Title of Invention


Abstract A cDNA molecule that encodes a protein designated Labyrinthin (Lab) is isolated and its nucleotide sequence is determined. The protein, or peptides derived from the protein, is markers useful to define novel case of cancers. Diagnostic assays for these cancers use antibodies to Lab or nucleotide probes that hyberdize with the lab gene or a fragment therefrom. Vaccines useful either to prevent initial recurrence of cancers in subjects who test positive for Lab (or lab), or to prevent initial occurrence of cancer, use proteins or peptides derived from Lab. Expression of Lab via immunogenic assays is used to monitor effects of cancer treatments. Antisense molecules against lab are used in treatments. Sense molecules of lab are used to restore lab function is diseased normal cells, for example, gland cells.
The invention relates to a gene encoding a protein and peptides therefrom that
includes an epitope, a cancer associated antigen, useful as a marker that is not
restricted to previously defined histological classes of cancer. Antigenic peptides are useful as a vaccine for treatment and prevention of cancer, and for the preparation of new, specific, monoclonal antibodies. Antisense molecules are useful in pharmaceutical compositions and are useful for diagnosis and treatment.
Cancer1 is a leading cause of death in men and women throughout the world.
In the United States alone, over I million new cases are diagnosed each year, and over 0.5 million deaths are reported annually (Landis, et al., 1998). Historically, tumors are grouped and treated, based in part by the tissues in which they arise, e.g.- breast cancer, colon cancer, and lung cancer, and the like. Yet, within lung cancer, for example, it is well recognized that these tumors are a very heterogeneous group of neoplasms. This is also true for tumors arising in other tissues. In part, because of
this heterogeneity, there are complex and inconsistent classification schemes which
are used for human tumors. Previous attempts to treat cancer have been hampered by :
1) the arbitrary classification of tumors arising within given tissues, and 2) by using
microscopic methods based on how these tumors look (histological classification).
Although existing classifications for
Terminology used herein is as follows, "cancer is a malignant tumor, wherein a
"tumor" is an abnormal mass of tissue, that need not be malignant. "Neoplasm"
is a form of new growth.
various tumor types have some prognostic value, almost all of the
classifications fail to predict responsiveness to treatments and likelihood of
cure or disease course. Improved classification schemes based on the
biological constitution of these neoplasms is required to significantly alter the
survival statistics of humans who have cancer. One approach to solving
these problems is to locate molecules specific to tumors, preferably antigens
in molecules that are markers for cancer cells. (A "marker" is defined herein
as any property which can be used to distinguish cancer from normal tissues
and from other disease states.) The markers' presence is then a basis for
Monoclonal antibodies (MCAs) prepared by somatic cell hybridization
techniques, usually in mice, are useful molecular probes for the detection and
discrimination of cellular antigens, and therefore have great potential for
detecting cancer associated antigens. These antibodies bind to specific
antigens and the binding is detectable by well known methods. When
binding occurs, the inference is made that a specific antigen is present.
Those cancer associated antigens which are exposed to the cell surface or
found in the cancer mass, are molecular targets for the immune systems
(including host antibodies) of the host. Recent findings suggest that cancer
patients who have antibodies against their tumors, do better than those who
do not mount this type of immune response (Livingston, et a/., 1994).
Therefore, natural, induced, or administered antibodies are a promising
therapeutic approach.
The humanization of non-human MCAs (the process by which
non-human MCA reactive sites are shuttled into cloned human antibodies
and expressed) results in reduced immunogenicity of the foreign antibodies
without the loss of their specific binding in in vivo and in ex vivo applications.
MCAs can be used as In vivo imaging aganti, diagnostic taati, and for
inarapy (Radesevteh, etat. 1988,1990 Resen, etal. 1988).
Vaccine therapy is a well established approach directed at inducing an
immune response without exposure to the causative agent of a disease or
condition. Many vaccines are available, for example, to stimulate a response
in a host to bacterial and viral agents. The use of tumor associated antigens
(markers) in a vaccine could prevent primary cancer occurrence, and couid
also provide a means to prevent recurrence of the disease.
Gene therapy is a means by which the genetic make-up of cells is
modified to express the gene of interest. There are many forms of gene
therapy including: gene replacement, antisense suppression therapy, and
surrogate gene expression. Discovering genes encoding cancer-associated,
preferably cancer-specific antigens (markers) opens the door to genetic
intervention against cancer cell proliferation. The accurate and consistent
use of a cancer marker to differentiate cancerous from normal tissue, not only
has diagnostic potential, but is also desirable for treatment and prognosis.
Therefore, such markers have been sought.
Recent studies have shown that the enzyme encoding human aspartyl
beta-hydroxylase (HAAH) is overexpressed in some human adenocarcinoma
cell fines, and in primary hepatocetlular cancers, therefore could be a marker.
The gene said to encode HAAH has been cloned and sequenced (Gronke,
et al., 1989, 1990; Wang, et al., 1991; Jia, et al, 1992, 1994; Korioth, et al.,
1994; Lavaissiere, et al., 1996). However, little is known about HAAH
expression in human tumors in general (Lavaissiere, et al, 1996).
The study of the HAAH enzyme grew out of the study of its bovine
counterpart (Gronke, etaL, 1989, 1990; Wang, etaL, 1991; Jia, et al, 1992).
Bovine aspartyl beta-hydroxylase is an intracellular, glycosylated protein,
localized in the rough endoplasmic reticulum. The protein has been reported
to have three major species of molecules; a 85 kilodalton form, and two active
forms with molecular weights of 56 and 52 kilodaJtons respectively
(Lavaissiere, et al., 1996).
Using standard biochemical methods, bovine aspartyl
beta-hydroxylese (bAAH) has been purified and characterized (Gronke, et al.
1990; Wang, etaL, 1991). The activity of the enzyme has been shown to be
correlated with the 52 and 56 kilodalton species which were purified
Immunologically, a related higher molecular weight form (85-90 kilodalton) was also
observed. As part of the purification, bAAH is bound to Con A sepharose, which is
consistent with the conclusion that the enzyme is glycosylated. (Subsequent reports
on the DNA sequence show three possible glycosylation sites, with one site being
very close to the known active enzyme domain.) The protein is very acidic in nature,
and a detergent is not required to solubilize the active fraction. The active enzyme
site is dependent from the biochemically isolated bovine protein (bAAH) on the
presence of histidine at position 675 (Jia, et al, 1994).
