Author: Hubert Bishop
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D. D. Schoof,I.2·' J. W. Smith If"., Mary L. Disis,' P. Brant-Zawadski,' W. Wood,' T. Doran,' E. Johnson,' and W. J. Urba'·' 'The Robert W. Franz Cancer Research Center Earle A. Chiles Research Institute Providence Portland Medical Center Portland, Oregon 97213 20regon Cancer Center Oregon Health Sciences University Portland, Oregon 97201 'University of Washington

INTRODUCTION Tumor-assocla,ted antigens have been identified on a variety of human neoplasms. Each of these antigens may be able to serve as a target for an immune respOf.lse, the result of which would be eliminatjon of the tumor celL An essential component of this immune response is the presentation of antigen to potential effector cells. This can be accom­ plished via host professional antigen~presenting cells (APe)! such as dendritic cells, or via

the tumor itself. The lack of information about tumor~associated antigens and their appar­ ent lack of immunogenicity in vivo complicate the induction of immune responses against breast cancers. We have undertaken the effort to increase the antigen-presenting cell (APe) function of a breast cancer line that expresses at least one tumor-associated anti­ gen, Her2/neu. The her2/neu gene encodes a ] 85-kd transmembrane tyrosine kinase recep­ tor that shares homology with the epidermal growth factor receptor. Previous studies indicated that Her2ineu was overexpressed by 20-30%1 of breast and ovarian tumors and its overexpression has been associated with a poor prognosis [1,2]. Studies have a)so sug­ gested that Her2/neu can also function as a tumor-associated antigen. Cytotoxic T lympho­ cytes (CTL) isolated from ovarian tumors specifically recognizes Her2lneu-derived peptides and can kill Her2!neu+ tumors but not Her2!neu- tumors [3, 4]. Antibodies to Corresponding author: Pl:cne: 503 2; 5·6588; Fax: 503 215-6841.

Gene Therap"" afCa"ct'l". edited OJ' \VaJdcn eJ at. Plenum Press, :";ew York, : 998,



D. D. Schoof C't uf.

Her2meu can be found in the sera of patients tvith metastatic breast cancer but not in con­ trol subjects [5, 6]. In addition, rats immunized w;th Her2!neu peptide, developed CD4+ I cell immune responses as well as antibody re:5ponses [7] and mice immunized with Her2/neu pep tides developed Her2/neu-specific tumor immunity [8]. Ihese data suggC5[ that vaccine strategies that :arget Her2!neu expressed by tumor cells are feasible but it is not known if immune responses to Her2/neu can be generated in humans in response to vaccination by antigen-bearing tumor cells. The induction of antigen-specific T-cell immune responses is further complicated by [he fact that T-cell activation is dependent on a series of activation signals initialed aIld or­ chestrated by an antigen·presenting ce:L The professiona~ APC delivers an antigen-spe­ cific signal to the T-cell antigen receptor through complexes ofantigen:c peptide and HLA Class I molecules as well as an obligatory nonspecific cost;mu]atory signal that can be de­ livered to the I Cell CD28 receptor through CDSO (B7.1) [9]. Costimlliation through B7 induces IL-2 production and T-cell proliferation in both naive and memory T helper cells. Ihe coexpression of B7 and LFA-3 may provide optimal APC function since they can induce AP-l and KF-kappn B, transcription factors involved in the induction of several cy­ tokine gene promoters, that play an important rolt: in the regulation ofIL-2 gene transcrip­ tion [10j. Murine tumor models confirmed the importance ofB7-mcdiated costimulation in the generation of ami-tumor immune responses, \Vhen B7 was transfected into murine meianoma ceils, tumors grew poorly and could protect the host from J. subsequent chal­ lenge by the unmoditled parental tumor cell line [II, i2J. Depletion of CD8+ I Cells in vivo rEstored the growth rate comparable to the parenlal tumor. Since in vivo depietion of CD4+ T cells did not diminish the growth rate of the B7~transfectcd tumor cell line it is possible that B7 costimulated CD8+ CTL and bypassed the need for CD4-mediatcd T cell help. However, not aU [Umor:5 modified to express B7 can induce immunity A series of nonimmunogenic B7-modified tumors did not provide protective immunity nor did they augmenr CTL functiun, suggesting that tumor immunogenic~ry is a critical parameter in the development of tumor immunity [13). The majodty of solid human tumors tested to date does not express 87 and are :herefore unable to deliver the costimulaLOry signals re­ quired for full I-cell activation. In the present srudy, we have genetically modified the hreast cancer eel! line MDA~M8-231 w express CD80. The CD80~rnodjfied ceHline was administered with G!v1-CSF as adjuvant [14J to patients with metastatic breast cancer to determine the toxicities of the vaccination protocol and nature of the immune response.

