U.S. patent application number 13/482406 was filed with the patent office on 2013-01-24 for dual specific immunotoxin for brain tumor therapy.
This patent application is currently assigned to The United States Government as represented by the Secretary, Department of Health and Human Service. The applicant listed for this patent is Darell Bigner, Chien-Tsun Kuan, Ira H. Pastan, Charles N. Pegram. Invention is credited to Darell Bigner, Chien-Tsun Kuan, Ira H. Pastan, Charles N. Pegram.
Application Number | 20130022598 13/482406 |
Document ID | / |
Family ID | 41215227 |
Filed Date | 2013-01-24 |
United States Patent
Application |
20130022598 |
Kind Code |
A1 |
Bigner; Darell ; et
al. |
January 24, 2013 |
Dual Specific Immunotoxin for Brain Tumor Therapy
Abstract
We tested the in vitro and in vivo efficacy of a recombinant
bispecific immunotoxin that recognizes both EGFRwt and
tumor-specific EGFRvIII receptors. A single chain antibody was
cloned from a hybridoma and fused to toxin, carrying a C-terminal
peptide which increases retention within cells. The binding
affinity and specificity of the recombinant bispecific immunotoxin
for the EGFRwt and the EGFRvIII proteins was measured. In vitro
cytotoxicity was measured. In vivo activity of the recombinant
bispecific immunotoxin was evaluated in subcutaneous models and
compared to that of an established monospecific immunotoxin. In our
preclinical studies, the bispecific recombinant immunotoxin,
exhibited significant potential for treating brain tumors.
Inventors: |
Bigner; Darell; (Durham,
NC) ; Kuan; Chien-Tsun; (Cary, NC) ; Pastan;
Ira H.; (Potomac, MD) ; Pegram; Charles N.;
(Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bigner; Darell
Kuan; Chien-Tsun
Pastan; Ira H.
Pegram; Charles N. |
Durham
Cary
Potomac
Durham |
NC
NC
MD
NC |
US
US
US
US |
|
|
Assignee: |
The United States Government as
represented by the Secretary, Department of Health and Human
Service
Potomac
MD
Duke University
Durham
NC
|
Family ID: |
41215227 |
Appl. No.: |
13/482406 |
Filed: |
May 29, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12418975 |
Apr 6, 2009 |
|
|
|
13482406 |
|
|
|
|
61044190 |
Apr 11, 2008 |
|
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Current U.S.
Class: |
424/135.1 ;
424/136.1; 435/7.1; 530/387.3; 530/391.3; 530/391.7 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 47/6829 20170801; C07K 2317/622 20130101; A61K 47/6849
20170801; C07K 2317/624 20130101; C07K 2319/04 20130101; A61K 38/00
20130101; C07K 2317/565 20130101; C07K 16/2863 20130101; A61K
2039/505 20130101; C07K 2319/55 20130101; C07K 2317/56 20130101;
C07K 14/21 20130101; C07K 2317/92 20130101; A61K 2039/545 20130101;
A61K 39/39558 20130101 |
Class at
Publication: |
424/135.1 ;
530/391.3; 530/391.7; 424/136.1; 530/387.3; 435/7.1 |
International
Class: |
C07K 16/30 20060101
C07K016/30; G01N 33/53 20060101 G01N033/53; A61P 35/00 20060101
A61P035/00; C07K 16/46 20060101 C07K016/46; A61K 39/395 20060101
A61K039/395 |
Claims
1. A single chain variable region antibody which binds with a
binding affinity that is at least 5.times.10.sup.8 M.sup.-1 as
measured by surface plasmon resonance to both (a) EGFR found on
normal human cells and (b) EGFR variant III mutant.
2. The single chain variable region antibody of claim 1 which is
cloned from a hybridoma producing a monoclonal antibody selected
from the group consisting of D2C7, F2A2, B10B11, and H11.
3. The single chain variable region antibody of claim 1 which is
covalently linked to a cytotoxic agent selected from the group
consisting of a toxin, a chemotherapeutic agent, and a
radionuclide.
4. The single chain variable region antibody of claim 3 wherein the
agent is a toxin which is produced as a fusion protein with the
single chain variable region antibody.
5. The single chain variable region antibody of claim 4 wherein the
monoclonal antibody is D2C7.
6. The single chain variable region antibody of claim 3 wherein the
agent is a form of Pseudomonas exotoxin A.
7. The single chain variable region antibody of claim 6 further
comprising a KDEL peptide.
8. The single chain variable region antibody of claim 1 which has a
VH sequence as shown in FIG. 1A
9. The single chain variable region antibody of claim 1 which as a
VL sequence as shown in FIG. 1B.
10. The single chain variable region antibody of claim 1 which has
CDR1, CDR2, and CDR3 regions as shown in FIGS. 1A and 1B.
11. The single chain variable region antibody of claim 1 wherein
said binding affinity is at least 6.times.10.sup.8 M.sup.-1.
12. The single chain variable region antibody of claim 1 wherein
said binding affinity is between 5.times.10.sup.8 M.sup.-1 and
5.times.10.sup.9 M.sup.-1.
13. The single chain variable region antibody of claim 1 which has
an IC50 of less than 2 ng/ml for human cells expressing EGFR as
found on normal cells or EGFRvIII.
14. The single chain variable region antibody of claim 1 which has
an IC.sub.50 of less than 1.5 ng/ml for human cells expressing EGFR
as found on normal cells or EGFRvIII.
15. The single chain variable region antibody of claim 1 which has
an IC.sub.50 of less than 1 ng/ml for human cells expressing EGFR
as found on normal cells or EGFRvIII.
16. The single chain variable region antibody of claim 1 which has
an IC.sub.50 of less than 0.5 ng/ml for human cells expressing EGFR
as found on normal cells or EGFRvIII.
17. A method of treating a tumor in a human, comprising:
administering a single chain variable region antibody according to
claim 1 to the human, whereby tumor cells are killed.
18. The method of claim 17 wherein the tumor is a squamous cell
head and neck tumor.
19. The method of claim 17 wherein the tumor is a brain tumor.
20. The method of claim 17 wherein the tumor is a breast tumor.
21. The method of claim 17 wherein the tumor is a glioblastoma
multiforme.
22. The method of claim 17 wherein the tumor is an astrocytoma.
23. The method of claim 17 wherein the tumor contains an EGFRvIII
allele.
24. The method of claim 17 wherein the administering is directly to
the central nervous system.
25. The method of claim 17 wherein the administering is directly to
the brain.
26. The method of claim 17 wherein the administering is directly to
a surgically-created tumor resection cavity.
27. The method of claim 17 wherein the administering is directly to
a natural tumor cyst.
28. The method of claim 17 wherein the administering is directly to
tumor parenchyma.
29. A monoclonal antibody which binds with a binding affinity that
is at least 5.times.10.sup.8 M.sup.-1 to both (a) EGFR found on
normal human cells and (b) EGFR variant III mutant, wherein said
antibody has a VH sequence as shown in FIG. 1A, a VL sequence as
shown in FIG. 1B, or CDR1, CDR2, and CDR3 regions as shown in FIGS.
1A and 1B.
30. The monoclonal antibody of claim 29 which is designated
D2C7.
31. The monoclonal antibody of claim 29 which comprises a human
IgG.
