terapia cellulare a bersaglio molecolare anti-ebv per il...
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Terapia cellulare a bersaglio molecolare anti-EBV per il carcinoma
del rinofaringe
Terapia cellulare a bersaglio Terapia cellulare a bersaglio molecolare molecolare antianti--EBVEBV per il carcinoma per il carcinoma
del del rinofaringerinofaringe
Paolo Pedrazzoli
Cagliari, 24 giugno 2005
• Epstein-Barr Virus e malattie associate
• Linfociti T citotossici (CTL) anti EBV
• Terapia Cellulare del carcinoma indifferenziato del rinofaringe (NPC)
•• EpsteinEpstein--BarrBarr Virus e malattie associateVirus e malattie associate
•• Linfociti T Linfociti T citotossicicitotossici (CTL) (CTL) antianti EBVEBV
•• Terapia Cellulare del carcinoma Terapia Cellulare del carcinoma indifferenziato del indifferenziato del rinofaringerinofaringe (NPC)(NPC)
Epstein-Barr Virus - associated diseaseEpsteinEpstein--BarrBarr Virus Virus -- associatedassociated diseasedisease
BurkittBurkitt lymphoma (1964)lymphoma (1964)Infectious Mononucleosis (1968)Infectious Mononucleosis (1968)NasopharingealNasopharingeal carcinoma (1970) carcinoma (1970) LymphoproliferativeLymphoproliferative diseases in hosts with diseases in hosts with impaired Timpaired T--cell immunity (1982 cell immunity (1982 →→))TT--cell Lymphoma (1988)cell Lymphoma (1988)Hodgkin disease (1989)Hodgkin disease (1989)
Epstein-Barr virusEpsteinEpstein--BarrBarr virusvirus
Member of the Member of the HerpesvirusHerpesvirus family, EBV infects family, EBV infects over 90% of humans persisting for the lifetime of over 90% of humans persisting for the lifetime of the personthe personInfection occurs by contact with oral secretionsInfection occurs by contact with oral secretionsEBV directly infects epithelial cells of the EBV directly infects epithelial cells of the oropharynxoropharynx and resting B cellsand resting B cellsResting memory B cells are thought to be the site Resting memory B cells are thought to be the site of persistence of EBV within the bodyof persistence of EBV within the bodyHLA restricted HLA restricted CTLsCTLs are important in controlling are important in controlling EBVEBV–– many of the many of the CTLsCTLs responses against latent proteins are responses against latent proteins are
targeted to the EBNA3 proteinstargeted to the EBNA3 proteins
Model of EBV infection in humansModel of EBV Model of EBV infectioninfection in in humanshumans
LatentLatent membrane membrane proteinsproteinsLMP 1 and 2LMP 1 and 2
EBV EBV nuclearnuclear antigensantigensEBNA 1,2,3EBNA 1,2,3
EBV EBV encodedencoded RNARNAEBEREBERBARF 0BARF 0
CytokinesCytokinesBHRF, BCRF1, BARF1 BHRF, BCRF1, BARF1
Model of progression for EBV-associated LDModel of Model of progressionprogression forfor EBVEBV--associatedassociated LDLD
From: JC Barrett. Herpes 2000; 7:4.
EBV latency programsEBV EBV latencylatency programsprograms
EBNAs:1, 2, 3a, 3b, 3c, LPEBERS
LMPs: 1, 2a, 2b
EBV-LCL
Prog. EBV proteins Disease
I EBNA 1(+ EBER RNAs)
Burkitt’s lymphoma
II EBNA 1, EBERLMP-1, 2a, 2b
Hodgkin’s diseaseNasopharyngeal ca.
III EBNA 1, 2,3a,3b,3c
LPLMP-1, 2a, 2b
Infectiousmononucleosis
EBV-LPD
Cellular immunotherapy - EBV-related lymphomasCellular immunotherapy - EBV-related lymphomas
Infusion of DLI was proved to be able to induce remission of EBV-related high-grade non-Hodgkin lymphoma (Papadopoulos et al., NEJM 1994)Infusion of EBV-specific CTL produced resolution of EBV-related high-grade non-Hodgkin lymphoma (Rooney et al., Lancet 1995 and Blood 1998)Treatment with EBV-specific CTL induced regression of relapsed EBV positive Hodgkin disease (Roskrow et al., Blood 1998)Infusion of EBV-specific CTL prevented development of EBV-related post-transplant lymphoproliferative d. (Comoli et al., Blood 2002)
EBV DNA concentrations before and after CTL infusionsEBV DNA concentrations before and after CTL infusionsEBV DNA concentrations before and after CTL infusions
CTL infusion
EB
V D
NA
cop
y nu
mbe
r(x1
03)
4
3
2
1
0-12 -10 -8
8
7
6
5
-1 0 1 2 3 4 8 12 16 20 24
Time (weeks) Comoli et al. Blood 2002
Generation of EBVGeneration of EBV--specific CTLsspecific CTLs
1ststep
Immortalized B-cell(EBV-LCL)PBMC
+ EBV antigen-presenting cell:4 - 6 weeks
PBMC+ EBV-LCL
2ndstep
CTL expansion:4 - 6 weeks+ IL-2
EBV-CTLsInfuse QA/QCCryopreserveTest for
specificity, phenotype,sterility, HLA type
Polyclonal EBVPolyclonal EBV--specific CTLs
specific CTLs
EBNA 1
LMP-1, 2a, 2b
EBNA 3
Burkitt’s lymphoma
Hodgkin’s diseaseNasopharyngeal ca.
