Determinant Spreading Associated with Clinical Response in Dendritic Cell-based ... · Determinant Spreading Associated with Clinical Response in Dendritic Cell-based Immunotherapy

Determinant Spreading Associated with Clinical Response inDendritic Cell-based Immunotherapy forMalignant Melanoma1

Lisa H. Butterfield, Antoni Ribas,Vivian B. Dissette, Saral N. Amarnani,Huong T. Vu, Denise Oseguera, He-Jing Wang,Robert M. Elashoff, William H. McBride,Bijay Mukherji, Alistair J. Cochran,John A. Glaspy, and James S. Economou2

Department of Surgery, Division of Surgical Oncology [L. H. B.,A. R., V. B. D., S. N. A., J. S. E.], Department of Medicine, Divisionof Hematology/Oncology [A. R., H. T. V., D. O., J. A. G.],Department of Biomathematics [H-J. W., R. M. E.], Department ofExperimental Radiation Oncology [W. H. M.], Department ofPathology and Laboratory Medicine [A. J. C.], and Department ofMicrobiology, Immunology and Molecular Genetics [J. S. E.],University of California Los Angeles Jonsson Comprehensive CancerCenter, University of California at Los Angeles, Los Angeles,California 90095-1782, and Department of Medicine, University ofConnecticut Health Center, Farmington, Connecticut [B. M.]

ABSTRACTPurpose: The purpose of this study was to determine the

toxicity and immunological effects of three different dosesand two routes of administration of autologous dendriticcells (DCs) pulsed with the MART-127–35 immunodominantepitope.

Experimental Design: Eighteen HLA-A*0201-positivesubjects with stage III-IV melanoma received three biweeklyi.v. or intradermal injections of ex vivo generated myeloidDCs pulsed with MART-127–35 epitope. Repeated bloodsamples were processed to obtain peripheral blood mononu-clear cells for immunological analysis using IFN-� ELIS-POT, MHC class I tetramer, intracellular cytokine staining,and microcytotoxicity assays.

Results: The frequency of MART-1/Melan-A (MART-1)antigen-specific T cells in peripheral blood increased in all doselevels as assessed by ELISPOT and MHC class I tetramerassays, but without a clear dose-response effect. The intrader-mal route generated stronger MART-1 immunity comparedwith the i.v. route. MART-1-specific immunity did not corre-late with clinical outcome in any of the four immunologicalassays used. However, analysis of determinant spreading toother melanoma antigens was noted in the only subject withcomplete response to this single-epitope immunization.

Conclusions: Intradermal immunization with MART-1peptide-pulsed DCs results in an increase in circulatingIFN-�-producing, antigen-specific T cells. The frequency ofthese cells did not correlate with response. In contrast,spreading of immune reactivity to other melanoma antigenswas only evident in a subject with a complete response,suggesting that determinant spreading may be an importantfactor of clinical response to this form of immunotherapy.

INTRODUCTIONDeterminant spreading is a phenomenon best described in

models of autoimmunity in which autoreactive T-cell responses,initiated by a single antigenic epitope, evolve into multiepitopicresponses. In some models, such as experimental allergic en-cephalomyelitis, priming with a single encephalitogenic peptideof the proteolipid protein generates T cells reactive with addi-tional peptide epitopes from the same proteolipid protein (in-tramolecular) and to other myelin antigens [intermolecular (1–3)]. In such models, the progression of autoimmune diseaseappears to depend on the acquisition of these new determinantspecificities because induction of tolerance to these latter anti-gens abrogates relapses (4–7). Based on these models, it isthought that determinant spreading plays an important role inthe pathogenesis of human autoimmune diseases such as mul-tiple sclerosis, systemic lupus erythematosus, and insulin-dependent diabetes (8–12).

It is thought that the initiating self-antigen is presented byMHC class II molecules at the surface of professional APCs3

within the host tissue. This MHC class II epitope activatesCD4� T cells that cause chronic inflammation and target celldeath, the debris from which contains secondary epitopes thatare taken up by APCs and cross-presented. This phenomenonhas traditionally been viewed as largely CD4� T-cell-restricted

Received 8/16/02; revised 10/28/02; accepted 10/29/02.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by NIH/National Cancer Institute Grants ROI CA 77623,RO1 CA 79976, T32 CA75956, and K12 CA 79605 and the MonkarshFund, Naify Fund, and Stacy and Evelyn Kesselman Research Fund (allto J. S. E.). In addition, the support of UCLA General Clinical ResearchCenter (USPHS Grant M01-RR-0865) and the UCLA Jonsson Compre-hensive Cancer Center Flow Cytometry Core Facility (NIH Grant CA16042) is gratefully acknowledged. A. R. is a recipient of an AmericanSociety of Clinical Oncology Career Development Award and NIHGrant K23 CA93376.2 To whom requests for reprints should be addressed, at Division ofSurgical Oncology, 54-140 CHS, UCLA Medical Center, 10833 LeConte Avenue, Los Angeles, CA 90095-1782. Phone: (310) 825-2644;Fax: (310) 825-7575; E-mail: [email protected].

3 The abbreviations used are: APC, antigen-presenting cell; DC, den-dritic cell; PBMC, peripheral blood mononuclear cell; GM-CSF, gran-ulocyte/macrophage colony-stimulating factor; IL, interleukin; NED, noevidence of disease; AFP, �-fetoprotein; i.d., intradermal; UCLA, Uni-versity of California Los Angeles; NIAID, National Institute of Allergyand Infectious Disease; PE, phycoerythrin.

