U.S. patent application number 15/342889 was filed with the patent office on 2017-05-25 for antibody-endostatin fusion protein and its variants.
The applicant listed for this patent is UNIVERSITY OF MIAMI. Invention is credited to Sherie Morrison, Joseph Rosenblatt, Seung-Uon Shin.
Application Number | 20170145110 15/342889 |
Document ID | / |
Family ID | 51259389 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170145110 |
Kind Code |
A1 |
Shin; Seung-Uon ; et
al. |
May 25, 2017 |
ANTIBODY-ENDOSTATIN FUSION PROTEIN AND ITS VARIANTS
Abstract
Methods of inhibiting the growth of tumors comprising
administering chimeric fusion molecules comprising endostatin
mutants and all or a portion of anti-Her2 or anti-EGFR
antibodies.
Inventors: |
Shin; Seung-Uon; (Miami,
FL) ; Rosenblatt; Joseph; (Miami, FL) ;
Morrison; Sherie; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MIAMI |
Miami |
FL |
US |
|
|
Family ID: |
51259389 |
Appl. No.: |
15/342889 |
Filed: |
November 3, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14254333 |
Apr 16, 2014 |
9611313 |
|
|
15342889 |
|
|
|
|
12665007 |
Aug 30, 2010 |
|
|
|
PCT/US2008/068434 |
Jun 26, 2008 |
|
|
|
14254333 |
|
|
|
|
60946245 |
Jun 26, 2007 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
G01N 2333/71 20130101; C07K 14/78 20130101; A61K 38/39 20130101;
C07K 16/32 20130101; A61K 2300/00 20130101; G01N 2333/70596
20130101; A61K 39/39558 20130101; A61K 45/06 20130101; C07K 2319/00
20130101; C07K 16/2863 20130101; A61K 31/00 20130101; G01N 33/57492
20130101; A61K 31/00 20130101; A61K 2039/505 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; C07K 14/78 20060101 C07K014/78; G01N 33/574 20060101
G01N033/574; A61K 39/395 20060101 A61K039/395; A61K 38/39 20060101
A61K038/39; C07K 16/28 20060101 C07K016/28; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of inhibiting the formation or growth of blood vessels
in a tumor comprising the steps of administering to an individual
who has been diagnosed with a tumor, a therapeutically effective
amount of a pharmaceutical composition comprising an isolated
chimeric fusion molecule, wherein the chimeric fusion molecule
comprises an anti-tumor antigen binding domain from an anti-HER2 or
anti-EGFR antibody and at least one human endostatin protein or
fragment thereof, wherein the at least one human endostatin protein
or fragment thereof comprises a proline to alanine amino acid
substitution at position 125 of human endostatin, wherein
administration of the pharmaceutical composition results in
reduction in the blood vessels in the tumor.
2. The method of claim 1, wherein the antigen binding domain
specifically binds one or more tumor antigens.
3. The method of claim 1, wherein the human endostatin protein or
fragment thereof comprise one or more NGR motifs (Asn-Gly-Arg)
and/or RGD (Arg-Gly-Asp) motifs.
4. The method of claim 3, wherein the one or more NGR motifs
(Asn-Gly-Arg) and RGD (Arg-Gly-Asp) motifs are located at the amino
(NH.sub.2--) terminal, and/or carboxy terminal (COOH--) and/or
amino acid positions 93-133 of human endostatin protein or fragment
thereof.
5. The method of claim 3, wherein the one or more NGR motifs
(Asn-Gly-Arg) and RGD (Arg-Gly-Asp) motifs are located at amino
acid positions 126-128 following the proline or alanine at position
125 of the human endostatin protein or fragment thereof.
6. The method of claim 1, wherein the antibody or fragment thereof,
is IgA, IgM, IgG, IgE, or IgD.
7. The method of claim 1, wherein the chimeric fusion protein is
administered to a patient, simultaneously and/or in separate
treatments with one or more of: cytoximab, sunitinib, sorafenib,
celebrex, MTOR inhibitors, AKT inhibitors, P13K inhibitors,
bevacizumab (Avastin), signal transduction inhibitors, tamoxifen,
toremifen, raloxifene, droloxifene, iodoxyfene, megestrol acetate,
anastrozole, letrazole, borazole, exemestane, flutamide,
nilutamide, bicalutamide, cyproterone acetate, goserelin acetate,
luprolide, finasteride, herceptin, methotrexate, 5-fluorouracil,
cytosine arabinoside, doxorubicin, daunomycin, epirubicin,
idarubicin, mitomycin-C, dactinomycin, mithramycin, cisplatin,
carboplatin, melphalan, chlorambucil, busulphan, cyclophosphamide,
ifosfamide, nitrosoureas, thiotephan, vincristine, taxol, taxotere,
etoposide, teniposide, amsacrine, irinotecan, topotecan, an
epothilone; a tyrosine kinase inhibitor, Iressa or OSI-774; an
angiogenesis inhibitor; an EGF inhibitor; a VEGF inhibitor; a CDK
inhibitor; a Her1/2 inhibitor and monoclonal antibodies directed
against growth factor receptors.
8. The method of claim 1, wherein the chimeric fusion protein is
administered in combination with and/or in separate treatments, one
or more antibodies comprising cytoximab, sunitinib, sorafenib,
celebrex, MTOR inhibitors, AKT inhibitors, P13K inhibitors,
bevacizumab (Avastin), signal transduction inhibitors, anti PDL1,
anti CTLA4, and anti her2 antibodies.
9. The method of claim 1, wherein the chimeric fusion protein is
administered in combination with and/or in separate treatments, one
or more anti-angiogenic factors comprising sunitinib, sorafenib,
and angiostatin.
10. A method of identifying treatment option for an individual
diagnosed with a tumor comprising the steps of: a) obtaining a
biopsy sample of the tumor; b) determining if a chimeric fusion
molecule comprising an anti-tumor antigen binding domain from an
anti-HER2 antibody or anti-EGFR antibody and at least one human
endostatin protein or fragment thereof, wherein the at least one
human endostatin protein or fragment thereof comprises a proline to
alanine amino acid substitution at position 125 of human
endostatin, inhibits vasculogenic mimicry in the tumor cells; and
c) if inhibition of vasculogenic mimicry is observed, then
identifying the chimeric fusion molecule as a useful treatment
option for the individual.
11. The method of claim 10, comprising a further step of
determining if the tumor cells exhibit vasculogenic mimicry between
steps a) and b).
12. A method of treatment of an individual diagnosed with a tumor
comprising the steps of combining: a) removing the tumor using
surgical or radiation treatment; and b) administration of a
chimeric fusion molecule comprising an anti-tumor antigen binding
domain from an anti-HER2 antibody or anti-EGFR antibody and at
least one human endostatin protein or fragment thereof, wherein the
at least one human endostatin protein or fragment thereof comprises
a proline to alanine amino acid substitution at position 125 of
human endostatin, wherein the removing the tumor is done before,
during or after the administration of the chimeric fusion molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/254,333, filed on Apr. 16, 2014, which in
turn is a continuation-in-part of U.S. patent application Ser. No.
12/665,007, filed on Dec. 16, 2009 (now abandoned), as a U.S.
National Stage under 35 U.S.C. 371 application of international
application No. PCT/US08/68434, filed on Jun. 26, 2008, which
claims the benefit of U.S. provisional application No. 60/946,245
filed Jun. 26, 2007, the disclosures of which are all herein
incorporated by reference in their entireties.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0002] This application contains, as a separate part of disclosure,
a Sequence Listing in computer-readable form (filename:
50556B_SeqListing.txt: 1,792 bytes, created Nov. 3, 2016), which is
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to compositions and methods for
targeting and modulating the activity of tumor cells. In
particular, the invention relates to chimeric fusion molecules
which have a tumor antigen targeting domain and an anti-tumor
effector function domain.
BACKGROUND
[0004] Anti-angiogenic tumor therapies have recently attracted
intense interest because of their broad-spectrum action, low
toxicity, and absence of drug resistance. Endostatin is a recently
characterized anti-angiogenic agent. Although the mechanism of
action of endostatin is not clear yet, the anti-tumor activity of
endostatin may be associated with inhibiting the proliferation and
migration of endothelial cells. In addition, endostatin may
down-regulate VEGF expression in tumor cells.
[0005] A number of animal experiments and human clinical trials
have been performed to assess the anti-tumor effect of endostatin.
Systemic administration of endostatin at 10 mg/kg suppressed the
growth of human renal cell cancer in a nude mouse xenograft model.
In early human phase I trials, endostatin administration at high
dose levels (240 mg/m.sup.2/day) in the range of active levels
established in tumor xenograft studies did not show any significant
detectable changes in biologic endpoints, such as urinary excretion
levels of VEGF and basic FGF. However, modest clinical benefit was
observed in three out of 15 patients. One patient with a pancreatic
neuroendocrine tumor had a minor tumor reduction, and disease in
two other patients briefly stabilized. Another human phase I trial
demonstrated that endostatin was well tolerated and did not induce
dose-limiting toxicity at dose-levels up to 600 mg/m.sup.2/day, but
little anti-tumor activity was seen in 25 patients, even at
circulating levels beyond those previously noted to be effective in
mouse models. Two patients (one with sarcoma, one with melanoma)
demonstrated minor and short-lived anti-tumor activity. The first
two phase I clinical trials proved that endostatin is a very safe
drug in a variety of dose schedules. However these results did not
demonstrate substantial endostatin anti-tumor activity. The dose
and schedules may have been suboptimal, and/or bulky disease in
late stage patients may not be optimally responsive to recombinant
human endostatin.
[0006] Anti-angiogenic gene therapy has been proposed as an
alternative way to continuously provide high concentrations of the
anti-angiogenic factors. Gene transfection of anti-angiogenic
agents using a viral vector can inhibit the growth of tumor in
several mouse models. Viral vectors, however, may cause
inflammation and immunological response on repeated injection, and
toxicity/safety considerations may preclude their use in humans in
the near future. Furthermore, use of gene-transduced hematopoietic
stem cells has been ineffective in an animal model, despite
sustained production of endostatin. Furthermore dosing of biologic
products using gene vectors is very difficult to standardize due to
variation in vector titer, transduction efficiency and expression
levels. There is, thus, a need in the art for improved anti-tumor
therapies.
SUMMARY
[0007] The invention provides methods and compositions for
targeting a chimeric molecule containing both (1) anti-angiogenic
agent and (2) a carrier domain such as all or a portion of an Ig
molecule to a tumor.
[0008] The advantages of the chimeric fusion molecules provide many
therapeutic advantages. For example, an increase in the half-life
of the endostatin molecule, ease of administration, presentation of
endostatin as a dimer, versatile targeting, increased activity
against lower her2 expressing tumors and duration/response rate
against 3+ tumors, increase in efficacy of endostatin and Herceptin
alike, treatment of tumors which have become resistant to
traditional treatments, e.g. Herceptin, Trastuzumab and any
chemotherapeutic agents. The therapeutic effector domain, e.g.
anti-angiogenic domain can be fused to provide alternate
specificities, e.g. anti-CD20, MUC-1, EDB and EGFR.
[0009] In a preferred embodiment, a method of treating, including
therapeutic treatment and prophylactic or preventative measures, a
tumor in an animal subject, comprises the step of administering to
the animal subject a chimeric molecule fusion composition,
comprising an anti-HER2 antigen binding domain and an endostatin
protein, peptide, mutants, variants or fragments thereof in a
therapeutically effective dose. Preferably, the antigen binding
domain comprises an isolated antibody, fragments thereof, or
aptamers. In one embodiment, a vector encoding the chimeric fusion
protein is administered to a patient.
[0010] In another preferred embodiment, the endostatin molecule in
the chimeric fusion molecule for treating a patient with a tumor or
cancer, comprises one or more mutations at amino acid positions
6-49, 50-92, 93-133 and 134-178 and/or integrin or integrin type
motifs, e.g. NGR, RGD and the like.
[0011] In another preferred embodiment, the chimeric fusion
molecule for treating cancer in a patient comprises endostatin
having one or more mutations at amino acid positions 93-133.
[0012] In another preferred embodiment, the chimeric fusion
molecule for treating cancer in a patient comprises a mutant
endostatin having an amino acid substitution at position 125 of
human endostatin.
[0013] In a preferred embodiment, the substitution comprises any
natural or non-natural, analog or variant amino acid.
[0014] In another preferred embodiment, the substitution at
position 125 is a proline to alanine (P125A).
[0015] In yet another preferred embodiment, the chimeric fusion
molecule for treating cancer in a patient comprises an antigen
binding domain comprising an isolated antibody, fragments thereof
or aptamers wherein the antigen binding domain specifically binds
one or more tumor antigens.
[0016] In a preferred embodiment, the tumor antigen comprises HER2,
phosphatase and tensin homolog (PTEN), phosphatidylinositol (PI)
kinase, variants, alleles and homologs thereof.
[0017] In another preferred embodiment, the chimeric fusion
molecule for treating cancer in a patient comprises an endostatin
molecule and mutants thereof comprising one or more NGR motifs
(Asn-Gly-Arg) and/or RGD (Arg-Gly-Asp) motifs. The endostatin
mutant molecules in addition to these integrin or integrin type
motifs comprises one or more mutations at amino acid position 125
of the endostatin molecule, at one or more mutations at amino acid
positions 6-49, 50-92, 93-133 and 134-178.
[0018] In another preferred embodiment, the one or more NGR motifs
(Asn-Gly-Arg) and RGD (Arg-Gly-Asp) motifs are located at the amino
(NH2-) terminal, and/or carboxy terminal (COOH--) and/or amino acid
positions 93-133.
[0019] In another preferred embodiment, the one or more NGR motifs
(Asn-Gly-Arg) and RGD (Arg-Gly-Asp) motifs are located at amino
acid positions 126-128 following the proline or alanine at position
125.
[0020] In another preferred embodiment, the chimeric fusion
molecule for treating cancer in a patient comprises an antigen
specific domain comprising an antibody or fragments thereof,
wherein the antibody or fragments thereof comprise IgA, IgM, IgG,
IgE, or IgD.
[0021] In another preferred embodiment, the chimeric fusion protein
is administered to a patient, simultaneously and/or in separate
treatments with one or more of: cytoximab, sunitinib, sorafenib,
celebrex, MTOR inhibitors, AKT inhibitors, P13K inhibitors,
bevacizumab (Avastin), signal transduction inhibitors, tamoxifen,
toremifen, raloxifene, droloxifene, iodoxyfene, megestrol acetate,
anastrozole, letrazole, borazole, exemestane, flutamide,
nilutamide, bicalutamide, cyproterone acetate, goserelin acetate,
luprolide, finasteride, herceptin, methotrexate, 5-fluorouracil,
cytosine arabinoside, doxorubicin, daunomycin, epirubicin,
idarubicin, mitomycin-C, dactinomycin, mithramycin, cisplatin,
carboplatin, melphalan, chlorambucil, busulphan, cyclophosphamide,
ifosfamide, nitrosoureas, thiotephan, vincristine, taxol, taxotere,
etoposide, teniposide, amsacrine, irinotecan, topotecan, an
epothilone; a tyrosine kinase inhibitor, Iressa or OSI-774; an
angiogenesis inhibitor; an EGF inhibitor; a VEGF inhibitor; a CDK
inhibitor; a Her1/2 inhibitor and monoclonal antibodies directed
against growth factor receptors.
[0022] In another preferred embodiment, a method of treating a
patient either prophylactically and/or therapeutically comprises
administering the chimeric fusion protein in combination with
and/or in separate treatments, one or more antibodies comprising
cytoximab, sunitinib, sorafenib, celebrex, MTOR inhibitors, AKT
inhibitors, P13K inhibitors, bevacizumab (Avastin), signal
transduction inhibitors, and anti her2 antibodies, one or more
anti-angiogenic factors comprising sunitinib, sorafenib, and
angiostatin.
[0023] In preferred embodiments, the therapeutically effective
doses are administered under a metronomic regimen.
[0024] In a preferred embodiment, a pharmaceutical composition
comprises a chimeric fusion molecule, wherein the chimeric fusion
molecule comprises an anti-tumor antigen binding domain and at
least one human endostatin protein, peptide, mutants, variants or
fragments thereof. The specificity of the fusion molecule can be
directed to any desired antigen by a fusion peptide comprising an
antigen binding domain specific for a desired antigen.
[0025] In another preferred embodiment, the chimeric fusion
molecule comprises one or more mutations at amino acid positions
6-49, 50-92, 93-133 and 134-178 of the endostatin molecule and
include integrin or integrin-like motifs, e.g. NGR, RGD and the
like.
[0026] In another preferred embodiment, the chimeric fusion
molecule comprises endostatin having one or more mutations at amino
acid positions 93-133.
[0027] In another preferred embodiment, the endostatin is a mutant
endostatin at amino acid position 125. The mutant can be a
substitution, deletion, variant and the like. In one aspect the
mutant endostatin comprises an amino acid substitution at position
125 of human endostatin.
[0028] In another preferred embodiment, the substitution at
position 125 is a proline to alanine (P125A).
[0029] In another preferred embodiment, the endostatin is
multimeric.
[0030] In another preferred embodiment, the multimers comprise one
or more endostatin molecules, one or more mutant endostatin
molecules and/or combinations thereof.
[0031] In another preferred embodiment, the antigen binding domain
comprises an isolated antibody or fragments thereof, or
aptamers.
[0032] In yet another embodiment, the antigen binding domain binds
to HER2/neu tumor antigens, tumor antigens, receptor/ligand
complex; or receptors. Preferably, the receptor is a receptor
involved in angiogenesis.
[0033] In another preferred embodiment, the antigen binding domain
specifically binds to antigens comprising HER2/neu tumor antigens,
phosphatase and tensin homolog (PTEN), phosphatidylinositol (PI)
kinase, receptor/ligand complex; or receptors.
[0034] In another preferred embodiment, the receptor, and ligands
thereof, is a receptor involved in angiogenesis and modulates
angiogenesis such as for example, receptor tyrosine kinases (RTKs),
vascular endothelial growth factor (VEGF) and its angiogenic
receptor (KDR); ang-1/2; thrombospondin-1 (TSP1) protein and its
receptor, integrins .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5 and .alpha..sub.5.beta..sub.1.
[0035] In another preferred embodiment, the chimeric fusion
molecule modulates embryogenesis, neovascularization and
tumorigenesis.
[0036] In another preferred embodiment, the receptor, and ligands
thereof comprises a protein-tyrosine kinase receptor. For example,
the Eph receptor family. This family is the largest family of
receptor tyrosine kinases identified to date. The Eph receptors and
their membrane-anchored ligands, ephrins, mediate bi-directional
signaling.
[0037] In yet another embodiment, the receptor involved in cellular
hyperproliferation comprises Granulocyte Colony-stimulating Factor
receptor (G-CSF-R), epidermal growth factor receptor (EGF-R),
vascular endothelial growth factor receptor (VEGF-R), brain derived
growth factor receptor, transforming growth factor receptor
(TGF-R), fibroblast growth factor receptor (bFGF-R),
platelet-derived growth factor receptor (PDGF-R), nerve growth
factor receptor (NGF-F), colony stimulating factor 1 receptor
(CSF1-R), insulin-like growth factor 1 receptor (IGF1-R) and
erythropoietin receptor (EPO-R), and the like, regulators, and
ligands thereof.
[0038] In another preferred embodiment, the receptor comprises a
signal transduction receptor including transmembrane, intracellular
and cell surface receptors. For example, G-protein coupled
receptors, e.g., chemokine receptors; receptor tyrosine kinases,
e.g., growth factor receptors; integrins; Toll-like receptors and
the like.
[0039] In another preferred embodiment, the chimeric fusion
molecule comprises an antigen specific binding domain wherein the
domain comprises an antibody or fragments thereof, comprising IgA,
IgM, IgG, IgE, or IgD.
[0040] In another preferred embodiment, the antibody or fragment
thereof is IgG1, IgG2, IgG3, and IgG4.
[0041] In another preferred embodiment, the antibody domain or
fragment thereof is any single chain, two-chain, diabody, minibody,
bispecific, multi-chain proteins and glycoproteins of polyclonal,
monoclonal, chimeric, and hetero immunoglobulins.
[0042] In another preferred embodiment, the antibody or fragment is
human or humanized antibody.
[0043] In another preferred embodiment, the isolated antibody or
immunoglobulin variable region comprise one or more of: Fab, Fab',
F(ab').sub.2, and Fv fragments.
[0044] In another preferred embodiment, the endostatin protein,
P125A endostatin protein, peptides, mutants, alleles, variants or
fragments thereof are fused to 3' end of an anti-HER2 antigen
binding domain.
[0045] In another preferred embodiment, a nucleic acid encoding the
chimeric molecule comprises an antigen specific binding domain and
a therapeutically effective domain.
[0046] In one preferred embodiment, the antigen binding domain
specifically binds tumor antigens comprising Her2, Her3, VEGF
receptors, PI Kinase receptors, PTEN, EGF receptors, Muc-1, PSMA,
CD20, Cd21, CD22, CD23, TAA, wt-1, Eph, alleles, mutants and
variants thereof.
[0047] In another preferred embodiment, the nucleic acid encodes a
therapeutically effective domain comprising endostatin, P125
endostatin, mutants, variants, fragments and alleles thereof.
[0048] In another preferred embodiment, chimeric fusion protein
comprises an anti-tumor antigen binding domain and an endostatin
protein, peptide, mutants, variants or fragments thereof or a
plurality of the endostatin molecules.
[0049] In another preferred embodiment, the antigen binding domain
binds to HER2/neu tumor antigens, tumor specific antigens,
receptor/ligand complex; or receptors.
[0050] In another preferred embodiment, the chimeric fusion protein
comprises a mutant endostatin having an amino acid substitution at
positions 6-49, 50-92, 93-133 and 134-178 of the endostatin
molecule. The amino acids comprise any natural, non-natural,
variant, analog, substituted molecule. The molecule further
comprises one or more substitutions comprising integrins or
integrin like motifs comprising NGR, RGD and the like.
[0051] In another preferred embodiment, the chimeric fusion protein
comprises a mutant endostatin having an amino acid substitution at
position 125 of human endostatin.
[0052] In another preferred embodiment, the substitution at
position 125 is a proline to alanine (P125A).
[0053] In another preferred embodiment, the endostatin is
multimeric.
[0054] In another preferred embodiment, the multimers comprise one
or more endostatin molecules, one or more mutant endostatin
molecules and/or combinations thereof.
[0055] In another preferred embodiment, a chimeric fusion protein
comprises an anti-tumor antigen binding domain and an endostatin
protein, peptide, mutants, variants or fragments thereof or a
plurality of the endostatin molecules.
[0056] In another preferred embodiment, the chimeric fusion protein
comprises an antigen specific binding domain having specificity
for: a receptor involved in angiogenesis, a protein-tyrosine kinase
receptor, a receptor involved in hyperproliferation, a signal
transduction receptor, alleles, mutants, fragments and variants
thereof.
[0057] In another preferred embodiment, the antigen binding domain
comprises an isolated antibody, fragments thereof, or aptamers,
wherein the antibody or fragments thereof, is IgA, IgM, IgG, IgE,
or IgD. In one aspect, the antibody or fragment thereof is IgG1,
IgG2, IgG3, and IgG4.
[0058] In another preferred embodiment, the antibody or fragment is
human or humanized antibody.
[0059] In another preferred embodiment, a method of treating a
patient with a tumor expressing low to undetectable levels of Her2,
comprises administering to a patient a therapeutically effective
amount of a chimeric fusion molecule comprising an anti-HER2
specific binding domain and an endostatin molecule having an
alanine substituted for proline at position 125.
[0060] In another preferred embodiment, a method of targeting
endostatin to a tumor cell in an animal subject, the method
comprising the step of administering to the animal subject a
composition comprising a chimeric molecule comprising an endostatin
domain and an antigen specific domain.
[0061] In another preferred embodiment a kit comprises a chimeric
fusion molecule comprising an anti-HER2 antigen binding domain and
an endostatin protein, peptide, mutants, variants or fragments
thereof.
[0062] In another preferred embodiment, an isolated cell comprises
a polynucleotide acid encoding a chimeric molecule comprising an
antigen specific binding domain and a therapeutically effective
domain.
[0063] In another preferred embodiment, the polynucleotide encodes
an antigen binding domain wherein the antigen binding domain
specifically binds tumor antigens comprising Her2, Her3, VEGF
receptors, PI Kinase receptors, PTEN, EGF receptors, Muc-1, PSMA,
CD20, Cd21, CD22, CD23, TAA, wt-1, Eph, alleles, mutants and
variants thereof.
[0064] In another preferred embodiment, the polynucleotide encodes
a therapeutically effective domain, wherein the therapeutically
effective domain comprises endostatin, P125A endostatin, mutants,
variants, fragments and alleles thereof.
[0065] In another preferred embodiment, the polynucleotide encodes
multimers of endostatin. In some embodiments the multimers are
dimers and/or trimers.
[0066] In another preferred embodiment, the multimers comprise one
or more endostatin molecules, one or more mutant endostatin
molecules and/or combinations thereof.
[0067] In another preferred embodiment, an isolated cell comprises
a vector or polynucleotide encoding a chimeric molecule comprising
an anti-tumor antigen binding domain and an endostatin protein,
peptide, mutants, variants or fragments thereof or a plurality of
the endostatin molecules.
[0068] In another preferred embodiment, the isolated cell
comprising a vector or polynucleotide encoding a chimeric molecule,
wherein the antigen binding domain binds to HER2/neu tumor
antigens, tumor specific antigens, receptor/ligand complex; or
receptors.
[0069] In another preferred embodiment, the isolated cell
comprising a vector or polynucleotide encoding a chimeric molecule,
wherein the mutant endostatin comprises an amino acid substitution
at position 125 of human endostatin. In one aspect, the
substitution at position 125 is a proline to alanine (P125A). In
another aspect of the invention, the endostatin molecule comprises
amino acid substitutions comprising any natural, non-natural,
variant, analog, substituted molecule. The molecule further
comprises one or more substitutions comprising integrins or
integrin like motifs comprising NGR, RGD and the like.
[0070] In another preferred embodiment, the isolated cell
comprising a vector or polynucleotide encoding a chimeric molecule,
wherein the polynucleotide encodes multimers of endostatin. In some
embodiments the multimers are dimers and/or trimers.
[0071] In another preferred embodiment, the multimers comprise one
or more endostatin molecules, one or more mutant endostatin
molecules and/or combinations thereof.
[0072] In another preferred embodiment, the isolated cell
comprising a vector or polynucleotide encoding a chimeric molecule,
wherein the antigen binding domain binds to a receptor comprising a
receptor involved in angiogenesis, a protein-tyrosine kinase
receptor, a receptor involved in hyperproliferation, a signal
transduction receptor, alleles, mutants, fragments and variants
thereof. The antigen binding domain comprises an isolated antibody,
fragments thereof, aptamers or integrins.
[0073] In one aspect the antibody or fragment thereof, is IgA, IgM,
IgG, IgE, or IgD.
[0074] In another aspect, the antibody or fragments thereof
comprise IgG1, IgG2, IgG3, and IgG4.
[0075] In another preferred embodiment, the antibody or fragment is
human or humanized antibody.
[0076] In one embodiment, the receptor is a protein-tyrosine kinase
receptor.
[0077] In another preferred embodiment, the receptor is a receptor
involved in hyperproliferation.
[0078] In another preferred embodiment, the antibody or fragment
thereof, comprises IgA, IgM, IgG, IgE, or IgD. Preferably, the
antibody or fragment thereof is IgG1, IgG2, IgG3, and IgG4.
[0079] In another preferred embodiment, the antibody or fragment
thereof is any single chain, two-chain, diabody, minibody,
bispecific, multi-chain proteins and glycoproteins of polyclonal,
monoclonal, chimeric, and hetero immunoglobulins.
[0080] In another preferred embodiment, the antibody or fragment is
human or humanized antibody.
[0081] In another preferred embodiment, the isolated immunoglobulin
variable region comprise Fab, Fab', F(ab').sub.2, and Fv
fragments.
[0082] In another preferred embodiment, the endostatin protein,
peptide or fragments thereof are fused to 3' end of an anti-HER2
antigen binding domain.
[0083] In another preferred embodiment, the antigen binding domain
binds to HER2/neu tumor antigens, tumor antigens, receptor/ligand
complex; or receptors.
[0084] In another preferred embodiment, a method of targeting
endostatin to a tumor cell in an animal subject, the method
comprising the step of administering to the animal subject a
composition comprising a chimeric molecule comprising an endostatin
domain and an Ig domain.
[0085] In another preferred embodiment, a method of treating a
tumor in an animal subject, the method comprising the step of
administering to the animal subject a chimeric molecule fusion
composition, comprising an anti-HER2 antigen binding domain and an
endostatin protein, peptide, mutants, variants or fragments
thereof; and, administration of the composition ameliorates the
tumor in the animal subject.
[0086] In another preferred embodiment, the chimeric fusion protein
is administered to a patient, simultaneously and/or in separate
treatments with one or more of: tamoxifen, toremifen, raloxifene,
droloxifene, iodoxyfene, megestrol acetate, anastrozole, letrazole,
borazole, exemestane, flutamide, nilutamide, bicalutamide,
cyproterone acetate, goserelin acetate, luprolide, finasteride,
herceptin, methotrexate, 5-fluorouracil, cytosine arabinoside,
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin, mithramycin, cisplatin, carboplatin, melphalan,
chlorambucil, busulphan, cyclophosphamide, ifosfamide,
nitrosoureas, thiotephan, vincristine, taxol, taxotere, etoposide,
teniposide, amsacrine, irinotecan, topotecan, an epothilone; a
tyrosine kinase inhibitor, Iressa or OSI-774; an angiogenesis
inhibitor; an EGF inhibitor; a VEGF inhibitor; a CDK inhibitor; a
Her1/2 inhibitor and monoclonal antibodies directed against growth
factor receptors.
[0087] In another preferred embodiment, the chimeric fusion protein
is administered in combination with and/or in separate treatments
one or more antibodies comprising cetuximab, bevacizumab (Avastin),
and anti her2 antibodies.
[0088] In another preferred embodiment, the chimeric fusion protein
is administered in combination with and/or in separate treatments
one or more anti-angiogenic factors comprising sunitinib,
sorafenib, and angiostatin.
[0089] In another preferred embodiment, a kit comprises a chimeric
fusion molecule comprising an anti-HER2 antigen binding domain and
an endostatin protein, peptide, mutants, variants or fragments
thereof.
[0090] In one embodiment, this disclosure provides a method for
inhibiting the formation and/or growth of blood vessels in a tumor.
The method comprises administering to an individual who has a tumor
an effective amount of a chimeric molecule fusion composition,
comprising an anti-HER2 antigen binding domain and an endostatin
protein or fragments thereof, or an anti-EGFR antigen binding
domain and an endostatin protein or fragments thereof. In one
embodiment, the endostatin protein or fragments have a proline to
alanine substitution at the 125 position of human endostatin.
[0091] In one embodiment, this disclosure provides a method for
inhibiting vasculogenic mimicry in a tumor. The method comprises
administering to an individual who has a tumor an effective amount
of a chimeric molecule fusion composition, comprising an anti-HER2
antigen binding domain and an endostatin protein or fragments
thereof, or an anti-EGFR antigen binding domain and an endostatin
protein or fragments thereof. In one embodiment, the endostatin
protein or fragments have a proline to alanine substitution at the
125 position of human endostatin. In one embodiment, the tumor is
known to display vasculogenic mimicry--either in vitro or in
vivo.
[0092] In one embodiment, this disclosure provides a method for
identifying treatment options for an individual diagnosed with a
tumor comprising determining if the tumor exhibits vasculogenic
mimicry (such as in vitro or in vivo) and if so, determining if the
fusion proteins of the present disclosure (such as
anti-Her2-huEndoP125A or anti-EGFR-huEndoP125A) inhibit
vasculogenic mimicry. If inhibition of vasculogenic mimicry is
observed, treatment options can be devised for the individual that
employ administration of the fusion proteins.
[0093] In one embodiment, this disclosure provides a method for
identifying treatment options for an individual diagnosed with a
tumor comprising determining if the fusion proteins of the present
disclosure (such as anti-Her2-huEndoP125A or anti-EGFR-huEndoP125A)
inhibit vasculogenic mimicry in the tumor (such as in vitro or in
vivo). If inhibition of vasculogenic mimicry is observed, treatment
options can be devised for the individual that employ
administration of the fusion proteins.
[0094] In another embodiment, the administration of the fusion
proteins is combined with other modalities of treatment including
surgical removal of the tumor or radiation treatment. The fusion
proteins may be administered before, after or during the same time
as the other modalities.
[0095] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0096] The invention is pointed out with particularity in the
appended claims. The above and further advantages of this invention
may be better understood by referring to the following description
taken in conjunction with the accompanying drawings, in which:
[0097] FIG. 1A is a schematic diagram showing anti-HER2 IgG3-human
endostatin fusion proteins. The endostatin domains (orange) are
indicated by an arrow and expression of anti-HER2
IgG3-CH3-endostatin fusion proteins (wild type and the mutant type
P125A). FIG. 1B are scans of photographs showing secreted human
endostatin fusion proteins labeled with [.sup.35S]methionine and
immunoprecipitated with Protein A and analyzed under non-reducing
and reducing conditions. Anti-HER2 IgG3-C.sub.H3-murine endostatin
fusion was used as a control.
[0098] FIGS. 2A-2F is a series of FACS scans shows binding of
anti-HER2 human endostatin fusion proteins to HER2 antigen and
HUVECs, and recognition by anti-human endostatin antibody. Human
breast cancer cells, SK-BR-3 (FIG. 2A, FIG. 2D), murine mammary
tumor cells, EMT6-HER2 (FIG. 2B, FIG. 2E) and EMT6 (FIG. 2C), and
HUVECs (FIG. 2F) were incubated with aHER2-huEndo (thin black line,
filled with red), aHER2-huEndo-P125A (thick black line, unfilled),
aHER2 IgG3 (thick green line, unfilled), human endostatin (thick
blue line), or isotype control (anti-dansyl IgG3, thin black line,
filled with gray). The unfilled, thin black line is unstained (the
secondary reagents only). The bound fusion proteins were identified
with either anti-human IgG-FITC conjugated (FIGS. 2A-2C), or
recognized with biotinylated anti-human endostatin antibody and
secondarily stained with a streptavidin-PE conjugate (FIGS.
2D-2F).
[0099] FIGS. 3A-3K show the effects of anti-HER2 IgG3-huEndo fusion
proteins on EC tube formation and EC proliferation. FIGS. 3A-3I:
HUVECs (4.times.10.sup.4 cells) were resuspended in 300 ul of full
endothelial cell growth medium and treated with the various
aHER2-huEndo fusion proteins before plating onto the
Matrigel-coated plates. After 16-20 hr of incubation, tube
formation was observed through an inverted photomicroscope. Full
media was used as negative control (FIG. 3A). Tube formation with
aHER2 IgG3 (FIG. 3B. 45.46 nM) and huEndo (FIG. 3C. 45.46 nM) were
compared to those with aHER2-huEndo (FIGS. 3D-3F, 4.55, 22.73,
45.46 nM, respectively) and aHER2-huEndo-P125A (FIGS. 3G-3I, 4.55,
22.73, 45.46 nM, respectively). Experiments repeated at least
twice. FIGS. 3J and 3K: HUVECs (4.times.10.sup.3 cells) were
treated with increasing concentrations of the endostatin fusion
proteins and proliferation measured at 72 hrs. FIG. 3J. VEGF: HUVEC
proliferation induced by VEGF (10 ng/ml). FIG. 3K. FGF: HUVEC
proliferation induced by bFGF (10 ng/ml). The data are presented as
the mean of triplicate determinations .+-.SD. Experiments were
repeated twice.
