AD_________________

Award Number: W81XWH-12-1-0223 TITLE: Innovative Strategies for Breast Cancer Immunotherapy PRINCIPAL INVESTIGATOR: Feng Wang-Johanning CONTRACTING ORGANIZATION: M.D. ANDERSON CANCER CENTERHouston, TX 77030 REPORT DATE: September 2013 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

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2. REPORT TYPE Annual

3. DATES COVERED 1 September 2012 - 31 August 2013

4. TITLE AND SUBTITLE

Innovative Strategies for Breast Cancer Immunotherapy 5a. CONTRACT NUMBER

5b. GRANT NUMBER

W81XWH-12-1-0223 5c. PROGRAM ELEMENT NUMBER

6. AUTHOR(S)

Feng Wang-Johanning 5d. PROJECT NUMBER

5e. TASK NUMBER

5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)

M.D. ANDERSON CANCER CENTERHouston, TX 77030AND ADDRESS(ES)

8. PERFORMING ORGANIZATION REPORT NUMBER

9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command

Fort Detrick, Maryland 21702-5012

11. SPONSOR/MONITOR’S REPORT

NUMBER(S)

12. DISTRIBUTION / AVAILABILITY STATEMENT

Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES

14. ABSTRACT In our grant application, we detected HERV-K viral particles by transmission electron microscopy (TEM) in sera from an invasive ductal carcinoma (IDC) patient and in BC cell culture media. In fact, HERV-K viral like particles have been found in a variety of tumor cells, as reported by others. These viral particles would be expected to have RT activity. The RT activity of various breast cell culture media was compared, and we found that all cancer cells had higher RT, compared with MCF-10AT cells. In addition, two IBC cell lines (KPL-4 and SUM149) had increased RT activity

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19a. NAME OF RESPONSIBLE PERSON USAMRMC

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Table of Contents

Page

Project 1…………………………………………………………….………..………4

Project 2………………………………………………………………………………6

Project 3……………………………………………………………………………..10

Project 4………………………………………………………………………………11

Project 5………………………………………………………………………………..13

Pr 1.tishutismIBno

1.Insepaexthlindeno

Wobca(frsigw

roject 1: T

1 Expresssues: Pruman endossues and c

mRNA (FigBC3) or prormal brea

2 We willn our grantera from aarticles haxpected tohat all cancnes (KPL-ensity gradot DNA u

We also foubtained froarcinoma ifractions 6gnificantlith fractio

Figure 1 (A)Acc185, Accexpression ohuman breas(green fluore

A

A

To identif

sion of Hrior to deteogenous recell lines bg. 1A) andrimary BCast tissues

l determint applicati

an invasiveave been fo have RTcer cells h-4 and SUdient fracsing SYT

und increaom variouin situ (D

6 to 10) wely higher ions obtain

) The expressc168, and Acof HERV-K est cells by Weescence) was

fy infectio

HERV-K eerminationetrovirus tyby RT-PCd protein (C biopsiess (ND263

ne reverseion, we dee ductal cfound in a

T activity. had higher

UM149) hations (DG

TO RNA s

ased RT aus donors CIS), or nere compain fraction

ned from n

sion of HERVcc204), but noenv protein western blot us

s demonstrate

ous retrov

env mRNn of reversype K (HE

CR, western(Fig. 1B as (Acc177and ND2

e transcripetected HEarcinoma

a variety o The RT ar RT, comad increas

GF) 5 to 6 elected st

activity in including

normal femared betwn A (Fig. 3normal fem

V-K env typeot in normal

was detected ising anti-HEed on malign

B

viruses in p

NA or proe transcripERV-K) enn blot, andand Fig. 17, Acc185273).

ptase (RTERV-K v(IDC) pa

of tumor cactivity of

mpared witsed RT acin both ce

tain.

blood plag patients male dono

ween norm3A) or B (male dono

e 1 or/and typbreast tissuein BC cells (MRV-K mAb (ant cells (KP

B

B

patient sp

tein in huptase (RT)nv RNA ad immunofC) are exp, Acc168,

T) activityiral partic

atient and ells, as repf various bth MCF-1

ctivity (Figell lines. I

asma sampwith invaors was de

mal donors(Fig. 3B)ors. In add

pe 2 mRNA es (ND263 anMCF-7, SUM(6H5). β-acti

PL-4, MDA-I

pecimens:

