U.S. patent application number 11/704714 was filed with the patent office on 2007-10-25 for page-4, an x-linked gage-like gene expressed in normal and neoplastic prostate, testis and uterus, and uses therefor.
This patent application is currently assigned to The Gov. of the USA as represented by the Secretary, Dept. of health and Human Services. Invention is credited to Ulrich Brinkmann, Byungkook Lee, Ira H. Pastan, George Vasmatzis.
Application Number | 20070248972 11/704714 |
Document ID | / |
Family ID | 22271898 |
Filed Date | 2007-10-25 |
United States Patent
Application |
20070248972 |
Kind Code |
A1 |
Pastan; Ira H. ; et
al. |
October 25, 2007 |
Page-4, an X-linked gage-like gene expressed in normal and
neoplastic prostate, testis and uterus, and uses therefor
Abstract
PAGE-4 is a gene preferentially expressed in normal male and
female reproductive tissues, prostate, testis, fallopian tube,
uterus and placenta, as well as in prostate cancer, testicular
cancer and uterine cancer. This expression pattern makes it a
target for diagnosis and for vaccine based therapy of neoplasms of
prostate, testis and uterus. The invention provides immunogenic
compositions comprising PAGE-4 protein or immunogenic peptides
thereof, methods of inhibiting the growth of malignant cells
expressing PAGE-4, and methods of inducing an enhanced immune
response to PAGE-4-expressing cancers.
Inventors: |
Pastan; Ira H.; (Potomac,
MD) ; Brinkmann; Ulrich; (Weilheim, DE) ;
Vasmatzis; George; (Byron, MN) ; Lee; Byungkook;
(Potomac, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET
SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Gov. of the USA as represented
by the Secretary, Dept. of health and Human Services
|
Family ID: |
22271898 |
Appl. No.: |
11/704714 |
Filed: |
February 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09763393 |
Jul 30, 2001 |
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PCT/US99/20046 |
Aug 31, 1999 |
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11704714 |
Feb 9, 2007 |
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60098993 |
Sep 1, 1998 |
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Current U.S.
Class: |
435/6.14 ;
435/243; 435/252.33; 435/320.1; 435/325; 536/23.5 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 39/00 20130101; C07K 14/4748 20130101; A61P 35/00
20180101 |
Class at
Publication: |
435/006 ;
435/243; 435/252.33; 435/320.1; 435/325; 536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/00 20060101 C07H021/00; C12N 1/00 20060101
C12N001/00; C12N 1/21 20060101 C12N001/21; C12N 15/00 20060101
C12N015/00; C12N 5/00 20060101 C12N005/00 |
Claims
1-49. (canceled)
50. An isolated nucleic acid molecule encoding (a) a polypeptide
comprising the amino acid sequence set forth as SEQ ID NO: 1; (b) a
polypeptide consisting of 8 to 11 contiguous amino acids of SEQ ID
NO: 1, wherein the polypeptide binds major histocompatibility
complex (MHC).
51. The isolated nucleic acid molecule of claim 50, wherein the
nucleic acid encodes a polypeptide comprising the amino acid
sequence set forth as SEQ ID NO: 1.
52. The isolated nucleic acid molecule of claim 50, wherein the
isolated nucleic acid encodes a polypeptide consisting of the amino
acid sequence set forth as SEQ ID NO: 1.
53. A vector comprising the isolated nucleic acid molecule of claim
1.
54. The vector of claim 53, wherein the vector is a viral
vector.
55. The vector of claim 53, wherein the vector is a bacterial
vector.
56. An isolated host cell transformed with the vector of claim
53.
57. The isolated host cell of claim 56, wherein the host cell is a
prokaryote.
58. The isolated host cell of claim 57, wherein the host cell is an
E. coli.
59. The isolated host cell of claim 56, wherein the host cell is a
mammalian cell.
60. A composition comprising an effective amount of the nucleic
acid molecule of claim 50 in a carrier.
61. A method for detecting the presence of a prostate cell, a
prostate cancer cell, or both in a biological sample, comprising
contacting isolated nucleic acids from the biological sample or
amplified from the biological sample with the nucleic acid molecule
of claim 1 or the complement thereof under conditions for specific
hybridization; and detecting hybridization between the nucleic
acids from the biological sample with the nucleic acid molecule of
claim 1, wherein the presence of hybridization indicates the
presence of a prostate cell, the prostate cancer cell, or both.
62. The method of claim 61, wherein the method comprises contacting
nucleic acids amplified from the biological sample with the nucleic
acid molecule of claim 1, and where the nucleic acid amplified from
the biological sample are amplified by polymerase chain
reaction.
63. A kit, comprising (i) the isolated nucleic acid of claim 1 or
the complement thereof; and (ii) instructions printed on a tangible
medium, describing the detection of nucleic acid molecules in a
sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/763,393 filed on Jul. 30, 2001, which is
incorporated herein by reference. U.S. patent application Ser. No.
09/763,393 is a .sctn. 371 U.S. national stage of PCT Application
No. PCT/US99/20046, filed Aug. 31, 1999, which was published in
English under PCT Article 21(2), which in turn claims the benefit
of U.S. Provisional Application 60/098,993 filed Sep. 1, 1998,
which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] ESTs are sequences derived from randomly selected clones
from various cDNA libraries (Adams, M. D. et al. Science
252:1651-1656 (1991); Adams, M. D. et al. Nature 377:3-174 (1995);
Adams, M. D. et al., Nature 355:632-634 (1992); Emmert-Buck et al.,
Science 274:998-1101 (1996); Krizman, D. B. et al., Cancer Res.
56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet.
16:415-516 (1997)). Each cDNA clone is generated from a transcript,
and the frequency and distribution of the many different
transcripts in any given tissue depends on the tissue specific
activity of the genes. The translation of transcript frequency and
distribution into frequency and distribution of EST sequences
depends not only on the specificity and magnitude of mRNA
expression, but also on other factors such as mRNA stability and
clonability of these EST sequences. Therefore, a specificity or
frequency analysis of ESTs only provides a guide for the prediction
of expression patterns. Nevertheless, ESTs provide a valuable
source of information that may be utilized to predict the
expression patterns of specific genes in different tissues.
[0003] The recently developed NCl Cancer Genome Anatomy Project
(CGAP) uses microdissection and laser-capture techniques to
generate defined and tissue/tumor specific EST libraries
(Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et
al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al.,
Nat. Genet. 16:415-516 (1997)). CGAP has already accumulated a vast
number of tissue-specific sequences and the CGAP sequence data base
is rapidly growing with the continuous addition of sequences from
different tissues and tumor types. There are many ways by which the
EST sequence data can be processed to cluster, sort and filter the
cDNA sequences, in order to identify genes that are specifically
expressed in certain tissues. Database "mining" for cDNAs that are
preferentially or exclusively expressed in defined tissues, or in
malignant/neoplastic tissues provides lists of potential target
genes for cancer therapy (Emmert-Buck et al., Science 274:998-1101
(1996); Krizman, D. B. et al., Cancer Res. 56:5380-5383 (1996);
Strausberg, R. L. et al., Nat. Genet. 16:415-516 (1997); Vasmatzis,
G. et al., Proc. Natl. Acad. Sci., USA 95:300-304 (1998)). Although
in many cases these "candidate genes", which appear tissue specific
in database analyses, cannot be confirmed in their specificity by
experimental techniques (e.g. Northern blots or PCR), a reasonable
number of candidate genes remain for which the predicted and
desired expression pattern can be experimentally confirmed
(Vasmatzis, G. et al., Proc. Natl. Acad. Sci., USA 95:300-304
(1998); He, W. W. et al., Genomics 43:69-77 (1997)). These
specifically expressed genes are of interest because of their
functions in cell or tumor biology and may also be directly used as
markers for cancer diagnosis and as the basis for a variety of
methods of therapy.
[0004] One method of therapy is to use the gene product as a
vaccine to enhance the patient's immune response to the cancer. T
cells play an important role in tumor regression in most murine
tumor models. Tumor infiltrating lymphocytes ("TIL") that recognize
unique cancer antigens can be isolated from many murine tumors. The
adoptive transfer of these TIL plus interleukin-2 can mediate the
regression of established lung and liver metastases (Rosenberg, S.,
et al., Science 233:1318-1321 (1986). In addition, the secretion of
IFN-.gamma by injected TIL significantly correlates with in vivo
regression of murine tumors suggesting activation of T-cells by the
tumor antigens. (Barth, R., et al., J. Exp. Med. 173:647-658
(1991)). In humans, the ability of tumor TIL to mediate the
regression of metastatic cancer in 35 to 40% of melanoma patients
when adoptively transferred into patients with metastatic melanoma
attests to the clinical importance of the antigens recognized
(Rosenberg, S., et al., N Engl J Med 319:1676-1680 (1988);
Rosenberg S., J. Clin. Oncol. 10:180-199 (1992)).
[0005] T cell receptors on CD8+ T cells recognize a complex
consisting of an antigenic peptide (9-10 amino acids for Human
Leukocyte Antigen ("HLA")-A2), .beta.-2 microglobulin, and class I
major histocompatibility complex ("MHC") heavy chain (HLA-A, B, C,
in humans). Peptides generated by digestion of endogenously
synthesized proteins are transported into the endoplasmic
reticulum, bound to class I MHC heavy chain and .beta.-2
microglobulin, and finally expressed in the cell surface in the
groove of the class I MHC molecule. Therefore, T cells can detect
molecules that originate from proteins inside cells, in contrast to
antibodies that detect molecules expressed on the cell surface.
Antigens recognized by T cells thus may be particularly useful for
inhibiting the progression of cancer.
[0006] Strong evidence that an immune response to cancer exists in
humans have been provided by, for example, the existence of
lymphocytes within melanoma deposits. These lymphocytes, when
isolated, are capable of recognizing specific tumor antigens on
autologous and allogeneic melanomas in an MHC restricted fashion.
(Kawakami, Y., et al., J. Immunol. 148:638-643 (1992); Hom, S., et
al., J. Immunother. 13:18-30 (1993)). TIL from patients with
metastatic melanoma recognize shared antigens including
melanocyte-melanoma lineage specific tissue antigens in vitro
(Kawakami, Y., et al., J. Immunother. 14:88-93 (1993); Anichini, A.
et al., J. Exp. Med. 177:989-998 (1993)).
[0007] Although several tumor associated antigens ("TAA") have been
found for melanoma, there is a need to identify tissue specific
genes whose expression is associated with cancers of other
tissues.
SUMMARY OF THE INVENTION
[0008] This invention provides a new class of proteins which are
preferentially expressed by cells of reproductive tissues,
including, but not limited to, the prostate gland, testis, uterus,
fallopian tubes, and placenta. The proteins are found in both
normal and cancerous reproductive tissues. These proteins share
some homology with the GAGE and MAGE family of proteins.
[0009] In particular, the invention provides the PAGE-4 protein.
The PAGE-4 protein and immunogenic peptides thereof can be used as
immunogenic compositions to raise cytotoxic T lymphocyte responses
against cells expressing PAGE-4 in vitro or in vivo. Such cells
include cancers of the prostate, testis, and uterus. Nucleic acids
encoding the protein or an immunogenic peptide thereof can also as
immunogenic compositions.
[0010] In addition to the uses as immunogenic compositions, the
present invention also provides for methods of detecting the
presence of the PAGE-4 protein in cells in cell samples or body
tissues. The detection can be performed by detecting the protein,
typically by using antibodies. Detection of the presence of the
protein can also be accomplished by detecting nucleic acids that
encode the proteins. Conveniently, this can be done by using
detectable probes complementary to all or a portion of sequences
encoding PAGE-4. The presence of PAGE-4 in tissues not related to
reproduction could be indicative of the spread of cancerous
reproductive tissue.
[0011] In addition to diagnostic and vaccine uses, PAGE-4 protein
and the nucleic acids encoding it can be used in therapeutic
applications. PAGE-4 protein can be used to raise antibodies which
can be used not only in the diagnostic assays described above, but
also as the targeting moiety of immunoconjugates. The toxic moiety
of the immunoconjugates can include, but is not limited to, toxins
such as ricin, abrin, diphtheria toxin and subunits thereof, as
well as botulinum toxins A through F, and Pseudomonas exotoxin
(PE).
DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1: Similarity of PAGE-4, GAGE and MAGE: (A) The
predicted PAGE-4 reading frame (SEQ ID NO: 1) is derived from the
full length PAGE-4 EST clone nh32c06. The GAGE and MAGE sequences
are from SW: GGE1 (SEQ ID NO: 2), GGE2 (SEQ ID NO: 3), GGE3 (SEQ ID
NO: 4), GGE4 (SEQ ID NO: 5), GGE5 (SEQ ID NO: 6), GGE6 (SEQ ID NO:
7), MAG5 (SEQ ID NO: 8) and MAG8_HUMAN (SEQ ID NO: 9). Note that
the "MAGE-alignment" matches amino acids that occur in MAGE5 and/or
MAGE8, which are similar to PAGE-4 and/or GAGE1-6; the homologies
between single members of the MAGE and PAGE and GAGE protein
families are weaker. (B) Alignment of PAGE-1 (SEQ ID NO: 10) with
other PAGEs. PAGE-2 (SEQ ID NO: 11) was translated from the EST
ai61a04 EST-cluster and PAGE-3 from om29f08. PAGE-3 (SEQ ID NO: 12)
was translated from one single EST and it is possible that the
truncated amino terminus results from a sequence artifact (the
homology extends further to the N-terminus in another reading
frame). Several other so far undefined EST clusters were found that
have homology to PAGE as well as to GAGE. These clusters do not
have the striking similarities that the other GAGE family members
have to each other, but they are also not significantly more
similar to PAGE than to GAGE. Representatives of some of these cDNA
clusters are the ESTS yd88e11 (fetal liver/spleen), yw86a06
(placenta) and yi21h01 (placenta).
[0013] FIG. 2: Hybridization analysis of PAGE-4 expression: (A) A
MTN Dot blot (left) and Northern blots (middle to right) were
probed with a 140 bp .sup.32P labeled PAGE-4 probe under very
stringent hybridization conditions (50% formamide, 55.degree. C.).
Specific PAGE-4 signals were observed in prostate, testis, placenta
and uterus, but not in other tissues (Table 1 legend lists the
analyzed tissues). Because the hybridization probe had some
similarity with another PAGE-4-like EST cluster that is expressed
in testis (PAGE-2 represented by the EST zv62h08, Table 2), we
additionally used a probe with minimal homology to zv62h08 to
confirm that the signal in testis corresponds to the expression of
the authentic PAGE-4. (B) Blots containing 20 .mu.g/lane total RNA
from normal or malignant ovary (right), fallopian tube (middle) and
uterus (left) were hybridized under stringent conditions. PAGE-4 is
expressed in fallopian tube, uterus and uterine cancer, but not in
ovary and ovarian cancer.
[0014] FIG. 3: Relation of the sequences of MAGE, GAGE, PAGE and
other so far uncharacterized EST clusters: The GCG program "PILEUP"
was used to compare the multiple protein sequences of the GAGE and
PAGE protein family. The dendrogram shows that PAGE proteins are a
separate group of proteins that are less related to GAGE
proteins.
