to the postitions in the respective proteins of Oct-T1 
and tyrptase-L are synthesized and used in a dose response-chromium 
release assay as described above. This experiment permits the 
determination of the specificity of the Oct-T1 and/or tryptase-L derived 
TRAs, such that TRAs which selectively provoke lysis of cells which 
express Oct-T1 and/or tryptase-L, but not homologous genes, can be 
selected. 
Example 9 
Normal cells are not lysed by CTLs which lyse cells which express leukemia 
associated genes 
This example describes CTL lysis experiments with various cell lines with 
or without incubation with the leukemia associated gene derived peptides 
determined above. SIAX leukemic cells, normal B cells from patient SIAX 
transformed with EBV (SIAX-EBV) and normal peripheral blood lymphocytes 
from the same patient (SIAX-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 SIAX leukemic cells by the CTLs, demonstrates that SIAX-EBV and 
SIAX-PBL cells are not recognized by the CTLs because such cells do not 
normally express the tumor rejection antigen derived from preproTRH, 
tryptase-L and/or Oct-T1 proteins. 
It is next determined whether these cells would be lysed by CTL if pulsed 
with a peptide derived from preproTRH, tryptase-L and/or Oct-T1. The 
peptides selected on the basis of the experiments above are tested for the 
ability to induce cell lysis of SIAX leukemic cells, SIAX-EBV cells, and 
non-autologous cells which express the appropriate HLA by CTL cells in a 
dose response assay as in previous examples. SIAX-EBV and SIAX-PBL pulsed 
with preferred peptides are not lysed by CTLs, but SIAX leukemic cells and 
the non-autologous cells pulsed with preferred peptides are lysed by CTLs. 
Other aspects of the invention will be clear to the skilled artisan and 
need not be repeated here. 
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. 
A sequence listing is presented followed by what is claimed: 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 16 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1581 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 106..831 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CGCCCGGGGTCCTCAGCGCTGCAGACTCCTGACCTGCCGACTGCGGATCCCGAGTCCCCG60 
GATCCCGGACCCATCCTGTGGAGCCCACTCCTGGCAGACGCCGCGATGCCCGGC114 
MetProGly 
CCTTGGTTGCTGCTCGCTCTGGCTTTGACCCTGAACCTGACCGGTGTC162 
ProTrpLeuLeuLeuAlaLeuAlaLeuThrLeuAsnLeuThrGlyVal 
51015 
CCCGGCGGCCGTGCTCAGCCAGAGGCGGCCCAGCAGGAGGCAGTGACG210 
ProGlyGlyArgAlaGlnProGluAlaAlaGlnGlnGluAlaValThr 
20253035 
GCCGCGGAGCATCCGGGCCTGGATGACTTCCTGCGCCAGGTGGAGCGC258 
AlaAlaGluHisProGlyLeuAspAspPheLeuArgGlnValGluArg 
404550 
CTCCTCTTCCTCCGGGAAAACATCCAGCGGCTGCAAGGGGACCAGGGT306 
LeuLeuPheLeuArgGluAsnIleGlnArgLeuGlnGlyAspGlnGly 
556065 
GAGCACTCCGCGTCCCAGATCTTTCAATCTGACTGGCTCTCCAAACGT354 
GluHisSerAlaSerGlnIlePheGlnSerAspTrpLeuSerLysArg 
707580 
CAGCATCCAGGCAAAAGAGAGGAGGAGGAGGAAGAGGGAGTTGAAGAA402 
GlnHisProGlyLysArgGluGluGluGluGluGluGlyValGluGlu 