A partial amino acid sequence was obtained for HAAH. DNA probes (a DNA
probe is a molecule having a nucleotide sequence that is capable of binding to a
specified nucleotide sequence under certain conditions) deduced from this amino acid
sequence was used to screen a bovine cDNA library (Jia, et al, 1992). (A cDNA
library contains the sections of DNA that encode for gene products, e.g. peptides as
opposed to genomic DNA). Several overlapping cDNA sequences in the library
contained a 764 amino acid open reading frame (ORF) sequence which will be
expected to encode an 85 kilodalton protein. Also present in this ORF sequence were
rwo other possible start codons, that is, locations at which encoding begins. The most
3' start codon was preceded by a ribosome binding site. Translation of the clone
having this sequence resulted in a protein that was about 85 kilodaltons. Antiserum
was raised to the membrane fraction of human MG-63 calls and was used to
immunoscreen a cDNA library made from MG-63 cells. Data on one clone was
reported which could encode a 757 amino acid protein, and, by sequence analysis, was
found to have strong N-terminal homology with bAAH (Korioth, et al, 1994). When
this clone was used in an in vitro translation system (an artificial cocktail of normal
cell cytoplasm used to convert mRNA into protein), a 56 kilodalton protein was
produced. It was suggested that this was due to posttranslational cleavage.
The HAAH enzyme is responsible for the modification of specific aspartic
acid residues within the epidermal growth factor-like domains of
proteins. It has been hypothesized that these modified aspartic acid residues
allow the epidermal growth factor-like domains to become calcium binding
domains. (Gronke, et al, 1989,1990; Wang, et al., 1991; Jia, et al, 1992,
1994; Korioth, et al, 1994; Lavaissiere, etai, 1996).
An enzyme related to HAAH, asparty! beta-hydroxylase (AAH), was
first studied because it specifically modified select aspartic acid or
asparagine residues in a group of biologically important proteins including the
vitamin K-dependent coagulation factors VII, IX, and X. Other proteins like C,
S, and 2 also have this modification (Gronke, et al, 1989, 1990; Wang, et al,
1991; Jia, et al, 1992,1994; Korioth, ef a/., 1994; Lavaissiere, et a/., 1996).
Aspartic acid and asparagine residues have been shown to be modified by
HMH in proteins containing epidermal growth factor-like domains. The
function of the beta-hydroxyasparfic and beta-hydroxyasparagine residues is
unknown, however, it-has been speculated that this modification is required
for calcium binding in the epidermal growth factor EGF-iike domains of
selected proteins.
Antibodies were raised to human hepatocellular carcinoma FOCUS
cells (Lavaissiere, et a/., 1990). One MCA reacted with an antigen that was
highly expressed in hepatocellular carcinomas (Lavaissiere, et al., 1996).
fmmunoscreening using this antibody and a lambda gt11 HepG2 library
resulted in the isolation of a partial cDNA, which was subsequently used to
isolate a larger clone.
A human adenocarcinoma cell line designated A549 was reported as
having very high levels of HMH activity (Lavaissiere, et al., 1996). A mouse
monoclonal antibody designated MCA 44-3A6 (U.S. Patent No. 4,816,402)
was produced against the human adenocarcinoma cell line A549 (ATCC
accession number CCL 185) (Radosevich, et a/,, 1985). The antibody
recognized a cell surface, non-glycosylated antigenic protein having an
estimated apparent molecular weight of 40 kDa),
The antigen was expressed by A549 cells, and was found to be a good
adenocarcinoma marker; that is, it was frequently expressed by cancers
which looked like adenocarcinomas when examined histologically
(Radosevich, etal., 1990a; Lee, etal., 1985). MCA44-3A6 is unique in that it
is the first monoclonal antibody which has this binding specificity. The results
from an International Workshop for Lung cancer confirmed other related
published findings on MCA44-3A6 (Stahel, 1994).
The antibody designated MCA 44-3A6 has clinical utility because it
differentiates antigens associated with adenocarcinomas. The normal and
fetal tissue distribution of the antigen is restricted to some glandular tissues
(Radosevich, et a/., 1991). Detection can occur on formalin fixed-paraffin
embedded tissue (Radosevich, etal., 1985, 1988, 1990a, 1990b; Lee, etal.,
1985,1986; Piehl, et al 1988; Combs, etal., 1988b, 1988c; Banner, etal.,
1985). The antibody has a restricted binding pattern within human pulmonary
tumors (Lee, etal., 1985; Banner, et al., 1985; Radosevich, et al, 1990a,
In a study of over two hundred pulmonary cancers, MCA 44-3A6 was
found to react with all of the adenocarcinomas tested, many of the large cell
carcinomas, as well as with subsets of intermediate neuroendocrine small cell
lung cancers, well-differentiated neuroendocrine small cell carcinomas,
carcinoids, but not mesotheliomas. MCA 44-3A6 does not react with
squamous cell carcinoma, bronchioloalveolar carcinoma, or small cell
carcinoma (Lee, et al, 1985). MCA44-3A6 is useful in distinguishing
adenocarcinomas that are metastatic to the pleura from mesothelioma (Lee,
et a/., 1986). The antibody has selected reactivity among adenocarcinomas
and in large cell carcinomas (Piehl, et al, 1988; Radosevich, et al., 1990b).
In a study of over 40 cases of lung cancer comparing cytological and
histological findings, MCA 44-3A6 was found to be useful in cytological
diagnosis and was consistent with the histological finding (Banner, et al,
1985). Histology is the study of tissues (which are made of cells). Cytology
li mo itudy ef cam which have been removed frem the organizational context
which is commonly referred to as tissue. Cells removed from tissues do not
always behave the same as if they were in the tissue from which they were
derived. Fortunately, the antigen detected by MCA 44-3A6 expressed in
adenocarcinoma cells in tissue behaves in the same ways as
adenocarcinoma cells removed from tissues. This is a very diagnostically
important characteristic. Similar correlations using cytologically prepared cell
blocks of pulmonary carcinomas, as well as ACs presenting in body fluids
from other sites throughout the body were demonstrated (Lee, et al/., 1985;
Spagnolo, et al., 1991; Combs, et al., 1988c). Also, MCA44-3A6 binds to
adenocarcinomas from sites other than lung cancer. The expression of the
antigen in primary and metastatic lesions was also reported ( Combs, et al.,
1988a). The utility of the MCA antibody in differentiating cancer from benign
lesions in human breast tissue was also noted (Duda, et al, 1991).
