U.S. patent application number 10/235236 was filed with the patent office on 2003-05-08 for immunogenic compositions to the cck-b/gastrin receptor and methods for the treatment of tumors.
Invention is credited to Caplin, Martyn, Grimes, Stephen, Michaeli, Dov, Watson, Susan A..
Application Number | 20030086941 10/235236 |
Document ID | / |
Family ID | 21942134 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030086941 |
Kind Code |
A1 |
Michaeli, Dov ; et
al. |
May 8, 2003 |
Immunogenic compositions to the CCK-B/gastrin receptor and methods
for the treatment of tumors
Abstract
The invention concerns immunogens, immunogenic compositions and
method for the treatment of gastrin-dependent tumors. The
immunogens comprise a peptide from the CCK-B/gastrin-receptor
conjugated to a spacer and to an immunogenic carrier. The
immunogens are capable of inducing antibodies in vivo which bind to
the CCK-B/gastrin-receptor in tumor cells, thereby preventing
growth stimulating peptide hormones from binding to the receptors,
and inhibiting tumor cell growth. The immunogens also comprise
antibodies against the CCK-B/gastrin-receptor for passive
immunization. The invention also concerns diagnostic methods for
detecting gastrin-dependent tumors in vivo or from a tissue biopsy
using the antibodies of the invention.
Inventors: |
Michaeli, Dov; (Larkspur,
CA) ; Caplin, Martyn; (London, GB) ; Watson,
Susan A.; (Edwalton, GB) ; Grimes, Stephen;
(Davis, CA) |
Correspondence
Address: |
WHITE & CASE LLP
PATENT DEPARTMENT
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Family ID: |
21942134 |
Appl. No.: |
10/235236 |
Filed: |
September 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10235236 |
Sep 4, 2002 |
|
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09076372 |
May 12, 1998 |
|
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60046201 |
May 12, 1997 |
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Current U.S.
Class: |
424/185.1 ;
530/350 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 2039/6037 20130101; A61K 39/001102 20180801; A61K 38/00
20130101; C07K 16/2869 20130101; C07K 16/30 20130101; A61K 39/0005
20130101; A61P 43/00 20180101; C07K 2317/77 20130101; C07K 14/72
20130101; C07K 2317/34 20130101; A61P 35/00 20180101; G01N 33/57488
20130101 |
Class at
Publication: |
424/185.1 ;
530/350 |
International
Class: |
A61K 039/00; C07K
014/705 |
Claims
We claim:
1. An immunogen comprising a peptide from the
CCK-B/gastrin-receptor conjugated to an Immunogenic carrier.
2. The immunogen of claim 1, wherein the peptide has the amino acid
sequence KLNRSVQGTGPGPGASL (SEQ ID NO.: 1 in the Sequence Listing)
or GPGAHRALSGAPISF (SEQ ID NO.: 2 in the Sequence Listing).
3. The immunogen of claim 1 or 2, further comprising a spacer
peptide sequence.
4. The immunogen of claim 3, wherein the spacer peptide sequence is
SSPPPPC (SEQ ID NO.: 3 in the Sequence Listing).
5. The immunogen of claim 1, wherein the immunogenic carrier is
selected from the group consisting of Diphtheria toxoid, tetanus
toxoid and bovine serum albumin.
6. A method for treating a malignant condition caused by
gastrin-dependent malignant cell growth comprising administering to
an animal in need of such treatment an effective amount of an
anti-CCK-B/gastrin-receptor immunogen.
7. The method of claim 6, wherein the immunogen comprises a peptide
from the CCK-B/gastrin-receptor.
8. The method of claim 7, wherein the peptide has the amino acid
sequence KLNRSVQGTGPGPGASL (SEQ ID NO.: 1 in the Sequence Listing)
or GPGAHRALSGAPISF (SEQ ID NO.: 2 in the Sequence Listing).
9. The method of claim 8, wherein the immunogen further comprises a
spacer peptide sequence.
10. The method of claim 9, wherein the spacer peptide sequence is
SSPPPPC (SEQ ID NO.:3 in the Sequence Listing).
11. The method of claim 10, further comprising an immunogenic
carrier selected from the group consisting of Diptheria toxoid,
tetanus toxoid and bovine serum albumin.
12. A method for treating a gastrin-dependent tumor comprising
administering to an animal in need of such treatment an effective
amount of anti-CCK-B/gastrin antibodies which recognize and bind to
the CCK-B/gastrin-receptors in the tumor cells.
13. The method of claim 12, wherein the antibodies are selected
from the group consisting of chimeric, monoclonal and humanized
antibodies.
14. The method of claim 12, wherein the anti-CCK-B/gastrin-receptor
antibodies recognize and bind the amino acid sequence
KLNRSVQGTGPGPGASL (SEQ ID NO.: 1 in the Sequence Listing) or
GPGAHRALSGAPISF (SEQ ID NO.: 2 in the Sequence Listing) of the
receptor.
15. The method of claim 12 or 14, wherein the antibodies are
further conjugated to a cytotoxic molecule.
16. The method of claim 15, wherein the cytotoxic molecule is a
toxin or a radioactive molecule.
17. The method of claim 16, wherein the toxin is cholera toxin.
18. The method of claim 16, wherein the radioactive molecule is
labeled with .sup.125I or .sup.131 I.
19. A method for detecting a gastrin-responsive tumor containing
CCK-B/gastrin-receptors, comprising exposing an
anti-gastrin-receptor antibody to cells isolated from a tumor
biopsy sample and detecting the CCK-B/gastrin-receptor in the
sample.
20. The method of claim 19, wherein the anti-gastrin-receptor
antibody is specific for an amino-terminal peptide of the
CCK-B/gastrin-receptor.
21. The method of claim 20, wherein the peptide is in the
amino-terminal region of the CCK-B/gastrin-receptor comprises amino
acid residues 5-21.
22. An immunogenic composition comprising an
anti-CCK-B/gastrin-receptor immunogen.
23. The composition of claim 22, wherein the immunogen comprises a
peptide from the CCK-B/gastrin-receptor.
24. The compostion of claim 22, wherein the immunogen comprises
antibodies against the CCK-B/gastrin-receptor.
25. A method for diagnosing a gastrin-dependent tumor, comprising
administering radiolabeled anti-CCK-B/gastrin-receptor antibodies
to a patient possessing a colorectal tumor and imaging the tumor by
scintigraphic scanning.
26. The method of claim 25, wherein the anti-CCK-B/gastrin-receptor
antibodies are radiolabeled with .sup.111Indium or .sup.90Yttrium.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e), of U.S. Provisional Application No. 60/046,201 filed
on May 12, 1997.
BACKGROUND OF THE INVENTION
[0002] The hormone gastrin binds to a gastrin/cholecystokinin
(CCK)-B receptor with high affinity via its 5 carboxy-terminal
amino acids. The CCK- B/gastrin receptor is a cytoplasmic membrane
protein which is coupled via a G protein to intracellular signal
transduction pathways that in turn control the expression of
various genes.
[0003] Gastrin is a peptide hormone which occurs in two forms,
tetratriacontagastrin (G34) and heptadecagastrin (G17), and is
synthesized and secreted by specialized cells, G cells, that are
located in the stomach antrum. The hormone is secreted into the
circulating blood and binds to specific cells in the stomach,
namely, enterochromaffin-like (ECL) and parietal cells, that
indirectly or directly affect stomach acid output. Historically,
gastrin hormones have been associated with the stimulation of
gastric acid secretion (Edkins, J. S. 1905). (The full citations
for the references cited herein are provided in the Reference
section preceding the claims.) In recent years, evidence has
accumulated that gastrin may act as a trophic factor within the
gastrointestinal tract (Johnson, L. 1997) and that it can promote
the growth of gastrointestinal cancers (Watson et al. 1989,
Dickinson, C. J. 1995), as well as non-gastrointestinal cancers
including small cell carcinoma of the lung (Rehfeld et al. 1989).
In the post-translational processing of gastrin, it is the "mature"
carboxy-amidated form that binds to the gastrin/CCK-B receptor via
the carboxy terminus (Kopin et al. 1992).
[0004] It has been shown that several types of tumors, e.g.,
colorectal, stomach, pancreatic and hepatocellular adenocarcinomas
possess CCK-B/gastrin receptors in their plasma membranes and that
they respond to gastrin with powerful cellular proliferation
(Rehfeld, J. F. 1972, Upp et al. 1989 and Watson et al. 1993).
Furthermore, more recently it has been discovered that many of
these cancer cells also secrete gastrin and thus effect an
autonomous proliferative pathway (Van- Solinge et al. 1993, Nemeth
et al. 1993 and Seva et al. 1994).
