Opthea Ltd — Regulatory Filings 2009

Apr 21, 2009

ASX and Media release

22 April 2009

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Circadian partner ImClone Systems demonstrates VEGFR-3 antibody improves anti-tumour effects of chemotherapy in mouse tumour models

• Data presented at prestigious American Association for Cancer Research 2009 conference

• Combination of VEGFR-3 antibody with cisplatin or docetaxel significantly better than either agent alone in inhibiting tumour growth of lung or head and neck cancers

• Positive indications support the ongoing therapeutic development of VEGFR-3 antibody as anti-cancer agent

Scientists from Circadian Technologies’ licensee, ImClone Systems, a wholly-owned subsidiary of Eli Lilly and Company, have today released data from preclinical animal studies demonstrating that a monoclonal antibody targeting the Vascular Endothelial Growth Factor Receptor 3 (VEGFR-3) protein combines effectively with standard chemotherapy treatments to inhibit the growth of tumours in subcutaneous mouse tumour models.

The data presented today at the prestigious American Association for Cancer Research (AACR) annual conference demonstrate that the combination of a VEGFR-3 antibody with the standard anti-cancer chemotherapy agents for lung cancer – cisplatin – or for head and neck cancer – docetaxel – gave significantly better results in tumour inhibition than either agent alone as demonstrated in subcutaneous mouse tumour models.

The data is shown in the presentation entitled “Potentiation of cytotoxic therapy in subcutaneous xenograft models with an antagonist monoclonal antibody to VEGFR-3” which follows.

Mr Robert Klupacs, Managing Director of Circadian said “This is exciting data that shows VEGFR-3 antibodies may have the potential to improve existing chemotherapy treatments. We believe that this data supports the potential of VEGFR-3 antibodies as a novel and promising cancer treatment.”

ImClone Systems and Circadian announced in October 2008 that the VEGFR-3 antibody, IMC-3C5, has been designated as a formal pre-clinical development candidate for oncology indications. IMC-3C5 is an antibody which inhibits VEGFR-3. Peer-reviewed publications have shown that VEGFR-3 plays an important role in the development of blood vessels and lymphatic vessels supporting tumours. Blocking the VEGFR-3 pathway may have the potential to therefore block tumour growth and metastasis by “starving” tumours of blood supply and preventing the spread of cancerous cells through the lymphatic system.

ImClone Systems is developing IMC-3C5 under an exclusive worldwide license from Circadian (through a wholly owned subsidiary) in return for annual license fees and royalties on potential future product sales.

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Level 1, 10 Wallace Avenue, Toorak, Victoria 3142, Australia P: +61 (3) 9826 0399 F: +61 (3) 9824 0083 www.circadian.com.au

Circadian Technologies Limited ABN 32 006 340 567

Circadian is highly focused on the development of antibody drugs to treat cancer. In addition to the product being developed by licensee ImClone, Circadian has under development a very promising pipeline of three pre-clinical drugs VGX-100, VGX-200 and VGX-300, two of which are antibody drugs. Circadian also has one other partnered program with Ark Therapeutics Group plc (LSE:AKT) relating to a novel treatment, Trinam®, to improve quality of care and quality of life for kidney dialysis patients which has commenced Phase 3 clinical trials.

For more information

Company enquiries Robert Klupacs Managing Director - Circadian Tel: +61 (0) 3 9826 0399 or robert.klupacs@circadian.com.au

Media enquiries Rebecca Wilson Principal - Buchan Tel: +61 (0) 417 382 391

About Circadian Technologies Limited

Circadian (ASX:CIR) is a biologics drug developer utilising the significant intellectual property portfolio around Vascular Endothelial Growth Factor (VEGF) C and D that it has accumulated in its unlisted wholly owned subsidiary Vegenics. The applications for the VEGF technology, which functions in regulating blood supply, are substantial and broad. Circadian’s internal product development programs are focussed on novel anti-cancer therapeutics for large unmet needs. Circadian has also licensed rights to some parts of its intellectual property portfolio for the development of other products to UK company Ark Therapeutics Group plc (LSE: AKT) and ImClone Systems (a wholly owned subsidiary of Eli Lilly & Company - NYSE: LLY). Ark is developing Trinam®, a treatment for vascular grafts associated with renal dialysis based upon Circadian intellectual property which has commenced Phase 3 clinical trials. ImClone Systems is currently developing an antibody-based drug targeting VEGFR-3 for the treatment of solid tumours.

