johnson revised supplemental.vf lj...

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Supplemental Materials

Methods References Supplemental Figure 1. Evaluation of hypoxic response in HUVECs. Supplemental Figure 2. In vitro characterization of two anti-EGFL7 antibodies. Supplemental Figure 3. In vitro characterization of the humanized anti-EGFL7 antibody 18F7. Supplemental Figure 4. Activity of anti-VEGF and anti-EGFL7 in the H1299 NSCLC xenograft model. Supplemental Figure 5. Response rates following long-term treatment in the KrasG12D; p53Frt/Frt NSCLC GEMM. Supplemental Figure 6. Response rates comparing short- versus long-term treatment in the KrasG12D; p53Frt/Frt NSCLC GEMM. Supplemental Figure 7. Characterization of CPCs. Supplemental Figure 8. Study designs for the Phase Ia and Ib studies for anti-EGFL7. Supplemental Figure 9. Assessment of biological variability of CPCs in blood from healthy donors and cancer patients.

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Supplemental Methods

Hypoxic response in HUVEC

HUVECs were treated with vehicle (DMSO) or the hypoxic mimetics DFX (Sigma Cat# D9533)

at 400 µM or CoCl2 (Sigma Cat# 15862) at 200 µM and cultured under normoxic (21% O2) or true

hypoxic (1% O2) conditions. After overnight incubation, cells were harvested and cell lysates

prepared for western blot or quantitative RT-PCR analysis of several HIF1α target genes and

the house keeping gene Rpl19. Anti-HIF1α (BD, Cat# 610968, 1:500) and γ-tubulin (Sigma Cat#

T5325, 1:5000) antibodies were used for western blotting against equal amount of total proteins

from lysates prepared from HUVECs treated with the aforementioned conditions. Quantitative

RT-PCR was performed using the following oligos from Applied Biosystems: Vegf:

Hs00900055_m1, Angptl4: Hs01101127_m1, Plgf: Hs00182176_m1, Slc2A1: Hs00892681_m1,

Ndrg1: Hs00608387_m1. Oligos for Rpl19 were synthesized at Genentech and the sequences are:

forward primer - ACC CCA ATG AGA CCA ATG AAA TC, reverse primer - ATC TTT GAT

GAG CTT CCG GAT CT, and Taqman probe - AAT GCC AAC TCC CGT CAG. Expression of

the HIF1α target genes was expressed as a ratio relative to the house-keeping gene Rpl19. Two

independent experiments were performed with the qRT-PCR measurements, and four

independent experiments were performed with the western blot measurement. DFX and CoCl2

were found to up-regulate HIF1α protein and HIF1α target genes in all independent

experiments.

Cell adhesion assay

Recombinant mouse or human EGFL7 protein (Genentech (1)), or Fibronectin (#11051407001,

Roche) were diluted in Sodium Carbonate buffer (pH 9.6) to 5 µg/ml concentration. 100 µl/well

were added to NUNC 96 well flat-bottom Immuno plates (MaxiSorp N/Ster 439454, VWR

62409-002) and incubated overnight at 4ºC. The next morning, plates were blocked with 1% BSA

in PBS (Sigma A9418) for at least 30 minutes (min) and then washed 3 times with PBS. HUVECs

3

were added at 20,000 cells/well in appropriate cell growth medium (EGM-2 Lonza #2CC-3162),

with and without testing antibodies at a range of concentrations. Each condition was evaluated

with 5 technical replicates. In separate tubes, the amount of cells seeded in 5 wells was added

and set aside for calculating cell seeding later. Plates with cells were spun down for 5 min at 140

g to synchronize contact of cells with culture substrate. Plates were then incubated in a tissue

culture incubator for 30 min, and then washed 3 times with PBS. All liquid from wells was

removed and the plates were frozen at –80ºC for at least 2 hours, and then thawed at room

temperature (RT) and 200 µl/well of CyQuant buffer (Molecular Probes, C7026) was added.

One ml of buffer was also added to the separate tube containing 5 times the cells seeded per

tube; after careful mixing, 200 µl from this tube was added to 3 empty wells each on the assay

plate to give a reading of cells seeded. After 10 min incubation, optical densities (OD) were read

using a plate reader (following manufactures instruction). Cell adhesion was measured as the

ratios between the # of cells in the wells used for adhesion assay relative to # of cells in the

wells used as seeding control. Each experiment was repeated at least 3 times, and results were

similar. Representative experiments are presented in Supplemental Figures 2 and 3.