The cellular localization of the antigen detected by MCA 44-3A6 was
determined. By using live cell radioimmunoassays (a radioactive antibody
test directed at determining binding of the antibody to live cells),
immunofluorescence, and live cell fluorescence activated cell sorter (FACS)
analysis, the antigen detected by MCA 44-3A6, was shown to be on the
outside surface of the cell (Radosevich, etal., 1985). Additional studies
using immunogold-electron microscopy and FACS analysis have
demonstrated that this antigen is non-modulated (that is not internalized by
the cancer cell when bound by an antibody), is expressed on the extracellular
surface of the plasma membrane, and is not cell cycle specific that is, the cell
makes protein all the time it is going through the process of cell replication,
and also when it is not dividing (Radosevich, et a/., 1991). The antigen is not
found in the serum of normal or tumor bearing patients, and is not shed into
the culture media by positive cell lines (that is, cancer cells are known to bleb
off portions of their cell membranes and release them into the surrounding
fluid.) (Radosevich, et at., 1985). Recently 3 of 27 randomly tested
adenocarcinoma patients were found to have naturally occurring antibodies
to the antigen. In addition, radiolabeled MCA 44-3A6 was used to localize
A549 tumors growing in nude mice. A douxorubicin immunoconjugate
MCA 44-3A6 is selectively toxic in vitro (Sinkule, ef a/., 1991).
Determination of the nucleotide and amino acid sequences of the antigen
detected by MCA 44-3A6 would enhance the usefulness of this antigen in cancer
diagnosis, treatment and prevention.
The antigen detected by the antibody MCA 44-3A6 as described in the
Background is now designated as "Labyrinthin." A gene (designated labyrinthin;
abbreviated lab) characterized by a unique nucleotide sequence that encodes the
antigen detected by MCA 44-3A6 was isolated and characterized, (lab notation
signifies the nucleic DNA/RNA forms; "Lab" notation refers to the protein which is
encoded by the lab DNA/RNA).
The invention described herein used the antibody MCA 44-3A6 as a tool to
done the gene encoding Lab. In addition, an epitope (the necessary binding site for
an antibody found on the antigen) for MCA 44-3 A6 was identified on the Lab protein
expressed by the clone to be PTGEPQ.2 The epitope represents an important
immunodominent sequence; that is, when injected into animals, the animals readily
produce antibodies to this sequence.
An aspect of the invention is the use of lab DNA in the sense3 expression
mode for: 1) the marking of human tumors by nucleotide probes; 2) the detection of
DNA and mRNA expression of lab in cells and tissues; 3) the transformation of cells
into a glandular-like cell type; 4) the production of Lab antigen in vivo for
immunization; 5) the ex vivo expression of Lab for immunization to produce
antibodies; and 6) production of Lab in vitro. Use of an antisense molecule, e.g. by
production of a mRNA or DNA strand in the reverse orientation to a sense molecule,
to suppress the growth of labyrinthin-expTessing (cancerous) cells is another aspect of
the invention.
Standard abbreviations for amino acids.
The normal transcription of a DNA sequence which proceeds from the 3' to the
5' end to produce a mRNA strand from the sense strand of DNA, the mRNA
being complementary to the DNA.
An aspect of the invention is a vector comprising a DNA molecule with a
nucleotide sequence encoding at least an epitope of the Lab antigen, and suitable
regulatory sequences to allow expression in a host cell.
Another aspect of the invention is an ammo acid sequence deduced from the
protein coding region of the lab gene. Selected regions of the sequence were found
via immunological methods, to produce effects corresponding to effects from both
naturally occurring (from cancer cells), chemically produced (synthetically produced
peptides), and expression products of the cloned lab gene.
Another aspect of the invention is the use of the entire deduced ammo acid
sequence of Lab, peptides derived from Lab, or chemically produced (synthetic) Lab
peptides, or any combination of these molecules, for use in the preparation of vaccines
to prevent human cancers and/or to treat humans with cancer. For purposes of the
present invention, "humans with cancer" are those persons who have the Lab antigen
detected in their cells. These preparations may also be used to prevent patients from
ever having these tumors prior to their first occurrence.
Monoclonal antibodies directed to the Lab protein, or antigenic components or
derivatives of Lab proteins, are useful for detection of Lab and for other purposes.
Monoclonal antibodies which are made in species other than those which react with
the Lab antigen can be modified by a number of molecular cloning methods such that
they retain their binding with the Labyrinthin peptides, yet are not immunogenic in
humans (Sastry, et a/., 1989; Sambrook, et al, 1990). In brief, this is done by
replacing the binding site sequence of a cloned human antibody gene, with the
binding site sequence of the non-human monoclonal antibody of interest. These
"humanized" MCAs are used as therapeutic and diagnostic reagents, in vivo, ex vivo,
and in vitro.
The use of the Lab protein or antigenic peptides derived therefrom in
diagnostic assays for cancer is a way to monitor patients for the presence and amount
of antibody that they have in their blood or other body fluids or tissue. This detection
is not limited to cancers of a class or classes previously defined, but is useful for
cancer cells that have the Lab marker
antigen. The degree of seroconversion, as measured by techniques known to those of
skill in the art [e.g., ELISA (Engrail and Perlmann, 1971)] may be used to monitor
treatment effects.
Treatment with antisense molecules to lab or antibodies to Lab in a
pharmaceutical composition is an approach to treat patients who have Lab in, or on,
their cancer cells.
FIG. 1 is the nucleic acid sequence of the lab gene.
FIG. 2 is the amino acid sequence for Lab, deduced from the lab gene.
