DTIC ADA423741: Engineering Protease-Specific Human Antibodies and Discovering Novel Serine Proteases Expressed in Prostate Cancer Using Phage Display

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ber: DAMDI?-03-1-2030

‘TYY.E: Sngineering Proteace-specific Human antibodies and
piscovewing Novel Serine Proteases Expressed in Pontate
Cancer Using Phage Display

PALICL2AY Jeongaoor Sur, PLD.

versity of California, San Francisco
San Francisco, Calizozris 94243-0862

emary 2004

‘YEE OF REEURT: anneal Suemary

FREPARED FOR: 0.8, Army Medica! Researcy and Materiel Command
Part Detsick, Maryland 22702-8012

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Enginroring vreteass-speci ‘la Horan Actibadies anc Dexpt7-p2-1~1030

Discovering Novel Serine F=oteagsa Expt
Canser ERong Phage tiepley

Feonghoon Son, hb.

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Quankitativo RI-FCR and immnshistochematry (INC) experiments cevealed that menbcaue-type
serine proleene 1 (MI-SPL}, a murber of the ITSP Larivy, ia apacitieelly overeroressed te
epitoel'si cance: coins and treave sarpion= it waa tepocted that in advamced senges oF
proskste, beast, and overien cancers Mivdel ig aot Tegslatad by ine endoyerooe mhé¥iter
HAI“L. WHE) playa duveise clue by accivatizg pro-nPA, prOvHGe, wad Fake2, Taken
togatiner, Wish! my be 3 kay opstzean £9etor dnvsived in’ tho Sell fenadeling plaandiogen
etivotion cascade and in signal transduction exeoades invelved in cel ceanaFoemstion,
Dysregulsted MI-s61 expression eczid prowole the proteolytic ectivity of MIcael resulting
An a hore invasive phecolype, le postulated char inhibition of ile seateolyeie activity
Bight “ausLei: tho invasive plenotype. a MI-GE1 1¢ 3 eel: eusface peotein, vo chose 20
tery the proteuse sith antioWP-SFl wing-> chazn worotlocel omtibedies(aeFos that we
Asolatad using phage display, Tasca aaFvs gotenrly ang selecLively inhibit tae provease
detivdty of MY-Cel, Our izitiel in vive stidies ‘uvetind the alzes 22 xenagratsed hun
Gvarinn tuiocs in aude mice were eigaifisantly requese. following treacuent with eee
Sonibinory serve, indicasing the thevapeut2 yotentis! of these ecive for the tceatwent of
epithelial cancers an ovary, prosLate and brosst

TRUSTE ERT

Pratiake canter, monoclons: snbibesies, nenbrane-tyes gerine 2
proteaee, phag? eiapiay

{7 SECURY CERSSREATION | TE SEGURTV CLASERCATION | TH: SECURITY CINEEEREATON —| BO UMTATE OF RaSTARCT

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Table of Contents

cover...

SF 298,

Table of Contents.

Introduction
Body. a
Kay Research Accomplishments. 8

Roportable Outcomes.

Conclusions...

References.

Appendices...
Introduction

Abtreviuidons: scF¥, single chain variable fragment; ECM, extaecellulur matrix. enzyinestinked
jmmunosorbeat assay, DMT-SPL-P, protease damsin of buman MJ-SHI; MT-SP1, membrane-type
serine prolease 1; INC, imanaohistochomisay, TS, Type U transmembrane serine prucease; RT-PCR,
reverse Ianscrptase polymetose can restion.

