U.S. patent application number 10/894359 was filed with the patent office on 2005-03-03 for immunogenic cancer peptides and uses thereof.
Invention is credited to Calenoff, Emanuel, Ditlow, Charles.
Application Number | 20050048588 10/894359 |
Document ID | / |
Family ID | 24905675 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050048588 |
Kind Code |
A1 |
Calenoff, Emanuel ; et
al. |
March 3, 2005 |
Immunogenic cancer peptides and uses thereof
Abstract
This invention relates to novel general methods and compositions
that provide cancer-specific or highly cancer-associated antigens
useful for diagnosis and treatment of cancer.
Inventors: |
Calenoff, Emanuel; (Chicago,
IL) ; Ditlow, Charles; (Chicago, IL) |
Correspondence
Address: |
BARNES & THORNBURG
P.O. BOX 2786
CHICAGO
IL
60690-2786
US
|
Family ID: |
24905675 |
Appl. No.: |
10/894359 |
Filed: |
July 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10894359 |
Jul 19, 2004 |
|
|
|
09723307 |
Nov 27, 2000 |
|
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Current U.S.
Class: |
435/7.23 ;
530/350 |
Current CPC
Class: |
G01N 33/57484 20130101;
G01N 33/57492 20130101; A61K 38/02 20130101; C07K 14/4748
20130101 |
Class at
Publication: |
435/007.23 ;
530/350 |
International
Class: |
C12Q 001/68; G01N
033/574; C07K 014/47 |
Claims
1. (canceled)
2. A cancer-specific or highly cancer-associated peptide comprising
the following structure: (a) an amino acid sequence of a length
from 3-1000 amino acids; (b) a net hydrophilic character; and (c)
at least one glycosylatable amino acid located at a position in the
amino acid sequence no further than 3 amino acids away from the
amino acid adjacent to either end of the peptide, wherein for cells
with normal growth patterns the amino acid is the site of
glycosylation, but in cancer cells the site is missing entirely, so
that the glycosylation site confers a cancer-specific or highly
cancer-associated immunogenicity or marker function to the
peptide.
3. The peptide of claim 2 further defined as immunogenic.
4. The peptide of claim 2, wherein the glycosylatable amino acid is
asparagine.
5. The peptide of claim 2, further comprising a plurality of
deglycosylated amino acids, and wherein each deglycosylated amino
acid is separated from the deglycosylated amino acid nearest to it
by no more than 6 unmodified amino acids.
6. The peptide of claim 5 further comprising: (a) a chemical
modification of at least one of the deglycosylated amino acids
wherein the chemical modification confers upon the peptide an
additional cancer-specific or highly cancer-associated
immunogenicity than that due to glycosylation; and (b) an amino
acid sequence wherein no more than 3 unmodified amino acids are
located on either side of a modified amino acid or amino acid that
has a glycosylation site removed.
7. (canceled).
8. The peptide of claim 2, wherein the peptide is produced
synthetically.
9. The peptide of claim 2, produced by the method of claim 1.
10. A composition comprising a peptide of claim 2.
11. An immunogenic composition capable of inducing a mammal to
produce antibodies specific for an epitope on a cancer cell,
wherein the immunogenic composition comprises a peptide of claim
2.
12-23. (canceled)
24. A therapeutic construct comprising a peptide of claim 2 and (a)
adjuvant/peptide conjugates comprising the peptide coupled to
molecule which facilitates enhanced immunogenicity; and (b)
neomolecules created by recombinant techniques containing a peptide
with adjuvant molecular sequences which promote increased
immunogenicity of the peptide of claim 2.
25-26. (canceled)
27. The peptide of claim 2 having a sequence selected from the
group consisting of:
4 uNu uNuu Nuuu uNuuu uuNuu uuNuuu uuuNuuu uNuMuuu uuNuMuuu
uuuNuMuuu uuuNMMuuu uuuNuMMuuu uuuNMMMuuu uNuuMuu uNuuMuuu
uuNuuMuuu uuuNuuMuuu uuNuuMuuNuu uuuNuuMuuNuuu uuuMuuNuuMuuu
[uuNuuN]n [uuuNuuuN]n [uuuNuuuuN]n [uuuNuuuuuN]n [uuuNuuuuuN]n
[uuuNuuuuuuN]n [uuuNuuuuuuNu]n [uuuNuuuuuuNuu]n [uuuNuuuuuuNuuu]n
[uuMuuN]n
wherein u is an unmodified amino acid, N is a deglycosylated amino
acid, and M is a modified amino acid.
Description
[0001] This invention relates to novel general methods and
compositions that provide cancer-specific or highly
cancer-associated antigens useful for diagnosis and treatment of
cancer.
BACKGROUND
[0002] The utilization of cancer-specific antigens and molecular
markers in the diagnosis and treatment of malignant tumors is a
goal of medical professionals. The realization of this goal has
been advanced by the use of in vivo animal and in-vitro model
systems in order to map out the relevant steps of a cancer-specific
immune response and also the steps required for its use in cancer
therapy. Methods which utilize cancer-specific and/or
cancer-associated markers for diagnosis and therapy have been
reported, but the principal shortcoming preventing the
implementation of these methods has been the paucity of
cancer-specific or highly cancer-associated antigens and other
markers of cancer in humans.
[0003] Some progress in obtaining candidates for cancer-specific or
highly cancer associated antigens for cancer diagnosis and
treatment includes the construction of synthetic peptides, for
example, for the production of antibodies specific for the
peptides, where the peptides are potentially useful as markers. For
example, different epitopes have been found to be associated with
mucins from malignant cells, in contrast to mucins in non-malignant
cells. Aberrant glycosylation has been found in some peptides from
tumors.
[0004] Vaccines and immunotherapies using specific domains of
membrane proteins have been reported to be more effective than
vaccines and immunotherapy using entire glycoproteins.
[0005] At present, not enough cancer antigens or markers are
available for use in implementing robust cancer diagnostic or
therapeutic methods in humans. Human cancer antigens and markers
described to date are either inadequate or too few in number to
provide useful clinical tools.
[0006] For example, the MAGE family of antigens described by Boon
et al. (1994) are reported to be cancer-associated antigens.
Cancer-associated antigens are those expressed in greater quantity
in molecules in or on, or derived from cancer cells, but are also
concurrently expressed in molecules from normal cells. This duality
complicates therapeutic utility of the antigens for vaccines and
antibodies where positive effects are dependent upon reaching a
therapeutic dose before a toxic dose level is realized. Other
limitations of the MAGE antigens are that they are also
intracellular cancer antigens thus greatly diminishing their
utility for cancer cell targeting which is more effective for cell
surface antigens. Intracellular antigens serve as poor localizing
targets for immunotherapy, targeted cytotoxic therapeutic agents,
cell receptor blocking agents, other cell-surface disruptive
agents, and for diagnostic imaging. They are poor immunogenic
targets for eliciting a measurable immune response. Their release
for direct quantification is unpredictable because cancer cell
disruption is required.
[0007] Cheever et al.(1997a,b) have described the potential
diagnostic and therapeutic use of oncogenic proteins which are
expressed by both cancer and normal cells. They describe using
oncogenic proteins with site-specific mutations as the
cancer-specific antigens. However, the oncogenic proteins cited by
Cheever, designated the p21 proteins, are intracellular and thus
share the drawbacks of other intracellular antigens, that is,
cannot the detected on cell surfaces. Furthermore, mutated
expression is not always manifested by expressed oncogenic proteins
in all cancer cells, thus leaving some cells to express oncogenic
proteins which are subject to self-recognition and are thus poorly
immunogenic.
[0008] Cheever's other example, the erbB-2 epidermal growth factor
receptor, also known as HER-2/neu, is used to support the
hypothesis that breaking self-recognition offers a novel
therapeutic pathway (Disis et al., 1998a, b; 1999) although that
method is not commonly accepted by most immunologists. The erbB-2
molecule is a transmembrane receptor with a significant
extracellular portion. Its extracellular domain is commonly
believed to be structurally similar for both cancer cells and
normal cells. Thus, the advantages it possesses over intracellular
antigen candidates is minimized because of its susceptibility to
down regulation of any specific immune response on the basis of
self recognition.
[0009] Use of derivatives of bombesin, an amphibian protein, was an
attempt to inhibit growth of tumor cells that respond to bombesin
(Knight et al., 1997). Bogden and Moreau attempted to treat human
cancer by administering analogs of a biologically active peptide to
a patient. However, these attempts used molecules that did not
differentiate normal from cancer cells.
[0010] The deglycosylated mucins described by Barratt et al., 1998
and Henderson et al., 1998 are another example of a class of
cancer-associated antigens with epitopes detectable outside of the
cell. Mucins are large secreted and/or transmembrane glycoproteins
with greater than 50% of their molecular weight derived from
O-linked carbohydrates attached to serine and theonine. Their
cancer specificity depends on a greater degree of altered structure
rather than on numerical over-expression. The loss or diminution of
carbohydrate side chains emanating from a central core protein
makes the Muc proteins more immunogenic. Finn et al. ascribes this
immunogenity as a result of significant altered molecular folding
made possible by a release from molecular rigidity conferred by the
many projecting glycoside chains found in mucin molecules in
non-cancerous cells. The alteration in folding creates neo-epitopes
which help break immune self-recognition and also separately
facilitates stimulation of a cellular immune response. Problems
with the Muc antigens include insufficient diversity needed to
provide wide enough antigenic coverage for many cancers, and their
rapid cellular release rate as a consequence of Muc antigens being
secreted proteins, as opposed to functional cell membrane proteins
such as receptor molecules, receptor-like molecules, or cell
adhesion molecules. The latter attribute makes Muc antigens less
effective therapeutic and imaging targets.