FIG. 3 is an illustration of the lab gene and how it is related to the HAAH
Molecular Biology QfJLabyrinthin: To demonstrate that the epitope MCA
44-3A6 is encoded by a protein sequence, high molecular weight DNA from the cell
line A549 was isolated. This DNA was co-precipitated (via calcium) with a plasmid
(pSVneo), and used to transfect a mouse cell line designated B78H1 cells (Albino, el
al, 1985). This mouse cell line is negative for the expression of the epitope and was
reported to have a high frequency of incorporation and expression of any human DNA
sequences. If a given B78H1 cell was in a state to take up DNA, it would be expected
to have taken up both human DNA and the plasmid DNA. The plasmid DNA makes
the cell resistant to G418 (a normally toxic drug). Therefore, if a cell normally
sensitive to G418 growth inhibitor grows in G418, it had to have taken up the
plasmid, and may also have taken up one or more A549 DNA sequences. After G418
selection (a way of choosing only cells which have resistance to growth in G418 by
the uptake/expression of the Neo gene on pSVneo plasmid, and therefore representing
cells that were in a state to uptake other DNA at the same time), approximately 15 of
IxlO5 clones were detected using immunoselection with MCA 44-3A6. This finding
is consistent
with a conclusion that human A549 ceils have DMA that encodes Lab and
possesses the regulatory sequences necessary for the expression of Lab.
Comparison of UAAH and Labvrinthirr, Because the DNA sequence of
lab was determined as an aspect of the present invention, HAAH and lab
could be compared. HAAH and the lab nucleotide sequences have some
internal fragment similarities, but are different on either side of the fragment,
and are related to different products. This conclusion is based in part by the
analysis and homology of the DNA sequences reported for these two genes.
Specifically, the lab 5' region has no homology with HAAH. The protein
coding region of lab has about a 99.6% homology with an internal segment of
the proposed protein coding region for HAAH. The 3' region has no
homology with the HAAH reported sequence. Virtually all of the other data
comparing HAAH and labyrinthin are different, for example: 1) molecular
weights of the proteins, 2) cellular localization, 3) chromosome localization,
4) histological presentation in normal tissues and tumors, 5) northern blot
expression, 6) immunological findings.
Although the protein coding region of lab is identical to an internal
region of the sequence reported for HAAH, the 5' untranslated region of
HAAH is different, and part of the 5' translated protein coding region of HAAH
is missing from that found in the lab clone. From both HAAH and lab clones,
the deduced protein would be expected to be very acidic in nature, and
therefore would run anomolously in SDS gels. As predicted, the Lab protein
migrates anomolously in SDS gels. What was cloned and disclosed in the
present invention migrates identically to the native protein found in several
cell lines. Convincing evidence that the correct gene fragment encoding the
antigen detected by MCA 44-3A6 has been cloned (mRNA) is that when the
recombinant protein is made, that recombinant protein should act (in this
case - have an apparent molecular weight) the same as independent
biologically derived source of that protein. Lab provided from clones has the
characteristics of Lab from cells.
The deduced amino acid sequence encoded by HAAH requires the use of an
open reading frame which would produce a protein that is 85-90 kilodaltons, and does
not take into account that there are several start codons and other shorter open reading
frames. The deduced HAAH protein (biochemically) is glycosylated and the reported
sequence has glycosylation sites ( Korioth, et al, 1994; Lavaisslere, et al, 1996). To
the contrary, Lab is not glycosylated, nor does it have predicted glycosylated sites.
The deduced HAAH amino acid sequence contains a region shared by the Lab
amino acid sequence which is predicted to be very hydrophobic. Lab requires strong
detergents in order to be soluble; HAAH does not, The increased expression of
HAAH (by enzyme activity measurements) in the same cell line (A549) which was
used to clone and study lab extensively, suggests that both of these gene products may
be important to the AC phenotype and that at least A549 cells make both functional
HAAH and Lab. Successful transfections of the antisense to lab into A549 resulted in
a marked decrease in expression of lab and in the growth rate of the cells. The
expression of a sense lab construct in NIH-3T3 cells (normal mouse fibroblasts)
resulted in a marked change in phenotype, a phenotype consistent with that of ACs.
Therefore, lab expression is associated with conversion of normal cells to cancerous
cells. Lab and HAAH have potential calcium binding domains in common.
cDNA Library Contraction and Cloning: A cDNA lambda gtl 1 phage library
was constructed using mRNA which was isolated from actively growing A549 cells
(Sambrook, et alt 1990), This oligo(dT)-primed cDNA was cloned into the Eco RI
site using Eco RI linkers. The library has about 83% clear (containing an insert)
plaques with a liter of 1.2 x 10'°/ml representing a minimum of 1.46 x 106
independent plaques which, by Polymerase Chain Reaction, have insert sizes ranging
from 0.6 to 5 kilobases. Since Lab is a 40 kiJodalton integral protein, (a protein which
is embedded in the plasma membrane) the theoretical full length mRNA encoding this
protein, including a potential leader sequence is estimated to be about 1.1 kilobases.
This library was
immunoscreened using the antibody MCA 44-3A6. Eight independently derived
phage stocks (identical phage which are from the same plaque) were isolated. These
have all been plaque purified by repeated cycles of immunoscreening/isolation. Upon
Eco RI digestion of these eight isolates, inserts of about 2kb were seen. The largest
insert was isolated (2A1A1) and the Eco RI fragment was cloned into the pGEM-3Z
Sequencing and Sequence Analysis: The DNA fragment designated 2A1A1
was found to have an insert of 2442 base pairs in length (FIG. 1), containing a 5'
untranslated region, a ribosome binding site, and a start codon which would be
expected to encode a 255 amino acid protein (FIG. 2). The 3' untranslated region is
remarkable in that it contains only four instability sequences; ATTTA (Xu, et al,
\ 997). In addition there are sequences found in the very 3' end of mRNA's which
result in adenylation of the mRNA (Sambrook, et al.t 1990). The Jab sequence
contains both a sub-optimal (ATTAAA) and optimal (AATAAA) poly-adenylation
site. These are sequences found in the very 3' end of mRNA's which result in
adenylatlon of the mRNA. This finding provides molecular data which supports the
cellular and biochemical data that has been outlined herein. (The HAAH clone has a
poly A signal, but the whole 3' region has not been sequenced.)
A calcium binding site motif is noted in the Lab amino and sequence (FIG. 2),
however, it is out of the known required structural context to be a binding site. In this
case, the calcium limiting sequence is there, but it is not in a protein sequence context
that is known to make it work as a binding site. Homology was noted with lab and an
EST clone (designated #05501) which represented only a portion of the 3'
untranslated region and independently confirmed this portion of the sequence. Some
internal fragment homology is also noted with HAAH, but the 5' untranslated and part
of the 5' translated region is different (58 amino acids), as well as a major portion of
the 3' coding region is missing in lab (FIG. 3).
