ANTI-ELTD1 MONOCLONAL ANTIBODY THERAPY ELTD1 Epidermal growth factor, and seven-transmembrane domain- containing 1 • Highly expressed in vascular endothelial and smooth muscle cells

Favara DM et al., Biochem Soc Trans 2014; 42: 1658-64. Glioblastoma Multiforme (GBM)

Background Available Treatment • of the brain or spinal cord • Surgical resection; • Tumors arise from cell types with neural • Radiation with temozolomide (TMZ); stem cell-like properties • Adjuvant chemotherapy with temozolomide • Typically very aggressive tumors (TMZ) • Treatment options are poor • Upon recurrence, bevacizumab (humanized VEGF mAb) has been approved to treat GBM Bevacizumab

• Approved in 2009 as a second line treatment for GBM • Humanized Vascular Endothelial Growth Factor monoclonal antibody (VEGF mAb) • Anti-angiogenic treatment i.e. “tumor starving” • Side effects include hemorrhage, blood clots, and GI perforation ELTD1 is associated with VEGFR2

Endothelial Cells Glioma Cells

ELTD1 (red), VEGFR2 (green) ELTD1 is a IHC Biomarker for High-Grade Gliomas

ELTD1 is highly expressed in high-grade gliomas ELTD1 is associated with angiogenesis 50 HGG patients, 25 low-grade glioma patients. (HG) ELTD1 (red), CD31 (endothelial cells; green) High Grade (LG) Low Grade Towner et al., Neurosurgery, 2013. Anti-VEGF Ab treatment reduced expression of ELTD1 & VEGF (IHC)

n=5 OMRF used a commercially available Polyclonal Ab against ELTD1 in our proof of concept studies Polyclonal Anti-ELTD1 Treatment Significantly Reduces Expression of ELTD1 and VEGFR2 (IHC)

Data suggests that ELTD1 pAb treatment is targeting the tumor more effectively i.e VEGF Receptor and ELTD1 on the tumor surface than VEGF mAb treatment that targets the ligand (VEGF) and the tumor (ELTD1) Orthotopic GBM Models

• Human G55 xenograft model has many attributes associated with human GBM, and is of high interest for angiogenesis studies

Human G55 xenograft in nude mouse Stereotaxic orthotopic intracerebral implantation of cells

7.0 Tesla Small Animal MRI Polyclonal Anti-ELTD1 Antibody Therapy does not cause Hemorrhaging G55

UT Anti-ELTD1 Anti-VEGF

n=5

Anti-ELTD1 antibody therapy does not cause hemorrhaging, as seen in anti-VEGF treatment

Ziegler et al., Neuro Oncol, 2017. OMRF developed a proprietary monoclonal Ab against ELTD1, which is the therapeutic candidate for further development and humanization Anti-ELTD1 Monoclonal Antibody Therapy: Human G55 Gliomas

Tumor Volumes: Targeting ELTD1 with a mAb is effective in a G55 orthotopic glioma model (human G55 xenograft) Anti-ELTD1 antibody therapy (optimized monoclonal) is more effective than the pAb (non-optimized polyclonal) Anti-ELTD1 Monoclonal Antibody Therapy: Human G55 Gliomas

Survival: Targeting ELTD1 with a mAb is effective in a G55 orthotopic glioma model (human G55 xenograft) Anti-ELTD1 antibody therapy (optimized monoclonal) is more effective than the pAb (non-optimized polyclonal)

pAb anti-ELTD1 treatment was not found to be as effective in a G55 tumor model, Anti-ELTD1 Antibody Therapy Decreases Microvascularity

mAb Anti-ELTD1 therapy is more effective at decreasing micro-vascularity than pAb therapy, as measured by perfusion MRI (relative cerebral blood flow [rCBF]) mAb Anti-ELTD1 restores vascularity to normal Anti-ELTD1 Antibody Therapy Decreases Hemorrhaging Untreated Polyclonal

20X 20X Monoclonal Avastin

20X 20X mAb Anti-ELTD1 therapy is more effective at decreasing hemorrhaging than Avastin, as measured by histological iron staining. Avastin causes hemorrhaging. mAb Anti-ELTD1 has no associated hemorrhaging Anti-ELTD1 Antibody Therapy Decreases Notch mAb Anti- UT pAb ELTD1 therapy is more 20X 20X effective at mAb Normal decreasing Notch than Avastin, as measured by 20X 20X IHC staining. Avastin

