Deg

rada

tion

of h

yalu

rona

n (H

A) b

y PE

GPH

20 p

rom

otes

ant

i-tum

or

imm

unity

and

enh

ance

s th

e ef

fect

of c

heck

poin

t blo

ckad

e in

an

HA

-ac

cum

ulat

ing

mou

se s

ynge

neic

tum

or m

odel

B

enja

min

J. T

hom

pson

, Ren

ee C

lift,

Trev

or K

imbl

er, J

esse

D. B

ahn,

Chu

nmei

Zha

o, B

arba

ra B

louw

, Cur

tis B

. Tho

mps

on, S

anna

Ros

engr

enH

aloz

yme

Ther

apeu

tics,

Inc.

, San

Die

go, C

A, U

SA

SUM

MA

RY

#P49

6

INTR

OD

UC

TIO

NR

ESU

LTS,

con

tinue

d

MET

HO

DS

REF

EREN

CES

AC

KN

OW

LED

GEM

ENTS

RES

ULT

S

The

glyc

osam

inog

lyca

nhy

alur

onan

(HA

)is

abun

dant

inm

any

solid

tum

ors,

and

itsac

cum

ulat

ion

isof

ten

asso

ciat

edw

ithpo

orpa

tient

outc

omes

1 .D

egra

datio

nof

HA

byin

trave

nous

lyad

min

iste

red

PE

Gyl

ated

reco

mbi

nant

hum

anhy

alur

onid

ase

PH

20(P

EG

PH

20)

rem

odel

sth

etu

mor

stro

ma,

redu

ces

intra

tum

oral

pres

sure

,dec

ompr

esse

stu

mor

bloo

dve

ssel

s,an

dfa

cilit

ates

drug

deliv

ery2

,3.R

ecen

tly,P

EG

PH

20w

assh

own

toen

hanc

etu

mor

grow

thin

hibi

tion

indu

ced

byth

eim

mun

ech

eckp

oint

inhi

bito

ran

ti-P

D-L

1in

HA

-acc

umul

atin

gpa

ncre

atic

and

orth

otop

icbr

east

syng

enei

ctu

mor

mod

els,

and

toen

hanc

etu

mor

T-ce

llin

filtra

tion4

,5.

Her

e,th

eH

Aco

nten

tof

com

mon

lyus

edsy

ngen

eic

mod

els

was

syst

emat

ical

lyst

udie

d,an

dM

C38

(Cha

rles

Riv

erLa

bora

torie

s,C

RL)

was

iden

tifie

dto

furth

erev

alua

teth

eco

mbi

nato

rial

effe

ctof

PE

GP

H20

and

anti-

PD

-L1

inH

A-a

ccum

ulat

ing

tum

ors.

HAELISA

:Tu

mor

sw

ere

dige

sted

with

prot

eina

seK

and

supe

rnat

ants

assa

yed

forH

Aus

ing

Hya

luro

nan

Duo

Set

ELI

SA

(R&

DS

yste

ms)

.QuantitativePCR

:Tu

mor

segm

ents

wer

ely

sed

usin

gFa

stP

rep.

Tota

lRN

Aw

asis

olat

edus

ing

RN

easy

Lipi

dTi

ssue

Min

iK

its(Q

IAG

EN

)an

dre

vers

etra

nscr

ibed

usin

gTa

qman

Rev

erse

Tran

scrip

tion

Kits

(The

rmoF

ishe

r).P

CR

was

perfo

rmed

byVi

iA7

Rea

l-tim

eP

CR

(The

rmoF

ishe

r)us

ing

apa

nelo

fTa

qman

prim

er/p

robe

sth

atin

clud

edim

mun

esu

rface

mar

kers

,cy

toki

nes,

chem

okin

es,

imm

une

chec

kpoi

nts,

and

imm

unom

odul

ator

yen

zym

es.