32. A monoclonal antibody which binds with a binding affinity that
is at least 1.times.10.sup.8 M.sup.-1 to both (a) EGFR found on
normal human cells and (b) EGFR variant III mutant, wherein said
antibody is selected from the group consisting of F2A2 and
B10B11.
33. A method of treating a tumor in a human, comprising:
administering an antibody according to claim 29, 30, 31, or 32, to
the human whereby tumor cells are killed.
34. A method of determining therapeutic plan to treat a tumor in a
human, comprising: contacting tissue of the tumor with an antibody
according to claim 1, 29, 30, 31, or 32; determining amount of
cells in the tissue which bind to the antibody, wherein greater
amounts of cells which bind are a positive factor to recommend
using the antibody therapeutically for the patient.
Description
[0001] This application claims the benefit of provisional
application Ser. No. 61/044,190 filed Apr. 11, 2008, the contents
of which are expressly incorporated herein.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of anti-tumor
immunotoxins. In particular, it relates to an immunotoxin specific
for two tumor-associated antigens.
BACKGROUND OF THE INVENTION
[0003] Gliomas are the most common primary tumors of the central
nervous system (CNS) {Louis, 1995}. Glioblastoma multiforme (GBM)
is the most frequent and the most malignant type of glioma. There
is a much higher incidence of GBM in adults than in children.
According to the Central Brain Tumor Registry of the United States
statistical report, GBM accounts for about 20% of all brain tumors
in the USA (CBTRUS, 1998-2002). Current treatment for patients with
GBM include, surgery followed by radiation and chemotherapy.
Despite intensive research the median survival for GBM patients
until the early 1990s was less than a year {Walker, 1978}. The
single most important advance in the treatment of these tumors over
the past 30 years has been the introduction of temozolomide,
initially in combination with external beam irradiation, and then
followed by repetitive cycles of temozolomide alone {Stupp, 2007}.
However, this has increased the overall median survival by only 75
days. Clearly, new and more efficient therapeutic approaches are
needed to improve GBM patient survival. Monoclonal antibodies
(mAbs), either armed (fused to immunotoxin [IT] or radioisotopes)
or unarmed, are presently a rapidly growing category of new drug
entities. This is well demonstrated by the large number of
mAb-based clinical trials currently in progress for brain tumor
patients. Boskovitz, A., Wikstrand, C. J., Kuan, C. T., Zalutsky,
M. R., Reardon, D. A., and Bigner, D. D. Monoclonal antibodies for
brain tumour treatment. Expert Opin Biol Ther, 4: 1453-1471, 2004.
More recently, genetically engineered single-chain variable-region
antibody fragments (scFvs), consisting of the heavy- and
light-chain variable regions (V.sub.H and V.sub.L) fused to toxins
and targeting antigens expressed specifically by brain tumor, are
under investigation. Archer, G. E., Sampson, J. H., Lorimer, I. A.,
McLendon, R. E., Kuan, C. T., Friedman, A. H., Friedman, H. S.,
Pastan, I. H., and Bigner, D. D. Regional treatment of epidermal
growth factor receptor vIII-expressing neoplastic meningitis with a
single-chain immunotoxin, MR-1. Clin Cancer Res, 5: 2646-2652,
1999. Because it is small, an scFv-IT fusion protein should have
greater tumor penetration than an intact IgG and therefore lead to
enhanced therapeutic efficacy {Pastan, 1995}.
[0004] The epidermal growth factor receptor (EGFR) is a 170-kDa,
transmembrane receptor tyrosine kinase (RTK). It is stimulated by
binding of its ligands, such as transforming growth factor
(TGF)-.alpha. or EGF, to its extracellular domain. Ligand binding
induces receptor dimerization and activates a tyrosine-specific
protein kinase activity {Ushiro, 1980} involved in controlling
epithelial cell growth and proliferation. Ultimately, the
receptor-ligand complexes are internalized, and the EGFR signal is
terminated. EGFR overexpression is frequently observed in a wide
variety of human cancers, including breast {Klijn, 1992; Osaki,
1992}, lung {Pavelic, 1993}, head and neck {Rubin Grandis, 1996},
prostate {Fox, 1994}, bladder {Chow, 2001}, colorectal {Yasui,
1988}, and ovarian carcinoma {Bartlett, 1996}, as well as brain
tumors {Arita, 1989; Libermann, 1984}. In contrast, the level of
EGFR in normal brain is undetectable or extremely low. EGFR is the
most frequently amplified gene in GBM {Fuller, 1992}. Correlating
with the gene amplification, the protein is overexpressed in about
60% to 90% of GBM cases. In the absence of gene amplification,
protein overexpression has also been observed in 12% to 38% of GBM
patients {Chaffanet, 1992}, which could be due to aberrant
translational and post-translational mechanisms. Preclinical
studies have shown that EGFR activation, in addition to protecting
cells from apoptosis, also induces several tumorigenic processes,
including proliferation, angiogenesis, and metastasis {Huang,
1999}.
[0005] EGFR gene amplification is often associated with gene
rearrangements. Several EGFR deletion mutants have been identified
{Rasheed, 1999}, the most common one being EGFRvIII, which is
present in 20% to 50% of GBMs with EGFR amplification. Wikstrand,
C. J., Fung, K. M., Trojanowski, J. Q., McLendon, R. E., and
Bigner, D. D. Antibodies and molecular immunology:
immunohistochemistry and antigens of diagnostic significance. In:
D. D. Bigner, R. E. McLendon, and J. M. Bruner (eds.), Russell and
Rubinstein's Pathology of the Nervous System, 6th edition, pp.
251-304. New York: Oxford University Press, 1998. The mutant
EGFRvIII contains a deletion of exon 2-7 of the EGFR gene, which is
characterized by an in-frame deletion of 801 base pairs of the
coding region {Sugawa, 1990}. This deletion creates a novel glycine
residue at the fusion junction at position 6, between amino acid
residues 5 and 274, generating a tumor-specific protein sequence
that is expressed specifically on tumor cells but not on normal
tissues. EGFRvIII is a constitutively active RTK which is not
further activated by EGFR ligands. Batra, S. K., Castelino-Prabhu,
S., Wikstrand, C. J., Zhu, X., Humphrey, P. A., Friedman, H. S.,
and Bigner, D. D. Epidermal growth factor ligand-independent,
unregulated, cell-transforming potential of a naturally occurring
human mutant EGFRvIII gene. Cell Growth Differ, 6: 1251-1259, 1995.
EGFRvIII is widely expressed in malignant gliomas {Humphrey, 1990}
and carcinomas. including head and neck {Sok, 2006} and breast.
Wikstrand, C. J., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey,
P. A., Kurpad, S, N., McLendon, R. E., Moscatello, D., Pegram, C.
N., Reist, C. J., and et al. Monoclonal antibodies against EGFRvIII
are tumor specific and react with breast and lung carcinomas and
malignant gliomas. Cancer Res, 55: 3140-3148, 1995. Overexpression
of EGFRvIII induces resistance in glioma cells to commonly used
chemotherapeutic agents {Nagane, 1998}.
[0006] Monoclonal antibodies targeting either the wild-type EGFR
(EGFRwt) or EGFRvIII have been developed. One of them, D2C7, a
murine IgG1.kappa., was developed by our group. The D2C7 hybridoma
recognizes both the EGFRwt and the tumor-specific EGFRvIII
receptors {Boskovitz, 2005}.