[0011] Hudziak et al. (1998a, b) describes the therapeutic utility
of monoclonal antibodies specific for the extracellular domain of
the normal HER-2/neu receptor (also known as erbB-2). The basis of
this therapeutic method is described as the inhibition of the
cancer-proliferative function of the receptor caused by the binding
of a specific monoclonal antibody to the outer domain of the
receptor thereby preventing the binding of circulating epidermal
growth factor and other ligands to the receptor. Decreased or
absent growth factor stimulation results in cancer cell death
through apoptosis. This method relies on higher expression of
Her-2/neu on cancer cells as compared to normal cells. Therapy is
dose dependent. Sufficient blocking antibody must be administered
so as to block enough cancer cell HER-2/neu molecules required to
affect cancer cell death without causing normal cell death or
normal cell toxicity. Adequate therapeutic dosing is not possible
for all patients who express HER-2/neu on the their tumor cells.
Some cancer patients express adequate amounts of HER-2/neu; some
express low amounts; and yet others express none. Consequently,
this therapeutic method works marginally, or not at all for most
patients. Occasionally, when patient circumstances are appropriate,
this method is capable of affecting total cancer remission. This
limited result illustrates the basic soundness of a therapeutic
method provided that a large repertoire of cancer-specific or
cancer-associated functional targets were made available. However,
more and better cancer-specific and cancer-associated antigens are
needed to make these approaches clinically useful.
[0012] A method of preparing phosphorylated tumor specific peptides
was reported by Calenoff (1998).
[0013] There are suggestions of expression of cancer-specific or
cancer-associated molecules, as well as over-expression or
under-expression of the molecules in or on cancer cells. For
example, many receptor-like adhesion proteins found on the surface
of cells have been described. Some of these adhesion proteins are
reported to facilitate tumor migration and invasion (Zheng et al.,
1999; Rabinovitz et al., 1995; Friedl et al., 1998) or metastatic
spread (Romanov et al., 1999). Others are reported to facilitate
essential functioning for both cancer cells and tissues and for
normal cells and tissues (Ekblom et al, 1998; Fleischmajer et al.,
1998; Bonkoff, 1998; Fujiwara et al., 1998; Lohi, 1998). Blocking
certain functions facilitated by receptor-like adhesion molecules
is suggested to provide new therapeutic modalities for eradicating
or controlling cancer (Ruoslahti et al., 1997). Although various
adhesion molecule isotypes are reported to be over-expressed
(Damiano et al., 1999; Liapis et al., 1996; Begum et al., 1995;
Katsura et al., 1998) or underexpressed (Furakawa et al., 1994;
Damjanovich et al., 1997; Luguki et al., 1999) on cancer cells as
compared to normal cells, none have been described which possess
the cancer-specific or highly cancer-associated structural
modifications of the present invention.
SUMMARY OF THE INVENTION
[0014] The invention relates to general methods and compositions
that provide cancer-specific or highly cancer associated antigens
useful for cancer diagnosis and treatment. An aspect of the
invention is algorithms for determining, selecting and/or
constructing synthetic peptides that are candidates for producing a
cancer-specific or cancer-associated immune response useful in the
diagnosis and treatment of cancer.
[0015] The invention also relates peptides selected by the methods
of the present invention. The peptides are preferably small, e.g.
from 3 to about 1000 amino acids in length, and are centered around
amino acids that are generally glycosylated in non-cancerous cells,
and are on the cell surface, but are not glycosylated in cancer
cells. More preferred lengths of the peptides are from 3-7 amino
acids or 3-10, or 5-10, although peptides up to about 25 or to 1000
amino acids in length, are also within the scope of the invention.
The peptides are also hydrophilic. The peptides or fragments
thereof include any variation in the amino acid sequence, whether
by conservative amino acid substitution, deletion, or other
processes, provided that the polypeptides are in accord with the
criteria of the present invention. More specifically, more than one
peptide, the sequences of which are in accord with the criteria of
the present invention, are preferably present to enhance the
discriminatory power of the immunoassays and therapies disclosed
herein. That is, a plurality of antigenic peptides forms an array
(or repertoire) of molecules suitable for diagnosis and treatment
of cancer.
[0016] A peptide of the present invention contains both unmodified
and modified amino acids. It is recognized that the conversion of a
normal to a cancerous cell type likely involves many steps. At some
point, a cell (more precisely, a group of cells--for example, a
tumor) becomes distinguishable as a "cancer cell". If at that
point, an amino acid differs in its state from that in
non-cancerous cells, it is defined herein as "modified." Not all
the cells in a cancerous tissue necessarily have the modification.
For purposes of the present invention, it suffices that the
modification allows some cancer cells to be distinguished from
normal cells by detection of the modification or modifications.
[0017] On the external domain of proteins of cells with normal
growth patterns, asparagine is the most frequent site of
glycosylation, but in cancer cells the peptides of the present
invention are missing a glycosidic complex altogether. The absence
of the glycosidic complex is expected to confer a cancer-specific
or highly cancer-associated immunogenicity to the altered peptide
region. Deglycosylation is expected to remove steric hindrance
present in non-cancerous cells, to phosphorylation or other
modifications of the neighboring amino acids. Removal of steric
hindrance allows available phosphorylases to add phosphate groups
to amino acids usually under the glycosidic umbrella. Addition of
phosphate groups facilitated by deglycosylation provides an
additional cancer-specific or cancer-associated molecular structure
to be detected.
[0018] The immunogenic peptides of the present invention may
include one or more of the constituent amino acids that are
chemically modified, either in the natural state of the cancer
cells, or synthetic, and the chemical modification confers upon the
peptide a cancer-specific or highly cancer-associated
immunogenicity or structured uniqueness that is different from, and
may be independent of, the specificity or association related to
the altered (deglycosylated) glycosylation sites.
[0019] Following the steps outlined in Table 1, peptides suitable
for the practice of the invention result in peptides with the
formulas shown in Table 2.
1TABLE 1 Steps in Obtaining Cancer Specific of Cancer-Associated
Antigenic Peptides Step 1: obtain amino acid sequence of the
extracellular domain of a candidate molecule e.g. a receptor or
receptor-like molecule. Step 2: map hydrophilic regions of the
domain by analyzing the amino acid sequence of the domain of step 1
employing the rolling sum analysis of 7 consecutive residues. Step
3: identify the hydrophilic regions of step 2 that are glycosylated
in non-cancerous (normal) cells, but are deglycosylated in cancer
cells. The deglycosylated regions of the peptide are candidates for
being cancer-specific or cancer associated peptide antigens. Step
4: look for amino acids to either side of the deglycosylated amino
acids identified in step 3 that are susceptible to alteration in
the absence of steric hinderance by glycoside chains. Step 5:
synthesize candidate peptides that fit the criteria obtained in
steps 3 or 4 and label the peptides at one end e.g. with biotin.
Step 6: use synthesized peptides as source antigens in immunoassays
used to measure peptide-specific antibody in biological fluids
(i.e. serum) from cancer patients and biological fluids from
control subjects. Peptides which specifically complex with antibody
in cancer patient fluids but not in control fluids are
cancer-specific antigens. Peptides which complex with antibodies in
cancer patient fluids more frequently than the complex with
antibodies from control fluids from (non cancerous patient, or at
least not know to be cancer patients) are designated
cancer-associated antigens. Peptides which complex with antibody in
both cancer patient fluids and also control fluids, or with
neither, are neither cancer-specific nor cancer-associated.
[0020] The invention is generally directed to immunogenic peptides
which include a sequence of three or more amino acids, possess a
net hydrophilic character, and contain at least one amino acid that
is glycosylated in normal cells (generally an asparagine residue)
but deglycosylated in cancer cells. As can be seen for the general
forms in Table 2, the deglycosylated amino acid is located no
further than:
[0021] 1. 3 unmodified amino acids away from a fourth unmodified
amino acid on either side of the deglycosylated amino acid;
[0022] 2. 3 amino acids away from the most distal modified amino
acid found on either side of the deglycosylated amino acid, where
distal refers to a location from a deglycosylated amino acid;
[0023] 3. 6 amino acids away from another deglycosylated amino acid
(if there are no modified amino acids in between the two adjacent
deglycosylated amino acids).
[0024] Arrays include differentiating pluralities of peptides of
the present invention, to diagnose cancer.
[0025] An aspect of the invention is immunoassays employing
immunogenic peptides to measure specific peptide-reactive
antibodies in biological fluids, more specifically: an aspect of
the invention is monoclonal antibodies and antibody-like molecules
such as Fab2 and FAb fragments, known to those skilled in the art,
and recombinant proteins thereof, which are specifically reactive
with the immunogenic peptides of the present invention.
Immunoassays employing these antibodies or antibody-like molecules
of the present invention are used to measure in biological fluids,
molecules containing altered peptide regions which correspond in
vivo to the immunogenic peptides of the present invention.
[0026] Cancer imaging reagents are developed using labeled
molecules of the present invention including antibodies or
antibody-like molecules, directed toward cancer specific or
cancer-associated peptides of the present invention. Suitable
labels include radioisotopes, a paramagnetic label, and a water
density label. The labels complexed with the antibodies or
antibody-like molecules target cancer cells and tissues and respond
to image detectors to identify the location of the cancer.
[0027] A therapeutic vaccine containing one or more immunogenic
peptides of the present invention, and prepared by methods known to
those skilled in vaccine development, is an aspect of the
invention. Adjuvent/peptide conjugates including the immunogenic
peptides coupled to molecules which facilitate enhanced
immunogenicity, are used to stimulate the host immune system to
facilitate the killing of cancer cells and thereafter maintain
immune surveillance in case of cancer recurrence.
[0028] Vaccines created by recombinant techniques containing
immunogenic peptides together with adjuvant molecular sequences
which promote increased immunogenicity of the immunogenic peptides
to stimulate the host immune system to facilitate the killing of
cancer cells and thereafter maintain immune surveillance in case of
cancer recurrence, are also within the scope of the invention.