Dat

aw

ere

anal

yzed

usin

gth

e2-∆∆

Ctm

etho

d,an

dno

rmal

ized

to-

actin

(Actb)

expr

essi

on.

Immunohistochem

istry(IHC):

Form

alin

fixed

tissu

ese

ctio

nsw

ere

anal

yzed

usin

gH

aloz

yme’

sH

Aex

plor

ator

ypr

otot

ype

assa

yus

ing

abi

otin

ylat

edH

Aaf

finity

prob

e(H

TI-6

01)

and

visu

aliz

edw

ithD

AB

.Se

ctio

nsw

ere

coun

ters

tain

edw

ithhe

mat

oxyl

in.

HA

accu

mul

atio

nw

asqu

antif

ied

bypo

sitiv

epi

xelc

ount

usin

gdi

gita

lim

age

anal

ysis

.Tumor

studies:

All

tum

orgr

owth

inhi

bitio

nan

dim

mun

ophe

noty

ping

stud

ies

wer

epe

rform

edin

the

MC

38co

lon

tum

orm

odel

atC

RL.

Tum

ors

wer

eim

plan

ted

subc

utan

eous

ly,an

dP

EG

PH

20(3

7.5

µg/k

g)w

asad

min

iste

red

intra

veno

usly

24h

prio

rto

anti-

PD

-L1

(10F

.9G

2,5

mg/

kgi.p

.)us

ing

abi

wee

kly

dosi

ngre

gim

en.

Trea

tmen

tsw

ere

initi

ated

whe

ntu

mor

sre

ache

d80

-120

mm

3 .Tu

mor

sw

ere

mea

sure

dbi

wee

kly

usin

gca

liper

s.Fo

rim

mun

ophe

noty

ping

stud

ies,

mic

ew

ere

treat

edfo

rtw

ow

eeks

befo

reha

rves

ting

the

tum

ors,

whi

chw

ere

ship

ped

over

nigh

tto

Hal

ozym

ein

MA

CS

Tiss

ueS

tora

geS

olut

ion

(Milt

enyi

Bio

tec)

at4°

Cfo

rflo

wcy

tom

etric

anal

ysis

.Flow

cytometry

:Tu

mor

sw

ere

diss

ocia

ted

with

Libe

rase

DH

and

DN

ase

I(b

oth

from

Roc

he)

usin

gG

entle

MA

CS

Oct

oD

isso

ciat

ors

with

heat

ers

(Milt

enyi

Bio

tec)

.S

ingl

ece

llsu

spen

sion

sw

ere

stai

ned

with

avi

abili

tydy

ean

dth

efo

llow

ing

fluor

ochr

ome-

conj

ugat

edan

tibod

ies:

CD

45(3

0-F1

1),

CD

8b(H

35-

17.2

),C

D4

(RM

4-5)

,CD

25(P

C61

),Fo

xP3

(FJK

-16s

),C

D64

(X54

-5/7

.1),

Ly6G

(1A

8),

Ly6C

(HK

1.4)

MH

CII

(M5/

114.

15.2

),C

D11

c(N

418)

,C

D11

b(M

1/70

),N

K1.

1(P

K13

6)an

dC

D10

3(2

E7)

.D

ata

wer

eac

quire

don

aN

ovoC

yte

cyto

met

eran

dan

alyz

edw

ithN

ovoE

xpre

ssso

ftwar

e(A

CE

AB

iosc

ienc

es,I

nc.).

Cel

lsw

ere

defin

edas

follo

ws:

CD

8+tu

mor

-infil

tratin

gly

mph

ocyt

es(T

ILs)

,C

D45

+C

D8b

+C

D4-

CD

44hi

gh;

CD

4+TI

Ls,

CD

45+

CD

8b-C

D4+

CD

44hi

gh;

tum

oras

soci

ated

mac

roph

ages

(TA

Ms)

,CD

45+

Ly6G

- CD

11b+

CD

64+

Ly6C

- ;de

ndrit

icce

lls(D

C),

CD

45+

Ly6G

-C

D64

-M

HC

II+C

D11

c+;

and

NK

cells

,C

D45

+Ly

6G-

CD

64-M

HC

II-N

K1.