Humanization of murine anti-EGFL7 antibodies

CDR grafts, generated by cloning VL positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) and VH

positions 26-35 (H1), 49-65 (H2) and 95-102 (H3) into the human kappa I and VH3 consensus

framework sequences, were displayed monovalently as Fab on phage (2). Important framework

vernier positions were rapidly identified using a framework toggle phage library that offered

either the murine or human amino acid at vernier position 87 in VL, and positions 48, 67, 69, 71,

73, 75, 76, 78 and 80 in VH (2). Affinity maturation was achieved using a pool of 76 single

position libraries with each library containing all possible 20 amino acids at a single CDR

position.

Multiple forms of antigen were tested for phage selections. Full length or truncated

EGFL7 (5 µg/ml) was immobilized in 50 mM sodium bicarbonate pH 9.6 on MaxiSorp

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microtiter plates (Nunc) overnight at 4oC. EMI1 and p5 peptides were either biotinylated

through their free cysteine (using maleimide PEO2-bitin; Pierce) or through the free amine on

their amino terminus (using NHS-LC-biotin, Pierce) using a 2-fold molar excess of biotin

reagent in PBS. Biotinylated EMI1 and p5 peptides were captured on neutravidin (2 µg/ml) that

had been immobilized in 50 mM sodium bicarbonate pH 9.6 on MaxiSorp microtiter plates

(Nunc) overnight at 4oC. All plates were blocked for at least 1 h using Casein Blocker (Pierce).

Phage were harvested from the culture supernatant and suspended in PBS containing

5% powdered milk and 0.05% Tween 20. Following addition of the phage library for 1 hr,

microtiter wells were washed extensively with PBS containing 0.05% Tween 20 (PBST) and

bound phage were eluted by incubating the wells with 20 mM HCl, 500 mM KCl for 30 min.

Eluted phage were neutralized with 1 M Tris, pH 8 and amplified using XL1-Blue cells and

M13/KO7 helper phage and grown overnight at 37 oC in 2YT, 50 µg/ml carbenacillin. The titers

of phage eluted from a target containing well were compared to titers of phage recovered from

a non-target containing well to assess enrichment. Selection stringency was increased by both

capturing phage that bound to decreasing concentrations of biotinylated p5 peptide in solution

followed by capture on netravidin for 10 min (on-rate selection) and by increasing the washing

time and temperature to allow weak binding phage to be washed away (off-rate selection).

Antibody-antigen affinity determinations

Affinity determinations were performed by surface plasmon resonance using a BIAcoreTM-2000.

Truncated EGFL7 or p5 peptide was immobilized (approximately 50 – 200 RU) in 10 mM

Sodium Acetate pH 4.8 on a CM5 sensor chip. Purified 18F7 IgG variants were injected (using a

2-fold serial dilution of 0.5 to 1000 nM in PBST) at a flow rate of 30 µL/min. Each sample was

analyzed with 3-minute association and 3.5-minute disassociation. After each injection the chip

was regenerated using 10 mM Glycine pH 1.7. Binding response was corrected by subtracting a

control flow cell from 18F7 variant IgG flow cells. A 1:1 Languir model of simultaneous fitting

of kon and koff was used for kinetics analysis.

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MECA32 immunofluorescence staining on RIP-TβAg tumor sections

Pancreases were dissected out from euthanized mice and fixed in 10% neutral buffered formalin

overnight at RT. Fixed pancreases were transferred to 70% ethanol and stored for several days.

