CLINICAL OBSERVATIONS,INTERVENTIONS,AND THERAPEUTIC TRIALS
歪脑袋木头桩读后感Treatment of nasopharyngeal carcinoma with Epstein-Barr virus–specific丫蛋儿
T lymphocytes
Karin C.M.Straathof,Catherine M.Bollard,Uday Popat,M.Helen Huls,Teresita Lopez,M.Craig Morriss,Mary V.Gresik,Adrian P.Gee, Heidi V.Rusll,Malcolm K.Brenner,Cliona M.Rooney,and Helen E.Heslop
Conventional treatment for nasopharyn-geal carcinoma(NPC)frequently fails and is accompanied by vere long-term side effects.Since virtually all undifferentiated NPCs are associated with Epstein-Barr virus(EBV),this tumor is an attractive candidate for cellular immunotherapy tar-geted against tumor-associated viral anti-gens.We now demonstrate that EBV-specific cytotoxic T-cell(CTL)lines can readily be generated from individuals with NPC,notwithstanding the patients’prior exposure to chemotherapy/radiation.A
total of10patients diagnod with ad-
vanced NPC were treated with autolo-
gous CTLs.All patients tolerated the
CTLs,although one developed incread
swelling at the site of pre-existing dis-
ea.At19to27months after infusion,4
patients treated in remission from locally
advanced dia remain dia free.Of
6patients with refractory dia prior to
treatment,2had complete respons,and
remain in remission over11to23months
after treatment;1had a partial remission
that persisted for12months;1has had
stable dia for more than14months;
and2had no respon.The results
demonstrate that administration of EBV-
specific CTLs to patients with advanced
土木工程认识实习NPC is feasible,appears to be safe,and
can be associated with significant antitu-
mor activity.(Blood.2005;105:1898-1904)
©2005by The American Society of Hematology
Introduction
Nasopharyngeal carcinoma(NPC)occurs worldwide and is the third most common malignancy in Southern China,where the incidence is as high as50per100000.1NPC is a radionsitive tumor and local control rates of more than80%can be obtained. However,a significant number of patients relaps
e,particularly when dia is advanced at diagnosis—the most common pren-tation due to a lack of early symptoms.2Moreover,radiation and chemotherapy are accompanied by vere short-and long-term side effects including condary malignancies.3Hence,there is a need for therapies that will improve dia-free survival and that may be associated with reduced toxicity.
Epstein-Barr virus(EBV)is prent in virtually all poorly and undifferentiated nonkeratinizing NPCs regardless of geographic origin,4and the viral antigens expresd by the tumor provide potential target antigens for immunotherapy.Adoptive transfer of cytotoxic T cells(CTLs)specific for EBV antigens has proved safe and effective as prophylaxis and treatment for EBV-associated lymphoproliferative dia in bone marrow and solid organ transplant recipients.5-11The highly immunogenic lymphomas express all latent EBV antigens,including the immunodominant EBV nuclear antigens(EBNA)3A,3B,and3C,and are therefore ideal targets for immunotherapy.By contrast,NPC express a restricted t of less immunogenic viral antigens,namely EBNA1, and latent proteins(LMPs)1and2.EBNA1is expresd in all NPCs,and although its processing through the HLA class I pathway is inhibited by a glycine-alanine repeat,peptides derived from incompletely translated proteins may be prented to CD8ϩT cells.12-15Expression of LMP1and/or LMP2is detectable in at least 50%of NPC tumors.16,17Since NPCs also express major histocom-patibility co
mplex(MHC)class I molecules as well as the peptide transporters TAP1and TAP2,they are capable of processing and prenting the antigens in the context of HLA class I molecules for recognition by CTLs.18LMP1-and LMP2-specific T cells are indeed prent in the peripheral blood of NPC patients,albeit at lower frequency than in healthy donors,19,20and could potentially be activated and expanded for immunotherapeutic strategies.