[0005] The CCK-B/gastrin receptor belongs to a family of G
protein-coupled receptors with seven transmembrane domains with
equal affinity for both CCK and gastrin (Soll et al. 1984). This
receptor was named a CCK type-B receptor because it was found
predominantly in the brain (Wank et al. 1992). The receptor was
subsequently found to be identical to the peripheral CCK/gastrin
receptor in the parietal and ECL cells of the stomach (Nakata et
al. 1992). This receptor has been well characterized in a number of
normal (Fourmy et al. 1984, Grider et al. 1990) and tumor tissues
(Singh et al. 1990, Watson et al. 1993), and extensively studied
using the rat pancreatic adenocarcinoma cell line AR42J (Scemama et
al. 1987). The AR42J CCK-B/gastrin receptor cDNA has been cloned
and sequenced, and it is more than 90% homologous in DNA sequence
to the CCK-B/gastrin receptor in rat and human brain, and more than
84% homologous in sequence to the canine parietal cell
CCK-B/gastrin receptor cDNA (Wank, S. A. 1995), demonstrating a
high sequence homology even between species.
[0006] The peptide hormones G17 and G34 bind to the CCK- B/gastrin
receptor on the cell membrane of normal cells. However, it has been
found that G17, and not G34, stimulates the growth of
gastrin-dependent cancer cells. Serum-associated G17, in
particular, has the potential to stimulate the growth of colorectal
tumors in an endocrine manner mediated by CCK-B/gastrin receptors
(Watson et al. 1993) in the tumor cells. Gastrin-17 appears to be
particularly implicated in stimulating the growth of colorectal
adenocarcinomas due to a possible increased affinity for the
CCK-B/gastrin receptor on the tumor cells, over other gastrin
hormone species (Rehfeld 1972 and 1993). The CCK-B/gastrin
receptors were found to be expressed in a high affinity form on
56.7% of human primary colorectal tumors (Upp et al. 1989). It has
been postulated that a potential autocrine loop may also exist due
to endogenous production of precursor gastrin peptides by such
tumors (Van-Solinge et al. 1993 and Nemeth et al. 1993). The
resulting G17 ligand/receptor complex stimulates cell growth by way
of secondary messengers for regulating cell function (Ullrich et
al. 1990). The binding of G17 to the CCK- B/gastrin receptor leads
to activation of phosphatidyl inositol breakdown, protein kinase C
activation with a resultant increase in intracellular calcium ion
concentration, as well as the induction of c-fos and c-jun genes
via mitogen-activated protein kinase, which has been implicated in
the regulation of cell proliferation (Tadisco et al. 1995).
Additionally, gastrin binding to the CCK-B/gastrin receptor has
been associated with the subsequent increase in phosphorylation by
a tyrosine kinase, pp125FADK (focal adhesion kinase), which may
also have a role in the transmission of mitogenic signals
(Tanaguchi et al. 1994).
[0007] A number of high affinity CCK-B/gastrin receptor antagonists
have been evaluated therapeutically both in vitro and in vivo in a
number of experimental gastrointestinal cancers. For example,
proglumide, a glutamic acid derivative (Seva et al. 190; Harrison
et al. 1990 and Watson et al. 1991a); Benzotript, an N-acyl
derivative of tryptophan; L-365,260, a derivative of Aspercillin
(Bock et al. 1989), and CI-988 a molecule that mimics the
C-terminal pentapeptide sequence of CCK (Hughes et al. 1990) have
been shown to effectively neutralize the effects of exogenous
gastrin on gastrointestinal tumor growth both in vitro and in vivo
(Watson et al. and Romani et al. 1994). However, these antagonists
have severe toxic side effects and lack specificity as they block
the action of all potential ligands of the receptor such as G34 and
CCK in normal cells. Recently, highly potent and selective
CCKB/gastrin receptor antagonists such as YM022 (Yuki et al., 1997)
and YF476 (Takinami et al., 1997) have been also described.
[0008] Proglumide and Benzotript have been widely assessed in the
pre-clinical studies. The main problem with these compounds is
their lack of potency, with relatively high concentrations required
to displace G17 (Watson et al., 1992a; Watson et al., 1992b).
Despite this, proglumide and benzotript inhibited the basal and
gastrin-stimulated proliferation of a number of cell lines (Seva et
al., 1990; Watson et al., 1991 a). In addition, proglumide
increased the survival of xenograft mice bearing the
gastrin-sensitive mouse colon tumor, MC26 to 39 days in the treated
animals from 25 days in the control animals.
[0009] Due to the low specificity of this class of gastrin
antagonising agents for the gastrin/CCKB receptor, the inhibition
of tumor growth may not be effectively control with gastrin
antagonists. Moreover, the cellular receptors which recognize and
bind the gastrins do not bind all the inhibitors tested (Seva et
al. 1994). Thus, if complete inhibition of gastrin binding to the
receptor does not occur in the autocrine growth cascade, then the
gastrin antagonists may be unable to block this mechanism of tumor
growth promotion.
SUMMARY OF THE INVENTION
[0010] The present invention provides immunogenic compositions and
immunological methods for the treatment of gastrin-dependent
tumors. The method comprises the active or passive immunization of
a patient with an anti-CCK-B/gastrin receptor immunogen or
anti-CCK-B/gastrin receptor antibodies. The antibodies produced by
the immunogens are specific against the CCK-B/gastrin receptor on
tumor cells and block the growth-promoting effects of gastrin on
the receptors. The antibodies prevent the peptide hormones from
binding to the CCK-B/gastrin receptors on gastrin-dependent tumor
cells; thus, the growth of the tumor is arrested. Furthermore,
surprisingly, the antibodies specific to the NH.sub.2-terminal end
of the receptor, upon binding to the receptor, are internalized and
rapidly translocated into the cytoplasm and into the nucleus of the
tumor cells. This internalization can occur as early as 10 seconds
after exposing the cells to the antibody. This rapid
internalization of the antibody/receptor complex in turn causes the
affected tumor cells to undergo apoptosis or suicide.
[0011] The immunogens of the invention comprise natural or
synthetic peptides derived from the human CCK-B/gastrin receptor,
as the immunomimic portion of the immunogen. The immunogens may
also comprise a spacer peptide sequence attached to an end of the
immunomimic peptide. The immunogen may also be conjugated to a
protein carrier, such as Diphtheria toxoid, tetanus toxoid, bovine
serum albumin and the like.
[0012] In one embodiment, the method of immunization against the
CCK-B/gastrin receptor comprises active immunization, wherein a
patient is immunized with an immunogen of the invention. The
immunogen stimulates the production of antibodies against the
CCK-B/gastrin receptor on tumor cells.
[0013] The antibodies produced by the anti-CCK-B/gastrin receptor
immunogens bind to the CCK-B/gastrin receptors on tumor cells and
effectively prevent the binding of the peptide hormones to the
receptors, thereby inhibiting the autocrine growth-stimulatory
pathway of tumor cell division and ultimately the growth of the
tumor.
[0014] In another embodiment of the invention, the method of
treatment comprises passive immunization, whereby antibodies
against the CCK-B/gastrin receptor are administered to a patient in
a sufficient concentration to bind to the CCK-B/gastrin receptors
of the tumor cells, and the antibodies block the binding of the
peptide hormones to the receptor. The prevention of binding of the
hormones to their receptor inhibits the growth-stimulus pathway of
the tumor cells, thereby inhibiting the growth of the
hormone-dependent tumors. In a preferred embodiment of this aspect
of the invention, the antibodies for human therapy may be chimeric,
humanized, or human monoclonal antibodies which may be produced by
methods well known in the art. In addition, the anti-CCK-B/gastrin
receptor antibodies may be further conjugated to cytotoxic
molecules such as cholera toxin, or to radioactive molecules
labeled with a radionuclide, such as .sup.125I and .sup.131I, to
enhance the killing of the tumor cells.
[0015] The invention also provides a method for diagnosing a
gastrin-responsive tumor, comprising the immunochemical detection
of gastrin-dependent (CCK-B/gastrin-containing) tumors from a
tissue biopsy using the antibodies of the invention. The specific
anti-CCK-B/gastrin receptor antibodies of the invention can be
labeled with a detection system utilizing compounds such as biotin,
horseradish peroxidase and fluorescein to detect the CCK-B/gastrin
receptors in the tumor tissue using standard immunochemical
procedures.
[0016] The invention also provides a method for diagnosing a
gastrin-dependent tumor, comprising the in vivo detection of
gastrin-dependent (CCK-B/gastrin receptor-containing) tumors, using
the anti-CCK-B/gastrin receptor antibodies. The method comprises,
administering to a patient possessing a colorectal tumor an
effective dose of radiolabeled anti-CCK-B/gastrin receptor
antibodies via an intravenous injection, and imaging or detecting
tumor cells having anti-CCK-B/gastrin receptor antibodies bound to
their cell membranes by standard scintigraphic scanning procedures.
