Trial of Interleukin-2 in Combination With Pembrolizumab for Patients With Unresectable or Metastatic Melanoma
Phase: PHASE2
Conditions: Metastatic Melanoma, Stage III Mucosal Melanoma of the Head and Neck, Stage IIIA Skin Melanoma, Stage IIIB Skin Melanoma, Stage IIIC Skin Melanoma, Stage IV Skin Melanoma, Stage IVA Mucosal Melanoma of the Head and Neck, Stage IVB Mucosal Melanoma of the Head and Neck, Stage IVC Mucosal Melanoma of the Head and Neck
Sponsor: Rutgers, The State University of New Jersey

Otficial Title:
A Phase 1blllTrial of lnterleukin-2 in Combination With
Pembrolizumab for Patients With Unrespectable or
Metastatic Melanoma
NCT number:
027 48564
Document Type:
Protoco! - SAP p.74
Date of the
Document:
0e12L12021,

Product: Peurbrolizurncb
ProtoeoUAmrrdmetrt No. :
I1.O APPENDICES
ll.1 ECOG Pcrforurnnte Stsm$
Grade
Description
0
I"[onuat rctivity. Fully active, oble to carr] orr all pre-disease
oerfomrance witlrout restriction"
I
Slnnptorns, btrt anrbuletory. Restricted in physically streutrous
activity. but ambulatory aud able to catry out work of a liglrt or
sedentarv uatrue (e.c..licht housework, office rvork)-
2
fn Uea <50% of the tiure. Arubulatory and cap*ble of all self'care. but
unable to cany out auy work activities. Up and about uore than 50o/o
of wakiuc hotus.
3
In bed >5096 of the tiure. Capable of only limited self-care, coufiued
to bed or chair nrore than 50olo of waking hours'
4
tOOi,i, bedriddeu. Courpletely disabled. Cannot cary on auy selieare-
Totally confined to bed or chair.
5
Dead.
-AsptrtliiffiAt" Am f Ciin. Oncol.: Oken, M.M., Cr.sech, R.H", TornE, D.C., I{ot7on, J., Datis, 7.E..
W*adden, 8.7", Carbafic, P.P..' llo;icifr,{nrl{espax,}e Ct'ite;r'ia Of Tlrc Eaxlern Cooperativa On6orq&}"
G1ttp. *n J Ctin Oncol 5.'d49-di5, 198J. Tlre Eastent Cooperative OncoloSry Group, Rsb€rt Conrh
M.D.. Group Chair.
ll.2 Common Terminology Criteris for Adverse Events V't-O (CTCAE)
The descriptions and grfldilg scales foturd iu the reliised NCI Commou Tenniuology Criteria
far Adverse Events (CTCAE) versiou 4.0 will be utilized for adverse event reporting.
Websife: htt$:llctep.c*ucer. gu8be$ortindctc.hhtl
11.3 Rerponse Evsluotion Critorir in Solid Tumor* (RECIST) 1.1 Criteria for
Evnlucting Response iu $olid Tumors
RECIST versios t.l will be rxed in this study for assessureut of huuor response' with one
modificatiou. To sccou*t for hunor ps*udo-propessicn tlrat cau sometime$ occur with
iuuuuotlrernpy, it is required that RECIST PD be confirmed on a repeat scan before n patient
is removed from shrdy (see Section 7.1.3.5 far detoils).
For ftlll details, refer to the rnacuscript as pubtished in the Etuupean Joumal of Cancer [43]:
8."4. Eisealr*uer, P" Therasse, J. Bogaerts, L.H. Schwadz. D. Sargerrt. R. Ford. J.
Dancey, S. Arbuck, S. Gwytber, M. Moouey, L. Rubinstein, L. Shankar, L. Dodd, R. Kaplan,
D. Lacombe, J. Verweij. New respoose evaltmtiou criteria in solid &rmors: Revised RECIST
guideline (rrercion t.l). Er$ J Catreet. 2009 Jeo;45(?):?28a7.
90

91
Product: Pentbrolianmab
Protocol/Amendment No.:
t 1.3,I &Ireruroble Bire*st
Measu-able lesiols are defined as those thnt can be accurately rueasured in at least ote
d"imension (longest diameter to be recorded) as > 20 nun by chest x-tay, as > l0 mm with cT
sca& or: lb 111}1 with calipers by cliuical exafir. All truuor nlea$u€meuts unrst be recorded i$
millimeters.
NOTE: Trxnor lesions that are sihrated in a previously irradiated area are not considered
ureasursble"
NOTE: Maligoant Lymph Nodes. To be considered pathologically enlarged and meastuable,
a lymph nodJurust Ue I t S nun in short axis when assessed by CT scau (CT scarr slice thickness
recommended to be no gpeater than .5 mrn). At baselire and in follow-up. only the short axis
will lrc rueasured aud followed.
If .3.2 Non-rnessurnbls Disenre
All other lesions (or sites of disease), including small lesious (lotgest diameter < 15 rrrrn short
axis), are considered uon-illeasurable disease. Boue lesions. leptonreningeal disease, a$cite$,
pleuaypericardial effusious. arrd trymphangitis cutis/pulnrouitis, are considered as non-
measrusble. No,-mes$uable also ineludes lesions tbat are < ?0 mnr by chest x-ray.
NOTE: Cystic lesions that meet ths criteria for radiographically defined simple-cysts *hould
not be considered as malignant lesions (neither measurnbl* tror $ofi$leasru:able) since they nre,
by definition, sirnple cysts.
'Cystic lesions' thought to represent cystic rnetastsses ca.u be cousidered as measuable lesiots,
if ihey meet the definition of measrxability described above. However. if uon-cysiic lesious
are present iri tbe same patient, these are preferred for selectiou ss target lesions.
11.3.$ Target Lesions
All ureasurable lesions up to a uraxinnnu of ? lesions per orgao and 5 lesions in total.
representative of all involved organ$1 shorrld be identifietl os txrget leliols nnd recorded and
measured af baselile. Target lesions should be selected on the basis of their size (lesions with
the lougest diar:refed, be representative of all involved orgall$, but in addition should be those
that leud themselves to reproducible repeated rneasuremetts. It may be the c*se that, ou
occasion, tbe largest lesion does not leud itself to reproducible ureasuement in which
circumstance the *ext l*rgest lesion which ca$ be rneasruEd reproducibly should be selected'
A sum of the diarueters (longest for non-nodal lesions, short axis for uodal lesions) for all target
lesiorx lvill be calculated and reported as the baseline suur dismeters. If lynrph nodes are to be
ilcluded in the sum, then ouly ihe shott axis is added into the suru. The baseline sun of the
t{iameters will be used as refereuce to firther characterizE any olrjective hrruor regression in
the ureasuable diruension of the disease.