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by virtue of the exogenous acquisition of antigen (13, 14). It isbecoming clearer, however, that exogenous antigen can be takenup by DCs and be loaded onto MHC class I (15–17) and thatdeterminant spreading can explain the acquisition of new CD8-specific CTL responses (18–20).

We report herein a clinical trial in which melanoma pa-tients were immunized with immature myeloid DCs pulsed witha single HLA-A*0201-restricted epitope (MART-127–35) withthe following observations: (a) pathological evidence of a com-plete response was observed in a patient in whom both intramo-lecular (MHC class I and class II) and intermolecular (differentantigens) determinant spreading were generated; (b) low DCdoses (105 DCs/vaccination) could generate detectable antigen-specific T-cell responses; (c) immature DCs stimulated IFN�-producing T cells; and (d) i.d. delivery was superior to i.v.delivery by immunological analysis using ELISPOT and MHCclass I tetramers.

MATERIALS AND METHODSStudy Design and Eligibility Criteria

Eighteen patients with histologically proven malignantmelanoma were enrolled in a Phase I, dose-escalating study toevaluate the toxicity and possible immunological effects ofMART-127–35 peptide-pulsed autologous DCs. Increasing num-bers of MART-1 peptide-pulsed DCs (105, 106, and 107) weregiven to groups of three patients either i.d or i.v. Patientsreceived three biweekly vaccinations. Eligibility included pa-tients with stage II-IV melanoma with or without measurabledisease; MART-1 expression in the tumor by immunohisto-chemistry or reverse transcription-PCR; expression of the HLA-A*0201 allele; a Karnofsky performance status of �70%; ade-quate hematopoietic, hepatic, and renal function; and immunecompetence demonstrated by a positive skin delayed hypersen-sitivity test to at least one recall antigen (Candida, tetanustoxoid, or mumps). Exclusion criteria included prior therapy �4 weeks before trial entry, untreated central nervous systemlesions, pregnancy, or concurrent immunosuppressive condi-tions or therapy. All subjects provided signed informed consent,and this trial was approved by the Institutional Review Boardand the Internal Scientific Peer Review Committee at UCLA(IRB #95-08-375), and the Food and Drug Administration (BBIND #7122). Additionally, this trial underwent ad hoc reviewsby the Jonsson Comprehensive Cancer Center Quality Assur-ance Committee.

Peptide Synthesis and CharacterizationClinical grade MART-127–35 (AAGIGILTV) peptide was

synthesized at the UCLA Peptide Synthesis Facility. Clinicalgrade peptides were subjected to identity, purity, and potencytesting (mass spectral analysis, microsequence analysis, highperformance capillary electrophoresis, quantification of residualsolvents, and freedom from adventitious infectious agents). Po-tency was tested by the ability to generate HLA-A*0201-restricted MART-1-specific T-cell responses.

Peptides used in vitro for immunological monitoring includedcontrol non-melanoma epitope AFP549–557 (KQEFLINLV; Ref.21) and the MHC class II peptide MART-151–73 (RNGYRALM-DKSLHVGTQCALTRR; Ref. 22) from the UCLA Peptide Syn-

thesis Facility and gp100280–288 (YLEPGPVTA; Refs. 23 and 24),tyrosinase1–9 (MLLAVLYCL; Ref. 25), and MAGE3271–279 (FL-WGPRALV; Ref. 26) synthesized at the University of ConnecticutHealth Center (Farmington, CT).

Vaccine Preparation and AdministrationPatients underwent a single unmobilized leukapheresis

processing 1 plasma volume. PBMCs were isolated by Ficoll-Hypaque (Amersham-Pharmacia, Piscataway, NJ) gradient cen-trifugation and cryopreserved in RPMI 1640 (Life Technolo-gies, Inc., Rockville, MD)/20% heat-inactivated autologousserum/10% DMSO (Sigma, St. Louis, MO). Median PBMCyield was 8 � 109 (range, 5 � 109 to 1.4 � 1010).

DCs were generated from adherent monocytes using GM-CSF and IL-4 following the procedure of Romani et al. (27).Briefly, PBMCs were thawed for each immunization, washed,and plated at 2.5–5 � 106 cells/ml in RPMI 1640/5% autologousserum/1entamicin. After adherence for 2 h at 37°C, nonadherentcells were gently removed, and adherent cells were cultured for7 days with recombinant human GM-CSF (800 units/ml; Im-munex, Seattle, WA) and recombinant human IL-4 (500 units/ml; Schering-Plough, Kenilworth, NJ). On the day of immuni-zation, DCs were harvested, washed, and pulsed with 10–50�g/ml MART-127–35 peptide in serum-free RPMI 1640 for 1–2h. The MART-127–35 peptide, the leukapheresis product, and allreagents used for vaccine preparation were tested for Myco-plasma, endotoxin, and sterility. Samples of each DC vaccinewere stained for cell surface markers CD86 (B7-2), HLA-DR,and CD14. Large, granular lymphocytes were gated on byforward and side scatter, and mean fluorescence intensity wasdetermined for the entire DC population. The mean fluorescenceand range were as follows: (a) CD86, 1129 (range, 199-2283;11–32-fold over background); (b) HLA-DR, 3226 (range, 816-8508; 7–50-fold over background); and (c) CD14, 1158 (43–6186; 38-fold over background). The percentage of DCs in thetotal cell population was determined by the percentage ofCD86�/HLA-DR� large, granular lymphocytes of the totalevents over the threshold size. Based on these criteria, mean DCcontent of the vaccines was 32% (range, 8–70%). After con-firming sterility results, DCs were prepared for immunization bywashing and resuspending the appropriate numbers (105, 106,and 107) in 0.2 ml of saline for i.d. injection in the lowerabdominal area or in 5.0 ml of saline for i.v. injection. Patientsreceived pretreatment with 50 mg of diphenhydramine and 650mg of acetaminophen (both p.o.) and were monitored for 2 hafter immunization in the UCLA General Clinical ResearchCenter.