[0100] FIGS. 4A and 4B are graphs showing the anti-tumor efficacy
of anti-HER2 IgG3-huEndo fusion proteins. SCID mice (n=5) were s.c.
inoculated with 2.times.10.sup.6 SK-BR-3 on the right flank back on
day 0, then the mice were i.v. injected with anti-HER2 IgG3-huEndo
fusion proteins (42 lig), antiHER2 IgG3 (34.9 lig), human
endostatin (8 lig), or PBS every other day (indicated with arrow
starting on day 5). FIG. 4A: Tumor growth was measured with
calipers. Tumor volume was calculated as
4/3.times.3.14.times.{(long axis+short axis)/4}.sup.3. The values
represent mean.+-.SEM of tumor volume (mm.sup.3) of 5 mice. FIG.
4B: Survival of mice per treatment group. Mice with greater than
2000 mm.sup.3 tumor volume were euthanized.
[0101] FIGS. 5A to 5F are graphs showing anti-tumor activity of
aHER2-huEndo fusion-P125A protein in a syngeneic mouse model.
BALB/c mice (n=3-8 per group) were implanted s.c. contralaterally
with EMT6 (FIGS. 5A-5C) and EMT6-HER2 (FIGS. 5D-5F)
(1.times.10.sup.6 cells per mouse), followed on day 6 by equimolar
injections every other day (11 times) of aHER2-huEndo-P125A (42
lig), human endostatin (8 lig), or PBS. FIG. 5A: Individual tumor
measurements of mice treated with aHER2-huEndo-P125A fusion protein
are presented. FIG. 5B: Comparison of tumor growth between
untargeted and targeted tumors on day 16. EMT6 and EMT6-HER2 tumor
measurements of individual mice are paired and presented. The thick
red lines represent average measurements.
[0102] FIG. 6A-6D shows the analysis of tumor vascularity. BALB/c
mice (n=4 per group) were implanted s.c. contralaterally with EMT6
and EMT6-HER2 (1.times.10.sup.6 cells per mouse), followed on day 4
by equimolar injections every other day (7 time treatments) of
aHER2-huEndo-P125A (42 lig), or PBS. On day 12, two mice were
sacrificed for the blood vessel analysis after four treatments.
Histologic sections of tumors from the sacrificed mice were
analyzed using immunofluorescent staining for PECAM (II-IV,
VI-VIII; green color). DAPI (I, III, V, VII; blue color) was used
for counter-staining of the nucleus. Representative
immunofluorescent staining of EMT6-HER2 tumors treated with PBS
(I-IV) or aHER2-huEndo-P125A (V-VIII) is presented. Magnification:
50.times. (I-III, V-VII) or 100.times. (IV, VIII). Bars: 500 i.tm
for I-III and V-VII, 200 i.tm for IV and VIII.
[0103] FIG. 7 is a schematic representation showing the endostatin
signaling network.
[0104] FIG. 8. Schematic diagram of human IgG3-human mutant
endostatin fusion proteins. Human mutant endostatin at 125 (Proline
to Alanine: P125A) is represented as huEndo-P125A, anti-HER2
IgG3-human mutant endostatin as aHER2-huEndo-P125A, and anti-EGFR
IgG3-human mutant endostatin as aEGFR-huEndo-P125A.
[0105] FIGS. 9A-9H. (FIG. 9A-9G) Binding of anti-HER2 human
endostatin fusion proteins to HER2 antigen and HUVECs, and
recognition by anti-human endostatin antibody. Human breast cancer
cells, SK-BR-3 (FIG. 9A-, FIG. 9E), marine mammary tumor cells,
engineered to express HER2, EMT6-HER2 (FIG. 9B, FIG. 9F) and EMT6
(FIG. 9C), or human umbilical vein endothelial cells (HUVECs) (FIG.
9D, FIG. 9G) were incubated with aHER2-huEndo (filled with gray),
aHER2-huEndo-P125A (red line), aHER2 IgG3 (green line), human
endostatin (blue line), human endostatin-P125A (red line filled
with pink), or isotype control (anti-dansyl IgG3, black line). The
bound fusion proteins were identified with either anti-human
IgG-FITC conjugated (FIGS. 9A-9D), or with biotinylated anti-human
endostatin antibody and secondarily stained with a streptavidin-PE
conjugate (FIGS. 9E-9G). (FIG. 9H) Serum clearance of human
endostatin fusion proteins following intravenous administration to
mice. aHER2-huEndo (open circle), aHER2-huEndo-P125 (filled
circle), huEndo (open triangle), and huEndo-P125A (filled triangle)
were injected via tail vein of BALB/c mice (n=3). At the indicated
time points, serum samples were assayed by an ELISA which detects
human endostatin. The data are presented as the mean.+-.95% C.I. A
representative experiment is shown.
[0106] FIGS. 10A-10I. Effects of anti-HER2 IgG3-huEndo fusion
proteins on HUVEC tube formation. HUVECs were resuspended in
endothelial cell growth medium and treated as indicated before
plating onto the Matrigel-coated plates. Following 16 hr of
incubation, tube formation was observed through an inverted
photomicroscope. Tube formation with media alone (FIG. 10A), 45.46
nM aHER2 IgG3 (FIG. 10B), 45.46 nM trastuzumab (FIG. 10C), 45.46 nM
huEndo (FIG. 10D), and 45.46 nM huEndo-P125A (FIG. 10GG) were
compared to those with 22.73 and 45.46 nM aHER2-huEndo (FIGS.
10E-10F) and 22.73 and 45.46 nM aHER2-huEndo-P125A (FIG.
10H-10I).
[0107] FIGS. 11A-11B (FIG. 11A) Anti-tumor efficacy of anti-HER2
IgG3-huEndo fusion proteins. SCID mice (n=5) were implanted s.c.
with 2.times.10.sup.6 SK-BR-3 on day 0, then i. v. injected with
aHER2-huEndo fusion proteins (42 lig), aHER2 IgG3 (34 lig), huEndo
(8 lig), or PBS every other day (indicated with arrows) starting on
day 5. Tumor growth was measured as described above. The values
represent mean.+-.95% CI of tumor volume (mm.sup.3) of 5 mice.
Experiments were repeated three times with similar results. A
representative experiment is shown. (FIG. 11B) Survival of mice per
treatment group. aHER2-huEndo-P125A (42 lig), huEndo (8 ug),
huEndo-P125A (8 ug), aHER2 IgG3 (34 ug), trastuzuman (30 ug), or
PBS every other day (indicated with arrows) starting on day 6. Mice
with greater than 2500 mm.sup.3 tumor volume were euthanized. The
proportion surviving in each mouse group (%) is indicated. N. Sig.:
not significant. (This is separate experiment from that shown in
FIG. 11A.)
[0108] FIGS. 12A-12B. Recognition by anti-human endostatin antibody
and binding of anti-EGFR human endostatin fusion proteins to EGFR
antigen. (FIG. 12A) To identify human endostatin on the fusion
proteins, the fusion proteins were detected by western blotting
with a biotinylated anti-human endostatin antibody/avidin-HRP and
anti-human IgG-HRP. (FIG. 12B) To test binding to EGFR antigen,
EGFR+A431 tumor cells were incubated with antibodies and bound
antibodies were detected by anti-human IgG-FITC for detection of
the human IgG domain and a biotinylated anti-human endostatin
antibody/avidin-FITC for detection of the endostatin domain.
[0109] FIGS. 13A-13C. Effects of antibody-human endostatin fusion
proteins on HUVEC tube formation. (FIGS. 13A and 13B) Effects of
anti-EGFR IgG3-human endostatin (aEGFR-huEndo) fusion proteins on
EC tube formation: HUVECs were resuspended in endothelial cell
growth medium and treated as indicated before plating onto the
Matrigel-coated plates. Following 16 hr of incubation, tube
formation was observed through an inverted photomicroscope. (FIG.
13A) Tube formation with media alone and 45.46 nM aEGFR IgG3 were
compared to those with 2.84-45.46 nM aEGFR-huEndo and
aEGFR-huEndo-P125A (two preparation A7 and H8). (FIG. 13B)
Quantitation of `tube` formation: iTF.sub.50 is represented as the
50% inhibition of tube formation on treated cells vs. on controls.
Each completed `circle` was counted as `one` tubular structure.
(FIG. 13C) Effects of anti-HER2 IgG3-human endostatin
(aHER2-huEndo) fusion proteins on EC tube formation: (FIG. 13C)
Tube formation with aHER2-huEndo and aHER2-huEndo-P125A were
compared at the concentrations of 2.05-136.38 nM. Tube formation
was quantified as described in FIG. 13B.
[0110] FIGS. 14A-14D. Effects of antibody-human endostatin fusion
proteins on vasculogenic mimicry. (FIG. 14A) Human triple negative
breast cancer cells, MDA-MB-231, were resuspended in endothelial
cell growth medium and treated as indicated before plating onto
Matrigel-coated plates. Following 16 hr of incubation, tube
formation was observed through an inverted photomicroscope.
Vasculogenic mimicry with media alone, control antibodies (45.5
nM), human endostatin (huEndo, 45.5 or 454.6 nM), and human
endostatin-P125A (huEndo-P125A, 45.5 or 454.6 nM), and aEGFR IgG3
(45.5 nM) were compared to those with aHER2-huEndo-P125A (11.4-45.5
nM) and aEGFR-huEndo-P125A (11.4-45.5 nM). (FIG. 14B and 14C) Tube
formation by human ovarian cancer cells, SKOV3 (B) and PEO-1 (FIG.
14C). Experiments were performed as described above in FIG. 14A.
(FIG. 14D) Tube formation by human uveal melanoma cells, MUM-2B,
was tested as shown above in FIG. 14A.
[0111] FIG. 15. Antitumor efficacy of aHER2 IgG3-huEndo-P125A
fusion protein on human ovarian cancer xenografts, SKOV3. NSG mice
(n=5-9) were implanted s.c. with SKOV3 (10.sup.6 cells), then i. v.
injected with aHER2 IgG3-huEndo-P125A fusion protein (42 lag), or
PBS twice a week (arrows) starting on day 8. Tumor growth was
measured and the values represent mean.+-.SEM of tumor volume
(mm.sup.3) of 5-9 mice.
[0112] FIGS. 16A-16D. FIG. 16A: To test binding to EGFR antigen,
EGFR+A431 tumor cells were incubated with antibodies and bound
antibodies were detected by anti-human IgG-FITC. FIG. 16B. Purified
aEGFR-huEndo fusion proteins (arrows) detected with Coomassie blue
staining. FIG. 16C. To identify human endostatin on the fusion
proteins, the fusion proteins were detected by western blotting
with a biotinylated anti-human endostatin antibody/avidin-HRP and
antihuman IgG-HRP. FIG. 16D. Effects of anti-EGFR-huEndo fusion
proteins on EC tube formation. HUVECs were resuspended endothelial
cell growth medium and treated as indicated before plating onto the
Matrigel-coated plates. Following 16 hr of incubation, tube
formation was observed through an inverted photomicroscope. Tube
formation with media alone and aEGFR IgG3 (45.46 nM) were compared
to those with aEGFR-huEndo-P125A (45.46 nM).
DETAILED DESCRIPTION
[0113] The invention provides methods and compositions for
targeting a therapeutic chimeric molecule containing both (1) an
active agent and (2) a carrier domain such as all or a portion of
an immunoglobulin (Ig) molecule, aptamer, to a tumor. The active
agent can be modulatory, for example, anti-angiogenic, or
cytolytic. Targeting anti-angiogenic proteins using antibody fusion
proteins would improve clinical activity of anti-HER2 antibody and
endostatin alike, and provides a versatile approach that can be
applied to other tumor targets with alternative antibody
specificities or using other anti-angiogenic or cytolytic
domains.
[0114] The below described preferred embodiments illustrate
adaptations of these compositions and methods. Nonetheless, from
the description of these embodiments, other aspects of the
invention can be made and/or practiced based on the description
provided below.
[0115] Methods involving conventional biological techniques are
described herein. Such techniques are generally known in the art
and are described in detail in methodology treatises such as
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates). Immunological methods (e.g.,
preparation of antigen-specific antibodies, immunoprecipitation,
and immunoblotting) are described, e.g., in Current Protocols in
Immunology, ed. Coligan et al., John Wiley & Sons, New York,
1991; and Methods of Immunological Analysis, ed. Masseyeff et al.,
John Wiley & Sons, New York, 1992.
DEFINITIONS
[0116] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
that will be used hereinafter.
[0117] As used herein, the singular forms "a", "an" and "the"
include plural referents unless the context clearly dictates
otherwise.
[0118] As used herein, "antibody" refers to single chain,
two-chain, and multi-chain proteins and glycoproteins belonging to
the classes of polyclonal, monoclonal, chimeric, and hetero
immunoglobulins (monoclonal antibodies being preferred); it also
includes synthetic and genetically engineered variants of these
immunoglobulins. "Antibody fragment" includes Fab, Fab',
F(ab').sub.2, and Fv fragments, as well as any portion of an
antibody having specificity toward a desired target epitope or
epitopes.
[0119] As used herein, the term "immunoglobulin" or "antibody" are
used interchangeably and refer to a protein consisting of one or
more polypeptides substantially encoded by immunoglobulin genes.
The recognized immunoglobulin genes include the .kappa., .lamda.,
.alpha., .gamma. (IgG1, IgG2, IgG3, IgG4), .delta., .epsilon. and
.mu. constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 KDa or 214 amino acids) are encoded by a variable region
gene at the NH.sub.2-terminus (about 110 amino acids) and a kappa
or lambda constant region gene at the COOH-terminus. Full-length
immunoglobulin "heavy chains" (about 50 KDa or 446 amino acids),
are similarly encoded by a variable region gene (about 116 amino
acids) and one of the other aforementioned constant region genes,
e.g., gamma (encoding about 330 amino acids). One form of
immunoglobulin constitutes the basic structural unit of an
antibody. This form is a tetramer and consists of two identical
pairs of immunoglobulin chains, each pair having one light and one
heavy chain. In each pair, the light and heavy chain variable
regions are together responsible for binding to an antigen, and the
constant regions are responsible for the antibody effector
functions. In addition to antibodies, immunoglobulins may exist in
a variety of other forms including, for example, Fv, Fab, and
F(ab').sub.2, as well as bifunctional hybrid antibodies (e.g.,
Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single
chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,
5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988),
which are incorporated herein by reference). (See, generally, Hood
et al., "Immunology", Benjamin, N.Y., 2nd ed. (1984), and
Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are
incorporated herein by reference). The antibody can be human,
humanized or from any desired species.
[0120] As used herein, "humanized antibody" refers to an antibody
derived from a non-human antibody, typically murine, that retains
or substantially retains the antigen-binding properties of the
parent antibody but which is less immunogenic in humans. This may
be achieved by various methods including (a) grafting only the
non-human CDRs onto human framework and constant regions with or
without retention of critical framework residues, or (b)
transplanting the entire non-human variable domains, but "cloaking"
them with a human-like section by replacement of surface residues.
Such methods as are useful in practicing the present invention
include those disclosed in Morrison et al., Proc. Nat'l Acad. Sci.
USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan,
Mol. Immunol., 28:489-498 (1991); Padlan, Mol. Immunol.,
31(3):169-217 (1994).
[0121] As used herein "chimeric molecule" comprises a targeting
sequence such as for example, an aptamer, antibody sequence and a
therapeutic effector molecule, e.g. endostatin and mutants thereof,
genetically fused to the targeting, e.g. antibody fragment. For
example, a chimeric molecule comprises endostatin genetically fused
to an anti-HER2/neu IgG3 heavy chain at the end of C.sub.H3, and
expressed with an anti-HER2/neu K light chain.
[0122] As used herein, "variant" in addition to its understood
meaning as a term of art includes any changes in a molecule from
its wild-type form. For example, alleles, fragments, mutations,
substitutions with natural or analog compounds, splice variants,
glycosylations, species variants, and the like. The term is not
limited to any one type of change or deviation from the wild type
form or "normal" molecule.
[0123] The phrase "specifically (or selectively) binds" to an
antibody or aptamer or "specifically (or selectively)
immunoreactive with," refers to a binding reaction that is
determinative of the presence of the protein in a heterogeneous
population of proteins and other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to a
particular protein at least two times the background and do not
substantially bind in a significant amount to other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, polyclonal
antibodies raised to marker "X" from specific species such as rat,
mouse, or human can be selected to obtain only those polyclonal
antibodies that are specifically immunoreactive with marker "X" and
not with other proteins, except for polymorphic variants and
alleles of marker "X". This selection may be achieved by
subtracting out antibodies that cross-react with marker "X"
molecules from other species. A variety of immunoassay formats may
be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive
with a protein (see, e.g., Harlow & Lane, Antibodies, A
Laboratory Manual (1988), for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity). Typically a specific or selective reaction will
be at least twice background signal or noise and more typically
more than 10 to 100 times background.
[0124] As used herein, a "pharmaceutically acceptable" component is
one that is suitable for use with humans and/or animals without
undue adverse side effects (such as toxicity, irritation, and
allergic response) commensurate with a reasonable benefit/risk
ratio.
[0125] As used herein, the term "safe and effective amount" or
"therapeutic amount" refers to the quantity of a component which is
sufficient to yield a desired therapeutic response without undue
adverse side effects (such as toxicity, irritation, or allergic
response) commensurate with a reasonable benefit/risk ratio when
used in the manner of this invention. By "therapeutically effective
amount" is meant an amount of a compound of the present invention
effective to yield the desired therapeutic response. For example,
an amount effective to delay the growth of or to cause a cancer,
either a sarcoma or lymphoma, or to shrink the cancer or prevent
metastasis. The specific safe and effective amount or
therapeutically effective amount will vary with such factors as the
particular condition being treated, the physical condition of the
patient, the type of mammal or animal being treated, the duration
of the treatment, the nature of concurrent therapy (if any), and
the specific formulations employed and the structure of the
compounds or its derivatives.
[0126] As used herein, "cancer" refers to all types of cancer or
neoplasm, benign or malignant tumors found in mammals, including,
but not limited to: leukemias, lymphomas, melanomas, carcinomas and
sarcomas. Examples of cancers are cancer of the brain, breast,
pancreas, cervix, colon, head and neck, kidney, lung, non-small
cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach,
prostate, testicles, uterus and medulloblastoma.
[0127] Additional cancers which can be treated the chimeric fusion
molecule according to the invention include, for example, Hodgkin's
Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,
breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung
tumors, primary brain tumors, stomach cancer, colon cancer,
malignant pancreatic insulanoma, malignant carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer,
lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,
genitourinary tract cancer, malignant hypercalcemia, cervical
cancer, endometrial cancer, adrenal cortical cancer, and prostate
cancer.
[0128] "Regression" refers to the reduction of tumor mass and size
as measured using standard techniques.
[0129] "Diagnostic" or "diagnosed" means identifying the presence
or nature of a pathologic condition. Diagnostic methods differ in
their sensitivity and specificity. The "sensitivity" of a
diagnostic assay is the percentage of diseased individuals who test
positive (percent of "true positives"). Diseased individuals not
detected by the assay are "false negatives." Subjects who are not
diseased and who test negative in the assay, are termed "true
negatives." The "specificity" of a diagnostic assay is 1 minus the
false positive rate, where the "false positive" rate is defined as
the proportion of those without the disease who test positive.
While a particular diagnostic method may not provide a definitive
diagnosis of a condition, it suffices if the method provides a
positive indication that aids in diagnosis.
[0130] The terms "patient" or "individual" are used interchangeably
herein, and refers to a mammalian subject to be treated, with human
patients being preferred. In some cases, the methods of the
invention find use in experimental animals, in veterinary
application, and in the development of animal models for disease,
including, but not limited to, rodents including mice, rats, and
hamsters; and primates.
[0131] "Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology or symptoms
of a disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already with the disorder as well as
those in which the disorder is to be prevented. In tumor (e.g.,
cancer) treatment, a therapeutic agent may directly decrease the
pathology of tumor cells, or render the tumor cells more
susceptible to treatment by other therapeutic agents, e.g.,
radiation and/or chemotherapy. As used herein, "ameliorated" or
"treatment" refers to a symptom which is approaches a normalized
value (for example a value obtained in a healthy patient or
individual), e.g., is less than 50% different from a normalized
value, preferably is less than about 25% different from a
normalized value, more preferably, is less than 10% different from
a normalized value, and still more preferably, is not significantly
different from a normalized value as determined using routine
statistical tests.
[0132] The "treatment of cancer or tumor cells", refers to an
amount of chimeric fusion molecule, described throughout the
specification and in the Examples which follow, capable of invoking
one or more of the following effects: (1) inhibition, to some
extent, of tumor growth, including, (i) slowing down (ii)
inhibiting angiogenesis and (ii) complete growth arrest; (2)
reduction in the number of tumor cells; (3) maintaining tumor size;
(4) reduction in tumor size; (5) inhibition, including (i)
reduction, (ii) slowing down or (iii) complete prevention, of tumor
cell infiltration into peripheral organs; (6) inhibition, including
(i) reduction, (ii) slowing down or (iii) complete prevention, of
metastasis; (7) enhancement of anti-tumor immune response, which
may result in (i) maintaining tumor size, (ii) reducing tumor size,
(iii) slowing the growth of a tumor, (iv) reducing, slowing or
preventing invasion and/or (8) relief, to some extent, of the
severity or number of one or more symptoms associated with the
disorder.
[0133] As used herein, "metronomic" therapy refers to the
administration of continuous low-doses of a therapeutic agent
and/or chimeric fusion molecule described herein.)
Fusion Molecules
[0134] In general, the invention provides antigen-binding fusion
proteins with a therapeutically effective domain which can be a
modulatory or cytolytic moiety having a significant serum half-life
(t.sub.1/2) beyond that of either antibody or modulatory/cytolytic
moiety alone. Modulatory and cytolytic antigen-binding fusion
proteins have more than an antigen-binding site activity or
function. A modulatory or cytolytic moiety on the fusion
antigen-binding protein will impart upon the protein certain or all
of the modulatory or cytolytic attributes of the fusion partner or
partners.
[0135] Accordingly, the invention is directed to chimeric fusion
molecules comprising single or multivalent therapeutically active
domains which can be modulatory and/or cytolytic and an
antigen-binding domain; compositions of single-chain and
multivalent modulatory and cytolytic antigen-binding fusion
proteins, methods of making and purifying single-chain and
multivalent modulatory and cytolytic antigen-binding fusion
proteins, and uses for single-chain and multivalent modulatory and
cytolytic antigen-binding fusion proteins. The invention provides a
modulatory or cytolytic antigen-binding fusion protein having at
least one single-chain antigen-binding protein molecule. Each
single-chain antigen-binding molecule has a first polypeptide and a
second polypeptide and can be joined by a linker. Each of the
polypeptides has the binding portion of the variable region of an
antibody heavy or light chain or other antigen specific moiety such
as an aptamer. Other binding moieties include integrin motifs and
NGR motifs. FIG. 1A is a schematic representation, which is not
meant to be limiting or construed as such, shows one embodiment of
the chimeric molecule.
[0136] In a preferred embodiment, a composition is provided
comprising a therapeutically effective anti-tumor molecule fused to
a targeting moiety such as for example, an antigen specific binding
domain of an antibody. By way of illustration, the composition
comprises an anti-tumor antibody specific, for example, the
HER2/neu tumor antigen, in which endostatin and/or mutants thereof,
is fused to the 3' end of a humanized or human anti-HER2 IgG3
antibody.
[0137] In a preferred embodiment, the endostatin molecule comprises
a mutation at amino acid 125 whereby the proline is substituted
with alanine. Introduction of a point mutation into human
endostatin at position 125 (proline to alanine; huEndo-P125A)
enhances endothelial cell binding, anti-angiogenic activity, and
anti-tumor activity as compared to the wild type endostatin
molecule. The mutant .alpha.HER2-huEndo-P125A fusion variant
inhibited tube formation of HUVEC in vitro and tumor growth in vivo
more effectively than .alpha.HER2-huEndo.
[0138] In another preferred embodiment, the endostatin molecule and
mutants thereof comprise one or more NGR motifs (Asn-Gly-Arg).
Human endostatin comprises an NGR motif (Asn-Gly-Arg) at position
126-128 following the proline at position 125. In a preferred
embodiment, the endostatin molecule comprises one or more NGR
motifs at the amino (NH.sub.2--) terminal, and/or carboxy terminal
(COOH--) and/or a repeating string of NGR molecules following the
NGR motif at position 126-128 or preceding the proline or alanine
at position 125.
[0139] In another preferred embodiment, the endostatin molecule
comprises an RGD motif preceding or following the proline or
alanine at amino acid position 125.
[0140] In another preferred embodiment, the endostatin molecule
comprises a mutant endostatin having an amino acid substitution at
positions 6-49, 50-92, 93-133 and 134-178 of the endostatin
molecule. The amino acids comprise any natural, non-natural,
variant, analog, substituted molecule. The molecule further
comprises one or more substitutions comprising integrins or
integrin like motifs comprising NGR, RGD and the like. These motifs
can be at one or more amino acid positions 6-49, 50-92, 93-133 and
134-178 of the endostatin molecule. Thus, one of skill in the art
would understand that the chimeric fusion molecule can contain
multimers of endostatin molecules and these molecules can be
combinations of the same endostatin molecules or comprise
combinations of endostatin molecules, molecules with amino acid
substitutions, molecules with amino acid substitutions and integrin
and integrin like motifs and various positions in the endostatin
molecule.
[0141] In another preferred embodiment, the chimeric molecule
comprises an integrin motif e.g. RGD, in addition to or in place of
the NGR motif.
[0142] The chimeric fusion molecule can be fused genetically, i.e.
the molecules are operably linked in frame so the chimeric fusion
molecule is encoded from the nucleic acid molecule. The molecule
can be fused at the amino acid level, such as described in detail
in the examples which follow.
[0143] In another preferred embodiment, the molecule comprises a
label for detecting the fusion molecule in vivo and to monitor the
effects of the chimeric molecule during therapy.
[0144] "Detectable moiety" or a "label" refers to a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, .sup.35S, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA),
biotin-streptavidin, dioxigenin, haptens and proteins for which
antisera or monoclonal antibodies are available, or nucleic acid
molecules with a sequence complementary to a target. The detectable
moiety often generates a measurable signal, such as a radioactive,
chromogenic, or fluorescent signal, that can be used to quantify
the amount of bound detectable moiety in a sample. Quantitation of
the signal is achieved by, e.g., scintillation counting,
densitometry, or flow cytometry.
[0145] In a preferred embodiment, the chimeric fusion molecule
comprises a polynucleotide sequence encoding an anti-HER2
antibody-human endostatin P125A molecule, variants, mutations,
alleles, substitutes, fragments and analogs thereof.
[0146] In another preferred embodiment, the chimeric fusion
molecule comprises a polypeptide comprising an anti-HER2 specific
antibody fused to human endostatin P125A. The endostatin can be a
monomer, however, multimers such as for example, a dimer and trimer
of endostatin and mutants thereof are preferred.
[0147] In another preferred embodiment, the multimer comprises an
endostatin molecule and a mutant form of endostatin, for example,
P125A endostatin and combinations thereof. Thus, if the multimer is
a trimer, then the molecule can comprise a wild type endostatin
molecule and two mutant forms of endostatin, or two wild type
endostatin molecules and one mutant endostatin molecule; or three
wild type endostatin molecules or three mutant forms of endostatin
molecules.
[0148] In another preferred embodiment, the dimer comprises a
mutant form of endostatin, for example, P125A endostatin and normal
or wild type endostatin; or combinations thereof, variants,
mutations, alleles, substitutes, fragments and analogs thereof.
[0149] In another preferred embodiment, the endostatin is human
endostatin, variants and alleles thereof. However, endostatin can
be derived from any species.
[0150] In another preferred embodiment, the endostatin molecule
comprises a polynucleotide having mutations at one or more
nucleotides encoding mutant endostatin, for example, P125A
endostatin.
[0151] In all embodiments, the molecules can be in any
stereoisomeric form, for example, enantiomers, diastereomers,
tautomers and the like. In all embodiments, the fusion molecule or
parts thereof includes all variants, mutations, alleles,
substitutes, fragments and analogs thereof.
[0152] In another preferred embodiment, the endostatin comprises
one or more amino acid at position 125. In one embodiment, proline
to alanine is preferred. However, other amino acid substitutions
can be made, for example, any of the 20 common, genetically-encoded
amino acids such as: tryptophan, valine, leucine, isoleucine. Other
amino acids include those classified as having, for example:
charged polar side chains (Arg, His, Lys etc); uncharged polar side
chains (Thr, Asn, Gln etc).
[0153] In another preferred embodiment, the amino acid mutations
can occur at any one or more amino acid positions 4 to 49, 50-92,
93-133 and 134-178. Preferably, the mutations are in one or more
nucleic acids encoding amino acid at positions 93-133 and/or at the
amino acid level at amino acid positions 93-133.
[0154] The mutations can be introduced at the nucleic acid level or
at the amino acid level. With respect to particular nucleic acid
sequences, because of the degeneracy of the genetic code, a large
number of functionally identical nucleic acids encode any given
protein. For instance, the codons GCA, GCC, GCG and GCU all encode
the amino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations,"
which are one species of conservatively modified variations. If
mutations at the nucleic acid level are introduced to encode a
particular amino acid, then one or more nucleic acids are altered.
For example proline is encoded by CCC, CCA, CCG, CCU; thus, one
base change, e.g. CCC (proline) to GCC gives rise to alanine. Thus
by way of example every natural or non-natural nucleic acid
sequence herein which encodes a natural or non-natural polypeptide
also describes every possible silent variation of the natural or
non-natural nucleic acid. One of skill will recognize that each
codon in a natural or non-natural nucleic acid (except AUG, which
is ordinarily the only codon for methionine, and TGG, which is
ordinarily the only codon for tryptophan) can be modified to yield
a functionally identical molecule or a different molecule.
Accordingly, each silent variation of a natural and non-natural
nucleic acid which encodes a natural and non-natural polypeptide is
implicit in each described sequence.
[0155] As to amino acid sequences, individual substitutions,
deletions or additions to a nucleic acid, peptide, polypeptide, or
protein sequence which alters, adds or deletes a single natural and
non-natural amino acid or a small percentage of natural and
non-natural amino acids in the encoded sequence, the alteration
results in the deletion of an amino acid, addition of an amino
acid, or substitution of a natural and non-natural amino acid with
a chemically similar amino acid. Conservative substitution tables
providing functionally similar natural amino acids are well known
in the art. Such conservatively modified variants are in addition
to and do not exclude polymorphic variants, interspecies homologs,
and alleles of the methods and compositions described herein.
[0156] In another preferred embodiment, the mutant endostatin
comprises one or more non-natural or analogs of amino acids.
[0157] A "non-natural amino acid" refers to an amino acid that is
not one of the 20 common amino acids or pyrolysine or
selenocysteine. Other terms that may be used synonymously with the
term "non-natural amino acid" is "non-naturally encoded amino
acid," "unnatural amino acid," "non-naturally-occurring amino
acid," and variously hyphenated and non-hyphenated versions
thereof. The term "non-natural amino acid" includes, but is not
limited to, amino acids which occur naturally by modification of a
naturally encoded amino acid (including but not limited to, the 20
common amino acids or pyrrolysine and selenocysteine) but are not
themselves incorporated, without user manipulation, into a growing
polypeptide chain by the translation complex. Examples of
naturally-occurring amino acids that are not naturally-encoded
include, but are not limited to, N-acetylglucosaminyl-L-serine,
N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.
Additionally, the term "non-natural amino acid" includes, but is
not limited to, amino acids which do not occur naturally and may be
obtained synthetically or may be obtained by modification of
non-natural amino acids.
[0158] In some cases, the non-natural amino acid substitution(s) or
incorporation(s) will be combined with other additions,
substitutions, or deletions within the polypeptide to affect other
chemical, physical, pharmacologic and/or biological traits. In some
cases, the other additions, substitutions or deletions may increase
the stability (including but not limited to, resistance to
proteolytic degradation) of the polypeptide or increase affinity of
the polypeptide for its appropriate receptor, ligand and/or binding
proteins. In some cases, the other additions, substitutions or
deletions may increase the solubility of the polypeptide. In some
embodiments sites are selected for substitution with a naturally
encoded or non-natural amino acid in addition to another site for
incorporation of a non-natural amino acid for the purpose of
increasing the polypeptide solubility following expression in
recombinant host cells. In some embodiments, the polypeptides
comprise another addition, substitution, or deletion that modulates
affinity for the associated ligand, binding proteins, and/or
receptor, modulates (including but not limited to, increases or
decreases) receptor dimerization, stabilizes receptor dimers,
modulates circulating half-life, modulates release or
bio-availability, facilitates purification, or improves or alters a
particular route of administration. Similarly, the non-natural
amino acid polypeptide can comprise chemical or enzyme cleavage
sequences, protease cleavage sequences, reactive groups,
antibody-binding domains (including but not limited to, FLAG or
poly-His) or other affinity based sequences (including but not
limited to, FLAG, poly-His, GST, etc.) or linked molecules
(including but not limited to, biotin) that improve detection
(including but not limited to, GFP), purification, transport thru
tissues or cell membranes, prodrug release or activation, size
reduction, or other traits of the polypeptide.
[0159] The methods and compositions described herein include
incorporation of one or more non-natural amino acids into a
polypeptide. One or more non-natural amino acids may be
incorporated at one or more particular positions which does not
disrupt activity of the polypeptide. This can be achieved by making
"conservative" substitutions, including but not limited to,
substituting hydrophobic amino acids with non-natural or natural
hydrophobic amino acids, bulky amino acids with non-natural or
natural bulky amino acids, hydrophilic amino acids with non-natural
or natural hydrophilic amino acids) and/or inserting the
non-natural amino acid in a location that is not required for
activity.
[0160] A variety of biochemical and structural approaches can be
employed to select the desired sites for substitution with a
non-natural amino acid within the polypeptide. Any position of the
polypeptide chain is suitable for selection to incorporate a
non-natural amino acid, and selection may be based on rational
design or by random selection for any or no particular desired
purpose. Selection of desired sites may be based on producing a
non-natural amino acid polypeptide (which may be further modified
or remain unmodified) having any desired property or activity,
including but not limited to agonists, super-agonists, partial
agonists, inverse agonists, antagonists, receptor binding
modulators, receptor activity modulators, modulators of binding to
binder partners, binding partner activity modulators, binding
partner conformation modulators, dimer or multimer formation, no
change to activity or property compared to the native molecule, or
manipulating any physical or chemical property of the polypeptide
such as solubility, aggregation, or stability. For example,
locations in the polypeptide required for biological activity of a
polypeptide can be identified using methods including, but not
limited to, point mutation analysis, alanine scanning or homolog
scanning methods. Residues other than those identified as critical
to biological activity by methods including, but not limited to,
alanine or homolog scanning mutagenesis may be good candidates for
substitution with a non-natural amino acid depending on the desired
activity sought for the polypeptide. Alternatively, the sites
identified as critical to biological activity may also be good
candidates for substitution with a non-natural amino acid, again
depending on the desired activity sought for the polypeptide.
Another alternative would be to make serial substitutions in each
position on the polypeptide chain with a non-natural amino acid and
observe the effect on the activities of the polypeptide. Any means,
technique, or method for selecting a position for substitution with
a non-natural amino acid into any polypeptide is suitable for use
in the methods, techniques and compositions described herein.
