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¡¡ JAMES W.
FETT & KAREN A. OLSON
Research
Activities Tumor
Therapy Experiments Investigations
to assess efficacy of angiogenin (Ang) antagonists for prevention of both establishment
and metastatic dissemination of human tumors implanted into athymic mice were continued. i) Therapy
of human prostate cancer. Metastatic
tumors. Combination of antiAng therapy with another
antiangiogenesis agent. The model of
metastatic prostate cancer, well-established in the laboratory was used which involves the
injection of the human prostate tumor cell line, PC-3M, into the surgically exposed
prostates of athymic mice. Metastasis
subsequently develops in the local iliac lymph nodes.
We had previously shown using this model that both the Ang neutralizing mAb
26-2F and the Ang antisense JF2S inhibit metastasis in 50% of the mice treated with either
of these agents. Increasing the dose of
either agent does not produce a significant increase in efficacy. We also wished to
determine if combination therapy directed against both Ang and another angiogenic factor,
vascular endothelial growth factor (VEGF), could show either an additive or synergistic
protection above that seen by either agent alone. The
first such experiment Ang antisense JF2S was administered along with an antisense directed
against VEGF, JF17S, at a dose previously found to be optimal for JF2S. Interestingly,
while the JF17S was not very protective by itself, addition of that dose of JF17S to the
optimal dose of JF2S produced at least an additive increase in protection above that
afforded by JF2S alone. However, in two
subsequent experiments, the VEGF antisense JF17S effectiveness in inhibiting metastasis
was found to be actually only slightly below that of JF14S, when given at the most optimal
dose for JF2S. Repeating the combination of
the two antisense each at this optimal dose did not produce an increased efficacy above
either agent alone as had been previously observed. When
both antisense were given alone at a dose previously determined for JF2S to produce a
decreased protection from metastasis, both agents exhibited similar low levels of
antimetastatic activity. Combination of these
two antisense (at half of this dose for each) did, however, not show the expected additive
effect. As a result this particular
combinatorial approach was abandoned. However,
experiments are underway to examine the effects of combining antiAng agents (e.g.,
antibody and antisense) in this model. Efficacy of
Ang antisense JF2S in preventing metastases in mice with established prostate tumors. In the abovementioned prior work Ang antisense
JF2S was shown to prevent metastasis in 50% of the treated mice in the orthotopic
prostrate cancer model while all diluent, sense and scrambled [S]ODN controls developed
metastasis. In these experiments the
treatments were given systemically one hour after the tumor cells were injected. We, therefore, wished to determine if the
antisense agent is effective in reducing metastasis when treatment is delayed until a
primary tumor is established in the prostate and metastasis is histologically observed in
a known percentage of the mice. Two
experiments were performed to first ¡¡
determine i)
the time at which primary tumors are macroscopically observable following tumor cell
injection and ii) the percentage of mice which exhibit histologically observable
metastasis at different time points following tumor cell injection into the prostate. We found that primary tumors were present in all
mice by day six. The percentage of mice
exhibiting metastasis was somewhat variable between the two experiments, but by day 32,
ten days before the usual sacrifice date, 80% of the mice exhibited metastasis. Based on these results two experiments were begun
to test the capacity of JF2S to inhibit metastasis when treatment is delayed until day 14,
28 or 32. At each of these time points,
additional untreated mice injected with tumor cells will be sacrificed to determine the
percentage of mice at these points which harbor lymph node metastasis. ii) Therapy of human breast cancer. Primary
tumors. In the previous reporting period we examined the
efficacy of the Ang antisense JF2S in preventing primary tumor growth in an orthotopic
model of estrogen-independent breast cancer in which the human breast cancer cell line
MDA-MB-435 is injected into the surgically exposed mammary fat pad of athymic mice. In two experiments using this model we observed
that JF2S prevented 100% of the mice from developing primary tumors. In the current reporting period this study was
repeated twice more, in order to obtain sufficient numbers for statistical analysis, with
the same results - 100% of the mice treated with Ang antisense failed to develop tumors. In all four experiments, all mice injected with
tumor cells and treated either with diluent, the Ang sense or the Ang scrambled [S]ODN
control material developed tumors. Metastatic
tumors. Orthotopic model. Previously we had tested a new, highly metastatic
human breast cancer cell line, MDA-MB-435L2 (from Dr. Price, M.D. Anderson Cancer Center),
in the orthotopic model of lung metastasis in which the cells are injected into the
surgically exposed mammary fat pad of athymic mice with subsequent metastases developing
at 12 weeks. With the use of this cell line the time required for the mice to form
metastasis was reduced from the 20 weeks needed when the parental cell line (MDA-MB-435)
was used. As was done previously with other
cell lines used in various tumor models, the effect of in vitro treatment of the
MDA-MB-435L2 cells with Ang antisense JF2S was investigated. JF2S both reduced the in vitro secretion of
Ang by these cells and compromised the capacity of these ex vivo treated tumor
cells to form primary tumors following their s.c. injection into athymic mice. In our previous report we presented preliminary
data which showed that systemic treatment with either mAb 26-2F or Ang antisense JF2S
decreased the incidence of lung metastases (based on macroscopic observation) by 25% and
43%, respectively. We also wished to
determine the extent of metastasis in those mice which were not completely
protected by antisense or antibody treatment. The
measurements of lung weights are usually used in the field for this purpose, but we were
concerned that weights as a measure of extent of metastasis were not sufficiently
sensitive to be reliable for this purpose. Therefore four experiments were initiated in
order to compare the use of lung weights to macroscopic and/or microscopic observation as
a means of evaluating the efficacy of the Ang antisense JF2S in preventing metastases. In addition, several experiments were performed in
order to determine the optimum time of sacrifice which would result in reproducible,
quantifiable metastases in control mice. Thus
far it appears that either macroscopic or microscopic observation at sacrifice at ten
weeks is optimal. Experiments thus far show a
trend toward reduction in number of metastases in JF2S-treated mice. We are currently testing different treatment
protocols with JF2S and mAb 26-2F to optimize the protective effect of these agents and to
obtain results that are statistically significant.