Product: Feurbroliaunab
Protocol/Amendrneut So.:
f f3.4 lton-t rget Lsions
All other lesioff (or *ites of disecse) ircluding my mefisumble lexistts over atrd above the 5
target lesiorl$ should be identilied as non-tnrget Imions aad should also be recorded at
baseline. Measurements of these lesions are not required- bul the pre$ence or abseuce of
uneq*ivocal progres*ion of each should be octed througbout fullow*rp.
11.3.3 Evalnstion of Target Lerions
Complete Respouse
(CR)
Disappearance of all target lesions. Any pathological lyuryh
nades (whether target or nnu-targe$ nrurt have reduction ia short
axis to < I0 urm.
Partial Response (PR)
At least a 30o,'ir decrcase in the sturr of the diaureters of target
lesions, taking as refereuce the baseline sum diameters
Progressive Disease
rD)
At least a ?09ir increase in the stun of tlre diamefers of target
lesious, taking as reference the srla/Ies, srrrrl on s/rdy (this
inchrdes the baselire strm if that is the surallest on study). In
additio* to the relative increase of 209o, the sum urust also
demoush'ate au absolute increase of at least 5 uun.
Note: the appearance of one or utore unequivocal new lesions is
also considered progression.
Stable Disease (SD)
Neither sufficient sluink*ge ro qratifu for PR uor sufficient
increasE to qualify for PD, taking *s refbrence lhe surallest surr
diameters vrhile on stndy,
1I.3.6 Evalugtion of Non-T*rget Lesions
9?
Complete Resporse
(CR)
Disappearaace of all non-tatget lesions. Alt tynrph todes must be
rron-pathological iu size (<10 nrm shcrt aris)
Progpessive Dise*se
(PD)
Appearance of one or more uew lesions andlor urequivocal
prCIpnession of existing non-target lesions. UnequirCIeal
progression should not nonually hruup target lesiou status. It
rru$t be representative of overall d.iseose stafirs change, not a
sin$le lesiou increase.
Non-CRNon-PD
Pemisteace of oue or rnol? uon-target lesion(s).

93
Product: Pelnbroliarmab
Protocol/Amenelment Ns. :
l13.? Evaluation sf Ncw Lesionr
The appe*r*uce ofnew lesions cgostitutes Propessive Disease (PD).
A growing lp:rph uode that did not rneet the criteria for teportins *s fl measuralle or $on-
m&ura5il iy,"gU node at baselfuie *hould only be report*d as a new lesion (cnd there re
prrgr**i.- &seare) if it a) i$ueffies in size to > 15 rnm iu the short axis' orb) tbere is new
pattologic*l coufinuation that it is disease (regardless of size).
See RECIST 1.1 mauuscript for fiuther details on what is evidence of a rrew lesion.
11.3.8 Evaluntion of Best Overall Response
The best overall re$ponse is the best rcspon$e recorded &om the stafi of the treahneot until
ai***** progressionlrec,6rre$ce or uon-protocol therapy (tnkiug as reftrence for progressive
disease the snrallest mea$utfirents recorded sinc.e the treafiuent started). The patiefif's best
resporse as*igpment will depend on the achievement of both measureruent and confirmation
criteria.
Target
lesions
Non*target
lesious
Nerv lesions
Overall respcnse
CR
CR
No
CR
CR
Non-CB"/nan-
PD
No
PR
CR
Not evaluated
No
PR
PR
Not-PD or uot
all evaluated
No
PR
SD
Non-PD or not
all evaluated
No
SD
Not all
evalurted
Non-PD
No
NE
PD
Atty
Yes or No
PD
Any
PD
Yes or No
PD
Any
AnY
Yes
PD
11.4 Events of Clinic*l Intere*t Guidsnce Document
Please see coruprurion documert, "Pembrolizumab Prograur (h/[d-3475) Event of Clinical
Interest Guidance Docrunent, Versio* 5^0,'" fbt clinical guidarrce on the identification ard
managemert of imnlure-related adverxe etenfs and their reporting requirernents. including
which ECI must be reported within 24 homs'

94
Product: Peurbroliarmab
PrototoUAmendmeut l{o.:
11.5 processing mil $hipping of Tumor Tissur snd Correlutive Science Blood Samples
11.5.1 Tistue Collettion
1I.S.LI Collettion of peripheral blood
a. peripheral blood *itt U. collected by venipryrctue irrto slx veusus blood collection
hfues (two RedTop tubes withno additive; BD vacutainers catalog# 366430 andfour
6ree[Top tub*s with sodirun heparin; BD vacutainers catalo6# 3664q0)."
b. The sarnple *iU U* lra&sported'at room teu4perah$e (18'C to 25"C) in a double
confairei from the colleetion site to the sauryle processing lnboratory'
c. Red Top tubes for $erun processing should b1 $lt sittiug uprtght at room
terrrprr*iure for at least 30 rninutes and at most 60 urinutes prior to processifig to
allow clotting. If fhe bloqd is not imnrediately proccssed after the clottiug period,
then hbes *froJA be stored (after the 30-60 urinutes of clotting time) *t 4"C for no
louger than 4 h.
d. Gr; Top tnbe* fbr periplreral blssd monouuclear cell (PtsMC) r99eryinp should
be kept on a rocker-at ioom tenrperahue until processed (to avoid ctrotfing) md
processed as soorl as possible (withrn 4 h nnaximrun) afler blood collection.
e. the collectiou tubes *itt t. labeled with the patient ID, date, and tirne of vetto*s
blood draw.
f. Saurplns will be haudled one patient at a tiure to avoid nix-ups.
f 1.5.X.2 Ctlkction of tumor tissue lnd hsndling of lrchivol tumor tissue
a. Fresh ftrmCIr tissue will be collected by biopsy and inrmersed (complrtely) in sterile
cold (2-7"c) HBSS in a sterile specimen crrp (for exa$rple. rwe catalo8# 15704-
0gg) on ice (care mtrst be takeu to not inuneme the cup/tissue irl ice to avoid &eezing
the tissue).
b. The speciruetr cup will be labeled with the patient ID, date, and tinre of h[uor tissne
collection.
c. The sa*rple will be trausported on icr firom the collection site to the satnple
processirrg laborntory.
d. Tissue prr****ing should be doue as soou as possible afier biopry (and within 3 h
rnaximum) after tissue collectioa-
e. Samples will be lraudled one patieut at a tirue to avoicl mix-ups.
f. ArcSival hyusr tissue (for exanrple, in portffur blocks or pathology slides) sbould be
kept at room temperature until shipping'