[0161] The structure and activity of naturally-occurring mutants of
a polypeptide that contain deletions can also be examined to
determine regions of the protein that are likely to be tolerant of
substitution with a non-natural amino acid. Once residues that are
likely to be intolerant to substitution with non-natural amino
acids have been eliminated, the impact of proposed substitutions at
each of the remaining positions can be examined using methods
including, but not limited to, the three-dimensional structure of
the relevant polypeptide, and any associated ligands or binding
proteins. X-ray crystallographic and NMR structures of many
polypeptides are available in the Protein Data Bank (PDB,
www.rcsb.org), a centralized database containing three-dimensional
structural data of large molecules of proteins and nucleic acids,
one can be used to identify amino acid positions that can be
substituted with non-natural amino acids. In addition, models may
be made investigating the secondary and tertiary structure of
polypeptides, if three-dimensional structural data is not
available. Thus, the identity of amino acid positions that can be
substituted with non-natural amino acids can be readily obtained.
Exemplary sites of incorporation of a non-natural amino acid
include, but are not limited to, those that are excluded from
potential receptor binding regions, or regions for binding to
binding proteins or ligands may be fully or partially solvent
exposed, have minimal or no hydrogen-bonding interactions with
nearby residues, may be minimally exposed to nearby reactive
residues, and/or may be in regions that are highly flexible as
predicted by the three-dimensional crystal structure of a
particular polypeptide with its associated receptor, ligand or
binding proteins.
[0162] A wide variety of non-natural amino acids can be substituted
for, or incorporated into, a given position in a polypeptide. By
way of example, a particular non-natural amino acid may be selected
for incorporation based on an examination of the three dimensional
crystal structure of a polypeptide with its associated ligand,
receptor and/or binding proteins, a preference for conservative
substitutions
[0163] Other Therapeutic Effector Domains:
[0164] In another preferred embodiment, the modulatory domain
comprises endostatin, angiogenin, angiostatin, chemokines,
angioarrestin, angiostatin (plasminogen fragment),
basement-membrane collagen-derived anti-angiogenic factors
(tumstatin, canstatin, or arrestin), anti-angiogenic antithrombin
III, signal transduction inhibitors, cartilage-derived inhibitor
(CDI), CD59 complement fragment, fibronectin fragment, gro-beta,
heparinases, heparin hexasaccharide fragment, human chorionic
gonadotropin (hCG), interferon alpha/beta/gamma, interferon
inducible protein (IP-10), interleukin-12, kringle 5 (plasminogen
fragment), metalloproteinase inhibitors (TIMPs),
2-methoxyestradiol, placental ribonuclease inhibitor, plasminogen
activator inhibitor, platelet factor-4 (PF4), prolactin 16 kD
fragment, proliferin-related protein (PRP), various retinoids,
tetrahydrocortisol-S, thrombospondin-1 (TSP-1), transforming growth
factor-beta (TGF-b), vasculostatin, vasostatin (calreticulin
fragment) and the like. These molecules include all forms,
variants, mutations, alleles, substitutes, fragments and analogs
thereof.
[0165] In another preferred embodiment, the modulatory domain of
the chimeric fusion molecule comprises endostatin, angiostatin,
tumstatin, arrestin and canstatin, variants, mutations, alleles,
substitutes, fragments and analogs thereof.
[0166] In another preferred embodiment, the molecule comprises
combinations of one or more of endostatin, angiostatin, tumstatin,
arrestin and canstatin, variants, mutations, alleles, substitutes,
fragments and analogs thereof.
[0167] In another preferred embodiment, the targeting domain
comprises antibody, aptamer, a ligand for a receptor (e.g. VEGF),
diabodies, peptides, lipopolysaccharides, integrins and the
like.
[0168] Other Specificities of the Antigen Binding Domain:
[0169] In another preferred embodiment, the chimeric fusion
molecules comprise an antigen binding domain specific for other
tumor antigens. The antigen binding domain can be an antibody or
aptamer, receptor, ligand etc.
[0170] In one preferred embodiment, the invention provides for
antibody fusion molecules comprising: F.sub.c region, C.sub.H1,
C.sub.H2 and/or C.sub.H3, Fab, Fab', F(ab').sub.2, single chain Fv
(S.sub.cFv) and Fv fragments, as well as any portion of an antibody
having specificity toward a desired target epitope or epitopes.
Also preferred are antibodies or antibody fragments or to single
chain, two-chain, and multi-chain proteins and glycoproteins
belonging to the classes of polyclonal, monoclonal, chimeric,
bispecific and hetero immunoglobulins (monoclonal antibodies being
preferred); it also includes synthetic and genetically engineered
variants of these immunoglobulins.
[0171] In another preferred embodiment, the antigen binding domain
is an aptamer. In the preferred embodiment, the chimeric molecule
comprises an aptamer fused to the endostatin molecule, variants,
mutants and fragments thereof. The aptamer can be specific for any
one or more tumor antigens.
[0172] As used herein, the term "aptamer" or "selected nucleic acid
binding species" refers to short strands of nucleic acid sequences,
DNA or RNA, that are designed to bind to a target molecule
specifically and with high affinity. The nucleic acid sequences
include non-modified or chemically modified RNA or DNA. The method
of selection may be by, but is not limited to, affinity
chromatography and the method of amplification by reverse
transcription (RT) or polymerase chain reaction (PCR), iterative
rounds using SELEX aptamer techniques and the like.
[0173] Many tumor antigens are well known in the art. See for
example, Van den Eynde B J, van der Bruggen P. Curr Opin Immunol
1997; 9: 684-93; Houghton A N, Gold J S, Blachere N E. Curr Opin
Immunol 2001; 13: 134-140; van der Bruggen P, Zhang Y, Chaux P,
Stroobant V, Panichelli C, Schultz E S, Chapiro J, Van den Eynde B
J, Brasseur F, Boon T. Immunol Rev 2002; 188: 51-64, which are
herein incorporated by reference in their entirety. Alternatively,
many antibodies directed towards tumor antigens are commercially
available.
[0174] In a preferred embodiment, the tumor antigens comprise HER2,
HER3, Muc-1, EGFR, PSMA, CD20, CD22, CD23, TAA, GDR antigens, VEGFR
and the like.
[0175] Other non-limiting examples of tumor antigens, include,
tumor antigens resulting from mutations, such as: alpha-actinin-4
(lung carcinoma); CASP-8 (head and neck squamous cell carcinoma);
beta-catenin (melanoma); Cdc27 (melanoma); CDK4 (melanoma);
Elongation factor 2 (lung squamous carcinoma);
LDLR-fucosyltransferaseAS fusion protein (melanoma); overexpression
of HLA-A2.sup.d (renal cell carcinoma); hsp70-2 (renal cell
carcinoma); KIAAO205 (bladder tumor); MART2 (melanoma); MUM-1f
(melanoma); MUM-2 (melanoma); MUM-3 (melanoma); neo-PAP (melanoma);
Myosin class I (melanoma); OS-9g (melanoma); PTPRK (melanoma).
Examples of differentiation tumor antigens include, but not limited
to: CEA (gut carcinoma); gp100/Pmel17 (melanoma); Kallikrein 4
(prostate); mammaglobin-A (breast cancer); Melan-A/MART-1
(melanoma); PSA (prostate carcinoma); TRP-1/gp75 (melanoma); TRP-2
(melanoma); tyrosinase (melanoma). Over or under-expressed tumor
antigens include but are not limited to: CPSF (ubiquitous); EphA3;
G250/MN/CAIX (stomach, liver, pancreas); HER-2/neu; Intestinal
carboxyl esterase (liver, intestine, kidney); alpha-foetoprotein
(liver); M-CSF (liver, kidney); MUC1 (glandular epithelia); p53
(ubiquitous); PRAME (testis, ovary, endometrium, adrenals); PSMA
(prostate, CNS, liver); RAGE-1 (retina); RU2AS (testis, kidney,
bladder); survivin (ubiquitous); Telomerase (testis, thymus, bone
marrow, lymph nodes); WT1 (testis, ovary, bone marrow, spleen);
CA125 (ovarian). Antigens that are preferentially expressed on the
tumor cell membrane represent preferred targets.
[0176] In another preferred embodiment, the present invention
features a compound having a plurality of binding moieties, wherein
at least two binding moieties have specificity for different
binding sites on the same target. In preferred embodiments, the
plurality of binding moieties includes a polypeptide. In other
preferred embodiments, the targeting moieties are all binding
polypeptides which bind to different sites on the desired target.
In certain preferred embodiments, the target is a protein, a
receptor, or a receptor/ligand complex and the binding polypeptides
bind to different epitopes on the protein, the receptor, or the
receptor/ligand complex.
[0177] In another preferred embodiment, the target is a receptor
involved in angiogenesis, hyperproliferative disorders or wound
healing. In another embodiment the target includes a family of
receptors, such as, for example, protein-tyrosine kinase receptors.
In a particularly preferred embodiment, the target is Flt-1 and
KDR, VEGF (VEGF-1, -2 or -3) or the KDR/VEGF and Flt-1/VEGF
complexes, and the binding moieties, particularly binding peptides,
bind to different epitopes on Flt-1 and KDR or the KDR/VEGF and
Flt-1/VEGF complexes. For example, VEGFR-2/KDR, VEGFR-1/Flt-1 and
VEGFR-3/Flt-4.
[0178] In connection with solid tumor treatment, the present
invention may be used in combination with classical approaches,
such as surgery, radiotherapy, chemotherapy, and the like. The
invention therefore provides combined therapies in which the
therapeutic compositions are used simultaneously with, before, or
after surgery or radiation treatment; or are administered to
patients with, before, or after conventional chemotherapeutic,
radiotherapeutic or other anti-angiogenic agents, or targeted
immunotoxins or coaguligands.
[0179] In another preferred embodiments, the chimeric molecule
comprises one or more cytolytic or other effector molecules.
Cytolytic molecules that can be used to fuse to an antibody or
fragment thereof, include, but are not limited to TNF-.alpha.,
TNF-.beta., suitable effector genes such as those that encode a
peptide toxin--such as ricin, abrin, diphtheria, gelonin,
Pseudomonas exotoxin A, Crotalus durissus terrificus toxin,
Crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique
toxin. (Hughes et al., Hum. Exp. Toxicol. 15:443, 1996; Rosenblum
et al., Cancer Immunol. Immunother. 42:115, 1996; Rodriguez et al.,
Prostate 34:259, 1998; Mauceri et al., Cancer Res. 56:4311;
1996).
[0180] ALSO suitable are genes that induce or mediate
apoptosis--such as the ICE-family of cysteine proteases, the Bcl-2
family of proteins, Bax, BclXs and caspases (Favrot et al., Gene
Ther. 5:728, 1998; McGill et al., Front. Biosci. 2:D353, 1997;
McDonnell et al., Semin. Cancer Biol. 6:53, 1995). Another
potential anti-tumor agent is apoptin, a protein that induces
apoptosis even where small drug chemotherapeutics fail (Pietersen
et al., Adv. Exp. Med. Biol. 465:153, 2000). Koga et al. (Hu. Gene
Ther. 11: 1397, 2000) propose a telomerase-specific gene therapy
using the hTERT gene promoter linked to the apoptosis gene
Caspase-8 (FLICE).
[0181] Also of interest are enzymes present in the lytic package
that cytotoxic T lymphocytes or LAK cells deliver to their targets.
Perforin, a pore-forming protein, and Fas ligand are major
cytolytic molecules in these cells (Brandau et al., Clin. Cancer
Res. 6:3729, 2000; Cruz et al., Br. J. Cancer 81:881, 1999). CTLs
also express a family of at least 11 serine proteases termed
granzymes, which have four primary substrate specificities (Kam et
al., Biochim. Biophys. Acta 1477:307, 2000). Low concentrations of
streptolysin 0 and pneumolysin facilitate granzyme B-dependent
apoptosis (Browne et al., Mol. Cell Biol. 19:8604, 1999).
[0182] Other suitable effectors encode polypeptides having activity
that is not itself toxic to a cell, but renders the cell sensitive
to an otherwise nontoxic compound--either by metabolically altering
the cell, or by changing a non-toxic prodrug into a lethal drug.
Exemplary is thymidine kinase (tk), such as may be derived from a
herpes simplex virus, and catalytically equivalent variants. The
HSV tk converts the anti-herpetic agent ganciclovir (GCV) to a
toxic product that interferes with DNA replication in proliferating
cells.
[0183] If desired, although not required, factors may also be
included, such as, but not limited to, chemokines, cytokines, e.g.
interleukins, e.g. IL-2, IL-3, IL-6, and IL-11, as well as the
other interleukins, the colony stimulating factors, such as GM-CSF,
interferons, e.g. .gamma.-interferon, erythropoietin.
Combination Therapies
[0184] In another preferred embodiment, the invention provides
administering the chimeric fusion molecule with a cocktail of one
or more compounds such as for example, endostatin, angiogenin,
angiostatin, chemokines, angioarrestin, angiostatin (plasminogen
fragment), basement-membrane collagen-derived anti-angiogenic
factors (tumstatin, canstatin, or arrestin), anti-angiogenic
antithrombin III, cartilage-derived inhibitor (CDI), CD59
complement fragment, fibronectin fragment, gro-beta, heparinases,
heparin hexasaccharide fragment, human chorionic gonadotropin
(hCG), interferon alpha/beta/gamma, interferon inducible protein
(IP-10), interleukin-12, kringle 5 (plasminogen fragment),
metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placental
ribonuclease inhibitor, plasminogen activator inhibitor, platelet
factor-4 (PF4), prolactin 16 kD fragment, proliferin-related
protein (PRP), various retinoids, tetrahydrocortisol-S,
thrombospondin-1 (TSP-1), transforming growth factor-beta (TGF-b),
vasculostatin, vasostatin (calreticulin fragment) and the like.
[0185] In another preferred embodiment, the chimeric fusion
molecules of the invention are administered with one more compounds
comprising signal transduction inhibitors, bevacizumab (Avastin),
antiangiogenic compounds such as for example, sunitinib, sorafenib,
celebrex, MTOR inhibitors, AKT inhibitors, P13K and the like. One
of ordinary skill in the art would identify which other therapeutic
compounds could be administered in conjunction with a therapy
comprising a regimen of chimeric fusion molecule.
[0186] In another preferred embodiment, one or more types of
chimeric fusion molecules can be administered to a patient. For
example a chimeric fusion molecule wherein the therapeutic effector
domain comprises endostatin, angiogenin, angiostatin, chemokines,
angioarrestin, angiostatin (plasminogen fragment),
basement-membrane collagen-derived anti-angiogenic factors
(tumstatin, canstatin, or arrestin), anti-angiogenic antithrombin
III, signal transduction inhibitors, cartilage-derived inhibitor
(CDI), CD59 complement fragment, fibronectin fragment, gro-beta,
heparinases, heparin hexasaccharide fragment, human chorionic
gonadotropin (hCG), interferon alpha/beta/gamma, interferon
inducible protein (IP-10), interleukin-12, kringle 5 (plasminogen
fragment), metalloproteinase inhibitors (TIMPs),
2-methoxyestradiol, placental ribonuclease inhibitor, plasminogen
activator inhibitor, platelet factor-4 (PF4), prolactin 16 kD
fragment, proliferin-related protein (PRP), various retinoids,
tetrahydrocortisol-S, thrombospondin-1 (TSP-1), transforming growth
factor-beta (TGF-b), vasculostatin, vasostatin (calreticulin
fragment) and the like. These molecules include all forms,
variants, mutations, alleles, substitutes, fragments and analogs
thereof.
[0187] Metronomic Therapy:
[0188] In accordance with the invention, the chimeric fusion
molecule composition is administered to a patient in combination
with metronomic therapy. For example, administration of continuous
low-doses of the chimeric fusion molecule and one or more
therapeutic agents. Therapeutic agents can include, for example,
chemotherapeutic agents such as, cyclophosphamide (CTX, 25
mg/kg/day, p.o.), taxanes (paclitaxel or docetaxel), busulfan,
cisplatin, cyclophosphamide, methotrexate, daunorubicin,
doxorubicin, melphalan, cladribine, vincristine, vinblastine, and
chlorambucil. Metronomic therapy can also include administering the
antibody-fusion molecule with a cocktail of one or more compounds
such as for example, endostatin, angiogenin, angiostatin,
chemokines, angioarrestin, angiostatin (plasminogen fragment),
basement-membrane collagen-derived anti-angiogenic factors
(tumstatin, canstatin, or arrestin), anti-angiogenic antithrombin
III, cartilage-derived inhibitor (CDI), CD59 complement fragment,
fibronectin fragment, gro-beta, heparinases, heparin hexasaccharide
fragment, human chorionic gonadotropin (hCG), interferon
alpha/beta/gamma, interferon inducible protein (IP-10),
interleukin-12, kringle 5 (plasminogen fragment), metalloproteinase
inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease
inhibitor, plasminogen activator inhibitor, platelet factor-4
(PF4), prolactin 16 kD fragment, proliferin-related protein (PRP),
various retinoids, tetrahydrocortisol-S, thrombospondin-1 (TSP-1),
transforming growth factor-beta (TGF-b), vasculostatin, vasostatin
(calreticulin fragment), signal transduction inhibitors and the
like. Other examples include antibodies that target signal
transduction receptors, ligands and/or complexes thereof.
[0189] Examples of signal transduction inhibitors include, but not
limited to: Gleevec (target Bcr-abl), Herceptin (monoclonal
antibody--target Her-2 (neu)), Iressa (small molecule
inhibitor--EGFR), Erbitux (monoclonal antibody--EGFR), Tarceva
(small molecule inhibitor) Ras inhibitor R11577 (farnesyl
transferase inhibitor), mTOR inhibitor Rapamune (rapamycin)
Ruboxistaurin (small molecule inhibitor), Avastin (monoclonal
antibody), PTK787/ZK 222584, Neovastat, ABX-EGF (monoclonal
antibody), TheraClM (monoclonal antibody), Mixed lineages
kinases--CEP 1347 (small molecule inhibitor), Tyrosine kinase--CEP
701 (small molecule inhibitor), Cyclin dependent
kinase--Flavopiridol (small molecule inhibitor), VEGF Trap (decoy
receptor).
[0190] In another preferred embodiment, the chimeric fusion protein
molecules can be administered with one or anti-cancer compounds.
The compounds of this invention may also be useful in combination
(administered together or sequentially) with known anti-cancer
treatments such as radiation therapy or with cytostatic or
cytotoxic agents, such as for example, but not limited to, DNA
interactive agents, such as cisplatin or doxorubicin; topoisomerase
II inhibitors, such as etoposide; topoisomerase I inhibitors such
as CPT-11 or topotecan; tubulin interacting agents, such as
paclitaxel, docetaxel or the epothilones; hormonal agents, such as
tamoxifen; thymidilate synthase inhibitors, such as 5-fluorouracil;
and anti-metabolites, such as methotrexate; tyrosine kinase
inhibitors such as Iressa and OSI-774 (Tarceva.TM.); bevacizumab
(Avastin), Herceptin, angiogenesis inhibitors; EGF inhibitors; VEGF
inhibitors; CDK inhibitors; Her1/2 inhibitors and monoclonal
antibodies directed against growth factor receptors such as erbitux
(EGF) and herceptin (Her2) or against angiogenic factors.
[0191] Examples of tyrosine kinase inhibitors include inhibitors of
the tyrosine kinase enzyme: Abl, CDK's, EGF, EMT, FGF, FAK,
Flk-1/KDR, HER-2, IGF-R, IR, LCK, MET, PDGF, Src, and VEGF. In
general, inhibitors could act as reversible cytostatic agents which
may be useful in the treatment of any disease process which
features abnormal cellular proliferation, e.g., benign prostatic
hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis,
atherosclerosis, pulmonary fibrosis, arthritis, psoriasis,
glomerulonephritis, restenosis following angioplasty or vascular
surgery, hypertrophic scar formation, inflammatory bowel disease,
transplantation rejection, endotoxic shock, and fungal
infections.
[0192] In another preferred embodiment, the treatment of abnormal
cell growth or cancer comprises a combination treatment in which
endostatin fusion molecules of the invention are administered to a
subject in combination with radiation therapy and/or
chemotherapy.
[0193] The language "chemotherapeutic agent" is intended to include
chemical reagents which inhibit the growth of proliferating cells
or tissues wherein the growth of such cells or tissues is
undesirable. Chemotherapeutic agents are well known in the art (see
e.g., Gilman A. G., et al. The Pharmacological Basis of
Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically
used to treat neoplastic diseases, tumors, and cancers.
[0194] The language "radiation therapy" is intended to include the
application of a genetically and somatically safe level of X-rays,
both localized and non-localized, to a subject to inhibit, reduce,
or prevent symptoms or conditions associated with undesirable cell
growth. The term X-rays is intended to include clinically
acceptable radioactive elements and isotopes thereof, as well as
the radioactive emissions therefrom. Examples of the types of
emissions include alpha rays, beta rays including hard betas, high
energy electrons, and gamma rays. Radiation therapy is well known
in the art (see e.g., Fishbach, F., Laboratory Diagnostic Tests,
3rd Ed., Ch. 10: 581-644 (1988)), and is typically used to treat
neoplastic diseases, tumors, and cancers.
[0195] Examples of chemotherapeutic agents include: bleomycin,
docetaxel (Taxotere), doxorubicin, edatrexate, etoposide,
finasteride (Proscar), flutamide (Eulexin), gemcitabine (Gemzar),
goserelin acetate (Zoladex), irinotecan (Campto/Camptosar),
ondansetron (Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar),
pilocarpine hydrochloride (Salagen), porfimer sodium (Photofrin),
interleukin-2 (Proleukin), rituximab (Rituxan), topotecan
(Hycamtin), trastuzumab (Herceptin), tretinoin (Retin-A), Triapine,
vincristine, and vinorelbine tartrate (Navelbine). Other examples
of chemotherapeutic agents include alkylating drugs such as
Nitrogen Mustards (e.g., Mechlorethamine (HN.sub.2),
Cyclophosphamide, Ifosfamide, Melphalan (L-sarcolysin),
Chlorambucil, etc.); ethylenimines, methylmelamines (e.g.,
Hexamethylmelamine, Thiotepa, etc.); Alkyl Sulfonates (e.g.,
Busulfan, etc.), Nitrosoureas (e.g., Carmustine (BCNU), Lomustine
(CCNU), Semustine (methyl-CCNU), Streptozocin (streptozotocin),
etc.), triazenes (e.g., Decarbazine (DTIC;
dimethyltriazenoimi-dazolecarboxamide)), Alkylators (e.g.,
cis-diamminedichloroplatinum II (CDDP)), etc.
[0196] Other examples of chemotherapeutic agents include
antimetabolites such as folic acid analogs (e.g., Methotrexate
(amethopterin)); pyrimidine analogs (e.g., fluorouracil
('5-fluorouracil; 5-FU); floxuridine (fluorode-oxyuridine); Fudr;
Cytarabine (cyosine arabinoside), etc.); purine analogs (e.g.,
Mercaptopurine (6-mercaptopurine; 6-MP); Thioguanine
(6-thioguanine; TG); and Pentostatin (2'-deoxycoformycin)), etc.
Other examples of chemotherapeutic agents also include vinca
alkaloids (e.g., Vinblastin (VLB) and Vincristine); topoisomerase
inhibitors (e.g., Etoposide, Teniposide, Camptothecin, Topotecan,
9-amino-campotothecin CPT-11, etc.); antibiotics (e.g.,
Dactinomycin (actinomycin D), adriamycin, daunorubicin,
doxorubicin, bleomycin, plicamycin (mithramycin), mitomycin
(mitomycin C), Taxol, Taxotere, etc.); enzymes (e.g.,
L-Asparaginase); and biological response modifiers (e.g.,
interferon-; interleukin 2, etc.). Other chemotherapeutic agents
include cis-diaminedichloroplatinum II (CDDP); Carboplatin;
Anthracendione (e.g., Mitoxantrone); Hydroxyurea; Procarbazine
(N-methylhydrazine); and adrenocortical suppressants (e.g.,
Mitotane, aminoglutethimide, etc.). Other chemotherapeutic agents
include adrenocorticosteroids (e.g., Prednisone); progestins (e.g.,
Hydroxyprogesterone caproate; Medroxyprogesterone acetate,
Megestrol acetate, etc.); estrogens (e.g, diethylstilbestrol;
ethenyl estradiol. etc.); antiestrogens (e.g. Tamoxifen, etc.);
androgens (e.g., testosterone propionate, Fluoxymesterone, etc.);
antiandrogens (e.g., Flutamide); and gonadotropin-releasing hormone
analogs (e.g., Leuprolide).
[0197] In terms of surgery, any surgical intervention may be
practiced in combination with the present invention. In connection
with radiotherapy, any mechanism for inducing DNA damage locally
within tumor cells is contemplated, such as .gamma.-irradiation,
X-rays, UV-irradiation, microwaves and even electronic emissions
and the like. The directed delivery of radioisotopes to tumor cells
is also contemplated, and this may be used in connection with a
targeting antibody or other targeting means.
[0198] Cytokine therapy also has proven to be an effective partner
for combined therapeutic regimens. Various cytokines may be
employed in such combined approaches. Examples of cytokines include
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, TGF-.beta., GM-CSF, M-CSF, G-CSF,
TNF.alpha., TNF.beta., LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF,
OSM, TMF, PDGF, IFN-.alpha., IFN-.beta., IFN-.gamma.. Cytokines are
administered according to standard regimens, consistent with
clinical indications such as the condition of the patient and
relative toxicity of the cytokine. Uteroglobins may also be used to
prevent or inhibit metastases (U.S. Pat. No. 5,696,092;
incorporated herein by reference). In one embodiment, the
administration of fusion proteins of the present disclosure can be
combined with the administration of anti CTLA4 or Anti PDL1.
[0199] In another preferred embodiment, the fusion molecules
described herein can also be administered in combination with one
or more anti-angiogenic factors. A wide variety of other
anti-angiogenic factors may also be utilized within the context of
the present invention. Representative examples include Platelet
Factor 4 (Sigma Chemical Co., #F1385); Protamine Sulphate
(Clupeine) (Sigma Chemical Co., #P4505); Sulfated Chitin
Derivatives (prepared from queen crab shells), (Sigma Chemical Co.,
#C3641; Murata et al. Cancer Res. 51:22-26, 1991); Sulfated
Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this
compound may be enhanced by the presence of steroids such as
estrogen, and tamoxifen citrate); Staurosporine (Sigma Chemical
Co., #S4400); Modulators of Matrix Metabolism, including for
example, proline analogs {[(L-azetidine-2-carboxylic acid (LACA)
(Sigma Chemical Co., #A0760)), cishydroxyproline,
d,L-3,4-dehydroproline (Sigma Chemical Co., #D0265), Thiaproline
(Sigma Chemical Co., #T0631)], .alpha.,.alpha.-dipyridyl (Sigma
Chemical Co., #D7505), .beta.-aminopropionitrile fumarate (Sigma
Chemical Co., #A3134)]}; MDL 27032
(4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Merion Merrel Dow
Research Institute); Methotrexate (Sigma Chemical Co., #A6770;
Hirata et al. Arthritis and Rheumatism 32:1065-1073, 1989);
Mitoxantrone (Polverini and Novak, Biochem. Biophys. Res. Comm.
140:901-907); Heparin (Folkman, Bio. Phar. 34:905-909, 1985; Sigma
Chemical Co., #P8754); Interferons (e.g., Sigma Chemical Co.,
#13265); 2 Macroglobulin-serum (Sigma Chemical Co., #M7151);
ChIMP-3 (Pavloff et al. J. Bio. Chem. 267:17321-17326, 1992);
Chymostatin (Sigma Chemical Co., #C7268; Tomkinson et al. Biochem
J. 286:475-480, 1992); .beta.-Cyclodextrin Tetradecasulfate (Sigma
Chemical Co., #C4767); Eponemycin; Camptothecin; Fumagillin (Sigma
Chemical Co., #F6771; Canadian Patent No. 2,024,306; Ingber et al.
Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Sigma:G4022; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446,
1987); (D-Penicillamine ("CDPT"; Sigma Chemical Co., #P4875 or
P5000(HCl)); .beta.-1-anticollagenase-serum; .alpha.2-antiplasmin
(Sigma Chem. Co.:A0914; Holmes et al. J. Biol. Chem.
262(4):1659-1664, 1987); Bisantrene (National Cancer Institute);
Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid
disodium or "CCA"; Takeuchi et al. Agents Actions 36:312-316,
1992); Thalidomide; Angostatic steroid; AGM-1470;
carboxynaminolmidazole; metalloproteinase inhibitors such as
BB94.
[0200] Although the above anti-angiogenic factors have been
provided for the purposes of illustration, it should be understood
that the present invention is not so limited. In particular,
although certain anti-angiogenic factors are specifically referred
to above, the present invention should be understood to include
analogues, derivatives and conjugates of such anti-angiogenic
factors. For example, paclitaxel should be understood to refer to
not only the common chemically available form of paclitaxel, but
analogues (e.g., taxotere, as noted above) and paclitaxel
conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or
paclitaxel-xylos). In another preferred embodiment, the fusion
molecules of the invention can also be targeted for treating
angiogenesis-associated diseases. The term "angiogenesis-associated
disease" is used herein, for purposes of the specification and
claims, to mean certain pathological processes in humans where
angiogenesis is abnormally enhanced/prolonged. Such
angiogenesis-associated diseases include diabetic retinopathy,
chronic inflammatory diseases, rheumatoid arthritis, dermatitis,
psoriasis, stomach ulcers, and most types of human solid
tumors.
[0201] Mutant Anti-Angiogenic Agents:
[0202] The anti-angiogenic agent can be an intact molecule, a
functional fragment of the agent, or a naturally occurring or
man-made mutant of the agent. For example, endostatin domains
useful in the invention include any molecule derived from a native
endostatin that shares a functional activity of endostatin, e.g.,
the ability to inhibit VEGF production or new vessel formation. The
endostatin domain can be a native endostatin or a fragment of a
native endostatin that retains a functional activity of a native
endostatin. The endostatin domain can also be a non-naturally
occurring form of endostatin (e.g., a mutant form created by amino
acid substitution) that retains a functional activity of a native
endostatin. A preferred embodiment is a mutant endostatin which has
an amino acid change at position 125. The mutant endostatin
molecules comprising the fusion molecule of the invention comprises
natural, non-natural, modified, derived amino acids etc.
[0203] Thus, the definition of the term "endostatin" includes
modifications or mutations of the protein, its subunits and peptide
fragments. Such mutations include substitutions of naturally
occurring amino acids at specific sites with other molecules,
including but not limited to naturally and non-naturally occurring
amino acids. Such substitutions may modify the bioactivity of the
anti-angiogenic protein and produce biological or pharmacological
agonists or antagonists. Modifications can also include modified
amino acids within the protein sequence, or modifications to the
intact protein sequence that inhibit protease activity, or
otherwise enhance the stability of the protein and decrease protein
degradation. Such modifications are well-known to those of skill in
the art.
[0204] Modified proteins are also referred to herein as derivative
proteins, or analogs. The term "derivative" or "analog" includes
any protein/polypeptide having an amino acid residue sequence
substantially identical to a sequence specifically shown herein in
which one or more residues have been conservatively substituted
with a functionally similar residue and which displays the activity
as described herein. Examples of conservative substitutions include
the substitution of one non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another, the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine,
between threonine and serine, the substitution of one basic residue
such as lysine, arginine or histidine for another, or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid for another.
[0205] The phrase "conservative substitution" also includes the use
of a chemically derivatized residue in place of a non-derivatized
residue provided that such polypeptide displays the requisite
inhibition activity.
[0206] "Chemical derivative" refers to a subject polypeptide having
one or more residues chemically derivatized by reaction of a
functional side group. Such derivatized molecules include for
example, those molecules in which free amino groups have been
derivatized to form amine hydrochlorides, p-toluene sulfonyl
groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types
of esters or hydrazides. Free hydroxyl groups may be derivatized to
form O-acyl or O-alkyl derivatives. The imidazole nitrogen of
histidine may be derivatized to form N-imbenzylhistidine. Also
included as chemical derivatives are those peptides which contain
one or more naturally occurring amino acid derivatives of the
twenty standard amino acids. For examples: 4-hydroxyproline may be
substituted for proline; 5-hydroxylysine may be substituted for
lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted for serine; and ornithine may be
substituted for lysine. Polypeptides of the present invention also
include any polypeptide having one or more additions and/or
deletions of residues relative to the sequence of a polypeptide
whose sequence is shown herein, so long as the requisite activity
is maintained.
[0207] The present invention also contemplates amino acid residue
sequences that are analogous to sequences of the proteins described
herein, and the nucleic acid sequences encoding these proteins. It
is well known in the art that modifications and changes can be made
without substantially altering the biological function of the
protein. In making such changes, substitutions of like amino acid
residues can be made on the basis of relative similarity of
side-chain substituents, for example, their size, charge,
hydrophobicity, hydrophilicity and the like. Alterations of the
type described may be made to enhance the peptide's potency or
stability to enzymatic breakdown or pharmacokinetics. Thus,
sequences deemed as within the scope of the invention, include
those analogous sequences characterized by a change in amino acid
residue sequence or type wherein the change does not alter the
fundamental nature and biological activity of the aforementioned
anti-angiogenic proteins, derivatives, mutants fragments and/or
fusion proteins.
[0208] It will be appreciated that the term "endostatin" includes
shortened proteins or peptides, referred to herein as fragments,
wherein one or more amino acid is removed from either or both ends
of endostatin, or from an internal region of the protein, yet the
resulting molecule retains endothelial proliferation inhibiting
activity. The term "endostatin" also includes lengthened proteins
or peptides wherein one or more amino acid is added to either or
both ends of endostatin, or to an internal location in the protein,
yet the resulting molecule retains endothelial proliferation
inhibiting activity. Such molecules, for example with tyrosine
added in the first position are useful for labeling such as
radioiodination with 125-iodine (.sup.125I) for use in assays.
Labeling with other radioisotopes may be useful in providing a
molecular tool for destroying the target cell containing endostatin
receptors. Other labeling with molecules such as ricin may provide
a mechanism for destroying cells with anti-angiogenic protein
receptors.
[0209] Anti-angiogenic fusion proteins of the present invention
encompass a protein comprising one or more of the proteins,
mutants, derivatives or fragments described herein as well as other
anti-angiogenic molecules known to those of skill in the art. For
example, a fusion protein encompassed by the present invention can
be encoded by a polynucleotide encoding endostatin linked to
another endostatin or endostatin mutant wherein the expressed
fusion protein comprises activity of both endostatin and endostatin
mutant, resulting in a reasonable increase of the biological
activity of the fusion protein over either monomeric wild-type
endostatin or endostatin mutant. Another type of fusion protein
encompassed by the present invention can be a fusion protein
encoded by a polynucleotide encoding two endostatin molecules in
tandem, endostatin mutants or combinations thereof optionally
linked by a linker. Again, it is reasonable to predict that the
fusion protein would have higher activity than the monomeric
endostatin.
[0210] Other examples of anti-angiogenic fusion proteins of the
present invention include conjugates of the proteins. Such fusion
proteins may or may not be capable of being cleaved into the
separate proteins from which they are derived. As used herein, the
term "conjugate of an anti-angiogenic protein" means an
anti-angiogenic protein chemically coupled to another protein to
form a conjugate. Examples of conjugates include a protein fragment
coupled to albumin or to a peptide fragment from another
anti-angiogenic protein.
[0211] As used herein, the term "anti-angiogenesis activity" refers
to the capability of a molecule to inhibit the growth of blood
vessels. As used herein, the term "endothelial inhibiting activity"
refers to the capability of a molecule to inhibit angiogenesis in
general and, for example, to inhibit the growth or migration of
bovine capillary endothelial cells in culture in the presence of
fibroblast growth factor or other known growth factors. An
anti-angiogenic protein, mutant, derivative, fragment or fusion
protein of the present invention may be characterized on the basis
of potency when tested for its "endothelial inhibiting activity".
Other measures of endothelial inhibiting activity are described
herein.
