Note: Descriptions are shown in the official language in which they were submitted.
CA 02826627 2013-09-04
SPECIFIC BINDING PROTEINS AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to specific binding members, particularly
antibodies and
fragments thereof, which bind to amplified epidermal growth factor receptor
(EGFR)
and to the de2-7 EGFR truncation of the EGFR. In particular, the epitope
recognized
by the specific binding members, particularly antibodies and fragments
thereof, is
enhanced or evident upon aberrant post-translational modification. These
specific
binding members are useful in the diagnosis and treatment of cancer. The
binding
members of the present invention may also be used in therapy in combination
with
chemotherapeutics or anti-cancer agents and/or with other antibodies or
fragments
thereof.
BACKGROUND OF THE INVENTION
The treatment of proliferative disease, particularly cancer, by
chemotherapeutic
means often relies upon exploiting differences in target proliferating cells
and other
normal cells in the human or animal body. For example, many chemical agents
are
designed to be taken up by rapidly replicating DNA so that the process of DNA
replication and cell division is disrupted. Another approach is to identify
antigens on
the surface of tumor cells or other abnormal cells which are not normally
expressed in
developed human tissue, such as tumor antigens or embryonic antigens. Such
antigens can be targeted with binding proteins such as antibodies which can
block or
neutralize the antigen. In addition, the binding proteins, including
antibodies and
fragments thereof, may deliver a toxic agent or other substance which is
capable of
directly or indirectly activating a toxic agent at the site of a tumor.
CA 02826627 2013-09-04
The EGFR is an attractive target for tumor-targeted antibody therapy because
it is
over-expressed in many types of epithelial tumors (27,28). Moreover,
expression of
the EGFR is associated with poor prognosis in a number of tumor types
including
stomach, colon, urinary bladder, brEtast, prostate, endometrium, kidney and
brain
glioma). Consequently, a number of EGFR antibodies have been reported in the
literature with several undergoing clinical evaluation (18, 19, 29). Results
from
studies using EGFR mAbs in patients with head and neck cancer, squamous cell
lung
cancer, brain gliomas and malignant astrocytomas have been encouraging. The
anti-
tumor activity of most EGFR antibodies is enhanced by their ability to block
ligand
binding (30, 31). Such antibodies may mediate their efficacy through both
modulation
of cellular proliferation and antibody dependent immune functions (e.g.
complement
activation). The use of these antibodies, however, may be limited by uptake in
organs
that have high endogenous levels of EGFR such as the liver and skin (18, 19).
A significant proportion of tumors containing amplifications of the EGFR gene
(i.e.,
multiple copies of the EGI.1& gene) also co-express a truncated version of the
receptor
(13) known as de2-7 EGFR, AEGFR, or A2-7 (terms used interchangeably herein)
(2).
The rearrangement seen in the de2-7 EGFR results in an in-frame mature mRNA
lacking 801 nucleotides spanning exons 2-7 (6-9). The corresponding EGFR
protein
has a 267 amino acid deletion comprising residues 6-273 of the extracellular
domain
and a novel glycine residue at the fusion junction (9). This deletion,
together with the
insertion of a glycine residue, produces a unique junctional peptidc at the
deletion
interface (9). The de2-7 EGFR (2) has been reported in a number of tumor types
including glioma, breast, lung, ovarian and prostate (1-4). While this
truncated
receptor does not bind ligand, it possesses low constitutive activity and
imparts a
significant growth advantage to glioma cells grown as tumor xenografts in nude
mice
(10) and is able to transform N1H3T3 cells (11) and MCF-7 cells. The cellular
mechanisms utilized by the de2-7 EGFR in glioma cells are not fully defined
but are
reported to include a decrease in apoptosis (12) and a small enhancement of
proliferation (12).
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As expression of this truncated receptor is restricted to tumor cells it
represents a
highly specific target for antibody therapy. Accordingly, a number of
laboratories
have reported the generation of both polyclonal (14) and monoclonal (3, 15,
16)
antibodies specific to the unique peptide of de2-7 EGFR. A series of mouse
mAbs,
isolated following immunization with the unique de2-7 peptide, all showed
selectivity
and specificity for the truncated receptor and targeted de2-7 EGFR positive
xenografts grown in nude mice (3, 25, 32).
However, one potential shortcoming of de2-7 EGFR antibodies is that only a
proportion of tumors exhibiting amplification of the EGLI( gene also express
the de 2-
7 EGFR (5). The exact percentage of tumors containing the de2-7 EGFR is not
completely established, because the use of different techniques (i.e. PCR
versus
imrnunohistochemistry) and various antibodies, has produced a wide range of
reported value' s for the frequency of its presence. Published data indicates
that
approximately 25-30% of gliomas express de2-7 EGFR with expression being
lowest
in anaplastic astrocytomas and highest in glioblastoma multiforme (6,13,17).
The
proportion of positive cells within de2-7 EGFR expressing gliomas has been
reported
to range from 37-86% (1). 27% of breast carcinomas and 17% of lung cancers
were
found to be positive for the de2-7 EGFR (1, 3, 13, 16). Thus, de2-7 EGFR
specific
antibodies would be expected to be useful in only a percentage of EGFR
positive
tumors.
= Thus, while the extant evidence of activity of EGFR antibodies is
encouraging, the
observed limitations on range of applicability and efficacy reflected above
remain.
Accordingly, it would be desirable to develop antibodies and like agents that
demonstrate efficacy with a broad range of tumors, and it is toward the
achievement
of that objective that the present invention is directed.
The citation of references herein shall not be construed as an admission that
such is
prior art to the present invention.
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CA 02826627 2013-09-04
SUMMARY OF THE INVENTION
In a broad aspect, the present invention provides an isolated specific binding
member,
particularly an antibody or fragment thereof, which recognizes an EGFR epitope
which does not demonstrate any amino acid sequence alterations or
substitutions from
wild type EGFR and which is found in tumorigenic, hyperproliferative or
abnormal
cells and is not detectable in normal or wild type cells (the term "wild type
cell" as
used herein contemplates a cell that expresses endogenous EGFR but not the de
2-7
EGFR and the term specifically excludes a cell that overexpresses the EG14R
gene;
the term "wild type" refers to a genotype or phenotype or other characteristic
present
in a normal cell rather than in an abnormal or tumorigenic cell). In a further
aspect,
the present invention provides a specific binding member, particularly an
antibody or
fragment thereof, which recognizes an EGFR epitope which is found in
tumorigenie,
hyperproliferative or abnormal cells and is not detectable in normal or wild
type cells,
wherein the epitope is enhanced or evident upon aberrant post translational
modification or aberrant expression. In a particular nonlimiting
exemplification
provided herein, the EGFR epitope is enhanced or evident wherein post-
translational
modification is not complete or full to the extent seen with normal expression
of
EGFR in wild type cells. In one aspect, the EGFR epitope is enhanced or
evident
upon initial or simple carbohydrate modification or early glycosylation,
particularly
high mannose modification, and is reduced or not evident in the presence of
complex
carbohydrate modification.
The specific binding member, which may be an antibody or a fragment thereof,
such
as an immunogenic fragment thereof, does not bind to or recognize normal or
wild
type cells containing normal or wild type EGFR epitope in the absence of
aberrant
expression and in the presence of normal EGFR post-translational modification.
More particularly, the specific binding member of the invention, may be an
antibody
or fragment thereof, which recognizes an EGFR epitope which is present in
cells
overexpressing EGFR (e.g., EGFR gene is amplified) or expressing the de2-7
EGFR,
particularly in the presence of aberrant post-translational modification, and
that is not
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CA 02826627 2013-09-04
detectable in cells expressing EGFR under normal conditions, particularly in
the
presence of normal post-translational modification.
The present inventors have discovered novel monoclonal antibodies, exemplified
herein by the antibody designated mAb 806, which specifically recognize
aberrantly
expressed EGFR. In particular, the antibodies of the present invention
recognize an
EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal
cells
and is not detectable in normal or wild type cells, wherein the epitope is
enhanced or
evident upon aberrant post-translational modification. The antibodies of the
present
invention are further exemplified by the antibodies mAb 124 and mAb 1133
described
herein. The novel antibodies of the invention also recognize amplified wild
type
EGFR and the de2-7 EGFR, yet bind to an epitope distinct from the unique
junctional
peptide of the de2-7 EGFR mutation. The antibodies of the present invention
specifically recognize aberrantly expressed EGFR, including amplified EGFR and
mutant EGFR (exemplified herein by the de2-7 mutation), particularly upon
aberrant
post-translational modification. Additionally, while mAb 806 does not
recognize the
EGFR when expressed on the cell surface of a glioma cell line expressing
normal
amounts of EGFR, it does bind to the extracellular domain of the EGFR (sEGFR)
immobilized on the surface of EITSA plates, indicating the recognition of a
conformational epitope. MAb 806 binds to the surface of A431 cells, which have
an
amplification of the EGFR gene but do not express the de2-7 EGFR. Importantly,
mAb 806 did not bind significantly to normal tissues such as liver and skin,
which
express levels of endogenous, wild type (wt) EGFR that are higher than in most
other
normal tissues, but wherein EGFR is not aberrantly expressed or amplified.
The antibodies of the present invention can specifically categorize the nature
of EGFR
tumors or tumorigenic cells, by staining or otherwise recognizing those tumors
or
cells wherein aberrant EGFR expression, including EGFR amplification and/or
EGFR
mutation, particularly de2-7EGFR, is present. Further, the antibodies of the
present
invention, as exemplified by mAb 806, demonstrate significant in vivo anti-
tumor
CA 02826627 2013-09-04
activity against tumors containing amplified Bag< and against de2-7 EGFR
positive
xenografts.
The unique specificity of mAb 806, whereby mAb 806 binds to the de2-7 EGFR and
amplified EGFR but not the normal, wild type EGFR, provides diagnostic and
therapeutic uses to identify, characterize and target a number of tumor types,
for
example, head and neck, breast, or prostate tumors and glioma, without the
problems
associated with normal tissue uptake that may be seen with previously known
EGFR
antibodies.
Accordingly, the invention provides specific binding proteins, such as
antibodies,
which bind to the de2-7 EGI-R. at an epitope which is distinct from the
junctional
peptide but which do not bind to EGFR on normal cells in the absence of
amplification of the EGFR gene. By amplification, it is meant to include that
the cell
comprises multiple copies of the EGFR gene.
Preferably the epitope recognized by mAb 806 is located within the region
comprising
residues 273-501 of the mature normal or wild type EGFR sequence. Therefore,
also
provided are specific binding proteins, such as antibodies, which bind to the
de2-7
EGFR at an epitope located within the region comprising residues 273-501 of
the
EGFR sequence. The epitope may be determined by any conventional epitope
= mapping techniques known to the person skilled in the art. Alternatively,
the DNA
sequence encoding residues 273-501 could be digested, and the resultant
fragments
expressed in a suitable host. Antibody binding could be determined as
mentioned
above.
In a preferred aspect, the antibody is one which has the characteristics of
the antibody
which the inventors have identified and characterized, in particular
recognizing
aberrantly expressed EGFR, as found in amplified EGFR and de2-7EGFR. In a
particularly preferred aspect the antibody is the mAb 806, or active fragments
thereof.
In a further preferred aspect the antibody of the present invention comprises
the VH
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CA 02826627 2013-09-04
and VL amino acid sequences depicted in Figures 14 (SEQ ID NO:2) and Figures
15
(SEQ lD NO:4) respectively.
In another aspect, the invention provides an antibody capable of competing
with the
806 antibody, under conditions in which at least 10% of an antibody having the
VH
and VL sequences of the 806 antibody is blocked from binding to de2-7EGFR by
competition with such an antibody in an ELISA assay. In particular, anti-
idiotype
antibodies are contemplated and are exemplified herein. The anti-idiotype
antibodies
LMI-1-11, LM1-1-12 and LMH-13 are provided herein.
An isolated polypeptide consisting essentially of the epitope comprising
residues 273-
501 of the mature normal, wild type EGFR (residues 6-234 of mature de2-7 EGFR)
forms another aspect of the present invention. The peptide of the invention is
particularly useful in diagnostic assays or kits and therapeutically or
prophylactically,
including as a tumor or anticancer vaccine. Thus compositions of the peptide
of the
present invention include pharmaceutical compositions and immunogenic
compositions.
The binding of an antibody to its target antigen is mediated through the
complementarity-determining regions (CDRs) of its heavy and light chains, with
the
role of CDR3 being of particular importance. Accordingly, specific binding
members
=
based on the CDR3 regions of the heavy or light chain, and preferably both, of
mAb806 will be useful specific binding members for in vivo therapy. The CDRs
of
the mAb 806 antibody are shown in Figures 16 and 17.
Accordingly, specific binding proteins such as antibodies which are based on
the
CDRs of the mAb 806 antibody identified, particularly the CDR 3 regions, will
be
useful for targeting tumors with amplified EGFR regardless of their de2-7 EGFR
status. As mAb 806 does not bind significantly to normal, wild type receptor,
there
would be no significant uptake in normal tissue, a limitation of EGFR
antibodies
currently being developed (18, 19).
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CA 02826627 2013-09-04
In the accompanying drawings, the nucleic acid sequence (SEQ ID NO:1) and
translation (SEQ JD NO:2) thereof of the 806 VH gene is shown in Figure 14.
The
VL gene of the 806 antibody is shown as Figure 15 as nucleic acid sequence
(SEQ ID
NO:3) and predicted amino acid sequence (SEQ ID NO:4). In Figures 16 and 17,
depicting the VH and VL polypeptide sequences of mAb 806, the CDRs are
indicated
in boxes.
In a further aspect, the present invention provides an isolated specific
binding member
capable of binding an antigen, wherein said specific binding member comprises
a
polypeptide binding domain comprising an amino acid sequence substantially as
set
out as residues 93-102 of SEQ II) NO:2, The invention further provides said
isolated
specific binding member which further comprises one or both of the polypeptide
binding domains substantially as set out as residues 26-35A and 49-64 of SEQ
ID
NO:2, preferably both. One example of such an embodiment is the sequence
substantially as shown in SEQ ID NO:2, In a preferred embodiment, the binding
domains are carried by a human antibody framework.
In another aspect, the invention provides an isolated specific binding member
capable
of binding a tumor antigen, wherein said specific binding member Comprises a
polypeptide binding domain comprising a heavy chain sequence comprising at
least
the CDR3 sequence of SEQ JD NO:2, together with a light chain comprising CDRs
whose amino acid sequences are substantially as found within SEQ ID NO:4. One
example of such an embodiment is the sequence substantially as shown in SEQ ID
NO:4. In a preferred embodiment, the CDRs are carried by a human antibody
framework.
In further aspects, the invention provides an isolated nucleic acid which
comprises a
sequence encoding a specific binding member as defined above, and methods of
preparing specific binding members of the invention which comprise expressing
said
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CA 02826627 2013-09-04
nucleic acids under conditions to bring about expression of said binding
member, and
recovering the binding member.
Yet a further aspect of the invention are compositions of such binding
proteins with
additional binding proteins, such as binding proteins which bind to EGFR,
preferably
inhibiting ligand binding thereto. Such compositions can be "one pot"
cocktails, kits,
and so forth, preferably formulated for ease of administration.
Specific binding members according to the invention may be used in a method of
treatment or diagnosis of the human or animal body, such as a method of
treatment of
a tumor in a human patient which comprises administering to said patient an
effective
amount of a specific binding member of the invention.
The present invention also relates to a recombinant DNA molecule or cloned
gene, or
a degenerate variant thereof, which encodes an antibody of the present
invention;
preferably a nucleic acid molecule, in particular a recombinant DNA molecule
or
cloned gene, encoding the antibody VH which has a nucleotide sequence or is
complementary to a DNA sequence shown in FIGURE 14 (SEQ ID NO:1). In
another embodiment, the present invention also relates to a recombinant DNA
molecule or cloned gene, or a degenerate variant thereof, preferably a nucleic
acid
molecule, in particular a recombinant DNA molecule or cloned gene, encoding
the
antibody VL which has a nucleotide sequence or is complementary to a DNA
sequence shown in FIGURE 15 (SEQ lD NO:3).
The present invention also includes polypeptides or antibodies having the
activities
noted herein, and that display the amino acid sequences set forth and
described above
and in Figures 14 and 15 hereof and selected from SEQ ID NO:2 and 4.
In a further embodiment of the invention, the full DNA sequence of the
recombinant
DNA molecule or cloned gene provided herein may be operatively linked to an
expression control sequence which may be introduced into an appropriate host.
The
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CA 02826627 2013-09-04
invention accordingly extends to unicellular hosts transformed with the cloned
gene
or recombinant DNA molecule comprising a DNA sequence encoding the present VH
and/or VL, or portions thereof, of the antibody, and more particularly, the VH
and/or
VL set forth above and in SEQ lD NO:1 and 3.
The present invention naturally contemplates several means for preparation of
the
antibodies and active fragments thereof, including as illustrated herein known
recombinant techniques, and the invention is accordingly intended to cover
such
synthetic or chimeric antibody preparations within its scope. The isolation of
the
cDNA and amino acid sequences disclosed herein facilitates the reproduction of
the
antibody of the present invention by such recombinant techniques, and
accordingly,
the invention extends to expression vectors prepared from the disclosed DNA
sequences for expression in host systems by recombinant DNA techniques, and to
the
resulting transformed hosts.
The present invention provides drugs or other entities, including antibodies
such as
anti-idiotype antibodies, that are capable of binding to the antibody thereby
modulating, inhibiting or potentiating the antibody activity. Thus, anti-
idiotype
antibodies to mAb806 are provided and exemplified herein. Such anti-idiotype
antibodies would be useful in the development of drugs that would specifically
bind
the antibodies such as mAb806 or its epitope or that would potentiate its
activity.
The diagnostic utility of the present invention extends to the use of the
antibodies of
the present invention in assays to characterize tumors or cellular samples or
to screen
for tumors or cancer, including in vitro and in vivo diagnostic assays.
In an immunoassay, a control quantity of the antibodies, or the like may be
prepared
and labeled with an enzyme, a specific binding partner and/or a radioactive
element,
and may then be introduced into a cellular sample. After the labeled material
or its
binding partner(s) has had an opportunity to react with sites within the
sample, the
CA 02826627 2013-09-04
resulting mass may be examined by known techniques, which may vary with the
nature of the label attached.
Specific binding members of the invention may carry a detectable or functional
label.
The specific binding members may carry a radioactive label, such as the
isotopes 3H,
14C, 32p, 35s, 36C1,
"Cr, 57CO, "CO, "Fe, 90Y, 1211, 124/, 125j, 1311, 111/n, 211m, 198Au,
CO, 225AC, 213lidi 99TC and 186Re. When radioactive labels are used, known
currently
available counting procedures may be utilized to identify and quantitate the
specific
binding members. In the instance where the label is an enzyme, detection may
be
accomplished by any of the presently utilized colorimetric,
spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques known in the
art.
The radiolabelled specific binding members, particularly antibodies and
fragments
thereof, are useful in in vitro diagnostics techniques and in in vivo
radioim4ng
techniques. In a further aspect of the invention, radiolabelled specific
binding
members, particularly antibodies and fragments thereof, particularly
rachoimmunoconjugates, are useful in radioimmunotherapy, particularly as
radiolabelled antibodies for cancer therapy. In a still further aspect, the
radiolabelled
specific binding members, particularly antibodies and fragments thereof, are
useful in
radioirarnuno-guided surgery techniques, wherein they can identify and
indicate the
presence and/or location of cancer cells, precancerous cells, tumor cells, and
hyperproliferative cells, prior to, during or following surgery to remove such
cells.
Immunoconjugates or antibody fusion proteins of the present invention, wherein
the
specific binding members, particularly antibodies and fragments thereof, of
the
present invention are conjugated or attached to other molecules or agents
further
include, but are not limited to binding members conjugated to a chemical
ablation
agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic
agent or
drug.
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CA 02826627 2013-09-04
The present invention includes an assay system which may be prepared in the
form of
a test kit for the quantitative analysis of the extent of the presence of, for
instance,
amplified EGFR or de2-7EGFR. The system or test kit may comprise a labeled
component prepared by one of the radioactive and/or enzymatic techniques
discussed
herein, coupling a label to the antibody, and one or more additional
immunochemical
reagents, at least one of which is a free or immobilized components to be
determined
or their binding partner(s).
In a further embodiment, the present invention relates to certain therapeutic
methods
which would be based upon the activity of the binding member, antibody, or
active
fragments thereof, or upon agents or other drugs determined to possess the
same
activity. A first therapeutic method is associated with the prevention or
treatment of
cancer, including but not limited to head and neck, breast, prostate and
glioma.
In particular, the binding members and antibodies of the present invention,
and in a
particular embodiment the 806 antibody whose sequences are presented in SEQ ID
NOS: 2 and 4 herein, or active fragments thereof, and chimeric (bispecific) or
synthetic antibodies derived therefrom can be prepared in pharmaceutical
compositions, including a suitable vehicle, carrier or diluent, for
administration in
instances wherein therapy is appropriate, such as to treat cancer. Such
pharmaceutical
compositions may also include methods of modulating the half-life of the
binding
members, antibodies or fragments by methods known in the art such as
pegylation.
Such pharmaceutical compositions may further comprise additional antibodies or
therapeutic agents.
Thus, a composition of the present invention may be administered alone or in
combination with other treatments, therapeutics or agents, either
simultaneously or
sequentially dependent upon the condition to be treated. In addition, the
present
invention contemplates and includes compositions comprising the binding
member,
particularly antibody or fragment thereof, herein described and other agents
or
therapeutics such as anti-cancer agents or therapeutics, anti-EGFR agents or
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CA 02826627 2013-09-04
antibodies, or immune modulators. More generally these anti-cancer agents may
be
tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-
translational
modulators, cell growth or division inhibitors (e.g. anti-mitotics), PDGFR
inhibitors
or signal transduction inhibitors. Other treatments or therapeutics may
include the
administration of suitable doses of pain relief drugs such as non-steroidal
anti-
inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or
opiates
such as morphine, or anti-emetics. Thus, these agents may be anti-EGFR
specific
agents, such as A01478, or may be more general anti-cancer and anti-neoplastic
agents, non limiting examples including doxorubicin, carboplatin and
cisplatin. In
addition, the composition may be administered with immune modulators, such as
interleukins, tumor necrosis factor (TNF) or other growth factors, cytolcines
or
hormones such as dexamethasone which stimulate the immune response and
reduction
or elimination of cancer cells or tumors. The composition may also be
administered
with, or may include combinations along with other anti-EGFR antibodies,
including
but not limited to the anti-EGFR antibodies 528; 225; SC-03; 108 (ATCC HB9764)
U.S. Patent No. 6,217,866; 14E1 (U.S. Patent No. 5,942,602); DH8..3; L8A4;
Y10;
HuMAX-EGFr (Genmab/Medarex); ICR62; and ABX-EGF (Abgenix).
The present invention also includes binding members, including antibodies and
fragments thereof, which are covalently attached to or otherwise associated
with other
molecules or agents. These other molecules or agents include, but are not
limited to,
molecules (including antibodies or antibody fragments) with distinct
recognition
characteristics, toxins, ligands, and chemotherapeutic agents.
Other objects and advantages will become apparent to those skilled in the art
from a
review of the ensuing detailed description, which proceeds with reference to
the
following illustrative drawings, and the attendant claims.
BRIEF DESCRIPTION OF THE DRAWINGS
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Figure 1 presents the results of flow cytometric analysis of glioma cell
lines.
U87M0 (light gray histograms) and U87MG.A2-7 (dark gray histograms) cells were
stained with either an irrelevant IgG2b antibody (open histograms), DJ8.3
(specific
for de2-7 EGFR), MAb 806 or 528 (binds both wild type and de2-7 EGFR) as
indicated.
Figures 2A-C present the results of EI ISA of MAb 806, DH8.3 and 528
antibodies.
(A) binding of increasing concentrations of MAb 806 (A) DH8.3 (4) or 528 (a)
antibody to sEGFR coated FT ISA plates. (B) inhibition of MAb 806 and 528
binding
to sEGI-.R. coated ELISA plates by increasing concentrations of sEGFR in
solution.
(C) binding of increasing concentrations of DH8.3 to the de2-7 junctional
peptide
illustrates binding curves for inAb 806 and 528 antibodies to immobilized wild-
type
sEGFR.
Figures 2D and 2E graphically present the results of BIAcore binding studies
using
C-terminal biotinylated peptide and including a naonoclonal antibody of the
invention,
along with other known antibodies, among them the L8A4 antibody which
recognizes
the junction peptide of the de2-7 EGFR mutant, and controls.
Figure 3 depicts the internalization of MAb 806 and the DH8.3 antibody.
U87MG.A2-7 cells were pre-incubated with MAb 806 (A) or DH8:3 (.)at 4 C,
transferred to 37 C and internalization determined by FACS. Data represents
mean
internalization at each time point SE of 3 (DH8.3) or 4 (MAb 806) separate
experiments.
Figures 4A and 4B illustrate biodistribution (% lD/g tumor) of radiolabeled
(a) 1251-
MAb 806 and (b) 131I-DH8.3 in nude mice bearing U87MG and U87MG.62-7
xenografts, Each point represents the mean of 5 mice SE except for 1 hr
where n =
4.
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Figures 5A and 5B illustrate biodistribution of radiolabeled 1251-MAb 806
(open bar)
and 1311-DH8.3 (filled bar) antibodies expressed as (a) tumor: blood or (b)
tumor:liver
ratios in nude mice bearing U87MG.A2-7 xenografts. Each bar represents the
mean
of 5 mice SE except for 1 hr where n = 4
Figure 6 illustrates flow cytometric analysis of cell lines containing
amplification of
the EGER gene. A431 cells were stained with either MAb 806, DH8.3 or 528
(black
histograms) and compared to an irrelevant IgG2b antibody (open histogram)
Figures 7A and 7B illustrate biodistribution (% ED/g tumor) of radiolabeled
(a) 1251-
MAb 806 and (b) 1311-528 in nude mice bearing U87MG.A2-7 nd A431 xenografts.
Figures 84 - 81) illustrate biodistribution of radiolabeled125I-MAb 806 (open
bar)
and 1311-528 (filled bar) and antibodies expressed as (a,b) tumor.blood or
(c,d)
twaionliver ratios in nude mice bearing (ac) U87MGA2-7 and (b,d) A431
xenografts.
Figure 9 illustrates anti-tumor effect of mAb 806 on A) U87MG and B) U87MG.A2-
7 xenograft growth rates in a preventative model. 3 x 106 U87MG or U87MG.A2-7
cells were injected s.c. into both flanks of 4 ¨ 6 week old BALB/c nude mice,
(n=5) at
day 0. Mice were injected i.p. with either 1 mg of mAb 806 (9); 0.1 mg of raAb
806
(A); or vehicle (o) starting one day prior to tumor cell inoculation.
Injections were
given three times per week for two weeks as indicated by the arrows. Data are
expressed as mean tumor volume S.E.
Figure 10 illustrates the anti-tumor effect of mAb 806 on A) U87MG, B)
U87MG.A2-7 and C) U87MG.wtEGPR. xenografts in an established model. 3 x 106
U87MG, U87MG.A2-7, or U87MG.wtEGFR cells, were injected s.c. into both flanks
of 4 ¨ 6 week old BALB/c nude mice, (n=5). Mice were injected i.p. with either
1 mg
doses of inAb 806 (es); 0.1 mg doses of mAb 806 (k); or vehicle (o) starting
when
tumors had reached a mean tumor volume of 65 - 80 mm3. Injections were given
three times per week for two weeks as indicated by the arrows. Data are
expressed as
mean tumor volume S.E.
CA 02826627 2013-09-04
Figure 11 illustrates anti-tumor effect of mAb 806 on A431 xenogafts in A)
preventative and B) established models. 3 x 106 A431 cells were injected s.c.
into
both flanks of 4 ¨ 6 week old BALB/c nude rnice (n=5). Mice were injected i.p.
with
either 1 mg doses of mAb 806 (.); or vehicle (o), starting one day prior to
twaior cell
inoculation in the preventative model, or when tumors had reached a mean tumor
volume of 200 mm3. Injections were given three times per week for two weeks as
indicated by the arrows. Data are expressed as mean tumor volume S.E.
Figure 12 illustrates the anti-tumor effect of treatment with mAb 806 combined
with
treatment with AG1478 on A431 xenografts in a preventative model. Data are
expressed as mean tumor Volume S.E.
Figure 13 depicts antibody 806 binding to A431 cells in the presence of
increasing
concentrations of A01478 (0.5uM and 5uM).
Figure 14 illustrates the nucleic acid sequence and the amino acid translation
thereof
of the 806 VH gene (SEQ ID NO:1 and SEQ ID NO:2, respectively).
Figure 15 illustrates the nucleic acid sequence and the amino acid translation
thereof
of the 806 VL gene (SEQ ID NO:3 and SEQ ID NO:4, respectively).
Figure 16 shows the VII sequence numbered according to Kabat, with the CDRs
boxed. Key residues of the VH are 24, 37, 48, 67 and 78.
Figure 17 shows the VL sequence numbered according to Kabat, with the CDRs
boxed. Key residues of the VL are 36, 46, 57 and 71.
Figure 18A ¨ 18D shows the results of in vivo studies designed to determine
the
therapeutic effect of combination antibody therapy, particularly mAb806 and
the 528
antibody. Mice received inoculations of U87MG.D2-7 (A and B), U87MG.DK (C),
or A431 (D) cells.
16
CA 02826627 2013-09-04
Figure 19 A-D Analysis of internalization by electron microscopy. U87MG.A7-7
cells were pre-incubated with MAb 806 or DH8.3 followed by gold conjugated
anti-
mouse IgG at 4 C, transferred to 37 C and internalization examined at various
time
points by electron microscopy. (A) localization of the DH8.3 antibody to a
coated pit
(arrow) after 5 min; (B) internalization of MAb 806 by macropinocytosis
(arrow) after
2 min; (C) localization of DH8.3 to lysosomes (arrow) after 20 min; (D)
localization
of MAb 806 to lysosomes (arrow) after 30 min. Original magnification for all
images
is X30,000.
Figure 20 Autoradiography of a U87MG.A.2-7 xenograft section collected 8 hr
after
injection of 125I-MAb 806.
Figure 21 Flow cytometric analysis of cell lines containing amplification of
the
EGFR gene. HN5 and MDA-468 cells were stained with an irrelevant IgG2b
antibody
(open histogram with dashed line), MAb 806 (black histogram) or 528 (open
histogram with closed lines). The DH8.3 antibody was completely negative on
both
cell lines (data not shown).
Figure 22 Immunoprecipitation of EGFR from cell lines. The EGFR was
immunoprecipitated from" S-labeled U87MG.A2-7 or A431 cells with MAb 806, sc-
03 antibody or a IgG2b isotype control. Arrows at the side indicate the
position of the
de2-7 and wt EGFR. Identical banding patterns were obtained in 3 independent
experiments.
Figure 23 Autoradiography of an A431 xenograft section collected 24 hr after
injection of 125I-MAb 806, areas of localization to viable tissue are
indicated (arrows).
Figure 24 A and B, extended survival of nude mice bearingintracranial U87
MG.AEGFR (A) and LN-Z308.AEGFR (B) xenografts with systemic mAb 806
treatment. U87 MG.AEGFR cells (1 x 105) or LN-Z308.AEGFR cells (5 X 105) were
17
CA 02826627 2013-09-04
implanted into nude mice brains, and the animals were treated with either mAb
806,
PBS, or isotype IgG from postimplantation days 0 through 14. C and D, growth
inhibition of intracranial tumors by mAb 806 treatment. Nude mice (five per
group),
treated with either inAb 806 or the isotype IgG control, were euthanized on
day 9 for
U87 MG.AEGFR (C) and on day 15 for LN-Z308.AFGFR (D), and their brains were
harvested, fixed, and sectioned. Data were calculated by taking the tumor
volume of
control as 100%. Values are mean SD. P < 0.001; control versus mAb 806.
Arrowheads, tumor issue. E, extended survival of nude mice bearing
intracranial U87
MG.AEGFR xenografts with intratumoral mAb 806 treatment. U87 MG.AEGFR cells
were implanted as described. Ten mg of mAb 806 or isotype IgG control in a
volume
of 5 u.1 were injected at the tumor-injection site every other day starting at
day 1 for
five times.
Figure 25 mAb 806 extends survival of mice with U87 MG.wtEGFR brain tumors
but not with U87 MG.DK. or U87 MG brain tumors. U87 MG (A), U8'7 MG.DK (B),
or 1J87 MG.wtEGFR (C) cells (5 X 105) were implanted into nude mice brains,
and
the animals were treated with mAb 806 from postimplantation days 0 through 14
followed by observation after discontinuation of therapy.
Figure 26 A, FACS analysis of mAb 806 reactivity with U87 MG cell lines. U87
MG, U87 MG.AEGFR, U87 MG. DK, and U87 MG.wtEGFR cells were stained with
anti-EGFR mAbs 528, EGFR.1, and anti-AEGFR antibody, inAb 806. Monoclonal
EGFR.1 antibody recognized wtEGFR exclusively and monoclonal 528 antibody
reacted with both wtEGFR and AFGFR. mAb 806 reacted intensively with U87
MG.AEGFR and U87 MG. DK and weakly with U87 MG.wtEGFR. Bars on the
abscissa, maximum staining of cells in the absence of primary antibody.
Results were
reproduced in three independent experiments. B, mAb 806 immunoprecipitation of
EGER forms. Mutant and wtEGFR were imrnunoisolated with anti-EGFR antibodies,
528, or EGFR.1, or anti-AFGFR antibody, mAb 806, from (Lane 1) U87 MG, (Lane
2) U87A.EGFR_, (Lane 3) U87 MG. DK, and (/ni,e 4) U87 MG.wtEGFR cells and
were then detected by Western blotting with anti-pan EG141( antibody, C13.
18
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Figure 27 Systemic treatment with mAb 806 decreases the phosphorylation of
AFGFR and Bc1-XL expression in U87 MG.AEGFR brain tumors. U87 MG.AEGFR
tumors were resected at day 9 of mAb 806 treatment, immediately frozen in
liquid
nitrogen and stored at -80 C before tumor lysate preparation. A, Western blot
analysis
of expression and the degree of autophosphorylation of AEGFR. Thirty ug of
tumor
lysates were subjected to SDS-polyacrylamide gels, transferred to
nitrocellulose
membranes, and probed with antiphosphotyrosine mAb, then were stripped and
reprobed with anti-EGER antibody, C13. B, Western blotting of Bc1-XL by using
the
same tumor lysates as in A. Membranes were probed with antihuman Bc1-X
polyclonal antibody. Lanes 1 and 2, U87 MG.AEGFR brain tumors treated with
isotype control; Lanes 3 and 4, U87 MG.AEGFR brain tumors treated with tnAb
806.
Figure 28 MAb 806 treatment leads to a decrease in growth and vasculogenesis
and
to increases in apoptosis and accumulating macrophages in U87 MG.AEGFR tumors.
Tumor sections were stained for Ki-67. Cell proliferative index was assessed
by the
percentage of total cells that were Ki-67 positive from four randomly selected
high-
power fields (X400) in intracranial tumors from four mice of each group. Data
are the
mean SE. Apoptotic cells were detected by TUNEL assay. Apoptotic index was
assessed by the ratio of TUNEL-positive cells:total number of cells from four
randomly selected high-power fields (X400) in intracranial tumors from four
mice of
each group. Data are the mean SE. Tumor sections were immunostained with
anti-
CD31 antibody. MVAs were analyzed by computerized image analysis from four
randomly selected fields (X200) from intracranial tumors from four mice of
each
group. Peritumoral infiltrates of macrophages in mAb 806-treated U87MG.LEGFR
tumors. Tumor sections were stained with anti-F4/80 antibody.
Figure 29 Flow cytometric analysis of parental and transfected U87 MG glioma
cell
lines. Cells were stained with either an irrelevant IgG2b antibody (open
histograms)
or the 528 antibody or mAb 806 (filled histograms) as indicated.
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CA 02826627 2013-09-04
Figure 30 Immunoprecipitation of EGFR from cell lines. The EGFR was
imrnunoprecipitated from 35S-labeled U87 MG.wtEGFR, U87 MG. A2-7, and A431
cells with mAb806 (806), sc-03 antibody (c-term), or a IgG2b isotype control
(con).
Arrows, position of the de2-7 and wt EGFR.
Figure 31 Representative H&F-stained paraffin sections of U87 MG.A2-7 and
U87MG.vv-tEGFR xenografts. U87 MG.A2-7 (collected 24 days after tumor
inoculation) and U87 MG.wtEGFR (collected 42 days after tumor inoculation)
xenografts were excised from mice treated as described in Fig. 10 above, and
stained
with H&E. Vehicle-treated U87 MG.A2-7 (collected 18 days after tumor
inoculation)
and U87 MG.wtEGFR (collected 37 days after tumor inoculation) xenografts
showed
very few areas of necrosis (left panel), whereas extensive necrosis (arrows)
was
observed in both U87 MG.A2-7 and U87 MG.wtEGFR xenografts treated with mAb
806 (right panel).
Figure 32 Immunohistochemical analysis of EGFR expression in frozen sections
derived from U87 MG, U87 MG.A2-7, and U87 MG.wtEGFR xenografts. Sections
were collected at the time points described in Fig. 31 above. Xenograft
sections were
immunostained with the 528 antibody (left panel) and inAb 806 (right panel).
No
decreased immunoreactivity to either wt EGFR, amplified EGFR, or de2-7 EGFR
was observed in xenografts treated with rnAb 806. Consistent with the in vitro
data,
parental U87 MG xenografts were positive for 528 antibody but were negative
for
mAb 806 staining.
Figure 33. Schematic representation of generated bicistronic expression
constructs.
Transcription of the chimeric antibody chains is initiated by Elongation
Factor-1
promoter and terminated by a strong artificial termination sequence. TRES
sequences
were introduced between coding regions of light chain and NeoR and heavy chain
and
dhfr gene.
CA 02826627 2013-09-04
Figure 34. Biodistribution analysis of the ch806 radiolabeled with either A)
1251 or B)
111In was performed in BALB/c nude mice bearing U87MG-de2-7 xenograft tumors.
Mice were injected with 5 ug of radiolabeled antibody and in groups of 4 mice
per
time point, sacrificed at either 8, 28, 48 or 74 hours. Organs were collected,
weighed
and radioactivity measured in a gamma counter.
Figure 35. Depicts (A) the % ID gram tumor tissue and (B) the tumor to blood
ratio.
Indium-111 antibody shows approximately 30% ED/gram tissue and a tumor to
blood
ratio of 4Ø
= Figure 36 depicts the therapeutic efficacy of chimeric antibody ch806 in
an
= established tumor model. 3x106 U87MG.42-7 cells in 100u1 of PBS were
inoculated
s.c. into both flanks of 4-6 week old female nude mice. The mAb806 was
included as
a positive contra Treatment was started when tumors had reached a mean volume
of
50 mm3 and consisted of 1 mg of ch806 or rnAb806 given i.p. for a total of 5
injections on the days indicated. Data was expressed as mean tumor volume +/-
S.E.
for each treatment group.