Membrane unchored serine proteases as new targers for treatm of cancer. In tbe eney phases of
toner, the groath of mor cells russ ccutinel 2 their aiginal issue hourdaries. Tuw2ver, apo.
becoming malignant, cqacer cells invade surrounding tissue hy migeting fom their issue compartment
tnd disseminating turouguout the bedy, ‘To penetrate theoogh velluer bacriens and metastasize, tumors
feamploy invasion machinery & hallmark of die machinery i Uysreyulaled wcateia-depiading proteases
‘Dut digest exteacelne austix (ECM) proteins by cleaving peptide bund of specific amino acid
sequences (And-easen at al, 1997; Hapahgn ot al. 2U0U; Funimow ell. 1999; Wallespp eth, 2000), An
lnceeasing amotit of roscae' as rosnely focused on a subgroup 0° dhe trgpsinsike serine proteases
tha. a dsecty a chered ta plasma membrane on cel surTuce(Tape> el al, 2001: Stabo cc al, 2003;
[Netel-Amet tl, 2103). These manbruneanchered proteases ure anchored via a C-terminal
trepamenbrae domain (ype }, via a glyouyl-phospbatidyinosiel Unkege, or via an N-cmainal
uansmembrane darn (Type Tor TSP). Disrupion or mutation ofthe genes encoding these
proteases ace astosiatod with cance: While these proteases show Ughtly eogulased expression in norm
cols, dey a dysregulated and ovorexprosod during lurar propessiou. Memb:ane anchored seins
pcteates therefore represent promising new ond ux upped Sou-ce of poeatial targets for the
development of now cause tisrapeutcs

_MT-SPI, To idowttysoring proweascs expres i Ihe PC2 calls, RT-PCR was porformed with
‘egonerateofigonuslemide primers thu were desigued isiug eaaserved proccin sequences from the
‘typein fold af sorin pruksais. Five independent serine protease cDNAs doriveo from HC-3 mRNA
‘ere secuenoa, und a novel sere proteate that se naried remibranc-fype serine proteune | (MT-SPL)
sravidenlfiel, The exzyuse wes iadepenc ently ideniied by anther group und named maiplase (Li et
AL 1997), MT-SP] isa mosaic pootsn of 855 amine acids that contuine a bursmeaubine signal anchor,
lwo CUB domalas, fon LDLR repests, mda serine protaute domuin. knmunobloting and
iuunuaohistochemice! analyses veri the proses of M'T-SPI in epithelial issue uf prostate tumor
(Takoushi tal, 1999; Sun cal, 2103) and breast teomor {Lin el al. 1999). As MT-ST is highly
expcessed in ovarian, breast, and prosatecureer cells aml appears to be lnvolved inranr invasion, i ix
‘potential turget for cancer diggs und eatnea: (Takeuchi etl. 199% Ooers ct at, 2181). im
addition to PC3 coll, MCP? and OVCARS cells express signilfeaat amounts (10 —50 fold compared to
normal calapof MT-SP1 mRNA (Rubee! Bok, personal comicstion), MT-SPI has been shovwn to be
‘overenprested iu huanen ovarian cancer sperimiens (Talroueh tal, 1909; "tanimow eal 1999). The
serine protetsedceaain has been clonsd ito tt, col expessien vector and lhe ecumbinant version of
‘the protein has boon exprassed, puritied, and vfolgod to produce active enzytue (Takench el. 1999).

MT-SPI may sorve asa hey upstean activator in the ECM remoting plascainagan sctvation cascade.
srhas boon shown to activate Laton urokinase plusninogen uclivator (uPA) al pro-human grovath
faotor HGR) in vir (Ler otal. 2000). uHA parlisipules in the plasnil-madiated remodeling ofthe
cetracellusr mete (ACM) surrourling uuu, the wetivation of lateal HGF. HGF contribute inthe
stimulation of cancer cell growth and mails (Corps eal £997; Sowter ot al, 1999) WPA contributes to
rivasian; the binding aC uPA, ieell a serine proteas, tots eeeepter incresscs ho proifeationaf cancer
cells in vtro (Fischer a al. 1898). Inkbtionar uPA by an antisense ollyonrteotcesignificenly
rednces the inuaperitomeal ypraud of ovarian cancer mouse xeuopraftiacols (Wilholm cal. 1995)
‘Anather in vitro substeue of MT-SP1 aya idasified as protease ativatod receptor 2 (PAR2). Recent
sds :sveal thst PAR-? stination was asociated ilk the unsientpespaonylaion of MAP lenase
(Uikunaea eal, 2008), Tho highly sisiur PAK- und 3 were uot acivated by MIT-SPI, suggesting thet
‘MTSE! could bs specific rogulalary proteuse that affects signal transduction of cll protectin
(Takecchi et al. 2000)

