Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TREATMENT OF TUMORS WITH OLIGONUCLEOTIDES DIRECTED TO
INSULIN-LIKE GROWTH FACTOR-I RECEPTOR
FIELD OF THE INVENTION
The present application is directed to inducing resistance to or regression of
tumor growth in humans using implanted diffusion chambers containing tumor
cells treated
with oligonucleotides directed to IGF-IR.
BACKGROUND OF THE INVENTION
Traditional methods of treating tumors in mammals include procedures such
as, for example, surgical removal of the tumor, injection or implantation of
toxic treatments
or syngeneic tissue samples, chemotherapy, and irradiation: The ultimate goal
of each of
these procedures is to reduce the growth of existing tumors, preferably
abrogating tumor
growth to the point of complete regression, and/or to induce resistance to
future tumor growth.
These procedures have numerous effects on tumor cells, as well as on non-tumor
cells.
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Tumors and other neoplastic tissues are known to undergo apoptosis
spontaneously or in response to treatment. Examples include several types of
leukemia, non-
Hodgkin's lymphoma, prostate tumor, pancreatic cancer, basal and squamous cell
carcinoma,
mammary tumor, breast cancer, and fat pad sarcoma. Several anti-cancer drugs
have been
S shown to induce apoptosis in target cells. Buttyam, et al., Mol. Cell.
Biol., 1989, 9, 3473-
3481; Kaufinann, CancerRes.,1989, 49, 5870-5878; and Barry, etal.,Biochem.
Pharmacol.,
1990, 40, 2353-2362, all of which are incorporated herein by reference in
their entirety.
Certain mildly adverse conditions can result in the injured cell dying by
programmed cell
death, including hyperthermia, hypothermia, ischemia, and exposure to
irradiation, toxins, and
chemicals. It should be noted that many of these treatments will also result
in necrosis at
higher doses, suggesting that mild injury to a cell might induce cell suicide,
perhaps to prevent
the inheritance of a mutation, while exposure to severe conditions leads
directly to cell death
by necrosis. However, the death process is difficult to observe due to the
rapidity of the
process and the reduced amount of inflammation. For these reasons,
quantification of
I S apoptosis is often difficult. A method of measuring the duration of the
histologically visible
stages of apoptosis (3 hours in normal rat liver) and a formula by which to
calculate the cell
loss rate by apoptosis is set forth by Bursch, et al., Carcinogenesis,
1990,11, 847-853.
Evidence is also rapidly accumulating that growth factors and their receptors
play a crucial role in the establishment and maintenance of transformed
phenotypes. It is well
established that growth factors play a crucial role in the establishment and
maintenance of the
transformed phenotype. Mouse embryo cells with a targeted disruption of IGF-IR
genes
cannot be transformed by SV40 T antigen and/or an activated Ha-ras oncogene
that easily
transform embryo cells generated from their wild-type littermates. Sell, et
al., Proc. Natl.
Acad. Sci. USA, 1993, 90, 11217-11221; Sell, et al., Mol. Cell. Biol.,
1994,14, 3604-3612;
Valentinis, et al., Oncogene,1994, 9, 825-831; and Coppola, et al., Mol. Cell.
Biol.,1994, l4"
4588-4595. Expression of an antisense RNA to the IGF-IR RNA in C6 rat
glioblastoma cells
not only abrogates tumorigenesis in syngeneic rats, but also causes complete
regression of
established wild type tumors. Resnicoff, et al., Cancer Res.; 1994x, 54, 2218-
2222 and
Resnicoff, et al., CancerRes.,1994b, 54, 4848-4850. Related to this finding is
also the report
by Harrington, et al. (EMBO J., 1994,13, 3286-3295), that IGF-I (and PDGF)
protect cells
from c-myc induced apoptosis. A decrease in cell death rate in tumors could
certainly be an
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important mechanism for tumor growth. Baserga, T he Biology of Cell
Reproduction, Harvard
University Press, Cambridge, MA,1985. Cells expressing an antisense RNA to the
IGF-IR
RNA or cells pre-incubated with antisense oligonucleotides to the IGF-IR RNA
completely
lose their tumorigenicity when injected in either syngeneic or nude mice.
Resnicoff et al.,
i 994a,1994b. The inj ected cells were suspected of undergoing apoptosis or,
at any rate, some
form of cell death. Dying cells, however, are very difficult to demonstrate,
because dying
cells, especially in vivo, disappear very rapidly, and one is left with
nothing to examine.
The importance of the IGF-IR in the control of cell proliferation is also
supported by considerable evidence: 1) many cell types in culture are
stimulated to grow by
IGF-I (Goldring, et al., Crit. Rev. Eukaryot. Gene E,~pr.,1991, l; 301-326 and
Baserga, et al.,
Crit. Rev Eukaryot. Gene Expr., 1993, 3, 47-61), and these cell types include
human diploid
fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, myeloid
cells, chondrocytes,
osteoblasts as well as the stem cells of the bone marrow; 2) interference with
the function of
the IGF-IR leads to inhibition of cell growth -- which has been demonstrated
using antisense
expression vectors or antisense oligonucleotides to the IGF-IR RNA; the
antisense strategy
was successful in inhibiting cellular proliferation in several normal cell
types and in human
tumor cell lines (Baserga, et al., 1994, supra.); and 3) growth can also be
inhibited using
peptide analogues of IGF-I (Pietrzkowski, et al., Cell Growth & Due, 1992x,
3,199-205 and
Pietrzkowski, et al., Mol. Cell. Biol., 1992b, 12, 3883-3889), or a vector
expressing an
antisense RNA to the IGF-I RNA (Trojan, et al., 1993, supra.). The IGF
autocrine or
paracrine loop is also involved in the growth pramoting effect of other growth
factors,
hormones (for instance, growth hormone and estrogens), and oncogenes like SV40
T antigen
and c-myb, and in tumor suppression, as in the case of WT1. Baserga, et al.,
1994, supra.
Inducing resistance to tumor growth is also disclosed in, for example, U.S.
Patent No.
5,714,170, which is incorporated herein by reference in its entirety. A review
of the role of
IGF-IR in tumors is provided in Baserga et al., Vitamins and Hormones, 1997,
53, 65-98,
which is incorporated herein by reference in its entirety.
Testing agents such as, for example, growth factors and growth factor
receptors
for their ability to maintain or suppress transformed phenotypes remains
difficult. In order
to obtain an accurate account of the tumor suppressive ability, testing should
be performed
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in vivo. The present invention provides a method of inducing resistance to or
regression of
tumor growth with markedly reduced side effects to the patient.
SUMMARY OF THE INVENTION
The present invention is directed to a method of inducing resistance to tumor
growth in a human comprising contacting a tumor cell in vitYO or ex vivo with
an
oligonucleotide complementary to IGF-IR, and implanting a diffusion chamber
containing the
treated tumor cell into the rectos sheath of the human fox a therapeutically
effective time,
thereby inducing resistance to tumor growth.
The present invention is also directed to a method of inducing regression of a
tumor in a human comprising contacting a tumor cell in vitro or ex vivo with
an
oligonucleotide complementary to IGF-IR, and implanting a diffusion chamber
containing the
treated tumor cell into the rectos sheath of the human for a therapeutically
effective time,
thereby inducing regression of the tumor.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a table summarizing the patients enrolled in the clinical
trial.
Figure 2 is a Western blot irnmunostained with apolyclonal antibody anti-IGF-
IR j3 subunit (upper arrow), then re-stained with a monoclonal antibody to the
focal adhesion
kinase (lower arrow). Lanes 1: T98G human glioblastoma cells; 2: Patient 1
before treatment;
3: Patient 1 + LR 4437-002A (IGF-IR antisense oligonucleotide) at I mg; 4:
Patient 1 + LR
4437-002A at 2 mg; 5: C6 rat glioblastoma cells; 6: T98G human glioblastoma
cells; 7:
Patient 8 before treatment; 8: Patient 8 + LR 4437-002A at 2 mg; 9: T98G human
glioblastoma cells;10: Patient 9 before treatment; l 1: Patient 9 + LR 4437-
002A at 2 mg;12:
Patient 10 before treatment; 13: Patient 10 + LR 4437-002A at 2 mg; and 14:
T98G human
glioblastoma cells.
Figures 3A and 3B show a table showing post-treatment analysis ofthe patients
enrolled in the clinical trial.
Figure 4 shows MRI scans of test case {Patient l, a-c} and case control (d-fj
of patients originally diagnosed with malignant glioma originating in the
dominant left
temporal lobe undergoing lobectomy and radiation therapy with failure and
progression into
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the deep gray matter and ipsilateral frontal lobe. At failure, pathology
revealed viable tumor
without evidence of radiation necrosis in both case (time 0). The test case
received image-
guided re-resection and IGF-IR antisense treatment and the case control
received image-
guided re-resection and GLIADEL~ implantation. Numbers in white ovals
represent time in
weeks after treatment.