Figure 37. CDC Activity on Target A) U87MG.de2-7 and B) A431 cells for anti-
EGFR chimeric IgG1 antibodies ch806 and control cG250. Mean (bars; + SD)
percent cytotoxicity of triplicate determinations are presented.
Figure 38. ADCC on target A) U87MG.de2-7 and B) A431 cells at Effector: Target
cell ratio of 50:1 mediated by ch806 and isotype control cG250 (0-10 ug/m1).
Results
are expressed as mean (bars; + SD) percent cytotoxicity of triplicate
determinations.
Figure 39. ADCC mediated by 1 ug/ml parental mAb 806 and ch806 on target
U87M0.de2-7 cells over a range of Effector: Target ratios. Mean (bars; + SD)
of
triplicate determinations are presented.
21
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Figure 40. Twenty-five hybridomas producing antibodies that bound ch806 but
not
huIgG were initially selected. Four of these anti-ch806 hybridomas with high
affinity
binding (clones 3E3, 5B8, 9D6 and 4D8) were subsequently pursued for clonal
expansion from single cells by limiting dilution and designated Ludwig
Institute for
Cancer Research Melbourne Hybridorna (LMI-1) -11, -12, -13 and -14,
respectively.
In addition, two hybridomas that produced mAbs specific for hulgG were also
cloned
and characterized further: clones 2C10 (L1VIH-15) and 2B8 (LMII-16).
Figure 41. After clonal expansion, the hybridoma culture supernatants were
exmined in triplicate by FT ISA for the ability to neutralize ch806 or mAb 806
antigen binding activity with sEG.L.R621. Mean (- SD) results demonstrated
the
antagonist activity of anti-idiotype mAbs LMEI-11, -12, -13 and -14 with the
blocking
in solution of both ch806 and murine mAb 806 binding to plates coated with
sEGFR
(LMH-14 not shown).
Figure 42. Microtitre plates were coated with 10 ug/m1purified A) LMH-11, B)
LMH-12 and C) LME-13. The three purified clones were compared for their
ability
to capture ch806 or mAb 806 in sera or 1% FCS/Media and then detect bound
ch806
or mAb806. Isotype control antibodies hu3S193 and m3S193 in serum and 1%
FCS/Media were included in addition to controls for secondary conjugate avidin-
HRP
and ABTS substrate. Results are presented as mean ( SD) of triplicate samples
using
biotinylated-L1VEH-12 (10 ug/m1) for detection and indicate LMH-12 used for
capture
and detection had the highest sensitivity for ch806 in serum (3 nem with
negligible
background binding.
Figure 43. Validation of the optimal pharmacokinetic FT ISA conditions using 1
ug/m1 anti-idiotype LM11-12 and 1 ug/mlbiotinylated LMH-12 for capture and
detection, respectively. Three separate ET .TSAs were performed in
quadruplicate to
measure ch806 in donor serum (0) from three healthy donors or 1% BSA/media (N)
with isotype control hu3S193 in serum (A) or 1% BSA/media (V). Controls for
secondary conjugate avidin-HRP (4) and ABTS substrate (hexagon) alone were
also
22
CA 02826627 2013-09-04
included with each MBA. Mean ( SD) results demonstrate highly reproducible
binding curves for measuring ch806 (2 u.g/m1¨ 1.6 ng/ml) in sera with a 3
ng/m1 limit
of detection. (n=12; 1-100 ng,/ml, Coefficient of Variation < 25%; 100 ng/ml-
5
1.tg/m1, Coefficient of Variation < 15%). No background binding was evident
with any
of the three sera tested and negligible binding was observed with isotype
control
hu3S193.
Figure 44 depicts an immunoblot of recombinant sEGFR expressed in CHO cells,
blotted with mAb806. Recombinant sEGFR was treated with PNGaseF to remove N-
linked glycosylation (deglycosylated), or untreated (untreated), the protein
was run on
SDS-PAGE, transferred to membrane and immunoblotted with mAb 806.
Figure 45 depicts immunoprecipitation of EGFR from 35S-labelled cell lines
(U87MG
A2-7, U87MG-wtEGFR, and A431) with different antibodies (SC-03, 806 and 528
antibodies).
Figure 46 depicts immunoprecipitation of EGFR from different cells (A431 and
U87MGA2-7) at different time points (time 0 to 240 minutes) after pulse-
labelling
with 35S raethioninekysteine. Antibodies 528 and 806 are used for
immunoprecipitation.
Figure 47 depicts immunoprecipitation of EGFR from various cell lines (U87MGA2-
7, U87MG-wtEGFR and A431) with various antibodies (SC-03, 806 and 528) in the
absence of (-) and after Endo H digestion (+) to remove high mannose type
carbohydrates.
Figure 48 depicts cell surface iodination of the A431 and U87MGA2-7 cell lines
followed by immunoprecipitation with the 806 antibody, and with or without
Endo H
digestion, confirming that the EGFR bound by mAb 806 on the cell surface of
A431
cells is an EndoH sensitive form.
23
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DETAILED DESCRIPTION
In accordance with the present invention there may be employed conventional
molecular biology, microbiology, and recombinant DNA techniques within the
skill
of the art. Such techniques are explained fully in the literature. See, e.g.,
Sambrook
et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in
Molecular Biology" Volumes [Ausubel, R. M., ed. (1994)]; "Cell Biology: A
Laboratory Handbook" Volumes [J. E. Celis, ed. (1994))); "Current Protocols
in
Immunology" Volumes I-B1 [Coligan, J. E, ed. (1994)]; "Oligonucleotide
Synthesis"
(M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Haines & S.J. Higgins
eds.
(1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds.
(1984)];
''Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And
Enzymes"
[1RL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning"
(1984).
Therefore, if appearing herein, the following terms shall have the definitions
set out
below.
A. l'ERMINOLOGY
The term "specific binding member"describes a member of a pair of molecules
which
have binding specificity for one another. The members of a specific binding
pair may
be naturally derived or wholly or partially synthetically produced. One member
of the
pair of molecules has an area on its surface, or a cavity, which specifically
binds to
and is therefore complementary to a particular spatial and polar organisation
of the
other member of the pair of molecules. Thus the members of the pair have the
property of binding specifically to each other. Examples of types of specific
binding
pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-
ligand,
enzyme-substrate. This application is concerned with antigen-antibody type
reactions.
The term "aberrant expression" in its various grammatical forms may mean and
include any heightened or altered expression or overexpression of a protein in
a tissue,
24
CA 02826627 2013-09-04
e.g. an increase in the amount of a protein, caused by any means including
enhanced
expression or translation, modulation of the promoter or a regulator of the
protein,
amplification of a gene for a protein, or enhanced half-life or stability,
such that more
of the protein exists or can be detected at any one time, in contrast to a non-
overexpressed state. Aberrant expression includes and contemplates any
scenario or
alteration wherein the protein expression or post-translational modification
machinery
in a cell is taxed or otherwise disrupted due to enhanced expression or
increased
levels or amounts of a protein, including wherein an altered protein, as in
mutated
protein or variant due to sequence alteration, deletion or insertion, or
altered folding is
expressed.
It is important to appreciate that the term "aberrant expression" has been
specifically
chosen herein to encompass the state where abnormal (usually increased)
quantities/levels of the protein are present, irrespective of the efficient
cause of that
abnormal quantity or level. Thus, abnormal quantities of protein may result
from
overexpression of the protein in the absence of gene amplification, which is
the case
e.g. in many cellular/tissue samples taken from the head and neck of subjects
with
cancer, while other samples exhibit abnormal protein levels attributable to
gene
amplification.
In this latter connection, certain of the work of the inventors that is
presented herein to
illustrate the invention includes the analysis of samples certain of which
exhibit
abnormal protein levels resulting from amplification of EFGR. This therefore
accounts for the presentation herein of experimental findings where reference
is made
= to amplification and for the use of the terms "amplification/amplified"
and the like in
describing abnormal levels of .Et.GR. However, it is the observation of
abnormal
quantities or levels of the protein that defines the environment or
circumstance where
clinical intervention as by resort to the binding members of the invention is
contemplated, and for this reason, the present specification considers that
the term
"aberrant expression" more broadly captures the causal environment that yields
the
corresponding abnormality in EFGR levels. =
CA 02826627 2013-09-04
Accordingly, while the terms "overexpression" and "amplification" in their
various
grammatical forms are understood to have distinct technical meanings, they are
to be
considered equivalent to each other, insofar as they represent the state where
abnormal 1-1-,GR protein levels are present in the context of the present
invention.
Consequently, the term "aberrant expression" has been chosen as it is believed
to
subsume the terms "overexpression" and "amplification" within its scope for
the
purposes herein, so that all terms may be considered equivalent to each other
as used
herein.
The term "antibody "describes an immunoglobulin whether natural or partly or
wholly synthetically produced. The term also covers any polypeptide or protein
having a binding domain which is, or is homologous to, an antibody binding
domain.
CDR grafted antibodies are also contemplated by this term.
As antibodies can be modified in a number of ways, the term "antibody" should
be
construed as covering any specific binding member or substance having a
binding
domain with the required specificity. Thus, this term covers antibody
fragments,
derivatives, functional equivalents and homologues of antibodies, including
any
polypeptide comprising an irrununoglobulin binding domain, whether natural or
wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin
binding domain, or equivalent, fused to another polypeptide are therefore
included..
Cloning and expression of chimeric antibodies are described in EP-A-0120694
and
EP-A-0125023 and U.S. Patent Nos. 4;816,397 and 4,816,567.
It has been shown that fragments of a whole antibody can perform the function
of
binding antigens. Examples of binding fragments are (i) the Fab fragment
consisting
of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and
CHI
domains; (iii) the Fv fragment consisting of the VL and VH domains of a single
antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature 341, 544-546
(1989))
which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab)2
fragments, a
26
CA 02826627 2013-09-04
bivalent fiagment comprising two linked Fab fragments (vii) single chain Fv
molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide
linker which allows the two domains to associate to form an antigen binding
site (Bird
et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883,
1988);
(viii) multivalent antibody fragments (scFv dimers, trimers and/or tetramers
(Power
and Hudson, J Immunol. Methods 242: 193-204 9 (2000))(ix) bispecific single
chain
Fv dimers (PC1/US92/09965) and (x) "diabodies", multivalent or multispecific
fragments constructed by gene fusion (W094/13804; P. Holliger et al Proc.
Natl.
Acad. Sci. USA 90 6444-6448, (1993)).
An "antibody combining site" is that structural portion of an antibody
molecule
comprised of light chain or heavy and light chain variable and hypervariable
regions
that specifically binds antigen,
The phrase "antibody molecule" in its various grammatical forms as used herein
contemplates both an intact immunoglobulin molecule and an immunologically
active
portion of an immunoglobulin molecule.
Exemplary antibody molecules are intact immunoglobulin molecules,
substantially
intact immunoglobulin molecules and those portions of an immunoglobulin
molecule
that contains the paratope, including those portions known in the art as Fab,
Fab',
F(ab')2 and F(v), which portions are preferred for use in the therapeutic
methods
described herein.
Antibodies may also be bispecific, wherein one binding domain of the antibody
is a
specific binding member of the invention, and the other binding domain has a
different specificity, e.g. to recruit an effector function or the like.
Bispecific
antibodies of the present invention include wherein one binding domain of the
antibody is a specific binding member of the present invention, including a
fragment
thereof, and the other binding domain is a distinct antibody or fragment
thereof,
including that of a distinct anti-EGFR antibody, for instance antibody 528
(U.S.
27
CA 02826627 2013-09-04
Patent No. 4,943,533), the chimeric and humanized 225 antibody (U.S. Patent
No.
4,943,533 and W0/9640210), an anti-<1e2-7 antibody such as DH8.3 alls, D. et
al
(1995) Int. J. Cancer 63(4).537-543), antibody L8A4 and Y10 (Reist, CJ et al
(1995)
Cancer Res. 55(19):4375-4382; FouIon CF et al. (2000) Cancer Res. 60(16):4453-
4460), ICR62 (Modjtahedi H et al (1993) Cell Biophys. Jan-Jun;22(1-3):129-46;
Modjtahedi et al (2002) P.A.A.C.R. 55(14):3140-3148, or the antibody of
Wilcstrand
et al (Wikstrand C. et al (1995) Cancer Res. 55(14)3140-3148). The other
binding
domain may be an antibody that recognizes or targets a particular cell type,
as in a
neural or glial cell-specific antibody. In the bispecific antibodies of the
present
=
invention the one binding domain of the antibody of the invention may be
combined
with other binding domains or molecules which recognize particular cell
receptors
and/or modulate c,ells in a particular fashion, as for instance an imm.une
modulator
(e.g., interieulcin(s)), a gnowth modulator or cytolcine (e.g. turnor necrosis
factor
(TNF), and particularly, the 'INF bispecific modality demonstrated in U.S.S.N.
60/355,838 filed February 13, 2002) or a
toxin (e.g.,
ricin) or anti-mitotic or apoptotic agent or factor.
Fab and F(ala.)2 portions of antibody imolecules may be prepared by the
proteolytic
reaction of papain and pepsin, respectively, on substantially intact antibody
molecules
by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to
Theofilopolous et al. Fab' antibody molecule portions are also well-known and
are
produced from F(ab1)2 portions followed by reduction of the disulfide bonds
'Juicing
the two heavy chain portions as with mercaptoethanol, and followed by
ancylation of
the resulting protein mercaptan with a reagent such as iodoacetamide. An
antibody
containing intact antibody molecules is preferred herein.
The phrase "monoclonal antibody" in its various grauunatical forms refers to
an
antibody having only one species of antibody combining site capable of
inununoreacting with a particular antigen. A monoclonal antibody thus
typically
displays a single binding affinity for any antigen with which it
inamunoreacts. A
monoclonal antibody may also contain an antibody molecule having a plurality
of
28
CA 02826627 2013-09-04
antibody combining sites, each imrnunospecific for a different antigen; e.g.,
a
bispecific (chimeric) monoclonal antibody.
The term "antigen binding domain" describes the part of an antibody which
comprises
the area which specifically binds to and is complementary to part or all of an
antigen.
Where an antigen is large, an antibody may bind to a particular part of the
antigen
only, which part is termed an epitope. An antigen binding domain may be
provided
by one or more antibody variable domains. Preferably, an antigen binding
domain
comprises an antibody light chain variable region (VL) and an antibody heavy
chain
variable region (VH).
"Post-translational modification" may encompass any one of or combination of
modification(s), including covalent modification, which a protein undergoes
after
translation is complete and after being released from the ribosome or on the
nascent
polypeptide cotranslationally. Post-translational modification includes but is
not
limited to phosphorylation, myristylation, ubiquitination, glycosylation,
coenzyme
attachment, methylation and acetylation. Post-translational modification can
modulate or influence the activity of a protein, its intracellular or
extracellular
destination, its stability or half-life, and/or its recognition by ligands,
receptors or
other proteins Post-translational modification can occur in cell organelles,
in the
nucleus or cytoplasm or extracellularly.
The term "specific" may be used to refer to the situation in which one member
of a
specific binding pair will not show any significant binding to molecules other
than its
specific binding partner(s). The term is also applicable where e., . an
antigen binding
domain is specific for a particular epitope which is carried by a number of
antigens, in
which case the specific binding member carrying the antigen binding domain
will be
able to bind to the various antigens carrying the epitope.
The term "comprise"generally used in the sense of include, that is to say
permitting
the presence of one or more features or components.
29
CA 02826627 2013-09-04
The term "consisting essentially of' refers to a product, particularly a
peptide
sequence, of a defined number of residues which is not covalently attached to
a larger
product. In the case of the peptide of the invention referred to above, those
of skill in
the art will appreciate that minor modifications to the N- or C- terminal of
the peptide
may however be contemplated, such as the chemical modification of the terminal
to
add a protecting group or the like, e.g. the arnidation of the C-terminus.
The term "isolated"refers to the state in which specific binding members of
the
invention, or nucleic acid encoding such binding members will be, in
accordance with
the present invention. Members and nucleic acid will be free or substantially
free of
material with which they are naturally associated such as other polypeptides
or
nucleic acids with which they are found in their natural environment, or the
environment in which they are prepared (e.g. cell culture) when such
preparation is by
recombinant DNA technology practised in vitro or in vivo. Members and nucleic
acid
may be formulated with diluents or adjuvants and still for practical purposes
be
isolated - for example the members will normally be mixed with gelatin or
other
carriers if used to coat microtitre plates for use in immunoassays, or will be
mixed
with pharmaceutically acceptable carriers or diluents when used in diagnosis
or
therapy. Specific binding members may be glycosylated, either naturally or by
systems of heterologous eukaryotic cells, or they may be (for example if
produced by
expression in a prokaryotic cell) unglycosylated.
Also, as used herein, the terms "glycosylation" and "glycosylated" includes
and
encompasses the post-translational modification of proteins, termed
glycoproteins, by
addition of oligosaccarides. Oligosaccharides are added at glycosylation sites
in
glycoproteins, particularly including N-linked oligosaccharides and 0-linked
oligosaccharides. N-linked oligosaccharides are added to an Asn residue,
particularly
wherein the Asn residue is in the sequence N-X-S/T, where X cannot be Pro or
Asp,
and are the most common ones found in glycoproteins. In the biosynthesis of N-
linked glycoproteins, a high mannose type oligosaccharide (generally comprised
of
CA 02826627 2013-09-04
dolichol, N-Acetylglucosamine, mannose and glucose is first formed in the
endoplasmic reticulum (ER). The high mannose type glycoproteins are then
transported from the ER to the Golgi, where further processing and
modification of
the oligosaccharides occurs. 0-linked oligosaccharides are added to the
hydroxyl
group of Ser or Thr residues. In 0-linked oligosaccharides, N-
Acetylglucosamine is
first transferred to the Ser or Thr residue by N-Acetylglucosaminyltransferase
in the
ER. The protein then moves to the Golgi where further modification and chain
elongation occurs. 0-linked modifications can occur with the simple addition
of the
0G1cNAc monosaccharide alone at those Ser or Thr sites which can also under
different conditions be phosphorylated rather than glycosylated.
As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "lig" mean
microgram, "mg" means milligram, "ul" or "0" mean microliter, "ml" means
milliliter, "1" means liter.
The terms "806 antibody", "mAb806", "ch806" and any variants not specifically
listed, may be used herein interchangeably, and as used throughout the present
application and claims refer to proteinaceous material including single or
multiple
proteins, and extends to those proteins having the amino acid sequence data
described
herein and presented in SEQ ID NO:2 and SEQ JD NO:4, and the chimeric antibody
ch806 which is incorporated in and forms a part of SEQ ID NOS: 7 and 8, and
the
profile of activities set forth herein and in the Claims. Accordingly,
proteins
displaying substantially equivalent or altered activity are likewise
contemplated.
These modifications may be deliberate, for example, such as modifications
obtained
through site-directed mutagenesis, or may be accidental, such as those
obtained
through mutations in hosts that are producers of the complex or its named
subunits.
Also, the terms ''806 antibody", "mAb806" and "ch806" are intended to include
within their scope proteins specifically recited herein as well as all
substantially
homologous analogs and allelic variations.
=
31
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The amino acid residues described herein are preferred to be in the "L"
isomeric form.
However, residues in the "D" isomeric form can be substituted for any L-amino
acid
residue, as long as the desired fuctional property of immunoglobulin-binding
is
retained by the polypeptide. NH2 refers to the free amino group present at the
amino
terminus of a polypeptide. COOH refers to the free carboxy group present at
the
carboxy terminus of a polypeptide. In keeping with standard polypeptide
nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid
residues are shown in the following Table of Correspondence:
TABI F OF CORRESPONDENCE
SYMBOL AMINO ACID
1-Letter 3-Letter
Tyr tyrosine
Gly glycine
Phe phenyla1anine
Met methionine
A Ala alanine
Ser serine
Be isoleucine
Leu leucine
Thr threonine
V Val valine
Pro proline
Lys lysine
His histidine
Gln glutamine
Glu glutamic acid
Trp tryptophan
Arg arginine
Asp aspartic acid
Asn asparagine
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Cys cysteine
It should be noted that all amino-acid residue sequences are represented
herein by
formulae whose left and right orientation is in the conventional direction of
amino-
terminus to carboxy-terminus. Furthermore, it should be noted that a dash at
the
beginning or end of an amino acid residue sequence indicates a peptide bond to
a
further sequence of one or more amino-acid residues. The above Table is
presented to
correlate the three-letter and one-letter notations which may appear
alternately herein.
A ''replicon" is any genetic element (e.g., plasmid, chromosome, virus) that
functions
as an autonomous unit of DNA replication in vivo; i.e., capable of replication
under its
own control.
A "vector" is a replicon, such as plasinid, phage or cosmid, to which another
DNA
segment may be attached so as to bring about the replication of the attached
segment.
A "DNA molecule" refers to the polymeric form of deoxyribonucleotides
(adenine,
guanine, thymine, or cytosine) in its either single stranded form, or a double-
stranded
helix. This term refers only to the primary and secondary structure of the
molecule,
and does not limit it to any particular tertiary forms. Thus, this term
includes double-
stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction
fragments),
viruses, plasrnids, and chromosomes. In discussing the structure of particular
double-
stranded DNA molecules, sequences may be described herein according to the
normal
convention of giving only the sequence in the 5' to 3' direction along the
nontranscribed strand of DNA (i.e., the strand having a sequence homologous to
the
mRNA).
An "origin of replication" refers to those DNA sequences that participate in
DNA
synthesis.
33
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A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed
and translated into a polypeptide in vivo when placed under the control of
appropriate
regulatory sequences. The boundaries of the coding sequence are determined by
a
start codon at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl)
teiminus. A coding sequence can include, but is not limited to, prokaryotic
sequences, cDNA from eukaryotic niRNA, genomic DNA sequences from eukaryotic
(e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation
signal and transcription termination sequence will usually be located 3' to
the coding
sequence.
Transcriptional and translational control sequences are DNA regulatory
sequences,
such as promoters, enhancers, polyadenylation signals, terminators, and the
like, that
provide for the expression of a coding sequence in a host cell.
=
A "promoter sequence" is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream (3'
direction) coding
sequence. For purposes of defining the present invention, the promoter
sequence is
bounded at its 3' terminus by the transcription initiation site and extends
upstream (5'
direction) to include the minimum number of bases or elements necessary to
initiate
transcription at levels detectable above background. Within the promoter
sequence
will be found a transcription initiation site (conveniently defined by mapping
with
nuclease S1), as well as protein binding domains (consensus sequences)
responsible
for the binding of RNA polymerase. Eukaryotic promoters will often, but not
always,
contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-
Dalganio sequences in addition to the -10 and -35 consensus sequences.
An "expression control sequence" is a DNA sequence that controls and regulates
the
transcription and translation of another DNA sequence. A coding sequence is
"under
he control" of transcriptional and translational control sequences in a cell
when RNA
polymerase transcribes the coding sequence into mRNA, which is then translated
into
the protein encoded by the coding sequence.'
34
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A "signal sequence" can be included before the coding sequence. This sequence
encodes a signal peptide, N-terminal to the polypeptide, that communicates to
the host
cell to direct the polypeptide to the cell surface or secrete the polypeptide
into the
media, and this signal peptide is clipped off by the host cell before the
protein leaves
the cell. Signal sequences can be found associated with a variety of proteins
native to
prokaryotes and eukaryotes.
The term "oligonucleotide," as used herein in referring to the probe of the
present
invention, is defined as a molecule comprised of two or more ribonucleotides,
preferably more than three. Its exact size will depend upon many factors
which, in
turn, depend upon the ultimate function and use of the oligonucleotide.
The term "primer" as used herein refers to an oligonucleotide, whether
occurring
naturally as in a purified restriction digest or produced synthetically, which
is capable
of acting as a point of initiation of synthesis when placed under conditions
in which
synthesis of a primer extension product, which is complementary to a nucleic
acid
strand, is induced, i.e., in the presence of nucle,otides and an inducing
agent such as a
DNA polymerase and at a suitable temperature and pH. The primer may be either
single-stranded or double-stranded and must be sufficiently long to prime the
synthesis of the desired extension product in the presence of the inducing
agent. The
exact length of the primer will depend upon many factors, including
temperature,
source of primer and use of the method. For example, for diagnostic
applications,
depending on the complexity of the target sequence, the oligonucleotide primer
typically contains 15-25 or more nucleotides, although it may contain fewer
nucleotides.
The primers herein are selected to be "substantially" complementary to
different
= strands of a particular target DNA sequence. This means that the primers
must be
sufficiently complementary to hybridize with their respective strands.
Therefore, the
primer sequence need not reflect the exact sequence of the template. For
example, a
CA 02826627 2013-09-04
non-complementary nucleotide fragment may be attached to the 5 end of the
primer,
with the remainder of the primer sequence being complementary to the strand.
Alternatively, non-complementary bases or longer sequences can be interspersed
into
the primer, provided that the primer sequence has sufficient complementarity
with the
sequence of the strand to hybridize therewith and thereby form the template
for the
synthesis of the extension product.
As used herein, the terms "restriction endonucleases" and "restriction
enzymes" refer
to bacterial enzymes, each of which cut double-stranded DNA at or near a
specific
nucleotide sequence.
A cell has been "transformed" by exogenous or heterologous DNA when such DNA
has been introduced inside the cell. The transforming DNA may or may not be
integrated (covalently linked) into chromosomal DNA making up the genome of
the
cell. In prokaryotes, yeast, and mammalian cells for example, the transforming
DNA
may be maintained on an episomal element such as a plasmid. With respect to
eukaryotic cells, a stably transformed cell is one in which the transforming
DNA has
become integrated into a chromosome so that it is inherited by daughter cells
through
chromosome replication. This stability is demonstrated by the ability of the
eukaryotic cell to establish cell lines or clones comprised of a population of
daughter
cells containing the transforming DNA. A "clone" is a population of cells
derived
from a single cell or common ancestor by mitosis. A "cell line" is a clone of
a
primary cell that is capable of stable growth in vitro for many generations.
Two DNA sequences are "substantially homologous" when at least about 75%
(preferably at least about 80%, and most preferably at least about 90 or 95%)
of the
nucleotides match over the defined length of the DNA sequences. Sequences that
are
substantially homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a Southern
hybridization
experiment under, for example, stringent conditions as defined for that
particular
system. Defining appropriate hybridization conditions is within the skill of
the art.
36
CA 02826627 2013-09-04
See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic
Acid
Hybridization, supra.
It should be appreciated that also within the scope of the present invention
are DNA
sequences encoding specific binding members (antibodies) of the invention
which
code for e.g. an antibody having the same amino acid sequence as SEQ ID NO:2
or
SEQ ID NO:4, but which are degenerate to SEQ ID NO:2 or SEQ NO:4. By
"degenerate to" is meant that a different three-letter codon is used to
specify a
particular amino acid. It is well known in the art that the following codons
can be
used interchangeably to code for each specific amino acid:
Phenylalanine (Phe or F) UUU or UUC
Leucine (Leu or L) UUA or LTUG or CUU or CUC or CUA or CUG
Isoleucine ale or 1) AUU or AUC or AUA
Methionine (Met or M) AUG
Valine (Val or V) GUU or GUC of GUA or GUG
Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC
Proline (Pro or P) CCU or CCC or CCA or CCG
Threonine (Thr or T) ACU or ACC or ACA or ACG
=
Alanine (Ala or A) GCU or GCG or GCA or GCG
Tyrosine (Tyr or Y) UAU or UAC
Histidine (His or H) CAU or CAC
Glutamine (Gln or Q) CAA or CAG
Asparagine (Asn or N) AAU or AAC
Lysine (Lys or K) AAA or AAG
Aspartic Acid (Asp or D) GAU or GAC
Glutamic Acid (Glu or E) GAA or GAG
Cysteine (Cys or C) UGU or UGC
Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG
Glycine (Gly or G) GGU or GGC or GGA or GGG
Tryptophan (Trp or W) UGG
37
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Termination codon UAA (ochre) or UAG (amber) or UGA (opal)
It should be understood that the codons specified above are for RNA sequences.
The
corresponding codons for DNA have a T substituted for U.
Mutations can be made in SEQ ID NO:2 or SEQ ID NO:4 such that a particular
codon
is changed to a codon which codes for a different amino acid. Such a mutation
is
generally made by making the fewest nucleotide changes possible. A
substitution
mutation of this sort can be made to change an amino acid in the resulting
protein in a
non-conservative manner (i.e., by changing the codon from an amino acid
belonging
to a grouping of amino acids having a particular size or characteristic to an
amino acid
belonging to another grouping) or in a conservative manner (i.e., by changing
the
codon from an amino acid belonging to a grouping of amino acids having a
particular
size or characteristic to an amino acid belonging to the same grouping). Such
a
conservative change generally leads to less change in the structure and
function of the
resulting protein. A non-conservative change is more likely to alter the
structure,
activity or function of the resulting protein. The present invention should be
considered to include seguences containing conservative changes which do not
significantly alter the activity or binding characteristics of the resulting
protein.
The following is one example of various groupings of amino acids:
Amino acids with nonpolar R groups
Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan,
Methionine
Amino acids with uncharged polar R groups
Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine
Amino acids with charged polar R groups (negatively charged at Ph 6.0)
Aspartic acid, Glutamic acid
38
CA 02826627 2013-09-04
Basic amino acids (positively charged at pH 6.0)
Lysine, Arginine, Histidine (at pH 6.0)
Another grouping may be those amino acids with phenyl groups:
Phenylalanine, Tryptophan, Tyrosine
Another grouping may be according to molecular weight (i.e., size of R
groups):
Glycine 75
Alanine 89
Serine 105
Proline 115
Valine 117
Threonine 119
Cysteine 121
Leucine 131
Isoleucine 131
Asparagine 132
Aspartic acid 133
Glutamine 146
Lysine 146
Glutamic acid 147
Methionine 149
Histidine (at pH 6.0) 155
Phenylalanine 165
Arginine 174
Tyrosine = 181
Tryptophan 204
Particularly preferred substitutions are:
- Lys for Arg and vice versa such that a positive charge may be maintained;
- Glu for Asp and vice versa such that a negative charge may be maintained;
39
CA 02826627 2013-09-04
- Ser for Thr such that a free -OH can be maintained; and
- Gln for Asn such that a free NH, can be maintained.
Amino acid substitutions may also be introduced to substitute an amino acid
with a
particularly preferable property. For example, a Cys may be introduced a
potential
site for disulfide bridges with another Cys. A His may be introduced as a
particularly
"catalytic" site (i.e., His can act as an acid or base and is the most common
amino acid
in biochemical catalysis). Pro may be introduced because of its particularly
planar
structure, which induces= f3-turns in the protein's structure.
Two amino acid sequences are "substantially homologous" when at least about
70%
of the amino acid residues (preferably at least about 80%, and most preferably
at least
about 90 or 95%) are identical, or represent conservative substitutions.
A "heterologous" region of the DNA construct is an identifiable segment of DNA
within a larger DNA molecule that is not found in association with the larger
molecule in nature. Thus, when the heterologous region encodes a mammalian
gene,
the gene will usually be flanked by DNA that does not flank the mammalian
genomic
DNA in the genome of the source organism. Another example of a heterologous
coding sequence is a construct where the coding sequence itself is not found
in nature
(e.g., a cDNA where the genomic coding sequence contains introns, or synthetic
sequences having codons different than the native gene). Allelic variations or
naturally-occurring mutational events do not give rise to a heterologous
region of
DNA as defined herein.
The phrase "pharmaceutically acceptable" refers to molecular entities and
compositions that are physiologically tolerable and do not typically produce
an
allergic or similar untoward reaction, such as gastric upset, dizziness and
the like,
when administered to a human.
CA 02826627 2013-09-04
The phrase "therapeutically effective amount" is used herein to mean an amount
sufficient to prevent, and preferably reduce by at least about 30 percent,
preferably by
at least 50 percent, preferably by at least 70 percent, preferably by at least
80 percent,
preferably by at least 90%, a clinically significant change in the growth or
progression
or mitotic activity of a target cellular mass, group of cancer cells or tumor,
or other
feature of pathology. For example, the degree of EGFR activation or activity
or
amount or number of EGFR positive cells, particularly of antibody or binding
member reactive or positive cells may be reduced.
A DNA sequence is "operatively linked" to an expression control sequence when
the
expression control sequence controls and regulates the transcription and
translation of
that DNA sequence. The term "operatively linked" includes having an
appropriate
start signal (e.g., ATG) in front of the DNA sequence to be expressed and
maintaining
the correct reading frame to permit expression of the DNA sequence under the
control
of the expression control sequence and production of the desired product
encoded by
the DNA sequence. If a gene that one desires to insert into a recombinant DNA
molecule does not contain an appropriate start signal, such a start signal can
be
inserted in front of the gene.
The term "standard hybridization conditions" refers to salt and temperature
conditions
substantially equivalent to 5 x SSC and 65 C for both hybridization and wash.
However, one skilled in the art will appreciate that such "standard
hybridization
conditions" are dependent on particular conditions including the concentration
of
sodium and magnesium in the buffer, nucleotide sequence length and
concentration,
percent mismatch, percent formarnide, and the like. Also important in the
determination of "standard hybridization conditions" is whether the two
sequences
hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization
conditions are easily determined by one skilled in the art according to well
known
formulae, wherein hybridization is typically 10-20 C below the predicted or
determined Tm with washes of higher stringency, if desired.
41
CA 02826627 2013-09-04
B. DETAILED DISCLOSURE.
The present invention provides a novel specific binding member, particularly
an
antibody or fragment thereof, including immunogenic fragments, which
recognizes an
EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal
cells
wherein the epitope is enhanced or evident upon aberrant post-translational
modification and not detectable in normal or wild type cells. In a particular
but non-
limiting embodiment, the binding member, such as the antibody, recognizes an
EGFR
epitope which is enhanced or evident upon simple carbohydrate modification or
early
glycosylation and is reduced or not evident in the presence of complex
carbohydrate
modification or glycosylation. The specific binding member, such as the
antibody or
fragment thereof, does not bind to or recognize normal or wild type cells
containing
normal or wild type EGFR epitope in the absence of overexpression and in the
presence of normal EGFR post-translational modification.
The invention relates to a specific binding member, particularly an antibody
or a
fragment thereof, which recognizes an EGPR epitope which is present in cells
expressing amplified EGFR or expressing the de2-7 EGFR and not detectable in
cells
expressing normal or wild type EGFR, particularly in the presence of normal
post-
translational modification.
It is further noted and herein demonstrated that an additional non-limiting
observation
or characteristic of the antibodies of the present invention is their
recognition of their
epitope in the presence of high mannose groups, which is a characteristic of
early
glycosylation or simple carbohydrate modification. Thus, altered or abetrant
glycosylation facilitates the presence and/or recognition of the antibody
epitope or
comprises a portion of the antibody epitope.
Glycosylation includes and encompasses the post-translational modification of
proteins, termed glycoproteins, by addition of oligosaccarides.
Oligosaccharides are
added at glycosylation sites in glycoproteins, particularly including N-linked
42
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oligosaccharides and 0-linked oligosaccharides. N-linked oligosaccharides are
added
to an Asn residue, particularly wherein the Asn residue is in the sequence N-X-
S/T,
where X cannot be Pro or Asp, and are the most common ones found in
glycoproteins.
In the biosynthesis of N-linked glycoproteins, a high mannose type
oligosaccharide
(generally comprised of dolichol, N-Acetylglucosamine, mannose and glucose is
first
formed in the endoplasmic reticulum (ER). The high mannose type glycoproteins
are
then transported from the ER to the Golgi, where further processing and
modification
of the oligosaccharides normally occurs. 0-linked oligosaccharides are added
to the
hydroxyl group of Ser or Thr residues. In 0-linked oligosaccharides, N-
Acetylglucosarnine is first transferred to the Ser or TM- residue by N-
Acetylgucosaminyltransferase in the ER. The protein then moves to the Golgi
where
further modification and chain elongation occurs.
In a particular aspect of the invention and as stated above, the present
inventors have
discovered novel monoclonal antibodies, exemplified herein by the antibody
designated mAb 806 and its chimeric ch806, which specifically recognize
amplified
wild type EGFR and the de2-7 EGFR, yet bind to an epitope distinct from the
unique
junctional peptide of the de2-7 EGFR mutation. The antibodies of the present
invention specifically recognize overexpressed EGFR, including amplified EGFR
and
mutant EGFR (exemplified herein by the de2-7 mutation), particularly upon
aberrant
post-translational modification. Additionally, while mAb 806 does not
recognize the
normal, wild type EGFR expressed on the cell surface of glioma cells, it does
bind to
the extracellular domain of the EGFR immobilized on the surface of FT ISA
plates,
indicating a conformational epitope with a polypeptide aspect. Importantly,
mAb 806
did not bind significantly to normal tissues such as liver and skin, which
express
levels of endogenous wt EGFR that are higher than in most other normal
tissues, but
wherein EGFR is not overexpre,ssed or amplified. Thus, mAb806 demonstrates
novel
and useful specificity, recognizing de2-7 EGFR and amplified EGFR, while not
recognizing normal, wild type EGFR or the unique junctional peptide which is
characteristic of de2-7 EGFR.
43
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In a preferred aspect, the antibody is one which has the characteristics of
the antibody
which the inventors have identified and characterized, in particular
recognizing
amplified EGFR and de2-7EGFR. In a particularly preferred aspect the antibody
is
the mAb 806, or active fragments thereof. In a further preferred aspect the
antibody
of the present invention comprises the VH and VL amino acid sequences depicted
in
Figure 14 (SEQ ID NO:2) and Figure 15 (SEQ ID NO:4) respectively.
Preferably the epitope of the specific binding member or antibody is located
within
the region comprising residues 273-501 of the mature normal or wild type EGFR
sequence. Therefore, also provided are specific binding proteins, such as
antibodies,
which bind to the de2-7 EGER at an epitope located within the region
comprising
residues 273-501 of the EGFR sequence. The epitope may be determined by any
conventional epitope mapping techniques known to the person skilled in the
art.
Alternatively, the DNA sequence encoding residues 273-501 could be digested,
and
the resultant fragments expressed in a suitable host. Antibody binding could
be
determined as mentioned above.
In particular, the member will bind to an epitope comprising residues 273-501
of the
mature normal or wild type EGFR. However other antibodies which show the same
or a substantially similar pattern of reactivity also form an aspect of the
invention.
This may be determined by comparing such members with an antibody comprising
the VH and VL domains shown in SEQ ID NO:2 and SEQ ID NO:4 respectively.
The comparison will typically be made using a Western blot in which binding
members are bound to duplicate blots prepared from a nuclear preparation of
cells so
that the pattern of binding can be directly compared.