[0212] The anti-angiogenic proteins of the present invention are
effective in treating diseases or processes that are mediated by,
or involve, angiogenesis. The present invention includes the method
of treating an angiogenesis-mediated disease with an effective
amount of an anti-angiogenic fusion protein produced by the methods
described herein, or a biologically active mutant, derivative,
fragment or fusion protein thereof, or combinations of proteins
that collectively possess anti-angiogenic activity, or the activity
of anti-angiogenic agonists and antagonists.
[0213] As used herein, the term "angiogenesis" means the generation
of new blood vessels into a tissue or organ, and involves
endothelial cell proliferation. Under normal physiological
conditions, mammals (humans or animals) undergo angiogenesis only
in very specific restricted situations. For example, angiogenesis
is normally observed in wound healing, fetal and embryonal
development, and formation of the corpus luteum, endometrium and
placenta. The term "endothelium" means a thin layer of flat
epithelial cells that lines serous cavities, lymph vessels, and
blood vessels.
[0214] As used herein, the term "angiogenesis-associated factor"
means a factor which either inhibits or promotes angiogenesis. An
example of an angiogenesis-associated factor is an angiogenic
growth factor, such as basic fibroblastic growth factor (bFGF),
which is an angiogenesis promoter. Another example of an
angiogenesis associated factor is an angiogenesis inhibiting factor
such as angiostatin.
[0215] As used herein, the term "growth factor" means a molecule
that stimulates the growth, reproduction, or synthetic activity of
cells.
[0216] The angiogenesis mediated diseases include, encompassed
herein but are not limited to, solid tumors; blood born tumors such
as leukemias; tumor metastasis; benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular
angiogenic diseases, for example, diabetic retinopathy, retinopathy
of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis;
Osler-Webber Syndrome; myocardial angiogenesis; plaque
neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; and wound granulation, intestinal adhesions, Crohn's
disease, atherosclerosis, scleroderma, and hypertrophic scars,
i.e., keloids. The anti-angiogenic proteins described herein can
also be used as a birth control agent by preventing vascularization
required for embryo implantation. The proteins are also useful in
the treatment of diseases that have angiogenesis as a pathologic
consequence such as cat scratch disease (Rochele minalia quintosa)
and ulcers (Helicobacter pylori).
[0217] Radiolabeling:
[0218] In another preferred embodiment, the fusion molecule of the
invention can be radiolabeled. Uses include therapeutic and imaging
for diagnostic purposes. The label may be a radioactive atom, an
enzyme, or a chromophore moiety. Methods for labeling antibodies
have been described, for example, by Hunter and Greenwood, Nature,
144:945 (1962) and by David et al. Biochemistry 13:1014-1021
(1974). Additional methods for labeling antibodies have been
described in U.S. Pat. Nos. 3,940,475 and 3,645,090. Methods for
labeling oligonucleotide probes have been described, for example,
by Leary et al. Proc. Natl. Acad. Sci. USA (1983) 80:4045; Renz and
Kurz, Nucl. Acids Res. (1984) 12:3435; Richardson and Gumport,
Nucl. Acids Res. (1983) 11:6167; Smith et al. Nucl. Acids Res.
(1985) 13:2399; and Meinkoth and Wahl, Anal. Biochem. (1984)
138:267.
[0219] The label may be radioactive. Some examples of useful
radioactive labels include .sup.32P, .sup.125I, .sup.131I, and
.sup.3H. Use of radioactive labels have been described in U.K.
2,034,323, U.S. Pat. No. 4,358,535, and U.S. Pat. No.
4,302,204.
[0220] Some examples of non-radioactive labels include enzymes,
chromophores, atoms and molecules detectable by electron
microscopy, and metal ions detectable by their magnetic
properties.
[0221] Some useful enzymatic labels include enzymes that cause a
detectable change in a substrate. Some useful enzymes and their
substrates include, for example, horseradish peroxidase (pyrogallol
and o-phenylenediamine), .beta.-galactosidase (fluorescein
.beta.-D-galactopyranoside), and alkaline phosphatase
(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The
use of enzymatic labels has been described in U.K. 2,019,404, EP
63,879, and by Rotman, Proc. Natl. Acad. Sci. USA, 47, 1981-1991
(1961).
[0222] Useful chromophores include, for example, fluorescent,
chemiluminescent, and bioluminescent molecules, as well as dyes.
Some specific chromophores useful in the present invention include,
for example, fluorescein, rhodamine, Texas red, phycoerythrin,
umbelliferone, luminol.
[0223] The labels may be conjugated to the antibody or nucleotide
probe by methods that are well known in the art. The labels may be
directly attached through a functional group on the probe. The
probe either contains or can be caused to contain such a functional
group. Some examples of suitable functional groups include, for
example, amino, carboxyl, sulfhydryl, maleimide, isocyanate,
isothiocyanate. Alternatively, labels such as enzymes and
chromophores may be conjugated to the antibodies or nucleotides by
means of coupling agents, such as dialdehydes, carbodiimides,
dimaleimides, and the like.
[0224] The label may also be conjugated to the probe by means of a
ligand attached to the probe by a method described above and a
receptor for that ligand attached to the label. Any of the known
ligand-receptor combinations is suitable. Some suitable
ligand-receptor pairs include, for example, biotin-avidin or
biotin-streptavidin, and antibody-antigen.
[0225] In another preferred embodiment, the chimeric fusion
molecules of the invention can be used for imaging. In imaging
uses, the complexes are labeled so that they can be detected
outside the body. Typical labels are radioisotopes, usually ones
with short half-lives. The usual imaging radioisotopes, such as
.sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.99mTc, .sup.186Re,
.sup.188Re, .sup.64Cu, .sup.67Cu, .sup.212Bi, .sup.213Bi,
.sup.67Ga, .sup.90Y, .sup.111In, .sup.18F, .sup.3H, .sup.14C,
.sup.35S or .sup.32P can be used. Nuclear magnetic resonance (NMR)
imaging enhancers, such as gadolinium-153, can also be used to
label the complex for detection by NMR. Methods and reagents for
performing the labeling, either in the polynucleotide or in the
protein moiety, are considered known in the art.
Domain Molecules
[0226] As described supra, the invention provides chimeric
molecules that include both an anti-angiogenic agent domain and
carrier domain. The anti-angiogenic agent domain reduces tumor
growth (e.g., by inhibiting angiogenesis), while the carrier domain
confers a functional attribute to the chimeric molecule. For
instance, where the carrier domain is an Ig domain, it can function
to target the chimeric molecule to a particular site (e.g., the
antigen-binding portion of the antibody binds to an antigen
expressed by a target cell and/or the Fc portion of the Ig domain
can target the chimeric molecule to an Fc receptor-bearing cell);
to increase stability of the chimeric molecule (e.g., for in vitro
storage or in vivo delivery); to impart an effector function to the
chimeric molecule (e.g., immune response-stimulating, cytotoxicity,
etc.); or to facilitate purification of the chimeric molecule.
[0227] The carrier domain can be any substance that imparts a
function to the chimeric molecule. For example, a carrier domain
can be a molecule that increases the stability of the chimeric
molecule (e.g., for in vitro storage or in vivo delivery);
introduces an effector function to the chimeric molecule (e.g.,
immune response-stimulating, cytotoxicity, etc.); or facilitates
purification of the chimeric molecule. For increasing the stability
of the chimeric molecule, the carrier domain can be a protein that
has been shown to stabilize molecules in an in vitro storage or in
vivo delivery setting. For example, carrier domains for increasing
the stability of the chimeric molecule include one or more domains
from an Ig molecule (e.g., a CH.sub.2--CH.sub.3 fragment). Other
carrier domains that can be used to stabilize the chimeric molecule
can be identified empirically. For instance, a molecule can be
screened for suitability as a carrier domain by conjugating the
molecule to anti-angiogenic agent and testing the conjugated
product in in vitro or in vivo stability assays.
[0228] In another preferred embodiment, carrier domains within the
invention facilitate purification of the chimeric molecule. Any
molecule known to facilitate purification of a chimeric molecule
can be used. Representative examples of such carrier domains
include antibody fragments and affinity tags (e.g., GST, HIS, FLAG,
and HA). Chimeric molecules containing an affinity tag can be
purified using immunoaffinity techniques (e.g., agarose affinity
gels, glutathione-agarose beads, antibodies, and nickel column
chromatography). Chimeric molecules that contain an Ig domain as a
carrier domain can be purified using immunoaffinity chromatography
techniques known in the art (e.g., protein A or protein G
chromatography).
[0229] Other carrier domains within the invention that can be used
to purify the chimeric molecule can be readily identified by
testing the molecules in a functional assay. For instance, a
molecule can be screened for suitability as a carrier domain by
fusing the molecule to an anti-angiogenic agent and testing the
fusion for purity and yield in an in vitro assay. The purity of
recombinant proteins can be estimated by conventional techniques,
for example, SDS-PAGE followed by the staining of gels with
Coomassie-Blue.
[0230] A number of other carrier domains can be used to impart an
effector function to the chimeric molecule. These include other
cytotoxins, drugs, detectable labels, targeting ligands, and
delivery vehicles. Examples of these are described in U.S. Pat. No.
6,518,061 and U.S. published patent application number
20020159972.
[0231] A preferred carrier domain for use in the chimeric molecule
is an Ig or portion of an Ig. The Ig domain may take the form of a
single chain antibody (e.g., a scFV), an Fab fragment, an
F(ab').sub.2 fragment, an Ig heavy chain, or an Ig in which one or
more of the constant regions has been removed. The Ig domain can be
derived from any Ig class including IgG, IgM, IgE, IgA, IgD and any
subclass thereof. In some applications, it is preferred that the Ig
domain includes a large hinge region, e.g., one from IgG3.
[0232] In another preferred embodiment, the Ig domain is a
minibody. A small protein scaffold called a "minibody" was designed
using a part of the Ig VH domain as the template (Pessi et al.,
Nature, 362:367-69 (1993)). Minibodies with high affinity
(dissociation constant (K.sub.d) about 10.sup.-7 M) to
interleukin-6 were identified by randomizing loops corresponding to
CDR1 and CDR2 of VH and then selecting mutants using the phage
display method (Martin et al., EMBO J. 1994 Nov. 15; 13(22):
5303-5309). These experiments demonstrated that the essence of the
antibody function could be transferred to a smaller system. Thus,
the chimeric fusion molecule may comprise a minibody Ig domain.
[0233] Chimeric molecules can be prepared using conventional
techniques in molecular biology or protein chemistry. Where the
chimeric molecule is a fusion protein, molecular biology methods
can be used to join two or more genes in frame into a single
nucleic acid. The nucleic acid can then be expressed in an
appropriate host cell under conditions in which the chimeric
molecule is produced. A carrier domain might also be conjugated
(e.g., covalently bonded) to an anti-angiogenic agent domain by
other methods known in the art for conjugating two such molecules
together. For example, the anti-angiogenic agent domain can be
chemically derivatized with a carrier domain either directly or
using a linker (spacer). Several methods and reagents (e.g.,
cross-linkers) for mediating this conjugation are known. See, e.g.,
catalog of Pierce Chemical Company; and Means and Feeney, Chemical
Modification of Proteins, Holden-Day Inc., San Francisco, Calif.
1971; "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic
Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine,
Academic Press, pp. 168-190 (1982); Waldmann (1991) Science, 252:
1657; and U.S. Pat. Nos. 4,545,985 and 4,894,443.
[0234] An anti-angiogenic agent domain may be fused or conjugated
to a carrier domain in various orientations. For example, the
carrier domain may be joined to either the amino or carboxy termini
of an anti-angiogenic agent domain. The anti-angiogenic agent
domain may also be joined to an internal region of the carrier
domain, or conversely, the carrier domain may be joined to an
internal location of the anti-angiogenic agent domain.
[0235] In some circumstances, it is desirable to free the carrier
domain from the anti-angiogenic agent domain when the chimeric
molecule has reached its target site. Therefore, chimeric
conjugates featuring linkages that are cleavable in the vicinity of
the target site may be used when one of the domains is to be
released at the target site. Cleaving of the linkage to release the
carrier domain from the anti-angiogenic agent domain may be
prompted by enzymatic activity or conditions to which the conjugate
is subjected either inside the target cell or in the vicinity of
the target site. When the target site is a tumor, a linker which is
cleavable under conditions present at the tumor site (e.g. when
exposed to tumor-associated enzymes or acidic pH) may be used. A
number of different cleavable linkers are known to those of skill
in the art. See, e.g., U.S. Pat. Nos. 4,618,492; 4,542,225; and
4,625,014. The mechanisms for release of an agent from these linker
groups include, for example, irradiation of a photolabile bond and
acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example,
includes a description of immunoconjugates comprising linkers which
are cleaved at the target site in vivo by the proteolytic enzymes
of the patient's complement system. In view of the large number of
methods that have been reported for attaching a variety of
radiodiagnostic compounds, radiotherapeutic compounds, drugs,
toxins, and other agents to proteins one skilled in the art will be
able to determine a suitable method for attaching a given carrier
domain to an anti-angiogenic agent domain.
Bispecific Chimeric Molecules
[0236] In another preferred embodiment, chimeric molecules
comprising a modulatory or cytolytic domain is fused to a
bispecific antibody domain or fragments thereof. In one aspect of
the invention, the bispecific antibody comprises two monoclonal
antibodies. However, the bispecific antibody can comprise two
polyclonal antibodies or an engineered bispecific antibody.
[0237] Preferably, each of the specificities of the bispecific
antibody are directed to one or more tumor antigens and/or specific
cell or tissue. Antibodies can be raised against any tumor antigen
from a patient. Thus the targeting of the chimeric molecule can be
individually tailored as the tumor displays different antigens.
[0238] Bispecific antibodies may be constructed by hybrid-hybridoma
techniques, by covalently linking specific antibodies or by other
approaches, like the diabody approach (Kipriyanow et al., Int. J.
Cancer 77 (1998), 763-773). In one aspect of the invention, the
bispecific antibody is a single chain antibody construct.
[0239] For tracking purposes, the bispecific antibody can be
directly labeled or a second antibody specific for a region of the
bispecific antibody is labeled. Detection of the localization of
the chimeric molecule is preferably through cell sorting techniques
such as flow cytometry. For example, wherein samples are taken at
different time intervals after administration of the chimeric
molecule for imaging and diagnostic purposes.
[0240] In accordance with the invention, the bispecific antibody,
targets chimeric molecules to a specific location in vivo. For
example, the location can be to myocardial tissues, breast, liver,
spleen, ovaries, testis, hepatocyte, kidneys and the like. The
bispecific antibody determines the specific antigen to which the
chimeric molecule is targeted.
[0241] As described above, the specificity of the antibody domain
can be directed to a specific tissue antigen wherein the tumor has
been detected coupled with specificity for that particular tumor
antigen. Alternatively, the bispecific antibody domain is directed
to two tumor antigens that are expressed by the tumor. The
bispecific domain can be fused to any modulatory or cytolytic
domain discussed above.
[0242] In another embodiment of the invention, the bispecific
antibody (BiAb) construct is a bispecific antibody that binds to
one or more tumor antigens as a first or second antigen and a cell
or tissue specific antigen as a second antigen. The antibody may be
covalently bound to the a modulatory or cytolytic molecule and the
chimeric molecule may be constructed by chemical coupling,
producing a fusion protein or a mosaic protein from the antibody
and from a modified or unmodified prokaryotic or eukaryotic
modulatory or cytotoxic molecule. Furthermore, the antibody may be
joined to modulatory or cytotoxic molecule via multimerization
domains.
[0243] In another embodiment of the invention, the chimeric
polypeptide of the invention, e.g., a endostatin construct, is a
fusion construct of a modified or an unmodified endostatin with a
modified or an unmodified modulatory or cytotoxic molecule. The
construct may be bound in vitro and/or in vivo, e.g., by a
multimerization domain, to bispecific antibody domain. The chimeric
molecule constructs may, inter alia, result from chemical coupling,
may be recombinantly produced, or may be produced as a fusion
protein as described above. In one aspect, the moiety specifically
binds to at least one tumor antigen.
[0244] The compositions of the invention can comprise any cytotoxic
agent as described infra. For example, in one aspect, the toxin may
be a polypeptide toxin, e.g., a Pseudomonas exotoxin, like PE38,
PE40 or PE37, or a truncated version thereof, or a ribosome
inactivating protein gelonin (e.g., Boyle (1996) J. Immunol.
18:221-230), and the like. The compositions of the invention can be
conjugated to any cytotoxic pharmaceuticals, e.g., radiolabeled
with a cytotoxic agents, such as, e.g., 0.1311 (e.g., Shen (1997)
Cancer 80(12 Suppl.): 2553-2557), copper-67 (e.g., Deshpande (1988)
J. Nucl. Med. 29:217-225).
[0245] In one embodiment, the chimeric molecule construct is a
fusion (poly)peptide or a mosaic (poly)peptide. The fusion
(poly)peptide may comprise merely the domains of the constructs as
described herein, as well as (a) functional fragment(s) thereof.
However, it is also envisaged that the fusion (poly)peptide
comprises further domains and/or functional stretches. Therefore,
the fusion (poly)peptide can comprise at least one further domain,
this domain being linked by covalent or non-covalent bonds. The
linkage as well as the construction of such constructs, can be
based on genetic fusion according to the methods described herein
or known in the art (e.g., Sambrook et al., loc. cit., Ausubel,
"Current Protocols in Molecular Biology", Green Publishing
Associates and Wiley Interscience, N.Y. (1989)) or can be performed
by, e.g., chemical cross-linking as described in, e.g., WO
94/04686. The additional domain present in the construct may be
linked by a flexible linker, such as a (poly)peptide linker,
wherein the (poly)peptide linker can comprises plural, hydrophilic,
peptide-bonded amino acids of a length sufficient to span the
distance between the C-terminal end of the further domain and the
N-terminal end of the peptide, (poly)peptide or antibody or vice
versa. The linker may, inter alia, be a Glycine, a Serine and/or a
Glycine/Serine linker. Additional linkers comprise oligomerization
domains. Oligomerization domains can facilitate the combination of
two or several antigens or fragments thereof in one functional
molecule. Non-limiting examples of oligomerization domains comprise
leucine zippers (like jun-fos, GCN4, E/EBP; Kostelny, J. Immunol.
148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Sci. USA 94 (1997),
3673-3678, Williams, Genes Dev. 5 (1991), 1553-1563; Suter, "Phage
Display of Peptides and Proteins", Chapter 11, (1996), Academic
Press), antibody-derived oligomerization domains, like constant
domains C.sub.H1 and C.sub.L (Mueller, FEBS Letters 422 (1998),
259-264) and/or tetramerization domains like GCN.sub.4-LI
(Zerangue, Proc. Natl. Acad. Sci. USA 97 (2000), 3591-3595).
[0246] In another preferred embodiment, the linker comprises
integrin motifs such as for example, one or more of NGR motifs
(Asn-Gly-Arg) and/or RGD (Arg-Gly-Asp) and combinations thereof.
Furthermore, the chimeric fusion construct to be used in the
present invention, as described herein, may comprise at least one
further domain, inter alia, domains which provide for purification
means, like, e.g. histidine stretches. The further domain(s) may be
linked by covalent or non-covalent bonds.
[0247] The linkage can be based on genetic fusion according to the
methods known in the art and described herein or can be performed
by, e.g., chemical cross-linking as described in, e.g., WO
94/04686. The additional domain present in the construct may be
linked by a flexible linker, such as a polypeptide linker to one of
the binding site domains; the polypeptide linker can comprise
plural, hydrophilic or peptide-bonded amino acids of a length
sufficient to span the distance between the C-terminal end of one
of the domains and the N-terminal end of the other of the domains
when the polypeptide assumes a conformation suitable for binding
when disposed in aqueous solution.
Immune Activating Chimeric Fusion Molecules
[0248] It is also envisaged that the constructs disclosed for uses,
compositions and methods of the present invention comprises (a)
further domain(s) which may function as immunomodulators. The
immunomodulators comprise, but are not limited to cytokines,
lymphokines, T cell co-stimulatory ligands, etc. Preferably, the
chimeric fusion molecule targets and delivers a modulatory or
cytolytic molecule to the tumor cell and also recruits immune cells
and/or activated immune cells to the tumor.
[0249] Adequate activation resulting in priming of naive T-cells is
critical to primary immune responses and depends on two signals
derived from professional APCs (antigen-presenting cells) like
dendritic cells. The first signal is antigen-specific and normally
mediated by stimulation of the clonotypic T-cell antigen receptor
(TCR) that is induced by processed antigen presented in the context
of MHC class-I or MHC class-II molecules. However, this primary
stimulus is insufficient to induce priming responses of naive
T-cells, and the second signal is required which is provided by an
interaction of specific T-cell surface molecules binding to
co-stimulatory ligand molecules on antigen presenting cells (APCs),
further supporting the proliferation of primed T-cells. The term
"T-cell co-stimulatory ligand" therefore denotes in the light of
the present invention molecules, which are able to support priming
of naive T-cells in combination with the primary stimulus and
include, but are not limited to, members of the B7 family of
proteins, including B7-1 (CD80) and B7-2 (CD86), 4-1BB ligand, CD40
ligand, OX40 ligand.
[0250] The chimeric fusion molecule construct described herein may
comprise further receptor or ligand function(s), and may comprise
immune-modulating effector molecule or a fragment thereof. An
immune-modulating effector molecule positively and/or negatively
influences the humoral and/or cellular immune system, particularly
its cellular and/or non-cellular components, its functions, and/or
its interactions with other physiological systems. The
immune-modulating effector molecule may be selected from the group
comprising cytokines, chemokines, macrophage migration inhibitory
factor (MIF; as described, in Bernhagen (1998), Mol Med 76(3-4);
151-61 or Metz (1997), Adv Immunol 66, 197-223), T-cell receptors
and soluble MHC molecules. Such immune-modulating effector
molecules are well known in the art and are described, inter alia,
in Paul, "Fundamental immunology", Raven Press, New York (1989). In
particular, known cytokines and chemokines are described in Meager,
"The Molecular Biology of Cytokines" (1998), John Wiley & Sons,
Ltd., Chichester, West Sussex, England; (Bacon (1998). Cytokine
Growth Factor Rev 9(2):167-73; Oppenheim (1997). Clin Cancer Res
12, 2682-6; Taub, (1994) Ther. Immunol. 1(4), 229-46 or Michiel,
(1992). Semin Cancer Biol 3(1), 3-15).
[0251] Immune cell activity that may be measured include, but is
not limited to: (1) cell proliferation by measuring the DNA
replication; (2) enhanced cytokine production, including specific
measurements for cytokines, such as IFN-.gamma., GM-CSF, or
TNF-.alpha.; (3) cell mediated target killing or lysis; (4) cell
differentiation; (5) immunoglobulin production; (6) phenotypic
changes; (7) production of chemotactic factors or chemotaxis,
meaning the ability to respond to a chemotactin with chemotaxis;
(8) immunosuppression, by inhibition of the activity of some other
immune cell type; and, (9) apoptosis, which refers to fragmentation
of activated immune cells under certain circumstances, as an
indication of abnormal activation.
Modified Chimeric Molecules
[0252] The constructs of the present invention may comprise domains
originating from one species, e.g., from mammals, such as human.
However, chimeric and/or human and/or humanized constructs are also
envisaged and within the scope of the present invention.
[0253] Furthermore, the polynucleotide/nucleic acid molecules of
the invention may comprise, for example, thioester bonds and/or
nucleotide analogues. The modifications may be useful for the
stabilization of the nucleic acid molecule, e.g., against endo-
and/or exonucleases in the cell. These nucleic acid molecules may
be transcribed by an appropriate vector containing a chimeric gene
which allows for the transcription of the nucleic acid molecule in
the cell. The polynucleotide/nucleic acid molecules of the
invention may be a recombinantly produced chimeric nucleic acid
molecule comprising any of the aforementioned nucleic acid
molecules either alone or in combination. The polynucleotide may
be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a
recombinantly produced chimeric nucleic acid molecule comprising
any of those polynucleotides either alone or in combination. The
polynucleotide can be part of a vector, e.g., an expression vector,
including, e.g., recombinant viruses. The vectors may comprise
further genes, such as marker genes, that allow for the selection
of the vector in a suitable host cell and under suitable
conditions.
[0254] In one aspect, the polynucleotides of the invention are
operatively linked to expression control sequences allowing
expression in prokaryotic or eukaryotic cells. Expression of the
polynucleotide comprises transcription of the polynucleotide into a
translatable mRNA. Regulatory elements ensuring expression in
cells, including eukaryotic cells, such as mammalian cells, are
well known to those skilled in the art. They usually comprise
regulatory sequences ensuring initiation of transcription, and,
optionally, poly-A signals ensuring termination of transcription
and stabilization of the transcript. Additional regulatory elements
may include transcriptional as well as translational enhancers,
and/or naturally-associated or heterologous promoter regions.
Exemplary regulatory elements permitting expression in prokaryotic
host cells comprise, e.g., the PL, lac, trp or tac promoter in E.
coli, and examples for regulatory elements permitting expression in
eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the
CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer,
SV40-enhancer or a globin intron in mammalian and other animal
cells. The nucleic acids of the invention can also comprise, in
addition to elements responsible for the initiation of
transcription, other elements, such regulatory elements and
transcription termination signals, such as the SV40-poly-A site or
the tk-poly-A site (termination sequences are typically downstream
of the polynucleotide coding sequence). Furthermore, depending on
the expression system used, nucleic acid sequences encoding leader
sequences capable of directing the polypeptide to a cellular
compartment, or secreting it into the medium, may be added to the
coding sequence of the polynucleotide of the invention; such leader
sequences are well known in the art. The leader sequence(s) is
(are) assembled in appropriate phase with translation, initiation
and termination sequences. In one aspect, the leader sequence is
capable of directing secretion of translated chimeric protein, or a
portion thereof, into the periplasmic space or extracellular
medium. Optionally, the heterologous sequence can encode a fusion
protein including an N-terminal identification peptide imparting
desired characteristics, e.g., stabilization or simplified
purification of expressed recombinant product; see supra. In this
context, suitable expression vectors are known in the art such as
Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8,
pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (GIBCO BRL).
Expression control sequences can be eukaryotic promoter systems in
vectors capable of transforming or transfecting eukaryotic host
cells; control sequences for prokaryotic hosts may also be used.
Once the vector has been incorporated into the appropriate host,
the host can be maintained under conditions suitable for high level
expression of the nucleotide sequences; and, as desired, the
collection and purification of the polypeptide of the invention may
follow.
[0255] As described above, the polynucleotide of the invention can
be used alone or as part of a vector (e.g., an expression vector or
a recombinant virus), or in cells, to express the chimeric fusion
molecules of the invention. The polynucleotides or vectors
containing the DNA sequence(s) encoding any one of the chimeric
fusion molecules of the invention can be introduced into the cells,
which in turn produce the polypeptide of interest.
[0256] The present invention is directed to vectors, e.g.,
plasmids, cosmids, viruses and bacteriophages, or any expression
system used conventionally in genetic engineering, that comprise a
polynucleotide encoding a chimeric fusion molecule of the
invention. The vector can be an expression vector and/or a gene
transfer or targeting vector. Expression vectors derived from
viruses such as retroviruses, vaccinia virus, adeno-associated
virus, herpes viruses, or bovine papilloma virus, may be used for
delivery of the polynucleotides or vectors of the invention into
targeted cell populations. Methods which are well known to those
skilled in the art can be used to construct recombinant vectors;
see, for example, the techniques described in Sambrook, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989)
N.Y. and Ausubel, Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley Interscience, N.Y. (1989).
Alternatively, the polynucleotides and vectors of the invention can
be reconstituted into liposomes for delivery to target cells. The
vectors containing the polynucleotides of the invention can be
transferred into the host cell by well-known methods, which vary
depending on the type of cellular host. For example, calcium
chloride transfection is commonly utilized for prokaryotic cells,
whereas calcium phosphate treatment or electroporation may be used
for other cellular hosts; see Sambrook, supra.
[0257] Once expressed, the chimeric fusion molecules of the present
invention can be purified according to standard procedures of the
art, including ammonium sulfate precipitation, affinity columns,
column chromatography, gel electrophoresis and the like; see,
Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982). In
alternative aspects, the invention is directed to substantially
pure chimeric polypeptides of at least about 90% to about 95%
homogeneity; between about 95% to 98% homogeneity; and about 98% to
about 99% or more homogeneity; these "substantially pure"
polypeptides can be used in the preparation of pharmaceuticals.
Once purified, partially or to a homogeneity as desired, the
polypeptides may then be used therapeutically (including
extracorporeally) or in developing and performing assay
procedures.
[0258] In a still further embodiment, the present invention relates
to a cell containing the polynucleotide or vector of the invention,
or to a host cell transformed with a polynucleotide or vector of
the invention. In alternative aspects, the host/cell is a
eukaryotic cell, such as a mammalian cell, particularly if
therapeutic uses of the polypeptide are envisaged. Of course, yeast
and prokaryotic, e.g., bacterial cells, may serve as well, in
particular, if the produced polypeptide is used for
non-pharmaceutical purposes, e.g., as in diagnostic tests or kits
or in screening methods.
[0259] The polynucleotide or vector of the invention that is
present in the host cell may either be integrated into the genome
of the host cell or it may be maintained extrachromosomally, e.g.,
as an episome.
[0260] The term "prokaryotic" is meant to include all bacteria that
can be transformed or transfected with DNA or RNA molecules for the
expression of a polypeptide of the invention. Prokaryotic hosts may
include gram negative as well as gram positive bacteria such as,
for example, E. coli, S. typhimurium, Serratia marcescens and
Bacillus subtilis. The term "eukaryotic" is meant to include yeast,
higher plant, insect and mammalian cells. Depending upon the host
employed in a recombinant production procedure, the chimeric fusion
molecules of the present invention may be glycosylated or may be
non-glycosylated. Chimeric fusion molecules of the invention may
also include an initial methionine amino acid residue. A
polynucleotide coding for a polypeptide of the invention can be
used to transform or transfect the host using any of the techniques
commonly known to those of ordinary skill in the art.
[0261] In one aspect, the nucleic acids encoding the chimeric
polypeptide of the invention (including those sequences in vectors,
e.g., plasmid or virus) further comprise, genetically fused
thereto, sequences encoding an epitope tag, e.g., an N-terminal
FLAG-tag and/or a C-terminal His-tag. In one aspect, the length of
the FLAG-tag is about 4 to 8 amino acids; or, is about 8 amino
acids in length. Methods for preparing fused, operably linked genes
and expressing them in, e.g., mammalian cells and bacteria are
well-known in the art (Sambrook, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989). The genetic constructs and methods described therein can be
utilized for expression of the polypeptide of the invention in
eukaryotic or prokaryotic hosts. In general, expression vectors
containing promoter sequences which facilitate the efficient
transcription of the inserted polynucleotide are used in connection
with the host. The expression vector typically contains an origin
of replication, a promoter, and a terminator, as well as specific
genes which are capable of providing phenotypic selection of the
transformed cells. Furthermore, transgenic non-human animals, such
as mammals (e.g., mice, goats), comprising nucleic acids or cells
of the invention may be used for the large scale production of the
chimeric polypeptides of the invention.
[0262] In a further embodiment, the invention is directed to a
process for the preparation of a polypeptide of the invention
comprising cultivating a (host) cell of the invention under
conditions suitable for the expression of the chimeric fusion
molecule construct and isolating the polypeptide from the cell or
the culture medium. The transformed hosts can be grown in
fermentors and cultured according to techniques known in the art to
achieve optimal cell growth. The produced constructs of the
invention can then be isolated from the growth medium, cellular
lysates, or cellular membrane fractions. The isolation and
purification of the expressed polypeptides of the invention (e.g.,
microbially expressed) may be by any conventional means such as,
e.g., preparative chromatographic separations and immunological
separations, such as those involving the use of monoclonal or
polyclonal antibodies directed against, e.g., a tag of the
polypeptide of the invention or as described in the appended
examples.
[0263] Depending on the host cell, renaturation techniques may be
required to attain proper conformation. If necessary, point
substitutions seeking to optimize binding may be made in the DNA
using conventional cassette mutagenesis or other protein
engineering methodology such as is disclosed herein. Preparation of
the polypeptides of the invention may also be dependent on
knowledge of the amino acid sequence (or corresponding DNA or RNA
sequence) of bioactive proteins such as enzymes, toxins, growth
factors, cell differentiation factors, receptors, anti-metabolites,
hormones or various cytokines or lymphokines. Such sequences are
reported in the literature and available through computerized data
banks. The present invention further relates to a chimeric
polypeptide, encoded by a polynucleotide of the invention or
produced by the method described hereinabove.
[0264] Additionally, the present invention provides for
compositions comprising the polynucleotide, the vector, the host
cell, and a chimeric fusion molecule, as described herein.
[0265] The term "composition", in context of this invention,
comprises at least one polynucleotide, vector, host cell, chimeric
polypeptide of the invention, as described herein. The composition,
optionally, further comprises other molecules, either alone or in
combination, such as molecules which are capable of modulating
and/or interfering with the immune system. The composition may be
in solid, liquid or gaseous form and may be, inter alia, in a form
of a powder(s), a tablet(s), a solution(s) or an aerosol(s). In
alternative embodiments, the composition comprises at least two, at
least three, at least four, or more than four, compounds of the
invention. The composition can be a pharmaceutical composition
further comprising, optionally, a pharmaceutically acceptable
carrier, diluent and/or excipient.
Humanized Antibodies
[0266] In a preferred embodiment, antibodies of the invention
comprise human or humanized antibodies. Humanized antibodies are
antibodies whose light and heavy chain genes have been constructed,
typically by genetic engineering, from immunoglobulin variable and
constant region genes belonging to different species. For example,
the variable segments of the genes from a mouse monoclonal antibody
may be joined to human constant segments, such as gamma 1 and gamma
3. A typical therapeutic chimeric antibody is thus a hybrid protein
composed of the variable or antigen-binding domain from a mouse
antibody and the constant or effector domain from a human antibody,
although other mammalian species may be used.
[0267] As used herein, the term "humanized" immunoglobulin refers
to an immunoglobulin comprising a human framework region and one or
more CDR's from a non-human (usually a mouse or rat)
immunoglobulin. The non-human immunoglobulin providing the CDR's is
called the "donor" and the human immunoglobulin providing the
framework is called the "acceptor." Constant regions need not be
present, but if they are, they must be substantially identical to
human immunoglobulin constant regions, i.e., at least about 85-90%,
preferably about 95% or more identical. Hence, all parts of a
humanized immunoglobulin, except possibly the CDR's, are
substantially identical to corresponding parts of natural human
immunoglobulin sequences. A "humanized antibody" is an antibody
comprising a humanized light chain and a humanized heavy chain
immunoglobulin, e.g., the entire variable region of a chimeric
antibody is non-human. One says that the donor antibody has been
"humanized", by the process of "humanization", because the
resultant humanized antibody is expected to bind to the same
antigen as the donor antibody that provides the CDR's.
[0268] It is understood that the humanized antibodies may have
additional conservative amino acid substitutions which have
substantially no effect on antigen binding or other immunoglobulin
functions. By conservative substitutions are intended combinations
such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys,
Arg; Phe, and Tyr.
[0269] Humanized immunoglobulins, including humanized antibodies,
have been constructed by means of genetic engineering. Most
humanized immunoglobulins that have been previously described have
comprised a framework that is identical to the framework of a
particular human immunoglobulin chain, the acceptor, and three
CDR's from a non-human donor immunoglobulin chain.