Tissues were then embedded into paraffin blocks according to standard protocol, and 5 µm

sections were cut and placed on glass slides. Before staining, tissue sections were submerged in

the following solutions for 5 min each: Xylene; 100% Ethanol; 95% Ethanol; 85% Ethanol; 75%

Ethanol; distilled water. Antigen Retrieval was performed using Dako Target Retrieval Solution

10X Concentrate (Dako, catalog #S1699) according to manufacturer’s instructions. Cool slides at

room temperature for 20 minutes. Wash three times with PBS. Incubate slides in 5% Goat Serum

+ 0.2% Triton X-100 in PBS for 1 hour at room temperature to block non-specific IgG binding

sites. Anti-MECA32 antibody (BD Pharmingen, catalog #550563) was used at 1:100 diluted in

2% goat serum / 0.2% Triton X-100 in PBS and incubated overnight at 4ºC. Slides were washed

with 6 changes of 0.2% Triton X-100 in PBS. Biotinylated Donkey anti Rat IgG (Jackson

Immunoresearch Laboratories, catalog #712-065-153) was used at 1:250 diluted in 2% goat

serum / 0.2% Triton X-100 in PBS and incubated at room temperature for 2 hours. Slides were

washed with 6 changes of 0.2% Triton X-100 in PBS. Strepavidin-Alexa 488 (Molecular Probes,

catalog #S11223) was used at 1:250 diluted with 2% goat serum / 0.2% Triton X-100 in PBS and

incubated at room temperature for 2 hours. Slides were washed with 6 changes of PBS. A

duplicate set of slides was processed without the primary antibody (anti-MECA32) but

included all the subsequent treatments to serve as negative controls. Slides were incubated in

Sudan Black B Solution (Sigma, catalog #3801-200mL) for 10 min at RT to eliminate red blood

cell autofluorescence, and then washed with 6 changes of PBS. Mount slides with Fluoromount-

G (Southern Biotechnology, catalog #0100-01).

Microvascular density analysis of the RIP-TβAg carcinomas

Vessel density analysis was performed in a semi-automated fashion utilizing Definiens

Developer, version 7.0.7 (Definiens AG, Munich). Digital whole slide images of immunostained

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RIP-TβAg pancreas tissue sections were acquired on an Applied Imaging Ariol system, and islet

tumors were identified using custom algorithms. Accurate segmentation was confirmed by

visual inspection, and any errors were corrected manually. Tumors in each group confirmed as

invasive carcinomas by visual inspection were included in the subsequent analysis. Tissue that

positively immunostained for the endothelial marker MECA32 within each invasive islet

carcinoma was isolated by threshold based segmentation and classified as tumor vasculature.

These segmentation results were also manually verified. Vessel length was then calculated

using the length to width ratio of each segment derived from a bounding box approximation,

expressed as

#P" # $" , where

#P" is the total number of pixels contained in the bounding

box

P" , and

"# is the length/width ratio of an image object

". The length of each vessel

segment was then summed and normalized to the tumor area. For each tumor, microvascular

density (MVD) was calculated as the “sum of vessel lengths normalized to tumor area”.

To examine differences in the mean MVD values in the different treatment groups, the

raw data from all animals and tumors was plotted. A mixed-effects model was fit to the MVD

response, with the six treatments as fixed effects, plus random animal effects. Theoretically, the

tumors within one animal are not independent, and combining them as such (ignoring the

random animal effects) could lead to incorrect conclusions. The fixed treatment effects

(estimated mean MVDs) were compared by calculating 95% confidence intervals from the

mixed-effects model. Data from this study is presented in Figure 2.

Electron microscopic analysis of tumor vessels in the RIP-TβAg carcinomas

Tumors (2 mm diameter) from 2-3 mice per treatment were fixed in half-strength Karnovsky

fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 0.025% CaCl2.2H2O and 0.1 M sodium

cacodylate buffer, pH 7.4) for 2-3 hours at room temperature. After rinsing, the tumor tissue

blocks were postfixed with 1% OsO4 and 1.5% K3Fe(CN)6, in 0.07 M Na-cacodylate, stained en

bloc with 0.5% aqueous uranyl acetate, dehydrated in ethanol and embedded in Epon. Ultrathin

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sections were stained with uranyl acetate and lead citrate. Per tumor, an area of 35000 µm2, at a

minimum distance of 150 µm from the tumor margin, was screened and the following items

were quantified: number of blood vessel (BV) transections, number of fibrin clots, number of

BVs associated with a fibrin clot, number of BVs with at least 1 EC with apoptotic features

(condensed nucleoplasm and cytoplasm), or with early necrotic features (distended RER

cisterns and/or nuclear envelope), number of EC nuclei, and number of apoptotic EC nuclei.