20X Avastin has no effect on Notch levels. mAb Anti-ELTD1 restores Notch levels to normal (contralateral) Summary: Anti-ELTD1 Therapy

Anti-ELTD1 monoclonal antibody therapy:  Is effective in a highly aggressive (G55) orthotopic rodent xenograft model of GBM  Reduces tumor volumes  Increases animal survival  Decreases microvascularity  Decreases tumor Notch levels  Decreased microvascularity indicate:

 ELTD1 treatment is anti-angiogenic (similar to VEGF mAb)

 Unlike anti-VEGF treatments, tumor vasculature returns to normal suggesting that the tumor microenvironment is altered  Unlike bevacizumab (VEGF mAb), using an antibody against ELTD1 doesn't cause hemorrhage (either pAb or mAb)  Anti-ELTD1 mAb could also treat other types of Next Steps Scientific: • Further studies planned in using scFV fragment of mAb: • Molecular-targeted MRI to assess binding affinity • Efficacy • Survival • Tumor volumes • Microvasculature (perfusion imaging, MVD) MR image of a G55 tumor-bearing Molecular-targeted MRI with mAb for ELTD1 xenograft mouse MR image of a G55 tumor-bearing xenograft Molecular-targeted MRI with scFV-mAb for mouse ELTD1 SA-HRP

20X

H&E

20X SA-HRP

20X

H&E

20X Anti-ELTD1 Antibody scFv Fragment has High Binding Affinity in G55 GBM Tumor MRI probe either has mAb, pAb or scFv fragment or non- of mAb, or specific IgG non-specific IgG bound to MRI contrast agent construct

mAb Anti-ELTD1 scFv fragment effectively binds to a G55 GBM tumor in a mouse xenograft model mAb Anti-ELTD1 scFv fragment binds to GBM tumors Anti-ELTD1 Antibody scFv Fragment has High Binding Affinity in G55 GBM Tumor Binding affinity kinetics MRI probe either has mAb, pAb or scFv fragment of mAb, or non-specific IgG bound to MRI contrast agent construct

mAb Anti-ELTD1 scFv fragment effectively binds to a G55 GBM tumor in a mouse xenograft model mAb Anti-ELTD1 scFv fragment binds to GBM tumors effectively Anti-ELTD1 mAb Fragment Therapy: Human G55 Gliomas

Survival: Targeting ELTD1 with a mAb fragment is effective in a G55 orthotopic glioma model (human G55 xenograft) Anti-ELTD1 antibody therapy (optimized mAB fragment) is more effective than the pAb (non- optimized polyclonal)

mAb fragment anti-ELTD1 treatment was found to be as effective as full mAb in a G55 tumor model Anti-ELTD1 mAb Fragment Therapy: Human G55 Gliomas

Tumor Volumes: Targeting ELTD1 with a mAb fragment seems to be effective in a G55 orthotopic glioma model (human G55 xenograft), but was not significant Anti-ELTD1 antibody therapy (optimized mAb fragment) shows a decreasing trend to be effective in reducing tumor volumes, but due to low animal no., was not significant, compared to UT Anti-ELTD1 mAb Fragment Therapy Decreases Microvascularity

mAb fragment Anti-ELTD1 therapy is as effective at decreasing micro-vascularity as pAb therapy, as measured by perfusion MRI (relative cerebral blood flow [rCBF]) mAb fragment Anti-ELTD1 restores vascularity to near normal Next Steps Scientific: • Preliminary data (RNA seq) suggest that ELTD1 treatment could help address other disease indications (MS, ophthalmic conditions, other cancers, wound healing etc) by impacting known genetic pathways implicated in these diseases • Further studies planned in: • Multiple Sclerosis (refractory MS) • Ophthalmology indications: AMD & Retinopathy • Other cancers • Wound healing & anti-hemorrhage Business: • Collaborate with a commercialization partner/investor to advance the asset Next Steps Multiple Sclerosis (MS) – mouse EAE (experimental autoimmune encephomyelitis) model

or non- specific IgG ng nd i 1 b i D T

L mAb Anti-ELTD1 MRI probe efficiently binds to various E brain regions in an EAE mouse model for MS, compared to non-selective control MRI contrast agent ELTD1 levels are elevated in EAE mice Next Steps Multiple Sclerosis (MS) – mouse EAE (experimental autoimmune encephomyelitis) model