uman infl) activity iand proteinfluorescenpressed in, and Acc2

y in blood cles by train BC celported bybreast cel10A or MCg. 2). TheImportant

ples fromasive breasetermineds vs. canceobtained

dition, RT

(ty1 and ty2)nd ND273) byM-149, and Min was used aIBC3, and SU

C

lammatorn blood orn was evalnce (IFS).n IBC cell204), but

or tissue ansmissionll culture m

y others2-5.l culture mCF-10AT highest R

tly, RT act

m breast cast cancer (

d (Fig.3). Fer donors from BC

T activity w

) was detectey RT-PCR. β

MDAMB231as control. (CUM149) by I

C

ry breastr tissue samluated in a We founls (SUM-1not in MC

samples: n electronmedia. In . These vimedia was

T cells. In RT activitytivity was

ancer patie(BC), or cFraction A(BC or Dor DCIS pwas comp

ed in invasiveβ-actin was u) but not exp

C) The surfacFS using 6H

Fig. 2 (comparefrom theSUM14murine l(StratagcalibratoRT activRNA, noRNA se

t cancer (Imples, exp

additional bnd that HE149, KPLCF-10A c

(months 0n microsco

fact, HERiral particls compareaddition, y was dems demonst

ents. RT acancer patA (fractionCIS). RT patient pl

pared in tu

e ductal carciused as contropressed in MCce expression

H5. mIgG was

(A) RT activied in cell culte IBC cell lin9. Serial diluleukemia viruene) were usors (data not vity was demot DNA usinlected stain.

IBC) cellpression obreast can

ERV-K enL-4, and M

ells and in

0 to 48) opy (TEMRV-K virales woulded, and wetwo IBC c

monstratedtrated in R

activity intients withns 1 to 5) units werasma, com

umor tissu

inoma (Acc1ol. (B) The CF-10A benin of HERV-Ks used as con

ity was ture media

nes KPL-4 anutions of us RT

sed as shown). (B)

monstrated in ng SYTO

ls and of ncer (BC) v

MDA-n

M) in al-like

d be e found cell d in

RNA,

n DGF h ductal and B

re mpared ues vs.

77,

gn K ntrol.

nd

underein

InofbydedeH

1.3

ninvolvedemonstrateported RTncluding IB

n addition,f cell cultuy Westernetected in ensity of eERV-K.

3 Identifi

Fig. 3 RT patients or BC (N=49) normal donopatients (P=color) and u(red color) t

Fig. 4 Immbottom of gwas used a

A

A

d normal bed in mosT activity BC cells;

, HERV-Kure median blot usin

only BC peach fracti

cation of

activity wastissues. RTand DCIS (Nors (Nl), in F

=0.0063) and uninvolved brthan in match

munoblot wagel. A) IDC

as positive co

breast tissust tumor tiin our grawe are pr

K viral enva or patienng anti-HEpatient plion was la

infectious

s compared iT activity wasN=20). RT acFraction A (fr

DCIS patienreast tissues (hed uninvolve

as used to detpatient plasm

ontrol.

ues obtainissues comant applicreparing a

v mRNA nt plasma wERV-K enasma (Figabeled. Ou

s viruses:

in plasma obs compared inctivity was siractions 1 to 5nts (P=0.0003(green color)ed breast tiss

tect HERV-Kma (left pane

B

ned from tmpared wiation, and

a manuscri

or proteinwas detec

nv monoclg. 4A), buur data ind

(months 6

btained fromn plasma fromignificantly h5; left panel)3) in Fraction) was comparsues.

K env proteinel). B) Norma

the same dith matched verified ipt to repo

ns with RTcted by RTlonal antib

ut not in pldicate tha

6 to 42)

m normal femm various femhigher in BC). Compared tn B (fractionsred (right pan

n in each fractal female don

B

RF

U

5

10

1.5

donors. Sied uninvothis incre

ort all of t

T activityT-PCR usbody (6Hlasma from

at fractions

male donorsmale donors patients (P=to normal dos 6 to 10; midnel). Signific

tion. The denor plasma (r

51 57 73 75

0

500000

000000

5100 6

****

****

****

ignificantolved normeased activthese resul

localizeding HERV5; Fig. 4).m normals with hig

, BC patientincluding no

=0.0009) and onors, RT actddle panel). Rantly higher

ensity of the fright panel). H

C RT Assay i

P

78 79 82 83 87 90

Tumor TissNormal Tis

****

tly higher mal breastvity in addlts.

d in gradieV-K prime. HERV-Kl female dgher RT ac

ts, and ductaormal donors

DCIS patientivity was alsRT activity inRT activity w

fraction accoHERV-K rec

in human tissu

atient Tissue ID

0 98 109

110

124

127

128

suesssues

*****

****

**

RT activit tissues (Fditional sa

ent centrifers (data nK viral endonors (Figctivity ind

al carcinoma(N=25), and

nts (P=0.0002so significantn BC tumor twas observed

mpanies eachcombinant fu

es

D

813

113

413

614

714

915

5

****

***

ity was Fig. 3C). Wamples he

fugation frnot shown

nv proteinsg. 4B). Thdeed expre

a in situ (DCd patients with2), comparedtly higher in Btissues (red d in tumor tis

h band at theusion protein

55 166

177

190

***

We ere,

fractions n), or s were he ess

CIS) h

d to BC

ssues

e

Wfracocawocotovoat inThfirdade Pr 2-InH(F

2.HEpase

We determiactions aftompared toancer patieomen with

ontaining A proceed.

olume) (6 37°C. Ce

ntegrated inhis technirst to demata providevelopmen

roject 2: T

-1 Evaluatn our grantERV-K v

Fig.6A) an

2 DetermERV-K watients withera (Fig. 6B

A

ned the syter ultraceno fractionsents with hhout canceAlexaFluoThen viruμl) were ulls were wnto target que in Fig

monstrate tde strong ent and tum

To evalua

tion of ant applicati

viral mRNnd even vi

mine the viwas quantit

h differentB).