EGFL7 and MECA32 or CD31 immunofluorescent staining on frozen RIP-TβAg pancreatic and

KrasG12D; p53Frt/Frt lung sections

RIP-TβAg transgenic mice were perfused with PBS and pancreas tissue was cryoprotected in

30% sucrose and then snap frozen in OCT. KrasG12D; p53Frt/Frt mice were perfused with 4%

paraformaldehyde and lung tissue frozen in OCT. For pancreas, 12 µm thick tissue sections

were dried at room temperature (RT) for 5 hours, rehydrated with PBS, and then blocked with

blocking buffer #1 (10% normal goat serum and 0.2% Triton X100 in PBS) for 2 hours. For lungs,

10 µm thick tissue sections were dried at RT for 2 hours and then incubated for 30 minutes at

room temperature in blocking buffer #2 (5% normal donkey serum, 2.5% bovine serum albumin

in PBS). Anti-MECA32 (BD Pharmingen, catalog #550563) was diluted to 2.5 µg/ml, anti-CD31

(BD Pharmingen, catalog #553370) was diluted to 10 µg/ml, and two Armenian-hamster

monoclonal antibodies, 1C8 and 5H7, that recognize different epitopes of EGFL7 were each

diluted to 5 µg/ml (pancreas) or 7.5 µg/ml (lung) in blocking buffers #1 and #2 for pancreas

and lung, respectively, and incubated overnight at 4ºC. Slides were washed with 3 changes of

PBS and incubated for 30 minutes with anti-Armenian hamster IgGs conjugated to CY3 (Jackson

Immunoresearch Laboratories, catalog #127-165-160) and donkey anti-rat conjugated to Alex

488 fluorochrome (Invitrogen, catalog #A21208) diluted 1:400 and 1:300, respectively, in PBS.

Slides were washed with 6 changes of PBS, rinsed with deionized water, and cover slipped with

8

Dako aqueous mounting medium (Dako, catalog #S3023) with 15 µg/ml DAPI (Molecular

Probes, catalog #D3571).

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References

1. Parker LH, Schmidt M, Jin SW, Gray AM, Beis D, Pham T, Frantz G, Palmieri S, Hillan K,

Stainier DY, et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube

formation. Nature. 2004;428(6984):754-8.

2. Baca M, Presta LG, O'Connor SJ, and Wells JA. Antibody humanization using monovalent

phage display. The Journal of biological chemistry. 1997;272(16):10678-84.

3. Singh M, Couto SS, Forrest WF, Lima A, Cheng JH, Molina R, Long JE, Hamilton P, McNutt

A, Kasman I, et al. Anti-VEGF antibody therapy does not promote metastasis in genetically

engineered mouse tumour models. The Journal of pathology. 2012;227(4):417-30.

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Supplemental Figure 1

A HIF1 Western Blot using HUVEC Lysates

B Quantitative RT-PCR analysis of HIF1 target genesVegf Angptl4 Plgf

Slc2A1 Ndrg1

MW MW

151 kD

97 kD

64 kD

51 kD

39 kD

151 kD

97 kD

64 kD

51 kD

39 kD

HIF1 HIF1

TubulinTubulin

DMSO

DFXCoCl2

21% O2

1% O2

******

**

***

***

******

***

***

******

*** ***

*** ***

*** p<0.001 ** p<0.005

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Supplemental Figure 1 Evaluation of hypoxic response in HUVECs. (A & B) HUVECs were either treated with vehicle (DMSO) or the hypoxia mimetics DFX or CoCl2, or cultured under normoxic (21% O2) or hypoxic (1% O2) conditions. (A) Protein lysates were generated from HUVECs and analyzed by

western blot for HIF1α and γ-Tubulin expression. Shown here is a representative result from four independent experiments. (B) RNA was extracted from HUVECs and analyzed by quantitative RT-PCR for the expression of five hypoxia-responsive genes. Each dot in the graphs represents data derived from a single well of HUVECs and is the mean of three technical PCR replicates. Error bars represent standard deviations amongst three biological replicates. P values indicate statistically significant differences between the treated samples and the control for each gene, and were calculated using unpaired, two-tailed Student’s t-test. Shown here is a representative result from two independent experiments. Both hypoxia and hypoxia mimetics

increased the expression of HIF1α protein (A) and hypoxia-responsive genes (B).