We hypothesized that ex vivo expansion of EBV-specific CTLs in the abnce of tumor inhibitory factors21,22and the subquent adoptive transfer of the cells may be of benefit to patients with EBV-positive NPC.Here we confirm the feasibility of this ap-proach,and in10patients show evidence for safety and activity. Patients,materials,and methods
Study entry criteria and patient details
This protocol was approved by the institutional review board(IRB)at Baylor College of Medicine and the Food and Drug Administration(FDA). Patients were eligible for study if they had stage III or IV nasopharyngeal carcinoma at diagnosis(according to American Joint Committee for Cancer
From the Center for Cell and Gene Therapy,Departments of Pediatrics, Radiology,Pathology,and Medicine,Molecular Virology and Microbiology, Baylor College of Medicine,Houston,TX;The Methodi
st Hospital,Houston, TX;and Texas Children’s Hospital,Houston TX.
Submitted August2,2004;accepted November1,2004.Prepublished online as Blood First Edition Paper,November12,2004;DOI10.1182/blood-2004-07-2975.
Supported by The Ter Meulen Fund,The Dutch Cancer Society,and the Ank van Vlissingen Foundation(KCMs),the General Clinical Rearch Center (GCRC)at Baylor College of Medicine(RR00188),the Methodist Foundation,and a Doris Duke Distinguished Clinical Scientist Award(H.E.H.).
An Inside Blood analysis of this article appears in the front of this issue. Reprints:Helen E.Heslop,Center for Cell and Gene Therapy,1102Bates St, Suite1120,Houston,TX77030;e-mail:du.
The publication costs of this article were defrayed in part by page charge payment.Therefore,and solely to indicate this fact,this article is hereby marked‘‘advertiment’’in accordance with18U.ion1734.
©2005by The American Society of Hematology
1898BLOOD,1MARCH2005⅐VOLUME105,NUMBER5总是放屁还很臭是怎么回事
Staging and End-Results Reporting staging system199723)and were either in remission or had refractory or relapd dia,and if their tumor was EBV-positive as determined by in situ hybridization or polymera chain reaction(PCR)amplification for Epstein-Barr virus–encoded RNA(EBER). Patients were treated on3escalating do levels and received either2dos of2ϫ107CTLs/m2(do level1)or one do of2ϫ107CTLs/m2and1 do of1ϫ108CTLs/m2(do level2)or1do of1ϫ108CTLs/m2and 1do of2ϫ108CTLs/m2(do level3).CTLs/m2were given intrave-nously with a2-week interval between each do.Peripheral blood was obtained before and at multiple time points after CTL infusion for evaluation of toxicity and EBV immunity.
Generation of EBV-transformed B-cell lines
and EBV-specific CTLs
After informed connt,peripheral blood(40-60mL)from patients with EBV-positive NPC was ud to generate both EBV-transformed lymphoblas-toid B-cell lines(LCLs)and EBV-specific CTL lines.24Briefly,for LCL generation,5ϫ106peripheral blood mononuclear cells(PBMCs)were incubated with concentrated supernatant of B95-8cultures,in the prence of1g/mL cyclosporin A(Sandoz,Vie
nna,Austria)to establish an LCL. Subquently,PBMCs(2ϫ106per well of a24-well plate)were stimulated with LCLs irradiated at4000rads at an effector-stimulator(E/S)ratio of 40:1.After9to12days,viable cells were restimulated with irradiated LCLs (at4:1E/S ratio).Subquently,CTLs were expanded by weekly stimula-tions with LCLs(at4:1E/S ratio)in the prence of recombinant human interleukin-2(rhIL-2,Proleukin;Chiron Emeryville,CA)(40-100U/mL). After expansion,CTLs were tested for sterility,HLA identity,immunophe-notype,and EBV specificity and cryoprerved.Specificity was tested in a 4-hour Cr51relea assay.In8lines,the CTLs showed a significantly higher killing of the autologous LCLs(mean,56.6%;range,38%-92%)compared with HLA antigen–mismatched LCLs(mean, 6.1%;range,0%-27%; PϽ.0001)or to HSB-2(mean,21.5%;range,6%-55%;PϽ.005)at an effector-target ratio of20:1.In2CTL lines,lysis of the HLA-mismatched LCLs was obrved,which was significantly reduced by depletion of T-cell receptor␥␦(TCR␥␦)–positive cells.Autoreactivity was excluded by the abnce of lysis of autologous phytohemagglutinin(PHA)–stimulated lymphoblasts in all10CTL lines.