ynthesis ofntrifugatios having thhigh or lower were usor 546 – dUuses from pused to infwashed and

cells wereg.5 allowsthat HERVevidence fmorigenes

ate anti-vi

nti-HERV ion, RT-P

NAs were iiral particl

iral load atated by qRt stages of

f infectiouon of plasmhe same dew RT actived as contUTP and oplasma (0.fect target d incubatee observeds us to visV-K virusfor infectisis.

ral antibo

antibodiePCR was uindeed preles are pre

 

as a potenRT-PCR uf breast ca

1

Co

py

nu

mb

ers

us viruses ima obtaineensity as thvity. Plasmtrols. Viruother reage2% of thecells (C3.5d in regula

d between ualize the

ses isolateion and ac

odies and

es as biomused to coesent in caesent in B

ntial biomusing HERancer. The

Control D103

104

105

106

107

108

109

101 0

py

B

in density ed from Bhat obtain

ma samplesses were lents neces

e fraction v555 felinear medium8 hr and 9

e viral gened from BCctivation o

viral RNA

markers foonfirm viraancer pati

BC patient

arker for R-K env SU

viral load

DCIS I

*** ***

Samp

gradient BC patientsned from ots obtainedlabeled in sary for th

volume) (0e cells) in mm for 3h, 896 hr (Fig.nome onceC patientsof HERV-

A as detec

or BC: (mal load in ient sera. Ot sera, esp

early detU primers

d was comp

IIA IIB

*****

ples

s, and ther

d from buffer

he endogen0.6 μl) or fmedia con

8h, or 96 h5C and Fie it is delis with IDC-K virus in

ction and

months 3 tosera by qOur data iecially in

tection: (mfrom RNA

pared with

IIIA

*** Fide(1ELgcoB)nufrofrocacodestawi

nous reverfrom BC tintaining 4 h after infeig.5D). ivered in tC are ablen BC, whi

progress

o 46) qRT-PCR,indicate thpatients w

months 6 toA isolatedh viral load

Fig. 5 Virusgradient fraca patient witwere labeledthe cell. Higwith 5dUTPvarious timethat infectedconfocal mic8 hr post-infvirus fractioinfection; Cefraction fromRight panel:plasma, 96 hDGF obtainedonor was uinfection; Levirions weremicroscope

ig. 6 A) Antietected at hig:200 dilutionLISA using Hg/ml), compaontrols. ) Viral load umber) was dom patient seom patients wancer were asopy numbers emonstrated iage IIA, stagith normal fe

rse transcrissues (2%μg of Polyction. Viru

target celle to infect ich may im

ion bioma

demonstrhat HERVwith DCIS

o 42) d from dond in norma

ses isolated fctions (DGFsth IDC havind to evaluate gh RT fractio

P and used fore points post-d the cells wecroscopy. Lefection; Left-

on from plasmenter-right pam plasma, 8 h: BC virus frahr post-infected from a no

used as controeft panel). HEe detected by(red color).

i-HERV-K engher levels in n) at various sHERV-K envared with nor

(HERV-K mdetermined inera by qRT-Pwith various ssayed. Signiof HERV-K

in patients wie IIB, and sta

emale donors

ription (en% of the fraybrene/ml uses that

ls. We aretarget cel

mpact BC

arkers:

rating thaV-K antiboS.

nor sera froal female c

from density s) of plasma fng RT activity

virus entry inons were laber infection. A-infection, virere observed eft panel: con-center panelma, 3 hr post-anel: BC viruhr post-infectaction from tion. The samrmal female ol (8 hr post-ERV-K incom confocal

nv antibodieBC patient sstages of dise

v fusion protermal female

mRNA copy n RNA obtainPCR. Sera obstages of breficantly highviral RNAs ith DCIS, staage IIIA, com.

ndo-RT) action for 3 h

e the lls. Our

C

at odies

om control

from y nto eled At ruses using

ntrol, : BC -us tion;

me

ming

s were sera ease by ein (10

ned btained east her were

age I, mpared

Seancodisy

erum antibntigen peptompared nscriminate

ynthetic pe

Fig. 7 Ain situ,includigroup i

bodies wertides (MA

normal feme between eptides can

Anti-HERV- TIS) and noing MAPs 95is also a cont

re also detAPs), whichmale donor

TIS and cn be used i

-K antibodiesormal female 5-96 (P=0.03trol group (fe

tected as mh are proters and paticontrols (Finstead of

s were detectcontrol sera 3), 75-76 (P=emale women

markers of ease and pients with Fig. 7) as wf HERV-K

ted using vari(1:200 diluti

=0.0136), 92-n without bre

f viral loadpeptidase rtumor in s

well as ourK proteins f

ious HERV-Kion) by ELIS-93 (P=0.026east cancer).