In this aspect of the invention, the
anti-CCK-B/gastrin-receptor-antibodies should be labeled with a
radionuclide such as .sup.111Indium, .sup.90Yttrium, and
.sup.131I.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A and 1B illustrate schematic views of the
CCK-B/gastrin receptor and its 7 transmembrane domains.
[0018] FIG. 2 shows data from ELISA assays with antibodies raised
in rabbits immunized with an immunogen against Peptide 1 of the
CCK-B/gastrin receptor.
[0019] FIG. 3 shows data from ELISA assays with antibodies raised
in rabbits immunized with an immunogen against Peptide 4 of the
CCK-B/gastrin receptor.
[0020] FIG. 4 is a graph showing data obtained from an inhibition
ELISA used to assess the specificity of affinity-purified
antibodies raised against GRP1-DT immunogen.
[0021] FIG. 5 is a bar graph showing data on the inhibition of the
binding of .sup.125I-human G17 to AR42J cells by peptide
inhibitors.
[0022] FIG. 6 is a bar graph of the cellular distribution of
immunogold-labeled AR4-2J tumor cells.
[0023] FIG. 7 is a photograph of a Western blot analysis of protein
extracts from nuclear membranes of adenocarcinoma cells using
antibodies raised against Peptide 1.
[0024] FIG. 8 is a photograph of a Western blot analysis of protein
extracts from extranuclear and plasma membranes of adenocarcinoma
cells using antibodies raised against Peptide 1.
[0025] FIG. 9 is a plot graph illustrating the C170HM2 tumor weight
of control and anti-CCK-B/gastrin receptor-treated animals.
[0026] FIG. 10 is a plot graph illustrating the cross-sectional
area of C170HM2 tumors from control and anti-CCK-B/gastrin
receptor-treated animals.
[0027] FIG. 11 is a bar graph showing the mean C170HM2 tumor
weights of control and anti-CCK-B/gastrin receptor-treated
animals.
[0028] FIG. 12 is a bar graph showing the mean cross-sectional area
of C170HM2 tumors of control and anti-CCK-B/gastrin
receptor-treated animals.
[0029] FIG. 13 is a bar graph showing the mean number of C170HM2
tumors in control and anti-CCK-B/gastrin receptor-treated
animals.
[0030] FIG. 14 is a bar graph showing the median C170HM2 tumor
weight of liver metastases, of control and anti-CCK-B/gastrin
receptor-treated animals.
[0031] FIG. 15 is a bar graph showing the median cross-sectional
area of C170HM2 tumors from control and anti-CCK-B/gastrin
receptor-treated animals.
[0032] FIG. 16 is a bar graph showing the median C170HM2 tumor
number in control and anti-CCK-B/gastrin receptor-treated
animals.
[0033] FIG. 17 is a bar graph showing the mean and median liver
C170HM2 tumor number in control and
anti-CCK-B/gastrin-receptor-treated animals.
[0034] FIG. 18 is a bar graph showing the mean and median liver
C170HM2 tumor weight in control and
anti-CCK-B/gastrin-receptor-treated animals.
[0035] FIG. 19 is a bar graph showing the mean and median values
for the cross-sectional area of C170HM2 liver tumor metastases in
control and anti-CCK-B/gastrin-receptor-treated animals.
[0036] FIG. 20 depicts a graph showing the concentration of
radiolabeled .sup.125I-antibodies in C170HM2 liver tumor xenografts
of control (normal rabbit serum) and anti-GRP1-treated nude
mice.
[0037] FIG. 21 depicts a bar graph showing the mean C170HM2 liver
tumor number per liver of xenografts of control and
anti-GRP1-treated nude mice.
[0038] FIG. 22 depicts a bar graph showing the mean C170HM2 liver
tumor weight of liver xenografts of control and anti-GRP1-treated
nude mice.
[0039] FIG. 23 depicts Western blots of C170HM2 liver tumor
xenograft proteins of control and anti-GRP1-treated nude mice.
[0040] FIG. 24 is a photograph of a histological section taken with
a light microscope showing a hematoxylin/eosin-stained section of a
C170HM2 liver xenograft of a control mouse.
[0041] FIG. 25 is a photograph of a histological section taken with
a light microscope showing a hematoxylin/eosin stained section of a
C170HM2 liver xenograft from a mouse treated with rabbit
anti-GRP1antibodies.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The methods of the invention are directed to the treatment
of gastrin hormone-dependent tumors in animals, including humans,
and comprise administering to a patient an
anti-CCK-B/gastrin-receptor immunogen, which produces antibodies in
the immunized patient which bind to the CCK-B/gastrin-receptor on
the tumor cells, so as to prevent the binding of the hormone to the
receptor in order to inhibit the growthpromoting effects of the
hormone. More importantly, from a clinical point of view, the
receptor/anti GRP1 complex is rapidly internalized, traverses the
cytoplasm and enters the nucleus. This apparently triggers the
affected tumor cells to commit suicide (apoptosis).
[0043] The immunogens comprise natural or synthetic peptides of the
human CCK-B/gastrin-receptor which act as immunomimics. In
particular, two synthetic peptides have been developed as the
immunomimics. These peptides, developed from the amino acid
sequence of the CCK-B/gastrin-receptor, are immunogenic and
cross-reactive with the endogenous CCK-B/gastrin-receptor of tumor
cells both in vivo and in vitro. Peptide 1 consists of amino acids
5 through 21 of the CCK- B/gastrin-receptor sequence:
KLNRSVQGTGPGPGASL (Peptide 1, SEQ ID NO.: 1 in the Sequence
Listing). Peptide 1 constitutes the amino-terminal end of the
receptor and is located on the extracellular surface of the cell
membrane (see FIG. 1).
[0044] In another embodiment, the immunogen comprises Peptide 4,
which consists of the following amino acid sequence of the
CCK-B/gastrin-receptor: GPGAHRALSGAPISF (Peptide 4, SEQ ID NO.: 2
in the Sequence Listing). Peptide 4 is part of the fourth
extracellular domain of the receptor and it too is on the outer
side of the plasma membrane (see FIG. 1).
[0045] The immunogens may also comprise an extension or spacer
peptide suitable for projecting the immunomimic peptide away from
the protein carrier and enhancing its capacity to bind the
lymphocyte receptors. A suitable spacer peptide sequences the amino
acid sequence SSPPPPC (Serine (Ser) spacer, SEQ ID NO.:3 in the
Sequence Listing). However other spacer peptides would be suitable
as well. The immunomimic peptides, with or without the spacer, are
then conjugated to a protein carrier, such as Diphtheria toxoid,
via a cysteine residue at the carboxy terminal end. The spacer
peptides are not immunologically related to the
CCK-B/gastrin-receptor-derived peptides and should therefore
enhance, but not determine, the specific immunogenicity of the
receptor-derived peptides.
[0046] The presence and density of CCK-B/gastrin-receptors on tumor
cells in a patient can be determined by reacting labeled
anti-receptor antibodies with a sample of obtained from tumor
biopsy sample. The anti-receptor antibodies can be labeled with
either a radioactive tracer, a dye or a fluorescent label. In
addition, the responsiveness of the tumor cells to gastrin can be
evaluated in vitro from a tumor biopsy sample of the patient using
standard techniques. Patients having tumors with biopsy samples
positive for the CCK-B/gastrin-receptor antibody assay are typical
candidates for treatment by the methods of the invention.
[0047] An effective dosage ranging from 0.001 to 2 mg of the
immunogenic composition is administered to the patient for the
treatment of the gastrointestinal cancer. The effective dosage of
the immunogenic composition should be capable of eliciting an
immune response in a patient consisting of effective levels of
antibody titer against the CCK-B/gastrin-receptor 1-3 months after
immunization. Following the immunization of a patient, the
effectiveness of the immunogens is monitored by standard clinical
procedures, such as ultrasound and magnetic resonance imaging
(MRI), to detect the presence and size of tumors. The antibody
titer levels against the receptor may also be monitored from a
sample of blood taken from the patient. Booster immunizations
should be given as required to maintain an effective antibody
titer. Effective treatment of gastrin-dependent cancers, such as
stomach, liver, pancreatic and colorectal adenocarcinomas,
according to this method should result in inhibition of tumor
growth and a decrease in size of the tumor.
[0048] The antibodies raised by the anti-CCK-B/gastrin-receptor
immunogens of the present invention may have anti-trophic effects
against gastrin-dependent tumors by three potential mechanisms: (i)
inhibition of gastrin binding to its receptor, (ii) degradation or
disruption of the signal transduction pathway of tumor cell
proliferation; and (iii) induction of apoptosis (or cell suicide)
in cells where receptor/antibody complexes are internalized and
migrate into the nucleus.