Peptides
The following peptides were ud for analysis of EBV-specific T-cell populations according to the patients’HLA specificity:LMP1,HLA-A2:YLQQNWWTL, YLLEMLWRL;LMP2,HLA-A2:LLWTLVVLL,CL
GGLLTMV,FL Y A-LALLI,GLGTLGAAI,TVCGGIMFL,L TAGFLIFL,LIVDA VLQL;HLA-A11:SSCSSCPLSKI;HLA-A24:TYGPVFMCL;HLA-A23/24:PYLFW-LAAI;HLA-A68:FTASVSTVV,ASCFTASVSTVVTA T(15-mer);HLA-B27: RRRWRRL TV,RRWRRL TVCGGIMFL(15-mer),RRL TVCGGIMFL;HLA-B60:IEDPPFNSL;EBNA1,HLA-B35:HPVGEADYFEY;EBNA2,HLA-A2: DTPLIPL TIF;EBNA3,HLA-A2:LLDFVRFMGV;HLA-A3:RLRAEAQVK; HLA-A11:A VFDRKSDAK,IVTDFSVIK,LPGPQVTA VLLHHEES, DEP ASTEPVHDQLL,NPTQAPVIQL VHA VY;HLA-A24:RYSIFFDY,TYSA-GIVQI;HLA-B7:RPPIFIRLL,QPRAPIRPI;HLA-B27:RRIYDLIEL;HLA-B35:YPLHEQHGM,A VLLHEESM;HLA-B44:VEITPYKPTW,EGGVG-WRHW,EENLLDFVRF,KEHVIQNAF;BZLF1,HLA-B35:EPLPQGQL TA Y; BRLF1,HLA-A2:YVLDHLIVV;HLA-A11:A TIGTAMYK;HLA-A24:DYC-NVLNKEF;BMLF1,HLA-A2:GLCTLV AML;and BMRF1,HLA-A2: TLDYKPLSV(listed in Khanna and Burrows25;Houssaint et al26;and K.C.M.S., Ann Leen,M.H.H.,H.E.H.,C.M.R.,and C.M.B.,manuscript in preparation). HLA-A2–restricted cytomegalovirus pp65–derived peptide NL VPMV A TV was ud as a control.Peptides were synthesized by either Martin Campbell, Synthetic Antigen Laboratory,The University of Texas M.D.Anderson Cancer Center,Houston,TX,or Genemed Synthesis(South San Francisco,CA).In this paper,the peptides are referred to by thefirst3amino acids as underlined. Tetramer staining
To identify LMP1-and LMP2-specific T cells,a lection from the following tetramers was ud,as determined by the HLA type of the patient:LMP1:HLA-A*0201-YLQQNWWTL;and LMP2:HLA-A*0201-CLG-GLLTMV,HLA-A*0201-FLYALALLI,HLA-A*0201-LLWTLVVLL, HLA-A*0201-TVCGGIMFL,HLA-A*1101-SSCSSCPLSKI,HLA-A*2301-PYLFWLAAI,HLA-A24-TYGPVFMCL,HLA-A68-FTAS-VSTVV,HLA-B*2705-RRRWRRLTV,and HLA-B*2705-RRLTVCG-GIMF.Tetramers were prepared by the National Institute of Allergy and Infectious Dias(NIAID)tetramer core facility(Atlanta,GA)or by the Baylor College of Medicine Tetramer Core Facility(Houston,TX).CTLs or PBMCs(5-10ϫ105)were incubated at room temperature for30minutes in phosphate-buffered saline(PBS)/1%fetal calf rum(FCS)containing the phycoerythrin(PE)–labeled tetrameric complex.Samples were costained with anti-CD8fluorescein isothiocyanate(FITC)and anti-CD3peridinin chlorophyll-alpha protein(PerCP).Appropriate isotype controls were included.Stained cells werefixed in PBS containing0.5%paraformalde-hyde.For each sample,a minimum of100000cells was analyzed using a FACS Calibur with the Cell Quest Software(Becton Dickinson,San Jo,CA). Enzyme-linked immunospot(ELISPOT)assay
The frequency of EBV-and LMP2-specific T cells in the infusion product as well as in the peripheral blood before and at multiple time points after CTL infusion was measured using an interferon-␥(IFN-
␥)ELISPOT assay.The 96-wellfiltration plates(MultiScreen,no.MAHAS4510;Millipore,Bed-ford,MA)were coated overnight with10g/mL anti–IFN-␥antibody (Catcher-mAB91-DIK;Mabtech,Cincinnati,OH).PBMCs were thawed24 hours before the assay in complete media supplemented with50U/mL Benzona(Novagen,Madison,WI),rested overnight in complete media, and plated at1to2ϫ105cells/well and2to3rial dilutions for LCL targets and3to4ϫ105/well for peptide targets.CTLs were rested overnight in complete media and plated at1ϫ105cells/well and2rial dilutions.Cells were stimulated with either irradiated(40Gy)autologous LCLs(1ϫ105/well)or5g/mL