In another aspect, the invention provides an antibody capable of competing
with the
806 antibody, under conditions in which at least 10% of an antibody having the
VH
and VL sequences of the 806 antibody is blocked from binding to de2-7EGFR by
competition with such an antibody in an ELISA assay. As set forth above, anti-
idiotype antibodies are contemplated and are illustrated herein.
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CA 02826627 2013-09-04
An isolated polypeptide consisting essentially of the epitope comprising
residues 273-
501-of the mature wild type EGFR (residues 6-234 of mature de2-7 EGFR) forms
another aspect of the present invention. The peptide of the invention is
particularly
useful in diagnostic assays or kits and therapeutically or prophylactically,
including as
an anti-tumor or anti-cancer vaccine. Thus compositions of the peptide of the
present
invention include pharmaceutical composition and immunogenic compositions.
Diagnostic and Therapeutic Uses
The unique specificity of the specific binding members, particularly
antibodies or
fragments thereof, of the present invention, whereby the binding member(s)
recognize
an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal
cells
and not detectable in normal or wild type cells and wherein the epitope is
enhanced or
evident upon aberrant post-translational modification and wherein the
member(s) bind
to the de2-7 EGFR and amplified EGFR but not the wt EGFR, provides diagnostic
and therapeutic uses to identify, characterize, target and treat, reduce or
eliminate a
number of tumorigenic cell types and tumor types, for example head and neck,
breast,
lung, bladder or prostate tumors and glioma, without the problems associated
with
normal tissue uptake that may be seen with previously known EGFR antibodies.
Thus, cells overexpressing EGFR (e.g. by amplification or expression of a
mutant or
variant EGFR), particularly those demonstrating aberrant post-translational
modification may be recognized, isolated, characterized, targeted and treated
or
eliminated utilizing the binding member(s), particularly antibody(ies) or
fragments
thereof of the present invention.
The antibodies of the present invention can thus specifically categorize the
nature of
EGFR tumors or tumorigenic cells, by staining or otherwise re-cognizing those
tumors
or cells wherein EGFR overexpression, particularly amplification and/or EGFR
mutation, particularly de2-7EGFR, is present. Further, the antibodies of the
present
invention, as exemplified by inAb 806 and chimeric antibody ch806, demonstrate
CA 02826627 2013-09-04
significant in vivo anti-tumor activity against tumors containing amplified
EGI-R and
against de2-7 EGFR positive xenografts.
As outlined above, the inventors have found that the specific binding member
of the
invention recognises tumor-associated forms of the EGFR (de2-7 EGFR and
amplified EGFR) but not the normal, wild-type receptor when expressed in
normal
cells. It is believed that antibody recognition is dependent upon an aberrant
post-
translational modification (e.g., a unique glycosylation, acetyiation or
phosphorylation
variant) of the EGFR expressed in cells exhibiting overexpression of the EGFR
gene.
As described below, mAb 806 and ch806 have been used in therapeutic studies.
mAb
806 and ch806 are shown to inhibit growth of overexpressing (e.g. amplified)
EGFR
xenografts and human de2-7 EGFR expressing xenografts of human tumors and to
induce significant necrosis within such tumors.
Moreover, the antibodies of the present invention inhibit the growth of
intracranial
tumors in a preventative model. This model involves injecting glioma cells
expressing de2-7 EGFR into nude mice and then injecting the antibody
intracranially
either on the same day or within 1 to 3 days, optionally with repeated doses.
The
doses of antibody are suitably about 10p.g. Mice injected with antibody are
compared
to controls, and it has been found that survival of the treated mice is
significantly
increased.
Therefore, in a further aspect of the invention, there is provided a method of
treatment
of a tumor, a cancerous condition, a precancerous condition, and arty
condition related
to or resulting from hyperproliferative cell growth comprising administration
of a
specific binding member of the invention.
Antibodies of the present invention are designed to be used in methods of
diagnosis
and treatment of tumors in human or animal subjects, particularly epithelial
tumors.
46
CA 02826627 2013-09-04
These tumors may be primary or secondary solid tumors of any type including,
but
not limited to, glioma, breast, lung, prostate, head or neck tumors.
Binding Member and Antibody Generation
The general methodology for making monoclonal antibodies by hybridomas is well
known. Immortal, antibody-producing cell lines can also be created by
techniques
other than fusion, such as direct transformation of B lymphocytes with.
oncogenic
DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al.,
"Hybridoma Techniques" (1980); Hammerling et al., "Monoclonal Antibodies And T-
een Hybridomas¶ (1981); Kennett et al., "Monoclonal Antibodies" (1980); see
also
U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570;
4,466,917;
4,472,500; 4,491,632; 4,493,890.
Panels of monoclonal antibodies produced against FYGR can be screened for
various
properties; le., isotype, epitope, affinity, etc. Of particular interest are
monoclonal
antibodies that mimic the activity of BFGR or its subunits. Such monoclonals
can be
re,adily identified in specific binding member activity assays. High affinity
antibodies
are also useful when immunoaffirrity purification of native or recombinant
specific
binding member is possible.
Methods for producing polyclonal anti- EPGR antibodies are well-known in the
art.
See U.S. Patent No. 4,493,795 to Nestor et al. A monoclonal antibody,
typically
containing Fab and/or F(ab)2 portions of useful antibody molecules, can be
prepared
using the hybridoma technology described in Antibodies. - A Laborcrtory
Manual,
Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York (1988).
Briefly, to form the hybridoma from which the
monoclonal antibody composition is produced, a myeloma or other self-
perpetuating
cell line is fused with lymphocytes obtained from the spleen of a mammal
hyperimmunized with an appropriate BGFR.
47
CA 02826627 2013-09-04
Splenocytes are typically fused with myeloma cells using polyethylene glycol
(PEG)
6000. Fused hybrids are selected by their sensitivity to HAT. Hybridomas
producing
a monoclonal antibody useful in practicing this invention are identified by
their ability
to immunoreact with the present antibody or binding member and their ability
to
inhibit specified tumorigenic or hyperproliferative activity in target cells.
A monoclonal antibody useful in practicing the present invention can be
produced by
initiating a monoclonal hybridoma culture comprising a nutrient medium
containing a
hybridoma that secretes antibody molecules of the appropriate antigen
specificity.
The culture is maintained under conditions and for a time period sufficient
for the
hybridoma to secrete the antibody molecules into the medium. The antibody-
.
containing medium is then collected. The antibody molecules can then be
further
isolated by well-known techniques.
Media useful for the preparation of these compositions are both well-known in
the art
and commercially available and include synthetic culture media, inbred mice
and the
like. An exemplary synthetic medium is Dulbecco's minimal essential medium
(DMEM; Dulbecco et al., %Tirol. 8:396 (1959)) supplemented with 4.5 gtn/1
glucose,
20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is
the
Balb/e.
Methods for producing monoclonal anti-EGER antibodies are also well-known in
the
art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983).
Typically,
the EGFR or a peptide analog is used either alone or conjugated to an
immunogenic
carrier, as the immunogen in the before described procedure for producing anti-
EGFR
monoclonal antibodies. The hybridomas are screened for the ability to produce
an
antibody that imrnunoreacts with the EGFR present in tumorigenic, abnormal or
hyperproliferative cells. Other anti-EGFR antibodies include but are not
limited to
the HuMAX-EGFr antibody from Genmab/Medarex, the 108 antibody (ATCC
13E9764) and U.S. Patent No. 6,217,866, and antibody 14E1 from Schering AG
(U.S.
Patent No. 5,942,602).
48
CA 02826627 2013-09-04
Recombinant Binding Members, Chimerics, Bispecifics and Fragments
In general, the CDR3 regions, comprising amino acid sequences substantially as
set
out as the CDR3 regions of SEQ ID NO:2 and SEQ ID NO:4 will be carried in a
structure which allows for binding of the CDR3 regions to an tumor antigen. In
the
case of the CDR3 region of SEQ ID NO:4, this is preferably carried by the VL
region
of SEQ NO:4.
By "substantially as set out" it is meant that that CDR3 regions of the
invention will
be either identical or highly homologous to the specified regions of SEQ ID
NO:2 and
SEQ ID NO:4. By "highly homologous" it is contemplated that only a few
substitutions, preferably from 1 to 8, preferably from 1 to 5, preferably from
1 to 4, or
from 1 to 3 or 1 or 2 substitutions may be made in the CDRs.
The structure for carrying the CDR3s of the invention will generally be of an
antibody
heavy or light chain sequence or substantial portion thereof in which the CDR3
regions are located at locations corresponding to the CDR3 region of naturally
occurring VH and VL antibody variable domains encoded by rearranged
inimunoglobulin genes. The structures and locations of imrnunoglobulin
variable
domains may be determined by reference to ICabat, B.A. et al, Sequences of
Proteins
of Immunological Interest. 4th Edition. US Department of Health and Human
Services. 1987, and updates thereof, now available on the Internet
(http://immuno.bme.nwu.edu)).
Preferably, the amino acid sequence substantially as set out as residues 93-
102 of
SEQ 1D NO:2 is carried as the CDR3 in a human heavy chain variable domain or a
substantial portion thereof, and the amino acid sequences substantially as set
out as
residues 24-34, 50-56 and 89-97 of SEQ ID NO:4 are carried as the CDRs 1-3
respectively in a human light chain variable domain or a substantial portion
thereof.
49
CA 02826627 2013-09-04
The variable domains may be derived from any germline or rearranged human
variable domain, or may be a synthetic variable domain based on consensus
sequences of known human variable domains. The CDR3-derived sequences of the
invention, as defined in the preceding paragraph, may be introduced into a
repertoire
of variable domains lacking CDR3 regions, using recombinant DNA technology.
For example, Marks er al (Bio/Technology, 1992, 10:779-783) describe methods
of
producing repertoires of antibody variable domains in which consensus primers
directed at or adjacent to the 5' end of the variable domain area are used in
conjunction with consensus primers to the third framework region of human VH
genes to provide a repertoire of VH variable domains lacking a CDR3. Marks et
al
further describe how this repertoire may be combined with a CDR3 of a
particular
antibody. Using analogous techniques, the CDR3-derived sequences of the
present
invention may be shuffled with repertoires of VH or VL domains lacking a CDR3,
and the shuffled complete VH or VL domains combined with a cognate VL or VH
domain to provide specific binding members of the invention. The repertoire
may
then be displayed in a suitable host system such as the phage display system
of
W092/01047 so that suitable specific binding members may be selected. A
repertoire
may consist of from anything from 104 individual members upwards, for example
from 106 to 108 or lOwmembers.
Analogous shuffling or combinatorial techniques are also disclosed by Stemmer
(Nature, 1994, 370:389-391), who describes the technique in relation to a P-
lactamase
gene but observes that the approach may be used for the generation of
antibodies.
A further alternative is to generate novel VH or VL regions carrying the CDR3-
derived sequences of the invention using random mutagenesis of, for example,
the
mAb806 VH or VL genes to generate mutations within the entire variable domain.
Such a technique is described by Gram et al (1992, Proc. Natl. Acad. Sci.,
USA,
89:3576-3580), who used error-prone PCR.
CA 02826627 2013-09-04
Another method which may be used is to direct mutagenesis to CDR regions of VH
or
VL genes. Such techniques are disclosed by Barbas et al, (1994, Proc. Natl.
Acad.
Sci., USA, 91:3809-3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).
All the above described techniques are known as such in the art and in
themselves do
not form part of the present invention. The skilled person will be able to use
such
techniques to provide specific binding members of the invention using routine
methodology in the art.
A substantial portion of an immunoglobulin variable domain will comprise at
least the
three CDR regions, 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. Additional residues at
the
N-terminal or C-terminal end of the substantial part of the variable domain
may be
those not normally associated with naturally occurring variable domain
regions. For
example, construction of specific binding members of the present invention
made by
recombinant DNA techniques may result in the introduction of N- or C-terminal
residues encoded by linkers introduced to facilitate cloning or other
manipulation
steps. Other manipulation steps include the introduction of linkers to join
variable
domains of the invention to further protein sequences including
inununoglobulin
heavy chains, other variable domains (for example in the production of
diabodies) or
protein labels as discussed in more detail below.
Although in a preferred aspect of the invention specific binding members
comprising
a pair of binding domains based on sequences substantially set out in SEQ ID
NO:2
and SEQ ID NO:4 are preferred, single binding domains based on either of these
sequences form further aspects of the invention. in the case of the binding
domains
based on the sequence substantially set out in SEQ ID NO:2, such binding
domains
may be used as targeting agents for tumor antigens since it is known that
51
CA 02826627 2013-09-04
immunoglobulin VH domains are capable of binding target antigens in a specific
manner.
In the case of either of the single chain specific binding domains, these
domains may
be used to screen for complementary domains capable of forming a two-domain
specific binding member which has in vivo properties as good as or equal to
the
mAb806 antibody disclosed herein.
This may be achieved by phage display screening methods using the so-called
hierarchical dual combinatorial approach as disclosed in U.S. Patent 5,969,108
in
which an individual colony containing either an H or L chain clone is used to
infect a
complete library of clones encoding the other chain (L or H) and the resulting
two-
chain specific binding member is selected in accordance with phage display
techniques such as those described in that reference. This technique is also
disclosed
in Marks et al, ibid.
Specific binding members of the present invention may further comprise
antibody
constant regions or parts thereof. For example, specific binding members based
on
SEQ ED NO:4 may be attached at their C-terminal end to antibody light chain
constant
domains including human CI< or a chains, preferably CX, chains. Similarly,
specific
binding members based on SEQ ID NO:2 may be attached at their C-terminal end
to
all or part of an immunoglobulin heavy chain derived from any antibody
isotype, e.g.
IgG, IgA, IgE, IgD and IgM and any of the isotype sub-classes, particularly
IgGl,
IgG2b, and IgG4. IgG1 is preferred.
The advent of monoclonal antibody (mAb) technology 25 years ago has provide an
enormous repertoire of useful research reagents and created the opportunity to
use
antibodies as approved pharmaceutical reagents in cancer therapy, autoirnmune
disorders, transplant rejection, antiviral prophylaxis and as anti-thrombotics
(Glennie
and Johnson 2000). The application of molecular engineering to convert murine
mAbs
into chimeric mAbs (mouse V-region, human C-region) and humanised reagents
52
CA 02826627 2013-09-04
where only the rnAb complementarity-determining regions (CDR) are of murine
origin has been critical to the clinical success of mAb therapy. The
engineered ruAbs
have markedly reduced or absent immunogenicity, increased serum half-Life and
the
human Fc portion of the mAb increases the potential to recruit the immune
effectors
of complement and cytotoxic cells (Clark 2000). Investigations into the
biodistribution, pharmacolcinetics and any induction of an immune response to
clinically administered mAbs requires the development of analyses to
discriminate
between the pharmaceutical and endogenous proteins.
The antibodies, or any fragments thereof, may also be conjugated or
re,combinantly
fused to any cellular toxin, bacterial or other, e.g. pseudornonas exotoxin,
ricin, or
diphtheria toxin. The part of the toxin used can be the whole toxin, or any
particular
domain of the toxin. Such antibody-toxin molecules have successfully been used
for
targeting and therapy of different kinds of cancers, see e.g. Pastan, Biochim
Biophys
Acta. 1997 Oct 24;1333(2):C1-6; Kreitman et al., N Engl J Med. 2001 Jul
26;345(4):241-7; Schnell et al., Leukemia. 2000 Jan;14(1):129-35; Ghetie et
al., Mol
Biote,chnol. 2001 Jul;18(3):251-68.
Bi- and tri-specific inultimers can be formed by association of different scFv
molecules and have been designed as cross-linking reagents for T-cell
recruitment
into tumors (immunotherapy), viral retargeting (gene therapy) and as red blood
cell
agglutination reagents (immunodiagnostics), see e.g. Todorovska et al., J
Immunol
Methods. 2001 Feb 1;248(1-2):47-66; Tomlinson et al., Methods Enzymol.
2000;326:461-79; McCall et al., J Immunol. 2001 May 15;166(10):6112-7.
Fully human antibodies can be prepared by immunizing transgenic mice carrying
large portions of the human immunoglobulin heavy and light chains. These mice,
examples of such mice are the Xenomouserm (Abgenix, Inc.) ([JS Patent Nos.
6,075,181 and 6,150,584), the HuMAb-Mouserm (Medarex, Inc./GenPharm) (US
patent 5545806 and 5569825), the TransChromo Mouse m (Kirin) and the KM
Mouse m (Medarex/Kirin), are well known within the art. Antibodies can then be
53
CA 02826627 2013-09-04
prepared by, e.g. standard hybridoma technique or by phage display. These
antibodies will then contain only fully human amino acid sequences.
Fully human antibodies can also be generated using phage display from human
libraries. Phage display may be performed using methods well known to the
skilled
artisan, as in Hoogenboom et al and Marks et al (Hoogenboom HR and Winter G.
(1992) J Mol Biol. 227(2):381-8; Marks JD et al (1991) J Mol Biol. 222(3):581-
97;
and also U.S. Patents 5885793 and 5969108).
Therapeutic Antibodies and Uses
. .
The in vivo properties, particularly with regard to tumorblood ratio and rate
of
clearance, of specific binding members of the invention will be at least
comparable to
niAb806. Following administration to a human or animal subject such a specific
binding member will show a peak tumor to blood ratio of > 1:1. Preferably at
such a
ratio the specific binding member will also have a tumor to organ ratio of
greater than
1:1, preferably greater than 2:1, more preferably greater than 5:1. Preferably
at such a
ratio the specific binding member will also have an organ to blood ratio of <
1:1 in
organs away from the site of the tumor. These ratios exclude organs of
catabolism and
secretion of the administered specific binding member. Thus in the case of
scFvs and
Fabs (as shown in the accompanying examples), the binding members are secreted
via
the kidneys and there is greater presence here than other organs. In the case
of whole
IgGs, clearance will be at least in part, via the liver. The peak localisation
ratio of the
intact antibody will normally be achieved between 10 and 200 hours following
administration of the specific binding member. More particularly, the ratio
may be
measured in a tumor xenograft of about 0.2 - 1.0 g formed subcutaneously in
one
flank of an athymie nude mouse.
Antibodies of the invention may be labelled with a detectable or functional
label.
Detectable labels include, but are not limited to, racholabels such as the
isotopes 3H,
54
CA 02826627 2013-09-04
I4c, 32p, , 35-
S 360, 61Cr, 57CO, "CO, "Fe, 90Y, 1211, 124/, 1251, 131/, win, 211A,t, t98Au,
67Cu, 225AC, 213Bi, 99TC and 186Re, which may be attached to antibodies of the
invention using conventional chemistry known in the art of antibody imaging.
Labels
also include fluorescent labels and labels used conventionally in the art for
MRI-CT
imagine. They also include enzyme labels such as horseradish peroxidase.
Labels
further include chemical moieties such as biotin which may be detected via
binding to
a specific cognate detectable moiety, e.g. labelled avidin.
Functional labels include substances which are designed to be targeted to the
site of a
tumor to cause destruction of tumor tissue. Such functional labels include
cytotoxic
drugs such as 5-fluorouracil or ricin and enzymes such as bacterial
carboxypeptidase
or nitroreductase, which are capable of converting prodrugs into active drugs
at the
site of a tumor.
Also, antibodies including both polyclonal and monoclonal antibodies, and
drugs that
modulate the production or activity of the specific binding members,
antibodies
and/or their subunits may possess certain diagnostic applications and may for
example, be utilized for the purpose of detecting and/or measuring conditions
such as
cancer, precancerous lesions, conditions related to or resulting from
hyperproliferative
cell growth or the like. For example, the specific binding members, antibodies
or
their subunits may be used to produce both polyclonal and monoclonal
antibodies to
themselves in a variety of cellular media, by known techniques such as the
hybridoma
technique utilizing, for example, fused mouse spleen lymphocytes and myeloma
cells.
Likewise, small molecules that mimic or antagonize the activity(ies) of the
specific
binding members of the invention may be discovered or synthesized, and may be
used
in diagnostic and/or therapeutic protocols.
The radiolabelled specific binding members, particularly antibodies and
fragments
thereof, are useful in in vitro diagnostics techniques and in in vivo
radioimaging
techniques and in radioimmunotherapy. In the instance of in vivo imaging, the
specific binding members of the present invention may be conjugated to an
imaging
CA 02826627 2013-09-04
agent rather than a radioisotope(s), including but not limited to a magnetic
resonance
image enhancing agent, wherein for instance an antibody molecule is loaded
with a
large number of paramagnetic ions through chelating groups. Examples of
chelating
groups include EDTA, porphyrins, polyamines crown ethers and polyoximes.
Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium,
europium, lanthanium, holmium and ferbium. In a further aspect of the
invention,
radiolabelled specific binding members, particularly antibodies and fragments
thereof,
particularly radioimmunoconjugates, are useful in radioimmunotherapy,
particularly
as radiolabelled antibodies for cancer therapy. In a still further aspect, the
radiolabelled specific binding members, particularly antibodies and fragments
thereof,
are useful in rachoimmuno-guided surgery techniques, wherein they can identify
and
indicate the presence and/or location of cancer cells, precancerous cells,
tumor cells,
and hyperproliferative cells, prior to, during or following surgery to remove
such
cells. =
Immunoconjugates or antibody fusion proteins of the present invention, wherein
the
specific binding members, particularly antibodies and fragments thereof, of
the
present invention are conjugated or attached to other molecules or agents
further
include, but are not limited to binding members conjugated to a chemical
ablation
agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic
agent or
drug.
Radioimmunotherapy (RAIT) has entered the clinic and demonstrated efficacy
using
various antibody immunoconjugates. 1311 labeled humanized anti-
carcinoembryonic
antigen (anti-CEA) antibody hMN-14 has been evaluated in colorectal cancer
(Behr
TM et al (2002) Cancer 94(4Suppl):1373-81) and the same antibody with 90Y
label
has been assessed in medullary thyroid carcinoma (Stein R et al (2002) Cancer
94(1):51-61). Radioimmunotherapy using monoclonal antibodies has also been
assessed and reported for non-Hodgkin's lymphoma and pancreatic cancer
(Goldenberg DM (2001) Crit Rev Oncol Hematol 39(1-2):195-201; Gold DV et al
(2001) Crit Rev Oncol Hematol 39 (1-2) 147-54). Radioimmunotherapy methods
56
CA 02826627 2013-09-04
with particular antibodies are also described in U.S. Patent 6,306,393 and
6,331,175.
Raelioimmunoguided surgery (RIGS) has also entered the clinic and demonstrated
efficacy and usefulness, including using anti-CEA antibodies and antibodies
directed
against tumor-associated antigens (Kim JC et al (2002) Int J Cancer 97(4):542-
7;
t=
Schneebaum S et al (2001) World J Surg 25(12):1495-8; Avital S et (2000)
Cancer
89(8):1692-8; McIntosh DG et al (1997) Cancer Biothcr Racliophann 12 (4):287-
94).
Antibodies of the present invention may be administered to a patient in need
of
treatment via any suitable route, usually by injection into the bloodstream or
CSF, or
directly into the site of the tum.or. The precise dose wiIl depend upon a
number of
factors, including whether the antibody is for diagnosis or for treatment, the
size and
location of the tumor, the precise nature of the antibody (whether whole
antibody,
fragment, diabody, etc), and the nature of the detectable or functional label
attached to
the antibody. Where a radionuclide is used for therapy, a suitable maximum
single
dose is about 45 mCi/m2, to a maximum of about 250 ma/m2. Preferable dosage is
in
the range of 15 to 40 mCi, with a further preferred dosage range of 20 to 30
mCi, or
to 30 mCi. Such therapy may require bone marrow or stem cell replacement. A
typical antibody dose for either tumor imaging or tumor treanxient will be in
the range
of from 0.5 to 40 mg, preferably from 1 to 4 rag of antibody in F(ab')2 form.
Naked
antibodies are preferable ailininistered in doses of 20 to 1000 mg protein per
dose, or
to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is a dose
for a
single treatment of an adult patient, which may be proportionally adjusted for
children
and infants, and also adjusted for other antibody formats in proportion to
molecular
weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly
intervals, at the discretion of the physician.
These formulatiems may include a second binding protein, such as the EGFR
binding
proteins d.escribed supra. In an especially preferred form, this second
binding protein
is a monoclonal antibody such as 528 or 225, discussed infra_
= 57
CA 02826627 2013-09-04
Pharmaceutical and Therapeutic Compositions
Specific binding members of the present invention will usually be administered
in the
form of a pharmaceutical composition, which may comprise at least one
component in
addition to the specific binding member.
Thus pharmaceutical compositions according to the present invention, and for
use in
accordance with the present invention, may comprise, in addition to active
ingredient,
a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other
materials
well lmown to those skilled in the art. Such materials should be non-toxic and
should
not interfere with the efficacy of the active ingredient. The precise nature
of the
carrier or other material will depend on the route of administration, which
may be
oral, or by injection, e.g. intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule,
powder or liquid form. A tablet may comprise a solid carrier such as gelatin
or an
adjuvant. Liquid pharmaceutical compositions generally comprise a liquid
carrier
such as water, petroleum, animal or vegetable oils, mineral oil or synthetic
oil.
Physiological saline solution, dextrose or other saccharide solution or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, injection, or injection at the site of affliction, the active
ingredient
will be in the form of a parenterally acceptable aqueous solution which is
pyrogen-
free and has suitable pH, isotonicity and stability. Those of relevant skill
in the an are
well able to prepare suitable solutions using, for example, isotonic vehicles
such as
Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
Preservatives, stabilisers, buffers, antioxidants and/or other additives may
be
included, as required.
A composition may be administered alone or in combination with other
treatments,
therapeutics or agents, either simultaneously or sequentially dependent upon
the
58
CA 02826627 2013-09-04
condition to be treated. In addition, the present invention contemplates and
includes
compositions comprising the binding member, particularly antibody or fragment
thereof, herein described and other agents or therapeutics such as anti-cancer
agents
or therapeutics, hormones, anti-EGFR agents or antibodies, or immune
modulators.
More generally these anti-cancer agents may be tyrosine kinase inhibitors or
phosphorylation cascade inhibitors, post-translational modulators, cell growth
or
division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors.
Other
treatments or therapeutics may include the administration of suitable doses of
pain
relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin,
paracetamol,
ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. The
composition can be administered in combination (either sequentially (i.e.
before or
after) or simultaneously) with tyrosine kinase inhibitors (including, but not
limited to
A01478 and /1)1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide,
cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea,
5-
fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin,
carmustine,
lomustine, and/or other chemotherapeutic agents. Thus, these agents may be
anti-
EGFR specific agents, or tyrosine kinase inhibitors such as AG1478, G1)1839,
STI571, OSI-774, or SU-6668 or may be more general anti-cancer and anti-
neoplastic
agents such as doxombicin,cisplatin, temozolomide, nitrosoureas, procarbazine,
vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside,
cyclophosphamide,
epipodophyllotoxin, carmustine, or lomustine. In addition, the composition may
be
administered with hormones such as dexamethasone, immune modulators, such as
interleukins, tumor necrosis factor (TNF) or other growth factors or cytokines
which
stimulate the immune response and reduction or elimination of cancer cells or
tumors.
An immune modulator such as TNF may be combined together with a member of the
invention in the form of a bispecific antibody recognizing the 806 EGFR
epitope as
well as binding to TNF receptors. The composition may also be administered
with, or
may include combinations along with other anti-EGFR antibodies, including but
not
limited to the anti-EGBR antibodies 528, 225, SC-03, DR8.3, L8A4, Y10, ICR62
and
ABX-EGF.
59
CA 02826627 2013-09-04
Previously the use of agents such as doxorubicin and cisplatin in conjunction
with
anti-EGFR antibodies have produced enhanced anti-tumor activity (Fan et al,
1993;
Base1ga et al, 1993). The combination of doxorubicin and mAb 528 resulted in
total
eradication of established A43I xenografts, whereas treatment with either
agent alone
caused only temporary in vivo growth inhibition (Base1ga et al, 1993).
Likewise, the
combination of cisplatin and either mAb 528 or 225 also led to the eradication
of well
established A431 xenografts, which was not observed when treatment with either
agent was used (Fan et al, 1993).
Conventional Radiotherapy
In addition, the present invention contemplates and includes therapeutic
compositions
for the use of the binding member in combination with conventional
radiotherapy. It
has been indicated that treatment with antibodies targeting EGF receptors can
enhance
the effects of conventional radiotherapy (Milas et al., Clin Cancer Res.2000
Feb:6
(2):701 8, Huang et al., Clin Cancer Res. 2000 Jun: 6(6):2166 74).
As demonstrated herein, combinations of the binding member of the present
invention, particularly an antibody or fragment thereof, preferably the
mAb806,
ch806 or a fragment thereof, and anti-cancer therapeutics, particularly anti-
EGFR
therapeutics, including other anti-EGFR antibodies, demonstrate effective
therapy,
and particularly synergy, against xenografted tumors. In the examples, it is
demonstrated that the combination of AG1478 and mAb 806 results in
significantly
enhanced reduction of A431 xenograft tumor volume in comparison with treatment
with either agent alone. AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline)
is a
potent and selective inhibitor of the EGF receptor kinase and is particularly
described
in United States Patent No. 5,457,105,:
(see also, Liu, W. et al (1999)J. Cell Sci. 112:2409; Eguchi, S. et al (1998)
J. Biol.
Chem. 273:8890; L=evitsky, A. and Gazit, A. (1995) Science 267:1782). The
specification examples further demonstrate therapeutic synergy of the 806
antibody
with other anti-EGFR antibodies, particularly with the 528 anti-EUER antibody.
CA 02826627 2013-09-04
The present invention further contemplates therapeutic compositions useful in
practicing the therapeutic methods of this invention. A subject therapeutic
composition includes, in admixture, a pharmaceutically acceptable excipient
(carrier)
and one or more of a specific binding member, polypeptide analog thereof or
fragment thereof, as described herein as an active ingredient. In a preferred
embodiment, the composition comprises an antigen capable of modulating the
specific binding of the present binding member/antibody with a target cell.
The preparation of therapeutic compositions which contain polypeptides,
analogs or
active fragments as active ingredients is well understood in the art.
Typically, such
compositions are prepared as injectables, either as liquid solutions or
suspensions.
However, solid forms suitable for solution in, or suspension in, liquid prior
to
injection can also be prepared. The preparation can also be emulsified. The
active
therapeutic ingredient is often mixed with excipients which are
pharmaceutically
acceptable and compatible with the active ingredient. Suitable excipients are,
for
example, water, saline, dextrose, glycerol, ethanol, or the like and
combinations
thereof. In addition, if desired, the composition can contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH buffering
agents which
enhance the effectiveness of the active ingredient.
A polypeptide, analog or active fragment can be formulated into the
therapeutic
composition as neutralized pharmaceutically acceptable salt forms.
.Pharmaceutically
acceptable salts include the acid addition salts (formed with the free amino
groups of
the polypeptide or antibody molecule) and which are formed with inorganic
acids
such as, for example, hydrochloric or phosphoric acids, or such organic acids
as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free
carboxyl
groups can also be derived from inorganic bases such as, for example, sodiuna,
potassium, ammonium, calcium, or ferric hydroxide.s, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and
the
like.
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The therapeutic polypeptide-, analog- or active fragment-containing
compositions are
conventionally administered intravenously, as by injection of a unit dose, for
example.
The term ''unit dose" when used in reference to a therapeutic composition of
the
present invention refers to physically discrete units suitable as unitary
dosage for
humans, each unit containing a predetermined quantity of active material
calculated to
produce the desired therapeutic effect in association with the required
diluent; i.e.,
carrier, or vehicle.
The compositions are administered in a manner compatible with the dosage
formulation, and in a therapeutically effective amount. The quantity to be
administered depends on the subject to be treated, capacity of the subject's
immune
system to utilize the active ingredient, and degree of EFGR binding capacity
desired.
Precise amounts of active ingredient required to be administered depend on the
judgment of the practitioner and are peculiar to each individual. However,
suitable
dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and
more
preferably one to several, milligrams of active ingredient per kilogram body
weight of
individual per day and depend on the route of administration. Suitable regimes
for
initial administration and booster shots are also variable, but are typified
by an initial
administration followed by repeated doses at one or more hour intervals by a
subsequent injection or other administration. Alternatively, continuous
intravenous
infusion sufficient to maintain concentrations of ten nanomolar to ten
rnicromolar in
the blood are contemplated.
Pharmaceutical compositions for oral administration may be in tablet, capsule,
powder or liquid form. A tablet may comprise a solid carrier such as gelatin
or an
adjuvant. Liquid pharmaceutical compositions generally comprise a liquid
carrier
such as water, petroleum, animal or vegetable oils, mineral oil or synthetic
oil.
Physiological saline solution, dextrose or other saccharide solution or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol may be included.
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For intravenous, injection, or injection at the site of affliction, the active
ingredient
will be in the form of a parenterally acceptable aqueous solution which is
pyrogen-
free and has suitable pH, isotonicity and stability. Those of relevant skill
in the art are
well able to prepare suitable solutions using, for example, isotonic vehicles
such as
Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
Preservatives, stabilisers, buffers, antioxidants and/or other additives may
be
included, as required.
Diagnostic Assays
The present invention also relates to a variety of diagnostic applications,
including
methods for detecting the presence of stimuli such as aberrantly expressed
EGFR, by
reference to their ability to be recognized by the present specific binding
member. As
mentioned earlier, the EGFR can be used to produce antibodies to itself by a
variety
of known techniques, and such antibodies could then be isolated and utilized
as in
tests for the presence of particular EGFR activity in suspect target cells.
Diagnostic applications of the specific binding members of the present
invention,
particularly antibodies and fragments thereof, include in vitro and in vivo
applications
well known and standard to the skilled artisan and based on the present
description.
Diagnostic assays and kits for in vitro assessment and evaluation of EGFR
status,
particularly with regard to aberrant expression of EGFR, may be utilized to
diagnose,
evaluate and monitor patient samples including those known to have or
suspected of
having cancer, a precancerous condition, a condition related to
hyperproliferative cell
growth or from a tumor sample. The assessment and evaluation of EGFR status is
also useful in determining the suitability of a patient for a clinical trial
of a drug or for
the administration of a particular chemotherapeutic agent or specific binding
member,
particularly an antibody, of the present invention, including combinations
thereof,
versus a different agent or binding member. This type of diagnostic monitoring
and
assessment is already in practice utilizing antibodies against the HERZ
protein in
breast cancer (Hercep Test, Dako Corporation), where the assay is also used to
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evaluate patients for antibody therapy using Herceptin. In vivo applications
include
imaging of tumors or assessing cancer status of individuals, including'
radioimaging.
As suggested earlier, the diagnostic method of the present invention comprises
examining a cellular sample or medium by means of an assay including an
effective
amount of an antagonist to an EFGR /protein, such as an anti- EFGR antibody,
preferably an affinity-purified polyclonal antibody, and more preferably a
mAb. In
addition, it is preferable for the anti- EFGR antibody molecules used herein
be in the
form of Fab, Fab', F(ab')2 or F(v) portions or whole antibody molecules. As
previously discussed, patients capable of benefiting from this method include
those
suffering from cancer, a pre-cancerous lesion, a viral infection, pathologies
involving
or resulting from hyperproliferative cell growth or other like pathological
derangement. Methods for isolating EFGR and inducing anti- EFGR antibodies and
for determining and optimizing the ability of anti-EFGR antibodies to assist
in the
examination of the target cells are all well-known in the art.
Preferably, the anti-EFGR antibody used in the diagnostic methods of this
invention is
an affinity purified polyclonal antibody. More preferably, the antibody is a
monoclonal antibody (inAb). In addition, the anti-EFGR antibody molecules used
herein can be in the form of Fab, Fab', F(ab)2 or F(v) portions of whole
antibody
molecules.
As described in detail above, antibody(ies) to the EGFR can be produced and
isolated
by standard methods including the well known hybridoma techniques. For
convenience, the antibody(ies) to the EGFR will be referred to herein as Abi
and
antibody(ies) raised in another species as Ab2.
The presence of EGFR in cells can be ascertained by the usual in vitro or in
vivo
immunological procedures applicable to such determinations. A number of useful
procedures are known. Three such procedures which are especially useful
utilize
either the EGFR labeled with a detectable label, antibody Abi labeled with a
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CA 02826627 2013-09-04
detectable label, or antibody Ab2 labeled with a detectable label. The
procedures may
be summarized by the following equations wherein the asterisk indicates that
the
particle is labeled, and "R" stands for the EGFR:
A. R* + Abl = R*Abi
B. R + RAbi*
C. R + Abi + Ab2* RAb1Ab2*
The procedures and their application are all familiar to those skilled in the
art and
accordingly may be utilized within the scope of the present invention. The
"competitive" procedure, Procedure A, is described in U.S. Patent Nos.
3,654,090 and
3,850,752. Procedure C, the "sandwich" procedure, is described in U.S. Patent
Nos.
RE 31,006 and 4,016,043. Still other procedures are known such as the "double
antibody," or 'DASP" procedure.
In each instance above, the EGFR forms complexes with one or more
antibody(ies) or
binding partners and one member of the complex is labeled with a detectable
label.
The fact that a complex has formed and, if desired, the amount thereof, can be
determined by known methods applicable to the detection of labels.
It will be seen from the above, that a characteristic property of Ab2 is that
it will react
with Abi. This is because Abi raised in one mammalian species has been used in
another species as an antigen to raise the antibody Ab2. For example, Ab2 may
be
raised in goats using rabbit antibodies as antigens. Ab2 therefore would be
anti-rabbit
antibody raised in goats. For purposes of this description and claims, Abi
will be
referred to as a primary or anti-EGFR antibody, and Ab2 will be referred to as
a
secondary or anti-Abi antibody.
The labels most commonly employed for these studies are radioactive elements,
enzymes, chemicals which fluoresce when exposed to ultraviolet light, and
others.
CA 02826627 2013-09-04
A number of fluorescent materials are known and can be utilized as labels.
These
include, for example, fluorescein, rhodamine, auramine, Texas Red, ANICA blue
and
Lucifer Yellow. A particular detecting material is anti-rabbit antibody
prepared in
goats and conjugated with fluorescein through an isothiocyanate.
The EGER or its binding partner(s) such as the present specific binding
member, can
also be labeled with a radioactive element or with an enzyme. The radioactive
label
can be detected by any of the currently available counting procedures. The
preferred
14C, 32p, 35s, 36C1, 51-r,
C 57Co, 58Co, 59Fe, 9 Y,1211,
isotope may be selected from 3H,
124L 1251, 1311, 211At, 198Au, 67cu, 225m, 213,-,9
9 Tc and 186Re.
Enzyme labels are likewise useful, and can be detected by any of the presently
utilized colorimetric, spectrophotometric, fluorospectrophotometric,
amperometric or
gasometric techniques. The enzyme is conjugated to the selected particle by
reaction
with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde
and the
like. Many enzymes which can be used in these procedures are known and can be
utilized. The preferred are peroxidase,13-glucuronidase, 13-D-glucosidase, Li-
D-
galactosidase, urease, glucose oxidase plus peroxidase and alkaline
phosphatase. U.S.
Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of
example
for their disclosure of alternate labeling material and methods.
A particular assay system that may be advantageously utilized in accordance
with the
present invention, is known as a receptor assay. In a receptor assay, the
material to be
assayed such as the specific binding member, is appropriately labeled and then
certain
cellular test colonies are inoculated with a quantity of both the labeled and
unlabeled
material after which binding studies are conducted to determine the extent to
which
the labeled material binds to the cell receptors. In this way, differences in
affinity
between materials can be ascertained.
Accordingly, a purified quantity of the specific binding member may be
radiolabeled
and combined, for example, with antibodies or other inhibitors thereto, after
which
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CA 02826627 2013-09-04
binding studies would be carried out. Solutions would then be prepared that
contain
various quantities of labeled and unlabeled uncornbined specific binding
member, and
cell samples would then be inoculated and thereafter incubated. The resulting
cell
monolayers are then washed, solubilized and then counted in a gamma counter
for a
length of time sufficient to yield a standard error of <5%. These data are
then
subjected to Scatchard analysis after which observations and conclusions
regarding
material activity can be drawn. While the foregoing is exemplary, it
illustrates the
manner in which a receptor assay may be performed and utilized, in the
instance
where the cellular binding ability of the assayed material may serve as a
distinguishing characteristic.
An assay useful and contemplated in accordance with the present invention is
known
as a "cis/trans" assay. Briefly, this assay employs two genetic constructs,
one of
which is typically a plasmid that continually expresses a particular receptor
of interest
when transfected into an appropriate cell line, and the second of which is a
plasmid
that expresses a reporter such as luciferase, under the control of a
receptor/ligand
complex. Thus, for example, if it is desired to evaluate a compound as a
ligand for a
particular receptor, one of the plasmids would be a construct that results in
expression
of the receptor in the chosen cell line, while the second plasmid would
possess a
promoter linked to the luciferase gene in which the response element to the
particular
receptor is inserted. If the compound under test is an agonist for the
receptor, the
ligand will complex with the receptor, and the resulting complex will bind the
response element and initiate transcription of the luciferase gene. The
resulting
chemiluminesc,ence is then measured photometrically, and dose response curves
are
obtained and compared to those of known ligands. The foregoing protocol is
described in detail in U.S. Patent No. 4,981,784 and PCT International
Publication
No. WO 88/03168, for which purpose the artisan is referred.
In a further embodiment of this invention, commercial test kits suitable for
use by a
medical specialist may be prepared to determine the presence or absence of
aberrant
expression of EGFR, including but not limited to amplified EGER and/or an EGER
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CA 02826627 2013-09-04
mutation, in suspected target cells. In accordance with the testing techniques
discussed above, one class of such kits will contain at least the labeled EGFR
or its
binding partner, for instance an antibody specific thereto, and directions, of
course,
depending upon the method selected, e.g., "competitive," "sandwich," "DASP"
and
the like. The kits may also contain peripheral reagents such as buffers,
stabilizers, etc.
Accordingly, a test kit may be prepared for the demonstration of the presence
or
capability of cells for aberrant expression or post-translational modification
of EGFR,
comprising:
(a) a predetermined amount of at least one labeled itnmunochemically reactive
component obtained by the direct or indirect attachment of the present
specific
binding member or a specific binding partner thereto, to a detectable label;
(b) other reagents; and
(c) directions for use of said kit.
More specifically, the diagnostic test kit may comprise:
(a) a known amount of the specific binding member as described above (or a
binding partner) generally bound to a solid phase to form an immunosorbent, or
in the
alternative, bound to a suitable tag, or plural such end products, etc. (or
their binding
partners) one of each;
(b) if necessary, other reagents; and
(c) directions for use of said test kit.
In a further variation, the test kit may be prepared and used for the purposes
stated
above, which operates according to a predetermined protocol (e.g.
"competitive,"
"sandwich," "double antibody," etc.), and comprises:
(a) a labeled component which has been obtained by coupling the specific
binding
member to a detectable label;
(b) one or more additional irnmunochemical reagents of which at least one
reagent
is a ligand or an immobilized ligand, which ligand is selected from the group
consisting of:
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CA 02826627 2013-09-04
(i) a ligand capable of binding with the labeled component (a);
(ii) a ligand capable of binding with a binding partner of the labeled
component (a);
(iii) a ligand capable of binding with at least one of the component(s) to be
determined; and
(iv) a ligand capable of binding with at least one of the binding partners of
at least one of the component(s) to be determined; and
(c) directions for the performance of a protocol for the detection and/or
determination of one or more components of an immunochemical reaction between
the EFGR, the specific binding member, and a specific binding partner thereto.
In accordance with the above, an assay system for screening potential drugs
effective
=
to modulate the activity of the EFGR, the aberrant expression or pOst-
translational
modification of the EGFR, and/or the activity or binding of the specific
binding
member may be prepared. The receptor or the binding member may be introduced
into a test system, and the prospective drug may also be introduced into the
resulting
cell culture, and the culture thereafter examined to observe any changes in
the S-phase
activity of the cells, due either to the addition of the prospective drug
alone, or due to
the effect of added quantities of the known agent(s).
Nucleic Acids
The present invention further provides an isolated nucleic acid encoding a
specific
binding member of the present invention. Nucleic acid includes DNA and RNA. In
a
preferred aspect, the present invention provides a nucleic acid which codes
for a
polypeptide of the invention as defined above, including a polypeptide as set
out as
residues 93-102 of SEQ ID NO:2 or 26-35A, 49-64 and 93-102 of SEQ ID NO:2, a
polypeptide as set out in residues 24-34, 50-56 and 89-97 of SEQ 1D NO:4, and
the
entire polypeptides of SEQ JD NO:2 and SEQ ID NO:4.
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CA 02826627 2013-09-04
The present invention also provides constructs in the form of plasmids,
vectors,
transcription or expression cassettes which comprise at least one
polynucleotide as
above.
The present invention also provides a recombinant host cell which comprises
one or
more constructs as above. A nucleic acid encoding any specific binding member
as
provided itself forms an aspect of the present invention, as does a method of
production of the specific binding member which method comprises expression
from
encoding nucleic acid therefor. Expression may conveniently be achieved by
culturing under appropriate conditions recombinant host cells containing the
nucleic
acid. Following production by expression a specific binding member may be
isolated
and/or purified using any suitable technique, then used as appropriate.
Specific binding members and encoding nucleic acid molecules and vectors
according
to the present invention may be provided isolated and/or purified, e.g. from
their
natural environment, in substantially pure or homogeneous form, or, in the
case of
nucleic acid, free or substantially free of nucleic acid or genes origin other
than the
sequence encoding a polypeptide with the required function. Nucleic acid
according
to the present invention may comprise DNA or RNA and may be wholly or
partially
synthetic.
Systems for cloning and expression of a polypeptide in a variety of different
host cells
are well known. Suitable host cells include bacteria, mammalian cells, yeast
and
baculovirus systems. Mammalian cell lines available in the art for expression
of a
heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby
hamster kidney cells, NSO mouse melanoma cells and many others. A common,
preferred bacterial host is E.coli.
The expression of antibodies and antibody fragments in prokaryotic cells such
as
E.coli is well established in the art. For a review, see for example
Pliickthun, A.
Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is
also
CA 02826627 2013-09-04
available to those skilled in the art as an option for production of a
specific binding
member, see for recent reviews, for example Raff, M.E. (1993) Curr. Opinion
Biotech. 4: 573-576; Trill J.J. et al. (1995) Curr. Opinion Biotech 6: 553-
560.
Suitable vectors can be chosen or constructed, containing appropriate
regulatory
sequences, including promoter sequences, terminator sequences, polyadenylation
sequences, enhancer sequences, marker genes and other sequences as
appropriate.
Vectors may be plasmids, viral e.g. 'phage, or phagernid, as appropriate. For
further
details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition,
Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known
techniques and protocols for manipulation of nucleic acid, for example
in.preparation
of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into
cells
and gene expression, and analysis of proteins, are described in detail in
Short
Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John
Wiley &
Sons, 1992.
Thus, a further aspect of the present invention provides a host cell
containing nucleic
acid as disclosed herein. A still further aspect provides a method comprising
introducing such nucleic acid into a host cell. The introduction may employ
any
available technique. For eulcaryotic cells, suitable techniques may include
calcium
phosphate transfection, D13AB-Dextran, electroporation, liposome-mediated
transfection and transduction using retrovirus or other virus, e.g. vaccinia
or, for
insect cells, baculovirus. For bacterial cells, suitable techniques may
include calcium
= chloride transformation, electroporation and transfection using
bactetiophage.
The introduction may be followed by causing or allowing expression from the
nucleic
acid, e.g. by culturing host cells under conditions for expression of the
gene.
In one embodiment, the nucleic acid of the invention is integrated into the
genome
(e.g. chromosome) of the host cell. Integration may be promoted by inclusion
of
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CA 02826627 2013-09-04
sequences which promote recombination with the genome, in accordance with
standard techniques.
The present invention also provides a method which comprises using a construct
as
stated above in an expression system in order to express a specific binding
member or
polypeptide as above.
As stated above, the present invention also relates to a recombinant DNA
molecule or
cloned gene, or a degenerate variant thereof, which encodes a specific binding
member, particularly antibody or a fragment thereof, that possesses an amino
acid
sequence set forth in SEQ ID NO:2 and/or SEQ ID NO:4; preferably a nucleic
acid
molecule, in particular a recombinant DNA molecule or cloned gene, encoding
the
binding member or antibody has a nucleotide sequence or is complementary to a
DNA sequence provided in SEQ ID NO: 1 and/or SEQ 1D NO:3.
Another feature of this invention is the expression of the DNA sequences
disclosed
herein. As is well known in the art, DNA sequences may be expressed by
operatively
linking them to an expression control sequence in an appropriate expression
vector
and employing that expression vector to transform an appropriate unicellular
host.
Such operative linking of a DNA sequence of this invention to an expression
control
sequence, of course, includes, if not already part of the DNA sequence, the
provision
of an initiation codon, ATG, in the correct reading frame upstream of the DNA
sequence.
A wide variety of host/expression vector combinations may be employed in
expressing the DNA sequences of this invention. Useful expression vectors, for
example, may consist of segments of chromosomal, non-chromosomal and synthetic
DNA sequences. Suitable vectors include derivatives of SV40 and known
bacterial
plasmids, e.g., E. coli plasmids col El, pCR1, pl3R322, pA4B9 and their
derivatives,
plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage X,
e.g.,
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CA 02826627 2013-09-04
NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage
DNA; yeast plasmids such as the 2u plasmid or derivatives thereof; vectors
useful in
eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors
derived
from combinations of plasmids and phage DNAs, such as plasmids that have been
= modified to employ phage DNA or other expression control sequences; and
the like.
Any of a wide variety of expression control sequences -- sequences that
control the
expression of a DNA sequence operatively linked to it -- may be used in these
vectors
to express the DNA sequences of this invention. Such useful expression control
sequences include, for example, the early or late prornoters of SV40, CMV,
vaccinia,
polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC
system, the LTR system, the major operator and promoter regions of phage X,
the
control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase
or other
glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the
promoters of
the yeast -mating factors, and other sequences known to control the expression
of
genes of prokaryotic or eukaryotic cells or their viruses, and various
combinations
thereof.
A wide variety of unicellular host cells are also useful in expressing the DNA
sequences of this invention. These hosts may include well known eukaryotic and
prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus,
Streptomyces,
fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, R1.1,
B-W
and L-M cells, African Green Monkey kidney cells (e.g., COS 1, cos 7, BSC1,
BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells
in tissue
culture.
It will be understood that not all vectors, expression control sequences and
hosts will
function equally well to express the DNA sequences of this invention. Neither
will all
hosts function equally well with the same expression system. However, one
skilled in
the art will be able to select the proper vectors, expression control
sequences, and
hosts without undue experimentation to accomplish the desired expression
without
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CA 02826627 2013-09-04
departing from the scope of this invention. For example, in selecting a
vector, the
host must be considered because the vector must function in it. The vector's
copy
number, the ability to control that copy number, and the expression of any
other
proteins encoded by the vector, such as antibiotic markers, will also be
considered.
In selecting an expression control sequence, a variety of factors will
normally be
considered. These include, for example, the relative strength of the system,
its
controllability, and its compatibility with the particular DNA sequence or
gene to be
expressed, particularly as regards potential secondary structures. Suitable
unicellular
hosts will be selected by consideration of, e.g., their compatibility with the
chosen
vector, their secretion characteristics, their ability to fold proteins
correctly, and their
fermentation requirements, as well as the toxicity to the host of the product
encoded
by the DNA sequences to be expressed, and the ease of purification of the
expression
products.
Considering these and other factors a person skilled in the art will be able
to construct
a variety of vector/expression control sequence/host combinations that will
express
the DNA sequences of this invention on fermentation or in large scale anima/
culture.
It is further intended that specific binding member analogs may be prepared
from
nucleotide sequences of the protein complex/subunit derived within the scope
of the
present invention. Analogs, such as fragments, may be produced, for example,
by
pepsin digestion of specific binding member material. Other analogs, such as
muteins, can be produced by standard site-directed mutagenesis of specific
binding
member coding sequences. Analogs exhibiting "specific binding member activity"
such as small molecules, whether functioning as promoters or inhibitors, may
be
identified by known in viva and/or in vitro assays.
As mentioned above, a DNA sequence encoding a specific binding member can be
prepared synthetically rather than cloned. The DNA sequence can be designed
with
the appropriate codons for the specific binding member amino acid sequence. In
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CA 02826627 2013-09-04
general, one will select preferred codons for the intended host if the
sequence will be
used for expression. The complete sequence is assembled from overlapping
oligonucleotides prepared by standard methods and assembled into a complete
coding
sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science,
223:1299
(1984); Jay et al., J. Biol. Chem., 259:6311 (1984)..
Synthetic DNA sequences allow convenient construction of genes which will
express
specific binding member analogs or "muteins". Alternatively, DNA encoding
muteins
can be made by site-directed mutagenesis of native specific binding member
genes or
cDNAs, and muteins can be made directly using conventional polypeptide
synthesis.
A general method for site-specific incorporation of unnatural amino acids into
proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill,
Michael C.
Griffith, Peter G. Schultz, Science, 244:182-188 (April 1989). This method may
be
used to create analogs with unnatural amino acids.
The present invention extends to the preparation of antisense oligonucleotides
and
ribozymes that may be used to interfere with the expression of the EGFR at the
translational level. This approach utilizes antisense nucleic acid and
ribozymes to
block translation of a specific mRNA, either by masking that mRNA with an
antisense nucleic acid or cleaving it with a ribozyme. =
Antisense nucleic acids are DNA or RNA molecules that are complementary to at
least a portion of a specific mRNA molecule. (See Weintraub, 1990; Marcus-
Sekura,
1988.) In the cell, they hybridize to that mRNA, forming a double stranded
molecule.
The cell does not translate an mRNA in this double-stranded form. Therefore,
antisense nucleic acids interfere with the expression of mRNA into protein.
Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG
initiation codon will be particularly efficient, since they are easy to
synthesize and are
likely to pose fewer problems than larger molecules when introducing them into
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CA 02826627 2013-09-04
producing cells. Antisense methods have been used to inhibit the expression of
many
genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).
Ribozymes are RNA molecules possessing the ability to specifically cleave
other
single stranded RNA molecules in a manner somewhat analogous to DNA
restriction
endonucleases. Ribozymes were discovered from the observation that certain
mRNAs have the ability to excise their own introns. By modifying the
nucleotide
sequence of these RNAs, researchers have been able to engineer molecules that
recognize specific nucleotide sequences in an RNA molecule and cleave it
(Cech,
1988.). Because they are sequence-specific, only mRNAs with particular
sequences
are inactivated.
Investigators have identified two types of ribozymes, Tetrahymena-type and
"hammerhead"-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-type ribozymes
recognize four-base sequences, while "hammerhead"-type recognize eleven- to
eighteen-base sequences. The longer the recognition sequence, the more likely
it is to
occur exclusively in the target mRNA species. Therefore, hammerhead-type
ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a
specific
mRNA species, and eighteen base recognition sequences are preferable to
shorter
recognition sequences.
The DNA sequences described herein may thus be used to prepare antisense
molecules against, and ribozymes that cleave mRNAs for EI.GRs and their
ligands.
The invention may be better understood by reference to the following non-
limiting
Examples, which are provided as exemplary of the invention. The following
examples
are presented in order to more fully illustrate the preferred embodiments of
the
invention and should in no way be construed, however, as limiting the broad
scope of
the invention.
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CA 02826627 2013-09-04
EXAMPLE 1
ISOLATION OF ANTD3ODIES
MA __ IERIALS
Cell lines
For immunization and specificity analyses, several cell lines, native or
transfected
with either the normal, wild type or "wtEGFR" gene or the AFGFR gene carrying
the
A2-7 deletion mutation were used: Murine fibroblast cell line NR6, NR6AF.GFR
(transfected with AFG1,10 and NR60FR (transfected with wtEGFR), human
glioblastoma cell line U87MG (expressing low levels of endogenous wtEGFR),
U87MOvice0FR (transfected with wtEGFR), U87MGaa0m (transfected with AF,GER),
and human squamous cell carcinoma cell line A431 (expressing high levels of
wtEGER)[38]. Cell lines and transfections were described previously (Nishikawa
R.,
et al. (1994) Proc. Natl. Acad. Sci. 91(16):7727-7731).
The U87MG astrocytoma cell line (20), which endogenously expresses low levels
of
the wt EGFR, was infected with a retrovuus containing the de2-7 EGFR to
produce
the U87MG.A2-7 cell line (10). The transfected cell line U87MG.wtEGFR was
produced as described in Nagane et al 1996 (Cancer Res., 56: 5079-5086).
Whereas
U87MG cells express apiroximately 1x105EGI,R, U87MG.wtEGFR cells express
approximately 1x106EGPR, and thus mimic the situation seen with gene
amplification.
Human squamous carcinoma A431 cells were obtained from ATCC (Rockville, MD).
All cell lines were cultured in DMEM/F-12 with GlutaMAXTm (Life Technologies,
Melbourne, Australia) supplemented with 10% FCS (CSL, Melbourne, Australia).
Reagents
Biotinylated unique junctional peptides (Biotin-LEEKKGNYVVTDH (SEQ ID NO:
5) and l. FEKKGNYVVTDH-Biotin (SEQ ID NO: 6)) from de2-7 EGFR were
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CA 02826627 2013-09-04
synthesized by standard Fmoc chemistry and purity (>96% ) determined by
reverse
phase HPLC and mass spectral analysis (Auspep, Melbourne, Australia).
Antibodies used in studies
In order to compare our findings with other reagents, additional mAbs were
included
in our studies. These reagents were mAb 528 to the wtEGFR (Sato, J.D. et al.
(1983)
Mol. Biol. Med. 1(5):511-529) and DH8.3, which was generated against a
synthetic
peptide spanning the junctional sequence of the A2-7 EGFR deletion mutation.
The
DH8.3 antibody (IgG1) has been described previously (Hills et al, 1995, Int.
J. Cancer
63(4); 537-543) and was obtained following immunization of mice with the
unique
junctional peptide found in de2-7 EGFR (16). The 528 antibody, which
recognizes
both de2-7 and wild type EGFR, has been described previously (21) and was
produced in the Biological Production Facility (Ludwig Institute for Cancer
Research,
Melbourne) using a hybridoma obtained from ATCC BB-8509. SC-03 is an affinity
purified rabbit polyclonal antibody raised against a carboxy terminal peptide
of the
EGFR (Santa Cruz Biotechnology Inc.).
GENERATION OF MONOCLONAL ANTD3ODIES
The murine fibroblast line NR6AEGFR was used as iminunogen. Mouse hybridornas
were generated by immunizing BALB/c mice five times subcutaneously at 2- to 3-
week intervals, with 5x105 ¨ 2x106 cells in adjuvant. Complete Freund's
adjuvant
was used for the first injection. Thereafter, incomplete Freund's adjuvant
(Difco) was
used. Spleen cells from immunized mice were fused with mouse myeloma cell line
SP2/0. Supernatants of newly generated clones were screened in hemadsorption
assays for reactivity with cell line NR6, NR6,EGFR, and NR6AFT;FR and then
analyzed
by hemadsorption assays with human glioblastoma cell lines U87MG,
U87MG,,,,tEGFR,
and U87MGARGFR. Selected hybridoma supernatants were subsequently tested by
western blotting and further analyzed by immunohistochemistry. Newly generated
mAbs showing the expected reactivity pattern were purified.
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CA 02826627 2013-09-04
=
Five hybridomas were established and three, clones 124 (IgG2a), 806 (IgG2b)
and
1133 (IgG2a) were selected for further characterization based on high titer
with
NR6AEGFR and low background on NR6 and NR6õtEGFR cells in the hemagglutination
assay. In a subsequent hemagglutination analysis, these antibodies showed no
reactivity (undiluted supernatant <10%) with the native human glioblastoma
ceII line
U87MG and U87MG,IEGFR, but were strongly reactive with U87MGAEcFa; less
reactivity was seen with A431. By contrast, in FACS analysis, 806 was
unre,active
with native U87MG and intensively stained U87MGAEoFR and to a lesser degree
U87MGmEGFR indicating binding of 806 to both, AEGFR and wtEGFR (see below).
In Western blot assays, mAbs 124, 806 and 1133 were then analyzed for
reactivity
with wtEGFR and AEGFR. Detergent lysates were extracted from NR6AEGFR,
U87MG6,EGFR as well as from A431. All three mAbs showed a similar reactivity
pattern with cell lysates staining both the wtEGFR (170 kDa) and AFGFR protein
(140 kDa). As a reference reagent, niAb R.I known to be reactive with the
wtEGFR
(Waterfield M.D. et al. (1982) J. Cell Biochem. 20(2):149-161) was used
instead of
naAb 528, which is known to be non-reactive in western blot analysis. Mab R.I
showed reactivity with wt and AFG.P.K. All three newly generated clones showed
reactivity with AEGFR and less intense with wtEGFR. DH8.3 was solely positive
in
the lysate of U87MGApr,FR and NR6AEGFR.
The immunohistochemical analysis of clones 124, 806, and 1133 as well as mAb
528
and mAb DH8.3 on xenograft tumors U87MG, U87MGAE0FR, and A431 are shown in
Table 1. All mAbs showed strong staining of xenograft U87MGAEGFR. Only mAb 528
showed weak reactivity in the native U87MG xenograft. In A431 xenografts, mAb
528 showed strong homogeneous reactivity. MAbs 124, 806, and 1133 revealed
reactivity with mostly the basally located cells of the squamous cell
carcinoma of
A431 and did not react with the upper cell layers or the keratinizing
component.
DH8.3 was negative in A431 xenografts.
TABLE 1
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CA 02826627 2013-09-04
Immunohistochemical Analysis of Antibodies 528. D1-18.3. and 124, 806 and 1133
Mab xenog,raft xcnograft A431 xenograft1.187MG (native)
AIJ87MGAEGFR
528 = pos. pos. pos. (focal staining)
mAb-124 Po8- pos, (predominantly basal -
cells)
rnAb-806 pos. pos. (predominantly basal -
cells)
mAb-1133 pos. pos. (predominantly basal -
cells)
DH8.3 pos.
minor strornal staining due to detection of endogenous mouse antibodies
EXAMPLE 2
BINDING OF ANTIBODIES TO CELL LINES BY FACS
in order to determine the specificity of tnAb 806, its binding co t.187MG,
U87.1v1G.6.2-
7 and U87MG.wtEGFR cells was analyzed by flow activated cell sorting (FACS).
Briefly, cells were labelled with the relevant antibody (10 Wm') followed by
fluorescein-conjugated goat anti-mouse 1gG (1:100 dilution; Calbiochom San
Diego,
USA). FACS data was obtained on a Coulter Epics Elite ESP by observing a
minimum of 5,000 events Ian d analysed using EXPO (version 2) for Windows. An
irrelevant TgO2b was included as an isotype control for inAb 806 and the 528
antibody was included as it recoguize-s both the de2-7 and wt EGFR.
Only the 528 antibody was able to stain the parental U87MG cell line (Figure
I)
consistent with previous reports demonstrating that these cells express the wt
EGFR
(Nishilcawa et al, 1)94). MAb 806 and DH8.3 had binding levels similar to the
control
antibody, clearly demonstrating that they are unable to bind the Wt receptor
Olgure
1). Binding of the isotype control antibody to U87MG.A2-7 and U87MG.wtEG1R
cells was similar as that observed for the 1.187MG cells.
CA 02826627 2013-09-04
MAb 806 stained U87MG.A2-7 and U87MG.wtEGFR cells, indicating that mAb 806
specifically recognizes the de2-7 EGFR and amplified EGFR (Figure 1). DH8.3
antibody stained U87MG.A2-7 cells, confirming that DH8.3 antibody specifically
recognizes the de2-7 EGFR (Figure 1). As expected, the 528 antibody stained
both
the U87MG.A2-7 and U87MG.wtEGFR cell lines (Figure I). As expected, the 528
antibody stained U87MG.A2-7 with a higher intensity than the parental cell as
it binds
both the de2-7 and wild type receptors that are co-expressed in these cells
(Figure 1).
Similar results were obtained using a protein A mixed hemadsorption which
detects
surface bound IgG by appearance of Protein A coated with human red blood cells
(group 0) to target cells. Monoclonal antibody 806 was reactive with U87M0.A2-
7
cells but showed no significant reactivity (undiluted supernatant less than
10%) with
U87MG expressing wid type EGF-R.
EXAMPLE 3
BINDING OF ANTIBODIES IN ASSAYS
To further characterize the specificity of naAb 806 and the DH8.3 antibody,
their
binding was examined by ELLSA. Two types of ET ISA were used to determine the
specificity of the antibodies. In the first assay, plates were coated with
sEGFR (10
liWm1 in 0.1 M carbonate buffer pH 9.2) for 2 h and then blocked with 2% human
serum albumin (HSA)in PBS. sEGFR is the recombinant extracellular domain
(amino
acids 1-621) of the wild type EGFR), and was produced as previously described
(22).
Antibodies were added to wells in triplicate at increasing concentration in 2%
HSA in
phosphate-buffered saline (PBS). Bound antibody was detected by horseradish
peroxidase conjugated sheep anti-mouse IgG (Silenus, Melbourne, Australia)
using
ABTS (Sigma, Sydney, Australia) as a substrate and the absorbance measured at
405
RM.
Both inAb 806 and the 528 antibody displayed dose-dependent and saturating
binding
curves to immobilized wild type sEGFR (Figure 2A). As the unique junctional
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CA 02826627 2013-09-04
peptide found in the de2-7 EGFR is not contained within the sEGFR, mAb 806
must
be binding to an epitope located within the wild type EGFR sequence. The
binding of
the 528 antibody was lower than that observed for mAb 806. As expected the
DH8.3
antibody did not bind the wild type sEGFR even at concentrations up to 10
p.g/m1
(Figure 2A). While sEGFR in solution inhibited the binding of the 528 antibody
to
immobilized sEGFR in a dose-dependent fashion, it was unable to inhibit the
binding
of rnAb 806 (Figure 2B). This suggests that mAb 806 can only bind wild type
EGFR
once immobilized on FT JSA plates, a process that may induce conformational
changes. Similar results were observed using a BlAcore whereby mAb 806 bound
immobilized sEGER. but immobilized mAb 806 was not able to bind sEGFR in
solution (data not shown). The DH8.3 antibody exhibited dose-dependent and
saturable binding to the unique de2-7 EGFR peptide (Figure 2C).
In the second assay, the biotinylated de2-7 specific peptide (Biotin-
LEEKKGNYVVTDH (SEQ 1D NO: 5)) was bound to ELISA plates precoated with
streptavidin (Pierce, Rockford, Illinois). Antibodies were bound and detected
as in the
first assay. Neither mAb 806 nor the 528 antibody bound to the peptide, even
at
concentrations higher than those used to obtain saturation binding of DH8.3,
further
indicating that mAb 806 does not recognize an epitope determinant within this
peptide.
To further demonstrate that mAb 806 recognizes an epitope distinct from the
junction
peptide, additional experiments were performed C-terminal biotinylated de2-7
peptide (LEEKKGNYVVTDH-Biotin (SEQ ID NO: 6)) was utilized in studies with
mAb806 and mAb L8A4, generated against the de2-7 peptide (Reist, CJ et al
(1995)
Cancer Res. 55(19):4375-4382; Foulon CF et al. (2000) Cancer Res. 60(16):4453-
4460).
Reagents used in Peptide Studies:
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CA 02826627 2013-09-04
Junction Peptide: T FEKKGNYVVTDH-OH (Biosource, Camarillo, CA);
Peptide C: LEEKKGNYVVTDH(K-Biot)-OH (Biosource, Camarillo, CA);
sEGFR: CHO-cell-derived recombinant soluble extracellular domain (aa 1-621)
of the wild type EGFR (LICR Melbourne);
mAb 806: mouse monoclonal antibody, IgG2b (L1CR NYB); '
mAb L8A4: mouse monoclonal antibody, IgGI (Duke University);
IgGI isotype control mAb ;
IgG2b isotype control mAb
Peptide C was immobilized on a Streptavidin microsensor chip at a surface
density of
350RU (+/- 30RU). Serial dilutions of raAbs were tested for reactivity with
the
peptide. Blocking experiments using non-biotinylated peptide were performed to
assess specificity.
MAb L8A4 showed strong reactivity with Peptide C even at low antibody
concentrations (6.25 nM) (Figure 2D). mAb 806 did not show detectable specific
reactivity with peptide C up to antibody concentrations of 100nM (highest
concentration tested) (Figures 2D and 2E). It was expected that mAb L8A4 would
react with Peptide C because the peptide was used as the immunogen in the
generation of mAb L8A4. Addition of the Junction Peptide (non-biotinylated, 50
ug/ml) completely blocks the reactivity of niAb L8A4 with Peptide C,
confirming the
antibody's specificity for the junction peptide epitope.
In a second set of Bacore experiments, sEGFR was immobilized on a CM
raicrosensor chip at a surface density of ¨4000RU. Serial dilutions of mAbs
were
tested for reactivity with sEGFR.
MAb 806 was strongly reactive with denaturated sEGFR while niAb L8A4 did not
react with denaturated. sEGFR. Reactivity of mAb 806 with denaturated sEGFR
decreases with decreasing antibody concentrations. It was expected that mAb
L8A4
=
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CA 02826627 2013-09-04
does not react with sEGFR because mAb L8A4 was generated using the junction
peptide as the immunogen and sEG1,1( does not contain the junction peptide.
Dot-blot immune stain experiments were also performed. Serial dilutions of
peptide
were spotted in 0.5)11 onto a PVDF or nitrocellulose membranes. Membranes were
blocked with 2% BSA in PBS, and then probed with 806, L8A4, DH8.3 and control
antibodies. Antibodies L8A4 and DH8.3 bound to peptide on the membranes (data
not shown). Mab806 did not bind peptide at concentrations where L8A4 clearly
showed binding (data not shown). Control antibodies were also negative for
peptide
binding.
mAb 806 bound to the wtEGFR in cell lysates following irrununoblotting
(results not
shown). This is different from the results obtained with DH8.3 antibody, which
reacted with de2-7 EGFR but not wtEGFR. Thus, inAb 806 can recognize the
wtEGFR following denaturation but not when the receptor is in its natural
state on the
cell surface.
EXAMPLE 4
SCATCHARD ANALYSIS
A Scatchard analysis using U87MG.A2-7 cells was performed following correction
for immunoreactivity in order to determine the relative affinity of each
antibody.
Antibodies were labelled with 1251 (Amrad, Melbourne, Australia) by the
chloramine
T method and immunoreactivity determined by Lindmo assay (23). All binding
assays
were performed in 1% HSA/PBS on 1-2 x 106 live U87MG.A2-7 or A431 cells for 90
min at 4 C with gentle rotation. A set concentration of 10 ng,/m1125I-1abe1ed
antibody
was used in the presence of increasing concentrations of the appropriate
unlabeled
antibody. Non-specific binding was determined in the presence of 10,000-fold
excess
of unlabeled antibody. After the incubation was completed, cells were washed
and
counted for bound 1251-1abe1ed antibody using a COBRA II gamma counter
(Packard
Instrument Company, Meriden, USA).
84
CA 02826627 2013-09-04
=
Both mAb 806 and the DH8.3 antibody retained high imrnunoreactivity when
iodinated and was typically greater than 90% for mAb 806 and 45-50% for the
DH8.3
antibody. mAb 806 had an affinity for the de2-7 EGFR receptor of 1.1 x 109 M-1
whereas the affinity of DH8.3 was some 10-fold lower at 1.0 x 108 M.'. Neither
'25I-
radiolabeled mAb 806 nor the 125I-radiolabeled DH8.3 antibody bound to
parental
US7MG cells. mAb 806 recognized an average of 2.4 x 105 binding sites per cell
with
the DH8.3 antibody binding an average of 5.2 x 105 sites. Thus, there was not
only
good agreement in receptor number between the antibodies, but also with a
previous
report showing 2.5 x 105 de2-7 receptors per cell as measured by a different
de2-7
EGFR specific antibody on the same cell line (25).
EXAMPLE 5
INTERNALIZATION OF ANTIBODIES BY U87MG.A2-7 CELLS
The rate of antibody internalization following binding to a target cell
influences both
its tumor targeting properties and therapeutic options. Consequently, the
inventors
examined the internalization of inAb 806 and the DH8.3 antibody following
binding
to U87MG.A2-7 cells by FACS. U87MG.A2-7 cells were incubated with either mAb
806 or the DH8.3 antibody (10 ug/m1) for 1 h in DME/v1 at 4 C. After washing,
cells
were transferred to DMEM pre-warmed to 37 C and aliquots taken at various time
points following incubation at 37 C. Internalization was stopped by
immediately
washing aliquots in ice-cold wash buffer (I% HSA/PBS). At the completion of
the
time course cells were stained by FACS as described above. Percentage
internalization was calculated by comparing surface antibody staining at
various time
points to zero time using the formula: percent antibody internalized = (mean
fluorescence at time x background fluorescence )/(mean fluorescence at time 0 -
background fluorescence) x 100. This method was validated in one assay using
an
iodinated antibody (mAb 806) to measure internalization as previously
described (24).
Differences in internalization rate at different time points were compared
using
Student's t-test.
CA 02826627 2013-09-04
Both antibodies showed relatively rapid internalization reaching steady-state
levels at
min for mAb 806 and 30 min for DH8.3 (Figure 3). Internalization of DH8.3 was
significantly higher both in terms of rate (80.5% of DH8.3 internalized at 10
min
compared to 36.8% for mAb 806, p<0.01) and total amount internalized at 60 min
(93.5% versus 30.4%, p<0.001). mAb 806 showed slightly lower levels of
internalization at 30 and 60 min compared to 20 min in all 4 assays performed
(Figure 3). This result was also confirmed using an internalization assay
based on
iodinated mAb 806,
EXAMPLE 6
ELECTRON MICROSCOPY ANALYSIS OF ANTIBODY
INTERNALIZATION
Given the above noted difference in internalization rates between the
antibodies, a
detailed analysis of antibody intracellular trafficking was performed using
electron
microscopy.
U87MG.A2-7 cells were grown on gelatin coated chamber slides (Nunc,
Naperville,
IL) to 80% confluence and then washed with ice cold DMEM. Cells were then
incubated with mAb 806 or the DH8.3 antibody in DMEM for 45 min at 4 C. After
washing, cells were incubated for a further 30 min with gold-conjugated (20 nm
particles) anti-mouse IgG (BB1nternational, Cardiff, UK) at 4 C. Following a
further
wash, pre-warmed DMEM/10% FCS was added to the cells, which were incubated at
37 C for various times from 1-60 min. Internalization of the antibody was
stopped by
ice-cold media and cells fixed with 2.5% giutaraldehyde in PBS/0.1 % HSA and
then
post-fixed in 2.5% osmium tetroxide. After dehydration through a graded series
of
acetone, samples were embedded in Epon/ Araldite resin, cut as ultra-thin
sections
with a Reichert Ultracut-S microtome (Leica) and collected on nickel grids.
The
sections were stained with uranyl acetate and lead citrate before being viewed
on a
86
CA 02826627 2013-09-04
Philips CM12 transmission electron microscope at 80 kV. Statistical analysis
of gold
grains contained within coated pits was performed using a Chi-square test.
=
While the DH8.3 antibody was internalized predominantly via coated-pits, mAb
806
appeared to be internalized by macropinocytosis (Figure 19). In fact, a
detailed
analysis of 32 coated pits formed in cells incubated with rnAb 806 revealed
that none
of them contained antibody. In contrast, around 20% of all coated-pits from
cells
incubated with DH8.3 were positive for antibody, with a number containing
multiple
gold grains. A statistical analysis of the total number of gold grains
contained within
coated-pits found that the difference was highly significant (p<0.01). After
20-30 min
both antibodies could be seen in structures that morphologically resemble
lysosomes.
The presence of cellular debris within these structures was also consistent
with their
lysosome nature.
EXAMPLE 7
BIODISTRIBUTION OF ANTIBODIES IN TUMOR BEARING NUDE MICE
The biodistribution of inAb 806 and the DH8.3 antibody was compared in nude
mice
containing U87MG xenografts on one side and U87MG.A2-7 xenografts on the
other.
A relatively short time period was chosen for this study as a previous report
demonstrated that the DH8.3 antibody shows peak levels of tumor targeting
between
4-24 h (16).
Tumor xenografts were established in nude BALB/c mice by s.c. injection of 3 x
106
U87MG, U87MG.A2-7 or A431 cells. de2-7 EGFR expression in U87MG.A2-7
xenografts remained stable throughout the period of biodistribution. Also,
A431 cells
retained their mAb 806 reactivity when grown as tumor xenografts as determined
by
immunohistochemistry (data not shown). U87MG or A431 cells were injected on
one
side 7-10 days before U87MG.A2-7 cells were injected on the other side because
of
the faster growth rate observed for de2-7 EGFR expressing xenografts.
Antibodies
were radiolabeled and assessed for immunoreactivity as described above and
were
87
CA 02826627 2013-09-04
injected into mice by the retro-orbital route when tumors were 100-200 mg in
weight.
Each mouse received two different antibodies (2 jig per antibody): 2 Ci of
125I-
labeled mAb 806 and 2 Ci of 131I labelled DH8.3 or 528. Unless indicated,
groups of
mice were sacrificed at various time points post-injection and blood obtained
by
cardiac puncture. The tumors, liver, spleen, kidneys and lungs were obtained
by
dissection. All tissues were weighed and assayed for 1251 and 1311 activity
using a dual-
channel counting Window. Data was expressed for each antibody as % ID/g tumor
determined by comparison to injected dose standards or converted into tumor to
blood/liver ratios (i.e. % ID/g tumor divided by % ID/g blood or liver).