[0007] There is a continuing need in the art for effective means of
treating brain tumors and prolonging life of affected patients.
SUMMARY OF THE INVENTION
[0008] According to one embodiment of the invention a single chain
variable region antibody is provided. The antibody binds with a
binding affinity that is at least 5.times.10.sup.8 M.sup.-1 as
measured by surface plasmon resonance to both (a) EGFR found on
normal human cells and (b) EGFR variant III mutant.
[0009] According to another embodiment a method is provided of
treating a tumor in a human. A single chain variable region
antibody is administered to the human. The antibody binds with a
binding affinity that is at least 5.times.10.sup.8 M.sup.-1 as
measured by surface plasmon resonance to both (a) EGFR found on
normal human cells and (b) EGFR variant III mutant. Tumor cells are
thereby killed.
[0010] According to yet another embodiment of the invention a
monoclonal antibody is provided that binds with a binding affinity
that is at least 5.times.10.sup.8 M.sup.-1 to both (a) EGFR found
on normal human cells and (b) EGFR variant III mutant. The antibody
has a V.sub.H sequence as shown in FIG. 1A (SEQ ID NO: 1), a
V.sub.L sequence as shown in FIG. 1B (SEQ ID NO: 2), or CDR1, CDR2,
and CDR3 regions as shown in FIG. 1A (SEQ ID NO: 3, 4, 5) and lB
(SEQ ID NO: 6, 7, 8).
[0011] Yet another embodiment of the invention provides a
monoclonal antibody that binds with a binding affinity that is at
least 1.times.10.sup.8 M.sup.-1 to both (a) EGFR found on normal
human cells and (b) EGFR variant III mutant. The antibody is
selected from the group consisting of F2A2 and B10B11.
[0012] An additional embodiment of the invention provides a method
of determining a therapeutic plan to treat a tumor in a human.
Tissue of the tumor is contacted with an antibody that binds with a
binding affinity that is at least 1.times.10.sup.8 M.sup.-1 to both
(a) EGFR found on normal human cells and (b) EGFR variant III
mutant. The amount of cells in the tissue that bind to the antibody
is determined. Greater amounts of cells which bind are a positive
factor to recommend using the antibody therapeutically for the
patient.
[0013] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with methods and reagents for treating brain tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A-1B. Deduced amino acid sequence of D2C7 scFv cloned
from D2C7 hybridoma. V.sub.H (FIG. 1A) and V.sub.L (FIG. 1B)
antigen-binding regions of D2C7 scFv. Amino acid numbering and CDR
(underlined) delimitation were determined according to the IMGT
database.
[0015] FIG. 2. Schematic of D2C7 (scdsFv)-PE38 KDEL. The arrow
marks the proteolytic site of PE for activation. S--S shows the
disulfide bond linkage between the Fv fragments. II, PE domain II
for translocation; III, PE domain III for ADP-ribosylation of EF2,
KDEL, for increased endoplasmic reticulum retention.
[0016] FIG. 3A-3B. Biacore analysis of D2C7 (scdsFv)-PE38 KDEL.
Binding kinetics and affinity constants of D2C7 (scdsFv)-PE38 KDEL
for EGFRwt (FIG. 3A) and EGFRvIII (FIG. 3B) were determined by
surface plasmon resonance against bacterially expressed recombinant
EGFRwt or EGFRvIII extracellular domain proteins. The association
and dissociation rates from the sensogram were
KA=6.3.times.10.sup.8 M.sup.-1 and KD=1.6.times.10.sup.-9 M against
EGFRwt and KA=7.8.times.10.sup.8 M.sup.-1 and
KD=1.3.times.10.sup.-9 M against EGFRvIII.
[0017] FIG. 4A-4C. Flow cytometric analysis of D2C7 (scdsFv)-PE38
KDEL immunotoxin to determine reactivity of the D2C7 IT. (FIG. 4A)
Parental NR6 (NIH 3T3 murine fibroblast) cells used as control.
Indirect FACS analysis demonstrates the reactivity of D2C7
(scdsFv)-PE38 KDEL immunotoxin with cells expressing (FIG. 4B)
EGFRwt (NR6W) or (FIG. 4C) EGFRvIII (NREM). Cells were stained with
D2C7 (scdsFv)-PE38 KDEL (grey open peaks) or a non-specific scFv
(anti-Tac-PE38 KDEL) control (filled black peaks).
[0018] FIG. 5A-5C. In vitro cytotoxicity assay of D2C7
(scdsFv)-PE38 KDEL on (FIG. 5A) NR6, (FIG. 5B) NR6W, and (FIG. 5C)
NR6M cells. The cytotoxic effect of D2C7-PE38 (.tangle-solidup.)
and D2C7 (scdsFv)-PE38 KDEL ( ) was compared to that of an
established EGFRvIII-specific scFv immunotoxin, MR1-1-(scdsFv)-PE38
KDEL (x). A non-specific scFv anti-Tac-PE38 (.box-solid.) was used
as a control. At least three different assays were performed for
each cell line, and results from one representative experiment are
shown.
[0019] FIG. 6A-6E. In vitro cytotoxicity of F2A2 on A431P and
D270MG (FIG. 6A-6B) and on NR6, NR6W, and NR6M (FIG. 6C-6E).
[0020] FIG. 7A-7V. Expression of tumor antigens and CD133 analyzed
by flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
[0021] It is a discovery of the present inventors that antibodies
can bind with comparable and high affinities to both EGFR found on
normal cells and to EGFR variant III. By virtue of binding to both
forms of EGFR, these antibodies can induce cytotoxicity in a higher
percentage of cells within a tumor. Moreover, high affinity binding
of the antibodies to the cell surface receptors permits and
enhances antibody internalization, again increasing their cytotoxic
effect.
[0022] Antibodies which have been identified which have excellent
properties in this regard include D2C7, F2A2, B10B11, and H11 (Life
Span Biosciences, Inc. Seattle, Wash.). These are mouse monoclonal
antibodies produced by hybridomas. These antibodies can be
"converted" into other forms, for example, humanized, chimeric, and
single chain variable region antibodies. These conversions are well
known in the art and typically involve cloning of the antibody
encoding genes from the hybridomas which produced the mouse
monoclonal antibodies. If the VH or VL or the CDR region sequences
remain the same as in the original monoclonal antibody, then the
antibody may have retain binding specificity.
[0023] Such antibodies and "converted" antibodies can be bound,
either covalently or non-covalently, to other useful moieties. For
example, they can be conjugated to radionuclides or radioactive
moieties. They can be joined to a biological toxin, such as
Pseudomanas exotoxin A, ricin, or diphtheria toxin. They can be
conjugated to chemotherapeutic agents. They can be joined to other
antibodies. Attachments of the antibodies to other moieties can
occur by means of genetic engineering, if the other moiety is a
protein, so that a fusion protein is produced in a host cell. The
attachments may be done chemically, in vitro. The attachments may
be covalent or non-covalent. Non-covalent attachments preferably
use strong biological specific binding pairs to achieve strong
attachments. For diagnostic purposes, the antibodies can be
attached to chromophores, or other easily detectable moieties.