¡¡
iii) Therapy
using neomycin and neomycin derivatives to inhibit tumor growth.
Dr. Guo-fu Hu of the Center has determined that neomycin is a potent inhibitor of
Ang-induced angiogenesis. In collaboration
with him we have tested the capacity of neomycin to inhibit tumor growth in mouse models
of human prostate, breast and colon cancer: i) while neomycin appeared to cause some
inhibition of tumor appearance in a s.c. model of primary prostate tumor growth, technical
problems involving the interaction of neomycin with Matrigel basement membrane material
(necessary for the in vivo s.c. growth of prostate tumor cells) precluded further
testing in this model. ii) three experiments were also performed in which
neomycin was evaluated using the orthotopic breast cancer model with MDA-MB-435 cells. The results, however, were variable. In one experiment neomycin protected four mice
from developing tumors when given i.p., while there was no protection when neomycin was
administered s.c. In the second experiment there was no protection using either route, and
in the third experiment four out of ten and one out of nine mice were protected from tumor
development when given neomycin s.c. or i.p., respectively.
Treatment with a dose three times that used in these experiments was found to be
100% lethal. In the third experiment a derivative of neomycin, 6-aminoglucosamine,
comprising one quarter of the neomycin molecule, was also tested in a limited number of
mice. However, it failed to protect any mice
from tumor development, presumably due to its weak antiAng activity as determined by Dr.
Hu. iii) testing in a s.c. model of human
colon cancer was also begun during this reporting period.
Of major concern in performing these types of tumor experiments is the
stability/availability of neomycin and derivatives in vivo, since they are known to
be rapidly cleared. Slow-release
methodologies are therefore being investigated. However,
in the first colon tumor experiment, all mice died within five minutes after implantation
of a commercially-available neomycin pellet although the dose within the pellet was
theoretically within the tolerated dose range. Further
experiments will therefore be conducted to identify safe slow-release administration of
neomycin and related compounds.
Drug
Development i) Chimerized antibody. A report describing the construction and
anti-breast tumor activity of the chimerized version of murine mAb 26-2F appeared during
1998 in the Proceedings of the National Cancer Institute.
ii) Humanized antibodies. Since in some cases an immune response directed
against the mouse V region portions of chimeric antibodies can still occur in patients,
fully humanized versions of murine mAbs constructed by CDR grafting and other techniques
are emerging for use clinically. As part of
our drug development program, we will produce antiAng humanized antibodies in addition to
the chimeric antibody already available. Toward this end we are collaborating with Dr. K.
R. Acharya at the University of Bath, UK, an expert in x-ray crystallographic techniques
and protein modeling and a continuing collaborator with this Center. As an important step toward humanization,
Dr. Acharya has collected a complete data set for the complex between the Fab fragment of
mAb 26-2F and Ang and has nearly solved the structure using molecular replacement
techniques. This 3-dimensional structure, in
which all critical contact amino acids will be known, will be invaluable for designing
humanized antibodies as well as CDR-based peptides and mimetics. ¡¡
Immunohistochemical
Analysis of Ang in Human Tissue Specimens These
studies are being performed in collaboration with Dr. Marc E. Key, Vice President of
Laboratory Operations, Dako Corp. Dako Corp.