859095 
GAGGAAGAGGAAGAAGGGGGGGCTGTGGGACCCCACAAACGGCAGCAC450 
GluGluGluGluGluGlyGlyAlaValGlyProHisLysArgGlnHis 
100105110115 
CCTGGCCGACGAGAAGATGAGGCTTCATGGTCAGTCGATGTAACCCAG498 
ProGlyArgArgGluAspGluAlaSerTrpSerValAspValThrGln 
120125130 
CACAAGCGGCAGCATCCTGGCCGGCGCTCCCCCTGGCTTGCATATGCT546 
HisLysArgGlnHisProGlyArgArgSerProTrpLeuAlaTyrAla 
135140145 
GTCCCGAAGCGGCAGCACCCAGGCAGAAGGCTGGCAGATCCCAAGGCT594 
ValProLysArgGlnHisProGlyArgArgLeuAlaAspProLysAla 
150155160 
CAAAGGAGCTGGGAAGAAGAGGAGGAGGAGGAAGAGAGAGAGGAAGAC642 
GlnArgSerTrpGluGluGluGluGluGluGluGluArgGluGluAsp 
165170175 
CTGATGCCTGAAAAACGCCAGCATCCGGGCAAGAGGGCCCTGGGAGGC690 
LeuMetProGluLysArgGlnHisProGlyLysArgAlaLeuGlyGly 
180185190195 
CCCTGTGGGCCCCAGGGAGCCTATGGTCAAGCGGGCCTCCTGCTGGGG738 
ProCysGlyProGlnGlyAlaTyrGlyGlnAlaGlyLeuLeuLeuGly 
200205210 
CTCCTGGATGACCTGAGTAGGAGCCAGGGAGCTGAGGAAAAGCGGCAG786 
LeuLeuAspAspLeuSerArgSerGlnGlyAlaGluGluLysArgGln 
215220225 
CACCCTGGTCGGCGGGCAGCCTGGGTCAGGGAGCCCCTGGAGGAG831 
HisProGlyArgArgAlaAlaTrpValArgGluProLeuGluGlu 
230235240 
TGAACCCAGTTTTCCCTGAAGTCGAGTTTGTGGTCTAAGGATGTCTTGAGCCCTGTGTGC891 
CCCACCATTCATGACCTCTGTATTCTCTAGTTAGATCCCTGACCATAAGCCTGAGCCCCT951 
CCCTCCCAGCCCCATATTCACACACATCCCAGCCCCTGGCCTTGCCCTCTTCCTTTAGGC1011 
ATGTGAGAAAATCAGCCTAGCAGTTTAAACCCCACTTTCCTCCACTTAGCACCATAGGCA1071 
AGGGGGCAGATCCCAGAGCCCCTCTCACCCCCCCCACCACAGGCCTGCTCCTTCCTTAGC1131 
CTTGGCTAAGATGGTCCTTCTGTGTCTTGCAAAGACTCCCCAAGTGGGACAGGGAGCCCC1191 
TGGGAGGGCAGCCAGTGAGGGTGGGGTGGGACTGAAGCGTTGTGTGCAAATCCAGCTTCC1251 
ATCCCCTCCCCAACCTGGCAGGATTCTCCATGTGTAAACTTCACCCCCAGGACCCAGGAT1311 
CTTCTCCTTTCTGGGCATCCCTTTGTGGGTGGGCAGAGCCCTGACCCACAGCTGTGTTAC1371 
TGCTTGGAGAAGCATATGTAGGGGCATACCCTGTGGTGTTGTGCTGTGTCTGGCTGTGGG1431 
ATAAATGTGTGTGGGAATATTGAAACATCGCCTAGGAATTGTGGTTTGTATATAACCCTC1491 
TAAGCCCCTATCCCTTGTCGATGACAGTCATCCTAATGATAATAAAACCTGCATCCAGAT1551 
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1581 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 242 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetProGlyProTrpLeuLeuLeuAlaLeuAlaLeuThrLeuAsnLeu 
151015 
ThrGlyValProGlyGlyArgAlaGlnProGluAlaAlaGlnGlnGlu 
202530 
AlaValThrAlaAlaGluHisProGlyLeuAspAspPheLeuArgGln 
354045 
ValGluArgLeuLeuPheLeuArgGluAsnIleGlnArgLeuGlnGly 
505560 
AspGlnGlyGluHisSerAlaSerGlnIlePheGlnSerAspTrpLeu 
65707580 
SerLysArgGlnHisProGlyLysArgGluGluGluGluGluGluGly 
859095 
ValGluGluGluGluGluGluGluGlyGlyAlaValGlyProHisLys 
100105110 
ArgGlnHisProGlyArgArgGluAspGluAlaSerTrpSerValAsp 
115120125 
ValThrGlnHisLysArgGlnHisProGlyArgArgSerProTrpLeu 
130135140 
AlaTyrAlaValProLysArgGlnHisProGlyArgArgLeuAlaAsp 
145150155160 
ProLysAlaGlnArgSerTrpGluGluGluGluGluGluGluGluArg 
165170175 
GluGluAspLeuMetProGluLysArgGlnHisProGlyLysArgAla 
180185190 
LeuGlyGlyProCysGlyProGlnGlyAlaTyrGlyGlnAlaGlyLeu 
195200205 
LeuLeuGlyLeuLeuAspAspLeuSerArgSerGlnGlyAlaGluGlu 
210215220 
LysArgGlnHisProGlyArgArgAlaAlaTrpValArgGluProLeu 
225230235240 
