Supplementary Materials and Methods Exploratory toxicology...

Fully Human Anti-Hedgehog Antibodies

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Supplementary Materials and Methods

Exploratory toxicology study in male and female Wistar Rats Wistar (RCCHan) rats

received an intravenous injection (via lateral tail vein) of vehicle (phosphate buffered saline),

10 mg/kg or 50 mg/kg of MEDI-5304 at a dose volume of 10 mL/kg on either the first (day

0) or seventh day (day 7) of the study. Study evaluations included body weights, food

consumption, hematology, coagulation, and clinical chemistry measurements. All animals

were necropsied 7 days after final dose administration. Organs were weighed before fixation

and organ/body weight ratios were calculated (using the final body weight obtained prior to

necropsy), as well as organ/brain weight ratios. All major organs were immersion-fixed in

10% neutral buffered formalin (NBF). Lungs were fixed by trans-tracheal inflation prior to

immersion. Paired tissues were cut longitudinally (left) and transversely (right). The tissues

were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined

microscopically by a veterinary pathologist. Calcified bone was de-calcified and processed

as above. MedImmune is an Association for the Assessment and Accreditation of Laboratory

Animal Care (AAALAC) accredited facility. Necropsy support and histopathology slide

preparation were performed by Charles River Laboratories, Pathology Associates (PAI). The

Institutional Animal Care and Use Committee (IACUC) reviewed and approved the study

protocol in accordance with provisions of the USDA Animal Welfare Act and The Guide for

the Care and Use of Laboratory Animals (NRC1996).

Wistar (RCCHan) rats between 6-7 weeks of age from Harlan Laboratories (Indianapolis, IN)

were assigned to study. Animals were identified with individual ear tags and each cage was

be labeled with a cage card. Animals were group housed in polycarbonate cages, in a

controlled environment (18-26°C and 30-70% relative humidity). Animals were fed Certified


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Global Harlan Teklad Laboratory 2018 Diet and also given various cage-enrichment devices.

Animals were provided ad libitum access to filtered tap water via an automatic watering

system and/or water bottles. The water was routinely analyzed for contaminants and specific

microbes and met the EPA standards for drinking water.

Preliminary pharmacokinetics and nonclinical safety assessment study in Cynomolgus

Monkeys A preliminary pharmacokinetics and nonclinical safety assessment was conducted

at Charles River Laboratories (Reno, NV) in naive male and female chinese cynomolgus

monkeys that were between 2.5 to 3.9 years of age with body weights ranging from 2.3 to 2.7

kg. The parameters and end points evaluated in this study were clinical signs

(mortality/moribundity checks, cage side observations, and detailed clinical observations),

body weights, food consumption, skin biopsies, hematology, coagulation, and clinical

chemistry. Samples for serum drug levels and drug kinetic evaluations were also taken.

Skin punch biopsies were performed prestudy (day -4) and on day 4 following the first dose.

The skin biopsy area was cleansed with antiseptic and three punch biopsies of approximately

8 mm each were collected from the shaved dorsal area of each monkey. Skin samples were

collected into and stored in RNAlater prior to RNA isolation. The biopsy site was closed

with suture and/or tissue adhesive as needed, and local analgesics (i.e., lidocaine) were

administered. Animals were given an opiate analgesic and were allowed to recover in their

cages. Antesedan (a reversal agent for dexdomitor) was administered at veterinary discretion

after the procedure. No termination of the animals was done for the study. After study

completion, all animals were returned to the testing facility animal colony.


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Animals were housed individually in stainless-steel cages. Primary enclosures were as

specified in the United States Department of Agriculture (USDA) Animal Welfare Act (9

Code of Federal Regulations [CFR], Parts 1, 2, and 3) and as described in the Guide for the

Care and Use of Laboratory Animals. Temperatures of 64°F to 84°F (approximately 18°C to

29°C) with a relative humidity of 30% to 70% were maintained. A 12-hour light-12-hour

dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air

recirculation) were maintained in the animal room. Temperatures of 64°F to 84°F

(approximately 18°C to 29°C) with a relative humidity of 30% to 70% were maintained. A

12-hour light-12-hour dark cycle was maintained. Ten or greater air changes per hour with

100% fresh air (no air recirculation) were maintained in the animal room. Animals were fed

Purina Certified Primate Diet No. 5048 in amounts appropriate for the size and age of the

animals. This diet was supplemented with fruit or vegetables 2-3 times weekly. Animals

were provided reverse osmosis filter water that was passed through ultraviolet (UV) light

treatment. Processed water was available ad libitum to each animal via an automatic

watering device. The water was routinely analyzed for contaminants by Western

Environmental Testing Laboratory, Sparks, NV. No contaminants were known to be present

in the water at levels that would interfere with the results of this study. Animals were also

provided an enrichment device, food treat, and were periodically socialized to provide for

psychological enrichment. Socialization was limited to grooming bar access only.