[0015] FIG. 4: RT-PCR analysis of PAGE-4 expression: Ethidium
bromide stained 2.5% agarose gel; PAGE-4 cDNA was amplified with 5'
and 3'-end specific PAGE primers (40 cycles 94.degree.
C.-58.degree. C.-72.degree. C., 1 min each).
[0016] FIG. 5: Nucleotide sequence encoding PAGE-4 (SEQ ID NO: 13):
The open reading frame is in bold type and underlined.
DETAILED DESCRIPTION
I. Introduction
[0017] Here we describe the identification of an X-linked gene that
is expressed in normal and malignant male and female reproductive
tissues. This gene, PAGE-4 (which we originally called PAGE-1 and
have now renumbered to be consistent with other findings), is
homologous to a family of MAGE/GAGE like proteins and is expressed
in normal prostate, testis, uterus, fallopian tube and placenta, as
well as in prostate, testicular and uterine cancers.
[0018] Prostate, testicular, and uterine cancers, are usually
treated, in part, by the surgical removal of the affected organ.
The metastases may not, however, be susceptible to surgical
removal, or they may be too small to be readily detected. Enhancing
the patient's immune response to the cancer, and particularly
enhancing the response of cytotoxic T lymphocytes ("CTLs") to the
cancer, can aid in slowing or stopping the progress of the disease.
PAGE-4 and immunogenic peptides thereof can be used as a vaccine to
enhance a patient's immune response against PAGE-4-expressing
cancers. Vaccines of DNA encoding the protein or an immunogenic
portion thereof can also be used. The vaccines can be administered
with adjuvants which help induce a CTL response to the antigen.
Since healthy people have tissues which express PAGE-4, it is
contemplated that the vaccines of the invention would be used as
therapeutic vaccines, rather than as prophylactic vaccines. The
identification of this new gene therefore arms researchers and
clinicians with a new weapon with which to attack prostate cancer,
testicular cancer, and other disorders in which death of the
specific tissues might be beneficial to the patient.
[0019] Because the PAGE-4 gene is expressed in reproductive
tissues, the gene product can be used as a target for reagents
directed to cells of these organs. Reagents specific for the gene
or the gene product can be used in in vitro assays. For example,
the presence of PAGE-4-expressing tissue in a biopsy from a
non-reproductive organ may be indicative of the presence of a
metastasis from a prostate or uterine cancer. Reagents with
appropriate labels can also be used in in vivo assays for the same
purpose. For example, radiolabeled antibodies directed against
PAGE-4, or against PAGE-4 expressed in conjunction with molecules
of the major histocompatibility complex ("MHC"), can be
administered to a patient and their presence in various parts of
the body then detected to determine whether metastatic cells of a
prostate, testicular, ovarian, or other PAGE-4-expressing cancer
have invaded other organs or portions of the body.
[0020] In addition to these diagnostic uses, the invention permits
targeting cells which express PAGE-4 with reagents intended to kill
those cells or to modulate their activity. For example, antibodies
which specifically recognize PAGE-4, can be used as the targeting
moiety of immunotoxins. Since the normal tissues in which the gene
is expressed are not essential to the survival of the individual,
selective killing of the cells expressing the PAGE-4 gene can
eliminate cancerous cells without the severe systemic effects of
conventional cancer chemotherapeutic agents.
[0021] After defining some of the terms used herein, the discussion
below sets forth the discovery of the nature of the PAGE-4 gene and
its expression and significance. A discussion of vaccines is
provided, as are in vitro and in vivo uses of the invention. The
discussion then turns to the making of antibodies against the
PAGE-4 protein, the use of those antibodies to form
immunoconjugates, such as immunotoxins, and methods of linking
effector molecules (such as toxins) to targeting molecules (such as
antibodies). The discussion further describes pharmaceutical
compositions, such as those containing vaccines, antibodies or
immunoconjugates of the invention, as well as diagnostic kits using
antibodies or nucleic acids.
II. Definitions
[0022] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, nucleic acids are written left to right in 5' to 3'
orientation; amino acid sequences are written left to right in
amino to carboxy orientation. The headings provided herein are not
limitations of the various aspects or embodiments of the invention
which can be had by reference to the specification as a whole.
Accordingly, the terms defined immediately below are more fully
defined by reference to the specification in its entirety.
[0023] "PAGE-4" is a gene expressed in prostate and other tissues.
In particular, it is expressed in cancers of the prostate, ovaries,
and testicles. The sequence of the PAGE-4 gene is set forth in FIG.
5. As used herein, a "PAGE-4 protein" refers to the protein encoded
by the PAGE-4 gene. With respect to immunogenic compositions
comprising a PAGE-4 protein, it further refers to variations of
this protein in which there are conservative substitutions of one
or more amino acids of the protein, or deletions or insertions of
one or more amino acids, so long as the variations do not alter by
more than 20% the ability of the protein, when bound to a Major
Histocompatibility Complex class I molecule, to activate cytotoxic
T lymphocytes against cells expressing wild-type PAGE-4
protein.
[0024] The term "peptide" is used to designate a series of amino
acid residues, typically L-amino acids, connected to each other
typically by peptide bonds. The peptides of the invention are less
than about 30 residues in length, and usually consist of between
about 7 and 15 residues, preferably about 8 to 11 residues, and
most preferably are 9 or 10 residues.
[0025] As used herein, a "PAGE-4 peptide" is a series of contiguous
amino acid residues from the PAGE-4 protein between 7 and 20 amino
acids in length, preferably about 8 to 11 residues, and most
preferably 9 or 10 residues. With respect to immunogenic
compositions comprising a PAGE-4 peptide, the term further refers
to variations of these peptides in which there are conservative
substitutions of one or more amino acids, so long as the variations
do not alter by more than 20% the ability of the peptide, when
bound to a Major Histocompatibility Complex Class I molecule, to
activate cytotoxic T lymphocytes against cells expressing wild-type
PAGE-4 protein. Induction of CTLs using synthetic peptides and CTL
cytotoxicity assays are taught in, e.g., U.S. Pat. No.
5,662,907.
[0026] "Major Histocompatibility Complex" or "MHC" is a generic
designation meant to encompass the histocompatibility antigen
systems described in different species, including the human
leukocyte antigens ("HLA").
[0027] An "immunogenic peptide" is a peptide which comprises an
allele-specific motif or other sequence such that the peptide will
bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL")
response against the antigen from which the immunogenic peptide is
derived. Immunogenic peptides are conveniently identified using
sequence motifs or other methods, such as neural net or polynomial
determinations, known in the art (see discussion, infra).
Typically, algorithms are used to determine the "binding threshold"
of peptides to select those with scores that give them a high
probability of binding at a certain affinity and will be
immunogenic. The algorithms are based either on the effects on MHC
binding of a particular amino acid at a particular position or the
effects on binding of a particular substitution in a
motif-containing peptide. Within the context of an immunogenic
peptide, a "conserved residue" is one which appears in a
significantly higher frequency than would be expected by random
distribution at a particular position in a peptide. Typically, a
conserved residue is one where the MHC structure may provide a
contact point with the immunogenic peptide.
[0028] "negative binding residues" are amino acids which, if
present at certain positions (for example, positions 1, 3, or 7 of
a 9-mer), will result in a peptide being a nonbinder or poor binder
and in turn will fail to be immunogenic (i.e., will fail to induce
a CTL response).
[0029] The term "motif" refers to the pattern of residues in a
peptide of defined length, usually about 8 to about 11 amino acids,
which is recognized by a particular MHC allele. The peptide motifs
are typically different for each MHC allele and differ in the
pattern of the highly conserved residues and negative binding
residues.
[0030] As used herein, "immunogenic composition" refers to a
composition comprising a PAGE-4 protein or a peptide derived from a
PAGE-4 protein, which peptide, when bound to a MHC class I
molecule, induces a measurable CTL response against cells
expressing PAGE-4 protein (a "PAGE-4 peptide"). It further refers
to isolated nucleic acids encoding a PAGE-4 protein or a PAGE-4
peptide. For in vitro use, the immunogenic composition may consist
of the isolated protein or peptide. For in vivo use, the
immunogenic composition will typically comprise pharmaceutically
acceptable carriers or other agents. Vaccines are an especially
important embodiment of immunogenic compositions for in vivo use.
Any particular peptide, PAGE-4 protein, or nucleic acid can be
readily tested for its ability to induce a CTL response by
art-recognized assays taught further herein.
[0031] The term "isolated" refers to material which is
substantially or essentially free from components which normally
accompany it as found in its native state.
[0032] The term "residue" refers to an amino acid or amino acid
mimetic incorporated in an oligonucleotide by an amide bond or
amide bond mimetic.
[0033] As used herein, "antibody" includes reference to an
immunoglobulin molecule immunologically reactive with a particular
antigen, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies),
heteroconjugate antibodies (e.g., bispecific antibodies) and
recombinant single chain Fv fragments (scFv), disulfide stabilized
(dsFv) Fv fragments (See, U.S. Ser. No. 08/077,252, incorporated
herein by reference), or pFv fragments (See, U.S. Provisional
Patent Application 60/042,350 and 60/048,848, both of which are
incorporated herein by reference.) The term "antibody" also
includes antigen binding forms of antibodies (e.g., Fab',
F(ab').sub.2, Fab, Fv and rIgG. See also, Pierce Catalog and
Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.)).
[0034] An antibody immunologically reactive with a particular
antigen can be generated by recombinant methods such as selection
of libraries of recombinant antibodies in phage or similar vectors.
See, e.g., Huse, et al., Science 246:1275-1281 (1989); Ward, et
al., Nature 341:544-546 (1989); and Vaughan, et al., Nature
Biotech. 14:309-314 (1996).
[0035] The phrase "single chain Fv" or "scFv" refers to an antibody
in which the heavy chain and the light chain of a traditional two
chain antibody have been joined to form one chain. Typically, a
linker peptide is inserted between the two chains to allow for
proper folding and creation of an active binding site.
[0036] The term "linker peptide" includes reference to a peptide
within an antibody binding fragment (e.g., Fv fragment) which
serves to indirectly bond the variable heavy chain to the variable
light chain.
[0037] The term "contacting" includes reference to placement in
direct physical association.
[0038] The terms "conjugating," "joining," "bonding" or "linking"
refer to making two polypeptides into one contiguous polypeptide
molecule. In the context of the present invention, the terms
include reference to joining a ligand, such as an antibody moiety,
to an effector molecule (EM). The linkage can be either by chemical
or recombinant means. Chemical means refers to a reaction between
the antibody moiety and the effector molecule such that there is a
covalent bond formed between the two molecules to form one
molecule.
[0039] As used herein, "encoding" with respect to a specified
nucleic acid, includes reference to nucleic acids which comprise
the information for translation into the specified protein. The
information is specified by the use of codons. Typically, the amino
acid sequence is encoded by the nucleic acid using the "universal"
genetic code. However, variants of the universal code, such as is
present in some plant, animal, and fungal mitochondria, the
bacterium Mycoplasma capricolumn (Proc. Nat'l Acad. Sci. USA
82:2306-2309 (1985), or the ciliate Macronucleus, may be used when
the nucleic acid is expressed in using the translational machinery
of these organisms.
[0040] As used herein, "expressed" includes reference to
translation of a nucleic acid into a protein. Proteins may be
expressed and remain intracellular, become a component of the cell
surface membrane or be secreted into the extracellular matrix or
medium.
[0041] By "host cell" is meant a cell which can support the
replication or expression of an expression vector. Host cells may
be prokaryotic cells such as E. coli, or eukaryotic cells such as
yeast, insect, amphibian, or mammalian cells.
[0042] The terms "effective amount" or "amount effective to" or
"therapeutically effective amount" includes reference to a dosage
of a therapeutic agent sufficient to produce a desired result, such
as inhibiting cell protein synthesis by at least 50%, or killing
the cell.
[0043] The term "therapeutic agent" includes any number of
compounds currently known or later developed to act as
anti-neoplastics, anti-inflammatories, cytokines, anti-infectives,
enzyme activators or inhibitors, allosteric modifiers, antibiotics
or other agents administered to induce a desired therapeutic effect
in a patient.
[0044] The term "immunoconjugate" includes reference to a covalent
linkage of an effector molecule to an antibody. The effector
molecule can be an immunotoxin.
[0045] The term "toxin" includes, but is not limited to, reference
to abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT),
botulinum toxin, or modified toxins thereof, or other toxic agents
that directly or indirectly inhibit cell growth or kill cells. For
example, PE and DT are highly toxic compounds that typically bring
about death through liver toxicity. PE and DT, however, can be
modified into a form for use as an immunotoxin by removing the
native targeting component of the toxin (e.g., domain Ia of PE and
the B chain of DT) and replacing it with a different targeting
moiety, such as an antibody.
[0046] The term "chimeric molecule," as used herein, refers to a
targeting moiety, such as a ligand or an antibody, conjugated
(coupled) to an effector molecule.
[0047] "Targeting moiety" refers to a portion of a chimeric
molecule intended to provide the molecule with the ability to bind
specifically to the PAGE-4 protein. A "ligand" is a targeting
molecule specific for the PAGE-4 protein and is generally
synonymous with "targeting moiety." An antibody is one version of a
ligand.
[0048] The terms "effector molecule," "effector moiety," "EM"
"therapeutic agent" and "diagnostic agent," or similar terms, refer
to the portion of a chimeric molecule that is intended to have a
desired effect on a cell to which the chimeric molecule is
targeted. Therapeutic agents include such compounds as nucleic
acids, proteins, peptides, amino acids or derivatives,
glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant
viruses. Nucleic acid therapeutic and diagnostic moieties include
antisense nucleic acids, derivatized oligonucleotides for covalent
cross-linking with single or duplex DNA, and triplex forming
oligonucleotides. Alternatively, the molecule linked to a targeting
moiety, such as an anti-PAGE-4 antibody, may be an encapsulation
system, such as a liposome or micelle that contains a therapeutic
composition such as a drug, a nucleic acid (e.g. an antisense
nucleic acid), or another therapeutic moiety that is preferably
shielded from direct exposure to the circulatory system. Means of
preparing liposomes attached to antibodies are well known to those
of skill in the art. See, for example, U.S. Pat. No. 4,957,735; and
Connor, et al., Pharm. Ther. 28:341-365 (1985). Diagnostic agents
or moieties include radioisotopes and other detectable labels.
[0049] A "conservative substitution," when describing a peptide or
protein refers to a change in the amino acid composition of the
peptide or protein that does not substantially alter the protein's
activity, including its binding to an HLA allele of interest. Thus,
"conservatively modified variations" of a particular amino acid
sequence refers to amino acid substitutions of those amino acids
that are not critical for protein activity or substitution of amino
acids with other amino acids having similar properties (e.g.,
acidic, basic, positively or negatively charged, polar or
non-polar, etc.) such that the substitutions of even critical amino
acids do not substantially alter activity. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. The six groups in the following table each
contain amino acids that are conservative substitutions for one
another: [0050] 1) Alanine (A), Serine (S), Threonine (T); [0051]
2) Aspartic acid (D), Glutamic acid (E); [0052] 3) Asparagine (N),
Glutamine (Q); [0053] 4) Arginine (R), Lysine (K); [0054] 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and [0055]
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also,
Creighton, PROTEINS, W.H. Freeman and Company (1984).