1m normal pels, MT-SPI aoivty is tighly related by the endogenous inbibitarhepatovyte growes
‘ctor ueivetor inbibitor | (IAL-1) (Kitchhofer eta. 2008), HAI-1 is found precumiountly ia the
‘pihelium uf many issue (ypes and is up-Fegnlatedn injured or regenerative issues, TAT ney
suppress he groweh and moilty of ence sells by inbibiting the yensratior. of active uPA and sative
TIGE by MT-SPI, The expression of MT-SP1, HALA, urthe MT-SP-TIAL-1 mio correla withthe
slincal stage, histologics} grads, vstolngieal type, ur olivia ouleuny uf ovassa cancer pasinta (Oberst
‘al. 002). Advaaoed-tiage (land 1V} ovarian furs were found lo be moe likely co express M~
SPI in the ubserve of IAL. This inditas tha an imbalgnec inthe MT-SPI-TLAT-L ratio could be
important jn the development af vanced disease, Lnregulatod ur dysreguluted MT-SPI expression
‘unui prumole Ue proteolytic activity of MT-SPI, and, consequently, u more invasive phenotype. Taken
together, “hese data suggest MT-SPL may play a hey regulary vale inthe prucess af ovarian cancer
progression

_Anabodies os protease inhibins. Various prowuse inhibitors have beca developed for ramennus it
vitro and in vivo applications, Although (ily eflecive as toals to shy cancer pragression and.
metastasis scl moleuule inkibilors ore plagued by problems with resieamoe and toxicity, while
‘conventignal rueromoleeular ish ors can suifer from promiscuity and low uutvity (Cousens eh
2003}. The Ecol serine protease iahibitorceotn lise proven to bea valuable tool in the discovery ot
novel suteases, However is peetlnass iy vio is limited, as it eficis an mame response whea injected
inlo rsice. Momver, sive proteases ae Widcy cisibutsd in cxture wad wany vf den shace neatly
‘denteul uctive site elements, we wer coucenus that ccotin would nol be selective enough to
ttiatinguish anoag the closely relsted MI-SPs, ‘The antibedy soeffod is un acractive choice for the
‘development of highly porent and seloctive inkitbinors far Unis Racy sine itcan distinguish beswoen
telsaedy sels proteus, Fortormore, ancbedies are regululy used diagnose and therapcutie reagents.
-fsnecessi, this tsthod can be applied to ather erzyme Ennilies such as met metalloprotease
fauulies snd protein kinase Fain.

The focus of FYZ. To tasks ar assigned for FY2. The firs was to wanito inibition of MT-SPL to
determine is cole viv using he optimized veFv. Te was origintly proposed that this would be
perfoumed with SCID mie tanaplanted with PC-3 cella lo show that MT-SP1 inhibition can be critical
fn intervention af epithelial cuncenin posta, best, and evatian canoes, Based on quantitative RT-
PCR (Teqman"™) analyst wus evident that OVCARS cell ine produoed higher levela of MT-SPL
‘mina, In he inmuaosislochelnicel sniag sealvsis, MT-SP] overexpression i more specific ia
‘ovarian cauoer dsstes thar. prostate sancce sss (eerepars the Hy. Yio is report wilh the Fig. 3 inthe
YI report, Thus, the pllec oxperimsnes were pecformed in collaboration with Dr. Robert Bok in
‘Deparemeut of Medisins at UCSF sth nude mice xenogrulled wilh ovarian munors instead of prostate
unars 23.4 step 70 carfy tho vols of MY-SP1. ‘The secu ask wus to find previously undiscovered
set protsass that might bs involved prostate carcer using cDNA expression libraiss devived fom
prostate t™moresllfinos and K [-PCK. This project is uuderwey ond wil be coutinued, In adliion, the
‘mode oF inhibition of the most patent sePv, E2 has been kinetically characterized

