Folate Receptor as a Target for the Management of Human

Christopher P. Leamon, Ph.D. VP Research can principally be taken up by 2 distinct mechanisms: FR and RFC

The reduced carrier binds folates with a low affinity (Kd=10-5 M). Folic acid is a very poor substrate. Upon binding to the FR (Kd=10-10 M), folic acid 2 is internalized via endocytosis

Folic acid binds Most antifolates enter 1 to the FR with cells this way high affinity 3 FR releases folic acid inside the acidic endosome 4 Folic acid escapes endosome and enters cytosol

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FR recycles back to cell surface The Folate Receptor (FR)

• 38 kDa GPI-linked membrane protein ( and β isoforms)

• Enters cells via Endocytosis – Non-destructive pathway

• The FR is recycled, not destroyed

• Normal tissue expression – Kidney (PT), placenta, choroid plexus, lung, thymus *Can be exploited for therapy

• Recognized tumor-associated protein – Predominant on epithelial cancers – Also found on CML, AML and lymphomas

• Binds folic acid, some antifolates, and folate-drug conjugates with high affinity (Kd in low nM range) Exploiting the Folate Receptor

H2N N N O Site of H HO2C HN N conjugation N OH O NH

Folic Acid O Macromolecules attached to folate are delivered into cancer cells via the folate receptor

Endocytosis of folate-gold conjugates (analyzed by electron microscopy)

Binding to cell surface

Gold particles linked to folate bind to surface of the Within a few minutes, the folate-gold cell and are internalized via endocytosis conjugates are released into the cytoplasm of the cell

5 >1 million newly diagnosed cancer patients over-express the folate receptor (United States, Europe, and Japan)

100%

90% Est. % FR α Est. % FR β 80%

(1) (2) 70%

60% and

positive etarfolatide target - 50%

FR both FRα and FRβ 40%

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Folate Receptor Positive Cancers by Cancer Type

Source: American Cancer Society and Endocyte estimates based upon imaging studies and tissue biopsies. (1) Represents patients with at least one folate receptor positive lesion. (2) Data for some indications based on IHC of alpha form of folate receptor (e.g., testicular, bladder). New data suggest some cancers express the beta form which is also targeted by Endocyte’s SMDCs and companion diagnostic imaging agent etarfolatide. Prognostic impact of FR expression on human cancer

Toffoli et al. (1997) Int. J. Cancer (Pred. Oncol.): 74, 193-198.

Hartmann et al. (2007). Int. J. Cancer 121, 938-942

Toffoli ,et al , 1998 Int J of Cancer

Overall survival by intensity of FR staining Disease-free survival by intensity of FR staining Specificity of folate targeting Fluorescent imaging of metastatic NSCLC in the thoracic cavity of a mouse Folate conjugates are highly specific for malignant

FR-positive tissue in humans

Courtesy: Philip S. Low Philip Courtesy: Real-time, Non-Invasive Assessment of FR Expression Using Companion Imaging Agents

CO2H O H2N N N

H O HN N O N N CO2H N O H H Tc O N N NH2 S O CO2H

99mTc-etarfolatide (99mTc-EC20) Folate-targeted imaging agents specifically bind to FR-positive tissue

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Blood Heart Lung Liver Spleen Kidney Muscle Tumor Intestine Stomach

Image provided by Leamon et al. (2002) Bioconj. Chem. 13:1200; Reddy et al. (2004) J. Nucl. Med. 45:857 Dr. Cristina Muller

Folate-targeted agents are partially re-absorbed by FR- positive kidneys and transferred back into circulation

Blood vessel Folate Receptors: healthy vs. cancer

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Primary mass

Cancer-free Advanced Patient Ovarian Cancer Imaging brain lesions

Brain Pituitary mets adenoma

Courtesy: Drs. Nelson Oyesiku and James Galt Emory University Targeted SMDC Therapy

Small Molecule Drug Conjugates

Current therapeutic approaches in oncology

arletuzumab F

Leamon et al. (2013) Pharmacogenomics and Personalized Medicine 6, 113-125. Our SMDC approach is modular in design

Drug (Module 4)

-Highly potent -Critical mechanism -Derivatizable Releasable Linker (Module 3)