[0056] The terms "substantially similar" in the context of a
peptide indicates that a peptide comprises a sequence with at least
90%, preferably at least 95% sequence identity to the reference
sequence over a comparison window of 10-20 amino acids. Percentage
of sequence identity is determined by comparing two optimally
aligned sequences over a comparison window, wherein the portion of
the polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0057] The terms "conjugating," "joining," "bonding" or "linking"
refer to making two polypeptides into one contiguous polypeptide
molecule. In the context of the present invention, the terms
include reference to joining an antibody moiety to an effector
molecule (EM) and to joining a lipid or other molecule to a protein
or peptide to increase its half-life in the body. The linkage can
be either by chemical or recombinant means. Chemical means refers
to a reaction between the antibody moiety and the effector molecule
such that there is a covalent bond formed between the two molecules
to form one molecule.
III. Identification of the PAGE-4 Gene
[0058] A. Database "Mining"
[0059] We identified the gene through a computer screening strategy
we established specifically to identify genes that are
preferentially expressed in normal prostate and in prostate cancer
(Vasmatzis, G. et al., Proc. Natl. Acad. Sci., USA 95:300-304
(1998)). Using this approach in combination with experimental
verification, we have found several candidate genes that are
preferentially expressed in the prostate and are evaluating these
genes as targets for the diagnosis or therapy of prostate cancer.
PAGE-4 was identified by relaxing the specificity requirements for
candidate ESTs in our screening procedure. Instead of removing or
giving low ranking to EST clusters which are expressed in
non-prostate tissues, we have allowed ESTs that occur in tumors and
in a limited number of non-essential normal tissues. Our rationale
for this approach is that the expression of a gene in a
nonessential tissue and in more than one type of tumor does not
exclude it as a target for therapy. In fact, expression in several
types of tumors is desirable because this broadens the application
of reagents that are developed based upon such targets.
[0060] The database analysis was performed on the complete human
EST sequence set in the dbEST database (NCBI dbEST/CGAP
(Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et
al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al.,
Nat. Genet. 16:415-516 (1997)) as of Apr. 25, 1998, which included
1,001,294 ESTs in 656 different libraries. The majority of the ESTs
(>650,000 ESTs, >64% of the total ESTs) came from Soares
libraries and/or the NCl Cancer Genome Anatomy Project (CGAP).
[0061] Our EST database clustering and filtering program,
originally designed to identify genes that are very specifically
expressed in prostate and prostate cancer (Vasmatzis, G. et al.,
Proc. Natl. Acad. Sci., USA 95:300-304 (1998)) was updated with the
additional EST data. We "relaxed" the specificity requirement for
the selection of potentially useful EST clusters because we
observed candidate genes on our search list which were not entirely
prostate specific, that might still be acceptable and useful as
targets for the diagnosis or therapy of prostate cancer. For
example, EST clusters which show several "expression-hits" in
non-prostate/cancer tissues are still interesting if the
non-prostate expression specificity is found in libraries other
than prostate that come from tumors or nonessential tissues.
Therefore, in selecting candidates for further, experimental
processing, we "tolerated" the occurrence of ESTs from candidate
clusters in a limited number of normal tissues. These were
placenta, other gender specific tissues and fetal tissues. In
identifying target antigens for tumor therapy, the expression of a
gene in more than one type of tumor is not an impediment to the
applicability of such targets; in fact it may be desirable because
expression of a given protein in multiple tumors will broaden the
application of reagents that are developed based upon such targets.
The expression of "tumor"--proteins in certain normal tissues may
be neglected if the expression is in reproductive tissues.
Expression in uterus, ovary or placenta is not relevant for males,
and prostate or testis expression is not relevant for females.
[0062] The output of our recent database analysis is a list of
clones that occur frequently in prostate, prostate cancer, as well
as in other tumors, and that may also be present in some normal
tissues. We sorted this list according to EST-frequency in prostate
and prostate tumors. Since the EST frequency in libraries of
defined tissues approximately correlates with the level of tissue
specific expression of the corresponding gene, this tissue-specific
ranking may identify genes that are preferentially expressed in
prostate and prostate cancer.
[0063] B. Detection of a cDNA Cluster that Encodes a X-Linked GAGE
Like Protein Predominantly Represented in Prostate and Prostate
Cancer Libraries
[0064] One of the cDNA clusters present on the database search list
was observed to be preferentially present in prostate and prostate
tumor libraries, and additionally, in placenta and in a mixed
pooled library which contained mRNA from uterus. The computational
analysis portion of Table 1 lists the distribution of individual
EST sequences that correspond to this cDNA cluster in several
different EST libraries. ESTs from this cluster are most abundantly
found in libraries from prostate and prostate cancer where they
represent 0.022% (prostate) and 0.031% (prostate cancer) of the
total cDNA sequence population. They were also represented in
placenta libraries (0.016%) and in a library pool that contained
cDNA from uterus (0.013%). Homology analyses showed that the
sequence of this cDNA cluster is similar to a family of GAGE like
proteins (Van den Eynde et al., J. Exp. Med. 182:689-698 (1995);
Lucas, S. et al., Cancer Res. 4:743-752 (1998); Old, L. J. et al.,
J. Exp. Med. 187:1163-1167 (1998)). An alignment of the protein
sequence that is predicted from its reading frame with the
sequences of members of the GAGE family is shown in FIG. 1A. The
homology to GAGE is highly significant but it is not as pronounced
as that of the other GAGE proteins to each other, and we observed
some weaker similarity to MAGE proteins (Old, L. J. et al., J. Exp.
Med. 187:1163-1167 (1998); van der Bruggen, P. et al., Science
254:1643-1647 (1991); Stockert, E., Jager, E. et al., J. Exp. Med.
187:1349-1354 (1998); De Plaen, E., et al. Immunogentics 40:360-369
(1994); Takahashi, K., et al., Cancer Res. 55:3478-3482 (1995);
Boel, P. et al., Immunity 2:167-175 (1995)). Because this novel
member of the MAGE/GAGE protein family appears to be strongly
expressed in prostate and placenta we named it PAGE-4. Further
database searches identified additional EST clusters with
significant similarity to PAGE-4 and less similarity to GAGE and
MAGE. This suggests that PAGE-4 is a member of a family of related
proteins like MAGE and GAGE. An alignment of PAGE-4 with sequences
of PAGE-2 (predominantly in testis) and PAGE-3 (one EST from a
pooled, testis containing library) is shown in FIG. 1. In addition,
we identified several other EST clusters with homology to PAGE as
well as to GAGE, but which do not have the striking similarities
that the other GAGE family members have to each other.
Representatives of some of these cDNA clusters are the ESTS yd88e11
(fetal liver/spleen), yw86a06 (placenta) and yi21h01 (placenta).
The relation of the sequences of GAGE and PAGE is shown in a graph
form (dendrogram) in FIG. 3. There are two sequence stretches in
PAGE-4 that contain Arg-Gly-Asp (RGD) motifs, and the surrounding
sequence is similar to a RGD containing sequence present in the
metabotropic glutamate receptor 6 (Hashimoto, T. et al., Eur. J.
Neurosci. 9:1226-1235 (1997)). RGD motifs are frequently found in
cell adhesion proteins, and it has been suggested that RGD
sequences in several receptor molecules are involved in cell-cell
interactions (Papadopoulos, G. K. et al., Int. J. Biol. Macromol.
1:51-57 (1998)).
[0065] Several GAGE/MAGE like proteins have recently been described
as CT antigens, i.e., proteins that are expressed preferentially in
cancers and testis (Old, L. J. et al., J. Exp. Med. 187:1163-1167
(1998); van der Bruggen, P. et al., Science 254:1643-1647 (1991);
Stockert, E., Jager, E. et al., J. Exp. Med. 187:1349-1354 (1998);
De Plaen, E., et al. Immunogentics 40:360-369 (1994); Takahashi,
K., et al., Cancer Res. 55:3478-3482 (1995); Boel, P. et al.,
Immunity 2:167-175 (1995)). It has been shown that many MAGE genes
are positioned in at least two clusters on the human X chromosome
(Lucas, S. et al., Cancer Res. 4:743-752 (1998); Old, L. J. et al.,
J. Exp. Med. 187:1163-1167 (1998); Lurquin, C. et al., Genomics
3:397-408 (1997); Muscatelli, F. et al., Proc. Natl. Acad. Sci. USA
92:4987-4991 (1995)). We have found, by radioactive hybridization
of a Somatic Cell Hybrid Southern blot, that the PAGE-4 gene is
also located on the X-chromosome (data not shown). A recent
database deposit of mapped X chromosomal transcript sequences
confirms the X chromosome mapping of PAGE-4 and places PAGE-4 at
position Xpl 1.23 (Strom, T. M. et al., Genbank AJ005894
(1998)).
[0066] C. PAGE-4 is Expressed in Normal Prostate, Testis, Uterus,
Fallopian Tube and Placenta, and in Prostate and Uterine
Cancers.
[0067] To evaluate experimentally the specificity of expression of
PAGE-4, which was suggested by the database analysis to be
expressed preferentially in prostate and prostate tumors, we
hybridized Dot blots and Northern blots of mRNAs from different
tissues with a radioactive labeled PAGE-4 probe. The results of
these experiments, which were done with a 140 bp probe under very
stringent hybridization conditions, are shown in FIG. 2 and
summarized in Table 1. Dot blot hybridizations (FIG. 2A) show a
significant level of PAGE-4 expression in normal prostate. We also
found weaker expression in testis, and very strong expression in
normal placenta. Additional signals were observed in uterus and a
very weak signal in ovary. The very strong placental expression
which was stronger than prostate and the expression in ovary and
testis was not predicted from the results of the database analysis.
The expression in uterus is congruent with the appearance of PAGE-4
ESTs in a library that was derived from pooled mRNAs, including
uterus. Among the 58 normal tissues and cancer cell lines that we
tested, only prostate, testis, placenta, uterus and ovary showed
PAGE-4 hybridization signals in dot blots. The signal with ovary
was very weak in Dot blots.
[0068] In Northern blots, prostate, testis, placenta and uterus,
but none of the other tissues, displayed a clear .about.500 b band
that hybridized with the PAGE-4 probe (FIG. 2A). Further analyses
of Northern blots with mRNA preparations from different uterine
cancer samples showed that PAGE-4 is in uterine cancers. FIG. 2B
(first panel) is a Northern blot containing total RNA from
different uterine tumors (Ins. 1, 3, 5, 7) and corresponding normal
uterus (Ins. 2, 4, 6, 8). The PAGE-4 signal is apparent in all
normal uterus and uterine cancer samples; and in some instances the
PAGE-4 signal in mRNA from uterine cancer is stronger than in the
adjacent normal tissue (FIG. 2B). On the other hand, expression of
PAGE-4 in ovary, which we tested because of the weak dotblot
hybridization signal, could not be confirmed. FIG. 2B (3.sup.rd
panel) shows that Northern blots containing mRNA from ovarian
cancer and adjacent normal ovary showed no evidence of PAGE-4
expression; however we did find PAGE-4 in mRNA from normal
fallopian tube (FIG. 2B, middle panel).
[0069] To confirm the presence of PAGE-4 transcripts experimentally
in malignant prostate we analyzed a prostate cancer cDNA
preparation (from Invitrogen) by RT-PCR with primers that
specifically amplify a full length PAGE-4 cDNA fragment. FIG. 4
shows that PAGE-4 mRNA can be detected by PCR in cDNA samples from
malignant prostate, as well as in normal prostate and in a
testicular tumor. The PAGE-4 fragment was also obtained by PCR from
a placental cDNA library, but not from libraries from human muscle
or liver. In the prostate tumor cell lines LnCAP and PC3, PAGE-4
expression could be detected by hybridization analyses of RT-PCR
products (data not shown) but not by Northern blots or RT-PCR using
ethidium-bromide stained agarose gels. These experimental
observations, combined with the fact that PAGE-4 is present in CGAP
cDNA libraries derived from different prostate cancers (Table 1),
indicates that PAGE-4 is predominantly expressed in normal and
neoplastic male and female reproductive tissues and particularly in
prostate, testis and uterus.
IV. Significance of the PAGE-4 Gene
[0070] PAGE-4 is a human X-linked gene that is strongly expressed
in prostate and prostate cancer, but is also expressed in other
male and female reproductive tissues: testis, fallopian tube,
placenta, uterus and uterine cancer. PAGE-4 shows similarity with
the GAGE protein family, but it diverges significantly from members
of the family so that it appears to belong to a separate family.
This, and the existence of other genes, PAGE-2 and PAGE-3, that
share more homology with PAGE-4 than with members of the GAGE
family indicates that the PAGE proteins constitute a separate
protein family.
[0071] The specificity of PAGE-4 expression in normal and malignant
tissues that are associated with male and female reproductive
function coincides with the localization of this gene on the X
chromosome. This observation provides a link between
sex-chromosomal genes and reproductive functions. For example, it
is known that a testis defining gene (SRY) is located on the Y
chromosome (McElreavey, K. et al., Heredity 6:599-611 (1995)), and
other sex determining genes are predicted to be positioned on the X
chromosome (Ogata T. et al., Acta Paediatr Jpn, 4: 390-398 (1996);
Dabovi, B. et al., Mamm Genome 9:571-580 (1995)). The family of
MAGE proteins is located on Xp21 and Xp28 and is expressed in
testis and tumors. PAGE-4 also is located on the X chromosome and
is expressed in male as well as female specific tissues. It is
interesting to speculate that MAGE and/or PAGE proteins are
involved in sex determination. One important question in this
context is in which cells of the reproductive tissues PAGE-4 is
expressed. Because of the high homology of the various members of
the GAGE family, this question can probably not be solved by in
situ hybridizations, but instead specific antibodies will be
required. The presence of PAGE-4 ESTs in microdissected CGAP tumor
libraries suggests that PAGE-4 is probably expressed in the
epithelial cells from which most tumors originate.
V. Uses of the PAGE-4 Protein and Gene
[0072] In addition to the interesting basic questions about the
molecular function of PAGE such as its cellular localization and
the function of the RGD sequences, the expression pattern of PAGE-4
opens the possibility of its usefulness in tumor diagnosis and in
therapy. In males, PAGE-4 is found in prostate and testis, as well
as in prostate and testicular cancer. Obviously, expression in
placenta, fallopian tube or uterus is irrelevant for therapy of
males. Conversely, testicular and prostate expression can be
neglected in females, as well as the high expression in normal
placenta.
[0073] In an important group of embodiments, all or part of the
PAGE-4 gene product can be used as a vaccine to enhance the immune
system's ability to eliminate PAGE-4 containing cells (Old, L. J.
et al., J. Exp. Med. 187:1163-1167 (1998); van der Bruggen, P. et
al., Science 254:1643-1647 (1991); Stockert, E., Jager, E. et al.,
J. Exp. Med. 187:1349-1354 (1998); De Plaen, E., et al.
Immunogenetics 40:360-369 (1994); Takahashi, K., et al., Cancer
Res. 55:3478-3482 (1995); Boel, P. et al., Immunity 2:167-175
(1995)). The relation to MAGE and GAGE proteins strongly suggests
that PAGE-4 is processed or presented by cells, and thus is
suitable as a vaccine, (Old, L. J. et al., J. Exp. Med.