Differences
between groups were analysed by Student's t-test.
In terms of % 1D/g tumor, mAb 806 reached its peak level inIJ87MG.A2-7
xenografts of 18.6 % ni/g tumor at 8 h (Figure 4A), considerably higher than
any
other tissue except blood. While DH 8.3 also showed peak tumor levels at 8 h,
the
level was a statistically (p<0.001) lower 8.8 % m/g tumor compared to mAb 806
(Figure 4B). Levels of both antibodies slowly declined at 24 and 48 h. Neither
antibody showed specific targeting of U87M0 parental xenografts (Figure 4A,B).
With regards to tumor to blood/liver ratios, mAb 806 showed the highest ratio
at 24 h
for both blood (ratio of 1.3) and liver (ratio of 6.1) (Figure 5A,B). The
DH8.3
antibody had its highest ratio in blood at 8 h (ratio of 0.38) and at 24 h in
liver (ratio
of 1.5) (Figure 5 A,B), both of which are considerably lower than the values
obtained
for mAb 806.
As described above, levels of mAb 806 in the tumor peaked at 8 hours. While
this
peak is relatively early compared to many tumor-targeting antibodies, it is
completely
consistent with other studies using de2-7 EGFR specific antibodies which all
show
peaks at 4-24 hours post-injection when using a similar dose of antibody (16,
25, 33).
Indeed, unlike the earlier reports, the 8 h time point was included on the
assumption
that antibody targeting would peak rapidly. The % ID/g tumor seen with mAb 806
was similar to that reported for other de2-7 EGER specific antibodies when
using
standard iodination techniques (16, 24, 32). The reason for the early peak is
probably
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CA 02826627 2013-09-04
two-fold. Firstly, tumors expressing the de2-7 EGFR, including the transfected
1J87M0 cells, grow extremely rapidly as tumor xenografts. Thus, even during
the
relatively short period of time used in these biodistribution studies, the
tumor size
increases to such an extent (5-10 fold increase in mass over 4 days) that the
%
tumor is reduced compared with slow growing tumors. Secondly, while
internalization of mAb 806 was relatively slow compared to DH8.3, it is still
rapid
with respect to many other tumor antibody/antigen systems. Internalized
antibodies
undergo rapid proteolysis with the degradation products being excreted from
the cell
(34). This process of internalization, degradation and excretion reduces the
amount of
iodinated antibody retained within the cell. Consequently, internalizing
antibodies
display lower levels of targeting than their non-internalizing counterparts.
The
electron microscopy data reported herein demonstrates that intemalind mAb 806
is
rapidly transported to lysosomes where rapid degradation presumably occurs.
This
observation is consistent with the swift expulsion of iodine from the cell.
The previously described L8A4 monoclonal antibody directed to the unique
junctional
peptide found in the de2-7 EGFR, behaves in a similar fashion to mAb 806 (35).
Using U87MG cells transfected with the de2-7 EGFR, this antibody had a similar
internalization rate (35% at 1 hour compared to 30% at 1 hour for niAb 806)
and
displayed comparable in vivo targeting when using 3T3 fibroblasts transfected
with
de2-7 EGFR (peak of 24 % [Dig tumor at 24 hours compared to 18 % IDig tumor at
8
hours for mAb 806) (25). Interestingly, in vivo retention of this antibody in
tumor
xenografts was enhanced when labeled with N-succinimidyl 5-iodo-3-pyridine
=
carboxylate (25). This labeled prosthetic group is positively charged at
lysosmal pH
and thus has enhanced cellular retention (33). Enhanced retention is
potentially useful
when considering an antibody for raciioimmunotherapy and this method could be
used
to improve retention of iodinated mAb 806 or its fragments.
EXAMPLE 8
BINDING OF inAb 806 TO CELLS CONTAINING AMPLIFIED EGFR
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CA 02826627 2013-09-04
To examine if mAb 806 could recognize the EGFR expressed in cells containing
an
amplified receptor gene, its binding to A431 cells was analysed. As described
previously, A431 cells are human squamous carcinoma cells and express high
levels
of wtEGFR. Low, but highly reproducible, binding of mAb 806 to A431 cells was
observed by FACS analysis (Figure 6). The DH8.3 antibody did not bind A431
cells,
indicating that the binding of mAb 806 was not the result of low level de2-7
EGFR
expression (Figure 6). As expected, the anti-EGFR 528 antibody showed strong
staining of A431 cells (Figure 6). Given this result, binding of mAb 806 to
A431 was
characterized by Scatchard analysis. While the binding of iodinated mAb 806
was
comparatively low, it was possible to get consistent data for Scatchard. The
average
of such experiments gave a value for affinity of 9.5 x 107 /V1-1 with 2.4 x
105 receptors
per cell. Thus the affinity for this receptor was some 10-fold lower than the
affinity
for the de2-7 EGFR. Furthermore, mAb 806 appears to only recognize a small
portion
of EGFR found on the surface of A431 cells. Using the 528 antibody, the
inventors
measured approximately 2 x 106 receptors per cell which is in agreement with
numerous other studies(26).
Recognition of the wild type sEGFR by mAb 806 clearly requires some
denaturation
of the receptor in order to expose the epitope. The extent of denaturation
required is
only slight as even absorption of the wild type sEGFR on to a plastic surface
induced
robust binding of mAb 806 in FT ISA assays. As mAb 806 only bound
approximately
10% of the EGFR on the surface of A431 cells, it is tempting to speculate that
this
subset of receptors may have an altered conformation similar to that induced
by the
de2-7 EGFR truncation. Indeed, the extremely high expression of the EGFR
mediated by gene amplification in A431 cells, may cause some receptors to be
incorrectly processed leading to altered conformation. Interestingly, semi-
quantitative
immunoblotting of A431 cell lysates with rnAb 806 showed that it could
recognize
most of the A431 EGF receptors following SDS-PAGE and western transfer. This
result further supports the argument that mAb 806 is binding to a subset of
receptors
on the surface of A431 cells that have an altered conformation. These
observations in
A431 cells are consistent with the immunohistocheinistry data demonstrating
that
CA 02826627 2013-09-04
rnAb 806 binds gliomas containing amplification of the EGFR gene. As mAb 806
binding was completely negative on parental U87MG cells it would appear this
phenomenon may be restricted to cells containing amplified EGFR although the
level
of "denatured" receptor on the surface of U87MG cells may be below the level
of
detection. However, this would seem unlikely as iodinated mAb 806 did not bind
to
U87MG cell pellets containing up to 1 x 107 cells.
EXAMPLE 9
IN VIVO TARGETING OF A431 CELLS BY MAb 806
A second biodistribution study was performed with mAb 806 to determine if it
could
target A431 tumor xenografts. The study was conducted over a longer time
course in
order obtain more information regarding the targeting ofU87MG./..12-7
xenografts by
mAb 806, which were included in all mice as a positive control. In addition,
the anti-
EGFR 528 antibody was included as a positive control for the A431 xenografts,
since
a previous study demonstrated low but significant targeting of this antibody
to A431
cells grown in nude mice (21).
During the first 48 h, mAb 806 displayed almost identical targeting properties
as those
observed in the initial experiments (Figure 7A compared with Figure 4A). In
terms
of % 1D/g tumor, levels of mAb 806 in U87MG.L12-7 xenografts slowly declined
after
24 h but always remained higher than levels detected in normal tissue. Uptake
in the
A431 xenografts was comparatively low, however there was a sniall increase in
%
, .
ID/g tumor during the first 24 h not observed in normal tissues such as liver,
spleen,
kidney and lung (Figure 7A). Uptake of the 528 antibody was very low in both
xenografts when expressed as % ID/g tumor (Figure 7B) partially due to the
faster
clearance of this antibody from the blood., In terms of tumor to blood ratio
mAb 806
peaked at 72 h for U87MG.A2-7 xenografts and 100 h for A431 xenografts (Figure
8A,B). While the tumor to blood ratio for mAb 806 never surpassed 1.0 with
respect
to the A431 turnor, it did increase throughout the entire time course (Figure
8B) and
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CA 02826627 2013-09-04
was higher than all other tissues examined (data not shown) indicating low
levels of
targeting.
The tumor to blood ratio for the 528 antibody showed a similar profile to mAb
806
although higher levels were noted in the A431 xenografts (Figure 8A,B). mAb
806
had a peak tumor to liver ratio in U87MG.A2-7 xenografts of 7.6, at 72 h,
clearly
demonstrating preferential uptake in these tumors compared to normal tissue
(Figure
8C). Other tumor to organ ratios for mAb 806 were similar to those observed in
the
liver (data not shown). The peak tumor to liver ratio for mAb 806 in A431
xenografts
was 2.0 at 100 h, again indicating a slight preferential uptake in tumor
compared with
normal tissue (Figure 8D).
EXAMPLE 10
THERAPY STUDIES
The effects of mAb806 were assessed in two xenograft models of disease ¨ a
preventative model and an established tumor model.
Xenograft Models
Consistent with previous reports (Nishikawa et al, Proc. Natl. Acad. Sci. USA,
91(16); 7727-7731) U87MG cells transfected with de2-7 EGFR grew more rapidly
than parental cells and U87MG cells transfected with the wt EGFR. Therefore,
it was
not possible to grow both cell types in the same mice.
3x106 tumor cells in 100 ml of PBS were inoculated subcutaneously. into both
flanks
of 4 ¨ 6 week old female nude mice (Animal Research Centre, Western Australia,
Australia). Therapeutic efficacy of mAb 806 was investigated in both
preventative
and established tumor models. In the preventative model, 5 rnice with 2
xenografts
each were treated intraperitoneally. with either 1 or 0.1 mg of mAb 806 or
vehicle
(PBS) starting the day before tumor cell inoculation. Treatment was continued
for a
total of 6 doses, 3 times per week for 2 weeks. In the established model,
treatment
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was started when tumors had reached a mean volume of 65 6.42 mm3 (U87MG.A2-
7), 84 9.07 inm3 (U87MG), 73 7.5 mm3 (1J87M0.wtEGFR) or 201 19.09 rnm3
(A43I tumors). Tumor volume in inm3 was determined using the formula (length x
width2)/2, where length was the longest axis and width the measurement at
right
angles to the length (Scott et al, 2000). Data was expressed as mean tumor
volume
S.E. for each treatment group. Statistical analysis was performed at given
time points
using Student's t test. Animals were euthanased when the xenografts reached an
approximate volume of 1.5 cm3 and the tumors excised for histological
examination.
This research project was approved by the Animal Ethics Committee of the
Austin
and Repatriation Medical Centre.
Histological Examination of Tumor Xenografts
Xenografts were excised and bisected. One half was fixed in 10% formalin/PBS
before being embedded in paraffin. Four micron sections were then cut and
stained
with haematoxylin and eosin (H&E) for routine histological examination. The
other
half was embedded in Tissue Tek OCT compound (Sakura Finetek, Torrance, CA),
frozen in liquid nitrogen and stored at -80 C. Thin (5 micron) cryostat
sections were
cut and fixed in ice-cold acetone for 10 min followed by air drying for a
further 10
min. Sections were blocked in protein blocking reagent (Lipshaw Imrnunon,
Pittsburgh U.S.A.) for 10 min and then incubated with biotinylated prinaary
antibody
(1 mg/ml), for 30 min at room temperature (RT). All antibodies were
biotinylated
using the ECL protein biotinylation module (Amersham, Baulkham HiLls,
Australia),
as per the manufactures instructions. After rinsing with PBS, sections were
incubated
with a streptavidin horseradish peroxidase complex for a further 30 min
(Silenus,
Melbourne, Australia). Following a final PBS wash the sections were exposed to
3-
amino-9-ethylcarbozole (AEC) substrate (0.1 M acetic acid, 0.1 M sodium
acetate,
0.02 M AEC (Sigma Chemical Co., St Louis, MO)) in the presence of hydrogen
peroxide for 30 min. Sections were rinsed with water and counterstained with
hematoxylin for 5 min and mounted.
Efficacy of mAb 806 in Preventative Model
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MAb 806 was examined for efficacy against U87MG and U87MG.A2-7 tumors in a
preventative xenograft model. Antibody or vehicle were administered i.p. the
day
before tumor inoculation and was given 3 times per week for 2 weeks. MAb 806
had
no effect on the growth of parental U87MG xenografts, which express the wt
EGFR,
at a dose of 1 mg per injection (Figure 9A). In contrast, niAb 806
significantly
inhibited the growth of U87MG.A2-7 xenografts in a dose dependent manner
(Figure
9B). At day 20, when control animals were sacrificed, the mean tumor volume
was
1637 178.98 mm3 for the control group, a statistically smaller 526 94.74
mm3 for
the 0.1 mg per injection group (p< 0.0001) and 197 42.06 mm3 for the 1 mg
injection group (p< 0.0001). Treatment groups were sacrificed at day 24 at
which
time the mean tumor volumes was 1287 243.03 mm3 for the 0.1 mg treated group
and 492 100.8 mm3 for the 1 mg group.
Efficacy of mAb 806 in Established Xenograft Model
Given the efficacy of mAb 806 in the preventative xenograft model, its ability
to
inhibit the growth of established tumor xenografts was then examined. Antibody
treatment was as described in the preventative model except that it commenced
when
tumors had reached a mean tumor volume of 65 6.42 mm3 for the U87MG.A2-7
xenografts and 84 9.07 mm3 for the parental U87MG xenografts. Once again,
mAb
806 had no effect on the growth of parental U87MG xenografts at a dose of 1 mg
per
injection (Figure 10A). In contrast, mAb 806 significantly inhibited the
growth of
U87MG.A2-7 xenografts in a dose dependent manner (Figure 10B). At day 17, one
day before control animals were sacrificed, the mean tumor volume was 935
215.04
nun3 for the control group, 386 57.51 mm3 for the 0.1 mg per injection group
(p<
0.01) and 217 58.17 nun3 for the 1 mg injection group (p< 0.002).
To examine whether the growth inhibition observed with mAb 806 was restricted
to
cell expressing de2-7 EGER, its efficacy against U87MG.wtEGFR tumor xenografts
was examined in an established model. These cells serve as a model for tumors
containing amplification of the EGFR gene without de2-7 EGER expression. MAb
806 treatment commenced when tumors had reached a mean tumor volume of 73
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CA 02826627 2013-09-04
7.5 mm3. MAb 806 significantly inhibited the growth of established
U87M0.wtEGFR xenografts when compared to control tumors treated with vehicle
(Figure 10C). On the day control animals were sacrificed, the mean tumor
volume
was 960 268.9 mm3 for the control group and 468 78.38 mrn3 for the group
treated with 1 mg injections (p< 0.04).
Histological and Immunohistochemical Analysis of Established Tumors
To evaluate potential histological differences between mAb 806-treated and
control
U87MG.A2-7 and U87MG.wtEGFR xenografts (collected at days 24 and 42
respectively), formalin-fixed, paraffin embedded sections were stained with
H&F.
Areas of necrosis were seen in sections from both U87MG.A2-7 (collected 3 days
after treatment finished), and U87MG.wtEGFR xenografts (collected 9 days after
treatment finished) treated with mAb 806. This result was consistently
observed in a
number of tumor xenografts (n=4). However, analysis of sections from
xenografts
treated with control did not display the same areas of necrosis seen with mAb
806
treatment. Sections from niAb 806 or control treated U87MG xenografts were
also
stained with H&E and revealed no differences in cell viability between the two
groups, further supporting the hypothesis that mAb 806 binding induces
decreased
cell viability/necrosis within tumor xenografts.
An immunohistochemical analysis of U87MG, U87MG.A2-7 and U87MG.wtEGER
xenograft sections was performed to determine the levels of de2-7 and wt EGFR
expression following inAb806 treatment. Sections were collected at days 24 and
42
as above, and were immunostained with the 528 or 806 antibodies. As expected
the
528 antibody stained all xenograft sections with no obvious decrease in
intensity
between treated and control tumors. Staining of U87MG sections was
undetectable
with the mAb 806, however positive staining of U87MG.A2-7 and U87MG.wtEG1R.
xenograft sections was observed. There was no difference in mAb 806 staining
density between control and treated U87MG.6.2-7 and U87MG.wtEGFR xenografts
suggesting that antibody treatment does not down regulate de2-7 or wt EGFR
expression.
CA 02826627 2013-09-04
Treatment of A431 Xenouafts with mAb 806
To demonstrate that the anti-tumor effects of mAb 806 were not restricted to
U87MG
cells, the antibody was administered to mice with A431 xenografts. These cells
contain an amplified EGBR gene and express approximately 2 x 106 receptors per
cell.
As described above, mAb 806 binds about 10% of these EGFR and targets A431
xenografts. MAb 806 significantly inhibited the growth of A431 xenografts when
examined in the previously described preventative xenograft model (Figure
11A). At
day 13, when control animals were sacrificed, the mean tumor volume was 1385
147.54 mm3 in the control group and 260 60.33 mm3 for the 1 mg injection
treatment group (p< 0.0001).
In a separate experiment, a dose of 0.1 mg inAb also significantly inhibited
the
growth of A431 xenografts in a preventative model.
Given the efficacy of mAb 806 in the preventative A431 xenograft model, its
ability
to inhibit the growth of established tumor xenografts was examined. Antibody
treatment was as described in the preventative model except it was not started
until
tumors had reached a mean tumor volume of 201 19.09 inna3. MAb 806
significantly
inhibited the growth of established tumor xenografts (Figure 11B). At day 13,
when
control animals were sacrificed, the mean tumor volume was 1142 120.06 mm3
for
the control group and 451 65.58 mm3 for the 1 mg injection group (p<0.0001).
In summary, the therapy studies with mAb 806 described here clearly
demonstrated
dose dependent inhibition of U87MG.A2-7 xenograft growth. In contrast, no
inhibition of parental U87MG xenografts was observed despite the fact they
continue
to express the wt EGFR in vivo. MAb 806 not only significantly reduced
xenograft
volume, it also induced significant necrosis within the tumor. This is the
first report
showing the successful therapeutic use of such an antibody in vivo against a
human
de2-7 EGFR expressing glioma xenografts.
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Gene amplification of the EGFR has been reported in a number of different
tumors
and is observed in approximately 50% of gliomas (Voldberg et al, 1997). It has
been
proposed that the subsequent EGFR over-expression mediated by receptor gene
amplification may confer a growth advantage by increasing intracellular
signalling
and cell growth (Filmus et al, 1987). The U87MG cell line was transfe-cted
with the
wt EGFR in order to produce a glioma cell that mimics the process of EGFR gene
amplification. Treatment of established U87MG.wtEGFR xenografts with mAb 806
resulted in significant growth inhibition. Thus, mAb 806 also mediates in vivo
anti-
tumor activity against cells containing amplification of the EGFR gene.
Interestingly,
mAb 806 inhibition of U87MG.wtEGFR xenografts appears to be less effective
than
that observed with U87MG.A2-7 tumors. This probably reflects the fact that mAb
806 has a lower affinity for the amplified EGFR and only binds a small
proportion of
receptors expressed on the cell surface. However, it should be noted that
despite the
small effect on U87MG.wtEGFR xenograft volumes, mAb 806 treatment produced
large areas of necrosis within these xenografts. To rule out the possibility
that mAb
806 only mediates inhibition of the U87MG derived cell lines we tested its
efficacy
against A431 xenografts. This squamous cell carcinoma derived cell line
contains
significant EGFR gene amplification which is retained both in vitro and in
vivo.
Treatment of A431 xenografts with mAb 806 produced significant growth
inhibition
in both a preventative and established model, indicating the anti-tumor
effects of mAb
806 are not restricted to transfected U87MG cell lines.
EXAMPLE 11
COMBINATION THERAPY TREATMENT OF A431 XENOGRAFTS WITH
MAb806 AND AG1478
The anti-tumor effects of mAb 806 combined with AG1478 was tested in mice with
A431 xenografts. AG1478 (4-(3-Chloroanilino)-6,7-dimethoxyquinazoline) is a
potent and selective inhibitor of the EGFR kinase versus HER2-neu and platelet-
derived growth factor receptor ldnase (Calbiochem Cat. No. 658552). Three
controls
were included: treatment with vehicle only, vehicle + mAb806 only and vehicle
+
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AG1478 only. The results are illustrated in Figure 12. 0.1mg mAb806 was
administered at 1 day prior to xenograft and 1, 3, 6, 8 and 10 days post
xenograft. 400
pg AG1478 was administered at 0, 2, 4, 7, 9, and 11 days post xenograft.
Both AG1478 and rnAb806, when administered alone produced a significant
reduction of tumor volume. However, in combination, the reduction of tumor
volume
was greatly enhanced.
In addition, the binding of mAb806 to EGFR of A431 cells was evaluated in the
absence and presence of AG1478. Cells were placed in serum free media
overnight,
then treated with AG1478 for 10 min at 37 C, washed twice in PBS then lysed in
1%
Triton and lysates prepared. The lysates were prepared as described in Example
20
herein. Lysate was then assessed for 806 reactivity by an ELISA is a modified
version of an assay described by Schooler and Wiley, Analytical Biochemistry
277,135-142 (2000). Plates were coated with 10 ug/ml of rnAb 806 in PBS/EDTA
ovemight at room temperature and then washed twice. Plates were then blocked
with
10% serum albumin/PBS for 2 hours at 37 C and washed twice. A 1:20 cell lysate
was added in 10% serum albumin/PBS for 1 hour at 37 C, then washed four times.
Anti-EGFR (SC-03 (Santa Cruz Biotechnology Inc.)) in 10% serum albumin/PBS was
reacted 90 min at room temperature, the plate washed four times, and anti-
rabbit-HRP
(1:2000 if from Silenus) in 10% serum albumin/PBS was added for 90 min at room
temperature, washed four timed, and color developed using ABTS as a substrate.
It
= was found that mAb806 binding is significantly increased in the presence
of
increasing amounts of AG1478 (Figure 13).
EXAMPLE 12
INIMUNOREACTIVITY IN HUMAN GLIOBLASTOMAS PRE-TYPED FOR
EGFR STATUS
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Given the high incidence of EGFR expression, amplification and mutation in
glioblastomas, a detailed immunohistochemical study was performed in order to
assess the specificity of 806 in tumors other than xenografts. A panel of 16
glioblastomas was analyzed by immunohistochernistry. This panel of 16
glioblastomas was pre-typed by RT-PCR for the presence of amplified wild-type
EGFR and de2-7 EGFR expression. Six of these tumors expressed only the wt EGFR
transcript, 10 had wtEGFR gene amplification with 5 of these showing wild-type
EGFR transcripts only, and 5 both wild-type EGFR and de2-7 gene transcript.
Itrununohistochemical analysis was performed using 5mm sections of fresh
frozen
tissue applied to histology slides and fixed for 10 minutes in cold acetone.
Bound
primary antibody was detected with biotylated horse anti-mouse antibody
followed by
an avidin-biotin-complex reaction. Diaminobenzidine tetra hydrochloride (DAB)
was
used as chromogen. The extent of the immunohistochemical reactivity in tissues
was
estimated by light microscopy and graded according to the number of
immunoreactive
cells in 25% increments as follows:
Focal = less than 5%
+ = 5-25%
++ = 25-50%
+++ = 50-75%
I _____ =>75%
The 528 antibody showed intense reactivity in all tumors, while DH 8.3
immunostaining was restricted to those tumors expressing the de2-7 EGFR (Table
2).
Consistent with the previous observations in FACS and rosetting assays, mAb
806 did
not react with the glioblastomas expressing the wtEGFR transcript from non-
amplified EGFR genes (Table 2). This pattern of reactivity for mAb 806 is
similar to
that observed in the xenograft studies and again suggests that this antibody
recognizes
the de2- 7 and amplified EGFR but not the wtEGFR when expressed on the cell
surface.
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TABLE 2
Immunoreactivity of MAbs 528, DH8.3 and 806 on glioblastomas pretyped for the
presence of wild type EGFR and mutated de2-7 EGFR and for their. amplification
status.
Amplification de2-7 EGNR 528 DH 8.3 806
Expression
No itil - -
No -F-F++ - -*
No +-H-4- - -
No -H- - -
No .4-i---1- - -
No iiii - -
Yes No ++++ - +-H-1-
Yes No +-H-+ - +
Yes No -H--i- - +-1-+
Yes No -F+++ - -4-+-H-
Yes No +-H-+ - + - +++-F
'
Yes Yes -1-4-1-+ ++++ ++++
Yes Yes -H-++ 1111 ++++
Yes Yes +-H-+ ++++ ++-1--F
Yes Yes 4--H-+ -H-++ -F-F++
Yes Yes 1411 ++ ++
* focal staining
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EXAMPLE 13
EGER INLYIUNOREACTIVITY IN NORMAL TISSUE
In order to determine if the (102-7 EGFR is expressed in normal tissue, an
immunohistochernical study with mAli 806 and DH8.3 was conducted in a panel of
25
tissues. There was no strong imnaunoreactivity with either triAb 806 or DH8.3
in any
tissue tested suocesting that the de2-7 EGFR is absent in normal tissues
(Table 3).
There was some variable staining present in tonsils with mAb 806 that was
restricted
to the basal cell layer of the epidermis and mucosal squamous cells of the
epithelium.
In placenta, occasional hnmunostaining of the trophoblast epithelium was
observe&
Interestingly, two tissues that express high endogenous levels of wtEGFR, the
liver
and skin, failed to show any significant inAh 806 reactivity. No reactivity
was
observed with the liver samples at all, and only weak and inconsistent focal
reactivity
was detected occasionally (in no more than 10% of all samples studied) in
basal
keratinoeytes in skin samples and In the squamous epithelium of the tonsil
mucosa,
further demonstrating that this antibody does not bind the wtEGFR expressed on
the
surfam of cells to imy significant extent (Table 3). All tissues were positive
for the
wtEGFR as evideaiced by the universal staining seen with the 528 antibody
(Table 3).
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TABLE 3
Reactivity of 582, DH8.3 and 806 on normal tissues.
TISSUE 528 DH8-3 806
Esophagus pos
Stomach pos
Duodenum pos
Small intestine/duodenum pos
Colon Fos
Liver pos
Salivary glands (parotid) pos
= Kidney pos
Urinary bladder pos
Prostate pos
Testis pos
Uterus (cx/endom) pos ..*
Fallopian tube pos
Ovary pos
Breast pos -*
Placenta pos
Peripheral nerve pos
Skeletal muscle pos
Thyroid gland pos
Lymph node pos
Spleen pos
Tonsil pos - occ. weak
reactivity of
basal layer of squamous
epithelium
Heart pos
Lung pos
Skin pos - occ. weak
reactivity of
basal layer of squamous
epithelium
* some stromal staining in various tissue
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EXAMPLE 14
EGFR IMNIUNOREACTIVITY IN VARIOUS TUMORS
The extent of de2-7 EGER in other tumor types was examined using a panel of 12
different malignancies. The 528 antibody showed often homogeneous staining in
many tumors analysed except melanoma and seminoma. When present, DH8.3
immunoreactivity was restricted to the occasional focal tumor cell indicating
there is
little if any de2- 7 EGER expression in tumors outside the brain using this
detection
system (Table 4). There was also focal staining of blood vessels and a varying
diffuse
staining of connective tissue with the DH8.3 antibody in some tumors (Table
4). This
staining was strongly dependent on antibody concentration used and was
considered
nonspecific background reactivity . The mAb 806 showed positive staining in
64% of
head and neck tumors and 50% of lung carcinomas (Table 4). There was little
mAb
806 reactivity elsewhere except in urinary tumors that were positive in 30% of
cases.
Since the head and neck and lung cancers were negative for the DH8.3 antibody
the
reactivity seen with the mAb in these tumors maybe associated with EGFR gene
amplification.
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TABLE 4
Monoclonal ainibodies 528, DI183 and 806 073 tumor panel.
Tumor 528 DJ-I8.3 806
iSelil I iguan ( melanoma 0/10 0/10 0./10
metastases
Urinary bladder (tcc,sqcc, 10)10 0/10*
adeno) (7x4-4-44,2x+++,1x4-) , (2x++++,1x++)
Mammary gland 6/10 1/10 1/10
(3x++-H-,3x++) . (lx+) (foc)._
Head + neck cancer (sqcc) T11/11 (/11* 7/11
lx-1-I-1- - 10x-I-E-H-) (3x+-l-1-4-,3x4-1-4-,1x+)
Lung (sqcc, adeno, neuroend) 12/12 0/12* G12
10x-1-1-1-1--170-i-t-) (3xi-+++ 3x+++)
' Leioinyosarcoma = 5/5 0/5 0/5
______________________ . (4x1-1-1-1-,1X+)
"Liposarcoma 5/5 0/5 0/5*
(2x + 3x ii+)
Synovial sarcoma 4/5* 0/5 015*
(4x ++++)
'
Mfh Malignant fibrous; hih-tiouyforna 4/5* 0/5* 0/5* -
_
Colonic carcinoma 10/10 0/10* 0/10
(9x4-1-1-4-, lx+) _
seminamit 1/10* 1/10''' 0/10
Ovary (serous-papillary) , 4/5 0/5* 0/5
,
- (3x 1 1 i 1, lx+)
*focal staining
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EXAMPLE 15
IMMUNOREACTIVITY IN HUMAN GLIOBLASTOMAS UNSELECTED
FOR EGFR STATUS
In order to confirm the unique specificity and to evaluate the reactivity of
mAb 806, it
was compared to the 528 and DH8.3 antibodies in a panel of 46 glioblastomas
not
preselected for their EGFR status. The 528 antibody was strongly and
homogeneously
positive in all samples except two (No.27 and 29) (44/46, 95.7%). These two
cases
were also negative for mAb806 and mAb DH8.3. The mAb 806 was positive in 27/46
(58.7% ) cases, 22 of which displayed homogeneous inununoreactivity in more
than
50% of the tumor.. The DH8.3 antibody was positive in 15/46 (32.6%)
glioblastomas,
9 of which showed homogeneous immunoreactivity. The immunochemical staining
of these unselected tumors is tabulated in Table 5.
There was concordance between mAb 806 and DH8.3 in every case except one
(No.35).
A molecular analysis for the presence of EGFR amplification was done in 44
cases
(Table 5). Of these, 30 cases co-typed with the previously established mAb 806
immunoreactivity pattern: e.g. 16 mAb 806-negative cases revealed no EGFR
amplification and 14 EGFR-amplified cases were also mAb 806 immunopositive.
However, 13 cases, which showed 806 immunoreactivity, were negative for EGFR
amplification while 1 EGFR-amplified case was mAb 806 negative. Further
analysis
of the mutation status of these amplification negative and 806 positive cases
is
described below and provides explanation for most of the 13 cases which were
negative for EGFR amplification and were recognized by 806.
Subsequently, a molecular analysis of the deletion mutation by RT-PCR was
performed on 41/46 cases (Table 5). Of these, 34 cases co-typed with DH8.3
specific
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for the deletion mutation: 12 cases were positive in both RT-16CR and
inununohistochemistry and 22 cases were negative/negative. Three cases (#2,
#34,
#40) were DH8.3 positive/RT-PCR negative for the deletion mutation and three
cases
(#12, #18, #39) were DH8.3 negative/RT-PCR positive. As expected based on our
previous specificity analysis, rnAb 806 immunoreactivity was seen in all DH8.3-
positive tissues except in one case (#35).
=
Case #3 also revealed a mutation (designated A2 in Table 5), which included
the
sequences of the de2-7 mutation but this did not appear to be the classical
de2-7
deletion with loss of the 801 bases (data not shown). This case was negative
for
DH8.3 reactivity but showed reactivity with 806, indicating that 806 may
recognize
an additional and possibly unique EGFR mutation.
TABLE 5
Imrnunohistochernical analysis of 46 unselected glioblastomas with mAbs 528,
806,
and DH8.3
# 528 806 I DH8.3 EGFR 5' MUT
Amp.*
1 ++++ ++++ ++ A 5' MUT
2 ++++ --+--+ i N WT
3 ++-F+ +++1- neg. N A2
(det.)
4 +-H-1- iiis neg. N WT
= 5 ++++ ++++ -f¨H-+ 5' MUT
6 ++++ -1-1--H- neg. A WT
7 4-1-1-4- -1-+-1-1- N 5' MUT
8 ++++ -1-+-1-+ -H¨H- A 5' MUT
9 ++++ neg. A WT
i neg. neg. N WT
11 ++ -H- ++ A 5' MUT
12 ++++ ++ neg. A 5' MUT
13 lilt ++-1--1- neg N WT
14 ++ neg. neg. Nd nd
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15 ++ ++ neg N WT
16 + neg. neg. N nd
-
17 ++++ neg. neg. N WT
18 ++-t-+ ++++ neg. A 5 MUT
19 i ili ++++ neg. N WT
20 ++-H- neg. neg N WT
21 ++++ ++++ neg. N WT _
22 -H-+ , neg. neg. N WT
23 ++++ ++++ ++ N 5' MUT _
24 ++++ -H-1-+ neg. A WT
-
25 -H-++ neg. neg. N WT .
-
26 ++++ ++++ ++-F A 5' MUT
27 neg. neg. neg. N WT
28 -H-+ neg. neg. N WT
29 neg. neg. neg. N WT
30 , flIl Nil neg. N WT
31 -F+++ neg. neg. N nd
par det .
32 ++ -H-+ -F-F N 5' MUT
33 +++ ++++ -i--++ A 5' MUT
34 ++++ -+++ ++++ N WT
35 +-1-++ neg. ++++ A 5' MUT
__________________________________________ _
36 +++ ++ +++ A 5' MUT
,
37 ++-H- + + A 5' MUT
_
38 ++++ neg. neg. N WT
_
39 ++ neg. neg. N 5' MUT
40 ++++ ++++ + A WT
41 ++ neg. i neg. N WT
42 ++-H- -H-i-+ neg. A WT
43 ++++ neg. neg. nd nd
_
44 -H-H- neg. neg. N WT
-
45 +-i-H- neg. neg. N WT
46 ++++ neg. neg. N _ nd
* N-= not amplified, A-al:uplifted,
+WT = wildtype, 5'-mut
nd = not done
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=
The 806 antibody reactivity co-typed with amplified or de2-7 mutant EGFR in
19/27
or over 70% of the cases. It is notable that 2 of these 8 cases were also
D1i8.3
reactive.
EXAMPLE 16
SYSTEMIC TREATMENT AND ANALYSIS OF DITRACRANIAL GLIOMA
TUMORS
To test the efficacy of the anti-AEGFR monoclonal antibody, mAb 806, we
treated
nude mice bearing intracranial AEGFR-overexpressing glioma xenografts with
intraperitoneal injections of mAb806, the isotype control IgG or PBS.
The human glioblastoma cell lines U87MG, LN-Z308 and A1207 (gift from Dr. S.
Aaronson, Mount Sinai Medical Center, New York, NY) were infected with AEGFR,
kinase-deficient AEGFR (DK), or wild-type EGFR (wtEGFR) viruses. Populations
expressing similar high levels of EGFRs were selected by fluorescence-
activated cell
sorting and designated as U87MG.AFGER, U87MG.DK, U87MG.wtEGFR, LN-
Z308.AEGFR, LN-Z308.DK, LN-Z308. wtEGFR, A1207.AEGFR, A1207.DK and
A1207.wtEGFR, respectively. Each was maintained in medium containing G418
(U87MG cell Lines, 400 ug/m1; LN-Z308 and A1207 cell lines, 800 ug/m1).
U87MG.AEGFR cells were implanted intracranially into nude mice and the
treatments began on the same day. 105 cells in Sul PBS were implanted into the
right
corpus striatum of nude mice brains. Systemic therapy with mAb 806, or the
IgG2b
isotype control, was accomplished by i.p. injection of 1 mg of mAbs in a
volume of
100 I every other day from post- implantation day 0 through 14. For direct
therapy
of intracerebral U87MG.AEGFR tumors, 10 Kg of mAb 806, or the IgG2b isotype
control, in a volume of 5 p.l were injected at the tumor-injection site every
other day
starting at day 1 for 5 days.
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Animals treated with PBS or isotype control IgG had a median survival of 13
days,
whereas mice treated with mAb 806 had a 61.5% increase in median survival up
to 21
days (P4.001).
Treatment of mice 3 days post-implantation, following tumor establishment,
also
extended the median survival of the mAb 806 treated animals by 46.1% (from 13
days
to 19 days; P<0.01) compared to that of the control groups.
To determine whether these antitumor effects of mAb 806 extended beyond
U87MG.AEGFR xenografts, similar treatments were administered to animals
bearing
other glioma cell xenografts of LN-Z308.dEGFR and A 1207.AEGFR The median
survival of mAb 806 treated mice bearing LN-Z308.AFGFR xenografts was extended
from 19 days for controls to 58 days (P<0.001). Remarkably, four of eight mAb
806
treated animals survived beyond 60 days. The median survival of animals
bearing
A1207.AEGFR xenografts was also extended from 24 days for controls to 29 days
(P<0.01).
MAb 806 Treatment Inhibits AEGFR-overexpressing Brain Tumor Growth.
Mice bearing U87MG.AEGFR and LN-Z308.AEGFR xenografts were euthanized at
day 9 and day 15, respectively. Tumor sections were histopathologically
analyzed and
tumor volumes were determined. Consistent with the results observed for animal
survival, mAb 806 treatment significantly reduced the volumes by about 90%
ofU87MG.AEGFR.(P<0.001) andLN-Z308.AEGFR by more than 95% (P< 0.001)
xenografts in comparison to that of the control groups. Similar results were
obtained
for animals bearing A1207.AEGFR tumors (65% volume reduction, P<0.01).
Intratumoral Treatment with mAb 806 Extends Survival of Mice Bearing
TJ87MG.AEGFR Brain Tumors.
The efficacy of direct intratumoral injection of mAb 806 for the treatment of
U87MG.AEGFR xenografts was also determined. Animals were given intratumoral
injections of rnAb 806 or isotype control IgG one day post-implantation.
Control
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animals survived for 15 days, whereas mAb 806 treated mice remained alive for
18
days (P<0.01). While the intratumoral treatment with mAb 806 was somewhat
effective, it entailed the difficulties of multiple intracranial injections
and increased
risk of infection. We therefore focused on systemic treatments for further
studies.
MAb 806 Treatment Slightly Extends Survival of Mice Bearing U87MG.wtEGFR but
not U87MG or U871v10.DK Intracranial Xenografts.