-Stable in blood Hydrophilic -Cleaves in the endosome Spacer -Self-immolative (Module 2)

-Separates the ligand from the drug payload -Provides water solubility Ligand (Module 1)

-High affinity -Small size Folate-SMDCs enter cells via the FR

The reduced folate carrier 2 Binds folates with a low affinity (Kd=10-5 M). Folate conjugates Upon binding to the folate receptor will not enter cell through the (Kd=10-10 M), the conjugate is reduced folate carrier. internalized via endocytosis most antifolates enter cells this way 1 Folate-conjugate binds the folate receptor 3 The drug is cleaved inside endosome 4 Drug escapes endosome and exerts activity on cell

Folate receptor recycles 5 back to cell surface

Vintafolide (EC145) A folate-vinca alkaloid conjugate

HO N

HN NH2 2' NH N N H H CO2CH3 CO2H CO2H O O O O H H H N N N CO2H HO O N N N N H H H HNHNOC H N O O O OH HN N O S S CH CO2H CO2H 3 H O H2N N N

Folic acid Peptide spacer Bio-releasable DAVLBH (tumor-targeting ligand) (hydrophilicity) linker (active warhead) Vintafolide receptor specific activity

Activity of Vintafolide: 2 h/72 h Assay

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V IC50 ~ 9 nM FR-Negative Vintafolide Conc. (nM)a Cell Kill % 40 Cell Line A549 100 nM 0% 20 Vintafolide Vintafolide + XS FA 4T1 100 nM 0% 0 -10 -9 -8 -7 -6 -5 HUVEC 100 nM 0% Concentration (Log M) LNCaP 100 nM 0% a2h pulse, 72 h total incubation

Leamon et al. (2007) Int. J. Cancer 121:1585 Vintafolide clears quickly from circulation

Vintafolide PK in M109 Tumor-Bearing Balb/c Mice: 9.6 mg/kg i.v. bolus dose

50000.0 45000.0 40000.0 35000.0 30000.0 25000.0 Stable 20000.0 t1/2 ~ 20 min

Concentration 15000.0 10000.0 5000.0 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Time

*Concentration (ng/mL); Time (h); n=3

* No released DAVLBH identified in sera Vintafolide is curative, specific and well-tolerated in vivo; the untargeted DAVLBH drug is not

100-fold excess Re:etarfolatide

1600 15 Vintafolide + competitor 1400 (No cures)

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800 0 Untargeted Drug 600 @ MTD (No cures) -5 400 Change Weight %

Tumor Volume (mm Volume Tumor Vintafolide 100% cures -10 200 -15 0 8 15 22 29 36 11 21 31 41 51 61 PTI (days) PTI (days)

*Vintafolide and DAVLBH tested at 1 µmol/kg, TIW 2 wk -regimen is MTD for DAVLBH and 1/5th MTD for Vintafolide Co-injected vintafolide blocks 99mTc-etarfolatide uptake in FR-positive tissues

99mTc-etarfolatide 99mTc-etarfolatide + xs vintafolide

Balb/c-derived nu/nu mice bearing subcutaneous KB tumor xenografts. 500 nmol/kg of 99mTc-etarfolatide i.v. without (panel A) or with (panel B) 50 µmol/kg of vintafolide. SPECT/CT performed on euthanized mice approximately 18 h post injection to identify tissue uptake. Green arrows, tumor; white arrows, kidneys. Residual radioactivity in the lower abdomen represents bowel clearance. Key finding

99mTc-etarfolatide predicts what tissues (lesions) will accumulate vintafolide FR expression, DAVLBH sensitivity, and vintafolide response in tumor models

Species Tumor Tissue origin FR Quantity Host Sensitive to Typical In Vivo Line (pmol/mg protein)1 DAVLBH response to Vintafolide A549 Lung 1.1 nu/nu Yes Unresponsive Ov90 Ovarian 4.1 nu/nu Yes Durable PR; CR; cures Human IGR-OV1 Ovarian 8.7 nu/nu Yes Durable PR KB Nasopharyngeal 38 nu/nu Yes Curative 4T1 Breast 0.3 Balb/c Yes Unresponsive J6456 Lymphoma Not determined2 DBA Yes Curative Mouse M109 Lung 20 Balb/c Yes Curative L1210A Lymphocytic leukemia 95 DBA No Unresponsive

