HER2/neu Peptide-Based Vaccines, with GM-CSF as an Adjuvant, in Patients with Advanced-Stage HER2/neu.. Expressing Cancers
Kathy Schiffman and Nfary L. Disis
Rationale Despite advances in surgery, chemo
merapy, and radiation, paciems wim solid mmors such as breast, ovarian, or lung cancer may relapse because of residual microscopic disease. The identification ofseveral tumor amigens, such as HER2! neu and p53, and an increased understanding of the role rhey play in malignam cransformation, have led to clinical studies evaluaring antigen-specific' cancer V;lccines chat target biologically relevant proteins. l This approach holds meoretic promise for potential eradicatio n of residual subclinical disease through active . ..lmmuruzanon.
The HER2/neu prmem is a rumor antigen. It consiscs of a large cysteine-rich extraCellular domain (EeD) which probably funccions in ligand binding, a shorr transmembrane domain, and a small cytoplasmic domain ,,:,iili tyrosine kinase a.crivity. In some nonna! adult tissue cells, me HER2/neu gene is present as a single capy.l Amplificacion of the gene or overe..xpressiol1 by posnranscriptiol1al medlal1isms leads W overexpression of the associated p[Qrein and, rhus, plays a role in malignanr transformarion by conrriburing to the uncontrolled growm of cancer cells. HER2/ neu overexpression has been described in a variety of differenc tumor types, including breast,
ovarian, non-small-ce!llung, renal cd!, prost<ue, pancreas, colon, gastric, salivary, bladder, and oral squamous cell carcinomas. HER2/neu overexpression, in general, is regarded as a poor prognostic factor. In addition, HER21 neu overexpression appears w be a prediccive factor ror resistance m some chemomerapeucic agencs)-G
Studies from our laboratory and others have demonstrated that: some patients with HER2!nro-over=pressing cancers have existent antibody and T-cell responses to the protein. 7 In particular, patients with HER2/neu-overexpressing non-small-cell lung cancers can have a derecrable antibody immunity to HER1/ neu.S HER1!ne-u-specific cytoroxic T lymphocytes (CTL) have been identified in patienrs with a variety of HER11 neuoverexpressing malignancies, including bteast, ovarian, and lung cancers. 9- 11
Additionally, helper T-cell proliferation specific for the HER2lneu protein and selected peptide epitopes has been detected in parienrs with HER2/neu-overexpressing breast cancers. 12
Once HER2!neu nad been defined as a human rumor ancigen, an immunization strategy ro augmenc immunity ill pa~iencs with a preexistent: immune response or co generate immunity in patients with no detectable HER2lmu
Division. of O~cojogy, Universiry ofW:lShlngmn., 5e:mJe, Washingm n
Submicred: Mar. [5, 2000, Revised, Jul. 15, 2000; Acc<:p<od: Jul. 20, 2000
Clinical Lung G1nC~ Vol. 2. No. l, 74-77, 2000
imm\,;niry needed co be defined. E;;,rly investigations had shown mac rats could not be immunized wi<:h ra~ neu pro1:ein. 13 We quescioned whether thIS rolerance to HER2/neu, a self-p rocein. could be circumvented by immunizacion with peptide-based vaccines. Immunization co foreign proteins normally generates immunity only to the subser of dominanr epitopes, whereas ocher, porencially immunogenic, subdominant epiropes are ignored. Indeed, a neu peptide-based vaccine incorporating subdominanr epitopes from rat neu proved to be the most effective construcr in generating neu
specific immunity in the rat. The addi· tion ofgranulocyte-macrophage colonystimulatiilg facwr (GM-CSF) (Q the pepcide-based vaccines, as a vaccine adjuvant, resulted i11 the in£!ux of effective antigen-presenting celis (APCl and augmenra~jon of che immune respomes genetated after imrnunUa.(ion_ i4
Our clinical erial srrategy is to creat:e or augment immunicy in patients whose tumors overexpress HER2/Ile<.t, using vaccinaEion with pep tides derived from HER2/nL"u tbar have been predicted by computer modeling to be potencial subdominant pepcide epitopes in humans. 12 The parienrs enrolled In this study receive one of rnree vaccine formularion5; each formulation comprises three pep tides, 15-18 amino acids in lengili, derived r£'om elmer cfl.e fCD or intracellular domain (ICD) of HER2/ neu. In addition, the third formulation consists of helper eplmpes mat encompass shorrer nine amino acid human
Addres' for correspondellce: M'lrY L. Dis;s, Box 356527, Oncology, University ofWashingron. leukocyre antigen (HL\)-Al bindingSeattle. WA 98195-6327 fax, ~OG-G85-31 ~s
peprides derived from rhe HER2/ neu
741 August 2000
procein sequence. Each vaccine formubcion is admixed wim 125 flg ofGM-CSt:
,IS an adjuv:ln<.