Genomic DNA Cloning and Analysis: Using a PCR fragment representing the
protein coding region of lab as a probe, a genomic lambda
FIX II library made from the human pulmonary fibroblast cell line WI-38 was
screened. Ten primary plaques were isolated out of approximately 1x106
screened plaques. Using seven of these as target DNA, Polymerase Chain
Reaction conditions were established with primers for the protein coding
region, producing a 765 base pair fragment, the expected protein coding
region for lab. On Northern blots (a method used to qualitatively assess
mRNA) lab only detects one band noted at 2.7 kilobases. The recombinant
protein made from the lab clone, when tested on Western blots (a method
used to qualitatively define proteins) using MCA 44-3A6, has the same
relative mobility as the Lab protein when made by A549 cells.
Lab and HAAH genes give different results in the proteins they
encode. HAAH consistently gives two bands on Northern blot analysis (2.6
and 4.3 kilobases) suggesting that the 2.6 kilobase band is due to alternative
splicing, i.e. the cell cuts and splices the mRNA. Also, if lab and HAAH are
the same gene, HAAH should be detected in all tissues and cancer cell lines
in which Lab is found. However, Lab is not seen on Northern blots of cell
lines EMT6 or QU-DB, nor is there immunoreactivity in these cells; indicating
that Lab mRNA is not made, and that Lab protein is not produced in these
cells. Lab protein is rarely expressed in normal cells, where both the HAAH
mRNA and HAAH protein have been reported to be expressed in almost
every tissue studied.
mRNA Analysis: Northern blot analysis of the DNA fragment from the
A549 cell line using lab cDNA as a probe identified a single band of about 2.7
kilobases. This is expected based on the cDNA (2442 base pairs) and a
poly-A tail of about 300 base pairs. Northern blot analysis of the mouse cell
line, EMT6, and of the human large cell carcinoma cell line, QU-DB, confirm
that no transcript for lab is produced by these cells. This is consistent with
immunoassays which are negative for lab expression on these cells.
Antlsense end Sense eDNA Expression* The plasmld (pBK-CMV)
(Sambrook, ef a/., 1990) may carry either the sense or antisense full length
cDNA lab into A549 and NIH 3T3 cells. An antisense molecule can be, for
example, a complementary sequence to a sense molecule that hybridizes
with the sense molecule, preventing its expression. Using the MTT assay
(Siddique, ef a/., 1992) to assess the growth rate of A549 cells expressing
antisense to lab, a marked reduction in growth rate was noted. The antisense
transfected A549 cells appear to have a greater degree of contact inhibition,
A detectable amount of Lab is reduced in these antisense transfected cells.
N1H-3T3 cells convert from a fibroblast-!ike cell type morphology (large, thin
spindle shaped) to a large, adenocarcinoma appearing cells (very round,
plump) when sense expression occurs,
Chromosome Localization: The chromosome localization for lab, using
full length cDNA as a probe via in situ hybridization (Sambrook, ef a/. 1990) is
tentatively on chromosome 2q12-14, with possibly some reactivity to
chromosomes 4 and 8. Using the same probe (the full length cDNA
sequence of lab) and FACS sorted chromosomes (Lebo, et al. 1985) staining
was also noted on chromosome 2, with weak staining on 4 and none on 8.
The use of genomic clones will be of particular value in resolving these data
because higher stringency hybridization conditions than that allowable for the
cDNA, can be used, thereby reducing background signals. This is yet
another proof that the correct gene was cloned and that the results are not
due to a method artifact. There may be mutations in the genomic DMA of
tumors and for the present invention, DMA was cloned from tumor cells
(A549). Therefore, a mutated gene could have been cloned. However, that
is not the case because the genomic DNA from a normal cell (DMA) produced
the same sequence as what cloned as described herein. Therefore, a normal
gene was cloned from A549 cells. The weak signals on chromosomes 4 and
8 are consistent with a pseudogene or a related gene. For example, HAAH
has been reported to be on chromosome 8q12 by in situ hybridization, so this
result on chromosome 8 could reflect the HAAH and lab sequence homology.
Protein Meleqular CheraetarltBtlon of Labvrlnthliv. Previous work using
Western blot analysis (a qualitative assay to assess antigens) has shown that
the Lab antigen is a 40 kilodalton (by relative mobility) protein detectable in
A549 cells (Radosevich, et al., 1985). The epitope does not appear to be modulated
or blocked by lectins, and is selectively expressed on the cell surface, primarily
localized to the plasma membrane. (Radosevich, et al, 1985,1991). Lab is sensitive
to proteases, but not lipid or carbohydrate altering reactions (Radosevich, et al.,
1985). The biochemical properties of Lab are consistent with Lab being an integral
membrane protein.
Having a deduced amino acid sequence from the lab gene of the present
invention, allows further characterization of the Lab protein. Extensive computer
analysis of Lab has identified a eukaryotic leader-like sequence and theoretical
cleavage site, 3 myristylation sequence sites, a weak membrane anchoring domain
(MAD I), and a strong membrane anchoring domain (MAD II) (FIG. 2). [(In the
HAAH sequence, there are 58 (theoretical) amino acids followed by a sequence
homology in the Lab protein coding sequence and an additional 445 amino acid 3' to
the lab sequence.)]
When Lab is expressed as a fusion protein in a bacteria! GST fusion
expression system (pGEMEX-2T) (Amereham Pharmacia Biotech, Inc., Piscataway,
New Jersey, 08854, USA), and subjected to Western blot analysis using the antibody
MCA 44-3 A6, the resulting blots demonstrate that the expressed cleaved fusion
protein has the same relative mobility as the protein detected in A549 cells. The
deduced molecular weight for Lab is 28.8 kilodaltons and on Western blots it has a
relative mobility identical to the form expressed by A549 cells (apparent relative
mobility = 40 kilodaltons). The 55 glutamic and 27 aspartic acid residues a (82
residues combined) are almost uniformly distributed throughout the protein (255
amino acids total; 228 no leader sequence), except for the leader sequence and the
strongest membrane anchoring domain (MAD II). These data suggest that Lab
migrates anomelously in SDS gels. Cell lines other than A549 (e.g. adenocarcinomas
DU-145, ATCC #HTB-81; ZR-75-1, ATCC # CRL-1504, and so forth) have an
antigen detected with the same molecular weight antigen as Lab. Neither a 85-90
kilodalton molecular weight species, nor a
52 and 56 kilodalton molecular weight species is noted when probing
Western blots for Lab,
Fpitnpp Mapping Using *HA Antibody M^ 44.3AR and Vaccine
Feasibility of Lab: Using Polymerase Chain Reaction and the GST fusion
protein system, subclones of the protein coding region were made, and
epitopes mapped the binding of MCA 44-3A6 to six amino acids (PTGEPQ)
representing amino acids #117-122 of Lab ("P" peptide). In order to
determine this epitope, the entire coding region was divided into regions,
Polymerase Chain Reaction primers were designed to amplify each region,
and the subsequent expression of Polymerase Chain Reaction products were
cloned and tested by Western blot analysis using the antibody MCA 44-3A6.