Veterinary care was available throughout the course of the study and animals were examined

by the veterinary staff as warranted by clinical signs or other changes.

Determination of serum MEDI-504 levels in cynomolgus monkeys. Concentrations of

MEDI-5304 in cynomolgus monkey serum were determined using a qualified ELISA

method. The ELISA plates were coated with 1.0 μg/ml of sheep anti-human IgG (H+L


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specific, The Binding Site, Cat # A4003CUS01) in phosphate buffered saline (PBS)

overnight at 2°C to 8°C, washed with 0.05% Tween 20 in PBS, and blocked for at least 1 h at

room temperature with I-Block Buffer (Tropix, Bedford, MA). MEDI-5304 reference-

standard, quality-control (QC) and test sample dilutions were prepared in 10% cynomolgus

monkey serum (assay matrix) and added to blocked plates for 2 h at room temperature. Plates

were washed as above and incubated an additional 1 h at room temperature with 1:8000

dilution of HRP-goat anti human IgG (H+L-specific, Bethyl Labs, Cat# A80-319P). Unbound

detection antibody was removed by washing, and 100 μl of TMB substrate was added to

wells for 5 minutes. The development of color was stopped by addition of 50 μL of 2M

H2SO4. Plates were read within 15 minutes at 450 nm with SpectraMax Plus 384 plate reader

(Molecular Devices, Sunnyvale, CA). Concentrations of MEDI-5304 in QC and test-sample

dilutions on each plate were interpolated from the reference-standard curve for that plate. The

range of the assay for quantification of MEDI5304 in 100% cynomolgus monkey serum was

38 to 960 ng/ml.


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Supplementary Figures

Supplementary Figure S1. Binding kinetics of hedgehog antibodies to human and mouse

hedgehog proteins using surface plasmon resonance.


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A

B

Supplementary Figure S2. Hedgehog ligands stimulate mGLI1 reporter activity and

osteoblast differentiation in a dose-dependent manner.


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A

B

C

Supplementary Figure S3. Selective inhibition of mGLI1 or mPTC1 induction in C3H10T1/2

cells by hedgehog antibodies.


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vehicle MEDI-53040.0

0.5

1.0

1.5

hGli1mGli1

***

Rel

Exp

rn(v

. h

um

or

mu

HP

RT

1)

A

B

Supplementary Figure S4. MEDI-5304 inhibits GLI1 expression in Colo205 xenograft

stromal cells but not tumor cells.


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A

B

sHH

IHH

GLI-2

GLI-3

PTCH1

SMO

DYRK1

SUFU0

5

10

15

20

25

30

35R

ela

tiv

e e

xp

res

sio

n(r

ati

o C

SC

s:n

on

-CS

Cs

)

0

5

10

15

20

25

30

Sph

ere

num

ber

Supplementary Figure S5. Hedgehog pathway component RNA expression in primary

pancreatic tumor explant model P479 and effects of MEDI-5304 on tumorspheres derived

from pancreatic explant model 947.


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A

B

0 5 10 15 20 25 30-2

-1

0

1

2

3

1 mg/kg10 mg/kg30 mg/kg

Days post dosing

Ser

um

[M

ED

I-53

04]

log

(μg

/ml)

DoseCmax

(µg/ml)AUCall

(µg*d/ml)AUCINF

(µg*d/ml)CL

(ml/kg/d)t1/2

(d)

1 mg/kg 13.7 ± 4.6 26.7 ± 6.0 26.9 ± 6.0 38.6 ± 7.2 2.7 ± 0.3

10 mg/kg 137 ± 44 923 ± 106 1015 ± 156 10.0 ± 1.6 8.5 ± 2.7

30 mg/kg 650 ± 114 3396 ± 268 3968 ± 279 7.6 ± 0.5 10.4 ± 1.1

AUCALL: area under the concentration-time curve from time zero to the last measurable time point

AUCINF: area under the concentration-time curve from time zero to infinityCL: systemic clearanceCmax: observed peak concentration t1/2: terminal phase elimination half-life

Supplementary Figure S6. Pharmacokinetics of MEDI-5304 in rats.