[0029] Definitions
[0030] The term "antigen presenting cell" (APC) includes
"professional antigen presenting cells" that constitutively express
MHC class II molecules (e.g., B lymphocytes, monocytes, dendritic
cells, Langerhans cells, and activated T cells in humans) as well
as other antigen presenting cells that are capable of presenting
antigen to T cells. APCs can express the appropriate combination of
MHC molecules and costimulatory and/or adhesion molecules known in
the art to be sufficient for presentation of antigen to T cells or
can be induced or engineered to express such molecules.
[0031] As used herein, the term "immune response" includes T cell
mediated and/or B cell mediated immune responses that are
influenced by modulation of T cell costimulation. Exemplary immune
responses include T cell responses, e.g., proliferation, cytokine
production, and cellular cytotoxicity. In addition, the term
"immune response" includes immune responses that are indirectly
effected by T cell activation, e.g., antibody production (humoral
responses) and activation of cytokine responsive cells, e.g.,
macrophages.
[0032] "Unmodified amino acids" are those found in the
non-cancerous state--that is, as the amino acids exist in normal
cells i.e. non-cancerous cells. Modified amino acids are those that
exist in altered states in cancerous cells.
[0033] The term "markers," as used herein, includes any molecule
which is detectable in a biological sample and indicates the
presence of another molecule of interest. Some markers are
antigenic. Markers are useful because their presence is associated
with a disease or condition of interest. Markers of interest herein
are those whose presence is associated with cancer.
[0034] The single letter code for amino acids, well known to those
of skill in the art, is used herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 presents the amino acid sequence (SEQ ID NO: 67) of
the human epidermal growth factor receptor (EGFR); larger letters
depict extracellular portions of human epidermal growth factor
receptor (EGFR); the bold N denotes normally glycosylated
asparagine residues on the EGFR extracellular portions; underlined
amino acid sequences=hydrophilic peptide regions on extracellular
portion of EGFR.
[0036] FIG. 2 presents amino acid sequence (portions of SEQ ID NO:
67) position numbers that indicate regions of
cancer-specific/highly cancer-associated immunogenic peptides from
the sequence shown in FIG. 1; N depicts deglycosylated asparagine
in cancer-specific or highly cancer-associated immunogenic peptide
regions; an underlined S, T or Y respectively depicts serine,
threonine, and tyrosine amino acids which can become aberrantly
phosphorylated because absent polysaccharide complexes emanating
from the highlighted asparagines no longer sterically prevent
various phosphorylases from approaching phosphorylateable amino
acids and attaching a phosphate group; the addition of a phosphate
group creates a novel immunogenic peptide region centered by the
phosphorylated amino acid(s) as well as the deglycosylated
asparagine(s).
[0037] FIG. 3(a) graphically indicates screening results using the
EGFR peptide (portion of SEQ ID NO: 67) rNvs; the x-axis shows
results for 2 groups of serum samples:
[0038] a. from patients with squamous cell carcinoma (dark
circles);
[0039] b. samples from patients not known to have any cancer (open
circles);
[0040] a dotted line shows the control (non-cancerous) population
mean background+2.5 standard deviations antibody levels (serum IgG)
shown on the y-axis were below the mean background+2.5 standard
deviations of the mean (+2.5 SD) for all samples from persons not
known to have cancer, whereas 2 of 45 of the samples from persons
with squamous cell carcinoma, had antibody levels above the same
mean+2.5 SD; this indicates that this peptide region of the
epidermal growth factor receptor erbB-1, in altered form, likely
serves as a cancer-specific immunogen or target.
[0041] FIG. 3(b) graphically indicates screening results using the
EGFR peptide (portion of SEQ ID NO: 67) rNvSrgr; the x-axis shows
results for 2 groups of serum samples:
[0042] a. samples from patients with squamous cell carcinoma (dark
circles);
[0043] b. samples from patients not known to have any cancer (open
circles);
[0044] antibody levels (serum IgG) shown on the y-axis) were below
the mean background+2.5 SD for the serum samples from persons not
known to have any cancer, whereas 3 of 45 serum antibody levels
were above the same mean+2.5 SD for the serum samples for persons
with squamous cell carcinoma; although the peptide antigen used to
elicit these results is structurally related to the peptide
(portion of SEQ ID NO: 67) rNvsr, the serum antibody levels
elicited for the peptide (portion of SEQ ID NO: 67) rNvSrgr are
much higher thus indicating that adding an aberrantly
phosphorylated extension offers a neoantigen which complexes with
specific serum antibody in excess to that afforded by the (portion
of SEQ ID NO: 67) rNvsr peptide alone; this, too, indicates that
this peptide region of the epidermal growth factor receptor erbB-1,
in a second altered form, likely serves as a cancer-specific
immunogen or target.
[0045] FIG. 4 graphically indicates screening results using the
TROP1 peptide with the amino acid sequence (SEQ ID NO: 68) aemNgSk;
the x-axis shows 2 groups of results:
[0046] a. serum samples from persons with squamous cell cancer
(dark circles); and
[0047] b. serum samples from persons not known to have cancer (open
circles);
[0048] the y-axis shows IgG antibody levels; 6 of 45 sera from
cancer patients were above the mean background level from the
controls+2.5 SD, whereas only one serum from the control population
was above that level; this indicates that this peptide region of
the TROP1 cell surface molecule in altered form, likely serves as a
highly cancer-associated immunogen or target; the single positive
result within the control population may also be indicative of
silent (clinically undetectable) cancer presence in the affected
subject.
[0049] FIG. 5 graphically illustrates serum antibody levels
obtained with a plurality of 4 biotinylated peptides used as test
antigens; the x-axis shows 2 groups:
[0050] a. samples from persons with squamous cell cancer (dark
circles);
[0051] b. samples from persons not known to have cancer (open
circles); the y-axis shows IgG antibody levels; 11 of 45 sera from
cancer patients were above the control mean background level+2.5
SD, whereas only one serum from the control population was above
that level; this graph illustrates the positive summative effect of
using a sufficiently large number of non-homologous synthetic
peptides corresponding to the humorally antigenic peptide regions
of cancer cell receptors and/or receptor-like molecules; by having
enough suitable antigenic peptides in the antigen mix of the
described immunoassay method, a point is reached where enough
antigenic peptides are available to provide the immunoassay with a
sensitivity approaching 100 percent while maintaining high
specificity.
[0052] FIG. 6 graphically illustrates serum antibody levels
obtained with 9 biotinylated peptides used as test antigens; the
x-axis shows 7 groups:
[0053] a. samples from persons with Stage I prostate cancer (dark
circles);
[0054] b. samples from persons with Stage II prostate cancer (open
circles);
[0055] c. samples from persons with Stage III prostate cancer (dark
squares);
[0056] d. samples from persons with Stage IV prostate cancer (open
squares);
[0057] e. samples from persons with benign prostatic hypertrophy
(BPH), a non-malignant enlargement of the prostate (diamonds with
crosses inside);
[0058] f. samples from men not known to have cancer or BPH (open
diamonds);
[0059] g. samples from women not known to have cancer (dark
triangles).
[0060] The y-axis shows IgG antibody levels; 4 of 7 sera (57%) from
Stage I prostate cancer patients were above the mean background
level+2.5SD; 3 of 7 sera (43%) from Stage II prostate cancer
patients were above that level; 2 of 3 sera (67%) from Stage III
prostate cancer patients were above that level; and 0 of 1 sera
(0%) from Stage IV prostate cancer patients were above that level;
whereas, only one serum (3.6%) from the BPH population and none of
the normal males or females were above the threshold level; this
graph also illustrates the positive summative effect of using a
sufficiently large number of non-homologous synthetic peptides
corresponding to the humorally antigenic peptide regions of cancer
cell receptors and/or receptor-like molecules.
[0061] FIG. 7 is a diagram showing a glycosylated (CHO) amino acid
(dark circle) in a peptide (chain of circles) in a non-cancerous
cell and an outer membrane protein of a receptor--or receptor like
molecule (OMP) with a transmembrane region (dashed line through a
cell membrane (CM)) attached to an inner cell portion (small dotted
circle).
[0062] FIG. 8 is a diagram of the same structure as in FIG. 7 with
the exception that the glycosylated amino acid in FIG. 7 (dark
circle) is now deglycosylated, as in a cancerous cell.
[0063] FIG. 9 is a diagram of the same of structure as in FIG. 8
except that one of the amino acids (shaded circles) in the peptide
is phosphorylated (P), another modification in a cancer cell in
addition to the deglycosylation shown in FIG. 8.
DESCRIPTION OF THE INVENTION
[0064] The invention relates to methods and compositions for
obtaining cancer specific or cancer associated antigens (generally
antigenic peptides) for use in diagnosis and treatment of cancer.
An aspect of the invention is algorithms for determining, selecting
and/or constructing antigens that are suitable for use in
diagnostic tests for cancer, for producing cancer-specific or
cancer-associated antibodies for use in diagnosis or treatment of
cancer, and for producing immunogenic constructs for treatment of
cancer. Aspects of the invention include a large repertoire (array)
of cancer-specific and cancer-associated peptide antigens located
on the surface of externally expressed cellular receptors or
receptor-like molecules.
[0065] An algorithm for a peptide of the present invention directs
among other things, that in the amino acid sequence, no more than 3
unmodified amino acids are located on either side of a modified
amino acid (see Tables 1 and 2). Amino acid modification may
include phosphorylation, conjugation of oxidized radicals (Tsimikas
et al., 1999; Brame et al., 1999), and/or conjugation of
glycosides(Shamsi et al., 1998) which differ from the glycosides
which are normally attached to the peptide.