95
Product: Pembrolinunab
ProtocoUAmendment No. :
f 1.5.2 Tissue Pracessing
11.5"2"1 Proc,esring of p*riphrrd blod
a. peripherd bff fu* n"U Top tubes will be used for se*m acquisition and
il&*[i 
uUoO &our green top tubes will be rxed for PBMC acquisition. Please
refer to th* Postirocissing and Stripping Table for post-procesoing ha$dlfug
details.
b. Red Top hrber will be processed as follows:
i. 
Red Top tubes will be ceutrifuged fur 20 minutes at 1100-1300 g at room
temPeratwe.
ii. Using n pipette. senun fiour bosr Red Top tubes will be transferred irrto a
50 m}conical tube aududxed'
iii. f1,g **** will thea be pipetted iuto the l*beled cryovials at an aliquof
voltuue of 250-500 pl per tutre. The caps os the vials will be closed ti$htly.
iv. This process shouldbe completed witlrin I horu of centrifugation'
v. Care ruust be take6 to pot picf up red blood cells when aliquoting. This can
be dore by keeping the pipet ubou* the red blood cell layer and leaving a
surall amotmt of senxu in the t$be'
vi. All al^iquots will be placed upright in 1 specimen box or rack i$ ao -80"C or
"oU* h*zer. AII sp*ciurens should rimoin at -80"C or colder prior to
shrppiug. ll.lre saruple,s slrould not be thawed prior to shipping.
c. Greeu Top hrtres will lre processed as follows:
i. 
Oreeu Top tubes *ltt t . mixed by inverling the tube gently 6 to I tinres'
ii. Up io l0 url of peripheral blood will be added to l0 ml of PBS in a 50 ml
Falco$ tube andmixedby inverfillg the hrbe gently 6 to 8 times'
iii. fft* iA nr! peripherol blCIod/pBS mixture will be overlayed by slow earefitl
prpetting onto a 20 ml layer of Ficotrl in s 50 ml trfie'
iv. fn*-purIpU**l btood/flisfficoll hrbe will be csrtrifugated for 2$ min at
1750 rpur at roour temperahue without the bseak'
v. Usilj a transfer pipeite the clear top plasrua layer will be reuroved a$d
discarded as biological waste'
vi. Using s re!!/ nausLr pipette, the cloudy PBMC layer will be transfered to
a 50 nrl eo$ical fube.
.oii. Care must be takeu aot to disnrpt the erythlocyte layer druiflg the transfer
by *i"t gpntle prpettirrg above the Ficoll layer and keepiug the tube
stationarY.
viii. The cells wiil be counted (using a standard heurocytoureter) aod the total
cell corurt recorded.
ix. ptsS rvillbe added to the PBMC tube up to the 50 ml urark end the tube will
be ceutrifugaled fur 5 uri* at l?50 ryrlat rcom tenrperatrre with the break.
x. Tlre supenratant will be disearded and tlre pellet dissolved ir Cryopreserue
solution (t ml Cryopreserve snhrtion per 1 ml cf blaod) and hansferred to
cryovials'(l ml per cryovia$. The cell coucentration aud soluliou vohmre
will be recorded.

96
Product: Pembrcrlianmab
ProtocsUAmendment lfo.:
xi. All aliquots will be placed upriglrt iu a in a Mr. Frosty (Thenno Scieutific
catalogi#S100-0001i at 4og irtlnp to 24 hours.and thel in -80"C or colder
freezer i1a speciureu box. All specinrens should remain at -80oC or colder
prior to -fnppi,,g The saurples ihould uot 5e thawed prior to shipping'
ff.5"1.2 Proc*s*ing of tumor tisrur
a. Tumor tissue witl tx sec ioned fufo tllree equal speci$eus' The RNA stabiliuatior
procedrue *tro*,ld rr* *"*pr*hd first. Fle*se refer to the Post-proces*itrg aild
'sfrippi"g Table for post-processing haudliug details'
b. eftei sitionin; tf,g tlri.* **o.plis will bi preserved- in HypoThennosol$-FRS
Hypothenni" stiag. and shipping rrredia Grrs-rns) for flow cytouretry analysis'
fonuali& fixed forllnmul1ohi*io.n**ittry $IC) mralysis, and stored iu an RNA
slsliliving reageut for array aunlysis-
c. HTS-FRS Prese.rratiot
i. 
Sterile f"r--p- *fl be used to gently place the specimen in a sterile labeled
cryovial or specim"rr coutaiuer-(whicliever is large euough to hold the-tissue
fullsubmerg"a*osru:otmdbycoldt2-s"C][[rS-FRssolution.Additoual
HTs-FRa ,iitt t * sdded with the vol ooe iequired to fully submerge the
tissue in the solution and fill the viaUcontaiuer completely without air
Pockets-
ii" 
-Care 
should lx taken to avoid cnrshing arlifacts and a separate forcep* ttlust
be used for each speciruen to avoid ct'oss cout*utination.
iii. 
The cryoviavspecimen contniner lid will be sec,ured
iv. The cryoviauspecimen coatciner must be kept at 2-8"C and shipped
inurrediatelytlresarnetlay(rrraxinrtuuwithin?4hows)"
d. Fonualiu {ixatiorr for IHC
i. 
Specimens intended for fonnelfui frxatior should tre processed after the
completion of other &esh tissue procedxres, *uch *s tlaxh freeziug and
strbmersiou in RNA stabilizing reageut'
ii. 
The tissue saurple should be trir:rued such tbat it is urnximally 0.5 cm in
thickness. Specinrens that exceed this diruension uray not allorv fnr
adequate perfirsiou of the fomralin'
iii. an.r tri**irrg, the specimeu lvill be transfeued to a cortainer with
fonualin gsirg-u forceps. To avoid cros$-contaurination, a separate {opeps
should be usel far each tissue sanrple. The vohune of fonnalin should be a
miriruun of 15-20 times the volurue of the tissue sarnple - 8.S., ?0 ml of
formalin per I cm3 of tis$re" "4. l5 ml sterile ce*trifuge or eodcal tube or
alternntively. a specimeu eup can be used'
iv. The specinen rr1rst be courpletely submerged in the formaliu {ixative and
dre contaifier lid secruely tighfeued to avoid any spill*ge
v. Tle tissue rvill be frxed at rooru teupemfure for a uritiuxuu of 24 ltotus, at
which poiot it will be kausferred to PBS in preparation for shipping.
vi. Formaiin slrould ust be discarded dowrr the drain. The chemical disposal
plan at the insfittrtion will be followed for an *ppropriate method of
disposal"

97
Product: Penlbrolianmab
ProtocsllAlnendment No.:
e" RNA $tebilizing for army nu*lysi*
i. 
Rt{A preservatiol reagent (RNAlater o,r TRIZOL} will be trans{brred into
* ,t*il" speciruen.oiaioer. A rniniururn of l0 volunres of RNA reagent is
required 6i "*"f, I mg of tissge - e.g., l0 I reagent per I rng of tissue (o1for
a specimep that is 0.5 cnr3" 5 rul of reagent rvill be used in a gterile container
that is of sufflcient size to allow for complete submersion of the speciraeu).
A lS ml sterile ce*trif,rge or couical tube is suflicient. Prefilled tubes cau
also be pruch*sed from tlre msnu&cttuer'
ii. 
Efforts must be made to expedite tle trsnsfer of harvested tissue to the
sterile pre-fi[led corrtsigers of RNA reagelt a* quickly ar possible after
han'est.
iii. 
It is irnperative that beight ancl wiclth of tlre speciruen rrot exceed 0'5 x 0'5
clr, {rs ihi* tir*u" pertiriiou of the RNA stabilizing reagent is siryrtic*utly
irupaired i, f*rg"i specimeus. However, specirnens can exceed 0'5 cul in
length.
iv. Using sterile forceps, tlre tissue will be placed iuto tlre tu56 gs6lsining the
RMA reag; *A l"i"pt tely submerged" A separate forceps will be used
for each specimet to avoid cross coutaurination'
v" The tissue fu] RNA reagent will be incubated ovemig$rt at 2-8"C' Care. [rust
be taken to keep the hrie irr an upright position into order to keep specinrens
sgbr:eerged. Even trausient exposrre of the specirnens to air can resuh iI
DNA degradatiou.
vi. After a -*ioi**" of 12 hourx of refrigeration, tlre specimen will be
transferred ssing a sterile forceps to a sterile l*beled cryovial-
ldi. The cryoviat wilt tfueu be trans{e$ed to a liquid nitrogen freezer or a
mechauical -80oC freezer for long-ternr storage'
Shipping Trble
Tissue
{processing endPoint}
Storage before
shipping
Storage
during
shipping
Time allowed between
procesaing and shiPPing
Blood (serum from Red ToP
tubes)
Room
Temperuture
Room
Temperature
Shipped same daY as
obtained (rnax <24h)
Blood (PBMCs from Green
Top tubes)
Room
Temperature
Room
Temperature
Shipped same daY as
obtained (max <24h)
Tumor { I{TS-FRS Preserved
frorn fresh tissue)
2-8'C
lce packs
Shipped sarne daY as
obtained {max <24h}
Turnor {formalin fixed from
fresh tissue)
Roorn
temperature
Room
ternperature
Shipped same daY as
obtained imax <?4h)
Tumor (Archival tissue:
blocks or slides)
Room
ternperature
Room
temperature
Shipped same daY as
obtained (max <24h)