[0024] Other moieties which can be attached to the antibodies
include those to provide additional beneficial properties. For
example, a KDEL (lys-asp-glu-leu) tetra-peptide can be added at the
carboxy-terminus of the protein to provide retention in the
endoplasmic reticulum. Variants such as DKEL, RDEL, and KNEL which
function similarly can also be used.
[0025] Binding affinities can be measured by any means known in the
art. One particular method employs surface plasmon resonance.
Binding affinities within one log for wild-type and mutant EGFRvIII
are desirable, as are those within 50%, 75%, and 90% of each other.
Binding to cells can be measured, for example by flow cytometry.
Association and dissociation rates can be determined. Affinity
constants can be calculated. The kinetics of binding may be a
significant factor in cytotoxicity in the body. Binding affinities
may be at least 1.times.10.sup.8 M.sup.-1, at least
5.times.10.sup.8 M.sup.-1, at least 1.times.10.sup.9 M.sup.-1, or
at least 5.times.10.sup.9 M.sup.-1. Techniques can be used to
"affinity mature" i.e., improve affinity of, candidate
antibodies.
[0026] Tumors which can be treated include those in which at least
one EGFRvIII allele is present. These may be found in breast, head
and neck, brain, glioblastoma multiforme, astrocytoma, lung, or
other tumors. It may be desirable to determine the presence of such
an allele prior to therapy. This can be done using a
oligonucleotide-based technique, such as PCR, or using an
immunological technique, such as immunohistochemistry. It may be
desirable to determine the amount, fraction, ratio, or percentage
of cells in the tumor which express EGFR and/or EGFRvIII. The more
cells which express EGFR on their surfaces, the more beneficial
such antibody therapy is likely to be.
[0027] Antibodies and antibody constructs and derivatives can be
administered by any technique known in the art. Compartmental
delivery may be desirable to avoid cytotoxicity for normal tissues
that express EGFR. Suitable compartmental delivery methods include,
but are not limited to delivery to the brain, delivery to a
surgically created tumor resection cavity, delivery to a natural
tumor cyst, and delivery to tumor parenchyma.
[0028] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
Materials and Methods
[0029] Cell Lines.
[0030] Cell lines expressing EGFRwt used were the human epidermoid
carcinoma cell line A431 {Merlino, 1984} and the murine Swiss 3T3
mouse fibroblast cell line EGFRwt transfectant NR6W {Batra, 1995}.
Cell lines transfected to express EGFRvIII included the murine
Swiss 3T3 mouse fibroblast cell line-derived transfectant NREM
{Batra, 1995}. The parental murine Swiss 3T3 mouse fibroblast cell
line, NR6, was used as control. All cell lines were cultured in
complete zinc option-10% fetal bovine serum (FBS) (Richter's zinc
option; Invitrogen, San Diego, Calif.) and passed at confluence
using 0.05% Trypsin-EDTA (Invitrogen).
[0031] Disaggregation of Xenograft Tumor Samples.
[0032] Xenograft tissues from malignant glioma (D256MG, D270MG,
D2159MG) obtained under sterile conditions from the Duke animal
facility were prepared for cell culture in a laminar flow hood with
a sterile technique. Tumor material was finely minced with scissors
and added to a trypsinizing flask with approximately 10 ml of 100
.mu.g Liberase (Roche Indianapolis, Ind.). This mixture was stirred
at 37.degree. C. for 10 min, and a cell-rich supernatant was
obtained. The dissociated cells were filtered through a steel sieve
with 100 mesh wire. The reaction was inhibited with the addition of
the ovomucoid solution. The cells were washed with complete medium
and pelleted at 1000 rpm for 5 min. The cell suspension was further
treated with Ficoll-Hypaque to remove any red blood cells and then
washed once in complete media. The cells were cultured and passaged
until sufficient numbers were obtained (first adherent population,
p0; subsequent passages, p1, p2, and so forth). The attached cells
were harvested with 0.05% Trypsin-EDTA.
[0033] Cloning of Variable Heavy (V.sub.H) and Variable Light
(V.sub.1) Domains of the D2C7 mAb.
[0034] Total cellular mRNA was isolated from 10.sup.6 hybridoma
cells by using a Dynabeads, mRNA direct kit (Invitrogen). V.sub.H
and V.sub.L cDNAs of the D2C7 mAb were obtained by a RACE method
using a SMART RACE cDNA amplification kit (Clontech, Palo Alto,
Calif.). In brief, adaptor-ligated cDNA was generated from 300 ng
of the mRNA by using PowerScript Reverse Transcriptase and SMART II
A oligonucleotide (Clontech), along with 12 .mu.M each of 3' end
primers designed to anneal the heavy-chain (HC) and light-chain
(LC) constant region sequence of immunoglobulin (mouselgGl/2:
5'-CTGGACAGGGATCCAGAGTTCCA-3' (SEQ ID NO: 9) and mouseLC:
5'-CTCATTCCTGTTGAAGCTCTTGAC-3'; SEQ ID NO: 10). The primers covered
the constant region sequences registered in the Kabat database. The
prepared cDNAs were used as the templates for PCR reactions between
5' end primer which binds to the adaptor sequence and the
immunoglobulin HC- and LC-specific 3' end primer specified above.
The obtained sequences were aligned and verified according to the
Kabat alignment scheme. The V.sub.H domain was fused to the V.sub.L
domain by a 15-amino-acid peptide (Gly.sub.4Ser).sub.3 (SEQ ID NO:
11) linker by PCR. The D2C7 scFv fragment was cloned into pRK79
vector using a T4 DNA ligase kit (Pierce Biotechnology, Rockford,
Ill.). The D2C7 (scdsFv) construct was obtained by mutating
residues 44 of V.sub.H and 100 of V.sub.L by site-directed
mutagenesis using a QuickChange Multi-Site Directed Mutagenesis Kit
(Stratagene, La Jolla, Calif.). The D2C7-(scdsFv)-PE38 KDEL IT was
obtained by ligating the D2C7 (scdsFv) PCR fragment into pRB199
vector, and the sequence was verified. The MR1-1-(scdsFv)-PE38 KDEL
IT was obtained by ligating the MR1-1 (scdsFv) PCR fragment into
pRB 199 vector, and the sequence was verified.
[0035] Preparation of Recombinant Immunotoxins.
[0036] The different D2C7 ITs were generated by fusing the specific
scFv with the sequences for domains II and III of Pseudomonas
exotoxin A (PE38) according to the protocol described previously
{Buchner, 1992}. The specific scFv IT was expressed under control
of the T7 promoter in E. coli BL21 (.lamda. DE3) (Stratagene, La
Jolla, Calif.). All recombinant proteins accumulated in the
inclusion bodies. The ITs were then reduced, refolded, and further
purified as a monomer (64 kDa) by ion exchange and size exclusion
chromatography to greater than 95% purity.
[0037] Surface Plasmon Resonance (Biacore Analysis).
[0038] Binding kinetic profiles of purified D2C7-(scdsFv)-PE38 KDEL
IT were measured by surface plasmon resonance by using a Biacore
3000 biosensor system (Pharmacia, Uppsala, Sweden). As an antigen,
either EGFRwt extracellular domain (ECD) or EGFRvIII ECD proteins
were immobilized on the surface of the CM5 sensor chip at pH 5.5.
