Sommaire du brevet 2874032 - Base de données sur les brevets canadiens (2024)

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COMBINATION THERAPY INVOLVING ANTIBODIES AGAINST CLAUDIN 18.2
FOR TREATMENT OF CANCER
Cancers of the stomach and the esophagus (gastroesophageal; GE) are among the
malignancies
with the highest unmet medical need. Gastric cancer is the second leading
cause of cancer death
worldwide. The incidence of esophageal cancer has increased in recent decades,
coinciding with
a shift in histological type and primary tumor location. Adenocarcinoma of the
esophagus is now
more prevalent than squamous cell carcinoma in the United States and Western
Europe, with
most tumors located in the distal esophagus. The overall five-year survival
rate for GE cancer is
20-25%, despite the aggressiveness of established standard treatment
associated with substantial
side effects.
The majority of patients presents with locally advanced or metastatic disease
and have to be
subjected to first-line chemotherapy. Treatment regimens are based on a
backbone of platinum
and fluoropyrimidine derivatives mostly combined with a third compound (e.g.
taxane or
anthracyclines). Still, median progression free survival of 5 to 7 months and
median overall
survival of 9 to 11 months are the best that can be expected.
The lack of a major benefit from the various newer generation combination
chemotherapy
regimens for these cancers has stimulated research into the use of targeted
agents. Recently, for
Her2/neu-positive gastroesophageal cancers Trastuzumab has been approved.
However, as only
¨20% of patients express the target and are eligible for this treatment, the
medical need is still
high.
The tight junction molecule Claudin 18 splice variant 2 (Claudin 18.2
(CLDN18.2)) is a member
of the claudin family of tight junction proteins. CLDN18.2 is a 27.8 kDa
transmembrane protein
comprising four membrane spanning domains with two small extracellular loops.
In normal tissues there is no detectable expression of CLDN18.2 by RT-PCR with
exception of
stomach. Immunohistochernistry with CLDN18.2 specific antibodies reveals
stomach as the only
positive tissue.
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CLDN18.2 is a highly selective gastric lineage antigen expressed exclusively
on short-lived
differentiated gastric epithelial cells. CLDN18.2 is maintained in the course
of malignant
transformation and thus frequently displayed on the surface of human gastric
cancer cells.
Moreover, this pan-tumoral antigen is ectopically activated at significant
levels in esophageal,
pancreatic and lung adenocarcinomas. The CLDN18.2 protein is also localized in
lymph node
metastases of gastric cancer adenocarcinomas and in distant metastases
especially into the ovary
(so-called Krukenberg tumors).
The chimeric IgG1 antibody IMAB362 which is directed against CLDN18.2 has been
developed
by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular
domain (ECD1)
of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to any
other claudin
family member including the closely related splice variant 1 of Claudin 18
(CLDN18.1).
IMAB362 shows precise tumor cell specificity and bundles four independent
highly potent
mechanisms of action. Upon target binding IMAB362 mediates cell killing by
ADCC, CDC and
induction of apoptosis induced by cross linking of the target at the tumor
cell surface and direct
inhibition of proliferation. Thus, IMAB362 lyses efficiently CLDN18.2-positive
cells, including
human gastric cancer cell lines in vitro and in vivo. Mice bearing CLDN18.2-
positive cancer cell
lines have a survival benefit and up to 40% of mice show regression of their
tumor when treated
with IMAB362.
The toxicity and PK/TK profile of IMAB362 has been thoroughly examined in mice
and
cynomolgus monkeys including dose range finding studies, 28-day repeated dose
toxicity studies
in cynomolgus and a 3-month repeated dose toxicity study in mice. In both mice
(longest
treatment duration weekly administration for 3 months, highest dose levels 400
mg/kg) and
cynomolgus monkeys (up to 5 weekly applications of up to 100 mg/kg) repeated
doses of
IMAB362 i.v. are well tolerated. No signs of systemic or local toxicity are
induced. Specifically,
no gastric toxicity has been observed in any toxicity study. IMAB362 does not
induce immune
activation and cytokine release. No adverse effects on male or female
reproductive organs were
recorded. IMAB362 does not bind to tissues lacking the target. Biodistribution
studies in mice
indicate that the reason for lack of gastric toxicity is most likely
compartimentalization of tight
junctions at the luminal site in healthy gastric epithelia, which appears to
impair accessibility of
the IMAB362 epitope profoundly. This compartimentalization is lost upon
malignant
transformation rendering the epitope drugable by IMAB362.
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IMAB362 is in early clinical testing. A phase I clinical study has been
conducted in human. 5
dose cohorts (33 mg/m2, 100 mg/m2, 300 mg/m2, 600 mg/m2, 1000 mg/m2) of 3
patients each
have received a single intravenous administration of IMAB362 and have been
observed for 28
days. IMAB362 was very well tolerated, with no relevant safety observation in
the patients. In
one patient all measured tumor markers decreased significantly within 4 weeks
after treatment.
In an ongoing phase Ha clinical study IMAB362 is given repetitively.
Here we present data demonstrating that chemotherapeutic agents can stabilize
or increase
expression of CLDN18.2 on the surface of cancer cells resutling in an enhanced
drugability of
CLDN18.2 by an anti-CLDN18.2 antibody such as IMAB362. A synergistic effect of
an anti-
CLDN18.2 antibody such as IMAB362 with particular chemotherapeutic regimens,
in particular
chemotherapeutic regimens used for gastric cancer treatment or treatment of
human solid cancers
was observed. Human cancer cells pre-treated with chemotherapy are more
susceptible to
antibody-induced target-specific killing. In mouse tumor models, tumor control
with an anti-
CLDN18.2 antibody plus chemotherapy is superior to that with an anti-CLDN18.2
antibody as
single agent.
Furthermore, the data presented herein indicate that bisphosphonates such as
zoledronic acid
(ZA), in particular when administered in conjunction with recombinant
interleukin-2 (IL-2),
further augment the activity of an anti-CLDN18.2 antibody such as IMAB362. The
underlying
mechanism is activation and expansion of a highly cytotoxic immune cell
population (y982 T
cells).
SUMMARY OF THE INVENTION
The present invention generally provides a combination therapy for effectively
treating and/or
preventing diseases associated with cells expressing CLDN18.2, including
cancer diseases such
as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as
non small cell lung
cancer (NSCLC), ovarian cancer, colon cancer, hepatic cancer, head-neck
cancer, and cancer of
the gallbladder and metastases thereof, in particular gastric cancer
metastasis such as Krukenberg
tumors, peritoneal metastasis and lymph node metastasis. Particularly
preferred cancer diseases
are adenocarcinomas of the stomach, the esophagus, the pancreatic duct, the
bile ducts, the lung
and the ovary.
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In one aspect, the present invention provides a method of treating or
preventing a cancer disease
comprising administering to a patient an antibody having the ability of
binding to CLDN18.2 in
combination with an agent stabilizing or increasing expression of CLDN18.2.
Expression of
CLDN18.2 is preferably at the cell surface of a cancer cell. The agent
stabilizing or increasing
expression of CLDN18.2 may be administered prior to, simultanously with or
following
administration of the antibody having the ability of binding to CLDN18.2, or a
combination
thereof.
The agent stabilizing or increasing expression of CLDN18.2 may be a cytotoxic
and/or cytostatic
agent. In one embodiment, the agent stabilizing or increasing expression of
CLDN18.2
comprises an agent which induces a cell cycle arrest or an accumulation of
cells in one or more
phases of the cell cycle, preferably in one or more phases of the cell cycle
other than the GI -
phase. The agent stabilizing or increasing expression of CLDN18.2 may comprise
an agent
selected from the group consisting of anthracyclines, platinum compounds,
nucleoside analogs,
taxanes, and camptothecin analogs, or prodrugs thereof, and combinations
thereof. The agent
stabilizing or increasing expression of CLDN18.2 may comprise an agent
selected from the
group consisting of epirubicin, oxaliplatin, cisplatin, 5-fluorouracil or
prodrugs thereof such as
capecitabine, docetaxel, irinotecan, and combinations thereof The agent
stabilizing or increasing
expression of CLDN18.2 may comprise a combination of oxaliplatin and 5-
fluorouracil or
prodrugs thereof, a combination of cisplatin and 5-fluorouracil or prodrugs
thereof, a
combination of at least one anthracycline and oxaliplatin, a combination of at
least one
anthracycline and cisplatin, a combination of at least one anthracycline and 5-
fluorouracil or
prodrugs thereof, a combination of at least one taxane and oxaliplatin, a
combination of at least
one taxane and cisplatin, a combination of at least one taxane and 5-
fluorouracil or prodrugs
thereof, or a combination of at least one camptothecin analog and 5-
fluorouracil or prodrugs
thereof. The agent stabilizing or increasing expression of CLDN18.2 may be an
agent inducing
immunogenic cell death. The agent inducing immunogenic cell death may comprise
an agent
selected from the group consisting of anthracyclines, oxaliplatin and
combinations thereof. The
agent stabilizing or increasing expression of CLDN18.2 may comprise a
combination of
epirubicin and oxaliplatin. In one embodiment, the method of the invention
comprises
administering at least one anthracycline, at least one platinum compound and
at least one of 5-
fluorouracil and prodrugs thereof. The anthracycline may be selected from the
group consisting
of epirubicin, doxorubicin, daunorubicin, idarubicin and valrubicin.
Preferably, the anthracycline
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is epirubicin. The platinum compound may selected from the group consisting of
oxaliplatin and
cisplatin. The nucleoside analog may be selected from the group consisting of
5-fluorouracil and
prodrugs thereof The taxane may be selected from the group consisting of
docetaxel and
pacl*taxel. The camptothecin analog may be selected from the group consisting
of irinotecan and
topotecan. In one embodiment, the method of the invention comprises
administering (i)
epirubicin, oxaliplatin and 5-fluorouracil, (ii) epirubicin, oxaliplatin and
capecitabine, (iii)
epirubicin, cisplatin and 5-fluorouracil, (iv) epirubicin, cisplatin and
capecitabine, or (v) folinic
acid, oxaliplatin and 5-fluorouracil.
In one embodiment, the method of the invention further comprises administering
an agent
stimulating y6 T cells. In one embodiment, the y6 T cells are Vy9V62 T cells.
In one
embodiment, the agent stimulating y6 T cells is a bisphosphonate such as a
nitrogen-containing
bisphosphonate (aminobisphosphonate). In one embodiment, the agent stimulating
y6 T cells is
selected from the group consisting of zoledronic acid, clodronic acid,
ibandronic acid,
pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic
acid, incadronic
acid and salts thereof In one embodiment, the agent stimulating y6 T cells is
administered in
combination with interleukin-2.
The method of the invention may further comprise administering at least one
further
chemotherapeutic agent which may be a cytotoxic agent.
The antibody having the ability of binding to CLDN18.2 may bind to native
epitopes of
CLDN18.2 present on the surface of living cells. In one embodiment, the
antibody having the
ability of binding to CLDN18.2 binds to the first extracellular loop of
CLDN18.2. In one
embodiment, the antibody having the ability of binding to CLDN18.2 mediates
cell killing by
one or more of complement dependent cytotoxicity (CDC) mediated lysis,
antibody dependent
cellular cytotoxicity (ADCC) mediated lysis, induction of apoptosis and
inhibition of
proliferation. In one embodiment, the antibody having the ability of binding
to CLDN18.2 is a
monoclonal, chimeric or humanized antibody, or a fragment of an antibody. In
one embodiment,
the antibody having the ability of binding to CLDN18.2 is an antibody selected
from the group
consisting of (i) an antibody produced by and/or obtainable from a clone
deposited under the
accession no. DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM
ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM
ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) an

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antibody which is a chimerized or humanized form of the antibody under (i),
(iii) an antibody
having the specificity of the antibody under (i) and (iv) an antibody
comprising the antigen
binding portion or antigen binding site, in particular the variable region, of
the antibody under (i)
and preferably having the specificity of the antibody under (i). In one
embodiment, the antibody
is coupled to a therapeutic agent such as a toxin, a radioisotope, a drug or a
cytotoxic agent.
In one embodiment, the method of the invention comprises administering the
antibody having
the ability of binding to CLDN18.2 at a dose of up to 1000 mg/m2. In one
embodiment, the
method of the invention comprises administering the antibody having the
ability of binding to
CLDN18.2 repeatedly at a dose of 300 to 600 mg/m2.
In one embodiment, the cancer is CLDN18.2 positive. In one embodiment, the
cancer disease is
selected from the group consisting of gastric cancer, esophageal cancer,
pancreatic cancer, lung
cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, cancer
of the gallbladder
and the metastasis thereof. The cancer disease may be a Krukenberg tumor,
peritoneal metastasis
and/or lymph node metastasis. In one embodiment, the cancer is an
adenocarcinoma, in
particular an advanced adenocarcinoma. In one embodiment, the cancer is
selected from the
group consisting of cancer of the stomach, cancer of the esophagus, in
particular the lower
esophagus, cancer of the eso-gastric junction and gastroesophageal cancer. The
patient may be a
HER2/neu negative patient or a patient with HER2/neu positive status but not
eligible to
trastuzumab therapy.
According to the invention, CLDN18.2 preferably has the amino acid sequence
according to
SEQ ID NO: 1.
In a further aspect, the present invention provides a medical preparation
comprising an antibody
having the ability of binding to CLDN18.2 and an agent stabilizing or
increasing expression of
CLDN18.2. The medical preparation of the present invention may further
comprise an agent
stimulating 76 T cells. The antibody having the ability of binding to CLDN18.2
and the agent
stabilizing or increasing expression of CLDN18.2, and optionally the agent
stimulating 1/43 T
cells, may be present in the medical preparation in a mixture or separate from
each other. The
medical preparation may be a kit comprising a first container including the
antibody having the
ability of binding to CLDN18.2 and a container including the agent stabilizing
or increasing
expression of CLDN18.2, and optionally a container including the agent
stimulating y8 T cells.
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The medical preparation may further include printed instructions for use of
the preparation for
treatment of cancer, in particular for use of the preparation in a method of
the invention.
Different embodiments of the medical preparation, and, in particular, of the
agent stabilizing or
increasing expression of CLDN18.2 and the agent stimulating y8 T cells are as
described above
for the method of the invention.
The present invention also provides the agents described herein such as the
antibody having the
ability of binding to CLDN18.2 for use in the methods described herein, e.g.
for administration
in combination with an agent stabilizing or increasing expression of CLDN18.2,
and optionally
an agent stimulating y8 T cells.
Other features and advantages of the instant invention will be apparent from
the following
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Effect of chemotherapy on gastric cancer cells. Cultivation of
Katoill cells for 96
hours leads to a cell cycle arrest in the GO/G1-Phase and downregulation of
CLDN18.2.
Cytostatic compounds resulting in a cell cycle arrest in different phases of
the cell cycle (S-
phase (5-FU) or G2-phase (epirubicin)) stabilize CLDN18.2-expression.
Figure 2. Effect of chemotherapy on gastric cancer cells. a/b: Effect of
chemotherapy on
transcript and protein levels of CLDN18.2 in gastric cancer cells. c: Flow
cytometry of
extracellular IMAB362 binding on gastric cancer cells treated with
chemotherapeutic agents
Figure 3. Effect of chemotherapy on gastric cancer cells. Cytostatic
compounds resulting in
a cell cycle arrest in different phases of the cell cycle (S/G2-phase
(Irinotecan) or G2-phase
(Docetaxel)).
Figure 4. IMAB362-induced ADCC mediated killing of gastric cancer cells
after
pretreatment with chemotherapeutic agents
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Figure 5. Effect of chemotherapy on gastric cancer cells. a: Cells treated
with Irinotecan,
Docetaxel or Cisplatin exhibit a lower level of viable cells compared to
medium cultivated target
cells. b: CLDN18.2 expression in cells treated with Irinotecan, Docetaxel or
Cisplatin is
increased compared to medium cultivated cells. c/d: Treatment of cells with
Irinotecan,
Docetaxel or Cisplatin augments the potency of IMAB362 to induce ADCC.
Figure 6. Effects of chemotherapy on IMAB362-induced CDC
Figure 7. Effects of chemotherapy on effector cells
Figure 8. Expansion of PBMCs in ZA/IL-2 supplemented cultures
Figure 9. Enrichment of Vy9V=52 T cells in ZA/IL-2 supplemented PBMC
cultures
Figure 10. Enrichment of Vy9V82 T cells in medium supplemented with ZA and
an
increasing IL-2 dose
Figure 11. Expansion and cytotoxic activity of Vy9V62 T cells upon co-
incubation with ZA-
pulsed monocytes and human cancer cells
Figure 12. ZA-dependent development of different cell types in PBMC
cultures
Figure 13. Display of surface markers on Vy9V62 T-cells after ZA/IL-2
treatment
Figure 14. ADCC activity of Vy9V52 T cells with IMAB362 on CLDN18.2-
positive NUGC-
4 gastric cancer cells
Figure 15. ADCC of IMAB362 using Vy9V82 T cells as effector cells
Figure 16. Effects of ZA on surface localization of CLDN18.2 on target
cells
Figure 17. Effects of chemotherapy and ZA/IL-2 treatment on effector cells
Figure 18. Biodistribution studies with conjugated antibodies in mice
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Figure 19. Early treatment of HEK293¨CLDN18.2 tumor xenografts
Figure 20. Treatment of advanced HEK293¨CLDN18.2 tumor xenografts
Figure 21. Effect of IMAB362 on subcutaneous tumor growth of gastric cancer
xenografts
Figure 22. Effects of immunotherapy with IMAB362 on NCI-N87¨CLDN18.2
gastric
carcinoma xenografts
Figure 23. Effects of combination therapy with IMAB362 and EOF regimen on
NCI-
N87¨CLDN18.2 xenografts
Figure 24. Effects of combination therapy with IMAB362 and EOF regimen on
NUGC-
4¨C LDN18.2 xenografts
Figure 25. Effect of ZA/IL-2 induced Vy9V62 T cells on control of
macroscopic tumors by
IMAB362 in NSG mice
Figure 26. Effects of combination therapy with IMAB362 and EOF regimen on
CLS-
103¨cldn18.2 allograft tumors
DETAILED DESCRIPTION OF THE INVENTION
Although the present invention is described in detail below, it is to be
understood that this
invention is not limited to the particular methodologies, protocols and
reagents described herein
as these may vary. It is also to be understood that the terminology used
herein is for the purpose
of describing particular embodiments only, and is not intended to limit the
scope of the present
invention which will be limited only by the appended claims. Unless defined
otherwise, all
technical and scientific terms used herein have the same meanings as commonly
understood by
one of ordinary skill in the art.
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In the following, the elements of the present invention will be described.
These elements are
listed with specific embodiments, however, it should be understood that they
may be combined
in any manner and in any number to create additional embodiments. The
variously described
examples and preferred embodiments should not be construed to limit the
present invention to
only the explicitly described embodiments. This description should be
understood to support and
encompass embodiments which combine the explicitly described embodiments with
any number
of the disclosed and/or preferred elements. Furthermore, any permutations and
combinations of
all described elements in this application should be considered disclosed by
the description of the
present application unless the context indicates otherwise.
Preferably, the terms used herein are defined as described in "A multilingual
glossary of
biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B.
Nagel, and H.
KOlbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
The practice of the present invention will employ, unless otherwise indicated,
conventional
methods of chemistry, biochemistry, cell biology, immunology, and recombinant
DNA
techniques which are explained in the literature in the field (cf , e.g.,
Molecular Cloning: A
Laboratory Manual, 2'd Edition, J. Sambrook et al. eds., Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor 1989).
Throughout this specification and the claims which follow, unless the context
requires otherwise,
the word "comprise", and variations such as "comprises" and "comprising", will
be understood to
imply the inclusion of a stated member, integer or step or group of members,
integers or steps
but not the exclusion of any other member, integer or step or group of
members, integers or steps
although in some embodiments such other member, integer or step or group of
members, integers
or steps may be excluded, i.e. the subject-matter consists in the inclusion of
a stated member,
integer or step or group of members, integers or steps. The terms "a" and "an"
and "the" and
similar reference used in the context of describing the invention (especially
in the context of the
claims) are to be construed to cover both the singular and the plural, unless
otherwise indicated
herein or clearly contradicted by context. Recitation of ranges of values
herein is merely
intended to serve as a shorthand method of referring individually to each
separate value falling
within the range. Unless otherwise indicated herein, each individual value is
incorporated into
the specification as if it were individually recited herein. All methods
described herein can be
performed in any suitable order unless otherwise indicated herein or otherwise
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contradicted by context. The use of any and all examples, or exemplary
language (e.g., "such
as"), provided herein is intended merely to better illustrate the invention
and does not pose a
limitation on the scope of the invention otherwise claimed. No language in the
specification
should be construed as indicating any non-claimed element essential to the
practice of the
invention.
Several documents are cited throughout the text of this specification. Each of
the documents
cited herein (including all patents, patent applications, scientific
publications, manufacturer's
specifications, instructions, etc.), whether supra or infra, are hereby
incorporated by reference in
their entirety. Nothing herein is to be construed as an admission that the
invention is not entitled
to antedate such disclosure by virtue of prior invention.
The term "CLDN18" relates to claudin 18 and includes any variants, including
claudin 18 splice
variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin
18.2 (CLDN18.2)).
The term "CLDN18.2" preferably relates to human CLDN18.2, and, in particular,
to a protein
comprising, preferably consisting of the amino acid sequence according to SEQ
ID NO: 1 of the
sequence listing or a variant of said amino acid sequence.
The term "CLDN18.1" preferably relates to human CLDN18.1, and, in particular,
to a protein
comprising, preferably consisting of the amino acid sequence according to SEQ
ID NO: 2 of the
sequence listing or a variant of said amino acid sequence.
The term "variant" according to the invention refers, in particular, to
mutants, splice variants,
conformations, isoforms, allelic variants, species variants and species
hom*ologs, in particular
those which are naturally present. An allelic variant relates to an alteration
in the normal
sequence of a gene, the significance of which is often unclear. Complete gene
sequencing often
identifies numerous allelic variants for a given gene. A species hom*olog is a
nucleic acid or
amino acid sequence with a different species of origin from that of a given
nucleic acid or amino
acid sequence. The term "variant" shall encompass any posttranslationally
modified variants and
conformation variants.
According to the invention, the term "CLDN18.2 positive cancer" means a cancer
involving
cancer cells expressing CLDN18.2, preferably on the surface of said cancer
cells.
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"Cell surface" is used in accordance with its normal meaning in the art, and
thus includes the
outside of the cell which is accessible to binding by proteins and other
molecules.
CLDN18.2 is expressed on the surface of cells if it is located at the surface
of said cells and is
accessible to binding by CLDN18.2-specific antibodies added to the cells.
According to the invention, CLDN18.2 is not substantially expressed in a cell
if the level of
expression is lower compared to expression in stomach cells or stomach tissue.
Preferably, the
level of expression is less than 10%, preferably less than 5%, 3%, 2%, 1%,
0.5%, 0.1% or 0.05%
of the expression in stomach cells or stomach tissue or even lower.
Preferably, CLDN18.2 is not
substantially expressed in a cell if the level of expression exceeds the level
of expression in non-
cancerous tissue other than stomach by no more than 2-fold, preferably 1,5-
fold, and preferably
does not exceed the level of expression in said non-cancerous tissue.
Preferably, CLDN18.2 is
not substantially expressed in a cell if the level of expression is below the
detection limit and/or
if the level of expression is too low to allow binding by CLDN18.2-specific
antibodies added to
the cells.
According to the invention, CLDN18.2 is expressed in a cell if the level of
expression exceeds
the level of expression in non-cancerous tissue other than stomach preferably
by more than 2-
fold, preferably 10-fold, 100-fold, 1000-fold, or 10000-fold. Preferably,
CLDN18.2 is expressed
in a cell if the level of expression is above the detection limit and/or if
the level of expression is
high enough to allow binding by CLDN18.2-specific antibodies added to the
cells. Preferably,
CLDN18.2 expressed in a cell is expressed or exposed on the surface of said
cell.
According to the invention, the term "disease" refers to any pathological
state, including cancer,
in particular those forms of cancer described herein. Any reference herein to
cancer or particular
forms of cancer also includes cancer metastasis thereof. In a preferred
embodiment, a disease to
be treated according to the present application involves cells expressing
CLDN18.2.
"Diseases associated with cells expressing CLDN18.2" or similar expressions
means according
to the invention that CLDN18.2 is expressed in cells of a diseased tissue or
organ. In one
embodiment, expression of CLDN18.2 in cells of a diseased tissue or organ is
increased
compared to the state in a healthy tissue or organ. An increase refers to an
increase by at least
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10%, in particular at least 20%, at least 50%, at least 100%, at least 200%,
at least 500%, at least
1000%, at least 10000% or even more. In one embodiment, expression is only
found in a
diseased tissue, while expression in a healthy tissue is repressed. According
to the invention,
diseases associated with cells expressing CLDN18.2 include cancer diseases.
Furthermore,
according to the invention, cancer diseases preferably are those wherein the
cancer cells express
CLDN18.2.
As used herein, a "cancer disease" or "cancer" includes a disease
characterized by aberrantly
regulated cellular growth, proliferation, differentiation, adhesion, and/or
migration. By "cancer
cell" is meant an abnormal cell that grows by a rapid, uncontrolled cellular
proliferation and
continues to grow after the stimuli that initiated the new growth cease.
Preferably, a "cancer
disease" is characterized by cells expressing CLDN18.2 and a cancer cell
expresses CLDN18.2.
A cell expressing CLDN18.2 preferably is a cancer cell, preferably of the
cancers described
herein.
"Adenocarcinoma" is a cancer that originates in glandular tissue. This tissue
is also part of a
larger tissue category known as epithelial tissue. Epithelial tissue includes
skin, glands and a
variety of other tissue that lines the cavities and organs of the body.
Epithelium is derived
embryologically from ectoderm, endoderm and mesoderm. To be classified as
adenocarcinoma,
the cells do not necessarily need to be part of a gland, as long as they have
secretory properties.
This form of carcinoma can occur in some higher mammals, including humans.
Well
differentiated adenocarcinomas tend to resemble the glandular tissue that they
are derived from,
while poorly differentiated may not. By staining the cells from a biopsy, a
pathologist will
determine whether the tumor is an adenocarcinoma or some other type of cancer.