peptide.In HLA-A2–positive patients,the cytomegalovirus(CMV)pp65–encoded HLA-A2–restricted peptide NLVP-MV ATV was ud as control.After18to24hours,the plates were washed and incubated with the condary biotin conjugated anti–IFN-␥monoclonal antibody(Detector-mAB[7-B6-1-Biotin];Mabtech).After incubation with avidin–biotinylated horradish peroxida complex(Vectastain Elite ABC Kit[Standard],no.PK6100;Vector Laboratories,Burlin-game,CA),plates were developed with3-amino-9-ethylcarbazole (AEC)substrate(Sigma,St Louis,MO).Plates were nt for evaluation to Zellnet Consulting(New York,NY).Spot-forming units(SFCs)per 1ϫ105CTLs or per1ϫ106PBMCs were calculated by linear regression analysis when rial dilutions were performed and sub-quent subtraction of background of nonstimulated T cells.If an epitope-specific T-cell population had been identified in the infusion product,EBV-and LMP2-specific immunity was monitor
ed in patient peripheral blood using this IFN-␥ELISPOT assay and,when enough PBMCs were available and HLA type was informative,by tetramer staining. PCR for EBV load in PBMCs
PBMCs were isolated from peripheral blood on a Ficoll(Lymphoprep; Axis-Shield,Oslo,Norway)gradient and washed with PBS.DNA was isolated from3to5ϫ106PBMCs using an anion exchange column (Qiagen,Valencia,CA).DNA(500ng)was then ud for real-time polymera chain reaction(PCR)to quantitate EBV genome copy number and was reported as copies(cp)/g DNA.27
Results
Patient characteristics
A total of10patients were enrolled in the study,and all had poorly differentiated or undifferentiated nasopharyngeal carci-noma(WHO II/III)at diagnosis.At the time of CTL infusion,4 patients at high risk for relap were in remission and6patients had failed multiple rounds of radiotherapy and chemotherapy T-CELL THERAPY FOR EBVϩNASOPHARYNGEAL CARCINOMA1899
BLOOD,1MARCH2005⅐VOLUME105,NUMBER5
and had relapd/refractory dia.Patient characteristics and previous treatment are summarized in Table1.
CTL lines contain LMP2-specific T-cell populations Autologous LCLs and EBV-specific CTLs were successfully generated from10of10NPC patients.The phenotype of the CTL lines is shown in Table2.The prence of LMP1-and LMP2-specific T cells within the CTL lines was evaluated by IFN-␥ELISPOT after stimulation with LMP1/2peptides.In8of9CTL lines for which informative peptides were available bad on HLA type,T cells specific for at least1LMP2epitope were detected (Table3).In addition,in1of5CTL lines evaluable for LMP1 specificity an LMP1-YLL–specific T-cell population was identi-fied.As measured by tetramer staining,up to5.5%of the total CD8ϩpopulation was specific for a single LMP2epitope(data not shown).In4lines,T cells specific for multiple(up to5)different LMP2epitopes were prent;in2cas the were restricted through different HLA alleles.Such T-cell respons targeted toward multiple tumor antigen-derived epitopes are important to reduce the risk of tumor escape through antigen deletion.Overall the T-cell respons against the subdominant LMP antigens were weaker than tho against epitopes derived from the immunodomi-nant lytic and EBNA3latent antigens(Table3),but in the same range as detected in LCL-reactivated CTL lines from healthy donors.28Moreover,the identified T-cell populations specific for in
dividual peptides reflect the minimum LMP2specificity prent and likely underestimate the total number of LMP2-specific T cells.