[0049] In another embodiment of the invention,
anti-CCK-B/gastrin-receptor antibodies are administered to a
patient possessing a CCK-B/gastrin-receptor-responsive tumor. The
antibodies specifically bind to the CCK-B/gastrin-receptors on the
tumor cells. The binding of the antibodies to the receptors
prevents the binding of gastrin to its ligand in the membranes of
cells and, therefore, the growth signal for the gastrin-dependent
tumor cells is inhibited and the growth of the tumor is arrested.
The antibodies are preferably chimeric or humanized antibodies, or
fragments thereof, which effectively bind to the target receptor
and may be produced by standard techniques such as those disclosed
in U.S. Pat. Nos. 5,023,077, 5,468,494, 5,607,676, 5,609,870,
5,688,506 and 5,662,702, the disclosures of which are hereby
incorporated by reference. These exogenously produced antibodies
may also be useful for killing tumor cells that bear the
CCK-B/gastrin-receptor on their plasma membranes by virtue of their
inhibiting the growth of the tumor cells or delivering a toxic
substance to the tumor cell. Preferred anti-CCK-B/gastrin
antibodies for therapy are those reactive with extracellular
domains 1 and 4 of the receptor protein shown in FIG. 1 as GRP-1
and GRP-4, respectively. Particularly preferred are, antibodies
which specifically recognize and bind amino acid sequences of the
receptor protein corresponding to Peptides 1 and 4. The inhibition
of tumor growth in this method of immunization is also monitored by
ultrasound imaging and MRI and repeated immunizations are
administered as required by the patient.
[0050] The effectiveness of the antibodies in inhibiting tumor cell
growth and killing of tumor cells can be enhanced by conjugating
cytotoxic molecules to the anti-CCK-B/gastrin antibodies. The
cytotoxic molecules can be toxins, for example, cholera toxin,
ricin, .alpha.-amanitin, or radioactive molecules labeled, for
example with .sup.125I or .sup.131I, or chemotherapeutic agents,
for example, cytosine arabinoside or 5-fluorouridine.
[0051] In addition to antibodies radiolabeled with .sup.125I and
.sup.131I, the anti-CCK-B/Gastrin-receptor antibodies can also be
labeled with radionuclide such as .sup.111Indium and
.sup.90Yttrium. In this aspect of the invention the antibodies are
useful for the detection and diagnosing of CCK-B/gastrin-receptor
possessing tumors in vivo, by administering these antibodies to the
patient, and detecting bound antibodies on
CCK-B/gastrin-receptor-containing tumor cells. After allowing the
radio-labeled anti-CCK-B/gastrin antibodies to reach the tumor,
about 1-2 hours after injection, the radioactive, "hot spots" are
imaged using standard scintigraphic procedures as previously
disclosed (Harrison's Principles of Internal Medicine, Isselbacher
et al. eds. 13.sup.th Ed. 1994).
[0052] The compositions in which the immunogens are administered
for the treatment of gastrin-dependent tumors in patients may be in
a variety of forms. These include, for example, solid, semi-solid
and liquid dosage forms, such as powders, liquid solutions,
suspensions, suppositories, and injectable and infusible solutions.
The preferred form depends on the intended mode of administration
and therapeutic applications. The compositions comprise the present
immunogens and suitable pharmaceutically acceptable components, and
may include other medicinal agents, carriers, adjuvants,
excipients, etc. Suitable adjuvants may include nor- muramyl
dipeptide (nor-MDP, Peninsula Labs., Calif.), and oils such as
Montanide ISA 703 (Seppic, Inc., Paris, France), which can be mixed
using standard procedures. Preferably, the compositions are in the
form of unit dose. The amount of active compound administered for
immunization or as a medicament at one time, or over a period of
time, will depend on the subject being treated, the manner and form
of administration, and the judgment of the treating physician.
[0053] The anti-CCK-B/gastrin-receptor antibodies of the invention
for passive immunization are preferably administered to a patient
intravenously using a pharmaceutically acceptable carrier, such as
a saline solution, for example, phosphate-buffered saline.
EXAMPLE 1
[0054] Preparation of GRP1-DT and GRP4-DT Conjugates
[0055] CCK-B/gastrin-receptor peptides were prepared by standard
solid state peptide synthesis. To make immunogens capable of
inducing specific immune responses each of Peptide 1 and 4 was
synthesized containing the spacer sequence SSPPPPC (SEQ ID NO.:3 in
the Sequence Listing) at its carboxy terminus. These peptides were
subsequently conjugated to amino groups present on the carrier,
Diphtheria toxoid ("DT"), via the terminal peptide amino acid
residue cysteine of the spacer utilizing a heterobifunctional
linking agent containing a succinimidyl ester at one end and
maleimide at the other end of the linking agent by either of Method
A or Method B as described below.
[0056] Method A: As previously described in U.S. Pat. No.
5,023,077, the linking of Peptide 1 or 4 above and the carrier is
accomplished as follows. Dry peptide was dissolved in 0.1 M Sodium
Phosphate Buffer, pH 8.0, with a thirty-fold molar excess of
dithiothreitol ("DTT"). The solution was stirred under a water
saturated nitrogen gas atmosphere for four hours. The peptide
containing reduced cysteine was separated from the other components
by chromatography over a G10 Sephadex column equilibrated with 0.2
M acetic acid. The peptide was lyophilized and stored under vacuum
until used. The carrier was activated by treatment with the
heterobifunctional linking agent e.g. Epsilon- maleimidocaproic
acid N-hydroxysuccinimide ester, ("EMCS"), in proportions
sufficient to achieve activation of approximately 25 free amino
groups per 105 molecular weight of carrier. In the specific
instance of diphtheria toxoid, this amounted to the addition of
6.18 mg of EMCS (purity 75%) to each 20 mg of diphtheria
toxoid.
[0057] Activation of diphtheria toxoid was accomplished by
dissolving each 20 mg aliquot of diphtheria toxoid in 1 ml of 0.2 M
Sodium Phosphate Buffer, pH 6.45. Aliquots of 6.18 mg EMCS were
dissolved into 0.2 ml of Dimethyl Formamide ("DMF"). Under darkened
conditions, the EMCS was added dropwise in 50 microliter (".mu.l")
amounts to the DT with stirring. After 2 hours of incubation in
darkness, the mixture was chromatographed on a G50 Sephadex column
equilibrated with 0.1 M Sodium Citrate buffet, pH 6.0, containing
0.1 mM EDTA.
[0058] Fractions containing the EMCS activated diphtheria toxoid
were concentrated over a PM 10 ultrafiltration membrane under
conditions of darkness. The protein content of the concentrate was
determined by either the Lowry or Bradford methods. The EMCS
content of the carrier was determined by incubation of the
activated carrier with cysteine-HCl followed by reaction with 10 mM
of Ellman's Reagent 5,5'dithio-bis (2-nitrobenzoic acid) 10 mM. The
optical density difference between a blank tube containing
cysteine-HCl and the sample tube containing cysteine-HCl and
carrier was translated into EMCS group content by using the molar
extinction coefficient of 13.6.times.10.sup.3 for
5-thio-2-nitrobenzoic acid at 412 nm.
[0059] The reduced cysteine content (--SH) of the peptide was also
determined utilizing Ellman's Reagent. Approximately 1 mg of
peptide was dissolved in 1 ml of nitrogen gas saturated water and a
0.1 ml aliquot of this solution was reacted with Ellman's Reagent.
Utilizing the molar extinction coefficient of 5-thio-2-nitrobenzoic
acid (13.6.times.10.sup.3, the free cysteine --SH was calculated.
An amount of peptide containing sufficient free --SH to react with
each of 25 EMCS activated amino groups on the carrier was dissolved
in 0.1 M Sodium Citrate Buffer, pH 6.0, containing 0.1 mM EDTA, and
added dropwise to the EMCS activated carrier under darkened
conditions. After all the peptide solution had been added to the
carrier, the mixture was incubated overnight in the dark under a
water-saturated nitrogen gas atmosphere.
[0060] The conjugate of the peptide linked to the carrier via EMCS
was separated from other components of the mixture by
chromatography over a G50 Sephadex column equilibrated with 0.2 M
Ammonium Bicarbonate. The conjugate eluted in the column void
volume was lyophilized and stored desiccated at 20.degree. C. until
used.
[0061] The resulting conjugate may be characterized as to peptide
content by a number of methods known to those skilled in the art
including weight gain, amino acid analysis, etc. Conjugates of
Peptides 1 and 4 with spacer and diphtheria toxoid produced by this
method were determined to have an effective peptide/carrier ratio
of 5-35 moles of peptide per 100 KD MW of carrier and all were
considered suitable as immunogens for immunization of test animals.