To determine whether the growth inhibition by mAb 806 was selective for tumors
expressing AEGFR, we treated animals bearing U87MG, U87MG.DK (kinase-
deficient AEGFR) and U87MG.wtEGFR brain xenografts. MAb 806 treatment did not
extend survival of mice implanted with U87MG tumors which expressed a low
level
of endogenous wild-type EGFR (wtEGFR), or animals bearing U87MG.DK
xenografts which overexpressed a lcinase-deficient AEGFR in addition to a low
level
of endogenous wtEGFR. The mAb 806 treatment slightly extended the survival of
mice bearing U87MG.wtEGFR tumors (P<0.05,median survival 23 days versus 26
days for the control groups) which overexpressed wtEGFR
MAb 806 Reactivity Correlates with In Vivo Anti-tumor Efficacy.
To understand the differential effect of mAb 806 on tumors expressing various
levels
or different types of EGFR, we determined mAb 806 reactivity with various
tumor
cells by FACS analysis. Consistent with previous reports, the anti-EGFR
monoclonal
antibody 528 recognized both AEGFR and wtEGFR, and demonstrated stronger
staining for U87MG.AEGFR cells compared to U87MG cells. In contrast, antibody
EGFR.I reacted with wtEGFR but not AEGFR, as U87MG.AFGFR cells were as
weakly reactive as U87MG cells. This EGFR.1 antibody reacted with
U87M0.wtEGFR more intensively than U87MG cells, as U87MG.wtEGFR cells
overexpressed wtEGFR. While mAb 806 reacted intensely with U87MG.AFGFR and
U87MG.DK cells and not with U87MG cells, it reacted weakly with
U87MG.wtEGFR, indicating that mAb 806 is selective for AFGFR with a weak
cross-activity to overexpressed wtEGFR. This level of reactivity with
U87MG.wtEGFR was quantitatively and qualitatively similar to the extension of
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survival mediated by the antibody treatment.
We further determined mAb 806 specificity by immunoprecipitation. EGFRs in
varions cell lines were inarnunoprecipitated with antibody 528, EGFR.1 and mAb
806.
Blots of electrophoretically-separated proteins were then probed with the anti-
EGFR
antibody, C13, which recognizes wtEGFR as well as AEGFR and DK. Consistent
with the FACS analysis antibody 528 recognized wtEGFR and mutant receptors,
while antibody EGFR.1 reacted with wtEGFR but not the mutant species.
Moreover,
the levels of mutant receptors in U87MG.AFGFR and U87MG.DK cells are
comparable to those of wtEGFR in the U87MG.wtEGFR cells. However, antibody
mAb 806 was able to precipitate only a small amount of the wtEGFR from the
U87M0.wtEGFR cell lysates as compared with the larger amount of mutant
receptor
precipitated from U87MG.AEGFR and U87M0.DK cells, and an undetectable
amount from the U87M0 cells. Collectively, these data suggest that mAb 806
recognizes an epitope in AFGFR which also exists in a small fraction of wtEGFR
only when it is overexpressed on cell surface.
MAb 806 Treatment Reduces AEGFR Autophosphorylation and Down- regulates
Bc1.X Expression in U87MG.AEGFR Brain Tumors.
We next investigated the mechanisms underlying the growth inhibition by mAb
806.
Since the constitutively active kinase activity and autophosphorylation of the
carboxyl
terminus of AEGFR are essential for its biological functions we determined
AEGFR
phosphorylation status in tumors from treated and control animals. It was
found that
mAb 806 treatment dramatically reduced AEGER autophosphorylation, even though
receptor levels were only slightly decreased in the mAb 806 treated
xenografts. We
have previously shown that receptor autophosphorylation causes up- regulation
of the
antiapoptotic gene, Bc1-XL, which plays a key role in reducing apoptosis of
AEGFR
overexpressing tumors. Therefore, we next determined the effect of mAb 806
treatment on Bc1-XL, expression. AEGFR tumors from raAb 806 treated animals
did
indeed show reduced levels of Bc1-XL.
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MAb 806 Treatment Decreases Growth and Angiogenesis, and Increases Apoptosis
in
U87MG.AEGFR Tumors.
In light of the in vivo suppression caused by mAb 806 treatment and its
biochemical
effects on receptor signaling, we determined the proliferation rate of tumors
from
control or treated mice. The proliferative index, measured by Ki-67.staining
of the
mAb 806-treated tumors, was significantly lower than that of the control
tumors (P<
0.00 1). In addition, analysis of the apoptotic index through TUNEL staining
demonstrated a significant increase in the number of apoptotic cells in mAb
806
treated tumors as compared with the control tumors (P< 0.001). The extent of
tumor
vascularization was also analyzed by immunostaining of tumors from treated and
control specimens for CD31. To quantify tumor vascularization, microvascular
areas
(MVA) were measured using computerized image analysis. MAb 806 treated tumors
showed 30% less MVA than control tumors (P<0.001). To understand whether
interaction between receptor and antibody may elicit an inflammatory response,
we
stained tumor sections for the macrophage marker, F4/80, and the NK cell
marker,
asialo GM1. Macrophages were identified throughout the tumor matrix and
especially
accumulated around the mAb 806 treated-U87MG.AEGFR tumor periphery. We
observed a few NK cells infiltrated in and around the tumors and no
significant
difference between mAb 806 treated and isotype-control tumors.
EXAMPLE 17
COMBINATION IMEMUNOTHERAPY WITH mAb806 AND mAb528
Are you sure this works. The example is in a somewhat different format to the
others
preceding it
The experiments set forth herein describe in vivo work designed to determine
the
efficacy of antibodies in accordance with this invention.
Female nude mice, 4-6 weeks old, were used as the experimental animals. Mice
received subscutaneous inoculations of 3 x 106 tumor cells in each of their
flanks.
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The animals received either U87MG.D2-7, U87MG.DK, or A431 cells, all of which
are described, supra. Therapy began when tumors had grown to a sufficient
size.
Mice then received injections of one of (i) phosphate buffered saline, (ii)
mAb 806
(0.5mg/injection), (iii) mAb 528 (0.5mg/injection), or (iv) a combination of
both
rnAbs. With respect to "(iv)," different groups of mice received either
0.5mg/injection of each mAb, or 0.25mg,/injection of each mAb.
The first group of mice examined were those which had received U87MG.D2-7
injections. The treatment protocol began 9 days after inoculation, and
continued, 3
times per week for 2 weeks (i.e., the animals were inoculated 9, 11, 13, 16,
18 and 20
days after they were injected with the cells). At the start of the treatment
protocol, the
average tumor diameter was 115mm3. Each group contained 50 mice, each with two
tumors.
Within the group of mice which received the combination of antibodies (0.5
mg/injection of each), there were three complete regressions. There were no
regressions in any of the other groups. Figure 18A shows the results
graphically.
In a second group of mice, the injected materials were the same, except the
combination therapy contained 0.25mg of each antibody per injection. The
injections
were given 10, 12, 14, 17, 19 and 21 days after inoculation with the cells. At
the start
of the therapy the average tumor size was 114mm3. Results are shown in Figure
18B.
The third group of mice received inoculations of U87MG.DK. Therapeutic
injections
started 18 days after inoculation with the cells, and continued on days 20,
22, 25, 27
and 29. The average tumor size at the start of the treatment was 107mm3.
Figure
18C summarizes the results. The therapeutic injections were the same as in the
first
group.
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Finally, the fourth group of mice, which had been inoculated with A431 cells,
received injections as in groups I and IR, at 8, 10, 12 and 14 days after
inoculation.
At the start, the average tumor size was 71mm3. Results are shown in Figure
18D.
The results indicated that the combination antibody therapy showed a
synergistic
effect in reducing tumors. See Figure 18A. A similar effect was seen at a
lower
dose, as per Figure 18B, indicating that the effect is not simply due to
dosing levels.
The combination therapy did not inhibit the growth of U87MG.DK (Figure 18C),
indicating that antibody immune function was not the cause for the decrease
seen in
, Figures 18A and 188.
It is noted that, as shown in Figure 18D, the combination therapy also
exhibited
synergistic efficacy on A431 tumors, with 4 doses leading to a 60%.complete
response rate. These data suggest that the EGI-it molecule recognized by
mAb806 is
functionally different from that inhibited by 528.
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EXAMPLE 18
NOVEL MONOCLONAL ANTIBODY SPECIFIC FOR ilik; DE2-7
EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) THAT ALSO
RECOGNIZES THE EGFR EXPRESSED IN CELLS CONTAINING
AMPLIFICATION OF THE EGFR GENE
The following experiments were presented in Johns et al, (2002) Int. J.
Cancer, 98,
The monoclonal antibody mAb
806 was studied and additional data respecting its binding characteristics as
to the
EGF receptor were developed, which is in addition to and corroborative of the
data
presented earlier herein. Accordingly, the following represents a review and
presentation of the material set forth in the patent application and
corresponding
publication.
Monoclonal antibody (MAb 806) potentially overcomes the difficulties
associated
with targeting the EGFR expressed on the surface of tumor cells. MAb 806 bound
to
de2-7 altit transfected U87MG glioma cells (U87MG.A2-7) with high affinity (-1
x
109 M '1 ), but did not bind parental cells that express the wild type EGFR.
Consistent
with this observation, MAb 806 was unable to bind a soluble version of the
wild type
EGFR containing the extra.cellular domain. In contrast, immobilization of this
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extracellular domain to FI ISA plates induced saturating and dose response
binding of
MAb 806, suggesting that MAb 806 can bind the wild type EGFR Under certain
conditions. MAb 806 also bound to the surface of A431 cells, which due to an
amplification of the EGFR gene express large amounts of the EGFR.
Interestingly,
MAb 806 only recognized 10% of the total EGFR molecules expressed by A431
cells
and the binding affinity was lower than that determined for the de2-7 EGFR.
lvlAb
806 specifically targeted U87MGA2-7 and A431 xenografts grown in nude mice
with
peak levels in U87M0.A2-7 xenografts detected 8 h after injection. No specific
targeting of parental U87MG xenografts was observed. Following binding to
U87MGA2-7 cells, MAb 806 was rapidly internalized by macropinocytosis and
subsequently transported to lysosomes, a process that probably contributes to
the early
targeting peak observed in the xenografts. Thus, MAb 806 can be used to target
tumor
cells containing amplification of the EGFR gene or de2-7 EGFR but does not
bind to
the wild type EGER when expressed on the cell surface.
As discussed above, MAb 806 is specific for the de2-7 EGFR yet binds to an
epitope
distinct from the unique junctional peptide. Interestingly, while MAb 806 did
not
recognize the wild type EGFR expressed on the cell surface of glioma cells, it
did
bind to the extracellular domain of the wild type EGFR immobilized on the
surface of
ELISA plates. Furthermore, MAb 806 bound to the surface of A431 cells, which
have
an amplification of the EGFR gene but do not express the de2-7 EGFR.
Therefore, it
is possible that MAb 806 could be used to specifically target tumors with
amplified
EGFR regardless of their de2-7 EGFR status, although our results suggest
tumors
coexpressing the mutated receptor would still show preferential targeting. As
MAb
806 does not bind wild type receptor in the absence of gene amplification,
there
would be no uptake in normal tissue, a potential problem associated with EGFR
antibodies currently being developed. 18'19
MATERIAL AND METHODS
MAbs and cell lines
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The U87MG astrocytoma cell line has been described in detail previous1y.2
This cell
line was infected with a retrovirus containing the de2-7 EGFR to produce the
U87MG.A2-7 cell line.10 Human squamous carcinoma A431 cells were obtained
from ATCC (Rockville, MD). These cell lines were cultured in DMEM/F-12 with
GlutaMAXTm (Life Technologies, Melbourne, Australia) supplemented with 10%
FCS (CSL, Melbourne, Australia). The murine pro-B cell line BaF/3, which does
not
express any lcnown EGFR related molecules, was transfected with de2-7 EGFR as
described above. The DH8.3 antibody (IgGI) has been described previously and
was
obtained following immunization of mice with the unique junctional peptide
found in
de2-7 EGFR.16MAb 806 (IgG2b) was produced following immunization of mice with
NR6 mouse fibroblasts transfected with the de2-7 EGFR. It was selected for
further
characterization as hemagglutination assays showed a high titer against
NR6.dEGFR
cells but low backgrounds on NR6.wtEGFR cells. The 528 antibody, which
recognizes both de2-7 and wild type EGFR, has been described previously21 and
was
produced in the Biological Production Facility (Ludwig Institute for Cancer
Research,
Melbourne) using a hybridoraa obtained from ATCC. The polyclonal antibody sc-
03
directed to the COOH-terminal domain of the EGFR was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
Other reagents
The recombinant extracellular domain (amino acids 1-621) of the wild type EGFR
(sEGFR) was produced as previously described. 22 The biotinylated unique
junctional
peptide (Biotin-LEEKKGNYVVIDH) from de2-7 EGFR was synthesized by
standard flmoc chemistry and purity (>96%) determined by re-verse phase HPLC
and
mass spectral analysis (Auspep, Melbourne, Australia).
FACS analysis
Cells were labeled with the relevant antibody (10 tig/ral) followed by
fluorescein-
conjugated goat anti-mouse IgG (1:100 dilution; Calbiochem, San Diego, CA).
FACS
data was obtained on a Coulter Epics Elite ESP by observing a minimum of 5,000
events and analyzed using EXPO (version 2) for Windows.
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ELISA assays
Two types of ELISA were used to determine the specificity of the antibodies.
In the
first assay, plates were coated with Segfr*(10 lig/rn1 in 0.1 M carbonate
buffer pH 9.2)
for 2 hr and then blocked with 2% human serum albumin (HSA) in PBS. Antibodies
were added to wells in triplicate at increasing concentration in 2% HSA in
phosphate-
buffered saline (PBS). Bound antibody was detected by horseradish peroxidase
conjugated sheep anti-mouse IgG (Silenus, Melbourne, Australia) using ABTS
(Sigrna, Sydney, Australia) as a substrate and the absorbance measured at 405
nm. In
the second assay, the biotinylated de2-7 specific peptide was bound to ELISA
plates
precoated with streptavidin (Pierce, Rock-ford, Illinois). Antibodies were
bound and
detected as in the first assay.
Scatchard analysis
Antibodies were labeled with 125 I (Amrad, Melbourne, Australia) by the
chloramine
T method and immunoreactivity determined by Lindmo assay.23 All binding assays
were performed in I% HSA/PBS on 1-2 X 106 live U87MG.L12-7 or A431 cells for
90 min at 4 C with gentle rotation. A set concentration of 10 ng/ml 1251-
1abeled
antibody was used in the presence of increasing concentrations of the
appropriate
unlabeled antibody. Non-specific binding was determined in the presence of
10,000-
fold excess of unlabeled antibody. Neither 125 I-radiolabcled MA.b 806 or the
DH8.3
antibody bound to parental U87MG cells. After the incubation was completed,
cells
were washed and counted for bound 1251-1abe1ed antibody using a COBRA gamma
counter (Packard Instrument Company, Meriden, CT). Scatchand analysis was done
following correction for immunoreactivity.
Internalization assay
U87MG.A2-7 cells were incubated with either MAb 806 or the DH8.3 antibody (10
Wm!) for I hr in DMEM at 4 C. After washing, cells were transfened to DMEM
pre-warmed to 37 C and aliquots taken at various time points following
incubation at
37 C. Internalization was stopped by immediately washing aliquots in ice-cold
wash
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buffer (1% HSA/PBS). At the completion of the time course cells were stained
by
FACS as described above. Percentage internalization was calculated by
comparing
surface antibody staining at various time points to zero time using the
formula:
= percent antibody internalized ===-- (mean fluorescence at time, -
background
fluorescence)/(mean fluorescence at time _ background fluorescence) X 100.
This
method was validated in 1 assay using an iodinated antibody (MAb 806) to
measure
internalintion as previously described.24 Differences in internalization rate
at
different time points were compared using Student's t-test.
Electron microscopy of 1,57MG.A2-7 cells
U87MG.62-7 cells were grown on gelatin coated chamber slides (Nunc,
Naperville,
IL) to 80% confluence and then washed with ice cold DMEM. Cells were then
incubated with MAb 806 or the D118.3 antibody in DMEM for 45 min at 4 C. After
washing, cells were incubated for a further 30 min with gold-conjugated (20 nm
particles) anti-mouse IgG (BBInternational, Cardiff, UK) at 4 C. Following a
further
wash, pre-warmed DMEM/10% PCS was added to the cells, which were incubated at
37 C for various times from 1-60 min. Internalization of the antibody was
stopped by
ice-cold media and cells fixed with 2.5% glutaraldehyde in PBS/0.1% HSA and
then
postfixed in 2.5% osmium tetroxide. After dehydration through a graded series
of
acetone, samples were embedded in Bpon/Araldite resin, cut as ultrathin
sections with
a Reichert Ultracut-S microtome (Leica) and collected on nickel grids. The
sections
were stained with uranyl acetate and lead citrate before being viewed on a
Philips
CM12 transmission electron microscope at 80 kV. Statistical analysis of gold
grains
contained within coated pits was performed using a e test.
lmmunoprecipitation studies
Cells were labeled for 16 hr with 100 Ci/m1 of Tran35S-Label (ICN
Biomedicals,
CA) in DMEM without methionine/cysteine supplemented with 5% dialyzed FCS.
After washing with PBS, cells were placed in lysis buffer (I% Triton*X-100, 30
niM
HEPES, 150 mM aC1, 500 M AEBSF, 150 nM aprotinin, 1 M E-64 protease
inhibitor, 0.5 mMEDTA and 1 M leupeptin, pH 7.4) for 1 hr at 4 C. Lysates
were
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clarified by centrifugation for 10 min at 12,000g, then incubated with 5 ug of
appropriate antibody for 30 min at 4 C before the addition of Protein A-
Sepharose
Imrnunoprecipitate,s were washed 3 times with lysis buffer, mixed with SDS
sample
buffer, separated by gel electrophoresis using a 4-20% Tris/glycine gel that
was then
dried and exposed to X-ray film.
Biodistribution in tumor bearing nude mice
Tumor xenografts were established in nude BAL13/c mice by s.c. injection of 3
X 106
U87MG, U87MG.A2-7 or A431 cells. de2-7 EGFR expression in U87MG,A2-7
xenografts remains stable throughout the period of biodistribution as measured
by
inununohistochemistry at various time points (data not shown). A431 cells also
retaine,d their MAb 806 reactivity when grown as tumor xenografts as
determined by
immunohistochemistry. U87MG or A431 cells were injected on 1 side 7-10 days
before U87MG.A2-7 cells were injected on the other side because of the faster
growth
rate observed for de2-7 EGFR expressing xenografts. Antibodies were
radiolabeled
and assessed for inununoreactivity as described above and were injected into
mice by
the retro-orbital route when tumors were 100-200 mg in weight. Each mouse
received
2 different antibodies (2 gg per antibody): 2 tiCi of 125 I-labeled MAb 806
and 2 põCi
of 131 I-labeled DH8.3 or 528. Unless indicated, groups of 5 mice were
sacrificed at
various time points post-injection and blood obtained by cardiac puncture. The
tumors, live,r, spleen, kidneys and lungs were obtained by dissection. All
tissues were
weighed and assayed for 125 I and 131 I activity using a dual-channel counting
window.
Data was expressed for each antibody as percentage injected dose per gram
tumor (%
133/g tumor) determined by comparison to injected dose standards or converted
into
tumor to blood/liver ratios (i.e., % ID/g tumor + % ID/g blood or liver).
Differences
between groups were analyzed by Student's t-test. After injection of
radiolabeled
MAb 806, some tumors were fixed in fOrma.lin, embedded in paraffin, cut into 5
gm
sections and then exposed to X-ray film (AGFA, Mortsel, Belgium) to determine
antibody localization by autonidiography.
RESULTS
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Binding of antibodies to cell lines
In order to confirm the specificity of MAb 806 and the DH8.3 antibody, binding
to
U87MG and U87MG.A2-7 cells was analyzed by FACS. An irrelevant murine IgG2b
was included as an isotype control for MAb 806 and the 528 antibody was
included as
it recognizes both the de2-7 and wild type EGFR. Only the 528 antibody was
able to
stain the U87MG cell line (Figure 1) consistent with previous reports
demonstrating
that these cells express the wild type EGFR.1 Both MAb 806 and the DH8.3
antibody
had binding levels similar to the irrelevant antibody, clearly demonstrating
they are
unable to bind the wild type receptor (Figure 1). Binding of the isotype
control
antibody to U87MG.A2-7 cells was similar as that observed for the U87MG cells.
MAb 806 and the DH8.3 antibody immunostained U87MG.A2-7 cells, indicating that
these antibodies specifically recognize the de2-'7 EGFR (Figure 1). The 528
antibody
stained U87MG.A2-7 with a higher intensity than the parental cell as it binds
both the
de2-7 and wild type receptors that are co-expressed in these cells (Figure 1).
Importantly, MAb 806 also bound the BaF/3.A2-7 cell line, demonstrating that
the co-
expression of wild type EGFR is not a requirement for MAb 806 reactivity
(Figure 1
but data not shown herein).
Binding of antibodies in ELISA assays
To further characterize the specificity of MAb 806 and the DH8.3 antibody,
their
binding was examined by ELISA. Both MAb 806 and the 528 antibody displayed
dose-dependent and saturating binding curves to immobilized wild type sEGFR
(Figure 2A). As the unique junctional peptide found in the de2-7 EGFR is not
contained within the sEGFR, MAb 806 must be binding to an epitope located
within
the wild type EGFR sequence. The binding of the 528 antibody was probably
lower
than that observed for MAb 806 as it recognizes a conformational determinant.
As
expected the DH8.3 antibody did not bind the wild type sEGFR even at
concentrations up to 10 jig/ml (Figure 2A). Although sEGFR in solution
inhibited the
binding of the 528 antibody to immobilized sEGFR in a dose-dependent fashion,
it
was unable to inhibit the binding of MAb 806 (Figure 2B). This suggests that
MAb
806 can only bind wild type EGFR once immobilized on ELISA plates, a process
that
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may induce conformational changes. Similar results were observed using a
BlAcore
whereby MAb 806 bound immobilized sEGFR but immobilized MAb 806 was not
able to bind sEGFR in solution (data not shown). Following denaturation by
heating
for 10 min at 95 C, sEGFR in solution was able to inhibit the binding of MAb
806 to
immobilized sEGI4R (Figure 2C but data not shown herein), confirming that MAb
806 can bind the wild type EGFR under certain conditions. Interestingly, the
denatured sEGFR was unable to inhibit the binding of the 528 antibody (Figure
2C
but data not shown herein), demonstrating that this antibody recognizes a
conformational epitope. The DH8.3 antibody exhibited dose¨dependent and
saturable
binding to the unique de2-7 EGFR peptide (Figure 2D). Neither MAb 806 or the
528
antibody bound to the peptide, even at concentrations higher than those used
to obtain
= saturation binding of DH8.3, further indicating MAb 806 does not
recognize an
epitope determinant within this peptide.
Scatchard analysis of antibodies
A Scatchard analysis was performed using U87MG.A2-7 cells in order to
determine
the relative affinity of each antibody. Both MAb 806 and the DH8.3 antibody
retained
high immunoreactivity when iodinated and was typically greater than 90% for
MAb
806 and 45-50% for the DH8.3 antibody. MAb 806 had an affinity for the de2-7
EGFR receptor of 1.1 X 109 M -I whereas the affinity of DH8.3 was some 10-fold
lower at 1.0 X 108 M -I. Neither iodinated antibody bound to U87MG parental
cells.
MAb 806 recognized an average of 2.4 X 105 binding sites per cell with the
DH8.3
antibody binding an average of 5.2 X 105 sites. Thus, there was not only good
agreement in receptor number between the antibodies, but also with a previous
report
showing 2.5 X 105 de2-7 receptors per cell as measured by a different de2-7
EGFR
specific antibody on the same cell line.25
Internalization of antibodies by U87MG.A2-7 cells
The rate of antibody intemali7ation following binding to a target cell
influences both
its tumor targeting properties and therapeutic options. Consequently, we
examined the
internalization of MAb 806 and the DH8.3 antibody following binding to
U87M0.A.2-
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7 cells by FACS. Both antibodies showed relatively rapid internalization
reaching
steady-state levels at 10 min for MAb 806 and 30 min for DH8.3 (Figure 3).
Internalization of DH8.3 was significantly higher both in terms of rate (80.5%
of
DH8.3 internalized at 10 min compared to 36.8% for MAb 806, p < 0.01) and
total
amount internalized at 60 min (93.5% vs. 30.4%, p < 0.001). MAb 806 showed
slightly lower levels of internalization at 30 and 60 min compared to 20 min
in all 4
assays performed (Figure 3). This result was also confirmed using an
internalization
assay based on iodinated MAb 806 (data not shown).
Electron microscopy analysis of antibody internalization
Given this difference in internalization rates between the antibodies, a
detailed
analysis of antibody intracellular trafficking was performed using electron
microscopy. Although the DH8.3 anti-body was internalized predominantly via
coated-pits (Figure 19A), MAb 806 appeared to be internalized by
macropinocytosis
(Figure 19B). In fact, a detailed analysis of 32 coated pits formed in cells
incubated
with MAb 806 revealed that none of them contained antibody. In contrast,
around
20% of all coated-pits from cells incubated with DH8.3 were positive for
antibody,
with a number containing multiple gold grains. A statistical analysis of the
total
number of gold grains contained within coated-pits found that the difference
was
highly significant (p < 0.01). After 20-30 min both antibodies could be seen
in
structures that morphologically resemble lysosomes (Figure 19C). The presence
of
cellular debris within these structures is also consistent with their lysosome
nature.
Biodistribution of antibodies in tumor bearing nude mice
The biodistribution of MAb 806 and the DH8.3 antibody was compared in nude
mice
containing U87M0 xenografts on 1 side and U87MG.A2-7 xenografts on the other.
A
relatively short time period was chosen for this study as a previous report
demonstrated that the DH8.3 antibody shows peak levels of tumor targeting
between
4-24 hr.16 In terms of % ID/g tumor, MAb 806 reached its peak level in
U87MG.A2-7
xenografts of 18.6% ID/g tumor at 8 hr (Figure 4A), considerably higher than
any
other tissue except blood. Although DH 8.3 also showed peak tumor levels at 8
hr, the
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level was a statistically (p < 0.001) lower 8.8 % TD/g tumor compared to MAb
806
(Figure 4B). Levels of both antibodies slowly declined at 24 and 48 hr.
Autoractiography of U87MG.A2-7 xenograft tissue sections collected 8 hr after
injection with 1251-labeled MAb 806 alone, clearly illustrates localization of
antibody
to viable tumor (Figure 20). Neither antibody showed specific targeting of
U87MG
parental xenografts (Figure 4A, 4B). With regards to tumor to blood/liver
ratios,
MAb 806 showed the highest ratio at 24 hr for both blood (ratio of 1.3) and
liver
(ratio of 6.1) (Figure 5A, 5B). The DH8.3 antibody had its highest ratio in
blood at 8
hr (ratio of 0.38) and at 24 hr in liver (ratio of 1.5) (Figure 5A, 5B), both
of which are
considerably lower than the values obtained for MAb 806.
Binding of MAb 806 to cells containing amplified EGFR
To examine if MAb 806 could recognize the EGFR expressed in cells containing
an
amplified receptor gene, its binding to A431 cells was analyzed. Low, but
highly
reproducible, binding of MAb 806 to A431 cells was observed by FACS analysis
(Figure 6). The DH8.3 antibody did not bind A431 cells, indicating that the
binding
of MAb 806 was not the result of low level de2-7 EGER expression (Figure 6).
As
expected, the anti-EGFR 528 antibody showed strong staining of A431 cells
(Figure
6). The average of 3 such experiments gave a value for affinity of 9.5 X 107M -
1 with
2.4 X 105 receptors per cell. Thus the affinity for this receptor was some 10-
fold
lower than the affinity for the de2-7 EGFR. Furthermore, MAb 806 appears to
only
recognize a small portion of EGFR found on the surface of A431 cells. Using
the 528
antibodyapproximately 2 X 106 receptors per cell were measured, which is in
agreement with numerous other studies.26 To ensure that these results were not
simply restricted to the A43I cell line, MAb 806 reactivity was exarnined in 2
other
cells lines exhibiting amplification of the EGFR gene. Both the BN5 head and
neck
cell line 27 and the MDA-468 breast cancer cell line 28 have been reported to
contain
multiple copies of the EGFR gene. Consistent with these reports, the 528
antibody
displayed intense staining of both cell lines (Figure 21). As with the A431
cell line,
the MAb 806 clearly stained both ceLl lines but at a lower level than that
observed
with the 528 antibody (Figure 21). Thus, MAb 806 binding is not simply
restricted to
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A431 cells but appears to be a general observation for cells containing
amplification
of the EGFR gene.
Immunoprecipitations
MAb 806 reactivity was further characterized by irnmunoprecipitation using 35
S-
labeled cells. The sc-03 antibody (a commercial polyclonal antibody specific
for the
c-terminal domain of the EGFR) immunoprecipitated 3 bands from U87MG.A2-7
cells; a doublet corresponding to the 2 de2-7 EGFR bands observed in these
cells and
a higher molecular weight band corresponding to the wt EGFR (Figure 22). In
contrast, while MAb 806 immunoprecipitated the 2 de2-7 EGFR bands, the wt EGFR
was completely absent. The sc-03 antibody immunoprecipitated a single band
corresponding to the wt EGFR from A431 cells (Figure 22). The MAb 806 also
immunoprecipitated a single band corresponding to the wt EGFR from A431 cells
(Figure 22) but consistent with the FACS and Scatchard data, the amount of
EGFR
immunoprecipitated by MAb 806 was substantially less than the total EGFR
present
on the cell surface. Given that MAb 806 and the sc-03 immunoprecipitated
similar
amounts of the de2-7 EGFR, this result supports the notion that the MAb 806
antibody only recognizes a portion of the EGFR in cells overexpressing the
receptor.
An irrelevant IgG2b (an isotype control for MAb 806) did not immunoprecipitate
EGFR from either cell line (Figure 22). Using identical conditions, MAb 806
did not
immunoprecipitate the EGFR from the parental U87MG cells (data not shown).
In vivo targeting of A431 cells by MAb 806
A second biodistiibution study was performed with MAb 806 to determine if it
could
target A431 tumor xenografts. The study was conducted over a longer time
course in
order to obtain more information regarding the targeting of U87MG.A2-7
xenografts
by MAb 806, which were included in all mice as a positive control. In
addition, the
anti-EGFR 528 antibody was included as a positive control for the A431
xenografts,
since a previous study demonstrated low but significant targeting of this
antibody to
A431 cells grown in nude mic,e.21 During the first 48 hr, MAb 806 displayed
almost
identical targeting properties as those observed in the initial experiments
(Figure
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CA 02826627 2013-09-04
7Acompared with Figure 4A). In terms of % ID/g tumor, levels of MAb 806 in
U87MG.A2-7 xenografts slowly declined after 24 hr but always remained higher
than
levels detected in normal tissue. Uptake in the A431 xenografts was
comparatively
low, however, there was a small increase in % 113/g tumor during the first 24
hr not
observed in normal tissues such as liver, spleen, kidney and lung (Figure 7A).
Uptake
of the 528 antibody was low in both xenografts when expressed as % ID/g tumor
(Figure 7B). Autoradiography of A431 xenograft tissue sections collected 24 hr
after
injection with 1251-labeled MAb 806 alone, clearly illustrates localization of
antibody
to viable tumor around the periphery of the tumor and not central areas of
necrosis
(Figure 23). In terms of tumor to blood ratio MAb 806 peaked at 72 hr for
U87MG.A2-7 xenografts and 100 hr for A431 xenografts (Figure 8A, 8B). Although
the tumor:blood ratio for MAb 806 never surpassed 1.0 with respect to the A431
tumor, it did increase through-out the entire time course (Figure 8B) and was
higher
than all other tissues examined (data not shown) indicating low levels of
targeting.
The tumor to blood ratio for the 528 antibody showed a similar profile to MAb
806
although higher levels were noted in the A431 xenografts (Figure 8A, 8)3). MAb
806
had a peak tumor to liver ratio in U87MG.A2-7 xenografts of 7.6 at 72 hr,
clearly
demonstrating preferential uptake in these tumors compared to normal tissue
(Figure
8C). Other tumor to organ ratios for MAb 806 were similar to those observed in
the
liver (data not shown). The peak tumor to liver ratio for MAb 806 in A431
xenografts
was 2.0 at lod hr, again indicating a slight preferentially uptake in tumor
compared
with normal tissue (Figure 8D).
DISCUSSION
The previously described L8A4 monoclonal antibody directed to the unique
junctional
peptide found in the de2-7 EGFR, behaves in a similar fashion to MAb 806.38
Using
U87MG cells transfected with the de2-7 EGFR, this antibody had a similar
internalization rate (35% at 1 hr compared to 30% at 1 hr for MAb 806) and
displayed
comparable in vivo targeting when using 3T3 fibroblasts transfe,cted with de2-
7 EGFR
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(peak of 24% 11D/g tumor at 24 hr compared to 18% ID/g tumor at 8 hr for MAb
806).25
Perhaps the most important advantage of MAb 806 compared to current EGFR
antibodies, is that MAb 806 can be directly conjugated to cytotoxic agents.
This
approach is not feasible with current EGFR specific antibodies as they target
the liver
and cytotoxic conjugation would almost certainly induce severe toxicity.
Conjugation
of cytotoxic agents such as drugs 41 or radioisotopes 42 to antibodies has the
potential
to improve efficacy and reduce the systemic toxicity of these agents. The
ability of a
conjugated antibody to mediate tumor kill is dependent upon its potential to
be
internalized. Thus, the rapid internalization observed with MAb 806
intI87MG.A2-7
cells, suggests MAb 806 is an ideal candidate for this type of approach.
MAb 806 is novel in that it is the first de2-7 EGFR specific antibody directed
to an
epitope not associated with the unique junctional peptide. It has superior
affinity and
better tumor targeting properties than DH8.3, a previously described de2-7
EGIR
antibody. An important property, however, is its ability to recognize a subset
of EGFR.
molecules expressed on the surface of tumor cells exhibiting amplification of
the
EGFR gene. This suggests that MAb 806 may possess a unique clinical property;
the
ability to target both de2-7 and amplified EGFR but not wild type receptors.
If proven
correct, this antibody would not target organs such as liver and therefore
would be
more versatile than current antibodies directed to the EGFR," which cannot be
used
for the coupling of cytotoxic agents. Finally, MAb 806 may be a useful reagent
for
analyzing the conformational changes induced by the truncation found in de2-7
EGFR. =
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19. Faillot T, Magdelenat H, Mady E, et al. A phase I study of an anti-
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20. Ponten J, Macintyre EH. Long term culture of normal and neoplastic human
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22. Domagala T, Konstantopoulos N, Smyth F, et al. Stoichiometry, kinetic and
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23. Lindmo T, Boven E, Cuttitta F, et al. Determination of the irnmunoreactive
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infinite antigen excess. J Immunol Methods 1984;72:77-89.
24. Huang HS, Nagane M, Klingbeil CK, et al. The enhanced tumorigenic activity
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25. Reist CJ, Archer GE, Wilcstrand CJ, et al. Improved targeting of an anti-
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28. Filmus J, Pollak MN, Cailleau R, et al. MDA-468, a human breast cancer
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32. Sturgis EM, Sacks PG, Masui H, et al. Effects of antiepidennal growth
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42. DeNardo SJ, Kroger LA, DeNardo GL A new era for radiolabeled antibodies in
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EXAMPLE 19
GROWTH SUPPRESSION OF INTRACRANUL XENOGRAFTED
GLIOBLASTOMAS OVEREXPRESSING MUTANT EPIDERMAL GROWTH
FACTOR RECEYTORS BY SYSTEMIC ADMINISTRATION OF
MONOCLONAL ANTIBODY (mAb) 806, A NOVEL MONOCLONAL
ANTIBODY DIRECTED TO THE RECEPTOR
This example presents the evaluation of mAb 806 on the growth of intracranial
xenografted gnomes in nude mice. The following corresponds to and was
presented
in Mishima et al, (2001) Cancer Research, 61:5349-5354.
The data and finding of Mishima et al. are set
forth below.
Systemic treatment with mAb 806 significantly reduced the volume of tumors and
increased the survival of mice bearing xenografts of U87 Madl3GFR, LN-
Z308.dEGFR, or A1207 gliomas, each of which expresses high levels of AEGFR. In
contrast, atAb 806 treatment was ineffective with mice bearing the parental
U87 MG
tumors, which expressed low levels of endogenous wild-type ECiFR, or U87 MG.DK
tumors, which expressed high levels of Icinase-deficient AEGFR. A.slight
increase of
survival of mice xenografted with a wild-type EGFR-overex-pressing
U87 MG glioma (U87 MG.wtEGFR) was effected by mAb 806 concordant with its
weak cross-reactivity with such cells. Treatment of U87 MG.dEGFR tannors in
mice
with mikb 806 caused decreases in both tumor growth and angiogenesis, as well
as
increased apoptosis. Mechanistically, in vivo mAb 806 treatment resulted in
reduced
phosphorylation of the constitutively active AEGFR and caused down-regulated
expression of the apoptotic protector, Bc1-XL . These data provide preclinical
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evidence that mAb 806 treatment may be a useful biotherapeutic agent for those
aggressive gliomas that express AEGFR.
The present example demonstrates that systemic treatment with the novel AEGFR-
specific mAb, rnAb 806, causes reduced phosphorylation of the constitutively
active
AEGFR and thereby suppresses growth of intracranially implanted gliomas
overexpressing this mutant receptor in nude mice and extends their survival.
The
inhibition of tumor growth was mediated by a decrease in proliferation and
angiogenesis and increased apoptosis of the tumor cells. This suppression
affected
active signaling by AEGFR because intracranial xenografts that were derived
from
cells overexpressing kinase-deficient AFGFR (DK), which are recognized equally
well by mAb 806, were not significantly suppressed after the same therapy.
MA1ERIALS AND METHODS
Cell Lines. Because primary explants of human glioblastomas rapidly lose
expression
of amplified, rearranged receptors in culture, no existing glioblastoma cell
lines
exhibit such expression. To force maintenance of expression levels comparable
with
those seen in human tumors, U87 MG, LN-Z308, and A1207 (gift from Dr. S.
Aaronson, Mount Sinai Medical Center, New York, NY) cells were infected with
AFGER, kinase-deficient AEGFR (DK), or wtEGFR viruses which also conferred
resistance to G4I8 as described previously (21). Populations expressing
similar levels
of the various EGFR alleles (these expression levels correspond approximately
to an
amplification level of 25 gene copies; human glioblastomas typically have
amplification levels from 10 to 50 gene copies of the truncated receptor) were
selected by FAGS as described previously (21) and designated as U87 MG.AEGFR,
U87 MG.DK, U87 MG.wtEGFR, LN-Z308.AEGFR, LN-Z308.DK, LN-
Z308.wtEGFR, A1207.AEGFR, A1207.DK, and A1207.wtEGFR, respectively. Each
was maintained in medium containing G418 (U87 MG cell lines, 400 mg/ml; LN-
Z308 and A1207 cell lines, 800 mg/m1). naAbs. mAb 806 (IgG2b, IC), a AEGFR
specific mAb, was produced after immunization of mice with NR6 mouse
fibroblasts
expressing the AEGFR. It was selected from several clones because
hemagglutination
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assays showed that it had a high reactivity against NR6.AEGFR cells, low
reactivity
for NR6.1.vtEGFR cells, and none for NR6 cells.