EXPERIMENTAL PROCEDURES/CLINICAL TRIAL DESIGN Patient Population Breast cancer patients who Tested HLA-A2 positive and had metastatic disease were eligible for this study following s.igned informed consent, The restricrion of eligibility to HLA-A2+ patients is to permit the planned analysis of HLA-A2-restricred I cell immune responses following vaccmation. Patients must not have received more :hail. tvlO chemo~ therapy treatments for their n:etastatic disease !lad must have recove;:cd from ail side ef~ feets from the last chemo!herapy treatment.

Study Design This study is designed to enroll 30 patients into one of six cohorts (5 patients/co­ ho:"t), Table 1 summarizes the composition of each cobort. The right anterior lhigh ~s \'ac~

Immunization of 1\1.


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3 4







"Toml CO$~ of or 12 hotlt;;. for "7 d;

cinated once wi~ dose of GM-CSF admin;stered bot anteri(}r thigh is van1 alone or nor with blue dye 1(. moved from the each patient set' crosspresentatior ministrarion (cot (given only once blood mononucle Tumor mea vaccination and 1 off study. Patienr until there is evid

Vaccine Cell I The tumor AICC [15]. It e, cell line Waf' tral Fenton) [16]. Ali bulk culture \vas based on its grow to develop our mE

Skin Tests The parenta diated (10.000 cC albumin. Graded anterior forearm ( ured for erythema test site was perf1 bJocks before imn

~ S;,:hOQf el al.

in con­ ,dCD4+T 1:zed with .ta suggest Ie but it is

a" Immunization On'lefastatic Breast Cancer Patienh 'toil/; CD80·ModHie-d Breast Cancer Cells




)licated by ed and ot'­ ~t:gen-spe­

,and HLA can be de­ 1rough B7 ,Iper cells. ~ they can

;everal cy­ transcrip· timulation Ito murine uent chal­

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II tine it 1S lted T cell \. series of 'r did they rameter in s tested to signais re­ ,dlfied the II line was cancer to ~ponse.


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I. Vaccinat:on protocol



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10" 3 + ; O~ 4 IOOmcgim" 100 m...:g/m~ IQ' S 250 !C'.cg"Cm 2 10" 6 + + -:·C:T,-m-:"-d-:lli-"-0-:'-:G-:-M-:-.-:-C~SF-:--,-.J-~-:-i:-:,;-s:-u-d:-'-o.--'-:II-y-p'-_-'-'-ho-o-"-:;-do-,-,-b-,-p'-il-h-h-,-If-,,--O-,-nn-p-1m":-,-od-12-S-m-,-g-,'r:;:, C"i?f} 12 hours for 7 days



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cinated once 'A'ith graded numbers of CD80-modjfied tumors with or withom a graded dose of G'vlCSF or BCG. GM-CSF (kindly provided by Immunex Corporation, Seattle) is administered both IlltradermaJ:y and subcutaneously every ! 2 hours for 7 days. The left anterior thigh is not injected with tumor. However, this control site is injected with adju­ vant alone or nothing depending on the cohort. Ten days later the vaccine sire is injected with blue dye to label the superficial inguinal lymph node(s). One-three nodes are fC­