maintains a bank of paraffin-embedded human normal and malignant tissues. In order to examine the incidence and distribution
of Ang, Dr. Key has developed sensitive and specific staining techniques for detecting
tissue-associated Ang protein. Using
mAb 26-2F he has begun screening clinical samples of normal and malignant prostate tissue. In this continuing study, Dr. Key and associates
have examined approximately 20 types of human tissues, both normal and malignant. Several tumor tissues (e.g., breast, prostate,
kidney, colon) stain positively for the presence of Ang whereas normal tissues, for the
most part, are not stained. Ang, therefore,
may play a role in a diverse set of human cancers. Presently,
contacts are being made with pathology core facilities of the Dana-Farber/Harvard Cancer
Center in order to examine tissue specimens from a greater number of patients of one
particular tumor in order to correlate the presence of Ang with such clinical features as
diagnosis, stage, grade, outcome, etc. It
is possible that Ang may serve as a useful biomarker for human cancers. Differential
Expression of Ang Protein and mRNA in Cancer i) As
alluded to above, Ang may be useful as a biomarker for certain human cancers. As an adjunct to the immunohistochemical studies
proposed above, we are obtaining serum and urine samples from prostate cancer patients
from Drs. Kenneth Falchuk and Jerome Richie, both of the Brigham and Women¡¯s Hospital. These are being tested by ELISA for the presence
of Ang and compared to PSA levels. Very early
results indicate that Ang may be elevated, along with PSA in patients with late-stage
disease. These studies are ongoing. ii)
Molecular profiling of tumor tissue for the presence of Ang mRNA together with expression
other angiogenesis-related genes has been initiated utilizing commercially-available
microarrays. Dr. Martinelli, Millennium
Pharmaceuticals, serves as a consultant for these investigations. For initial studies RNA has been isolated from two
prostate cancer cell lines grown in vitro - one more metastatic than the other. Differential analysis indicates that Ang message
is upregulated in the more metastatic cell type. Thus,
Ang may play a role in the metastatic phenotype of prostate cancer. Similar studies are underway examining different
human tumor cell lines grown orthotopically in athymic mice. Future plans call for
examining human tumor species in a similar fashion. Collaborators/Consultants
K. Ravi
Acharya, Ph.D.
University of Bath H. Randolph
Byers, M.D., Ph.D.
Boston
University School of Medicine Kenneth H.
Falchuk, M.D.
CBBSM & Brigham and Women's Hospital Jack T.
Johansen, Ph.D.
Boston BioSystems Mark E. Key,
Ph.D.
Dako Corp. Richard
Martinelli, Ph.D. Millennium Pharmaceuticals Janet E.
Price, Ph.D.
M.D. Anderson Cancer Center Jerome
Richie, M.D. Brigham and Women¡¯s Hospital Publications
- James W. Fett & Karen A. Olson Verselis
S.J., Olson K.A., Fett J.W. (1999) Regulation of Angiogenin Expression in Human HepG2
Hepatoma Cells by Mediators of the Acute Phase Response. Biochem. Biophys. Res. Commun. 259:
178-184. Olson K.A.,
Byers H.R., Key M.E., Fett J.W. Prevention
of Human Prostate Tumor Metastasis in Athymic Mice by Antisense Targeting of Human
Angiogenin. Submitted. Support Department
of Defense. RP951418. ¡°Novel Antiangiogenic/Cytotoxic Therapies for
Advanced Breast Cancer.¡± 09/01/96 -
08/31/00. $453,433 direct costs. James W. Fett, P.I.; Karen A. Olson, 30% effort Award - CaP
CURE Association. ¡°Angiogenin and Prostate Cancer: Therapeutic Opportunities and
Proteomic/Transcriptional Profiling¡± 1999. $100,000.
James W. Fett & Karen A. Olson - CoPIs Teaching Advances in
Medical Biology Seminar (CEM 4105). Leader of
weekly HMS/BWH-accredited seminars in the Center which fulfill type I credits for
Continuing Medical Education; 20-40 scientists, physicians, graduate and medical students,
and visiting professors. 10-20 hours/year. James
W. Fett & Karen A. Olson Meetings
attended 90th
Annual Meeting of the American Association for Cancer Research, Philadelphia, PA, April
10-14, 1999. James W. Fett &
Karen A. Olson 6th
Annual CaP CURE Annual Retreat, Lake Tahoe, NV, October 14-17, 1999. James W. Fett & Karen A. Olson Invited
presentations - James
W. Fett ¡°Angiogenin:
Opportunities for Research and Clinical Studies¡±.
Symposium - Tools for Drug Discovery, The Angiogenesis Model. Sponsored by Chemicon Intl., Inc., Philadelphia,
PA. April 9, 1999. ¡°Angiogenin
- Therapeutic Opportunities¡±. 5th
International Meeting on Ribonucleases, Warrenton, VA.
May 13, 1999. ¡°Angiogenin:
Role(s) in Prostate Cancer¡±. 6th
Annual CaP CURE Annual Retreat, Lake Tahoe, NV, October 14, 1999. Other
- James W. Fett Served on
the Pathobiology 4 Study Section, Department of Defense Breast Cancer Research Program. Vienna, VA, August 29-31, 1999. |