Adenocarcinomas can arise in many tissues of the body due to the ubiquitous
nature of glands
within the body. While each gland may not be secreting the same substance, as
long as there is
an exocrine function to the cell, it is considered glandular and its malignant
form is therefore
named adenocarcinoma. Malignant adenocarcinomas invade other tissues and often
metastasize
given enough time to do so. Ovarian adenocarcinoma is the most common type of
ovarian
carcinoma. It includes the serous and mucinous adenocarcinomas, the clear cell
adenocarcinoma
and the endometrioid adenocarcinoma.
By "metastasis" is meant the spread of cancer cells from its original site to
another part of the
body. The formation of metastasis is a very complex process and depends on
detachment of
13

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malignant cells from the primary tumor, invasion of the extracellular matrix,
penetration of the
endothelial basem*nt membranes to enter the body cavity and vessels, and then,
after being
transported by the blood, infiltration of target organs. Finally, the growth
of a new tumor at the
target site depends on angiogenesis. Tumor metastasis often occurs even after
the removal of the
primary tumor because tumor cells or components may remain and develop
metastatic potential.
In one embodiment, the term "metastasis" according to the invention relates to
"distant
metastasis" which relates to a metastasis which is remote from the primary
tumor and the
regional lymph node system. In one embodiment, the term "metastasis" according
to the
invention relates to lymph node metastasis. One particular form of metastasis
which is treatable
using the therapy of the invention is metastasis originating from gastric
cancer as primary site. In
preferred embodiments such gastric cancer metastasis is Krukenberg tumors,
peritoneal
metastasis and/or lymph node metastasis.
Krukenberg tumor is an uncommon metastatic tumor of the ovary accounting for
1% to 2% of all
ovarian tumors. Prognosis of Krukenberg tumor is still very poor and there is
no established
treatment for Krukenberg tumors. Krukenberg tumor is a metastatic signet ring
cell
adenocarcinoma of the ovary. Stomach is the primary site in most Krukenberg
tumor cases
(70%). Carcinomas of colon, appendix, and breast (mainly invasive lobular
carcinoma) are the
next most common primary sites. Rare cases of Krukenberg tumor originating
from carcinomas
of the gallbladder, biliary tract, pancreas, small intestine, ampulla of
Vater, cervix, and urinary
bladder/urachus have been reported. The interval between the diagnosis of a
primary carcinoma
and the subsequent discovery of ovarian involvement is usually 6 months or
less, but longer
periods have been reported. In many cases, the primary tumor is very small and
can escape
detection. A history of a prior carcinoma of the stomach or another organ can
be obtained in only
20% to 30% of the cases.
Krukenberg tumor is an example of the selective spread of cancers, most
commonly in the
stomach-ovarian axis. This axis of tumor spread has historically drawn the
attention of many
pathologists, especially when it was found that gastric neoplasms selectively
metastasize to the
ovaries without involvement of other tissues. The route of metastasis of
gastric carcinoma to the
ovaries has been a mystery for a long time, but it is now evident that
retrograde lymphatic spread
is the most likely route of metastasis.
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Women with Krukenberg tumors tend to be unusually young for patients with
metastatic
carcinoma as they are typically in the fifth decade of their lives, with an
average age of 45 years.
This young age of distribution can be related in part to the increased
frequency of gastric signet
ring cell carcinomas in young women. Common presenting symptoms are usually
related to
ovarian involvement, the most common of which are abdominal pain and
distension (mainly
because of the usually bilateral and often large ovarian masses). The
remaining patients have
nonspecific gastrointestinal symptoms or are asymptomatic. In addition,
Krukenberg tumor is
reportedly associated with virilization resulting from hormone production by
ovarian stroma.
Ascites is present in 50% of the cases and usually reveals malignant cells.
Krukenberg tumors are bilateral in more than 80% of the reported cases. The
ovaries are usually
asymmetrically enlarged, with a bosselated contour. The sectioned surfaces are
yellow or white;
they are usually solid, although they are occasionally cystic. Importantly,
the capsular surface of
the ovaries with Krukenberg tumors is typically smooth and free of adhesions
or peritoneal
deposits. Of note, other metastatic tumors to the ovary tend to be associated
with surface
implants. This may explain why the gross morphology of Krukenberg tumor can
deceptively
appear as a primary ovarian tumor. However, bilateralism in Krukenberg tumor
is consistent
with its metastatic nature.
Patients with Krukenberg tumors have an overall mortality rate that is
significantly high. Most
patients die within 2 years (median survival, 14 months). Several studies show
that the prognosis
is poor when the primary tumor is identified after the metastasis to the ovary
is discovered, and
the prognosis becomes worse if the primary tumor remains covert.
No optimal treatment strategy for Krukenberg tumors has been clearly
established in the
literature. Whether a surgical resection should be performed has not been
adequately addressed.
Chemotherapy or radiotherapy has no significant effect on prognosis of
patients with Krukenberg
tumors.
By "treat" is meant to administer a compound or composition or a combination
of compounds or
compositions to a subject in order to prevent or eliminate a disease,
including reducing the size
of a tumor or the number of tumors in a subject; arrest or slow a disease in a
subject; inhibit or
slow the development of a new disease in a subject; decrease the frequency or
severity of
symptoms and/or recurrences in a subject who currently has or who previously
has had a disease;

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and/or prolong, i.e. increase the lifespan of the subject.
In particular, the term "treatment of a disease" includes curing, shortening
the duration,
ameliorating, preventing, slowing down or inhibiting progression or worsening,
or preventing or
delaying the onset of a disease or the symptoms thereof.
The term "patient" means according to the invention a subject for treatment,
in particular a
diseased subject, including human beings, nonhuman primates or another
animals, in particular
mammals such as cows, horses, pigs, sheeps, goats, dogs, cats or rodents such
as mice and rats.
In a particularly preferred embodiment, a patient is a human being.
The term "agent stabilizing or increasing expression of CLDN18.2" refers to an
agent or a
combination of agents the provision of which to cells results in increased RNA
and/or protein
levels of CLDN18.2, preferably in increased levels of CLDN18.2 protein on the
cell surface,
compared to the situation where the cells are not provided with the agent or
the combination of
agents. Preferably, the cell is a cancer cell, in particular a cancer cell
expressing CLDN18.2, such
as a cell of the cancer types desribed herein. The term "agent stabilizing or
increasing expression
of CLDN18.2" refers, in particular, to an agent or a combination of agents the
provision of which
to cells results in a higher density of CLDN18.2 on the surface of said cells
compared to the
situation where the cells are not provided with the agent or the combination
of agents.
"Stabilizing expression of CLDN18.2" includes, in particular, the situation
where the agent or the
combination of agents prevents a decrease or reduces a decrease in expression
of CLDN18.2, e.g.
expression of CLDN18.2 would decrease without provision of the agent or the
combination of
agents and provision of the agent or the combination of agents prevents said
decrease or reduces
said decrease of CLDN18.2 expression. "Increasing expression of CLDN18.2"
includes, in
particular, the situation where the agent or the combination of agents
increases expression of
CLDN18.2, e.g. expression of CLDN18.2 would decrease, remain essentially
constant or
increase without provision of the agent or the combination of agents and
provision of the agent
or the combination of agents increases CLDN18.2 expression compared to the
situation without
provision of the agent or the combination of agents so that the resulting
expression is higher
compared to the situation where expression of CLDN18.2 would decrease, remain
essentially
constant or increase without provision of the agent or the combination of
agents.
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According to the invention, the term "agent stabilizing or increasing
expression of CLDN18.2"
includes chemotherapeutic agents or combinations of chemotherapeutic agents
such as cytostatic
agents. Chemotherapeutic agents may affect cells in one of the following ways:
(1) Damage the
DNA of the cells so they can no longer reproduce, (2) Inhibit the synthesis of
new DNA strands
so that no cell replication is possible, (3) Stop the mitotic processes of the
cells so that the cells
cannot divide into two cells.
According to the invention, the term "agent stabilizing or increasing
expression of CLDN18.2"
preferably relates to an agent or a combination of agents such a cytostatic
compound or a
combination of cytostatic compounds the provision of which to cells, in
particular cancer cells,
results in the cells being arrested in or accumulating in one or more phases
of the cell cycle,
preferably in one or more phases of the cell cycle other than the G1 - and GO-
phases, preferably
other than the Gl-phase, preferably in one or more of the G2- or S-phase of
the cell cycle such as
the Gl/G2-, S/G2-, G2- or S-phase of the cell cycle. The term "cells being
arrested in or
accumulating in one or more phases of the cell cycle" means that the
precentage of cells which
are in said one or more phases of the cell cycle increases. Each cell goes
through a cycle
comprising four phases in order to replicate itself. The first phase called G1
is when the cell
prepares to replicate its chromosomes. The second stage is called S, and in
this phase DNA
synthesis occurs and the DNA is duplicated. The next phase is the G2 phase,
when the RNA and
protein duplicate. The final stage is the M stage, which is the stage of
actual cell division. In this
final stage, the duplicated DNA and RNA split and move to separate ends of the
cell, and the cell
actually divides into two identical, functional cells. Chemotherapeutic agents
which are DNA
damaging agents usually result in an accumulation of cells in the G1 and/or G2
phase.
Chemotherapeutic agents which block cell growth by interfering with DNA
synthesis such as
antimetabolites usually result in an accumulation of cells in the S-phase.
Examples of these drugs
are 6-mercaptopurine and 5-fluorouracil.
According to the invention, the term "agent stabilizing or increasing
expression of CLDN18.2"
includes anthracyclines such as epirubicin, platinum compounds such as
oxaliplatin and
cisplatin, nucleoside analogs such as 5-fluorouracil or prodrugs thereof,
taxanes such as
docetaxel, and camptothecin analogs such as irinotecan and topotecan, and
combinations of
drugs such as combinations of drugs comprising one or more of anthracyclines
such as
epirubicin, oxaliplatin and 5-fluorouracil such as a combination of drugs
comprising oxaliplatin
and 5-fluorouracil or other drug combinations described herein.
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In one preferred embodiment, an "agent stabilizing or increasing expression of
CLDN18.2" is an
"agent inducing immunogenic cell death".
In specific circ*mstances, cancer cells can enter a lethal stress pathway
linked to the emission of
a spatiotemporally defined combination of signals that is decoded by the
immune system to
activate tumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140:
798-804). In such
scenario cancer cells are triggered to emit signals that are sensed by innate
immune effectors
such as dendritic cells to trigger a cognate immune response that involves
CD8+ T cells and IFN-
y signalling so that tumor cell death may elicit a productive anticancer
immune response. These
signals include the pre-apoptotic exposure of the endoplasmic reticulum (ER)
chaperon
calreticulin (CRT) at the cell surface, the pre-apoptotic secretion of ATP,
and the post-apoptotic
release of the nuclear protein HMGB1. Together, these processes constitute the
molecular
determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and
y irradiation are
able to induce all signals that define ICD, while cisplatin, for example,
which is deficient in
inducing CRT translocation from the ER to the surface of dying cells - a
process requiring ER
stress - requires complementation by thapsigargin, an ER stress inducer.
According to the invention, the term "agent inducing immunogenic cell death"
refers to an agent
or a combination of agents which when provided to cells, in particular cancer
cells, is capable of
inducing the cells to enter a lethal stress pathway which finally results in
tumor-specific immune
responses. In particular, an agent inducing immunogenic cell death when
provided to cells
induces the cells to emit a spatiotemporally defined combination of signals,
including, in
particular, the pre-apoptotic exposure of the endoplasmic reticulum (ER)
chaperon calreticulin
(CRT) at the cell surface, the pre-apoptotic secretion of ATP, and the post-
apoptotic release of
the nuclear protein HMGB1.
According to the invention, the term "agent inducing immunogenic cell death"
includes
anthracyclines and oxaliplatin.
Anthracyclines are a class of drugs commonly used in cancer chemotherapy that
are also
antibiotics. Structurally, all anthracyclines share a common four-ringed
7,8,9,10-
tetrahydrotetracene-5,12-quinone structure and usually require glycosylation
at specific sites.
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Anthracyclines preferably bring about one or more of the following mechanisms
of action: 1.
Inhibiting DNA and RNA synthesis by intercalating between base pairs of the
DNA/RNA strand,
thus preventing the replication of rapidly-growing cancer cells. 2. Inhibiting
topoisomerase II
enzyme, preventing the relaxing of supercoiled DNA and thus blocking DNA
transcription and
replication. 3. Creating iron-mediated free oxygen radicals that damage the
DNA and cell
membranes.
According to the invention, the term "anthracycline" preferably relates to an
agent, preferably an
anticancer agent for inducing apoptosis, preferably by inhibiting the
rebinding of DNA in
topoisomerase II.
Preferably, according to the invention, the term "anthracycline" generally
refers to a class of
compounds having the following ring structure
0 OH
0 OH
including analogs and derivatives, pharmaceutical salts, hydrates, esters,
conjugates and prodrugs
thereof.
Examples of anthracyclines and anthracycline analogs include, but are not
limited to,
daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin,
rhodomycin,
pyrarubicin, valrubicin, N-trifluoro-acetyl doxorubicin-14-valerate,
aclacinomycin,
morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin
(cyanomorpholino-
DOX), 2-pyrrolino-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone and
aclacinomycin A (aclan.ibicin). Mitoxantrone is a member of the anthracendione
class of
compounds, which are anthracycline analogs that lack the sugar moiety of the
anthracyclines but
retain the planar polycylic aromatic ring structure that permits intercalation
into DNA.
Particularly preferred as anthracyline according to the invention is a
compound of the following
formula:
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Ri
0 OH 0
*IWO OH
R2 0 OH ONH2
R4
CH3
wherein
R1 is selected from the group consisting of H and OH, R2 is selected from the
group consisting of
H and OMe, R3 is selected from the group consisting of H and OH, and R4 is
selected from the
group consisting of H and OH.
In one embodiment, R1 is H, R2 is OMe, R3 is H, and R4 is OH. In another
embodiment, R1 is
OH, R2 is OMe, R3 is H, and R4 is OH. In another embodiment, R1 is OH, R2 is
OMe, R3 is OH,
and R4 is H. In another embodiment, R1 is H, R2 is H, R3 is H, and R4 is OH.
Specifically contemplated as anthracycline in the context of the present
invention is epirubicin.
Epirubicin is an anthracycline drug which has the following formula:
OHO
0 OH
"Os"0
0 0 OH 0,,O,,..CH3
I-13%a"
NH2
and is marketed under the trade name Ellence in the US and Pharmorubicin or
Epirubicin Ebewe
elsewhere. In particular, the term "epirubicin" refers to the compound
(8R,10S)-10-
[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,1 1 -dihydroxy- 8 -
(2-
hydroxyacety1)- 1 -methoxy- 8 -methyl-9, 1 0-dihydro-7H-tetracen-5, 1 2-dion.
Epirubicin is favoured
over doxorubicin, the most popular anthracycline, in some chemotherapy
regimens as it appears
to cause fewer side-effects.