Immunological MonitoringTetramer Analysis. MART-127–35/HLA-A*0201 tet-

ramers were used for the detection of CD8� T cell with theability to recognize the MHC class I-restricted MART-127–35

peptide epitope (28). Initial preparations of MART-127–35 tet-ramers were provided by Drs. Mark Davis and Peter P. Lee atStanford University (Stanford, CA), and early patient PBMCsamples were independently tested in both laboratories. Subse-quent tetramer preparations were obtained from the TetramerFacility sponsored by the NIAID (NIAID, Emory University at

999Clinical Cancer Research



Yerkes, Atlanta, GA). Each new MART-127–35/tetramer prepa-ration was titrated using MART-1-restricted T-cell populationsto determine the optimal staining concentration. PBMCs fromeach time point were thawed simultaneously, and 106 cells werestained with the tetramers and CD8-FITC (Caltag, Burlingame,CA) and antibodies used to gate out non-CD8� T lymphocytes(tricolor-conjugated CD4, CD13, and CD19; Caltag). Stainingwas performed at room temperature for 30 min in the dark. Thelymphocytes were gated on by forward and side scatter,and cells positive for CD4/CD13/CD19 were gated out. Thepositive cells are a distinct population of CD8-FITC�/tetramer-PE� cells.

IFN-� ELISPOT. To determine the frequency ofpeptide-specific cytokine-producing cells, the ELISPOT tech-nique was used as described previously (29–31). PBMCs werethawed as described above, and T-cell restimulation was per-formed overnight with 1–2 � 106 PBMCs incubated with 1 �105 JY cells pulsed with specific (MART-127–35) or nonspecific(AFP549–557) peptides. Unpulsed JY cells also served as anegative control. For determinant spreading studies, PBMCswere pulsed with MART-151–73, gp100280–288, or tyrosinase1–9

epitopes overnight and then added to precoated ELISPOTplates. Primary antibody (BD PharMingen, San Diego, CA)-coated plates (Millipore, Bedford, MA) were incubated withrestimulated cells (in duplicate at three dilutions) at 37°C for24 h. After washes and staining with matched secondary anti-body, the plate was developed, and colored spots, representingcytokine-producing cells, were counted once or twice manuallyand unblinded under a dissecting microscope. There were nospots detected for JY cells plated alone. In several studies,PBMCs were plated with JY cells without peptides, and “allo-specific” spots were routinely detected. Background spots tonegative control AFP549–557 epitope were factored out. Thiscontrol was necessary to account for variation between PBMCsamples obtained at different times during the treatment of thepatients. Measurement of MART-127–35-specific responses overtime was generally performed once, due to limited PBMCsupply. In 16 samples, the results were repeated with similarresults. Determinant spreading in patient E1 was confirmed insix separate experiments with similar results.

Intracellular Cytokine Staining. To detect both cellsurface phenotype and cytokine synthesis in a single cell,FastImmune analysis was performed (32). Two million thawedPBMCs were rested overnight and then restimulated for 6 h withspecific (MART-127–35) or nonspecific (AFP549–557) peptideson 1 � 105 JY cells. The cells were treated with intracellulartransport inhibitor brefeldin A (Sigma) for 4 h to increase levelsof cytokines in the cytoplasm. Cells were stained for CD3-PerCP for 15 min at room temperature, followed by permeabi-lization and intracellular staining with IL-4-PE and IFN-�-FITCaccording to the manufacturer’s instructions. Controls includednonrestimulated lymphocytes, cells maximally stimulated withphorbol 12-myristate 13-acetate (Sigma) and calcium ionophore(Sigma), and samples stained with isotype control antibodies(PharMingen). Background staining to AFP peptide restimula-tion and isotype antibodies was subtracted to determine MART127–35-specific IFN-� and IL-4 staining.

Cytotoxicity Assay. The presence of MART-127–35

peptide-specific cytotoxicity was assessed on PBMCs after two

rounds of ex vivo restimulation as described previously (31, 33,34). Briefly, thawed patient PBMCs were pulsed with MART-127–35 peptide, washed, and cultured on day 0 with IL-7 (10–25ng/ml) and keyhole limpet hemocyanin (5 �g/ml) in RPMI1640/10% AB serum at 3 � 106 cells/1.5 ml/well. Cells wererestimulated weekly for 2 weeks. JY target cells were chromatedand peptide-pulsed at 10 �g/ml for 2 h, and then target cellswere washed, diluted to 5 � 104 cells/ml, and plated withvarious ratios of CTL. To control for nonspecific lysis, a 10–50-fold excess of unchromated K562 was added to target pop-ulations before adding to CTL. After 4–6 h at 37°C, superna-tants were harvested and counted in a gamma counter. Triplicatewells were averaged, and the percentage of specific lysis wascalculated as follows: [sample � spontaneous release]/[maxi-mum release � spontaneous release]. Any background lysis ofAFP549–557-pulsed targets was subtracted to determine MART-127–35-specific lysis.