Product: Peutbroliaunab
PrctotollArnentlmeut l$o. :
can be sent to
must be made so that one coPY
iiiForu cart be lnaittlatue,i ai lhe
h.
storage coutainers)' 
D^^* nranacoi,,* anrl Qhinnino Talrle fr 
lld
Please refer to the Post-processiug and Shipping Table for post-proces$ing a
haildling details.
Just prior to shipment, storage containers with sarnples lrorn indivitlual patients sholld
be placett in the shippilrg coitainer/s aud the conterts of the package shouldbe matched
to the shipping **if"*t" Both cnpies of the shipping rusnifelt should be sigped ar:d
date.d auiorr* *opy of the shipping mauifest aud record should be placediu thq bo1
The box sbould be sealed and a shipping label attached snto the outside of the shipping
container. The coutainer should be labeled as contaioiug biohazardous speciureus.
98
II.5.3 Shipping
collectionsileM
g6nrples &or1 each patielt that are shipped rmder that sarue shipphg conditiol should
j.

99
Product: Penbroliannrab
Protocol/Amendment lio.:
r1.5.4.r Innmunophenotypic arrlysis of immnne cells nrd markers by
multipcrameter flow cYtometrY
Phenotypic analysis of imrnture cells rvill be co[ducted to detennine cell populntion
ratios. activation, differentiation, nnd suppressive and fiutctional poteutial:f T:*]!'
a. Such phenofypic analyses will utilize l5-pararnete$o* cytometry with au LSR-fi
Ilow cytometer, ,vlrereby cells rvill be staiued r.vith fluorescent autibodies to tlre cell
markers furuchased priruarily frour eBioscieuce)"
b. Cell poprilatiog ratios witl include identifying cells as cytotoxic CD8+ T cells (Tcs)'
CD4i helper T cell (Th) subsets (in conjimciion with transcriptiou factors defining
such s6bsets), regrrlatory CD4+ T cells (Tregs), MDSCs, B cells, NK cells, and
DCs.
c. phenofypic analyxis for cell subsets that csn be distinguished urost accurately by
cytokine producion and trarxgiptiou fnctors (iocluding' IT--SIIFNI with r-bet for
rtrt*; IL-a with GATA-3 for Thits; IL-l? with RoRyt for Thl7s, IL-22 for Th22s'
11-10 with FoxP3 for Tregs) will be perfornred using a six-hour stirnul*tion assay
with PMAllonoruyciu (as per uranufacturer instmctions)'
d. Activation, diffeientiaiiori, suppressive stahtslfi.mctional potential will include
early and lnte markeg of activation; rnarkers of nalve. effector, meulory (central'
"dtnr, 
and resident) cells" Autibodies to be used inchde: HLA class I (A,B,C)
attd II {DP, DQ, DR), CD3, CD4, CD8, CDllb. cDl lc, cDl4, cD16, CDl9,
cD25, bpg:, ibrq,'cn+s. CD45RA, cD4sRo, cD56, cD57, cD6?L' cD69'
cD107a, CDl27. perforin, Granztrme B, Foxp3, CITR, CCR7, HLA-DR,ICOS,
ILT-?, ILT-3, ILT-4, ILT-s. PD-l . Tim3, Lag3. KIR?DL l, KIR2DL2, KIR3DLI.
e. To deielnine whether tissues express HLA-A3 and thus can be utilized in tetramer
studies iuvestigating anti-specifrc CD8+ T cell respollse$, stairiing and a$alysis of
HLA-A? ou CD.{5+ PBMCs will be cottlucted'
f. pBMCs frou Green Top tubes or extracted frour flash frozen huuor tissue will be
used to charactedze imuruue resporrses- Specilically:
i" Flash frozen truuor tissue will be quickly thawed by irumersioa of the bottom
909o of the cryovial in a 37oC watsr bath and processed after thawing as
follows:

t00
Produch Pembroliauuab
Protocol/Amendrnent l{o.:
ii. 
The thawed tissue will be wsshed CIuce kl PBS and rscCIustituted iu a solution
of DNAse t+o ps/$r$ ancl collagenase (1 m#rnl) in 1 l'}l of IIBSS in a
Miltenyi C-tube.
iii. The h[nor tissue will be mechafiically dissociated ruing a GeutleMACs
OctoDissociator (Miltenyi) using the B-01 protraul. fiuther dissociated ill a
shaking incubator for jO minutes at ,50 rpm, alrd flrtber nreclrardcally
dissociatedu*ing the B-01 proglam of tlre OctoDissociator.
iv. Tl,* hugor tissue wifi Uren be pa ssd through a ?0 pm screeu -(BD
Bioscie*ces) to ***" a gingls **il *t"p*otion' and wasbed in PBS (with
eenfrifugatio* for 5 rnin at 1500 rym antl4"C)'
v. Tu[or tissue will be recoustituted iu PBS (2x106 iu 50 -pl PBS, and the
remainder fiozeu down. as described previotrsly at 2x106 iu ctyopreserve
solutiou.
b. PBMCs from Green Top hrbes and processed tissues will be thawed quickly by
inunErsion of the bottourgO% af ttre cryovial in a 3 7"C water beth afid washed once
iu pBS {with ceutriftrgatiou and recanstitution of the cel} pellet in 50 pl PBS per
sample iu a 96 well pl-ate) prior to flow cytometric autibody staining'
c. For extracellular ma*ert:
i. 
plates wittr cells to be stained will be centriftiged at standard conditions (4oC'
1500 rpm, 5 uiin) and decarted by iuversiou' .
ii. 
Extracellular nrarker arrtibodies ivill be added at titrated conce$trations (for
exanrple, 0.5 pl antibody ill 50 pl of PBS for antibodies used at 1:I00)'
iii. nre ptate wili be incub*ted in tlre dark for 30 mintrtes at room tempemhue
nnd then centriftiged at standard conditious and washed once with PBS.
iv. Cegs uot reryririig inkacellular stairring (ICS) rvill be analyzed irnmediately
by flow cytometr{or fixed in 50/50 v/vlOg fonualin iu PBS, kept in tlr: {*k
ui 4.C, ,od *uly."d within 24 hotus. Prior to analysis, 5 pl of Countbright
beads will be *Aa*a to each well (and the bead csucentration rccorded from
the Countbrjght bottle).
For inkacellular rnarkers:
i. 
cells requiriry ICS, will be centrifugated at standard couditions and
recolstitutea riittr lgb pl of BD Pe*'/fix per well, ilc*bated at 4"C for 15
urin, receive 100 pl of 10910 PennWash to each well, and ceatrifuged
immediately at standard couditions.
ii. 
The plare will be then decaflted arrd a 100 pl ICS stain nrixtrue (rvifh
altibidies) added to each rvell i, a 109'i, Pe,*Wash mixture.
iii" Tlre plate will be iucirbated at 4oC for 30 urinrtes, centrifuged at standard
coudifions, decante4 wa*hed 2x with ?00 pl of l0% Perm$/ash per well'
ceatrifuged at stagdald colditions, decanted, and analyzed I}y flow cytomeffy
or fixed in 10% fonnalin for up to 24 horrs'
iv. Prior to analysis, 5 pl of Corurtbright beads will be added to each well (and
the bead conce,ntration recorded fi'om the Corurtbright bottle).
Flow cytonrehy pauels rvill be based ol1 optimized Multicolor
Imrrrtmoflu*r"**"o". Panel (OMIPs) ptlblished iu the iorrmal Cytometry Part A'
d.
e.