1+.

RES

ULT

S, c

ontin

ued

1 Shepa

rd et a

l. Fron

t.Oncol. 5:192

, 201

5 2 Tho

mpson

 et a

l. Mol Can

cer T

her. Nov;9(11):305

2‐64

, 20

103 Provenzan

o et al. Ca

ncer Cell. 21

(3): 41

8‐29

, 201

2.

4 Clift e

t al. AA

CR #64

1, 201

75 Tho

mpson

 et a

l. AA

CR Tum

or Im

mun

olog

y an

d Im

mun

otherapy #B3

8, 201

7

Figu

re1.

Scre

enin

gof

mou

sesy

ngen

eic

tum

orm

odel

sfo

rH

Aco

nten

tan

dim

mun

eac

tivity

.(A

)In

dica

ted

mou

sece

lllin

esw

ere

impl

ante

din

tosy

ngen

eic

mic

eei

ther

subc

utan

eous

ly(S

C)o

rint

ram

uscu

larly

(IM)a

ndas

saye

dfo

rtot

alH

Aco

nten

tby

ELI

SA

.MC

38-

CR

Lac

cum

ulat

edth

em

ostH

Aam

ong

the

mod

els

test

ed.

Dat

ade

pict

the

mea

nH

Aco

nten

tof

indi

vidu

altu

mor

s±

SE

M.(

B)T

hesa

me

tum

orm

odel

sas

in(A

)wer

esc

reen

edby

qPC

Rfo

rthe

expr

essi

onof

gene

sas

soci

ated

with

imm

une

activ

ityan

dre

gula

tion.

Row

sde

pict

rela

tive

expr

essi

onle

vels

ofin

divi

dual

gene

s,gr

oupe

dby

unsu

perv

ised

clus

terin

g.R

edin

dica

tes

high

erex

pres

sion

;gr

een

indi

cate

slo

wer

expr

essi

on.

Col

umns

depi

ctin

divi

dual

tum

ors.

Due

tohi

ghle

vels

ofH

Aac

cum

ulat

ion

and

rela

tivel

yhi

ghex

pres

sion

ofim

mun

e-re

late

dge

nes,

the

MC

38-

CR

Lm

odel

was

sele

cted

forf

urth

erst

udie

sw

ithP

EG

PH

20an

dan

ti-P

D-L

1.

Figu

re2.

PEG

PH20

decr

ease

dH

Aco

nten

tin

the

tum

orm

icro

envi

ronm

ent.

Rep

rese

ntat

ive

imag

esof

MC

38-C

RL

tum

ors

treat

edw

ith(A

)ve

hicl

eor

(B)

PE

GP

H20

at37

.5μg

/kg

and

stai

ned

for

HA

cont

entb

yIH

C24

haf

ter

asi

ngle

treat

men

t.E

ach

imag

ede

pict

san

indi

vidu

altu

mor

.(C

)Tu

mor

sw

ere

anal

yzed

byIH

C24

haf

ter

1,2,

or3

dose

sof

PE

GP

H20

;dat

ade

pict

mea

npo

sitiv

epi

xelc

ount

±S

EM

of3

indi

vidu

altu

mor

sfo

reac

hco

nditi

on.

4T1

OT

LLC

IMSC

KLN205

IMSC

B16F10

IMSC

Pan0

2IM

SCEM

T6SC

CT26 IM

MC3

8SC

0

500

1000

1500

2000

Syng

enei

c tu

mor

mod

el

MC38-CRLTumorsAccum

ulateHAandContain

ImmuneActivity

AB

Vehi

cle

PEG

PH20

(37.