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B

C

Antibody Recombinant EGFL7 Protein ka (1/Ms) kd (1/s) KD (nM)

10G9 Human 2.62e5 2.88e-4 1.10 Murine 5.83e5 1.07e-3 1.83

18F7 Human 1.41e5 5.78e-5 0.411 Murine 5.37e5 1.02e-4 0.191

A

0 2 5 15 45 135405 0 2 5 15 45 13

5405 0 2 5 15 45 13

5405

0

25

50

75

100Fibronectin human EGFL7 murine EGFL7

18F7 Antibody Concentration (nM)

% Cell B

ound

0 2 5 15 45 135405 0 2 5 15 45 13

5405 0 2 5 15 45 13

5405

0

25

50

75

100Fibronectin human EGFL7 murine EGFL7

10G9 Antibody Concentration (nM)

% Cell B

ound

Supplemental Figure 2 In vitro characterization of two anti-EGFL7 antibodies. (A) Binding affinities of two anti-EGFL7 antibodies 10G9 and 18F7 were measured by BIACORE at 25ºC against recombinant human and mouse EGFL7 proteins. Each antibody bound human and mouse antigens with comparable affinities. 18F7 is a higher affinity antibody than 10G9. (B & C) HUVEC adhesion to Fibronectin (open bars), recombinant human EGFL7 (grey bars) or murine EGFL7 (black bars) protein was

13

measured in the presence of increasing concentrations of anti-EGFL7 antibodies 18F7 (B) or 10G9 (C). Both antibodies inhibited cell adhesion to recombinant EGFL7 proteins in an antibody concentration dependent manner, but had no effect on cell adhesion to Fibronectin. Each bar represents the mean of five replicates. Error bars represent standard deviations.

14


A

B

10

20

30

40

50

60

0.001 0.01 0.1 1 10 100

IC50 humanized 18F7 = 2.35 µg/ml

IC50 mouse 18F7 = 3.14 µg/ml

18F7 Antibody Concentration ( g/ml)

% Cell Bound

Supplemental Figure 3 In vitro characterization of the humanized anti-EGFL7 antibody 18F7. (A) Binding affinities of the original anti-EGFL7 antibody murine 18F7 and its humanized derivative were measured by BIACORE at 37ºC against recombinant human, cynomolgus monkey, rat, and mouse EGFL7 proteins. Binding affinities to the EGFL7 proteins of four different species were comparable. Note that binding affinities in this experiment were lower than reported in Supplemental Figure 2 because the assay temperatures were different in these two experiments. (B) HUVEC adhesion to recombinant murine EGFL7 protein was measured in the presence of increasing concentrations of murine 18F7 (m18F7, dashed line) or humanized 18F7 (h18F7, solid line). Both antibodies inhibited HUVEC adhesion with similar potency. Each bar represents mean of five replicates. Error bars represent SEM.

15


0 22 44 66 88

0

200

400

600

800

1000

H1299 Xenograft Model

Days on study

Mean tumor volumes (mm3)

V, 10 mg/kg (12)

VE, 10 + 0.2 mg/kg (12)

VE, 10 + 2.0 mg/kg (12)

VE, 10 + 20 mg/kg (12)

Supplemental Figure 4 Combination of anti-EGFL7 plus anti-VEGF inhibits the growth of H1299 NSCLC xenograft tumors. Mean tumor volume plots of mice harboring established tumors and treated with the indicated regimens. Anti-VEGF (B20-4.1) was dosed at 10 mg/kg and anti-EGFL7 (h18F7) at 0.2, 2.0, or 20.0 mg/kg. Both antibodies were dosed i.p., 1x/wk, until tumors reached 1000 mm3. The study was terminated on day 84. Tumors that reached endpoint before day 84 were continuously plotted at the end point volume of 1000 mm3. N = 12 mice per group.