101
Produtt: Perrtbrolianmab
Protocol/Alneudment No. :
11.5.{.2AnclysirofPDligrndexpression(Qudtekanalysis)
a. Fornrali*-fixed tissue will be utilize<l fbr analysis of PD-LI and PD-L2 trsing
couuuercially available reagents fi'oru the Dako Envisio[ FLEX+ HRP-PolSrner
kit (DAKO XgOtZ; Dako. Carpinteria, CA) and the 22C3 a'tibody.
b. nriefly, slides will be baked at bg'C. deparaffiuized in xyleoes, andrehyd'ated rvith
graded etlranols to distilled waler-
c. iotig*, retyieval will be p*{fbr111ed using the Dako EnVision F'LEX Target
Retrieval Solution, low pH isupplied ** t *itrut.-b"sed Suffered solution, pH 6'I)
in a rteamer.
d. Cooled slicles will be staiued usiug the automated TechMate IHC staiuing platfonn
includiBg an additional Proteil}asi-K antigen retrieval step (Dako #s30?0
e. The 22C3 autibody rvill be diluted at 2 ltg/rnl ln Dako Priumry Antibody Diluenf
for osplatfnnu ovemiglrf inflil)atiou in a dark humidifred chamber'
f. The EuVision Fl,EXi'Polyruer reagents will be utilized for priurary 'tttody
detectiou including the mouse liukei HRP-polymer, DAB cluomogeu aad DAB
enlraacer.
g. ErrVisiorr FLEX+ wash buffer will be used between ilctrbatio$ $teps, with slides
couuterstained rvith heruatoxylil'
h. Slides will then be rj-used in ciistilled water aud subjected to an etharol dehydratiott
series and xyleue chnnges before coverslipping'
1f.5.4.3 llilelrurement of xntigen-specilic T cell* rnd evelustion of cellulnr
furctionslity by irtrrcellubr cytokine ard effector malecule staining
pBMCs derived fionr Greer Top tubes aud flnsh frozen tumor tissue will be trtilized fbr
evaluatiou of cellular frrnctiogaiity by cytokirre detecfiou alter stirnulation. Briefly.
a. Altigeu preseuf ing cells (APCs; urouorytes, DCs. etc., Ix 105 cells) rvill be obtained
from pBMCs isoiited *our 6rsen Toptubes by HLA-DR positive bead selectiou
(Ivfiltenyi Biotec), washed iu Aim v nredia nnd pulsed at 3?oC in a 596 COz,
lnuniclifying ine$bator in Airn v metlia for I hour with tuuror autigen peptides of
iuterest igplOO, Mart-l, tlnosilase) apd waslted 3x Airl V media.
b. Nou-ApCs fioru the pBMCs (S0-90% T cells) will then be culturecl with these
loaded cells AFCs for 24 hours at 37'C in a 50..,6 COn. tnnnidifyitg incubator (or
asti*CD3 stirnulatiou attibody at lurg'lurl [positive control] or rxrloaded APCs or
no APCs [negative controlsJ. Duriu$ the last 6 lrorrs in incubation, Golgi Plug
[Pharmingen] will be added.
c. ite.r stiiulation cells will be washed 3x iu PBS and stained fur extracellular'
a*tibodies identi$igg CD8+ cells subsets and intracelltrlar antibodies for cytokines
and efrecto, *ol".uL. of interest (IFNy, TMa. It-2. pauzyrne B, perforin, and
CDl07), accordirtg to protocols described iu the "Iuutll
irrunure cells and ruarliers by rmrltipalarueter tlow cytornetry'
lrttp:liqap?.ontinelitr$ry.wiley.cor#jpu$al/ I 0' t 002/ilSSI''{) I 552:
49 3 0/hou:epaselinforma ti<xr*orr*oruips'lrtm

102
Product: Peurbroliaunab
Protocol/Amtndment 5'o. :
d. Cells will 5e nrralyzed by flow cytouretry. fuitigerr-*peci{ic T cellswill lle defured
as cells pro*""irg *y ooe *f tU* cyfokiues or effector nrolecules of iuterest' while
flruetionality witi U,g. defined Uy ilre ability to co-produce combinations of the
cytokines or effector molecules of interest'
11.S.{.{ Stlessur*ment of antigon-rpecilic T cclls by MIIC dextr*mer si*ining
To measrue tumor a3tigen,specific responses PBMCs frorn Green Top tubes or exhacted
&om flash frozen tuurolr tissue ofpatieats founcl to be HlA-A2-positive by protocols in
the .okuurmr"pU*otypic analysis'of iuunune cells and urarkers by unrllip*rameter flow
cyto*retly,. section',"will be siained with MHC dexkarners (incttrcli$g those identifying
""ll* *pu.ifi* against melanoma-speciflc afitigens gpl00.' MART-I, and tyrosinaset or
positive ao{ olgutive control antigeus CMV, EnV, hrfluenza, HfV, and empty)'
Specifically:
s" pBMCs hom Green Top tr$es and procersed tissues witl be thawed quickly by
inulersiogof the bottorn9096 of the cryovial io I37"C water bath and rryaslred once
h pBS (with centrifiigation and reconstitutiou of the cell pellet in 50 pl PBS per
sample) prior to dextraurer $16irtirg'
b. As per granufhch$er instrustioffi."tO-AO pl of tetramer will be added to the cells
and iucubsted for 3s min iII the dark af room temperntrue.
c. ffrercafter, tUr iCS protocol described above willbe followed to detenniue CD8+T
cells (md exchrde B, monocytelmaerophage. aud deudritic cells frour dexhanrer
aualYsis)'
11.S.4.S Serological a$sey$ to evsluste antibody titers to tumor-assoriated ontigens
Serum froru Red Top hrbes will be thawed for analysis of antibody titers to hrmar associated
antigens. BrieflY:
Tumor-associated antigens (gpl00, MART-I. and fylosinase) will bg.p'?o*q
rvirhin PBS for ptrare w'eli *o*iiog ?00 pl per well of 96 well ptate) and iucubated
at 37 oC fur 30 rrrin., or itcubated (cover-ed) overuig$t at 4 oC'
The coating solution will be removed and the plate washed 3x with PBS prior to
the aclditional blocking step (30 min" 50,6 BSA-PBS) Dilute the monoclonal priruary
a*tibody iD pBS. ttreiptimat clilution shnuld be tletennined usiug a titratiou-assay'
Then 0.2 ml of the diluted monoclonal antibody rvitl be added to each well- The
uegative coutrol will be a species- ald isotlpe-matched. non-specific
iurmunoglobulin diltrted iu PBS'
The plate will be inctrbated at rootn temperahue for-2 horus, waslted 3x irr PBS'
followed by additioD of the secondary a0tigen (0'2-"1per well)'
fh" p*e *iU U* incub*ted at rooul ternper*ture for { hoyl' waslred 3x in PBS,
a$d ihe &eshly prepar.ed erzyme substrate will be added (0.? ml of the fi'eshly
prepared sttbstrate to each well)'
c.
d.
e.
b.