The DMA fragment representing the positive Western blot result was
then further subdivided. Polymerase Chain Reaction products were
generated and cloned, expressed, and tested via Western blot. Constructs
were made in this way both from the 5' end and the 3' end and the intervals of
the number of amino acids were reduced upon each round. This resulted in
the last round representing a one amino acid difference from the previous
round (in both directions), such that one could deduce the exact binding site
of the MCA 44-3A6. This demonstrates that at least these six amino acids
are exposed to the external cell surface. To further prove the point, the DMA
encoding only these six amino acids have been cloned and the fusion protein
is positive by Western blot analysis. Synthetically prepared "P" peptide can
be specifically detected by MCA 44-3A6, and the synthetic peptide was
immunogenic in 5 of 5 mice tested. Computer analysis/ modelling also
predicted that this epitope would be very immunogenic using computer
assisted analysis (GCG programs) (Genetics Computer Group, Madison, Wl
Vaccine Preparation: A vaccine is a preparation of antigen(s), which
when given to a host, results in the host producing antibodies against the
antigen(s). The host response results in the host being immune to the
disease to which the vaccine was directed. Vaccine treatment therefore,
prevents the clinical presentation of a disease, without the host being exposed to the
disease causing agents. Lab has all the characteristics of a preferred cancer vaccine.
The lab gene is frequently expressed by tumors which look like adenocarcinomas, is
expressed an the outside of the cells, is expressed by all of the cells within a given
cancer, is expressed at all times by these cancer cells, and is infrequently expressed by
normal cells. Lab protein (peptides) can be produced by any number of methods
using molecular cloning techniques, and can be produced in large quantities, thus
making it a practical antigen to use as a vaccine. After the Lab protein has been
purified so that it is suitable for injection into humans, it is administered to individuals
intradermally, subcutaneously, or by other routes, so as to challenge the immune
system to produce antibodies against this protein (peptides).
The use of molecular modeling and computer assisted analysis GCG programs
(Genetics Crystal Group, Madison, WJ 53703) allows the identification of small
portions of a molecule, slightly larger than an epitope (six to seven amino acids for
proteins), which are expected to be on the surface of a protein molecule. In addition,
the degree of hydrophobicity or hydrophilicity of a given sequence, and how
immunogenic the sequence would be in animals, can be determined (Genetics Crystal
Group, Madison, WI 53703). After defining which sequences meet these criteria, the
peptides are synthetically made, or produced by a number of standard methods. One
or more of these peptides can then be formulated to be used as a vaccine, and
administered to the host as outlined above, as a vaccine.
A vaccine comprising a molecule having an amino acid sequence selected
from the group of sequences encoded by the cDNA of FIG. 1, sequences of FIG. 2,
encoded by the cDNA, the peptides APPEDNPVED, EEQQEVPPDT,
DGPTGEPQQE, and EQENPDSSEPV, and any fragments or combinations thereof.
A given vaccine may be administered once to a host, or may be administered
many times. In order for some patients to recognize a given
vaccine, an acjuvant may also need to be administered with the peptides. Adjuvants
are nonspecific immune stimulators which heighten the immune readiness and aid in
the conversion of the host from not having detectable serum antibodies to having very
high titer serum antibodies. It is this high level (titer) of antibodies, which effectively
protects the host from the diseases or conditions to which the antibodies are directed,
Functional Studies: Studies directed at understanding the cellular function(s)
of Lab are extensions of cell localization/characterization studies (Siddique, el al.,
1992). Changes in levels of Lab in response to extracellular exposure to various
response to extracellular exposure to various cations (Ca-H-, Mg++, CU-H-, and Fe-H-)
were undertaken. Lab expression in A549 cells was only modulated by Ca++. Using
the highly specific fluorescent Fura-2/AM Ca-H- method of measuring cytosolic
Ca++, (Molecular Probes Inc., Eugene, OR 97402) it was demonstrated that; 1) the
internal Ca-H- concentration is higher in A549 cells than in QU-DB cells, and 2) that
the A549 cell line responds to various external Ca-H- levels (Siddique, et al, 1992).
Since pH can modulate intracellular free Ca-H- levels, external pH manipulations
should result in changes in the expression levels of Lab. Extracellular pH changes (in
the presence of normal Ca-H- concentrations) result in 1) a parallel change in
intracellular pH as measured by SNARF-1 AM/FACS, (Molecular Probes Inc.,
Eugene, OR 97402) 2) transcript levels increase for Lab (when compared to GAPDH
expression via Northern blot); and that 3) Lab protein also increases (using
Western/Slot blot analysis). The intracellular changes in pH (due to external changes)
for A549 cells are Identical to those reported for normal cells. The increased
expression of lab is also not due to cell death (as measured by MTT assays)
(Siddique, el a/., 1992). In addition, incubation of recombinant Lab at various pH
solutions does not alter immunoreactivity. Preliminary data suggests that when these
experiments are conducted on A549 cells grown in reduced Ca-H-, the induced
expression of lab is blunted.
Methods of Diagnosing Cancer Cells in a Sample of Cells: Biological samples
from a subject are used to determine whether cancer cells are
present in the subject. Examples of suitable samples include blood and
biopsy material. One method of diagnosis is to expose DMA from cells in the
sample to a labeled probe that is capable of hybridizing to the lab gene, or a
fragment thereof, under stringent conditions, e.g. 6x ssc; O.OSx blotto; 50%
formamtde; 42°C (Sambrook, et a/., 1990). Of course, the hybridizing
conditions are altered to achieve optimum sensitivity and specificity
depending on the nature of the biological sample, type of cancer, method of
probe preparation, and method of tissue preparation.