GluGlu 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2259 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 8..577 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGCCAGGATGCTGAGCCTGCTGCTGCTGGCGCTGCCCGTCCTGGCGAGC49 
MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSer 
1510 
CCGGCCTACGTGGCCCCTGCCCCAGGCCAGGCCCTGCAGCAAACGGGC97 
ProAlaTyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGly 
15202530 
ATTGTTGGGGGGCAGGAGGCCCCCAGGAGCAAGTGGCCCTGGCAGGTG145 
IleValGlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnVal 
354045 
AGCCTGAGAGTCCGCGGCCCATACTGGATGCACTTCTGCGGGGGCTCC193 
SerLeuArgValArgGlyProTyrTrpMetHisPheCysGlyGlySer 
505560 
CTCATCCACCCCCAGTGGGTGCTAACCGCGGCGCACTGCGTGGAACCG241 
LeuIleHisProGlnTrpValLeuThrAlaAlaHisCysValGluPro 
657075 
GACATCAAGGATCTGGCCGCCCTCAGGGTGCAACTGCGGGAGCAGCAC289 
AspIleLysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHis 
808590 
CTCTACTACCAGGACCAGCTGCTGCCGGTCAGCAGGATCATCGTGCAC337 
LeuTyrTyrGlnAspGlnLeuLeuProValSerArgIleIleValHis 
95100105110 
CCACAGTTCTACATCATCCAGACCGGGGCGGACATCGCCCTGCTGGAG385 
ProGlnPheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGlu 
115120125 
CTGGAGGAGCCCGTGAACATCTCCAGCCACATCCACACGGTCACGCTG433 
LeuGluGluProValAsnIleSerSerHisIleHisThrValThrLeu 
130135140 
CCCCCTGCCTCGGAGACCTTCCCCCCGGGGATGCCGTGCTGGGTCACT481 
ProProAlaSerGluThrPheProProGlyMetProCysTrpValThr 
145150155 
GGCTGGGGCGACGTGGACAATAATGGTGGGTGTTGGGGACAGCGGGAG529 
GlyTrpGlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGlu 
160165170 
GCCGGGCCAGGTGGGCACCAAGTCACAGCCACAGGCCAGTCCGTGGGG577 
AlaGlyProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly 
175180185190 
TGACAGGGTCCCTCAGGGCGGCTCAGGGAGGGGGACTGTGGAGGCCAGGATGGATGGAGC637 
AGGCGGTGGCGAGAGGCAGCAGGTGCCCTGAGCAGAGACGGTGAGTCCAAAGGGCCTGGG697 
CGTCCCCCACCCCAGGGGTTTGGAGAGTCCCTTAGCACCTCCGTGCCTCGGTTTCCCCTT757 
GCCTGAAAGGGTGCATCAAAAGTTTGTACGTCACGGACTTGCTATGTGGAGAGAGAAATC817 
ACACGGGGGTCTTGCTGGAAGGAGAGAGACCGGTGCTGGGATGAGACCTGCCTGCCCTCC877 
ATCCCTGTGCTACAGACAAGGCAGGGGCCTGGGAATCGGGGTCGTGGCAGTGCTGTGGGG937 
GGCTGGACGAAGCTCACTGTGGCCCTCCACGAGGCACATTTTCACTTCTAGAAGGTCTTG997 
TCCCCATTTTATCCACAATTCAGAGCAAAGCTTTGGGGTACAGCCTGACGCAACCCTGGG1057 
CTGTGACCTCTGGGTCACTCCAGAAGGGGCCTGAGCCACTGTCCCGCTATTCCGCCCCAC1117 
ACAGCGGGGAAGCTGAGCCCAGCGCCCTGTGTTCCCCTCGGCTAGGGCCAACCGTGGACC1177 
ATGGGCCTAGCCCAGACGAAAGTCAGCTGAGCCCAGGGGGAGACACGGGTCGGGCTCTGC1237 
ACCCCCGTGCCATGGAGCCCAGCTTGGCAACCTCCAGGGCCCTCCCCTCCCTTCCCCAGA1297 
TGGGGCTTAAATGAGGCCAGGGACCCAGGACCAGCCTCAGCGGAGGGGCCTGGACTGCAT1357 
TCACCGCCCCTTCCCCGGGGCTGCAGGCACAGAACAGCACTGGGCCCATGGTGCCATCTC1417 
CCCTGCCCGTGACTCTGCCACCAAGTCCACGAAGCAGCACCCAGCCGGCCCCAGACCCGG1477 
CTCCACGCCCCCCTCCGCCCCCAGTGCACCTGCCGCCGCCATACCCGCTGAAGGAGGTGG1537 
AAGTCCCCGTAGTGGAAAACCACCTTTGCAACGCGGAATATCACACCGGCCTCCATACGG1597 
GCCACAGCTTTCAAATCGTCCGCGATGACATGCTGTGTGCGGGGAGCGAAAATCACGACT1657 
CCTGCCAGGGTGACTCTGGAGGGCCCCTGGTCTGCAAGGTGAATGGCACCTAACTGCAGG1717 
CGGCCGTGGTCAGCTGGGAGGAGAGCTGTGCCCAGCCCAACCGGCCTGGCATCTACACCC1777 
GTGTCACCTACTACTTGGACTGGATCCACCACTATGTCCCCAAGAAGCCCTGACCCAGGC1837 
CTGGGTTGTCCACCCGGGTCACTGGAGGGCCAGCCCCTCCTGTCCAAACCACCACTGCTT1897 