Immunoprecipitation and Western Blot Analysis. Cells were lysed with lysis
buffer containing 50 mM HEPES (pH 7.5), 150 mM NaC1, 10% glycerol, 1% Triton
X-100, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM sodium PP; ,1
mM phenylmethlsulfonyl fluoride, 2 mM Na3 v04 ,5 ng/m1 leupeptin, and 5 ng /m1
aprotinin. Antibodies were incubated with cell lysates at 4 C for 1 h before
the
addition of protein-A and -G Sepharose. Immuno-precipitates were washed twice
with
lysis buffer and once with HNTG buffer [50 inM BEPES (pH 7.5), 150 mM NaC1,
0.1% Triton X-100, and 10% glycerol], electrophoresed, and transferred to
nitrocellulose membranes. Blots were probed with the anti-EGFR antibody, C13,
and
proteins were visualized using the ECL chemilmnineseent detection system
(Aniersham Pharmacia Biotech.). The mAbs used for precipitation were mAb 806,
anti-EGFR mAb clone 528 (Oncogene Research Products, Boston, MA), or clone
EGFR.1 (Oncogene Research Products). A mAb, C13, used for detection of both
wild-type and AEGFR on immunoblots was provided by Dr. G. N. Gill (University
of
California, San Diego, CA). Antibodies to Bcl-X (rabbit poly-clonal antibody;
Transduction Laboratories, Lexington, KY) and phosphotyrosine (4010, Upstate
Biotechnology, Lake Placid, NY) were used for Western blot analysis as
described
previously (26).
Flow Cytometry Analysis. Cells were labeled with the relevant antibody
followed by
fluorescein-conjugated goat antimouse IgG (1:100 dilution; Becton-Dickinson
PharMingen, San Diego, CA) as described previously (21). Stained cells were
analyzed with a FACSCalibur using Cell Quest software (Becton-Dickinson
PharMingen). For the first antibody, the following mAbs were used: mAb 806,
anti-
EGFR mAb clone 528, and clone EGFR.I. Mouse IgG2a or IgG2b was used as an
isotype control.
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Tumor Therapy. U87 MG.AEGFR cells (1 X 105 ) or 5 X 105 IN-Z308.AEGFR,
A1207. AEGFR, U87 MG, U87 MG.DK, and U87 MG.wtEGFR cells in 5 ul of PBS
were implanted into the right corpus striatum of nude mice brains as described
previously (27). Systemic therapy with niAb 806, or the IgG2b isotype control,
was
accomplished by i.p. injection of 1 p.g of mAbs in a volume of 100 ul every
other day
from postimplantation day 0 through 14. For direct therapy of intracerebral
U87
MG.AEGFR tumors, 10 ug of mAb 806, or the IgG2b isotype control, in a volume
of
ul were injected at the tumor-injection site every other day starting at day 1
for 5
days.
Inununohistochemistry. To assess angiogenesis in tumors, they were fixed in a
solution containing zinc chloride, paraffin embedded, sectioned, and
immunostained
using a monoclonal rat antimouse CD31 antibody (Becton-Dickinson PharMingen;
1:200). Assessment of tumor cell proliferation was performed by Ki-67
immunohistochernistry on fomialin-fixed paraffin-embedded tumor tissues. After
deparaffinization and rehydration, the tissue sections were incubated with 3%
hydrogen peroxide in methanol to quench endogenous peroxidase. The sections
were
blocked for 30 min with goat serum and incubated overnight with the primary
antibody at 4 C. The sections were then washed with PBS and incubated with a
biotinylated secondary antibody for 30 min. After several washes with PBS,
products
were visualized using streptavidin horseradish peroxidase with
diaminobenzidine as
chromogen and hematoxylin as the counterstain. As a measure of proliferation,
the
Ki-67 labeling index was determined as the ratio of labeled:total nuclei in
high-power
(3400) fields. Approximately 2000 nuclei were counted in each case by
systematic
random sampling. For macrophage and NK cell staining, frozen sections, fixed
with
buffered 4% paraformaldehyde solution, were immunostained using bio-tinylated
rnAb F4/80 (Serotec, Raleigh, NC) and polyclonal rabbit antiasialo GM1
antibody
(Dako Chemicals, Richmond, VA), respectively. Angiogenesis was quantitated as
vessel area using computerized analysis. For this purpose, sections were
immunostained using anti-CD31 and were analyzed using a computerized image
analysis system without counterstain. MVAs were determined by capturing
digital
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CA 02826627 2013-09-04
images of the sections at 3200 magnification using a CCD color camera as
described
previously (27). Images were then analyzed using Image Pro Plus version 4.0
software (Media Cybernetics, Silver Spring, MID) and MVA was determined by
measuring the total amount of staining in each section. Four fields were
evaluated for
each slide. This value was represented as a percentage of the total area in
each field.
Results were confirmed in each experiment by at least two observers (K. M., H-
J. S.
H.).
TUNEL Assay. Apoptotic cells in tumor tissue were detected by using the TUNEL
method as described previously (27). TUNEL-positive cells were counted at
X400.
The apoptotic index was calculated as a ratio of apoptotic cell number:total
cell
number in each field.
Statistical Analysis. The data were analyzed for significance by Student's t
test,
except for the in vivo survival assays, which were analyzed by Wilcoxon
analysis.
=
RESULTS
Systemic Treatment of mAb 806 Extends the Survival of Mice Bearing AEGFR-
overexpressing Intracranial Glioma Tumors.
To test the efficacy of the anti-AEGFR mAb, mAb 806, we treated nude mice
bearing
intracranial AEGFR-overexpressing glioma xenografts with i.p. injections of
mAb
806, the isotype control IgG, or PBS. U87 MG.AEGFR cells were implanted
intracranially into nude mice, and the treatments began on the same day as
described
in "Materials and Methods."
Animals treated with PBS or isotype control IgG had a median survival of 13
days,
whereas mice treated with mAb 806 had a 61.5% increase in median survival up
to 21
days (P < 0.001;Figure 24A). Treatment of mice 3 days postimplantation, after
tumor
establishment, also extended the median survival of the mAb 806-treated
animals by
46.1% (from 13 days to 19 days; P < 0.01) compared with that of the control
groups
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(data not shown). To determine whether these antitumor effects of mAb 806
extended
beyond U87 MG.AEGFR xenografts, we also did similar treatments of animals
bearing other glioma cell xenografts of LN-Z308.AEGFR and A1207.AEGFR. The
median survival of mAb 806-treated mice bearing LN-Z308.AFGFR xenografts was
extended from 19 days for controls to 58 days (P < 0.001; Figure 24B).
Remarkably,
four of eight mAb 806-treated animals survived beyond 60 days (Figure 24B).
The
median survival of animals bearing A1207.A.EGFR xenografts was also extended
from 24 days for controls to 29 days (P < 0.01; data not shown).
inAb 806 Treatment Inhibits AEGFR-overexpressitjg Brain Tumor Growth.
Mice bearing U87 MG.AEGFR and LN-Z308. AEGFR xenografts were killed at day
9 and day 15, respectively. Tumor sections were histopathologically analyzed,
and
tumor volumes were determined as described in "Materials and Methods."
Consistent
with the results observed for animal survival, mAb 806 treatment significantly
reduced the volumes of U87 MG.AEGFR by 90% (P < 0.001; Figure 24C), and of
LN-Z308.AFGFR by .95% (P <0.001; Figure 24D), of xenografts in comparison
with those of the control groups. Similar results were obtained for animals
bearing
A1207.AEGFR tumors (65% volume reduction; P <0.01; data not shown).
Intrahunoral Treatment with mAb 806 Extends Survival of Mice Bearing U87
MG.AEGFR Brain Tumors.
We also determined the efficacy of direct intratumoral injection of mAb 806
for the
treatment of U87 MG.AEGFR xenografts. Animals were given intratumoral
injections
of mAb 806 or isotype control IgG at 1 day postimplantation, as described in
"Materials and Methods." Control animals survived for 15 clays, whereas mAb
806
treated mice remained alive for 18 days (P < 0.01; Figure 24E). Although the
intratumoral treatment with mAb 806 was somewhat effective, it entailed the
difficulties of multiple intracranial injections and of increased risk of
infection. We,
therefore, focused on systemic treatments for additional studies.
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inAb 806 Treatment Slightly Extends Survival of Mice Bearing U87
MG.wtEGFR but not of Mice Bearing U87 MG or U87 MG.DK Intracranial
Xenografts.
To determine whether the growth inhibition by mAb 806 was selective for tumors
expressing AEGFR, we treated animals bearing U87 MG, U87 MG.DK (kinase-
deficient AFGFR) or U87 MG.wtEGFR brain xenografts. mAb 806 treatment did not
extend the survival of mice implanted with U87 MG tumors (Figure 25A), which
expressed a low level of endogenous wtEGFR (22), or of animals bearing U87
MG.DK xenografts, which overexpressed a lcinase-deficient AEGFR in addition to
a
low level of endogenous wtEGFR (Figure 25B). The mAb 806 treatment slightly
extended the survival of mice bearing U87 MG.wtEGFR tumors (P < 0.05; median
survival, 23 days versus 26 days for the control groups), which overexpressed
wtEGFR (Figure 25C).
mAb 806 Reactivity Correlates with in Vivo Antitumor Efficacy.
To understand the differential effect of mAb 806 on tumors expressing various
levels
or different types of EGFR, we determined inAb 806 reactivity with various
tumor
cells by FACS analysis. Consistent with previous reports (21), the anti-EGFR
mAb
528 recognized both AEGFR and wtEGFR and demonstrated stronger staining for
U87 MG.AEGFR cells compared with U87 MG cells (Figure 26A, 528). In contrast,
antibody EGFR.1 reacted with wtEGFR but not with AFGFR (21), because U87
MG.AEGFR cells were as weakly reactive as U87 MG cells (Flgure 26A, panel
EGFR 1). This EGFR.1 antibody reacted with U87 MG.wtEGFR more intensively
than with U87 MG cells, because U87 MG.wtEGFR cells overexpressed wtEGFR
(Figure 26A, panel EGFR.1). Although mAb 806 reacted intensely.with U87
MG.A.EGFR and U87 MG.DK cells and not with U87 MG cells, it reacted weakly
with U87 MG.wtEGFR, which indicated that mAb 806 is selective for AEGFR with a
weak cross-activity to overexpressed wtEGFR (Figure 26A, panel mAb 806). This
level of reactivity with U87 MG.wtEGFR was quantitatively and qualitatively
similar
to the extension of survival mediated by the antibody treatment (Figure 25C)
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We further determined mAb 806 specificity by immunoprecipitation. EGFRs in
various cell lines were immunoprecipitated with antibody 528, EGFR.1, and mAb
806. Blots of electrophoretically separated proteins were then probed with the
anti-
EGFR antibody, C13, which recognizes wtEGFR as well as AFGFR and DK (22).
Consistent with the FACS analysis, antibody 528 recognized wtEGFR and mutant
receptors (Figure 26B-panel IP: 528), whereas antibody EGFR.1 reacted with
wtEGFR but not with the mutant species (Figure 26B, panel IP: EGF1'.1).
Moreover,
the levels of mutant receptors in U87 MG.AEGFR and U87 MG.DK cells are
comparable with those of wtEGFR in the U87 MG.wtEGER cells (Figure 26B, panel
IP: 528).
However, antibody rnAb 806 was able to precipitate only a small amount of the
wtEGFR from the U87 MG.wtEGFR cell lysates as compared with the larger amount
of mutant receptor precipitated from U87 MG.AEGFR and U87 MG.DK cells, and an
undetectable amount from the U87 MG cells (Figure 26B, panel IP: mAb 806).
Collectively, these data suggest that mAb 806 recognizes an epitope in AEGFR
that
also exists in a small fraction of wtEGFR only when it is overexpressed on the
cell
surface.
mAb 806 Treatment Reduces AEGFR Autophosphorylation and Down-Regulates
BcI-XL Expression in U87 MG.AEGFR Brain Tuniors.
The mechanisms underlying the growth inhibition by mAb 806 were next
investigated. Because the constitutively active kinase activity and
autophosphorylation of the COOH terminus of AEGFR are essential for its
biological
functions (21, 22, 28, 29), AEGFR phosphorylation status was determined in
tumors
from treated and control animals. As shown in Figure 27A, mAb 806 treatment
dramatically reduced AEGFR autophosphorylation, although receptor levels were
only slightly decreased in the mAb 806-treated xenografts. We have previously
shown
that receptor autophosphorylation causes up-regulation of the antiapoptotic
gene, Bcl-
X L, which plays a key role in reducing apoptosis of AEGFR -overexpressing
tumors
(28, 29). Therefore, the effect of mAb 806 treatment on Bel-X ',expression was
next
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CA 02826627 2013-09-04
determined. AEGFR tumors from mAb 806-treated animals did indeed show reduced
levels of Bc1-)CL (Figure 27A).
inAh 806 Treatment Decreases Growth and Angiogenesis and Increases
Apoptosis in U87 MG.AEGFR Tumors.
In light of the in vivo suppression caused by mAb 806 treatment and its
biochemical
effects on receptor signaling, we determined the proliferation rate of tumors
from
control or treated mice. The proliferative index, measured by I(i-67.staining
of the
mAb 806-treated tumors, was significantly lower than that of the control
tumors (P <
0.001; Figure 28). In addition, analysis of the apoptotic index through TUNEL
staining demonstrated a significant increase in the number of apoptotic cells
in mAb
806-treated tumors as compared with the control tumors (P < 0.001; Figure 28).
The
extent of tumor vascularization was also analyzed by immunostaining of tumors
from
treated and control specimens for CD31. To quantify tumor vascularization,
MVAs
were measured using computerized image analysis. mAb 806-treated tumors showed
30% less MVA than did control tumors (P <0.001; Figure 28). To understand
whether interaction between receptor and antibody may elicit an inflammatory
response, we stained tumor sections for the macrophage marker, F4/80, and the
NK
cell marker, asialo GM1. Macrophages were identified throughout the tumor
matrix
and especially accumulated around the mAb 806-treated-U87 MG. AEGFR ¨tumor
periphery (Figure 28). We observed few NK cells infiltrated in and-around the
tumors
and no significant difference between mAb 806-treated and isotype-control
tumors
(data not shown).
DISCUSSION
AEGFR appears to be an attractive potential therapeutic target for cancer
treatment of
gliomas. It is correlated with poor prognosis (25), whereas its genetic or
pharmacological inhibition effectively suppresses growth of AEGFR -
overexpressing
cells both in vitro and in vivo (29, 30). Because this mutant EGFR is
expressed on the
cell surface, it represents a potential target for antibody-based therapy,
and, here, we
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tested the efficacy of a novel anti- AEGFR mAb, mAb 806, on the treatment of
intracranial xenografts of AEGFR ¨overexpressing gliomas of different cellular
backgrounds in nude mice. The systemic administration of mAb 806 inllibite,cl
tumor
growth and extended animal survival. The effect of mAb 806 was evident for
each
cell line and was independent of the p53 status of the tumors, because U87 MG.
APGFR and A1207. AEGFR expressed wild-type p53, whereas LN-Z308. AEGFR
was p53-null.
The enhanced turnorigenicity of AEGFR is mediated through its constitutively
active
kinase activity and tyrosine autophosphorylation at the COOH terminus (22, 28,
29).
Phosphorylation of AEGFR in mAb 806-treated tumors was significantly
decreased,
= proliferation was reduced, and apoptosis was elevated, which suggests
that the
antitumor effect of mAb 806 is, at least in part, attributable to the
inhibition of the
intrinsic function of the receptor. The AEGFR signaling caused up-regulation
of the
antiapoptotic gene, Bcl-X L (28), and treatment with mAb 806 resulted in down-
regulation of Bcl-X L expression, which further suggests that the antitumor
effect of
mAb 806 is mediated through the inhibition of AEGFR signaling. The level of
AEGFR in the mAb 806-treated tumors was also slightly reduced (Figure 27A),
but
not to a degree that was consistent with the degree of dephosphorylation of
the mutant
receptor or sufficient to explain the magnitude of its biological effect. The
antitumor
effect of mAb 806 is likely to result, at least in part, from the inhibition
of the intrinsic
signaling function of AEGFR. This assertion is also supported by the lack of
antitumor effects on DK tumors, which bind to the antibody but are kinase
deficient.
Intratumoral injection of a different anti- AEGFR antibody, mAbY10, inhibited
the
growth of AEGFR -expressing B16 melanoma tumors in mouse brains through a
Fc/Fc receptor-dependent mechanism (31). In conjunction with this, mAbY10 was
shown to mediate antibody-dependent macrophage cytotoxicity in vitro with both
murine and human effector cells (17), although it had little effect with macro-
phage
infiltration found in our niAb 806-treated tumors raises the question as to
whether the
antitumor effect of mAb 806 may be accomplished by macrophage-mediated
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cytotoxicity. We believe this to be unlikely, because macrophage infiltration
also
occurred on mAb 806 treatment of U87 MG.DK (kinase-deficient AEGFR) tumors, in
which it was ineffective in regulating tumor growth.
mAb 806 appears to be selective for AEGFR with a weak cross-reactivity with
overexpressed wtEGFR. Consistent with the in vitro specificity, mAb 806
treatment
was very effective in AEGFR -over-expressed tumors, whereas it showed a much
less
robust, but reproducible, growth inhibition for tumors overexpressing wtEGFR.
However, the simple interaction between mAb 806 and its target molecules is
insufficient to inhibit tumor growth because, although mAb 806 is capable of
binding
equally well to kinase-deficient AEGFR (DK) receptors and AEGFR, it is
ineffective
in affecting DK-expressing tumor growth. The inability of mAb 806 to interact
with
the low-level of wtEGFR normally present in cells suggests a large therapeutic
window for AEGFR -overexpressed as well as, to a lesser extent, wtEGFR-
overexpressed cancers when compared with normal tissues.
Although the mAb 806 treatment was effective for suppression of intracranial
xenografts, it should be noted that the AEGFR-tumors eventually grew, and
durable
remissions were not achieved. This may have resulted from inefficient
distribution of
antibody in the tumor mass. mAbs in combination with other therapeutic
modalities
such as toxins, isotopes or drugs, for cancer treatments have been shown to be
more
effective than antibody alone in many cases (2, 3, 32-34). Chemotherapeutic
drugs
such as doxorubicin and cisplatin in conjunction with wtEGFR antibodies have
also
shown enhanced antitumor activity (35, 36). Combination treatments targeted at
tumor growth as well as angiogenic development have more effectively inhibited
glioblastoma growth than either treatment alone (27). This raises the
possibility that
mAb 806 in combination with chemotherapeutic drugs or compounds modulating
angiogenesis may be even more effective than mAb 806 alone.
=
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and Huang, H. J. A mutant epidermal growth factor receptor conunon in human
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(Baltimore), 45: 1442-1453,1999.
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KAIVATLE 20
MONOCLONAL ANTIBODY 806 INHIBITS THE GROWTH OF TUMOR
rE'NOGRAFTS EXPRESSING EITITER THE DE2-7 OR AMPLIFIED
EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) JITUT NOT WILD-
TYPE EGFR
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The following example presents findings by the present inventors that is also
set forth
in Luwor et al., (2001) Cancer Research, 61:5355-5361. The disclosure of this
publication is incmporated herein in its entirety with cross referencing to
the Figures
herein where approptiate.and made apart hereof.
The monoclonal antibody (mAb) 806 was raised against the delta2-7 epidermal
growth factor receptor (de2-7 EGFR or EGFRAE), a truncated version of the EGFR
commonly expressed in gliorna. Unexpectedly, mAb 806 also bound the EGFR
expressed by cells exhibiting amplification of the EGFR gene but not to cells
or
normal tissue expressing the wild-type receptor in the absence of gene
amplification.
The unique specificity of mAb 806 offers an advantage over current EGFR
antibodies, which all display significant binding to the liver and skin in
humans.
Therefore, we examined the antitumor activity of mAb 806 against human rumor
xe-
nogafts grown in nude mice. The growth of U87 MG xenografts, a glioma cell
line
that endogenously expresses ¨105 EGFRs in the absence of gene amplification,
was
not inhibited by mAb 806. In contrast, mAb 806 significantly inhibited the
growth of
U87 MG xenogafts transfected with the de.2-7 EGFR. in a dose-dependent manner
using both preventative and established tumor models. Significantly, U87 MG
cells
transfected With the wild-type EGFR, which increased expression to
¨106EGFRs/cell
and mimics the situation of gene amplification, were also inhibited by mAb 806
when
grown as xenogafts in nurics371iC0. Xenografts treated with mAb 806 all
displayed
large areas of necrosis that were absent in control tumors. This reduced
xenograft
viability was not mediated by receptor down-regulation or clonal selection
because
levels of antigen expression were similar in control and treated groups. The
antitumor
effect Of mAb 806 was not resnicted to U87 MG cells because the antibody
inhibited
the growth of new and established A431 xenografts, a cell line expressing >106
EGFRs/cell. This study demonstrates that mAb 806 possesses significant
antitumor
activity.
The de2-7 EGFR specific mAb 806 was produced after immunization of mice with
NR6 mouse fibroblasts expressing the truncated de2-7 EGFR. mAb 806 binds the
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U87 MG glioma cell line transfected with the de2-7 EGFR but not the parental
U87
MG cell line, which expresses the wt EGFR without gene amplification.3 Similar
results were observed in vivo with mAb 806 showing specific targeting of de2-7
EGFR expressing U87 MG xenografts but not parental U87 MG tumors.3
Interestingly, mAb 806 was capable of binding an EGFR subset (-10%) on the
surface of the A431 cell line, which contains an amplified EGFR gene.
Therefore,
unlike all other de2-7 EGFR-specific antibodies, which recognize the unique
peptide
junction that is generated by the de2-7 EGFR truncation, mAb 806 binds to an
epitope also found in overexpressed wt EGFR. However, it would appear that
this
epitope is preferentially exposed in the de2-7 EGFR and a small proportion of
receptors expressed in cells containing wt EGFR gene amplification.
Importantly,
normal tissues that expresses high levels of endogenous wt EFGR, such as liver
and
skin, show no significant mAb 806 binding. On the basis of the unique property
of the
mAb 806 to bind both the de2-7 and amplified wt EGFR but not the native wt
EGFR
when expressed at normal levels, we decided to examine the efficacy of inAb
806
against several tumor cell lines grown as xenografts in nude mice.
MATERIALS AND METHODS
Cell Lines and Monoclonal Antibodies. The human glioblastoma cell line U87 MG,
which endogenously expresses the wt EGFR, and the transfected cell lines U87
=
MG.A2-7 and U87 MG.wtEGFR, which express the de2-7 EGFR and overexpress
the wt EGFR, respectively, have been described previously (16,23). The
epidermoid
carcinoma cell line A431 has been described previously (24).
All cell lines were maintained in DMEM (DMEM/F12; Life Technologies, Inc.,
Grand Island, NY) containing 10% FCS (CSL, Melbourne, Victoria, Australia), 2
mlvf
glutamine (Sigma Chemical Co., St. Louis, MO), and peni-cillin/ streptomycin
(Life
Technologies, Inc., Grand Island, NY). In addition, the U87 MG.D2-7 and U87
MG.wtEGFR cell lines were maintained in 400 mg/ml of geneticin (Life
Technologies, Inc., Melbourne, Victoria, Australia). Cell lines were grown at
37 C in
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a umidified atmosphere of 5% CO2. The InAb 806 (IgG2b) was produced after
immunization of mice with NR6 mouse fibroblasts expressing the de2-7 EGFR. mAb
806 was selected after rosette assays showed binding to NR6 cells, which
overexpressed the de2-7 EGFR (titer of 1:2500). mAb 528, which recognizes both
de2-7 and wt EGFR, has been described previously (10) and was produced in the
Biological Production Facility (Ludwig Institute for Cancer Research,
Melboume,
Victoria, Australia) using a hybridoma obtained from American Type Culture
Collection (Rockville, MD). The DH8.3 mAb, which is specific for the de2-7
EGFR,
was kindly provided by Prof. William Gullick (University of Kent and
Canterbury,
Kent, United Kingdom) (19). The polyclonal antibody sc-03 directed to the COOH-
terminal domain of the EGFR was purchased from Santa Cruz Bio-technology
(Santa
Cruz Biotechnology, Santa Cruz, CA).
FACS Analysis of Receptor Expression. Cultured parental and trans-fected U87
MG cell lines were analyzed for wt and de2-7 EGFR expression using the 528,
806,
and DH8.3 antibodies. Cells (1 3 10 6) were incubated with 5 rngiml of the
appropriate antibody or an isotype-matched negative control in PBS containing
1%
HSA for 30 min at 4 C. After three washes with PBS/1% HSA, cells were
incubated
an additional 30 min at 4 C with H1C-coupled goat antirnouse antibody (1:100
dilution; Calbiochem, San Diego, CA). After three subsequent washes, cells
were
analyzed on an Epics Elite ESP (Beckman Coulter, Hialeah, FL) by observing a
minimum of 20,000 events and analyzed using EXPO (version 2) for Windows.
Scatchard Analysis. The mAb 806 was labeled with 125 I (Amrad, Mel-bourne,
Victoria, Australia) by the Chloramine T method. All binding assays were
performed
in 1% HSA/PBS on 1-2 X 106 live U87 MG.A2-7 or A431 cells for 90 min at 4 C
with gentle rotation. A set concentration of 10 ng/m1 125 I-labeled mAb 806
was used
in the presence of increasing concentrations of unlabeled antibody.
Nonspecific
binding was determined in the presence of 10,000-fold excess of unlabeled
antibody.
After incubation, cells were washed and counted for bound 125 I-labeled mAb
806
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using a COBRA II gamma counter (Packard Instrument Company, Meriden, CT).
Scatchard analysis was done after correction for immunoreactivity.
Immunoprecipitation Studies. Cells were labeled for 16 h with 100 mCi/m1 of
Tran
35 S-Label (ICN Biomedicals, Irvine, CA) in DMEM without methionine/cysteine
supplemented with 5% dialyzed FCS. After washing with PBS, cells were placed
in
lysis buffer (1% Triton X-100, 30 rtiM HEPES, 150 mM NaCI, 500 mM 4-(2-
aminoethyl) benzenesulfonylfluoride, 150 nM aprotinin, 1 mM E-64 protease
inhibitor, 0.5 mM EDTA, and 1 mM leupeptin, pH 7.4) for 1 h at4 C. Lysates
were
clarified by centrifugation for 10 min at 12, 000 3 g and then incubated with
5 mg of
appropriate antibody for 30 min at 4 C before the addition of protein A-
Sepharose.
Immunoprecipitates were washed three times with lysis buffer, mixed with SDS
sample buffer, separated by gel electrophoresis using a 7.5% gel that was then
dried,
and exposed to X-ray Elm.
Xenograft Models. Consistent with previous reports (23, 25), U87 MG cells
transfected with de2-7 EGFR grew more rapidly then parental cells and U87 MG
cells transfected with the wt EGFR. Tumor cells (3 X 106 )in 100m1 of PBS were
inoculated s.c. into both flanks of 4-6-week-old, female nude naice (Animal
Research
Center, Western Australia, Perth, Australia). Therapeutic efficacy of mAb 806
was
investigated in both preventative and established tumor models. In the
preventative
model, five mice with two xenografts each were treated i.p. with either 0.1 or
1 mg of
mAb 806 or vehicle (PBS) starting the day before tumor cell inoculation.
Treatment
was continued for a total of six doses, three times per week for 2 weeks. In
the
established model, treatment was started when tumors had reached a mean volume
of
65 mm 3 (U87 MG.A2-7), 84 mm 3 (U87 MG), 73 mm 3 (U87 MG.wtEGFR), or 201
min 3 (A431 tumors). Tumor volume in mm 3 was determined using the formula
(length 3 width 2 )/2, where length was the longest axis and width the
measurement
at right angles to the length (26). Data were expressed as mean tumor volume 6
SE fo
each treatment group. This research project was approved by the Animal Ethics
Committee of the Austin and Repatriation Medical Centre.
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Ifistological Examination of Tumor Xenografts. Xenografts were excised at the
times indicated and bisected. One half was fixed in 10% formalin/PBS before
being
embedded in paraffin. Four- mra sections were then cut and stained with ME for
routine histological examination. The other half was embedded in Tissue Tek
OCT
compound (Salcura Finetek, Torrance, CA), frozen in liquid nitrogen, and
stored at
280 C. Thin (5- mm) cryostat sections
were cut and fixed in ice-cold acetone for 10 min, followed by air drying for
an
additional 10 min. Sections were blocked in protein blocking reagent (Lipshaw
Immunon, Pittsburgh, PA) for 10 min and then incubated with biotinylated
primary
antibody (1 mg/m1) for 30 min at room temperature. All antibodies were
biotinylated
= using the ECL protein biotinyla.tion module (Amersham, BaulkharnIls, NSW,
Australia), as per the manufacturer's instructions. After rinsing with PBS,
sections
were incubated with a streptavidin- horseradish peroxidase complex for an
additional
30 min (Silenus, Melbourne, Victoria, Australia). After a final PBS wash, the
sections
were exposed to 3-amino-9-ethylcarbazole substrate [0.1 M acetic acid, 0.1 M
sodium
acetate, 0.02 M 3-amino-9-ethylcarbazole (Sigma Chemical Co., St. Louis, MO)]
in
the presence of hydrogen peroxide for 30 min. Sections were rinsed. with water
and
counterstained with hematoxylin for 5 min and mounted.
Statistical Analysis. The in vivo tumor measurements in mm 3 are ex-pressed as
the..
mean 6 SE. Differences between treatment groups at given time points were
tested for
statistical significance using Student's (test.
RESULTS
Binding of Antibodies to Cell Lines. To determine the specificity of mAb 806,
its
binding to U87 MG, U87 MGD2-7, and U87 MG.wtEGFR cells was analyzed by
PACS. An irrelevant IgG2b (mAb 100.-310 directed to the human antigen A33) was
= included as an isotype control for mAb 806, and the 528 antibody was
included
because it recognizes both the do2-7 and wt EGFR. Only the 528 antibody was
able
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to stain the parental U87 MG cell line (Figure 29), consistent with previous
reports
demonstrating that these cells express the wt EGFR (16). mAb 806 had binding
levels
similar to the control antibody, clearly demonstrating that it is unable to
bind the wt
EGFR (Figure 29). Binding of the isotype control antibody to the U87 MG.D2-7
and
U87 MG.wtEGFR cell lines was similar to that observed for the U87 MG cells.
mAb
806 stained U87 MG.D2-7 and U87 MG. wtEGFR cells, indicating that mAb 806
specifically recognized the de2-7 EGFR and a subset of the overexpressed EGFR
(Figure 29). As expected, the 528 antibody stained both the U87 MG.D2-7 and
U87
MG.wtEGFR cell lines (Figure 29). The intensity of 528 antibody staining on
U87
MG.wtEGFR cells was much higher than mAb 806, suggesting that mAb 806 only
recognizes a portion of the overexpressed EGFR. The mAb 806 reactivity
observed
with U87 MG.wtEGFR cells is similar to that obtained with A431 cells, another
cell
line that over expresses the wt EGFR.3
A Seatchard analysis was performed using U87 MG.D2-7 and A431 cells to
determine the relative affinity and binding sites for mAb 806 on each cell
line. mAb
806 had an affinity for the de2-7 EGFR receptor of 1.1 X 109 /,,{1 and
recognized an
average (three separate experiments) of 2.4 X 105 binding sites/cell. In
contrast, the
affinity of mAb 806 for the wt EGFR on A431 cells was only 9.5 X 107M-1.
Interestingly, mAb 806 recognized 2.3 X 105 binding sites on the surface of
A431,
which is some 10-fold lower than the reported number of EGFR found in these
cells.
To confirm the number of Bail{ on the surface of our A431 cells, we performed
a
Scatchard analysis using 125I-1abeled 528 antibody. As expected, this antibody
bound
to approximately 2 X 106 sites on the surface of A431 cells. Thus, it appears
that mAb
806 only binds a portion of the EGFR receptors on the surface of A431 cells.
Importantly, 1251-labeled mAb 806 did not bind to the parental U87 MG cells at
all,
even when the number of cells was increased to 1 X 107.
Inununoprecipitations. We further characterized mAb 806 reactivity in the
various
cell lines by immunoprecipitation after 35 S-Iabeling using rnAb 806, sc-03 (a
commercial polyclonal antibody specific for the COOH-terminal domain of the
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EGFR) and a IgG2b isotype control. The sc-03 antibody irnmunoprecipitated
three
bands from U87 MG.A2-7 cells, a doublet corresponding to the two de2-7 EGFR
bands observed in these cells and a higher molecular weight band corresponding
to
the wt EGFR (Figure 30). In contrast, although mAb 806 immunoprecipitated the
two
de2-7 EGFR bands, the wt EGFR was completely absent (Figure 30). The pattern
seen in U871\,{G.wtEGER and A431 cells was essentially identical. The sc-03
antibody irnmunoprecipitated a single band corresponding to the wt EGFR from
both
cell lines (Figure 30). The mAb 806 also immunoprecipitated a single band
corresponding to the wt EGFR from both U87 MG.wtEGFR and A431 cells (Figure
30). Consistent with the FACS and Scatchard data, the amount of EGFR
immunoprecipitated by mAb 806 was substantially less than the total EGFR
present
on the cell surface. Given that mAb 806 and the sc-03 immunoprecipitated
similar
=
amounts of the de2-7 EGFR, this result supports the notion that the mAb 806
antibody only recognizes a portion of the EGFR in cells overexpressing the
receptor.
Comparisons between mAb 806 and the 528 antibody showed an identical pattern
of
reactivity (data not shown). An irrelevant IgG2b (an isotype control for mAb
806) did
not imtnunoprecipitate EGFR from any of the cell lines (Figure 30). Using
identical
conditions, mAb 806 did not immunoprecipitate the EGFR from the parental 1J87
MG
cells (data not shown).
Efficacy of mAb 806 in Preventative Models. mAb 806 was examined for efficacy
against U87 MG and U87 MG.A2-7 tumors in a preventative xenograft model.
Antibody or vehicle was administered i.p. the day before tumor inoculation and
was
given three times per week for 2 weeks (see "Materials and Methods"). At a
dose of 1
mg/injection, mAb 806 had no effect on the growth of parental U87 MG
xenografts
that express the wt EGFR (Figure 9A). In contrast, mAb 806 inhibited
significantly
the growth of U87 xenografts in a dose-dependent manner (Figure
9B).
Twenty days after tumor inocu-lation, when control animals were sacrificed,
the mean
tumor volume was 1600 180 mm3for the control group, a significantly smaller
500
95 mm3 for the 0.1 mg/injection group (P < 0.0001) and 200 42 mm3 for the 1
mg/injection group (P< 0.0001). Treatment groups were sacrificed at day 24, at
which
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time the mean tumor volumes were 1300 240 mm 3 for the 0.1 mg treated group
and 500 100 mm3 for the 1 mg group (P <0.005).
Efficacy of mAb 806 in Established Xenograft Models. Given the efficacy of mAb
806 in the preventative xenograft model, its ability to inhibit the growth of
established
tumor xenografts was examined. Antibody treatment was as described in the
preventative model, except that it commenced when tumors had reached a mean
tumor volume of 65 mm3 (10 days after implantation) for the U87 MG.A2-7
xenografts and 84 mm3 (19 days after implantation) for the parental U87 MG
xenografts. Once again, mAb 806 had no effect on the growth of parental U87 MG
xenografts, even at a dose of 1 mg/injection (Figure 10A). In contrast, mAb
806
significantly inhibited the growth of U87 MG.A2-7 xenografts in a dose-
dependent
manner (Figure 10B). At day 17, 1 day before control animals were sacrificed,
the
mean tumor volume was 900 200 mm3 for the control group, 400 60 mm3 for
the
0.1 mg/injection group (P < 0.01), and 220 60 mm3 for the 1 mg/injection
group (P
<0.002). Treatment of U87 MG.A2-7 xenografts with an IgG2b isotype control had
no effect on tumor growth (data not shown).
To examine whether the growth inhibition observed with mAb 806 Was restricted
to
cells expressing de2-7 EGER, its efficacy against the U87 MG.wtEGFR xenografts
was also examined in an established model. These cells serve as a model for
tumors
containing amplifi-cation of the EGFR gene without de2-7 EGFR expression. mAb
806 treatment commenced when tumors had reached a mean tumor volume of 73 min
3 (22 days after implantation). mAb 806 significantly inhibited the growth of
established U87 MG.wtEGFR xenografts when compared with control tumors treated
with vehicle (Figure 10C). On the day control animals were sacrificed, the
mean
tumor volume was 1000 300 mm3 for the control group and 500 - 80 mm3 for
the
group treated with 1 mg/injection (P <0.04).
Histological and Immunohistochemical Analysis of Established
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Tumors. To evaluate potential histological differences between mAb 806-treated
and
control U87 MG.A2-7 and U87 MG.wtEGFR xenografts, formalin-fixed, paraffin-
embedded sections were stained with H&E (Figure 31). Areas of necrosis were
seen
in sections from mAb 806-treated U87 MG.A2-7 (mAb 806-treated xenografts were
collected 24 days after tumor inoculation and vehicle treated xenografts at 18
days),
and U87 MG.wtEGFR xenografts (mAb 806 xenografts were collected 42 days after
tumor inoculation and vehicle treated
xenografts at 37 days; Figure 31). This result was consistently observed in a
number
of tumor xenografts (n 5 4 for each cell line). However, sections from U87
MG.A2-7
and U87 MG.wtEGFR xenografts treated with vehicle (n 5 5) did not display the
same
areas of necrosis seen after mAb 806 treatment (Figure 31). Vehicle and mAb
806-
treated xenografts removed at identical times also showed these differences in
tumor
necrosis (data not shown). Thus, the increase in necrosis observed was not
caused by
the longer growth periods used for the mAb 806-treated xenografts.
Furthermore,
sections from inAb 806-treated U87 MG xenografts were also stained with H&F,
and
did not reveal any areas of necrosis (data not shown), further supporting the
hypothesis that mAb 806 binding induces decreased cell viability, resulting in
increased necrosis within tumor xenografts.
