EntreMed Scientists Report Potent Antiangiogenic Effects in Preclinical Studies of Recombinant Human Endostatin and Angiostatin Protein At the American Association for Cancer Research Annual Meeting.

ROCKVILLE, Md.--(BW HealthWire)--April 14, 1999--

Recombinant Human Endostatin(TM) Manufacturing Process Validated

for the Production of Material for Human Trials

EntreMed, Inc. (NASDAQ:ENMD) scientists presented several preclinical studies demonstrating the efficacy of Endostatin (TM) protein and Angiostatin (R) protein in inhibiting tumor growth this week at the 90th Annual Meeting of the American Association for Cancer Research (AACR) in Philadelphia, Pa.

EntreMed also reported its manufacturing process for the production of recombinant human (rhu) Endostatin (TM) protein yields soluble, well characterized product in quantities sufficient for early clinical trials.

In all, thirteen abstracts were presented on Endostatin(TM) protein and Angiostatin (R) protein by EntreMed scientists and other researchers from around the world. Endostatin (TM) protein and Angiostatin (R) protein are potent, naturally-occurring, angiogenesis inhibitors that specifically inhibit endothelial cell proliferation, angiogenesis and tumor growth.

In an AACR mini-symposium on angiogenesis, EntreMed's Vice President for Preclinical Research, Dr. B. Kim Lee Sim today presented an abstract entitled "Potent Inhibition of Experimental Metastases and Primary Tumors by Recombinant Human Endostatin(TM) That is Suitable for Human Use."

Dr. Sim reported that GMP-Endostatin(TM) protein, (clinical grade Endostatin(TM) for human use) was manufactured in quantity and shipped to Children's Hospital, Boston where it was used to treat human prostate cancer xenografts transplanted into mice.

The human GMP Endostatin(TM) protein had the same activity and efficacy as EntreMed's non-GMP material used in earlier preclinical studies in the United States and Europe, and are the first data confirming the efficacy of EntreMed's clinical grade production in a preclinical model.

In a second EntreMed abstract presented at the AACR mini-symposium on angiogenesis, entitled "Recombinant Human Angiostatin(R): Dose-Dependent Inhibition of Early and Late Stage Established Metastases in Mice," EntreMed scientists presented data showing that their recombinant Angiostatin (R) protein markedly inhibited malignant melanoma in both early stage and late stage metastatic cancer in the lungs of mice.

EntreMed researchers cloned the human Angiostatin (R) protein and now produce it in scale-up fermentation using the yeast Pichia pastoris.

In total, EntreMed researchers gave nine presentations of the Company's own research on its antiangiogenic product candidates and their respective effects in preclinical cancer models at the AACR Annual Meeting, including two from the Company's internal discovery program - "Enantiomeric, Dependent Antimetastatic Activity of a Stable Thalidomide Analog," and "Tissue Factor Pathway Inhibitor (TFPI) Is an Inhibitor of Angiogenesis." A complete list of EntreMed's scientific study abstracts is attached.

Dr. Joanna C. Horobin, EntreMed's Senior Vice President of Commercial Development, commented on the presentations : "EntreMed is pleased to provide progress reports on our antiangiogenic product portfolio at this prestigious scientific meeting. We remain on schedule to commence Phase I safety trials of Endostatin(TM) protein later this year."

The AACR is a professional society with more than 14,000 laboratory and clinical scientists engaged in cancer research in the United States, Canada and 60 other countries.

Its principal activities include fostering advances in cancer and biomedical research through programs that promote scientific education and communication, meetings to present significant new discoveries in cancer and the publishing of four major peer-reviewed journals including Cancer Research, Clinical Cancer Research, Cell Growth & Differentiation and Cancer Epidemiology, Biomarkers & Prevention.

Rockville, Md.-based EntreMed, Inc., The Angiogenesis Company(TM), is a leader in the field of antiangiogenesis research, which studies the inhibition of abnormal blood vessel growth recently associated with a broad range of diseases.

The Company's strategy is to accelerate development of its core technologies through collaborations and sponsored research programs with university medical departments, research companies and government laboratories. For further information, please visit the EntreMed web site at www.entremed.com.

Statements herein that are not descriptions of historical facts are forward-looking and subject to risk and uncertainties.