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According to the invention, the term "platinum compound" refers to compounds
containing
platinum in their structure such as platinum complexes and includes compounds
such as
cisplatin, carboplatin and oxaliplatin.
The term "cisplatin" or "cisplatinum" refers to the compound cis-
diamminedichloroplatinum(II)
(CDDP) of the following formula:
H3N
C I
CV'
Pt%
-NH3
The term "carboplatin" refers to the compound
cis-diammine(1, 1 -
cyclobutanedicarboxylato)platinum(II) of the following formula:
0
H3N\
Pt
H3N,e \
0
0
The term "oxaliplatin" refers to a compound which is a platinum compound that
is complexed to
a diaminocyclohexane carrier ligand of the following formula:
H2
N\ /
Pt
H2
In particular, the term "oxaliplatin" refers to the compound R1R,2R)-
cyclohexane-1,2-
diamineRethanedioato-0,0')platinum(II). Oxaliplatin for injection is also
marketed under the
trade name Eloxatine.
The term "nucleoside analog" refers to a structural analog of a nucleoside, a
category that
includes both purine analogs and pyrimidine analogs. In particular, the term
"nucleoside analog"
refers to fluoropyrimidine derivatives which includes fluorouracil and
prodrugs thereof.
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The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the
brand names Adrucil,
Carac, Efudix, Efudex and Fluoroplex) is a compound which is a pyrimidine
analog of the
following formula:
H
ONO ..,,,,,..
HNF
In particular, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-
dione.
The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent
that is a prodrug
that is converted into 5-FU in the tissues. Capecitabine which may be orally
administered has the
following formula:
HO OH
( 0),____N
0
H3C"'"*C0)."'"N
..õ.
y...,
N
H
F
In particular, the term refers to the compound pentyl [1-(3,4-dihydroxy-5-
methyltetrahydrofuran-
2-y1)-5-fluoro-2-oxo- 1 H-pyrimidin-4-yl] carb amate.
Taxanes are a class of diterpene compounds that were first derived from
natural sources such as
plants of the genus Taxus, but some have been synthesized artificially. The
principal mechanism
of action of the taxane class of drugs is the disruption of microtubule
function, thereby inhibiting
the process of cell division. Taxanes include docetaxel (Taxotere) and
pacl*taxel (Taxol).
According to the invention, the term "docetaxel" refers to a compound having
the following
formula:
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cH3
H3C 0
HO OH
H3C o H3C
H3C CH3
0NH 0
T 0H3 111
0
s...
0µµ
HO
110 OH0- CH
0 0 3
According to the invention, the term "pacl*taxel" refers to a compound having
the following
formula:
1111 OCH3
0 0
0 NH 0H3C C H
AL- CH
6-1-1 3 di
o' w CH
0
OH : H
0 1-r CH3
0
110
According to the invention, the term "camptothecin analog" refers to
derivatives of the
compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy- 1 H-p yrano [3 ',4': 6,7]
indolizino [ 1,2-b]
quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analog"
refers to
compounds comprising the following structure:
0
/ 0
H3C---'ss'OH
According to the invention, preferred camptothecin analogs are inhibitors of
DNA enzyme
topoisomerase I (topo I). Preferred camptothecin analogs according to the
invention are
irinotecan and topotecan.
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Irinotecan is a drug preventing DNA from unwinding by inhibition of
topoisomerase I. In
chemical terms, it is a semisynthetic analogue of the natural alkaloid
camptothecin having the
following formula:
0
H3C /
N
G 0
-0--cc N NHO 00
0
H3C
In particular, the term "irinotecan" refers to the compound (S)-4,11-diethy1-
3,4,12,14-tetrahydro-
4-hydroxy-3,14-dioxo1H-pyrano [3 ',4' :6,7] -indolizino [ 1,2-13] quinolin-9-
yl- [1,4 ' bipiperidine] -1' -
carboxylate.
Topotecan is a topoisomerase inhibitor of the formula:
0
H3C.
_ 1 N 0
N
H3C /
HO 411. N HO 0
H3C
In particular, the term "topotecan" refers to the compound (S)-10-
[(dimethylamino)methy1]-4-
ethyl-4,9-dihydroxy-1H-pyrano [3 ',4':6,7] indolizino [1,2-b] quinoline-
3,14(4H,12H)-dione
monohydrochloride.
According to the invention, an agent stabilizing or increasing expression of
CLDN18.2 may be a
chemotherapeutic agent, in particular a chemotherapeutic agent established in
cancer treatment
and may be part of a combination of drugs such as a combination of drugs
established for use in
cancer treatment. Such combination of drugs may be a drug combination used in
chemotherapy,
and may be a drug combination as used in a chemotherapeutic regimen selected
from the group
consisting of EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF
chemotherapy, FLO chemotherapy, FOLFOX chemotherapy, FOLFIRI chemotherapy, DCF

chemotherapy and FLOT chemotherapy.
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The drug combination used in EOX chemotherapy comprises of epirubicin,
oxaliplatin and
capecitabine. The drug combination used in ECF chemotherapy comprises of
epirubicin,
cisplatin and 5-fluorouracil. The drug combination used in ECX chemotherapy
comprises of
epirubicin, cisplatin and capecitabine. The drug combination used in EOF
chemotherapy
comprises of epirubicin, oxaliplatin and 5-fluorouracil.
Epirubicin is normally given at a dose of 50 mg/m2, cisplatin 60 mg/m2,
oxaliplatin 130 mg/m2,
protracted venous infusion of 5-fluorouracil at 200 mg/m2/day and oral
capecitabine 625 mg/m2
twice daily, for a total of eight 3-week cycles.
The drug combination used in FLO chemotherapy comprises of 5- fluorouracil,
folinic acid and
oxaliplatin (normally 5-fluorouracil 2,600 mg/m2 24-h infusion, folinic acid
200 mg/m2 and
oxaliplatin 85 mg/m2, every 2 weeks).
FOLFOX is a chemotherapy regimen made up of folinic acid (leucovorin), 5-
fluorouracil and
oxaliplatin. The recommended dose schedule given every two weeks is as
follows: Day 1:
Oxaliplatin 85 mg/m2 IV infusion and leucovorin 200 mg/m2 IV infusion,
followed by 5-FU 400
mg/m2 IV bolus, followed by 5-FU 600 mg/m2 IV infusion as a 22-hour continuous
infusion;
Day 2: Leucovorin 200 mg/m2 IV infusion over 120 minutes, followed by 5-FU 400
mg/m2
bolus given over 2-4 minutes, followed by 5-FU 600 mg/m2 IV infusion as a 22-
hour continuous
infusion.
The drug combination used in FOLFIRI chemotherapy comprises of 5-fluorouracil,
leucovorin,
and irinotecan.
The drug combination used in DCF chemotherapy comprises of docetaxel,
cisplatin and 5-
fluorouracil.
The drug combination used in FLOT chemotherapy comprises of docetaxel,
oxaliplatin, 5-
fluorouracil and folinic acid.
The term "folinic acid" or "leucovorin" refers to a compound useful in
synergistic combination
with the chemotherapy agent 5-fluorouracil. Folinic acid has the following
formula:

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NH
N)NN rcI
H2NNN OH
OH
In particular, the term refers to the compound (2S)-2- f[4-[(2-amino-5-formy1-
4-oxo-5,6,7,8-
tetrahydro-1H-pteridin-6-yl)methyl amino]benzo yl] amino pentanedioic acid.
y6 T cells (gamma delta T cells) represent a small subset of T cells that
possess a distinct T cell
receptor (TCR) on their surface. A majority of T cells have a TCR composed of
two glycoprotein
chains called a- and 13-TCR chains. In contrast, in y6 T cells, the TCR is
made up of one 7-chain
and one 6-chain. This group of T cells is usually much less common than aj3 T
cells. Human y6 T
cells play an important role in stress-surveillance responses like infectious
diseases and
autoimmunity. Transformation-induced changes in tumors are also suggested to
cause stress-
surveillance responses mediated by y6 T cells and enhance antitumor immunity.
Importantly,
after antigen engagement, activated y6 T cells at lesional sites provide
cytokines (e.g. INFy,
TNFa) and/or chemokines mediating recruitment of other effector cells and show
immediate
effector functions such as cytotoxicity (via death receptor and cytolytic
granules pathways) and
ADCC.
The majority of y6 T cells in peripheral blood express the Vy9V62 T cell
receptor (TCR76).
Vy9V62 T cells are unique to humans and primates and are assumed to play an
early and
essential role in sensing "danger" by invading pathogens as they expand
dramatically in many
acute infections and may exceed all other lymphocytes within a few days, e.g.
in tuberculosis,
salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis,
toxoplasmosis, and malaria.
-y6 T cells respond to small non-peptidic phosphorylated antigens
(phosphoantigens) such as
pyrophosphates synthesized in bacteria and isopentenyl pyrophosphate (IPP)
produced in
mammalian cells through the mevalonate pathway. Whereas IPP production in
normal cells is
not sufficient for activation of y6 T cells, dysregulation of the mevalonate
pathway in tumor cells
leads to accumulation of IPP and y6 T cell activation. IPPs can also be
therapeutically increased
by aminobisphosphonates, which inhibit the mevalonate pathway enzyme farnesyl
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pyrophosphate synthase (FPPS). Among others, zoledronic acid (ZA, zoledronate,
ZometaTm,
Novartis) represents such an aminobiphosphonate, which is already clinically
administered to
patients for the treatment of osteoporosis and metastasic bone disease. Upon
treatment of
PBMCs in vitro, ZA is taken up especially by monocytes. IPP accumulates in the
monocytes and
they differentiate to antigen-presenting cells stimulating development of y8 T
cells. In this
setting, the addition of interleukin-2 (IL-2) is preferred as growth and
survival factor for
activated y8 T cells. Finally, certain alkylated amines have been described to
activate Vy9V82 T
cells in vitro, however only at millimolar concentrations.
According to the invention, the term "agent stimulating y8 T cells" relates to
compounds
stimulating development of 78 T cells, in particular Vy9V82 T cells, in vitro
and/or in vivo, in
particular by inducing activation and expansion of y8 T cells. Preferably, the
term relates to
compounds which in vitro and/or in vivo increase isopentenyl pyrophosphate
([PP) produced in
mammalian cells, preferably by inhibiting the mevalonate pathway enzyme
farnesyl
pyrophosphate synthase (FPPS).
One particular group of compounds stimulating 78 T cells are bisphosphonates,
in particular
nitrogen-containing bisphosphonates (N-bisphosphonates; aminobisphosphonates).
For example, suitable bisphosphonates for use in the invention may include one
or more of the
following compounds including analogs and derivatives, pharmaceutical salts,
hydrates, esters,
conjugates and prodrugs thereof:
[1 -hydroxy-2 -(1H-imidazol-1 -ypethane-1 ,1 -diy1] bis (pho sphoni c acid),
zoledronic acid, e.g.
zoledronate;
(dichloro-phosphono-methyl)phosphonic acid, e.g. clodronate
{ 1 -hydroxy-3- [methyl (pentyl)amino] prop ane-1,1 - diyl bis (pho sphonic
acid), ibandronic acid,
e.g. ibandronate
(3-amino-l-hydroxypropane-1,1-diy1)bis (phosphonic acid), pamidronic acid,
e.g. pamidronate;
(1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid, risedronic acid,
e.g. risedronate;
(1 -Hydroxy-2 - imidazo [1,2 -a] pyridin-3 -y1-1 -pho sphono ethyl)pho sphoni
c acid, minodronic acid;
[3 -(dimethylamino)-1-hydroxypropane-1, 1 -diyl]bis(phosphonic acid),
olpadronic acid.
[4-amino-l-hydroxy-1-(hydroxy-oxido-phosphory1)-butyl]phosphonic acid,
alendronic acid, e.g.
alendronate;
[(Cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid;
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(1-hydroxyethan-1,1-diy1)bis(phosphonic acid), etidronic acid, e.g.
etidronate, and
{[(4-chlorophenyl)thio]methylenelbis(phosphonic acid), tiludronic acid.
According to the invention, zoledronic acid (INN) or zoledronate (marketed by
Novartis under
the trade names Zometa, Zomera, Aclasta and Reclast) is a particularly
preferred
bisphosphonate. Zometa is used to prevent skeletal fractures in patients with
cancers such as
multiple myeloma and prostate cancer, as well as for treating osteoporosis. It
can also be used to
treat hypercalcemia of malignancy and can be helpful for treating pain from
bone metastases.
In one particularly preferred embodiment, an agent stimulating y5 T cells
according to the
invention is administered in combination with IL-2. Such combination has been
shown to be
particularly effective in mediating expansion and activation of y952 T cells.
Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule
in the immune
system. It is a protein that attracts lymphocytes and is part of the body's
natural response to
microbial infection, and in discriminating between foreign (non-self) and
self. IL-2 mediates its
effects by binding to IL-2 receptors, which are expressed by lymphocytes.
The IL-2 used according to the invention may be any IL-2 supporting or
enabling the stimulation
of y5 T cells and may be derived from any species, preferably human. I1-2 may
be isolated,
recombinantly produced or synthetic IL-2 and may be naturally occurring or
modified IL-2.
In one embodiment of the present invention, the standard chemotherapy
according to the EOX
regimen in combination with an antibody having the ability of binding to
CLDN18.2, in
particular IMAB362 is administered for max. 8 cycles. The doses and schedules
may be as
follows:
= 50 mg/m2 Epirubicin will be administered i.v. as 15 minute infusion on
day 1 of each
cycle during the EOX phase.
= 130 mg/m2 Oxaliplatin will be administered i.v. as 2 h infusion on day 1
of each cycle
during the EOX phase.
= 625 mg/m2 Capecitabine are taken p.o. twice daily for 21 days in the
morning and in the
evening starting with the evening of day 1 of each cycle during the EOX phase.
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= 1000 mg/m2 antibody will be administered i.v. as a 2 h infusion on day 1
of cycle 1.
Thereafter 600 mg/m2 antibody will be administered i.v. as 2 h infusion on day
1 of each other
cycle after infusion of Oxaliplatin is completed.
= After termination of chemotherapy, the patient will continue with 600
mg/m2 antibody as
2 h infusion every 3 or 4 weeks.
In one embodiment of the present invention, the standard chemotherapy
according to the EOX
regimen in combination with ZA/IL-2 and an antibody having the ability of
binding to
CLDN18.2, in particular IMAB362 is administered for up to 8 cycles (24 weeks).
The term "antigen" relates to an agent such as a protein or peptide comprising
an epitope against
which an immune response is directed and/or is to be directed. In a preferred
embodiment, an
antigen is a tumor-associated antigen, such as CLDN18.2, i.e., a constituent
of cancer cells which
may be derived from the cytoplasm, the cell surface and the cell nucleus, in
particular those
antigens which are produced, preferably in large quantity, intracellular or as
surface antigens on
cancer cells.
In the context of the present invention, the term "tumor-associated antigen"
preferably relates to
proteins that are under normal conditions specifically expressed in a limited
number of tissues
and/or organs or in specific developmental stages and are expressed or
aberrantly expressed in
one or more tumor or cancer tissues. In the context of the present invention,
the tumor-associated
antigen is preferably associated with the cell surface of a cancer cell and is
preferably not or only
rarely expressed in normal tissues.
The term "epitope" refers to an antigenic determinant in a molecule, i.e., to
the part in a molecule
that is recognized by the immune system, for example, that is recognized by an
antibody. For
example, epitopes are the discrete, three-dimensional sites on an antigen,
which are recognized
by the immune system. Epitopes usually consist of chemically active surface
groupings of
molecules such as amino acids or sugar side chains and usually have specific
three dimensional
structural characteristics, as well as specific charge characteristics.
Conformational and non-
conformational epitopes are distinguished in that the binding to the former
but not the latter is
lost in the presence of denaturing solvents. An epitope of a protein such as
CLDN18.2 preferably
comprises a continuous or discontinuous portion of said protein and is
preferably between 5 and
100, preferably between 5 and 50, more preferably between 8 and 30, most
preferably between
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and 25 amino acids in length, for example, the epitope may be preferably 8, 9,
10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
The term "antibody" refers to a glycoprotein comprising at least two heavy (H)
chains and two
light (L) chains inter-connected by disulfide bonds, and includes any molecule
comprising an
antigen binding portion thereof. The term "antibody" includes monoclonal
antibodies and
fragments or derivatives of antibodies, including, without limitation, human
antibodies,
humanized antibodies, chimeric antibodies, single chain antibodies, e.g.,
scFv's and antigen-
binding antibody fragments such as Fab and Fab' fragments and also includes
all recombinant
forms of antibodies, e.g., antibodies expressed in prokaryotes, unglycosylated
antibodies, and
any antigen-binding antibody fragments and derivatives as described herein.
Each heavy chain is
comprised of a heavy chain variable region (abbreviated herein as VH) and a
heavy chain
constant region. Each light chain is comprised of a light chain variable
region (abbreviated
herein as VL) and a light chain constant region. The VH and VL regions can be
further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR),
interspersed with regions that are more conserved, termed framework regions
(FR). Each VH
and VL is composed of three CDRs and four FRs, arranged from amino-terminus to
carboxy-
terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable
regions of the heavy and light chains contain a binding domain that interacts
with an antigen.
The constant regions of the antibodies may mediate the binding of the
immunoglobulin to host
tissues or factors, including various cells of the immune system (e.g.,
effector cells) and the first
component (Clq) of the classical complement system.
The antibodies described herein may be human antibodies. The term "human
antibody", as used
herein, is intended to include antibodies having variable and constant regions
derived from
human germline immunoglobulin sequences. The human antibodies described herein
may
include amino acid residues not encoded by human germline immunoglobulin
sequences (e.g.,
mutations introduced by random or site-specific mutagenesis in vitro or by
somatic mutation in
vivo).
The term "humanized antibody" refers to a molecule having an antigen binding
site that is
substantially derived from an immunoglobulin from a non-human species, wherein
the remaining
immunoglobulin structure of the molecule is based upon the structure and/or
sequence of a
human immunoglobulin. The antigen binding site may either comprise complete
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domains fused onto constant domains or only the complementarity determining
regions (CDR)
grafted onto appropriate framework regions in the variable domains. Antigen
binding sites may
be wild-type or modified by one or more amino acid substitutions, e.g.
modified to resemble
human immunoglobulins more closely. Some forms of humanized antibodies
preserve all CDR
sequences (for example a humanized mouse antibody which contains all six CDRs
from the
mouse antibody). Other forms have one or more CDRs which are altered with
respect to the
original antibody.
The term "chimeric antibody" refers to those antibodies wherein one portion of
each of the amino
acid sequences of heavy and light chains is hom*ologous to corresponding
sequences in
antibodies derived from a particular species or belonging to a particular
class, while the
remaining segment of the chain is hom*ologous to corresponding sequences in
another. Typically
the variable region of both light and heavy chains mimics the variable regions
of antibodies
derived from one species of mammals, while the constant portions are
hom*ologous to sequences
of antibodies derived from another. One clear advantage to such chimeric forms
is that the
variable region can conveniently be derived from presently known sources using
readily
available B-cells or hybridomas from non-human host organisms in combination
with constant
regions derived from, for example, human cell preparations. While the variable
region has the
advantage of ease of preparation and the specificity is not affected by the
source, the constant
region being human, is less likely to elicit an immune response from a human
subject when the
antibodies are injected than would the constant region from a non human
source. However the
definition is not limited to this particular example.
The terms "antigen-binding portion" of an antibody (or simply "binding
portion") or "antigen-
binding fragment" of an antibody (or simply "binding fragment") or similar
terms refer to one or
more fragments of an antibody that retain the ability to specifically bind to
an antigen. It has
been shown that the antigen-binding function of an antibody can be performed
by fragments of a
full-length antibody. Examples of binding fragments encompassed within the
term "antigen-
binding portion" of an antibody include (i) Fab fragments, monovalent
fragments consisting of
the VL, VH, CL and CH domains; (ii) F(abD2 fragments, bivalent fragments
comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments
consisting of the
VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a
single arm of
an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546),
which consist of a
VH domain; (vi) isolated complementarity determining regions (CDR), and (vii)
combinations of
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two or more isolated CDRs which may optionally be joined by a synthetic
linker. Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they
can be joined, using recombinant methods, by a synthetic linker that enables
them to be made as
a single protein chain in which the VL and VH regions pair to form monovalent
molecules
(known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:
423-426; and Huston
et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain
antibodies are also
intended to be encompassed within the term "antigen-binding fragment" of an
antibody. A
further example is binding-domain immunoglobulin fusion proteins comprising
(i) a binding
domain polypeptide that is fused to an immunoglobulin hinge region
polypeptide, (ii) an
immunoglobulin heavy chain CH2 constant region fused to the hinge region, and
(iii) an
immunoglobulin heavy chain CH3 constant region fused to the CH2 constant
region. The
binding domain polypeptide can be a heavy chain variable region or a light
chain variable region.
The binding-domain immunoglobulin fusion proteins are further disclosed in US
2003/0118592
and US 2003/0133939. These antibody fragments are obtained using conventional
techniques
known to those with skill in the art, and the fragments are screened for
utility in the same manner
as are intact antibodies.
The term "bispecific molecule" is intended to include any agent, e.g., a
protein, peptide, or
protein or peptide complex, which has two different binding specificities. For
example, the
molecule may bind to, or interact with (a) a cell surface antigen, and (b) an
Fc receptor on the
surface of an effector cell. The term "multispecific molecule" or
"heterospecific molecule" is
intended to include any agent, e.g., a protein, peptide, or protein or peptide
complex, which has
more than two different binding specificities. For example, the molecule may
bind to, or interact
with (a) a cell surface antigen, (b) an Fc receptor on the surface of an
effector cell, and (c) at
least one other component. Accordingly, the invention includes, but is not
limited to, bispecific,
trispecific, tetraspecific, and other multispecific molecules which are
directed to CLDN18.2, and
to other targets, such as Fc receptors on effector cells. The term "bispecific
antibodies" also
includes diabodies. Diabodies are bivalent, bispecific antibodies in which the
VH and VL
domains are expressed on a single polypeptide chain, but using a linker that
is too short to allow
for pairing between the two domains on the same chain, thereby forcing the
domains to pair with
complementary domains of another chain and creating two antigen binding sites
(see e.g. ,
Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak,
R. J., et al. (1994)
Structure 2: 1121-1123).
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An antibody may be conjugated to a therapeutic moiety or agent, such as a
cytotoxin, a drug
(e.g., an immunosuppressant) or a radioisotope. A cytotoxin or cytotoxic agent
includes any
agent that is detrimental to and, in particular, kills cells. Examples include
taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,
mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine,
lidocaine, propranolol, and puromycin and analogs or hom*ologs thereof.
Suitable therapeutic
agents for forming antibody conjugates include, but are not limited to,
antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-
fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil,
melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol,
streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC), and
anti-mitotic agents (e.g., vincristine and vinblastine). In a preferred
embodiment, the therapeutic
agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the
therapeutic agent is
an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-
CSF. In a
preferred embodiment, the therapeutic agent is doxorubicin, cisplatin,
bleomycin, sulfate,
carmustine, chlorambucil, cyclophosphamide or ricin A.
Antibodies also can be conjugated to a radioisotope, e.g., iodine-131, yttrium-
90 or indium-111,
to generate cytotoxic radiopharmaceuticals.
The antibody conjugates of the invention can be used to modify a given
biological response, and
the drug moiety is not to be construed as limited to classical chemical
therapeutic agents. For
example, the drug moiety may be a protein or polypeptide possessing a desired
biological
activity. Such proteins may include, for example, an enzymatically active
toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria
toxin; a protein
such as tumor necrosis factor or interferon-y; or, biological response
modifiers such as, for
example, lympholcines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"),
granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte
colony stimulating
factor ("G-CSF"), or other growth factors.
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Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g.,
Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds. ), pp. 243-56
(Alan R. Liss,
Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled
Drug Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers
Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies
'84: Biological
And Clinical Applications, Pincheraet al. (eds. ), pp. 475-506 (1985);
"Analysis, Results, And
Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer
Therapy", in
Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16
(Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic
Properties Of
Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).
As used herein, an antibody is "derived from" a particular germline sequence
if the antibody is
obtained from a system by immunizing an animal or by screening an
immunoglobulin gene
library, and wherein the selected antibody is at least 90%, more preferably at
least 95%, even
more preferably at least 96%, 97%, 98%, or 99% identical in amino acid
sequence to the amino
acid sequence encoded by the germline immunoglobulin gene. Typically, an
antibody derived
from a particular germline sequence will display no more than 10 amino acid
differences, more
preferably, no more than 5, or even more preferably, no more than 4, 3, 2, or
1 amino acid
difference from the amino acid sequence encoded by the germline immunoglobulin
gene.
As used herein, the term "heteroantibodies" refers to two or more antibodies,
derivatives thereof,
or antigen binding regions linked together, at least two of which have
different specificities.
These different specificities include a binding specificity for an Fc receptor
on an effector cell,
and a binding specificity for an antigen or epitope on a target cell, e.g., a
tumor cell.
The antibodies described herein may be monoclonal antibodies. The term
"monoclonal antibody"
as used herein refers to a preparation of antibody molecules of single
molecular composition. A
monoclonal antibody displays a single binding specificity and affinity. In one
embodiment, the
monoclonal antibodies are produced by a hybridoma which includes a B cell
obtained from a
non-human animal, e.g., mouse, fused to an immortalized cell.
The antibodies described herein may be recombinant antibodies. The term
"recombinant
antibody", as used herein, includes all antibodies that are prepared,
expressed, created or isolated
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by recombinant means, such as (a) antibodies isolated from an animal (e.g., a
mouse) that is
transgenic or transchromosomal with respect to the immunoglobulin genes or a
hybridoma
prepared therefrom, (b) antibodies isolated from a host cell transformed to
express the antibody,
e.g., from a transfectoma, (c) antibodies isolated from a recombinant,
combinatorial antibody
library, and (d) antibodies prepared, expressed, created or isolated by any
other means that
involve splicing of immunoglobulin gene sequences to other DNA sequences.
Antibodies described herein may be derived from different species, including
but not limited to
mouse, rat, rabbit, guinea pig and human.
Antibodies described herein include polyclonal and monoclonal antibodies and
include IgA such
as IgAl or IgA2, IgGl, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In
various
embodiments, the antibody is an IgG1 antibody, more particularly an IgGl,
kappa or IgG1 ,
lambda isotype (i.e. IgGl, K, X.), an IgG2a antibody (e.g. IgG2a, K, 2), an
IgG2b antibody (e.g.
IgG2b, K, k), an IgG3 antibody (e.g. IgG3, K, X) or an IgG4 antibody (e.g.
IgG4, K, X.).
The term "transfectoma", as used herein, includes recombinant eukaryotic host
cells expressing
an antibody, such as CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant
cells, or fungi,
including yeast cells.
As used herein, a "heterologous antibody" is defined in relation to a
transgenic organism
producing such an antibody. This term refers to an antibody having an amino
acid sequence or an
encoding nucleic acid sequence corresponding to that found in an organism not
consisting of the
transgenic organism, and being generally derived from a species other than the
transgenic
organism.
As used herein, a "heterohybrid antibody" refers to an antibody having light
and heavy chains of
different organismal origins. For example, an antibody having a human heavy
chain associated
with a murine light chain is a heterohybrid antibody.
The invention includes all antibodies and derivatives of antibodies as
described herein which for
the purposes of the invention are encompassed by the term "antibody". The term
"antibody
derivatives" refers to any modified form of an antibody, e.g., a conjugate of
the antibody and
another agent or antibody, or an antibody fragment.