[0270] A principle is that as an acceptor, a framework is used from
a particular human immunoglobulin that is unusually homologous to
the donor immunoglobulin to be humanized, or use a consensus
framework from many human antibodies. For example, comparison of
the sequence of a mouse heavy (or light) chain variable region
against human heavy (or light) variable regions in a data bank (for
example, the National Biomedical Research Foundation Protein
Identification Resource) shows that the extent of homology to
different human regions varies greatly, typically from about 40% to
about 60-70%. By choosing as the acceptor immunoglobulin one of the
human heavy (respectively light) chain variable regions that is
most homologous to the heavy (respectively light) chain variable
region of the donor immunoglobulin, fewer amino acids will be
changed in going from the donor immunoglobulin to the humanized
immunoglobulin. Hence, and again without intending to be bound by
theory, it is believed that there is a smaller chance of changing
an amino acid near the CDR's that distorts their conformation.
Moreover, the precise overall shape of a humanized antibody
comprising the humanized immunoglobulin chain may more closely
resemble the shape of the donor antibody, also reducing the chance
of distorting the CDR's.
[0271] Typically, one of the 3-5 most homologous heavy chain
variable region sequences in a representative collection of at
least about 10 to 20 distinct human heavy chains will be chosen as
an acceptor to provide the heavy chain framework, and similarly for
the light chain. Preferably, one of the 1-3 most homologous
variable regions will be used. The selected acceptor immunoglobulin
chain will most preferably have at least about 65% homology in the
framework region to the donor immunoglobulin.
[0272] In many cases, it may be considered preferable to use light
and heavy chains from the same human antibody as acceptor
sequences, to be sure the humanized light and heavy chains will
make favorable contacts with each other. Regardless of how the
acceptor immunoglobulin is chosen, higher affinity may be achieved
by selecting a small number of amino acids in the framework of the
humanized immunoglobulin chain to be the same as the amino acids at
those positions in the donor rather than in the acceptor.
[0273] Humanized antibodies generally have advantages over mouse or
in some cases chimeric antibodies for use in human therapy: because
the effector portion is human, it may interact better with the
other parts of the human immune system (e.g., destroy the target
cells more efficiently by complement-dependent cytotoxicity (CDC)
or antibody-dependent cellular cytotoxicity (ADCC)); the human
immune system should not recognize the framework or constant region
of the humanized antibody as foreign, and therefore the antibody
response against such an antibody should be less than against a
totally foreign mouse antibody or a partially foreign chimeric
antibody.
[0274] Antibodies can also be genetically engineered. Particularly
preferred are humanized immunoglobulins that are produced by
expressing recombinant DNA segments encoding the heavy and light
chain CDR's from a donor immunoglobulin capable of binding to a
desired antigen, such as the tumor antigens e.g. HER2, attached to
DNA segments encoding acceptor human framework regions.
[0275] The DNA segments typically further include an expression
control DNA sequence operably linked to the humanized
immunoglobulin coding sequences, including naturally-associated or
heterologous promoter regions. Preferably, the expression control
sequences will be eukaryotic promoter systems in vectors capable of
transforming or transfecting eukaryotic host cells, but control
sequences for prokaryotic hosts may also be used. Once the vector
has been incorporated into the appropriate host, the host is
maintained under conditions suitable for high level expression of
the nucleotide sequences, and, as desired, the collection and
purification of the humanized light chains, heavy chains,
light/heavy chain dimers or intact antibodies, binding fragments or
other immunoglobulin forms may follow (see, S. Beychok, Cells of
Immunoglobulin Synthesis, Academic Press, New York, (1979), which
is incorporated herein by reference).
[0276] Human constant region DNA sequences can be isolated in
accordance with well known procedures from a variety of human
cells, but preferably immortalized B-cells. The CDR's for producing
preferred immunoglobulins of the present invention will be
similarly derived from monoclonal antibodies capable of binding to
the predetermined antigen, such as the human T cell receptor CD3
complex, and produced by well known methods in any convenient
mammalian source including, mice, rats, rabbits, or other
vertebrates, capable of producing antibodies. Suitable source cells
for the constant region and framework DNA sequences, and host cells
for immunoglobulin expression and secretion, can be obtained from a
number of sources, such as the American Type Culture Collection
("Catalogue of Cell Lines and Hybridomas," sixth edition (1988)
Rockville, Md., U.S.A., which is incorporated herein by
reference).
[0277] Other "substantially homologous" modified immunoglobulins to
the native sequences can be readily designed and manufactured
utilizing various recombinant DNA techniques well known to those
skilled in the art. For example, the framework regions can vary at
the primary structure level by several amino acid substitutions,
terminal and intermediate additions and deletions, and the like.
Moreover, a variety of different human framework regions may be
used singly or in combination as a basis for the humanized
immunoglobulins of the present invention. In general, modifications
of the genes may be readily accomplished by a variety of well-known
techniques, such as site-directed mutagenesis (see, Gillman and
Smith, Gene, 8, 81-97 (1979) and S. Roberts et al., Nature, 328,
731-734 (1987), both of which are incorporated herein by
reference).
[0278] Substantially homologous immunoglobulin sequences are those
which exhibit at least about 85% homology, usually at least about
90%, and preferably at least about 95% homology with a reference
immunoglobulin protein.
[0279] Alternatively, polypeptide fragments comprising only a
portion of the primary antibody structure may be produced, which
fragments possess one or more immunoglobulin activities (e.g.,
complement fixation activity). These polypeptide fragments may be
produced by proteolytic cleavage of intact antibodies by methods
well known in the art, or by inserting stop codons at the desired
locations in vectors known to those skilled in the art, using
site-directed mutagenesis.
[0280] As stated previously, the DNA sequences can be expressed in
hosts after the sequences have been operably linked to (i.e.,
positioned to ensure the functioning of) an expression control
sequence. These expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers, e.g., tetracycline or neomycin resistance, to
permit detection of those cells transformed with the desired DNA
sequences (see, e.g., U.S. Pat. No. 4,704,362, which is
incorporated herein by reference).
[0281] E. coli is one prokaryotic host useful particularly for
cloning the DNA sequences of the present invention. Other microbial
hosts suitable for use include bacilli, such as Bacillus subtilis,
and other enterobacteriaceae, such as Salmonella, Serratia, and
various Pseudomonas species. In these prokaryotic hosts, one can
also make expression vectors, which will typically contain
expression control sequences compatible with the host cell (e.g.,
an origin of replication). In addition, any number of a variety of
well-known promoters will be present, such as the lactose promoter
system, a tryptophan (trp) promoter system, a beta-lactamase
promoter system, or a promoter system from phage lambda. The
promoters will typically control expression, optionally with an
operator sequence, and have ribosome binding site sequences and the
like, for initiating and completing transcription and
translation.
[0282] Other microbes, such as yeast, may also be used for
expression. Saccharomyces is a preferred host, with suitable
vectors having expression control sequences, such as promoters,
including 3-phosphoglycerate kinase or other glycolytic enzymes,
and an origin of replication, termination sequences and the like as
desired.
[0283] In addition to microorganisms, mammalian tissue cell culture
may also be used to express and produce the polypeptides of the
present invention (see, Winnacker, "From Genes to Clones," VCH
Publishers, New York, N.Y. (1987), which is incorporated herein by
reference). Eukaryotic cells are actually preferred, because a
number of suitable host cell lines capable of secreting intact
immunoglobulins have been developed in the art, and include the CHO
cell lines, various COS cell lines, HeLa cells, preferably myeloma
cell lines, etc, and transformed B-cells or hybridomas. Expression
vectors for these cells can include expression control sequences,
such as an origin of replication, a promoter, an enhancer (Queen et
al., Immunol. Rev., 89, 49-68 (1986), which is incorporated herein
by reference), and necessary processing information sites, such as
ribosome binding sites, RNA splice sites, polyadenylation sites,
and transcriptional terminator sequences. Preferred expression
control sequences are promoters derived from immunoglobulin genes,
SV40, Adenovirus, cytomegalovirus, Bovine Papilloma Virus, and the
like.
[0284] The vectors containing the DNA segments of interest (e.g.,
the heavy and light chain encoding sequences and expression control
sequences) can be transferred into the host cell by well-known
methods, which vary depending on the type of cellular host. For
example, calcium chloride transfection is commonly utilized for
prokaryotic cells, whereas calcium phosphate treatment or
electroporation may be used for other cellular hosts. (See,
generally, Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, (1982), which is incorporated herein by
reference.)
[0285] Once expressed, the whole antibodies, their dimers,
individual light and heavy chains, or other immunoglobulin forms of
the present invention, can be purified according to standard
procedures of the art, including ammonium sulfate precipitation,
affinity columns, column chromatography, gel electrophoresis and
the like (see, generally, R. Scopes, "Protein Purification",
Springer-Verlag, N.Y. (1982)). Substantially pure immunoglobulins
of at least about 90 to 95% homogeneity are preferred, and 98 to
99% or more homogeneity most preferred, for pharmaceutical uses.
Once purified, partially or to homogeneity as desired, the
polypeptides may then be used therapeutically (including
extracorporeally) or in developing and performing assay procedures,
immunofluorescent staining, and the like. (See, generally,
Immunological Methods, Vols. I and II, Lefkovits and Pernis, eds.,
Academic Press, New York, N.Y. (1979 and 1981)).
[0286] In general, the subject humanized antibodies are produced by
obtaining nucleic acid sequences encoding the variable heavy and
variable light sequences of an antibody which binds a tumor
antigen, preferably HER2/neu, identifying the CDR's in the variable
heavy and variable light sequences, and grafting such CDR nucleic
acid sequences onto human framework nucleic acid sequences.
[0287] Preferably, the selected human framework will be one that is
expected to be suitable for in vivo administration, i.e., does not
exhibit immunogenicity. This can be determined, e.g., by prior
experience with in vivo usage of such antibodies and by studies of
amino acid sequence similarities. In the latter approach, the amino
acid sequences of the framework regions of the antibody to be
humanized, are compared to those of known human framework regions,
and human framework regions used for CDR grafting are selected
which comprise a size and sequence most similar to that of the
parent antibody, e.g., a murine antibody which binds HER2/neu.
Numerous human framework regions have been isolated and their
sequences reported in the literature. See, e.g., Kabat et al.,
(Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et
al., Sequences of protein of immunological interest (1991)). This
enhances the likelihood that the resultant CDR-grafted "humanized"
antibody, which contains the CDR's of the parent (e.g., murine)
antibody grafted onto the selected human framework regions
significantly retain the antigen binding structure and thus the
binding affinity of the parent antibody.
[0288] Methods for cloning nucleic acid sequences encoding
immunoglobulins are well known in the art and are described in
detail in the Examples which follow. Such methods generally involve
the amplification of the immunoglobulin sequences to be cloned
using appropriate primers by polymerase chain reaction (PCR).
Primers suitable for amplifying immunoglobulin nucleic acid
sequences, and specifically murine variable heavy and variable
light sequences have been reported in the literature. After such
immunoglobulin sequences have been cloned, they are sequenced by
methods well known in the art. This will be effected in order to
identify the variable heavy and variable light sequences, and more
specifically the portions thereof which constitute the CDR's and
FRs. This can be effected by well known methods.
[0289] Once the CDRs and FRs of the cloned antibody sequences which
are to be humanized have been identified, the amino acid sequences
encoding CDRs are then identified (deduced based on the nucleic
acid sequences and the genetic code and by comparison to previous
antibody sequences) and the corresponding nucleic acid sequences
are grafted onto selected human FRs. This may be accomplished by
use of appropriate primers and linkers. Methods for selecting
suitable primers and linkers to provide for ligation of desired
nucleic acid sequences is well within the purview of the ordinary
artisan and include those disclosed in U.S. Pat. No. 4,816,397 to
Boss et al. and U.S. Pat. No. 5,225,539 to Winter et al.
[0290] After the CDRs are grafted onto selected human FRs, the
resultant "humanized" variable heavy and variable light sequences
will then be expressed to produce a humanized chimeric fusion
molecule which binds, for example, HER2/neu. The humanized variable
heavy and/or variable light sequences will be expressed as a fusion
protein so that an intact chimeric fusion molecule which binds, for
example, HER2/neu is produced.
[0291] In another preferred embodiment, the variable heavy and
light sequences can also be expressed in the absence of constant
sequences to produce a humanized Fv chimeric fusion molecule which
binds, for example, HER2/neu. However, fusion of human constant
sequences to the humanized variable region(s) is potentially
desirable because the resultant humanized antibody which binds, for
example, HER2/neu will then possess human effector functions such
as complement-dependent cytotoxicity (CDC) and antibody-dependent
cellular cytotoxicity (ADCC) activity.
[0292] The following references are representative of methods and
vectors suitable for expression of recombinant immunoglobulins
which may be utilized in carrying out the present invention. Weidle
et al., Gene, 51:21-29 (1987); Dorai et al., J. Immunol.,
13(12):4232-4241 (1987); De Waele et al., Eur. J. Biochem.,
176:287-295 (1988); Colcher et al., Cancer Res., 49:1738-1745
(1989); Wood et al., J. Immunol., 145(a):3011-3016 (1990); Bulens
et al., Eur. J. Biochem., 195:235-242 (1991); Beggington et al.,
Biol. Technology, 10:169 (1992); King et al., Biochem. J.,
281:317-323 (1992); Page et al., Biol. Technology, 9:64 (1991);
King et al., Biochem. J., 290:723-729 (1993); Chaudary et al.,
Nature, 339:394-397 (1989); Jones et al., Nature, 321:522-525
(1986); Morrison and Oi, Adv. Immunol, 44:65-92 (1988); Benhar et
al., Proc. Natl. Acad. Sci. USA, 91:12051-12055 (1994); Singer et
al., J. Immunol., 150:2844-2857 (1993); Cooto et al., Hybridoma,
13(3):215-219 (1994); Queen et al., Proc. Natl. Acad. Sci. USA,
86:10029-10033 (1989); Caron et al., Cancer Res., 32:6761-6767
(1992); Cotoma et al., J. Immunol. Meth., 152:89-109 (1992).
Moreover, vectors suitable for expression of recombinant antibodies
are commercially available. The vector may, e.g., be a bare nucleic
acid segment, a carrier-associated nucleic acid segment, a
nucleoprotein, a plasmid, a virus, a viroid, or a transposable
element.
[0293] Host cells known to be capable of expressing functional
immunoglobulins include, e.g.: mammalian cells such as Chinese
Hamster Ovary (CHO) cells; COS cells; myeloma cells, such as NSO
and SP2/0 cells; bacteria such as Escherichia coli; yeast cells
such as Saccharomyces cerevisiae; and other host cells. SP2/0 cells
are one of the preferred types of host cells useful in the present
invention.
[0294] After expression, the antigen binding affinity of the
resulting humanized antibody will be assayed by known methods,
e.g., Scatchard analysis. In a particularly preferred embodiment,
the antigen-binding affinity of the humanized antibody will be at
least 50% of that of the parent antibody, e.g., anti-HER2/neu, more
preferably, the affinity of the humanized antibody will be at least
about 75% of that of the parent antibody, more preferably, the
affinity of the humanized antibody will be at least about 100%,
150%, 200% or 500% of that of the parent antibody.
[0295] In some instances, humanized antibodies produced by grafting
CDRs (from an antibody which binds, for example, a tumor antigen
such as, for example, HER/neu) onto selected human framework
regions may provide humanized antibodies having the desired
affinity to HER2/neu. However, it may be necessary or desirable to
further modify specific residues of the selected human framework in
order to enhance antigen binding. This may occur because it is
believed that some framework residues are essential to or at least
affect antigen binding. Preferably, those framework residues of the
parent (e.g., murine) antibody which maintain or affect
combining-site structures will be retained. These residues may be
identified by X-ray crystallography of the parent antibody or Fab
fragment, thereby identifying the three-dimensional structure of
the antigen-binding site. Also, framework residues involved in
antigen binding may potentially be identified based on previously
reported humanized murine antibody sequences. Thus, it may be
beneficial to retain such framework residues or others from the
parent murine antibody to optimize, for example, HER2/neu binding.
Preferably, such methodology will confer a "human-like" character
to the resultant humanized antibody thus rendering it less
immunogenic while retaining the interior and contacting residues
which affect antigen-binding.
[0296] The present invention further embraces variants and
equivalents which are substantially homologous to the humanized
antibodies and antibody fragments set forth herein. These may
contain, e.g., conservative substitution mutations, i.e. the
substitution of one or more amino acids by similar amino acids. For
example, conservative substitution refers to the substitution of an
amino acid with another within the same general class, e.g., one
acidic amino acid with another acidic amino acid, one basic amino
acid with another basic amino acid, or one neutral amino acid by
another neutral amino acid. What is intended by a conservative
amino acid substitution is well known in the art.
Methods of Delivering a Chimeric Molecule to a Cell
[0297] The invention also provides a method of delivering an
anti-angiogenic agent-carrier chimeric molecule to a cell. The
chimeric molecules of the invention can be delivered to a cell by
any known method. For example, a composition containing the
chimeric molecule can be added to cells suspended in medium.
Alternatively, a chimeric molecule can be administered to an animal
(e.g., by a parenteral route) having a cell expressing a receptor
that binds the chimeric molecule so that the chimeric molecule
binds to the cell in situ. For example, the chimeric molecules of
this invention that feature an Ig domain that is specific for
HER2/neu are particularly well suited as targeting moieties for
binding tumor cells that over express HER2/neu, e.g., breast cancer
and ovarian cancer cells.
Administration of Compositions to Animals
[0298] For targeting a tumor cell in situ, the compositions
described above may be administered to animals including human
beings in any suitable formulation. For example, compositions for
targeting a tumor cell may be formulated in pharmaceutically
acceptable carriers or diluents such as physiological saline or a
buffered salt solution. Suitable carriers and diluents can be
selected on the basis of mode and route of administration and
standard pharmaceutical practice. A description of exemplary
pharmaceutically acceptable carriers and diluents, as well as
pharmaceutical formulations, can be found in Remington's
Pharmaceutical Sciences, a standard text in this field, and in
USP/NF. Other substances may be added to the compositions to
stabilize and/or preserve the compositions.
[0299] The compositions of the invention may be administered to
animals by any conventional technique. The compositions may be
administered directly to a target site by, for example, surgical
delivery to an internal or external target site, or by catheter to
a site accessible by a blood vessel. Other methods of delivery,
e.g., liposomal delivery or diffusion from a device impregnated
with the composition, are known in the art. The compositions may be
administered in a single bolus, multiple injections, or by
continuous infusion (e.g., intravenously). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form. In preferred embodiments, the
compositions are administered intravenously, parenterally, intra
muscularly and the like.
Methods of Treatment
[0300] In a preferred embodiment, a method of treating a patient
with cancer comprises administering to a patient a chimeric fusion
molecule comprising the chimeric fusion molecules described
herein.
[0301] In a preferred embodiment, the chimeric fusion molecule
comprises a polypeptide comprising an anti-HER2 binding domain and
an endostatin molecule having a proline substituted with alanine at
amino acid position 125.
[0302] The chimeric molecules described herein have been found to
exhibit superior anti-angiogenic properties as compared to
endostatin or Herceptin alone. For example, treatment of tumors
with Herceptin alone results in Herceptin resistance and the tumors
continue to proliferate. Furthermore, these chimeric molecules
treat tumors termed Her2.sup.- which have low levels of Her and
have been shown to be untreatable with Herceptin. The chimeric
fusion molecules are thus useful in treating Her2 refractory
tumors.
[0303] Clinical experience with endostatin has been disappointing
(Thomas J P, et al. J Clin Oncol 2003; 21(2):223-31; Hansma A H, et
al. Ann Oncol 2005; 16(10):1695-701; Kulke M H, et al. J Clin Oncol
2006; 24(22):3555-6). In early human Phase I trials, huEndo
administration at variable dose levels and schedules was feasible
and safe. However, no consistent evidence for anti-tumor activity
or biological activity was demonstrated. In a Phase II study in
forty-two patients with advanced pancreatic neuroendocrine tumors
or carcinoid tumors treated with huEndo administered as a twice a
day subcutaneous injection, huEndo was associated with minimal
toxicity. However, no patient achieved a partial response and only
two patients had a biochemical response. Therefore, although
initial clinical trials proved that endostatin is a very safe drug
delivered in a variety of dose schedules, they did not demonstrate
comparable anti-tumor activity compared to that seen in murine
models. In contrast, the chimeric fusion molecules described
herein, are therapeutically effective. Details of the results are
shown in the examples which follow.
[0304] In a preferred embodiment, the chimeric fusion molecule can
be targeted to any tumor specific antigen. Examples include, but
not limited to HER2/neu tumor antigens, phosphatase and tensin
homolog (PTEN), phosphatidylinositol (PI) kinase and receptor
thereof, Eph, VEGF and receptors thereof, receptor/ligand
complexes; ligands, receptors, mutants, fragments, alleles and
variants thereof.
[0305] In another preferred embodiment, the chimeric fusion
molecules described herein are administered metronomically either
alone or in combination with one or more other compounds. These
compounds include, for example, anti-angiogenic compounds,
chemotherapeutic compounds, cell cycle arresting compounds,
chemokines, cytokines and the like. Examples include, but not
limited to: angiogenin, angiostatin, chemokines, angioarrestin,
angiostatin (plasminogen fragment), basement-membrane
collagen-derived anti-angiogenic factors (tumstatin, canstatin, or
arrestin), anti-angiogenic antithrombin III, signal transduction
inhibitors, cartilage-derived inhibitor (CDI), CD59 complement
fragment, fibronectin fragment, gro-beta, heparinases, heparin
hexasaccharide fragment, human chorionic gonadotropin (hCG),
interferon alpha/beta/gamma, interferon inducible protein (IP-10),
interleukin-12, kringle 5 (plasminogen fragment), metalloproteinase
inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease
inhibitor, plasminogen activator inhibitor, platelet factor-4
(PF4), prolactin 16 kD fragment, proliferin-related protein (PRP),
various retinoids, tetrahydrocortisol-S, thrombospondin-1 (TSP-1),
transforming growth factor-beta (TGF-b), vasculostatin, vasostatin
(calreticulin fragment) and the like. These molecules include all
forms, variants, mutations, alleles, substitutes, fragments and
analogs thereof.
[0306] In another preferred embodiment, the chimeric fusion
molecules care administered with one or more compounds comprising:
with one more compounds comprising signal transduction inhibitors,
bevacizumab (Avastin), antiangiogenic compounds such as for
example, sunitinib, sorafenib, celebrex, MTOR inhibitors, AKT
inhibitors, P13K and the like, anti-PDL1 and/or CTLA4. One of
ordinary skill in the art would identify which other therapeutic
compounds could be administered in conjunction with a therapy
comprising a regimen of chimeric fusion molecule.
Administration of Compositions to Animals
[0307] For targeting a tumor cell in situ, the compositions
described above may be administered to animals including human
beings in any suitable formulation. For example, compositions for
targeting a tumor cell may be formulated in pharmaceutically
acceptable carriers or diluents such as physiological saline or a
buffered salt solution. Suitable carriers and diluents can be
selected on the basis of mode and route of administration and
standard pharmaceutical practice. A description of exemplary
pharmaceutically acceptable carriers and diluents, as well as
pharmaceutical formulations, can be found in Remington's
Pharmaceutical Sciences, a standard text in this field, and in
USP/NF. Other substances may be added to the compositions to
stabilize and/or preserve the compositions.
[0308] The compositions of the invention may be administered to
animals by any conventional technique. The compositions may be
administered directly to a target site by, for example, surgical
delivery to an internal or external target site, or by catheter to
a site accessible by a blood vessel. Other methods of delivery,
e.g., liposomal delivery or diffusion from a device impregnated
with the composition, are known in the art. The compositions may be
administered in a single bolus, multiple injections, or by
continuous infusion (e.g., intravenously). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form.
Formulations
[0309] While it is possible for an antibody or fragment thereof to
be administered alone, it is preferable to present it as a
pharmaceutical formulation. The active ingredient may comprise, for
example, from 0.001% to 100% w/w, e.g., from 1% to 50% by weight of
the formulation, although it may comprise as much as 99.9999% w/w
of the formulation.
[0310] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of where treatment is required, such as
liniments, lotions, creams, ointments or pastes, and drops suitable
for administration to the eye, ear, or nose. Drops according to the
present invention may comprise sterile aqueous or oily solutions or
suspensions and may be prepared by dissolving the active ingredient
in a suitable aqueous solution of a bactericidal and/or fungicidal
agent and/or any other suitable preservative, and preferably
including a surface active agent. The resulting solution may then
be clarified and sterilized by filtration and transferred to the
container by an aseptic technique. Examples of bactericidal and
fungicidal agents suitable for inclusion in the drops are
phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride
(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for
the preparation of an oily solution include glycerol, diluted
alcohol and propylene glycol.
Kits
[0311] Kits according to the present invention include frozen or
lyophilized chimeric fusion molecules, humanized or human
antibodies or humanized or human antibody fragments to be
reconstituted, respectively, by thawing (optionally followed by
further dilution) or by suspension in a (preferably buffered)
liquid vehicle. The kits may also include buffer and/or excipient
solutions (in liquid or frozen form)--or buffer and/or excipient
powder preparations to be reconstituted with water--for the purpose
of mixing with the fusion molecules or antibody fragments to
produce a formulation suitable for administration. Thus, preferably
the kits containing the chimeric fusion molecules, humanized
antibodies or humanized antibody fragments are frozen, lyophilized,
pre-diluted, or pre-mixed at such a concentration that the addition
of a predetermined amount of heat, of water, or of a solution
provided in the kit will result in a formulation of sufficient
concentration and pH as to be effective for in vivo or in vitro use
in the treatment or diagnosis of cancer. Preferably, such a kit
will also comprise instructions for reconstituting and using the
chimeric fusion molecules, humanized antibody or humanized antibody
fragment composition to treat or detect cancer. The kit may also
comprise two or more component parts for the reconstituted active
composition. For example, a second component part--in addition to
the chimeric fusion molecule, humanized antibodies or humanized
antibody fragments--may be bifunctional chelant, bifunctional
chelate, or a therapeutic agent such as a radionuclide, which when
mixed with the humanized antibodies or humanized antibody fragments
forms a conjugated system therewith. The above-noted buffers,
excipients, and other component parts can be sold separately or
together with the kit.
[0312] It will be recognized by one of skill in the art that the
optimal quantity and spacing of individual dosages of a chimeric
fusion molecule, antibody or antibody fragment of the invention
will be determined by the nature and extent of the condition being
treated, the form, route and site of administration, and the
particular animal being treated, and that such optima can be
determined by conventional techniques. It will also be appreciated
by one of skill in the art that the optimal course of treatment,
i.e., the number of doses of an antibody or fragment thereof of the
invention given per day for a defined number of days, can be
ascertained by those skilled in the art using conventional course
of treatment determination tests.
Anti-Cancer and Chimeric Fusion Molecule Cocktails
[0313] The subject chimeric fusion molecules, including the
humanized chimeric fusion molecules may also be administered in
combination with other anti-cancer agents, e.g., other antibodies
or drugs. Also, the subject chimeric molecules or fragments may be
directly or indirectly attached to effector having therapeutic
activity. Suitable effector moieties include by way of example
cytokines (IL-2, TNF, interferons, colony stimulating factors,
IL-1, etc.), cytotoxins (Pseudomonas exotoxin, ricin, abrin, etc.),
radionuclides, such as .sup.90Y, .sup.131I, .sup.111In, .sup.125I,
among others, drugs (methotrexate, daunorubicin, doxorubicin,
etc.), immunomodulators, therapeutic enzymes (e.g.,
beta-galactosidase), anti-proliferative agents, etc. The attachment
of antibodies to desired effectors is well known. See, e.g., U.S.
Pat. No. 5,435,990 to Cheng et al. Moreover, bifunctional linkers
for facilitating such attachment are well known and widely
available. Also, chelators (chelants and chelates) providing for
attachment of radionuclides are well known and available.
[0314] The subject chimeric fusion molecules may be used alone or
in combination with other antibodies, e.g. anti-HER2/neu.
Effects on Angiogenesis and Vasculogenic Mimicry
[0315] In one embodiment, the present fusion molecules can be used
for interfering with angiogenesis in tumors. The method comprises
administering to an individual who has a tumor an effective amount
of a chimeric fusion molecule composition, comprising an anti-HER2
antigen binding domain and an endostatin protein or fragments, or
an anti-EGFR antigen binding domain and an endostatin protein or
fragments. In one embodiment, the endostatin protein or fragments
have a proline to alanine substitution at the 125 position of human
endostatin. Exemplary fusion molecules are anti-HER2-huEndoP125A
and anti-EGFR-huEndoP125A.
[0316] Formation of blood vessels is considered to be important for
the growth of tumors. The ability of certain tumor cells to exhibit
formation of tube like structures can be demonstrated in vitro or
in vivo and is generally referred to as vasculogenic mimicry. For
example, vasculogenic mimicry can be demonstrated as the ability to
form tube-like structures in a 3-D matrix in vitro. A commonly used
3-D matrix in culture is matrigel. It is considered that this in
vitro ability correlates with vasculogenic mimicry in vivo (See
Misra et al., Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo:
Critical Role of HIF-1.alpha.-Neuropilin-1 Axis. PLoS ONE 7(11):
e50153.0.) In the present disclosure, it was observed that while
huEndo or huEndoP125A alone did not have any effect on vasculogenic
mimicry, the fusion molecules of this disclosure (such as anti-HER
2-EndostatinP125A and anti-EGFR-EndostatinP125A) had a greater
effect on vasculogenic mimcry than similar fusion molecules that do
not have the P125A substitution. While not intending to be bound by
any particular theory, it is considered that due to the formation
of the fusion protein, a dimeric form of the mutant endostatin can
be delivered to the cells. It is considered that the delivery of a
dimeric form of huEndoP125A by anti-HER 2 or anti-EGFR antibody may
contribute to the unexpected enhanced inhibition of vasculogenic
mimictry. Thus, in one embodiment, this disclosure provides a
method for inhibiting vasculogenic mimicry in tumors comprising
administering to an individual a chimeric fusion molecule
composition, comprising an anti-HER2 antigen binding domain and an
endostatin protein or fragments, or an anti-EGFR antigen binding
domain and an endostatin protein or fragments. In one embodiment,
the endostatin protein or fragments have a proline to alanine
substitution at the 125 position of human endostatin. Exemplary
fusion molecules are anti-HER2-huEndoP125A and
anti-EGFR-huEndoP125A.
[0317] In one embodiment, this disclosure provides a method of
inhibiting angiogenesis as well as vasculogenic mimcry (for tumors
that exhibit vasculogenic mimcry) comprising administering to an
individual who has a tumor an effective amount of a chimeric fusion
molecule composition comprising the fusion proteins of the present
disclosure (such as anti-HER 2-huEndoP125A or
anti-EGFR-huEndoP125A). The ability of the fusion molecules of the
present disclosure to inhibit both angiogenesis and vasculogenic
mimicry is unexpected and should lead to an enhanced anti-tumor
effect.
[0318] In one embodiment, this disclosure provides a method for
reducing the formation of metastatic foci (metastasis) or
recurrence of tumors comprising the steps of administering to an
individual who has or had a tumor an effective amount of a chimeric
fusion molecule composition comprising the fusion proteins of the
present disclosure (such as anti-HER 2-huEndoP125A or
anti-EGFR-huEndoP125A).
[0319] In one embodiment, this disclosure provides a method for
identifying treatment options for an individual diagnosed with a
tumor comprising determining if the tumor exhibits vasculogenic
mimicry (such as in vitro or in vivo) and if so, determining if the
fusion proteins of the present disclosure (such as anti-HER
2-huEndoP125A or anti-EGFR-huEndoP125A) inhibit vasculogenic
mimicry. If inhibition of vasculogenic mimicry is observed,
treatment options can be devised for the individual that employ
administration of the fusion proteins. The ability of tumors to
exhibit vasculogenic mimicry can be evaluated in vitro (by
morphological analysis of cells in culture) or by
immunohistochemical techniques in vivo. For example, tumor cells
can be obtained in a biopsy and suspended in suitable culture
medium (such as endothelial cell growth medium (EGM) or other cell
culture media including serum-free media). The cells can be plated
on matrigel coated plates. Following a suitable period of growth
(such as 10-20 hours at 37.degree. C. in a humidified atmosphere
generally containing 5% CO.sub.2), the plates can be examined for
tube formation. The ability of tumor cells to exhibit vasculogenic
mimicry may also be inferred by evaluating for the presence of
angiogenic markers including Neuropilin-1 (NRP-1) (See Misra et al.
2012, PLoS ONE 7(11): e50153.o.)
[0320] In another embodiment, the administration of the fusion
proteins is combined with other modalities of treatment including
surgical removal of the tumor or radiation treatment. For example,
in one embodiment, the fusion proteins of the present disclosure
(such as anti-HER2-huEndoP125A or anti-EGFR-huEndoP125A) can be
administered to an individual following surgical removal of the
tumor. In one embodiment, the fusion proteins of the present
disclosure (such as anti-HER2-huEndoP125A or anti-EGFR-huEndoP125A)
are administered to an individual following radiation treatment,
which may be combined with surgical removal. It is considered that
administration of the fusion proteins may reduce metastatic lesions
or recurrence of the tumors. In one embodiment, the fusion
molecules of the present disclosure (such as anti-HER 2-huEndoP125A
or anti-EGFR-huEndoP125A) can be administered to an individual
before, during or after surgical removal of the tumor or before,
during or after radiation treatment.
[0321] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
offered by way of illustration, not by way of limitation. While
specific examples have been provided, the above description is
illustrative and not restrictive. Any one or more of the features
of the previously described embodiments can be combined in any
manner with one or more features of any other embodiments in the
present invention. Furthermore, many variations of the invention
will become apparent to those skilled in the art upon review of the
specification.
[0322] All publications and patent documents cited in this
application are incorporated by reference in pertinent part for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, Applicants do not admit any
particular reference is "prior art" to their invention.
EXAMPLES
[0323] The present invention is further illustrated by the
following specific examples. The examples are provided for
illustration only and are not to be construed as limiting the scope
or content of the invention in any way.
[0324] Materials & Methods
[0325] Cell Lines and Animals:
[0326] CT26, CT26-HER2, human embryonic kidney (HEK) 293, and
transfected Sp2/0 cells were cultured in Iscove's Modified
Dulbecco's Medium (IMDM; Cellgro, Mediatech, Inc., Herndon, Va.)
with 5% calf serum (GIBCO, Invitrogen Corp. Carlsbad, Calif.).
Female BALB/c mice (4-6 weeks) and SCID mice (4-6 weeks) were
purchased from the Jackson Laboratory (Bar Harbor, Me.). Animal
care and use procedures were performed in accordance with standards
described in the National Institutes of Health Guide for Care and
Use of Laboratory Animals.
[0327] Construction, Expression, and Characterization of
Anti-HER2/neu IgG3-Endostatin Fusion Protein:
[0328] Experimental murine endostatin gene originated from
pFLAG-CMV-1-endostatin by PCR using primers
5'-CCCCTCGCGATATCATACTCATCAGG-ACTTTCAGCC-3' (SEQ ID NO 1) and
5'-CCCCGAATTCGTTAACCTTTGGAGAAAGAGGTCATGAAG-C-3' (SEQ ID NO 2). PCR
products were subcloned into p-GEM-T Easy Vector (Promega, Madison,
Wis.), then sequenced for verification. The EcORV-EcOR1 fragment of
the subcloned endostatin gene was ligated to the carboxyl end of
the heavy chain constant domain (C.sub.H3) of human IgG3 in the
vector pAT135, as previously described (Shin S U et al. J Immunol.