103
Product: Peutbroliaunab
Protocol/Alnendmeut l{o. :
t Colnr dnveloprnent will be obseflred (expected after 30 miu of iucubatiou at room
tegrp) *rd abiorbauce will be read {at 405 rrrn) irnmediately in a microplate retder
or the reactinu will be stopped with 50 pl per well of the appropriate st'opping
reagent and absorbance readwithin 2 hotus'
11.5.4-6 *{ultiplex cytakine nuclysis
Senus from Red Top tubes wilt be thasred for analysis of cytokiues usirg the Bio-Plex
Cytokine Assay (BioRad). BrieflY:
a. Tbawed $enur saurples will be prepared for by diluting I volurue of the seruul
snmple witb 3 vohxnes ofthe Bio-Plex lnunau serum sanrple diluent. Exhemely
[6]i; samples should lrc fi]tered throug[ aA.22 pnr filter to preveut clogging'
b. rrr* .ytor.ii* staudard dilutions will be prepared a$ !et- nrantrfactruer
inshrctious from the msfiufacturer's saster rtaudard stock, hr the cytokines of,
inter*st, including IFNuo IrN$,IEN1, IL-lp. IL'z,IL'4. IL'6,IL-8,IL-10, IL-
12P?0, and THF-aIPh*.
e. a mitiplex bead working solution will be prepared from the yticytokine
*o"iusuL; isx beads and the multiplex plate will be prepared using Bio-Plex
assay and wash buffers"
d. Diluted staudard cr serum-derived sarnples will be pipetted (50 pl each) per
well, sealed within &e plate, and incub*ted on a microplate shaker for 30 urin
at room temPerahue-
e. The wells wilt be washed with Bio-Flex wash buffer {I00 pl; 3x), aud detection
a*tibody solution (0.5 pllwell) will be added to the wells. sealed wiihin tlre plate'
uoC io..rb"ted on a uricroplate shaker for 30 miu (1.100 rpm for 30 sec and
f. 300 rpru at room teruperature for the renainder of the i[cubation).
g. me *utt* will be washed with Bio-Plex wash buffer (100 pl; 3x), and pre-
diluteJ strept*r,idin-P€ {lx 50 pllwell) will be added to each well, sealed
within the plate, a$d incubated sn a microplate shaker for l0 min (1,100 rpm
for 30sec *A fOO rpm*troourteuperahue &rtheremairder of theincubatiou)
at room tenrPerahre'
h. The wells *ltt U* wnshed with Bio-Plex w*sh buffer (100 pl; 3x), and Bio-Plex
assay buffer will be added (125 pVwelt) to the wells. sealed within the plate,
and incubated on a uricroplate shaker $or 30 seconds (1,100 tpm) at room
tenrPerahre.
i. The plat* will be anntyzed on a Bio-Plex array reader, s P€r the manufachuer
inskuctions within I horu of last incubatiou'
f 1.5.4.? Nouostring Gene Expression Analyris
RNA &our preservatiou media will be utilized for gene expression aualysis using the
Nanostring nCounter Systeru. Briefly:
a. Extracted total RNA will be fiurher prrified using
(Qiagerr) according to tlte uranu{bchu'er's ptoiocol" The
coluuurs

t04
Produtt: Feutbrolianmab
PrototollAmendmert l{o.:
b.
c.
of the total RNA preparation will be detenninecl by nreasruing alrsorba[ce ot 260
*a pgo tmusiug-the Nauodrop syste$ (Thenno licientific).
Ench hylxidizatian re*ctio* *iit require iOO-tSO ag of total RNA. The integrity of
the total RNA preparation will be verified on a Bioanalyzer (urodel ?100, Agilent
f..motogies)Lefore proceeding with the Sybridization reaction. RNA should be
uliqoot"aito avoid {ieezelthaw iycles)-agd stored fi:r up to several years at -80'C.
Uyiniair"iion of the target "rRNi 
to the gene-specific-fr9!1-nairs rvill be carried
o.,1 ; kiplicate with eih saurple containing 5 pl RNf---------------' (150 ng)' 10 pl.reporter
probe (final a0 pr*ll, s pl captt[e probe lrurat zoo pM), al}d l0 pl hybridizntion
Luffer (5x SPPE, pH ?.5, rvith 0.lo.4 Tween'?0)'
U*;ot u th.,r*"y.ler, tle lrybridization reactiott will be couducted will be at 55"C
for oltiural$ tOb (at le*st 12 h but not urore than 30 h)'
Lt*i tt * nytriAizailon reactious arc completed, post-lybridization processilg lvill
io""*.fiut*fy coutinue usiug the nColnter Prep Station- as per the ruanufacfixtr
iustruetions on trsiug tlte instnrment-
efier post-hybridizition processiug, the nCou[ter Digital Analyzer will be used to
;;qd t,"-gs of the imrnobitizei iluoresceutiy labeled target nrIlNA molecules
; th- ***pi* cafiridge uuing a CCD carners ald a microscope objective lens ald
[*"*pr"*rios levetiio g.i* will be measured and tabulated i' a CSV fonuat by
the system.
U**i Excel, rhe individual data files will be irnported iuto a collector {ile teurplate
pro*-i?ed by the analyzer uranttfachuer. The aburdance of target urRNA urolecules
*iU t " 
compared *irox multiple dit't'erent samples aud thus nonnalized for a'll
t*rg"i g*o"rl ail saurples baseO on the positive spike-in cottrols to account for
difierences ill hy]:rtdieation effrciency andpost-hybridization ptocessing, including
purificatiou and inunobilization of camplexes'.
h. to detennine if the uonualized, backpgotmd-subtracted couots are statistically
above l:ackpryound, a Studeut's t test rvill be perfonued agaiust .1$t lnxnan
,retative "orit 
olr. A gene witl be considered to be above background if the average
coturt {br the tar.get lerre is greater thau tlte arierage couuts for the eight negative
contol gene$ and if the P value of the t test is less fhau 0.05.
i. T1e relative *h*g*, in tlre abuudance of target rRNA molecules will be calculated
ruing the normaiized, backgronud-zubtmcted corurts for one or lnole reference
genes iucluded mauufachtrer set.
f 1.5.4.S Othrr .A,ndYses
If samples are available, soule or all of the follorviug aualyses may also be perfiomred:
G*netic studien, proteomics, ard grafting of fiesh dssue iufo experirneutal a*imals using
blood and tunror tissue. The experimeatal protocol will foltow laboratnry SOP$ and
published procedwes.
e.
f.