[0066] The antigens of the present invention generally have the
following attributes and characteristics:
[0067] a. the antigens possess cancer-specific or cancer-associated
alterations which confer antigenic and/or structural specificity
upon the extracellular domains of commonly expressed receptor or
receptor-like molecules;
[0068] b. the receptors and receptor-like molecules are potentially
expressed by all cancer types thereby providing broad-based
antigenic diversity and significant quantitative expression for
most cancers;
[0069] c. the receptors and receptor-like molecules can serve as
cell surface cancer-specific or cancer-associated antigens or as
cell surface cancer markers;
[0070] d. the receptors and receptor-like molecules are found as
early as Stage I as well as in Stages II, III, and IV of cancer
progression;
[0071] f. because the receptor and receptor-like repertoire is
significantly large, diminished expression among different cancers
of some receptor or receptor-like molecules of the array is
compensated by the standard expression or over-expression of other
receptors or receptor-like molecules of the repertoire thereby
providing sufficient antigenic and/or marker coverage. This varying
molecular expression allows diagnostic discrimination of individual
cancer types;
[0072] g. because the receptor and receptor-like repertoire is
significantly large, enough receptor or receptor-like molecules are
available which either remain affixed to the outer surface of
cancer cells thereby serving as ideal antigenic or marker targets
for diagnosis or therapy, or are predictably released into the
peripheral circulation or biological fluids which bath the cancer
cells thereby serving as shed cancer antigens to be measured for
diagnostic purposes.
[0073] The antigen repertoire of this invention is different from
the antigens reported by others in the following ways:
[0074] a. the MAGE antigens of Boon (1994) expressed in cancer
cells are not structurally unique compared to MAGE concomitantly
expressed in normal cells. MAGE antigens are intracellularly
expressed and therefore require cancer cell damage or fragmentation
for reliable extracellular expression;
[0075] b. the antigens described by Cheever (1997) possess
cancer-specific structural alterations but are intracellular, or
are over-expressed on the outer surface of cancer cells, but lack
cancer-specific structural alterations which would confer
immunogenic and/or marker specificity;
[0076] c. the Muc antigens described by Finn (1998 a, b) possess
cancer-associated structural specificity that confers antigenic
and/or marker specificity but are excreted and thereby poorly
retained on the cancer cell surface. The structural alterations of
Muc antigens are different from the alterations described for the
antigens of the present invention. The antigenic/marker sites of
peptides for this invention are small and are therefore less
affected by a twisting-type of conformational change but rely more
on peptide denuding and upon the modification of amino acids which
are normally hidden by attached glycosides. The Muc antigen
repertoire is numerically insufficient to provide across the board
coverage for adenocarcinomas which express Muc antigens;
[0077] d. the antigenic site employed in the method of Hudziak
(1998 a,b) to target the HER-2/neu epidermal growth factor receptor
is described as being structurally similar to HER-2/neu expressed
by non-cancerous cells.
2TABLE 2 Examples of formulas for suitable candidate cancer
antigenic peptides (where u = unmodified amino acid; N =
deglycosylated amino acid; M = modified amino acid and [ ]n
symbolizes n number of repeats of a basic unit in brackets) are:
uNu uNuu Nuuu uNuuu uuNuu uuNuuu uuuNuuu uNuMuuu uuNuMuuu uuuNuMuuu
uuuNMMuuu uuuNuMMuuu uuuNMMMuuu uNuuMuu uNuuMuuu uuNuuMuuu
uuuNuuMuuu uuNuuMuuNuu uuuNuuMuuNuuu uuuMuuNuuMuuu [uuNuuN]n
[uuuNuuuN]n [uuuNuuuuN]n [uuuNuuuuuN]n [uuuNuuuuuN]n [uuuNuuuuuuN]n
[uuuNuuuuuuNu]n [uuuNuuuuuuNuu]n [uuuNuuuuuuNuuu]n [uuMuuN]n
[0078]
3TABLE 3 Comparison of Peptides Present in Serum From Cancer
Patients to Peptide Sequences Cancer Tissue (SEQ ID NOS 1-66,
respectively, in order of appearance) Presence of Cell Surface
Prostate Cancer Absence of CSP Proteins Serum Test in Prostate
(CSP) Tested CSP Peptides Result Cancer E-Cadherin vkNst (SEQ ID
NO: 1) + + N-Cadherin dkNls (SEQ ID NO: 2) + + CD9 nnNNss (SEQ ID
NO: 3) - - CD38 dkNst (SEQ ID NO: 4) - - CD40 gtNkt (SEQ ID NO: 5)
+ + CD44 drNgt (SEQ ID NO: 6) + + NhSeg (SEQ ID NO: 7) + spNhs (SEQ
ID NO: 8) - CD46 drNht (SEQ ID NO: 9) - - CD53 sdNst (SEQ ID NO:
10) - - CD55 fcNrs (SEQ ID NO: 11) - CD63 knNht (SEQ ID NO: 12) - -
CD66 saNrs (SEQ ID NO: 13) + + trNdt (SEQ ID NO: 14) - skNqs (SEQ
ID NO: 15) - CD82 pgNrt (SEQ ID NO: 16) - - desmoglein 1 tkNgt (SEQ
ID NO: 17) - - desmoglein 2 kiNat (SEQ ID NO: 18) - - erbB-1 daNkt
(SEQ ID NO: 19) + + perNrt (SEQ ID NO: 20) - crNvs (SEQ ID NO: 21)
- erbB-2 dtNrs (SEQ ID NO: 22) + + erbB-3 heNct (SEQ ID NO: 23) - +
erbB-4 aeNct (SEQ ID NO: 24) - - pdNct (SEQ ID NO: 25) - fgfr1
esNrt (SEQ ID NO: 26) - + fgfr2 ekNgs (SEQ ID NO: 27) - + fgfr4
diNss (SEQ ID NO: 28) - - hepatocyte gfr vgNks (SEQ ID NO: 29) - +
pdgfr alpha eeNns (SEQ ID NO: 30) - + pdgfr beta kdNrt (SEQ ID NO:
31) - - trNvs (SEQ ID NO: 32) - ICAM 1 hkNqt (SEQ ID NO: 33) - +
integrin alpha 1 qrNit (SEQ ID NO: 34) - - seNas (SEQ ID NO: 35) -
integrin alpha 2 drNhs (SEQ ID NO: 36) - - integrin alpha 3 meNkt
(SEQ ID NO: 37) + + leNht (SEQ ID NO: 38) - rmNit (SEQ ID NO: 39) -
integrin alpha 5 kaNts (SEQ ID NO: 40) - - lrNes (SEQ ID NO: 41) -
integrin alpha 6 raNhs (SEQ ID NO: 42) - + integrin alpha 9 qkNqt
(SEQ ID NO: 43) - - kgNcs (SEQ ID NO: 44) integrin alpha (v) qdNkt
(SEQ ID NO: 45) + + kaNtt (SEQ ID NO: 46) - teNqt (SEQ ID NO: 47) -
ekNdt (SEQ ID NO: 48) - integrin beta I nkNVt (SEQ ID NO: 49) + +
vtNrs (SEQ ID NO: 50) - kNvtNrs (SEQ ID NO: 51) - keNss (SEQ ID NO:
52) - eqNct (SEQ ID NO: 53) integrin beta 3 skNfs (SEQ ID NO: 54) -
- integrin beta 5 rcNgs (SEQ ID NO: 55) - - pdNqt (SEQ ID NO: 56) -
integrin beta 6 qkNss (SEQ ID NO: 57) - - evNss (SEQ ID NO: 58) -
protein-tyrosine seNdt (SEQ ID NO: 59) - - phosphatase PCP-2
protein-tyrosine keNdt (SEQ ID NO: 60) - - phosphatase kappa ghNes
(SEQ ID NO: 61) - gdNrt (SEQ ID NO: 62) - tgfr beta type II deNit
(SEQ ID NO: 63) + + eyNts (SEQ ID NO: 64) + insulin-like gf pdNdt
(SEQ ID NO: 65) - + receptor rnNtt (SEQ ID NO: 66) -
[0079] Table 3 column 2 to the right is a compilation of
cancer-modified peptide regions to be found on 41 receptors,
receptor-like molecules, or adhesion molecules reported in the
literature (CSP). This table illustrates the diversity of tissue
and organ types which possess receptors, receptor-like molecules,
or adhesion molecules able to present with cancer-specific or
highly cancer-associated structural alterations.
[0080] Table 3 third column illustrates the positive or negative
reactivity between peptides representing modified peptide regions
of cancer cell surface protein molecules with antibodies in sera
from prostate cancer patients. The presence or absence in prostate
cancer cells of the 41 tested molecules is shown as + or - in
column four. Table 3 illustrates that reactivity exists between
most of the modified peptides of prostate cancer cell surface
molecules but shows no reactivity with modified peptide regions of
molecules not found in prostate cancer, thus illustrating the
ability to serologically determine cancer type by having first
mapped out the peptide antigen repertoire needed to identify each
cancer. These results support feasibility of using the peptides of
the present invention, in particular a plurality of peptides, for
cancer diagnosis.
[0081] An aspect of the invention is monoclonal antibodies and
antibody-like molecules such as Fab2 and FAb fragments, known to
those skilled in the art, and recombinant proteins (Hussain et al.,
1996).
[0082] A cancer imaging reagent is developed using molecules
including labeled antibodies or antibody-like molecules directed to
antigenic peptides of the present invention. Suitable labels
include a radioisotopic label for the cancer imaging reagents
which, upon binding to the cells that form a cancerous tumor,
highlight the presence of the tumor when scanned with a nuclear
medicine scanner (Goldenberg, 1993; 1999).
[0083] Another suitable label is a paramagnetic label which, upon
binding to the cells of a cancerous tumor, highlights the presence
of the tumor when scanned with a nuclear magnetic resonance (NMR)
scanner (To et al., 1992).
[0084] Another suitable label comprises a water density label
which, upon binding to the cells of a cancerous tumor, highlights
the presence of the tumor when scanned with a CAT scanner.
[0085] Cancer therapeutic reagents developed using the molecules
including antibodies or antibody-like molecules directed to the
peptides of the present invention, have at least one of the
following characteristics: they (1) bind to a cancer cell and
promote lysis of that cell; (2) bind to and block the function of a
receptor or receptor-like molecule on a cancer cell, thereby
promoting a reduction or cessation of cancer cell growth or
promoting cancer cell death; and (3) carry a radioisotope or a
toxin which upon binding to a cancer cell damages or promotes
cancer cell death (Goldenberg, 1993).