1997; 158(10):4797-804). To complete the construct, the
IgG3-endostatin heavy chain constant region (AgeI-BamHI) was then
joined to an anti-HER2/neu variable region of a recombinant
humanized monoclonal antibody 4D5-8 (rhuMAb HER2, Herceptin;
Genentech, San Francisco, Calif.) in the expression vector
(pSV2-his) containing HisD gene for eukaryotic selection
(Challita-Eid P M. et al. J Immunol 1998; 160(7):3419-26; Coloma M
J. et al, J. Immunol. Methods 1992; 152:89-104). The finished
anti-HER2/neu heavy chain IgG3-endostatin construction vector was
transfected by electroporation into Sp2/0 cells stably expressing
the anti-HER2/neu K light chain in order to assemble entire
anti-HER2/neu IgG3-endostatin fusion proteins. Transfected cells
were selected with 5 mM histidinol and transfectomas producing the
fusion proteins were identified by a enzyme-linked immunosorbent
assay (ELISA) using anti-human IgG antibody coated plates and an
anti-human kappa detection antibody (Sigma, Saint Louis, Mo.). The
anti-HER2/neu IgG3-endostatin fusion proteins were biosynthetically
labeled with [.sup.35S]methionine (Amersham Biosciences,
Piscataway, N.J.) and analyzed by SDS-PAGE on 5% sodium phosphate
buffered polyacrylamide gels without reduction or on 12.5%
Tris-glycine buffered polyacrylamide gels following treatment with
0.15 M .beta.-mercaptoethanol at 37.degree. C. for 30 min. The
fusion protein was purified from culture supernatants using protein
A immobilized on Sepharose 4B fast flow (Sigma, Saint Louis,
Mo.).
[0329] To obtain active endostatin, a mouse endostatin expression
vector (pFLAG-CMV-1-endostatin) was co-transfected with pcDNA3.1
(CLONTECH, Palo Alto, Calif.) into human embryonic kidney (HEK) 293
cells, and G418 (0.6 .mu.g/ml)-resistant cells selected. Secreted
endostatin was harvested from serum-free conditioned medium and
purified in a heparin-Sepharose CL-6B column. Purity was assessed
by Coomassie blue staining of the SDS-PAGE gels. For Western blot
analysis, the endostatin fusion proteins were treated with
.beta.-mercaptoethanol, fractionated by SDS-PAGE and transferred
onto a membrane. Rabbit anti-endostatin (BodyTech, Kangwon-Do,
Korea) was used as the primary antibody and mouse anti-rabbit IgG
conjugated with HRP (Sigma, St. Louis, Mo.) was used as the
secondary antibody. Goat anti-human IgG conjugated with HRP (Sigma,
Saint Louis, Mo.) was used to detect human antibody.
[0330] Chorioallantoic Membrane (CAM) Assay:
[0331] The ability of anti-HER2/neu IgG3-endostatin to block
VEGF/bFGF-induced angiogenesis was tested by CAM assay, which
employed Leghorn chicken embryos (Charles River SPAFAS, Wilmington,
Mass.) at 12-14 days of embryonic development. Vitrogen gel pellets
(Collagen Biomaterials, Palo Alto, Calif.) were supplemented with
(a) vehicle (0.1% DMSO) in PBS alone (negative control); (b)
VEGF/bFGF (100 ng and 50 ng/pellet, respectively; positive
control); or (c) VEGF/bFGF and either of anti-HER2/neu IgG3,
anti-HER2/neu IgG3-endostatin, or endostatin, at various
concentrations (0.5-10 mg/pellet) and were allowed to polymerize at
37.degree. C. for 2 h. Pellets were then placed on a nylon mesh
(pore size 250 .mu.m; Tetko Inc., USA) and polymerized mesh was
placed onto the outer region of the chorioallantoic membrane of the
embryo and incubated for 24 hours as described (Iruela-Arispe M L,
et al. Thromb. Haemost. 1997; 78(1):672-7; Iruela-Arispe M L, et
al. Circulation 1999; 100(13):1423-31). To visualize vessels, 400
.mu.l of fluorescein isothiocyanate-dextran (100 .mu.g/ml, Sigma,
USA) was injected in the chick embryo blood stream. After 5-10 min
of incubation, the chick embryo was topically fixed with 3.7%
formaldehyde for 5 min. The implanted mesh was then dissected and
mounted on slides. Fluorescence intensity was analyzed with a
computer-assisted image program (NIH Image 1.59).
[0332] Pharmacokinetic and Biodistribution of Anti-HER2/neu
IgG3-Endostatin:
[0333] Anti-HER2/neu IgG3 (100 .mu.g), anti-HER2/neu
IgG3-endostatin (100 .mu.g), anti-dansyl IgG3 (100 .mu.g), and
endostatin (100 .mu.g) were iodinated with 0.5 mCi of [.sup.125I]
(Amersham Biosciences, Piscataway, N.J.) by the chloramine T method
(Pardridge W M, et al. Proc. Natl. Acad. Sci. USA 1995; 92:5592.).
BALB/c mice (4-6 weeks of age) were injected s.c. with either
1.times.10.sup.6 CT26-HER2 or CT26 cells or left uninjected. Groups
of three mice with either CT26-HER2 or CT26 tumors or no tumor were
injected i.v. with either 32 .mu.Ci of [.sup.125I]-anti-HER2/neu
IgG3, 30 .mu.Ci of [.sup.125I]-anti-HER2/neu IgG3-endostatin, 32
.mu.Ci of [.sup.125I]-anti-dansyl IgG3, or 24 .mu.Ci of
[.sup.125I]-endostatin. Blood samples were serially obtained at
various intervals ranging from 15 min to 96 hours from the
retro-orbital plexus of mice injected with either the anti-HER2/neu
IgG3, anti-HER2/neu IgG3-endostatin, or anti-dansyl IgG3. Mice
injected with endostatin alone were bled within 15 second to 60 min
after the i.v. injection. The TCA precipitable radioactivity in
each blood sample was measured in a .gamma.-counter. The
pharmacokinetic parameters were calculated by fitting plasma
radioactivity data to a bi-exponential equation as described
previously (Shin S U, et al. J Immunol. 1997; 158(10):4797-804,
Yoshikawa T et al. J Pharmacol. Exp. Ther. 1992; 263:897; Penichet
M L. et al. J Immunol. 1999; 163(8):4421-6).
Cp(t)=A.sub.1e-.sup.K.sub.1.sup.t+A.sub.2e.sup.-K.sub.2.sup.t
[0334] The equation was fitted to plasma data using derivative free
nonlinear regression analysis (PARBMDP, Biomedical Computer P
series Program developed at UCLA Health Sciences Computing
Facilities). Data were weighed using
weight=1/(concentration).sup.2, where concentration was either
count per minute (cpm) per microliter (.mu.l) or % ID (percentage
of injected dose) per milliliter. Area under the plasma
concentration curve (AUC) and mean residence time (MRT) were
calculated from the slopes and intercept of the bi-exponential
equation. The volume of distribution (V.sub.D) of the antibodies
was determined from the ratio of disintegrations per minute per
gram of organ divided by disintegrations per minute per microliters
of corresponding plasma at each time after injection. The organ
permeability-surface area product (Ki) of the antibodies was
calculated from:
Ki=[V.sub.D-V.sub.0]C.sub.P(T)/AUC(t)
[0335] where Cp(T) is the terminal plasma concentration and V.sub.0
is the organ plasma volume. The organ delivery of the antibodies
was determined from:
% ID/g=Ki.times.AUC(t)
[0336] where Ki and AUC(t) correspond to the 1, 48, or 96 hour time
period after injection.
[0337] The pharmacokinetic parameters were calculated by fitting
plasma TCA-precipitable radioactivity data to a bi-exponential
equation as described previously (Shin S U, et al. J. Immunol.
1997; 158(10):4797-804, Yoshikawa T et al. J Pharmacol. Exp. Ther.
1992; 263:897; Penichet M L. et al. J Immunol. 1999; 163(8):4421-6;
Gibaldi M. et al. Pharmacokinetics, Marcel Dekker, Inc., New York.
1982; Pardridge W M. et al. J. Pharm. Sci. 1995; 84:943-8). Plasma
clearance, the initial plasma volume, systemic volume of
distribution, steady state area under the plasma concentration
curve (AUC.sub.0-.infin.), and mean residence time were also
determined.
[0338] Following the pharmacokinetic experiments, mice were
exsanguinated by perfusion with 20 ml PBS for measurements of the
tissue distribution of .sup.125I-labeled antibody-endostatin fusion
protein. The heart, lung, liver, spleen, kidney, muscle, and tumor
were removed, weighed, .gamma.-counted and the percent of injected
dose per gram of tissue calculated. Specific tumor targeting is
expressed as the radiolocalization index (the % ID/g in tumor
divided by the % ID/g in blood).
[0339] To determine the preferential distribution and localization
of the .sup.125I-labeled proteins in mice simultaneously implanted
with CT26 and CT26-HER2 tumors on opposite flanks, groups of three
mice were injected i.v. with either 5 .mu.Ci
[.sup.125I]-anti-HER2/neu IgG3-endostatin fusion protein or 5
.mu.Ci [.sup.125I]-anti-HER2/neu IgG3. The animals were sacrificed
at different times (6, 24, and 96 hours) after injection of labeled
protein and organs (e.g., lung, liver, kidney, spleen, muscle, CT26
tumor, CT26-HER2 tumor, blood, and urine) were isolated after
perfusion of the mouse with PBS, weighed, and counted in a gamma
scintillation counter. The percentage of injected dose per gram (%
ID/g) for each organ was determined as above.
[0340] In vivo Anti-Tumor Effects:
[0341] The in vivo anti-tumor efficacy of anti-HER2/neu
IgG3-endostatin was examined using a CT26-HER2 BALB/c syngeneic
mouse model and a SK-BR-3 human breast xenograft SCID mouse model.
To determine targeting and efficacy of anti-HER2/neu
IgG3-endostatin, BALB/c (8/group, 4-6 weeks of age) mice were
injected s.c. in the right flank with 1.times.10.sup.6 CT26-HER2
cells and control CT26 cells injected on the left flank. On day
seven, mice (8 mice/group) were injected i.v. with the
anti-HER2/neu IgG3-endostatin fusion proteins (42 .mu.g/injection,
2.times.10.sup.-10 mole, equimolar to 8 .mu.g of endostatin),
anti-HER2/neu IgG3 alone (34 .mu.g/injection, 2.times.10.sup.-10
mole), endostatin alone (8 .mu.g/injection, 4.times.10.sup.-10
mole), the combination of anti-HER2/neu IgG3 (34 .mu.g) and
endostatin (8 .mu.g), or PBS as a control. All mice received seven
treatments at 2-day intervals. Tumor size and growth rates were
recorded and calculated using the following equation:
Tumor Volume (mm.sup.3)=4/3.times.3.14.times.{(Long axis+Short
axis)/4}.sup.3
[0342] Human breast cancer SK-BR-3 xenografts in SCID mice were
also used to evaluate anti-tumor activity of anti-HER2/neu
IgG3-endostatin fusion protein. SK-BR-3 (1.times.10.sup.6 cells per
mouse) was implanted on the flank of SCID mice. On day 15, mice (8
mice/group) were injected i.v. with the anti-HER2/neu
IgG3-endostatin fusion proteins (42 .mu.g), anti-HER2/neu IgG3 (34
.mu.g), the combination of anti-HER2/neu IgG3 (34 .mu.g) and
endostatin (8 .mu.g), or endostatin (8 .mu.g). This treatment was
repeated every other day. Visible tumors were measured using a
caliper and the tumor growth rate analyzed as described above.
[0343] Immunohistochemistry and Image Analysis of Blood Vessel
Formation:
[0344] Mice were killed at the end of the experiments. Tumors were
placed in OCT Compound (Tissue-Tek, Elkhart, Ind.) and snap frozen
in isopentane chilled with liquid nitrogen. Frozen tumors were
stored at -80.degree. C. until further use. For conventional
immunohistochemistry, five-.mu.m tissue sections were cut using a
cryostat (Shandon, Pittsburgh, Pa.) and placed on positively
charged slides (Fisher Scientific, Pittsburgh, Pa.). Tumor sections
were air-dried and fixed with 4% paraformaldehyde for 10 min. To
analyze the microvessel formation in tumors, sections were stained
with a rat anti-mouse platelet-endothelial cell adhesion molecule 1
(PECAM-1; CD31) MAb (PharMingen, San Diego, Calif.) and
subsequently with the ABC (Vector Lab, Burlingame, Calif.) method.
HER2/neu expression on tumors has been examined with staining tumor
sections with anti-HER2/neu IgG3-endostatin fusion antibody. All
sections were counter-stained with hematoxylin (Sigma, St. Louis,
Mo.). Positively stained vascular endothelial cells (brown) were
visualized and imaged using a digital camera attached to a Zeiss
microscope (Carl Zeiss, Thornwood, N.Y.).
[0345] For confocal microscopic analysis, thirty-.mu.m cryosections
were cut and stained with a rat anti-mouse CD31 monoclonal
antibodies. Blood vessels have been visualized with anti-rat
IgG-Alexa 594 (Molecular Probes, Eugene, Oreg.) and a coverslip was
placed on top of the piece of sections with anti-fade mounting
media (Vectorshield: Vector Lab, Burlingame, Calif.). These
fluorescent blood vessels were then viewed via LSM5 confocal
microscope (Carl Zeiss, Thornwood, N.Y.), and 14-21 digital images
were obtained per section. These digital images have been composed
as one image per each section to measure blood vessel density, and
blood vessel area (pixel) was then computed from the composite
images and averaged to measure blood vessel density per tumor.
Images were analyzed using NIH ImageJ v1.31 software by color image
to form a binary image of the tumor blood vessels.
[0346] Statistical Analysis:
[0347] Antiangiogenic activity, pharmacokinetics, biodistribution,
and tumor growth are presented as the mean.+-.SEM. Two-sided
Student's t test was used to determine the significance of
differences between two group means. Differences were considered
statistically significant at P<0.05. All statistical tests were
two-sided.
[0348] Focus Formation Assay:
[0349] Focus formation assay was used to determine whether
anti-HER2/neu antibody-endostatin fusions protein exerted
antiproliferative effects on tumor bearing HER2/neu antigens. In
vitro SK-BR-3, BT474, MCF7-HER2 (positive tumor cells) and MCF7
(negative tumor cell) were treated with different concentrations of
anti-HER2/neu antibody-endostatin fusion proteins (0.1, 1, 10
.mu.g/ml). One thousand of tumor cells were plated in 60-mm dishes
in 1.5 ml of medium containing 0.33% agar, which were overlaid onto
solidified 0.5% agar medium. The medium used for soft agar assays
was DMEM containing 10% fetal calf serum, and contained the
endostatin fusion proteins. The soft agar plates were fed with 0.5
ml of medium every 5-7 days, and after 14 days, the cells were
stained overnight (at 37.degree. C. and 5% CO.sub.2) with the vital
dye p-iodonitrotetrazolium violet (Sigma), and counted. The
resulting foci are stained with crystal violet and counted.
[0350] MTT Assay:
[0351] If tumor cells did not grow properly on soft agar assays,
MTT assays were used to evaluate the antiproliferative effect of
anti-HER2/neu antibody-endostatin fusion proteins on tumor
expressing HER2/neu. Tumor cells such as SK-BR-3, BT474, MCF7 and
MCF7-HER2, CT26 and CT-HER2/neu, or EMT6 and EMT6-HER2/neu were
treated with different concentrations (0.1, 1, 10 .mu.g/ml) of
anti-HER2/neu antibody-endostatin fusion proteins or controls.
Briefly, tumor cells were plated out at 2-5.times.10.sup.3
cells/well on 96 well plates and allowed to adhere overnight. The
following day tumor cells were treated with various concentrations
of endostatin fusion proteins or control, and incubated for a
further 48-72 hours. To determine cell growth, 20 pl of 10 mg/ml
MTT (3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
Sigma) was added to each well and the plates were incubated at
37.degree. C. in 5% CO.sub.2 for a further three hours. The
supernatant was removed and the formed crystals dissolved in 200
.mu.l dimethyl sulphoxide. The plates were then quantitated by
determining their absorbance at 595 to 600 nm in a microplate
reader. Growth inhibition was calculated by expressing the
differences in optical densities between treatment wells and
control wells as a percentage of the control. Each assay was
performed in triplicate.
[0352] Effect on VEGF Secretion:
[0353] The following cell lines were tested for effects of
anti-HER2/neu IgG3 control and/or endostatin, and for identifying
informative cell lines that responded to endostatin, anti-HER2/neu
antibody, or both proteins. To investigate effect of anti-HER2/neu
antibody-endostatin fusion proteins on VEGF family expression,
tumor cells such as SK-BR-3, BT474, MCF7 and MCF7-HER2, CT26 and
CT-HER2/neu, or EMT6 and EMT6-HER2/neu were treated with
anti-HER2/neu antibody-endostatin fusion proteins. 5.times.10.sup.5
tumor cells/well were seeded in 24-well plates (Falcon). Cells were
allowed to adhere overnight, and then treated with different
concentrations (0.1, 1, 10, 100 .mu.g/ml) of endostatin fusion
proteins, endostatin, or antibody. Cells were removed by
centrifugation at different time points (24, 48, 96 hours), and the
supernatants filtered using a 0.22-.mu.m pore size filter. Secreted
VEGF levels were analyzed by a sandwich ELISA (R&D Systems,
Minneapolis, Minn., USA) that detected all VEGF spliced forms.
Human recombinant VEGF165 (R&D Systems, Minneapolis, Minn.,
USA) served as the standard.
[0354] Endothelial Cell Proliferation Assay:
[0355] The antiproliferative effect of anti-HER2/neu
antibody-endostatin fusion proteins were tested using C-PAE cells.
The cells were plated in 24-well fibronectin (10 .mu.g/ml)-coated
plates at 12,500 cells/well in 0.5 ml of DMEM containing 2% FBS.
After a 24-h incubation at 37.degree. C., the medium was replaced
with fresh DMEM and 2% FBS containing 3 ng/ml of bFGF (R & D
systems, Minneapolis, Minn., USA) with or without endostatin fusion
proteins and endostatin (1, 10, or 100 .mu.g/ml). The cells were
pulsed with 1 .mu.Ci of [.sup.3H]thymidine for 24 h. Medium was
aspirated, cells were washed three times with PBS, and then
solubilized by addition of 1.5 N NaOH (100 .mu.l/well) and
incubated at 37.degree. C. for 30 min. Cell-associated
radioactivity was determined with a liquid scintillation
counter.
[0356] Migration Assay:
[0357] To determine the ability of anti-HER2/neu
antibody-endostatin fusion proteins to block migration of human
endothelial cells (ECV304) toward bFGF, a migration assay was
performed using 12-well Boyden chemotaxis chambers (Neuro Probe,
Inc.) with a polycarbonate membrane (25.times.80-mm, PVD free,
8-.mu.m pores; Poretics Corp., Livermore, Calif.). The nonspecific
binding of growth factor to the chambers was prevented by coating
the chambers with a solution containing 0.5% gelatin, 1 mM
CaCl.sub.2, and 150 mM NaCl at 37.degree. C. overnight. ECV304
cells were grown in 10% FBS containing 5 ng/ml
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiIC18; Molecular Probes, Eugene, Oreg.) overnight and washed with
PBS containing 0.5% BSA. After trypsinization, the cells were
counted and diluted to 300,000 cells/ml in medium containing 0.5%
FBS. The lower chamber was filled with medium containing 25 ng/ml
bFGF. The upper chamber was seeded with 15,000 cells/well with
different concentrations of endostatin fusion protein (1, 10, 100
.mu.g/ml). Cells were allowed to migrate for 4 h at 37.degree. C.
At that time, the cells on the upper surface of the membrane were
removed with a cell scraper, and the (migrated) cells on the lower
surface are fixed in 3% formaldehyde and washed in PBS. Images of
the fixed membrane were obtained using fluorescence microscopy at
550 nM with a digital camera, and the number of cells on each
membrane is determined.
[0358] In Vitro Matrigel Assay:
[0359] Capillary tube formation assay in Matrigel is a useful in
vitro assay to determine the branching morphogenesis of endothelial
cells, which is a complex developmental program that regulates the
formation of the blood vessels. Matrigel (Becton Dickinson,
Franklin Lakes, N.J.) was used to coat a 24-well plate at 4.degree.
C. and allowed to polymerize at 37.degree. C. for 30 min. HUVECs
are seeded (5.times.10.sup.4 cells/well) on Matrigel-coated plates.
Cells were incubated with VEGF (15 ng/ml) with or without
endostatin fusion proteins or endostatin (1, 10, 100 .mu.g/ml) in
endothelial cell basal medium containing 2% FBS. After cells were
incubated for 24 hrs or 96 hrs at 37.degree. C., capillary tube
formation was examined visually under a phase-contrast microscope
and photographed. The intact tube number in six random views of
.times.100 magnification was counted.
[0360] Apoptotic Activities of Anti-HER2/Neu Antibody-Endostatin
Fusion Protein:
[0361] To analyze the mechanism of endostatin fusion protein action
on endothelial cells and nonendothelial cells, C-PAE cells or
HUVECs were tested for apoptosis by measuring annexin V-FITC
staining with FACS and terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling assay (TUNEL staining).
As addition of endostatin lead to a reduction in the antiapoptotic
proteins Bcl-2 and Bcl-XL, expression of these antiapoptotic
proteins in the presence of anti-HER2/neu antibody-endostatin
fusion proteins were monitored by Western blot analysis.
[0362] Annexin V-FITC Staining Assay:
[0363] Annexin V, a calcium-dependent phospholipid-binding protein
with a high affinity for phosphatidylserine (PS) was used to detect
early stage apoptosis. C-PAE cells or HUVECs (2.times.10.sup.5)
were plated onto a fibronectin-coated 6-well plate in DMEM
containing 2% FCS and 3 ng/ml b-FGF. Different concentrations
(1-100 .mu.g/ml) of antibody-endostatin fusion proteins, control
antibodies, or endostatin were added to each well, and cells were
harvested and processed 18 h after treatment. For the time course
study, 10 .mu.g/ml antibody-endostatin fusion proteins, control
antibodies, or endostatin were added and cells were processed after
3, 4, 6, 12, and 18 h. Human recombinant TNF-.alpha. (40 ng/ml) was
used as a positive control. The cells were washed in PBS and
resuspended in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM
NaCl, 2.5 mM CaCl.sub.2). Annexin V-FITC was added to a final
concentration of 100 ng/ml, and the cells were incubated in the
dark for 10 min, then washed again in PBS and resuspended in 300
.mu.l of binding buffer. 10 .mu.l of propidium iodide (PI) was
added to each sample before flow cytometric analysis. The cells are
analyzed using a Becton Dickinson FACStar plus flow cytometer. In
each sample, a minimum of 10,000 cells were counted and stored in
listmode. Data analysis was performed with standard Cell Quest
software (Becton-Dickinson).
[0364] Microscopic Detection of TUNEL Staining:
[0365] C-PAE cells or HUVECs were seeded at a density of 5,000
cells/well on fibronectin-coated (10 .mu.g/ml) Lab-Tek chamber
slides and grown in 0.4 ml of DMEM with 10% FCS. After 2 days, the
old medium was aspirated and fresh DMEM with 2% FCS was added, and
the cells were starved overnight. The following day, 0.36 ml of new
medium (with 2% FCS) containing 3 ng/ml b-FGF were added along with
antibody-endostatin fusion proteins, control antibodies, or
endostatin (10 .mu.g/ml) or TNF-.alpha. (20 ng/ml). For control
samples, fresh medium (2% FCS) containing bFGF (3 ng/ml) was added.
Following induction (24 h), the slides were washed twice with PBS,
and subsequently fixed in fresh 4% formaldehyde/PBS at 4.degree. C.
for 25 min. The slides were washed in PBS and the cells
permeabilized in 0.2% Triton X-100/PBS for 5 min on ice, then
washed with fresh PBS twice for 5 min each at room temperature, and
the TUNEL assay was performed as described in the ApoAlert DNA
fragmentation assay kit (CLONTECH), except that the final
concentration of propidium iodide (Sigma) used was 1 .mu.g/ml.
After the assay, a drop of anti-fade solution is added, and the
treated portion of the slide was covered with a glass coverslip
with the edges sealed with clear nail polish. Slides were viewed
immediately under a fluorescent microscope using a dual filter set
for green (520 nm) and red fluorescence (>620 nm). The images
were captured using a digital camera. Images are imported into
NHImage 1.59, and measurements of fluorescence intensity are
obtained as positive pixels. For all samples except the positive
control (TNF-.alpha.: 5 fields), 15 random fields were chosen, and
the number of green and red cells per field were counted.
[0366] Western Blotting Analysis of Expression of Antiapoptotic
Proteins, Bcl-2 or Bcl-XL:
[0367] C-PAE cells and HUVECs (1.times.10.sup.6) were seeded in
10-cm Petri dishes precoated with fibronectin (10 .mu.g/ml) in the
presence of 2% FCS containing 3 ng/ml b-FGF. Antibody-endostatin
fusion proteins, control antibodies, or endostatin were added at 10
.mu.g/ml, and cells were harvested at 12, 24, and 48 h after
treatment. Cells are washed three times in PBS buffer, pH 7.4, and
the cells are resuspended in 1 ml of 1.times.EBC buffer (50 mM
Tris-HCl, pH 8.0, 120 mM NaCl, 1% Nonidet P-40) containing freshly
added complete protease inhibitor tablet (Boehringer Mannheim), 100
.mu.g/ml Pefabloc, 1 .mu.g/ml pepstatin. The protein concentration
in whole cell lysate was measured by the bicinchoninic acid (BCA)
method (Pierce). 30 .mu.g of whole cell extract was loaded onto a
4-15% gradient polyacrylamide gel. Transfer was performed using a
semi-dry transblot apparatus (Bio-Rad). The membrane was blocked in
wash buffer (1.times.Tris-buffered saline) with 5% nonfat dry milk
and incubated at 37.degree. C. for 1 h. Goat antibody against human
Bcl-2 and mouse polyclonal antibody against Bax and Bcl-XL (Santa
Cruz Biotechnology, Santa Cruz, Calif.) were used as primary
antibodies. Polyclonal anti-actin antibody (Sigma) was used to
normalize for protein loading. Secondary antibodies were anti-goat,
mouse and rabbit immunoglobulin conjugated to HRP (Amersham
Pharmacia Biotech). The immunoreactivity was detected with an
enhanced chemiluminescence reagent (Pierce). Images were scanned
using a flat bed scanner and quantitated by the NIH image 1.59
software. Normalization was calculated by dividing the Bcl-2 signal
by that of actin within each experiment.
[0368] In Vivo Evaluation of the Antiangiogenic Properties of
Anti-HER2/Neu Antibody-Endostatin:
[0369] For in vivo antiangiogenesis, effects of the fusion were
tested in Matrigel angiogenesis model in mice using VEGGF or VEGF
with endostatin fusion proteins or endostatin alone. BALB/c mice
(6-8 wk, n=3) were subcutaneously injected with 0.5 ml Matrigel
(9-10 .mu.g/ml) containing 150 ng/ml VEGF, with or without
endostatin fusion proteins, antibody or endostatin (1 to 100
.mu.g/ml), near the abdominal midline by using a 26-gauge needle.
One week after Matrigel injection, mice were sacrificed, and the
Matrigel plug, along with overlying skin and peritoneal membrane,
was removed and fixed in 4% buffered formaldehyde in PBS. Plugs
were embedded in paraffin, sectioned, and stained by incubation
overnight at 4.degree. C. with antibodies (DAKO Corporation,
Carpenteria, Calif.) for endothelium-specific antigens such as
PECAM (CD31) and proliferation cell nuclear antigen (PCNA) or Ki67
(MIB-1) to access endothelial cell proliferation. Thereafter,
sections were treated with biotinylated antibody (ABC kit) for
40-45 min at room temperature, followed by a 45-min incubation with
the avidin-biotin-peroxidase complex (ABC kit). The
antigen-antibody complex was visualized by incubation with freshly
prepared 3,3'-diaminobenzidine (DAB kit, Vector Laboratories).
Sections were counterstained with hematoxylin-eosin. Ten fields
were randomly selected using a microscope at 400.times.
magnification, and photographed using a digital camera. The number
of PECAM-1-positive, PCNA-positive, Ki67-positive cells were
counted.
[0370] Antiangiogenic Activity of Anti-HER2/Neu
Antibody-Endostatins in Tumors by Immunohistochemical Staining:
[0371] BALB/c or BALB/c BCDM were injected s.c. in the flank region
with 106 EMT6-HER2/neu or CT26-HER2/neu cells on the right flank
and control 106 EMT6 or CT26 on the left flank, respectively. In
addition, SCID mice were injected s.c. in the flank region with 106
MCF7-HER2/neu, SK-BR-3, or BT474 cells on the right flank and
control 106 MCF7 on the left flank.
[0372] On the seventh day, mice were injected i.v. with the
antibody-endostatin fusion proteins (42 .mu.g), equimolar control
antibodies (34 .mu.g) or equimolar endostatin (8 .mu.g). This
treatment was repeated 7 times every other day. Visible tumors,
along with overlying skin and surrounding tissue, were removed at
various time points (2 days, 8 days, 16 days after treatments, 3
mice/time point), and tissue sections are immunohistochemically
stained with mouse antibody specific for endothelium-specific
antigens such as PECAM (CD31, DAKO Corporation, Carpenteria,
Calif.), and doubly stain tissue with mouse antibody specific for
proliferation cell nuclear antigen (PCNA, DAKO Corporation,
Carpenteria, Calif.) or Ki67 (MIB-1, DAKO Corporation, Carpenteria,
Calif.) to access endothelial cell proliferation.
[0373] Tissue sections were fixed in 4% paraformaldehyde/PBS pH
7.4, dipped in a quenching solution (3% hydrogen peroxide/60%
methanol) to remove endoperoxidase activity, and then placed in 10%
normal blocking serum (ABC kit, Vector Laboratories, Inc.,
Burlingame, Calif.) for 20 min before incubation overnight at
4.degree. C. with mouse antibody for PECAM-1, Ki67, or PCNA, or
with mouse IgG as the control. Thereafter, tissue sections were
treated with biotinylated antibody (ABC kit) for 40-45 min at room
temperature, followed by a 45-min incubation with the
avidin-biotin-peroxidase complex (ABC kit). The antigen-antibody
complex was visualized by incubation with freshly prepared
3,3'-diaminobenzidine (DAB kit, Vector Laboratories), and the
tissue was counterstained with hematoxylin. Ten fields were
randomly selected using a microscope at 400.times. magnification,
and photographed using a digital camera. The number of
PECAM-1-positive, PCNA-positive, Ki67-positive cells are
counted.
[0374] Antiangiogenic Activity of Anti-HER2/Neu Antibody-Endostatin
on VEGF Expression and Neovascularization in Tumors:
[0375] Anti-tumor activity of endostatin was associated with a
down-regulation of VEGF expression. The experiment shown above was
repeated in human breast cancer xenografts (SK-BR-3, BT474, MCF7
and MCF7-HER2) in SCID mice (n=3) to evaluate antiangiogenic
activity of the antibody-endostatin fusion proteins (42 .mu.g),
equimolar control antibodies (34 .mu.g) or equimolar endostatin (8
.mu.g). This treatment was repeated 7 times every other day.
Visible tumors, along with overlying skin and surrounding tissue,
were removed at various time points (2 days, 8 days, 16 days after
treatments, 3 mice/time point). Tissues were stained for
endothelial cell proliferation using PCNA or Ki67 as described
above, and the tissue sections were also stained with specific
antibodies (VEGF-A, Neomarker; VEGF-C and VEGF-D, Santa Cruz
Biotechnology, Santa Cruz, Calif.) for VEGF family.
[0376] Serum was collected at various times (before treatment, 2
days, 8 days, 16 days after treatments) for VEGF ELISA (R&D
Systems, MN) to measure antiangiogenic abilities of endostatin
fusion proteins as described above. In disease states, VEGF can be
detected in various tumor cells and five different VEGF isoforms,
with 121, 145, 165, 189, and 206 amino acids, can be generated as a
result of alternative splicing from the single VEGF gene. These
isoforms differ in their molecular mass and in their biological
properties, such as their ability to bind to heparin or
heparan-sulphate proteoglycans and to different VEGF receptors
(VEGFRs). The splice forms VEGF.sub.121, VEGF.sub.145, and
VEGF.sub.165 are secreted, whereas VEGF.sub.189 is tightly bound to
cell surface heparan-sulphate and VEGF.sub.206 is an integral
membrane protein. In contrast to the other forms, VEGF.sub.121,
does not bind to heparin or extracellular matrix proteoglycans. The
signaling tyrosine kinase receptors VEGFR-1 (flt-1, fms-like
tyrosine kinase 1) bind VEGF.sub.121 and VEGF.sub.165, and VEGFR-2
(KDR, kinase domain region/flk-1, fetal liver kinase 1)
additionally VEGF.sub.145 (apart from certain VEGF-related
peptides).
[0377] mRNA expression of VEGF isoforms was determined in excised
tumors by RT-PCR. For RT-PCR, frozen samples (1 g) were crushed in
an achate mortar under liquid nitrogen; RNA was isolated by the
phenol-guanidinium thiocyanate method and purified by isopropanol
and repeated ethanol precipitation; and contaminating DNA was
destroyed by digestion with RNase-free DNase 1 (20 min at
25.degree. C.). After inactivation of the DNase (15 min at
65.degree. C.), cDNA was generated with 1 .mu.l (20 pmol) of oligo
(dT) 15 primer (Amersham) and 0.8 .mu.l of superscript RNase
H-reverse transcriptase (Gibco) for 60 min at 37.degree. C. For
PCR, 4 .mu.l of cDNA was incubated with 30.5 .mu.l of water, 4
.mu.l of 25 mM MgCl.sub.2, I PI of dNTP, 5 .mu.l of 10.times.PCR
buffer, 0.5 .mu.l (2.5 U) of platinum Taq DNA polymerase (Gibco),
and the following primers (2.5 .mu.l each containing 10 pmol):
non-selective for all VEGF splice variants
5'-ATG-GCA-GAA-GGG-CAG-CAT-3' (sense) (SEQ ID NO: 3) and
5'-TTG-GTG-AGG-TTT-GAT-CCG-CAT-CAT3' (antisense)(SEQ ID NO: 4)
yielding a 255 bp fragment (40 cycles, annealing temperature
55.degree. C.); selective for VEGF splice variants
5'-CCA-TGA-ACT-TTC-TGC-TGT-CTT-3' (sense) (SEQ ID NO: 5) and
5'-TCG-ATC-GTT-CTG-TAT-CAG-TCT-3' (antisense) (SEQ ID NO: 6)
yielding a different fragment size for each variant (40 cycles,
annealing temperature 55.degree. C.). With selective primers, the
526 bp product corresponds to VEGF.sub.121, the 598 bp product to
VEGF.sub.145, the 658 bp product to VEGF.sub.165, the 730 bp
product corresponds to VEGF.sub.189, and the 781 bp product
corresponds to VEGF.sub.206.
Example 1: Targeted Delivery of Anti-HER2 Antibody-Human Endostatin
P125A Protein Results in Enhanced Anti-Tumor Efficacy in Murine and
Human Breast Tumor Models
[0378] Two anti-HER2 human endostatin fusion proteins were
generated by fusing human wild type or a mutant form of human
endostatin (huEndo-P125A) to the 3' end of a humanized anti-HER2
IgG3 antibody. HuEndo-P125A antibody fusion protein
(.alpha.HER2-huEndo-P125A) inhibited VEGF and bFGF induced
endothelial cell proliferation, and capillary formation in vitro,
to a greater degree than wild type endostatin fusion protein
(.alpha.HER2-huEndo), endostatin alone, or anti-HER2 antibody
(.alpha.HER2 IgG3). Treatment of SKBR-3 breast cancer xenografts
with anti-HER2 IgG3-huEndo-P125A fusion resulted in complete
regression, and improved survival, compared to either .alpha.HER2
IgG3, human endostatin, or anti-HER2 IgG3-huEndo treated mice.