Safety of EBV-specific CTLs
Upon administration of EBV-specific CTLs,no immediate or long-term toxicity was obrved in the4patients without detect-able dia and in5of6patients with refractory/relapd dia (Table4).However,in one patient(P845)with bulky dia, pre-existing facial swelling incread markedly2days after infusion of thefirst do of CTLs(2ϫ107/m2)requiring a tracheostomy.A needle biopsy of this mass showed tumor cells and no inflammatory cells suggesting tumor progression as the caus-ative factor,but a contributory effect from CTL cannot be excluded.
Changes in EBV immunity after CTL administration
Viral load and the frequency of EBV-specific T cells were monitored in the peripheral blood at multiple time points after
M indicates male;RT,radiotherapy;5-FU,5-fluoruracil;F,female;and MTX,methotrexate.
Stage according to American Joint Committee for Cancer Staging and End-Results Reporting staging system1997.23研究报告模板
臧雅菲
喝水简笔画VP16indicates etoposide;CTP-11is irinotecan.
*This patients received additional dos of1ϫ108CTLs/m2at6months,9months,and12months after the initial CTL infusions.
1900STRAATHOF et al BLOOD,1MARCH2005⅐VOLUME105,NUMBER5
CTL infusion to evaluate in vivo persistence and activity of the infud CTLs.Of9patients with a detectable amount of EBV-DNA in PBMCs prior to CTL infusion,EBV load fell within6weeks after infusion in6patients(Table5).A decrea in EBV viral load in the peripheral blood likely reflects the lysis of EBV-infected B cells, and therefore demonstrates in vivo activity of the infud EBV-specific CTLs.
In9of10patients,the low normal frequency of EBV-specific T cells in the peripheral blood(mean:274,ra
nge:197-384 SFCs/1ϫ105PBMCs),as measured by IFN-␥cretion of PBMCs upon stimulation with autologous LCLs,remained unchanged after CTL infusion(data not shown).In one patient (P845)with a low number of circulating EBV-specific CTLs prior to CTL infusion(24SFCs/1ϫ105PBMCs),a transient 3-fold increa in the number of EBV-specific CTLs was measured.In addition,the LMP2-specific T-cell populations identified in the infusion product were monitored in the peripheral blood after CTL infusion.In5HLA-A2ϩpatients, using IFN-␥ELISPOT analysis,the number of T cells specific for a cytomegalovirus pp65–derived epitope was determined at the same time points to control for natural variations in viral immunity.In4of8evaluated patients,the number of T cells specific for LMP2epitopes incread more than2-fold,whereas the pp65-specific immunity remained stable over this time period(Table5).However,this increa in LMP2immunity was transient as the number of LMP2-specific T cells was similar to baline6weeks after CTL infusion in3of the4patients. Additional tetramer analysis of the frequency of LMP2-specific T cells in the peripheral blood after CTL infusion in3patients failed to detect a persistent increa in LMP2immunity(data not shown).
Clinical respons after CTL therapy indicate antitumor activity Clinical respons were evaluated from computed tomography and magnetic resonance imaging scans before and after CTL therapy,using the international criteria propod by the Re-spon Evaluation Criteria in Solid Tumor
s Committee.29All4 patients who were in remission at the time of enrollment on the study remained in complete remission19to27months after CTL therapy(Table4).Of the6patients with refractory/ relapd dia,2patients had no respon,1patient has had stable dia for more than14months without additional therapy,1patient had a partial respon sustained for12months, and2patients attained complete remission(CR).One of the patients who attained CR(P389)with refractory relapd dia had a24%reduction in tumor size after the initial2CTL infusions on do level2.Becau of this partial respon,this patient received3additional dos of1ϫ108CTLs/m2at6 months,9months,and12months after the initial CTL infusions with IRB and FDA approval.During this period,the patient did not receive other treatment and showed continuing respon. Positron emission tomography imaging at15months after the first CTL infusion showed normal isotope uptake consistent with a complete respon and residualfibrosis.In the cond patient who had a CR(P894),a biopsy of the nasopharynx prior to CTL infusion showed poorly differentiated EBER-positive NPC. Multiple biopsies taken6months after CTL therapy were all negative for tumor indicating a complete remission(Figure1). Of the2patients who had no direct respon to CTL infusion,1 (P845)came off study at2weeks becau of progressive dia,but subquently developed a partial respon to palliative chemotherapy(gemcitabine and carboplatin),to which the dia had been previously unresponsive.The condition of this patient remained stable for4months until the tumor again progresd.