[0086] Examples of cancer therapeutic methods which can be
formulated using a suitable cancer antigens/markers array are (see
Materials and Methods for details and citations):
[0087] a. passive immunization using constructs such as engineered
antigen presenting cells and production of antigen presenting
dendritic cells able to stimulate the host immune system to
recognize and kill cancer cells;
[0088] b. active immunization using cancer vaccines including
recombinant fusion proteins, vaccine compositions containing
adjuvants, vaccine compositions containing nucleic acid molecules,
recombinant microorganisms which express cancer antigens,
antigen/antibody conjugates wherein the antibody acts as a delivery
vehicle for targeting the antigen onto antigen presenting cells,
and heat shock protein/antigen complexes;
[0089] c. cell lytic therapeutic antibodies, cell adhesion blocking
antibodies, and growth factor receptor blocking antibodies.
[0090] Therapeutic methods using the non-phosphorylated peptide
antigens of the present invention, either their amino acid
sequences or the corresponding nucleic acid sequences that encode
the peptides include the following:
[0091] a. passive immunization using constructs such as engineered
antigen presenting cells and production of antigen presenting
dendritic cells able to stimulate the host immune system to
recognize and kill cancer cells;
[0092] b. active immunization using cancer vaccines including
recombinant fusion proteins, vaccine compositions containing
adjuvants, vaccine compositions containing nucleic acid molecules,
recombinant microorganisms which express cancer antigens,
antigen/antibody conjugates wherein the antibody acts as a delivery
vehicle for targeting the antigen onto antigen presenting cells,
and heat shock protein/antigen complexes.
[0093] Criteria for an antigen array suitable for passive
immunotherapy specific for cancerous cells include the
following:
[0094] 1. each type of cancer possesses antigens such as protein,
peptide, carbohydrate, or lipid molecules which are structurally
unique as compared to non-cancerous cells and are also
immunogenic;
[0095] 2. the cancer antigens are located on the cell surface so
they are sufficiently accessible for targeting T cells;
[0096] 3. the cancer antigens are present at the earliest stages of
cancer progression as well as during later stages;
[0097] 4. each cancer cell must have on its surface a sufficient
number of specific antigens to serve as an adequate target for an
effective cellular-mediated immune response;
[0098] 5. the cancer antigens are retained on the surface of the
cancer cells for a time sufficient for the therapeutic T cells to
find their target and also to retain the bound T cells for a time
sufficient to affect cancer cell death.
[0099] Criteria, for an antigen array suitable for developing
effective constructs for active immunotherapy include the
following:
[0100] 1. each cancer type possesses antigens such as protein,
peptide, carbohydrate, or lipid molecules which are structurally
unique as compared to non-cancerous cells and are also
immunogenic;
[0101] 2. the cancer antigens are located on the cell surface to be
sufficiently accessible and thus more easily recognized by the host
immune system;
[0102] 3. the cancer antigens are present at the earliest stages of
cancer progression as well as during later stages;
[0103] 4. each cancer cell has on its surface a sufficient number
of specific antigens that serve as an adequate target for a humoral
and/or cellular-mediated immune response;
[0104] 5. the cancer antigens are retained on the surface of the
cancer cells for a time sufficient for the therapeutic effector
cells, and antibodies elicited by the immunostimulatory constructs
to find their target and also to retain the effector cells and
antibodies for a time sufficient to affect cancer cell death.
[0105] Criteria for a marker array including an antigen suitable
for developing cell-lytic therapeutic antibodies include the
following:
[0106] 1. each cancer type possesses markers such as protein,
peptide, carbohydrate, or lipid molecules which are structurally
unique as compared to non-cancerous cells;
[0107] 2. the cancer markers are available in sufficient numbers on
the surface of cancer cells to provide an adequate therapeutic
target at the earliest stages of cancer progression as well as
during later stages;
[0108] 3. the cancer markers are retained on the surface of the
cancer cells for a time sufficient for the therapeutic antibodies
to find their target and also to retain the bound antibody for a
time sufficient to affect cancer cell death.
[0109] Criteria for a marker array suitable for developing growth
factor receptor blocking antibodies include:
[0110] 1. each cancer type possesses markers such as protein,
peptide, carbohydrate, or lipid molecules which are structurally
unique as compared to non-cancerous cells;
[0111] 2. the cancer markers are available in sufficient numbers on
the surface of cancer cells to provide an adequate therapeutic
target at the earliest stages of cancer progression as well as at
later stages;
[0112] 3. the cancer markers are retained on the surface of the
cancer cells for a time sufficient for the growth factor receptor
blocking antibodies to find their target and also to retain the
bound antibody for a time sufficient to affect cancer cell
death;
[0113] Criteria for a marker array suitable for developing cell
surface adhesion blocking antibodies include:
[0114] 1. each cancer type possesses receptor-like adhesion
molecules which are structurally unique as compared to
non-cancerous cells;
[0115] 2. the adhesion molecules are available in sufficient
numbers on the surface of cancer cells to provide an adequate
therapeutic target at the earliest stages of cancer progression as
well as at later stages;
[0116] 3. the cancer markers are retained on the surface of the
cancer cells for a time sufficient for adhesion blocking antibodies
to find their target and also to retain the bound antibody for a
time sufficient to prevent cancer cell attachment, migration,
de-differentiation or other function essential for cancer cell
survival or metastasis.
[0117] Those of skill in the art recognize that identification of
Stage I cancer generally provides a 90 percent or greater cure rate
through the use of currently available cancer therapies (DeVita et
al., 1985). Therefore, diagnostic assays for early stage cancer are
extremely important.
[0118] Examples of cancer diagnostic methods which can be
formulated using a suitable cancer antigen/marker repertoire are:
cancer-specific antibody assays, cancer-specific antigen assays,
and in-vivo cancer imaging.
[0119] Criteria for an antigen array suitable for developing
cancer-specific antibody assays include:
[0120] 1. each cancer type possesses antigens such as protein,
peptide, carbohydrate, or lipid molecules which are structurally
unique as compared to non-cancerous cells and are also
immunogenic;
[0121] 2. the cancer antigens are located on the cell surface to be
sufficiently accessible and thus more easily recognized by the host
immune system;
[0122] 3. enough cancer cells have on their surface a sufficient
number of specific antigens to elicit an immune response capable of
being measured at the earliest stages of cancer progression as well
as at later stages and among most affected patients.
[0123] Criteria for a marker array suitable for developing
cancer-specific antigen-capture immunoassays include the
following:
[0124] 1. each cancer type possesses markers such as protein,
peptide, carbohydrate, or lipid molecules which are structurally
unique as compared to non-cancerous cells;
[0125] 2. the cancer markers are predictably secreted or otherwise
released into the pericellular fluids to be reliably measured;
[0126] 3. enough cancer cells shed enough specific marker from
within a cancerous tumor to be reliably measured at the earliest
stages of the tumor's progression and during later stages of most
affected patients.
[0127] Criteria for a marker array suitable for developing
cancer-specific imaging reagents include the following:
[0128] 1. each cancer type possesses markers such as protein,
peptide, carbohydrate, or lipid molecules which are structurally
unique as compared to non-cancerous cells;
[0129] 2. the cancer markers are available in sufficient numbers on
the surface of cancer cells to provide an adequate imaging target
at the earliest stages of cancer progression as well as during
later stages;
[0130] 3. the cancer markers are retained on the surface of the
cancer cells for a time sufficient for the imaging agents to find
their target and also to retain the bound imaging agent for a time
sufficient to record the presence and location of the cancer.
[0131] Possible Outcomes for Peptides Screened as Antigens in Serum
Antibody Assays:
[0132] 1. A positive result indicating the presence of a
peptide-specific antibody in cancer patient biological fluid
samples, absent evidence of antibody in samples from subjects
without cancer (FIG. 3) indicates the tested peptide is a
cancer-specific peptide (immunogen).
[0133] 2. A significantly higher positive prevalence of a
peptide-specific antibody in cancer patient biological fluid
samples as compared to samples from subjects without cancer (FIG.
4) indicates either that the tested peptide is cancer specific and
that the few control positives have asymptomatic cancer or that the
peptide serves as a highly cancer-associated antigen.
[0134] 3. No difference in positive antibody levels between cancer
patients and subjects without cancer. Biotinylated peptides
producing these results are neither cancer specific nor highly
cancer-associated.
EXAMPLES
[0135] The following examples illustrate embodiments of the
invention.
Example 1
Use of the EGFR Peptide on Serum from Cancer Patients and
Controls
[0136] Using immunoassay 2 (see Materials and Methods) the
following results were obtained.
[0137] FIG. 3(a) graphically indicates screening results using the
EGFR peptide (portion of SEQ ID NO: 67) rNvs; the x-axis shows
results for 2 groups of serum samples:
[0138] a. from patients with squamous cell carcinoma;
[0139] b. samples from patients not known to have any cancer;
antibody levels (serum IgG) were below the mean background plus 4
standard deviations of the mean (+2.5 SD) for all samples from
persons not known to have cancer, whereas 2 of 45 of the samples
from persons with squamous cell carcinoma, had antibody levels
above the mean+4SD.
[0140] FIG. 3(b) graphically indicates screening results using the
EGFR peptide (portion of SEQ ID NO: 67) rNvSrgr; the x-axis shows
results for 2 groups of serum samples:
[0141] a. samples from patients with squamous cell carcinoma;
[0142] b. samples from patients not known to have any cancer;
antibody levels (serum IgG) were below the median background plus
2.5 SD for the serum samples from persons not known to have any
cancer, whereas 3 of 45 serum levels were above the median+2.5 SD
for the serum samples for persons with squamous cell carcinoma.
Example 2
Use of the TROP1 Peptide on Serum Samples from Cancer Patients
Compared to Controls
[0143] FIG. 4 graphically indicates screening results using the
TROP1 peptide with the amino acid sequence (portion of SEQ ID NO:
68) emNgSk; the x-axis shows 2 groups of results:
[0144] a. serum samples from persons with squamous cell cancer;
and
[0145] b. serum samples from persons not known to have cancer; the
y-axis shows IgG antibody levels; 6 of 45 serum from cancer
patients were above the median background level+2.5 SD, whereas
only one serum from the control population was above that
level.