d. We syntresistant, asitu (TIS). r HERV-Kfor detecti

K multiple anA. Significa

66), and 73 (P

thesized aas early de

The MAK protein-bng serum

ntigen peptidantly higher tP=0.006), bu

and tested etection maPs-based abased assaantibodies

des (MAPs) (titers were obut not in MAP

several HEarkers of Bassays werays, sugges in ELISA

(10 g/ml) inbserved in TIPs 108 and 82

ERV-K mBC. We re able to sting that A assays.

n BC patient IS patients 2-83. The PC

multiple

(tumor

C+HC

2.3ReOnanexW(Npr

2.Mexblincoprov

3 Determecently, sne group

nd the othxpression

Wilcoxon rN=21) hadredictive b

4 DetermMCF-10ATxpression oot (Fig. 9A

ncreased prompared inroliferationverexpress

Fig. 9 HER10AT cells expression HERV-K inHERV-K enwith pLVXproliferationobserved insignificantlytransfected

A

A

mine whethera taken was metaer group (of HERV

rank-sum d higher exbiomarker

mine whethT cells werof HERV-A). Cell prroliferationn the cells n and transsion promo

RV-K env cDto generate pof HERV-K n MCF-10ATnv protein w

X vector only n was compa

n MCF-10ATy higher numwith vector o

her viral lfrom two

astatic BC (NED) sho

V-K in MBtest (Fig. xpressionr of metas

her HERVre transfec-K env RNroliferationn (p=0.030transfecte

sformationotes tumor

NA isolated pLVX-Kenv.env RNA wa

T cells transfewas increased

cells (1.83 foared in cells cT cells transfember of colononly.

load can bo groups o

patients (owed no e

BC (N=568). These

n than NEDstatic BC.

V-K virusted with p

NA and pron and tran07) and coed pLVX. n. Our nexrigenicity

from infectio. MCF-10ATas compared

fected with Kin MCF-10A

old increasedcultured for 3ected with pLnies (p<0.000

Cell prolif

Cell n

um

bers

/ w

ell

500

1000

1500

be used asof BC pati(MBC) whevidence o

6) over NBe results vaD (N=21)Sites of m

ses promopLVX-Kenotein in bo

nsformationolony numThese resuxt plannedin vivo in

ous viruses wT cells transfe

between pLVKenv, comparAT cells transd by Image J 3 days post-trLVX-Kenv th01) in MCF-1

B

B

feration of Kenv tby cell number

pLVX0

00

00

00 p

s a biomarents at diaho develoof disease

BC (N=56alidate pre (P = 0.03

metastasis

ote tumor nv or emptoth cell linn was dete

mber (p<0.0ults indica

d set of expmouse mo

was cloned inected with pLVX and pLVred with cellssfected with panalysis), byransfection. Ahan in cells tr10AT cells tr

transduced MCF-counting (Day 3)

pLVX-Kenv

p=0.0307

rker for pagnosis woped metae after 3 y6; Fig. 8A)evious fin34), and ps of these M

growth: ty vector (

nes was deermined in0001) wasate that HEperimentsodels.

nto a pLVX LLVX-Lenti v

VX-Kenv, ands transfected pLVX-Kenv

y Western bloA significantransfected wiransfected wi

-10AT cells)

predictingwere matchastatic breayears. Sign) was dem

ndings frorovide strMBC are

(pLVX), totermined b

n both cells observedERV-K en will be to

Lentivirus (Lector only (pd there was swith vector o

v, in comparisot using anti-tly increased ith pLVX onith pLVX-Ke

FeswsHbp

g BC metahed for ERast cancernificantly monstratedm a pilot

rong evideshown in

o overexprby RT-PCl lines (Figd in MCF-nv gene proo determin

Lenti) vector pLVX) were usignificantly ionly (P=0.00son to MCF--HERV-K mAcell prolifera

nly. (C) A sofenv than in th

Fig. 8 (A) Sienv RNAs waera with MB

without metasites from MB

Higher metasbrain, lung, bpleura.

C

astasis: (mR, PR andr 3 years a(P = 0.04

d in our lastudy, wh

ence that Hn Fig. 8B.

ress HERVCR, qRT-Pg. 9B and 910ATpLVomotes no

ne whether

and transfectused as contrincreased exp

002). The exp10AT cells trAb (6H5). (Bation (p=0.03ft agar assay he same cell l

gnificantly has demonstra

BC compared stasis (NBC)BC patients wtasis sites weone, and othe

months 12 d HER2 stafter diagn15) increa

ab using thhere MBCHERV-K

V-K. The CR, and W9C). Signi

VX-Kenv on-cancer cr HEDRV-

ted into MCFrol. (A) The pression of pression of ransfected

B) Cell 307) was revealed a line

higher HERVated in patien

in patient se). (B) Metastawere compareere found in ers than liver

to 48) atus. nosis, ased he

C is a

Western ificantly

cell -K

F-

V-K nt era asis ed.

r and

2. A.

AfHEtratustacodeinthboobshsh

deustai 2.TootwBCexMstap5evexan

5 Determ

.

fter we obERV-K inansfected w

umor growably transontrol shRemonstratendicate thahese mice both groupsbserved in hRNAenv hRNAc th