Actual results could differ materially from those currently anticipated due to a number of factors, including those set forth in the Company's Securities and Exchange Commission filings under "Risk Factors," including risks relating to the early stage of products under development; uncertainties relating to clinical trials; dependence on third parties; future capital needs; and risks relating to the commercialization, if any, of the Company's proposed products (such as marketing, safety, regulatory, patent, product liability, supply, competition and other risks). -0-

Attachment to
Press Release
EntreMed AACR Abstracts
4-14-99

No. 2554 Mouse pharmacokinetics and pharmacological basis for drug
development of recombinant human Endostatin(TM).
    Tomaszewski, JE, Schweikart, KM, Covey, JM, Turner, N, Tosca, PJ,
Simpson, B, Fogler, WE and Sim, BKL. Toxicology and Pharmacology
Branch, DTP, NCI, Bethesda, MD 20892, Battelle Memorial Institute,
Columbus, OH 43201, CytImmune Sciences, Inc., College Park, MD 20740
and EntreMed, Inc., Rockville, MD 20850.

    Recombinant human Endostatin(TM) produced in the yeast Pichia
pastoris has Been shown to be effective in both the primary Lewis lung
carcinoma and the metastatic B16-BL6 tumor models after multiple
subcutaneous (sc) injections. No toxicity has been observed in the
efficacy studies performed to date. Thus, a pharmacological basis for
drug development was sought in the event that toxicity could not be
established in preclinical toxicology studies. Since mice were
injected with either 1.5 or 50 mg/kg (4.5 or 150 mg/m(squared)) of
human Endostatin(TM) by the sc route in the B16-BL6 and Lewis lung
models, respectively, these doses levels were evaluated in
pharmacokinetic studies. Peak serum levels and AUCs that were
associated with efficacy in these models were established. The 1.5
mg/kg sc dose produced peak serum levels of 161 ng/mL and an AIC of 16
ug(dot)min/mL; while the 50 mg/kg dose produced 4582 ng/mL and 700
ug(dot)min/mL, respectively. Bioavailability was determined to be
essentially 100% at both dose levels when the AUC was compared to that
produced by comparable intravenous doses. Thus, safety and toxicity
studies will target these levels in an attempt to define a therapeutic
index for human clinical trials. Supported by NCI Contract No.
N01-CM-87028.


No. 2745 Endostatin(TM)-induced inhibition of endothelial cell
migration correlates with in-vivo anti-tumor activity.
    Fortier, A.H., Nelson, B., Vu, H., Plum, S., Fogler, W.E., and
Sim, B.K.L. EntreMed, Inc., Rockville, MD 20850.

    We analyzed the ability of recombinant human Endostatin(TM) to
block bFGF-stimulated human umbilical vein endothelial cell (HUVEC)
migration. Confluent cultures of HUVEC were established on
gelatin-coated plates and an area was wounded with a sterile razor
blade. The cells were then cultured for 24 hr with 2ng/mL bFGF in the
presence or absence of Endostatin. In the absence of Endostatin(TM),
HUVEC migrated into the wounded area in response to bFGF stimulation.
In ten individual experiments, Endostatin(TM) protein preparations
inhibited bFGF-induced HUVEC migration in a dose-related manner with a
range of IC(50), 0.5-35 ug/mL (0.025 -2 ug/mL). Over an identical
concentration range, a control protein preparation consisting of the
kringle 1 of plasminogen fused to kringle 2 of tissue-plasminogen
activator through a factor Xa cleavage site (K1XaK2tPA) had no effect
on HUVEC migration. The ability of Endostatin(TM) preparations to
inhibit HUVEC migration was then compared to in vivo anti-tumor
activity (range of T/C= 0.06-0.53) against B16BL6 metastases. A
coefficient of correlation for these two activities was o.78 (p less
than 0.05). Thus, inhibition of endothelial cell migration by
Endostatin(TM) was predictive of in vivo inhibition of metastatic
disease.

No. 2553 Pharmacokinetics and range-finding toxicity studies of
recombinant human Endostatin(TM) in cynomolgus monkeys.
    Tomaszewski, JE, Schweikart, KM, Covey, JM, Turner, N, Tosca, PJ,
Hassler, C, Simpson, B, Foger, WE, and Sim, BKL. Toxicology &
Pharmacology Branch, DTP, NCI, Bethesda, MD 20892, Battelle Memorial
Institute, Columbus, OH 43201, CytImmune Sciences, Inc., College Park,
MD 20740 and EntreMed, Inc., Rockville, MD 20850.