Preferably, the range of the peptide from 10-30 moles per 100 KD MW
of DT produced an effective immune response.
[0062] Method B: In a preferred method, conjugates comprising GRP1
coupled to DT and GRP4 peptide coupled to DT were prepared at room
temperature as follows. Purified DT (400 mg) was dissolved in 20 ml
of 0.5 M phosphate buffer, pH=6.6, saturated with nitrogen gas to
give a DT solution of 20 mg/ml. The DT solution was placed in a 60
ml dark amber glass bottle (serving as a reaction vessel and
filtration reservoir). EMCS coupling reagent (123.6 mg) was
dissolved in 2.0 ml of dimethylformamide. The EMCS solution was
added dropwise to the DT solution over a 15 minute period with
continuous stirring. The bottle was capped, and the mixture was
stirred at room temperature for an additional 1 hour 45 minutes, to
form activated DT (M-DT). The M-DT was then purified by
diafiltration using an Amicon Model TFC10 Thin-Channel
Ultrafiltration System per operating manual 1-113G with a XM50
diaflow ultrafiltration membrane. The M-DT was washed twice against
volumes of 420 ml phosphate buffer, concentrating to 20 ml each
time, then washed once against 420 ml of 0.1 M sodium citrate
buffer, pH=6.0, containing 0.1 M EDTA, and concentrating the
solution down to 20 ml.
[0063] To make GRP1-DT conjugate, 2.02 ml of M-DT solution
(containing 22.3 mg M-DT) was placed in a 10 ml dark amber glass
vial, then 13 mg of GRP1 peptide was dissolved in the citrate
buffer to give 40 mg/ml peptide and added dropwise to the M-DT
solution with stirring. To make GRP4-DT conjugate, 2.21 ml of M-DT
solution (containing 24.4 mg M-DT) was placed in a 10 ml dark amber
glass vial, then 13 mg of GRP4 peptide was dissolved in the citrate
buffer to give 40 mg/ml peptide and added dropwise to the M-DT
solution with stirring.
[0064] The reactions were allowed to proceed overnight in the dark.
Each conjugate was removed from the reaction vessels and separately
dialyzed in 12,000-14,000 MW cutoff dialysis tubing against 5
changes 500 ml of 0.1 M ammonium bicarbonate solution. Each
conjugate was lyophilized. The conjugates were then analyzed by
amino acid analysis and their peptide to DT substitution ratios
were determined to be 21.8 peptides per 10.sup.5 MW of DT for
GRP1-DT and 21.1 peptides per 10.sup.5 MW of DT for GRP4-DT.
[0065] Conjugates of Peptides 1 and 4 with spacer and DT produced
by this method have an effective peptide/carrier ratio of 5-35
moles of peptide per 100 KD MW of carrier and all are considered
suitable as immunogens. A preferred ratio range for producing an
effective immune respose is from 10-25 moles of peptide per 100 KD
MW of DT.
[0066] Preparation of Immunogens
[0067] The present immunogens containing either Peptide 1 or
Peptide 4 with spacer conjugated to DT as described above were used
to immunize rabbits. Immunogens were prepared as follows: Conjugate
was dissolved in 0.15 M Sodium phosphate buffered saline, pH 7.3 to
a concentration of 3.79 mg/ml. The conjugate solution was added to
Montanide ISA (703) Adjuvant (Seppic, Inc.) in a 30:70 (wt:wt)
ratio of conjugate solution to Montanide ISA 703, then the mixture
was homogenized using a Silverson Homogenizer for 3 minutes at
8,000 RPM to form an emulsion containing 1 mg/ml of conjugate.
[0068] Immunization and Sample Collection
[0069] Rabbits were injected intramuscularly with 0.1 ml of
immunogen consisting of 0.1 mg of either GRP1-DT, or GRP4-DT
conjugate. Each rabbit was given injections of immunogen at 0 and 4
weeks. Blood was collected from each rabbit at 6 and 8 weeks of the
experiment. Serum was prepared from each blood sample and stored at
-20.degree. C. until utilized in assays to determine the presence
of anti-CCK-B/gastrin-receptor antibodies.
[0070] Enzyme-Linked Immunosorbent Assay (ELISA)
[0071] A solid-phase ELISA was used to screen for reaction or
cross-reaction of antisera raised against Peptide 1 and Peptide 4
of each immunized rabbit. The ELISA was carried out by coating
polystyrene 96 well plates (IMMULON II, Dynatech) with 25
.mu.l/well of 10 .mu.g/ml of Peptide 1 linked to bovine serum
albumin (BSA) ("GRP1-BSA"), or Peptide 4 linked to BSA ("GRP4-BSA")
antigen in 0.1 M Glycine-HCl, pH 9.5 buffer. The plates were
incubated overnight at 4.degree. C., and subsequently washed in
buffer.
[0072] Antisera obtained from the immunized rabbits were serially
diluted to a range of 10.sup.-1 to 10.sup.-8 in 1% BSA-FTA
hemagglutination buffer, pH 7.2. Twenty five .mu.l of test
antiserum per well was incubated with each test peptide for 1 hr at
room temperature. After incubation, the plates were washed
thoroughly with buffer to remove any unbound antibody. Each well
was treated with 25 .mu.l of biotinylated goat anti-rabbit IgG
(H+L) diluted 1: 1000 in 1% BSA-FTA dilution buffer for 1 hour at
room temperature. After washing the plates to remove unbound
anti-rabbit reagent, each well was incubated for 1 hour at room
temperature with 25 .mu.l of avidin-alkaline phosphatase conjugate
diluted 1:1000 in 1% BSA-FTA buffer. The plates were washed
thoroughly to remove unbound avidin-alkaline phosphatase reagent,
and incubated with 25 .mu.l of 1 mg/ml of p-nitrophenylphosphate
("PNPP") in 10% diethanolamine buffer containing 0.01%
MgCl.sub.2.6H.sub.2O, pH 9.8. The plates were allowed to develop
until the absorbance of the reaction at 490 nm wavelength reached
an optical density between 0.8 to 1.5. To test the specificity of
the antisera produced by the rabbits, rabbits were also immunized
with DT and for ELISA testing, plates were coated with DT as
antigen to determine the reactivity of the antisera produced
against the carrier.
[0073] FIG. 2 shows the ELISA results using Peptide 1/GRP1 and FIG.
3 shows the ELISA results using Peptide 4/GRP4 as the antigen. As
seen in FIG. 2, the ELISA results show that the rabbits immunized
with Peptide 1-spacer-DT conjugate produced high antibody titers
which specifically bind to Peptide 1, as indicated by the antibody
binding Peptide 1 even at high (1: 100,000) dilutions of the
antiserum. Similarly, FIG. 3 shows that rabbits immunized with
Peptide 4-spacer-DT conjugate produced high titers of anti-Peptide
4 antibodies. As seen in FIGS. 2 and 3, the rabbits immunized
against each peptide produced antibodies which bound specifically
to each peptide at small antisera concentration. The data indicate
that the anti-Peptide 1 and anti-Peptide 4 antibodies have a large
capacity for binding Peptides 1 and 4 of the
CCK-B/gastrin-receptor. The data also shows that immunization of
rabbits with the present conjugates elicits powerful immune
responses against Peptide 1 and Peptide 4, respectively. In
addition, rabbits immunized with either Peptide-1 or Peptide-4
conjugate appeared and behaved normal and did not exhibit any
symptoms of disease or pathologies during the experiments.
EXAMPLE 2
[0074] The following experiments were performed to establish the
specificity of antibodies raised in rabbits against the GRP1-DT
peptide containing Ser spacer described Example 1 using Method B. A
series of tests were conducted to assess the specificity of rabbit
antibodies induced by immunization with the GRP1-DT and affinity
purified by immunoadsorption over a GRP1-Ser Sepharose column.
[0075] An inhibition ELISA was used to assess the specificity of
the affinity purified antibodies for GRP1-Ser peptide. The assays
were run as follows: GRP1-Ser-BSA conjugate was coated onto 96 well
plates (Immulon U bottom) by overnight incubation of 50 .mu.l of a
2 .mu.g/ml solution of conjugate in glycine buffer (0.1M, pH=9.5)
at 4.degree. C. Affinity purified anti-GRP1 Ab (at a final
concentration of 10 ng/ml) was combined with various inhibitors (in
1:10 dilution series) and incubated for 1 hour at room temperature.