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The antibodies described herein are preferably isolated. An "isolated
antibody" as used herein, is
intended to refer to an antibody which is substantially free of other
antibodies having different
antigenic specificities (e.g., an isolated antibody that specifically binds to
CLDN18.2 is
substantially free of antibodies that specifically bind antigens other than
CLDN18.2). An isolated
antibody that specifically binds to an epitope, isoform or variant of human
CLDN18.2 may,
however, have cross-reactivity to other related antigens, e.g., from other
species (e.g., CLDN18.2
species hom*ologs). Moreover, an isolated antibody may be substantially free of
other cellular
material and/or chemicals. In one embodiment of the invention, a combination
of "isolated"
monoclonal antibodies relates to antibodies having different specificities and
being combined in
a well defined composition or mixture.
The term "binding" according to the invention preferably relates to a specific
binding.
According to the present invention, an antibody is capable of binding to a
predetermined target if
it has a significant affinity for said predetermined target and binds to said
predetermined target in
standard assays. "Affinity" or "binding affinity" is often measured by
equilibrium dissociation
constant (KD). Preferably, the term "significant affinity" refers to the
binding to a predetermined
target with a dissociation constant (KD) of 10-5 M or lower, 10-6 M or lower,
le M or lower, 10"
8M or lower, le M or lower, 1040M or lower, 1041M or lower, or 1042M or lower.
An antibody is not (substantially) capable of binding to a target if it has no
significant affinity for
said target and does not bind significantly, in particular does not bind
detectably, to said target in
standard assays. Preferably, the antibody does not detectably bind to said
target if present in a
concentration of up to 2, preferably 10, more preferably 20, in particular 50
or 100 1./g/m1 or
higher. Preferably, an antibody has no significant affinity for a target if it
binds to said target
with a KD that is at least 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, or
106-fold higher than
the KD for binding to the predetermined target to which the antibody is
capable of binding. For
example, if the KD for binding of an antibody to the target to which the
antibody is capable of
binding is le M, the KD for binding to a target for which the antibody has no
significant affinity
would be is at least 10-6 M, 10-5 M, 104 M, 10-3 M, 10-2 M, or 104 M.
An antibody is specific for a predetermined target if it is capable of binding
to said
predetermined target while it is not capable of binding to other targets, i.e.
has no significant
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affinity for other targets and does not significantly bind to other targets in
standard assays.
According to the invention, an antibody is specific for CLDN18.2 if it is
capable of binding to
CLDN18.2 but is not (substantially) capable of binding to other targets.
Preferably, an antibody
is specific for CLDN18.2 if the affinity for and the binding to such other
targets does not
significantly exceed the affinity for or binding to CLDN18.2-unrelated
proteins such as bovine
serum albumin (BSA), casein, human serum albumin (HSA) or non-claudin
transmembrane
proteins such as MHC molecules or transferrin receptor or any other specified
polypeptide.
Preferably, an antibody is specific for a predetermined target if it binds to
said target with a KD
that is at least 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, or 106-fold
lower than the KD for
binding to a target for which it is not specific. For example, if the KD for
binding of an antibody
to the target for which it is specific is 10-7 M, the KD for binding to a
target for which it is not
specific would be at least 10-6 M, 10-5 M, 104 M, 10-3 M, 10-2 M, or 10-1 M.
Binding of an antibody to a target can be determined experimentally using any
suitable method;
see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In
Fundamental
Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis
Immunology,
W. H. Freeman and Company New York, N Y (1992), and methods described herein.
Affinities
may be readily determined using conventional techniques, such as by
equilibrium dialysis; by
using the BIAcore 2000 instrument, using general procedures outlined by the
manufacturer; by
radioimmunoassay using radiolabeled target antigen; or by another method known
to the skilled
artisan. The affinity data may be analyzed, for example, by the method of
Scatchard et al., Ann
N.Y. Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-
antigen
interaction can vary if measured under different conditions, e.g., salt
concentration, pH. Thus,
measurements of affinity and other antigen-binding parameters, e.g., KD, IC50,
are preferably
made with standardized solutions of antibody and antigen, and a standardized
buffer.
As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1)
that is encoded by
heavy chain constant region genes.
As used herein, "isotype switching" refers to the phenomenon by which the
class, or isotype, of
an antibody changes from one Ig class to one of the other Ig classes.
The term "naturally occurring" as used herein as applied to an object refers
to the fact that an
object can be found in nature. For example, a polypeptide or polynucleotide
sequence that is
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present in an organism (including viruses) that can be isolated from a source
in nature and which
has not been intentionally modified by man in the laboratory is naturally
occurring.
The term "rearranged" as used herein refers to a configuration of a heavy
chain or light chain
immunoglobulin locus wherein a V segment is positioned immediately adjacent to
a D-J or J
segment in a conformation encoding essentially a complete VH or VL domain,
respectively. A
rearranged immunoglobulin (antibody) gene locus can be identified by
comparison to germline
DNA; a rearranged locus will have at least one recombined heptamer/nonamer
hom*ology
element.
The term "unrearranged" or "germline configuration" as used herein in
reference to a V segment
refers to the configuration wherein the V segment is not recombined so as to
be immediately
adjacent to a D or J segment.
According to the invention an antibody having the ability of binding to
CLDN18.2 is an antibody
capable of binding to an epitope present in CLDN18.2, preferably an epitope
located within the
extracellular domains of CLDN18.2, in particular the first extracellular
domain, preferably amino
acid positions 29 to 78 of CLDN18.2. In particular embodiments, an antibody
having the ability
of binding to CLDN18.2 is an antibody capable of binding to (i) an epitope on
CLDN18.2 which
is not present on CLDN18.1, preferably SEQ ID NO: 3, 4, and 5, (ii) an epitope
localized on the
CLDN18.2-loopl, preferably SEQ ID NO: 8, (iii) an epitope localized on the
CLDN18.2-loop2,
preferably SEQ ID NO: 10, (iv) an epitope localized on the CLDN18.2-loopD3,
preferably SEQ
ID NO: 11, (v) an epitope, which encompass CLDN18.2-loopl and CLDN18.2-loopD3,
or (vi) a
non-glycosylated epitope localized on the CLDN18.2-loopD3, preferably SEQ ID
NO: 9.
According to the invention an antibody having the ability of binding to
CLDN18.2 preferably is
an antibody having the ability of binding to CLDN18.2 but not to CLDN18.1.
Preferably, an
antibody having the ability of binding to CLDN18.2 is specific for CLDN18.2.
Preferably, an
antibody having the ability of binding to CLDN18.2 preferably is an antibody
having the ability
of binding to CLDN18.2 expressed on the cell surface. In particular preferred
embodiments, an
antibody having the ability of binding to CLDN18.2 binds to native epitopes of
CLDN18.2
present on the surface of living cells. Preferably, an antibody having the
ability of binding to
CLDN18.2 binds to one or more peptides selected from the group consisting of
SEQ ID NOs: 1,
3-11, 44, 46, and 48-50. Preferably, an antibody having the ability of binding
to CLDN18.2 is
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CA 02874032 2014-11-19
WO 2013/174510 PCT/EP2013/001504
specific for the afore mentioned proteins, peptides or immunogenic fragments
or derivatives
thereof. An antibody having the ability of binding to CLDN18.2 may be obtained
by a method
comprising the step of immunizing an animal with a protein or peptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46,
and 48-50, or a
nucleic acid or host cell expressing said protein or peptide. Preferably, the
antibody binds to
cancer cells, in particular cells of the cancer types mentioned above and,
preferably, does not
bind substantially to non-cancerous cells.
Preferably, binding of an antibody having the ability of binding to CLDN18.2
to cells expressing
CLDN18.2 induces or mediates killing of cells expressing CLDN18.2. The cells
expressing
CLDN18.2 are preferably cancer cells and are, in particular, selected from the
group consisting
of tumorigenic gastric, esophageal, pancreatic, lung, ovarian, colon, hepatic,
head-neck, and
gallbladder cancer cells. Preferably, the antibody induces or mediates killing
of cells by inducing
one or more of complement dependent cytotoxicity (CDC) mediated lysis,
antibody dependent
cellular cytotoxicity (ADCC) mediated lysis, apoptosis, and inhibition of
proliferation of cells
expressing CLDN18.2. Preferably, ADCC mediated lysis of cells takes place in
the presence of
effector cells, which in particular embodiments are selected from the group
consisting of
monocytes, mononuclear cells, NK cells and PMNs. Inhibiting proliferation of
cells can be
measured in vitro by determining proliferation of cells in an assay using
bromodeoxyuridine
bromo-2-deoxyuridine, BrdU). BrdU is a synthetic nucleoside which is an
analogue of thymidine
and can be incorporated into the newly synthesized DNA of replicating cells
(during the S phase
of the cell cycle), substituting for thymidine during DNA replication.
Detecting the incorporated
chemical using, for example, antibodies specific for BrdU indicates cells that
were actively
replicating their DNA.
In preferred embodiments, antibodies described herein can be characterized by
one or more of
the following properties:
a) specificity for CLDN18.2;
b) a binding affinity to CLDN18.2 of about 100 nM or less, preferably, about 5-
10 nM or less
and, more preferably, about 1-3 nM or less,
c) the ability to induce or mediate CDC on CLDN18.2 positive cells;
d) the ability to induce or mediate ADCC on CLDN18.2 positive cells;
e) the ability to inhibit the growth of CLDN18.2 positive cells;
f) the ability to induce apoptosis of CLDN18.2 positive cells.
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In a particularly preferred embodiment, an antibody having the ability of
binding to CLDN18.2
is produced by a hybridoma deposited at the DSMZ (Mascheroder Weg lb, 31824
Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig,
Germany) and
having the following designation and accession number:
a. 182-D1106-055, accesssion no. DSM ACC2737, deposited on October 19, 2005
b. 182-D1106-056, accesssion no. DSM ACC2738, deposited on October 19, 2005
c. 182-D1106-057, accesssion no. DSM ACC2739, deposited on October 19, 2005
d. 182-D1106-058, accesssion no. DSM ACC2740, deposited on October 19, 2005
e. 182-D1106-059, accesssion no. DSM ACC2741, deposited on October 19, 2005
f. 182-D1106-062, accesssion no. DSM ACC2742, deposited on October 19, 2005,
g. 182-D1106-067, accesssion no. DSM ACC2743, deposited on October 19, 2005
h. 182-D758-035, accesssion no. DSM ACC2745, deposited on Nov. 17, 2005
i. 182-D758-036, accesssion no. DSM ACC2746, deposited on Nov. 17, 2005
j. 182-D758-040, accesssion no. DSM ACC2747, deposited on Nov. 17, 2005
k. 182-D1106-061, accesssion no. DSM ACC2748, deposited on Nov. 17, 2005
1. 182-D1106-279, accesssion no. DSM ACC2808, deposited on Oct. 26, 2006
m. 182-D1106-294, accesssion no. DSM ACC2809, deposited on Oct. 26, 2006,
n. 182-D1106-362, accesssion no. DSM ACC2810, deposited on Oct. 26, 2006.
Preferred antibodies according to the invention are those produced by and
obtainable from the
above-described hybridomas; i.e. 37G11 in the case of 182-D1106-055, 37H8 in
the case of 182-
D1106-056, 38G5 in the case of 182-D1106-057, 38H3 in the case of 182-D1106-
058, 39F11 in
the case of 182-D1106-059, 43A11 in the case of 182-D1106-062, 61C2 in the
case of 182-
D1106-067, 26B5 in the case of 182-D758-035, 26D12 in the case of 182-D758-
036, 28D10 in
the case of 182-D758-040, 42E12 in the case of 182-D1106-061, 125E1 in the
case of 182-
D1106-279, 163E12 in the case of 182-D1106-294, and 175D10 in the case of 182-
D1106-362;
and the chimerized and humanized forms thereof.