[0146] Materials and Methods
[0147] A Method for Selecting Cancer-Specific or Highly
Cancer-Associated Immunogenic Peptides and/or Markers
[0148] The identification and validation (or confirmation) of
cancer-specific and cancer-associated antigenic peptide regions
and/or marker peptide regions found on the extracellular domain of
receptors or receptor-like molecules is performed through the use
of a algorithms such as the following:.
[0149] First, the amino acid sequence of the extracellular domain
of a receptor or receptor-like molecules is obtained. For example,
the human epidermal growth factor receptor (EGFR), erbB-1, as
illustrated in FIG. 1.
[0150] Second, the amino acid sequence is analyzed employing
rolling sum analysis of 7 consecutive residues (Hopp et al., 1981;
Parker et al., 1986; Fauchere et al., 1983; Taragu et al., 1990) in
order to map out peptide regions which are hydrophilic and
therefore apt to be expressed on the outer surface of the protein.
For example, the hydrophilic regions of the EGFR outer domain are
underlined in FIG. 1.
[0151] Third, hydrophilic peptide regions containing amino acids
which are normally glycosylated are identified. These amino acids,
illustrated by a bold capital letter N in FIG. 1, are apt to be
totally (or partially) deglycosylated in cancer cells. The absence
or truncation of the glycoside chain results in peptide structures
which are structurally distinct for cancer cell proteins. The
distinctly structured peptide regions can serve as a tumor-specific
antigenic site if this alteration is not expressed in normal cells,
or can serve as a cancer-associated antigen by cancer cells. The
ability of a peptide to serve as an antigen depends on the host's
immune system being able to process and recognize the peptide as an
antigen. The processing and recognition of an antigen is dependent
on individual MHC genotypes. If an altered peptide cannot serve as
a cancer-specific or cancer-associated antigen, it may still be
useful as a molecular marker of cancer on the basis of its
cancer-specific or cancer-associated molecular alteration.
[0152] Fourth, the deglycosylated peptide regions are evaluated for
the inclusive presence of amino acids that are susceptible to
alteration in the absence of glycoside chains which normally would
sterically restrict the contact of enzyme or other agents with the
amino acids susceptible to molecular modification. The amino acid
modification confers a second order alteration on the affected
peptide which can result in a new and distinct peptide structure
with specific antigenic and/or marker properties. Examples of such
an amino acid modification include aberrant phosphorylation of
serine, threonine, and tyrosine residues, malondialdehyde (MDA)
modification of lysine residues, aberrant glycosylation of arginine
residues, and the like.
[0153] Fifth, candidate peptides which fit the criteria of the
cancer-modified peptide regions described in Steps 1 through 4 are
synthesized and biotin labels are attached at either end of each
peptide.
[0154] Sixth, employing the immunoassays disclosed herein, each
biotinylated peptide is screened as an antigen against sera or
other relevant biological fluids containing antibodies taken from
one of the following groups: cancer patients, patients with benign
lesions or inflammatory conditions, and healthy subjects. Peptides
can also be screened using tumor-infiltrating lymphocytes or
peripheral blood-born lymphocytes from cancer patients. Candidate
peptides we consider cancer-specific or cancer-associated depend on
whether they fit the following definitions.
[0155] Potentially useful peptides prepared in accord with steps
1-5 preferably possess the following attributes:
[0156] a. contain no more than 3 unmodified amino acids attached on
either side of a deglycosylated amino acid or a modified amino
acid. Peptides containing both deglycosylated amino acids and
modified amino acids have no more than 2 unmodified amino acids
between the deglycosylated amino acids and the modified amino
acids. Peptides with more unmodified amino acids on either side of
a deglycosylated amino acid become antigenically less
differentiating for cancer as the respective unmodified amino acid
numbers increase. The EGFR peptide (portion of SEQ ID NO: 67)
daNktg in FIG. 2 represents a peptide suitable for the practice of
the invention, with a single deglycosylated amino acid at its
central portion. The EGFR peptide (portion of SEQ ID NO: 67)
daNkTglk in FIG. 2 represents a suitable peptide with both a
modified amino acid and a deglycosylated amino acid in its central
portion.
[0157] b. a plurality of deglycosylated amino acids and modified
amino acids providing that the modified amino acids proximal to a
deglycosylated amino acid are no further than the third amino acid
position from the nearest deglycosylated amino acid and that 2 or
more deglycosylated amino acids in a peptide are connected by no
more than 6 unmodified amino acids.
[0158] Synthesis of Deglycosylated Peptides and Phosphorylated,
Modified Forms
[0159] Methods of synthesis of peptides and their corresponding,
encoding nucleic acid molecules are well know in the art and can be
obtained commercially from U.S. companies such as the American
peptide Company (Sunnyvale, Calif. 94086) and Commonwealth
Biotechnologies, Inc. (Richmond, Va. 23235).
[0160] Immunoassay Method 1: Used to Detect Serum IgA, IgD, IgE,
IgG, and IgM Antibodies Specific for Individual Peptide
Antigens
[0161] Materials:
[0162] Neutravidin coated microtiter plates manufactured as per
Example 4.
[0163] Wash Buffer: 20 mM Tris-HCl+150 mM NaCl+0.05% Triton
X405+0.2 mg/mL thimerosal, pH 7.4.
[0164] Biotinylated peptide solution containing 1.5 g/mL peptide in
20 mM Tris-HCl+600 mM NaCl+30 mg/mL polyethylene glycol 4000
(PEG-4000, Mallinckrodt Chemical H273-61)+0.05% Triton X405+0.2
mg/mL thimerosal, pH 7.4.
[0165] Anti-IgA/alkaline phosphatase (Kirkegaard and Perry
075-1001)+anti-IgG/alkaline phosphatase (Kirkegaard and Perry
075-1002) solution: 0.3 g/mL of each conjugate in solution
containing 20 mM Tris-HCl+600 mM NaCl+30 mg/mL polyethylene glycol
4000 (PEG-4000, Mallinckrodt Chemical H273-61)+0.05% Triton
X405+0.2 mg/mL thimerosal, pH 7.4.
[0166] 4-methylumbelliferyl phosphate (4-MUP) fluorescing substrate
solution:
[0167] 25.2 mg 4-MUP (Sigma M-8883)/mL solution containing 180 mM
2-amino-2-methyl-1-propanol+123 mM magnesium chloride, pH 9.5.
[0168] Fluorolite 1000 microtiter plate fluorometer (Dynatech) with
excitation set at 365 nm and emission at 450nm.
[0169] Procedure:
[0170] 1) Adsorb neutravidin (NA) reactive antibodies and biotin
reactive antibodies from serum samples by adding one
neutravidin-conjugated paper disc to every 25 mL of serum and 1
biotin-conjugated paper disc to every 200 mL serum. Allow
disc/serum mixture to incubate for 26 to 18 hours at room
temperature, under gentle agitation.
[0171] 2) Mix 75 mL of adsorbed serum together with 75 mL of
peptide solution.
[0172] 3) Vortex mixture and let incubate at room temperature for
40 minutes.
[0173] 4) Aspirate well contents and wash microtiter wells of
neutravidin plate (275 mL wash buffer/well).times.6.
[0174] 5) Add 100 mL biotinylated peptide/serum solution to
corresponding well and incubate for 3.5 minutes.
[0175] 6) Aspirate well contents and wash microtiter wells (275 mL
wash buffer/well).times.6.
[0176] 7) Add 100 mL anti-IgA/alkaline
phosphatase+anti-IgG/alkaline phosphatase conjugate solution and
incubate for 40 minutes.
[0177] 8) Aspirate well contents and wash microtiter wells (275 mL
wash buffer/well).times.6.
[0178] 9) Add 100 mL 4-MUP substrate solution.
[0179] 10) Read derived fluorescence using microtiter plate
fluorometer at 5, 10, 20, 30, and 60 minutes.
[0180] Immunoassay Method 2: Used to Detect Serum IgA, IgD, IgE,
IgG, and IgM Antibodies Specific for Individual Peptide
Antigens.
[0181] Materials:
[0182] NeutrAvidin.sup.a conjugated paper disc, 6 mm.
[0183] Serum diluent: 10 mM sodium phosphate, pH 7.20, with 150 mM
sodium chloride, and 0.20 mg/mL sodium azide.
[0184] NeutrAvidin.sup.a coated white microtiter plate, stored in
10 mM Tris-HCl, pH 7.50, containing 600 mM sodium chloride and 0.2
mg/mL thimerosal.
[0185] Plate blocking solution: 10 mM sodium phosphate, pH 7.20,
containing 150 mM sodium chloride, 100 mg/mL Triton X-405, and 0.2
mg/mL thimerosal.
[0186] Plate wash buffer: 20 mM Tris chloride, pH 7.4, containing
150 mM sodium chloride, 0.5 mg/mL Triton X-405 and 0.2 mg/mL
thimerosal.
[0187] Peptide solution: 0.06 .mu.g/mL peptide dissolved in 20 mM
Tris chloride, pH 7.4, containing 600 mM sodium chloride, 30 mg/mL
polyethylene glycol 4000, 1 mM ethylenediaminetetraacetic acid, 1
mM ethylene glycol-bis(.sctn.-aminoethyl
ether)N,N,N',N'-tetraacetic acid, 0.5 mg/mL Triton X-405 and 0.2
mg/mL thimerosal.
[0188] Control peptide solution: 0.013 .mu.g/mL control peptide
dissolved in 20 mM Tris chloride, pH 7.4, containing 600 mM sodium
chloride, 30 mg/mL polyethylene glycol 4000, 1 mM
ethylenediaminetetraacetic acid, 1 mM ethylene
glycol-bis(.sctn.-aminoethyl ether)N,N,N',N'-tetraacetic acid, 0.5
mg/mL Triton X-405 and 0.2 mg/mL thimerosal.