Test samples were diluted in running buffer (10 mM HEPES/150 mM
NaCl/3.4 mM EDTA, pH 7.4) and passed over the chip at
concentrations from 25 to 200 nM. The association and dissociation
rate constants and the average affinity were determined by using
the nonlinear curve-fitting BIAevaluation software (Pharmacia).
[0039] Flow Cytometry.
[0040] Indirect FACS analysis was performed with D2C7-(scdsFv)-PE38
KDEL IT. Briefly, 1.times.10.sup.6 cells (NR6, NR6W, or NREM) were
suspended in 500 .mu.l of PBS (Invitrogen) containing 5% FBS
(Invitrogen) (5% FBS/PBS). The D2C7-(scdsFv)-PE38 KDEL IT or
negative control, anti-Tac(scFv)-PE38 (a gift from Dr. Ira Pastan),
was added to the cells at a concentration of 10 .mu.g/ml, and the
samples were incubated for 40 min. After washing, cells were
incubated with rabbit anti-Pseudomonas exotoxin A antibody (Sigma,
St. Louis, Mo.) followed by labeling with FITC-conjugated goat
anti-rabbit IgG antibody (Zymed, South San Francisco, Calif.). To
prevent internalization of target antigens during assays, all the
reagents and buffers were kept on ice, and experiments were
performed at 4.degree. C. Stained cells were analyzed on a Becton
Dickinson FACSort instrument equipped with CellQuest software
(Becton Dickinson, San Jose, Calif.).
[0041] In Vitro Cell Killing Assay.
[0042] The cytotoxicity of the ITs on cultured cell lines and cells
isolated from xenografts was assayed by inhibition of protein
synthesis as described previously {Beers, 2000}. Cells were seeded
in 96-well plates at a density of 2.times.10.sup.4 cells per well
in 200 .mu.l of complete zinc option medium 24 h before the assay.
Immunotoxins were serially diluted to achieve a final concentration
of 0.01 to 1000 ng/ml in PBS containing 0.2% bovine serum albumin
(BSA; 0.2% BSA/PBS), and 10 .mu.l of diluted toxin was added to
each well. Plates were incubated for 20 h at 37.degree. C. and then
pulsed with 1 .mu.Ci/well of L-[4,5-.sup.3H]leucine (Amersham
Biosciences, Buckinghamshire, UK) in 25 .mu.l of 0.2% BSA/PBS for 3
h at 37.degree. C. Radiolabeled cells were captured on filter-mats
and counted in a MicroBeta scintillation counter (PerkinElmer,
Shelton, Conn.). The cytotoxic activity of an IT was defined by
IC.sub.50, which was the toxin concentration that suppressed
incorporation of radioactivity by 50% as compared to the
radioactivity measured in cells that were not treated with
toxin.
[0043] Determination of Nonspecific Toxicity in Mice.
[0044] The single-dose mouse LD.sub.40 was determined by using
female BALB/c mice (6-8 weeks old, 20 g), which were given a single
intraperitoneal (i.p.) injection of different doses of
D2C7-(scdsFv)-PE38 KDEL IT (0.25 to 1.25 mg/kg) diluted in 200
.mu.l of PBS containing 0.2% human serum albumin (PBS-HSA). Mice
were observed for 2 weeks following IT injection.
[0045] In Vivo Tumor Model.
[0046] Female athymic nude mice (approximately 20 g body weight,
4-6 weeks of age) were injected subcutaneously (s.c.) in the right
flank with 3.times.10.sup.6 A431 or NREM cells suspended in 50
.mu.l of PBS. A total of 8 to 10 mice per arm were randomly
selected for inoculation when the implanted tumors reached a median
tumor volume of 200 to 300 mm.sup.3. Mice were treated with three
doses of 0.3 mg/kg of D2C7-(scdsFv)--PE38 KDEL IT or
MR1-1-(scdsFv)-PE38 KDEL IT diluted in 0.2% PBS-HSA, by i.p.
injections every other day. The control mice were handled in the
same manner and treated with 0.2% PBS-HSA. Tumors were measured
twice weekly with a handheld vernier caliper, and the tumor volumes
were calculated in cubic millimeters by using the formula:
([length].times.[width.sup.2])/2. Animals were tested out of the
study when tumor volume met both of the following criteria: 1)
larger than 1000 mm.sup.3, and 2) 5 times its original treatment
size.
[0047] Assessment of Response.
[0048] The response of the s.c. xenografts was assessed by delay in
the growth of tumor in mice treated with drug as compared with
growth in control mice (T-C). The growth delay was the difference
between the median times required for tumors in treated (T) and
control (C) mice to reach five times the size at the initiation of
therapy and at least greater than 1000 mm.sup.3. Tumor regression
was defined as a decrease in tumor volume over two successive
measurements.
[0049] Statistical analysis was performed using the Wilcoxon
rank-sum test for growth delay and Fisher's exact test for tumor
regressions as previously described {Friedman, 1994}.
Example 2
Cloning of the V.sub.H and V.sub.L Domain of D2C7IgG1.kappa.
[0050] V.sub.H and V.sub.L cDNAs were isolated from the D2C7
hybridoma by a RACE method as described in "Materials and Methods."
The heavy-chain and light-chain variable domains were cloned and
sequenced. The amplified V.sub.H and V.sub.L fragments were
approximately 360 and 321 bp, respectively. The deduced amino acid
sequences of the D2C7 V.sub.H and V.sub.L domains are shown in FIG.
1. Sequence analysis of the V.sub.H and V.sub.L amino acids, using
the database of germ-line genes
(http://www.ncbi.nlm.nih.gov/igblast/), revealed that the sequences
were derived from different germ-line V genes with a similarity of
70% to 75%.
Example 3
Construction, Expression, and Purification of D2C7-(scdsFv)-PE38
KDEL Immunotoxin
[0051] The carboxyl terminus of the D2C7 V.sub.H domain was
connected to the amino terminus of the V.sub.L domain by a
15-amino-acid peptide (Gly.sub.4Ser).sub.3 linker (SEQ ID NO: 11).
In order to obtain a stable IT, it is essential to ensure that
during renaturation V.sub.H is positioned near V.sub.L. This was
achieved by mutating a single key residue in each chain to
cysteine, for the stabilizing disulfide bond to form. On the basis
of predictions using molecular modeling and empirical data with
other dsFv-recombinant ITs, we chose one amino acid in each chain
to mutate to cysteine {Reiter, 1996}. These are residues 44 in the
framework region 2 (FR2) of V.sub.H and 100 in the FR4 of V.sub.L
(according to the Kabat numbering). Thus, we prepared an Fv that
contains both a peptide linker and a disulfide bond generated by
cysteine residues that replace Ser44 of V.sub.H and Gly100 of
V.sub.L. The D2C7 (scdsFv) PCR fragment was then fused to DNA for
domains II and III of Pseudomonas exotoxin A. The version of
Pseudomonas exotoxin A used here, PE38 KDEL, has a modified C
terminus which increases its intracellular retention, in turn
enhancing its cytotoxicity. The D2C7-(scdsFv)-PE38 KDEL (FIG. 2)
was expressed in E. coli under the control of T7 promoter and
harvested as inclusion bodies. The IT was refolded and purified as
described in "Materials and Methods."