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Preferred chimerized antibodies and their sequences are shown in the following
table.
chimerized
clone mAb Isotype variable region antibody
heavy
chain 43A11 182-D1106-062 IgG2a SEQ ID NO:29 SEQ ID NO:14
163E12 182-D1106-294 IgG3 SEQ ID NO:30 SEQ ID NO:15
125E1 182-D1106-279 IgG2a SEQ ID NO:31 SEQ ID NO:16
166E2 182-D1106-308 IgG3 SEQ ID NO:33 SEQ ID NO:18
175D10 182-D1106-362 IgG1 SEQ ID NO:32 SEQ ID NO:17
45C1 182-D758-187
IgG2a SEQ ID NO:34 SEQ ID NO:19
light
chain 43A11 182-D1106-062 IgK SEQ ID NO:36
SEQ ID NO:21
163E12 182-D1106-294 IgK SEQ ID NO:35
SEQ ID NO:20
125E1 182-D1106-279 IgK SEQ ID NO:37
SEQ ID NO:22
166E2 182-D1106-308 IgK SEQ ID NO:40
SEQ ID NO:25
175D10 182-D1106-362 IgK SEQ NO:39 SEQ
ID NO:24
45C1 182-D758-187 IgK SEQ ID NO:38
SEQ ID NO:23
45C1 182-D758-187 IgK SEQ ID NO:41
SEQ ID NO:26
45C1 182-D758-187 IgK SEQ ID NO:42
SEQ ID NO:27
45C1 182-D758-187 IgK SEQ ID NO:43
SEQ ID NO:28
In preferred embodiments, antibodies, in particular chimerised forms of
antibodies according to
the invention include antibodies comprising a heavy chain constant region (CH)
comprising an
amino acid sequence derived from a human heavy chain constant region such as
the amino acid
sequence represented by SEQ ID NO: 13 or a fragment thereof. In further
preferred
embodiments, antibodies, in particular chimerised forms of antibodies
according to the invention
include antibodies comprising a light chain constant region (CL) comprising an
amino acid
sequence derived from a human light chain constant region such as the amino
acid sequence
represented by SEQ ID NO: 12 or a fragment thereof. In a particular preferred
embodiment,
antibodies, in particular chimerised forms of antibodies according to the
invention include
antibodies which comprise a CH comprising an amino acid sequence derived from
a human CH
such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment
thereof and which
comprise a CL comprising an amino acid sequence derived from a human CL such
as the amino
acid sequence represented by SEQ ID NO: 12 or a fragment thereof.
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In one embodiment, an antibody having the ability of binding to CLDN18.2 is a
chimeric
mouse/human IgG1 monoclonal antibody comprising kappa, murine variable light
chain, human
kappa light chain constant region allotype Km(3), murine heavy chain variable
region, human
IgG1 constant region, allotype Glm(3).
In certain preferred embodiments, chimerised forms of antibodies include
antibodies comprising
a heavy chain comprising an amino acid sequence selected from the group
consisting of SEQ ID
NOs: 14, 15, 16, 17, 18, 19, and a fragment thereof and/or comprising a light
chain comprising
an amino acid sequence selected from the group consisting of SEQ ID NOs: 20,
21, 22, 23, 24,
25, 26, 27, 28, and a fragment thereof.
In certain preferred embodiments, chimerised forms of antibodies include
antibodies comprising
a combination of heavy chains and light chains selected from the following
possibilities (i) to
(ix):
(i) the heavy chain comprises an amino acid sequence represented by SEQ ID NO:
14 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 21 or a fragment thereof,
(ii) the heavy chain comprises an amino acid sequence represented by SEQ ID
NO: 15 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 20 or a fragment thereof,
(iii) the heavy chain comprises an amino acid sequence represented by SEQ ID
NO: 16 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 22 or a fragment thereof,
(iv) the heavy chain comprises an amino acid sequence represented by SEQ ID
NO: 18 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 25 or a fragment thereof,
(v) the heavy chain comprises an amino acid sequence represented by SEQ ID NO:
17 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 24 or a fragment thereof,
(vi) the heavy chain comprises an amino acid sequence represented by SEQ ID
NO: 19 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 23 or a fragment thereof,
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(vii) the heavy chain comprises an amino acid sequence represented by SEQ ID
NO: 19 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 26 or a fragment thereof,
(viii) the heavy chain comprises an amino acid sequence represented by SEQ ID
NO: 19 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 27 or a fragment thereof, and
(ix) the heavy chain comprises an amino acid sequence represented by SEQ ID
NO: 19 or a
fragment thereof and the light chain comprises an amino acid sequence
represented by SEQ ID
NO: 28 or a fragment thereof.
"Fragment" or "fragment of an amino acid sequence" as used above relates to a
part of an
antibody sequence, i.e. a sequence which represents the antibody sequence
shortened at the N-
and/or C-terminus, which when it replaces said antibody sequence in an
antibody retains binding
of said antibody to CLDN18.2 and preferably functions of said antibody as
described herein, e.g.
CDC mediated lysis or ADCC mediated lysis. Preferably, a fragment of an amino
acid sequence
comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of
the amino acid
residues from said amino acid sequence. A fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, and 28
preferably relates to said sequence wherein 17, 18, 19, 20, 21, 22 or 23 amino
acids at the N-
terminus are removed.
In a preferred embodiment, an antibody having the ability of binding to
CLDN18.2 comprises a
heavy chain variable region (VH) comprising an amino acid sequence selected
from the group
consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, and a fragment thereof.
In a preferred embodiment, an antibody having the ability of binding to
CLDN18.2 comprises a
light chain variable region (VL) comprising an amino acid sequence selected
from the group
consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, and a fragment
thereof.
In certain preferred embodiments, an antibody having the ability of binding to
CLDN18.2
comprises a combination of heavy chain variable region (VH) and light chain
variable region
(VL) selected from the following possibilities (i) to (ix):
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(i) the VH comprises an amino acid sequence represented by SEQ ID NO: 29 or a
fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
36 or a
fragment thereof,
(ii) the VH comprises an amino acid sequence represented by SEQ ID NO: 30 or a
fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
35 or a
fragment thereof,
(iii) the VH comprises an amino acid sequence represented by SEQ ID NO: 31 or
a fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
37 or a
fragment thereof,
(iv) the VH comprises an amino acid sequence represented by SEQ ID NO: 33 or a
fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
40 or a
fragment thereof,
(v) the VH comprises an amino acid sequence represented by SEQ ID NO: 32 or a
fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
39 or a
fragment thereof,
(vi) the VH comprises an amino acid sequence represented by SEQ ID NO: 34 or a
fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
38 or a
fragment thereof,
(vii) the VH comprises an amino acid sequence represented by SEQ ID NO: 34 or
a fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
41 or a
fragment thereof,
(viii) the VH comprises an amino acid sequence represented by SEQ ID NO: 34 or
a fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
42 or a
fragment thereof,
(ix) the VH comprises an amino acid sequence represented by SEQ ID NO: 34 or a
fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO:
43 or a
fragment thereof.
In a preferred embodiment, an antibody having the ability of binding to
CLDN18.2 comprises a
VH comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3
selected
from the following embodiments (i) to (vi):
(i) CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID
NO: 14,
CDR3: positions 116-125 of SEQ ID NO: 14,
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(ii) CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID
NO: 15,
CDR3: positions 116-126 of SEQ ID NO: 15,
(iii) CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID
NO: 16,
CDR3: positions 116-124 of SEQ ID NO: 16,
(iv) CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ ID
NO: 17,
CDR3: positions 116-126 of SEQ ID NO: 17,
(v) CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID
NO: 18,
CDR3: positions 115-125 of SEQ ID NO: 18, and
(vi) CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID
NO: 19,
CDR3: positions 117-128 of SEQ ID NO: 19.
In a preferred embodiment, an antibody having the ability of binding to
CLDN18.2 comprises a
VL comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3
selected
from the following embodiments (i) to (ix):
(i) CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQ ID
NO: 20,
CDR3: positions 115-123 of SEQ ID NO: 20,
(ii) CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID
NO: 21,
CDR3: positions 110-118 of SEQ ID NO: 21,
(iii) CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID
NO: 22,
CDR3: positions 109-117 of SEQ ID NO: 22,
(iv) CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID
NO: 23,
CDR3: positions 115-123 of SEQ ID NO: 23,
(v) CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID
NO: 24,
CDR3: positions 115-123 of SEQ ID NO: 24,
(vi) CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID
NO: 25,
CDR3: positions 115-122 of SEQ ID NO: 25,
(vii) CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID
NO: 26,
CDR3: positions 115-123 of SEQ ID NO: 26,
(viii) CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID
NO: 27,
CDR3: positions 115-123 of SEQ ID NO: 27, and
(ix) CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 of SEQ ID
NO: 28,
CDR3: positions 109-117 of SEQ ID NO: 28.

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In a preferred embodiment, an antibody having the ability of binding to
CLDN18.2 comprises a
combination of VH and VL each comprising a set of complementarity-determining
regions
CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix):
(i) VH: CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ
ID NO: 14,
CDR3: positions 116-125 of SEQ ID NO: 14, VL: CDR1: positions 49-53 of SEQ ID
NO: 21,
CDR2: positions 71-73 of SEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO:
21,
(ii) VH: CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ
ID NO: 15,
CDR3: positions 116-126 of SEQ ID NO: 15, VL: CDR1: positions 47-58 of SEQ ID
NO: 20,
CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO:
20,
(iii) VH: CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ
ID NO: 16,
CDR3: positions 116-124 of SEQ ID NO: 16, VL: CDR1: positions 47-52 of SEQ ID
NO: 22,
CDR2: positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO:
22,
(iv) VH: CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQ
ID NO: 18,
CDR3: positions 115-125 of SEQ ID NO: 18, VL: CDR1: positions 47-58 of SEQ ID
NO: 25,
CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO:
25,
(v) VH: CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ
ID NO: 17,
CDR3: positions 116-126 of SEQ ID NO: 17, VL: CDR1: positions 47-58 of SEQ ID
NO: 24,
CDR2: positions 76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO:
24,
(vi) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ
ID NO: 19,
CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID
NO: 23,
CDR2: positions 76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO:
23,
(vii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ
ID NO: 19,
CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID
NO: 26,
CDR2: positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO:
26,
(viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of
SEQ ID NO:
19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ
ID NO:
27, CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID
NO: 27,
and
(ix) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ
ID NO: 19,
CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-52 of SEQ ID
NO: 28,
CDR2: positions 70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO:
28.
In further preferred embodiments, an antibody having the ability of binding to
CLDN18.2
preferably comprises one or more of the complementarity-determining regions
(CDRs),
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preferably at least the CDR3 variable region, of the heavy chain variable
region (VH) and/or of
the light chain variable region (VL) of a monoclonal antibody against
CLDN18.2, preferably of a
monoclonal antibody against CLDN18.2 described herein, and preferably
comprises one or more
of the complementarity-determining regions (CDRs), preferably at least the
CDR3 variable
region, of the heavy chain variable regions (VH) and/or light chain variable
regions (VL)
described herein. In one embodiment said one or more of the complementarity-
determining
regions (CDRs) are selected from a set of complementarity-determining regions
CDR1, CDR2
and CDR3 described herein. In a particularly preferred embodiment, an antibody
having the
ability of binding to CLDN18.2 preferably comprises the complementarity-
determining regions
CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and/or of the
light chain
variable region (VL) of a monoclonal antibody against CLDN18.2, preferably of
a monoclonal
antibody against CLDN18.2 described herein, and preferably comprises the
complementarity-
determining regions CDR1, CDR2 and CDR3 of the heavy chain variable regions
(VH) and/or
light chain variable regions (VL) described herein.
In one embodiment an antibody comprising one or more CDRs, a set of CDRs or a
combination
of sets of CDRs as described herein comprises said CDRs together with their
intervening
framework regions. Preferably, the portion will also include at least about
50% of either or both
of the first and fourth framework regions, the 50% being the C-terminal 50% of
the first
framework region and the N-terminal 50% of the fourth framework region.
Construction of
antibodies made by recombinant DNA techniques may result in the introduction
of residues N-
or C-terminal to the variable regions encoded by linkers introduced to
facilitate cloning or other
manipulation steps, including the introduction of linkers to join variable
regions of the invention
to further protein sequences including immunoglobulin heavy chains, other
variable domains (for
example in the production of diabodies) or protein labels.
In one embodiment an antibody comprising one or more CDRs, a set of CDRs or a
combination
of sets of CDRs as described herein comprises said CDRs in a human antibody
framework.
Reference herein to an antibody comprising with respect to the heavy chain
thereof a particular
chain, or a particular region or sequence preferably relates to the situation
wherein all heavy
chains of said antibody comprise said particular chain, region or sequence.
This applies
correspondingly to the light chain of an antibody.
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The term "nucleic acid", as used herein, is intended to include DNA and RNA. A
nucleic acid
may be single-stranded or double-stranded, but preferably is double-stranded
DNA.
According to the invention, the term "expression" is used in its most general
meaning and
comprises the production of RNA or of RNA and protein/peptide. It also
comprises partial
expression of nucleic acids. Furthermore, expression may be carried out
transiently or stably.
The teaching given herein with respect to specific amino acid sequences, e.g.
those shown in the
sequence listing, is to be construed so as to also relate to variants of said
specific sequences
resulting in sequences which are functionally equivalent to said specific
sequences, e.g. amino
acid sequences exhibiting properties identical or similar to those of the
specific amino acid
sequences. One important property is to retain binding of an antibody to its
target or to sustain
effector functions of an antibody. Preferably, a sequence which is a variant
with respect to a
specific sequence, when it replaces the specific sequence in an antibody
retains binding of said
antibody to CLDN18.2 and preferably functions of said antibody as described
herein, e.g. CDC
mediated lysis or ADCC mediated lysis.
It will be appreciated by those skilled in the art that in particular the
sequences of the CDR,
hypervariable and variable regions can be modified without losing the ability
to bind CLDN18.2.
For example, CDR regions will be either identical or highly hom*ologous to the
regions of
antibodies specified herein. By "highly hom*ologous" it is contemplated that
from 1 to 5,
preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in
the CDRs. In
addition, the hypervariable and variable regions may be modified so that they
show substantial
hom*ology with the regions of antibodies specifically disclosed herein.
For the purposes of the present invention, "variants" of an amino acid
sequence comprise amino
acid insertion variants, amino acid addition variants, amino acid deletion
variants and/or amino
acid substitution variants. Amino acid deletion variants that comprise the
deletion at the N-
terminal and/or C-terminal end of the protein are also called N-terminal
and/or C-terminal
truncation variants.
Amino acid insertion variants comprise insertions of single or two or more
amino acids in a
particular amino acid sequence. In the case of amino acid sequence variants
having an insertion,
one or more amino acid residues are inserted into a particular site in an
amino acid sequence,
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although random insertion with appropriate screening of the resulting product
is also possible.
Amino acid addition variants comprise amino- and/or carboxy-terminal fusions
of one or more
amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
Amino acid deletion variants are characterized by the removal of one or more
amino acids from
the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino
acids. The deletions
may be in any position of the protein.
Amino acid substitution variants are characterized by at least one residue in
the sequence being
removed and another residue being inserted in its place. Preference is given
to the modifications
being in positions in the amino acid sequence which are not conserved between
hom*ologous
proteins or peptides and/or to replacing amino acids with other ones having
similar properties.
Preferably, amino acid changes in protein variants are conservative amino acid
changes, i.e.,
substitutions of similarly charged or uncharged amino acids. A conservative
amino acid change
involves substitution of one of a family of amino acids which are related in
their side chains.
Naturally occurring amino acids are generally divided into four families:
acidic (aspartate,
glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine,
leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine,
asparagine,
glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine,
tryptophan, and
tyrosine are sometimes classified jointly as aromatic amino acids.
Preferably the degree of similarity, preferably identity between a given amino
acid sequence and
an amino acid sequence which is a variant of said given amino acid sequence
will be at least
about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or
identity is given
preferably for an amino acid region which is at least about 10%, at least
about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at
least about 80%, at least about 90% or about 100% of the entire length of the
reference amino
acid sequence. For example, if the reference amino acid sequence consists of
200 amino acids,
the degree of similarity or identity is given preferably for at least about
20, at least about 40, at
least about 60, at least about 80, at least about 100, at least about 120, at
least about 140, at least
about 160, at least about 180, or about 200 amino acids, preferably continuous
amino acids. In
preferred embodiments, the degree of similarity or identity is given for the
entire length of the
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reference amino acid sequence. The alignment for determining sequence
similarity, preferably
sequence identity can be done with art known tools, preferably using the best
sequence
alignment, for example, using Align, using standard settings, preferably
EMBOSS: :needle,
Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
"Sequence similarity" indicates the percentage of amino acids that either are
identical or that
represent conservative amino acid substitutions. "Sequence identity" between
two amino acid
sequences indicates the percentage of amino acids that are identical between
the sequences.
The term "percentage identity" is intended to denote a percentage of amino
acid residues which
are identical between the two sequences to be compared, obtained after the
best alignment, this
percentage being purely statistical and the differences between the two
sequences being
distributed randomly and over their entire length. Sequence comparisons
between two amino
acid sequences are conventionally carried out by comparing these sequences
after having aligned
them optimally, said comparison being carried out by segment or by "window of
comparison" in
order to identify and compare local regions of sequence similarity. The
optimal alignment of the
sequences for comparison may be produced, besides manually, by means of the
local hom*ology
algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the
local
hom*ology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by
means of the
similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci.
USA 85, 2444, or
by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA,
BLAST
P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics
Computer Group,
575 Science Drive, Madison, Wis.).
The percentage identity is calculated by determining the number of identical
positions between
the two sequences being compared, dividing this number by the number of
positions compared
and multiplying the result obtained by 100 so as to obtain the percentage
identity between these
two sequences.
The term "transgenic animal" refers to an animal having a genome comprising
one or more
transgenes, preferably heavy and/or light chain transgenes, or
transchromosomes (either
integrated or non-integrated into the animal's natural genomic DNA) and which
is preferably
capable of expressing the transgenes. For example, a transgenic mouse can have
a human light
chain transgene and either a human heavy chain transgene or human heavy chain