moved from the vaccine and control site and are cryopreserved for future studies, Since each patient serves as their own comrol~ it wiH be possible to determine if anrigen·· crosspresentatjon of endogenous breast cancer antigens occurs as a result of GM-CSF ad­ ministration (cohorts 1 VS. cohort 2; cohort 4 vs. cohort 5). The adjuvant effects of BCG (given only once; cohorts 3 Jnd 6) will be compared to GM-CSF. In addition, peripheral blood mononuclear celts are coHected and stored for study, Tumor measurements a!'e obtained before the first injection and just prior fo the 4th vaccination and every two months thereafter. Patients with progressive disease are taken off study. Patients who respond to therapy are given a monthly vaccination indefinirely until there is evidence of tumor progression,

Vaccine Cell Line The tumor cell line MOA-MB-231 is an epIthelial breast tumor obtained from the ATCC [15]. It expresses Her2/neu, HLA-A2, CDS4 (ICAM-l) and C058 (LFA-3). The cell line was transfected by Iipofeetion with pCMV-B7 (kindly provided by Dr. Robert Fenton) [16]. After transfection and se:ection in G418, the B7-modified MOA-MB-23I bulk culture was cloned by limiting dilution. One stably transfected clone was selected based on its growth raie l expression ofB7.1 (CDSO), lier2!oeu aod HLA-A2 and \Vas used to develop our master cell bank,

Skin Tests The parental and CD80-modified tumor ceUs used for s~jn testing were lethally irra­ diated (l 0,000 cGy), washed, counted and resuspended in 0.2 ml PBS+ 3% human serum albumin. Graded numbers ofceHs (10 4 ,10' and 10') were injected subcutaneously ll1to the anterior forearm of each patlent. Forty-eight hours later, each skin test reaction was meas­ ured for erythema and induration. 10 60me patients; a 6-mm punch biopsy th:-ough the skin test site waS performed. TIssue was placed in 10% formalin and embedded into paratTIn blocks before irnmunohistochemical analysis,



D, D. Schoof et Ill.

Antibody Responses An ELISA was used to detect Her2/neu protein-specific antibody responses as de­ scribed in deraiL Briefly, a capture antibody wilh specificity for the extracellular domain of Her2/neu was immobilized on 96-well Immulon plates) [6]. After washing and block­ ing, Her2!neu protein, prepared from the lysate of SKBR3, was added. After overnight in­ cubation, human sera were serially diluted from \:25, 1:50, 1:\00 and 1:200 added to each well. After incubation and thorough washing, sheep antihuman Ig(Fab'), horseradish per­ oxidase was added to each welL After incubation and waShing, the color reaction was de­ veloped with TMB. Positive results were defined as an OD greater than the mean+4SD of the normal population at a I :25 dilution, which detects a positive in approximately 1% of normals.

Immunohistochemistry Punch biopsies were fixed in formalin before 6-IJ.ID sections were prepared and placed on glass slides. Sections were incubated 0.03% H,o, and then with norn)ai serum. Slides were then incubated with primary antibody overnight at 4'C. After washing, cells were incubated with anti-CD3 for identification of T cells or anti-CD20 for identification of B cells. Appropriate secondary biotinylated antibodies were then applied for 60 minutes at room temperature followed by the avidin-biotin-complex reagent. The peroxidase reac­ lion was visualized by the chromogen 3,3'-diaminobenzidine (DAB).


biopsy sil patients h tients, hOI treatment

To c a DTH tYJ ated tumo cine skin statistical! shes that r tIte DTH , test site w infiltrates One specific ir the sera 0 treated wil antibodies the produc an associa body respc

RESULTS Ten patients have been enrolled in this trial and all ten were previously treated with chemotherapy andlor hormonal therapy. Their characteristics are listed in Table 2. Nearly all patients received two different chemotherapy regimens for metastatic disease prjor to study entry. While many patients had visceral metastases only four patients had metastatic disease confined to bnne' only. Patients enrolled in cohort one received 3, 5, 3, 2 and 3 vaccinations respectively before going off study due to progressive disease. Patients in co­ hort 2 received 3, 3, 6~ 3,5 vaccinations respectively.