105
ProducL Peulbroliaunab
Protocsl/Arntndrnent No,:
1f.5.5 $lfety Precnutions
Universal precaurions (i.e., a ruethod of infrction control in which all buman blpod and body
fluids are treated as ilthey are infectious for Hepatitis Vinrses, Human hrmrmodeficieucy
rrirus, nnd other kuown *ud unknowu isfectious agents) will be utilieed when haudling all
rmfixed cells atrd tissues.
a. Hepatiti* B and Hepatitis C vinrses may be trausmitted through blood and other
may n*as, and ar.e associated with acute hepatitis, cluanic liver disea*e, and
hepatocellular sarcicoura in humaus. The probability of seroconversion after
ueedlestick exposure is estirnated at 1s/a. Untreated virus can persist for up to one
week at room-tenrper*true. All staft who work with hlnnan tissue must provide
evideuce of Hep*titis B vaccination.
b. Hnmcn lnmtulodeficiency virus (HIV) is a retrovims that cause$ seYere
immmodesciency. Infection increases the risk of developing malignaucies,
infectiou Uy oppoit*istic organisms, aud death. The probability of seroconversion
after rreedLstick expos** ir estimated at 0.5?'o. Iufectivity of urheated virus
persisfs Ibr up to oue week at roou temperahue'
c. 'Other potelti*lly infectious ag€uts! trottr kuown and unknowu, po*e hazards to
those working with hxnao tissue. trneluded are tuberculosis, rITLVl.
coccidiCIruycoiis" creutzfeldt-Jacob disease, omongst others.
d. hrdividual "igstitutiouat *ud OSHA guideliues must be followod when lradling
humsu cells aud tissues, snd refened fo for additional informntiou on bloodborne
pathogens, laboratory urf*ty. chemical safety. aad biohazardous waste disposal'
Briefly:
i. 
persogal protective equiprnent {PPE) mtmt be used at all tiures while working
with hggras tissue. Tlrese irrciude disposable latex or nitrile gloves, face
shirld, proteetive splnsh-resistant laboratory coat (disposable preferred), and
covered Protective shoes'
ii. 
Gloves *U"*A be furmediately removed and replaced in the event that they
become tonr or perforated. Gloves must be renroved prior to leaviug the work
area, f,nd disposed of fur au appropriate waste disposal container. Hands rnust
be washetl it a "clefi$" sink after reuoval of gloves'
iii. 
Face shields, goggles and urasks should be wom wheuever a potential for
exposure to sptasles: $pl?y: splatter, droplets" aerosols of blood or fissue
fluid, or other potentiatty intlciious materials may be geuerat*d,-aild if there
is a patential fior eye, oo*u or mouth contamin*tian. They shorrld be wom at
all times while haldliug tissue in the for processing'
iv. protective lafu coats, preferably disposable types, nrust be donned while
workigg with tissue. Contaudnaied clothing rnust be removed prior to leaving
the woik area, and approprintely lcrurderid or discarded, as per individual
iustitutional guidelines.
v. All waste ulist be disposed of prior to leaving the work area- Biohazardorx
*norg* **i be propeily disposed of in an approved "$harp$" coulainer. AJI
otler uou-shary wnste '*n*i b. disposed of in an approved orallge or red
biohazardous waste disPosal bag'

Product: Penrbroliannalr
ProtoroUAmendmeut No.:
I06
vi. AIter completion of work lvith lnunan tissue. all work mrfhces mu$t be
disinfected rvith a prodtrct that has been deuonstrated to be effective against
bacteria, r.,intses. pseudourotal, hrberculosir and frrngi" Preiduct literahue
should be refurred to for appropriate use.
e. Any injnries or expailue to hunm tissue or potentially infectious biologic agents
nru*t be reported pronrptly as specifred in individr:al institutioual safety guidelines.

Product: Pembrolinunab
Protocol/Amendment lfo. :
u"0 nErEarNCEs
1. 
Welch" I{"G., S" Wolorhiu, end L.M. $chwartz, ,9HN }iopsy rflfas rffrd incidence al
welrrnorno: population based ecological srrldfy. BMJ, 2005.331(7515): p.4St.
2. 
Goldstein, A,M. *td M.A" Tucke4 Etiologt, eptulenriologt, risk factars, and public
health ismres atmelaxonrn. Cun Opin Oucol, 1993. 5(2): p" 358-63.
3. 
Steveus, N.G.. J.M. Lif{ and N.S. Wei*s^ Plantar tilslailofiw: is the incielence of
melanorna afthe sole atthefoot really higher in blacks that whfiesl lut J Cancer, 1990.
{5(a): p. 691-3"
4. 
Davies, I{., et a1., .ilflrfcffons af lils "8rL{.F. gaxa in lwman €$$cen Natru'e, 2002"
{17(6&9!}: p,949-e54.
Crulin, J.A., et al., Dtslinct Sets of Geuelic Alter*tions in Melanorna. Nerv Englaud
Jomnal of Mediciue, 2005. 353(20): p. 2135-2147.
American Cancer Society, A.C.S., Cancer Focts and Figure^r. 2010.
Ries, L.A., et al.. 17re onmml report to the raliorr an the. s/nlas af canc*r, lgV3-1997,
wifh a spacial sectiax an c*lorectol tarner. Carcer, 2000. 88(10): p. ?398-424.
Wong, J.R., et al., Incidence of Childhood a*d Adolescent Melanoma in the United
,Slater.' 1973-2009. Pedietrics, 2013. n1(5): p. 846-854.
9. 
K*rakousis, C., 'W. Goruras, and U. Rao, .dtil/aet node. disse.ction in ntr,lignanl
urul*noun. Am J $ug, 1991. 162: p. 20?-?07.
10. Overett, T.K. and M.H. Shiu, &n'gical ft'eiltilt€nt af distant met(rstittic melanom*
indi.catiotts and results. Carcer, 1985. 56(5): p. 1222-1?30.
11. Andersoa, C.M., A.C. Buzaid, and S.S. Legha, Sys/ernic Treatments for Advanced
Cnfanerus Melananw. Oncology (Williston Park), 1995. 9(l l): p. l149-1158.
12. Chapuran, P.8", et al., Phase III Multicenter Rondonfiied Trial of the Dartmsuth
.(egr;ner Irar,rrs Dacarbazine fu Patiettts Witlt J+{etastatic Melanoma. Jorwral of
Clinical Oucology, 1999. f7(9): p. 2745.
13. Wrg$er, J.S., et al., Current Therapv af Cutaneous Melanonm. Plastie and
Reconstnrctive Srugery. 2000. f 05(5): p. 17 7 4-17 99.
14. Middleton, I{.R., et al., Ranclamixd Phase. III Studv of Tenwzolowide Yersus
D*carb*ilne fu the Treatrnent al Patients With Advanmd lvletastatic Malignant
&{elanorna. Journal of Clinigal Oucology, 2S0S. 18(l): p. 158.
15. Rosenberg, S.A., et al., Irtea/rtent of J8"J canseantive patients wilh nrelastatic
tnelanonw or rensl call cancer asing high-dose bolns i*terleukiu J. JAMA, 199,+.
27r(13): p. 907-913.
16. Sparano, J.A., et al., Randomized phase III trial af teamer$ with high-dose
interlenkirx*Z either clote or ix corxbinatianlufth ixtederon alfu-2a in pntients with
advuxeed *rcla*oma. Jounral of Clinieal 0*cology, 1?93. 11(10): p. t969-77.
L7. "Atkins, ld.B., et *1., .flig&-Dosa Racombinant Interlr,,ki* 2 Tkerapv/ar P*tients With
Jl,fetixslfitic Melanoma: lnalysfs af 370 Parients Treatad deh*e.en 1985 and 1993.
Journal of Cliuical Oncology, 1999. f7(7): p- 2105.
18. Chaprnan, P.8.. ef al., Inrproved wwival with vemurafemi$ $n melanoma with BRA-F
V600E nnttarior,. N Engl J lr{ed, ?01l. 364(26): p. 2507-16.
107
6.
7.
5.
8.
19. Hodi. F.S., et al., Iarproved Sun:ival with lpilirnrnrub in Patients wiilr lfetastatic
lvlelnnorna. Nerv England Jorunal of Metticini, ?010. 363(8): p I