5 μg

/kg)

TumorHADegradation

byPEGPH20

AC

B

Tumor Volume (mm3) SEM

05

1015

0

200

400

600

800

1000

Stud

y D

ay

AP

I buf

fer/r

at Ig

G2b

^

PEG

PH20

/rat I

gG2b

^

API

buf

fer/a

nti-P

D-L

1

PEG

PH20

/ant

i-PD

-L1^

NSNS

Tumor Volume (mm3) SEM

020

4060

80050100

Day

s

Percent survival

AP

I buf

fer/P

BS

PEG

PH20

/PBS

AP

I buf

fer/a

nti-P

D-L

1PE

GPH

20/a

nti-P

D-L

1

010

2030

050100

Day

s

AP

I buf

fer/I

gG2b

PEG

PH20

/IgG

2bA

PI b

uffe

r/ant

i-PD

-L1

PEG

PH20

/ant

i-PD

-L1

Percent survival

API buffe

r/Rat

IgG2b

PEGPH20/R

at IgG2b

API buffe

r/anti-P

D-L1

PEGPH20/an

ti-PD-L1

Absolute cell # per g tumorMeanSEM

API buffe

r/Rat

IgG2b

PEGPH20/R

at IgG2b

API buffe

r/anti-P

D-L1

PEGPH20/an

ti-PD-L1

0

510

5

110

6

1.5

106

Absolute cell # per g tumorMeanSEM

API buffe

r/Rat

IgG2b

PEGPH20/R

at IgG2b

API buffe

r/anti-P

D-L1

PEGPH20/an

ti-PD-L1

Absolute cell # per g tumorMeanSEM

API buffe

r/Rat

IgG2b

PEGPH20/R

at IgG2b

API buffe

r/anti-P

D-L1

PEGPH20/an

ti-PD-L1

0

110

6

210

6

310

6

410

6

510

6

NS

NS

API buffe

r/Rat

IgG2b

PEGPH20/R

at IgG2b

API buffe

r/anti-P

D-L1

PEGPH20/an

ti-PD-L1

0.0

0.5

1.0

1.5

NS

API buffe

r/Rat

IgG2b

PEGPH20/R

at IgG2b

API buffe

r/anti-P

D-L1

PEGPH20/an

ti-PD-L1

020406080

% of CD45+ cellsMean SEM

NS

PEGPH20

Enhanced

Tumor

Growth

Inhibition

Inducedby

anti-PD-L1andExtendedSurvivalin

2of3Studies

Stud

y 1

Stud

y 2

Stud

y 3

A BSt

udy

1St

udy

2St

udy

3

Figu

re3.

Com

bina

tion

trea

tmen

tw

ithPE

GPH

20an

dan

ti-PD

-L1

sign

ifica

ntly

enha

nced

tum

orgr

owth

inhi

bitio

nan

dsu

rviv

alve

rsus

eith

erag

ent

alon

ein

2of

3st

udie

s.(A

)G

row

thcu

rves

ofM

C38

-CR

Ltu

mor

stre

ated

with

the

indi

cate

dag

ents

from

3in

depe

nden

tst

udie

s.E

ach

mea

sure

men

ttim

epo

int

depi

cts

the

mea

ntu

mor

volu

me

±S

EM

for

that

grou

p.D

ata

wer

ean

alyz

edus

ing

repe

ated

mea

sure

sA

NO

VA.

N=

10an

imal

spe

rgr

oup.

Whe

rein

dica

ted

(^),

1-3

tum

ors

wer

eex

clud

edfro

man

alys

isdu

eto

ulce

ratio

n.(B

)K

apla

n-M

eier

surv

ival

anal

ysis

from

the

sam

e3

stud

ies

show

nin

(A).

Dat

aw

ere

anal

yzed

usin

gth

eLo

g-ra

nkte

st.

*p<0

.05,

***p

<0.0

01,

****

p<0.

0001

,N

S=

not

sign

ifica

nt.