187:1163-1167 (1998); van der Bruggen, P. et al., Science
254:1643-1647 (1991); Stockert, E., Jager, E. et al., J. Exp. Med.
187:1349-1354 (1998); De Plaen, E., et al. Immunogentics 40:360-369
(1994); Takahashi, K., et al., Cancer Res. 55:3478-3482 (1995);
Boel, P. et al., Immunity 2:167-175 (1995)).
[0074] Many approaches to vaccination are known in the art. One
preferred option is direct vaccination with plasmid DNA (Donnelly,
J. J. et al., Annu. Rev. Immunol. 15:617-648 (1997)). We are able
to obtain good expression of PAGE-4 protein with mammalian
expression plasmids (data not shown), and it has been demonstrated
that DNA-immunization with such expression constructs leads to good
immune responses (Donnelly, J. J. et al., Annu. Rev. Immunol.
15:617-648 (1997); Chowdhury, P. et al., Proc. Natl. Sci USA
95:669-674 (1998)). Therefore, this method should generate
anti-PAGE-4 responses, and allow us to demonstrate that
"PAGE-4-vaccination" can eliminate PAGE-4 expressing cells, as a
therapeutic approach towards neoplasms of the prostate, testis and
uterus.
[0075] In some embodiments, only an immunogenic portion of the
PAGE-4 protein is used in forming the vaccine. Methods of
determining immunogenic portions of a protein and selecting
appropriate peptides which can activate T-cell responses to an
antigen of interest are known in the art.
[0076] The specific detection of PAGE-4 is expected to be valuable
for the diagnosis of prostate and testicular tumors, as well as
uterine tumors. There are sufficient differences between PAGE-4 and
other members of the PAGE and MAGE antibodies to produce specific
antibodies, and we have demonstrated the production of PAGE-4
antibodies. Analyses with such antibodies are expected to confirm
by immunohistology the expression specificity that is seen in
database and mRNA analyses. The antibodies can also be used to
detect the presence of PAGE-4 in biological samples. In in vitro
applications, the antibodies can be used in any of a number of
standard immunoassays. For example, the sample can be contacted
with mouse anti-PAGE-4 antibody, washed with buffer, and tested for
the presence of bound antibody using a goat anti-mouse antibody
(antisera from a goat which recognizes mouse antigens).
Alternatively, the presence of PAGE-4 can be determined by
detecting the presence of mRNA encoding PAGE-4, through PCR or any
of several other assays known in the art.
[0077] Since removal of normal prostate, testis or uterine tissue,
together with the cancerous lesions, is part of standard cancer
therapy, the detection of PAGE-4 in body tissues following removal
of the lesions and of the organs expected to express PAGE-4
indicates that not all of the cancerous tissue has been removed.
Accordingly, detection of PAGE-4 protein can be a valuable signal
that further steps may be needed to eliminate the cancer.
[0078] Specific targeting and elimination of PAGE-4 positive normal
and malignant tissue is expected to be a promising therapeutic
approach. In one embodiment, ligands to PAGE-4, or to portions of
PAGE-4 expressed in conjunction with MHC molecules, are used to
target therapeutic agents to cells or tissues expressing PAGE-4.
For example, anti-PAGE-4 antibodies can be coupled to conventional
chemotherapeutic agents to deliver them specifically to PAGE-4
containing cells, thus providing a high local concentration of the
agent while reducing the agent's systemic effect. Anti-PAGE-4
antibodies can also be coupled to and used to deliver other
therapeutic agents, such as radioisotopes or plant or bacterial
cytotoxins. The antibody portion of the molecule acts as a
targeting moiety and delivers the toxin to the cell recognized by
the antibody. The toxin, in turn, is engineered to lack portions
responsible for non-specific binding so that it affects primarily
only those cells to which it is delivered by the targeting
moiety.
[0079] In another embodiment, nucleic acids which are complementary
to RNA encoding PAGE-4 can be administered to a patient in need.
The use of synthetic oligonucleotides to bind to mRNA in a
sequence-specific manner has been found to block translation of the
encoded protein. Protocols for a number of clinical trials for
systemic administration of antisense oligonucleotides directed
against a variety of conditions are available in the literature and
provide those of skill in the art guidance on, among other things,
the construction of appropriate synthetic oligonucleotides, the
design of preclinical and clinical studies, methods of
administering such oligonucleotides to patients, and other
information relevant to use of antisense oligonucleotides as
therapeutic agents. See, e.g., Yacyshyn, Gastroenterology,
114:1133-1142 (1998); Bishop, J. Clin. Oncol., 14:1320-1326 (1996);
Morgan, Human Gene Ther. 7:1281-1306 (1996); Bayever, Antisense Res
Devel, 3:383-390 (1993). See also, Zon, Molec. Nueurobiol.
10:219-229 (1995). At least one antisense drug has been approved by
the FDA.
VI. Vaccine Strategies
[0080] A. Determination of Immunogenic Peptides
[0081] CTL response is based on the presentation to CD8+ T cells of
antigens in combination with molecules of HLA class I. One
difficulty in vaccine design has been the polymorphic nature of HLA
class I molecules, of which more than 100 alleles and isotypes are
known. In humans, MHC class I antigens are encoded by the HLA-A, B,
and C loci. HLA-A and B are expressed at the cell surface in
roughly equal densities, whereas HLA-C is expressed as
significantly lower density. Each of these loci has numerous
alleles.
[0082] For peptide based vaccines, it is preferable if the peptides
comprise motifs recognized by alleles having a wide distribution in
the human population. Since the MHC alleles occur at different
frequencies within different ethnic groups and races, the choice of
target MHC may depend upon the target population. For example, the
majority of the Caucasoid population can be covered by peptides
which bind to four HLA allele subtypes, HLA-A2.1, A1, A3.2, and
A24.1, while adding peptides binding to a first allele, HLA-A11.2,
encompasses the majority of the Asian population. See, e.g.,
International Publication No. WO 94/20127; Sidney, J., et al.,
"Broadly Reactive HLA Restricted T Cell Epitopes and their
Implications for Vaccine Design," in Kaufmann, ed., Concepts in
Vaccine Design (Walter de Gruyter, Berlin, 1996).
[0083] Analysis of numerous alleles has also permitted
characterization of the alleles by "supertypes," which recognize
antigen presented for recognition by the presence of certain amino
acids or types of amino acids in certain positions. For example,
the B7-like supertype recognizes proline in position 2 and
hydrophobic or aliphatic amino acids at the C-terminus, whereas the
A3-like supertype is defined by a shared preference for peptides
bearing (in single letter code) A, L, I, V, M, S, or T at position
2 and a positively charged residue at the C-terminus. See, Sidney,
supra. Binding motifs for alleles HLA-A1, A2.1, A3.2, A11.2, and
A24, for example, are well characterized (see, e.g., Rammensee, et
al., Ann. Rev. Immun. 11:213-244 (1993); Ruppert, et al., Cell,
74:929-937 (1993); Kubo, et al., J. Immun., 152:3913-3924 (1994));
these alleles are expressed in 90% of the Caucasian population. In
fact, the "sequence motifs" and "anchor residues of the various MHC
alleles have been analyzed in detail and hundreds of peptide
ligands for the more important class I molecules have been reported
(reviewed in Rammensee, H-G., et al., Immunogenetics, 41:178-228
(1995) ("Rammensee, 1995")). Rammensee, 1995 sets forth for each
allele the "anchor" residues at positions 2 and 9, any auxiliary
anchor positions for the particular allele, and preferred residues
at other positions.
[0084] The selection and screening of peptide epitopes for their
MHC binding capacity and in vitro and in vivo activation of CTLs
has also been closely studied and is taught in the literature. See,
e.g., Celis, E., et al., Cancer Biol., 6:329-336 (1995); Chesnut,
R., et al., "Design and Testing of Peptide-Based Cytotoxic T-Cell
Mediated Immunotherapeutics to Treat Infectious Diseases and
Cancer," in Powell, M. and Newman, M., eds., Vaccine Design: The
Subunit and Adjuvant Approach (Plenum Press, New York, 1995);
Celis, E., et al., Mol. Immunol., 31:1423-1430 (1994) (hereafter,
"Celis, 1994"); Lanzavecchia, A. Science, 260:937-943 (1993);
Celis, E., et al., Proc Natl Acad Sci USA 91:2105-2109 (1994);
Sinigaglia, F., and Hammer, J., J. Exp. Med. 181:449-451
(1995).
[0085] For example, Celis, et al., Mol. Immun. 31:1423-1430 (1994)
("Celis, 1994"), describes the identification of potential CTL
epitopes of MAGE-1. They screened the 309 amino acid sequence of
the MAGE-1 protein for the presence of peptides 9-10 residues in
length, containing binding motifs for HLA-A1, -A2.1, -A3.2, -A11,
and A24, and synthesized 170 such peptides. The peptides were
tested for their binding to purified MHC molecules by standard
assays and those with high or intermediate affinity to the purified
MHC molecules selected as potential epitopes for melanoma-specific
CTL. Peptides so selected are then candidates for testing for their
ability to activate CTLs. A variety of assays for this purpose are
known in the art, and include immunization of transgenic mice, in
vitro CTL studies using PBMC or tumor infiltrating lymphocytes
isolated from patients with cancers of the relevant type, and in
vitro inductions using PBMC from normal HLA-typed individuals. See,
e.g., Celis, 1994; Vitiello, et al., J. Exp. Med., 173:1007-1015
(1991); Traversari, et al., J. Exp. Med., 176:1453-1457 (1992);
Celis, et al., Proc Natl Acad Sci USA, 91:2105-2109 (1994). The
tumor associated antigens MAGE-2 and MAGE-3 have also been used as
the basis for the development of 9-mer and 10-mer synthetic
peptides which bind to a chosen HLA allele and which stimulate CTLs
against those antigens. See, U.S. Pat. No. 5,662,907.
[0086] Recent studies and advances in combinatorial peptide
chemistry have improved the ability to describe and to predict the
specificities of HLA molecules. See, e.g., Buus, S., Curr. Opin.
Immunol., 11:209-213 (1999); Schafer, J., et al., Vaccine,
16:1880-1884 (1998). Additional methods for predicting whether a
particular peptide will bind to a MHC molecule have been developed
and have been asserted to be more accurate than the use of sequence
motifs. For example, Gulukota, K., et al., J. Mol. Biol.,
267:1258-1267 (1997), teach what they style as neural net and
polynomial methods for predicting whether a particular peptide will
bind to a MHC molecule. The methods are complementary, with one
eliminating false positives and the other better at eliminating
false negatives.
[0087] B. Vaccines of the Invention
[0088] The present invention provides the recognition that the
PAGE-4 protein is expressed in cancers, especially those of the
prostate, testicles, and uterus. As noted in the preceding
sections, the relation to MAGE and GAGE proteins strongly suggests
that PAGE-4 is processed or presented by cells, and thus is
suitable as a vaccine.
[0089] The PAGE-4 protein itself can be used as a therapeutic
vaccine to enhance the patient's own immune response to tumor cells
expressing PAGE-4. The vaccine, which acts as an immunogen, may be
a cell, a lysate from cells transfected with a recombinant
expression vector, or a culture supernatant containing the
expressed protein. Alternatively, the immunogen can be a partially
or substantially purified recombinant PAGE-4 protein.
[0090] In an important class of embodiments, the immunogen is a
PAGE-4 peptide or analog thereof. As noted in the preceding
section, HLA alleles bind 9-mer or 10-mer amino acid sequences, but
different alleles preferentially bind particular amino acids at
particular positions. In most cases, high affinity peptides are
immunogenic. See, e.g., Sette, et al., J. Immunol., 153:5586-5592
(1994). Using the art-recognized sequence motifs, neural net and
polynomial methods discussed above, one of skill can readily select
sequences of the PAGE-4 protein which have high affinity for
alleles which are found in any given target human population. Given
the relation of PAGE-4 to MAGE proteins, it is expected that the
procedure used by Celis, 1994 to identify CTL epitopes for MAGE-1
and the procedures employed by Kubo, et al., to find synthetic
peptides activating CTLs against MAGE-2 and MAGE-3 (as taught in
U.S. Pat. No. 5,662,907), can be employed to find and test
potential CTL epitopes for PAGE-4.
[0091] The identification of motifs can be performed manually, or
by computer, using programs such as the "FINDPATTERNS" program
reported by Devereux et al., Nucl. Acids Res., 12:387-395 (1984).
The peptides will typically be between 7 and 30 amino acids in
length, are preferably between 8 and 20 amino acids in length, and
more preferably are between 8 and 15 amino acids in length. Most
preferably, the peptides are 9 or 10 amino acids in length, and
correspond to the length most useful for the particular allele to
which they are intended to bind. Typically, the peptides are
selected because they satisfy the binding preferences of one or
more MHC alleles found in a desired percentage of the target
population. The peptides selected can readily be tested for binding
to an HLA allele of choice and for activation of cytotoxic T
lymphocytes by the assays mentioned above (see, e.g., U.S. Pat. No.
5,662,907).
[0092] As noted, each allele has certain preferred residues at
certain positions. Thus, the binding affinity of a particular
peptide for a particular allele can in some cases be improved by
making a substitution of a preferred amino acid for the one present
in the native sequence of the original protein. The peptide can
then be tested by the assays discussed above to determine its
binding ability and to see if it retains the ability to induce a
CTL response against cells expressing the PAGE-4 protein.
[0093] Modified peptides or analogs thereof can also be substituted
for the natural amino acids to impart desired characteristics to
the immunogenic peptide. The modification can be, for example, the
substitution of L-amino acids by their D-isomers, or by derivatized
L-amino acids. Further descriptions of substitutions and
modifications are disclosed in, for example, International
Publication Nos. WO 94/20127 and WO 95/25122. The proteins or
peptides may be conjugated with other agents to increase their
immunogenicity or half-life in the body, or both. In some
embodiments, the peptides or proteins are conjugated to lipid or
lipoprotein or administered in liposomal form or with adjuvant.
Lipidation of the peptides or proteins, and appropriate adjuvants,
are discussed in the section on pharmaceutical compositions,
infra.
[0094] While the immunogenic peptides or proteins may to be
administered in a pure or substantially pure form, it is generally
preferable to present them as a pharmaceutical composition,
formulation or preparation. In the case of nucleic acids, however,
"naked" DNA (typically, DNA in a plasmid with a strong promoter)
can be introduced. See, e.g., Rappuoli and Del Giudice,
"Identification of Vaccine Targets," in Paolletti and McInnes,
eds., Vaccines, From Concept to Clinic, CRC Press, Boca Raton, Fla.
(1999). These authors state that vaccination by naked DNA is the
most effective method known to date for inducing a cytotoxic immune
response against an antigen. Id. In a preferred embodiment, the
nucleic acids are loaded onto gold microcarriers, which are then
introduced through the skin by a pulse of helium via the Helios.TM.