Example 4
Antigen Binding Characteristic of D2C7-(scdsFv)-PE38 KDEL
Antibody
[0052] The antigen-binding capability of the D2C7-(scdsFv)-PE38
KDEL IT was assessed by surface plasmon resonance (Biacore). The
purified D2C7-(scdsFv)-PE38 KDEL IT was applied to sensor chips
that were coated with either purified recombinant EGFRwt (FIG. 3A)
or EGFRvIII (FIG. 3B) ECD proteins. The D2C7-(scdsFv)-PE38 KDEL IT
bound to both the EGFRwt- and the EGFRvIII-ECD-protein-coated
chips. Values of the on rates and off rates were determined for
four different concentrations of the IT. The association and
dissociation constants of D2C7-(scdsFv)-PE38 KDEL IT on the EGFRwt-
and EGFRvIII-coated chips were K.sub.A=6.3.times.10.sup.8 M.sup.-1
and K.sub.D=1.6.times.10.sup.-9 M and K.sub.A=7.8.times.10.sup.8
M.sup.-1 and K.sub.D=1.3.times.10.sup.-9 M, respectively. Thus, the
cloned D2C7-(scdsFv)-PE38 KDEL IT binds with similar kinetics to
both the wild-type and the mutant EGFR proteins.
[0053] To determine whether the D2C7-(scdsFv)-PE38 KDEL IT is able
to bind to native EGFRwt and EGFRvIII proteins, indirect flow
cytometric analysis was performed as shown in FIG. 4. FACS analysis
revealed that the D2C7-(scdsFv)-PE38 KDEL IT is able to bind to
both the EGFRwt-expressing NR6W cells (FIG. 4B) and the
EGFRvIII-expressing NR6M cells (FIG. 4C). The parental NR6 cells
(FIG. 4A) were used as negative control, which confirmed the
binding specificity of this toxin. These results demonstrate that
the D2C7-(scdsFv)-PE38 KDEL IT is able to bind both to the purified
EGFRwt and the EGFRvIII proteins on a chip and to the native
protein molecules expressed on the surface of transfected
cells.
Example 5
Cytotoxicity of D2C7-(scdsFv)-PE38 KDEL IT on Transfected Cells and
Cancer Cell Lines
[0054] We next examined the effects of D2C7-(scdsFv)-PE38 KDEL IT
on EGFRwt- or EGFRvIII-transfected NR6W and NR6M mouse cell lines,
respectively. The ability of the D2C7-(scdsFv)-PE38 KDEL IT to
inhibit protein synthesis was used as a measure of its cytotoxic
effect. The cytotoxicity of D2C7-(scdsFv)-PE38 KDEL IT was compared
to that of a known EGFRvIII-specific IT, MR1-1-(scdsFv)-PE38 KDEL
(MR1-1) {Beers, 2000} and that of the parental D2C7-PE38 IT, which
lacks the disulfide-stabilized linkage and the endoplasmic
reticulum retention signal, KDEL. We initially evaluated the
cytotoxicity of the various ITs to the EGFRwt-expressing NR6W
cells. The IC.sub.50 of D2C7-(scdsFv)-PE38 KDEL IT on NR6W cells
(FIG. 5B) was more than 100-fold lower than that of the MR1-1 IT.
Even on the NR6M cells (FIG. 5C), a well-established model for
MR1-1 cytotoxicity {Beers, 2000}, D2C7-(scdsFv)-PE38 KDEL IT had a
1.4-fold lower IC.sub.50 value than that of MR-1-1 IT. The
IC.sub.50 of D2C7-(scdsFv)-PE38 KDEL IT was approximately 7-fold
lower than that of the parental D2C7-PE38 IT, against both NR6W and
NR6M cells (FIGS. 5B and 5C). All of the ITs exhibited no
cytotoxicity against the parental NR6 cells (FIG. 5A). The
cytotoxic effects of D2C7-(scdsFv)-PE38 KDEL IT were also tested on
various EGFRwt- and EGFRvIII-positive human cancer cell lines. The
A431P epidermoid carcinoma cell line overexpresses the wild-type
EGFR protein, and the three glioblastoma cell lines D2159MG, D270MG
and D256MG express both the EGFRwt and EGFRvIII proteins. As shown
in Table 1, the D2C7-(scdsFv)-PE38 KDEL IT was more effective than
the parental IT, D2C7-PE38, and the EGFRvIII-targeted IT, MR1-1, in
killing all the human cancer cell lines tested.
TABLE-US-00001 TABLE 1 Cytotoxicity of D2C7 and MR1-1 immunotoxins
toward various cell lines D2C7- MR1-1- (scdsFv)- (scdsFv)-
D2C7-PE38 PE38KDEL PE38KDEL Cell Line Cancer type IC.sub.50 ng/ml
IC.sub.50 ng/ml IC.sub.50 ng/ml A431 Epidermoid 1.0 0.170 8.0
D2159MG Glioblastoma 1.8 0.360 0.800 D270MG Glioblastoma 1.8 0.360
0.580 D256MG Glioblastoma 5.1 0.520 1.9 NOTE: All the cell lines
are of human origin. Cytotoxicity data are given as IC.sub.50
value, the concentration of immunotoxin that causes a 50%
inhibition of protein synthesis after a 20-h incubation with
immunotoxin.
Example 6
Nonspecific Toxicity of D2C7-(scdsFv)-PE38 KDEL IT in Mice
[0055] The nonspecific toxicity of D2C7-(scdsFv)-PE38 KDEL IT was
evaluated in BALB/C mice. Groups of 10 mice were given single i.p.
injections of escalating doses of the IT. The mice were monitored
for weight loss, signs of distress, or death for 14 days
postinjection. The mortality data is shown in Table 2. Almost all
of the deaths occurred within 72 h after treatment. We calculated
the LD.sub.10 of D2C7-(scdsFv)-PE38 KDEL IT to be approximately
0.3125 mg/kg. The observed toxicity in mice is due to nonspecific
uptake of the IT by the liver.
TABLE-US-00002 TABLE 2 Toxicity of D2C7-(scdsFv)-PE38KDEL
immunotoxin administered to BALB/C mice Dose (mg/kg) Mortality 0.25
0/10 0.5 4/10 0.75 8/10 1.0 9/10 1.25 10/10 NOTE: Groups of 10
BALB/C mice were injected i.p. with 200 .mu.l of escalating doses
of the IT diluted in 0.2% PBS-HSA. Animals were observed for 14
days. Mortality is expressed as number of dead mice/total number of
animals in treatment group.
Example 7
Anti-Tumor Activity of D2C7-(scdsFv)-PE38 KDEL IT In Vivo
[0056] To evaluate the anti-tumor activity of the ITs, animals
bearing A431 tumors were treated with three doses of either
D2C7-(scdsFv)-PE38 KDEL or MR1-1 (MR1-1 scFv binds with a lower
affinity to the wild-type EGFR, unpublished data) at 0.3 mg/kg
concentration. Relative to the control group, the
D2C7-(scdsFv)-PE38 KDEL-treated mice showed statistically
significant growth delay, where T-C was 22 days (p<0.001) (FIG.
6A and Table 3). In contrast, the growth delay in the MR1-1 treated
group was approximately 4 days (p=0.01) (FIG. 6A and Table 3). The
A431 tumors regressed in 7 of 8 mice treated with
D2C7-(scdsFv)-PE38 KDEL, whereas no tumor regression was observed
in the MR1-1-treated A431 group. In an in vivo xenograft model of
NREM tumors, both D2C7-(scdsFv)-PE38 KDEL and MR1-1 elicited
similar responses (FIG. 6B and Table 3). In comparison to the
control group, the D2C7-(scdsFv)-PE38 KDEL-treated group and the
MR1-1-treated group had a growth delay of 10 and 9 days,
respectively, with a p value of <0.001 (FIG. 6B and Table 3).