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transchromosome, such that the mouse produces human anti-CLDN18.2 antibodies
when
immunized with CLDN18.2 antigen and/or cells expressing CLDN18.2. The human
heavy chain
transgene can be integrated into the chromosomal DNA of the mouse, as is the
case for
transgenic mice, e.g., HuMAb mice, such as HCo7 or HCo12 mice, or the human
heavy chain
transgene can be maintained extrachromosomally, as is the case for
transchromosomal (e.g.,
KM) mice as described in WO 02/43478. Such transgenic and transchromosomal
mice may be
capable of producing multiple isotypes of human monoclonal antibodies to
CLDN18.2 (e.g.,
IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.
"Reduce", "decrease" or "inhibit" as used herein means an overall decrease or
the ability to cause
an overall decrease, preferably of 5% or greater, 10% or greater, 20% or
greater, more preferably
of 50% or greater, and most preferably of 75% or greater, in the level, e.g.
in the level of
expression or in the level of proliferation of cells.
Terms such as "increase" or "enhance" preferably relate to an increase or
enhancement by about
at least 10%, preferably at least 20%, preferably at least 30%, more
preferably at least 40%, more
preferably at least 50%, even more preferably at least 80%, and most
preferably at least 100%, at
least 200%, at least 500%, at least 1000%, at least 10000% or even more.
Mechanisms of mAb action
Although the following provides considerations regarding the mechanism
underlying the
therapeutic efficacy of antibodies of the invention it is not to be considered
as limiting to the
invention in any way.
The antibodies described herein preferably interact with components of the
immune system,
preferably through ADCC or CDC. Antibodies described herein can also be used
to target
payloads (e.g., radioisotopes, drugs or toxins) to directly kill tumor cells
or can be used
synergistically with traditional chemotherapeutic agents, attacking tumors
through
complementary mechanisms of action that may include anti-tumor immune
responses that may
have been compromised owing to a chemotherapeutic's cytotoxic side effects on
T lymphocytes.
However, antibodies described herein may also exert an effect simply by
binding to CLDN18.2
on the cell surface, thus, e.g. blocking proliferation of the cells.
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Antibody-dependent cell-mediated cytotoxicity
ADCC describes the cell-killing ability of effector cells as described herein,
in particular
lymphocytes, which preferably requires the target cell being marked by an
antibody.
ADCC preferably occurs when antibodies bind to antigens on tumor cells and the
antibody Fe
domains engage Fe receptors (FcR) on the surface of immune effector cells.
Several families of
Fe receptors have been identified, and specific cell populations
characteristically express defined
Fe receptors. ADCC can be viewed as a mechanism to directly induce a variable
degree of
immediate tumor destruction that leads to antigen presentation and the
induction of tumor-
directed T-cell responses. Preferably, in vivo induction of ADCC will lead to
tumor-directed T-
cell responses and host-derived antibody responses.
Complement-dependent cytotoxicity
CDC is another cell-killing method that can be directed by antibodies. IgM is
the most effective
isotype for complement activation. IgG1 and IgG3 are also both very effective
at directing CDC
via the classical complement-activation pathway. Preferably, in this cascade,
the formation of
antigen-antibody complexes results in the uncloaking of multiple Cl q binding
sites in close
proximity on the CH2 domains of participating antibody molecules such as IgG
molecules (Clq
is one of three subcomponents of complement Cl). Preferably these uncloaked
Clq binding sites
convert the previously low-affinity Cl q¨IgG interaction to one of high
avidity, which triggers a
cascade of events involving a series of other complement proteins and leads to
the proteolytic
release of the effector-cell chemotacticiactivating agents C3a and C5a.
Preferably, the
complement cascade ends in the formation of a membrane attack complex, which
creates pores
in the cell membrane that facilitate free passage of water and solutes into
and out of the cell.
Antibodies described herein can be produced by a variety of techniques,
including conventional
monoclonal antibody methodology, e.g., the standard somatic cell hybridization
technique of
Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell
hybridization procedures are
preferred, in principle, other techniques for producing monoclonal antibodies
can be employed,
e.g., viral or oncogenic transformation of B-lymphocytes or phage display
techniques using
libraries of antibody genes.
The preferred animal system for preparing hybridomas that secrete monoclonal
antibodies is the
murine system. Hybridoma production in the mouse is a very well established
procedure.
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Immunization protocols and techniques for isolation of immunized splenocytes
for fusion are
known in the art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also
known.
Other preferred animal systems for preparing hybridomas that secrete
monoclonal antibodies are
the rat and the rabbit system (e.g. described in Spieker-Polet et al., Proc.
Natl. Acad. Sci. U.S.A.
92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).
In yet another preferred embodiment, human monoclonal antibodies can be
generated using
transgenic or transchromosomal mice carrying parts of the human immune system
rather than the
mouse system. These transgenic and transchromosomic mice include mice known as
HuMAb
mice and KM mice, respectively, and are collectively referred to herein as
"transgenic mice."
The production of human antibodies in such transgenic mice can be performed as
described in
detail for CD20 in W02004 035607
Yet another strategy for generating monoclonal antibodies is to directly
isolate genes encoding
antibodies from lymphocytes producing antibodies of defined specificity e.g.
see Babco*ck et al.,
1996; A novel strategy for generating monoclonal antibodies from single,
isolated lymphocytes
producing antibodies of defined specificities. For details of recombinant
antibody engineering
see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-
89603-918-8
and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.
To generate antibodies, mice can be immunized with carrier-conjugated peptides
derived from
the antigen sequence, i.e. the sequence against which the antibodies are to be
directed, an
enriched preparation of recombinantly expressed antigen or fragments thereof
and/or cells
expressing the antigen, as described. Alternatively, mice can be immunized
with DNA encoding
the antigen or fragments thereof. In the event that immunizations using a
purified or enriched
preparation of the antigen do not result in antibodies, mice can also be
immunized with cells
expressing the antigen, e.g., a cell line, to promote immune responses.
The immune response can be monitored over the course of the immunization
protocol with
plasma and serum samples being obtained by tail vein or retroorbital bleeds.
Mice with sufficient
titers of immunoglobulin can be used for fusions. Mice can be boosted
intraperitonealy or
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intravenously with antigen expressing cells 3 days before sacrifice and
removal of the spleen to
increase the rate of specific antibody secreting hybridomas.
To generate hybridomas producing monoclonal antibodies, splenocytes and lymph
node cells
from immunized mice can be isolated and fused to an appropriate immortalized
cell line, such as
a mouse myeloma cell line. The resulting hybridomas can then be screened for
the production of
antigen-specific antibodies. Individual wells can then be screened by ELISA
for antibody
secreting hybridomas. By Immunofluorescence and FACS analysis using antigen
expressing
cells, antibodies with specificity for the antigen can be identified. The
antibody secreting
hybridomas can be replated, screened again, and if still positive for
monoclonal antibodies can be
subcloned by limiting dilution. The stable subclones can then be cultured in
vitro to generate
antibody in tissue culture medium for characterization.
Antibodies also can be produced in a host cell transfectoma using, for
example, a combination of
recombinant DNA techniques and gene transfection methods as are well known in
the art
(Morrison, S. (1985) Science 229: 1202).
For example, in one embodiment, the gene(s) of interest, e.g., antibody genes,
can be ligated into
an expression vector such as a eukaryotic expression plasmid such as used by
the GS gene
expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or
other expression
systems well known in the art. The purified plasmid with the cloned antibody
genes can be
introduced in eukaryotic host cells such as CHO cells, NS/0 cells, HEK293T
cells or HEK293
cells or alternatively other eukaryotic cells like plant derived cells, fungal
or yeast cells. The
method used to introduce these genes can be methods described in the art such
as
electroporation, lipofectine, lipofectamine or others. After introduction of
these antibody genes
in the host cells, cells expressing the antibody can be identified and
selected. These cells
represent the transfectomas which can then be amplified for their expression
level and upscaled
to produce antibodies. Recombinant antibodies can be isolated and purified
from these culture
supernatants and/or cells.
Alternatively, the cloned antibody genes can be expressed in other expression
systems, including
prokaryotic cells, such as microorganisms, e.g. E. coli. Furthermore, the
antibodies can be
produced in transgenic non-human animals, such as in milk from sheep and
rabbits or in eggs
from hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J.
Immunol. Meth. 216: 165-
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181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R.,
et al. (1999) Biol.
Chem. 380: 825-839.
Chimerization
Murine monoclonal antibodies can be used as therapeutic antibodies in humans
when labeled
with toxins or radioactive isotopes. Nonlabeled murine antibodies are highly
immunogenic in
man when repetitively applied leading to reduction of the therapeutic effect.
The main
immunogenicity is mediated by the heavy chain constant regions. The
immunogenicity of murine
antibodies in man can be reduced or completely avoided if respective
antibodies are chimerized
or humanized. Chimeric antibodies are antibodies, the different portions of
which are derived
from different animal species, such as those having a variable region derived
from a murine
antibody and a human immunoglobulin constant region. Chimerisation of
antibodies is achieved
by joining of the variable regions of the murine antibody heavy and light
chain with the constant
region of human heavy and light chain (e.g. as described by Kraus et al., in
Methods in
Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-
89603-918-8). In
a preferred embodiment chimeric antibodies are generated by joining human
kappa-light chain
constant region to murine light chain variable region. In an also preferred
embodiment chimeric
antibodies can be generated by joining human lambda-light chain constant
region to murine light
chain variable region. The preferred heavy chain constant regions for
generation of chimeric
antibodies are IgGl, IgG3 and IgG4. Other preferred heavy chain constant
regions for generation
of chimeric antibodies are IgG2, IgA, IgD and IgM.
Humanization
Antibodies interact with target antigens predominantly through amino acid
residues that are
located in the six heavy and light chain complementarity determining regions
(CDRs). For this
reason, the amino acid sequences within CDRs are more diverse between
individual antibodies
than sequences outside of CDRs. Because CDR sequences are responsible for most
antibody-
antigen interactions, it is possible to express recombinant antibodies that
mimic the properties of
specific naturally occurring antibodies by constructing expression vectors
that include CDR
sequences from the specific naturally occurring antibody grafted onto
framework sequences from
a different antibody with different properties (see, e.g., Riechmann, L. et
al. (1998) Nature 332:
323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al.
(1989) Proc. Natl.
Acad. Sci. U. S. A. 86: 10029-10033). Such framework sequences can be obtained
from public
DNA databases that include germline antibody gene sequences. These gerrnline
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differ from mature antibody gene sequences because they will not include
completely assembled
variable genes, which are formed by V (D) J joining during B cell maturation.
Germline gene
sequences will also differ from the sequences of a high affinity secondary
repertoire antibody at
individual evenly across the variable region.
The ability of antibodies to bind an antigen can be determined using standard
binding assays
(e.g., ELISA, Western Blot, Immunofluorescence and flow cytometric analysis).
To purify antibodies, selected hybridomas can be grown in two-liter spinner-
flasks for
monoclonal antibody purification. Alternatively, antibodies can be produced in
dialysis based
bioreactors. Supernatants can be filtered and, if necessary, concentrated
before affinity
chromatography with protein G-sepharose or protein A-sepharose. Eluted IgG can
be checked by
gel electrophoresis and high performance liquid chromatography to ensure
purity. The buffer
solution can be exchanged into PBS, and the concentration can be determined by
0D280 using
1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and
stored at -80 C.
To determine if the selected monoclonal antibodies bind to unique epitopes,
site-directed or
multi-site directed mutagenesis can be used.
To determine the isotype of antibodies, isotype ELISAs with various commercial
kits (e.g.
Zymed, Roche Diagnostics) can be performed. Wells of microtiter plates can be
coated with anti-
mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or
purified isotype
controls, at ambient temperature for two hours. The wells can then be reacted
with either mouse
IgGl, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated
probes. After
washing, the plates can be developed with ABTS substrate (1 mg/ml) and
analyzed at OD of
405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit
(Roche, Cat. No.
1493027) may be used as described by the manufacturer.
In order to demonstrate presence of antibodies in sera of immunized mice or
binding of
monoclonal antibodies to living cells expressing antigen, flow cytometry can
be used. Cell lines
expressing naturally or after transfection antigen and negative controls
lacking antigen
expression (grown under standard growth conditions) can be mixed with various
concentrations
of monoclonal antibodies in hybridoma supernatants or in PBS containing 1%
FBS, and can be
incubated at 4 C for 30 min. After washing, the APC- or A1exa647-labeled anti
IgG antibody can
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bind to antigen-bound monoclonal antibody under the same conditions as the
primary antibody
staining. The samples can be analyzed by flow cytometry with a FACS instrument
using light
and side scatter properties to gate on single, living cells. In order to
distinguish antigen-specific
monoclonal antibodies from non-specific binders in a single measurement, the
method of co-
transfection can be employed. Cells transiently transfected with plasmids
encoding antigen and a
fluorescent marker can be stained as described above. Transfected cells can be
detected in a
different fluorescence channel than antibody-stained cells. As the majority of
transfected cells
express both transgenes, antigen-specific monoclonal antibodies bind
preferentially to
fluorescence marker expressing cells, whereas non-specific antibodies bind in
a comparable ratio
to non-transfected cells. An alternative assay using fluorescence microscopy
may be used in
addition to or instead of the flow cytometry assay. Cells can be stained
exactly as described
above and examined by fluorescence microscopy.
In order to demonstrate presence of antibodies in sera of immunized mice or
binding of
monoclonal antibodies to living cells expressing antigen, immunofluorescence
microscopy
analysis can be used. For example, cell lines expressing either spontaneously
or after transfection
antigen and negative controls lacking antigen expression are grown in chamber
slides under
standard growth conditions in DMEM/F12 medium, supplemented with 10 % fetal
calf serum
(FCS), 2 mM L-glutamine, 100 IU/m1 penicillin and 100 jig/ml streptomycin.
Cells can then be
fixed with methanol or paraformaldehyde or left untreated. Cells can then be
reacted with
monoclonal antibodies against the antigen for 30 min. at 25 C. After washing,
cells can be
reacted with an A1exa555-labelled anti-mouse IgG secondary antibody (Molecular
Probes) under
the same conditions. Cells can then be examined by fluorescence microscopy.
Cell extracts from cells expressing antigen and appropriate negative controls
can be prepared and
subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.
After
electrophoresis, the separated antigens will be transferred to nitrocellulose
membranes, blocked,
and probed with the monoclonal antibodies to be tested. IgG binding can be
detected using anti-
mouse IgG peroxidase and developed with ECL substrate.
Antibodies can be further tested for reactivity with antigen by
Immunohistochemistry in a
manner well known to the skilled person, e.g. using paraformaldehyde or
acetone fixed
cryosections or paraffin embedded tissue sections fixed with paraformaldehyde
from non-cancer
tissue or cancer tissue samples obtained from patients during routine surgical
procedures or from
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mice carrying xenografted tumors inoculated with cell lines expressing
spontaneously or after
transfection antigen. For immunostaining, antibodies reactive to antigen can
be incubated
followed by horseradish-peroxidase conjugated goat anti-mouse or goat anti-
rabbit antibodies
(DAKO) according to the vendors instructions.
Antibodies can be tested for their ability to mediate phagocytosis and killing
of cells expressing
CLDN18.2. The testing of monoclonal antibody activity in vitro will provide an
initial screening
prior to testing in vivo models.
Antibody dependent cell-mediated cytotoxicity (ADCC):
Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear
cells or other
effector cells, from healthy donors can be purified by Ficoll Hypaque density
centrifugation,
followed by lysis of contaminating erythrocytes. Washed effector cells can be
suspended in
RPMI supplemented with 10% heat-inactivated fetal calf serum or, alternatively
with 5% heat-
inactivated human serum and mixed with 51Cr labeled target cells expressing
CLDN18.2, at
various ratios of effector cells to target cells. Alternatively, the target
cells may be labeled with a
fluorescence enhancing ligand (BATDA). A highly fluorescent chelate of
Europium with the
enhancing ligand which is released from dead cells can be measured by a
fluorometer. Another
alternative technique may utilize the transfection of target cells with
luciferase. Added lucifer
yellow may then be oxidated by viable cells only. Purified anti-CLDN18.2 IgGs
can then be
added at various concentrations. Irrelevant human IgG can be used as negative
control. Assays
can be carried out for 4 to 20 hours at 37 C depending on the effector cell
type used. Samples
can be assayed for cytolysis by measuring 51Cr release or the presence of the
EuTDA chelate in
the culture supernatant. Alternatively, luminescence resulting from the
oxidation of lucifer
yellow can be a measure of viable cells.
Anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations
to determine
whether cytolysis is enhanced with multiple monoclonal antibodies.
Complement dependent cytotoxicity (CDC):
Monoclonal anti-CLDN18.2 antibodies can be tested for their ability to mediate
CDC using a
variety of known techniques. For example, serum for complement can be obtained
from blood in
a manner known to the skilled person. To determine the CDC activity of mAbs,
different
methods can be used. 51Cr release can for example be measured or elevated
membrane
permeability can be assessed using a propidium iodide (PI) exclusion assay.
Briefly, target cells
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can be washed and 5 x 105/m1 can be incubated with various concentrations of
mAb for 10-30
min. at room temperature or at 37 C. Serum or plasma can then be added to a
final concentration
of 20% (v/v) and the cells incubated at 37 C for 20-30 min. All cells from
each sample can be
added to the PI solution in a FACS tube. The mixture can then be analyzed
immediately by flow
cytometry analysis using FACSArray.
In an alternative assay, induction of CDC can be determined on adherent cells.
In one
embodiment of this assay, cells are seeded 24 h before the assay with a
density of 3 x 104/well in
tissue-culture flat-bottom microtiter plates. The next day growth medium is
removed and the
cells are incubated in triplicates with antibodies. Control cells are
incubated with growth medium
or growth medium containing 0.2% saponin for the determination of background
lysis and
maximal lysis, respectively. After incubation for 20 min. at room temperature
supernatant is
removed and 20% (v/v) human plasma or serum in DMEM (prewarmed to 37 C) is
added to the
cells and incubated for another 20 min. at 37 C. All cells from each sample
are added to
propidium iodide solution (10 gimp. Then, supernatants are replaced by PBS
containing 2.5
g/m1 ethidium bromide and fluorescence emission upon excitation at 520 nm is
measured at
600 nm using a Tecan Safire. The percentage specific lysis is calculated as
follows: % specific
lysis = (fluorescence sample-fluorescence background)/ (fluorescence maximal
lysis-
fluorescence background) x 100.
Induction of apoptosis and inhibition of cell proliferation by monoclonal
antibodies:
To test for the ability to initiate apoptosis, monoclonal anti-CLDN18.2
antibodies can, for
example, be incubated with CLDN18.2 positive tumor cells, e.g., SNU-16, DAN-G,
KATO-III
or CLDN18.2 transfected tumor cells at 37 C for about 20 hours. The cells can
be harvested,
washed in Annexin-V binding buffer (BD biosciences), and incubated with
Annexin V
conjugated with FITC or APC (BD biosciences) for 15 min. in the dark. All
cells from each
sample can be added to PI solution (10 1.tg/m1 in PBS) in a FACS tube and
assessed immediately
by flow cytometry (as above). Alternatively, a general inhibition of cell-
proliferation by
monoclonal antibodies can be detected with commercially available kits. The
DELFIA Cell
Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic
immunoassay based on the
measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA
synthesis of
proliferating cells in microplates. Incorporated BrdU is detected using
europium labelled
monoclonal antibody. To allow antibody detection, cells are fixed and DNA
denatured using Fix
solution. Unbound antibody is washed away and DELFIA inducer is added to
dissociate
europium ions from the labelled antibody into solution, where they form highly
fluorescent
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chelates with components of the DELFIA Inducer. The fluorescence measured -
utilizing time-
resolved fluorometry in the detection - is proportional to the DNA synthesis
in the cell of each
well.
Preclinical studies
Monoclonal antibodies which bind to CLDN18.2 also can be tested in an in vivo
model (e.g. in
immune deficient mice carrying xenografted tumors inoculated with cell lines
expressing
CLDN18.2, e.g. DAN-G, SNU-16, or KATO-III, or after transfection, e.g. HEK293)
to
determine their efficacy in controlling growth of CLDN18.2-expressing tumor
cells.
In vivo studies after xenografting CLDN18.2 expressing tumor cells into
immunocompromised
mice or other animals can be performed using antibodies described herein.
Antibodies can be
administered to tumor free mice followed by injection of tumor cells to
measure the effects of the
antibodies to prevent formation of tumors or tumor-related symptoms.
Antibodies can be
administered to tumor-bearing mice to determine the therapeutic efficacy of
respective
antibodies to reduce tumor growth, metastasis or tumor related symptoms.
Antibody application
can be combined with application of other substances as cystostatic drugs,
growth factor
inhibitors, cell cycle blockers, angiogenesis inhibitors or other antibodies
to determine
synergistic efficacy and potential toxicity of combinations. To analyze toxic
side effects
mediated by antibodies animals can be inoculated with antibodies or control
reagents and
thoroughly investigated for symptoms possibly related to CLDN18.2-antibody
therapy. Possible
side effects of in vivo application of CLDN18.2 antibodies particularly
include toxicity at
CLDN18.2 expressing tissues including stomach. Antibodies recognizing CLDN18.2
in human
and in other species, e.g. mice, are particularly useful to predict potential
side effects mediated
by application of monoclonal CLDN18.2-antibodies in humans.
Mapping of epitopes recognized by antibodies can be performed as described in
detail in
"Epitope Mapping Protocols (Methods in Molecular Biology) by Glenn E. Morris
ISBN-089603-
375-9 and in "Epitope Mapping: A Practical Approach" Practical Approach
Series, 248 by
Olwyn M. R. Westwood, Frank C. Hay.
The compounds and agents described herein may be administered in the form of
any suitable
pharmaceutical composition.

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Pharmaceutical compositions are usually provided in a uniform dosage form and
may be
prepared in a manner known per se. A pharmaceutical composition may e.g. be in
the form of a
solution or suspension.
A pharmaceutical composition may comprise salts, buffer substances,
preservatives, carriers,
diluents and/or excipients all of which are preferably pharmaceutically
acceptable. The term
"pharmaceutically acceptable" refers to the non-toxicity of a material which
does not interact
with the action of the active component of the pharmaceutical composition.
Salts which are not pharmaceutically acceptable may used for preparing
pharmaceutically
acceptable salts and are included in the invention. Pharmaceutically
acceptable salts of this kind
comprise in a non limiting way those prepared from the following acids:
hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric,
formic, malonic,
succinic acids, and the like. Pharmaceutically acceptable salts may also be
prepared as alkali
metal salts or alkaline earth metal salts, such as sodium salts, potassium
salts or calcium salts.
Suitable buffer substances for use in a pharmaceutical composition include
acetic acid in a salt,
citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.
Suitable preservatives for use in a pharmaceutical composition include
benzalkonium chloride,
chlorobutanol, paraben and thimerosal.
An injectible formulation may comprise a pharmaceutically acceptable excipient
such as Ringer
Lactate.
The term "carrier" refers to an organic or inorganic component, of a natural
or synthetic nature,
in which the active component is combined in order to facilitate, enhance or
enable application.
According to the invention, the term "carrier" also includes one or more
compatible solid or
liquid fillers, diluents or encapsulating substances, which are suitable for
administration to a
patient.
Possible carrier substances for parenteral administration are e.g. sterile
water, Ringer, Ringer
lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated
naphthalenes and, in
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particular, biocompatible lactide polymers, lactide/glycolide copolymers or
polyoxyethylene/polyoxy- propylene copolymers.
The term "excipient" when used herein is intended to indicate all substances
which may be
present in a pharmaceutical composition and which are not active ingredients
such as, e.g.,
carriers, binders, lubricants, thickeners, surface active agents,
preservatives, emulsifiers, buffers,
flavoring agents, or colorants.
The agents and compositions described herein may be administered via any
conventional route,
such as by parenteral administration including by injection or infusion.
Administration is
preferably parenterally, e.g. intravenously, intraarterially, subcutaneously,
intradermally or
intramuscularly.
Compositions suitable for parenteral administration usually comprise a sterile
aqueous or
nonaqueous preparation of the active compound, which is preferably isotonic to
the blood of the
recipient. Examples of compatible carriers and solvents are Ringer solution
and isotonic sodium
chloride solution. In addition, usually sterile, fixed oils are used as
solution or suspension
medium.
The agents and compositions described herein are administered in effective
amounts. An
"effective amount" refers to the amount which achieves a desired reaction or a
desired effect
alone or together with further doses. In the case of treatment of a particular
disease or of a
particular condition, the desired reaction preferably relates to inhibition of
the course of the
disease. This comprises slowing down the progress of the disease and, in
particular, interrupting
or reversing the progress of the disease. The desired reaction in a treatment
of a disease or of a
condition may also be delay of the onset or a prevention of the onset of said
disease or said
condition.
An effective amount of an agent or composition described herein will depend on
the condition to
be treated, the severeness of the disease, the individual parameters of the
patient, including age,
physiological condition, size and weight, the duration of treatment, the type
of an accompanying
therapy (if present), the specific route of administration and similar
factors. Accordingly, the
doses administered of the agents described herein may depend on various of
such parameters. In
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the case that a reaction in a patient is insufficient with an initial dose,
higher doses (or effectively
higher doses achieved by a different, more localized route of administration)
may be used.
The agents and compositions described herein can be administered to patients,
e.g., in vivo, to
treat or prevent a variety of disorders such as those described herein.
Preferred patients include
human patients having disorders that can be corrected or ameliorated by
administering the agents
and compositions described herein. This includes disorders involving cells
characterized by an
altered expression pattern of CLDN18.2.
For example, in one embodiment, antibodies described herein can be used to
treat a patient with
a cancer disease, e.g., a cancer disease such as described herein
characterized by the presence of
cancer cells expressing CLDN1 8.2.
The pharmaceutical compositions and methods of treatment described according
to the invention
may also be used for immunization or vaccination to prevent a disease
described herein.
The present invention is further illustrated by the following examples which
are not be construed
as limiting the scope of the invention.
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EXAMPLES
Example 1: CLDN18.2 expression of human gastric cancer cell lines is
stabilized by in vitro
treatment with chemotherapeutic agents
KatoIII cells, a human gastric tumor cell line, was cultivated in RPMI 1640
medium (Invitrogen)
containing 20% FCS (Perbio) and 2 mM Glutamax (Invitrogen) at 37 C and 5% CO2,
with or
without cytostatic compounds. Epirubicin (Pfizer) was tested at a
concentration of 10 or 100
ng/ml, 5-FU (Neofluor from NeoCorp AG) was tested at a concentration of 10 or
100 ng/ml, and
oxaliplatin (Hospira) was tested at a concentration of 50 or 500 ng/ml. A
combination of all 3
compounds (EOF; epirubicin 10 ng/ml, oxaliplatin 500 ng/ml, 5-FU 10 ng/ml) was
also used.
8x105 KatoIII cells were cultivated for 96 hours without medium change or for
72 hours
followed by 24 hours cultivation in standard medium to release cells from cell
cycle arrest in a 6-
well tissue culture plate at 37 C, 5% CO2. Cells were harvested with
EDTA/trypsin, washed and
analysed.
For extracellular detection of CLDN18.2 cells were stained with the monoclonal
anti-CLDN18.2
antibody IMAB362 (Ganymed) or an isotyp-matched control antibody (Ganymed). As
secondary
reagent goat-anti-huIgG-APC from Dianova was used.
Cell cycle stages were determined based on measurement of cellular DNA
content. This allows
one to discriminate between cells in the G1 S- or G2-phase of the cell cycle.
In the S-phase
DNA duplication occurs whereas in the G2-phase cells grow and prepare for
mitosis. Cell cycle
analysis was done using the CycleTEST PLUS DNA Reagent Kit from BD Biosciences