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A B

0 7 1410

100

1000

10000

10 mg/kg50 mg/kg

days post dose

seru

m [

ME

DI-

5304

]( μ

g/m

l)

C

D

DoseCmax

(µg/ml)AUCINF

(µg*d/ml)CL

(ml/kg/d)t1/2

(d)

10 mg/kg 268 ± 1 833 ± 119 12.2 ± 1.8 6.1 ± 1.4

50 mg/kg 885 ± 5 6601 ± 866 7.7 ± 1.0 9.0 ± 1.0

vehicle MEDI-5304 50 mg/kg

Supplementary Figure S7. Exploratory toxicology of MEDI-5304 in rats


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ParametersGroup 2 Group 3 Group 4

0.3 mg/kg 3 mg/kga 1 mg/kg 10 mg/kga 3 mg/kg 75 mg/kga

n 4 4 4 4 4 4Cmax

(µg/mL)5.08 ± 0.54 89.7 ± 10.2 20.2 ± 1.0 290 ± 4.3 55.8 ± 4.9 1900 ± 270

AUClast

(day·µg/mL)16.7 ± 1.1 912 ± 84 80.6 ± 10.1 3400 ± 100 244 ± 25 22100 ± 2970

AUCinf

(day·µg/mL)18.5 ± 0.9 ND 98.2 ± 5.9 ND 342 ± 47 ND

t1/2

(d)4.12 ± 0.38 7.75 ± 0.32 5.76 ± 0.48 10.1 ± 0.7 7.74 ± 0.81 8.07 ± 0.48

CL(mL/kg/d)

16.4 ± 0.9 8.24 ± 1.08 10.3 ± 0.6 7.93 ± 0.34 9.45 ± 1.66 9.33 ± 1.47

A

Parameters are shown as mean ± SDa AUClast was calculated f rom the time interval 28 through 35 days post last dose on Day 28 and all PK parameters were derived f rom concentration-time prof ile data post last dose on Day 28.

B

0 7 14 21 28 35 420.1

1

10

100

1000

10000

Grp2 0.3mg/kgGrp3 1mg/kgGrp4 3mg/kgGrp2 3mg/kgGrp3 10mg/kgGrp4 75mg/kg

Time (day)

seru

m [

ME

DI-

5304

]( μ

g/m

l)

Supplementary Figure S8. Pharmacokinetics of MEDI-5304 from preliminary toxicology

study in cynomolgus monkeys.


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B

C

A

pretreated day 40.0

0.1

0.2

0.3

0.4

control3 mpk10 mpk75 mpk

** **

***Rel

Exp

rn G

LI1

(vs

cyn

o H

PR

T1)

pretreated day 40.0

0.1

0.2

0.3

0.4


* ** **

Rel

Exp

rn P

TC

H1

(vs

cyn

o H

PR

T1)

pretreated day 40

2.0×10-3

4.0×10-3

6.0×10-3

8.0×10-3

1.0×10-2


* ***

Rel

Exp

rn P

TC

H2

(vs

cyn

o H

PR

T1)

Supplementary Figure S9. Pharmacodynamics of MEDI-5304 in cynomolgus monkeys.


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Supplementary Figure Legends

Supplementary Figure S1. Representative sensograms of SPR experiments are depicted for

binding of human and mouse hedgehog proteins to anti-hedgehog monoclonal antibodies 6D7

and 3H8 affinity-captured or 5E1 directly immobilized onto the C1 chip. Dashed black lines

represent fits of the monoexponential binding model to the experimental data shown as full

gray lines. (A) 6D7 binding to human SHH (B) 6D7 binding to human/mouse IHH (C) 6D7

binding to mouse SHH (D) 3H8 binding to human SHH (E) 3H8 binding to human/mouse

IHH (F) 3H8 binding to mouse SHH (G) 5E1 binding to human SHH and (H) 5E1 binding to

human/mouse IHH.

Supplementary Figure S2. (A) Serially diluted human SHH C24II or human/mouse C28II

IHH was added to NIH3T3 cells stably expressing the mGLI1 luciferase reporter, and cells

were incubated for 24 hours. Fold stimulation in comparison to cells that did not receive

hedgehog ligand is plotted. (B) Serially diluted human SHH C24II or human/mouse C28II

IHH was added to C3H10T1/2 cells and incubated for 3 days. Alkaline phosphatase activity

was determined as a measure of osteogenesis.

Supplementary Figure S3. Hedgehog antibodies inhibit induction of endogenous mPTC1 by

hedgehog proteins. C3H10T1/2 cells were treated with 600 ng/ml (30 nM) SHH C24II (A) or

2 µg/ml (100 nM) IHH C28II (B) for 24 hours in the absence or presence of serially diluted

6D7, 3H8, or R347. Modulation of endogenous levels of mPTC1 RNA by hedgehog

stimulation and treatment with anti-hedgehog mAbs was determined by qRT-PCR (TaqMan)

using the 2- ∆∆Ct method. mPTC1 expression of each sample was normalized to mHPRT1

expression and fold stimulation was calculated relative to unstimulated cells in the absence of


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antibody. (C) C3H10T1/2 cells were treated with 600 ng/ml SHH C24II in the presence of

varying concentrations of 6D7 or 5E1 and treated for 72 h. Values plotted are per cent

mGLI1 levels (normalized to mHPRT1) induced by SHH treatment in the absence of

antibody. All values represent means ± SEM.