CCTACCCAGGTGGTGACTAAACCCCACACCTTCCCCCATCCTGAGTCCCCTCTCCCATCC1957 
TGAGCCCTGTCCCCTGTCCTGAGCCCCCTCCCCTTTCTTGATCCCCTCCCCCATCCTGAG2017 
CCCCCTCCCCCACCCTGAGCCCCCTCCCCTGTCTTGAGCCCCTCATCCATCCTGAGCCCC2077 
TCCTCCATCCTGAGCCCCCTCCCCCATCCTGGGCCCCCTCCCCTTTCTTGAGCCCCCTCC2137 
CCCACCCTGAGCCCCTTCCCCTTTCTTGAGCCCCTCCTCCACCCTCAGCCCCCTCCCCTT2197 
TCTTGAGCCCCTCCTCCACCCTCAGCCCCCTCCCCTTTCTTGAGCCCCTCCCCCACCCTG2257 
AG2259 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 190 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSerProAla 
151015 
TyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGlyIleVal 
202530 
GlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnValSerLeu 
354045 
ArgValArgGlyProTyrTrpMetHisPheCysGlyGlySerLeuIle 
505560 
HisProGlnTrpValLeuThrAlaAlaHisCysValGluProAspIle 
65707580 
LysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHisLeuTyr 
859095 
TyrGlnAspGlnLeuLeuProValSerArgIleIleValHisProGln 
100105110 
PheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGluLeuGlu 
115120125 
GluProValAsnIleSerSerHisIleHisThrValThrLeuProPro 
130135140 
AlaSerGluThrPheProProGlyMetProCysTrpValThrGlyTrp 
145150155160 
GlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGluAlaGly 
165170175 
ProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly 
180185190 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2218 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: NVB352/3 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 8..577 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GGCCAGGATGCTGAGCCTGCTGCTGCTGGCGCTGCCCGTCCTGGCGAGC49 
MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSer 
1510 
CCGGCCTACGTGGCCCCTGCCCCAGGCCAGGCCCTGCAGCAAACGGGC97 
ProAlaTyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGly 
15202530 
ATTGTTGGGGGGCAGGAGGCCCCCAGGAGCAAGTGGCCCTGGCAGGTG145 
IleValGlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnVal 
354045 
AGCCTGAGAGTCCGCGGCCCATACTGGATGCACTTCTGCGGGGGCTCC193 
SerLeuArgValArgGlyProTyrTrpMetHisPheCysGlyGlySer 
505560 
TTCATCCACCCCCAGTGGGTGCTAACCGCGGCGCACTGCGTGGAACCG241 
PheIleHisProGlnTrpValLeuThrAlaAlaHisCysValGluPro 
657075 
GACATCAAGGATCTGGCCGCCCTCAGGGTGCAACTGCGGGAGCAGCAC289 
AspIleLysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHis 
808590 
CTCTACTACCAGGACCAGCTGCTGCCGGTCAGCAGGATCATCGTGCAC337 
LeuTyrTyrGlnAspGlnLeuLeuProValSerArgIleIleValHis 
95100105110 
CCACAGTTCTACATCATCCAGACCGGGGCGGACATCGCCCTGCTGGAG385 
ProGlnPheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGlu 
115120125 
CTGGAGGAGCCCGTGAACATCTCCAGCCACATCCACACGGTCACGCTG433 
LeuGluGluProValAsnIleSerSerHisIleHisThrValThrLeu 
130135140 
CCCCCTGCCTCGGAGACCTTCCCCCCGGGGATGCCGTGCTGGGTCACT481 
ProProAlaSerGluThrPheProProGlyMetProCysTrpValThr 
145150155 
GGCTGGGGCGACGTGGACAATAATGGTGGGTGTTGGGGACAGCGGGAG529 
GlyTrpGlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGlu 
160165170 
GCCGGGCCAGGTGGGCACCAAGTCACAGCCACAGGCCAGTCCGTGGGG577 
AlaGlyProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly 