.alpha.HER2-huEndo fusion proteins specifically targeted tumors
expressing HER2 in mice simultaneously implanted with murine
mammary tumor cell line EMT6 and EMT6 engineered to express HER2
antigen (EMT6-HER2). .alpha.HER2 huEndo-P125A fusion antibody
showed enhanced anti-angiogenic and anti-tumor activity and
inhibited EMT6-HER2 growth more effectively than huEndo (p=0.003),
or .alpha.HER2-huEndo (p=0.004).
[0379] Materials and Methods
[0380] Cell Lines, Materials and Animals:
[0381] To produce a murine breast tumor expressing human HER2, the
murine mammary tumor cell line EMT6 was transduced by use of a
retroviral construct containing the cDNA encoding the human HER2
gene (EMT6-HER2). EMT6, EMT6-HER2, the human breast cancer cell
line SK-BR-3, and transfected Sp2/0 or P3X63Ag8.653 cells were
cultured in Iscove's modified Dulbecco's medium with 5% calf
serum.
[0382] Human umbilical vein endothelial cells (HUVEC), were
obtained from Clontech Lab, Inc. (Palo Alto, Calif.) and used
between passages 3 and 5 and maintained in EGM2-MV medium
(Clontech, Palo Alto, Calif.) that contained endothelial basal
medium 2 (EBM-2), supplemented with 5% fetal bovine serum (FBS),
gentamicin, amphotericin B, hydrocortisone, ascorbic acid, vascular
endothelial growth factor (VEGF), basic fibroblast growth factor
(bFGF), human epidermal growth factor, and insulin-like growth
factor I.
[0383] Human recombinant endostatin was purchased from Sigma
(E8154, St Louis, Mo.) and anti-CD31 antibody conjugated with
biotin from BD Biosciences (Franklin Lakes, N.J.). Alexa Fluor 488
was obtained from Invitrogen (Carlsbad, Calif.), and DAPI from
Molecular Probes (Carlsbad, Calif.).
[0384] Female BALB/c mice (4-6 weeks) and severe combined
immunodeficient (SCID) mice (4-6 weeks) were purchased from The
Jackson Laboratory (Bar Harbor, Me.) and used for in vivo tumor
growth and xenograft experiments (SK-BR-3) as indicated. All
experiments were conducted in compliance with the NIH Guides for
the Care and Use of Laboratory Animals and approved by the
University of Miami Institutional Animal Care and Use
Committee.
[0385] Construction, Expression, and Characterization of
.alpha.HER2-huEndo Fusion Protein:
[0386] The human endostatin (huEndo) gene was cloned from the human
collagen, type XVIII, alpha 1 gene by PCR using primers
5'-CCCCTCGCGATATCACAGCCACCGCGACTTCCAGCCG-3' (SEQ ID NO: 1) and
5'-CCCCGAATTCGTTAACCCTTGGAGGCAGTCATGAAGC-3' (SEQ ID NO: 2). PCR
products were subcloned into pCR-Blunt II-TOPO vector and
sequenced. A single-point mutant clone at a position 125 in
wild-type human endostatin was derived by site-directed mutagenesis
using PCR with phosphorylated primer, 5'p-GGCTCGGACGCCAACGGGCGC-3'
(SEQ ID NO: 7). An alanine residue was substituted for proline at
position 125 by site-directed mutagenesis. A point mutation in
human endostatin at position 125 (proline to alanine; huEndo-P125A)
has been reported to enhance endothelial cell binding and
anti-angiogenic activity. The subcloned huEndo and huEndo-P125A
genes were ligated in frame to the carboxyl end of the heavy chain
constant domain of human IgG3 in the vector pAT135 (Shin S U, et
al. J Immunol 1997; 158:4797-804). The endostatin heavy chain
constant region was joined to the anti-HER2 variable region of a
recombinant humanized monoclonal antibody 4D5-8 (trastuzumab;
Genentech, San Francisco, Calif.) in the expression vector
(pSV2-his) containing HisD gene for eukaryotic selection.
[0387] To obtain active endostatin fusion proteins, the
.alpha.HER2-huEndo fusion constructs were stably transfected into
SP2/0 or P3X63Ag8.653 myeloma cells expressing the anti-HER2 kappa
light chain by electroporation as described previously (Shin S U,
et al. Methods Enzymol 1989; 178:459-76). The .alpha.HER2-huEndo
fusion proteins were biosynthetically labeled with
[.sup.35S]methionine (Amersham Biosciences, Piscataway, N.J.),
immunoprecipitated using IgGSorb suspension (S. aureus cells), and
analyzed by SDS-PAGE. The endostatin fusion proteins were purified
from culture supernatants using protein A immobilized on Sepharose
4B fast flow (Sigma, St. Louis, Mo.) (Shin S U, et al. Methods
Enzymol 1989; 178:459-76).
[0388] Flow Cytometry:
[0389] To detect the binding of .alpha.HER2-huEndo fusion proteins
to HER2 antigen, human breast cancer cells, SK-BR-3, or murine
mammary tumor cells, EMT6 and EMT6-HER2, were incubated at
4.degree. C. with 1 .mu.g/ml of endostatin fusion proteins,
.alpha.HER2 IgG3, or isotype control. After 15 min the cells were
washed with PBS containing 0.1% BSA and 0.05% NaN.sub.3, and the
bound fusion proteins were identified with either FITC conjugated
anti-human IgG-, or the endostatin domain was recognized with
biotinylated anti-human endostatin antibody and secondarily stained
with a streptavidin-PE conjugate at 4.degree. C. After incubation,
the cells were washed twice and resuspended in 0.4 ml of PBS.
FACScan flow cytometry was used for data acquisition. Background
staining was estimated after incubation with the secondary FITC or
PE labeled antibody alone.
[0390] Matrigel Tube Formation Assay:
[0391] The matrigel tube formation assay was performed in 48-well
plates, as previously reported (Merchan J R, et al. Int J Cancer
2005; 113(3):490-8). Each well of pre-chilled 48-well cell culture
plates was coated with 100 .mu.l of unpolymerized Matrigel (7
mg/ml) and incubated at 37.degree. C. for 30-45 minutes. HUVECs
were harvested with trypsin, and 4.times.10.sup.4 cells were
resuspended in 300 .mu.L of full endothelial cell growth medium
(see above) and treated with endostatin, control antibody or the
various .alpha.HER2-huEndo fusion proteins before plating onto the
Matrigel-coated plates. After 16 hours of incubation, endothelial
cell tube formation was assessed with an inverted photomicroscope,
and microphotographs of the center of each well were taken at low
power (40.times.). Tube formation by untreated HUVECs in full
endothelial cell growth medium was used as a control.
[0392] HUVEC Proliferation Assay:
[0393] A total of 4.times.10.sup.3 cells in 100 .mu.l of the
endothelial basal medium with 1% FBS, penicillin (100 U/ml), and
streptomycin (100 .mu.g/ml) were placed into each well of a 96-well
plate, treated with .alpha.HER2-huEndo fusion proteins and
controls, and incubated at 37.degree. C. for 72 hours; control
cells were cultured in basal medium, 1% FBS, and antibiotics, as
above. VEGF (10 .mu.g/ml) or bFGF (10 .mu.g/ml) were added as
stimulants of endothelial cell proliferation. After the 72-hour
incubation, WST-1 (10 .mu.l, Roche, Indianapolis, Ind.) was added
to each well, and after a 3-hour incubation at 37.degree. C.,
absorbance at 450 nm was determined for each well with a microplate
reader (Bio-Rad Laboratories, Hercules, Calif.). Data presented are
the average of triplicate experiments which were repeated
twice.
[0394] In Vivo Tumor Growth Assays:
[0395] To evaluate anti-tumor activity of .alpha.HER2-huEndo fusion
proteins SK-BR-3 cells (2.times.10.sup.6 per mouse) were implanted
s.c. in the flank of SCID mice. On day 5, mice (5 per group) were
injected i.v. with equimolar amounts of purified .alpha.HER2-huEndo
fusion proteins (42 .mu.g), .alpha.HER2 IgG3 (34 .mu.g), or human
endostatin (8 .mu.g). This treatment was repeated every other day
for 13 doses. Tumor size was measured with calipers and growth
rates were recorded and calculated using the following equation:
tumor volume (mm.sup.3)=4/3.times.3.14.times.{(long axis+short
axis)/4}.sup.3.
[0396] Murine mammary tumor EMT6 cells were transduced with a
retroviral vector encoding human HER2 antigen as described
previously (Cho H M, Rosenblatt J D, et al. Mol Cancer Ther 2005;
4(6):956-67). The EMT6-HER2 cells that were used in these studies
proliferate at the same rate in vitro as parental EMT6 cells. The
in vivo anti-tumor efficacy and specificity of .alpha.HER2-huEndo
fusion proteins was examined using the EMT6 and EMT6-HER2 cell
lines simultaneously implanted contralaterally in syngeneic BALB/c
mice. To determine efficacy of .alpha.HER2-huEndo fusion proteins,
BALB/c mice (3-8 per group, 4-6 weeks) were injected s.c. with
1.times.10.sup.6 EMT6-HER2 cells in the right flank and/or control
EMT6 cells in the left flank. On day 6, mice were injected i.v.
with the .alpha.HER2-huEndo fusion proteins (42 .mu.g/injection,
2.times.10-10 mol, equimolar to 8 .mu.g human endostatin),
.alpha.HER2 IgG3 alone (34 .mu.g/injection, 2.times.10-10 mol),
endostatin alone (8 .mu.g/injection, 4.times.10-10 mol), or PBS as
a control. All mice received a total of eleven injections at 2-day
intervals and tumor growth analyzed as described above.
[0397] Immunofluorescent Staining:
[0398] To investigate blood vessel formation in tumors treated with
.alpha.HER2-huEndo-P125A fusion protein, EMT6 and EMT6-HER2 cell
lines (1.times.10.sup.6) were contralaterally implanted in
syngeneic BALB/c mice (n=4) as described above. On day 4, mice were
injected i.v. with .alpha.HER2-huEndo-P125A (42 .mu.g/injection),
or PBS as a control every two days. On day 12, two mice were
sacrificed for analysis of vascularity after four treatments. On
day 18, another two mice were sacrificed for analysis after seven
treatments. Tumors were excised from the sacrificed mice and frozen
in liquid nitrogen and the 8 .mu.m frozen sections were prepared.
The tumor sections were fixed with methanol for 10 min, washed with
PBS 3 times, and incubated with blocking solution for 1 hour in a
humidified chamber. Slides were then washed with PBS 3 times, for
10 min. For immunofluorescent staining, diluted primary antibodies
(anti-CD31 antibody conjugated with biotin: 1:200) in PBS were
added to each slide. After incubation at room temperature
overnight, the sections were incubated with diluted secondary
antibodies conjugated with Alexa Fluor 488 (1:500) with PBS, and
then with diluted DAPI (1:5000) in a humidified chamber and mounted
with Gel mounting media (Biomeda Corp. Foster City, Calif.). The
stained images were analyzed with a Zeiss microscope. The digital
tumor images from each treatment were measured as blood vessel area
(pixel) and averaged to measure blood vessel density per tumor
(n=5). Images were analyzed using NIH ImageJ software by color to
form a binary image of the tumor blood vessels.
[0399] Statistical Analysis:
[0400] Statistical analysis was carried out with Graphpad Prism 4
(GraphPad Software, Inc. La Jolla, Calif.). HUVEC proliferation and
anti-tumor efficacy in human breast cancer SK-BR-3 xenografts were
analyzed by two-way repeated measures (RM) analyses of variance
(ANOVA), followed by Bonferroni posttest. On Day 16, anti-tumor
efficacy in murine syngeneic tumor model was analyzed by Student's
t test (unpaired, two-tailed) within each treatment group, and was
analyzed among three different treatments by one-way ANOVA,
followed by Bonferroni's multiple comparison test. Blood vessel
density was analyzed by Student's t test (unpaired, two-tailed)
within each category. Graphs were expressed as the mean values with
95% confidence interval (CI). Differences were considered
statistically significant at P<0.05.
[0401] Results:
[0402] Construction and Purification of .alpha.HER2-huEndo Fusion
Proteins:
[0403] We previously demonstrated enhanced anti-tumor activity of
an anti-HER2 antibody-mEndo fusion protein, relative to that seen
with anti-HER2 antibody and/or murine endostatin delivered alone or
in combination. In preparation for potential human use and an
effort to reduce antigenicity, the fusion protein was "humanized"
through substitution of human endostatin sequences for the murine
endostatin fusion domain. The human endostatin (huEndo) gene was
cloned from the human collagen, type XVIII, alpha 1 gene by PCR.
Clones containing wild-type human endostatin were identified. A
point mutation in human endostatin at position 125 (proline to
alanine; huEndo-P125A) within a previously mapped angiogenic domain
of endostatin had increased endothelial cell binding and enhanced
anti-angiogenic activity. A P125A mutation was introduced into
human endostatin using site directed mutagenesis. The subcloned
huEndo and huEndo-P125A genes were ligated in frame to the carboxyl
end of the heavy chain constant domain of human IgG3 and the
endostatin heavy chain constant region was then joined to the
anti-HER2 variable region derived from the humanized monoclonal
antibody 4D5-8 (HER2, trastuzumab; Genentech) in the expression
vector (pSV2-his) containing HisD gene for eukaryotic selection. A
schematic of the resulting fusion proteins is shown in FIG. 1A.
[0404] The anti-HER2 IgG3-huEndo fusion protein constructs were
then stably transfected into SP2/0 or P3X63Ag8.653 myeloma cells
stably expressing the anti-HER2 kappa light chain in order to
assemble the entire anti-HER2 IgG3-huEndo fusion proteins,
anti-HER2 IgG3-huEndo (aHER2-huEndo) and anti-HER2
IgG3-huEndo-P125A (aHER2-huEndo-P125A) (FIG. 1A). The aHER2-huEndo
fusion proteins were biosynthetically labeled with
[.sup.35S]methionine and analyzed by SDS-PAGE. aHER2-huEndo fusion
proteins of the expected molecular weight were secreted as the
fully assembled H2L2 form (FIG. 1B). The secreted
[.sup.35S]methionine-labeled proteins had a molecular weight of
-220 kDa under non-reducing conditions, the size expected for a
complete antibody (170 kDa) with two molecules of endostatin (25
kDa each) attached (FIG. 1A). Following reduction, heavy and light
chains of the expected molecular weight were observed (85 kDa and
25 kDa, respectively) (FIG. 1B). The endostatin fusion proteins
were then purified from culture supernatants using a protein A
column.
[0405] Binding Ability of Anti-HER2 Human Endostatin Fusion
Proteins to HER2 Target Antigen and to HUVECs:
[0406] An investigation of whether the endostatin fusion proteins
could recognize the HER2 antigen was conducted (FIGS. 2A-2F). The
HER2 expressing human breast cancer cell line, SK-BR-3, and murine
mammary tumor cells, EMT6 and EMT6-HER2 were used to test binding
to HER2 antigen, using anti-human IgG antibody as a detection
antibody, aHER2 IgG3, aHER2-huEndo and aHER2-huEndo-P125A, bound to
the HER2+SKBR3 breast cancer cells and EMT6-HER2 cells (FIGS. 2A
and 2B, respectively), while the isotype control antibody
(anti-dansyl IgG3) did not bind to SK-BR-3 and EMT6-HER2. aHER2
IgG3, aHER2-huEndo or aHER2-huEndo-P125A did not bind to parental
EMT6 cells that did not express any HER2 antigen (FIG. 2C).
[0407] To investigate structural integrity of the human endostatin
moiety of the fusion proteins, anti-HER2 human endostatin fusion
proteins were incubated with SK-BR-3, EMT6-HER2 and EMT6. The human
endostatin domain of fusion proteins bound to SK-BR-3 and EMT6-HER2
was detected with biotinylated anti-human endostatin and stained
with streptavidin-PE conjugate. aHER2-huEndo and aHER2-huEndo-P125A
were both recognized following binding to SK-BR-3 and EMT6-HER2 by
the anti-human endostatin detection antibody (FIGS. 2D and 2E),
while aHER2 IgG3 and the isotype control antibodies were not
detected. Endostatin could be detected using either aHER2-huEndo or
ccHER2-huEndo-P125A as the primary antibody and both fusion
proteins bound to HER2+SK-BR-3 and EMT6-HER2 cells with similar
affinity.
[0408] To determine whether aHER2-huEndo fusion proteins could bind
to endothelial cells, HUVECs were treated with aHER2 IgG3, huEndo,
or aHER2-huEndo fusion proteins, and human endostatin domain bound
to HUVECs was detected with biotinylated anti-human endostatin and
stained with streptavidin-PE conjugate. Binding of human endostatin
and aHER2-huEndo fusion proteins to HUVECs was readily detected,
while the isotype control, or aHER2 IgG3 binding was not detected
(FIG. 2F). Of note aHER2-huEndo-P125A showed slightly greater
binding to HUVECs relative to either human endostatin or
aHER2-huEndo.
[0409] Inhibition of Endothelial Tube Formation by the aHER2-huEndo
Fusion Proteins:
[0410] To evaluate anti-angiogenic properties of the human
endostatin antibody fusion proteins, the effects of the
aHER2-huEndo fusion proteins were tested in an in vitro
angiogenesis assay in which human endothelial cells are plated on
Matrigel, and spontaneously aggregate and assemble into
multicellular capillary-like tubular structures in response to
vascular stimuli (e.g. bFGF, VEGF, FBS). Neither parental antibody
nor human endostatin alone showed appreciable inhibition of tube
formation. In contrast, aHER2-huEndo fusion protein treatment
strongly inhibited assembly into tubular structures, with cells
remaining dispersed and exhibiting a morphology resembling adherent
cells on plastic (scattered phenotype) in a dose dependent fashion
(FIGS. 3A-3I). The aHER2-huEndo and aHER2-huEndo-P125A fusion
proteins (FIGS. 3D-3I)) showed significantly greater inhibition of
HUVEC tube formation compared to aHER2 IgG3 (FIG. 3B) or to human
endostatin (FIG. 3C). The increased in vitro anti-angiogenic effect
of aHER2-huEndo fusions relative to native endostatin may be due to
presentation of endostatin as a dimer.
[0411] Inhibition of tubule assembly seen with aHER2-huEndo-P125A
(FIGS. 3G-3I) was significantly greater than that seen for
aHER2-huEndo (FIGS. 3D-2F) at comparable concentrations and
treatment of HUVEC at 45 nM resulted in complete disruption of
tubule formation and extensive morphologic changes (scatter) (FIGS.
3H-3I). Mutation of proline to alanine at amino acid position 125
of human endostatin in the fusion protein therefore increased
inhibition of tubule formation by endothelial cells compared to
either native endostatin (huEndo, FIG. 3C) or wild type endostatin
fusion protein aHER2-huEndo (FIGS. 3D-3F).
[0412] Proliferation of Endothelial Cells by the aHER2-huEndo
Fusion Proteins:
[0413] The effects of aHER2-huEndo fusion proteins were assessed on
endothelial cell (EC) proliferation. HUVECs were exposed to
increasing concentrations of the fusion proteins for 72 hrs in the
absence or presence of either VEGF or bFGF. Both wild type and
mutant antibody-endostatin fusion proteins markedly inhibited
endothelial cell proliferation induced by either VEGF (FIG. 3J) or
bFGF (FIG. 3K). HUVEC proliferation was more effectively inhibited
by aHER2-huEndo-P125A at comparable concentrations than by
aHER2-huEndo (p=0.0085 at the presence of VEGF, p=0.0034 at the
presence of bFGF) or by endostatin alone (p=0.0003 at the presence
of VEGF or bFGF) (FIGS. 3J and 3KB).
[0414] Anti-Tumor Efficacy in Human Breast Cancer SK-BR-3
Xenografts:
[0415] SK-BR-3 is a HER2-amplified human breast cancer cell line
which grows slowly in SCID mice. Trastuzumab, anti-HER2 IgG1, is
able to inhibit the growth of human breast cancer SK-BR-3
overexpressing HER2 alone or in combination with chemotherapy.
Anti-tumor activity of aHER2-huEndo fusion proteins was assayed
against human breast cancer SK-BR-3 xenografts in SCID mice. A
representative experiment is shown in FIGS. 4A and 4B. Equimolar
doses of protein were injected every other day for 4 weeks (FIGS.
4A and 4B). In FIG. 4A, endostatin and aHER2 IgG3 did not
significantly inhibit tumor growth relative to the non-treated
group (PBS, p value=0.1504) by day 29, and aHER2 IgG3 very
significantly inhibited tumor growth relative to the non-treated
group (PBS, p=0.0045), while treatment with aHER2-huEndo and
aHER2-huEndo-P 125A resulted in markedly greater inhibition of
growth (p<0.0001, respectively). Mice treated with
aHER2-huEndo-P125A significantly enhanced inhibition of tumor
growth compared to those treated with aHER2-huEndo (p=0.0161),
human endostatin (p=0.0343), or aHER2 IgG3 (p=0.0253) (FIG. 4A).
There was no significant difference in inhibition of growth among
the treatments with aHER2-huEndo, human endostatin, or aHER2
IgG3.
[0416] Treatment with aHER2-huEndo-P125A completely eradicated
tumors after 30 days and showed the highest degree of inhibition.
The proportion of tumor-free survivors was higher for the
ccHER2-huEndo-P125A group (5 of 5 in the experiment shown compared
to PBS (0 of 5), aHER2 IgG3 and human endostatin (1 of 5), and
aHER2-huEndo (2 of 5) (FIG. 4B). Mice treated with aHER2-huEndo-P
125A showed improved survival relative to those treated with
aHER2-huEndo, human endostatin alone, or aHER2 IgG3 alone (FIG.
4B). Similar results were seen in the duplicated experiment of
which a representative experiment is shown.
[0417] Anti-Tumor Efficacy Requires Presence of the HER2
Target:
[0418] To investigate whether the ability of ccHER2-huEndo-P125A
fusion protein to specifically target HER2 expressing tumors
enhanced efficacy, BALB/c mice were simultaneously implanted with
EMT6 and EMT6-HER2 tumors on opposite flanks. Mice were then
treated with either ccHER2-huEndo-P125A or human endostatin.
Equimolar administration of ccHER2-huEndo-P125A to mice showed
preferential growth inhibition of EMT6-HER2 on day 16, when
compared to parental EMT6 implanted on the contralateral flank
(FIG. 5A, mean tumor volume of EMT6=1391 mm.sup.3, mean tumor
volume of EMT6-HER2=360.8 mm.sup.3, difference between mean tumor
volumes=1030.+-.152.1, 95% CI=703.6 to 1356, p<0.0001). PBS
(FIG. 5C, mean tumor volume of EMT6=1677 mm.sup.3, mean tumor
volume of EMT6-HER2=1527 mm.sup.3, difference between mean tumor
volumes=140.7.+-.150.0, 95% CI=-275.7 to 150.0, p=0.4014) and
endostatin (FIG. 5D, mean tumor volume of EMT6=1169 mm.sup.3, mean
tumor volume of EMT6-HER2=877.8 mm.sup.3, difference between mean
tumor volumes=291.1.+-.191.3, 95% CI=-150.0 to '732.2, p=0.1665)
showed no significant difference on preferential inhibition of
EMT6-HER2 and EMT6 tumors. Among three different treatments,
ccHER2-huEndo-P125A (FIG. 5E, p<0.001) inhibited EMT6-HER2 tumor
growth more effectively than PBS (FIG. 5E, p<0.001 A), or
endostatin (p<0.001) on day 16. However, there was no
significant difference on inhibition of HER2 negative EMT6 parental
tumor among the treatments (FIG. 5F, p>0.05). Selective
targeting of HER2 expressing tumor was therefore required for
maximum efficacy.
[0419] Immunofluorescent Staining of Blood Vessel of Treated
Tumor:
[0420] To investigate the effects of ccHER2-huEndo-P125A fusion
protein on tumor angiogenesis, tumors were resected, histologic
sections of tumors were derived from treated and untreated mice
after 4 or 7 treatments, and tumor microvasculature was visualized
using anti-PECAM fluorescence immunostaining (FIGS. 6A-6D)
Immunofluorescent staining of EMT6-HER2 tumors demonstrated that
the antibody-endostatin fusion treated group showed thin, short,
and fragmented blood vessels on day 12 after 4 treatments (FIG.
6A), compared to those of the PBS treated group (FIG. 6B). The
total blood vessel density (Vd) was measured by determining the
area that was occupied by vessels. Treatment with
ccHER2-huEndo-P125A fusion protein caused a statistically
significant decrease in total Vd in tumors (FIG. 6C, mean total Vd
treated with ccHER2-huEndo-P125A fusion=32020 pixel, mean total Vd
treated with PBS=58560 pixel, difference between mean total
Vd=26540.+-.6574, 95% CI=11380 to 41700, p=0.0038). To investigate
average blood vessel area, total Vd was divides by vessel numbers.
Tumors treated with endostatin fusion showed an extremely
significant reduction of average Vd (FIG. 6D, mean average Vd
treated with ccHER2-huEndo-P125A fusion=44.07 pixel, mean average
Vd treated with PBS=112.1 pixel, difference between mean average
Vd=68.05.+-.13.25, 95% CI=37.50 to 98.60, p=0.0009). By day 18 (7
treatments), EMT6-HER2 tumor from one of two mice treated with
ccHER2-huEndo-P125A had completely regressed and the other in the
treated group demonstrated very small tumor without any clearly
stainable vessels, while vasculature was readily demonstrated in
PBS treated tumors.
[0421] Discussion:
[0422] Anti-angiogenic therapy with endostatin has been shown to
block tumor growth in mice with little or no evidence for emergence
of resistance despite multiple cycles of therapy, in a variety of
murine models. In several murine models, repeated treatment with
endostatin resulted in permanent eradication of tumors. However,
Phase I/II studies of human endostatin did not demonstrate the
levels of anti-tumor activity seen in murine models, although these
clinical trials evidenced that human endostatin is a very safe drug
when used at a variety of dose schedules (Hansma A H, Broxterman H
J, van der Horst I, et al. Ann Oncol 2005; 16(10):1695-701; Kulke M
H, Bergsland E K, Ryan D P, Enzinger P C, et al. J Clin Oncol 2006;
24(22):3555-61). We hypothesized that several of the logistical
disadvantages of the long-term treatment with high dosages of
endostatin could be overcome if the half-life of endostatin could
be extended and if endostatin could be specifically targeted to the
tumor, to achieve higher local concentrations and greater
specificity. In addition, we hypothesized that endostatin might be
more effective if delivered as a dimer in the context of an
antibody fusion protein. We had demonstrated that an anti-HER2
IgG3-C.sub.H3-murine endostatin fusion protein retained
anti-angiogenic activity, exhibited prolonged serum half-life and
stability, selectively targeted tumors bearing HER2, inhibited
blood vessel formation, and inhibited tumor growth more effectively
in vivo than either endostatin or anti-HER2 antibody alone or
delivered in combination (Cho H M, Rosenblatt J D, Kang Y S, et al.
Mol Cancer Ther 2005; 4(6):956-67). We demonstrated the ability of
such fusions to selectively localize to HER2+ tumors, and noted
enhanced efficacy in several murine models including the CT26-HER2,
EMT6-HER2 murine tumors which had been engineered to express human
HER2, and against SKBR3 xenografts that constitutively express high
levels of HER2.
[0423] In order to reduce the possible antigenicity of the murine
endostatin fusion domain in preparation for human application, we
have now constructed two new fusions based on human endostatin and
on a mutated form of endostatin with increased anti-angiogenic
properties. The aHER2-huEndo and aHER2-huEndo-P125A fusion proteins
markedly inhibited endothelial tube formation and proliferation of
HUVEC in vitro, and did so more efficiently than human endostatin.
The aHER2-huEndo-P125A fusion protein showed greater inhibition of
tube formation in vitro than either native endostatin or than wild
type aHER2-huEndo fusion. Treatment of established SK-BR-3
xenografts in SCID mice with the aHER2-huEndo-P125A fusion resulted
in greater inhibition of growth, compared to aHER2 IgG3, human
endostatin, or aHER2-huEndo fusion protein treated mice. The
aHER2-huEndo fusion protein specifically targeted tumors expressing
HER2 and inhibited tumor growth in syngeneic mice simultaneously
implanted with EMT6 and EMT6-HER2. aHER2-huEndo-P125A inhibited
EMT6-HER2 tumor growth more effectively than PBS, or human
endostatin (p value=0.003). Combining the targeting capability of
anti-HER2 antibody with the anti-angiogenic activity of human
endostatin presented in a dimer form in the context of a fusion
antibody improves the inhibition of endothelial tube formation and
proliferation of HUVEC in vitro and enhances anti-tumor activity in
vivo.
[0424] In the endothelial tube formation experiment, the human
endostatin fusion proteins led to profound morphologic changes in
HUVEC, and prevented tube formation. Human or murine endostatin
treatment inhibited HUVEC assembly into tubular structures in
vitro, with cells remain dispersed and exhibit a morphology
resembling adherent cells on plastic rather than aggregating into
characteristic capillary-like tubes. Dimers or trimers of
endostatin stimulated the motility of endothelial cells, but
endostatin monomers did not, which demonstrated that endostatin
oligomerization was important for the efficient inhibition of tube
formation activity. Since the aHER2-huEndo fusion proteins retain
two endostatin domains in a fusion protein, they may effectively
present endostatin as a dimer, and this may result in more
dispersed and scattered morphology of HUVECs seen in these
experiments. Dimerization of the endostatin domain of the fusion
proteins could further facilitate binding to integrins, perlecan,
and glypicans, and further increasing fusion protein activity. The
mutant ccHER2-huEndo-P125A fusion variant inhibited tube formation
of HUVEC in vitro and tumor growth in vivo more effectively than
aHER2-huEndo.
[0425] Linking endostatin to an antibody may significantly enhance
the anti-tumor activity of trastuzumab. Because the overall
response rates of HER2+ breast cancers to trastuzumab remain
relatively low (15-34%) (Fricke I, et al. Clin Cancer Res 2007;
13(16):4840-8; Baselga J, et al. J Clin Oncol 1996; 14:737-44;
Vogel C L, et al. J Clin Oncol 2002; 20(3):719-26; Burstein H J, et
al. J Clin Oncol 2003; 21(15):2889-95), this approach holds promise
for increasing both response rate and duration relative to
trastuzumab, and may expand the spectrum of anti-tumor activity of
trastuzumab given alone or in combination with other anti-tumor
strategies such as other cytotoxic agents (carboplatin, docetaxel),
and/or anti-angiogenic drugs (e.g. bevacizumab; anti-VEGF antibody,
thrombospondin-1). Since administration of endostatin appears to be
quite safe, antibody-endostatin fusion proteins may also be
suitable for use in the adjuvant setting as well. Indeed, we have
observed marked synergy when the anti-HER2 IgG3-marine endostatin
fusion and the anti-VEGF antibody bevacizumab were given in
combination to SK-BR-3 xenograft containing mice. This indicates
the antibody fusion may be useful when combined with other
anti-angiogenic approaches.
[0426] In addition to endostatin, other anti-angiogenic domains
could also be incorporated into fusions (e.g. angiostatin,
tumstatin, etc). Since endostatin is a powerful and global
regulator of angiogenic gene expression, we concentrated initial
experiments on endostatin as a candidate fusion. Finally, in
addition to the HER2 antigenic target, targeting anti-angiogenic
proteins using antibody is a versatile approach that could be
applied to other tumor targets (such as epidermal growth factor
receptor or prostate-specific membrane antigen) through
substitution with other antibody specificities/variable domains.
This approach could be used to enhance efficacy and utility of
antibodies directed to tumor antigens in which parental antibody
shows only modest efficacy (e.g. Cetuximab).
Example 2: Chimeric Molecules
[0427] Examples of Ig-endostatin chimeric molecules include
anti-HER2/neu scFv-Endo, anti-HER2/neu IgG3-C.sub.H1-Endo,
anti-HER2/neu IgG3-H-Endo, and Endo-anti-HER2/neu IgG3 fusion
proteins. In one method to produce antibody fusion proteins,
vectors that contained unique restriction sites at the 3' end of
the CH1 exon, immediately after the hinge, or at the 3' end of the
C.sub.H3 exon as well as on the variable domains of both human
kappa light chain and IgG3 heavy chain IgG3 were used. Using these
constructs, endostatin could be joined to anti-HER2/neu after
C.sub.H1 of anti-HER2/neu IgG3; and endostatin of Endo-IgG3 could
be joined at the amino terminus of the variable region with
flexible linker (Gly.sub.4-Ser).sub.3. The Fv genes of
anti-HER2/neu heavy (FvH) and light chain (FvL) variable region
genes could be cloned by PCR, and the cloned Fv gene fragments
joined with a flexible linker (Gly.sub.4-Ser).sub.3. Endostatin was
joined at the 3' end of the FvL-(Gly.sub.4-Ser).sub.3-FvH gene to
form scFv-Endo. The constructed fusion genes could be expressed in
myeloma cell line SP2/0. For example, transfectomas expressing
anti-HER2/neu IgG3-C.sub.H3-Endo fusion, endostatin, anti-HER2/neu
IgG3, and anti-dansyl IgG3 of unrelated control specificity have
been generated. To purify the fusion proteins produced by the host
cells, the proteins were isolated from culture medium through
protein A affinity chromatography for C.sub.H3-Endo and Endo-IgG3,
or using heparin affinity chromatography (which binds to the
endostatin moiety) for scFv-Endo, CH1-Endo, and H-Endo which lack a
protein A-binding site. For fusion proteins containing both heavy
and light chains, size and assembly into H.sub.2L.sub.2 form is
assessed using SDS-PAGE. Western blotting analysis with rabbit
anti-endostatin sera can be used to detect the attached endostatin
moiety. Expected characteristics of the fusion proteins are shown
below.
TABLE-US-00001 "Predicted" Properties of Fusion Proteins HER2/neu
Tumor Binding Serum Effector Recombinant Proteins Penetration
Ability Half-Life Function IgG3 Heavy Chain ++ ++++ +++ Yes Single
Chain Fv-Endo ++++ ++ + No (scFv-Endo) IgG3-C.sub.H1-Endo +++ +++
++ No (C.sub.HI-Endo) IgG3H-Endo ++ ++++ +++ No (H-Endo)
IgG3-C.sub.H3-Endo ++ ++++ +++ Yes (C.sub.H3-Endo) Endo-IgG3 ++
++++ +++ Yes
Example 3: Serum Stability Studies
[0428] To characterize the in vivo pharmacokinetic patterns of the
antibody-endostatin fusion protein, mice with/without implanted
tumors (CT26 or CT26-HER2/neu) were injected intravenously with
[.sup.125I] labeled anti-HER2/neu IgG3, anti-HER2/neu
IgG3-CH3-Endo, endostatin, and a control anti-dansyl IgG3 and
clearance of endostatin on fusion measured. [.sup.125I]-endostatin
was rapidly removed from the plasma compartment in mice
with/without tumors (T.sub.112.sup.2 elimination: 0.5-3.8 hrs),
while the rate of removal of [.sup.125I] labeled anti-HER2/neu
IgG3-C.sub.H3-Endo (T.sub.112.sup.2: 40.244.0 hrs) was similar to
those of [.sup.125I] labeled anti-HER2/neu IgG3 (T.sub.1/2.sup.2:
39.9-63.8 hrs) and control anti-dansyl IgG3 (T.sub.1/2.sup.2:
43.7-46.5 hrs).
[0429] To analyze the serum stability of [.sup.125I] labeled
anti-HER2/neu IgG3, anti-HER2/neu IgG3-C.sub.H3-Endo, endostatin
and anti-dansyl IgG3 plasma samples were TCA-precipitated and
counted. 96 hours following injection approximately 90% of the
anti-HER2/neu IgG3 and anti-HER2/neu IgG3-C.sub.H3-Endo in serum
remained intact. Endostatin was rapidly eliminated with little
remaining in the circulation by 60 min. For endostatin,
approximately 90% was intact 2 min after injection and only 55% of
the remaining circulating endostatin remained intact at 60 min. In
contrast anti-HER2/neu IgG3-C.sub.H3-Endo cleared much more slowly
with kinetics resembling anti-HER2/neu IgG3 and anti-dansyl IgG3.