Objectives The endpoints of the study J.re (I) to
Jdine the safeey of immunizing against:l
sdf-\Umor :lncigen and (2) co Jecermine whether immunity co HER2lm:u pwcein (111. be:: elicieed by immunizaeion \Vieh a
peptide::-based vaccine.
Toxicities The aneicipated toxicities of our vaccine
seraeegy include local injection Site re:lctions
mac would be expeaed \Vim immunizations
md cheorerica! systemic coxicicies rdaced
[0 development of immuniry [Q HfR21 l/I.'U, a self-protein. HER2/nne is expressed in high levels d~ring fecal growch and de. ,'-c::!opmenc and in low amounrs 011 a few
normal adult <issues, including breast,
merus. vagina. digestive tract epithelium.
bue and pancreanc ductS. kidney and ureter,
bronchi. and epidermis. The prorein has
noc be--..n detected in che circufacory. hema
cologie. musculoskeleral, endocrine, or
ceneral nervous sYStems. Preclinical dara
from our lab looked at the hisropamology of tissues expressing basallevds of rat
JIm protein in the animals who developed immune responses after pep(ide-bas~d
\";lccinaoon. There were no signs of micro
Kopic autoimmune damage.
All adverse events are scored by rhe
:-\acional Cancer lnsticute Common Toxicicy Criteria. Pacienc evaluations include
assessment ofany local reactions ac the site
of me injecrion (p;lin, eendemess, erymema,
and induration) and assessment of any
svstemic reaction to immunization (such
~ malaise or fever).' Because induction of
J.llcoimmunieyby HER2lneu immuniza·
tion would potencially cause damage to
tissues expressing basal levels ofHER2lnrn,
such as skin. digestive tract epithelium,
liver. and lung, pacienrs have extensive serologic chemistries performed before each
immunizarion 10 assess organ fi.mcrion as
well as a complete physical exam. Finally,
HERlJ11m 15 overexpresse-d in a noncancer
OilS sr:ue in the human fetus during the
third <rimester of pregnancy. It is possible
Lfa woman developed significant immunity
co HER2! I/I.'U [hat this immu nity may pre-
Tanle 1: Predided Response Roles
Observed -. .. .. .. . . • • Response ••
• I
. . •• . . Rate •
10% 2%-28% 2%-4%
20'1' 77f-40% 9"<-.\6%
30% 14%-51% 17$<---47%
22%-6[% 2jQ.;-0C
50% 30%-70% 34%-66%
GO'1 3991'-78% ~3<:t'·-5%
70% 49%-86% 54%-83%
60%-93s:f 6-tq ·91 q,80"<'
venc her from carrying a fetus to full eerm. For this reason female patientS enrolled
mllSr be finished with childbearing.
Treatment Plan Sixty patiems with breast. ovarian. and
non-smail-cell IWlg cancer will Se enrolled
in the srudy, 20 patients per vaccine formu
!arion. While 20 patients per arm is a larger number than expecred for a wxicity study,
rhis is the minimum number ofparients
studied mar would allow us co extrapolate
che results co a larger pop ulatio n. If a re
sponse rate (defined as eic..'J.er me generarion
of or the augmentation of an inunune response) is known for 20 patients, me ability ofa particular V<lccine formulaeion (Q elicit
these responses in a general population can
be prediCted. For example, from Table 1, if 20. pacien tS were entered onw a study
and [4 responses (70%) were observed, me ~rue response rare ar a 90% confidence in
terval is 490/0-86%.15 Progressing to a. ph= n scudy of efficacy would require that we
could expeCt mat the majority of patients
vaccinated would generate che appropriate antigen-specific immune response.
Table 2: Ehgibility Requirements
Patiencs wili receive eidler che fCD or
the leO formulation. a1cema,ing sequen
tially as they are enrolled. Those patients
who are HU..-A2 will r=ive che third fo,
mulacion, which coomins HL\·A2 binding
epitopc:s. All treatment ,,,ill be conducted
:Ie the Umversiry of Washingran, Seattle,
Washingron. Eligibility "'ill be determined
by rdeprlOne communication with che patient and her/his private physician prior
to the first visit.