20X 20X Molecular-targeted MR imaging for Immunohistochemistry staining for ELTD1 (streptavidin-HRP binds to biotin ELTD1 moiety of anti-ELTD1 probe) mAb Anti-ELTD1 MRI probe efficiently binds to endothelial cells in brain of an EAE mouse model for MS Immunohistochemistry staining for ELTD1 indicates high levels in endothelial cells of EAE mouse ELTD1 levels are elevated in EAE mice APPENDIX RNA seq. data and other disease indications mAb Anti-ELTD1 Therapy -Fold Changes: 15 Down-regulated >2-fold 7 Upregulated >2-fold Possible MOA Anti-angiogenic Gene Gene Protein SCN5A Sodium channel protein NTSR1 1 • BMP2 is pro-angiogenic type 5 subunit alpha BGLAP bone gamma- and increases vascular ADA Adenosine deaminase carboxyglutamate (gla) density CHRNA1 cholinergic receptor protein • APLN promotes nicotinic alpha 1 subunit MATN2 matrilin 2 angiogenesis, and may be L1CAM L1 cell adhesion CD74 CD74 molecule, major associated with TMZ Anti-ELTD1 molecule histocompatibility resistance F2RL3 coagulation factor II complex, class II invariant (thrombin) receptor-like chain • Inhibition of L1CAM 3 SLC14A1 solute carrier family 14 normalizes vasculature ASS1 argininosuccinate (urea transporter), • Decrease of VWA1 in EC synthase 1 member 1 (Kidd blood results in dysfunctional PCDH19 protocadherin 19 group) angiogenesis NCAM1 neural cell adhesion IKZF1 IKAROS family zinc finger 1 Effect on cell motility molecule 1 (Ikaros) • PRICKLE 1 plays a role in stanniocalcin 2 VWA1 von Willebrand factor A STC2 tumor cell motility; APLN apelin domain containing 1 SPNS2 spinster homolog 2 negative regulator of wnt BMP2 bone morphogenetic Effect on cell growth protein 2 • L1CAM required for growth ZNF469 zinc finger protein 469 of CD133+ glioma cells MME membrane metallo- endopeptidase PRICKLE1 prickle homolog 1 mAb Anti-ELTD1 Therapy Gene-Fold Changes: Down-regulated >2-fold MS Implications Gene >2-fold Protein Description decrease SCN5A -5.11 Sodium channel protein Protein mediates the voltage-dependent sodium ion permeability type 5 subunit alpha of excitable membranes

SCN5A encodes several sodium channels including NaV1.5. NaV1.5 is upregulated in EAE (experimental autoimmune encephalomyelitis) mouse model (Pappalardo et al., Neuroreport 2014; 25: 1208-15) ADA -4.53 Adenosine deaminase Catalyzes the hydrolytic deamination of adenosine and 2-deoxyadenosine Elevated adenosine deaminase in CSF of MS patients (Samuraki et al., Mult Scler Relat Disord 2017; 13: 44-46) APLN -2.81 apelin Gene encodes a peptide that functions as an endogenous ligand for the G protein coupled receptor APJ Serum apelin-13 levels are elevated in MS patients (Alpua et al., Ann Indian Acad Neurol 2018; 21: 126) SPNS2 -2.74 spinster homolog 2 SPNS2 has 2,740 functional associations with biological entities spanning 8 categories SPNS2 knock-out mice are protected against EAE (Donoviel et al., FASEB J 2015; 29: 5018-28) BMP2 -2.71 bone morphogenetic Gene encodes a secreted ligand of the TGF-beta (transforming protein 2 growth factor-beta) superfamily of High serum levels of BMP-2 related to failure of remyelination and neuro- regeneration in relapsing remitting MS patients (Pen et al., J Neuroimmunol 2017; 310: 120-128) mAb Anti-ELTD1 Therapy Gene-Fold Changes: Upregulated >2-fold MS Implications

