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