175180185190 
TGACAGGGTCCCTCAGGGCGGCTCAGGGAGGGGGACTGTGGAGGCCAGGATGGATGGAGC637 
AGGCGGTGGCGAGAGGCAGCAGGTGCCCTGAGCAGAGACGGTGAGTCCAAAGGGCCTGGG697 
CGTCCCCCACCCCAGGGGTTTGGAGAGTCCCTTAGCACCTCCGTGCCTCGGTTTCCCCTT757 
GCCTGAAAGGGTGCATCAAAAGTTTGTACGTCACGGACTTGCTATGTGGAGAGAGAAATC817 
ACACGGGGGTCTTGCTGGAAGGAGAGAGATCGGTGCTGGGATGAGACCTGCCTGCCCTCC877 
ATCCCTGTGCTACAGACAAGGCAGGGGCCTGGGAATCGGGGTCGTGGCAGTGCTGTGGGG937 
GGCTGGACGAAGCTCACTGTGGCCCTCCACGAGGCACATTTTCACTTCTAGAAGGTCTTG997 
TCCCCATTTTATCCACAATTCAGAGCAAAGCTTTGGGGTACAGCCTGAGCGGCAACCCTG1057 
GGCTGTGACTCTGGGTCACTCAGAAGGGGCCTGAGCCACTGTCCCGCTATTCCGCCCCAC1117 
ACAGCGGGGAAGCTGAGCCCAGCGCCCTGTGTTCCCCTCGGCTAGGGCCAACCGTGGACC1177 
ATGGGCCTAGCCCAGACGAAAGTCAGCTGAGCCCAGGGGGAGACACGGGTCGGGCTCTGC1237 
ACCCCCGTGCCATGGAGCCCAGCTTGGCAACCTCCAGGGCCCTCCCCTCCCTTCCCCAGA1297 
TGGGGCTTAAATGAGGCCAGGGACCCAGGACCAGCCTCAGCGGAGGGGCCTGGACTGCAT1357 
TCACCGCCCCTTCCCCGGGGCTGCAGGCACAGAACAGCACTGGGCCCATGGTGCCATCTC1417 
CCCTGCCCGTGACTCTGCCACCAAGTCCACGAAGCAGCACCCAGCCGGCCCCAGACCCGG1477 
CTCCACGCCCCCCTCCGCCCCCAGTGCACCTGCCGCCGCCATACCCGCTGAAGGAGGTGG1537 
AAGTCCCCGTAGTGGAAAACCACCTTTGCAACGCGGAATATCACACCGGCCTCCATACGG1597 
GCCACAGCTTTCAAATCGTCCGCGATGACATGCTGTGTGCGGGGAGCGAAAATCACGACT1657 
CCTGCCAGGTGGGCCCTCGCGTCCCCCACCCCAATCCCCGGAGCCTGGCCAGCGAGCGCA1717 
TCCCTCATCCTGACCCCCGAAGCCTGGCCAGCGAGCACTGACCTCTGACCTTCCCAGGGT1777 
GACTCTGGAGGGCCCCTGGTCTGCAAGGTGAATGGCACCTAACTGCAGGCGGGCGTGGTC1837 
AGCTGGGAGGAGAGCTGTGCCCAGCCCAACCGGCCTGGCATCTACACCCGTGTCACCTAC1897 
TACTTGGACTGGATCCACCACTATGTCCCCAAGAAGCCCTGAGCCAGGCCTGGGGTGTCC1957 
ACCCGGGTCACTGGAGGGCCAGCCCCTCCTGTCCAAACCACCACTGCTTCCTACCCAGGT2017 
GGTGACTGCCCCCCACACCTTCCCCCATCCTGAGTCCCCTCTCCCATCCTGAGCCCTGTC2077 
CCCTGTCCTGAGCCCCCTCCCCTTTCTTGATCCCCTCCCCCATCCTGAGCCCCCTCCCCC2137 
ACCCTGAGCCCCCTCCCCTGTCTTGAGCCCCTGCTCCATCCTGAGTCCCCTCCCCCACAC2197 
TGAGCCCCCTCCCCTTTCTTG2218 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 190 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSerProAla 
151015 
TyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGlyIleVal 
202530 
GlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnValSerLeu 
354045 
ArgValArgGlyProTyrTrpMetHisPheCysGlyGlySerPheIle 
505560 
HisProGlnTrpValLeuThrAlaAlaHisCysValGluProAspIle 
65707580 
LysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHisLeuTyr 
859095 
TyrGlnAspGlnLeuLeuProValSerArgIleIleValHisProGln 
100105110 
PheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGluLeuGlu 
115120125 
GluProValAsnIleSerSerHisIleHisThrValThrLeuProPro 
130135140 
AlaSerGluThrPheProProGlyMetProCysTrpValThrGlyTrp 
145150155160 
GlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGluAlaGly 
165170175 
ProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly 
180185190 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4524 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 174..1433 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CAGAGGGAGCGCCTGGCAGCAGCAGGAGCAGCAGCAGCAGCCCGCGGCGGGGCCGCCGCC60 