Body

‘My mela, Dz. Chatles Craik has provided outstanting raining end rich enviroxameat for legalng
rile biochemistry. Di. Mate Shuman and Dt, Robett Bok in the éeparament of Misicine in UCSF
raved the foclies for studying te biology’ of provewe cauver sd touting ovariun lumur xemogretted
suite mize with soF vs, have attended ens monthly Proteases in Cancer Projest meetings where [have
fed che opportunity ls peeseat daca aud intracc with a diverse g-oup of scientists including oncologists.
omatologits, itunologists, surucaal bolngists, and wectauistic enzyncologists. Other elpful
lurertctous included the weekly meetings hele be-ween Tr. Craik, Dr, Mare Sima, pextdoctoral
researchers in Dr. Shuman groip, aud axel, For my irtllectual development, | hove altende

ly seminar seres offered hy the depacuacars of pharraccuical cemsty, biochemistry and
Diophysies, ad cellule and molecular pharmacology, and prayeucsin chemistry and ehemiva biology.
td quaatitative biology and various azuatTeeturcships at UCSP. Thuve acended eae Kxpsrimental
Biology 2003 Ama: Moving, ASME sector (April 1-15, San Diego, CA). This moeting allowed ae
Co nterscr with other sential ase workng on similar areas ofreseersh, The US Army Medical

ath aul Material Command Prosare Cancer Rerer'ch Program was acknowledge

Inmuaohistoctendsry (HAC), To determine te efsasy of usmg the evs as innmnologteal ceagents,
THC seiuing as performed in evurian wed puosate nr tissues from randomly selotod pation
"These experialents 1ovesled that only une of te sx aati M{I-SP' sckvs, 8, stained tissues 1 an
appreciable extent, Noteworthy isthe prenence of antibody specific red chromuen in te glands
1xmen in steasiticovatian cancer tissue, suggesting he MT-SP] js overexpressed aud likey shed
From the surGie ofthe cauoerons cpithoix: ell Figure 1)

Qeantiatve HT-POK (Fegonan) anys
Ins GiG.ThewaNAS on he
Dike Orrian Tse Bank were obtained and
selativs mRNA lovcl were Bzated using
“Taquan analyse Ml of the paints (22724)
vwete go UC-1Y. Tours frm ive somal
vate: Wore used aa cunils. The relative
ai _ tnotsage lee! fee MT-SP i ota cancer was
Hiketonmteaten cin tetomncncesaanarane 9287 £0307 (a=
ag Canpoe an he oma tics PY
sez age consent th cur preliney data

Supression of senografied ovarian cancer by the aie MT-SPI sob'w: (Fig 2 and Fig 3). 80 in vin
pilot srady ate sen performel ta uses the efficacy of she antiMT-SPI scFes in xauografted mary uf
inde ree. Thin study oes considezed asa pilot study to clarify the sole of MT-SP1 before PC-3 cell
(earsooted mone experieiauls wars pero: Human
QVCARS cele were injevied into 10 mde moe subcutaneously
Aficr 14 cys, when wusrs were well established, the raive were
sopatated into a herapy gromp (5 mice) and excseal group (S
‘miee). An inhibitor cochai,« combination af tree seF (04
ug of E2, 1.2 uo of Sand OA go $4) was ijented into cach
‘iouse in che therapy group 3 Knnesa weeks subcutancousty. A
conical seEW SAS (2 pig) that either sinds nor inkbite MT-SPIL
‘eas injected ino sach mnouse ‘a the contra group. Befure the
snimal sal al of sos vere purified using FPLC and sanied
thee of endororin eontaminalion, Tumors were measured weedy

oe sing calipers and were dncumeuted photographically (Fig. 3)
[No overt toxceity way ubserved in any of mice. fier eeching