Post Transplantlymphoproliferative
diseases
X
RINOFARINGERINOFARINGETettoTetto: (base cranica) tonsilla faringea: (base cranica) tonsilla faringeaPareteParete anterioreanteriore: coane nasali: coane nasaliPareteParete posterioreposteriore: piano muscolare: piano muscolareParetiPareti lateralilaterali: tube di Eustachio e : tube di Eustachio e fossette del fossette del RosenmullerRosenmullerConfiniConfini inferioriinferiori: proiezione : proiezione posteriore del velo palatinoposteriore del velo palatino
CLASSIFICAZIONE ISTOPATOLOGICA WHO DEL CA DEL RINOFARINGE
TIPO II: NKCTIPO I: KC80-90%3-11% DIFFERENZIATO
(a cellule transizionali)
INDIFFERENZIATO(linfoepitelioma)
EBV-related nasopharyngeal carcinoma (NPC)EBVEBV--related nasopharyngeal carcinoma (NPC)related nasopharyngeal carcinoma (NPC)
Most important EBV-related neoplasm in health terms– 20-50/100,000 cases/year in Southeast Asia and Northern Africa– 1/100,000 cases / year in Europe
Overall survival 5 year survival rate is around 60%
Radiotherapy + Chemotherapy cure 80-90% of early stage
Outcome of patients with advanced stage disease at diagnosis or relapsing after first line therapy is poor
Once distant metastases have developed, only 15% of patients survive at 1 year
Second line therapies in refractory/relapsing patients have little effect on the natural history of the disease
EBER+: tumor biopsy
Cell therapy approach for EBV-related NPC: RATIONALECell therapy approach for EBVCell therapy approach for EBV--related NPC: related NPC: RATIONALERATIONALE
NPC tumor cells express a restricted number of viral proteins, namely EBNA1, LMP1 and LMP2 - Cohen: N Engl J Med 2000NPC cells show high levels of HLA class I alleles on the cell surface and have normal expression of the MHC-encoded putative peptide transporters TAP-1 and TAP-2, as well as of other components of the class I processing pathway - KhannaKhanna, , CancerCancer Res 1998Res 1998EBV-specific CTLs are present in patients with newly diagnosed NPC, with a specificity for EBV latent protein LMP2 -LeeLee, J , J ImmunolImmunol 20002000
NPC cells are capable of immunological processing and CTL recognition
Cell therapy strategies to control EBV-related NPCCell therapy strategies to control EBVCell therapy strategies to control EBV--related NPCrelated NPC
Problems
Activation of LMP-2 specificCTLs i.e. usingDendritic Cells
HLAHLA--matchedmatcheddonor
LMPLMP--2 best 2 best availableavailable targettarget–– EBNA1 EBNA1 isis notnot immunogenicimmunogenic and and
LMP1 LMP1 isis oftenoften mutatedmutated in TCin TC–– EBNA3 (immunodominant) not EBNA3 (immunodominant) not
present on NPC cells present on NPC cells
EBV specific TEBV specific T--cell immunity cell immunity is reduced in NPC is reduced in NPC patientspatients donor
Patient 1/allo: EBVPatient 1/allo: EBV--specific donor specific donor CTLsCTLs
Phenotype analysis Functional analysis
% positivecells
CD3 96
HLA-DR 98
CD8 70
CD4 26
CD56 4
CD8/CD56 80
B-LCLauto
+a class I Tumor cellspatient
% s
peci
ficly
sis
+a class I
E:T ratio 5:1
60
40
20
Cell therapy strategies to control EBV-related NPCCell therapy strategies to control EBVPatient 2 (M, age 50)
Cell therapy strategies to control EBV--related NPCrelated NPCPatient 2 (M, age 50)Patient 2 (M, age 50)
T3N1M0– CT (CDDP + 5FU) plus RT → CRRelapse (local + LN)– Bleo/MTX/CDDP plus RT → CRRelapse (local + LN)– CDDP + 5FU → SDProgression (intracranial / local)– Taxol → SDProgression (intracranial / local, LN)– CDDP + Doxo → SD– 4 weekly doses of CTLs (4x107 /dose) →→ SDSD