In aetermine wsed to deteil vein and

6 Determo establishther mechill continuC cell linexpression.

MDM2 andabilization53, MDM2valuate whxpression ntibodies (

Fig. 10 (Astably trancontrol shRSignifican= 0.0005).metastatic shRNA (shmRNA (sh

mine wheth

bserved incnvolvemenwith shRN

wth in NODsfected wiRNA (shRNed in MDAat HERV-bearing xes and the n

pieces of (bottom tan with sh

addition, Mwhether HEermine the d in situ, a

mine assoch that HERanisms byue to exples treated We foun

d c-Myc mn of the p

M2 and c-mhether HEof these g(Fig. 11B)

A) Tumor sizensfected with RNA (shRNA

ntly reduced t. (C) Lung titumor cells w

hRNAc), comhRNAenv).

her HERV

creased prnt in tumorNA targetinD/SCID gaith shRNA

RNAc) (FigA-MB-23K plays a

enografts onumber of f lung tissutwo panelhRNAenvMDA-MB-ERV-K ov changes i

and metast

ciations oRV-K is iy which Hlore our fiwith anti-

nd that knomRNAs. c-

53 proteinmyc in cancERV-K is genes at th). A possi

es were comp shRNA targAc) (Top, regtumor weightissues were cwere observempared to MD

V-K virus

roliferationrigenicity ng the HEamma femA targetingg. 10). Sig1 cells sta

an importaof cells trametastase

ue from mils). More mv (top two-231 or Mverexpressin growth asis to lun

of HERV-involved i

HERV-K tnding of p-HERV-Kockdown o-Myc indun 19. In thicer cell linaffecting

he proteinible pathw

pared betweegeting HERVgular light. Bts were evidecompared beted in MDA-MDA-MB-231

ses promo

B

n and transand metas

ERV-K envmale mice w

g HERV-Kgnificantlyably transfant role in ansfected wes formed wice injectemetastatic

o panels). MCF-7 brea

sion promorates and c

ng, liver, a

-K with thin oncogetriggers chp53 involv

K monocloof HERVuces the tuis grant, qnes stablysignaling

n level werway involv

en two groupV-K env mRNBottom underent in the HEtween the twMB-231 cells cells transfe

ote metast

B.

sformationstasis. Towv gene or cwas compaK env mR

y reduced tfected wittumorigen

with shRNwas determ

ed with MDcells were

ast cancer otes tumorcell transf

and other o

he p53 paenic signalhanges thavement inonal antib

V-K env wumor suppqRT-PCRy transfectg via the pre also detving HER

s of MDA-MNA (shRNAenr fluorescent

ERV-K knocko groups. Mas transfected ected with HE

tasis: (mon

n in MCF-ward this econtrol shared in mi

RNA (shRtumor sizeth shRNAnicity. FurNAenv or mined (FigDA-MB-2e also obs

cell lines r growth. Cformation. organs will

athway: (ling pathwat result inn HERV-Kbodies or s

with shRNApressor ARwas perfo

ted with sh14ARF/Mtermined

RV-K sign

MB-231 cells nv) or with light). (B)

kdown group any more with control

ERV-K env

nths 18 to

C.

-10AT cellend, MDAhRNA withice xenog

RNAenv), es (Fig. 10AAenv comprthermore,shRNAc.

g. 10C). S231cells traerved in M

will be traCell prolifAlso, thes

l be exami

(months 8ways, we pn the deveK signalinshRNA toA resultedRF, whichormed to dhRNA tar

MDM2/p5using flow

naling in c

(p

46)

ls (Fig. 9)A-MB-231 h a scrambrafted witcomparedA) and wepared with, lung met. Lung tisStronger gransfected w

MDA-MB-

ansfected wferation anse cells wiined.

to 47) propose toelopment ng, and foco down-regd in downh inhibits determinergeting the3 axis (Fiw cytomeancer is sh

, we furthecells were

bled insert th MDA-Md with miceights (Figh shRNActastasis wassues werereen fluorwith shRN-231 cells

with pLVXnd soft agaill be injec

o investigof BC. In cus on thegulate HE

n-regulatioMDM2 a

e changes e HERV-Kig. 11A). Ctry with vhown in F

er exploree stably (shRNAc

MB-231 cce injecteg.10B) werc. These ras comparee harvestedrescence wNAc than transfecte

X-Kenv toar assays wcted throug

gate p53 anthis Aim

e p53 pathERV-K on of H-Raand leads tin expresK env genChanges ivarious Fig. 11C.

ed

c). The cells d with re results ed in d from

was withn

ed with

o will be gh the

nd , we hway in

as, to sion of ne, to in

Pr 3.AnanortraCTcetarre

Fuanpa

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% S

pec

ific

rele

ase

20:1

0

50

100

150

**

A.

roject 3: H

1. Screenn HERV-

ntibody ger other donansfected wTL assaysells were crgeting HEsults indic

A.

urthermornd MDA-Matient (Fig

Fig. 11 (A) qRNAs in canRNA by shRshRNA. (B) Aexpression ofMYOD1 wer10A and MCcell lines. (C