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Supplemental Figure 5 In Vivo Tumor Burden (m

m2 )

Weeks on Study

2

3

4

5

10

25

50

75

100

150

200

300

0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6

Control, N=19 V (5 mpk), N=16 VE (5 + 0.1 mpk), N=20 VE (5 + 1.0 mpk), N=19 VE (5 + 25 mpk), N=30

BTreatment

# Mice Scanned at Baseline

Scan Timepoint (Weeks)

# Mice Scanned Deaths # PR # SD # PD % PR + SD^ % PD^

C 19 2 17 2 0 11 6 57.9% 42.1%

V (5 mpk) 16 2 15 1 2 13 0 93.8% 6.3%

VE (5 + 0.1 mpk) 20 2 19 1 2 17 0 95.0% 5.0%

VE (5 + 1.0 mpk) 19 2 19 0 1 18 0 100.0% 0.0%

VE (5 + 25 mpk) 30 2 30 0 1 28 1 96.7% 3.3%

C 19 4 13 6 0 2 11 10.5% 89.5%

V (5 mpk) 16 4 14 2 1 13 0 87.5% 12.5%

VE (5 + 0.1 mpk) 20 4 18 2 2 16 0 90.0% 10.0%

VE (5 + 1.0 mpk) 19 4 18 1 2 14 2 84.2% 15.8%

VE (5 + 25 mpk) 30 4 29 1 0 26 3 86.7% 13.3%

C 19 6 5 14 0 0 5 0.0% 100.0%

V (5 mpk) 16 6 13 3 0 10 3 62.5% 37.5%

VE (5 + 0.1 mpk) 20 6 13 7 0 8 5 40.0% 60.0%

VE (5 + 1.0 mpk) 19 6 17 2 0 15 2 78.9% 21.1%

VE (5 + 25 mpk) 30 6 24 6 1 14 9 50.0% 50.0%

A

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Supplemental Figure 5 Response rates following long-term treatment in the KrasG12D; p53Frt/Frt NSCLC GEMM. (A) In vivo x-ray micro-computed tomography (micro-CT) was used to monitor tumor growth in individual NSCLC mice for each treatment regimen examined. Thin blue (male) & pink (female) lines represent the interval between serial images at baseline, ~2 weeks, ~4 weeks, and ~6 weeks (if alive) post-study start. Thick black lines represent the fitted lines using a linear mixed effect model. The antibody doses (mg/kg = mpk) and number of animals (N) examined are depicted next to each treatment arm. All treatments resulted in significantly decreased tumor burden growth rates relative only to Control based upon linear mixed effect modeling (P<0.0005). Note: mice in the Control and V treatment cohorts represent a subset of mice reproduced with permission from (3), DOI: 10.1002/path.4053. (B) Quantitation of tumor burden response rates at ~2, 4, and 6 weeks post-first treatment dose. Tumor burden responses at each time point, all relative to the initial baseline micro-CT result, were classified as follows: partial response (PR) if there was a >30% reduction; stable disease (SD) if the percent change was between -30% to 100%; progressive disease (PD) if there was a >100% increase. ^Since very few PRs were observed, the number of PRs and SDs were pooled; the final PD% represents pooled numbers of PD and deaths. None of the VE combination treatments resulted in significant changes in response rates at any time point relative to V. For panels (A) and (B), all antibodies were dosed i.p.: control anti-ragweed IgG2a (C; 5 mg/kg (mpk) + 1.0 mpk, 2x/wk), anti-VEGF (V, B20-4.1.1; 5 mpk, 2x/wk), anti-EGFL7 (E, m18F7; 0.1, 1.0 or 25 mpk, 2x/wk). These data were used to generate the data presented in Table 1. For Figure 5, B-G, additional mice were included in the C, V, and VE (5 + 1.0 mpk) treatment cohorts; however, only those mice that were exposed to similar numbers of longitudinal micro-CT scans across all five treatment cohorts were used to calculate response rates.