Figure 5 shows MRI scans of test case (Patient 7, a-c) and case control (d-f)
of patients originally diagnosed with malignant glioma originating in the
dominant left
posterior temporal-parietal region, both with mild receptive aphasiac,
undergoing resection
and radiation therapy with failure and progression into the ipsilateral deep
gray matter and
frontal lobe. At failure, pathology revealed viable tumor without evidence of
radiation
necrosis in both case (time 0). The test case received image-guided re-
resection and IGF-IR
antisense treatment and the case control received image-guided re-resection
and GLIADEL~
implantation. Numbers in white ovals represent time in weeks after treatment.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
The present invention is directed, in part, to methods of inducing resistance
to
tumor growth, or inducing regression of a tumor, in a human comprising
treating a tumor cell
in vitro or ex vivo with a pro-apoptotic agent, placing the treated tumor
cells in a diffusion
chamber thereby producing a tumor cell-containing diffusion chamber, and
inserting or
implanting the tumor cell-containing diffusion chamber into the rectus sheath
of the human
for a therapeutically effective time thereby inducing resistance to tumor
growth or inducing
regression of the tumor.
An important advantage of the present invention is that toxic treatments to
the
tumor cells such as, for example, treatment with irradiation or
chemotherapeutic compounds,
are performed in vitro or ex vivo thereby eliminating toxicity to the patient.
In addition, tumor
cells can be placed into culture in a diffusion chamber and the chamber
directly implanted into
the patient, thus eliminating the possibility of physical spreading of the
tumor cells which can
be associated with direct injection of tumor cells into a patient.
Human tumors which are treatable with the methods of the present invention
can be primary or secondary, benign, malignant, metastatic, or micrometastatic
tumors in a
human patient. A human patient is an individual who is diagnosed with cancer,
an individual
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who has been diagnosed as having cancer and who is now cancer-free, or an
individual who
is suspected of having cancer. Tumors treatable with the methods of the
present invention
include, but are not limited to, melanoma, prostate, ovary; mammary,
pancreatic, lungs, colon,
and smooth muscle tumors, as well as cells from glioblastoma, bone marrow stem
cells,
hematopoietic cells, osteoblasts, epithelial cells, fibroblasts, as well as
any other tumor cells
which undergo apoptosis and induce resistance to or regression of tumor cells.
As used herein, the terms "tumor cell(s)," "tumor{s);" and "cancer cell(s),"
as
applied to cells which are within a diffusion chamber, are used
interchangeably throughout
the present application and include, but are not limited to, autografts,
allografts, syngeneic,
non-syngeneic and xenografts as well as cells derived therefrom. Tumor cells
include any
type of cell which upon apoptosis induces resistance to tumor growth or
induces regression
of a tumor including, but not limited to, naturally-occurring tumor cells or
tumor cell lines.
Tumor cells include, but are not limited to, melanoma, prostate, ovary,
mammary, pancreatic,
lungs, colon, and smooth muscle tumors, as well as cells from glioblastoma,
bone marrow
stem cells, hematopoietic cells, osteobIasts, epithelial cells, fibroblasts,
as well as any other
tumor cells which undergo apoptosis and induce resistance to or regression of
tumor cells.
Tumor cell lines include, but are not limited to, C6 rat glioblastoma cell
line, FO-1 human
melanoma cell line, BA 1112 rat rhabdomyosarcoma cell line, B 1792-F 10 mouse
melanoma,
B 16 mouse melanoma, and CaOV-3 human ovarian carcinoma. Tumorflus tissue,
tumors, or
tumor cells can also be excised from the human patient in which the diffusion
chamber will
be inserted or from another source which has been cultured in vitro.
Tumor cells used in the methods of the present invention are cultured in vitro
or ex vivo in a medium supplemented with a pro-apoptotic agent and
subsequently transferred
to a diffusion chamber. Alternatively, the tumor cells can be initially
cultured in a diffusion
chamber and treated with the pro-apoptotic agent therein. Preferably; the
diffusion chamber
contains tumor cells which are derived from the same type of tumor to which
resistance or
regression is induced. Alternatively, the tumor cells are placed in a
diffusion chamber are of
a different type than the tumor to which resistance or regression is induced.
The tumor cells are cultured in ultra or ex vivo and are supplemented with a
pro-apoptotic agent for a period of time, preferably 3 to 48 hours, mare
preferably 24 hours.
Prior to culture in vitro or ex vivo, the tumor cells can be gently
dissociated with trypsin and
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subsequently washed prior to implanting in a human. Pro-apoptotic agents which
supplement
the culture medium of the tumor cells in vitro or ex vivo are preferably
agents which induce
cell death in vivo. A pro-apoptotic agent, for purposes of the present
invention, is an agent
which causes death of the tumor cells in the diffusion chamber in vivo such
that the cell death
S has a tumor growth inhibiting effect or tumor regression effect, i.e., a
resistant effect or
regression effect, on a tumor ar tumors or tumor cells in the human in which
the diffusion
chamber is implanted. Such pro-apoptotic agents include, but are not limited
to, nucleic acid
molecules, proteins or peptides; non-protein or non-polynucleotide compounds;
and physical
conditions.
i 0 The pro-apoptatic agents used in the methods of the present invention
induce
cell death, or apoptosis, of the tumor cells in the diffusion chamber in vivo
or ex vivo.
Apoptosis, for purposes of the present invention, is defined as cell death and
includes, but is
not limited to, regression of primary and metastatic tumors. Apoptosis is a
programmed cell
death which is a widespread phenomenon that plays a crucial role in the myriad
of
15 physiological and pathological processes. Necroses, in contrast, is an
accidental cell death
which is the cell's response to a variety of harmful conditions and toxic
substances.
Apoptosis, morphologically distinct from necrosis, is a spontaneous farm of
cell death that
occurs in many different tissues under various conditions. This type of cell
death typically
occurs in scattered cells and progresses so rapidly it is difficult to
observe.
20 Pro-apoptotic agents, or apoptosis-inducing agents, which induce apoptosis
of
tumor cells in vivo include, for example, nucleic acid molecules. In one
embodiment of the
invention, the nucleic acid molecule is an oligonucleotide directed against
DNA or RNA of
a growth factor or growth factor receptor, such as, far example, IGF-IR. Most
preferably, the
oligonucleotide is directed against DNA or RNA of IGF-IR. The oligonucleotide
can be
25 directed to any portion of IGF-IR DNA or RNA. Preferably, the nucleotide
sequence of the
oligonucleotide includes, but is not limited to, nucleotide sequences
complementary to codons
1-309 of IGF-IR (SEQ ID NO:1), comprising either RNA or DNA. The antisense
oligonucleotides can also comprise nucleotide sequences complementary to
portions of
codons 1-309. In addition, mismatches within the nucleotide sequence of the
oligonucleotide
30 complementary to codons 1 to 309 are also within the scope of the
invention. An
oligonucleotide complementary to nucleotides -29 to -24 of the IGF-IR signal
sequence (SEQ
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ID N0:2) comprising DNA or RNA is also within the scope of the present
invention. The
signal sequence of IGF-IR is a 30 amino acid sequence. Contemplated by this
definition are
oligonucleotides complementary to the 30 amino acid signal sequence:
Alternatively,
fragments of oligonucleotides within SEQ ID NO:2 are also contemplated.
Additional
S oligonucleotides of the invention include, but are not limited to,
oligonucleotides comprising
the following nucleotide sequences: GGACCCTCCTCCGGAGCC (SEQ ID N0:3),
CCGGAGCCAGACTTCAT (SEQ ID N0:4), C TGCTCCTCCTCTAGGATGA (SEQ ID
NO:S), CCCTCCTCCGGAGCC (SEQ iD N0:6), TACTTCAGACCGAGGCC (SEQ ID
N0:7), CCGAGGCCTCCTCCCAGG (SEQ ID N0:8), and TCCTCCGGAGCCAGACTT
(SEQ ID N0:9). The oligonucleotides of the invention can comprise from about
10 to about
50 nucleotides, more preferably from about 14 to about 25 nucleotides, and
more preferably
from about 17 to about 20 nucleotides.
In another preferred embodiment of the invention, the nucleic acid molecule
is a vector which produces an oligonucleotide directed against DNA or RNA of a
growth
factor or growth factor receptor such as, for example, SEQ ID Numbers 1-9. The
nucleic acid
molecule complementary to a portion of IGF-IR RNA or DNA is inserted into an
appropriate
delivery vehicle, such as, for example, an expression plasmid, cosmid, YAC
vector, and the
like. Almost any delivery vehicle can be used for introducing nucleic acids
into tumor cells.