Analysis of serum samples by SDS-PAGE confirmed that the
anti-HER2/neu IgG3-C.sub.H3-Endo in circulation remained
intact.
Example 4: Biolocalization Studies
[0430] To measure biodistribution and biolocalization of the
endostatin fusion protein, purified endostatin fusion protein was
labeled with .sup.125I. 96 hours following an intravenous injection
into mice bearing tumors, the radiolocalization indices (the %
injected dose [ID]/g in tumor divided by the % ID/g in blood) of
anti-HER2/neu IgG3-C.sub.H3-Endo and anti-HER2/neu IgG3 were
similar. Anti-HER2/neu IgG3-C.sub.H3-Endo showed a tumor/blood
ratio of 3.76 for CT26-HER2/neu and a 0.50 tumor/blood ratio for
CT26; whereas anti-HER2/neu IgG3 showed 2.83 and 0.47 ratios for
CT26-HER2/neu and CT26, respectively. No enhanced targeting to
tumors was seen for endostatin alone. Therefore, both anti-HER2/neu
antibody and anti-HER2/neu antibody-endostatin fusion protein
retained the ability to localize to HER2/neu bearing tumors.
[0431] In mice simultaneously implanted with CT26 and CT26
expressing HER2/neu (CT26-HER2) tumors on opposite flanks,
.sup.125I-labeled anti-HER2/neu IgG3-endostatin fusion protein and
anti-HER2/neu IgG3 preferentially localized to CT26-HER2 tumors.
Specific tumor radiolocalization indices of anti-HER2/neu
IgG3-endostatin were actually greater than those of anti-HER2/neu
IgG3 in several separate experiments. This indicated relative
localization of targeted antibody-endostatin fusions to tumor due
to binding to HER2/neu target antigen.
TABLE-US-00002 Time CT26-HER2 Radiolocalization Treatment (Hrs)
CT26 (% ID/g) (% ID/g) Indices* Anti-HER21 6 3.51 .+-. 1.38 3.94
.+-. 1.83 1.12 neu IgG3 24 7.04 .+-. 3.48 14.95 .+-. 3.48 2.12 96
3.03 .+-. 0.63 7.88 .+-. 2.18 2.60 Anti-HER21 6 1.16 .+-. 0.38 6.20
.+-. 0.76 5.34 neu IgG3- 24 1.31 .+-. 0.60 9.72 .+-. 1.05 7.42
endostatin 96 0.33 .+-. 0.05 1.17 .+-. 0.07 3.55
*Radiolocalization. Indices represent the ratios of the % ID/g in
CT26-HER2 divided by the % ID/g in CT26.
Example 5: Anti-Tumor Studies
[0432] The ability of anti-HER2/neu IgG3-C.sub.H3-Endo,
anti-HER2/neu IgG3 and endostatin to preventing the growth of CT26
expressing HER2/neu in BALB/c mice was examined. BALB/c mice were
subcutaneously injected with 1.times.106 cells and tumor growth
measured. On day 7, most of mice developed palpable tumors (about 5
mm in diameter) and the treatment of mice bearing tumors (n=5 per
group) initiated every other day by intravenous injection (5 times)
of anti-HER2/neu IgG3-C.sub.H3-Endo, anti-HER2/neu IgG3,
anti-dansyl IgG3, endostatin, or PBS controls. Tumor growth in mice
treated with anti-HER2/neu IgG3 or endostatin was reduced relative
to anti-dansyl IgG3 or PBS controls. Treatment with anti-HER2/neu
IgG3-C.sub.H3-Endo resulted in additional reduction in tumor
volume. Anti-HER2/neu IgG3-C.sub.H3-Endo demonstrated significant
growth inhibition (p<0.05) compared with PBS, anti-HER2/neu IgG3
or endostatin administered at two-fold molar excess relative to
anti-HER2/neu IgG3-endostatin alone. A ten-fold increase in
endostatin alone further increased efficacy.
[0433] In mice simultaneously implanted with CT26, and
CT26-HER2/neu on opposite flanks, equimolar administration of
anti-HER2/neu IgG3-endostatin to mice showed preferential
inhibition of CT26-HER2/neu, compared to contralaterally implanted
CT26 parental tumor. Anti-HER2/neu IgG3-endostatin inhibited more
effectively than endostatin, anti-HER2/neu IgG3 antibody, or the
combination of antibody and endostatin (p<0.05).
[0434] Whether anti-HER2/neu IgG3-endostatin, endostatin,
anti-HER2/neu IgG3 antibody, or the combination of antibody and
endostatin would inhibit the growth of the human breast cancer
SK-BR-3 in SCID mice was examined. SCID mice (n=8 per group) were
subcutaneously injected with 1.times.10.sup.6 cells of SK-BR-3 and
tumor growth measured. By day 15, most mice developed palpable
tumors (about 5 mm in diameter) and treatment was initiated every
other day with intravenous injection (10 times) of anti-HER2/neu
IgG3-CH3-Endo, anti-HER2/neu IgG3, endostatin, or the combination
of antibody and endostatin. Mice treated with anti-HER2/neu
IgG3-C.sub.H3-Endo, endostatin, anti-HER2/neu IgG3 antibody, or the
combination of antibody and endostatin all showed inhibition of
tumor growth. Administration of anti-HER2/neu IgG3-C.sub.H3-Endo
consistently resulted in the greater reduction of tumor volume,
compared to either anti-HER2/neu antibody alone, endostatin alone,
or antibody and endostatin given in combination (p<0.05).
Example 6: Production and Characterization of Anti-HER2/Neu
IgG3-Endostatin
[0435] The anti-HER2/neu antibody-endostatin fusion protein of the
expected molecular weight was produced and secreted from the stably
transfected Sp2/0 cell lines as the fully assembled H.sub.2L.sub.2
form. The secreted .sup.355-methionine labeled anti-HER2/neu
IgG3-endostatin has a molecular weight of approximately 220 kDa
under non-reducing conditions, the size expected for a complete
antibody (170 kDa) with 2 molecules of endostatin (25 kDa)
attached. Following reduction, H and L chains of the expected
molecular weight were observed. To confirm that the endostatin
moiety was present in the anti-HER2/neu IgG3-endostatin protein,
purified anti-HER2/neu IgG3-endostatin and endostatin were resolved
under non-reducing conditions. Following Western blotting,
anti-HER2/neu IgG3-endostatin was identified at the molecular
weight of 220 kDa by both anti-human IgG or anti-endostatin
antibody. Following reduction, the predominant heavy chain band
from anti-HER2/neu IgG3-endostatin migrated at the expected size of
85 kDa.
Example 7: Antiangiogenic Activity of Anti-HER2/Neu
IgG3-Endostatin
[0436] The ability of endostatin to block VEGF/bFGF-induced
angiogenesis in vitro was tested using the chorioallantoic membrane
(CAM) assay. Pellets containing Vitrogen and VEGF/bFGF (100 ng and
50 ng/pellet, respectively) and either anti-HER2/neu IgG3 (0.5-10
mg/pellet: 2.95-59 pmol/pellet), anti-HER2/neu IgG3-endostatin
(0.5-10 mg/pellet: 2.25-45 pmol/pellet), or endostatin (0.5-10
mg/pellet: 20-400 pmol/pellet) were measured for invasion of new
capillaries. Two independent preparations of anti-HER2/neu
antibody-endostatin fusion protein were able to suppress the
angiogenic response mediated by VEGF/bFGF in a dose-dependent
manner with a specific activity similar to that seen with
endostatin. In contrast anti-HER2/neu IgG3 showed no
anti-angiogenic response. Therefore genetically engineered
anti-HER2/neu-IgG3-endostatin maintains the ability to inhibit the
angiogenic response mediated by VEGF/bFGF.
Example 8: Serum Clearance and Stability of Anti-HER2/Neu
IgG3-Endostatin
[0437] To characterize the pharmacokinetics of anti-HER2/neu
IgG3-endostatin, mice with/without implanted tumors (CT26 or
CT26-HER2) were injected intravenously with
[.sup.125I]-anti-HER2/neu IgG3, anti-HER2/neu IgG3-endostatin,
endostatin, or a control anti-dansyl IgG3 and clearance of injected
radiolabeled proteins measured. Representative results from mice
with implanted HER2/neu-expressing CT26 tumors and the
pharmacokinetic data for mice in all groups are summarized in Table
1. [.sup.125I]-endostatin was rapidly removed from the plasma
compartment in mice with or without tumors (T.sub.112.sup.2
elimination: 0.5-3.8 hrs), while the clearance rate of
[.sup.125I]-anti-HER2/neu IgG3-endostatin (T.sub.112.sup.2:
40.2-44.0 hrs) was similar to that of [.sup.125I]-anti-HER2/neu
IgG3 (T.sub.1/2.sup.2: 39.9-63.8 hrs) and anti-dansyl IgG3
(T.sub.1/2.sup.2: 43.746.5 hrs). Therefore endostatin fused with
antibody is cleared from the peripheral compartment much more
slowly than endostatin alone.
[0438] In mice bearing CT26-HER2 tumors (Table 1), the area under
the plasma concentration curve (AUC) of anti-HER2/neu
IgG3-endostatin was increased by a factor of 56 (13,100% IDmin/ml
vs. 233% IDmin/ml) compared to endostatin, as a consequence of both
a longer half-life of elimination (69 fold increase: 2,640 min vs.
38 min) and an increased "mean residence time" (MRT) (56 fold
increase: 2800 min vs. 50 min). Endostatin was very rapidly removed
from serum within 30 min, principally by glomerular filtration and
renal clearance, but anti-HER2/neu IgG3-endostatin demonstrated
much slower clearance from serum, similar to those of anti-HER2/neu
IgG3 and anti-dansyl IgG3.
[0439] To analyze the serum stability of [.sup.125I] labeled
anti-HER/neu IgG3, anti-HER2/neu IgG3-C.sub.H3-endostatin,
endostatin and anti-dansyl IgG3, plasma samples were
TCA-precipitated and counted. 96 hours following injection
approximately 90% of the anti-HER2/neu IgG3 and anti-HER2/neu
IgG3-endostatin in serum remained intact. For endostatin,
approximately 90% was intact 2 min after injection and only 55% of
the remaining circulating endostatin remained intact at 60 min. In
contrast anti-HER2/neu IgG3-endostatin cleared much more slowly
with kinetics resembling anti-HER2/neu IgG3 and anti-dansyl IgG3.
Analysis of serum samples by SDS-PAGE confirmed that the
anti-HER/neu IgG3-endostatin in circulation remained intact. Thus,
the antibody moiety of anti-HER2/neu IgG3-endostatin fusion protein
renders the genetically fused endostatin much more stable in the
blood stream.
Example 9: Biodistribution and Biolocalization of Anti-HER2/Neu
IgG3-Endostatin
[0440] Ninety-six hours following an intravenous injection of mice
bearing CT26-HER2 tumors, anti-HER2/neu IgG3 was found mainly in
the tumor and blood (5.67 and 2.10% ID/g, respectively). The
radiolocalization indices at 96 hours post injection (the % ID/g in
tumor divided by the % ID/g in blood) of anti-HER2/neu
IgG3-endostatin and anti-HER2/neu IgG3 were similar. Anti-HER2/neu
IgG3-endostatin showed a tumor/blood ratio of 3.76 for CT26-HER2
and a 0.50 for CT26, whereas anti-HER2/neu IgG3 showed tumor/blood
ratios of 2.83 and 0.47 for CT26-HER2 and CT26, respectively.
Therefore, both anti-HER2/neu antibody and anti-HER2/neu
antibody-endostatin fusion protein preferentially localized to
HER2/neu expressing tumors.
[0441] To measure localization of antibody-endostatin fusion
proteins to the antigenic target, mice simultaneously implanted
with CT26 and CT26-HER2 tumors on opposite flanks were injected
intravenously with either .sup.125I-labeled anti-HER2/neu
IgG3-endostatin fusion protein or .sup.125I-labeled anti-HER2/neu
antibody (Table 2). The biodistribution and biolocalization of the
labeled proteins was examined at different times (6, 24, and 96
hours) after injection of labeled proteins. Anti-HER2/neu
IgG3-endostatin fusion protein and anti-HER2/neu IgG3
preferentially localized to CT26-HER2 tumors. Specific tumor
radiolocalization indices of anti-HER2/neu IgG3-endostatin were
actually greater than those of anti-HER2/neu IgG3 (Table 2). This
indicated that the relative localization of targeted
antibody-endostatin fusions to tumor was due to binding to the
HER2/neu target antigen (Table 2).
Example 10: Anti-Tumor Activities of Anti-HER2/Neu IgG3-Endostatin
In Vivo
[0442] Murine colon adenocarcinoma CT26 cells were transduced with
the gene for HER2/neu antigen as previously described. The
CT26-HER2 cells have been used in these studies and proliferated at
the same rate in vitro as parental CT26 cells. Preliminary
experiments revealed that the CT26-HER2 tumors implanted in BALB/c
mice grew at the same rate as the parental CT26 tumors (Ref Lab
Animal).
[0443] The anti-tumor effects of anti-HER2/neu IgG3-endostatin,
anti-HER2/neu IgG3 and endostatin on the growth of CT26-HER2 in
BALB/c mice were studied. Tumor growth in mice treated with
anti-HER2/neu IgG3 or endostatin was reduced relative to an isotype
control anti-dansyl IgG3 or PBS control. Treatment with
anti-HER2/neu IgG3-endostatin resulted in additional reduction in
tumor volume. Anti-HER2/neu IgG3-endostatin demonstrated
significantly better growth inhibition when compared to PBS,
anti-HER2/neu IgG3 or endostatin administered. Genetic fusion of
endostatin to the anti-HER2/neu IgG3 initially appeared to inhibit
tumor growth more efficiently than either anti-HER2/neu IgG3 or
endostatin alone.
[0444] To confirm the preliminary experiments, mice were
simultaneously implanted with CT26, and CT26-HER2 on opposite
flanks. Administration of anti-HER2/neu IgG3-endostatin showed
preferential inhibition of CT26-HER2 growth, compared to
contralaterally implanted CT26 parental tumor. Anti-HER2/neu
IgG3-endostatin inhibited more effectively than endostatin,
anti-HER2/neu IgG3 antibody, or the combination of antibody and
endostatin p<0.05).
[0445] Herceptin, anti-HER2/neu IgG1, was able to inhibit the
growth of SK-BR-3 breast carcinoma cells, which overexpress
HER2/neu. It was next determined whether anti-HER2/neu
IgG3-endostatin, endostatin, anti-HER2/neu IgG3 antibody, or both
antibody and endostatin in combination would inhibit the growth of
human breast cancer SK-BR-3 xenografts in SCID mice. SK-BR-3 was
implanted on the flank of SCID mice. The treatment was repeated 10
times. Administration of anti-HER2/neu IgG3-endostatin resulted in
a greater reduction of tumor volume, compared to either
anti-HER2/neu antibody alone, endostatin alone, or antibody and
endostatin given in combination (p<0.05).
Example 11: Blood Vessel Formation in CT26-HER2 Tumors Treated with
the Anti-HER2/neu IgG3-Endostatin Fusion Protein
[0446] To better understand the mechanism of anti-tumor activity of
the anti-HER2/neu IgG3-endostatin fusion protein, blood vessel
formation in tumors was analyzed. Mice were simultaneously
implanted with CT26 and CT26-HER2 tumors on opposite flanks and
allowed to grow until the tumor diameter was 4-6 mm at which time
the mice were intravenously treated with either anti-HER2/neu
IgG3-endostatin fusion proteins or PBS. CT26-HER2 tumors grew
slower in mice treated with anti-HER2/neu IgG3-endostatin compared
to the others with kinetics similar to those above. After the fifth
treatments, the tumors were removed and cryosections of tumors were
immunohistochemically stained for endothelial cells with
anti-PECAM-1 antibody to visualize the blood vessel formation of
these tumors. The parental CT26 tumor tissue and the untreated
CT26-HER2 tumor tissue appeared to have more vessels than CT26-HER2
treated with endostatin fusion proteins.
[0447] To distinguish the blood vessel formation, the tumor
sections were stained with rat anti-mouse anti-PECAM antibody,
detected with anti-rat IgG-Alexa 594, and then analyzed through
confocal microscope. Confocal microscopic analysis for these tumors
revealed striking differences in the vasculature between CT26-HER2
tumors treated with anti-HER2/neu IgG3-endostatin and the others,
which may explain the altered tumor growth observed above. Images
composed of 14-21 digital microscopic images showed that blood
vessels in the parental CT26 tumors with/without endostatin fusion
treatments and in PBS-treated CT26-HER2 tumors appeared more
organized and branched than the blood vessels in the CT26-HER2
tumors treated with anti-HER2/neu IgG3-endostatin.
[0448] The alterations in vasculature were quantified by measuring
the blood vessel density. The blood vessel density was measured by
determining the area that was occupied by vessels, which provided
the amount of vascular area within each tumor. Using this measure,
the HER2/neu expressing tumors with endostatin fusion treatments
had significantly less vascular area (16%) than did the untreated
CT26-HER2 tumors (Table 3).
Example 12: Angiogenic Effects of VEGF Ischemic/Non-Ischemic
Tissues
[0449] Antiangiogenic effects of the antibody-endostatin fusion
proteins will be investigated using animal hindlimb models of
therapeutic angiogenesis. Rat or rabbit hindlimb ischemia models
are available. The ischemic levels in the rabbit model can be
manipulated as maximal, severe, or moderate ischemic
conditions.
[0450] Rabbit Hindlimb Ischemia Model:
[0451] The normal distribution of arteries and capillaries 1 h
after surgery (iliac tie and femoral excision), flow through the
iliac and femoral arteries was eliminated indicating ischemia.
Although there had been significant collateral development and
return of flow to the limb, flow through the femoral artery and its
associated vessels was still absent. Significant inflammation,
necrosis, or tissue loss was not detected despite the severe early
ischemia indicating that the muscle was significantly reperfused.
In contrast, the VEGF-treated limb recovered full flow to the
distal branches of the femoral artery. Quantitation of these
vessels from the original angiography revealed >10-fold more
collateral vessels with external diameter >100 um in the treated
limbs. The generation of new vessels in the VEGF treated limbs
could involve combinations of vasculogenesis, angiogenesis, and
arteriogenesis.
[0452] Angiogenic Effects of VEGF in Non Ischemia Model:
[0453] Neovascularization of non-ischemic tissues has been examined
at the rat subcutaneous peritoneal fat pad and mouse ear flap. In
both case sutures were tied into the tissues and 2.times.10.sup.9
pfu of Ad-CMV-VEGF or Ad-(3-Gal were injected around the sutures.
Tissues were analyzed after 3-weeks. New vessels were clearly
visible in both tissues injected with Ad-CMV-VEGF but not with the
(3-gal. The VEGF injected tissues also contained a visible red
blush indicative of leaky vessels. These results showed that VEGF
can activate angiogenesis/vasculogenesis in non-ischemic
tissue.
Example 13: Combination Treatments with Other Antiangiogenic
Strategies
[0454] PDGF Blockade:
[0455] Herceptin has been approved for the treatment of advanced
breast cancer and Gleevec (STI57, imatinib, Novartis Pharma AG) has
been approved for chronic myelogenous leukemia and gastrointestinal
stromal tumors. Imatinib disrupts the association of pericytes with
neovasculature in tumors through effects on PDGFR. While endostatin
inhibits early blood vessel formation, imatinib may affect
maturation by effects on pericytes. Initially MCF7 and MCF7-HER2
tumors subcutaneously implanted on the left and right flank,
respectively, will be treated with a combination of anti-HER2
IgG3-huEndo and fusion proteins and imatinib. Imatinib (50 mg/kg)
will be administered orally twice a day. The blood vessel formation
and tumor growth in tumors will be examined as outlined supra.
[0456] VEGF Blockade:
[0457] A humanized anti-VEGF antibody (bevacizumab, AVASTIN.TM.,
rhuMAb-VEGF; Genentech) has been approved for use in combination
with chemotherapy in a phase III trial in metastatic colon
carcinoma. AVASTIN.TM. has been reported to have clinical benefit
of 17% (complete and partial responses plus stable disease 6
months) in phase II trials in breast cancer. AVASTIN.TM. also has
activity in renal cell carcinoma, and has been reported to augment
taxane activity in a phase III breast cancer trial. AVASTIN.TM.
binds and neutralizes all of the major isoforms of VEGF-A,
decreases vascular volume, microvascular density, interstitial
fluid pressure and the number of viable, circulating endothelial
cells. Combining fusion proteins with AVASTIN.TM. may augment
activity of both approaches. SK-BR-3, or MCF7 and MCF7-HER2 tumors
in SCID mice will be treated in combination with AVASTIN.TM. (50
.mu.g/injection) and anti-HER2 antibody-huEndo fusion proteins (10,
50, and 250 .mu.g/injection, i.v., q.o.d.) or human endostatin, or
antibody alone.
[0458] Metronomic Therapy:
[0459] Proliferating endothelial cells forming new blood vessels
within tumors are sensitive to the cytotoxic effects of many
chemotherapeutics. Conventional chemotherapeutic regimes with
maximum tolerable doses require extended rest periods which allow
repair of the endothelial compartment. However, "metronomic"
therapy (i.e. administration of continuous low-doses) may sustain
antiangiogenic effects. MCF7/MCF7-HER2 tumors will be treated in
SCID mice in combination with various concentrations of anti-HER2
antibody-huEndo fusion proteins (10, 50, and 250 .mu.g/injection,
i.v., q.o.d.) and low dose cyclophosphamide (CTX, 25 mg/kg/day,
p.o.), 79-80 or alone. Repeated administration of low dose taxanes
(paclitaxel or docetaxel), using "metronomic" scheduling for the
treatment of cancers will also be tested.
Example 14: Construction, Purification, and Characterization of
Anti-EGFR igG3-huEndo Fusion Proteins
[0460] We obtained publically available amino acid sequences of
Cetuximab (disclosed in Patent Publication No. WO2008083949, which
is incorporated herein by reference in its entirety). The DNA
sequences of the heavy and light chains were deduced using the
Vector NTI program and DNA sequences were synthesized. The heavy
and light chain variable region genes were cloned into pUC57 vector
(EcoRV site). The anti-EGFR heavy chain variable region gene
fragment (EcoRV-Nhel) was cloned into the EcoRV-Nhel site of the
human IgG3 and human IgG3-huEndo-P125A expression vectors and the
anti-EGFR light chain variable region gene fragment (EcoRV-SalI)
was cloned into the EcoRV-SalI site of the human kappa expression
vector. Anti-EGFR IgG3 (aEGFR IgG3), anti-EGFR IgG3-huEndo
(aEGFR-huEndo) or anti-EGFR IgG3-huEndo-P125A (aEGFR-huEndo-P125A)
fusion genes were co-transfected into B cell myeloma, SP2/0 or P3
cells, by electroporation. aEGFR IgG3, aEGFR-huEndo and
aEGFR-huEndo-P125A fusion proteins were synthesized and purified
(FIG. 16). Binding of aEGFR IgG3, cetuximab, aEGFR-huEndo, and
aEGFR-huEndo-P125A fusions to EGFR on epidermoid carcinoma A431
cells were confirmed by flow cytometry (FIG. 16A). The purified
fusion protein has a molecular weight of 220 kDa under nonreducing
conditions (FIG. 16B). We confirmed the presence of endostatin in
the fusion proteins by western blotting with a biotinylated
anti-human endostatin antibody/avidin-HRP and anti-human IgG-HRP
(FIG. 16C). aEGFR-huEndo and aEGFR-huEndo-P125A were identified by
both anti-human IgG and anti-endostatin antibody (FIG. 16C).
aEGFR-huEndo fusion proteins inhibited VEGF and bFGF induced
endothelial cell tube formation in vitro, more efficiently than
endostatin alone, or parental aEGFR IgG3 (FIG. 16D).
Example 15: Effects on Vasculogenic Mimicry
[0461] This example describes inhibition of vasculogenic mimicry.
In these experiments, we tested two antibody-human mutant
endostatin fusions (aHER2-huEndo-P125A and aEGFR-huEndo-P125A) with
different antibody specificities for use in the treatment of human
tumors expressing the corresponding targets (FIG. 8).
aHER2-huEndo-P125A has enhanced antiangiogenic properties, relative
to those seen with wild type endostatin in the fusion molecule.
Enhanced antiangiogenic properties were initially noted using a
fusion with anti-HER2 specificity and endostatin-P125A. The
anti-HER2 IgG3-Endostatin-P125A fusion protein (aHER2-huEndo-P125A)
demonstrates strong binding to HER2 through the antibody domain,
and binding to HUVEC (human umbilical vein endothelial cells)
through the endostatin domain (FIGS. 9A-9G). The administration of
the aHER2-huEndo-P125A protein to mice in vivo demonstrated a
significantly prolonged half-life compared to either human
endostatin (huEndo) or huEndo-P125A (FIG. 9H).
[0462] We tested for the antiangiogenic properties of
aHER2-huEndo-P125A in vitro using an endothelial cell tube
formation assay. HUVECs were suspended in endothelial cell growth
medium (EGM) and plated on matrigel coated plates with EGM.
Following sixteen hours of incubation we assayed for tube formation
quantitatively using a microscope (FIGS. 10A-10I). Neither huEndo
nor huEndo-P125A used alone, showed significant effects on tube
formation (a surrogate assay for angiogenesis). Neither trastuzumab
nor parental anti-HER2 IgG3 (aHER2 IgG3) antibody had any effects
on tubule formation in contrast to anti-HER2 IgG3-endostatin fusion
using wild type endostatin (aHER2-huEndo) which significantly
inhibited angiogenesis. However, aHER2-huEndo-P125A had
significantly greater inhibitory affects on angiogenesis, resulting
in complete abrogation of tube formation at a concentration of
45.46 nM. Hence, the fusion protein incorporating huEndo-P125A had
significantly greater effects than either a wild type endostatin
fusion or than mutant endostatin (huEndo-P125A) alone. This is an
unexpected finding.
[0463] The in vitro effects on angiogenesis were then recapitulated
in vivo using a SKBR3 xenograft model for HER2+ breast cancer grown
in SCID mice. Although wild type aHER2-huEndo had greater activity
than antibody or endostatin administered alone (or combined), use
of the aHER2-huEndo-P125A fusion administered repeatedly
intravenously over a 30 day period resulted in complete elimination
of tumor growth which was statistically superior as compared to
wild type fusion alone (FIGS. 11A-11B). Hence, the
aHER2-huEndo-P125A fusion had unexpectedly greater effects against
breast cancer xenograft growth in vivo, than either a parental
antibody or antibody endostatin fusion with wild type
endostatin.
[0464] We developed additional novel fusion molecules incorporating
an anti-EGFR antibody domain. EGFR was chosen as a target due to
the high prevalence of EGFR expression among a variety of human
solid tumors including human colon, lung, head and neck, ovarian,
squamous cell carcinoma, bladder, and other tumors. In addition
EGFR is expressed on the surface of triple negative breast cancers,
for which anti-HER2 reagents are inactive. In clinical practice the
use of anti-EGFR antibodies alone is minimally effective in all of
the aforementioned tumors, while cetuximab, an anti-EGFR antibody,
has been combined with either chemotherapy or radiation in a
variety of clinical settings with modest incremental response. We
linked the mutant endostatin-P125A domain to an anti-EGFR antibody
in an effort to target EGFR+ tumors. The anti-EGFR IgG3 fusions
with wild type endostatin (aEGFR-huEndo) or endostatin-P125A
(aEGFR-huEndo-P125A) were constructed as shown in FIG. 1
substituting anti-EGFR sequences for anti-HER2 sequences. The
anti-EGFR fusion proteins retained anti-EGFR specificity, (FIG.
12B), and correct folding of the endostatin domain as demonstrated
by protein gel and western blotting (FIG. 12A). Anti-EGFR
specificity was demonstrated by flow cytometry, and anti-endostatin
antibodies recognized both wild type and mutant endostatin-P125A
domains within the fusions by western blot, or by flow cytometry
(FIGS. 12A and 12B).
[0465] We next tested for effects on endothelial tube formation
using HUVEC as shown in FIGS. 13A-13C. Human endostatin-P125A
(huEndo-P125A) had minimal effects on tubule formation. In contrast
increasing doses of anti-EGFR IgG3-human endostatin (aEGFR-huEndo)
fusion showed marked inhibition of tube formation, and near
complete abrogation of tube formation at the highest concentrations
used. Anti-EGFR IgG3-human endostatin-P125A (aEGFR-huEndo-P125A)
fusion resulted in significant inhibition of tube formation, which
was significantly more potent than that seen with wild type
endostatin fusion (aEGFR-huEndo) at all concentrations tested
(FIGS. 13A and 13B). No inhibition of tube formation was seen with
cetuximab or using media controls. In addition, no inhibition of
tube formation was seen with a control anti-EGFR IgG3 (aEGFR IgG3)
antibody. This once again indicated a significantly greater
antiangiogenic effect of the presently claimed aEGFR-huEndo-P125A
fusion. This was quantitated using a quantitative assay for an
inhibition of tube formation, in which we compared anti-EGFR
IgG3-wild type endostatin (aEGFR-huEndo) fusion to anti-EGFR
IgG3-human endostatin-P125A (aEGFR-huEndo-P125A) fusion. Wild type
endostatin fusion showed 50% inhibition of tube formation at a
concentration of 27.2 nM, while two different preparations of
anti-EGFR IgG3-human endostatin-P125A fusion demonstrated 50%
inhibition at concentrations of 10.5 and 5.5 nM, respectively (FIG.
13B). A comparison of anti-HER2 IgG3-huEndo using a wild type
endostatin (aHER2-huEndo) fusion domain to anti-HER2
IgG3-huEndo-P125A using a mutant endostatin-P125A domain
(aHER2-huEndo-P125A) shows enhanced antiangiogenic activity of the
mutant endostatin-P125A fusion (FIG. 13C). The markedly increased
anti-angiogenic activity of the aEGFR-huEndo-P125A fusion was
unexpected.
[0466] A variety of investigators have reported on the formation of
blood vessel like structures in vitro, so-called `vasculogenic
mimicry`, using several human tumors including human breast cancer
and uveal melanoma, as well as human ovarian cancer cultured using
angiogenic media on matrigel. Human triple negative breast cancer
(TNBC), as well as human melanoma and other solid tumors appear to
demonstrate unusual plasticity in vitro and are able to form
tube-like structures mimicking the vasculogenic tubular structures
formed by endothelial cells in vitro. This phenomenon, known as
`vasculogenic mimicry`, has been correlated with more aggressive
behavior in vivo especially in the setting of uveal melanoma and
HER2+ breast cancer. A variety of tumors have been demonstrated in
vivo to form vessel-like channels which may serve as conduits for
nutrients, as well as red blood cells, and may actually anastomose
with blood vessels, which actually serve to support tumor growth.
Hence, we tested if inhibition of vasculogenic mimicry is useful in
targeting tumor growth in vivo.
[0467] We tested a triple negative breast cancer cell (TNBC) line,
MDA-MB-231, for the ability to form vascular-like structures in
vitro. As demonstrated in FIG. 14A, MDA-MB-231 cells form tube-like
structures on endothelial cell growth media (EGM) in matrigel. Tube
formation was not inhibited by human endostatin, human mutant
endostatin-P125A, cetuximab, trastuzumab, or anti-HER2 IgG3
antibody. In contrast, when MDA-MB-231 cells were exposed to
increasing concentrations of the presently claimed antibody-human
endostatin-P125A fusions (aHER2-huEndo-P125A or
aEGFR-huEndo-P125A), dose dependent inhibition of tube formation
was observed. Tube formation was completely abrogated a
concentration of 45.5 nM. In contrast, commercially available
Avastin, an anti-VEGF IgG1 had no effects on tube formation (FIG.
14A). The unusually potent effect of antibody-endostatin-P125A
fusions (aHER2-huEndo-P125A or aEGFR-huEndo-P125A) on vasculogenic
mimicry was totally unexpected. Since endostatin and
endostatin-P125A were not known to directly affect `vasculogenic
mimicry,` nor known to directly interact with tumor cells, the
results obtained using the antibody-endostatin-P125A fusions in
inhibiting `vasculogenic mimicry` by tumor cells were completely
unexpected.
[0468] Similar experiments were performed with SKOV3 and with
PEO-1, both HER2+ ovarian cancer cell lines (FIG. 14B). These cell
lines also formed tubes in vitro, and exhibited similar
vasculogenic mimicry. Tube formation by either SKOV3 or PEO-1 was
completely inhibited using either anti-HER2 IgG3-human
endostatin-P125A (aHER2-huEndo-P125A) fusion protein or anti-EGFR
IgG3-human endostatin-P125A (aEGFR-huEndo-P125A) fusion in dose
dependent fashion (FIGS. 14B and 14C). As before, direct effects of
the antibody-endostatin-P125A fusions on vasculogenic mimicry by
ovarian cancer cells were completely unexpected. Since
aggressiveness and progress in ovarian cancer has been correlated
to ability to engage in vasculogenic mimicry, the observed effects
on vasculogenic mimicry are likely to have clinical
significance.
[0469] In addition to the aforementioned cell lines, we also tested
the ability of the endostatin fusions to inhibit vasculogenic
mimicry by human uveal melanoma cells. The uveal melanoma cell line
MUM-2B formed tube-like structures in vitro. Both the anti-HER2
IgG3-huEndo-P125A (aHER2-huEndo-P125A) and anti-EGFR
IgG3-huEndo-P125A (aEGFR-huEndo-P125A) fusions markedly reduced
tube formation as compared to endostatin, cetuximab, or
trastuzumab, respectively (FIG. 14D).
[0470] Finally, we investigated anti-tumor efficacy in human
ovarian cancer SKOV3 xenografts (FIG. 15). SKOV3 is a
HER2-amplified human ovarian cancer cell line which grows slowly as
a xenograft in NSG mice. We assayed for anti-tumor activity of
presently claimed aHER2-huEndo-P125A fusion protein against SKOV3
xenografts in NSG mice. In FIG. 15, aHER2-huEndo-P125A fusion
proteins were injected twice a week (FIG. 15). aHER2-huEndo-P125A
fusion protein significantly inhibited tumor growth relative to the
non-treated group (PBS, p<0.0001). This demonstrated anti-tumor
efficacy against HER2+ human ovarian tumors.
[0471] The data provided herein demonstrates that
anti-HER2-huEndo-P125A and anti-EGFR-huEndo-P125A inhibited both
normal endothelial angiogenesis and also of tumor cell vasculogenic
mimicry. The dual inhibition of angiogenesis and vasculogenic
mimicry is expected to result in improved anti tumor efficacy.
[0472] The data presented in the present application and herein
shows that the difficulties experienced in treatment of cancer can
surprisingly be overcome by the presently claimed compositions and
chimeric fusion molecules. These results are unexpected.
OTHER EMBODIMENTS
[0473] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention. Other aspects, advantages, and
modifications are within the scope of the following claims.
[0474] All references cited herein, are incorporated by reference
in their entirety.
Sequence CWU 1
1
7137DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 1cccctcgcga
tatcacagcc accgcgactt ccagccg 37237DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 2ccccgaattc gttaaccctt ggaggcagtc atgaagc
37318DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 3atggcagaag ggcagcat
18424DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 4ttggtgaggt
ttgatccgca tcat 24521DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
5ccatgaactt tctgctgtct t 21621DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 6tcgatcgttc tgtatcagtc t 21721DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 7ggctcggacg ccaacgggcg c 21
* * * * *
References