    Recombinant human Endostatin(TM) produced in the yeast Pichia
pastoris has been administered to monkeys as either a single
intravenous (iv) or subcutaneous (sc) bolus dose or as a one hour
continuous intravenous infusion in a series of pharmacokinetic (PK) or
range-finding (RF) toxicity studies prior to the design or
IND-directed toxicology studies. The doses used in the PK studies were
comparable to effective mouse doses on a mg/m(squared) basis. Kinetics
were determined to be dose-dependent between 4.5 and 150
mg/m(squared). Bio-availability of the sc doses was about 60%. The iv
monkey doses produced peak serum levels that were 12-340 times greater
than the effective levels observed in mice; while AUCs were about
1.3-56 fold higher. In separate RF studies, monkeys received single iv
bolus doses up to 100 mg/kg (1200 mg/m(squared)). These doses were
non-toxic and produced serum levels as high as 604 ng/mL 24 hours
after drug administration. A dose of 300 mg/kg administered as a 1 hr
iv infusion was also non-toxic and produced a 24 hr level of 244
ng/mL. Cardiovascular telemetry in this animal did not reveal any
acute or long-term changes in HR, BP, or EKG measurements. The results
of these studies indicate that Endostatin can be given safely in high
doses to monkeys and that IND-directed toxicology studies targeting
effective levels determined in the mouse can be readily designed and
are likely to be safe. Supported by NCI Contract No. N01-CM-87028.

No. 451 Tissue factor pathway inhibitor (TFPI) is an inhibitor of
angiogenesis.
    Hembrough, W.A., Papathanassiu, A.E., Swartz, G.M., Fogler, W.E.,
Lapcevich, R.K., Liang, H., LaValle, T.M., and Green, S.J. EntreMed,
Inc., Rockville, MD 20850

    An increasing amount of evidence suggests that proteins of the
blood coagulation and fibrinolytic systems play an essential role in
regulating neovascularization, the mechanism by which new capillaries
are formed from pre-existing blood vessels via migration and
proliferation of endothelial cells. Here, we report that TFPI, a
protein mainly known as the major physiological inhibitor of tissue
factor-induced blood coagulation, is also a potent inhibitor of
angiogenesis. TFPI specifically inhibited growth factor-induced
proliferation and migration of endothelial cells in vitro with an
IC(50) value between 150 and 300 nM. Administration of TFPI in
tumor-bearing animals delayed primary tumor growth and suppressed
hematogenous metastasis of B16BL6 melanoma cells. Our data indicate
that the antiangiogenic activity of TFPI is not related to its heparin
binding affinity, but requires the presence of the Kunitz-3 domain, a
region of the protein with no clear biological functions.

    No. 448 Enantiomeric, dependent antimetastatic activity of a
stable thalidomide analog.
    Swartz, G.M., Shah, J.H., Fogler, W.E., Papathanassiu, A.E.,
Treston, T., Madsen, J.M., and Green S.J. EntreMed, Inc., Rockville,
MD 20850

    A partially reduced and relatively stable analog of thalidomide,
2-phthalimidinoglutaric acid (2PGA) demonstrated potenti
antimetastatic activity when administered orally. Inhibition of
pulmonary metastases in an experimental B16-BL6 murine melanoma model
by 2PGA was dose dependent with an IC(50) value of 0.1 mg/kg and time
dependent with treatment being effective when initiated within the
first five days following tumor cell inoculation. To improve potential
antitumor activity and prevent racemization, various analogs of 2PGA
were synthesized. Substitution at the benzene ring of the
phthalimidino group led to inactivation or highly toxic products,
while methyl substitution at the chiral carbon maintained biological
activity and prevented racemization. Chiral separation of (R)- and
(S)-2-methyl-2-phthalimidinoglutaric acid revealed that only the
(S)-enantiomer demonstrated the ability to inhibit BL16-BL6
metastases, and therefore the antitumor activity of 2PGA is
enantiomeric dependent.


    No. 437 Measurement of circulating levels of endogenous
endostatin.
    Paciotti, G.F., Simpson, B., Soltysiak, K.A., Myer, L.D., Fogler,
W.E., Ruiz, A., Kough, E., Fortier, A.H. and Tamarkin, L. CytImmune
Sciences, College Park, MD 20740 and EntreMed, Inc., Rockville, MD
20850.

    Endostatin(TM) has been shown to cause a dramatic reduction of
primary and metastatic tumors in animal models. Although these data
support a potential therapeutic application for the protein, little is
known about its biology. To address this question two Accucyte(TM)
kits were developed; one to measure human and one to measure murine
endostatin. The assays were validated by parallelism, quantitative
recovery and crossreactivity studies for the detection of endogenous
endostatin in serum and plasma samples. Human endostatin was measured
in serum samples from normal volunteers, as well as pre- and
postoperative cancer patients. The murine assay was used to measure
serum levels of murine endostatin in control and B16 tumor burdened
C57BL mice. The results are expressed as the mean + or - s.e.m.
in ng/ml.