Toxicities and Response There have been no treatment-related deaths nor has any life threatening toxicity been observed. There have been grade 1 or 2 toxicities associated with the vaccination protocoL These are mamly flu-like symptoms and bone pain probably related to the ad­ ministration of OM-CSF. In addition, postoperative swelling and pain at the lymph node

Table 2. Characteristics of the study population Parameter

Cohort 1

Cohort 2

Cohort 3 .~-~.~.~.--.

Median age (range) Median K PS (range) # Tx with prior chemo # Pts with Jiver mets # Pts with bone mets only

55 (32-77) SO (7G----1OO) 415

U5 115

44 (41-74) 80 (70--l00)

2!5 21S 3/5

48 (32~77) 80 (70-100) 6/10 4/10 4/10

DIscm This breast can adjuvant ( sponses it


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in co-

Immunizathm of Metastatic Breast Canc(>r Patient.. with CDSO--Modifled Breast Cancer Cells

biopsy site has been observed but only one patient had persistent drainage, Eight of ten patients have had objective evidence of progression of their metastatic disease. Two pa­ tients, both with disease metastatic to the bone onIy have stable disease and continue on treatment. Three patients: have died, the other seven remain alive with disease,

Immune Monitoring Studies To determine the capacity of parental tumor and CDSO-modified tumor to stimulate a DTH type response, patients were injected subcutaneously with 10', 10' and 10' irradi­ ated tumor cells, Forty-eight hours later, skin test reactions were evaluated, The pre-vac­ cine skin test results for the first 10 patients are presented in Table 3, There are no statistically significant differences in the size of the skin test lesions betweeri skin test sites that received parental versus CDSO-modified tumor To determine the composition of the DTH reaction, punch biopsies were obtained in selected patients, Figure 1, The skin test site was characterized by an extensive infiltration of eosinophil,. Furthermore, T cell infiltrates were present but only a rare B cell was observed in the skin test site. One of the immunological endpoints of this study is the determination of Her2lneu­ specific immune responses. We have measured Her2/neu-specific antibody responses in the sera of patients enrolled in cohort 1, Table 4, Three of the first five patients to be treated with the CD80-modified tumor cell vaccine developed increased Her2ineu-specific antibodies as detected by ELISA, Additional patient data will be required to determine if the production of antibodies is the result of the vaccination procedure and whether there is an association with Her2/neu expression by the patient's tumor and the titer of the anti­ body response.

DISCUSSION This preliminary report of our ongoing vaccine study suggests that vaccination of breast cancer patients with CD80-modified tumor cells in combination with G!I,f-CSF as adjuvant can induce cellular immune responses as weB as antigen-specific -antibody re~ sponses in some 'patients. CeBular immune responses against the lIer2/neu receptor exTable 3. Comparison of pretreatment DTH skin test results Patient #

toxicity :cination I the ad­ ph node


Parental tumor"



19 x g

30 x 75




2 3 4

5 6

20)( 20

l'iR NR 32};. 45




14 x 14

9 10

13)( 10 5( 19 n)( It

ilOTH skin lest result! for pa:ients injccwd with IO~ pnrerJIII. or CD80~ modified tumor cells. The dimensions of the longes: p~rpend1Cular measurements On mm) were measurOO after 48 bNR , nOT\oreaelive. ~ND. flot dent.



D. D. Schoof (It al.

Figure I. Photograph of skin tests on rhe forearm of a breast cancer patient after completion of the vaccination protocol. The DTH~1ike reaction consisted of it prominent eosinophil rc~ sponse. top; a T~cel! infiltrate as detected with the pan~T cell marker ccn, middle; only rare B celis were observed using the B~cell marker CO:W, bottom,