AGCCGCCGCGACCGCCGCGGCTGCAGCCTCCGAAGGGAGGCCGGGTGAGCCGGCGTACGC120 
ACTTTCCCGCGGACTTTCGGAGTGTTTGTGGATATACATGCCAAGCCGCCACGATG176 
Met 
1 
ATGTCCATGAACAGCAAGCAGCCTCACTTTGCCATGCATCCCACCCTC224 
MetSerMetAsnSerLysGlnProHisPheAlaMetHisProThrLeu 
51015 
CCTGAGCACAAGTACCCGTCGCTGCACTCCAGCTCCGAGGCCATCCGG272 
ProGluHisLysTyrProSerLeuHisSerSerSerGluAlaIleArg 
202530 
CGGGCCTGCCTGCCCACGCCGCCGCTGCAGAGCAACCTCTTCGCCAGC320 
ArgAlaCysLeuProThrProProLeuGlnSerAsnLeuPheAlaSer 
354045 
CTGGACGAGACGCTGCTGGCGCGGGCCGAGGCGCTGGCGGCCGTGGAC368 
LeuAspGluThrLeuLeuAlaArgAlaGluAlaLeuAlaAlaValAsp 
50556065 
ATCGCCGTGTCCCAGGGCAAGAGCCATCCTTTCAAGCCGGACGCCACG416 
IleAlaValSerGlnGlyLysSerHisProPheLysProAspAlaThr 
707580 
TACCACACGATGAACAGCGTGCCGTGCACGTCCACTTCCACGGTGCCT464 
TyrHisThrMetAsnSerValProCysThrSerThrSerThrValPro 
859095 
CTGGCGCACCACCACCACCACCACCACCACCACCAGGCGCTCGAACCC512 
LeuAlaHisHisHisHisHisHisHisHisHisGlnAlaLeuGluPro 
100105110 
GGCGATCTGCTGGACCACATCTCCTCGCCGTCGCTCGCGCTCATGGCC560 
GlyAspLeuLeuAspHisIleSerSerProSerLeuAlaLeuMetAla 
115120125 
GGCGCGGGCGGCGCGGGCGCGGCGGCCGGCGGCGGCGGCGCCCACGAC608 
GlyAlaGlyGlyAlaGlyAlaAlaAlaGlyGlyGlyGlyAlaHisAsp 
130135140145 
GGCCCGGGGGGCGGTGGCGGCCCGGGCGGCGGCGGCGGCCCGGGCGGC656 
GlyProGlyGlyGlyGlyGlyProGlyGlyGlyGlyGlyProGlyGly 
150155160 
GGCGGCCCCGGGGGAGGCGGCGGTGGCGGCCCGGGGGGCGGCGGCGGC704 
GlyGlyProGlyGlyGlyGlyGlyGlyGlyProGlyGlyGlyGlyGly 
165170175 
GGCCCGGGCGGCGGGCTCCTGGGCGGCTCCGCGCACCCTCACCCGCAT752 
GlyProGlyGlyGlyLeuLeuGlyGlySerAlaHisProHisProHis 
180185190 
ATGCACAGCCTGGGCCACCTGTCGCACCCCGCGGCGGCGGCCGCCATG800 
MetHisSerLeuGlyHisLeuSerHisProAlaAlaAlaAlaAlaMet 
195200205 
AACATGCCGTCCGGGCTGCCGCACCCCGGGCTGGTGGCGGCGGCGGCG848 
AsnMetProSerGlyLeuProHisProGlyLeuValAlaAlaAlaAla 
210215220225 
CACCACGGCGCGGCAGCGGCAGCGGCGGCGGCGTCGGCCGGGCAGGTG896 
HisHisGlyAlaAlaAlaAlaAlaAlaAlaAlaSerAlaGlyGlnVal 
230235240 
GCAGCGGCATCGGCGGCGGCGGCCGTGGTGGGCGCAGCGGGCCTGGCG944 
AlaAlaAlaSerAlaAlaAlaAlaValValGlyAlaAlaGlyLeuAla 
245250255 
TCCATCTGCGACTCGGACACGGACCCGCGCGAGCTCGAGGCGTTCGCG992 
SerIleCysAspSerAspThrAspProArgGluLeuGluAlaPheAla 
260265270 
GAGCGCTTCAAGCAGCGGCGCATCAAGCTGGGCGTGACGCAGGCCGAC1040 
GluArgPheLysGlnArgArgIleLysLeuGlyValThrGlnAlaAsp 
275280285 
GTGGGCTCGGCGCTGGCCAACCTCAAGATCCCGGGCGTGGGCTCACTC1088 
ValGlySerAlaLeuAlaAsnLeuLysIleProGlyValGlySerLeu 
290295300305 
AGCCAGAGCACCATCTGCAGGTTCGAGTCGCTCACGCTCTCGCACAAC1136 
SerGlnSerThrIleCysArgPheGluSerLeuThrLeuSerHisAsn 
310315320 
AACATGATCGCGCTCAAGCCCATCCTGCAGGCGTGGCTCGAGGAGGCC1184 
AsnMetIleAlaLeuLysProIleLeuGlnAlaTrpLeuGluGluAla 
325330335 
GAGGGCGCCCAGCGCGAGAAAATGAACAAGCCTGAGCTCTTCAACGGC1232 
GluGlyAlaGlnArgGluLysMetAsnLysProGluLeuPheAsnGly 
340345350 
GGCGAGAAGAAGCGCAAGCGGACTTCCATCGCCGCGCCCGAGAAGCGC1280 
GlyGluLysLysArgLysArgThrSerIleAlaAlaProGluLysArg 
355360365 
TCCCTCGAGGCCTACTTCGCCGTGCAGCCCCGGCCCTCGTCCGAGAAG1328 
SerLeuGluAlaTyrPheAlaValGlnProArgProSerSerGluLys 
370375380385 
ATCGCCGCCATCGCCGAGAAACTGGACCTCAAAAAGAACGTGGTGCGG1376 
IleAlaAlaIleAlaGluLysLeuAspLeuLysLysAsnValValArg 
390395400 