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A

In Vivo Tu

mor Burde

n (m

m2 )

Weeks on Study

45

10

25

50

75

100

150

200

300

0 2 4 6 0 2 4 6

VE (5 + 1.0 mpk), N=46 V2E2 (5 + 1.0 mpk), N=28

BTreatment

# Mice Scanned at Baseline

Scan Timepoint (Weeks)

# Mice Scanned Deaths # PR # SD # PD % PR + SD^ % PD^

VE 46 2 43 3 4 39 0 93.5% 6.5%

V2E2 28 2 25 3 4 21 0 89.3% 10.7%

VE 46 4 42 4 2 37 3 84.8% 15.2%

V2E2 28 4 23 5 0 18 5 64.3% 35.7%

VE 46 6 37 9 1 30 6 67.4% 32.6%

V2E2 28 6 17 11 1 4 12 17.9% 82.1%

******

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CFo

ld Growth (L

og2)

Change in Volum

e (%)

SD

PD

PR

3

2

0

1

-2

-1

700%

0

300%

100%

-50%

-75%

2 Week CT Scan

D

Fold Growth (L

og2)

Change in Volum

e (%)

3

2

0

1

-2

-1

700%

0

300%

100%

-50%

-75%

SD

PD

PR

4 Week CT Scan

E

SD

PD

PRFold Growth (L

og2)

Change in Volum

e (%)

3

2

0

1

-2

-1

0

700%

300%

100%

-50%

-75%

6 Week CT Scan

V2E2 (5 + 1.0 mpk)VE (5 + 1.0 mpk)

20

Supplemental Figure 6 Response rates comparing short- versus long-term treatment in the KrasG12D; p53Frt/Frt NSCLC GEMM. (A) In vivo x-ray micro-computed tomography (micro-CT) was used to monitor tumor growth in individual NSCLC mice for each treatment regimen examined. Thin blue (male) & pink (female) lines represent the interval between serial images at baseline, ~2 weeks, ~4 weeks, and ~6 weeks (if alive) post-study start. Thick black lines represent the fitted lines using a linear mixed effect model. The antibody doses (mg/kg = mpk) and number of animals (N) examined are depicted next to each treatment arm. VE resulted in a significantly decreased tumor burden growth rate relative to V2E2 based upon linear mixed effect modeling (P<0.005. (B) Quantitation of tumor burden response rates at ~2, 4, and 6 weeks post-first treatment dose. Tumor burden responses at each time point, all relative to the initial baseline micro-CT result, were classified as follows: partial response (PR) if there was a >30% reduction; stable disease (SD) if the percent change was between -30% to 100%; progressive disease (PD) if there was a >100% increase. ^Since very few PRs were observed, the number of PRs and SDs were pooled; the final PD% represents pooled numbers of PD and deaths. VE resulted in a significant change in response rates relative to V2E2 at the 6-week treatment interval based upon Fisher’s exact test (***P<0.0005). (C-E) Waterfall plot depicting tumor growth rates as determined by in vivo micro-CT ~2 (C), 4 (D), and 6 (E) weeks post-first dose with anti-VEGF plus anti-EGFL7, either until the end of study (VE, blue) or for two weeks only (V2E2, purple). The left y-axis indicates the log2 of tumor fold growth relative to the baseline micro-CT result, whereas the right y-axis depicts the percent change in tumor volume. The dotted line indicates a -30% reduction in tumor burden volume. The vertical graphic on the right indicates the boundaries of the response rate classes defined above and quantified in (B). For panels (A-E), all antibodies were dosed i.p.: anti-VEGF (V, B20-4.1.1; 5 mpk) plus anti-EGFL7 (E, m18F7; 1.0 mpk); VE = 2x/wk until the end of study; V2E2 = 2x/wk for two weeks only and then discontinued. These data were used to generate the data presented in Table 2 and Figure 6, A and B. A subset of the mice in the VE cohort was used to support the studies in Table 1 and Supplemental Figure 5.

21


A B

Signal Intensity

1

4

16

64

256

1024

4096

16384

ALDHA1 CD133 CD117 VEGF VWF KDR ITGB3

CD45-

CECs

CPCs

Signal Intensity

WBC PB PBMC AML CLL CML

0

500

1000

1500

CD34+

CD105 PE-H VEGFR2 APC-H

C

Count

Count

Isotype PE-H Isotype APC-H

D Normal Human PBMC

CPC

CEC

Tumor Bearing Mouse Blood

CPC

CEC

Antibody

Dose (mpk)

(N)

16

32

64

128

256

512

1024

2048

4096E

# of CPCs / ml Whole Blood

RIP-T AG

C

5 + 10

(11)

V

5

(7)

VE (hu)

5 + 1.5

(3)