Fig. 12 Hspecific CA(ND#4274shRNA) orMDAMB2

Effector to targ

10:1

*

HERV-K

ning PBM-K specificenerated fnors (Fig.1with shRN to determ

cytotoxic toERV-K encate that H

re, HERVMB-435ebg.13A) sho

qRT-PCR wancer cells tranRNA targeting

Anti-HERV-f various prore determined

CF10AT), wit) Possible pa

ERV-K specAR T cells w

478; C), and wr control shR231 shRNA c

et cell ratio

5:1

2.5:

1

CAR+ : MDA-M

CAR+ : MDA-M

**

**

viral targ

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from anti-H12C) that wNA targetin

mine whethoward MDnv RNA, w

HERV-K sp

-K specifib1 BC celowed grea

as used to detnsfected withg led to down-K monoclonteins includind. Increased th the exceptiathways throu

cific CAR cytwere generatewere used fo

RNA (MDAMcells, were ki

MB-231cont

MB-231shRNA

% S

pec

ific

rele

ase

-

-

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gets for im

BC patiec antigen HERV-K were bearing HERV

her or not tDA-MB-23which dowpecific CA

B.

fic T cells lls servedater cytoto

termine the ch shRNA targn-regulation onal antibody (ng HERV-Kexpression oion of TP53Augh which an

totoxicity towed from PBMor CTL assayMB231 controilled by HER

Effec

20:1

-40

-20

0

20

40

60

mmunothe

ent bloodreceptor (

K 6H5 antibing the CA

V-K env RNthe cytoto31 cells tr

wn-regulateAR killing

were gend as target oxicity tow

hanges in exgeting HERVof H-Ras, MD(6H5; 10µg/m

K, Caspases 3,f multiple pr

AIP1 in MCFnti-HERV-K

ward breast cMC cells isola

s. MDA-MBol) were used

RV-K specific

tor to target ce

10:1 5:

1

erapy:

for T-cel(CAR) wabody. PB

AR were uNA (shRNxicity dep

reated withed HERV-

g depends o

nerated frocells (Fig

ward MD

xpression of sV-K env RNA

MDM2, and c-Mml) was used, 8, and 9, CIroteins was obF-10A. 6H5 antibody blo

cancer cells (ated from BCB-231 breast d as target cec CAR T cell

ell ratio

2.5:

1

CAR+ : MDA-M

CAR+ : MDA-M

ll responsas generat

BMCs fromused for adNA) or conpend on HEh control s-K env RNon target c

om BC (Fig.13). HERA-MB-43

several genesAs, relative toMyc, and upr

d to treat varioIDEA, TNFSbserved in Bthus has an e

ocks BC prol

MDA-MB-2C (Acc108; A

cancer cells ells for the CTls obtained fr

MB-231cont

MB-231shRNA

C.

ses: (Monted from am BC (Figdoptive thentrol shRNERV-K exshRNA, buNA exprescell expres

ig.13A) oRV-K spe35eb1 cell

s including Ho control shRregulation ofous breast ce

SF8, TNFRSFBC cell lines ceffect on expriferation and

231) was deteA), OC (Acc15

stably transfeTL assays. Orom BC, OC,

nths 3–42)an anti-HE.12A) or O

erapy (Fig.NA (cont) wxpression. ut not cellssion in MDssion of H

C.

r a normaecific T cels than did

HERV-K env,RNA. Down-rf Tp53 in canell lines in a sF10D, TP53,compared wiression of ca

d progression

ermined using53; B), and a

fected with HOnly MDA-M, and a norma

. ERV-K siOC patient. 12). MDAwere used HERV-K

s treated wDA-MB-2ERV-K.

al female dells generad T cells g

, H-Ras, MDMregulation of ncer cells transingle assay a TP53AIP1,

ith non-BC ceancer-relevann.

g a CTL assaa normal fem

HERV-K shRNMB-231 contral female don

ngle chaint blood (FiA-MB-23

d as target cK specific Cwith shRNA231 cells. O

donor (Figated from generated

M2, and Tp5f HERV-K ennsfected withand changes GML, and ell lines (MC

nt proteins in

ay. HERV-K male control

NA (MDAMrol cells, but nor.

n ig.12B) 1 cells cells for CAR T A Our

g.13B) a BC from a

53 nv h

in

CF-BC

MB231 not

nobyge

3.Tuunlyan 3.3wi• m• tSy• TTC• T• Tau Pr 4.acInTLofanar

Effec

% S

pec

ific

rele

ase

20:1

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20

40

60

80

ormal femy flow cytenerated in

A.