Tumor regression was seen in 10 of 10 mice in the
D2C7-(scdsFv)-PE38 KDEL-treated group, but only 6 of 10 mice in the
MR1-1-treated group displayed tumor regression.
TABLE-US-00003 TABLE 3 In vivo anti-tumor activity of
D2C7-(scdsFv)- PE38KDEL and MR1-1-(scdsFv)-PE38KDEL D2C7-(scdsFv)-
MR1-1-(scdsFv)- Tumor Control PE38KDEL PE38KDEL A431 T - C (days)
22.177 3.6875 P <0.001 0.01 Regressions 0/8 7/8 0/9 NR6M T - C
(days) 10.6565 9.1835 P <0.001 <0.001 Regressions 0/10 10/10
6/10 NOTE: Groups of 8 to 10 nude mice bearing A431 or NR6M tumors
were treated with 0.3 mg/kg of the IT diluted in 0.2% PBS-HSA. T -
C denotes the delay in tumor growth in mice treated with IT as
compared with control mice. Tumor regression was defined as a
decrease in tumor volume over two successive measurements.
Example 8
[0057] In this study, we have focused on the in vitro and in vivo
characterization of a dual-specificity (EGFRwt/EGFRvIII) IT,
D2C7-(scdsFv)-PE38 KDEL. The results from the in vitro cytotoxicity
assays showed that D2C7-(scdsFv)-PE38 KDEL is highly effective in
killing a variety of EGFRwt- or EGFRvIII-expressing human tumor
cell lines. In an animal model of EGFRwt tumors, when administered
every other day for a total of three doses at a concentration of
0.3 mg/kg, the IT inhibited tumor growth, leading to a decrease in
tumor volume. In addition, D2C7-(scdsFv)-PE38 KDEL was also found
to be as effective as that of an established, affinity-matured,
EGFRvIII-targeted IT, MR1-1, in both in vitro and in vivo assays.
To the best of our knowledge, this is the first report
demonstrating that a dually specific IT can target both the
wild-type EGFR and the mutant EGFRvIII.
[0058] Different versions of the D2C7 IT were constructed that
vastly improved its efficacy. The parental D2C7-PE38 IT had only a
15-amino-acid peptide linker between V.sub.H and V.sub.L. The
subsequent version of the IT had both a 15-amino-acid peptide
linker and a disulfide linkage between V.sub.H and V.sub.L
[D2C7-(scdsFv)-PE38]. The disulfide linkage helps the IT to fold
better, in turn providing improved stability. The
D2C7-(scdsFv)-PE38 IT showed a 2- to 4-fold decrease in IC.sub.50
values compared to the parental D2C7-PE38 IT (data not shown).
Further, when the endoplasmic reticulum retention signal KDEL was
engineered into the final product [D2C7-(scdsFv)-PE38 KDEL], it
decreased the IC.sub.50 values of the IT on all of the cell lines
tested by an additional 2-fold. Thus, increasing the stabilization
and adding an intracellular retention signal enhanced the
efficiency of D2C7-(scdsFv)--PE38 KDEL when compared with that of
the parental D2C7-PE38 IT.
[0059] In the in vitro cytotoxicity assays with either the
EGFRvIII-transfected NR6M cells or EGFRvIII-expressing GBM cells,
D2C7-(scdsFv)-PE38 KDEL outperformed MR1-1. The difference in
IC.sub.50 values between D2C7-(scdsFv)-PE38 KDEL and MR1-1 was
higher in the EGFRwt- and EGFRvIII-co-expressing GBM cells than in
the EGFRvIII-expressing NR6M cells. The presence of both the
wild-type and the mutant EGFR proteins on the GBM cells provides a
higher concentration of targets on the cell surface for
D2C7-(scdsFv)-PE38 KDEL as opposed to a single target for MR1-1.
Hence, on the cells that express both the EGFRwt and EGFRvIII
proteins, D2C7-(scdsFv)-PE38 KDEL exhibited better cytotoxicity
than MR1-1. Also, a competition assay by Biacore analysis with
EGFRvIII ECD protein revealed that D2C7-(scdsFv)-PE38 KDEL and
MR1-1 bind to different epitopes (data not shown). Thus, the
availability of the epitopes on the cell surface for the ITs to
bind might also play a role in the observed difference in
cytotoxicity between D2C7-(scdsFv)-PE38 KDEL and MR1-1.
[0060] Several anti-EGFR mAbs that have demonstrated antitumor
activity against EGFR-expressing human tumor cells in mouse
xenograft models and/or culture have been developed {Laskin, 2004}.
Some of these anti-EGFR mAbs are in clinical trials for a variety
of human cancers, including head and neck, colorectal, pancreatic,
lung, renal cell or prostate carcinoma, or high-grade glioma
{Boskovitz, 2004; Laskin, 2004}. Anti-EGFRwt mAbs EGFR1, H17E2, and
mAb 425 were the first to be introduced in targeted radiotherapy
trials that involved systemic injection of radiolabeled mAb in
patients with malignant glioma {Brady, 1992; Epenetos, 1985;
Kalofonos, 1989}. The anti-EGFR mAbs that are currently in Phase II
trials for patients with high-grade glioma include
.sup.125I-labeled mAb 425 in combination with surgery, radiation
therapy and chemotherapy (Protocol IDs: 12555, NCT00589706), which
has already demonstrated an increase in median survival {Quang,
2004}. Further, a recombinant EGFR ligand (transforming growth
factor-.alpha.) Pseudomonas exotoxin fusion protein (TP-38) has
also been tested in Phase I clinical trials for treating malignant
gliomas {Sampson, 2008}.
[0061] Because of the truly tumor-specific nature of EGFRvIII, both
polyclonal antibodies and mAbs directed against this mutant form of
EGFR have been developed {Humphrey, 1990; Wikstrand, 1995}. The
development of mAbs and single-chain fragment antibody constructs
specific for the mutant EGFRvIII, including L8A4, Y10, P14, X32,
MR1, MR1-1, and 14E1, has been well described {Beers, 2000; Kuan,
1999; Kuan, 2000; Reist, 1995; Schmidt, 1999; Wikstrand, 1995;
Wikstrand, 1997}. Among the various antibody constructs, the one
with enormous potential is the MR1 single-chain antibody fragment,
as well as its affinity-matured derivative MR1-1 {Beers, 2000;
Kuan, 1999; Kuan, 2000}. MR1-1, which differs from the parental MR1
by three amino acid residues (one in V.sub.LCDR3 [F92W] and two in
V.sub.HCDR3 [S98P, T99Y]), has a 15-fold greater K.sub.D
(1.5.times.10.sup.-9 M) for the extracellular domain of EGFRvIII
than does MR1 {Kuan, 2000}. A Phase I clinical study with the MR1-1
IT is currently underway for the treatment of patients with
EGFRvIII-overexpressing GBM tumors. Due to the recurrent nature of
malignant gliomas, as well as the diversity of antigens populating
the glioma cell surface, innovative therapies are needed.