Recombinant nucleic acid molecules (or recombinant vectors) include, for
example, plasmid
DNA vectors, cDNA-containing liposomes, artificial viruses, nanoparticles, and
the like. It
is also contemplated that vectors expressing the oiigonucleotides can be
injected directly into
the tumor cells.
The regulatory elements of the recombinant nucleic acid molecules of the
invention are capable of directing expression in mammalian tumor cells,
preferably human
tumor cells. The regulatory elements include a promoter and a polyadenylation
signal. In
addition, other elements, such as a Kozak region, may also be included in the
recombinant
nucleic acid molecule. Examples of polyadenylation signals useful to practice
the present
invention include, but are not limited to, SV40 polyadenylation signals and
LTR
polyadenylation signals. In particular, the SV40 polyadenylation signal which
is in pCEP4
plasmid (Invitrogen, San Diego, CA),.referred to as the SV40 polyadenylation
signal, can be
used.
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The promoters useful in constructing the recombinant nucleic acid molecules
of the invention may be constitutive or inducible. A constitutive promoter is
expressed under
all conditions of cell growth. Exemplary constitutive promoters include the
promoters for the
following genes: hypvxanthine phosphvribosyl transferase (HPRT), adenosine
deaminase,
S pyruvate kinase, (3-actin, human myosin, human hemoglobin, human muscle
creatine, and
others. In addition, many viral promoters fiznction constitutively in
eukaryotic cells, and
include, but are not limited to, the early and late promoters of SV40, the
Mouse Mammary
Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Malvney
leukemia
virus, Human Immunodeficiency Virus (HIV), Cytomegalovirus (CMV) immediate
early
promoter, Epstein Barr Virus (EBV); Rous Sarcoma Virus (RSV), and other
retroviruses, and
the thymidine kinase promoter of herpes simplex virus. Other promoters are
known to those
of ordinary skill in the art.
Inducible promoters are expressed in the presence of an inducing agent. For
example, the metallothionein promoter is induced to promote (increase)
transcription in the
presence of certain metal ions, and the Drosophila HSP70 promoter. Other
inducible
promoters are known to those of ordinary skill in the art.
Recombinant nucleic acid molecules comprising oligonucleotides of the
invention can be introduced into a tumor cell or "conta.cted" by a tumor cell
by, for example,
transfection yr transduction procedures. Transfection refers to the
acquisition by a cell of new
genetic material by incorporation of added nucleic acid molecules.
Transfection can occur by
physical or chemical methods. Many transfection techniques are known tv those
of ordinary
skill in the art including: calcium phosphate DNA co-precipitation; DEAE-
dextran DNA
transfection; electroporation; naked plasmid adsorption, and cationic liposome-
mediated
transfection. Transduction refers to the process of transferring nucleic acid
into a cell using
a DNA or RNA virus. Suitable viral vectors for use as transducing agents
include, but axe not
limited to, retroviral vectors, adeno associated viral vectors, vaccinia
viruses, and Semliki
Forest virus vectors.
In a preferred embodiment of the invention, recombinant vectors comprising
oligonucleotides directed to DNA or RNA of IGF-TR, which are described, for
example, in
Resnicoff, et al. (1994a,1994b, supra), both of which are incorporated herein
by reference,
are used. Briefly, plasmid HSP/IGF-IR AS expresses an antisense transcript 309
hp in length
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directed to IGF-IR RNA, under the control of a Drosophila HSP70 promoter. The
hepatitis
B polyadenylation signal sequence and a neomycin-resistance gene under the
control of the
SV40 promoter are present at the 3' termini of the 309 by IGF-IR fragment. One
skilled in
the art can readily prepare additional vectors producing any of the
oligonucleotides of the
invention described herein.
In other embodiments of the invention, the pro-apoptotic agents comprise
proteins or peptides such as, for example, associated dominant negative
mutants of IGF-IR
and MHC class I peptides. Dominant negative mutants of IGF-IR include, for
example,
soluble IGF-IR, described in D'Ambrosio, et al., Cancer Res., 1996, 56, 4013-
4020;
incorporated herein by reference, and myristylated C-terminus of IGF-IR
(MyCF). MHC
class I associated peptides include, for example, Tyr-Leu-Glu-Pro-Gly-Pro-Val-
Thr-Ala (SEQ
ID NO:10) recognized by melanoma-specific CTL lines (Cox, et al.,
Science,1994, 264, 716-
719), Leu-Leu-Asp-Gly-Thr-Ala-Thr-Leu-Arg-Leu (SEQ ID NO:11 ) derived from gp
100 and
involved in regression of human melanoma (Kawakami, et al., Proc. Natl. Aced.
Sci. USA,
1994, 91, 6458-6462), and Phe-Glu-Cys-Asn-Thr-Ala-Gln-Pro-Gly (SEQ ID N0:12)
derived
from connexin 37 and induces CTL responses against marine lung carcinoma
(Mandelbolm,
et al., Nature, 1994, 369, 67-71), and Tyr-Leu-Arg-Pro-Gly-Pro-Val-Thr-Ala
(SEQ ID
NO:15). In addition, inverted D-amino acid analogs of the above-identified
peptides, such as
Ala-Thr-Val-Pro-Gly-Pro-Glu-Leu-Tyr (SEQ ID NU:13) and Leu-Arg-Leu-Thr-Ala-Thr-
Gly-
Asp-Leu-Leu (SEQ ID NO:14), are also active. Amino acid substitutions are also
contemplated by the present invention. The peptides of the present invention
can be made
synthetically as is well known to those skilled in flee art.
In other embodiments of the invention, the pro-apoptotic agents comprise non-
protein or non-polynucleotide compounds such as, for example, chemotherapeutic
compounds
or synthetic chemical compounds. Preferably, chemotherapeutic compounds
include, for
example, etoposide, cisplatin, camptothecin, and tumor necrosis factor alpha
(TNF-a).
In other embodiments of the invention, the pro-apoptotic agents comprise
physical conditions such as, for example, hyperthermia, hypothermia,
ischernia, and ionizing
iiradiation. In embodiments where the tumor cells are exposed to such
conditions, the
condition is defined for purposes of the present invention as an agent, an
apoptosis-inducing
agent.
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Therapeutically effective doses of the pro-apoptotic agents or apoptotic-
inducing agents will be about that of the drugs alone; dosages will be set
with regard to
weight, and clinical condition of the patient. The proportional ratio of
active ingredient to
culture medium will naturally depend on the chemical nature, solubility, and
stability of the
compounds, as well as the dosage contemplated. The culture medium is also
pharmaceutically
acceptable. The apoptosis-inducing agents of the invention can be used alone
or in
combination with other apoptosis-inducing agents. Preferably, between about 10
p.g/10'cells
and 100 mg/10' cells of pro-apoptotic agent is used to treat the tumor cells
in vitro or ex vivo.
More preferably, between about 100 ~g/10'cells and 10 mg/10' cells of pro-
apoptotic agent
is used. More preferably, between about 1 rng/10'cells and 5 mg/10' cells of
pro-apoptotic
agent is used. Most preferably, about 2 mg/10' cells of pro-apoptotic agent is
used.
The present invention employs the use of a diffusion chamber, in which the
cells are contained. Cells are impermeable to a filter fitted on the diffusion
chamber; they
cannot leave or enter the chamber. The filter on the diffusion chamber has
pores in the size
I S range of about 0.25 ~.m or smaller, preferably about 0.1 ~m in diameter.
Lange, et al., J.
Immunol., 1994,153, 205-211 and Lanza, et al., Transplantation,1994, 57,1371-
1375, both
of which are incorporated herein by reference in their entirety. Accordingly,
cell death or
apoptosis, can be quantitatively determined. The use of a diffusion chamber
can be extended
to other cell lines, even non-syngeneic; and even from different species,
because of the
rapidity with which cell death occurs, about 24 hours, well before any immune
reaction could
be established.
Diffusion chambers useful in the present invention include any chamber which
does not allow passage of cells between the chamber and the patient in which
it is implanted,
however, permits interchange and passage of factors between the chamber and
the patient.
The chamber can allow for multiple and sequential sampling of the contents,
without
contamination and without harming the patient, therefore significantly
reducing the number
of implantation procedures performed on the patient. A preferred diffusion
chamber is
described in, for example, U.S. Patent No. 5,714,170. A representative
diffixsion chamber
comprises a chamber barrel having two ends, a first end and a second end. The
barrel may be
comprised of one or more rings secured together by non-toxic means. The
chamber is fitted
at each end with a f lter, a first filter and a second filter. The filters are
porous to factors such
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that the factors may pass between the chamber and the patient. The filter
pores size can be
about 0.25 pm or smaller, preferably about 0. i p,m. The filters can be made
of plastic, teflon,
polyester, or any inert material which is strong, flexible and able to
withstand chemical
treatments. The filters can be secured in position with rubber gaskets which
may also provide
a tighter seal. On the barrel portion of the chamber, an opening is provided
which can be
covered by a cap which is accessed from outside of the patient's body once the
chamber is
implanted, thus allowing the diffusion chamber to be refilled. The cap can be
a screw-on type
of self sealing rubber and fitted to the opening. Sampling of the chamber
contents can be
performed by accessing the opening by removing the cap on the outside of the
patient's body
and inserting an ordinary needle and syringe. The chamber can be made of any
substance,
such as and not limited to plastic, teflon, Lucite, titanium, or any inert
material which is non-
toxic to and well tolerated by humans. In addition, the chambers should be
able to survive
sterilization. Diffizsion chambers are preferably constructed from 14 mm
Lucite rings with
0.1 pm pore-sized hydrophilic Durapore membranes (Millipore, Bedford; MA). The
diffusion
chambers are preferably sterilized with ethylene oxide prior to use.