2. Developumor specninvolved ymphocytend banked

3. We wilill compamRNA withe anchorynthesis kiTCR geneCR constaThe PCR pThe anti-tuutologous

roject 4: V

1. Determctivation an this projeLR and Rf TLR inclnd IFS usire infected

Fig. 13 HE(ND#42747MB-435eb1patient (A), from BC papatient 2 we

ctor to target cell r

10:1 5:

1

male donortometry (Fn BC pati

pment of cimens fromtissue infi

es accordinfor future

ll generatere its antiill be isolar sequenceit (Clontec amplifica

ant region products wumor effeccancer cel

Viral activ

mine if HEand signaect, we in

RLR famililuding TLing their ad with HE

ERV-K specif78; B) pulsed 1) was determ

but not fromatients who exeeks post-pul

ratio

1.25

:1

#M

KM

r (Fig.13BFig.13C). ients, but n

tumor infm BC surgiltrating lyng to the Se use.

e TCRs thitumor effated from se-containinch). ation will tand the SM

will be cloncts of HERlls in vitro

vation of

ERV-K moling, and

nvestigate ies similar

LR3 (greenantibodiesERV-K vir

fic T cells wed with HERVmined using am a normal fexpress HERVlsing with HE

#277 T cells:MDA-MB-435eb1

K10 #277 T cells:MDA-MB-435eb1

B). CD3 anThese datnot in nor

B

filtrating gery patien

ymphocyteStatement o

hat targetfect with Hsorted, higng cDNA w

then be peMART olined into p

RV-K spec and in viv

the innate

odulationif so whicwhether or to exogen), 7 (red)

s. One resruses isola

ere generatedV-K fusion proa CTL assay. male donor (

V-K. C) PercERV-K fusio

nd CD8 spta indicatermal fema

.

lymphocynts were c

es (NIL) wof Work p

t the HLAHERV-K

ghly avid Cwill be syn

erformed oigonucleot

pCRII vectcific CARvo.

e immune

n effects (Tch TLRs aor not HEenous viru), and 9 (rsult is shoated from

d from PBMCotein (K10GOnly MDA-

(B). The datacentages of Con protein by

pecific T e that grea

ale donors

ytes for Tcollected awere culturprovided in

A-A*0201-K CARs sidCD8 T celnthesized

on the singtide. tor and the

R vs. HERV

e response

Toll-like rand RLRs

ERV-K hasuses and ored) in varwn in FigBC patien

C (isolated fr17) or contro-MB-435eb1a indicate thaCD8 (9.23%)

flow cytome

cells fromater numbs who do n

T-cell adopand tumor red. Monon “Support

-restrictedde-by-sidels using a from the m

gle-strande

e inserts wV-K specif

e in BC: (

receptor) s. s the abiliother endorious brea

g.14. We wnts who h

rom BC patieol KLH prote cells were k

at HERV-K sp and CD3 poetry.

m HERV-Kbers of HEnot expres

C

ptive therinfiltratingnuclear ce

rting Docu

d epitopese: (monthsOligotex D

mRNA ob

ed cDNA u

will be sequfic TCR w

months 3

TLR and

ity to activogenous dast cell linwill carry have eleva

ent Acc277; Aein. Cytotoxikilled by HERpecific T kill

ositive cells (7

K specificERV-K spss HERV-

C.

rapy: (mong lymphocells were cumentation

s of HERVs 12 to 46)Direct mR

btained wit

using prim

uenced. will be com

to 44)

d RIG-I-lik

vate the indanger signnes was de

out these ated RT ac

A), and a noricity toward bRV-K specifiling depends 70.7%) in PB

c T cells wpecific T c-K.

nths 4 to 4cytes (TILcultured ann”. These c

V-K env p)

RNA Minith a SMAR

mers comp

mpared sid

ke recepto

nnate immnals. Firstetermined

same assctivity.

rmal female cbreast canceric T cells obton the use o

BMC obtaine

were detercells were

44) L) or normnd expandcells were

protein, an

kit. RTer PCR

lementary

de-by-side

ors (RLR

mune respot, the exprby Westeays after c

control r cells (MDAained from a

of PBMC obtaed from a BC

rmined only

mal ed to stored

nd we

R cDNA

y to the

in

R)

onse ression ern blot cells

A-a BC ained

C

4.no• A• Esu• BAP 4.3im• IHEre• Tvi

CoMthTLbebe

2. We wilormal breAnalyze thExpress BupernatantsBlock HERPOBEC3

3. We wilmpact on TInvestigateERV-K knquired forThe involvtro and in

o-localizatMCF-10A ahe transformLR9. Our e required etween TL

ll assess theast: (monhe extent tst-2/tethers of tetherRV-K withrestriction

ll evaluateTLRs or Re the relatinockdownr HERV-Kvement of vivo.

tion (yelloand MDA-med but ndata demofor HERV

LR9 and H

he effect onths 4 to 45to which thrin in 293Trin-expressh siRNA t

n.

e changes RLRs: (mionship be

n on variouK to exert if Polo-like

ow; left pa-MB-231 c

non-tumorionstrate foV-K to exe

HERV-K in

of HERV-5) hese restriTcells, infsing cells to see if th

occurrinmonths 5 toetween BCus parametits effects kinases (P

anel) of TLcells (Fig.igenic breaor the first ert its effecn upcomin

-K on anti

iction factofect the cel

he effect on

g at very o 46) C stem cellters associin CSCs oPlks) as es

LR 9 (red; 15). Co-last cell lintime co-ex

cts on breang studies.

iretrovira

ors alter rells with HE

n restrictio

early stag

ls (CSCs) iated with or during mssential co

right panelocalizatio

ne MCF-10xpressionast tumorig

al genes an

etroviral reERV-K an

on is speci

ges of BC

and HERVmetastasi

metastasismponents

el) and HEon was pro0A, whichof TLR9 agenicity.