[0189] Conjugate solution: 0.100 .mu.g/mL alkaline phosphatase
conjugated polyclonal goat anti human IgG dissolved in 20 mM
Tris-HCl, pH 7.40, with 600 mM sodium chloride, 30.0 mg/mL PEG4000,
3.0 mg/mL BSA, 0.5 mg/mL Triton X-405 and 0.20 mg/mL
thimerosal.
[0190] Substrate solution: 25.2 .mu.g/mL 4-methylumbelliferyl
phosphate dissolved in 180 mM 2-amino-2-methyl-1-propanol, pH 9.50,
containing 123 .mu.M magnesium chloride.
[0191] Serum preparation:
[0192] 1. Add 100 .mu.L serum to 15 NeutrAvidin.sup.a coated paper
discs in a suitably sized test tube.
[0193] 2. Incubate with gentle mixing at ambient temperature for
16-20 hours.
[0194] 3. Add 7.900 mL of serum diluent and mix gently for 30
minutes.
[0195] 4. Vortex the tube gently to completely release the serum
from the discs.
[0196] 5. Remove the treated serum from the discs and transfer it
to a suitable storage tube.
[0197] 6. Store the treated serum at 4 EC.
[0198] Assay procedure:
[0199] 1. Two days before assay, aspirate the storage solution from
the NeutrAvidin.sup.a coated white microtiter plate and add 200
.mu.L plate blocking solution to each well.
[0200] 2. Cover the plate and incubate at ambient temperature for
16-20 hours.
[0201] 3. One day before assay. wash the blocked plate three times
with plate wash buffer, approximately 275 .mu.L per well per wash.
Aspirate the final wash and add 100 .mu.L peptide solution or 100
.mu.L control peptide solution to the appropriate wells of the
plate.
[0202] 4. Cover the plate and incubate with gentle mixing at
ambient temperature for 16-20 hours.
[0203] 5. Day of assay, wash the blocked plate three times with
plate wash buffer, approximately 275 .mu.L per well per wash.
Aspirate the final wash and add 100 .mu.L treated serum to the
appropriate wells of the plate.
[0204] 6. Cover the plate and incubate at 25 EC for 2 hours.
[0205] 7. Wash the blocked plate six times with plate wash buffer,
approximately 275 .mu.L per well per wash. Aspirate the final wash
and add 100 .mu.L conjugate solution to each assay well.
[0206] 8. Cover the plate and incubate at 25 EC for 1.5 hours.
[0207] 9. Wash the blocked plate six times with plate wash buffer,
approximately 275 .mu.L per well per wash. Aspirate the final wash
and add 100 .mu.L substrate solution to each assay well.
[0208] 10. Read the plate at 30 and 60 minutes in a fluorescence
microtiter plate reader set at 365 nm excitation and 450 nm
emission.
[0209] Biotinylation of Human Serum Albumin
[0210] Materials:
[0211] Human Serum Albumin: Sigma A 8763
[0212] Sulfosuccinimidyl 6-(biotinamido) Hexanoate: Pierce
21335
[0213] Tris base: Sigma T 1503
[0214] 20 mM sodium phosphate, pH 7.2
[0215] 100 mM sodium hydroxide solution
[0216] Procedure:
[0217] Human serum albumin is dissolved in phosphate buffer at a
concentration of approximately 40 mg/mL. The protein concentration
of the solution is determined by absorbance at 280 nm (1
mg/mL=OD280 of 0.58) or by the Lowry method.
[0218] Immediately prior to biotinylation, the pH of the albumin
solution is adjusted to 8.5 by the addition of sodium hydroxide.
Succinimidyl biotin is then added at a molar ratio of 50:1 (422 mg
succinimidyl biotin per mg albumin). The reaction mixture is
vortexed thoroughly and then mixed gently for 45 minutes at ambient
temperature.
[0219] Reaction byproducts and unreacted biotin are removed by
extensive dialysis against phosphate buffer. The biotinylated human
serum albumin is stored at 4 C.
[0220] Preparation of Covalent Ready Cyanogen Bromide (CNBr)
Activated Paper Discs
[0221] Materials:
[0222] Paper discs: Schleicher and Schuell 53870
[0223] CNBr solution: 20 gm CNBr (Sigma C6388)+600 mL distilled
water
[0224] 1M NaOH
[0225] 0.05M NaHCO3
[0226] 25%, 50%, 75%, and 100% acetone
[0227] Distilled water
[0228] Dessicant packets: Sigma S8394
[0229] Zip lock plastic bags
[0230] Procedure:
[0231] The following procedure is performed under a hooded, well
ventilated environment. 20 gm paper discs are swelled in 200 mL
distilled water at room temperature. Swelled paper discs are then
added to 600 mL of CNBr solution while stirring. Bring up the pH of
the stirring mixture to 10.5 and maintain at pH 10.5 until 100 mL
of 1M NaOH have been used up. Aspirate the resulting liquid and
wash discs with 500 mL of NaHCO3 buffer for 2 minutes at room
temperature. Repeat wash step.times.12. Rinse discs twice with 500
mL distilled water. Rinse discs twice with 500 mL 25% acetone.
Rinse discs twice with 500 mL 50% acetone. Rinse discs twice with
500 mL 75% acetone. Rinse discs twice with 500 mL 100% acetone.
Aspirate last acetone wash solution and allow discs to dry under a
running fume hood at room temperature. Store dried CNBr activated
paper discs in zip lock plastic bags containing dessicant
packettes.
[0232] Preparation of Neutravidin Conjugated Paper Discs and
Biotinylated Human Serum Albumin Conjugated Paper Discs
[0233] Materials:
[0234] Biotinylated human serum albumin: Prepared by method of
Example 1
[0235] Neutravidin: Pierce 31000
[0236] CNBr-activated paper discs: Prepared by method of Example
2
[0237] Modified Coca's buffer: 0.05M NaHCO3+0.15M NaCl. PH 7.2
[0238] 0.05M ethanolamine solution
[0239] 0.2M sodium acetate buffer, pH 4.0.
[0240] Paper disc incubation buffer: 0.05M sodium phosphate+0.1 5M
NaCl+0.05% NaN3
[0241] +0.5% Tween20
[0242] Procedure:
[0243] A 2.5 mg/mL solution of neutravidin is prepared in modified
Coca's buffer. A 2.5 mg/mL solution of biotinylated human serum
albumin is prepared in modified Coca's buffer. 50 CNBr-activated
discs are added to each mL of protein solution. Each protein/disc
mixture is agitated for 16 to 18 hours at room temperature. Each
solution surrounding the respective paper discs is aspirated and
each set of discs are washed.times.3 with modified Coca's buffer.
The washed discs are immersed in 0.05M ethanolamine solution and
agitated for 3 hours in order to block any unreacted CNBr sites.
Each set of paper discs is then washed.times.3 with the sodium
acetate buffer. During the third step, the paper discs are
incubated in the sodium acetate buffer for 30 minutes under gentle
agitation. Each set of paper discs is then washed.times.4 in Coca's
buffer and then stored in the paper disc incubation solution at 4
C.
[0244] Preparation of Neutravidin Coated Microtiter Plates
[0245] Materials:
[0246] Amino Polystyrene Microtiter Plates (White): Nunc 453686 or
the equivalent
[0247] Neutravidin: Pierce 31000
[0248] Disuccinimidyl suberate (DSS): Pierce 21555
[0249] Dimethyl sulfoxide (DMSO): Burdick and Jackson 081-1
[0250] 20 mM sodium phosphate, pH 5.50
[0251] 50 mM sodium carbonate, pH 9.6
[0252] PBS with sodium azide
[0253] Procedure:
[0254] Prepare a volume of neutravidin appropriate for the number
of plates to be coated. The coating solution contains 20 mg/mL
neutravidin in 20 mM sodium phosphate, pH 5.50.
[0255] Prepare a suitable volume of DSS, 1.22 mg/mL, in dry DMSO.
This solution must be used within 2 hours of preparation.
[0256] For each plate to be coated, add 60 mL DSS solution to each
well followed by 60 mL of 50 mM sodium carbonate, pH 9.6. Incubate
this mixture in the wells for 36 minutes at ambient temperature.
Aspirate the wells and wash twice with deionized water. Immediately
add 100 mL neutravidin solution. Cover the plate and incubate at
ambient temperature for 16-18 hours.
[0257] Aspirate the coating solution and add approximately 280 mL
PBS with azide to each well. Seal the plate with a foil cover.
Store the coated plates at about 4 C.
[0258] Vaccination Methods
[0259] Methods for preparing and administering a vaccine using a
peptide as an epitope have been reported. For example, Gilewski et
al. (2000) used a MUC1 peptide with a keyhole limpet hemocyonin
(KLM) in a conjugate to determine whether an immune response could
be generated against the MUCI peptide that would also bind with
tumor cells. An immunogenic response was reported.
[0260] Passive Immunization Constructs That the Host Immune System
Recognizes and Kills Cancer Cells
[0261] 1. Engineered Antigen Presenting Cells
[0262] See the "Detailed Description of the Invention" from U.S.
Pat. No. 5,851,320, incorporated by reference.
[0263] 2. Dendritic Cells
[0264] See the "Detailed Description of the Invention" and
"Examples 1-7" from U.S. Pat. No. 5,871,156 and the "Detailed
Description of the Invention" and "Examples 1-5" from U.S. Pat. No.
6,080,409, incorporated by reference.
[0265] Active Immunization Using Cancer Vaccines
[0266] 1. Recombinant Fusion Proteins
[0267] See the "Summary of the Invention" from U.S. Pat. No.
4,399,216 and U.S. Pat. Nos. 6,106,829 and 5,616,477 incorporated
by reference.
[0268] 2. Adjuvants
[0269] See U.S. Pat. Nos. 5,750,110; 5,876,966; 5,876,735;
6,013,268 and 6,080,399, incorporated by reference.