Figure 2 ingen pot in ted ey iy vi ng

Feetssbaitsciaredticnate sumasia ercera (ara spunteaeons cxpiteion),aninals were

socrifced end anors distectod aué weighed. Perineal suioss and organs ware examined for maeto+
aed misroscopie metastaes. The ve ‘seeed with ibibitory scFv survive fora meen of 38 days
‘tile the mice toated with De eouzol survived for a aean af 30 days (Fig. 2) The tice in Uae
controf p-onp develope larger subcuurevas luo and more sects (Hig, 2). These resulls indicate a
therapeutie efter fot these naPvuund stauely snggest te experiment be renewed with lager aueiber
‘of animals, Sines the wookly doves adkainisered wete mininnl, we conld inercase lhe dose and ijectioa
Trequeacy ofthe soFes, Rone wach Fy revealed a dilleeal ibibicou mechanism, binding ufinity

atl binding mode, the efficacy ofeach re should be iudividua!ly teste.

rs ate ay hae GP nee sarin cael) te
Urs Suhail doe crite team fart" ae srachian Suan Ti ang barre

Creating CDNA Hbrary derived from PC:3 and OVCAR-3 oats, Two eDNA library ar in the prucess
of beiag crested thconp’ reverse manseription of poly-A-curidied ee RNAS from burnae #C-¥ cell ard
OVCARS sills, The eDNA preducts were -ugged wilh exndom primed clongation and PCR
aungltiostion using x swndaedprotocal. The resting pool of inser: Wasselccteé bused un Ler size
Sivall-size INA inserts were preferentially amplified during a PCR reactor. ané the efficacy of wech
sop is heig eomitored by PCR. Once te biased ampi‘cstion problems 2¢ solved bibrary coaslereon
‘oan be continued. We huve bee fooused au charactovizing dos sot rather thun exploring new targets,
‘We would like te have an understanding a how este inbibitws felon beline we focus owe atencion
vn uller TSP,

Kinstio characterteation of the solv &2 (Hy 4). 02 unl a MT-SPI substrate spectroryme PA were
inenbsted Th ae protense camacin of humen MT-SPL {UMIT-SP1-F) was added to alin the progressive
‘ues of WMIT-SP!-P cotviy, This experimeal was perlormed at 10 diferent £2 enoexntrations an 4
Gitferentspectroryms tPA oneentutics, Eaca curve was tit i tho integrated Frl onder cale equation
that doseres hu ow establishment uf squisiwa betscou cazyne and iclihitor. The uation is

B81 Ga Pollo *) ae
whore P is product sia, Ve isthe iia strudy-stue velocity, Vis the final stenly-state velucity, tis
time, wad ki 5 the appre Fst order rat winslan. Thus, 40 dilleenr keys of HMIT-SPU-P ihibition by
E2 were obtained Cum 40 combinations of B2 and Speclrozeme IPA concentrations It wos pustulared
that dhe mechunisut would be ane step competitive ihibion as represented i the scheme

E+ Sa ESP

|

cy Sahm 1

k

Ve equation Linking fo Wi ke, analy i (ig. 4 Botton et)
hws kin k{TAD ESTER)

vwhate kis association rat and kis dissociation rte, Af fixed], here exist Lisa aatonahip
berwosn obs and (1. Frow: this reltiouship k,= 2.04 10'S" was abtaiaed, Py iting slope ofthis
graph ta fi + SR Kay 34 fj, The uesocntion eae KL = 2.48 < 10" 07" Pig. fi righ.

is inete characterization revcals that 2 ix exteeely lightbineing, competitive, rapidly associat
and slowing dnsuciating MT-$P1 inhibitor.

igor ket cmaranetanc be Ea) Prog mies SE iy a0 eof soersnme PD
EAT Ete ai toa oct tj Propmacic cane TSP! wa a 254 espemcesae PAC SE
‘Ecceraton sje ar 0H Sar nese smd 230, aul 694M df seameye PA coeaec (MH
ere earn crdpmaacpe Bl 76 bslne 125) oe 253 och S00 aa
shear te2 ha“t =

Key research accomplishments
+ Comper kinetis ehurucleriation of E2 suggesting that 2 iv en extemely tight-binding, competitive,
tapi assoriting and slowieg dissaciving MT-SPVinkihi.