HistologySpecimens were fixed in 10% neutral buffered formalin

and embedded in paraffin following standard procedures. Three-�m-thick sections were cut, and slides were deparaffinized inxylene and graded ethanol and brought to water and then stainedwith H&E (Fisher Scientific, Pittsburgh, PA). Immunohisto-chemical staining was performed following the DAKO EnVi-sion Systems. Endogenous peroxidase activity was quenched bytreating slides with 3% hydrogen peroxide in methanol for 10min. Heat-induced epitope retrieval was performed on the slidesusing 1 mM EDTA (pH 8; for MART-1) or using 0.01 M citratebuffer (pH 6; for the rest of antibodies) and a vegetable steamer(Black & Decker). After heating for 25 min, cooling, andwashing in 0.01 M PBS, slides were placed on a DAKO Au-tostainer (DAKO Corp., Carpenteria, CA) and then sequentiallyincubated in primary antibodies for 30 min. The followingantibodies were used: (a) MART-1, 1:50 (Biocare Medical,Walnut Creek, CA); (b) S-100, 1:1000 (DAKO Corp.); (c)gp100, 1:50 (HMB-45; DAKO Corp.); (d) tyrosinase, 1:100(T311; Novacastra Laboratories); (e) CD8, 1:50 (DAKO Corp.);(f) CD4, 1:100 (DAKO Corp.); (g) CD20, 1:1000 (DAKOCorp.); and (h) CD57, 1:30 (Becton Dickinson, San Jose, CA).Rabbit antimouse immunoglobulins (DAKO Corp.) were ap-plied for 15 min, followed by EnVision � rabbit peroxidase(DAKO Corp.) for 30 min. Diaminobenzidine and hydrogenperoxide were used as the substrates for 10 min at room tem-perature. After washing in tap water, the slides were counter-stained with hematoxylin, and coverslips were mounted. As anegative control, normal mouse serum (DAKO Corp.) was usedin place of primary antibodies.

Statistical AnalysisAnalysis of results of immunological assays was carried

out using pretreatment samples and samples from trial day �14,�28, �35, �56, and �112, with day 0 as the date of the firstimmunization. The maximal value for each subject and eachassay during days 14–56 was used as the measure for treatmentresponse because only 50% of the subjects had samples fromday �112. Analysis of mean pre- and posttreatment values foreach assay was analyzed by the Wilcoxon signed-rank test.

1000 Determinant Spreading in MART-1 Peptide/DC Trial



Spearman correlation coefficient was used to evaluate the cor-relation between the maximum responses by any two assays.Univariate analysis was performed using the Wilcoxon rank-sum or the Kruskal-Wallis test to examine the association of thetreatment response with each of the following factors: (a) gen-der (male versus female); (b) age (dichotomized as �60 years or�60 years); (c) dose (105, 106, and 107); (d) route (i.d. versusi.v.); (e) stage (III versus IV); (f) disease status (NED versusalive with disease); and (g) clinical response only for subjectswith measurable disease (response versus stabilization plus pro-gressive disease, or response plus stabilization versus progres-sive disease). A linear model was also developed to simulta-neously correlate the above factors with treatment response.Stepwise procedure was used for factor selection. All tests aretwo-sided. The Ps reported here were not adjusted for multipletesting. Due to the small sample size, the Ps are approximate.

RESULTSPatient Characteristics. Eighteen HLA-A*0201-posi-

tive patients with stage III or IV MART-1-positive melanomawere enrolled into this trial in which MART-127–35 peptide-pulsed DCs (105, 106, and 107) were administered i.d. or i.v. incohorts of three patients in a dose-escalation fashion. DCs weregenerated from GM-CSF/IL-4 differentiated monocyte precur-sors that were deemed immature by the following phenotypiccriterion: HLA-DR high; CD86 (B7-2) high; and CD14-positivelarge granular lymphocytes. Previously, we have shown that theDCs made by this methodology are immature but have highlevels of MHC class I and II antigens, CD80 and CD86, CD54,and CD40 and are more stimulatory in allogeneic and autolo-gous mixed lymphocyte reaction.

Patient characteristics are shown in Table 1. Mean age was60 years (range, 39–79 years). Twelve patients were male, andsix were female. Eight subjects had no measurable disease(seven subjects had stage III disease, and one subject had stageIV with NED after surgical excision). Ten subjects had stage IVdisease with measurable metastatic lesions at the followingsites: nodes (seven subjects); skin (six subjects); lung (threesubjects); liver (two subjects); intestine (one subject); breast(one subject); and adrenal glands (one subject). Pretreatment inthe seven subjects with stage III disease included surgery (sevensubjects), IFN-�2b (three subjects), biochemotherapy [cisplatin,1,3-bis(2-chloroethyl)-1-nitrosourea, dacarbazine, IFN-�2b, andIL-2; two subjects], irradiation (one subject), and other experi-mental vaccines (one subject). Pretreatment in 11 subjects withstage IV disease included surgery (11 subjects), recombinantcytokines (IL-2, IL-2 � histamine hydrochloride, or IFN-�2b; 7subjects), external beam irradiation (2 subjects), biochemo-therapy (2 subjects), chemotherapy (2 subjects), tamoxifen (2subjects), and other experimental vaccines (2 subjects).