Tumor cells can be placed in a diffusion chamber in varying amounts.
Preferably, about 1 x 104 to about S x 106 cells can be placed in the
diffusion chamber. More
preferably, about 1 x 1 O5 to about I .5 x 1 O6 cells can be placed in the
diffusion chamber. More
preferably; about S x 105 to 1 x 106 cells are placed in the chamber. Most
preferably, about
1 x 106 cells are placed in the diffusion chamber. The cells are placed in the
diffusion
chamber containing media: It is contemplated that any media known to those
skilled in the
art that supports the growth of cancer cells and which is compatible with a
human can be used.
The diffusion chamber can be implanted in a human in the following non-
limiting ways: subcutaneously, intraperitoneally, intracranially; or into the
rectus sheath, for
example. Most preferably, the diffusion chambers) is implanted into an
acceptor site of the
body having good lymphatic drainage and/or vascular supply such as the rectus
sheath. The
chamber can be removed about 24 to about 30 hours after implantation, if
desired.
Alternatively, a refillable chamber can be employed such that the diffusion
chamber can be
re-used for treatments and emptied following treatments. In addition, a
plurality of diffusion
chambers, preferably between five and 20, can be used in a single patient.
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A clinically proven surgical procedure for carrying out the invention in
humans
involves implantation of diffusion chambers containing the tumor cells treated
with the pro-
apoptotic agent, such as IGF-IR antisense oligonucleotide, as a suspension. A
preferred
procedure involves several surgical and/or in vitro tissue processing
objectives: l) harvesting
viable tumor tissue, free of fibrinogen and necrosis, from a tumor bed for ex-
vivo tumor cell
treatment with the IGF-IR antisense oiigonucieotide; 2) avoiding excessive or
unacceptable
operative morbidity, specifically post-operative neuroIogic deficit related to-
the procedure;
3) using image-directed tissue selection to achieve these first two
objectives; 4) using image-
directed surgical resection to maximize the resection of tumor and to minimize
residual tumor
burden, thereby minimizing any inflammatory process related to either
autologous tumor
rejection or related to a direct and cytotoxic effect on tumor cells in the
post-implantation
period; 5) selecting and creating an autoiogous acceptor site for the
generation of the
biological response; 6) processing human tissue expiants in vitro in order to
generate the
desired biological response upon re-implantation of the autologous treated
tumor cells within
20 hours; 7) in lieu of the previously stated six surgical objectives,
processing autologous,
homologous, or xenographic cells from established cell lines in order to
generate the desired
biological response upon implantation of the tumor cells within about 20
hours; and 8)
designing a specific implantation protocol for all of the above cell
preparations to generate the
resistance to or regression of tumor growth:
Successful clinical trials have applied the present invention to human brain
tumors known as glioma. Certain tumors, such as brainstem glioma, or deep;
and/or multiple
brain metastases, are surgically inaccessible and no tissue can be safely
harvested in these
patients. In such cases where tumors are surgically inaccessible, homologous
(human but not
host) cell lines, xenographic (non-human) established cell lines, or
homologous human
primary cell cultures can be utilized as a source to induce the resistance to
or regression of
tumor growth. To avoid or prevent the complication of deep venous thrombosis,
the
prophylactic use of low molecular weight heparin is recommended to carry out
the present
invention. The following examples are illustrative but are not meant to be
limiting of the
invention.
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EXAMPLES
Example 1: General Implantation Protocol
Diffusion chambers comprising either autologous implants ofharvested tumor
cells or established autologous, homologous, or xerographic cell lines treated
with antisense
IGF-IR oligonucleotides are implanted into a human in order to induce
resistance to or
regression of tumor growth. All such tumor cells have been treated with IGF-IR
antisense
oligonucleotides ex vivo. Use of IGF-IR antisense is made to target the IGF-IR
in host tumor
cell explants in vitro and to commit the tumor cells to undergo apoptosis in
vivo. The targeted
pre-treated cells are committed to undergo apoptosis in vivo and subsequently
generate
biological response modifiers which induce resistance to or regression of
tumor growth.
A preferred embodiment comprises a combination method involving in vitro
followed by in viyo procedures. First, this method targets IGF-IR in tumor
cells in vitro.
Tumor cells with decreased levels of IGF-IR are encapsulated in the diffusion
chambers and
implanted in vivo in the patient (i.e. subcutaneously, within the rectus
sheath, intracranially,
or in any other host location considered acceptable for implantation). Cells
with decreased
levels of IGF-IR undergo apoptosis in the diffusion chambers and release
diffusible biological
response modifiers which are believed to cause the elimination of human
malignant brain
tumors in the host patient.
One of two surgical protocols is preferably utilized to carry out the present
invention with respect to primary and rnetastatic brain tumors. In a preferred
protocol, a
craniotomy is employed for tumor resection and autologous cell implantation.
Another
protocol utilizes no craniotomy but requires the implantation of autologous
cell lines,
homologous cell lines, or xerographic cell lines in patients.
First, autologous tissue is harvested utilizing a surgical method for glioma.
Viable tumor tissue is harvested from the tumor bed (target between 0.5 to 2
grams; a
minimum of 500 mg is usually adequate) for ex vivo tumor cell treatment with
IGF-IR
antisense DNA. It is an objective ofthis protocol to avoid excessive or
unacceptable operative
morbidity, specifically post-operative neurologic deficit related to the
procedure. Image-
directed tissue selection is utilized to achieve viable tumor tissue harvest
and to avoid
excessive or unacceptable operative morbidity. Surgical resection is also
image-directed to
maximize tumor resection, to minimize residual tumor burden, and to minimize
subsequent
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inflammatory effects related to autologous tumor rejection in the post-
transplantation period.
An autologous acceptor site is selected, such that the acceptor site is
appropriate for
implantation of the diffusion chamber and fox the diffusion of the biological
modifier. Human
tissue explants are processed in vitro to generate the desired biological
response upon re-
implantation of the autologous treated tumor cells. The re-implantation
protocol is designed
to generate the biological response modifier trigger in humans.
The patient is first prepared for craniotomy with placement of fiducial
coordinates on the scalp. These fiducials will serve as surface registration
points for MRI-
based infra-operative image guidance. The patient is then taken to the MRI
scanner where
i 0 gadolinium is infused, and image sets in all three orthogonal planes are
obtained. The
obtained image sets are suitable for the infra-operative image guidance
computer software to
be utilized in the operating room. Use of gadolinium or similar contrast
agents serves to
create a tissue plane marker where viable acceptor tumor tissue cells are
located. The image-
directed localization of a viable tumor plane is key to a successful cell
harvest for processing.
Once the imaging phase is complete, the patient is taken to the operating room
and prepared for craniotomy. Prophylactic antibiotics are infused
intravenously, as are agents
to minimize cerebral edema, including steroids and mannitol with lasix. The
head is
immobilized in three-point head pin fixation and the infrared LED reference
block is applied
to the head pin fixation device with attention to the orientation of the
reference block to the
infrared camera. The reference block is positioned for both an unobstructed
link to the camera
and for sufficient distance from the operative field for unobstructed
operative access. A
frameless viewing wand is then registered to the scalp fiducials until an
acceptable ( s 1.5 mm}
registration error is obtained. The fiducial markers are removed and 40 scalp
points are
registered to ensure accurate registration over the entire operative f eld.
The registration is
checked on the computer screen by moving the wand over the scalp in all three
orthogonal
planes.
Prior to craniotomy, a preferred site for implantation (e.g. the abdomen) is
preferably examined and prepared. . For example, a preferred mode would
include an incision
just superolateral to the umbilicus. Other sites, designated above, would also
be acceptable.