following the manufacturer's protocol. Flow cytometry acquisition and analysis
were performed
by using BD FACS CantoII (BD Biosciences) and FlowJo (Tree Star) software.
The columns in Figure la and b show the respective percentage of cells in the
G1 S- or G2-
phase of the cell cycle. Medium cultivated KatoIII cells show a cell cycle
arrest predominantly in
the G1 -phase. Cells treated with 5-FU are blocked predominantly in the S-
phase. Epirubicin- or
EOF-treated KatoIII cells show a cell cycle arrest predominantly in the G2-
phase. Oxaliplatin
treated KatoIII cells show enrichment of cells predominantly in the G1 - and
G2-phases. As can
be seen in Figure 1 c, a cell cycle arrest in the S-phase or G2-phase results
in stabilization or
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upregulation of CLDN18.2. As soon as cells are released from any phase of the
cell cycle (Figure
lb) the expression of CLDN18.2 on the cell surface of KatoIII cells is
upregulated (Figure 1d).
NUGC-4 and KATO III cells were treated with 5-FU + OX (10 ng/ml 5-FU and 500
ng/ml
oxaliplatin), EOF (10 ng/ml epirubicin, 500 ng/ml oxaliplatin and 10 ng/ml 5-
FU) or FLO (10
ng/ml 5-FU, 50 ng/ml folinic acid and 500 ng/ml oxaliplatin) for 96 hours. RNA
of
chemotherapy pretreated NUGC-4 and KATO III cells was isolated and converted
to cDNA.
CLDN18.2 transcript level was analysed in quantitative real-time PCR. Results
are shown in
Figure 2a as relative expression in comparison to the transcript level of the
housekeeping gene
HPRT. Figure 2b shows a Western blot of CLDN18.2 and actin loading control of
untreated and
treated NUGC-4 cells. The intensity of the luminescence signal is shown in
relation to actin in
percent.
Pretreatment of NUGC-4 and KATO III cells with EOF, FLO as well as 5-FU + OX
combination
chemotherapies results in increased RNA and protein levels of CLDN18.2 as
shown by
quantitative real-time PCR (Figure 2a) and Western blot (Figure 2b).
IMAB362 binding on NUGC-4 and KATO III gastric cancer cells treated with EOF
(10 ng/ml
epirubicin, 500 ng/ml oxaliplatin and 10 ng/ml 5-FU) or FLO (10 ng/ml 5-FU, 50
ng/ml folinic
acid and 500 ng/ml oxaliplatin) for 96 hours by flow cytometry was analysed.
The amount of
CLDN18.2 protein targetable by IMAB362 on the surface of gastric cancer cell
lines is increased
as shown in Figure 2c. This effect was most prominent in cells pretreated with
EOF or FLO.
KatoIII cells were pretreated for 4 days with Irinotecan or Docetaxel and
analysed for
CLDN18.2 expression and cell cycle arrest. Treatment of cells with Irinotecan
resulted in a dose
dependent inhibition of cell growth and a cell cycle arrest in the S/G2-phase
(Figure 3).
Treatment of cells with Docetaxel resulted in a dose dependent inhibition of
cell growth and a
cell cycle arrest in the G2-phase (Figure 3).
Example 2: Pretreatment of human gastric cancer cells with chemotherapeutics
results in higher
efficiency of IMAB362-mediated ADCC
IMAB362-mediated ADCC was investigated using NUGC-4 gastric cancer cells as
target, which
were either pretreated with 10 ng/ml 5-FU and 500 ng/ml oxaliplatin (5-FU +
OX), 10 ng/ml

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epirubicin, 500 ng/ml oxaliplatin and 10 ng/ml 5-FU (EOF) or 10 ng/ml 5-FU, 50
ng/ml folinic
acid and 500 ng/ml oxaliplatin (FLO) for 96 hours (effector:target ratio 40:1)
or untreated. ECso
values were obtained from 7 healthy donors for untreated and EOF, FLO or 5-FU
+ OX
pretreated NUGC-4 cells.
As shown in Figure 4a, dose/response curves on pretreated cells shifted
upwards and to the left
compared to untreated target cells. This resulted in a higher maximal lysis
and in a decrease of
the EC50 values to one third of untreated cells (Figure 4b).
Peripheral blood mononuclear cells (PBMCs) including NK cells, monocytes,
mononuclear cells
or other effector cells from healthy human donors were purified by Ficoll
Hypaque density
centrifugation. Washed effector cells were seeded in X-Vivo medium. KatoIII
cells which
express CLDN18.2 endogenously and are of gastric origin were used as target
cells in this
setting. Target cells stably expressed luciferase, lucifer yellow, which is
oxidized by viable cells
only. Purified anti-CLDN18.2 antibody IMAB362 was added at various
concentrations and as an
isotype control antibody an irrelevant chim huIgG1 antibody was used. Samples
were assayed
for cytolysis by measuring luminescence resulting from the oxidation of
lucifer yellow which is a
value for the amount of viable cells left after IMAB362 induced cytotoxicity.
KatoIII pretreated
for 3 days with Irinotecan (1000 ng/ml), Docetaxel (5 ng/ml) or Cisplatin
(2000 ng/ml) were
compared to untreated medium cultivated target cells and IMAB362 induced ADCC
was
quantified.
KatoIII cells pretreated for 3 days with Irinotecan, Docetaxel or Cisplatin
exhibited a lower level
of viable cells compared to medium cultivated target cells (Figure 5a) and
claudin18.2
expression in cells pretreated with Irinotecan, Docetaxel or Cisplatin was
increased compared to
medium cultivated cells (Figure 5b).
Furthermore, pretreatment of KatoIII cells with Irinotecan, Docetaxel or
Cisplatin augmented the
potency of IMAB362 to induce ADCC (Figure 5c, d).
Example 3: Chemotherapy results in higher efficiency of IMAB362-induced CDC
Effects of chemotherapeutic agents on IMAB362-induced CDC were analyzed by
pretreating
KATO III gastric cancer cells with 10 ng/ml 5-FU and 500 ng/ml oxaliplatin (5-
FU + OX) for 48
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hours. Representative dose response curves of IMAB362-induced CDC using
chemotherapeutic
pretreated KATO III cells are shown in Figure 6. Pretreatment of tumor cells
for 48 hours
augmented the potency of IMAB362 to induce CDC, resulting in higher maximal
cell lysis of
pretreated tumor cells compared to untreated cells.
Example 4: Capability of immune effector cells to execute IMAB362-mediated
ADCC is not
compromised by treatment with chemotherapeutics
Chemotherapeutic agents used in EOF or FLO regimen are highly potent in
inhibition of target
cell proliferation. To investigate adverse effects of chemotherapy on effector
cells, PBMCs from
healthy donors were treated with 10 ng/ml epirubicin, 500 ng/ml oxaliplatin
and 10 ng/ml 5-FU
(EOF) or 10 ng/ml 5-FU, 50 ng/ml folinic acid and 500 ng/ml oxaliplatin (FLO)
for 72 hours
before application in ADCC assays. Figure 7a shows EC50 values of 4 healthy
donors and Figure
7b shows representative dose/response curves of IMAB362-induced ADCC using EOF
or FLO
pretreated effector cells. IMAB362-induced ADCC of NUGC-4 gastric carcinoma
cells is not
compromised due to EOF or FLO chemotherapies.
Example 5: A combination of ZA/IL-2 treatment results in optimized expansion
of peripheral
blood mononuclear cell (PBMC) cultures
The effect of ZA/IL-2 on proliferation of PBMC cultures was assessed in vitro.
PBMCs were
harvested from healthy human donors and cultures were treated with a single
dose of ZA. IL-2
was added every 3-4 days. Specifically, PBMC derived from 3 different healthy
human donors
(#1, #2, #3) were cultured in RPMI medium (1x106 cells/nil) for 14 days with 1
1.1M ZA plus
high (300 U/ml) or low (25 U/ml) doses of IL-2; cf. Figure 8a. PBMCs of the
same donors were
cultured additionally in RPMI medium for 14 days with 300 U/ml IL-2 plus ZA or
without ZA;
cf. Figure 8b. Increase in cell numbers was determined by counting living
cells on day 6, 8, 11
and 14.
In medium supplied with a high dose of IL-2 about 2 - 5fold more cells
expanded, as compared
to cultures supplied with a low IL-2 dose (Figure 8a). Expansion of cells in
medium without ZA
was approximately 2fold lower as compared to cells grown in medium with ZA
(Figure 8b).
These data show the necessity to apply both ZA and IL-2 compounds in
combination to ensure
proper expansion of the cells.
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Example 6: ZAJIL-2 treatment results in expansion of high amounts of Vy9V82 T-
cells in PBMC
cultures
PBMCs were cultivated for 14 days in RPMI medium supplemented with 300 U/ml IL-
2 and
with or w/o 1 tiM ZA. The percentage of V19+VS2+ T cells within the CD3+
lymphocyte
population (Figure 9a) and the percentage of CD16+ cells within the
CD3+Vy9+V.52+ T cell
population (Figure 9b) was determined by multicolor FACS on day 0 and day 14.
Results were
scored for each donor in the scatter plot. Figure 9c shows a scatter plot
displaying the increase
over time (enrichment) in the number of CD3+ Vy9+V.52+ and CD3+CD16+ Vy9+V62+
T cells
within the lymphocyte population. The amount of cells seeded on day 0 and the
amount of cells
harvested on day 14 were taken into account.
IL-2 addition in the PBMC cultures is required for survival and growth of
lymphocytes. They
efficiently expand in cultures supplied with 300 U/ml IL-2. FACS analysis
using V19 and V82
specific antibodies reveal that addition of ZA/IL-2 specifically induces the
accumulation of
Vy9V82 T cells (Figure 9a). After 14 days, the CD3+ lymphocyte population can
comprise up to
80% of Vy9V62 T cells. A portion of Vy9V62 T cells express CD16, whereas
enrichment of
these cells within the CD3+ lymphocyte population is 10-700fold, dependent on
the donor
(Figures 9b and 9c). Enrichment of the CD16+Vy9+V82+ T cells in the cultures
is 10-600fold
higher as compared to cultures grown without ZA (Figure 9c). We conclude that
ZA/IL-2
treatment of PBMCs in vitro results in the up-regulation of the ADCC-mediating
FcyIII receptor
CD16 in a significant proportion ofy8 T cells.
Example 7: IL-2 affects expansion of Vy9V82 T cells in a dose-dependent manner
Addition of ZA in the cultures is the most important factor to induce
development of V79V82 T
cells. It is well known, that IL-2 is required for growth and survival of T
cells.
PBMCs were cultivated for 14 days in RPMI medium supplemented with 1 M ZA and