An immunohistochemical analysis of U87 MG, U87 MG.A2-7, and U87
MG.wtEGFR xenograft sections was performed to determine the levels of de2-7
and
= wt EGFR expression after mAb 806 treatment (Figure 32). As expected, the
528
antibody stained all xenografts sections with no obvious decrease in intensity
between
treated and control tumors (Figure 32). Staining of U87 MG sections was
undetectable with the mAb 806; however, positive staining of U87 MG.A2-7 and
U87
MG.wtEGFR xenograft sections was observed (Figure 32). There was no difference
in mAb 806 staining intensity between control and treated U87 MG.62-7 and U87
MG.wtEGFR xenografts, suggesting that antibody treatment does not lead to the
selection of clonal variants lacking mAb 806 reactivity.
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CA 02826627 2013-09-04
Treatment of A431 Xenografts with mAb 806. To demonstrate that the antitumor
effects of mAb 806 were not restricted to 1J87 MG cells, the antibody was
administrated to mice containing A431 xe-nografts. These cells contain an
amplified
EGFR gene and express approximately 2 X 106 receptors/cells. We have
previously
shown that mAb 806 binds ¨10% of these EGFRs and targets A431 xenografts.(3)
mAb 806 significantly inhibited the growth of A431 xenografts when examined in
the
preventative xenograft model described previously (Figure 11A). At day 13,
when
control animals were sacrificed, the mean tumor volume was 1400 150 mm3 in
the
vehicle-treated group and 260 60 mm3 for the 1 mg/injection treatment group
(P <
0.0001). In a separate experiment, a dose of 0.1mg of mAb also inhibited
significantly
(P < 0.05) the growth of A431 xenografts in a preventative model (data not
shown).
Given the efficacy of mAb 806 in the preventative A431 xenograft model, its
ability
to inhibit the growth of established tumor xenografts was examined. Antibody
treatment was as described in the preventative model, except it was not
started until
tumors had reached a mean tumor volume of 200 20 nun3 . mAb 806
significantly
inhibited the growth of established A431 xenografts (Figure 11B). At day 13,
the day
control animals were sacrificed, the mean tumor volume was 1100 100 mm 3 for
the
control group and 450 70 mm3 for the 1 mg/injection group (P < 0.0001).
We have shown previously3 that mAb 806 targets both de2-7 EGFR-transfected U87
MG xenografts and A431 xenografts that over express the wt EGFR. mAb 806 did
not
target parental U87 MG cells, which express ¨105 EGFR3 (16). As assessed by
FACS,
immunohistochemistry, and immunoprecipitation, we now demonstrate that mAb 806
is also able to specifically bind U87 MG.wtEGFR cells, which express >106
EGFRs/cell. Thus, the previous observed binding of rnAb 806 to A431 cells is
not the
result of some unusual property of these cells but rather appears to be a more
general
phenomenon related to over expression of the wt EGFR.
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CA 02826627 2013-09-04
(a) We were unable to detect mAb 806 binding to the parental U87 MG cell line,
which expresses 1 X 105 wt EGFRs/cell (16), either by FACS,
immunoprecipitation,
immunohistochemistry, or with iodinated antibody. Indeed, iodinated mAb 806
did
not bind to U87 MG cell pellets containing 1 X 107 cells, which based on the
Scatchard data using 1 X 106 A431 cells, are conditions that should detect low
level
antibody binding (i.e., the total number of receptors being similar in both
cases).
(b) Scatchard analysis clearly showed that inAb 806 only bound to 10% of the
total
EGFR on the surface of A431 cells. If mAb 806 simply binds to the wt EGFR with
low affinity, then it should have bound to a considerably higher percentage of
the
receptor.
(c) Comparative immunopre,cipitation of the A431 and U87 MG. wtEGFR cell lines
with mAb 806 and the sc-03 antibody also supported the hypothesis that only a
subset
of receptors are recognized by mAb 806. Taken together, these results support
the
notion that mAb
806 recognizes a EGFR subset on the surface of cells overexpressing the EGIR.
We
are currently analyzing the EG1-.1( immunoprecipitated by mAb 806 to see if it
displays altered biochemical properties related to glycosylation or lcinase
activity.
The xenograft studies with mAb 806 described here demonstrate dose-dependent
inhibition of U87 MG.D2-7 xenograft growth. In contrast, no inhibition of
parental
U87 MG xenografts was observed, despite the fact that they continue to express
the
wt EGFR in vivo. mAb 806 not only significantly reduced xenograft volume, it
also
induced significant necrosis within the tumor. As noted above, other de2-7
EGFR-
specific mAbs have been generated (20 ¨22), but this is the first report
showing the
successful therapeutic use of such an antibody in vivo against a human de2-7
EGFR-
expressing glioma xenograft. A recent report demonstrated that the de2-7 EGFR-
specific Y10 mAb had in vivo antitumor activity against murine B16 melanoma
cells
transfected with a =nine homologue of the human de2-7 EGFR (33). Y10 mediated
in vitro cell lysis (>90%) of B16 melanoma cells expressing the de2-7 EGFR in
the
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CA 02826627 2013-09-04
absence of complement or effector cells. In contrast to their in vitro
observations, the
in vivo Y10 antibody efficacy was completely mediated through Fc function when
using B16 melanoma cells grown as xenografts in an immuno-competent model.
Thus, the direct effects observed in vitro do not seem to be replicated when
cells are
grown as tumor xenografts.
Overexpression of the EGFR has been reported in a number of different tumors
and is
observed in most gliomas (4, 14). It has been proposed that the subsequent
EGFR
over expression mediated by receptor gene amplification may confer a growth
advantage by increasing intracellular signaling and cell growth (34). The U87
MG
cell line was transfected with the wt EGFR to produce a glioma cell that
mimics the
process of EGFR gene amplification. Treatment of established U87 MG.wtEGFR
xenografts with mAb 806 resulted in significant growth inhibition. Thus, mAb
806
also mediates in vivo antitumor activity against cells overexpressing the
EGFR.
Interestingly, mAb 806 inhibition of U87 MG.wtEGFR xenografts was less
pronounced than that observed with U87 MG.A2-7 tumors. This probably reflects
the
fact that mAb 806 has a lower affinity for the overexpressed wt EGFR and only
binds
a small proportion of receptors expressed on the cell surface.(3) However, it
should
be noted that despite the small effect on U87 MG.wtEGFR xenograft volumes, mAb
806 treatment produced large areas of necrosis within these xenografts. To
exclude
the possibility that mAb 806 only mediates inhibition of the U87 MG-derived
cell
lines, we tested its efficacy against A431 xenografts. This squamous cell
carcinoma-
derived cell line contains significant EGFR gene amplification, which is
retained both
in vitro and in vivo. Treatment of A431 xenografts with InAb 806 produced
significant growth inhibition in both a preventative and established model,
indicating
the antitumor effects of mAb 806 are not restricted to transfected U87 MG cell
lines.
Complete prevention of A431 xenograft growth by antibody treatment has been
reported previously. The wt EGFR naAbs 528, 225, and 425 all prevented the
formation of A431 xenografts when administered either on the day or 1 day
after
tumor inoculation (9, 10). The
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CA 02826627 2013-09-04
reason for this difference in efficacy between these wt EOM anti-bodics and
mAb
806 is not known but may be related to the mechanism of cell growth
inhibition. The
wt EGFR antibodies function by blocking ligund binding to the EGFR, but this
is
probably not the case with mAb 806 because it only binds a small EGFR subset
on the
strrface of A431 cells. Thc significant efficacy of mAb 806 against U87 MG
cells
expressing the ligand-independent de2-7 EGFR further supports thc notion that
this
antibody mediates its antitumor activity by a mechanism not involving ligand
blockade. Therefore, we arc currently investigating the noniramunological and
immunological mechanisms that contribute to the antitumor effects of mAb 806,
Nonimmunological mechanisms may include subtle changes in receptor levels,
blockade of signaling, or Induction of inappropriate signaling.
=
Previously, agents such as doxorubicin and cisplatin in conjunction with wt
EGFR
antibodies have produced enhanced antitumor activity (35,36). The combination
of
doxorubicin and mAb 528 resulted in total eradication of established A431
xenowafts, whereas treatment with either agent alone caused only temporary in
vivo
growth inhibition (36). Likewise, the combination of cisplatin and either mAb
528 or
225 also led to the eradication of well-established A43I xenografts, which was
not
observed when treatment with either agenr was used (35). Thus, future studies
involving the combination of chemotherapeutic agents with mAb 806 are planned
using xenograft models.
Maybe the most important advantage of mAb 806 compared with current MFR.
=
antibodies is that it should be possible to directly conjugate cytotoxic
agents to .mAb
806. This approach is not feasible with current BM-specific antibodies because
they
target the liver and cytotoxic conjugation would almost certainly induce
severe
toxicity. Conjugation of cytotoxic agents such as drugs
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CA 02826627 2013-09-04
(37) or radioisotopes (38) to antibodies has the potential to improve efficacy
and
reduce the systemic toxicity of these agents. This study clearly
detrionstrates that mAb
806 has significant in vivo antitumor activity against de24 EGFR-positive
xenogafts
and tumors overexpressing the EGFR. The unique specificity of niAb 806
suggests
immunotberapeutic potential in targeting a number of tumor typeS, particularly
head
and neck tumors and glioma, without the restrictions associated with normal
tissue
uptake.
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EXAMPLE 21,
CONSTRUCTION, EXPRESSION AND ANALYSTS OF CIIIMERIC 806
ANTIBODY
=
Chimeric antibodies are a class of molecules in which heavy and light chain
variable
regions of for instance, a mouse, rat or other species are joined onto human
heavy and
light chain regions. Chimeric antibodies are produced necombinantly. One
advantage
of chimeric antibodies is that they can reduce xesnoantigenic effects, the
inherent
immunogenicity of non-human antibodies (for instance, mouse, rat or other
species).
In addition, recombinantly prepared chimeric antibodies can often be produced
in
large quantities, particularly when utilizing high level expression vectors.
For high level production, the most widely used mammalian expression system is
one
which utilizes the gene amplification procedure offered by clehydrofolate
neductage.
deficient ("dhfr-") Chinese hamster ovary cells. The system is well known to
the
skilled artisan. The system is basfyi upon the dehydrofbIate rectuctaso "dhfr"
gene,
which encodes the MFR. enzyme, which catalyzes conversion of dehydrofolate to
tetrahydrofolate. In order to achieve high production, dhfr- CHO cells are
transfccted
with an expression vector containing a functional DHFR gene, together with a
gene
that encodes a desired protein. In this case, the desin:d protein is
recombinant
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CA 02826627 2013-09-04
antibody heavy chain and/or light chain.
By increasing the amount of the competitive DI-IFR inhibitor methotrexatc
(MTX),
the recombinant cells develop resistance by amplifying the dhfr gene. In
standard
cases, the amplification unit employed is much larger than the si:4e of the
dhfr gene,
and as a result the antibody heavy chain is co-amplified.
When large scale production of the protein, such as the antibody chain, is
desired,
both the expression level, and the stability of the cells being employed, are
critical. In
long terrn culture, recombinant CHO cell populations iose homogeneity with
respect
to their specific antibody productivity during amplification, even theme' they
derive
from a single, parental eIone.
Bicistronic expression vectors were prepared for use in recombinant expression
of the
chirneric antibodies. These bicistronic expression vectors. employ an
"intexnal
ribosomal entry site" or `TRES." In these constructs for production of
chimeric
rainEGPR, the immunoglobulin chains and selectable markers cIDNAs are linked
via
an 1RE5. MPS are cis-acting elements that recruit the small ribosomal subunits
to an
internal initiator c.oclon in the rnRNA with the help of cellular trans-acting
factors.
IRES facilitate the expression of two or more proteins from a polycistronic
transcription unit in eulcaryotic cells. The use of bicistrouic expression
vectors in
which the selectable marker gene is translated in a cap dependent manner, and
the
gene of interest in an IRES dependent tnanner has been applied to a variety of
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CA 02826627 2013-09-04
experimental methods. IRES elements have been successfully incorporated into
vectors for cellular transformation, production of transgenic animals,
recombinant
protein production, gene therapy, gene trapping, and gene targeting.
Synopsis of Chimeric Antibody 806 (ch806) Construction
The chimeric 806 antibody was generated by cloning the VH and VL of the 806
antibody from the parental murine hybridoma using standard molecular biology
techniques. The VH and VL were then cloned into the pREN mammalian expression
vectors, the construction of which are set forth in SEQ ID NO:7 and SEQ ED
NO:8,
and transfected into CHO (DHFR -/-ve) cells for amplification and expression.
Briefly, following trypsinization 4 x 106 CHO cells were co-transferred with
10i.tg of
each of the LC and HC expression vectors using electroporation under standard
conditions. Following a 10min rest period at room temperature, the cells were
added
to 15ml medium (10%fetal calf serum, hypoxanthine/thymidine supplement with
additives) and transferred to 15x 10cm cell culture petri dishes. The plates
were then
placed into the incubator under normal conditions for 2 days. At this point,
the
addition of gentarnycin, 5n.M methotrexate, the replacement of fetal calf
serum with
dialyzed fetal calf serurnand the removal of hypoxanthine/thymidine, initiated
the
selection for clones that were successfully transfected with both the LC and
HC from
the medium. At day 17 following transfection, individual clones growing under
selection were picked and screened for expression of the chimeric 806
antibody. An
FIJSA was utilized for screening and consisted of coating an El ISA plate with
denatured soluble EGF receptor (denatured EGFR is known to allow 806 binding).
This assay allows for the screening of production levels by individual clones
and also
for the functionality of the antibody being screened. All clones were shown to
be
proding functional ch806 and the best producer was taken and expanded for
amplification. To amplify the level of ch806 being produced, the highest
producing
clone was subjected to reselection under a higher rnethotrexate concentration
(100nM
vs 5nM). This was undertaken using the aforementioned procedures.
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Clones growing at 100nM MTX were then passed onto the Biological Procution
Facility, Ludwig Institute, Melbourne, Australia for measurement of production
levels, weaning off serum, cell banking. The cell line has been shown to
stably
produce -10E12g/litre in roller bottles.
The nucleic acid sequence of the pREN ch806 LC neo vector is provided in SEQ
ID
NO:7. The nucleic acid sequence of the pREN ch806 HC DHFR vector is provided
in
SEQ NO:8.
Figure 33 depicts the vectors pREN-HC and pREN-LC, which employ an IRES. The
pREN bicistronic vector system has been described.
Ch806 was assessed by FACS analysis to demonstrate that the chimeric 806
displays
identical binding specificity to that of the =nine parental antibody..
Analysis was
. performed using wild type cells (1)87 MG parental cells), cells
overexpressing the
EGP receptor (A431 cells and 1JA87 wt EGFR cells) and UA87 A2-7 cells (data
not
shown). Similar binding specificity of Mab806 and ch806 was obtained using
cells
overexpressing EGPR and cells expressing the de2-'7130FR. No binding was
observed in wild type cells. Scatchard analysis revealed a binding atrmity for
radiolabeled ch806 of 6.4x109 M-1 using U87MGde2-7 cells (data not shown).
Biodistribution analysis of the ch806 antibody was performed in BALB/c nude
mice
beating U87MG-cle2-7 xenograft tumors and the results are shown in Mtge 34.
2vfice were injected with Sug of radiolabelled antibody and were sacrificed in
groups
of four per time point at 8, 24, 48 and 74 hours. Organs were collected,
weighed and
radioactivity n2easurexl in a gamma counter. 1251-labe11ed ch806 displays
reduced
targeting to the tumor compared to 1111n-labelled ch806, which has high tumor
uptake
and cumulative tumor rete,ntion over the 74 hour time period. At 74 hours, the
"11n-
labelled antibody displays approximately 30% ID/gram tissue and a tumor to
blood
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CA 02826627 2013-09-04
ratio of 4.0 (Figure 35). The "Ili-labelled ch806 shows some nonspecific
retention
in the liver, spleen and lcidneys. This is common for the use of this isotope
and
decreases with time, which supports that this binding is non-specific to ch806
and due
to '1 'In binding.
Chimeric antibody ch806 was assessed for theraeutic efficacy in an established
tumor
model. 3x106U87MG.A2-7 cells in 100u1 of PBS were inoculated s.c. into both
flanks of 4-6 week old female nude mice (Aminal Research Center, Western
Australia, Australia). The mAb806 was included as a positive control. The
results
are depicted in Figure 36. Treatment was started when tumors had reached a
mean
volume of 50 rrun3 and consisted of 1 mg of ch806 or mAb806 given i.p. for a
total of
injections on the days indicated. Tumor volume in mm3 was determined using the
formula (length x width2)/2, where length was the longest axis and width the
measurement at right angles to the length. Data was expressed as mean tumor
volume
+/- S.E. for each treatment group. The ch806 and mAb806 displayed nearly
identical
anti-tumor activity against U87MG.A2-7 xenografts.
Analysis of Ch806 Immune Effector Function
Materials and Methods
Antibodies and Cell lines:
Murine anti-de2-7 EGFR monoclonal mAb 806, chimeric antibody ch806 (IgGI) and
control isotype matched chimeric anti-G250 monoclonal antibody cG250 were
prepared
by the Biological Production Facility, Ludwig Institute for Cancer Research,
Melbourne,
Australia. Both complement-dependant cytotoxicity (CDC) and antibody-dependent
cellular- cytotoxicity (ADCC) assays utilised U87MG.de2-7 and A431 cells as
target
cells. The previously described U87MG.de2-7 cell line is a human astrocytoma
cell line
infected with a retrovirus containing the de2-7EGFR (Nishikawa, R. et al.
(1994) Proc
Nati Acad Sci U S A. 91: 7727-31). Human squamous carcinoma A431 cells were
purchased from the American Type Culture Collection (Manassas, VA). All cell
lines
were cultured in DMEM/F-12 with GlutamaxTm (Life Technologies, Melbourne,
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Australia) supplemented with 10% heat-inactivated FCS (CSL, Melbourne,
Australia),
100 units/ml penicillin and 100 ug/m1 streptomycin. To maintain selection for
retrovirally
transfected U87MG.de2-7 cells, 400 ug/ml G418 was included in the media.
Preparation of human peripheral blood mononuclear cells (PBMC) Effector Cells:
PBMCs were isolated from healthy volunteer donor blood. Heparinised whole
blood was
fractionated by density centrifugation on Picoll-Hypaqu:(ICN Biomedical Inc.,
Ohio,
USA). PBMC fractions was collected and washed three times with RPME+ 1640
supplemented with 100 U/ml penicillin and 100 ug/ml streptomycin, 2mM L-
glutamine,
containing 5% heat-inactivated FCS.
Preparation of Target Cells:
CDC and ADCC assays were performed by a modification of a previously published
method ((Nelson, D. L. et al. (1991) In: J. E. Colignan, A. M. Kruisbeek, D.
D. Margulies,
E. M. Shevach, and W. Strober (eds.), Current Protocols in Immunology, pp.
7.27.1. New
York: Greene Publishing Wiley Interscience). Briefly, 5 x 106 target U87MG.de2-
7 and
A431cells were labeled with 50 t.zCí 51Cr (Geneworks, Adelaide, Australia) per
1 x 106
cells and incubated for 2 hr at 37 C. The cells were then washed three time
with PBS
(0.05M, pH 7.4) and a fourth wash with culture medium. Aliquots (1 x 104
cells/50 ul) of
the labeled cells were added to each well of 96-well microtitre plates (NUNC,
Roskilde,
Denmark).
CDC Assay:
To 50 ul labeled target cells, 50 ul ch806 or isotype control antibody c03250
were added in
triplicate over the concentration range 0.00315 ¨ 10 ug/ml, and incubated on
ice 5 min.
Fifty ul of freshly prepared healthy donor complement (serum) was then added
to yield a
1:3 final dilution of the serum. The microtitre plates were incubated for 4 hr
at 37 C.
Following centrifugation, the released 'Cr in the supernatant was counted (Con
automated Gamma Counter, Canberra Packard, Melbourne, Australia). Peicentage
specific
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CA 02826627 2013-09-04
lysis was calculated from the experimental 5ICr release, the total (50 ul
target cells + 100
ul 10% Tween 20) and spontaneous (50 ul target cells + 100 ul medium) release.
ADCC Assay:
Ch806-mediated ADCC effected by healthy donor PBMCs was measured by two 4-hr
5ICr
release assays. In the first assay, labelled target cells were plated with the
effector cells in
96-well "U" bottom microplates (NUNC, Roskilde, Denmark) at effector/target
(E:T) cell
ratios of 50:1. For ADCC activity measurements, 0.00315-10 ug/ml (final
concentration)
test and control antibodies were added in triplicate to each well. In the
second ADCC
assay, the ADCC activity of ch806 was compared with the parental murine mAb
806 over
a range of Effector: Target cell ratios with the test antibody concentration
constant at 1
ug/ml. In both assays, micotitre plates were incubated at 37 C for 4 hours,
then 50 ul
supernatant was harvested from each well and released 5ICr was determined by
gamma
counting (Cobra II automated Gamma Counter, Canberra Packard, Melbourne,
Australia).
Controls included in the assays corrected for spontaneous release (medium
alone) and
total release (10% Tween20/PBS). Appropriate controls with the same subclass
antibody
were run in parallel.
The percentage cell lysis (cytotoxicity) was calculated according to the
formula:
Percentage Cytotoxicity = Sample Counts ¨ Spontaneous Release x 100
Total Release ¨ Spontaneous Release
The percent (%) cytotoxicity was plotted versus concentration of antibody
(i.kg/rn1).
Results
The results of the CDC analyses are presented in Figure 37. Minimal CDC
activity
was observed in the presence of up to 10 ug/m1 ch806 with CDC comparable to
that
observed with isotype control c0250.
Ch806 mediated ADCC on target U87MG.de2-7 and A431 cells at t:T ratio of 50:1
is
presented in Figure 38. Effective ch806 specific cytotoxicity was displayed
against
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CA 02826627 2013-09-04
target U87M0.de2-7 cells, but minimal ADCC was mediated by ch806 on A431
cells.
The levels of cytotoxicity achieved reflect the number of ch806 binding sites
on the
two cell populations. Target U87MG.de2-7 cells express ¨ 1 x 106 de2-7EGFR
which
are specifically recognised by ch806, while only a subset of the 1 x 106 wild-
type
EGFR molecules expressed on A431 cells are recognised by ch806 (see above
Examples).
Further ADCC analyses were performed to compare the ADCC mediated by 1 ug/nal
ch806 on target U87MG.de2-7 cells with that effected by 1 ug/ml parental
murine
mAb 806. Results are presented in Figure 39. Chimerisation of mAb 806 has
effected
marked improvement of the ADCC achieved by the parental murine mAb with
greater
than 30% cytotoxicity effected at E:T ratios 25:1 and 50:1.
The lack of parental murine inAb 806 immune effector function has been
markedly
improved upon chimerisation. Ch806 mediates good ADCC, but minimal CDC
activity.
EXAMPLE 22
GENERATION OF ANTI-LDIOTYPE ANTIBODIES TO CHIMERIC
ANTIBODY ch806
To assist the clinical evaluation of mAb806 or ch806, laboratory assays are
required
to monitor the serum pharmacokinetics of the antibodies and quantitate any
immune
responses to the mouse-human chimeric antibody. Mouse monoclonal anti-
idiotypic
antibodies (anti-ids) wet generated and characterised for suitability as ELBA
reagents for measuring ch806 in patient sera samples and use as positive
controls in
human anti-chimeric antibody immune response analyses. These antiidiotype
antibodies may also be useful as therapeutic or prophylactic vaccines,
generating a
natural anti-EGFR antibody response in patients.
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CA 02826627 2013-09-04
Methods for generating anti-idiotype antibodies are well known in the art
(Bhattacharya-Chatterjee, Chatterje,e et al. 2001, Uemura et al. 1994,
Steffens,
Boerman et al. 1997, Safa and Foon 2001, Brown and Ling 1988).
Mouse monoclonal anti-idiotypic antibodies (anti-ids) were, briefly, generated
as
follows. Splenocytes from mice immunized with ch806 were fused with SP2/0-AG14
plasmacytoma cells and antibody producing hybridomas were selected through
FT .ISA for specific binding to ch806 and competitive binding for antigen
(Figure 40).
Twenty-five hybridomas were initially selected and four, designated LM1-1-11, -
12 -13
and -14, secreted antibodies that demonstrated specific binding to ch806, mAb
806
and were able to neutralise ch806 or mAb 806 antigen binding activity (Figure
41).
The recognition of the ch806/mAb 806 idiotope or CDR region was demonstrated
by
lack of cross-reactivity with purified polyclonal human IgG.
In the absence of readily available recombinant antigen de2-7 EGFR to assist
with the
determination of ch806 in serum samples, the ability of the novel anti-
idiotype ch806
antibodies to concurrently bind 806 variable regions was exploited in the
development
of a sensitive, specific ELLSA for measuring ch806 in clinical samples (Figure
42).
Using LMH-12 for capture and Biotinylated ¨LMH-12 for detection, the validated
ELISA demonstrated highly reproducible binding curves for measuring ch806 (2
ug/ml ¨ 1.6 ng/ml) in sera with a 3 ng/ml limit of detection. (n=12; 1-100
ng/ml,
Coefficient of Variation <25%; 100 ng/ml- 5 ug/ml, Coefficient of Variation <
15%).
No background binding was evident with the three healthy donor.sera tested and
negligible binding was observed with isotype control hu3S193. The hybridoma
produces high levels of antibody LMH-12, and larger scale production is
planned to
enable the measurement of ch806 and quantitation of any immune responses in
clinical samples. (Brown and Ling 1988)
RESULTS
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CA 02826627 2013-09-04
Mice Immunization and hybridoma clone selection
Immunoreactivity of pre- and post-immunization sera samples indicated the
development of high titer mouse anti-ch806 and anti-huIgG mAbs. Twenty-five
hybridomas producing antibodies that bound ch806, but not hulgG,.were
initially
selected. The binding characteristics of some of these hybridomas are shown in
Figure 42A and 42B. Four of these anti-ch806 hybridomas with high affinity
binding
(clones 3E3, 5B8, 9D6 and 4D8) were subsequently pursued for clonal expansion
from single cells by limiting dilution and designated Ludwig Institute for
Cancer
Research Melbourne Hybridoma (LMH) -11,-12,-13, and -14, respectively (Figure
42).
Binding and Blocking Activities of Selected Anti-Idiotype Antibodies
The ability of anti-ch806 antibodies to concurrently bind two ch806 antibodies
is a
desirable feature for their use as reagents in an ET JSA for determining serum
ch806
levels. Clonal hybridomas, LME-11, -12, -13, and -14 demonstrated concurrent
binding (data not shown).
After clonal expansion, the hybridoma culture supernatants were examined by
ELISA
for the ability to neutralise ch806 or mAb 806 antigen binding activity with
sEGFR621. Results demonstrated the antagonist activity of anti-idiotype mAbs
LMH-
11, -12, -13, and -14 with the blocking in solution of both ch806 and murine
mAb 806
binding to plates coated with sEGFR (Figure 41 for LMH-11, -12, -13).
Following larger scale culture in roller bottles the binding specificity's of
the
established clonal hybridomas, LMH-11, -12, -13, and -14 were verified by
F.TSA.
L,MH-11 through -14 antibodies were identified as isotype IgGlx by mouse
monoclonal antibody isotyping kit.
ch806 in Clinical Serum Samples: Pharmacokinetic ELBA Assay Development
To assist with the determination of ch806 in serum samples, the ability of the
anti-
idiotype ch806 antibodies to concurrently bind the 806 variable region was
exploited
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CA 02826627 2013-09-04
in the development of a sensitive and specific FLTSA assay for ch806 in
clinical
samples. The three purified clones LMH-11, -12, and -13 (Figure 49 , B, and C
respectively) were compared for their ability to capture and then detect bound
ch806
in sera. Results indicated using LMH-12 (10 lig/m1) for capture and
biotinylated-
LMH-12 for detection yielded the highest sensitivity for ch806 in serum (3
ng/ml)
with negligible background binding.
Having established the optimal pharmacolcinetic FLISA conditions using 1
ptg/m1
anti-idiotype LMII-12 and 1 pg/mlbiotinylated LMH-12 for capture and
detection,
respectively, validation of the method was performed. Three separate FTISAs
were
performed in quadruplicate to measure ch806 in donor serum from three healthy
donors or 1% BSA/media with isotype control hu3S193. Results of the validation
are
presented in Figure 43 and demonstrate highly reproducible binding curves for
measuring ch806 (2 ttg/ml ¨ L6 ng/ml) in sera with a 3 ng/ml limit of
detection.
(n=12; 1-100 ng/ml, Coefficient of Variation < 25%; 100 nghnl- 5 gg/ml,
Coefficient
of Variation < 15%). No background binding was evident with any of the three
sera
tested and negligible binding was observed with isotype control hu3S193.
REFERENCES
These should really be incorporated with those at the end of Example 17 and
put to
the end of all the examples
Brown, G. and N. Ling (1988). Murine Monoclonal Antibodies. Antibodies, Volume
I. A Practical Approach. D. Catty. Oxford, England,1RL Press: 81-104,
Bhattacharya-Chatteijee, M., S. K. Chatterjee, et al. (2001). "The anti-
idiotype
vaccines for immunotherapy." Curr Opin Mol Ther 3(1): 63-9.
Domagala, T., N. Konstantopoulos, et al. (2000). "Stoichiometry, kinetic and
binding
analysis of the interaction between epidermal growth factor (EGF) and the
extracellular domain of the EGF receptor." Growth Factors 18(1): 11-29
Safa, M. M. and K. A. Foon (2001). "Adjuvant immunotherapy for melanoma and
colorectal cancers." Semin Oncol 28(1): 68-92.
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CA 02826627 2013-09-04
Uemura, H., E. Okajima, et al. (1994). "Internal image anti-idiotype
antibodies related
to renal-cell carcinoma- associated antigen G250." Int J Cancer 56(4): 609-14.
EXAMPLE 23
ASSESSMENT OF CARBOHYDRATE STRUCTURES AND
ANTIBODY RECOGNITION
Experiments were undertaken to further assess the role of carbohydrate
structures in
the binding and recognition of the EGFR, both amplified and de2-7 EGFR, by the
mAb806 antibody.
To determine if carbohydrate structures are directly involved in the rnAb 806
epitope,
the recombinant sEGFR expressed in CHO cells was treated with PNGase F to
remove N-linked glycosylation. Following treatment, the protein was run on SDS-
PAGE, transferred to membrane and iaununoblotted with mAb 806 (Fig-ure44). As
expected, the deglycosylated sEGER ran faster on SDS-PAGE, indicating that the
carbohydrates had been successfully removed. The mAb 806 antibody clearly
bound
the deglycosylated material demonstrating the antibody epitope is peptide in
nature
and not solely a glycosylation epitope.
Lysates, prepared from cell lines metabolically labelled with 35S, were
inununoprecipitated with different antibodies directed to the EGFR (Figure45).
As
expected, the 528 antibody immunoprecipitated 3 bands from U87MG.,62-7 cells,
an
upper band corresponding to the wild type (wt) EGFR and two lower bands
corresponding to the de2-7 EGFR. These two de2-7 EGFR bands have been reported
previously and are assumed to represent differential glycosylation (Chu et al.
(1997)
Biochem. J. Jun 15;324 (Pt 3):885-861). In contrast, mAb 806 only
imrnunopre,cipitated the two de2-7 EGFR bands, with the wt receptor being
completely absent even after over-exposure (data not shown). Interestingly,
mAb806
showed increased relative reactivity with the lower de2-7 EGFR band but
decreased
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CA 02826627 2013-09-04
reactivity with the upper band when compared to the 528 antibody. The SC-03
antibody, a commercial rabbit polyclonal antibody directed to C-terminal
domain of
the EGFR, immunoprecipitated the three EGFR bands as seen with the 528
antibody
although the total amount of receptor immunoprecipitated by this antibody was
considerably less. No bands were observed when using an irrelevant IgG2b
antibody
as a control for mAb 806.
The 528 antibody immunoprecipitated a single band from U87MG.wtEGFR cells
corresponding to the wt receptor (Figure 45). MAb 806 also immunoprecipitated
a
single band from these cells, however, this EGFR band clearly migrated faster
than
the 528 reactive receptor. The SC-03 antibody immunoprecipitated both EGFR
reactive bands from U87MG.wtEGFR cells, further confirming that the mAb 806
and
528 recognize different forms of the EGFR in whole cell lysates from these
cells.
As observed with U87MG.wtEGFR cells, the 528 antibody immunoprecipitated a
single EGFR band from A431 cells (Figure 45). The 528 reactive EGFR band is
very
broad on these low percentage gels (6%) and probably reflects the diversity of
receptor glycosylation. A single EGFR band was also seen following
immunoprecipitation with mAb 806. While this EGFR band did not migrate
considerably faster than the 528 overall broad reactive band, it was located
at the
leading edge of the broad 528 band in a reproducible fashion. Unlike U87MG.A2-
7
cell lysates, the total amount of EGFR immunoprecipitated by mAb 806 from A431
lysates was considerably less than with the 528 antibody, a result consistent
with our
Scatchard data showing mAb 806 only recognizes a portion of the EGFR on the
surface of these cells (see Example 4 above). Immunoprecipitation with SC-03
resulted in a single broad EGFR band as for the 528 antibody. Similar results
were
obtained with HN5 cells (data not shown). Taken together, this data indicates
that
mAb 806 preferentially reacts with faster migrating species of the EGFR, which
may
represent differentially glycosylated forms of the receptor.
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In order to determine at what stage of receptor processing mAb 806 reactivity
appeared a pulse/chase experiment was conducted. A431 and U87MG.A2-7 cells
were pulsed for 5 min with 35S methionine/cysteine, then incubated at 370C for
various times before immunoprecipitation with mAb 806 or 528 (Figure46). The
immunoprecipitation pattern in A431 cells with the 528 antibody was typical
for a
conformational dependent antibody specific for the EGFR. A small amount of
receptor was immunoprecipitated at 0 min (i.e.after 5 min pulse) with the
amount of
labelled EGFR increasing at each time point. There was also a concurrent
increase in
the molecular weight of the receptor with time. In contrast, the mAb 806
reactive
EGFR material was present at high levels at 0 min, peaked at 20 min and then
reduced
at each further time point. Thus, it appears that mAb 806 preferentially
recogniz,es a
form of the EGFR found at an early stage of processing.
The antibody reactivity observed in pulse-labelled U87MG.A2-7 cells was more
complicated. Immunoprecipitation with the 528 antibody at 0 min revealed that
a
small amount of the lower de2-7 EGFR band was labelled (Figure 46). The amount
of 528 reactive de2-7 EGFR lower band increased with time, peaking at 60 min
and
declining slowly at 2 and 4 h. No significant amount of the labelled upper
band of
de2-7 EGFR was detected until 60 min, after which the level continued to
increase
until the end of the time course. This clearly indicates that the upper de2-7
EGFR is a
more mature form of the receptor. MAb 806 reactivity also varied during the
time
course study, however mAb 806 preferentially precipitated the lower band of
the de2-
7 EGFR. Indeed, there were no significant levels of mAb 806 upper band seen
until 4
h after labelling.
The above experiments suggest that mab 806 preferentially reacts with a more
immature glycosylation form of the de2-7 and wt EGFR. This possibility was
tested
by immunoprecipitating the EGFR from different cells lines labelled overnight
with
35S methionine/cysteine and then subjecting the resultant precipitates to
Endoglycosidase H (Endo H) digestion. This enzyme preferentially removes high
mannose type carbohydrates (i.e. immature glycosylation) from proteins while
leaving
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complex carbohydrates (i.e. mature glycoslation) intact. Irnmunoprecipitation
and
digestion with Endo H of labelled U87MG.A2-7 cell lysates with 528, mAb 806
and
SC-03 gave similar results (Figure 47). As predicted, the lower de2-7 EGFR
band
was fully sensitive to Endo H digestion, migrating faster on SDS-PAGE after
Endo H
digestion, demonstrating that this band represents the high mannose form of
the de2-7
EGFR. The upper de2-7 EGFR band was essentially resistant to Endo H digestion,
showing only a very slight difference in migration after Endo H digestion,
indicating
that the majority of the carbohydrate structures are of the complex type. The
small
but reproducible decrease in the molecular weight of the upper band following
enzyme digestion suggests that while the carbohydrates on the upper de2-7 EGFR
band are predominantly of the complex type, it does possess some high mannose
structures. Interestingly, these cells also express low amounts of endogenous
wt
EGFR that is clearly visible following 528 immunopreciptitation. There was
also a
small but noticeable reduction in molecular weight of the wt receptOr
following Endo
H digestion, indicating that it also contains high mannose structures.
The sensitivity of the immunoprecipitated wt EGFR to Endo H digestion was
similar
in both U87MG.wtEGFR and A431 cells (Figure 47). The bulk of the material
precipitated by the 528 antibody was resistant to the Endo H enzyme although a
small
amount of the material was of the high mannose form. Once again there was a
small
decrease in the molecular weight of the wt EGFR following Endo H digestion
suggesting that it does contain some high mannose structures. The results
using the
SC-03 antibody were similar to the 528 antibody. In contrast, the majority of
the
EGFR precipitated by mAb 806 was sensitive to Endo H in both U87MG.wtEGFR
and A431 cells, confirming that mAb 806 preferentially recognizes the high
mannose
form of the EGFR. Similar results were obtained with HN-5 cells, wherein the
majority of the material precipitated by mAb 806 was sensitive to Endo H
digestion,
while the majority of the material precipitated by mAb528 and SC-03 was
resistant to
Endo H digestion (data not shown).
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Cell surface iodination of the A431 ceLlline, was performed with 123I followed
by
immtmoprecipitation with the 806 antibody. The protocol for surface iodination
was
as follows: The cell lysis, immunoptecipitation, Endo H digestion, SDS PAGE
and
autoradiography are as described above herein. For labeling, cells were grown
in
media with 10% FCS, detached with EDTA, washed twice with PBS then
resuspended in 400 ul of PBS (approx 2-3 x106 cells). To this was added 15 ul
of 1251
(100 mCilml stock), 100 ul bovine lactoperoxidase (1 mWrn1) stock, 10 ul H202
(0.1%
stock) and this was incubated for 5 min. A further 10 ul of H202 was then
added and
the incubation continued for a further 3 min. Cells were then washed again 3
times
with PBS and lysed in 1% Triton. ceu surface iodination of the A431 cell line
with
lactoperoxidase, followed by immunoprecipitation with the 806 antibody, showed
that, similar to the whole cell lysates described above, the predominant form
of the
EGFR recognized by 806 bound on the cell surface of A431 cells was sensitive
to
EndoH digestion (Figure 42). This confirms that the form of BIM bound by 806
on
the cell surface of A431 cells is an EndoH sensitive form and thus is the high
mannose type.
This invention may be embodied in other forrns or carried out in other ways
without
departing from the spirit or essential characteristics thereof. The present
disclosure is
therefore to be considered as in all aspects illustrated and not restrictive,
the scope of
the invention being indicated by the appended Claims, and all changes which
come
within the meaning and range of equivalency are intended to be embraced
therein.
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=
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