In this case, a four centimeter transverse incision is made with a #10 blade
after infiltration
with 1 % Lidocaine. The wound edge is retracted with a self retaining
retractor to minimize
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tissue margin disruption during pocket preparation. Sharp dissection with
Metzenbaum
scissors is performed, and the rectos sheath is exposed, with careful
attention to hemostasis
with electrocautery. Utilizing toothed forceps and Metzenbaum scissors, the
rectos sheath is
incised parallel to the skin incision, thereby exposing the rectos abdominus
muscle. A series
S of interconnected pockets are established utilizing blunt finger dissection
in the cephalad and
caudal extent of the wound between the rectos muscle and the sheath to allow
subsequent
implantation of up to 10 diffusion chambers, with each chamber measuring 1.$
crn in
diameter. Following sub-rectos sheath tunneling, the wound is copiously
irngated with
Bacitracin solution in normal saline, is closed in a single layer with a
running 3-0 Nylon
suture, and is appropriately dressed. Once the acceptor pocket has been
initially formed, the
craniotomy is performed using the wand to define an accurate scalp and bone
flap, if
necessary, and standard sterile technique is utilized to prepare the operative
field for surgery.
After infiltration with 1% Lidocaine, the scalp is incised with a #10 scalpel
blade and the scalp reflected with towel clips. The bone plate is removed, if
necessary, with
a combination of an acorn Midas Rex dissecting tool followed by the B-1
dissector with
footplate. Pre-existing bone plates are removed as appropriate. Prior to
opening dura, four
peripheral bone fiducials are made with the B-1 dissector to serve as
registration points for the
viewing wand during tumor resection. The dura is then opened, preferably in a
cruciate
fashion, utilizing a #IS scalpel blade and Gerald forceps, thereby exposing
the cortical
surface.
Tumor sample resection then proceeds utilizing infra-operative image
guidance. The computer monitor displays on a split screen each orthogonal
plane, as well as
a fourth view which features an axis at right anghs to the viewing wand.
Cortisectomy is
performed with bipolar electrocautery and a #9 or #I 1 Frazier sucker,
utilizing the wand at
each point to direct accurate sampling of the tumor bed. The viable tumor bed
margin is
featured on the monitor as an area of contrast enhancement. When viable tumor
tissue is
registered and judged clinically to be acceptable for oligonucleotide pre-
treatment (reducing
harvest of necrotic debris, inflammatory reaction, and fibrinogen), bayoneted
tumor forceps
are utilized to harvest tissue for frozen section histopathological
confirmation. When viable
neoplastic tissue is confirmed, preferably a minimum of 500 mg of tumor tissue
is harvested.
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Every effort is made to harvest viable tissue free of clot and necrotic debris
to improve the
success of subsequent effective disaggregation and viable cell plating:
After sample harvest; additional tumor debulking is performed until a judged
gross total or near-total resection is achieved utilizing image guidance. An
aggressive
resection is j udged to be important to minimize any post-implantation
inflammation secondary
to autoiogous tumor rejection.
When satisfied that an effective quantity of tissue has been obtained and an
appropriate resection performed, the resection cavity is inspected for any
residual bleeding.
Bleeding is contarolled f rst with thrombin-soaked cotton balls. Subsequently
bleeding is
controlled with bipolar electrocautery. When adequate hemostasis is achieved,
the resection
cavity is lined with thrombin-soaked surgicel. The dura is closed with 4-0
interrupted and
running sutures, and the bone plate is re-affixed with a titanium plating
system. The scalp is
closed in two layers: the galea with 2-0 vicryl interrupted buried sutures and
the scalp with
either 3-0 nylon or staples.
Preparation of Cells and Incubation of Cells with IGF IR Antisense DNA, Days
One and Two
Tumor tissue samples from the operating room are immediately sent for ex vivo
processing. Under sterile conditions; the viable tumor margin tissue is gently
disaggregated
first with a scalpel. Disaggregation is completed with collagenase and
protease treatment.
Single cell suspension is obtained after passage through a series of gradually
decreasing
internal diameter needles. Finally, the cells are washed and plated in culture
medium
supplemented with 10% serum (e.g. fetal calf or fetal bovine).
The cells are allowed to attach for four to six hours. The cells are carefully
washed and treated with up to 2 mg of IGF-IR antisense oligonucleotide in a
final volume of
20 ml of serum-free medium to avoid exposure to nucleases present in the
serum. The
oligonucleotide treatment ranges from a minimum of six hours to a maximum of
ten hours.
The oligonucleotide treatment dose has been established for 1x10' cells.
Proportionately more
oligonucleotide can be utilized fax more cells. For example, up to 3.6 mg of
IGF-IR is used
in 36 ml of serum-free medium for 18 million cells.
Following the overnight treatment, the cells are harvested and washed
carefully. The cells are re-suspended in phosphate-buffered saline (calcium
and magnesium-
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free), and they are placed in the diffusion chambers to a volume of 200
~,1/chamber and a
density of 1 X I06 cells/chamber. This volume/concentration relationship is
important to
avoid cell death due to hypoxia. In one embodiment, the chambers axe
irradiated with 5 Gy
just prior to implantation to comply with FDA regulations. Another preferred
embodiment
does not include irradiation. The chambers consist of small Lucite rings
measuring I.4 cm
in outer diameter and 0.8 cm in height, with a hydrophilic membrane at each
end. The
membranes have a 0.1 ~.m exclusion limit which impedes the exit or entry of
intact cells and
allows the diffusion of soluble factors.
Surgicallmplantation ofDiffusion Chambers, Days 2
On the second post-operative day, the abdominal acceptor site is prepared for
diffusion chamber implantation. The timing of this procedure relative to the
harvest of the
autologous tumor cells is non-obvious and critical. Tumor cells with a
targeted IGF-IR are
committed to undergo apoptosis under anchorage-independent conditions, such as
those in
vivo: To avoid the situation where the tumor cells treated with antisense IGF-
IR
oligonucleotides undergo apoptosis in the diffusion chambers before
implantation in the rectal
sheath of the patient, a time window of an hour (maximum) has been established
between
harvest of tumor cells and implantation of diffusion chambers. The time window
between cell
harvest and re-implantation of the autologous treated tumor cells, which
generate the
biological signal, preferably should not exceed one hour in order for the peak
of the triggering
process to occur in vivo. Preferably; the cell harvest and re-implantation of
pre-treated cells
is performed within as small a time window as possible.
At bedside, the patient is prepared for the procedure; the patient is sedated
with
10 mg of Versed, and 1 g ofAncef antibiotic is infusedfor wound infection
prophylaxis. The
previously prepared abdominal acceptor site is exposed using standard sterile
technique, and
0.5% Sensorcaine is injected into the abdominal incision. Utilizing Metzenbaum
scissors, the
acceptor site is opened, thereby exposing the rectus sheath which was incised
the previous
day. Excessive residual bleeding or presence of fibrinous exudate is to be
avoided as f brie
may interfere with signal generation and thereby reduce the effectiveness of
the procedure.
Care is taken once again to control any source ofhemorrhage, however small,
with a portable
heat cautery unit. Any residual fibrin material is removed with copious
irngation with a
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Bacitracin solution in normal saline. Up to 10 sterile diffusion chambers,
each with a volume
of 200 ~,1 of tumor cell suspension, are implanted in the previously described
rectos sheath
pockets. The biological dosimetry includes implantation of 1 x 106
cells/chamber in 10
chambers for a maximum of 1 x 10' cells. The diffusion chambers are implanted
so the broad
membrane side is flat against the rectos abdominus muscle in all cases. The
orientation of the
chambers is critical to biologically effective transmission of the triggering
signal. Selection
of this mode, which includes the rectos abdominus muscle as an acceptor site,
is important
because the lymphatic drainage of this area proceeds via the inguinal lymph
nodes to the peri-
aortic nodes, which, in turn, drain into the thoracic duct. This pathway of
drainage permits
diffusible substances derived from the transduced cells to encounter
peripheral lymphoid
tissue via regional lymph nodes, which are approximately three centimeters
from the
implantation site.
The rectos sheath is re-approximated with 2-0 vicryl interrupted sutures to
secure the diffusion chambers in the flat orientation. The acceptor site is
closed with a
running 3-0 nylon interlocking suture and is appropriately dressed.
In the first four patients treated with this protocol, a high rate of deep
venous
thrombosis occurred, possibly attributable to this treatment. Therefore, an
addition to the
protocol is the use of subcutaneously injected low-molecular weight heparin
(fractionated
heparin or LOVENOX~) at 40 mg a day. The LOVENOX~ is continued as a daily
subcutaneous injection for three months.
Surgical Removal of Diffusion Chambers, Day 3
At bedside, the patient is prepared for the procedure of surgical removal of
the
diffusion chambers. The patient is sedated with 10 mg of Versed; and 1 g of
Ancef antibiotic
is infused for wound infection prophylaxis. The previously prepared abdominal
acceptor site
is exposed and prepared with standard sterile procedure. The site is draped,
and 0.5%
Sensorcaine is injected into the abdominal incision. Utilizing Metzenbaum
scissors, the
wound is opened, thereby exposing the rectos sheath which was loosely
approximated at
bedside the previous day. -The~sterile diffusion chambers are retrieved from
the rectos sheath
pockets and taken back to the laboratory where they are carefully examined.