Table3.T-cell populations specific for EBV antigens(SFCs/1؋105
CTL lines were screened for the prence of T-cell populations specific for the indicated antigens by IFN-␥ELISPOT.The panel of peptides ud for stimulation was bad on the HLA type of the patient(Table1).The quence of the peptides referred to by thefirst3amino acids is listed in“Patients,materials,and methods.”
ND indicates not done,as no informative peptides were available.
For patient815,there were no informative peptides available.T-CELL THERAPY FOR EBVϩNASOPHARYNGEAL CARCINOMA1901
BLOOD,1MARCH2005⅐VOLUME105,NUMBER5
Discussion
Although patients with advanced,relapd NPC have been expod to intensive radiation and chemotherapy,EBV-specific CTLs can readily be reactivated from their PBMCs.Adoptive transfer of the CTL lines appears safe in this patient group,although caution may be required in patients with bulky dia.The infud lines contained cytotoxic T cells specific for LMP2(an EBV antigen usually expresd by NPC tumor cells),and were biologically active in vivo,reducing levels of EBV DNA in pe
ripheral blood mononuclear cells.Although there was no persistent ri in the frequency of circulating T cells specific for LMP2after infusion, the CTLs appeared to have significant antitumor activity.Of6 patients with dia that was resistant to,or had relapd after, intensive chemotherapy and radiation,2have had complete and sustained remissions.A third patient had a partial respon and a fourth has stable dia.All4patients who were in remission at the time of CTL infusion remained dia free after19to 27months.
The EBV-specific CTLs ud in this study were reactivated using LCLs that express all EBV latent antigens.LCLs are excellent antigen-prenting cells that are readily available for all patients,as only a limited amount of blood is required to establish an LCL line.As expected using this method,only a minority of the expanded T cells were specific for the subdominant antigen LMP2. However,upon encounter with NPC cells in vivo the LMP2-specific T cells may expand in number.Although such an increa in the frequency of LMP2-specific T cells was not detectable in the peripheral blood in the majority of patients using ELISPOT assays or tetramers,only a small number of T cells were infud (4-30ϫ107CTL/m2)and less than10%were LMP2specific.An expansion of veral logs would be required to detect a significant increa in the peripheral blood,and it may be that the infud T cells instead accumulate and expand at local sites of tumor antigen prentation rather than circulat
e in the periphery.In addition to LMP2-directed immune respons,immunity to other EBV anti-gens may have contributed to the tumor respons.Recent insights in the processing and prentation of EBNA1suggest that although a glycine-alanine repeat prevents the processing of the full-length protein,peptides derived from incompletely translated proteins may be available for T-cell recognition.12-15Of note,the CTL line from P894,who attained a complete respon,contains a relatively large T-cell population specific for an EBNA1-derived, HLA class I–restricted epitope(Table3).In addition,clinically relevant dos of chemotherapy can induce the expression of EBV lytic cycle antigens in NPC tumors in vivo.30Similarly,gamma-irradiation at clinically relevant dos can induce lytic EBV infection in EBV-positive B-cell tumors in vivo.31Patient845,who progresd2days after CTL therapy,received chemotherapy
N/A indicates not applicable;CR,complete remission;and PR,partial respon according to the international criteria propod by the Respon Evaluation Criteria in Solid Tumors Committee.
29
描写花朵的四字词语cp indicates copies;wk,weeks;ND,not done as not enough PBMCs or no informative peptide was available.
*At3months after CTLs,as a sufficient number of PBMCs was not available at6weeks after CTLs time point.
†At8weeks after CTLs,as a sufficient number of PBMCs was not available at6weeks after CTLs time point.
‡Pentadecamers containing minimum epitope were ud for stimulation.
1902STRAATHOF et al BLOOD,1MARCH2005⅐VOLUME105,NUMBER5