Gene Gun (Bio-Rad Laboratories, Inc., Hercules, Calif.).
VI. Ex vivo Uses of the Peptides or Protein
[0095] The compositions and methods of the invention can be used ex
vivo to augment an organism's immune response. In this regard, a
portion of the organism's lymphocytes are removed and cultured in
vitro with high doses of the immunogenic peptides or the PAGE-4
protein, providing a stimulatory concentration of peptide in the
cell medium in excess of that which could be achieved in the body.
Following treatment to stimulate the CTLs, the cells are returned
to the host, allowing the activated CTLs to attack
PAGE-4-expressing cells in the organism.
[0096] In one method, CTL responses to PAGE-4-expressing cells are
induced by incubating in tissue culture a patient's CTL precursor
cells together with a source of antigen presenting cells and the
appropriate immunogenic peptide or the PAGE-4 protein. After an
appropriate incubation period (which may be 1-4 weeks), the CTL
precursors are activated and mature and expand into CTLs. To
optimize in vitro conditions, the culture of stimulator cells is
typically maintained in an appropriate serum-free medium.
Peripheral blood lymphocytes are conveniently isolated following
simple venipuncture or leukopheresis of normal donors or patients
and used as the responder cell sources of CTL precursors. In one
embodiment, the appropriate APC are incubated with about 10-100
.mu.M of peptide in serum-free media for 4 hours under appropriate
culture conditions. The peptide-loaded APC are then incubated with
the responder cell populations in vitro for 5 to 10 days under
optimized culture conditions.
[0097] Positive CTL activation can be determined by assaying the
cultures for the presence of CTLs that kill radiolabeled target
cells, both specific peptide-pulsed targets as well as target cells
expressing endogenously processed form of the PAGE-4 protein from
which the peptide sequence was derived. Specificity and MHC
restriction of the CTL of a patient can be determined by a number
of methods known in the art. For instance, CTL restriction can be
determined by testing against different peptide target cells
expressing appropriate or inappropriate human MHC class I. The
peptides that test positive in the MHC binding assays and give rise
to specific CTL responses are identified as immunogenic peptides.
More details about the selection of CTLs and their separation from
antigen presenting cells are set forth in, e.g., U.S. Pat. No.
5,932,224. See also; WO 95/25122.
[0098] Antigen presenting cells may also be exposed to vectors
carrying nucleic acid sequences encoding the immunogenic peptides
or the PAGE-4 protein, or "naked" DNA encoding the peptides or
proteins can be introduced by the Helios.TM. Gene Gun (see previous
section) or other methods. Once dosed or transfected, the cells may
be propagated in vitro or returned to the patient. Conveniently,
the cells are propagated in vitro until they reach a predetermined
cell density, after which they are reintroduced into the host.
[0099] Return of cells to the host may be by any of several methods
well known in the art, and include procedures such as those
exemplified in U.S. Pat. No. 4,844,893 to Honsik and U.S. Pat. No.
4,690,915 to Rosenberg. Conveniently, the cells may be reintroduced
by intravenous infusion.
VII. Antibody Production
[0100] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably
isolated PAGE-4 protein or immunogenic peptides thereof are mixed
with an adjuvant and animals are immunized with the mixture. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. If desired, further fractionation of the antisera to
enrich for antibodies reactive to the polypeptide is performed.
See, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene,
NY (1991); and Harlow & Lane, supra, which are incorporated
herein by reference.
[0101] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Description of techniques for
preparing such monoclonal antibodies may be found in, e.g., Stites,
et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4TH ED.), Lange
Medical Publications, Los Altos, Calif., and references cited
therein; Harlow & Lane, supra; Goding, MONOCLONAL ANTIBODIES:
PRINCIPLES AND PRACTICE (2D ED.), Academic Press, New York, N.Y.
(1986); Kohler & Milstein, Nature 256:495-497 (1975); and
particularly Chowdhury, P. S., et al., Mol. Immunol. 34:9 (1997),
which discusses one method of generating monoclonal antibodies.
[0102] It is preferred here that monoclonal antibodies are made by
immunizing an animal with a nucleic acid sequence that encodes the
desired immunogen, in this case, PAGE-4. Immunization with
non-replicating transcription units that encode heterologous
protein(s) elicits antigen specific immune responses. After
translation into the foreign protein, the protein is processed and
presented to the immune system like other cellular proteins.
Because it is foreign, an immune response is mounted against the
protein and peptide epitopes that are derived from it (Donnelly, et
al., J. Immunol. Methods 176:145-152 (1994); and Boyer, et al., J.
Med. Primatol. 25:242-250 (1996)). This technique has two
significant advantages over protein-based immunization. One is that
it does not require the purification of the protein, which at best,
is time consuming and in cases of many membrane proteins, is very
difficult. A second advantage is that since the immunogen is
synthesized in a mammalian host, it undergoes proper
post-translational modifications and folds into the native
structure.
[0103] In preferred embodiments, the monoclonal antibody is a scFv.
Methods of making scFv antibodies have been described. See, Huse,
et al., supra; Ward, et al. Nature 341:544-546 (1989); and Vaughan,
et al., supra. In brief, mRNA from B-cells is isolated and cDNA is
prepared. The cDNA is amplified by well known techniques, such as
PCR, with primers specific for the variable regions of heavy and
light chains of immunoglobulins. The PCR products are purified by,
for example, agarose gel electrophoresis, and the nucleic acid
sequences are joined. If a linker peptide is desired, nucleic acid
sequences that encode the peptide are inserted between the heavy
and light chain nucleic acid sequences. The sequences can be joined
by techniques known in the art, such as blunt end ligation,
insertion of restriction sites at the ends of the PCR products or
by splicing by overlap extension (Chowdhury, et al., Mol. Immunol.
34:9 (1997)). After amplification, the nucleic acid which encodes
the scFv is inserted into a vector, again by techniques well known
in the art. Preferably, the vector is capable of replicating in
prokaryotes and of being expressed in both eukaryotes and
prokaryotes.
[0104] In a particularly preferred embodiment, scFv are chosen
through a phage display library. After antibody titers against the
antigen in the immunized animal reach their maximum, the animal is
sacrificed and the spleen removed. The procedure described above
for synthesizing scFv is followed. After amplification by PCR, the
scFv nucleic acid sequences are fused in frame with gene III (gIII)
which encodes the minor surface protein gIIIp of the filamentous
phage (Marks, et al., J. Biol. Chem. 267:16007-16010 (1992); Marks,
et al., Behring Inst. Mitt. 91:6-12 (1992); and Brinkmann, et al.,
J. Immunol. Methods 182:41-50 (1995)). The phage expresses the
resulting fusion protein on their surface. Since the proteins on
the surface of the phage are functional, phage bearing
PAGE-4-binding antibodies can be separated from non-binding or
lower affinity phage by panning or antigen affinity chromatography
(McCafferty, et al., Nature 348:552-554 (1990)).
[0105] In a preferred embodiment, scFv that specifically bind to
PAGE-4 protein or immunogenic peptides thereof are found by
panning. Panning is done by coating a solid surface with PAGE-4
protein or an immunogenic peptide thereof and incubating the phage
on the surface for a suitable time under suitable conditions. The
unbound phage is washed off the solid surface and the bound phage
is eluted. Finding the antibody with the highest affinity is
dictated by the efficiency of the selection process and depends on
the number of clones that can be screened and the stringency with
which it is done. Typically, higher stringency corresponds to more
selective panning. However, if the conditions are too stringent,
the phage will not bind. After one round of panning, the phage that
bind to PAGE-4 or immunogenic peptide coated plates are expanded in
E. coli and subjected to another round of panning. In this way, an
enrichment of 2000-fold occurs in 3 rounds of panning. Thus, even
when enrichment in each round is low, multiple rounds of panning
will lead to the isolation of rare phage and the genetic material
contained within which encodes the sequence of the highest affinity
antibody. The physical link between genotype and phenotype provided
by phage display makes it possible to test every member of a cDNA
library for binding to antigen, even with libraries as large as
100,000,000 clones.
VIII. Production of Immunoconjugates
[0106] Immunoconjugates include, but are not limited to, molecules
in which there is a covalent linkage of an effector moiety, such as
a therapeutic agent, to an antibody. A therapeutic agent is an
agent with a particular biological activity directed against a
particular target molecule or a cell bearing a target molecule. One
of skill in the art will appreciate that therapeutic agents may
include various drugs such as vinblastine, daunomycin and the like,
cytotoxins such as native or modified Pseudomonas exotoxin or
Diphtheria toxin, encapsulating agents, (e.g., liposomes) which
themselves contain pharmacological compositions, radioactive agents
such as .sup.125I, .sup.32P, .sup.14C, .sup.3H and .sup.35S and
other labels, target moieties and ligands.
[0107] The choice of a particular therapeutic agent depends on the
particular target molecule or cell and the biological effect is
desired to evoke. Thus, for example, the therapeutic agent may be a
cytotoxin which is used to bring about the death of a particular
target cell. Conversely, where it is merely desired to invoke a
non-lethal biological response, the therapeutic agent may be
conjugated to a non-lethal pharmacological agent or a liposome
containing a non-lethal pharmacological agent.
[0108] With the therapeutic agents and antibodies herein provided,
one of skill can readily construct a variety of clones containing
functionally equivalent nucleic acids, such as nucleic acids which
differ in sequence but which encode the same EM or antibody
sequence. Thus, the present invention provides nucleic acids
encoding antibodies and conjugates and fusion proteins thereof.
[0109] In preferred embodiments, the cell growth-inhibiting
molecules of the invention are prepared by cloning techniques.
Examples of appropriate cloning and sequencing techniques, and
instructions sufficient to direct persons of skill through many
cloning exercises are found in Sambrook, et al., MOLECULAR CLONING:
A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor
Laboratory (1989)), Berger and Kimmel (eds.), GUIDE TO MOLECULAR
CLONING TECHNIQUES, Academic Press, Inc., San Diego Calif. (1987)),
or Ausubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
Greene Publishing and Wiley-Interscience, NY (1987). Product
information from manufacturers of biological reagents and
experimental equipment also provide useful information. Such
manufacturers include the SIGMA chemical company (Saint Louis,
Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB
Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo
Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San
Diego, Calif., and Applied Biosystems (Foster City, Calif.), as
well as many other commercial sources known to one of skill.
[0110] Nucleic acids encoding native effector molecules or
anti-PAGE-4 antibodies can be modified to form the effector
molecule, antibodies, or immunoconjugates of the present invention.
Modification by site-directed mutagenesis is well known in the art.
Nucleic acids encoding effector molecule or anti-PAGE-4 antibodies
can be amplified by in vitro methods. Amplification methods include
the polymerase chain reaction (PCR), the ligase chain reaction
(LCR), the transcription-based amplification system (TAS), the
self-sustained sequence replication system (3SR). A wide variety of
cloning methods, host cells, and in vitro amplification
methodologies are well known to persons of skill.
[0111] In a preferred embodiment, immunoconjugates are prepared by
inserting the cDNA which encodes an anti-PAGE-4 scFv antibody into
a vector which comprises the cDNA encoding the effector molecule.
The insertion is made so that the scFv and the EM are read in frame
that is in one continuous polypeptide which contains a functional
Fv region and a functional EM region. In a particularly preferred
embodiment, cDNA encoding a diphtheria toxin fragment is ligated to
a scFv so that the toxin is located at the carboxyl terminus of the
scFv. In a most preferred embodiment, cDNA encoding PE is ligated
to a scFv so that the toxin is located at the amino terminus of the
scFv.
[0112] In addition to recombinant methods, the immunoconjugates,
effector molecules, and antibodies of the present invention can
also be constructed in whole or in part using standard peptide
synthesis. Solid phase synthesis of the polypeptides of the present
invention of less than about 50 amino acids in length may be
accomplished by attaching the C-terminal amino acid of the sequence
to an insoluble support followed by sequential addition of the
remaining amino acids in the sequence. Techniques for solid phase
synthesis are described by Barany & Merrifield, THE PEPTIDES:
ANALYSIS, SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE
SYNTHESIS, PART A. pp. 3-284; Merrifield, et al. J. Am. Chem. Soc.
85:2149-2156 (1963), and Stewart, et al., SOLID PHASE PEPTIDE
SYNTHESIS, 2ND ED., Pierce Chem. Co., Rockford, Ill. (1984).
Proteins of greater length may be synthesized by condensation of
the amino and carboxyl termini of shorter fragments. Methods of
forming peptide bonds by activation of a carboxyl terminal end
(e.g., by the use of the coupling reagent
N,N'-dicycylohexylcarbodiimide) are known to those of skill.
[0113] Once the nucleic acids encoding an EM, anti-PAGE-4 antibody,
or an immunoconjugate of the present invention are isolated and
cloned, one may express the desired protein in a recombinantly
engineered cell such as bacteria, plant, yeast, insect and
mammalian cells. It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of proteins including E. coli, other bacterial hosts,
yeast, and various higher eukaryotic cells such as the COS, CHO,
HeLa and myeloma cell lines. No attempt to describe in detail the
various methods known for the expression of proteins in prokaryotes
or eukaryotes will be made. In brief, the expression of natural or
synthetic nucleic acids encoding the isolated proteins of the
invention will typically be achieved by operably linking the DNA or
cDNA to a promoter (which is either constitutive or inducible),
followed by incorporation into an expression cassette. The
cassettes can be suitable for replication and integration in either
prokaryotes or eukaryotes. Typical expression cassettes contain
transcription and translation terminators, initiation sequences,
and promoters useful for regulation of the expression of the DNA
encoding the protein. To obtain high level expression of a cloned
gene, it is desirable to construct expression cassettes which
contain, at the minimum, a strong promoter to direct transcription,
a ribosome binding site for translational initiation, and a
transcription/translation terminator. For E. coli this includes a
promoter such as the T7, trp, lac, or lambda promoters, a ribosome
binding site and preferably a transcription termination signal. For
eukaryotic cells, the control sequences can include a promoter and
preferably an enhancer derived from immunoglobulin genes, SV40,
cytomegalovirus, and a polyadenylation sequence, and may include
splice donor and acceptor sequences. The cassettes of the invention
can be transferred into the chosen host cell by well-known methods
such as calcium chloride transformation or electroporation for E.
coli and calcium phosphate treatment, electroporation or
lipofection for mammalian cells. Cells transformed by the cassettes
can be selected by resistance to antibiotics conferred by genes
contained in the cassettes, such as the amp, gpt, neo and hyg
genes.
IX. Pseudomonas Exotoxin and Other Toxins
[0114] Toxins can be employed with antibodies of the present
invention to yield immunotoxins. Exemplary toxins include ricin,
abrin, diphtheria toxin and subunits thereof, as well as botulinum
toxins A through F. These toxins are readily available from
commercial sources (e.g., Sigma Chemical Company, St. Louis, Mo.).
Diphtheria toxin is isolated from Corynebacterium diphtheriae.
Ricin is the lectin RCA60 from Ricinus communis (Castor bean). The
term also references toxic variants thereof. For example, see, U.S.