[0062] The EGFRvIII mutation occurs in 52% of all human GBMs and is
co-expressed in 50% to 60% of tumors that have EGFRwt amplification
{Frederick, 2000; Wikstrand, 1995}. Thus, it would be advantageous
to have antibodies that could target both EGFRwt and EGFRvIII
antigens for GBM therapy. This could be achieved by co-targeting
these two antigens with a bispecific scFv antibody, an scFv
antibody that has dual specificity. This could promote increased
targeting of the tumor over antibodies specific for a single
antigen. No bispecific scFv that can target both EGFRwt and
EGFRvIII has been reported, although reports have been published
describing two mAbs, mAb 528 and mAb 806, with dual specificity for
the wild-type and mutant EGFR proteins expressed on different cell
lines. The mAb 528 is an IgG2a antibody that was raised against
EGFR by using the epidermoid carcinoma cell line A431 {Masui,
1984}. This antibody binds both the EGFRwt expressed on A431 cells
and EGFRvIII expressed on U87MG..DELTA.2-7 {Perera, 2005}.
Monoclonal antibody 528 competes with EGF binding to the receptor
and inhibits the growth of EGFR-expressing cells both in vitro and
in vivo {Masui, 1984}. The IgG2b mAb 806 is an EGFR-specific
antibody that was raised in mice immunized with NR6 cells
transfected with EGFRvIII {Johns, 2002}. The mAb 806 binds EGFRvIII
with high affinity and EGFRwt at a low percentage (10%) on A431
cells {Johns, 2002}. A humanized form of the mAb 806 (ch806) was
used in Phase I clinical trials for patients with diverse tumor
types expressing EGFR {Scott, 2007}. Combination therapy with the
mAbs 528 and 806 was performed with xenografts expressing EGFRwt or
EGFRvIII. A significant decrease in tumor volume was observed when
the mAbs were administered in combination {Perera, 2005}. In our in
vivo models, the tumor growth inhibition by D2C7-(scdsFv)-PE38 KDEL
at 3 doses was comparable to the response observed with the mAbs
528 and 806 together, for a total of 6 doses. Hence, the
dual-specificity IT is likely more efficacious than the combined
mAb treatment. Moreover, treating brain tumors that co-express
EGFRwt and EGFRvI1I with D2C7-(scdsFv)-PE38 KDEL IT will address
the concern that expression of EGFRvIII may cause resistance to
EGFR antibody therapy. Thus, a single antibody with specificity
against two different tumor antigens eliminates the necessity for
multiple therapeutics to treat tumor.
[0063] In conclusion, we have created a dual specific-scFv molecule
that is capable of mediating selective in vitro and in vivo tumor
targeting. Further, we believe this to be the first significant
evidence of enhanced tumor targeting with high selectivity and
specificity by an antibody specific for two tumor-associated
antigens. Taken together, our results suggest that the bispecific
antibody D2C7-(scdsFv)-PE38 KDEL may be efficacious in vivo against
brain tumors.
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Sequence CWU 1
1
131117PRTMus musculus 1Glu Val His Leu Gln Gln Ser Gly Pro Glu Leu
Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Ser Phe Thr Gly Tyr 20 25 30Asn Met Asn Val Lys Gln Ser Asn Gly
Lys Ser Leu Glu Trp Ile Gly 35 40 45Asn Ile Asp Pro Tyr Tyr Gly Asp
Thr Asp Tyr Asp Gln Lys Phe Lys 50 55 60Gly Thr Leu Thr Ala Asp Lys
Ser Ser Asn Thr Val Tyr Met Gln Leu65 70 75 80Gln Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly 85 90 95Ala His Arg Asp
Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110Val Thr
Val Ser Ser 1152107PRTMus musculus 2Asp Ile Gln Met Thr Gln Ser Pro
Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys
Arg Thr Ser Glu Asn Ile Tyr Ile Tyr 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Ala Lys Thr
Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Gln Phe Ser Leu Lys Ile Asn Gly Leu Gln Pro65 70 75 80Glu Asp
Phe Gly Gly Tyr Tyr Cys Gln Gln His Tyr Gly Thr Pro Tyr 85 90 95Thr
Phe Gly Gly Gly Thr Lys Leu Glu Lys Lys 100 10535PRTMus musculus
3Gly Tyr Asn Met Asn1 5417PRTMus musculus 4Asn Ile Asp Pro Tyr Tyr
Gly Asp Thr Asp Tyr Asp Gln Lys Phe Lys1 5 10 15Gly511PRTMus
musculus 5Gly Ala His Arg Asp Tyr Tyr Ala Met Asp Tyr1 5
10611PRTMus musculus 6Arg Thr Ser Glu Asn Ile Tyr Ile Tyr Leu Ala1
5 1077PRTMus musculus 7Asn Ala Lys Thr Leu Ala Glu1 589PRTMus
musculus 8Gln Gln His Tyr Gly Thr Pro Tyr Thr1 5923DNAMus musculus
9ctggacaggg atccagagtt cca 231024DNAMus musculus 10ctcattcctg
ttgaagctct tgac 241115PRTArtificial Sequencesynthetic linker 11Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
1512747DNAArtficial sequencesingle chain variable region fragment
of F2A2 12gaagtgcagc tggtagagtc tgggggaggc ttagtgaggc ctggagggtc
cctgaaactc 60tcctgtgcag cctctggatt cactttcagt gactattaca tgtattgggt
tcgccagact 120ccggaaaaga ggctggagtg ggtcgcaagc attagtggtg
gtgatgatta cacctactac 180tcagagagtg tgaaggggcg attcaccatc
tccagagaca atgccaagaa caccctgtgc 240ctccaaatga acagtctgaa
gtctgacgac acagccatgt attactgtgt tagaggagag 300gggaggaact
gggacgacta tgctatggac tattggggtc aaggaacttc agtcaccgtc
360tcctcgggtg gtggcggttc aggcggaggt ggctctggcg gtggcggatc
ggatattgtg 420atgacccaaa ctccactctc cctgcctgtc agtcttggag
atcaagcctc catctcttgc 480agatctagtc agagccttgt acacactcat
ggacacacct atttacattg gcacctgcag 540aagccaggcc agtctccaaa
gctcctgatc tataaagttt ccaaccgatt ttctggggtc 600ccagacaggt
tcagtggcag tggatcaggg acagatttca cactcaagat cagcagagtg
660gaggctgagg atctgggagt ttatttctgc tctcaaagta cacatgttcc
tcggacgttc 720ggtggaggca ccaagctgga aatcaaa 74713249PRTArtificial
Sequencesingle chain variable region fragment F2A2 13Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr
Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40
45Ala Ser Ile Ser Gly Gly Asp Asp Tyr Thr Tyr Tyr Ser Glu Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Cys65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Asp Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Val Arg Gly Glu Gly Arg Asn Trp Asp Asp Tyr Ala
Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Ile Val Met Thr Gln Thr 130 135 140Pro Leu Ser Leu Pro Val
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys145 150 155 160Arg Ser Ser
Gln Ser Leu Val His Thr His Gly His Thr Tyr Leu His 165 170 175Trp
His Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys 180 185
190Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
195 200 205Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp 210 215 220Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val
Pro Arg Thr Phe225 230 235 240Gly Gly Gly Thr Lys Leu Glu Ile Lys
245
* * * * *
References