Specifically, the
integrity of the membrane is inspected, and the contents of each chamber are
noted for the
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presence of fibrinogen. The volume recovered from each chamber is determined,
and this
volume should be same as the original recorded volume. The cellular contents
of each
chamber are carefully examined, and the rate ofviable cell recovery is
quantitated. The rectos
sheath is reapproximated with 2-0 vicryl interrupted sutures, and the
superficial fascia is
reapproximated with 2-0 vicryl interrupted horizontal mattress suttees. The
skin is
reapproximated with an interrupted vertical mattress suture utilizing 3-0
Nylon, and the
wound is then appropriately dressed.
Example 2: Implantation Of Autologous CeII Lines, Homologous CeII Lines, Or
Xerographic Celi Lines
Surgical candidates for this protocol are not candidates for craniotomy for
tumor removal. An example of patients in this group would include patients
with brainstem
gliomas - tumors often diagnosed radiographically without biopsy due to the
dangerous
location of these tumors. The surgical paradigm utilized is the same as that
described for days
two and three.
Day One
On day one, an appropriate cell line is selected and pre-treated according to
the
same specifications for the freshly harvested autologous samples. The in vitro
incubation
period and dose of oligonucleotide will also remain the same as for the
harvested autologous
samples described in detail above.
Post-Implantation Surveillance for Both Groups
After removal of the chambers, surveillance of the patient begins. Routine
vital signs and serial neurologic examinations proceed as per routine with
standard post-
craniotomy patients. MRI scans with and without gadolinium are performed at
the first and
second post-operative weeks and at each month thereafter.
Additional details of the method
In a preferred embodiment of the invention, twelve preferred steps are
performed. Image guided craniotomy (1 ) is performed to resect tumor. The
tumor is removed
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and placed in phosphate-buffered saline (2). Subsequently, tumor tissue is
transferred to a
petri dish, where the tumor is comminuted into fine pieces with scalpels (3).
Disaggregation
is continued by enzymatic digestion and single cells are plated in a T-25
tissue culture flask;
after 4-6 hours, once the cells have attached, they are incubated with 2 mg of
antisense DNA
to the IGF-IR in a final volume of 20m1 of serum-free medium for a minimum of
six hours
(4). Then, cells are detached and transferred to a 15m1 conical tube for
several washes (5).
Next, the cells are encapsulated in diffusion chambers (6). Before
implantation, the cells
encapsulated in the diffusion chambers are irradiated with 5 Gy of radiation
(7). Cells
encapsulated in diffusion chambers are surgically implanted into the rectos
sheath of a patient
{8), and apoptosis occurs in vivo within 20 hours. Then the chambers are
removed from the
patient's rectos sheath, and the wound is permanently closed (9). The chambers
are opened,
and the cells are recovered (9). Subsequently, the cells are transferred to an
Eppendorf tube
{10). Next, cells are washed (11), and the cells recovered from the diffusion
chambers are
analyzed for viability by trypan blue exclusion and they are quantitated in a
hemocytometer
(12).
The details of Day 1 of the procedure are as follows. Tumor tissue from the
operating room is transported in a 50 ec conical tube to a BL-2 facility. In
the BL-2 facility,
the tumor is washed to eliminate red blood cells, and it is transferred to a
petri dish and finely
minced with scalpels in PBS (I). Cells are transferred to a 1 Sml tube and
washed in PBS (2).
Then, cells are transferred to a petri dish and dissociated with repeated
cycles of enzymatic
digestion (3). Next, cells are transferred to a 15m1 conical tube and washed
(4). The cells are
plated in a T-25 tissue culture flask and allowed to attach (5).
Attachment is required before IGF-IR antisense DNA is added to the
incubation medium of 2 mg of IGF-IR antisense DNA in a final volume of 20m1 of
serum-free
medium for a minimum of six hours. On Day 2, cells are detached with trypsin;
and the cells
are transferred to a 15m1 conical tube and washed carefully (1 ). The effect
of trypsin is
stopped by adding medium supplemented with 10% serum; 106 cells suspended in
200~t1 are
loaded in each diffusion chamber, and the diffusion chambers are then
irradiated with 5 Gy
of radiation (2).
On Day 3, diffusion chambers are harvested from the patient's abdomen. Each
diffusion chamber is opened and cell contents are removed with an Eppendorf
pipette (1).
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Cells are then transferred to a lml Eppendorf tube and washed (2). The cells
are counted in
a hemocytometer (3}. Cells recovered from the diffusion chambers are re-plated
into a T-25
tissue culture flask to assess viability (4).
Observations in patients participating in a Phase 1 study
The observations in 12 patients participating in a Phase I study are
presented.
As described above, for each patient craniotomy was performed and an abdominal
acceptor
site was prepared for chamber implantation. Tumors resected at surgery were
disaggregated
in a BL-2 facility and attached cells in culture were incubated overnight with
an IGF-1R
antisense oligonucleotide. Within 24 hours of surgery, up to 10' treated cells
were
encapsulated in diffusion chambers, non-lethally irradiated, and implanted for
an additional
period of 24 hours. After chamber retrieval, patients were followed with
serial MRI and
clinical examinations.
Following antisense treatment, IGF-1R levels in the treated cells dropped to
< 10% as determined by Western blotting. Following 24 hour implantation, less
than 2% of
the antisense treated cells could be recovered. At follow-up, 6 out of 10
evaluable patients
revealed partial or complete radiographic and clinical responses. When
compassionate
re-treatment responses were included, 8 treatment responses were observed.
Other than deep
venous thrombosis in the first four patients, no other treatment-related side-
effects were noted.
This treatment was well-tolerated and yielded a high rate of radiographic and
clinical responses. Additionally, histologic observations in post-treatment
tumor samples
supported treatment-related effects. These data indicate that a non-toxic
systemic treatment
effect is achieved utilizing this paradigm.
Patients and Methods
This protocol was reviewed and approved by the FDA and the Institutional
Review Board at Thomas Jefferson University. Patients with a diagnosis of WHO
Grade III
or IV glioma who had failed conventional therapies including radiation therapy
were enrolled.
A total of twelve adult patients, 9 males and 3 females (ages 32 to 68 years,
Figure 1 ) were
enrolled. Four patients had anaplastic astrocytomas and 8 had glioblastoma,
and one patient
in each group had multifocal disease. Mean time interval between initial
diagnosis and this
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treatment intervention was 122 weeks. Tumor recurrence was associated with
clinical
symptoms in 9 patients and was documented by surveillance MRI in all 12
patients.
All patients underwent MRI-based image-guided tumor resections as
previously described. Where necessary, intraoperative speech and/ormotor
corticographywas
performed to preserve function of eloquent cortex. Prior to craniotomy, an
abdominal
acceptor site for chamber implantation was prepared, dividing the rectus
sheath and
establishing interconnected pockets between the rectus muscle and the sheath.
During image-guided resection, viable tumor tissue was confirmed by frozen
section, and sent to a BL-2 facility for subsequent disaggregatiori and
treatment. Tumor
samples were gently disaggregated first with a scalpel and then by enzymatic
digestion under
sterile conditions. Cells were plated in compete culture medium (DMEM
supplemented with
10% fetal bovine serum, penicillin, streptomycin, and glutamine). When the
cells attached,
they were carefully washed and shifted to serum-free medium {DMEM supplemented
with
1 ~M ferrous sulfate and 0. 1% BSA fraction ~l, and treated with an IGF-IR
antisense
oligonucleotide. An 18-mer phosphorothioate oligodeoxynucleotide
(GGACCCTCCTCCGGAGCC; SEQ ID N0:3) targeting the IGF-1R RNA and starting 6
nucleotides downstream from the initiating rnethionine was used. This
antisense
oligodeoxynucleotide was synthesized by Lynx 'Therapeutics (Hayward,
California), Lot
#LR4437-002A, the same Lot as previously described. After a minimum six hour
incubation
with a dose of 2 mg antisense/10' cells, the cells were harvested, carefully
washed with PBS,
calcium and magnesium-free, and placed in the diffusion chambers at a density
of 106
cells/200 pl/ chamber. The chambers are small Lucite rings (1.4 cm in diameter
with a 0.1m
pore-sized hydrophilic membrane at each end. They allow the passage of soluble
factors (such
as nutrients or peptides) impeding the exit or entry of intact cells.
On the first post-craniotomy day, the abdominal acceptor site was opened at
bedside for diffusion chamber implantation. A time window was selected so that
tumor cells
treated ex vivo with IGF-1R antisense oligodeoxynucleotide were detached,
encapsulated in
the chambers and non-lethally irradiated (S Gy) at the same time the acceptor
site was opened.
After local anesthesia and antibiotic prophyiaxis, the rectus sheath pockets
were exposed and
up to 10 sterile diffusion chambers were implanted.