increasing IL-2 concentrations. IL-2 was added on day 0 and day 4. The
enrichment of
CD16+Vy9+V62+ T cells within the CD3+ lymphocyte population was determined by
multicolor FACS staining on day 0 and day 14. To compare the different donors,
the amount of
CD16+Vy9+VS2+ T cells harvested after cultivation with 600 U/ml IL-2 was set
to 100%; cf.
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Figure 10, left. Furthermore, ADCC activity of the isolated cultures grown for
14 days in
increasing concentrations of IL-2 was tested; cf. Figure 10, right.
We confirmed by dose response analysis that IL-2 also stimulates growth and
survival of the
Vy9V82 T cell subset. By adding low IL-2 concentrations in the medium, a
correlation was
found between IL-2 dose and the percentage of CD16+Vy9V82 T cells within the
CD3+
lymphocyte population (Figure 10, left). ADCC activity of the cells grown in
higher IL-2
concentrations (150-600U/m1) is improved compared to cells grown in low IL-2
concentrations
(Figure 10, right).
Example 8: ZA induces IPP production in monocytes and cancer cells stimulating
both the
expansion of Vy9V62 T cells
Fresh PBMCs (Exp. #1) or 14 day ZA/IL-2 stimulated Vy9V62 T cell cultures
(Exp. #2-5) were
incubated either without monocytes (effector:monocyte ratio 1:0), with 0.2fold
(4:1) or 5fold
(ratio 1:4) the amount of monocytes + 1 tM ZA. The enrichment of Vy9V62 T
cells in the co-
cultures after 14 days was determined by multicolor FACS, whereas the
expansion of the culture
was considered in the calculation. The enrichment factor of V79VS2 T cells
cultured with
monocytes in a 1:4 ratio was set to 100% for each experiment. The increase of
monocytes in the
culture resulted in an enrichment of Vy9V62 T cells of more than 10fold. This
effect was clearly
ZA-dependent; cf Figure 1 la.
Furthermore, human stomach cancer cells (NUGC-4-luciferase) and murine stomach
cancer cells
(CLS103 - calcein stained) were pretreated with or w/o 5 1AM ZA for 2 days.
Human Vy9VS2 T
cells were MACS purified (day 14) and co-cultured with the cancer cells for 24
h. Cytotoxicity
of Vy9V52 T cells towards non-treated and ZA-treated target cells was
determined by measuring
remaining luciferase activity or calcein fluorescence; cf. Figure 11b. Target
cells (NUGC-4 and
CLS103) were pretreated with or w/o 5 [tM ZA for 2 days and subsequently
incubated for 4 h
with mitomycin c (50 MI) to stop proliferation. MACS purified human 14d old
resting V79VS2
T cells and 3H-thymidine were added to the target cells and co-cultures were
incubated for 48 h
at 37 C. Proliferation was determined by measuring 3H thymidine incorporation
in the DNA
using a MicroBeta scintillation counter. Proliferation of target cells not
treated with ZA and w/o
Vy9V.32 T cells was set to 100%; cf. Figure 11c.
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As shown in Figures 1 lb and 11c the ZA-pulsed human cancer cells activated
Vy9V82 T cells in
terms of cytotoxicity (5-10fold) and proliferation (1.4-1.8fold), whereas the
murine cancer cell
line CLS103 failed to elicit these effects on V19V82 T cells.
Example 9: ZA/IL-2 treatment affects composition of PBMC cultures
Growth and differentiation of specific cell types in PBMC cultures depends on
presence of
cytokines. These components are either added to the medium (e.g. growth
factors present in the
serum, IL-2) or secreted by the immune cells themselves. Which type of cells
evolves also
depends on the initial composition of the PBMCs and on genetic endowments. To
analyze the
overall increase in effector cells (NK cells and Vy9V52 T cells) PBMCs of 10
different donors
were grown in the presence of 300 U/ml IL-2 and with or w/o 1 1.1.M ZA for 14
days. The amount
of effector cells within the lymphocyte population was identified by
multicolor FACS staining
using CD3, CD16, CD56, Vy9 and Vo2 antibodies. CD3-CD56+CD16+ cells represent
NK cells
and CD3+V19+V82+ represent Vy9V82 T cells.
Multicolor FACS analysis revealed that upon IL-2 treatment mainly NK cells
develop, whereas
in ZA/IL-2 treated cultures Vy9V62 T cells are predominantly expanded (Figure
12).
Example 10: ZA/IL-2 treatment generates Vy9Vo2+ effector memory T cells
Subpopulations of T lymphocytes can be delineated with the help of two surface
markers, the
high m.w. isoform of the common lymphocyte antigen CD45RA and the chemokine
receptor
CCR7. CCR7+ naive and central-memory (CM) T cells are characterized by the
ability to
repeatedly circulate into lymph nodes and encounter antigen. In contrast,
effector-memory (EM)
and effector T lymphocytes RA+ (TEMRA) down-regulate CCR7 and appear
specialized in
migrating to peripheral nonlymphoid tissues e.g. to infected or tumor sites.
The EM cells can be
further subdivided based on differential CD27 and CD28 expression. Progressive
loss of CD28
and CD27 surface expression is concomitant with up-regulation of cytolytic
capacity of the cells.
In addition the level of CD57 correlates with the expression of granzymes and
perforMs and thus
represents a third marker displaying cytotoxicity/cell maturation.
PBMCs were cultivated with or w/o 1 1AM ZA and 300 U/ml IL-2 for 14 days. The
expression of
the different surface markers was determined by multicolor FACS analysis on
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and day 14. Naive cells are CD45RA+ CCR7+, central memory cells (CM) are
CD45RA-
CCR7+, TEMRA are CD45RA+ CCR7- and effector memory cells (EM) are negative for
both
markers; cf. Figure 13a. Furthermore, cytolytic activity of the Vy9V62 1-cells
was determined
by staining for CD27 and CD57 markers; cf. Figure 13b,c. In addition, the
development of NK
cell-like characteristics important for ADCC activity was analyzed by staining
CD3+ cells with
CD16 (antibody binding) and CD56 (adhesion); cf. Figure 13d.
Multicolor FACS analysis of the Vy9V62 T cells revealed that ZA/IL-2 treatment
clearly
stimulated development of Vy9V62 T cells of the EM type which are CD27- and
CD57+ (Figure
13b-c). In addition to enhanced cytolytic activity, an increase in the level
of CD16 and CD56,
which are known from NK cells (CD3-CD16+CD56+) to be involved in ADCC was
observed in
the CD3+ population (Figure 13d).
Taken together, these data imply that ZA treatment of PBMCs results in the
development of
CD16+Vy9+V62+ effector memory T cells, which are able to migrate to peripheral
nonlymphoid
tissues and which display markers of high cytolytic activity. In combination
with the IMAB362
tumor targeting antibody these cells are extremely well harnessed to migrate
to, target and kill
tumor cells.
Example 11: ZA/IL-2 expanded Vy9V62 T cells are potent effectors for IMAB362-
mediated
CLDN18.2 dependent ADCC
Similar to NK cells, the ZA/IL-2 expanded Vy9V62 T cells are positive for CD16
(see Figure 9
and 13), the FcyRIII receptor via which a cell-bound antibody triggers ADCC.
To evaluate
whether Vy9V62 T cells are capable of inducing potent ADCC in conjunction with
IMAB362 a
series of experiments has been performed.
PBMCs derived from 2 different donors (#1 and #2) were cultivated in medium
with 300 U/ml
IL-2 and with or w/o 1 iM ZA. After 14 days cells were harvested and added
with increasing
concentrations (0.26 ng/ml - 200 ii.g/m1) of IMAB362 to NUGC-4 cells
expressing CLDN18.2.
Specific killing was determined in luciferase assays; cf. Figure 14a. Figure
14b,c gives an
overview of ADCC assays performed with 27 donors grown in 300 U/ml IL-2 and
either with or
w/o ZA. NUGC-4 served as target cells. For each donor, the EC50 values (b)
calculated from the
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dose-response curves and the maximum specific killing rate at a dose of 200
g/m1 IMAB362 (c)
were scored in the scatter plots.
Strong IMAB362-dependent ADCC activity was observed against CLDN18.2-positive
NUGC-4
cells using PBMCs cultivated for 14 days with ZA/IL-2 (Figure 14a). Using
ZA/IL-2-treated
PBMC cultures, ADCC depends on the presence of V79V.32 T cells (Figures 12 and
15). If cells
are cultured without ZA, ADCC activity is reduced for most donors. In these
cultures, residual
ADCC activity is NK-cell dependent (Figures 11 and 14). By testing more than
20 donors,
ADCC assays reveal that ZA/IL-2 treatment of PBMCs improves the EC50 and
maximum
specific killing rates as compared to PBMCs cultured with IL-2 alone.
Furthermore, PBMCs of two different donors (#1 + #2) were cultured with 1 p,M
ZA and 300
Wm' IL-2. These effector cell cultures were used in ADCC assays with CLDN18.2-
positive
(NUGC-4, KATO III) and negative (SK-BR-3) human target cell lines (E:T ratio
40:1).
Increasing amounts (0.26 ng/ml - 200 gimp of IMAB362 antibody were added.
ADCC was
measured in luciferase assays; cf. Figure 15a. Same experiment as described in
(a) was
performed with NUGC-4 target cells and effector cells harvested from cultures
treated with
ZA/IL-2 at different time points; cf. Figure 15b. Same experiment as described
in (a) was
performed using NUGC-4 as target cells; cf. Figure 15c. The ZA/IL-2 expanded
cells were either
used directly, or Vy9V52 T cells were purified from the cultures using TCRy6
MACS sorting
(Miltenyi Biotech). A purity of more than 97.0% V19V62 T cells in lymphocytes
was obtained.
Strong ADCC activity against CLDN18.2-positive, but not CLDN18.2-negative
human tumor
cell lines has been observed (Figure 15a). Furthermore, no ADCC activity is
obtained with
isotype control antibodies (not shown). In the course of ZA/IL-2 treatment
ADCC lytic activity
increases over time for a fraction of donors (Figure 15b). The dose/effect
curve of IMAB362
shifts upward and to the left showing improved EC50 values and maximum lysis
rates over time.
Compared to unconditioned PBMC, the V79\762 effector T cells enriched by ZA/IL-
2 treatment
are capable of reaching a higher maximum killing rate of CLDN18.2-positive
target cells plus
they require lower concentrations of IMAB362 for the same killing rate.
To confirm that Vy9VS2 T cells are the reservoir for lytic activity, these
cells were isolated with
> 97% purity by magnetic cell sorting from ZA/IL-2-cultured PBMC populations
on day 14. The
ADCC activity in conjunction with IMAB362 is retained and partly improved due
to higher
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purity. These data confirm that V79\762 T cells are mainly responsible for the
ADCC activity
observed with 14 days old PBMC cultures (Figure 15c).
Example 12: Treatment of target cell lines with ZA/IL-2 does not affect
surface expression of
CLDN18.2
IMAB362 triggered modes of action are strictly dependent on the presence and
amount of
extracellular detectable CLDN18.2. Therefore the influence of ZA/IL-2
treatment on CLDN18.2
surface density has been analyzed by flow cytometry using endogenous CLDN18.2
expressing
NUGC-4 and KATO III cell lines. Specifically, flow cytometric analysis of
IMAB362 binding
on unpermeabilized NUGC-4 gastric cancer cells pretreated with ZA/IL-2 or
ZA/IL-2+EOF or
ZA/IL-2+5-FU/OX for 72 hours was performed.
ZA/IL-2 treatment in vitro reveals no change in amount of CLDN18.2 surface
localization; cf.
Figure 16.
Example 13: Augmentation of IMAB362-mediated ADCC by ZA/IL-2 treatment of
PBMCs is
not compromised by EOF pretreatment
Chemotherapeutic agents compromise cell proliferation. In contrast ZA/IL-2
treatment triggers
expansion of Vy9V82 T cells. To analyze the influences of these opposing
interactions on
effector cells, PBMCs of 6 healthy donors were cultured with ZA/IL-2 or ZA/IL-
2+EOF for 8
days before application in ADCC assays (E:T ratio 15:1). IMAB362
concentrations resulting in
50% ADCC mediated lysis of untreated NUGC-4 target cells (EC50) were
determined.
Augmentation of IMAB362 induced ADCC of NUGC-4 cells due to PBMC treatment
with
ZA/IL-2 is not significantly altered by combined treatment of PBMCs with EOF
(Figure 17).
Example 14: In vivo targeting of IMAB362 to CLDN18.2-positive tumors and
antitumoral
effects of IMAB362 on human tumor cell xenografts in nude mice
To investigate in vivo tumor cell targeting of IMAB362, 80n Dyelight 680-
labeled antibody
was administered intravenously to nude mice that were xenografted
subcutaneously with the
human gastric cancer cell line NUGC-4. NUGC-4 cells display surface expression
of CLDN18.2
as well as of HER2/neu (target of trastuzumab), but are negative for CD20.
Control studies were
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conducted injecting NUGC-4 engrafted groups of mice with either Dyelight 680-
labeled
trastuzumab (positive control group) or Dyelight 680-labeled rituximab
(negative control).
IMAB362 accumulates strongly and exclusively in the tumor xenografts, as
demonstrated by live
imaging of mice using a Xenogen fluorescence imaging system 24 hours after
i.v. injection of
antibodies (Figure 18). IMAB362 is efficiently retained in the target-positive
tumor and
detectable in comparable intensity even after 120 hours (Figure 18).
Trastuzumab is also detected
exclusively in the xenografts 24 hours after injection. The trastuzumab signal
is rapidly washed
out within 120 hours after injection. No signal is detected with rituximab.
Furthermore, IMAB362 was used to treat nude mice bearing CLDN18.2-positive
xenograft
tumors. Early treatment model studies (with administrations of IMAB362 as
early as 3 days after
tumor cells inoculation) were conducted. Moreover, advanced tumor treatment
experiments were
initiated up to 9 days after tumor cell inoculation when tumors had reached
volumes of about 60
-120 mm3.
Nude mice were subcutaneously inoculated with 1 x107 HEK293¨CLDN18.2
transfectants.
Treatment of 10 mice per group started 3 days after tumor inoculation. Mice
were treated with
200 jig IMAB362, infliximab as isotype control and PBS twice per week for 6
weeks alternating
intravenous and intraperitoneal routes of application. Whereas all mice in the
groups treated with
either PBS or isotype control died within 70 - 80 days, animals treated with
IMAB362 had a
survival benefit (Figure 19). Not only time to death was prolonged, but 4 of
10 mice survived the
entire observation period of 210 days.
Treatment of 9 to 10 mice per group was initiated when mean tumor volumes
reached 88 mm3
(62 - 126 mm3). Prior to treatment mice were stratified into test groups to
ensure comparable
tumor sizes in all groups. Mice were treated with 200 jig IMAB362, isotype
control or PBS
twice per week for 6 weeks alternating intravenous and intraperitoneal routes
of application. All
mice in the groups treated with either PBS or isotype control died within 50-
100 days. Animals
treated with IMAB362 had a survival benefit, with nearly a doubling of median
survival time (47
versus 25 days). Three of these mice survived the entire observation period
(Figure 20).
Importantly, antitumoral efficacy in vivo depends on the presence of the
target on the tumor
cells. No antitumoral effects of IMAB362 treatment were seen in mice engrafted
with
CLDN18.2-negative HEI(293 tumor cells.
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The NUGC-4 gastric tumor model was used to investigate the efficacy of IMAB362
against
cancer cells with endogenous expression of CLDN18.2. NUGC-4 cells grow
aggressively in
nude mice.
1x107 NUGC-4 gastric cancer cells were injected subcutaneous into the left
flank of athymic
nude mice (n = 9 for IMAB362 group; n = 8 for control groups). IMAB362 (200
ptg per
injection) and controls were applied twice weekly alternating i.v. and i.p.,
starting 6 days after
tumor inoculation with i.v. injection. Tumor sizes were monitored twice
weekly. Data presented
in Figure 21a are means with SEM. Tumor growth of mice treated with IMAB362
was
significant inhibited compared to mice treated with controls (* p<0.05).
Figure 21b shows tumor
volumes at day 21 after tumor inoculation. Tumor volumes of IMAB362 treated
mice were
significant smaller than tumors of control mice (* p<0.05).
When lx107 tumor cells are inoculated in mice, the median survival time of
untreated mice is not
longer than 25 days. Treatment with IMAB362, cetuximab, trastuzumab or isotype
and buffer
controls was initiated when tumor volumes reached a mean size of about 109 mm3
(63 - 135
mm3). Mice were stratified size-dependently into treatment groups (Figure 21).
IMAB362 was
shown to significantly reduce tumor growth rate. No significant reduction of
tumor growth as
compared to saline or antibody controls was observed for this aggressively
growing tumor
model. The delay in tumor growth was associated with a non-significantly
increased median
survival time of IMAB362 treated mice (31 days versus 25 days).
Antitumor activity of IMAB362 was examined with two human gastric carcinoma
xenograft
models using NCI-N87 or NUGC-4 cells with lentiviral transduction of IMAB362
target
CLDN18.2 (NCI-N87¨CLDN18.2 and NUGC-4¨CLDN18.2).
NCI-N87¨CLDN18.2 xenograft tumors were inoculated subcutaneously by injection
of 1x107
NCI-N87¨CLDN18.2 cells into the flank of 8 nude mice (female, 6 weeks old) per
treatment
group. Treatment started 5 days after tumor inoculation by intravenous
injection of 800 1.tg
IMAB362 or with 200 I 0.9% NaC1 for saline control group. Intravenous
administration was
continued weekly for the whole observation time. Tumor size and animal health
was monitored
semi-weekly. Figure 22a shows the effects of IMAB362 treatment on tumor
growth. The size of
s.c. tumors was measured twice weekly (mean + SEM, ***p<0.001). Figure 22b
shows Kaplan-
Meier survival plots. Mice were sacrificed, when tumor reached a volume of
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Thus, continuous IMAB362 treatment inhibited highly significant (p<0.001)
tumor growth of
NCI-N87¨CLDN18.2 gastric carcinoma xenografts (Figure 22a). The delay in tumor
growth was
associated with a significantly (p<0.05) longer survival time of IMAB362
treated mice (Figure
22b).
IMAB362 immunotherapy of rapid growing NUGC-4¨CLDN18.2 xenografts resulted in
significant (p<0.05) smaller tumor sizes at day 14 of treatment. After the
first two weeks of
IMAB362 treatment tumor progession of NUGC-4¨CLDN18.2 was very aggressive.
However,
the inhibition of NUGC-4¨CLDN18.2 tumor growth until day 14 of treatment
resulted in
significantly (p <0.05) longer survival of IMAB362 treated mice.
In summary, IMAB362 was highly effective in treatment of gastric carcinoma
xenografts
showing significant retardation of tumor progression and prolonged survival in
endogenous
CLDN18.2-positive tumor models. In very aggressive tumor model systems these
antitumoral
effects of IMAB362 are less prominent but nonetheless significant, emphasizing
the strong
antitumoral capacity of IMAB362.
Example 15: Antitumoral effects of IMAB362 combined with chemotherapy in mouse
tumor
models
In vitro, IMAB362-mediated ADCC is more efficient on human gastric cancer
cells pretreated
with combinations of chemotherapeutic agents including the EOF and 5-FU + OX.
Therefore,
antitumoral impact of combining these compounds with 11VIAB362 was
investigated in vivo in
mouse tumor models.
NCI-N87¨CLDN18.2 xenograft tumors were inoculated by injection of 1x107 NCI-
N87¨CLDN18.2 cells subcutaneous into the flank of 9 mice for each treatment
group. Tumor
bearing mice were treated according to EOF regimen with 1.25 mg/kg epirubicin,
3.25 mg/kg
oxaliplatin and 56.25 mg/kg 5-fluorouracil intraperitoneal on day 4, 11, 18
and 25 after tumor
inoculation, followed by intravenous injection of 800 ptg IMAB362 24 hours
after chemotherapy
administration. IMAB362 treatment was continued weekly. Tumor size and animal
health was
monitored semi-weekly. Figure 23a shows the effects of combined treatment on
tumor growth.
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The size of s.c. tumors was measured twice weekly (mean + SEM; * p<0.05).
Figure 23b shows
Kaplan-Meier survival plots. Mice were sacrificed, when tumor reached a volume
of 1400 mm3.
NCI-N87¨CLDN18.2 tumor bearing nude mice treated with IMAB362 or EOF regimen
showed
highly significant suppressed tumor growth compared to control mice.
Additional IMAB362
treatment in combination with EOF chemotherapy resulted in significantly
(p<0.05) higher tumor
growth inhibition than treatment with EOF regimen alone (Figure 23a). Median
survival of mice
in saline control group was 59 days. Weekly IMAB362 treatment of mice
prolonged significantly
median survival to 76 days similar to survival of mice in EOF group with a
median survival of
76 days, too. But combined treatment with IMAB362 and EOF augmented median
survival to 81
days (Figure 23b).
Xenograft tumors were inoculated by injection of 1x107 NUGC-4¨CLDN18.2 cells
subcutaneous into the flank of 10 nude mice (female, six weeks old) per
treatment group. Mice
were treated on day 3, 10, 17 and 24 with chemotherapeutic agents. IMAB362
treatment was
continued weekly. Figure 24a shows tumor growth curves of s.c. NUGC-4¨CLDN18.2

xenografts (mean + SEM). Figure 24b shows Kaplan-Meier survival plots (Log-
rank (Mantel-
Cox) Test, "p<0.01).
Subcutaneous NUGC-4¨CLDN18.2 xenograft tumors grow very aggressively.
Nevertheless
IMAB362 treatment of tumor bearing nude mice inhibited significantly the tumor
growth
compared to saline treated control group. In combined therapy with EOF,
IMAB362 effects on
NUGC-4¨CLDN18.2 tumor growth was masked by growth inhibition due to EOF
treatment,
showing no increased tumor growth inhibition compared to treatment with EOF
alone (Figure
24a). However, median survival of mice treated with IMAB362 and EOF regimen
was highly
significant (p<0.01) prolonged, compared to survival of mice treated with EOF
alone (Figure
24b).
Example 16: ZA/IL-2 expanded V79V82 T cells improve IMAB362-mediated control
of
advanced tumors in vivo
To investigate combined activity of IMAB362 and ZA/IL-2 generated 7,5 T cells
in mouse
systems, we resorted to NSG mice. NSG mice lack mature T cells, B cells,
natural killer (NK)
cells, multiple cytokine signaling pathways, and they have many defects in
innate immunity,
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whereas the niches in the primary and secondary immunological tissues are
permissive to
colonization by human immune cells.
NSG mice were inoculated subcutaneously with 1 x107 CLDN18.2-transfected
HEK293 cells.
On the same day, mice received 8x106 human PBMCs enriched for Vy9V62 T cells,
which were
cultured for 14 days in ZA-supplemented medium. Moreover, mice were injected
with 50 g/kg
ZA and 5000 U IL-2 (Proleukin). To maintain human T cells functional, IL-2 was
administered
semi-weekly and ZA weekly. When HEK293¨CLDN18.2 tumors became macroscopically
visible, semi-weekly treatment with 200 lig of IMAB362 was started. In
addition to 9 mice
treated as described, two control groups of mice were established. One group
did not receive
human 76 T cells, the other group was treated with an isotype control antibody
instead of
IMAB362. Outgrowth of CLDN18.2-positive tumors in mice treated with IMAB362 in
the
presence of human y.5 T cells and ZA was significantly inhibited and nearly
abrogated, whereas
in mice either treated with an isotype control antibody or lacking human T
cell effectors, tumors
grew aggressively and mice had to be terminated prematurely (Figure 25).
Example 17: Antitumoral effects of IMAB362 combined with chemotherapy in mouse
tumor
models
Antitumor activity of IMAB362 in combination with chemotherapy was examined in

subcutaneous gastric carcinoma allografts in immunocompetent outbred NMRI mice
using CLS-
103 cells with lentiviral transduction of murine cldn18.2 (CLS-103¨c1dn18.2).
CLS-103¨cldn18.2 allograft tumors were inoculated by injection of 1x106 CLS-
103¨cldn18.2
cells subcutaneous into the flank of 10 NMRI mice for each treatment group.
Tumor bearing
mice were treated with 1.25 mg/kg epirubicin, 3.25 mg/kg oxaliplatin and 56.25
mg/kg 5-
fluorouracil (EOF) intraperitoneal on day 3, 10, 17 and 24 after tumor
inoculation, followed by
intravenous injection of 800 ptg IMAB362 24 hours after each chemotherapy
administration. IL-
2 was administered semi-weekly by subcutaneous injection of 3000 IE. After end
of
chemotherapy, IMAB362 and IL-2 treatment was continued for the whole
observation period.
Tumor size and animal health were monitored semi-weekly. Mice were sacrificed,
when tumor
reached a volume of 1400 mm3 or tumors became ulcerous.
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As can be seen in Figure 26, CLS-103¨c1dn18.2 tumor bearing NMRI mice treated
with
IMAB362 or EOF alone showed no significant tumor growth inhibition compared to
saline
control group. In contrast, the combination of EOF chemotherapy and IMAB362
treatment
resulted in significantly higher tumor growth inhibition and in prolonged
survival of tumor
bearing mice. These observations indicate the existence of additive or even
synergistic
therapeutic effects by combination of EOF chemotherapy and IMAB362
immunotherapy. IL-2
treatment showed no effect on tumor growth.
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Applicant's or agent's International application No.
file reference
342-71 PCT
INDICATIONS RELATING TO DEPOSITED MICROORGANISM
OR OTHER BIOLOGICAL MATERIAL
(PCT Rule 1 3bis)
A. The indications made below relate to the deposited microorganism or other
biological material referred to in the description
on page flip_ , line 6
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an
additional sheet
Name of depositary institution
DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
Address of depositary institution (including postal code and country,)
Mascheroder Weg lb
38124 Braunschweig
DE
Date of deposit Accession Number
October 19, 2005 DSM ACC2737
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This
information is continued on an additional sheet
- Mouse (Mus musculus) myeloma P3X63Ag8U.1 fused with mouse (Mus musculus)
splenocytes
- Hybridoma secreting antibody against human claudin-18A2
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are
not /hr all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau
later (spec(t the general nature of the indications e.g., 'Accession
Number of Deposit")
___________________________________________ For receiving Office use only
For International Bureau use only
ElThis sheet was received with the international application El This sheet
was received by the International Bureau on:
Authorized officer Authorized officer
Fnmi PCT/R0/114 I99R' renrint Jarman/ 20(141

CA 02874032 2014-11-19
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New International Patent Application
Ganymed Pharmaceuticals AG, et al.
õCOMBINATION THERAPY INVOLVING ANTIBODIES AGAINST CLAUDIN 18.2 FOR
TREATMENT OF CANCER"
Our Ref.: 342-71 PCT
Additional Sheet for Biological Material
Identification of further deposits:
1) The Name and Address of depositary institution for the deposits (DSM
ACC2738, DSM
ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM
ACC-2745, DSM ACC2746, DSM ACC2747, DSM ACC2748) are:
DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
Mascheroder Weg lb
38124 Braunschweig
DE
2) The Name and Address of depositary institution for the deposits (DSM
ACC2808, DSM
ACC2809, DSM ACC2810) are:
DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
Inhoffenstr. 7 B
38124 Braunschweig
DE
Date of desposits Accession Numbers The indications made below
relate to the deposited
microorganism in the
description on the following
page(s)
October 19, 2005 DSM ACC2738 page 40, line 7
October 19, 2005 DSM ACC2739 page 40, line 8
October 19, 2005 DSM ACC2740 page 40, line 9
October 19, 2005 DSM ACC2741 page 40, line 10
October 19, 2005 DSM ACC2742 page 40, line 11
October 19, 2005 DSM ACC2743 page 40, line 12
November 17, 2005 DSM ACC2745 page 40, line 13
November 17, 2005 DSM ACC2746 page 40, line 14
November 17, 2005 DSM ACC2747 page 40, line 15
November 17, 2005 DSM ACC2748 page 40, line 16
October 26, 2006 DSM ACC2808 page 40, line 17
October 26, 2006 DSM ACC2809 page 40, line 18
October 26, 2006 DSM ACC2810 page 40, line 19
81

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Additional Indications for all above mentioned deposits:
- Mouse (Mus musculus) myeloma P3X63Ag8U.1 fused with mouse (Mus
musculus) splenocytes
- Hybridoma secreting antibody against human claudin-18A2
3) Depositor:
Al! above mentioned depositions were made by:
Ganymed Pharmaceuticals AG
FreiligrathstraBe 12
55131 Mainz
DE
82