Product: Pembrolizumab
ProtocoUAmendment No. :
108
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Robert, C., et al., Ipilimumab plus Dacarbazine for Previously tJntreated Metastotic
Me lanoma. New England Journal of Medicine, 2Ol I . 364(26): p. 25 17 -2526.
Lotze, M.T., et al., Lysis of Fresh and cultured Autologous Tumor by Human
Lymphocytes Cultured in T-Cell Growth Factor. Cancer Research, 1981. 41(l I Part l):
p.4420-4425.
Mazumder, A. and S.A. Rosenberg, Successful immunotherapy of natural killer-
resistant established pulmonary melanoma metastases by the intrqvenous adoptive
transfer of syngeneic lymphocytes activated in vitro by interleukin 2. The Journal of
Experimental Medicine, 1984. 159(2): p. 495-507.
Rosenberg, s.A., et al., Regression of established pulmonary metastases and
subcutaneous tumor mediated by the systemic administration of high-dose recombinant
interleukin 2. The Journal of Experimental Medicine, 1985. 161(5): p. I169-1188.
Rosenberg, S.A., et al., Observations on the Systemic Administration of Autologous
Lymphokine-Activated Killer Cells and Recombinant Interleukin-2 to Pstients with
Metastatic Cancer. New England Journal of Medicine, 1985.313(23): p.l485-1492.
Lotze, M.T., et al., High-dose recombinant interleukin 2 in the treotment of patients
with disseminated cancer: Responses, treatment-related morbidity, and histologic
findings. JAMA, 1986. 256(22): p. 3 I 17 -3 124.
Rosenberg, S.A., et al., A Progress Report on the Treatment of t5z Patients with
Advanced Cancer Using Lymphokine-Activated Killer Cells and Interleukin-2 or High-
Dose Interleukin-2 Alone. New England Journal of Medicine, 1987.316(15): p. 889-
897.
Cesana, G.C., et al., Characterization of CD4+CD25+ Regulatory T Cells in Patients
Treated With High-Dose Interleukin-2 for Metastatic Melanoma or Renal Cell
Carcinoma. Journal of Clinical Oncology, 2006.24(7): p. l169-1177 .
Sabatino, M., et al., Serum Vascular Endothelial Growth Factor and Fibronectin
Predict Clinical Response to High-Dose Interleukin-2 Therapy. Journal of Clinical
Oncology, 2009. 27 (l 6): p. 2645 -2652.
Ugurel, S., et al., Impact of the CCR5 gene polymorphism on the survival of metastatic
melanoma patients receiving immunotherapy. Cancer Immunology, Immunotherapy,
2008. 57(5): p. 685-691.
Yang, J.c., et al., Randomized comparison of high-dose and low-dose intravenous
interleukin-2 for the therapy of metastatic renal cell carcinoma: an interim report.
Journal of Clinical Oncology, 1994.12(8): p. 1572-6.
Yang, J.C., et al., Randomized Study of High-Dose and Low-Dose Interleukin-2 in
Patients With Metastatic Renal Cancer. Journal of Clinical Oncology, 2003.21(16): p.
3127-3132.
Rosenberg, S.A., et al., Durability of complete responses in patients with metastatic
cancer treated with high-dose interleukin-2: identification of the antigens mediating
response. Annals of Surgery, 1998.228(3): p.307-319.
Kammula, U.S., D.E. White, and S.A. Rosenberg, Trends in the safety of high dose
bolus interleukin-2 administration in patients with metastatic cancer. Cancer, 1998.
83(4): p.797-805.
Dutcher, J., et al., High dose interleukin-2 (Aldesleukin) - expert consensus on best
management practices-2}14. Journal for Immunotherapy of Cancer, 2014.2(l): p.26.
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35' Larkin, J', et al., Combined Nivolurlab and lpilimumab or Monotherapy in (Jntreated
Melanoma. New England Journal of Medicine.
36' West, E'E., et al., PD-LI blockade synergizes with IL-2 therapy in reinvigorating
exhaustedT cells. The Journalof Clinicallivestigation,20t3.tii61,p.2604-2615.
37' Maker, A.,.et al.,-Tumor Regression and Autiimmunity in patients Treated With
Cytotoxic T Lymphocyte-Assotciated Antigen 4 Blockade and Interleukin 2: A phase
I/II study. Annars of Surgical oncorogy, roos. tzltzl: p. r 005- l 0 I 6.
38' Prieto, P.A., et al., crLA-4 Blockadi iith lpihm)mlb; Long-term Follow-up of 177
Patients with Metastatic Melanoma. clinicul crn... Researc-h, 2012. lg(7): p.203g-
2047.
39' Gunturi, A', et al., A retrospective a.nalysis of high-dose aldesleukin (HD IL-2)
following immune checkpoint blockade itcry 
"in rietastatic melanoma (MM) and
metastatic renal cell carcinoma (mRCC). J Clin Oncol 33,2015 (suppl; abstr 3053).
40' McDermott, D.F., etal., Randomized Piase III Triat of Hign-oori titrr'triiir-z Versus
Subcutaneous Interleukin-2 and Interferon in Patints With Metastatic Renal Cell
Corcinoma. Journar of crinicar oncorogy, zoos.23(r): p. 133-14r.
41' Fang, s., et al., c-Reactive Protein As i'Marker oTiilanoma progression. lournal of
Clinical Oncology, 201 5.
42' Ribas, A., et_al., Association of response to programmed death receptor I (pD-l)
blockade with pemb.rolizumab (MK-3475) ,itl, in interferon-inflammatory immune
gene signature. J Clin Oncol,20l5.33: p. suppl; abstr 3001.
43' Eisenhauer, P.-A., et al., New response evaliation criteria in solid tumours; Revised
RECIST guid3line (version t. t). European Journal of Cancer, ZO0g. 4S(2): p.22g-247 .
44' 
Yuan Ji' et al., A modtled toxicity probability interval methodfor dose-finding trials.
Clinical Trials, 2010.7(6): p. 653_663.
45' Gunturi, A., et al., A retiospective- analysis of high-dose oldesleukin (HD IL-2)
following immune checkpoint blockade itcal in ietastatic melanoma (MM) and
metastatic renal cell carcinoma (%RCC). ASCO Meeting Abstracts, 2015.
33(15_suppl): p.3053.
46' Elizabeth Iannotti Buchbinder, J.P.D., Jessica C. Perritt, Joseph Clark, Shernan G.
Holtan' John M' Kirkwood, Brendan D. Curti, Christopher D. Lao, Howard Kaufman,
Mayer N' Fishman and David F. McDerm ott, A Proipective Analysis of High-Dose
Interleukin-2 (HD IL-2) following PD-t inhibiror theiapy in patients wirh metastatic
melanoma and renal cell corcinoma. ASC} Meeting Abstracts, 2016.34(15).
47' 
Robert' C', et.al., Efi.cacy and safety of retreatmen| with ipilimumab in)atients with
pretreated advanced melanoma who progressed ofter initially achieving disease
control. Clin Cancer Res, 2013. 19(S) : p. 2232-9.
48' Trissel, Lawrence A. (2015). Trissel's stability of compounded formulations. Washington,
D.C. :American pharmacists Association.