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On the second post-craniotomy day, the diffusion chambers were retrieved
from the rectus sheath pockets and the wound closed: The chambers were
transported to a
BL-2 facility for analysis after recovery. Post-implantation surveillance
included serial clinical
and MRI examination.
Compassionate Re-Treatment
Because of the observed anti-tumor effects, three patients who failed initial
treatment with IGF-IR antisense were re-treated with a higher biological dose.
With FDA
approval, the allowable number of chambers was increased to 20 at 1 O6
cells/chamber and
maintained the dose of antisense at 2 mg/10~ cells during ex vivo incubation.
E~cacy, Specificity, and Biosafety of the Treatment Paradigm: Ex vivo
Assessments prior to
Chamber Implantation
To control for the biological activity of the antisense oligodeoxynucleotide,
both efficacy and specificity of IGF-1 R targeting were assessed. The efficacy
of the antisense
oligodeoxynucleatide on IGF-1R targeting was determined by measuring the
effects on
IGF-1 R expression at the protein level by Western blotting (see Figure 2,
upper panel). When
excess tissues were available, samples were analyzed (N=6) and revealed the
presence of
IGF-1R with a z90% reduction ofIGF-1R levels after IGF-1R antisense treatment
(Figure 2,
upperpanel). The specificity ofthe antisense oligodeoxynucleotide targeting
was determined
by ruling out effects on the expression of other tyrosine kinase receptors at
the cell surface,
such as the focal adhesion kinase (see Figure 2, lower panel).
The absence of adventitious agents in the autologous tumor cells prior to
implantation was confirmed by the use of commercially available kits,
including Gram (Fisher
Diagnostics) and mycoplasma staining kits {Sigma Chemicals).
Ex viva Assessments-after Chamber Retrieval
Integrity of the diffusion chambers was determined by measuring the volume
before and after implantation. The volume originally loaded in each chamber
was 200 ml and
the volume recovered from each of the 146 implanted chambers was 198 ~ 2 ml.
CeII
recovery, determined after 24 hour implantation of the diffusion chambers, was
< 2% of the
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original cell number implanted. Meticulous microscopic analysis of the cells
recovered from
the chambers showed that only tumor cells (and mastly non-viable as indicated
by trypan blue
staining) could be recovered. No other cell types including lymphocytes,
monocytes,
macrophages, or dendritic cells could be identified. Also, the presence ofGFAP-
positive cells
(a marker for glioma cells) in samples obtained from the abdominal acceptor
site was ruled
out.
Enoxaparin Prophylaxis
Based on a higher than expected rate of DVT in the first four patients, a
regimen of DVT prophylaxis was established in a1I subsequent patients. On the
first
postcraniotomy day, a three month regimen of enoxaparin prophylaxis was
initiated which
involved a daily 40 mg subcutaneous injection. Non-invasive doppler studies
were performed
immediately before and after the completion of a 12 week course of enoxaparin
treatment.
Response assessments
All patients underwent serial MRI evaluation using a standard brain imaging
protocol consisting of T i and T2 weighted pre-contrast images and TI weighted
postcontrast
images in the sagittal, axial, and coronal planes. MRI scans were obtained
within 4$ hours
to document residual tumor at treatment inception. Progression, regression or
stability of
disease was determined by changes in the assessment of seven image
characteristics between
serial studies including: {1) local mass effect; (2) size of T2-weighted
abnormality (e.g.
edema, tumor, radiation change}; (3) size of the enhancing area; (4}
characteristics of the
enhancement (e.g, progression or regression of nodularity); {5) intensity of
enhancing area;
(6) invasion of the deep white matter tracts; and (7) distal progression.
Radiographic
evaluation was performed by assessing changes in any of these characteristics
when
comparable images from serial studies were evaluated. Each imaging
characteristic was rated
as increased, decreased, or without change. In all cases, MRI comparisons were
performed
for each patient by one neuroradiolagist. The first comparison evaluated
changes between an
MRI study performed immediately following treatment and a follow-up
examination
performed approximately 4 weeks after initiation of therapy. Subsequent
comparisons
evaluated changes at two month intervals and later at three month intervals.
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Serial clinical examinations were independently performed by a neurosurgeon
and a neurologist. Performance status was assessed according to the Karnofsky
performance
scale {KPS).
Response was assessed after treatment utilizing the following criteria:
complete response: improvement in all assessable imaging characteristics
between serial studies to either complete resolution or to a stable image with
features
consistent with post-operative and/or radiation changes, with improvement or
stability of
neurological and general physical examinations, offcorticosteroid medication,
for at least one
month.
partial response: improvement in two or more of assessable imaging
characteristics, with improvement or stability of neurological and physical
examinations, and
a stable or decreasing corticosteroid dose, for at least one month.
stable disease: no significant change in the imaging characteristics
between serial studies with improvement or stability of neurological and
physical
examinations, and a stable or decreasing corticosteroid dose, for at least one
month.
progressive disease: An increase in the imaging characteristics between
serial studies with deterioration of neurological and physical examinations,
and with a
stable or increasing corticosteroid dose.
Results
Twelve patients with operable malignant gliomas were safely treated utilizing
tumor tissue harvested at craniotomy with a mean follow-up of 67 t 7 weeks
(range 40 to 100
weeks). With the exception of compassionate re-treatment in three cases, these
patients
received no other treatment while under study except appropriate supportive
medical and/or
surgical therapy. No treatment-related toxicity was identified, and clinical
examinations were
supported with normal examinations of complete blood counts, liver function
studies, CD4+
and CD8+ counts, ANA, anti-ssDNA and anti-dsDNA antibodies.
Biosafety
In all 12 cases, the cells were assessed prior to implantation and found to be
free of adventitious agents, and in the post-implantation period there was no
clinical evidence
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of wound infection (N=12). Where evaluable, no glioma tumor seeding was
identified at the
implantation sites (N=5). A total of one hundred fifty-six diffusion chambers
were implanted
(Figures 3A and 3B), and no membrane disruptions were noted when recovered.
Only
autologous tumor cells were recovered, and in aII cases <2% of recovered cells
were viable,
as assessed by trypan blue staining.
The only treatment-related complication seen thus far is the high incidence of
deep venous thrombosis (DVT) in the first four patients (Patients 1-4; see
Figure 3A).
Subsequently a prophylactic regimen utilizing fractionated low-molecular
weight heparin
(enoxaparin) was initiated in all protocol patients treated thereafter; no
DVTs in 8 of the latter
10 patients have occurred. Both patients retreated on a compassionate basis
developed DVTs,
one while on enoxaparin, the other following selfdiscontinuation of
enoxaparin.
Treatment Response: Clinical and Radiographic Observations
In addition to the safety, anti-tumor effects were observed in 4 patients
treated
once and in an additional 3 patients treated twice, all of whom either
improved or stabilized
clinically. In ail cases, radiographic improvements extended beyond the
resection cavity and
throughout the ipsilaterai and, where applicable, contralateral hemisphere,
ans all responses
occurred at least two months after a previous conventional treatment judged a
failure. An
additional two patients improved clinically, one with a sustained complete
radiographic and
clinical response. A case-control group treated with matched treatments in the
same time
interval was identified and no comparable anti-tumor effects were observed. In
the present
series, radiographic improvements occurred by 14 weeks with intervals ranging
from eight
weeks (Patient 7) to 14 weeks (Patient 2) whereas control cases always
revealed persistent
tumor characterized by contrast-enhancement. Representative test responses and
case controls
are shown in Figures 4 and 5 and all cases are summarized in Figures 3A and
3B. As shown
in Figure 4, a Loss of enhancement and mass effect was observed in the test
case with
corresponding persistence of contrast-enhancement in the case control. In the
test case, the
patient had clinical improvement which corresponded to the radiographic
improvement. As
shown in Figure S, a loss of enhancement and mass effect with restitution of
ipsilateral trigone
was observed in the test case but corresponding persistence of contrast-
enhancement and
effacement of trigone in the case control. In the test case, the patient had
clinical
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improvement with return to all pre-morbid activities which corresponded to the
radiographic
improvement. This patient succumbed to recurrent disease in the contralateral
right
frontopolar region and an autopsy has active tumor at the site of recurrence
but only scattered
tumor cells at the primary site.
In three patients who deteriorated despite some initial radiographic
improvement, compassionate retreatment was granted by the FDA and the IRB.
After
re-treahnent, furtherradiographic loss ofnodular eilhancement remote from
surgical resection
occurred in all three with either clinical improvement or stabilization.
Serial tumor tissues in nine cases were analyzed before and after treatment.
These observations are summarized in Figures 3A and 3B. In ~1 I of 12 cases,
only viable
tumor at treatment was observed; without evidence of radiation changes
reflected as fibroid
necrosis. In two cases of prior GLIADEL~ implantation, viable tumor cells were
observed
contiguous to wafer remnants.