1Range for procured human OvCa specimens: 10-50 pmol/mg [Parker et al. (2005) Anal Biochem 338(2): 284-293.] 2J6456 cells are FR-positive; we have not yet measured FR levels in xenograft tissue

Two requirements for an vintafolide response

1) The tumor must be FR-positive (99mTc-etarfolatide) 2) The tumor must be sensitive to vinca alkaloids (DAVLBH) Vintafolide is effective against triple negative MDA-MB-231 tumors

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MDA-MB-231 Control {0,0,0} Vintafolide 2 mol/kg TIW x 2 wks {2,2,1} Drug Combination Studies

Vintafolide can safely be combined with a wide variety of standard chemotherapeutic agents

Vintafolide + Doxil® combination is more active against M109 tumors than the individual drugs alone

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0 -25 5 15 25 35 45 55 65 75 85 95 5 15 25 35 45 55 65 75 Days Post Tumor Implant Days Post Tumor Implantation

Vintafolide: 2 mol/kg TIW, 2 wk Doxil: 4 mg/kg, weekly x 2

*Supporting data for Endocyte’s PRECEDENT trial Vintafolide + Doxil combination is effective against KB tumors (human nasopharyngeal carcinoma)

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T -20 Vintafolide + Doxil 200 (3/5 cures) -30 0 7 19 31 43 55 67 79 91 PTI (days) Vintafolide + Doxil combination is effective against IGROV1 tumors (human OVCA)

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0 -30 6 26 46 66 86 106 126 146 PTI (days) Vintafolide combinations studies Additive activity with Avastin® and topotecan; synergistic activity with docetaxel and cisplatin

Combination cohorts in blue

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Tumor Volume (mm Volume Tumor 200 cures 0 0 7 19 31 43 55 67 79 91 7 17 27 37 47 57 PTI (days) PTI (days)

KB tumor model, nu/nu mice EC145 synergizes with Taxotere® (docetaxel) against FR-positive KB tumors in nu/nu mice

Activity Gross Toxicity 1600 30 1400

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400 100% % Weight Change -20 Tumor Volume (mm Volume Tumor cures 200 -30 0 7 14 21 28 35 42 49 56 PTI (days) KB Controls EC145 1 mol/kg, TIW x 2 wk (1/5th MTD): 1/5 cures Docetaxel 10 mg/kg TIW x 2 wk (MTD): 0/5 cures EC145 + Docetaxel: 5/5 cures

*Similar results with paclitaxel (weekly or biweekly) and abraxane 33 99mTc-Etarfolatide and Vintafolide

Clinical Utility

Selecting the right patient, for the right therapy, at the right time

99mTc-Etarfolatide: Companion Imaging

Vintafolide: Therapy HO N

HN NH2 2' NH N N H H CO2CH3 CO2H CO2H O O O O H H H N N N CO2H HO O N N N N H H H HNHNOC H N O O O OH HN N O S S CH CO2H CO2H 3 H O H2N N N 99mTc-Etarfolatide accumulates in FR-positive, not FR-negative lesions

FR+

FR- Folate Receptor-Positive Lesions

Whole Body Scan

FR(100%) FR(10-90%) FR(0%) All target lesions positive Some target lesions positive No positive target lesions ~40% of pts are FR(100%) ~40% of pts are FR(10-90%) ~20% of pts are FR(0%) Reasons 99mTc-etarfolatide (EC20) is better than IHC

1. Assessment of FR status a. EC20: Real-time analysis b. IHC: Archived tissue

2. Specimen analyzed a. EC20: Whole-body imaging b. IHC: Selected tissue specimen

3. Accessibility a. EC20: Reveals accessible tissue sites b. IHC: Indiscriminant (e.g. normal lung)

4. Functionality a. EC20: Binds only to functional FR b. IHC: Detects functional and non-functional FR

White Arrow: Normal alveolar cells of the alveolar wall CONFIDENTIAL Vintafolide clinical trials

• Single agent phase 2 studies – FV-02: Recurrent ovarian cancer – FV-03: Recurrent NSCLC