The inhibitors included GRP1-Ser, GRP1 EPT, Ser, human gastrin
17(1-9)-Ser spacer (hG17(9)-Ser), GRP1 EPT+Ser, and buffer (no
inhibitor). Incubation buffer consisted of PBS+0.5% BSA+0.05% Tween
20+0.02% NaN.sub.3. Subsequent steps used the same buffer without
BSA. The 96 well plates were washed free of nonbound GRP1-Ser-BSA,
and the Ab+inhibitor mixtures were added (50 .mu.l/well). After 1
hour, the plates were washed and a goat anti-rabbit Ig (H+L)
alkaline phosphatase conjugate (Zymed) was added (1:2000 dilution).
After 1 hour incubation, the plates were washed to remove nonbound
reagent, and 50 .mu.l/well of pNPP substrate (Sigma) solution (1
mg/ml) was added in substrate buffer (PBS+0.1 mg/ml MgCl.sub.2+10%
diethanolamine+0.02% NaN.sub.3). Following a 60 minute incubation,
absorbance was measured on a MRX reader (Dynatech Laboratories).
Samples were run in duplicate, and means were calculated for each
concentration. Background binding (established with affinity
purified rabbit anti-GnRH antibodies) was subtracted from all
values, and the % Inhibition relative to no inhibitor added
(anti-GRP1 Ab+buffer) was calculated for each inhibitor tested: %
Inhibition=(100)(1-((A.sub.uninhi-
bited-A.sub.inhibited)/A.sub.uninhibited), where A=Absorbance. The
results are shown in FIG. 4.
[0076] FIG. 4 presents the percent inhibition of antibody binding
as a function of inhibitor concentration. As can be seen in the
figure, the GRP1-Ser peptide fully inhibited antibody binding to
GRP1-Ser-BSA. Approximately 60% inhibition was attained with the
GRP1 EPT peptide, which does not contain the Ser spacer sequence,
and by an equimolar mixture of GRP1 EPT plus Ser spacer. The
failure of these peptides to produce full inhibition suggests that
a proportion of the antibodies were specific for an epitope(s)
comprising elements of both the GRP1 and the Ser spacer sequences.
No inhibition was obtained by either the Ser spacer sequence itself
or by an unrelated peptide bearing the Ser spacer ("hG17(9)-Ser",
consisting of the amino-terminal nine residues of hG17followed by
the Ser spacer). These ELISA results demonstrate that the affinity
purified antibody preparation was specific for the GRP1-Ser
peptide, and that 60% of the binding activity was directed against
the gastrin-receptor epitope component of the peptide.
EXAMPLE 3
[0077] AR42J tumor cells (European Collection of Animal Cell
cultures, Porton Down, UK) are derived from a rat pancreatic
adenocarcinoma and are known to have well characterized
CCK-B/gastrin-receptors. Thus AR42J were tested to confirm the
expression of CCK-B/gastrin-receptor and specificity of the
receptor for hG17 by radioligand inhibition. AR42J cells were
cultured at 37.degree. C. with 7% CO.sub.2 in complete RPMI 1640
(Sigma) supplemented with 10% FCS (Gemini Bioproducts), 2 mM
glutamine (JRH Biosciences), 1 mM sodium pyruvate (JRH B.) and 50
.mu.g/ml gentamicin (Gemini Bioproducts). The cells were harvested
from 175 cm.sup.2 T-flasks (Falcon Plastics) with PBS containing
0.25% EDTA, then washed twice with PBS (no EDTA) by centrifugation
(400 X g for 10 min). The cells were kept at 0-4.degree. C. for all
manipulations. A single cell suspension was prepared in buffer, and
the cell concentration was adjusted to 10.sup.6 cells/ml. Aliquots
of 1 ml of cell suspension were added to 12.times.75 mm culture
tubes, then the cells were centrifuged and the supernatants
discarded. The cells were resuspended in PBS (0.1 ml/tube)
containing human G17 (hG17), gonadotropin releasing hormone (GnRH),
or no peptide. The peptide concentrations were 1.0 ng/ml, 100 ng/ml
and 10 .mu.g/ml. An aliquot of 0. 1 ml of .sup.125I-hG17 (NEN),
containing approximately 26,300 CPM (specific activity, 2200
Ci/mmol), was added to each tube. The tubes were vortexed, then
incubated for 15 minutes. The cells were washed twice with PBS,
then counted in a y counter (Wallac). Samples were run in
duplicate. Background counts were subtracted, then the % inhibition
of .sup.125I-hG17 binding by each inhibitor was calculated using
the equation: % Inhibition=(100)(1-((CPM.s-
ub.uninhibited-CPM.sub.inhibited)/CPM.sub.inhibited).
[0078] The results of the radioligand binding inhibition tests are
shown in FIG. 5, which presents the means (.+-.SE) of the
individual values. As can be seen in the figure, binding of
.sup.125I-hG17 to AR42J cells was inhibited by hG17. The degree of
inhibition increased with the quantity of inhibitor added, to 32%
inhibition at 1 .mu.g hG17 per tube, the highest concentration of
peptide tested. Conversely, GnRH produced no inhibition at the two
highest concentrations tested (the 6% inhibition obtained with 100
pg GnRH was considered to be nonspecific), indicating that the
inhibition by hG17 was specific for gastrin. These results
confirmed the cell surface expression of gastrin-receptor by the
AR42J tumor cells.
EXAMPLE 4
[0079] Binding of the GRP1-Ser specific antibodies to AR42J cells
was assessed by immunofluorescence. AR42J cells were grown as in
the previous Examples and harvested with cell scrapers from 175
cm.sup.2 T-flasks and washed twice with buffer (PBS with 0.02%
NaN.sub.3) by centrifugation (400 X g for 7 min). The cells were
kept at 0-4.degree. C. for all manipulations. A single cell
suspension was prepared in buffer, and the cell concentration was
adjusted to 10.sup.6 cells/ml. The cell suspension was added to 1.5
ml microfuge tubes (1 ml/tube). The cells were pelleted by
centrifugation and supernatants were aspirated. The cells were
resuspended in buffer (0.1 ml/tube) containing peptide inhibitors
(1.0 mg/ml). The inhibitors included GRP1-Ser, GnRH, hG17(9)-Ser
and buffer (no inhibitor). Antibodies, including the rabbit
anti-GRP1-Ser (100 .mu.g/ml), affinity purified rabbit anti-DT
(negative control, 100 .mu.g/ml), mouse anti-AR42J antiserum
(positive control, 1:100 dilution, heat inactivated) or normal
mouse serum were added to the appropriate tubes and the contents
were mixed. The cells were incubated for 1 hour, with occasional
mixing. The cells were then washed three times with buffer, and 0.1
ml of fluorescein-labeled goat anti-rabbit IgG (Antibodies
Incorporated) (diluted 1:50) was added per tube. The cells treated
with mouse sera were developed with a fluorescein-anti-mouse IgG
reagent (Zymed). The cells were re-suspended by vortexing, then
incubated for 1 hour. The cells were again washed three times, then
re-suspended in glycerol: PBS (1:1, v:v), 50 .mu.l/tube. Wet mounts
were prepared with the contents of each tube, and the cells
examined using a Laborlux 12 fluorescent microscope (Leitz).
Fluorescence was scored on a scale of 0 to 4, with 0 representing
background fluorescence (obtained with the normal mouse serum) and
4 representing maximal fluorescence (obtained with the mouse
anti-AR42J positive control antiserum).
[0080] The results of the immunofluorescesce tests are presented in
Table 1. As can be seen in the Table, AR42J cells treated with
anti-GRP1-Ser antibodies in the absence of peptide inhibitors
fluoresced strongly, indicating that the antibody bound to the
cells. Rabbit anti-DT antibodies did not produce fluorescent
staining, demonstrating that the staining observed with the
anti-GRP1-Ser antibodies was not a consequence of non-specific cell
surface binding by rabbit immunoglobulin. Moreover, the binding was
shown to be specific for the GRP1-Ser peptide. Addition of GRP1-Ser
fully inhibited binding, whereas unrelated peptides, including
hG17(9)-Ser and GnRH, failed to inhibit. As the GRP1 epitope
comprises residues 5-21 of the gastrin-receptor, it was concluded
that the anti-GRP1-Ser antibodies were specific for the
gastrin-receptor expressed by AR42J cells.