GTGTGGTTTTGCAACCAGAGACAGAAGCAGAAGCGGATGAAATTCTCT1424 
ValTrpPheCysAsnGlnArgGlnLysGlnLysArgMetLysPheSer 
405410415 
GCCACTTACTGAGGGGGCTGGGAGGTGTCGGGCGGGACAGAATGGGGAG1473 
AlaThrTyr 
420 
CTGAGGAGGCATTTTTGGGGGGCTTTCCTCTGCTTGCCTCCCCTCGGATTTGGAGTGTCC1533 
GTTATCCTGCCTGCATTTGGGGAGTCCCTTCTCGCTCTCTTTCCTCCACCCATTCTCTGA1593 
TTTTCCTGCCTTTGCTGTCCCCTAGCCTTGAGGACTGGGGTGCTGGGTGTGGGGATTGGA1653 
GTATAGGGTAGGGGAGAAGGGGGGGAGCATTCGGGGGAGTGGGGAGTGGGGGGAAGGAAA1713 
GCGGAGACCCGAGCAGGGGTTTTAAGGAGCAGGATGGTTCTGGGGTTTGGGTGGGGGGAG1773 
ACGCGGGAAGGGTAGGAAAATGGACTGTTTCTGACCAGAGACACTTACCTAAATATCCTG1833 
GGGACCAAGGAACTATGTACAAAAACAAACCTACCAACCACCAAAAACTAGACAAATAAA1893 
GACAAACTAAAACAAAACAGAACAAAAGCAAAGGAAAATGCTTTAGAAATTTTAACTCCG1953 
GGGAGCCATAATCTGCAACTTCATTTTCCCCCATAGAAGAGAAAAAAGAGCACCACCATT2013 
ATTACCACCTCCCCAACCCTACACGCACGAACTGAGTCGAAAAACGAAAACCAAACGAGC2073 
GAGAAGTTGAAGTTCTGGGTATCAAAGCTAGTTGTTCTGTCTGCGTGTTTAATTTTTCCC2133 
TCTCTCACCTCCACCCCATCCATATCCTCTTTATTTCCTCCGTTCCAATGAGAGGCCTAT2193 
GGCTGCTCTCCAATCCCGGGAAGTGAGTGGGAGCACAGCTGAAAAGAGAGGGTCAGGGGG2253 
AGGCTGGCTGCTTGCTTAGGTGGAATCCAAGTTTTCCCGTGGCCCTGCCTATACTCTGGT2313 
GGCCTGGTCCTGTTGGGGTGGGGGTCTTTGGAGAGAAGGGCATAGTCTTTGAGCTACTAA2373 
AAAGCAGAATTCCGGAGCTTCGAGATATCTTATTCTAGGAAAATGAAACAATTTTAACAA2433 
CAGTTTTTTTTCCTCTTATGTCGAAGATCTAGTTTTAGACAATTTCAAAATAAGCTTTTC2493 
CCACTCATAGAACTTTAACTTGCCCTTTCAGTTTTATCTTTTTTTTAGAGAGAGGTTTAA2553 
ACTACTGATTTTTCCTGTTGATTCAAATAGACTAATGGGGTGAAAGTTATTAGGAGAGAT2613 
ACTCTCTCCTGTTTTCTCCACTGAACGAGACTCATCTTGCTCTTCTAGGTCCCGTTTCTT2673 
CCTCTCTTGGAGGACATGAAATTATAGAAATGTTGAGAAGTTCCTGCTTTCTTTTGCGGT2733 
AGGACTTGGCTGTGAGAAAATCACCTAAATCCCAGAAAAGAGGAAGACAGATTTAAAGTG2793 
CCCCCACCCCCATTTGTTTCAAAGAGGTCTGCATGTTGGGCGAAAACAGAACAACTGTGT2853 
TTCCTTTTACTTGTTCTTATTATTCAAGAGTCATTTATTACAGGGGATAAATGTTGGGTA2913 
GCAAGAACTTTAATTTGCACTACCAGTCTCCCAAATAGAAAATCATGTATAGTATTTCAT2973 
AGTAATAATCAGGTACCTTACAAGCTGCTGGTGGATTTTAAAAAATTAAGATAGTTGAAG3033 
GTGGTTAGGTAAAATGCCTGCTTTGTGTACAAGATACTCTTTGGATCTCTCGTAGAGATG3093 
GTTTGTTACCATCCTTTAATCATAACTAAAACATTGAAAACAGAACAAATGAGAAAAGAA3153 
AAAAAACCTGCCGATTAACAAGACTGAAATCATGCATGATCTGAAAGGTGTGGAAAGAAA3213 
CACAATTAGGTCTCACTCTGGTTAGGCATTATTTATTTAATTATGTTGTATATCATTGTT3273 
TGCAGGGCAAACATTCTATGCATTTGAAACTGAGCACTAAACTGGGCTAGCTTTCTGGTA3333 
GACCGTTTTGTGGCTAGTGCGATTTCACAGTCTACTGCCTGTTTCCACTGAAAACATTTT3393 
TGTCATATTCTTGTATTCAAAGAAAAAGGAAAAAAGATTATTGTAAATATTTTATTTAAT3453 
GCACACATTCACACAGTGGTAACAGACTGCCAGTGTTCATCCTGAAATGTCTCACGGATT3513 
GATCTACCTGTCCATGTATGTCTGCTGAGCTTTCTCCTTGGTTATGTTTTTTCTCTTTTA3573 
CCTTTCTCCTCCCTTACTTCTATCAGAACCAATTCTATGCGCCAAAATACAACAGGGGGA3633 
TGTGTCCCAGTACACTTACAAATAAAACATAACTGAAAGAAGAGCAGTTTTATGATTTGG3693 
GTGCGTTTTTGTGTTTATACTGGGCCAGGTCCTGGTAGAACCTTTCAACAAACAACCAAA3753 
CAAAAAGAAAACACAAAGAAATGGGGGGAGGGTAGGGTGTTGAAGGGGGACAAAAAGGGG3813 
AGAGATTGAGAATGATGTATTTTTTTGCTGAATCAGAATTCACTTTCAGATAACTCATGA3873 
AAAGTGGTGCTCTAAATAAAATGAATTCTATATTAGTTGCCTGTGTTTATAAAAGTTATT3933 
ATTTTTTAACTGCAGAAACTCTTAAACCACAGCACTTGATCCAGAGTGGTGAAAACCAAT3993 