2 Tsoltion uf mcauman MT-SDP1 scFv eit inhibi he protease activity of MY-SPX potenty and
specifcely

fe The scTv SL was seusitive p dewct spose overexpression of MIT-SP1 in normal ovary and ovarian
‘cancer sve

+ Treskneut of ovarian sur xenojge nice with seF¥ combination reveuled a pot of priveiple tha:
[ahibiing MT-SP! conid bean efficucious modality co teat epithelial cancer progression and aneuntasis.
‘A sizilae result i profit in prolate Lomurs where MT-SP1 is overoxpressed

Reportable outcomes

‘Previously 1 repo-ted thet tw revlls of FY 1 were ja the process of publication fm Biochemistry. Ihwas
published in FY2 (loonghoon Sun, Jaume Pons, uud Churles S. Craik, “Potent aud Selective Inhibition
‘ul Mambeaue-Type Serine Protease | by Tuman Single-Chain Snnibodiss”, Giochemisry, 2003, 892
900}. Lavtacled the zeprat, The US Amy Metical Reseurch and Material Command Prosta‘e Cancer
Research Program was acknowledged.

‘An FY2, Lvs inv a x speaker in Faperiimetal Biology 2083 Aarual Meeting, ASBMD secto:
{pi 1-15, Sau Diego, CA) and prevented the results af FY] and part of FY2. DOD PCRP was
fekuowledged, At die ond of this repost the absteel was attached

Conclusions

Gur pelitninary deta und reported esis of aches indent that MJ-SPL may bea erideal element inthe
progression of eptiexftumems af We prostate, breast, al ovary. Te gui insiyht inte Cue roles dat MT-
SPI plays in epithelial cancers, a genera custhod for selectively and potently Blacking ts protolytie,
activity was established. Since MT-SPI isa member of a very sloselyreluied tally of TSP itis
immportaneto sleotivelyimbibt unly MT-SP1 aztvity, while not affecting olber TTSPs tat frequently
feooupress und share cunszuoe. sbstuae binding siee The preferenve fur AryL ys inte $1 pocket ofthe
substrate binding ste exhibited by tis family of ever 20 enzymes limits fe snctess of small molecule
appenaches fo slective inhibition of MT-SP1 ina biological welling. Towever, the potential difiultiey
‘Were overcame by Geveloping hienan scFve as very specifie MT-SPI inhibitors. Using phage display
Iuinan Fs wate olatod, The sohvs inhibit proteulyuc activity of MT-SP1 potently and extremely
selectively. I was demonstrased tha nhiition of MT-SPI by these soFs resulted in suppressing
‘evar tunots in me where hummer avariun umes were xenogrfted, MT-SPT averexpression is
Cominon 19 ovarian and. provate aumens, We chose his xeagotie ovatian cansor model uso pit study
Donause of ts low baskground level u MT-SP{ inthe noemtal ovary a4 compared with he aoaal
roseat. The pil lucy wx peeformed in elatively smal mumber of onizals wi nininal doses of tie
tahihivory scFv. Thus, prevTinical ial ia abis model ané heman prostate wntor mode! in SCID mie
should be performed with staiscelysigoiicsnt number af anieal, increased dosas, optttized
‘ugenls, Saproved igection metheds, anc vacous tumor medels inching prostate canaortandels,

‘Thsve seFv-basesegin-eus could convenicrlly be develuped a» diggnostis and/or prognostica auch us
TIC aud ELISA ssageats to follow up individual cures of ovarizn cancer pation. Als those scPv
reagents cao be davelopad a8 inmmoblating or immunopeeciitation reagens tha could be a valuable
taal to exploe the colo 92 MT-SP1. Taken weber, duis approach will adgkess the program's youls ia
the seeas of precliniced development of targeted lherapeatic and tumor biolegy by eetablishing a general
method that penmitya thorough anulysis ofthe coneTiution that MT-SPL males lb u complex biologioa!
prosess of epithelial canoer, Furlennare, the proof of principle that antibodies van be poveal and
Ssclotive inibitar of protensewcivity can be applicd ta other memsbors of TSP, dke uber of which
isl increasing und ure found in vatious tuners in prostate, bess, volun, and ovacy

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