[0270] 3. Nucleic Acid Molecules
[0271] See U.S. Pat. Nos. 5,593,972; 5,817,637; 5,830,876;
6,063,384; 6,077,663; 5,981,505 and 5,942,235 incorporated by
reference.
[0272] 4. Recombinant Microorganisms which Express Cancer
Antigens
[0273] See U.S. Pat. No. 6,051,237 incorporated by reference.
[0274] 5. Antibody Delivers Antigen to Antigen Presenting Cells
[0275] See U.S. Pat. No. 5,194,254 incorporated by reference.
[0276] 6. Heat Shock Protein/Antigen Complexes
[0277] See U.S. Pat. Nos. 5,837,251; 5,981,706; 5,985,270;
5,997,873; 6,030,618 and 6,136,315 incorporated by reference.
[0278] 7. Cell Lytic Therapeutic Antibodies
[0279] See the section entitled "Therapy" in U.S. Pat. No.
4,585,742 incorporated by reference.
[0280] 8. Cell Adhesion Blocking Antibodies Such as Intergrin
Antagonists
[0281] (See Kerr et al., 2000).
[0282] 9. Growth Factor Receptor Blocking Antibodies
[0283] See U.S. Pat. No. 5,772,997 incorporated by reference.
[0284] Presence or Absence of 41 Molecules Listed in Table 3 in
Prostate Cancer Peptides
[0285] Information on the molecules in Table 3 were obtained from
the following sources:
[0286] Bryden et al., 1999;
[0287] Cress et al., 1995;
[0288] Dong et al., 1996;
[0289] Fudge et al., 1994;
[0290] Giri et al., 1999;
[0291] Grasso et al., 1997;
[0292] Kimura et al., 1996;
[0293] Kramer et al., 1995;
[0294] Luo et al., 1999;
[0295] Rokhlin et al., 1997;
[0296] Takahashi et al., 1998;
[0297] Tozawa, 1996;
[0298] Tronet. al., 1999;
[0299] Watanabe et al., 1999; and
[0300] Zheng et al., 2000.
[0301] Use of Peptides of the Present Invention on Microchips
[0302] Microchips that have oligonucleotides or peptides have been
developed by many groups or researches for various applications
e.g. determining whether genes are present in a biological sample
by determining whether DNA molecules in the sample hybridize under
conditions wherein hybridization implies a specific degree of
homology between a DNA molecule in a sample applied to the
microchip and a DNA molecule in the microchip. Microchips are
designed so that questions such as "Is the gene for the disease X
present in a person?" or "Does the patient have a particular
mutation?" or "Is there a specific antigen(s) present in the
sample?" can be answered by interpreting the hybridization pattern
in the chip, or in the case of antigen or antibody detection, the
pattern of antigen-antibody complexing on the microchip. Examples
of patents in the microchip area are U.S. Pat. No. 5,861,247 and
U.S. Pat. No. 5,770,721. Microchips are sold commercially by
Affymetrix, Hyseq and other companies. Licenses are available for
microchip technologies through Argonne National Laboratory.
Documents Cited
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Sequence CWU 1
1
68 1 5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 1 Val Lys Xaa Ser Thr 1 5 2 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 2 Asp
Lys Xaa Leu Ser 1 5 3 6 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 3 Asn Asn Xaa Xaa Ser Ser 1 5
4 5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 4 Asp Lys Xaa Ser Thr 1 5 5 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 5 Gly
Thr Xaa Lys Thr 1 5 6 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 6 Asp Arg Xaa Gly Thr 1 5 7 5
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 7 Xaa His Ser Glu Gly 1 5 8 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 8 Ser
Pro Xaa His Ser 1 5 9 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 9 Asp Arg Xaa His Thr 1 5 10
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 10 Ser Asp Xaa Ser Thr 1 5 11 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 11
Phe Cys Xaa Arg Ser 1 5 12 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 12 Lys Asn Xaa His Thr 1 5 13
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 13 Ser Ala Xaa Arg Ser 1 5 14 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 14
Thr Arg Xaa Asp Thr 1 5 15 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 15 Ser Lys Xaa Gln Ser 1 5 16
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 16 Pro Gly Xaa Arg Thr 1 5 17 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 17
Thr Lys Xaa Gly Thr 1 5 18 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 18 Lys Ile Xaa Ala Thr 1 5 19
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 19 Asp Ala Xaa Lys Thr 1 5 20 6 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 20
Pro Glu Arg Xaa Arg Thr 1 5 21 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 21 Cys Arg Xaa
Val Ser 1 5 22 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 22 Asp Thr Xaa Arg Ser 1 5 23 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 23 His Glu Xaa Cys Thr 1 5 24 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 24 Ala Glu Xaa
Cys Thr 1 5 25 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 25 Pro Asp Xaa Cys Thr 1 5 26 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 26 Glu Ser Xaa Arg Thr 1 5 27 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 27 Glu Lys Xaa
Gly Ser 1 5 28 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 28 Asp Ile Xaa Ser Ser 1 5 29 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 29 Val Gly Xaa Lys Ser 1 5 30 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 30 Glu Glu Xaa
Asn Ser 1 5 31 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 31 Lys Asp Xaa Arg Thr 1 5 32 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 32 Thr Arg Xaa Val Ser 1 5 33 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 33 His Lys Xaa
Gln Thr 1 5 34 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 34 Gln Arg Xaa Ile Thr 1 5 35 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 35 Ser Glu Xaa Ala Ser 1 5 36 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 36 Asp Arg Xaa
His Ser 1 5 37 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 37 Met Glu Xaa Lys Thr 1 5 38 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 38 Leu Glu Xaa His Thr 1 5 39 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 39 Arg Met Xaa
Ile Thr 1 5 40 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 40 Lys Ala Xaa Thr Ser 1 5 41 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 41 Leu Arg Xaa Glu Ser 1 5 42 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 42 Arg Ala Xaa
His Ser 1 5 43 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 43 Gln Lys Xaa Gln Thr 1 5 44 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 44 Lys Gly Xaa Cys Ser 1 5 45 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 45 Gln Asp Xaa
Lys Thr 1 5 46 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 46 Lys Ala Xaa Thr Thr 1 5 47 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 47 Thr Glu Xaa Gln Thr 1 5 48 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 48 Glu Lys Xaa
Asp Thr 1 5 49 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 49 Asn Lys Xaa Val Thr 1 5 50 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 50 Val Thr Xaa Arg Ser 1 5 51 7 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 51 Lys Xaa Val
Thr Xaa Arg Ser 1 5 52 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 52 Lys Glu Xaa Ser Ser 1 5 53
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 53 Glu Gln Xaa Cys Thr 1 5 54 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 54
Ser Lys Xaa Phe Ser 1 5 55 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 55 Arg Cys Xaa Gly Ser 1 5 56
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 56 Pro Asp Xaa Gln Thr 1 5 57 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 57
Gln Lys Xaa Ser Ser 1 5 58 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 58 Glu Val Xaa Ser Ser 1 5 59
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 59 Ser Glu Xaa Asp Thr 1 5 60 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 60
Lys Glu Xaa Asp Thr 1 5 61 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 61 Gly His Xaa Glu Ser 1 5 62
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 62 Gly Asp Xaa Arg Thr 1 5 63 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 63
Asp Glu Xaa Ile Thr 1 5 64 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 64 Glu Tyr Xaa Thr Ser 1 5 65
5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 65 Pro Asp Xaa Asp Thr 1 5 66 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 66
Arg Asn Xaa Thr Thr 1 5 67 1210 PRT Homo sapiens 67 Met Arg Pro Ser
Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu
Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35
40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly
Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser
Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile
Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln
Ile Ile Arg Gln Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu
Ala Val Leu Ser Asn Tyr Asp Ala Asp 115 120 125 Lys Thr Gly Leu Lys
Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala
Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165
170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp
Pro 180 185 190 Ser Cys Pro Asp Gly Ser Cys Trp Gly Ala Gly Glu Glu
Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys
Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His
Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser
Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys
Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr
Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290
295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu
Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro
Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys
Asp Ser Leu Ser Ile Asp 340 345 350 Ala Thr Asn Ile Lys His Phe Lys
Asp Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val
Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile
Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asp Arg Thr Asp 405 410
415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asp Ile Thr
Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp
Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr
Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys
Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala
Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys
Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asp 515 520 525 Val
Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535
540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asp Ile Thr Cys Thr Gly
Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp
Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly
Glu Asp Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His
Val Cys His Leu Cys His Pro Asp Cys 610 615 620 Thr Tyr Gly Cys Thr
Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Pro Lys
Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660
665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu
Leu 675 680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln
Ala Leu Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile
Lys Val Leu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys
Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val
Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn
Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp
Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780
Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785
790 795 800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu
Leu Asn 805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu
Glu Asp Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn
Val Leu Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe
Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His
Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875 880 Met Ala Leu
Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val
Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905
910 Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu
915 920 925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp
Val Tyr 930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp
Ser Arg Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser
Lys Met Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly
Asp Glu Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe
Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val
Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe 1010 1015
1020 Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser
Ala 1025 1030 1035 1040 Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp
Arg Asn Gly Leu Gln 1045 1050 1055 Ser Cys Pro Ile Lys Glu Asp Ser
Phe Leu Gln Arg Tyr Ser Ser Asp 1060 1065 1070 Pro Thr Gly Ala Leu
Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro 1075 1080 1085 Val Pro
Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser 1090 1095
1100 Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro
Ser 1105 1110 1115 1120 Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr
Ala Val Gly Asn Pro 1125 1130 1135 Glu Tyr Leu Asn Thr Val Gln Pro
Thr Cys Val Asn Ser Thr Phe Asp 1140 1145 1150 Ser Pro Ala His Trp
Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp 1155 1160 1165 Asn Pro
Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn 1170 1175
1180 Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg
Val 1185 1190 1195 1200 Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala
1205 1210 68 7 PRT Artificial Sequence Description of Artificial
Sequence synthetic peptide 68 Ala Glu Met Xaa Gly Ser Lys 1 5
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