VE (hu)

5 + 10

(5)

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Supplemental Figure 7 Characterization of circulating progenitor cells (CPCs). (A) Prevalence of EGFL7 gene expression in peripheral blood cells was evaluated in the Gene Logic database using the 218825_at probe set sequence present on the U133A Affymetrix genechip. Gene expression was evaluated in white blood cells (WBC, n=39), peripheral blood CD34+ cells (PB CD34+, n=8), peripheral blood mononuclear cells (PBMCs, n=10), and PBMCs from acute myeloblastic leukemia (AML, n=5), chronic lymphoid leukemia (CLL, n=38), and chronic myeloid leukemia (CML, n=5). Y-axis represents global normalized MAS 5.0 signal intensity. (B) Circulating endothelial cell (CEC) and circulating progenitor cell (CPC) populations were sorted from healthy donor PBMCs. As a negative control, CD45+ cells lacking CD31 or CD34 were also sorted from the same donors. RNA extracted from these cells was analyzed on Affymetrix U133Plus arrays. The y-axis represents global normalized MAS5.0 signal intensity. (C) CPCs (2x105/well) were allowed to differentiate on Fibronectin-coated 24-well plates in endocult medium for 21 days. Cells were fixed and incubated with primary antibodies against CD105-PE or VEGFR2-APC or isotype controls after which the stained cells were analyzed on the BD FACS canto cytometer. Data were analyzed on FACSDiva. (D) Comparison of CEC and CPC populations between peripheral blood obtained from either the MDA-MB231 xenograft tumor

model (tumor volumes ~ 300 mm3) or healthy human donors. (E) RIP-TβAg mice were treated with control (C) anti-ragweed (murine IgG2a) + anti-gD (human IgG1) or anti-VEGF (V, B20-4.1.1) either alone or in combination with anti-EGFL7 (E (hu), h18F7). All antibodies were dosed at the concentrations specified below each cohort via intraperitoneal (i.p.) injection, 1x/wk on days 1 and 8. Whole blood was collected on Day 15 (or earlier if the mice became moribund) via cardiac puncture for CPC enumeration. The total number of mice examined (N) is indicated below each cohort. The mean +/- SEM is depicted. Please note that the mice in the control (C) and anti-VEGF (V) groups are reproduced from Figure 7F for the purposes of a direct comparison. Additionally, some or all of the mice from these four cohorts were used to support Figure 2B.

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Phase Ib

anti-EGFL7 2 mg/kg bevacizumab 10 + paclitaxel*

3


9

Arm A

Q2 week dosing [h18F7] Arm B

Q2 week dosing [h18F7]

Expansion Cohort 5 mg/kg and 10 mg/kg bevacizumab 10 mg/kg

10

anti-EGFL7 2 mg/kg bevacizumab 10 mg/kg

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6


3

*paclitaxel 90 mg/m2 qwk

Start Arm

B


6

A

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Phase Ia

Q3 week dosing [h18F7]

anti-EGFL7 0.3 mg/kg 3

Expansion Cohort 3 mg/kg and 15 mg/kg

12 anti-EGFL7 15 mg/kg

6

anti-EGFL7 7.5 mg/kg 3

anti-EGFL7 3 mg/kg 3

anti-EGFL7 1 mg/kg 3

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Supplemental Figure 8 Study designs for the Phase Ia and Ib studies for anti-EGFL7 (h18F7). The number of patients examined per cohort is indicated in the upper right corner of each panel.

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Screen Pre-dose

% C

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Healthy Donors Phase I Patients

Supplemental Figure 9 Assessment of biological variability of CPCs in blood from healthy donors and cancer patients. (A) Longitudinal analysis of CPCs from healthy donor peripheral blood (N=4) collected weekly to evaluate biological variability over a one month period. The y-axis represents the% change from day 1. (B) Screen and pre-dose blood collections were performed on all patients in the Phase Ia and Ib studies to evaluate intra-patient biological variability. Data is shown as % change from pre-dose (Day 0) from N=32 patients. The range in % variability was 1.62 to -11.48 ± 6.58 (CI: 95%). This data was used to set a call for true change in CPCs to 30% (~3-fold away from average variability).

johnson revised supplemental.vf lj...

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