Supplementary Figure S4. (A) Colo205 colon tumor cells were implanted into athymic

mice and once tumors reached ~ 400 mm3 in size, tumor bearing animals were treated with

vehicle or 10 mg/kg MEDI-5304 for 24 h. RNA expression of human and mouse GLI1 was

determined by qRT-PCR using species-specific TaqMan primers/probes and normalized to

the corresponding expression of human or mouse HPRT1. Values represent means ± SEM.

Statistical analysis was performed using Student’s t test. A significance level of P < 0.001 is

indicated (***) when comparing antibody treatment to vehicle controls. (B) Animals bearing

Colo205 xenograft tumors received vehicle or 1, 3, or 10 mg/kg MEDI-5304 or 5E1, and

relative mGLI1 RNA levels were determined in excised tumors 24 h after dose administration

as described in (A). Statistical analysis was performed using one-way ANOVA and

Dunnett’s multiple comparison tests. Statistical significance of P < 0.01 is indicated (**).

Supplementary Figure S5. (A) Cancer stem cells from the P479 model were isolated by

FACS sorting from dissociated bulk tumor cells using the cell surface markers ESA, CD44,

and CD24. The RNA expression of various hedgehog pathway components was determined

in CSCs and bulk tumor cells with human specific TaqMan primers/probes. The ratio of

expression in CSCs to bulk tumors is shown. (B) Effect of MEDI-5304 on formation of

tumorspheres derived from the primary pancreatic model 947. Following FACS sorting,

single cell suspensions from patient pancreatic cancer xenografts were resuspended in

tumorsphere media. Cells were plated in the absence or presence of 5, 10 or 30 µg/ml MEDI-


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5304. Tumorspheres were cultured for 4 days and counted. No statistically significant

differences were observed.

Supplementary Figure S6. (A) Serum MEDI-5304 levels in rats. A single i.v. bolus dose of

1, 10, 30 mg/kg MEDI-5304 was administered to Hans Wistar rats. Each dosing group was

comprised of three males and three females. Blood samples were collected via tail vein

without anti-coagulant agent. Serum levels of MEDI-3504 were determined by ELISA

specific for human IgG. (B). Noncompartmental analysis of the preliminary rat PK study

depicted in (A) was performed on individual animal serum concentration data using

WinNonlin Professional (version 5.2, Pharsight Corp., Mountain View, CA).

Supplementary Figure S7. (A) The maxilla from a vehicle-treated rat whose incisor has a

normal odondoblast layer (boxed area). 20 x, HE. Inset: Higher magnification of the

odontoblast layer (arrow heads). 100 x, HE. (B) The maxilla from a rat treated with MEDI-

5304 (50 mg/kg) whose incisor exhibits disorganization of the odondoblast layer admixed

with dentin-like material (boxed area). 20 x, HE. Inset: Higher magnification of the

odontoblast layer (arrow heads). 100 x, HE. (C) MEDI-5304 pharmacokinetics. Serum levels

of MEDI-5304 were determined during the execution of the exploratory rat toxicology study.

Doses were given i.v. on day 0 and day 7 (arrows). Antibody levels were determined by

ELISA that detects total human IgG. (D). Noncompartmental analysis of the rat PK data was

conducted based on data collected post first dose on day 0 during the exploratory rat

toxicology study.

Supplementary Figure S8. (A) Serum levels of MEDI-5304 were determined during the

execution of the preliminary non-clinical pharmacokinetics safety study conducted in


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cynomolgus monkeys. Animals received either vehicle (Grp1), or 0.3 (Grp2), 1 (Grp3), or 3

mg/kg (Grp4) MEDI-5304. After 14 days, the dose each group received was increased to 3

(Grp2), 10 (Grp3), or 75 mg/kg (Grp4). These doses were given on days 14, 21, and 28.

Time of dose administration is denoted by the arrows. Pharmacokinetic parameters of

MEDI-5304 were determined in the varying dosing groups. (B). Noncompartmental analysis

of the monkey PK data collected during the monkey safety study.

Supplementary Figure S9. Pharmacodynamic effects of MEDI-5304 observed in skin of

cynomolgus monkeys. Skin punches were performed 4 days prior to antibody (pretreated)

dosing and on day 4. Three skin punches were obtained from each animal at time of

sampling. The effects of MEDI-5304 on GLI1 (A), PTCH1 (B) and PTCH2 (C) RNA levels

were determined with TaqMan primer/probe sets for the rhesus orthologs and validated to

have 100% amplification efficiency with cynomolgus brain RNA. Values are means ± SEM.

Statistical significance was evaluated using one-way ANOVA and Dunnett’s multiple

comparison tests. Significance levels of P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) are

indicated for comparisons with the vehicle control.

Supplementary Materials and Methods Exploratory toxicology...

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