pressed by tumor cells have been reported. These laboratory studies demonstrated that T cells copurified from solid ovarian tumor or malignant ascites could be serially stimulated to induce CTL function. Recently. these data were extended to include antibody responses in patients with breast cancer [6]. Although only 3/63 patients with tumors that expressed normalleve!s of Her2/neu had significant anti·Her2/neu antibody titers. 9/44 patients with tumors that overexpressed Her2!neu developed specific antireceptor antibodies. In this study. three of the first five patients vaccinated with CDSO-modified tumor registered in­ creased Her2!neu-specific antibodies although the antibody level in one patient was low­ titered. Further evidence of immune system stimulation was obtained from skjn test data. The majority of patient's skin tested prior to their first vaccination developed DTH-like re­ sponses to the CD80-modified tumor but not to the parental tumor. The results of punch biopsies obtained 48 hours after injection of CD80-modified tumor cells showed T-cell in­ fi]tration into the skin tesr site, In conrrast, B ce]]s were present but rare, Another feature

Table 4. Her2/neu antibody measurements· Patient #







0 0

0 1:100

0 150


3 4



*Pnt:f:nts vaccinated ... ilh CDSO-mcdified n:mQf


can de­

\-clop fintibody fcsp:m,-es aireCted ~gain$t rh:s retepl{lt. Re· ,ults show j:lre-Vilccma~ioll aria posHaccinatlon Her2!nel:­ specific antibody tilers.

, D, Schoof et al.

Immunization ofMetastaUe Breast Cancer Patients ~ith CD80-Modified Breast Cancer Cells


of the skin test site was the presence of eosinophils. T cells and eosinophils are well char­ acterized components of classical delayed-type hypersensitivity (DTH) reactions espe­ cially in allergen-induced late phase skin reactions [17]. It is not clear if the eosinophil infiltrate is a response to the fetal calf serum antigens in the media used to culture the tu­ mor cells, Comparisons of pre-vaccination and post-vaccination skin test reactions may clarify the role of the eosinophils in this reaction. Finally, the vaccine protocol appears to be safe and well tolerated. None of the patients enrolled in cohort I received all of the six planned vaccinations because in each instance, the development of progressive disease prevented them from completing the entire protocol.


eann of a breast

:nation protocol. :nt eosinophil re~

:h the pan-T cell

: observed uslng

,trated that T Iy stimulated ,dy responses ,at expressed patients with odies. In this registered in­ ent was low­ :kin test data. DTH-like re­ ults of punch ,ed T-cell in­ lother feature

This study has completed two of five cohorts and aecrued 10 of a total of 30 pa­ tients. The tOXIcities related to this treatment have been tolerable. The planned immune monitoring studics are not yet completed. Six of ren patients enrolled on this trial devel­ oped larger DTH reactions to CDSO-modified tumor relative to unmodified control but overall, no significant differences in DTH reaction sizes were noted. However, prelimi­ nary results suggest that in some patients, vaccination in combination with GM-CSF can induce antibody responses against the Her2ineu protein expressed by the vaccine cell line. At the completion of this stndy, we will determine the safety, tolerahility, and immu­ nologic effects of vaccination with CDBO-modified tumor cells + GM-CSF.

,I i





L Siamon, D,J., W. Godolphin, L.A, Jones. J,A. Holr, 8,0. Wong, D.E. Keith. W,J. Levin. S.O. Stuart. J. Udove, A. Ullrich, and a. et. Studies of the HER-21ncu proto-oncQgene in human breast and ovarian can­ cer. Science, 1989. 244(4905): p. 707-712. 2. Siamon, D.l. and G.M. Clark. Amplification of c-erbB-2 alld aggressive human breast tumors? Science, 1988. 240(486{), p. 1795·-1798. 3. Yoshino, t. G.E. Peoples, P.S. Goedegebuure, R. MazIarz, and T.J. Eberlein. Association of HER2!neu ex-.

pression wjth sensiti'vity to tumor-specific CTL in hwnan ovarian caneeL Journal of Immunology, 1994.

t52(5), p. 2393-2400.

4, ioannides, CG., B, Fisk. D, Fan, W,E. Biddison, J.T. Wharton, and CA. O'Brian. CyLotoxic T cells iso·

lated from ovarian malignant ascites recognize a peptide derived from the HER

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