Pat. Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin
(RCA) occurs in two forms designated RCA.sub.60 and RCA.sub.120
according to their molecular weights of approximately 65 and 120 kD
respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta
266:543 (1972)). The A chain is responsible for inactivating
protein synthesis and killing cells. The B chain binds ricin to
cell-surface galactose residues and facilitates transport of the A
chain into the cytosol (Olsnes, et al., Nature 249:627-631 (1974)
and U.S. Pat. No. 3,060,165).
[0115] Abrin includes toxic lectins from Abrus precatorius. The
toxic principles, abrin a, b, c, and d, have a molecular weight of
from about 63 and 67 kD and are composed of two disulfide-linked
polypeptide chains A and B. The A chain inhibits protein synthesis;
the B-chain (abrin-b) binds to D-galactose residues (see, Funatsu,
et al., Agr. Biol. Chem. 52:1095 (1988); and Olsnes, Methods
Enzymol. 50:330-335 (1978)).
[0116] In preferred embodiments of the present invention, the toxin
is Pseudomonas exotoxin (PE). The term "Pseudomonas exotoxin" as
used herein refers to a full-length native (naturally occurring) PE
or a PE that has been modified. Such modifications may include, but
are not limited to, elimination of domain Ia, various amino acid
deletions in domains Ib, II and III, single amino acid
substitutions and the addition of one or more sequences at the
carboxyl terminus such as KDEL (SEQ ID NO: 14) and REDL (SEQ ID NO:
15). See Siegall, et al., J. Biol. Chem. 264:14256 (1989). In a
preferred embodiment, the cytotoxic fragment of PE retains at least
50%, preferably 75%, more preferably at least 90%, and most
preferably 95% of the cytotoxicity of native PE. In a most
preferred embodiment, the cytotoxic fragment is more toxic than
native PE.
[0117] Native Pseudomonas exotoxin A (PE) is an extremely active
monomeric protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells.
The native PE sequence is provided as SEQ ID NO:1 of commonly
assigned U.S. Pat. No. 5,602,095, incorporated herein by reference.
The method of action is inactivation of the ADP-ribosylation of
elongation factor 2 (EF-2). The exotoxin contains three structural
domains that act in concert to cause cytotoxicity. Domain Ia (amino
acids 1-252) mediates cell binding. Domain II (amino acids 253-364)
is responsible for translocation into the cytosol and domain III
(amino acids 400-613) mediates ADP ribosylation of elongation
factor 2. The function of domain Ib (amino acids 365-399) remains
undefined, although a large part of it, amino acids 365-380, can be
deleted without loss of cytotoxicity. See Siegall, et al., J. Biol.
Chem. 264:14256-14261 (1989), incorporated by reference herein.
[0118] PE employed in the present invention includes the native
sequence, cytotoxic fragments of the native sequence, and
conservatively modified variants of native PE and its cytotoxic
fragments. Cytotoxic fragments of PE include those which are
cytotoxic with or without subsequent proteolytic or other
processing in the target cell (e.g., as a protein or pre-protein).
Cytotoxic fragments of PE include PE40, PE38, PE37, and PE35. PE40
is a truncated derivative of PE as previously described in the art.
See, Pai, et al., Proc. Nat'l Acad. Sci. USA 88:3358-62 (1991); and
Kondo, et al., J. Biol. Chem. 263:9470-9475 (1988). PE35 is a 35 kD
carboxyl-terminal fragment of PE composed of a met at position 280
followed by amino acids 281-364 and 381-613 of native PE. PE37,
another truncated derivative of PE, is described in U.S. Pat. No.
5,821,238. PE38 is a truncated PE pro-protein composed of amino
acids 253-364 and 381-613 which is activated to its cytotoxic form
upon processing within a cell (see U.S. Pat. No. 5,608,039,
incorporated herein by reference).
[0119] In a particularly preferred embodiment, PE38 is the toxic
moiety of the immunotoxin of this invention, however, other
cytotoxic fragments, such as PE35, PE37, and PE40, are contemplated
and are disclosed in U.S. Pat. Nos. 5,602,095; 5,821,238; and
4,892,827, each of which is incorporated herein by reference.
X. Detectable Labels
[0120] Antibodies of the present invention may optionally be
covalently or non-covalently linked to a detectable label.
Detectable labels suitable for such use include any composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means. Useful
labels in the present invention include magnetic beads (e.g.
DYNABEADS), fluorescent dyes (e.g., fluorescein isothiocyanate,
Texas red, rhodamine, green fluorescent protein, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or
.sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and calorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads.
[0121] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
illumination. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
XI. Conjugation of Effector Molecules to a Targeting Moiety
[0122] In a non-recombinant embodiment of the invention, effector
molecules, e.g., therapeutic, diagnostic, or detection moieties,
are linked to PAGE-4 targeting moieties, such as anti-PAGE-4
antibodies, using any number of means known to those of skill in
the art. Both covalent and noncovalent attachment means may be used
with anti-PAGE-4 antibodies or other ligands.
[0123] The procedure for attaching an effector molecule to an
antibody will vary according to the chemical structure of the EM.
Polypeptides typically contain variety of functional groups; e.g.,
carboxylic acid (COOH), free amine (--NH.sub.2) or sulfhydryl
(--SH) groups, which are available for reaction with a suitable
functional group on an antibody to result in the binding of the
effector molecule.
[0124] Alternatively, the antibody is derivatized to expose or
attach additional reactive functional groups. The derivatization
may involve attachment of any of a number of linker molecules such
as those available from Pierce Chemical Company, Rockford Ill.
[0125] A "linker", as used herein, is a molecule that is used to
join the antibody to the effector molecule. The linker is capable
of forming covalent bonds to both the antibody and to the effector
molecule. Suitable linkers are well known to those of skill in the
art and include, but are not limited to, straight or branched-chain
carbon linkers, heterocyclic carbon linkers, or peptide linkers.
Where the antibody and the effector molecule are polypeptides, the
linkers may be joined to the constituent amino acids through their
side groups (e.g., through a disulfide linkage to cysteine).
However, in a preferred embodiment, the linkers will be joined to
the alpha carbon amino and carboxyl groups of the terminal amino
acids.
[0126] In some circumstances, it is desirable to free the effector
molecule from the antibody when the immunoconjugate has reached its
target site. Therefore, in these circumstances, immunoconjugates
will comprise linkages which are cleavable in the vicinity of the
target site. Cleavage of the linker to release the effector
molecule from the antibody may be prompted by enzymatic activity or
conditions to which the immunoconjugate is subjected either inside
the target cell or in the vicinity of the target site. When the
target site is a tumor, a linker which is cleavable under
conditions present at the tumor site (e.g. when exposed to
tumor-associated enzymes or acidic pH) may be used.
[0127] In view of the large number of methods that have been
reported for attaching a variety of radiodiagnostic compounds,
radiotherapeutic compounds, drugs, toxins, and other agents to
antibodies one skilled in the art will be able to determine a
suitable method for attaching a given agent to an antibody or other
polypeptide.
XII. Pharmaceutical Compositions and Administration
[0128] Peptide and protein drugs are generally administered by
parenteral means. See, e.g., Banga, A., Parenteral Controlled
Delivery of Therapeutic Peptides and Proteins, in Therapeutic
Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster,
Pa., 1995. The peptide and protein vaccines of the invention will
typically be administered parenterally. Conveniently, this may be
done by i.v., s.c. or i.m. injection. Injection by s.c. and i.m.
procedures is preferable not only because they may permit a longer
duration of action, but also because they are exposed to dendritic
cells in the skin. These dendritic cells are important antigen
presenting cells. To extend the time during which the peptide or
protein is available to stimulate a response, the peptide or
protein can be provided as an implant, an oily injection, or as a
particulate system. The particulate system can be a microparticle,
a microcapsule, a microsphere, a nanocapsule, or similar particle.
See, e.g., Banja, supra.
[0129] A particulate carrier based on a synthetic polymer has been
shown to act as an adjuvant to enhance the immune response, in
addition to providing a controlled release. Aluminum salts may also
be used as adjuvants.
[0130] In preferred embodiments, the vaccines are administered in a
manner to direct the immune response to a cellular response (that
is, a CTL response), rather than a humoral (antibody) response. A
number of means for inducing cellular responses are known. Lipids
have been identified as agents capable of assisting in priming CTL
in vivo against various antigens. For example, as described in U.S.
Pat. No. 5,662,907, palmitic acid residues can be attached to the
alpha and epsilon amino groups of a lysine residue and then linked
(e.g., via one or more linking residues, such as glycine,
glycine-glycine, serine, serine-serine, or the like) to an
immunogenic peptide. The lipidated peptide can then be injected
directly in a micellar form, incorporated in a liposome, or
emulsified in an adjuvant. As another example, E. coli
lipoproteins, such as tripalmitoyl-5-glycerylcysteinlyseryl-serine
can be used to prime tumor specific CTL when covalently attached to
an appropriate peptide. See, Deres et al., Nature, 342:561-564
(1989). Further, as the induction of neutralizing antibodies can
also be primed with the same molecule conjugated to a peptide which
displays an appropriate epitope, the two compositions can be
combined to elicit both humoral and cell-mediated responses where
that is deemed desirable.
[0131] In yet another to inducing a CTL response to an immunogenic
peptide, a MHC class II-restricted T-helper epitope is added to the
CTL antigenic peptide to induce T-helper cells to secrete cytokines
in the microenvironment to activate CTL precursor cells. The
technique further involves adding short lipid molecules to retain
the construct at the site of the injection for several days to
localize the antigen at the site of the injection and enhance its
proximity to dendritic cells or other "professional" antigen
presenting cells over a period of time. See, e.g., Chesnut et al.,
"Design and Testing of Peptide-Based Cytotoxic T-Cell-Mediated
Immunotherapeutics to Treat Infectious Diseases and Cancer," in
Powell, et al., eds., Vaccine Design, the Subunit and Adjuvant
Approach, Plenum Press, New York, 1995.
[0132] In a preferred embodiment, the peptides or protein is mixed
with an adjuvant containing two or more of a stabilizing detergent,
a micelle-forming agent, and an oil. Suitable stabilizing
detergents, micelle-forming agents, and oils are detailed in U.S.
Pat. Nos. 5,585,103; 5,709,860; 5,270,202; and 5,695,770, all of
which are incorporated by reference.
[0133] By "stabilizing detergent" is meant a detergent that allows
the components of the emulsion to remain as a stable emulsion. Such
detergents include polysorbate, 80 (TWEEN)
(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl;
manufactured by ICI Americas, Wilmington, Del.), TWEEN 40, TWEEN
20, TWEEN 60, Zwittergent 3-12, TEEPOL HB7, and SPAN 85. These
detergents are usually provided in an amount of approximately 0.05
to 0.5%, preferably at about 0.2%.
[0134] By "micelle-forming agent" is meant an agent which is able
to stabilize the emulsion formed with the other components such
that a micelle-like structure is formed. Such agents preferably
cause some irritation at the site of injection in order to recruit
macrophages to enhance the cellular response. Examples of such
agents include polymer surfactants described by BASF Wyandotte
publications, e.g., Schmolka, J. Am. Oil. Chem. Soc. 54:110 (1977),
and Hunter et al., J. Immunol. 129:1244 (1981), PLURONIC L62LF,
L101, and L64, PEG1000, and TETRONIC 1501, 150R1, 701, 901, 1301,
and 130R1. The chemical structures of such agents are well known in
the art. Preferably, the agent is chosen to have a
hydrophile-lipophile balance (HLB) of between 0 and 2, as defined
by Hunter and Bennett, J. Immun. 133:3167 (1984). The agent is
preferably provided in an amount between 0.5 and 10%, most
preferably in an amount between 1.25 and 5%.
[0135] The oil is chosen to promote the retention of the antigen in
oil-in-water emulsion, i.e., to provide a vehicle for the desired
antigen, and preferably has a melting temperature of less than
65.degree. C. such that emulsion is formed either at room
temperature (about 20.degree. C. to 25.degree. C.), or once the
temperature of the emulsion is brought down to room temperature.
Examples of such oils include squalene, Squalane, EICOSANE,
tetratetracontane, glycerol, and peanut oil or other vegetable
oils. The oil is preferably provided in an amount between 1 and
10%, most preferably between 2.5 and 5%. The oil should be both
biodegradable and biocompatible so that the body can break down the
oil over time, and so that no adverse affects, such as granulomas,
are evident upon use of the oil.
[0136] In a particularly preferred embodiment, the adjuvant is a
mixture of stabilizing detergents, micelle-forming agent, and oil
available under the name Provax.RTM. (IDEC Pharmaceuticals, San
Diego, Calif.).
[0137] In another class of embodiments, the vaccine is in the form
of DNA encoding the immunogenic peptide or the PAGE-4 protein. As
noted elsewhere herein, one approach to vaccination is direct
vaccination with plasmid DNA. We have had good expression of PAGE-4
protein with mammalian expression plasmids. If desired, the PAGE-4
gene, or a nucleotide sequence encoding an immunogenic peptide, can
be placed under the control of a strong promoter to increase
expression of the molecule. Immunization by nucleic acid constructs
is well known in the art and taught, for example, in U.S. Pat. No.
5,643,578, which describes methods of immunizing vertebrates by
introducing DNA encoding a desired antigen to elicit a
cell-mediated or a humoral response, and U.S. Pat. Nos. 5,593,972
and 5,817,637, which describe operably linking a nucleic acid
sequence encoding an antigen to regulatory sequences enabling
expression.
[0138] U.S. Pat. No. 5,880,103 describes several methods of
delivery of nucleic acids encoding immunogenic peptides or other
antigens to an organism. The methods include liposomal delivery of
the nucleic acids (or of the synthetic peptides themselves), and
immune-stimulating constructs, or ISCOMS, negatively charged
cage-like structures of 30-40 nm in size formed spontaneously on
mixing cholesterol and Quil A (saponin). Protective immunity has
been generated in a variety of experimental models of infection,
including toxoplasmosis and Epstein-Barr virus-induced tumors,
using ISCOMS as the delivery vehicle for antigens (Mowat and
Donachie, Immunol. Today 12:383-385 (1991)). Doses of antigen as
low as 1 .mu.g encapsulated in ISCOMS have been found to produce
class I mediated CTL responses. Takahashi et al., Nature
344:873-875, (1990).
[0139] In another approach to using nucleic acids for immunization,
the PAGE-4 protein or an immunogenic peptide thereof can also be
expressed by attenuated viral hosts or vectors or bacterial
vectors. Recombinant vaccinia virus, AAV, herpesvirus, retrovirus,
or other viral vectors can be used to express the peptide or
protein, thereby eliciting a CTL response. For example, vaccinia
vectors and methods useful in immunization protocols are described
in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin) provides
another vector for expression of the peptides. BCG vectors are
described in, for example, Stover, Nature, 351:456-460 (1991).
[0140] In a preferred embodiment, nucleic acids encoding an
immunogenic peptide or the PAGE-4 protein are introduced directly
into cells. For example, the nucleic acids may be loaded onto gold
microspheres by standard methods and introduced into the skin by a
device such as Bio-Rad's HelioS.TM. Gene Gun. The nucleic acids can
be "naked," consisting of plasmids under control of a strong
promoter. Typically, the DNA is injected into muscle, although it
can also be injected directly into other sites, including tissues
in proximity to metastases. Dosages for injection are usually
around 0.5 .mu.g/kg to about 50 mg/kg, and typically are about
0.005 mg/kg to about 5 mg/kg. See, e.g., U.S. Pat. No.