The VEGF patent portfolio developed by LICR and Licentia has been assigned to Vegenics. Vegenics also has rights to CoGenesys Inc/Human Genome Sciences Inc’s VEGF-C intellectual property.

About VEGF Technology

In Cancer

The clinical and outstanding commercial success of Avastin®, an antibody that blocks the activity of VEGF-A, clinically validated anti-angiogenic drugs as an effective means of inhibiting solid tumour growth. By blocking the interaction of VEGF-A with its receptors, primarily VEGFR-2, the multi-billion dollar cancer therapeutic slows tumour growth by inhibiting blood vessel recruitment into the tumour, effectively starving tumours of essential nutrients and oxygen required for growth. Avastin, which is sold by Genentech, now part of Roche, and Hoffman-La Roche, had U.S. sales in 2007 of US$2.3 billion and worldwide sales in excess of US$6 billion.

VEGF-C and VEGF-D inhibitors, key therapeutics in the portfolio of Circadian’s unlisted subsidiary Vegenics, blocks the alternative ligands for VEGFR-2. As such, they have the potential to block blood vessel growth in tumours resistant to anti-VEGF-A therapy and, when used in combination with drugs like Avastin, may completely shut down angiogenesis (the growth of blood vessels) mediated by VEGFR-2, resulting in greater clinical efficacy.

VEGF-C and VEGF-D also bind and activate VEGFR-3 which drives lymphatic vessel and tumour-associated blood vessel growth. Inhibitors of VEGF-C, VEGF-D and VEGFR-3 thus have therapeutic potential to inhibit not only primary tumour growth through their anti-angiogenic activities, but to also inhibit tumour spread or metastasis via the lymphatic vessels - a mechanism of tumour dissemination that is often the deadliest aspect of many tumour types and a mechanism that is not effectively blocked by anti-VEGF-A or anti-VEGFR-2 therapeutics.

Other Disease Applications

VEGF Technology also has applications in other diseases, where shutting down angiogenesis and/or lymphatic vessel growth is important, such as eye diseases including age related macular degeneration and diabetic retinopathy.