(reduction of the intracranial tumor)
1989
1997
1999
2000
2001
CD8+ lymphs and TCR message on tumor biopsies
Pre-infusion
0
20000
40000
60000
80000
100000
120000
prepost
1 month post-infusion
vb1 vb4 vb15 vb18 vb24
Comoli et al. Ann Oncol 2004
Patient 2: Spectratyping analysis of TILsbefore and after immunotherapy
Vbeta families
CONCLUSIONS – ALLOGENEIC settingCONCLUSIONS CONCLUSIONS –– ALLOGENEIC settingALLOGENEIC setting
CTLs are able to exert specific killing of autologoustumor cells in vitro, and may have antitumor effect in vivo
SpectratypingSpectratyping analysisanalysis suggestsuggestss that the effects of the that the effects of the T cell infusion may be due either to a bystander T cell infusion may be due either to a bystander activation or to a direct effect of the CTL which induce activation or to a direct effect of the CTL which induce a release of tumor antigensa release of tumor antigens
AIMS of the protocol AIMS of the protocol –– AUTOLOGOUS AUTOLOGOUS CTLsCTLs
To generate and expand ex vivo autologous EBV-specific CTLs
To evaluate the safety of EBV-CTL infusions in EBV-positive, poor-prognosis NPC patients
To analyse the immunological and clinical efficacyof EBV-specific CTLs for treatment of EBV-positive NPC
To determine the survival of EBV-specific CTLs in treated NPC patients
PATIENTS – INCLUSION CRITERIAPATIENTS PATIENTS –– INCLUSION CRITERIAINCLUSION CRITERIA
Less than 70 years with istologically-confirmed EBV-related NPC
Disease in progression despite prior therapies and not amenable to complete surgical resection or further systemic or local conventional treatments
Measurable disease
Normal organ function
Informed consent
TREATMENT PLANTREATMENT PLANTREATMENT PLAN
4 escalating doses (EBV CTL/m2) – 20 x 106
– 40 x 106
– 60 x 106
– 80 x 106
DISEASE EVALUATION
Maintenance– 60 x 106 every 2-4 weeks
low-dose recombinant IL-2 in the last 5 patients
bi-weekly intervals
Main characteristics of treated patientsMain characteristics of treated patientsMain characteristics of treated patients
Nr. Age (yr)/ sex
Stage atdiagnosis
Site(s) of tumorinvolvement No of prior therapies * PS
1 40/M II(T2N1M0) Bone, bone marrow, liver > 2 lines of CT, RT 2
2 46/M III(T3N2M0) Lymph nodes RT, surgery
2 lines of CT 0
3 22/M III(T2N2M0)
Lymph nodes, softtissues, bone
> 2 lines of CT, RT, surgery of bonemetastasis 0
4 17/M Unknown Parapharyngeal tissue,lymph nodes > 2 lines of CT, RT surgery 0
5 70/M IV(T4N1M0) Lung, skull base 2 lines of CT, RT, surgery 1
6 63/M Unknown Primary tumor, skull base 2 lines of CT, RT, surgery 2
7 60M II Liver, lymph nodes > 2 lines of CT, RT 2
8 48/M IV(TXN2M1) Primary tumor, liver > 2 lines of CT, RT 0
9 54/M IV(T4N3M0)
Primary tumor, lymphnodes, skull base 2 lines of CT, RT 2
10 50/M III(T3N2M0) Lymph nodes. liver > 2 lines of CT, RT 0
Toxicity and OutcomeToxicity and OutcomeToxicity and OutcomeNr. No of CTL
infusions Toxicity Outcome
1 2 None PD
2 10 Inflammatory reaction in the disease site
PR
3 23 None SD (15 months)
4 14 None SD (4 months)
5 5 None PD
6 2 Side effects due to IL2 therapy PD
7 11 Side effects due to IL2 therapy PR
8 6 None PD
9 12 None SD (4 months)
10 17 Inflammatory reaction in the disease site
SD ( 8+ months)
ClinicalClinical efficacyefficacy of of EBVEBV--targetedtargeted CTLsCTLsCTCT--PET, PET, patientpatient # 7# 7
A B
C D
Comoli et al. 2005; submitted.