After contacting the sample with the probe, the next step is
determining whether the probe has hybridized with nucleotide sequences of
the DMA from the sample, from which the presence of the lab gene is
inferred, said presence being diagnostic of cancer.
Another diagnostic method is to obtain monoclonal antibodies
preferably labeled, either antibodies already existing, or new ones directed to
the antigenic peptides that are aspects of the present invention, and contact
a sample with these to detect the Lab antigen. These monoclonal antibodies
are useful in the development of very specific assays for the detection of Lab
antigen, and allow the tests to be carried out in many different formats;
resulting in a broader application in science and medicine.
The current invention is useful in that it describes a new gene which is
expressed on the surface of tumors, which was not previously reported. This
gene is not tissue specific, and therefore will allow the detection of tumors
regardless of the organ in which they arise. Likewise, the use of this gene to
produce a vaccine for these tumors, will have a very broad application.
Diagnostic tests will also have this broad tissue use, making the detection of
Lab//ai> a "pan-marker" for cancer, in particular for what have been
designated previously, adenocarcinomas.
Albino, AP, Graf, LH, Kontor, RRS, ef a/. DNA-mediated transfer of
human melanoma cell surface glycoprotein gp130: Identification of
transfectants by erythrocyte resetting. Mol. Cell. Biol. 5:692-697,1985.
Banner BF, Gould VE, Radosevich JA, ef a/. Application of
monoclonal antibody 44-3A6 in the cytodiagnosis and classification of
pulmonary carcinomas. Diag Cytopathol. 1:300-307,1985.
Brown, DT and Moore, M. Monoclonal antibodies against two human
lung carcinoma cell link. Br. J. Can. 46:794-801,1980.
Combs SG, Hidvegi DF, Ma Y, ef a/. Pleomorphic Carcinoma of the
Pancreas: A rare case report of combined histological features of
pleomorphic adenocarcinoma and giant cell tumor of the pancreas. Diag.
Cytopathol. 4:316-322,1988a.
Combs SG, Radosevich JA, Ma Y, ef a/. Expression of the Antigenic
Determinant Recognized by the Monoclonal Antibody 44-3A6 on Select
Human Adenocarcinomas and Normal Human Tissues. Tumor Biol.
9:116-122, 1988b.
Combs SG, Radosevich JA, and ST Rosen. Cytological expression of
the adenocarcinoma antigen marker in human body fluids. Tumor Biol.
Duda RB, August CZ, Radosevich JA and ST Rosen. Monoclonal
Antibody 44-3A6 as a Marker For Differentiation of Breast Cancer. Tumor
Biol. 12:254-260,1992.
Engvall, E and Perlmann, P. Enzyme linked immunosorbent assay
(ELISA): Quantitative assay of IgG. Immunochemistry. 8:87-874,1971.
Gronke RS, VanDusen WJ, Garsky VM, Jacobs JW, Sardana MK,
Stern AM, and PA Friedman. Aspartyl beta hydroxylase: In vitro
hydroxylation of a synthetic peptide based on the structure of the first growth
factor-like domain of human factor IX. PNAS. 86:3609-3613,1989.
Gronke RS, Weisch DJ, VanDusen WJ, Garsky VM, Sardana MK,
Stern AM, and PA Friedman, Partial purification and characterization of
bovine liver asparty! beta hydroxylase. J. Biol. Chem. 265:8558-8565,
Jia S, VanDusen WJ, Diehl RE, Kohl NE, Dixon RAF, Elliston KO,
Stern AM, and PA Friedman. cDNA cloning and expression of bovine
aspartyl (asparageinyl) beta-hydroxyiase. J. Biol. Chem. 267:14322-14327,
Jia S, McGinns K, VanDusen WJ, Burke CJ, Kuo A, Griffin PR,
Sardana MK, Elliston KO, Stern AM, and PA Friedman. A fully active
catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed
in Escherichia coli: Characterization and evidence for the identification of an
active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases.
PNAS 91:7227-7231, 1994.
Korioth F, Gieffers C, and J Frey. Cloning and characterization of the
human gene encoding aspartyl beta-hydroxylase. Gene 150:395-399, 1994.
Landis, S.H., Murray, T., Bolden S., and P.A. Wingo. Cancer
Statistics, 1998., CA 44:6-9.
Lavaissiere L, Jia S, Nishiyama M, de la Monte S, Stren AM, Wands
JR, and PA Friedman. Overexpression of human aspartyl (asparaginy!)
beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma. J.
Clin. Invest. 98:1313-1323, 1996.
Lebo, RV, Tolan, DR, Bruce, BD, Cheng, MC, and Kan, YW. Spot blot
analysis of sorted chromosomes assigns a fructose intolerance gene locus to
chromosomes. Cytometry. 6:476-483,1985.
Lee I, Radosevich, JA, Rosen, ST, ef a/. Immunohistochemistry of
lung carcinomas using monoclonal antibody 44-3A6. Can. Res.
45:5813-5817, 1985.
Lie I, Ridoiivieh JA, Chejfte Q, el a/. Malignant Mesethellemas:
Improved Differential Diagnosis From Lung Adenocarcinomas Using
Monoclonal Antibodies 44-3A6 and 624A12. Amer. J. Path. 123:497-507,
Livingston, PO, Wong, GYC, Adluri, S, Tao, Y, Padevan, M, Parente,
R, Hanlon, C, Calves, MJ, Helling, F, Ritter, G, Oettgen, HF, and Old, LJ.
Improved survival in AJCC stage I I I melanoma patients with GM2 antibodies:
A randomized trial of adjuvant vaccination with GM2 ganglioside. J. Clin.
Oncol., 12:1036-1044, 1994.
Piehl MR, Gould VE, Radosevich JA, et al. Immunohistochemical
Identification of Exocrine and Neuroendocrine Subsets of Large Cell Lung
Carcinomas. Path. Res. and Prac. 183:675-682,1988.
Radosevich JA, Ma Y, Lee I, et al. Monoclonal antibody 44-3A6 as a
probe for a novel antigen found human lung carcinomas with glandular
differentiation. Can. Res 45:5805-5812, 1985.