5,589,466.
[0141] In addition to the vaccines of the invention, the cell
growth inhibiting chimeric molecules of this invention (i.e., PE
linked to an anti-PAGE-4 antibody), can be prepared in
pharmaceutical compositions. They are particularly useful for
parenteral administration, such as intravenous administration or
administration into a body cavity or lumen of an organ. For
example, metastases of prostate or testicular cancers may be
treated by intravenous administration or by localized delivery to
the tissue surrounding the tumor. To treat ovarian cancers, the
pharmaceutical compositions of this invention can be administered
directly into the pleural or peritoneal cavities.
[0142] The compositions for administration will commonly comprise a
solution of the cell growth inhibiting chimeric molecules dissolved
in a pharmaceutically acceptable carrier, preferably an aqueous
carrier. A variety of aqueous carriers can be used, e.g., buffered
saline and the like. These solutions are sterile and generally free
of undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of fusion protein in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0143] Thus, a pharmaceutical composition of the present invention
for intravenous administration, such as an immunotoxin, would be
about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to
about 100 mg per patient per day may be used, particularly if the
drug is administered to a secluded site and not into the
circulatory or lymph system, such as into a body cavity or into a
lumen of an organ. Actual methods for preparing administrable
compositions will be known or apparent to those skilled in the art
and are described in more detail in such publications as
REMINGTON'S PHARMACEUTICAL SCIENCE, 19TH ED., Mack Publishing
Company, Easton, Pa. (1995).
[0144] The compositions of the present invention can be
administered for therapeutic treatments. In therapeutic
applications, compositions are administered to a patient suffering
from a disease, in an amount sufficient to cure or at least
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. An effective amount of the compound is that which provides
either subjective relief of a symptom(s) or an objectively
identifiable improvement as noted by the clinician or other
qualified observer.
[0145] Single or multiple administrations of the compositions are
administered depending on the dosage and frequency as required and
tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient. Preferably, the dosage is
administered once but may be applied periodically until either a
therapeutic result is achieved or until side effects warrant
discontinuation of therapy. Generally, the dose is sufficient to
treat or ameliorate symptoms or signs of disease without producing
unacceptable toxicity to the patient.
[0146] Controlled release parenteral formulations of the
compositions of the present invention can be made as implants, oily
injections, or as particulate systems. For a broad overview of
protein delivery systems see, Banga, A. J., THERAPEUTIC PEPTIDES
AND PROTEINS: FORMULATION, PROCESSING, AND DELIVERY SYSTEMS,
Technomic Publishing Company, Inc., Lancaster, Pa., (1995)
incorporated herein by reference. Particulate systems include
microspheres, microparticles, microcapsules, nanocapsules,
nanospheres, and nanoparticles. Microcapsules contain the
therapeutic protein as a central core. In microspheres the
therapeutic is dispersed throughout the particle. Particles,
microspheres, and microcapsules smaller than about 1 .mu.m are
generally referred to as nanoparticles, nanospheres, and
nanocapsules, respectively. Capillaries have a diameter of
approximately 5 .mu.m so that only nanoparticles are administered
intravenously. Microparticles are typically around 100 .mu.m in
diameter and are administered subcutaneously or intramuscularly.
See, e.g., Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J.
Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342
(1994); and Tice & Tabibi, TREATISE ON CONTROLLED DRUG
DELIVERY, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,
pp. 315-339, (1992) both of which are incorporated herein by
reference.
[0147] Polymers can be used for ion-controlled release of
compositions of the present invention. Various degradable and
nondegradable polymeric matrices for use in controlled drug
delivery are known in the art (Langer, R., Accounts Chem. Res.
26:537-542 (1993)). For example, the block copolymer, polaxamer 407
exists as a viscous yet mobile liquid at low temperatures but forms
a semisolid gel at body temperature. It has shown to be an
effective vehicle for formulation and sustained delivery of
recombinant interleukin-2 and urease (Johnston, et al., Pharm. Res.
9:425-434 (1992); and Pec, et al., J Parent. Sci. Tech. 44(2):58-65
(1990)). Alternatively, hydroxyapatite has been used as a
microcarrier for controlled release of proteins (Ijntema, et al.,
Int. J. Pharm. 112:215-224 (1994)). In yet another aspect,
liposomes are used for controlled release as well as drug targeting
of the lipid-capsulated drug (Betageri, et al., LIPOSOME DRUG
DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, Pa.
(1993)). Numerous additional systems for controlled delivery of
therapeutic proteins are known. See, e.g., U.S. Pat. Nos.
5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028 4,957,735 and
5,019,369, 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;
4,902,505; 5,506,206, 5,271,961; 5,254,342 and 5,534,496, each of
which is incorporated herein by reference.
[0148] Among various uses of the compositions of the present
invention, such as immunotoxins of anti-PAGE-4 antibodies and PE40,
are included a variety of disease conditions caused by specific
human cells that may be eliminated by the toxic action of the
fusion protein. One preferred application for the immunotoxins of
the invention is the treatment of malignant cells expressing
PAGE-4. Exemplary malignant cells include prostate, testicular, and
ovarian cancers.
XIII. Diagnostic Kits
[0149] In another embodiment, this invention provides for kits for
the detection of PAGE-4-containing cells or tissues in a biological
sample. A "biological sample" as used herein is a sample of
biological tissue that contains PAGE-4. Such samples include, but
are not limited to, tissue from biopsies, autopsies, and pathology
specimens. Biological samples also include sections of tissues,
such as frozen sections taken for histological purposes. A
biological sample is typically obtained from a multicellular
eukaryote, preferably a mammal, such as a rat, mouse, cow, dog,
guinea pig, or rabbit, more preferably from a primate, such as a
macaque or a chimpanzee, and most preferably from a human.
[0150] Kits for detecting the PAGE-4 protein will typically
comprise an anti-PAGE-4 antibody. In some embodiments, the
anti-PAGE-4 antibody will be an Fv fragment. For in vivo uses, the
antibody is preferably an scFv fragment.
[0151] In addition the kits will typically include instructional
materials disclosing means of use of an antibody of the present
invention (e.g. for detection of PAGE-4-containing cells in a
sample). The kits may also include additional components to
facilitate the particular application for which the kit is
designed. Thus, for example, the kit may additionally contain means
of detecting a label (e.g. enzyme substrates for enzymatic labels,
filter sets to detect fluorescent labels, appropriate secondary
labels such as a sheep anti-mouse-HRP, or the like). The kits may
additionally include buffers and other reagents routinely used for
the practice of a particular method. Such kits and appropriate
contents are well known to those of skill in the art.
[0152] In one embodiment of the present invention, the diagnostic
kit comprises an immunoassay. Although the details of the
immunoassays may vary with the particular format employed, the
method of detecting PAGE-4 protein in a biological sample generally
comprises the steps of contacting the biological sample with an
antibody which specifically reacts, under immunologically reactive
conditions, to PAGE-4. The antibody is allowed to bind under
immunologically reactive conditions, and the presence of the bound
antibody is detected directly or indirectly.
[0153] In an alternative set of embodiments, kits can be provided
for detecting nucleic acids encoding PAGE-4 protein (a "PAGE-4
nucleic acid) in a biological sample. For example, a tissue sample
from a biopsy can be tested to determine whether nucleic acids
encoding PAGE-4 protein are present. Typically, the kits will
provide primers which will amplify all or a portion of a PAGE-4
nucleic acid. Conveniently, the amplification is performed by
polymerase chain reaction (PCR). A number of other techniques are,
however, known in the art and are contemplated for use in the
invention.
[0154] For example, Marshall, U.S. Pat. No. 5,686,272, discloses
the amplification of RNA sequences using ligase chain reaction, or
"LCR." LCR has been extensively described by Landegren et al.,
Science, 241:1077-1080 (1988); Wu et al., Genomics, 4:560-569
(1989); Barany, in PCR Methods and Applications, 1:5-16 (1991); and
Barany, Proc. Natl. Acad. Sci. USA, 88:189-193 (1991). Or, the RNA
can be reverse transcribed into DNA and then amplified by LCR, PCR,
or other methods. An exemplar protocol for conducting reverse
transcription of RNA is taught in U.S. Pat. No. 5,705,365.
Selection of appropriate primers and PCR protocols are taught, for
example, in Innis, M., et al., eds., PCR Protocols 1990 (Academic
Press, San Diego Calif.).
[0155] In addition the kits will typically include instructional
materials disclosing means of use for the primers (e.g. for
detection of PAGE-4-containing cells in a sample). The kits may
also include additional components to facilitate the particular
application for which the kit is designed. The kits may
additionally include buffers and other reagents routinely used for
the practice of a particular method. Such kits and appropriate
contents are well known to those of skill in the art.
EXAMPLES
Example 1
Computer Analysis of EST Sequences
[0156] The NCBI dbEST/CGAP database (Emmert-Buck et al., Science
274:998-1101 (1996); Krizman, D. B. et al., Cancer Res.
56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet.
16:415-516 (1997)), was used as a source for cDNA sequences. The
ESTs from human tissues and tumors were downloaded from
ftp://ncbi.nlm.nih.gov/repository/dbEST. The EST sequences were
clustered and sorted as described before (Vasmatzis, G. et al.,
Proc. Natl. Acad. Sci., USA 95:300-304 (1998)). However, the
candidate gene list was updated by using the EST dataset of Apr.
25, 1998. This dataset contains 1,001,294 human EST sequences from
656 libraries. Two updated candidate lists were prepared, one with
the specificity cutoff for prostate of three as before and another
with the cutoff value of six.
Example 2
Molecular Biology Techniques
[0157] EST-plasmids were obtained from the IMAGE Consortium (Genome
Systems, Inc., St. Louis, Mo.). The identities of the sequences
were confirmed and extended by automated fluorescent DNA sequencing
using an Applied Biosystem's Rhodamine-terminator cycle sequencing
kit. PCR was performed on a Biometra Thermocycler using Boehringer
Mannheim high fidelity reagent kits and the Hot-Start Technique.
Northern blots containing 2 .mu.g poly A mRNA from various tissues
and cancer cell lines (CLONTECH Laboratories, Inc., Palo Alto,
Calif.), blots with 20 .mu.g/lane total tumor RNA (Invitrogen
Corp., Carlsbad, Calif.), mRNA dot blots (Clontech), and a Somatic
Cell Hybrid Southern Blot (Oncor, Inc., Gaithersburg, Md.) were
hybridized with random primed .sup.32P labeled DNA fragments. The
specific activity of the labeled probe was 1 mCi/.mu.g. The
membranes were blocked for 4 hrs in hybridization solution (50%
formamide, without probe), hybridized for 15 hrs with the probe at
55.degree. C., rinsed in 2.times.SSC/0.1% SDS, washed once with
2.times.SSC/0.1% SDS and twice with 0.2.times.SSC/0.1% SDS at
55.degree. C.
Example 3
Generation of Antibodies
[0158] An exemplary PAGE-4 peptide was selected to demonstrate that
the PAGE-4 protein could be used to generate antibodies. The
sequence used was, in single letter code, EGTPPIEERKVEGDC (SEQ ID
NO: 16).
[0159] The peptide was conjugated with keyhole limpet hemacyanin
and used to immunize two New England White rabbits. Immunizations
took place on days 0, 14, 28, 42, 56, and 70. For the first
immunization, the peptide was emulsified with complete Freund's
adjuvant and 200 .mu.g of peptide was injected in sufficient
adjuvant to make a total volume of 200 .mu.L. For all following
immunization, 100 .mu.g of peptide was emulsified in enough
incomplete Freund's adjuvant for a total injection volume of 200
.mu.l. Serum was collected at days 49, 63, and 77, and analyzed by
ELISA.
Example 4
Expression of Plasmids Encoding PAGE-4
[0160] PAGE-4 open reading frame was cloned in frame into pET 23a
vector with a His tag at the 3' end. The recombinant plasmid was
then transformed into E. coli BL 21 (DE3) cells. Cells were grown
in liquid culture and PAGE-4 expression was induced with
isopropyl-B-D-thiogalactopyranoside ("IPTG"). PAGE-4 protein was
purified from the cell lysate using a Ni(2+)-NTA-agarose gel column
(Invitrogen).
Example 5
Immunization with DNA Encoding PAGE-4 Protein
[0161] A group of 6 male and 6 female BALB/c mice is injected
intradermally with a pCR3.1 vector (Invitrogen) with a pCRPage4
plasmid. The injections comprise 15 .mu.g of DNA in 100 .mu.l of
saline solution (0.15 molar) per injection per mouse, and are given
every other week for a total of three injections. Sera is collected
two weeks after the third injection and tested by ELISA on
recombinant PAGE-4. If higher titer is desired, additional
injections will be given. If desired, CTLs from the immunized mice
can also be tested for activation against PAGE-4-expressing
cells.
[0162] Although the present invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims. All publications and patents mentioned in this
specification are herein incorporated by reference into the
specification to the same extent as if each individual publication
or patent was specifically and individually indicated to be
incorporated herein by reference.
Table 1
Comparison of the Distribution of PAGE-4 Sequences in EST Libraries
with PAGE-4 Northern Hybridization Signals.
[0163] The human cDNA sequence libraries (dataset of Apr. 25, 1998)
were processed by computer analyses as previously described
(Vasmatzis, Proc. Natl. Acad. Sci. USA 95:300-304 (1998)).
Individual PAGE-4 ESTs are nh24e10.s1, nc27g01.r1, nh24a11.s1,
nf19h11.s1, nr35f03.s1 (prostate), nh32c06.s1, nt72b09.s1,
nc33g02.s1/r1, nc79f08.s1/r1, nt78f01.s1, (prostate cancer),
EST81031, EST80996, C18969, C18137, yi82c07.s1/r1, yw73c12.s1/r1
(placenta) and zr65g11.s1/r1, aa07e08.s1 (uterus; see note marked
by asterisk (*)).
[0164] Note (*): The ESTs with uterus-specificity were part of
pooled uterus-containing libraries; the other tissues of this
library did not show any PAGE-4 expression in hybridization
analyses. % PAGE-4/tissue: the number of PAGE-4 ESTs was divided by
the total number of tissues specific ESTs. Original dot blot and
Northern results are shown in FIG. 2. (+, ++, +++) indicate the
signal intensity on the dot blots or Northern blots: signal, strong
signal and very strong signal, respectively. Ovary gave a very weak
signal after prolonged exposure of the autoradiograph.