Immunological effects of autologous EBV-targetedCTL therapy on patient EBV specific IFNγ production
Spot
s/10
5PB
MC A
Before CTL
After 4 weeks
After 8 weeks
0
50
100
150
P1 P2 P3 P4 P7P5 P6 P8 P9 P10 0
25
50
75
P1 P2 P3 P4 P7P5 P6 P8 P9 P10
B
Panel A: response to EBV LCL
Panel B: response to EBV LMP-2 protein peptide mix
IFNγ-secreting cells are represented as number of spots/105 PBMC (mean spots of triplicate experiments).
Comoli et al. 2005; submitted.
Therapy with EBV-specific CTLs in NPC: MI-PV EXPERIENCE
10 patients with refractory NPC treated with poly-specific CTLs
Clinical results: – feasible and safe– 2 documented PR, 4 SD
Immunological results:– increases frequency of EBV specific immunity– appearance of LMP2-specific response
Comoli et al. 2005; submitted
Therapy with EBV-specific CTLs in NPC HUSTON EXPERIENCE
10 patients treated with poly-specific CTLs (4 in remission)
Clinical results: – decrease of viral load– 2 documented CR, 1 PR, 1 SD
Immunological results:– anti-LMP2 activity present in the infused CTLs– no evidence of persistence of LMP2-specific T cells in the
peripheral bloodStraathof et al. Blood 2005; 105:1898.
FUTURE DIRECTIONSFUTURE DIRECTIONS
Cell therapy with EBV-targeted CTLsearlier in the course of NPC disease
Administration of higher CTL dosesfollowing lymphoablative chemotherapy
Increasing the number of LMP2 and/or LMP1-specific T cells in the infusion product
Ospedale Niguarda Ca’ Ospedale Niguarda Ca’ GrandaGranda -- MILANOMILANO
S. C. Divisione di Oncologia Medica S. C. Divisione di Oncologia Medica FalckFalck
P. Pedrazzoli, R. Schiavo, M. P. Pedrazzoli, R. Schiavo, M. MoroniMoroni, , O. O. CarminatiCarminati, S. Secondino, S. Siena, S. Secondino, S. Siena
IRCCS Policlinico S. Matteo - PAVIA
Laboratori Sperimentali Area Trapianti e Unità di Oncoematologia Pediatrica
P. Comoli, S. Basso, M. Labirio, C. Frasson,F. Locatelli, R. Maccario
Main clinical characteristics and clinical outcome of patients with NPC treated with EBV-targeted CTL therapy
Main clinical characteristics and clinical outcome of patients wMain clinical characteristics and clinical outcome of patients with ith NPC treated with EBVNPC treated with EBV--targeted CTL therapytargeted CTL therapy
SD ( 8+ months)
Single episode of inflammatory
reaction in the disease site
170RT, > 2 lines of CTLymph nodes. liverIII (T3N2M0)50/M10
SD(4 months)None122RT, 2 lines of CT Nasopharinx, lymph
nodes, skull baseIVB (T4N3M0) 54/M9
PDNone60RT, > 2 lines of CTPrimary tumor, liverIVC (T2N2M1)48/M8
PR(5 months)
Side effects due to IL2 therapy112RT, > 2 lines of CTLiver, lymph nodesIIB (T1N1M0)60M7
PDSide effects due to IL2 therapy22RT, 2 lines of CT,
surgeryNasopharinx, skull
baseIII (T1N2M0)63/M6
PDNone51RT, 2 lines of CT, surgeryLung, skull baseIVA (T4N1M0)70/M5
SD (4 months) None140RT, > 2 lines of CT
surgeryParapharyngeal
tissue, lymph nodesUnknown17/M4
SD(15 months) None23 0
RT, > 2 lines of CT, surgery of bone
metastasis
Lymph nodes, soft tissues, boneIII (T2N2M0)22/M3
PR(3 months)
Inflammatory reaction in the
disease site 10 0RT, 2 lines of CT,
surgeryLymph nodesIII (T3N2M0)46/M2
PDNone22RT, > 2 lines of CTBone, bone marrow, liverII (T2N1M0)40/M1
Response to therapy
(duration)Side effectsCTL
infusionsPS
(ECOG)Prior therapies Site(s) of tumor
involmenet at time of cell therapy
Stage at diagnosis
Age (yr) / sex
Patient N.
IMMUNOLOGICAL EFFECTS OF POLYCLONAL EBV-SPECIFIC CTLS
960960950950EBV LCLEBV LCL
252500LMP2LMP2
6633LMP2LMP2
IFNIFNγγ--producingproducing cellscells
111111100100EBV LCLEBV LCL
CTLpCTLp
PostPost--IV IV infusioninfusion
PrePre--infusioninfusion
0
20
40
60
Pre-infusion
+1 weekpost-
I infusion
% s
peci
ficly
sis
+2 monthspost-
IV infusion
+1 weekpost-
IV infusion
Comoli et al. Ann Oncol 2004