• Combination phase 2 studies – PRECEDENT: Platinum-resistant ovarian cancer (Doxil/Caelyx) – TARGET: NSCLC

• Combination phase 3 studies – PROCEED: Platinum-resistant ovarian cancer

*Dr. Josep M. del Campo will discuss these studies following this presentation SMDC vs. ADC

Current therapeutic approaches in oncology

arletuzumab F

Leamon et al. (2013) Pharmacogenomics and Personalized Medicine 6, 113-125. Many advantages for using small ligands versus receptor targeted antibodies

• SMDCs are rapidly cleared from body, Antibody reducing toxicity (e.g. non-specific uptake) 150,000 mw

• Advanced linkers allows attachment of wide variety of molecules

• Receptor docking efficiency

• Superior manufacturing: – Synthetic ease – Homogeneity – Stability in storage and in vivo – Lower manufacturing cost SMDC 2000 mw • Smaller size allows for better penetration of solid tumors

42 Better solid tumor penetration with smaller molecules

Tumor Uptake 1st 65 min (red)

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12 Large MW payload Large Molecule 9

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Rhodamine fluorescence (a.u.) fluorescence Rhodamine 0 0 10 20 30 40 50 60 70 80 90 100 Time (minutes)

Small Molecule (1 min p.i.) 43 Summary

• The folate receptor (FR) – Tumor biomarker – Expression is associated with aggressive disease • 99mTc-etarfolatide and vintafolide are highly specific for FR-expressing tissues • Unlike ADCs, SMDCs (e.g. like etarfolatide and vintafolide) penetrate deeply into solid tumor tissue • Vintafolide is very well tolerated – Can be combined with many standard anti-cancer agents due to its non-overlapping toxicity profile • 99mTc-etarfolatide is the best method for detecting active FR – Anatomically predicts where vintafolide will accumulate – Superior to alternative methodology (like IHC) – Real time detection – The only method for predicting possible response to vintafolide and other folate- based SMDCs Acknowledgements

• Philip S. Low, Ph.D. • R Wendel Naumann, M.D. • Iontcho Vlahov, Ph.D. • Jim Symanowski, Ph.D. • Joe Reddy, Ph.D. • Wael Harb, M.D. • June Lu, Ph.D. • Martin Edelman, M.D. • Pat Klein, Ph.D. • Chandra Lovejoy, M.S. • Marilynn Vetzel, B.S. • Rich Messmann, M.D. • Elaine Westrick, B.S. • Pat LoRusso, D.O. • Nikki Parker, B.S. • Ed Sauseville, M.D. • Le-Cun Xu, Ph.D. • P. Ron Ellis, M.B.A. • Hari Krishna Santhapuram, Ph.D. • Fei You, Ph.D. • Longwu Qi. Ph.D. • Paul Kleindl, M.S. Special thanks to our patients

Backup The Importance of Folate

1. Role in human health 2. Role in cancer 3. Why dietary folate doesn’t interfere with vintafolide Folate Oral folic acid is reduced and methylated to low affinity forms prior to entering the blood Bolus-administered SMDCs “displace” endogenous folates from the tumor surface

A c tiv ity o f 1 0 0 n M E C 1 4 5 Test Article Class Relative Fold Weaker 1 1 0 Binding Affinity Affinity 2 h /7 2 h a s s a y 1 0 0 Folic Acid F A Vitamer 1.00 1.0 9 0

(Pte-Glu) y t

i A v . P t. s e ru m l 8 0 5-formyl- i fo la te (2 7 n M )

tetrahydrofolate Vitamer 0.01 125.0 b a

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tetrahydrofolate Vitamer 0.07 14.3 e

C 5 0 (5MTHF) 5 M T H F EC20 Folate-based % 0.92 1.1 4 0 (etarfolatide) Imaging Agent EC145 3 0 Folate-SMDC 0.47 2.1 L V (vintafolide) 2 0 0 1 0 2 0 3 0 4 0 5 0 C o n c . o f V ita m e r (n M )

Leamon et al. (2008) Impact of high and low folate diets on tissue folate receptor levels and antitumor responses toward folate-drug conjugates. J Pharmacol Exp Ther 327, 918-25.