Radosevich JA, Lee I, Gould VE, and ST Rosen. Monoclonal antibody
assays for lung cancer. In: In vitro diagnosis of human tumors using
monoclonal antibodies. Kupchik HZ and N Rose (Eds.) Marcel Dekker
Radosevich JA, Combs SG, and ST Rosen. Immunohistochemica!
analysis of lung cancer differentiation markers. In: Lung Cancer
Differentiation. Lung Biology in Health and disease. L'Enfant C, Bernal S,
and Baylin S. (Eds.). Marcel Dekker, 1990a.
Radosevich JA, Noguchi M, Rosen ST, Y. Shimosato.
Immunocytochemical analysis of human adenocarcinomas and
bronchioloalveolar carcinomas of the lung using the monoclonal antibody
44-3A6. Tumor Biology. 11:181-188,1990b.
Radosevich JA, Combs SG, and ST Rosen. Expression of
MCA 44-3A6 in human fetal development. Tumor Biology 12:321-329,1991.
Radosevich JA, Siddique FS, Rosen ST, and WJ Kabat. Cell Cycle
and EM Evaluation of tne Adanooeroinoma Antigen Recognized by the
Monoclonal Antibody 44-3A6. Br. J. Can. 63:86-87,1991.
Rosen, ST, Mulshine, JL, Cuttitta, F, and Abrams, PG. Biology of
Lung Cancer. Marcel Dekker, Inc. New York, NY, Vol. 37, 1988.
Sambrook J, Fritsch EF, and T Maniatis. Molecular cloning: a
laboratory manual. 2nd Ed. Cold Spring Harbor Lab. Press., 1990.
Sastry, L, Alting-Mees, M, Huse, WD, Short, JM, Hay, BN, Janda, KD,
Benkovis, SJ, and Lerner. Cloning of the immunological repertoire in
Escherichia coli for generation of monoclonal catalytic antibodies:
Construction of a heavy chain variable region-specific cDNA library. PNAS.
86:5728-5732, 1989.
Siddique FS, Iqbal 2, and JA Radosevich. Changes in the expression
of the tumor-associated Antigen recognized by monoclonal antibody 44-3A6
in A549 cells due to calcium. Tumor Biol. 13:142-151,1992.
Sinkule J, Rosen ST, and JA Radosevich. MCA 44-3A6 Douxorubicin
(Adriamycin) Immunoconjugates: Comparative In Vitro Anti-Tumor Efficacy of
Different Conjugation Methods. Tumor Biol. 12:198-206,1991.
Spagnolo DV, Witaker D, Carrello S, et a/. The use of monoclonal
antibody 44-3A6 in cell blocks in the diagnosis of lung carcinoma, carcinomas
metastatic to lung and pleura, and pleural malignant mesothelioma. Am. J.
Clin. Path. 95:322-329, 1991
Stahel, RA (Chairman). Third International lASLC Workshop on Lung
Tumor and Differentiation Antigens. Inter. J. Cancer Suppl 8:6-26,1994.
Wang Q, VanDusen WJ, Petroski CJ, Garsky VM, Stern AM, and PA
Freidman. Bovine liver aspartyl beta-hydroxylase. J. Biol. Chem.
266:14004-14010, 1991.
Xu, N.f Chen, C-Y A, Shyu, A-B. Modulation of the fate of cytoplasmic
mRNA by AU-rich elements: Key sequence features controlling mRNA
deadenylation and decay. Mol. Cell. Biology. 17:4611-4621,1997.

1. A cDNA molecule which encodes a protein designated labyrinthin (Lab) with a nucleotide sequences as shown:
(Sequence Removed)
2. A cDNA molecule with a nucleotide sequence showing about 70%
homology to the nucleotide sequence as claimed in claim 1.
3. A cDNA molecule as claimed in claim 1, wherein the segment extends
from the start codon (ATG) to the stop codon TAA in the nucleotide sequence
and includes 765 base pairs.
4. A cDNA molecule as claimed in claim 2, wherein the segment extends from
the start codon (ATG) to the stop codon TAA, in the nucleotide sequence and
includes about 765 base pairs.
5. A cDNA as claimed in claim 1, wherein it is sufficient to encode in the Lab
protein detectable by the antibody MCA 44-3A6.
6. An amino acid sequence encoded by the cDNA segment as claimed in claim 1.
7. An amino acid sequence encoded by the cDNA segment as claimed in claim 2.
8. A cDNA molecule as claimed in claim 1, wherein a molecule with an amino
acid sequence selected from the group consisting of APPEDNPVED,
9. A cDNA molecule as claimed in claim 8, wherein the said molecule is with
the amino acid sequence PTGEPQ. '
10. A cDNA molecule as claimed in claim 1, wherein a peptide selected from
the group consisting of all sequences that are between 3 and 20 amino acids in
length, aligned as are the amino acids as shown below:

(Sequence Removed)
BOLD = Eukaryotic Leader Sequence
= Myristylation Site
lower case - Membrane Anchoring Domain (MAD)
wherein, the position of the first amino acid of the sequence of n amino acids is selected from the group consisting of position 1 to n-3 to n-20.
11. An antibody directed to a peptide having an amino acid sequence selected from the sequence as claimed in claim 6, 8, 9 or 10.

12. The antibody as claimed in claim 12, is a monoclonal antibody.
13. An antibody produced in mammals against an amino acid sequence encoded by the DNA molecule as claimed in claim 2, 3, 4, or 5 or a fragment thereof containing an epitope.
14. A vector comprising the isolated cDNA molecule as claimed in claim 2, 3, 4 or 5.
15. A vector comprising a cDNA molecule that encodes a molecule with an
amino acid sequence as claimed in claims 6, 8, 9 or 10.









in-pct-2000-00186-del-complete specification (granted).pdf




in-pct-2000-00186-del-description (complete).pdf



























Patent Number 224400
Indian Patent Application Number IN/PCT/2000/00186/DEL
PG Journal Number 44/2008
Publication Date 31-Oct-2008
Grant Date 14-Oct-2008
Date of Filing 12-Sep-2000
Name of Patentee IMMVARX, INC.
Applicant Address 7135 SENTINEL ROAD, ROCKFORD, ILLINOIS 61107, U.S.A.
# Inventor's Name Inventor's Address
PCT International Classification Number C12N 15/52
PCT International Application Number PCT/US99/05365
PCT International Filing date 1999-03-11
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 09/040,485 1998-03-17 U.S.A.