----------------------------------------------------------------------

                       Human Endostatin
                       ----------------

Normal                     Pre-Op                 Post-Op
------                     ------                 ------
21.4 + or - 3.5            123 + or - 39          258 + or - 53
n  = 9                     n = 10                 n = 9

----------------------------------------------------------------------

                      Murine Endostatin
                      -----------------

                Control            Tumor Burdened
                -------            --------------
               60.8 + or - 1.5     103.3 + or - 10.1
               n=3                 n = 4

----------------------------------------------------------------------

    Comparison of the endostatin levels with the levels of VEGF and
bFGF in normal volunteers revealed that endostatin concentrations were
5-20 times higher than the other angiogenic factors. The data support
the hypothesis that endostatin is present at basal (ng/ml) levels in
normals. Furthermore, endostatin levels may change in response to
conditions and diseases which affect angiogenesis, suggesting that it
may be may be an endogenous mediator of this process.


No. 4092 Potent inhibition of experimental metastases and primary
tumors by recombinant human Endostatin that is suitable for human use.
    Sim, B.K.L., Fogler, W.E., Zhou, X.H., Liang, H., Madsen, J.W.,
O'Reilly, M.S., Panigraphy, D., and Fortier A.H. EntreMed, Inc.,
Rockville, MD 20850; and Children's Hospital, 300 Longwood Avenue,
Boston, MA 02115.

    Endostatin, a potent endogenous inhibitor of angiogenesis,
regresses large primary tumors without induction of acquired drug
resistance in mice. We report that a soluble recombinant human
Endostatin produced in the yeast Pichia pastoris, with characteristics
of the native Endostatin protein, effectively inhibits the growth by
human Endostatin was dose dependent. Human recombinant Endostatin
administered at 1.5 mg/kg/day resulted in a 90% inhibition of early
(day 3) murine melanoma metastases. We extended this study to include
the treatment of late stage (day 11) lung metastases. Endostatin in
this model at doses of 4.5 mg/kg/day inhibited the growth of
macrometastases by 70-90%, as assessed by lung weight. Lewis lung
primary tumors are potently inhibited (80-90%) from growth upon
treatment with 50 mg/kg bid of human Endostatin. This recombinant
human Endostatin can be used to assess the effectiveness of Endostatin
in the clinic. Our production system yields soluble, potent, well
characterized recombinant human Endostatin at quantities sufficient
for human use.

No. 4098 Recombinant human Angiostatin(TM): Dose-dependent
inhibition of early and late stage established metastases in mice.
    Fogler, W.E., Grella, D., Vu, H., Plum, S., Chang, A., Lu, M.,
Fortier, A., Trail, P., Lewin, A., Sim, B.K.L. EntreMed, Inc., 9610
Medical Center Drive, Rockville, MD 20850; Bristol-Myers Squibb,
Princeton, NJ 08543.

    Angiostatin(TM) is a potent inhibitor of angiogenesis and tumor
growth. We had previously shown the inhibition of early stage (day 3)
B16BL6 pulmonary metastases following the administration of
recombinant human Angiostatin(TM) to mice. We now show that this
inhibition is dose-dependent, with greater than 90% inhibition
obtained after daily systemic administration of 4.5 mg/kg
Angiostatin(TM) to mice. Pharmacokinetic studies in mice with
effective anti-tumor doses demonstrated a dose-dependent increase in
circulating levels of Angiostatin(TM). Moreover, at 4.5 mg/kg
Angiostatin(TM) administration led to an approximate 65% inhibition of
late stage (day 11) macrometastases as assessed by lung weight.
Immunohistochemical analyses using antibody against vWf showed a
corresponding inhibition of neovascularization in lung metastases of
mice treated with Angiostatin(TM). Taken together, these data suggest
that Angiostatin(TM)-mediated inhibition of tumor growth was a direct
result of angiogenesis inhibition.

No. 3029 2-Methoxyestradiol does not require p53 in endothelial
cells.
    LaVallee, T.M., Hembrough, W.A., Papathanassiu, A.E., Pribluda,
V.S., Swartz, G. and Green, S.J. EntreMed, Inc., Rockville, MD 20850.

    2-Methoxyestradiol (2ME2) is an endogenous estrogen metabolite
with antitumor and antiangiogenic properties. 2ME2 inhibits
endothelial cell proliferation, migration and differentiation in vitro
and suppresses tumor growth in vivo. Although it has been reported
that presence of wild type p53 is necessary for the antiproliferative
effect of 2ME2 in tumor cell lines, it has not been addressed whether
2ME2 requires p53 in endothelial cells. Using three different
endothelial cell lines, HUVEC, ECV and HMVECD, we demonstrate that
2ME2 has a similar antiproliferative and apoptopic effect on all three
cell lines and inhibits migration of HUVEC and ECV cells; however,
2ME2 does not upregulate p53 in ECV cells. These data clearly indicate
that there is no correlation between the sensitivity