Safety and Toxicity. Forty-nine doses of vaccine wereadministered. Two subjects did not complete the vaccinationschedule, one due to the development of clinically evident brainmetastasis 1 week after the initial vaccination (C3), and one dueto rapid disease progression after leukapheresis (F1). One vac-cine administered i.v. caused hypertension requiring hospitaladmission; this preparation grew a Pseudomona species bacte-ria. DC immunizations resulted in no local reaction at the

injection site and no constitutional symptoms. Two subjects hadworsening of preexisting vitiligo (B2 and D2), but none of thesubjects developed vitiligo de novo. One subject (D1) developedalopecia 1 month after the final immunization but completelyrecovered within 2 months. This subject had completed adjuvantIFN-�2b therapy 3 months before enrollment and had normallevels of thyroid hormones.

Clinical Outcome. Median follow-up for the wholegroup is 18 months (range, 1–28 months). Of 10 subjects withmeasurable disease, 1 patient (E1) with �80 s.c. metastases hasa durable complete response (24� months), first noted 1 monthafter the third vaccination. Two subjects had disease stabiliza-tion of prior progressive metastases in lymph nodes (subject A1,12 months, died at 28 months) and lung (subject C1, 4 months,died at 23 months). One subject (F1) had rapid disease progres-sion after leukapheresis and before receiving any vaccination.The remaining six subjects (A2, A3, B1, B2, B3, and F3) haddisease progression after completing the full vaccine schedule,and all have died. Of eight subjects with NED at trial entry, one(C3) developed clinically significant central nervous systemmetastasis after the first immunization. Two subjects pro-gressed: patient D1 with stage III melanoma developed lungmetastasis at 8 months and is alive with disease at 27� months;and patient D2 with resected stage IV melanoma developednodal metastasis at 7 months and is alive at 25� months. Fivesubjects with stage III disease (C2, D3, E2, E3, and F2) havehad no evidence of relapse at a median of 24� months (range,18–27� months) of follow-up.

Immunological Analysis of MART-127–35-specific Re-sponses. PBMCs were obtained before, during, and after threebiweekly vaccinations and analyzed for MART-1 recognition byMHC class I tetramer, IFN-� ELISPOT, IFN-� and IL-4 intra-cellular cytokine staining, and short-term cytotoxicity. Theseassays were chosen to cover a wide range of functional assaysfor cytotoxicity and cytokine production (ELISPOT and intra-cellular staining) as well as simple enumeration of peptide-specific cells in peripheral blood. Intracellular staining waschosen as a secondary cytokine assay due to its ability to detect

Table 1 Patient characteristics

No. of patients 18Male 12Female 6

Median age (range) (yrs) 65 (39–79)Stage

III 7IV NED 1IV 10

Sites of diseaseSkin 6Lymph nodes 7Lung 2Liver 2Other visceral 4

Prior therapyAdjuvant IFN-�2b 5Chemotherapy 5Other immunotherapy 8Hormonotherapy 2Irradiation 3




cell surface (CD3) as well as type 1 and type 2 cytokine (IFN-�and IL-4), despite having lower repeated sensitivity comparedwith the ELISPOT.

Baseline MART-127–35 reactivity in peripheral blood waslow to undetectable in the majority of subjects, as determined bythe four immunological assays. Samples from postimmunizationtime points showed an increase in frequency of MART-127–35-reactive cells for the tetramer (Figs. 1 and 2) and IFN-� ELIS-POT assays (Fig. 3; P 0.0001 and 0.0012, respectively) but nooverall difference between mean pre- and postimmunizationvalues for IFN-� or IL-4 intracellular cytokine staining (Fig. 4)and microcytotoxicity assays (Fig. 5). Peak values did not havea clear relation with the number of immunizations, althoughthere was a general trend of an increase early after the immu-nizations and a subsequent decrease in MART-127–35 reactivityover time.

In selected subjects with accessible metastatic lesions, pre-and postvaccination samples of melanoma tissue and tumor-infiltrating lymphocytes were analyzed. In a subject with acomplete response (E1), postimmunization biopsies of s.c. me-tastasis revealed a dense lymphocytic infiltrate within the mel-anoma lesions (Fig. 6, A�F), consisting largely of CD8� Tcells with some CD4� cells (Fig. 6, G�J). In this subject,biopsy of a residual lesion 2 months after the final immunizationrevealed a complete disappearance of melanoma cells and alocal decrease in epidermal melanocytes (without clinically ev-ident vitiligo).

Univariate and multivariate analysis was conducted to de-termine the relation between the results of the immunologicalassays and several parameters that may be predictive for immu-nological responses. Univariate analysis of postvaccinationMART-127–35 reactivity was not correlated with gender, age,dose, initial stage, and disease status for either tetramer or

Fig. 1 MART-127–35/HLA-A*0201 tetramer analysis of patient E1. PBMCs were cryopreserved at prevaccination, 2 weeks after the first vaccine(day �14), 2 weeks after the second vaccine (day �28), and 1 week (day �35), 3 weeks (day �56), and 5 weeks (day �75) after the third vaccine.Cryopreserved samples from all time points were stained for CD8-FITC, MART-127–35/HLA-A*0201 tetramer-PE, and CD4-tricolor�CD13-tricolor�CD19-tricolor and analyzed for the presence of CD8�/tetramer� cells (in the box) after gating on lymphocytes (by forward and side scatter)and gating out tricolor� cells.