InS

tudi

es1

and

3,P

EG

PH

20si

gnifi

cant

lyen

hanc

edth

etu

mor

grow

thin

hibi

tion

indu

ced

byan

ti-P

D-L

1;in

the

sam

etw

ost

udie

s,co

mbi

natio

ntre

atm

ent

with

PE

GP

H20

and

anti-

PD

-L1

sign

ifica

ntly

prol

onge

dsu

rviv

alco

mpa

red

toei

ther

treat

men

talo

ne.

PEGPH20

Increased

Tumor

Accum

ulation

ofTandNKCells,and

Decreased

TAMFrequency

CD

8+TI

LA

bsol

ute

Num

ber

CD

4+TI

LA

bsol

ute

Num

ber

NK

Cel

lA

bsol

ute

Num

ber

TAM

Abs

olut

e N

umbe

rTA

M%

of C

D45

+D

C%

of C

D45

+

Figu

re4.

Imm

unop

heno

typi

ngan

alys

isof

tum

ors

trea

ted

with

PEG

PH20

alon

ean

din

com

bina

tion

with

anti-

PD-L

1.(A

-D)A

bsol

ute

num

bers

ofC

D8+

TIL

(A),

CD

4+TI

L(B

),N

Kce

lls(C

),an

dTA

M(D

)pe

rgr

amof

tum

ortis

sue.

(E-F

)Fr

eque

ncie

sof

TAM

(E)

and

DC

(F)

amon

gto

tall

ive

CD

45+

cells

.D

ata

wer

ean

alyz

edus

ing

one-

way

AN

OVA

with

Sid

ak’s

post

-hoc

test

.W

hen

nece

ssar

y,da

taw

ere

log

trans

form

edto

achi

eve

equa

lvar

ianc

eac

ross

grou

ps.

N=

6an

imal

spe

rgr

oup.

*p<0

.05,

**p<

0.01

,**

*p<0

.001

,**

**p<

0.00

01,

NS

=no

tsi

gnifi

cant

.A

sa

mon

othe

rapy

,PE

GP

H20

incr

ease

dth

eab

solu

tenu

mbe

rsof

CD

8+TI

L,C

D4+

TIL,

and

NK

cells

com

pare

dto

cont

rol-t

reat

edtu

mor

s.Li

kew

ise,

PE

GP

H20

inco

mbi

natio

nw

ithan

ti-P

D-L

1in

crea

sed

the

abso

lute

num

bers

ofth

esa

me

3po

pula

tions

com

pare

dto

anti-

PD

-L1

alon

e.A

mon

gto

talC

D45

+ce

lls,P

EG

PH

20de

crea

sed

TAM

frequ

ency

and

incr

ease

dD

Cfre

quen

cy.

AB

C

DE

F

M

C38

-CR

Ltu

mor

s,w

hich

are

imm

unol

ogic

ally

activ

ean

dse

nsiti

veto

PD-1

bloc

kade

aspr

evio

usly

desc

ribed

,con

tain

high

leve

lsof

HA

rela

tive

toot

hers

ynge

neic

mod

els.

D

egra

datio

nof

HA

byP

EG

PH

20en

hanc

edth

eef

fect

ofan

ti-P

D-L

1on

MC

38-C

RL

tum

orgr

owth

in2

of3

stud

ies.

C

ombi

natio

nof

PEG

PH

20w

ithan

ti-P

D-L

1pr

olon

ged

surv

ival

in2

of3

stud

ies.

P

EG

PH

20,a

lone

,and

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ing

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auth

ors

grat

eful

lyac

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ledg

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hery

lG

arro

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nnife

rSou

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a,S

usan

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mer

man

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eval

,and

the

stud

ype

rson

nel

atC

harle

sR

iver

Labo

rato

ries

for

thei

rco

ntrib

utio

ns.

PresentedattheSocietyforImmunotherapyofCancer(SITC)

meeting,NationalHarbor,MD,Nov8-122017.

****

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