The subject's eligibilicy and me treatment
plan are outlined in Tables 2 and 3, respec
tive!y. 'The components of rhe plan are
designed ro answer me follo"'mg questions.
Is There Any Baseline Organ Damage Which Would Preclude- Enrollment:'
A hisrory and physical, complere blood coum and diffi:renci:al, blood urea nitrogen,
se~ creatinine, alanine aminotransferase.
and tocal bilirubin are performed.
Does the Patient's Immu.ne System Function?
After informed consenr, delayed-type
hypersensitivity (DTH) to seven common
• Stage 1lI 0, IV di=
• Breo.> r, ovari~ n, 0r no O--<r:1 alI-cell lung Cance'
• HER21,..,. ovetexp=ion in primary mmo, or m""""rasis
• Complered srandMd of care m,a'ment for disease prior co enrollment
• No cytotoxic (hetapy or treacmenl dose of steroids for a minimum of 1 momh peiOt to enrollment
• No immufiomoduLJ.co<}' drug, Wfllie on study, :e. r"'Smzumab or other VaCCln~
• Age" IS yea", old
• Wh'te blood cell coune > 3500, pl-.1(ele< coum > 100,000. ",rum Creannll'" < !.5 mg'dL ur
c~kula,ed CreanOIO< clearance> GO cc.'mlO. b,l,rub,n < t.5 mgJdL
• Female P'''' ems m u" bt: posemenopamal
August 2000 I 75
Table 3: HER 2/neu Peptide Vaccine Treatment 5(hedule
Procedure
Vaccine Number
PhyslClI exam
Sj re ,n'peGion
V,t~l "";0,
Lab T,,~ts
cae Live r fllnetion
Pn:g rl.J.nc~- (esC
Renal funcroon, .kccro!;"tes
Immune S'udies
DTH '0 r"cdl an'igens
1<l.H I rnn~un Ll:;.l(lOn
OTH to HER21nm pepcides
Sk,n "'OF;" •An"gen-sp.,;iflc T,,,!!
and annbody assays
Pre
X
x
X
X
X
X
X
X
X j
I
X
X
X
x
X
X
X
KiH : kel'hole l,m~[ hemoc\"anin
recall antigens, reunus mxoid. diphtheria
roxoid. proreus, suepcoco=. cuberculjn.
candida alblCUls, and trichophyton (Mulci.
;:est CM!, Connaught Labs, Insritur,
!vlerieLL'l:, Lyon, France), IS placed. SubjectS
mLlSt have a DTH response of 2 mm o'r
greater co ;u least rwo recall amigens to
proceed on srudy. Ifconvenie~(, this test
can be placed by dIe printe ph.ysician to
determine digibiliry prior [Q coming to
tht: University of\Vashingwn_
Table 4: Patient Evaluation
Vaccine Schedule
3 4 6 POSt
X X X X X
X X X X
X X X X
x x x x X
X X X X X
X X X X X
X
X
X X X I X X
dda;-ed·eypt> hypersensicj",cy·:
Is There /111 Imlfllllll: Response to HER2lneu Peptides by DTH?
OTH {cst;ng co each peplidt: in the
vaccine ~ormubtlon. Glvl-CS F. ,md sterde
\VJter is performed pnor (0 J,dministr:lcion
ot' the £1m vaccina,lon. rnclufJ,{ion is read
at 48 hours. This is repe:l,ed 30 days after
cf:e l;J5t vaccine to :tssess ,he devdopmem
of In immune response. At rhar rime. skin
biopsies of DTH sites are obtained using
J 3-mm punch 10 be stained and ev:J.lua'ed
r'o r ,he presence of CD I a, H LA- 0 R, .1nd CD3- cells incorporaring both CD4and COB- Iympho(,.ltes
~re Thae Any ElLd-Organ Toxicities to Organs Expressing Basal Levels of HER2lneu?
Prior to each vacci ne, potenrial side
roxicities are reviewed. a brief physical is
performed, and complere blood coune and chemiscrics (as above) are evaluated and
moni(Ored for abnormalities (Q suggest
toXlciry.
Do Patients Gena-ate Measurable Immune Responses to HER2/neu Peptides and Pro(l:in Assessed by Lymphocyte and Antibody Assays?
Prior co rhe first vaccine, monthly,
and 30 days following [he last vaccine,
patierres donare 200 cc of blood ro obtain
peripheral blood mononuclear cells that
can be used for evalua,ing baseline and postvaccination immunity co rhe HER2! n...~ p<:ptides and protein. Parienes are eval
uatc:d for immunity every 3 monrns following their last visit so long as they demon
SIr<l.Ce lmmuniry.