Fig. 2 MART-127–35/HLA-A*0201 tetramer analysis of baseline andpostvaccination MART-127–35-specific CD8� T cells. Cryopreservedsamples from available time points were thawed and immediately ana-lyzed for the presence of CD8� T cells with affinity for MART-127–

35/HLA-A*0201 tetramers. X axis, patient identifiers. Y axis, CD8 andMART-127–35/HLA-A*0201 double positive events over the totalCD8� T-cell population. Z axis, time points of PBMCs.

Fig. 3 MART-127–35-specific IFN-�-producing cells by ELISPOT.Cryopreserved PBMCs were thawed and restimulated in vitro with JYcells pulsed with MART-127–35 or a control epitope (AFP549–557) over-night, and the frequency of MART-127–35-specific IFN-�-producingcells was analyzed in ELISPOT assays. Results are presented as a ratiobetween the specific (MART-127–35) and nonspecific (AFP549–557) IFN-�-producing cells, presented on the Y axis.




ELISPOT assays. The i.d. immunizations resulted in higherlevels of MART-127–35-reactive cells by the functional IFN-�ELISPOT (P 0.032), which was not confirmed by univariateanalysis of MHC class I tetramer data. However, multivariateanalysis using dose, route, and disease status as possible pre-dictive factors confirmed that i.d. administration of DC vaccinesresulted in an enhancement of MART-127–35 reactivity com-pared with i.v. delivery by both ELISPOT and tetramer (P 0.03 and P 0.07, respectively). The effect of the dose ofMART-127–35 peptide-pulsed DCs on immunological assaysrevealed discordant results, with the tetramer assay suggestingan increase in MART-127–35-specific effectors with increasingvaccine doses (Fig. 2, P 0.01), with a trend toward anopposite effect of vaccine dose when analyzed by IFN-� ELIS-POT (Fig. 3, P 0.07). In subjects with measurable melanomalesions at trial entry, the correlation between results of immu-nological assays and clinical outcome was studied. In this smallgroup of subjects receiving different doses of vaccines by tworoutes, no correlation could be drawn between these two vari-ables.

Overall conclusions from this extensive in vitro analysis ofMART-127–35 reactivity after MART-127–35/DC immunizationssuggest that the tetramer and ELISPOT assays were the most

instructive in following the immune activation in peripheralblood after immunizations, with the i.d. route resulting in higherlevels of circulating MART-127–35-specific cells. However, nei-ther of these assays served as a good surrogate for clinicalactivity in the current Phase I clinical trial.

Melanoma-specific Determinant Spreading. Determi-nant spreading is believed to be the main immunopathogenicevent in several systemic and organ-specific autoimmune dis-eases. Because detailed analysis of MART-127–35 reactivitycould not explain the differences between a subject with acomplete response in this trial and the other nine subjectswithout clinical benefit, we tested whether MART-127–35-specific lymphocytes served as a driver clone for a multideter-minant response. Cryopreserved PBMCs from day �75 post-vaccination were analyzed for reactivity to the HLA-DR4-restricted MART-151–73 epitope as evidence for intramoleculardeterminant spreading within the MART-1 tumor antigen pro-tein sequence. This is a MHC class II-binding epitope that isunlikely to be processed and presented by melanoma cells butmay have been cross-presented by host APCs in this DR4�patient. To study the possibility of intermolecular determinantspreading, PBMCs were also restimulated with the HLA-A*0201-binding melanoma epitopes gp100280–288 and tyrosin-ase1–9. Reactivity to these epitopes, which were not present inthe DC vaccines but were expressed by the patients’ melanomacells, could also be cross-presented by APCs from debris re-leased by killed melanoma cells. As a negative control, cellswere also restimulated with the HLA-A*0201-binding non-melanoma epitope AFP549–557. A series of seven ELISPOTstudies were performed analyzing patient E1 reactivity to theseadditional melanoma epitopes. The results are summarized inFig. 7. A consistent pattern was noted, with posttreatment pe-ripheral leukocytes from this subject producing IFN-� whenrestimulated with HLA-matched melanoma-derived epitopes forgp100, tyrosinase, and the MHC class II MART-1 epitope. No

Fig. 4 MART-127–35-specific intracellular cytokine staining. PBMCswere restimulated with MART-127–35 or control peptide-pulsed JY cellsfor 6 h, surface-stained with CD3 to allow gating on lymphocytes, andthen analyzed for intracellular content of IFN-� (A) or IL-4 (B).

Fig. 5 In vitro microcytotoxicity analysis. PBMCs were restimulatedin vitro with MART-127–35 peptide-pulsed autologous PBMCs. Aftertwo rounds of restimulation, cells were tested for MART-127–35-specificlysis of MART-127–35 or control peptide-pulsed, chromated JY cells inthe presence of 1:20 unlabeled K562 to inhibit nonspecific natural killeractivity.




such reactivity was noted in pretreatment samples. PBMC sam-ples were also tested from eight other subjects who did not havea clinical response, but who had metastatic disease (six of eightsubjects) or increased vitiligo (two of eight subjects) or receivedthe same DC dose and route (one of eight subjects). No otherpatient tested had a detectable response to the other epitopesbefore or after vaccination. Therefore, determinant spreadingcan occur in human subjects after DC-based single-epitopeimmunizations, which correlates with clinical response in thissmall series of subjects.