#4089 Potentiation of cytotoxic therapy in subcutaneous xenograft models with an antagonist monoclonal antibody to VEGFR-3 Marguerita O’Mahony, Hui Xiao, James R. Tonra, Marie Prewett, Rajiv Bassi, Chris Damoci, Kris Persaud and Bronislaw Pytowski ImClone Systems ImClone Systems 180 Varick Street, New York, NY 10014 a wholly owned subsidiary of Eli Lilly and Company ABSTRACT Efficacy of mF4-31C1 in Subcutaneous Xenograft Vascular density in areas of high tumor We performed studies to delineate the mechanism through which treatment with antagonist antibodies to the vascular endothelial growth factor receptor 3 (VEGFR-3) potentates the effect of chemotherapy in mouse tumor models. Vascular endothelial growth factor receptor 3 (VEGFR-3/Flt-4) is activated by VEGFs C and VEGF-D. Models in Combination with Chemotherapy proliferation (Ki+) Expression of this receptor in adult mammals is limited to lymphatic vessels and specialized fenestrated vascular endothelium. However, VEGFR-3 is also expressed by tumor endothelial cells, specifically at or near the growing regions of capillaries called endothelial tips. Antibody mediated inhibition of VEGFR-3 reduces tumor microvessel density and growth in human xenograft models in mice. Head and Neck Carcinoma Xenograft Model (Cal27) Cal27 NCI-H292 We speculated that targeting VEGFR-3 in tumors may increase the response of the tumor cells to cytotoxic agents as has been reported for other antiangiogenic agents. Immunodefficient mice bearing xenografts of various human tumor cell lines were treated with a highly specific monoclonal antibody against VEGFR-3 (mF4MOA Study Saline mF-31C14 Saline 31C1) in combination with various chemotherapeutic agents. VEGFR-3 inhibition increased the anti-tumor Rationale: VEGFR-3 (+) tip cells are likely found in mF4-31C1 effects of cytotoxic therapy in mouse xenograft models of human colorectal, non-small cell lung (NSCLC), 500 Effect on blood and lymphatic capillaries regions of tumor growth since tumor cells pancreatic and head and neck carcinomas, significantly reducing the rate of tumor growth. Best results was PBS mF4-31C1 obtained with the Cal27 head and neck carcinoma model in which T/C% values for monotherapy with mAb mF4Saline mF4-31C1 are a major source of angiogenic growth 31C1 and cisplatin (7 mg/kg, q7d) were 59 and 49 respectively, while the T/C% produced by the combination Cisplatin 400 Combined factors (VEGF, VEGF-C) therapy was 30. This was the only model in which combination therapy resulted in regression of the implanted tumors. Method: We focused further analysis on the Cal27 head and neck and the NCI-H292 NSCL carcinoma models. We T/C% RM ANOVA[[#]] 700 repeated the original studies but stopped the treatment at an earlier (d19) time point and collected tumor tissues 300 for immunohistological analysis. Combination treatment led to an increase in apoptosis within the tumors 55 p = 0.008 � VEGFR-3 vs. control 1. Using FITC illumination only, identify areas with only, identify areas with identify areas with significantly greater than that obtained with each monotherapy. As expected, treatment with mAb mF4-31C1 600 USP Saline (+) 39 p = 0.004 cisplatin vs. control cells significantly reduced the density of lymphatic vessels within the tumors and in the peritumoral tissues. Initial high density of Ki67 200 Ki 67 Meca32 (BV) Hoescht (Nuclear) 10X analysis of the density of blood vessels in the various xenografts failed to demonstrate a difference among the 13 p = 0.032 both vs. cisplatin 2. Record several images using FITC and Alexa546 various treatment groups. However, significant reduction in microvessel density was noted in the 500 Docetaxel Combination Saline chemotherapy and mF4-31C1 treated tumors when the analysis was limited to areas of viable tumor defined by illumination Saline 250 reactivity with antibodies to ki67. This result agrees with the previously reported localization of VEGFR-3 to 100 (+) 250 mF4-31C1 mF4-31C1 400 highly proliferative tumor endothelium that is most likely to be found in areas of active tumor growth. mF4-31C1 3. In Image Pro, draw areas with high density of Ki67 Docetaxol Cisplatin Combination tumor cells (A) Combination 300 0 4. Count the number of microvessels in that area and INTRODUCTION 200 Cisplatin 0 2 4 6 8 10 12 14 16 18 20 200 Days record area size (B) In postnatal animals, VEGFR-3 is primarily expressed on lymphatic endothelium and specialized fenestrated 200 Meca32 (BV) Lyve-1 (LV) Hoescht (Nuclear) 10X 10 vascular endothelia. Upon activation by its cognate ligands, VEGFs C and D (A), VEGFR-3 mediates crucial 5. Calculate density. early signals required for lymphangiogenesis, the formation of new lymphatic capillaries from preexisting 100 MF4-31C1 and 80 lymphatic vessels. This process can be potently inhibited in vivo with an antagonist monoclonal antibody 8 Saline 150 150 1 Cisplatin (mAb) mF4-31C1 directed to the murine VEGFR-3 . However, VEGFR-3 is expressed on tumor blood vessels and mF4-31C1 0 Cisplatin its inhibition results in reduction in tumor growth via an anti-angiogenic mechanism although to a lesser extent 6 Combination A B than corresponding inhibition of VEGR-22 (B). In addition, recent work has demonstrated that expression of 0 10 20 30 40 50 60 70 60 VEGFR-3 in proliferating blood capillaries is largely restricted to the tip cells that are key participants in the Random formation of angiogenic sprouts .3 Days Bars ± S.E.M. 4 100 tumor 100 40 The present investigation had two primary aims: 1) To investigate whether in vivo treatment with antagonist 2 regions mAbs to VEGFR-3 could be combined with chemotherapy to further reduce the rate of tumor growth and 2) To analyzed investigate the mechanism of this inhibition. We show that in two distinct tumor xenograft models, the 20 0 combination of anti-VEGFR-3 treatment with chemotherapy results in a significant combinatorial anti-tumor 50 Saline mF4-31C1 Cisplatin Combo 50 effect. The reduction in tumor growth rate is paralleled with increase in tumor cell apoptosis. Treatment with anti-VEGFR-3 dramatically reduced tumor lymphatic vessel density in agreement with previously published observations. However, contrast to previous studies, we were unable to demonstrate a reduction in the overall Chemicon ApopTag Red In Situ 0 tumor blood vessel density. However, a clear anti-angiogenic effect was documented when the analysis was Mean +/- SEM focused on areas of active tumor growth. Such regions of the tumor are likely to contain the highest density of 0 0 angiogenic sprouts that express VEGFR-3.