[0165] Note (#): Other tissues in the database analysis are those
that were represented by dbEST files in May 1998. Other tissues
that were tested by hybridization analyses were brain, spinal cord,
heart, aorta, skeletal muscle, colon, bladder, stomach, pancreas,
pituitary, adrenal, thyroid, salivary, mammary, kidney, liver,
small intestine, spleen, thymus, peripheral leukocyte, lymph node,
bone marrow, appendix, lung, trachea, fetal brain, fetal heart,
fetal kidney, fetal liver, fetal spleen, fetal thymus, fetal lung,
and cancer cell lines HL60, HeLa, K562, Molt4, Raji, SW480, A549
and G361. TABLE-US-00001 TABLE 1 Comparison of the distribution of
PAGE-1 sequences in EST libraries with PAGE-4 Northern
hybridization signals. Computational Analysis # of PAGE-4 % PAGE-4/
mRNA Analysis Tissue ESTs Tissue ESTs Dot Blot Northern RT-PCR
Prostate 5 5/22,334 (0.022%) ++ ++ + Prostate ca. 7 7/20,871
(0.031%) + Testis 0 0/31,263 + + Testis ca. 0 0/1,123 + Uterus 3*
0/22,333 (0.013%) + + Uterus ca. 0/1,112 ++ Ovary 0 0/5,573 (+) -
Ovary ca. 0 0/21,989 - Fallopian tube 0 0/0 ++ Placenta 8 8/49,467
(0.016%) +++ +++ + Other tissues/ca.# 0* 0/823,754 - - -
[0166] TABLE-US-00002 TABLE 2 Distribution of other members of
PAGE-4-like ESTs in the database. ESTs with homology to PAGE-4 were
identified by BLAST and FASTA (Altschul, S., et al., J. Mol. Biol.,
215:403-410 (1990); Pearson, W., et al., Proc. Natl. Acad. Sci.
USA, 85:2444- 2448 (1988). "/Ca." indicates ESTs from tumor
libraries. PAGE 2 and PAGE 3 are sequences that are more homologous
to PAGE than to any member of the GAGE protein family (see FIGS. 1
and 2). Tissue Distribution EST Cluster Prostate/Ca. Testis
Placenta Germ Cell/Ca. Pool (w/Uterus) Pool (w/Testis) PAGE 1
nh24e10 yw73c12 aa07e08 nc24a11 yi82c07 zr65g11 nh27g01 C18137
nf19h11 C18969 nr35f03 EST80996 nh32c06/Ca. EST81031 nc33g02/Ca.
nt72b09/Ca. nt78f01/Ca. nc79f08/Ca. PAGE-2 ai61a04 om69f10/Ca.
om13c03 zv62h08 oj89d1 aj29d06 zv58h12 PAGE 3 om29f08
[0167]
Sequence CWU 1
1
16 1 102 PRT Homo sapiens 1 Met Ser Ala Arg Val Arg Ser Arg Ser Arg
Gly Arg Gly Asp Gly Gln 1 5 10 15 Glu Ala Pro Asp Val Val Ala Phe
Val Ala Pro Gly Glu Ser Gln Gln 20 25 30 Glu Glu Pro Pro Thr Asp
Asn Gln Asp Ile Glu Pro Gly Gln Glu Arg 35 40 45 Glu Gly Thr Pro
Pro Ile Glu Glu Arg Lys Val Glu Gly Asp Cys Gln 50 55 60 Glu Met
Asp Leu Glu Lys Thr Arg Ser Glu Arg Gly Asp Gly Ser Asp 65 70 75 80
Val Lys Glu Lys Thr Pro Pro Asn Pro Lys His Ala Lys Thr Lys Glu 85
90 95 Ala Gly Asp Gly Gln Pro 100 2 117 PRT Homo sapiens 2 Met Ser
Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg Tyr 1 5 10 15
Val Glu Pro Pro Glu Met Ile Gly Pro Met Arg Pro Glu Gln Phe Ser 20
25 30 Asp Glu Val Glu Pro Ala Thr Pro Glu Glu Gly Glu Pro Ala Thr
Gln 35 40 45 Arg Gln Asp Pro Ala Ala Ala Gln Glu Gly Glu Asp Glu
Gly Ala Ser 50 55 60 Ala Gly Gln Gly Pro Lys Pro Glu Ala Asp Ser
Gln Glu Gln Gly His 65 70 75 80 Pro Gln Thr Gly Cys Glu Cys Glu Asp
Gly Pro Asp Gly Gln Glu Met 85 90 95 Asp Pro Pro Asn Pro Glu Glu
Val Lys Thr Pro Glu Glu Glu Met Arg 100 105 110 Ser His Tyr Val Ala
115 3 116 PRT Homo sapiens 3 Met Ser Trp Arg Gly Arg Ser Thr Tyr
Arg Pro Arg Pro Arg Arg Tyr 1 5 10 15 Val Glu Pro Pro Glu Met Ile
Gly Pro Met Arg Pro Glu Gln Phe Ser 20 25 30 Asp Glu Val Glu Pro
Ala Thr Pro Glu Glu Gly Glu Pro Ala Thr Gln 35 40 45 Arg Gln Asp
Pro Ala Ala Ala Gln Glu Gly Glu Asp Glu Gly Ala Ser 50 55 60 Ala
Gly Gln Gly Pro Lys Pro Glu Ala His Ser Gln Glu Gln Gly His 65 70
75 80 Pro Gln Thr Gly Cys Glu Cys Glu Asp Gly Pro Asp Gly Gln Glu
Met 85 90 95 Asp Pro Pro Asn Pro Glu Glu Val Lys Thr Pro Glu Glu
Gly Glu Lys 100 105 110 Gln Ser Gln Cys 115 4 118 PRT Homo sapiens
4 Met Asn Leu Ser Arg Gly Lys Ser Thr Tyr Tyr Arg Pro Arg Pro Arg 1
5 10 15 Arg Tyr Val Gln Pro Pro Glu Val Ile Gly Pro Met Arg Pro Glu
Gln 20 25 30 Phe Ser Asp Glu Val Glu Pro Ala Thr Pro Glu Glu Gly
Glu Pro Ala 35 40 45 Thr Gln Arg Gln Asp Pro Ala Ala Ala Gln Glu
Gly Glu Asp Glu Gly 50 55 60 Ala Ser Ala Gly Gln Gly Pro Lys Pro
Glu Ala Asp Ser Gln Glu Gln 65 70 75 80 Gly His Pro Gln Thr Gly Cys
Glu Cys Glu Asp Gly Pro Asp Gly Gln 85 90 95 Glu Met Asp Pro Pro
Asn Pro Glu Glu Val Lys Thr Pro Glu Glu Gly 100 105 110 Glu Lys Gln
Ser Gln Cys 115 5 117 PRT Homo sapiens 5 Met Ser Trp Arg Gly Arg
Ser Thr Tyr Tyr Arg Pro Arg Pro Arg Arg 1 5 10 15 Tyr Val Gln Pro
Pro Glu Met Ile Gly Pro Met Arg Pro Glu Gln Phe 20 25 30 Ser Asp
Glu Val Glu Pro Ala Thr Pro Glu Glu Gly Glu Pro Ala Thr 35 40 45
Gln Arg Gln Asp Pro Ala Ala Ala Gln Glu Gly Glu Asp Glu Gly Ala 50
55 60 Ser Ala Gly Gln Gly Pro Lys Pro Glu Ala Asp Ser Gln Glu Gln
Gly 65 70 75 80 His Pro Gln Thr Gly Cys Glu Cys Glu Asp Gly Pro Asp
Gly Gln Glu 85 90 95 Met Asp Pro Pro Asn Pro Glu Glu Val Lys Thr
Pro Glu Glu Gly Glu 100 105 110 Lys Gln Ser Gln Cys 115 6 117 PRT
Homo sapiens 6 Met Ser Trp Arg Gly Arg Ser Thr Tyr Tyr Arg Pro Arg
Pro Arg Arg 1 5 10 15 Tyr Val Gln Pro Pro Glu Val Ile Gly Pro Met
Arg Pro Glu Gln Phe 20 25 30 Ser Asp Glu Val Glu Pro Ala Thr Pro
Glu Glu Gly Glu Pro Ala Thr 35 40 45 Gln Arg Gln Asp Pro Ala Ala
Ala Gln Glu Gly Glu Asp Glu Gly Ala 50 55 60 Ser Ala Gly Gln Gly
Pro Lys Pro Glu Ala Asp Ser Gln Glu Gln Gly 65 70 75 80 His Pro Gln
Thr Gly Cys Glu Cys Glu Asp Gly Pro Asp Gly Gln Glu 85 90 95 Met
Asp Pro Pro Asn Pro Glu Glu Val Lys Thr Pro Glu Glu Gly Glu 100 105
110 Lys Gln Ser Gln Cys 115 7 117 PRT Homo sapiens 7 Met Ser Trp
Arg Gly Arg Ser Thr Tyr Tyr Arg Pro Arg Pro Arg Arg 1 5 10 15 Tyr
Val Gln Pro Pro Glu Val Ile Gly Pro Met Arg Pro Glu Gln Phe 20 25
30 Ser Asp Glu Val Glu Pro Ala Thr Pro Glu Glu Gly Glu Pro Ala Thr
35 40 45 Gln Arg Gln Asp Pro Ala Ala Ala Gln Glu Gly Glu Asp Glu
Gly Ala 50 55 60 Ser Ala Gly Gln Gly Pro Lys Pro Glu Ala Asp Ser
Gln Glu Gln Gly 65 70 75 80 His Pro Gln Thr Gly Cys Glu Cys Glu Asp
Gly Pro Asp Gly Gln Glu 85 90 95 Val Asp Pro Pro Asn Pro Glu Glu
Val Lys Thr Pro Glu Glu Gly Glu 100 105 110 Lys Gln Ser Gln Cys 115
8 124 PRT Homo sapiens 8 Met Ser Leu Glu Gln Lys Ser Gln His Cys
Lys Pro Glu Glu Gly Leu 1 5 10 15 Asp Thr Gln Glu Glu Ala Leu Gly
Leu Val Gly Val Gln Ala Ala Thr 20 25 30 Thr Glu Glu Gln Glu Ala
Val Ser Ser Ser Ser Pro Leu Val Pro Gly 35 40 45 Thr Leu Gly Glu
Val Pro Ala Ala Gly Ser Pro Gly Pro Leu Lys Ser 50 55 60 Pro Gln
Gly Ala Ser Ala Ile Pro Thr Ala Ile Asp Phe Thr Leu Trp 65 70 75 80
Arg Gln Ser Ile Lys Gly Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85
90 95 Thr Ser Pro Asp Pro Glu Ser Val Phe Arg Ala Ala Leu Ser Lys
Lys 100 105 110 Val Ala Asp Leu Ile His Phe Leu Leu Leu Lys Tyr 115
120 9 127 PRT Homo sapiens 9 Met Leu Leu Gly Gln Lys Ser Gln Arg
Tyr Lys Ala Glu Glu Gly Leu 1 5 10 15 Gln Ala Gln Gly Glu Ala Pro
Gly Leu Met Asp Val Gln Ile Pro Thr 20 25 30 Ala Glu Glu Gln Lys
Ala Ala Ser Ser Ser Ser Thr Leu Ile Met Gly 35 40 45 Thr Leu Glu
Glu Val Thr Asp Ser Gly Ser Pro Ser Pro Pro Gln Ser 50 55 60 Pro
Glu Gly Ala Ser Ser Ser Leu Thr Val Thr Asp Ser Thr Leu Trp 65 70
75 80 Ser Gln Ser Asp Glu Gly Ser Ser Ser Asn Glu Glu Glu Gly Pro
Ser 85 90 95 Thr Ser Pro Asp Pro Ala His Leu Glu Ser Leu Phe Arg
Glu Ala Leu 100 105 110 Asp Glu Lys Val Ala Glu Leu Val Arg Phe Leu
Leu Arg Lys Tyr 115 120 125 10 87 PRT Homo sapiens PAGE1 10 Met Ser
Ala Arg Val Arg Ser Arg Ser Arg Gly Arg Gly Asp Gly Gln 1 5 10 15
Glu Ala Pro Asp Val Val Ala Phe Val Ala Pro Gly Glu Ser Gln Glu 20
25 30 Glu Glu Pro Pro Thr Asp Asn Gln Gly Pro Asp Met Glu Ala Phe
Gln 35 40 45 Gln Glu Leu Asp Leu Glu Lys Thr Arg Ser Glu Arg Gly
Asp Gly Ser 50 55 60 Asp Val Lys Glu Lys Thr Pro Pro Asn Pro Lys
His Ala Lys Thr Lys 65 70 75 80 Glu Ala Gly Asp Gly Gln Pro 85 11
109 PRT Homo sapiens PAGE2 11 Met Ser Glu Leu Val Arg Ala Arg Ser
Gln Ser Ser Glu Arg Gly Asn 1 5 10 15 Asp Gln Glu Ser Ser Gln Pro
Val Gly Ser Val Ile Val Gln Glu Pro 20 25 30 Thr Glu Glu Lys Arg
Gln Gln Glu Glu Pro Pro Thr Asp Asn Gln Asp 35 40 45 Ile Glu Pro
Gly Gln Glu Arg Glu Gly Thr Pro Pro Ile Glu Glu Arg 50 55 60 Lys
Val Glu Gly Asp Cys Gln Glu Met Ala Leu Leu Lys Ile Glu Asp 65 70
75 80 Glu Pro Gly Asp Gly Pro Asp Val Arg Glu Gly Ile Met Pro Thr
Phe 85 90 95 Asp Leu Thr Lys Val Leu Glu Ala Gly Asp Ala Gln Pro
100 105 12 79 PRT Homo sapiens 12 Met Thr Ser Phe Asn Lys Thr Ala
Pro Pro Ile Glu Ser Gln Asp Tyr 1 5 10 15 Thr Pro Gly Gln Glu Arg
Asp Glu Gly Ala Leu Asp Phe Gln Val Pro 20 25 30 Ser Leu Ala Ala
Tyr Leu Trp Glu Leu Thr Arg Pro Lys Thr Gly Gly 35 40 45 Glu Arg
Gly Asp Gly Pro Asn Val Lys Gly Glu Ser Leu Pro Asn Leu 50 55 60
Glu Pro Val Lys Ile Pro Glu Ala Gly Glu Gly Gln Pro Ser Val 65 70
75 13 476 DNA Homo sapiens 13 gaagaattcg ccaggctctc tgctgactca
agttcttcag ttcacgatct tctagttgca 60 gcgatgagtg cacgagtgag
atcaagatcc agaggaagag gagatggtca ggaggctccc 120 gatgtggttg
cattcgtggc tcccggtgaa tctcagcaag aggaaccacc aactgacaat 180
caggatattg aacctggaca agagagagaa ggaacacctc cgatcgaaga acgtaaagta
240 gaaggtgatt gccaggaaat ggatctggaa aagactcgga gtgagcgtgg
agatggctct 300 gatgtaaaag agaagactcc acctaatcct aagcatgcta
agactaaaga agcaggagat 360 gggcagccat aagttaaaaa gaagacaagc
tgaagctaca cacatggctg atgtcacatt 420 gaaaatgtga ctgaaaattt
gaaaattctc tcaataaagt ttgagttttc tctgaa 476 14 4 PRT Homo sapiens
Carboxyl terminus 14 Lys Asp Glu Leu 1 15 4 PRT Homo sapiens
Carboxyl terminus 15 Arg Glu Asp Leu 1 16 15 PRT Homo sapiens
PAGE-4 peptide used to generate antibodies 16 Glu Gly Thr Pro Pro
Ile Glu Glu Arg Lys Val Glu Gly Asp Cys 1 5 10 15
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