DISCUSSIONThe results of this trial show that immunization with im-

mature DCs pulsed with a single MHC class I peptide cangenerate robust peptide-specific T-cell responses in most pa-tients, even with small numbers of DCs (105) and without sideeffects. The i.d. delivery route was superior to the i.v. deliveryroute, and IFN-� ELISPOT and MART-127–35/MHC class Itetramer assays were instructive as an immunological end pointof successful vaccination. However, T-cell responses to theimmunizing peptide epitope were not predictive of clinicalresponse, despite the four assays used and the numerous timepoints assessed during the treatment. Our results should beinterpreted with caution because the performance specifications

Fig. 6 Tumor regression and lymphocytic infiltrate in patient E1. Photographs were taken at the times shown to monitor the appearance of the s.c.tumor (A�C). Biopsies obtained were stained with H&E to identify tumor deposits and lymphocyte infiltration (D�F). To characterize the natureof the lymphocytic infiltrate, the biopsy at day �130 was stained for the cell surface markers shown (G�J).

Fig. 7 Determinant spreading. PBMCs were assayed for the presenceof IFN-�-producing cells when restimulated overnight with the HLA-DR4 MART-1151–73 epitope and the HLA-A*0201-binding melanomaepitopes gp100280–288 and tyrosinase1–9. As a negative control, cellswere also restimulated with the HLA-A*0201-binding non-melanomaepitope AFP549–557. Only subject E1 had detectable reactivity toMART-151–73, gp100280–288, and tyrosinase1–9 (P 0.11, not signifi-cant due to the small number of events).




for the immunological assays used in the current trial monitor-ing have not been established. Additionally, the limited numberof subjects, although adequate for determining safety and tox-icity in a Phase I trial, does not allow us to make inferences withadequate confidence about the treatment effects on immuneresponses, the correlation between vaccine schedule or dose andimmune responses, and the relationship between immune andclinical responses. The real value of our attempts to detect largedifferences in immunological outcomes is the exploration ofstrong trends that may lead to further investigation.

Whereas some recent immunotherapy trials have foundconcordance between immunological monitoring assays andclinical response (35, 36), most have not (37–41). Many clinicaltrial immunological monitoring studies have also been limitedby few PBMC samples and one or two assays performed, manyonly testing pretreatment and posttreatment time points. Amongstudies in which several assays were performed on multiple timepoint PBMC samples, one found a good correlation betweentetramer enumeration of peptide-specific T cells and their func-tion (42), one found no correlation between assays (43), anotherfound correlation between cytokine assays but not with delayed-type hypersensitivity (44). In this trial, we found limited con-cordance between the levels of MART-1 peptide-specific T cellsmeasured by the most sensitive assays, MHC class I tetramerand IFN-� ELISPOT. This is not unexpected because the tet-ramer enumerates circulating cells regardless of function, andthe ELISPOT detects peptide-specific cells that secrete IFN-�.Recently, a thorough analysis of three MART-1 peptide-immu-nized patients showed T-cell phenotype and activity fluctuationsover time, with some peptide-specific cells secreting IFN-� andGM-CSF, whereas others secreted only IL-2 (45); hence, asexpected, all active peptide-specific cells may not secrete asingle cytokine.

The complete responder (E1) in this series developedHLA-A*0201-restricted responses to two additional melanomaantigens (gp100 and tyrosinase) expressed by her tumor as wellas a HLA-DR4-restricted MART-1 epitope. Determinantspreading was not found in other patients with measurabledisease (B1, B2, B3, C1, and F3), increased vitiligo (B2 nd D2),and disease stabilization (C1) or in those receiving 107 DCs i.d.(E2). We hypothesize that the MART-127–35-specific driverclone in E1 generated melanoma cell lysis, resulting in endog-enous priming with new melanoma-derived epitopes (MART-1MHC class II epitope, gp100, and tyrosinase) by endogenousAPCs. Therefore, the driver clone activated a cascade of tumor-specific autoreactive T-cell clones. This study provides supportthat cancer vaccines may take advantage of the immunopatho-genic mechanisms that lead to serious autoimmune diseases togenerate clinical responses.

An example of intramolecular determinant spreading hasbeen described previously in a complete responder in anotherDC-based clinical trial (46). In this trial, subjects received s.c.and i.v. injections of DCs pulsed with the HLA-A*0201 mela-noma-derived epitopes MART127–35, gp100280–288, and tyro-sinase368–376D. Using the ELISPOT assay, Ranieri et al. (46)detected reactivity to two other HLA- A*0201-binding epitopes(gp100209–217 and tyrosinase1–9) and four HLA-DR4 class IIepitopes (MART151–73, gp10044–59, gp100615–633, and tyrosin-ase56–70) in the one patient tested. Also, intermolecular deter-

minant spreading has been reported in one subject immunizedwith MART127–35 and tyrosinase368–376 peptide-pulsed DCsand subsequent spreading to the gp100209–218 epitope (47),whereas another subject immunized with gp100209–2M emulsi-fied in incomplete Freund’s adjuvant showed subsequentspreading to MAGE-12170–178 (48). Taken together with theresults of our trial, determinant spreading may be an importantphenomenon in responders to DC-based immunotherapy. Wedid not test all known HLA-A*0201 epitopes, hence spreadingto other antigens or epitopes might have occurred in E1 or otherpatients.