AAATAACCAGGCACCCAAAAAAACATTTAAATTTAGGGTCAGGGACAGAGGAATTTGGAG4053 
GTTTAGATGTGATATTCTACCCTAAAAACACCTAGTAACTGAATGGCTTTTCTTTGGAGG4113 
GTAATACATTTTAAAACATTTAGTGTGCCACCTACTGCTCCACAGTGACTAGAGAGCCTC4173 
TATTTCTTGGTGACTTGGGACTAGATCAGATGCCAAATGTACAAAGTTTCTTAAGAGTTG4233 
AGATTATATCATCTGAAGTCATCTTATTTTAGCCAAATCTTTTTAATTTCACCGGCAAAT4293 
CTGTGAAGGAAAACACTTGATGTTCAAAAAGAATAGTACATTTTAAAAGCTGCGATTTTA4353 
AACAGTTGTTAATGTTAAAAAAAAAAAGCACTAGAGGTATTTTTAAACATAGAACTCTTC4413 
CATAAAAAGTTGATTTGTTTTTGCTGTTATTGACTTGAAACATCATCAGTTTTAAATAAA4473 
ATGCATTTGTAAAAAAACCGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA4524 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 420 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
MetMetSerMetAsnSerLysGlnProHisPheAlaMetHisProThr 
151015 
LeuProGluHisLysTyrProSerLeuHisSerSerSerGluAlaIle 
202530 
ArgArgAlaCysLeuProThrProProLeuGlnSerAsnLeuPheAla 
354045 
SerLeuAspGluThrLeuLeuAlaArgAlaGluAlaLeuAlaAlaVal 
505560 
AspIleAlaValSerGlnGlyLysSerHisProPheLysProAspAla 
65707580 
ThrTyrHisThrMetAsnSerValProCysThrSerThrSerThrVal 
859095 
ProLeuAlaHisHisHisHisHisHisHisHisHisGlnAlaLeuGlu 
100105110 
ProGlyAspLeuLeuAspHisIleSerSerProSerLeuAlaLeuMet 
115120125 
AlaGlyAlaGlyGlyAlaGlyAlaAlaAlaGlyGlyGlyGlyAlaHis 
130135140 
AspGlyProGlyGlyGlyGlyGlyProGlyGlyGlyGlyGlyProGly 
145150155160 
GlyGlyGlyProGlyGlyGlyGlyGlyGlyGlyProGlyGlyGlyGly 
165170175 
GlyGlyProGlyGlyGlyLeuLeuGlyGlySerAlaHisProHisPro 
180185190 
HisMetHisSerLeuGlyHisLeuSerHisProAlaAlaAlaAlaAla 
195200205 
MetAsnMetProSerGlyLeuProHisProGlyLeuValAlaAlaAla 
210215220 
AlaHisHisGlyAlaAlaAlaAlaAlaAlaAlaAlaSerAlaGlyGln 
225230235240 
ValAlaAlaAlaSerAlaAlaAlaAlaValValGlyAlaAlaGlyLeu 
245250255 
AlaSerIleCysAspSerAspThrAspProArgGluLeuGluAlaPhe 
260265270 
AlaGluArgPheLysGlnArgArgIleLysLeuGlyValThrGlnAla 
275280285 
AspValGlySerAlaLeuAlaAsnLeuLysIleProGlyValGlySer 
290295300 
LeuSerGlnSerThrIleCysArgPheGluSerLeuThrLeuSerHis 
305310315320 
AsnAsnMetIleAlaLeuLysProIleLeuGlnAlaTrpLeuGluGlu 
325330335 
AlaGluGlyAlaGlnArgGluLysMetAsnLysProGluLeuPheAsn 
340345350 
GlyGlyGluLysLysArgLysArgThrSerIleAlaAlaProGluLys 
355360365 
ArgSerLeuGluAlaTyrPheAlaValGlnProArgProSerSerGlu 
370375380 
LysIleAlaAlaIleAlaGluLysLeuAspLeuLysLysAsnValVal 
385390395400 
ArgValTrpPheCysAsnGlnArgGlnLysGlnLysArgMetLysPhe 
405410415 
SerAlaThrTyr 
420 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CCAGCGGCTGCAAGGGGACCA21 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TGCCCGCCGACCAGGGTGCT20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CCCAAGAAGCCCTGAGC17 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CCCTCAAGAAAGGGGAGGGGG21 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CTGATCTAGTCCCAAGTCACC21 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
ACAGCACTTGATCCAGAGTGG21 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GGCATCGTGATGGACTCCG19 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GCTGGAAGGTGGACAGCGA19 
__________________________________________________________________________