2
X
X
X
X
X
X
X
Abbr"v!arions: CBC ~ complNe blood coum; DTH ~
Can the Parinzrs Be Immunized to a IVontumor Antigen?
Pacienrs rec.:ive a single immWlizarion
wirh a foreign amigen, keyhOle limper
hemocyanin (KLH). KLH will s~['.-e as a
posiciv.:: control neuunmuillzacion ro assess
the paciem's abiliry m be immunized and
,0 generare an immune ~esponse. An ,i
boay and T-c<>Il responses co KLH will
be compared wirh responses to HER2 pepride '-accines.
Antigen-Specific Immune Responses An,ibody immut"llty
T he! per Lmmun iry
Cyto'o:<ic T-<:eJl immurury
Delayed-,,.,,. hypel'$"nswvirl"
Method ofAnalysis
Quamira"ve EUSA (mglmL) measwcing response ro re<ombinan( ECD, (CO proteins, HER2hTtlJ peprjdes. KLH, and eemnus toxoid
IgG an d IgA respo nses
19G ismj'pe :espoose'
PcolifeeanM by modIfied llmiein,g djlll(ian analv", eval"",i,,!)"
res po lise '0 HER2/neu peP"".s. ECD and ICD proteins. cancrol
ant:lgenSi li '
ELlSPOT and chromIUm rele-ase ossays using JucoJogous cugecs
OTH responses m [he Lmmunmn,g pep,ides. >tenie wa,~r. ~nd
G~[ -CSF Pee- and pos' -VaCC' ne sene,
Abbeev,a,ions: DTH ~ rlelaycd .eype h;:peeSe'l$lClv"y; ECD ~ excmcellular dom~Ln:
ELISA ~ e'lz;:me·linked Lrr.mlHlosorbem a.I'''~: ELISPOT ~ ~n"yme·[,nked lmmunmpnc; GM,CSf : granuloc:-te-m..r.:rophag~ colonY"Clmulacing facme; ICD ~ I nr,....cdllllar domaIn: KlH : keynole Jimf'<" nemocynn:n
76 I Augusr 2000
Immunologic Evaluation There is no gold sranchrd for measuring
rhe developmenr of rumor antigen-specific
immune responses. 16 Therefore, immuno
logic moniroring will include overlapping
merhods for directly and inferen [ially
measuring CD4 and CDB T cdls and anti
bodies (Table 4). In vivo T-cell responses
will be assessed by subjects undergoing
OTH testing- to rheir immunizing pep
rides. Prior co their fim vaccine, mon,hly,
and 30 days after their last vaccine, T· celt proliFeration a.ssays co their immuniz
ing pep,ides, reranus toXOId, KLH, and rhe HER2fnt'U ECD and [CD prote:ns are
pCdiJrmcd on thcir peripheral blood mono
nuckar cells. I , Ev:llu:uion of the gcnera
[ion ofcywwxic T cdls speciFic for HER2/
"<'fI .i, ass~s5ed b(J[h by cnzym c" lin ked im
munospor ~ay, which mC:J.Sures anogenspeci ric cywki nc re!<:;\.\e ,I ~ Jnd by rht: gen
erarion or" CTL lines in vitro which cJ.n lyse HL.l,.-marched HER2/neu overexpres
,ing: tumors,' 9.20 Antibody responses may
serve .1.\ a surrogat~ ror ,he developmem of:l T-c~lI response. parricuJarly !gG ,md 19A which require T-cell h.elp in Ig class :;wi[ching. Thus, quami[:lrivc an[ibody
subdJSS .lnd iso[ype analysis by ~nzymeIi rtk~d immunosorbenr assay is abo pcrformed.~1
Acknowledgements This work is supponed For Mary L.
Disis by grams from the NariollJI Insti{Utes of Healrh, &:lcional Cancer Institll(e
(K08 CA61334 and ROt CA75163), De
partmem of Defense Breast Cancer Pro
gram, and ilie Cancer Research Tre:l1:ment
Foundacioll.
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':0. Mu"on K.C:u,Ovp, Di,,, M. r",lanon ofHER-Cin'" (HER2) sp<:<:ine T <ell don'" from, brcm =.::r p>"cn, iUl]owmg Lmmuniz3fton WLch a HER:! pep rid.: ~".I:cdn~
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August 2000 I 77 I