Determinant spreading is a normal feature of protectiveimmune responses to infectious agents, allowing recognition ofmultiple antigenic targets (3). The immune system depends ondiversification to adequately protect against dangerous non-self.It is also possible that it may also play a role in protectionagainst dangerous self processes such as cancer. Therefore,immune responses leading to tumor regression may use thesame mechanisms involved in the immunopathogenesis of ag-gressive autoimmune diseases (1, 2). Most melanoma-derivedantigens are lineage-specific proteins, which are differentiallyrecognized by T cells when expressed by malignant as opposedto benign pigmented cell (49–52). A continuum exists betweenantitumor and anti-self responses because the development ofclinical vitiligo has been associated with clinical response toIL-2-based melanoma therapy (49).

In experimental models of autoimmune diseases, a spread-ing hierarchy has been defined, starting with a determinant thathas the largest pool of high-avidity T cells that serve as a driverclone for spreading to determinants with progressively smallerand low-avidity T-cell precursor clones (1, 53, 54). This phe-nomenon had been generally believed to be unique to CD4�T-cell responses after cross-priming by exogenous antigen pre-sented by host APCs. It is now clear that cross-priming caninduce CD8� T-cell responses (15), and the possibility of MHCclass I determinant spreading has been studied. In a model oftumor immunity, a single MHC class I tumor-derived epitopegenerated protective immunity that was clearly linked to in-tramolecular spreading to other determinants derived from thesame tumor (13). In HLA-A*0201 positive subjects with mel-anoma (as well as healthy donors), anti-MART127–35-reactive Tcells can be detected at higher frequencies compared with othermelanoma-derived epitopes (55), and the analysis of HLA-A*0201 restricted tumor-infiltrating lymphocytes from subjectswith spontaneous or IL-2-induced responses (56) suggests thatthe MART127–35 epitope is immunodominant within this prev-alent HLA subtype. Therefore, the observations in the respon-sive subject described in this report may reflect a similar spread-ing hierarchy from the high-avidity large T-cell pool ofMART127–35-reactive T cells to other lower avidity and lessfrequent T cells.

There was no attempt to mature DCs in this trial, which hada phenotype consistent with immature DCs. Other clinical trialshave clearly documented immunological and clinical responseswith antigen-loaded immature DCs in human subjects (57–68).Similarly, in vivo immunization with immature DCs generatesantitumor responses in murine models (69, 70). However, a trialattempting to immunize against influenza with peptide-pulsedimmature DCs raised the concern that immune tolerance might




be induced (71). Furthermore, another trial directly comparingcoinjection of immature and mature DCs loaded with differentantigens reported a consistent pattern of superior immune acti-vation with mature DCs (47). The discussion between the rela-tive value of mature and immature DCs is hampered by the lackof uniform criteria to define these populations, which develop ina continuum, and the multiple factors that may lead to function-ally different DCs. These include the type of tissue culturemedia; the presence or absence of serum; the use of autologous,allogeneic, or xenogeneic serum; the tissue culture flasks; andthe number of DC manipulations (data not shown; Ref. 72). It iswidely believed that matured DCs are superior to immature DCswhen attempting to generate human CTLs in vitro, given theclear superior antigen-presenting phenotype of mature DCs (73).The same rationale may not hold true when using DCs in vivo.The beneficial effects of ex vivo maturation may be counterbal-anced by a decreased ability to traffic to lymph nodes (74) ormay be superfluous after maturation signals from the trauma ofinjection, local inflammatory cells, or early encounter with CD4T cells in vivo. Hence, when used in vivo, there are too littlecomparative data and too many variables in DC preparation toconclude that a particular degree of maturation is required forantitumor effects.

In conclusion, we attempted to generate anti-melanomaresponses using autologous DCs pulsed with an epitope presentin the patient’s melanoma lesions. A clear expansion of MART-127–35-specific T cells was evident in most patients by tetramerand ELISPOT analysis. Detailed immunological and statisticalanalysis of epitope-specific responses failed to correlate withclinical responses. This may be due in part to a low frequencyof clinical responses in this pilot trial, which is common in mostimmunotherapy interventions, and the low numbers of subjectsreceiving the same dose and route of DCs. However, analysis ofintra- and intermolecular determinant spreading did provide anunambiguous tool to reliably differentiate a subject with acomplete response compared with other nonresponders.

ACKNOWLEDGMENTSWe thank Drs. Peter P. Lee and Mark Davis for MART-127–35

tetramers and training in their use; the AIDS Reagent Program, NIAIDTetramer Facility, NIH for additional tetramer manufacture; the UCLAPeptide Synthesis Facility (Joseph Reeve, Jr., Director); and the JonssonComprehensive Cancer Center Quality Assurance Committee members(Christine King, Director).

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2003;9:998-1008. Clin Cancer Res Lisa H. Butterfield, Antoni Ribas, Vivian B. Dissette, et al. MelanomaDendritic Cell-based Immunotherapy for Malignant Determinant Spreading Associated with Clinical Response in

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