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mF4-31C1 Saline mF4-31C1
Cisplatin
400
Combined factors (VEGF, VEGF-C)
Method:
T/C% RM ANOVA [[#]]
700
300
55 p = 0.008 � VEGFR-3 vs. control
1. Using FITC illumination only, identify areas with only, identify areas with identify areas with
USP Saline
600 (+)
39 p = 0.004 cisplatin vs. control cells
high density of Ki67
200 Ki 67 Meca32 (BV) Hoescht (Nuclear) 10X
13 p = 0.032 both vs. cisplatin
2. Record several images using FITC and Alexa546
500 Docetaxel Combination
Saline
illumination Saline 250
250
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(+)
mF4-31C1
400
3. In Image Pro, draw areas with high density of Ki67
mF4-31C1
Cisplatin
tumor cells (A)
Combination
300 0
4. Count the number of microvessels in that area and
200
Cisplatin 0 2 4 6 8 10 12 14 16 18 20
200
Days
record area size (B)
200 Meca32 (BV) Lyve-1 (LV) Hoescht (Nuclear) 10X
10
5. Calculate density.
100
MF4-31C1 and 80
8 Saline 150
150
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mF4-31C1
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40
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analyzed
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0
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Chemicon ApopTag Red In Situ 0
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Renal Carcinoma Xenograft
MOA Stud
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Rat IgG 80 mg/kg
DC101 10 mg/kg
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mF4-31C1 40 mg/kg UPS Saline Effect on blood and lymphatic capillaries
2500 mF4-31C1
1600 Saline mF4-31C1
Docetaxel
CONCLUSIONS
2000 2000
T/C% RM ANOVA [#] 1400 mF4-31C1 & Docetaxexl
1500
1800
74 p = 0.001 � VEGFR-3 vs. control USP Saline 1200
1000
1600
49 p < 0.001 Doc vs. control
1000
�
500 In the two xenograft models presented, VEGFR-3 inhibition significantly increased
1400 23 p < 0.001 Both vs. Doc
Mean +/- SEM
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0 mF4-31C1 Combination the anti-tumor effects of the cytotoxic therapy without increase in the toxicity of the
Cisplatin
1200
0 5 10 15 20 25 30 35
600
chemotherapy (no weight loss detected)
Days of Treatment
1000
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Docetaxel
800 200 � In both models, the extent of inhibition of tumor growth closely correlated with the
600 0 2 4 6 8 10 12 14 16 18
apoptotic index of tumor cells determined by immunohistochemistry
mF4-31C1
Days of Treatment
400 &
15
Meca32 (BV) Lyve-1 (LV) Hoescht (Nuclear) 10X
Docetaxel
200
Saline
200 �
The anti-angiogenic activity of the anti-VEGFR-3 mAb could be documented only
mF4-31C1
Docetaxol
0 Combination
when analysis was focused on regions of active tumor growth
10
150
�
0 5 10 15 20 25 30
Random
Days of Treatment tumor � The above correlates with the observations by others (reviewed by Lohela et al.,
100
5
regions Figure at right) that VEGFR-3 expression is localized to angiogenic sprouts
analyzed �
50
�
�
/6 (w x w x w )), where "w " represents the largest 11 2 2 1 Combinatorial effect of anti-VEGFR-3 mAb and chemotherapy cannot be explained
0
Saline mF4-31C1 Doc Combo solely by effects on capillary density in the tumor
0
Chemicon ApopTag Red In Situ Apoptosis Kit Mean +/- SEM
)
3
)
3
Mean Tumor Volume (mm
)
2
)
2
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Mean Tumor Volume (mm
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Mean Tumor Volume (mm3)
)
2
Percentage of apoptotic cells(%)
BloodVessel density(vessels/mm
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A B
VEGF C
VEGF D
PlGF
VEGF B
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3000
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VEGF
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NRP-1 NRP-2
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VEGFR-1 VEGFR-2 VEGFR-2 VEGFR-3 Mean +/- SEM
0
Lymphatic EC and
0 5 10 15 20 25 30 35
Vascular EC Tumor Vascular EC
Days of Treatment
1. Pytowski B, Goldman J, Persaud K, Wu Y, Witte L, Hicklin DJ, Skobe M, Boardman KC, Swartz MA. Complete and specific inhibition of adult lymphatic regeneration by
a novel VEGFR-3 neutralizing antibody. (2005) J. Natl. Cancer Inst. 97:14-21.
2. Laakkonen,P., Waltari,M., Holopainen,T., Takahashi,T., Pytowski,B., Steiner,P., Hicklin,D., Persaud,K., Tonra,J.R., Witte,L., and Alitalo,K. (2007). Vascular endothelial
growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer Res. 67 , 593-599.
3. Tammela,T., Zarkada,G., Wallgard,E., Murtomaki,A., Suchting,S., Wirzenius,M., Waltari,M., Hellstrom,M., Schomber,T., Peltonen,R., Freitas,C., Duarte,A., Isoniemi,H.,
Laakkonen,P., Christofori,G., Yla-Herttuala,S., Shibuya,M., Pytowski,B., Eichmann,A., Betsholtz,C., and Alitalo,K. (2008). Blocking VEGFR-3 suppresses angiogenic
sprouting and vascular Network formation. Nature 454 , 656-660.
MATERIALS AND METHODS
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1. Pytowski B, Goldman J, Persaud K, Wu Y, Witte L, Hicklin DJ, Skobe M, Boardman KC, Swartz MA. Complete and specific inhibition of adult lymphatic regeneration by a novel VEGFR-3 neutralizing antibody. (2005) J. Natl. Cancer Inst. 97:14-21. 2. Laakkonen,P., Waltari,M., Holopainen,T., Takahashi,T., Pytowski,B., Steiner,P., Hicklin,D., Persaud,K., Tonra,J.R., Witte,L., and Alitalo,K. (2007). Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer Res. 67 , 593-599.