Gene >2-fold Protein Description increase CD74 3.45 CD74 molecule, major Associates with class II major histocompatibility complex (MHC) and is histocompatibility complex, an important chaperone that regulates antigen presentation for immune class II invariant chain response. Also serves as for cytokine macrophage migration inhibitory factor (MIF) which, when bound to the encoded protein, initiates survival pathways and cell proliferation. MIF receptor CD74 is downregulated in B cells from early onset MS patients (Rijvers et al., Eur J Immunol 2018; in press) IKZF1 2.63 IKAROS family zinc finger 1 Gene encodes a transcription factor that belongs to the family of zinc- (Ikaros) finger DNA-binding proteins associated with chromatin remodeling. The expression of this protein is restricted to the fetal and adult hemo- lymphopoietic system, and it functions as a regulator of lymphocyte differentiation. IKZF1 plays a central regulatory role in controlling gene expression in the pathogenesis of MS (Liu et al., Mol Biol Rep 2013; 40: 3731-7) mAb Anti-ELTD1 Therapy Gene-Fold Changes: Down-regulated >2-fold Other Disease Indications

Retinopathy Gene >2-fold Protein Description decrease ADA -4.53 Adenosine deaminase Catalyzes the hydrolytic deamination of adenosine and 2-deoxyadenosine Causal role of ADA2 in inflammation; Increased ADA2 associated with diabetic retinopathy (Fulzele et al., Biomed Res Int 2015; 846501) APLN -2.81 apelin Gene encodes a peptide that functions as an endogenous ligand for the G protein coupled receptor APJ Apelin is associated with retinal neovascularization (McAnally et al., PLoS One 2018; 13(9): e0202436; Ishimaru et al., Sci Rep 2017; 7(1): 15062) Other Cancers Gene >2-fold Protein Description decrease SCN5A -5.11 Sodium channel protein Protein mediates the voltage-dependent sodium ion permeability type 5 subunit alpha of excitable membranes Voltage-gated Na+ channels (VGSC) are associated with malignancy progession in breast cancer cells (Mohammed et al., Int J Oncol 2016; 48(1): 73-83) VGSC enhances metastasis (motility, invasion and oncogenic expression) in breast cancer cells (Aktas et al., Acta Biochim Biophys Sin (Shanghai) 2015; 47(9): 680-6) L1CAM -3.79 L1 cell adhesion molecule Protein encoded by this gene is an axonal glycoprotein belonging to the immunoglobulin supergene family Overexpression of L1CAM associated with proliferation and metastasis of pancreatic cancer cells (Zuo et al., Pancreatology 2018; 18(3): 328- 333) mAb Anti-ELTD1 Therapy Gene-Fold Changes: Upregulated >2-fold Tissue Regeneration Gene >2-fold Protein Description increase BGLAP 3.52 bone gamma- BGLAP has 4,051 functional associations with biological entities carboxyglutamate (gla) spanning 8 categories (molecular profile, organism, chemical, functional protein term, phrase or reference, disease, phenotype or trait, structural feature, cell line, cell type or tissue, gene, protein or microRNA). Thought to stabilize healing phase (Ghiacci et al., Biomed Mater 2017; 12(4): 045016) MATN2 3.48 matrilin 2 Gene encodes member of von Willebrand factor A domain containing protein family. Thought to be involved in the formation of filamentous networks in the extracellular matrices of various tissues. Required for regeneration of muscle, nerve and other tissues in wound healing (Korpos et al., Neural Regen Res 2015; 10(6): 866-9) Matrilin 2 involved in peripheral nerve regeneration (Malin et al., J Cell Sci 2009; 122(Pt 7): 995-1004) VWA1 2.49 von Willebrand factor A Belongs to von Willebrand factor A (VWFA) domain superfamily of domain containing 1 ECM proteins and appears to play a role in cartilage structure and function Associated with fracture healing (Yuan and Cai, Mol Med Rep 2017; 16(4): 4529-36) Other Cancers Gene >2-fold Protein Description increase CD74 3.45 CD74 molecule, major Associates with class II MHC and involved in regulating antigen histocompatibility complex, presentation for immune response. Cell surface receptor for cytokine class II invariant chain macrophage MIF, involved in survival pathways and cell proliferation. Highly expressed in thyroid carcinoma (Cheng et al., Endocr Relat Cancer 2015; 22(2): 179-90) Rheal Towner, M.D., Ph.D. Associate Member

Advanced Magnetic Resonance Center