Treatment Response: Histopathologic Observations
Residual viable tumor cells were identified in all post-mortem examinations
and in all post-therapy surgical biopsies. Individual cases were observed
where the original
tumor site either lost endothelial cell proliferation (Patient 1), tumor cell
number and
pleamorphism (Patient 7), or acquired extensive areas of necrosis (Patient 8
after re-
treatment). Microthrombi limited only to tumor-associated blood vessels were
identified in
6 of 10 cases for which post-therapy tissue was available. Also, varying
degrees of tumor
perivasculax lymphocytic infiltration were identified in four cases in which
no lymphocytic
infiltrates were observed in pre-treatment tumor. In brain sections without
identifiable tumor
cells, inflammation, vasculitis, hemorrhage, necrosis, demyelination, or
vessel thrombosis was
not identified.
Summary
No systemic toxicity due to this process was noted other than a higher
incidence ofDVT which prompted initiation ofprophylactic anti-coagulation.
Radiographic
and clinical responses were documented, and these responses cannot be
attributed to either
surgical intervention or the escalation of steroid dose. In all cases the
radiographic
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improvements were either remote from the resection cavity or represented
continued
radiographic improvements in the residual tumor resection cavity without any
further
therapeutic intervention. Steroid doses were either unchanged or tapered
during periods of
clinical improvement.
S Pathologic examination of tumor corresponding to loss of enhancement on
MRI scans of Patients 2 and 3 demonstrated thrombosis of tumor blood vessels.
Patient 2
showed striking loss of enhancement of the entire tumor and pathologic
examination revealed
a complete absence of endothelial proliferation in the post-mortem specimen.
Endothelial
proliferation was broadly identified in pre-therapy tumor biopsy specimen from
Patient 2.
These findings raise the possibility that the radiologic response was related
to loss of tumor
vasculature attributable to this treatment.
Because of the inapparent toxicity and compelling responses, this treatment
appears to yield an extraordinary therapeutic advantage as a biologic
treatment of glioma. The
responses described herein were obtained when the antisense-treated tumor
cells were
encapsulated in diffusion chambers constructed with 0.1 m pore-sized
hydrophilic membranes.
This exclusion limit excludes cells and suggests that soluble factors released
by the
antisense-treated cells could be responsible for triggering these responses.
A number of studies have documented lymphocytic infiltrates (LI) as a fairly
common histologic feature of gliomas, but few have assessed Ll as a possible
response to
treatment. In one such study of200 patients, histologic specimens from 28
documented cases
of LI were reviewed for LI variations in successive biopsies obtained before
and after
intervening treatments such as radiation therapy. Eleven patients in this
series revealed LI at
initial biopsy with variable levels of LI at subsequent biopsies. Sixteen of
17 cases revealed
absence of LI at initial biopsy with continued absence through all subsequent
biopsies. In the
current series, the much higher frequency of newly identified LI in post-
treatment tumor
tissues suggests a treatment-related effect when compared to results in this
study. Previous
studies have found a favorable correlation between perivascular LI and
prognosis. In this
series, three of the 4 patients with perivascular LI manifested clinical and
radiographic
improvement after treatment.
Selective tumor vessel thrombosis may represent a mechanism independent of
an immune-mediated process. Tumor vessel throrrabosis was observed in 6 of 6
evaluable
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patients treated, and although a different mechanism of selective tumor vessel
thrombosis
remains unclear, this response has been associated with an unambiguous tumor
regression and
associated clinical improvement in all of these patients. Similar tumor
regressions have been
observed in an animal model after selective occlusion of tumor vasculature.
5 The expected incidence of DVT in brain tumor patients is around 40%. In the
pre-enoxaparin cohort, the incidence was 100% or 2.5-fold greater warranting
enoxaparin
prophylaxis. After enoxaparin treatment; the incidence dropped to 20%, or half
the expected
incidence, and in all cases without any associated hemorrhages. These findings
are consistent
with the safe and effective administration of enoxaparin in neurosurgical
patients. Recent
I 0 reports have reviewed the anti-thrombotic efficacy of enoxaparin in
prospective randomized
trials, and in one study a lower incidence of cancer-related mortality during
the study period
was shown. Despite this retrospective observation, a tumor lysis effect
secondary to
enoxaparin could be ruled out because responses were observed in three of the
first four
patients in whom enoxaparin treatment was not provided.
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SEQUENCE LISTING
<1 l 0> Thomas Jefferson University, et al.
< 120> Treatment Of Tumors With Oligonucleotides Directed To Insulin-Like
Growth Factor-I Receptor (IGF-
~~
<130> TJU-2386
<I40>
<141>
<150>60/096,354
<151>1998-08-13
<I50>60/I13,599
<15I>1998-I2-24
<160> 15
<210> 1
<211> 927
<212> DNA
<213> Homo sapien
<400> 1
cuuuguuuuc uuuucuuccu cacagaccuu 50
cgggcaagga ccuucacaag
ggaugcagua caugcucugg cugccguugc 100
ggaugaagcc cgaggggcac
uccugcaugc acucgccguc guggaucaca 150
aaccccucgg agucgcugcu
cucggcgcug aggauguugg cgcagaaguc 200
acgguccaca cagcgccagc
ccucaaaccu guagguguug ggcgggcagg 250
caggcacaca gacaccggca
uaguaguagu ggcggcaagc uacacaggcc 300
gugucguugu caggcgcgcu
gcagcugccc aggcacucgg gguggcagca 350
cucauuguuc ucggugcacg
cccgcuuccc acacgugcuu gggcacauuu 400
ucuggcagcg guuugugguc
cagcagcggu aguuguacuc auuguugaug 450
guggucuucu cacacaucgg
cuucuccucc auggucccug gacacagguc 500
cccacauucc uuugggggcu
uauuccccac aauguaguua uuggacaccg 550
cauccaggau cagggaccag
uccacagugg agagguaaca gaggucagca 600
uuuuucacaa uccugauggc
cccccgagua auguuccuca gguuguaaag 650
cccaauaucc uugagauugg
ucaucucgaa gaugaccagg gcguaguugu 700
agaagaguuu ccagccgcgg
augaccguga gguuggggaa gaggucuccg 750
aggcucucga ggccagccac
1
CA 02339858 2001-02-13
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ucggaacagc-agcaaguacu cgguaaugac cgugagcuug gggaagcggu 800
agcugcggua guccucggcc uuggagauga gcaggaugug gagguagccc 850
ucgaucaccg ugcaguucuc caggcgcuuc agcugcugau agucguugcg 900
gaugucgaug ccuggcccgc agauuuc 927
<210> 2
<2ii> i8
<2i2> DNA
<213> Artificial Sequence
<220>
<223> antisense oiigonucleotide
<400> 2
tcctccggag ccagactt 18
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> antisense oligonucleotide
<400> 3
ggacectcctccggagcc 18
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> antisense oligonucleotide
<400> 4
ccggagccag acttcat 17
<210> 5
<211> 20
<212> DNA
<2I3> Artificial Sequence
<220>
<223> antisense oligonucleotide
<400> 5
ctgctcctcc tctaggatga 20
z
CA 02339858 2001-02-13
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<210> 6
<211> 15
<212> DNA
<2I3> Artificial Sequence
<220>
<223> antisense oligonucleotide
<400> 6
ccctcctccg gagcc 15
<210> 7
<211> I7
<212> DNA
<213> Artificial Sequence
<220>
<223> antisense oligonucleotide
<400> 7
tacttcagac cgaggcc 17
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> antisense oligonucleotide
<400> 8
ccgaggcctc ctcccagg 18
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> antisense oligonucleotide
<400> 9
tcctccggag ccagactt 18
<210> 10
<2i1> 9
<212> PRT
<213> Artificial Sequence
3
CA 02339858 2001-02-13
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<220>
<223> MHC class I-associated peptide
<400> 10
Tyr Leu Glu Pro Gly Pro Val Thr Ala
1 5
<210> 11
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> MHC class I-associated peptide
<400> 11
Leu Leu Asp Gly Thr Ala Thr Leu Arg Lets
I 5 10
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> MHC class I-associated peptide
<400> 12
Phe Glu Cys Asn Thr Ala Gln Pro Gly
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> MHC class I-associated peptide
«100> 13
Ala Thr Val Pro Gly Pro Glu Leu Tyr
1 .5
<210> 14
<211> ZO
<212> PRT _
<213> Artificial Sequence
<220>
<223> MHC class I-associated peptide
4
CA 02339858 2001-02-13
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<400> 14'
Leu Arg Leu Thr Ala Thr Gly Asp Leu Leu
1 5 IO
<210> 15
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> MHC class I-associated peptide
<400> 15
Tyr Leu Arg Pro Gly Pro Val Thr Ala
1 5
5