1TABLE 1 Antibody Inhibitor Preparation GRP1-Ser hG17(9)-Ser GnRH
Buffer Rabbit anti-GRP1-Ser 0 3+ 2+ 3+ Rabbit anti-DT 0.5+ 0.5+
0.5+ 0.5+ Mouse anti-AR42J 4+ Normal Mouse Serum 0
EXAMPLE 5
[0081] AR42J cells, passage nos. 16-18 were cultured in RPMI-1640
medium containing 10% FCS and 2 mM glutamine. All cells were
maintained at 37.degree. C. in 5% CO.sub.2 in air at 100% humidity,
grown to 80% conflucency in T75 flasks (Falcon, London, UK) and
passage following a 0.02% EDTA treatment to bring adherent cells
into suspension. Cells were incubated for 10, 30 seconds, 30
minutes and 1 hour with anti-CCK-B/gastrin-receptor antibody (aGR)
generated in rabbits with a CCK-B/gastrin Peptide 1 receptor
immunogen of the invention as described in Example 1, which had
been purified by affinity chromatography in a column prepared with
Peptide 1.
[0082] The cells were fixed in 1% glutaraldehyde for one hour and
prepared for immunoelectron microscopy (ImmunoEM) studies using
standard techniques. The cell suspensions was centrifuged twice at
2000 rpm for 2 minutes and then the cell pellet resuspended in
phosphate buffered saline (PBS). The cell pellet was infiltrated
with LRwhite plastic resin. Ultrathin sections of 70-90 nm in
thickness were cut and place on Pioloform coated nickel grids. The
grids were placed in normal goat serum (Dako, High Wycombe, UK) in
0.1% bovine serum albumin (BSA) (Sigma, Poole, Dorset) and
incubated at room temperature for 30 minutes. Grids were rinsed in
PBS then incubated with a secondary antibody, biotin-conjugated
goat anti-rabbit antibody (gold-labelled), diluted 1:50 in 1% BSA,
for 1 hour at room temperature. Control experiments were performed
without secondary antibody. After final PBS wash, the grids were
counterstained in saturated aqueous uranyl acetate for 3 minutes
and Reynold's lead citrate for 3 minutes. Gold particles on the
cell membrane, in the cytoplasm, on the nuclear membrane and within
the nucleus were counted. Twenty-five cells/grid were counted by an
independent observer. For controls AR42J cells were exposed to
antibodies for less than 1 second, and liver cells which are devoid
of CCK-B/gastrin-receptor were used. AR42J cells exposed to normal
IgG were also used as controls for determining non-specific binding
of the anti-CCK-B/gastrin-receptor antibodies. The results of these
experiments are shown in Table 2 and FIG. 6.
2TABLE 2 Distribution of CCK-B/gastrin-receptor Immunogold
Particles Within AR42J cells Cell Cell Nuclear Nuclear membrane
matrix membrane matrix No. gold 14.2(.+-.0.97) 43.3(.+-.2.32)
9.3(.+-.0.81) 51.4(.+-.3.32) particles Percent 12% 36.6% 7.9% 43.5%
distribution within cell (mean .+-. SEM for 25 cells, repeated n =
5.)
[0083] As demonstrated in Table 2 and FIG. 6, immunogold-antibody
particles attached to the CCK-B/gastrin-receptor were localized on
plasma membrane, cytoplasm, nuclear membrane, and nuclear matrix of
the adenocarcinoma cells, further demonstrating that the
antibody/receptor complex is internalized by the cells.
[0084] As seen in Table 2, the immunoEM studies using an antiserum
directed against the amino-terminal end of the
CCK-B/gastrin-receptor shows that after one hour incubation, the
distribution of immunogold-labelled CCK-B/gastrin-receptor antibody
is quickly internalized as 12% of the antibody receptor complex is
associated with the cell membrane, 36.6% is within the cytoplasm,
7.9% is in the nuclear membrane and, quite surprisingly, 43.5% is
within the cell nucleus. Areas of intense CCK- B/gastrin-receptor
immunoreactivity within the nucleus are found on chromatin, which
may suggest specific binding sites for regulation of the DNA.
[0085] These electron microscopy studies with anti-immunoglobulin
conjugated to gold beads (immmunogold) reveal that an extremely
rapid turnover of the anti-receptor/receptor complex occurs in the
tumor cells; as early as 10 seconds after exposure to antibodies,
complexes are detectable in the cell nucleus as seen in FIG. 6.
EXAMPLE 6
[0086] Adenocarcinoma cell lines, namely AR42J, HCT116, C170HM2,
LoVo, ST16 and MGLVA1, were grown in vitro and harvested as
described in Examples 3. Cells from 30.times.T-75 flasks were
suspended in 5 ml of homogenization buffer (1 mM sodium hydrogen
carbonate, 2 mM magnesium chloride, 1 nM phenylmethylsulphonyl
fluoride, 40 mM sodium chloride, 10 .mu.l leupeptin, 1 .mu.M
pepstatin, 5 nM EDTA [Sigma]). Homogenization was carried out by 5
bursts of 5 second duration in a homogenizer. For extranuclear
membranes, tissue debris was pelleted by centrifugation at 500g, 7
minutes, 4.degree. C. The pellet was discarded and the supernatant
centrifuged at 500g, 4.degree. C. to remove further debris. The
supernatant was recentrifuged at 48,000g, 1 hour, 4.degree. C. The
pellet containing the extranuclear membrane preparation was
suspended in Tris/NP-40 solution (0.1 M TRIZMA, 0.5% NONIDET P40
[Sigma Chemical]).
[0087] For nuclear membrane preparations, following homogenization
in a second homogenization buffer (25 mM Tris-HCl, pH 7.4, 0.1%
TRITON 100, 0.32 M sucrose, 3 mM MgCl.sub.2, 2 mM EGTA, 0.1 mM
spermine tetrahydrochloride, 2 mM PMSF, 10 mM bezomidine
hydrochloride, 3 mM EGTA aminoacetonitrile hydrochloride [Sigma]),
tissue debris was pelleted by centrifugation at 400 g, for 10
minutes at 4.degree. C. The pellet was resuspended in 55% (0.2 M)
sucrose in HPLC water. This mixture was spun at 60,000 g for 1 hour
at 4.degree. C. The pellet was washed with 0.4% NONIDET P40 in
homogenization buffer without TRITON 100. The pellet was spun at
700 g for 15 min at 4.degree. C. and resuspended in homogenization
buffer without TRITON 100.
[0088] Protein content is determined by the Lowry method (using a
kit from Pierce). Samples containing 10-15 .mu.g of protein were
loaded onto a 8-16% Tris/glycine gradient polyacrylamide gel
electrophoresis PAGE (Novex R and D systems) in Tris/glycine buffer
and run for 90 minutes at 125 constant volts, 36 mA. The gel was
fixed in 10% glacial acetic acid for 1 hour and samples were
blotted onto nitrocellulose membrane. The membranes were incubated
in 1% BSA for 1 hour, followed by incubation with GRP-1 antiserum
(with and without preabsorption) for 1 hour. Antibody binding were
detected by the avidin:biotin-peroxidase complex method using
diamino-bezidene as the substrate. The Western blot analysis
results using Rabbit-antiserum raised against Peptide 1 (Rabbit
anti-GRP1 antiserum) are shown in FIG. 7 and FIG. 8.
[0089] As shown in FIG. 7, the protein molecular weight markers
range from 116, 66, 45 and 29 kDa. The blot shows a prominent
anti-Peptide 1 immunoreactive band localizing at about 43 kDa in
all adenocarcinoma cells studied, i.e., HCT116, C170HM2, LoVo, ST16
and MGLVA1, except one (AP5LV). This protein corresponds to a
truncated form of the CCK-B/gastrin-receptor. Some cell lines (HCT
116 and C170HM2) show at least 3 other bands, ranging in molecular
weight between 60 and 100 KDa. The data indicate that the
anti-CCK-B/gastrin-receptor antibodies can recognize and bind to
various isoforms of the CCK-B/gastrin-receptor in tumor cells.
[0090] FIG. 8 shows a Western blot from extranuclear (ENM) and
plasma membrane of C170HM2 and HCT 116 adenocarcinoma cells. As
shown in FIG. 8, adenocarcinoma cell lines tested for ENM
CCK-B/gastrin-receptors demonstrate the existence of two strongly
stained bands: one about 43 KDa and the other at about 66 KDa. When
only the plasma membrane fraction was stained, a single band at
about 66 KDa was present. Thus, the Western blot studies confirm
the immunoEM results that the CCK-B/gastrin-receptor is present in
adenocarcinoma tumor cells, although the immunoEM studies do not
distinguish between the isoforms of the CCK-B/gastrin-receptor. The
data indicate that the present immunogens elicit
anti-CCK-B/gastrin-receptor antibodies which can recognize and bind
various isoforms of the receptor, which would be advantageous for
the treatment of these tumors.
EXAMPLE 7
[0091] C170HM2 adenocarcinoma cells were injected intraperitoneally
into nude mice and tumors were allowed to grow in the liver.