Cells: Cal27 and NCI-H292 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured at 37°C in a 5 % CO atmosphere, routinely passaged by Trypsin-EDTA (Invitrogen) treatment. 2 Animal Studies: Mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed under pathogen-free conditions in microisolator cages with laboratory chow and water available ad libitum. All experiments and procedures were performed in accordance with the United States Department of Agriculture, Department of Health and Human Services and National Institute of Health policies regarding the humane care and use of laboratory animals. Subcutaneous xenografts were established by injecting cells in 50% Matrigel (Collaborative Research Biochemicals, Bedford, MA) subcutaneously(s.c.) into the a flank region of athymic mice. Subcutaneous tumors were allowed to reach a threshold volume and then mice were randomized by tumor volume into treatment groups . All treatments were administered by intraperitoneal (i.p.) injection. Tumors were measured approximately twice each week � with calipers and subcutaneous flank tumor volumes calculated by the formula ( /6 (w x w x w )), where "w " represents the largest 11 2 2 1 tumor diameter and "w " represents the smallest tumor diameter. Tumor volumes were analyzed using RM ANOVA in the JMP Statistical 2 Discovery package (v. 5.1; SAS Institute Inc., Cary, NC). At the end of the study tumors were resected and cut in half. One half was processed for paraffin embedding, sectioned, and stained for endpoint H&E histological evaluation. The other half was snap frozen for immunohistological analysis.

Lohela et al. Current Opinion in Cell Biology, 2009

Mean +/- SEM