nd retrov

eplication nd measur

ific for HE

and duri

V-K expreis, and dete. of antivir

ERV-K (grominent inh showed land HERVWe will fo

Fig. 15 Hfluorescendetected bMDA-MBthan in Mbreast celantibodieantibody.

iral restri

as a functe recovery

ERV-K an

ng BC pr

ession, evaermine wh

ral pathwa

reen; centen MDA-Mlow expresV-K in canfocus on ev

HERV-K (grence) and TLRby IFS in theB-231 to a gr

MCF-10A nonlls using theires. 6H5 is ant.

iction fact

tion of HEy of HERV

nd can reve

rogression

aluate the hether TLR

ays will be

er panel) wMB-231 BC

ssion of boncer cells.valuating i

Fig. 14TLR3 9 (red)IFS in MDAMMCF-10A u

een R 9 (red) were BC cell linereater extent n-tumorigenir respective ti-HERV-K e

tors in BC

ERV-K staV-K from

erse tether

n that hav

influence Rs or RLR

determine

was compaC cells but oth HERVTLRs ma

interaction

4 TLRs inclu(green), 7 (re

) were detectthe breast ce

MB231, MCF10AT, and Msing their ant

re e

c

env

C and

atus.

rin or

e an

of Rs are

ed in

ared in not in

V-K and ay thus ns

uding ed), and ted by ell lines F-7,

MCF-tibodies.

Pr 5.m InlabmCYanfu

• Uto 5.ef • Cex• W• Wan 5.3 • A• Maftu 

roject 5: F

1. We wilmemory B-

n this projeb (see Fig

memory B YTOX mnd C3 werurther emp

A.

Using stan these anti

2. We wilffector fun

Clone intoxpress in 2We will caWe will qunnexin V a

3. We wil

Anti-tumoMultiple after treatmeumorigenic

Fig. 16 (A) obtained fromscreening forproduced by

Fully hum

ll develop -cells of pr

ect, a micrg. 16A). Acells fromarker wasre selectedployed to

ndard singibodies.

ll carry ounctionality

o an Ig exp293T cells.arry out stuantify anand caspas

ll characte

or effects oassays willent with h

c potential

A nanowell m 6H5 hybrir their ability

y PCR using t

man anti-v

rapid scrre-screen

roengravinAnti-HERm BC paties used for d as depicconfirm th

le-cell RT

ut molecuy in vitro u

pression ve. andard ch

ntibody depse assays a

erize ther

of human al be carriedhuman or m of BC cel

screen has bedoma cells w

y to kill targetheir primers

viral antib

reening ofed BC pa

ng techniqRV-K mAbents. Singmeasurin

cted in Figheir seque

T-PCR tech

ular charausing BC

ector carry

aracterizatpendent cyand antibo

rapeutic a

anti-HERVd out to demurine antll lines (so

een developewere tested uset MDA-MB-, with the exc

body for B

f high-affiatients usin

que for vab 6H5 hyb

gle hybridong cytotoxg. 16A andences (dat

hniques w

acterizatiocell lines

ying the co

tion (ELISytotoxicitydy mediat

antibodies

V-K monoetermine thti-HERV-Koft agar ass

ed in our lab wsing this met-231 BC cellception in C3

BC:

inity, highng nanow

alidation obridoma coma cells

xicity. Singd PCR wata not show

B

we will amp

on of hum: (months

onstant reg

SA/Biacory (ADCC)ted inducti

in a mou

oclonal anthe relationK antibodisay).

which can rathod. The sins. (B) Heavy3, for which t

hly cytotowell screen

of cytotoxcells were were addgle 6H5 h

as carried wn).

B.

plify and c

man anti-H6 to 46)

gion of hu

re) (for aff and compion of apo

use model

tibodies (mnship betwies (or con

apidly screen ngle cells A1y chain and lithe light chai

xic humaning: (mon

xicity of ane used for ded singlehybridomaout as in F

clone the p

HERV-K e

uman γ1, C

finity measplement deptosis usin

for BC: (

mAbs) wilween the exntrol antib

for high-affi, A7, B3, B5ight chain frain is missing

an antibodnths 3 to 4

ntibodies testing ou MDA-Ma cells thaFig. 16B.

paired vari

env antibo

Ck, and Cλ

surement)ependent cng caspase

(months 12

ll be testedxpression odies) and

inity single c5, and C3 weragments from.

dies again40)

has been ur hypothe

MB-231 celat included

Sequenc

iable regio

odies and

λ, sequence

. cytotoxicite assays.

2 to 46)

d in vitro aof HERV

d cell grow

cell antibodiere selected us

m the above s

st BC fro

developedesis beforells and a d A1, A7,ce analysis

ons corresp

d quantify

e and tran

ty (CDC)

and in vivo-K env pro

wth, apopto

es. Single cellsing nanowelsingle cells w

m the

d in our e using

B3, B5, s was

ponding

siently

using

o. otein osis and

ls ll

were