Control mice received an infusion of phosphate buffer saline
solution (PBS) and experimental mice received an infusion of one
anti-CCK-B/gastrin-receptor antibodies. In Group 1, each mouse was
infused daily with 0.5 mg of Rabbit anti-CCK-B/gastrin-receptor
antibodies generated against Peptide 1 (Rabbit anti-Peptide 1,
Rbt@GRP1). In Group 2, each mouse received daily 0.5 mg of Rabbit
anti-CCK-B/gastrin-receptor antibodies generated against Peptide 4
(Rabbit anti-Peptide 4, Rbt@GRP4). The mice were studied for a
period of 40 days after antibody infusion, sacrificed and the
tumors removed for study. The weight, size and cross-sectional area
of the tumors were assessed by standard techniques. The results are
shown in FIGS. 9 and 10.
[0092] As seen in FIGS. 9 and 10, implantation of the colorectal
adenocarcinoma cancer cell line C170HM2 in mice without treatment
results in the rapid growth of large tumor masses, as determined by
tumor weight, or tumor size, and the tumor cross-sectional area of
the tumors. However, infusion of the animals with Rabbit
anti-Peptide 1 or Rabbit anti-Peptide 4 antibodies results in a
marked decrease in the number of animals having any detectable
tumor, as well as in the weight and size of tumors in animals
having them when compared to control. The same effect can be seen
when mean tumor weight, mean tumor size, or mean tumor number is
calculated. These data are shown in FIGS. 11, 12 and 13.
[0093] Further insight into the distribution within the population
is gained by calculating the medians of tumor numbers, weight and
size. The results are shown in FIGS. 14, 15 and 16. As seen in
these figures, the Rabbit anti-Peptide 1 immunogen was consistently
more effective than Rabbit anti-Peptide 4 in inhibiting tumor
growth. However, both Rabbit anti-Peptide 1 and Rabbit anti-Peptide
4 antibodies did exhibit powerful tumor inhibitory activity as
compared to the control treatment.
EXAMPLE 8
[0094] A larger tumor burden was generated in nude mice using the
colon cancer cell line C170HM2 by a method as described in Example
7, but with a higher initial cell innoculum. The C170HM2 is a
liver-invasive xenograft model. Control and experimental mice were
treated also as described in Example 7.
[0095] Forty days after antibody infusion, the mice were sacrificed
and liver tumors were removed and studied. FIGS. 17, 18 and 19 show
the results of these experiments. FIG. 17 shows the mean and median
liver tumor numbers of control and anti-CCK-B/gastrin-receptor
antibody treated animals. The data show that the
Rabbit-anti-CCK-B/gastrin-receptor antibodies ("Rabbit@GRP") are
effective in inhibiting the growth of the metastatic tumors in the
liver. There is a statistically significant decrease in mean liver
tumor numbers in mice livers using Rabbit anti- Peptide 1
(Student's T test), p=0.0084 and in the median liver tumor number,
p=0.0016 (Mann Whitney) when compared to controls. Mice treated
with anti-Peptide 4 antibodies also show a decrease in mean liver
tumor number; however, there was no difference in the mean liver
tumor number in this animals when compared to controls.
[0096] FIG. 18 shows that anti-Peptide 1 and anti-Peptide 4
antibodies were also capable of reducing the mean and median tumor
weights of liver metastases when compared to control animals. The
data in FIG. 19 show that anti-CCK-B/gastrin-receptor treated mice
also had a significant decrease in mean and median cross-sectional
area of the liver tumors when compared to control animals.
[0097] The data indicate that the anti-CCK-B/gastrin-receptor
antibodies are effective in controlling the spread and growth of a
gastrin-dependent colon cancer in the liver, which constitutes the
major site of metastatic spread of this cancer.
EXAMPLE 9
[0098] These studies we carried out to confirm GRP1
immunoreactivity on C170HM2 cells. The aim of the study was to
evaluate tumor localization of antiserum raised against GRP1 and to
determine its therapeutic effect on the growth of C170HM2 cells
within the liver of nude mice. C170HM2 cells were injected
intraperitoneally into nude mice as described in Examples 7 above.
GRP1 antiserum was raised in rabbits. The antiserum was
radiolabelled with .sup.125I and administered to nude mice with
established C170HM2 xenografts by a tail vein injection. Control
mice received .sup.125I radiolabelled normal rabbit serum. Mice
were terminated at increasing time points following injection of a
single dose of .sup.125I antibodies. Radioactivity was measured as
counts per minute per gram of (CPM/g) tissue and the liver/liver
tumor ratio calculated.
[0099] FIG. 20 is a graph which shows the radiolabeled rabbit
anti-GRP1 antibodies bound to liver tumors versus control. As seen
in the figure, more rabbit anti-GRP1 antibodies are bound to liver
tumor tissue when compared to controls. FIG. 20 also shows the
liver tumor/liver ratio on the y axis with increasing time on the x
axis for both radiolabeled normal rabbit serum and GRP1 antiserum.
The normal rabbit serum achieved a ratio of 1 from day 1 which
remained constant until day 5. This indicates the level of
radiolabel in the liver tumour and normal liver was equal. The
ratio for GRP1 antiserum accumulated exponentially approaching 2 by
day 5. This indicates radiolabeled GRP1 antiserum specifically
localizes within C170HM2 liver tumors.
EXAMPLE 10
[0100] Therapeutic Effect of GRP1 Antiserum on C170HM2
xenografts
[0101] The C170HM2 tumor xenografts were initiated by
intraperitoneal injections of cells. Three different cell inocula
were used to generate 3 levels of tumor burden. The GRP1 antiserum
was administered passively by tail vein injection daily from day 0.
Therapy was terminated on day 40.
[0102] Effect of GRP1 Antiserum on Tumor `Take Rate`
[0103] The initial parameter evaluated was mean tumor number within
the liver which is shown in FIG. 21. The normal rabbit antiserum
treated controls are grouped in increasing cell inocula. As seen in
FIG. 21, in the control groups the mean tumor number per liver was
between 1 and 3. In the GRP1 antiserum treated group the mean tumor
number per liver was less than 1 for all three cell inocula, which
was significant for all 3 experiments (one inoculum, n=18, p=0.003;
2 inocula, n=12, p=0.0001 and 3 inocula, n=20, p=0.0068, Mann
Whitney analysis).
[0104] Effect of GRP 1 Antiserum on Tumor Weight of Established
Tumors
[0105] FIG. 22 shows the mean tumor weight for the normal rabbit
serum treated controls on the left panel for the 3 increasing cell
innocula. The figure also shows the mean tumor weight of nude mice
following treatment with GRP1 antiserum the mean liver weight was
reduced by 60% with all 3 cell innocula, which was significant for
all 3 experiments (one inoculum, p=0.0016; 2 innocula, p=0.0084,
and 3 innocula, p=0.0001, Mann Whitney analysis).
[0106] GRP1 Immunoreactivity in C170HM2 xenografts as Determined by
Western Blotting
[0107] Extra-nuclear membrane proteins were prepared from C 170HM2
xenografts from 2/3 experiments. These were analyzed by Western
blotting using the GRP1 antiserum. FIG. 23 is a photograph of the
Western blot showing in the normal rabbit serum-treated xenografts
2 immunoreactive bands were present at 74 and 50 kDa, with the
former band showing the strongest immunoreactivity. In the GRP1
antiserum treated xenografts, there are 2 immuno-reactive bands
together with an intermediate band, not seen in the control
xenografts or cells grown in vitro. A 50 kDa band shows the
strongest immunoreactivity. This indicates that in the GRP1
antiserum treated xenografts a larger proportion of the
CCKB/gastrin-receptors may be present as an internalized form.
[0108] Histological Analysis of C170HM2 xenografts
[0109] FIG. 24 shows a microscopic view of a C170HM2 xenograft
invading a liver of a nude mouse. The tumor is generally composed
of a necrotic center with a viable leading edge which squashes the
hepatocytes as it invades the liver. The degree of apoptosis was
measured in the viable leading edge of C170HM2 tumors by the Tunel
method with positive cells visualized by in situ hybridization.
FIG. 25 shows that apoptotic cells were present in the viable tumor
cells in the GRP1 antiserum-treated xenografts, but not in the
normal rabbit serum-treated tumors.
[0110] The data show that antiserum raised against the amino
terminal epitope of the CCKB/gastrin-receptor selectively localizes
within liver-invasive C170HM2 tumors. Neutralization of the GRP1
epitope induced a significant effect on both tumor `take rate` and
gross tumor burden of tumors that did establish. This
tumor-inhibitory effect may be due to (a) a general cytostatic
effect induced by blocking the CCKB/gastrin-receptor and/or (b) an
indirect effect of targeting an antibody to the nucleus of the
cell, possibly resulting in apoptosis.
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Sequence CWU 1
1
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