CROAVIANO

Sviluppo di vaccini Sviluppo di vaccini idiotipiciidiotipici per per studi di fase I/II di studi di fase I/II di

immunoterapia "subsetimmunoterapia "subset--specifica" specifica" per pazienti con disordini per pazienti con disordini

linfoproliferativilinfoproliferativi a cellule B"a cellule B"

RiccardoRiccardo DolcettiDolcetti

Cancer BioCancer Bio--Immunotherapy UnitImmunotherapy UnitDept. of Medical OncologyDept. of Medical Oncology

C.R.O.C.R.O.–– IRCCS, National Cancer Institute,IRCCS, National Cancer Institute,Aviano (PN)Aviano (PN)

CROAVIANO

PROGRAMMA 3PROGRAMMA 3Trasferimento delle conoscenze allo sviluppo diTrasferimento delle conoscenze allo sviluppo di

interventi volti a prevenire, diagnosticare einterventi volti a prevenire, diagnosticare etrattare il cancrotrattare il cancro

Convenzione ACCConvenzione ACC--44

U.O.1U.O.1: Centro di Riferimento Oncologico : Centro di Riferimento Oncologico –– IRCCS, AvianoIRCCS, AvianoR. Dolcetti (Coordinamento)R. Dolcetti (Coordinamento)

U.O.2U.O.2: Istituto Oncologico Veneto, Padova: Istituto Oncologico Veneto, PadovaA. RosatoA. Rosato

U.O.3U.O.3: Istituto Nazionale Tumori : Istituto Nazionale Tumori ““Fondazione Fondazione PascalePascale””, Napoli, NapoliF.M. F.M. BuonaguroBuonaguro

Sviluppo di vaccini Sviluppo di vaccini idiotipiciidiotipici per studi di fase I/per studi di fase I/IIII di di immunoterapia "subsetimmunoterapia "subset--specifica" per pazienti con specifica" per pazienti con

disordini disordini linfoproliferativilinfoproliferativi a cellule B"a cellule B"

1.1. Attivazione di uno studio di fase I/II basato sullAttivazione di uno studio di fase I/II basato sull’’utilizzo utilizzo della proteina della proteina idiotipicaidiotipica VK3VK3--20 quale vaccino per il20 quale vaccino per iltrattamento di linfomi a basso grado di malignittrattamento di linfomi a basso grado di malignitàà esprimentiesprimenticatene leggere catene leggere molecolarmentemolecolarmente correlate.correlate.

2.2. Sviluppo di nuovi vaccini Sviluppo di nuovi vaccini idiotipiciidiotipici ricombinanti da applicarsiricombinanti da applicarsiin schemi di immunoterapia in schemi di immunoterapia ““subsetsubset--specificaspecifica”” per disordini per disordini linfoproliferativilinfoproliferativi a cellule B.a cellule B.

Obiettivi primariObiettivi primari

Gruppi di ricerca afferenti al DI:Gruppi di ricerca afferenti al DI:

-- U.O. U.O. BioBio--ImmunoterapiaImmunoterapia dei Tumori Umani, dei Tumori Umani, R. DolcettiR. Dolcetti-- U.O. Farmacologia Sperimentale e Clinica, U.O. Farmacologia Sperimentale e Clinica, V. De ReV. De Re-- U.O. Ematologia Sperimentale e Clinica, U.O. Ematologia Sperimentale e Clinica, V. V. GatteiGattei-- U.O. Oncologia Sperimentale 1, U.O. Oncologia Sperimentale 1, R. MaestroR. Maestro-- U.O. Oncologia Medica A, U.O. Oncologia Medica A, U. U. TirelliTirelli-- U.O. Anatomia Patologica, U.O. Anatomia Patologica, V. CanzonieriV. Canzonieri-- U.O. Epidemiologia, U.O. Epidemiologia, D. D. SerrainoSerraino

Gruppi di ricerca afferenti ad altre Istituzioni:Gruppi di ricerca afferenti ad altre Istituzioni:

-- U.O. U.O. ImmunobiologiaImmunobiologia dei Tumori Umani, Istituto Nazionale dei Tumori Umani, Istituto Nazionale Tumori, Milano. Tumori, Milano. A. A. AnichiniAnichini-- U.O. Immunoterapia Sperimentale, Istituto Superiore di U.O. Immunoterapia Sperimentale, Istituto Superiore di SanitSanitàà, Roma. , Roma. M. M. FerrantiniFerrantini

U.OU.O.1: C.R.1: C.R.O. .O. -- IRCCS AvianoIRCCS Aviano

Contributo dellContributo dell’’U.O.1: C.R.O.U.O.1: C.R.O.-- Aviano Aviano

Allestimento di un database dedicato comprendente le sequenzeAllestimento di un database dedicato comprendente le sequenzenucleotidiche e nucleotidiche e aminoacidicheaminoacidiche predette dei geni predette dei geni IgVHIgVH e e IgVLIgVL deideipazienti affetti da neoplasie linfoidi di interesse, inclusa unpazienti affetti da neoplasie linfoidi di interesse, inclusa un’’ampiaampiacasistica di CLL; casistica di CLL;

Identificazione di Ig Identificazione di Ig clonotipicheclonotipiche "subset"subset--specifiche"; specifiche";

CH2

CL1

CH2

CH3 CH3

CH1CH1 CL1

Contributo dellContributo dell’’U.O.1: C.R.O.U.O.1: C.R.O.-- Aviano Aviano

Allestimento di vettori di espressione adeguati alla purificazioAllestimento di vettori di espressione adeguati alla purificazione ene eproduzione (GMP) di Ig produzione (GMP) di Ig clonotipicheclonotipiche ricombinanti;ricombinanti;

Caratterizzazione dellCaratterizzazione dell’’immunogenicitimmunogenicitàà di Ig di Ig clonotipicheclonotipiche "subset"subset--specifiche" sia in termini di risposte umorali che specifiche" sia in termini di risposte umorali che cellulocellulo--mediatemediate;;

Identificazione e validazione di Identificazione e validazione di epitopiepitopi CTL e definizione dellCTL e definizione dell’’entitentitààdi risposte T memoria di risposte T memoria IdId ed ed epitopoepitopo--specifichespecifiche specifiche in donatorispecifiche in donatorisani e in pazienti con linfoma/leucemia;sani e in pazienti con linfoma/leucemia;

Definizione della frequenza, stadio Definizione della frequenza, stadio differenziativodifferenziativo e delle funzionie delle funzionieffettrici di linfociti T antigeneeffettrici di linfociti T antigene--specifici;specifici;

Definizione della capacitDefinizione della capacitàà di CTL di CTL IdId-- o o epitopoepitopo--specificispecifici didiriconoscere ed uccidere in modo specifico linee di linfoma/leucericonoscere ed uccidere in modo specifico linee di linfoma/leucemia mia esprimenti esprimenti IdId molecolarmentemolecolarmente correlati (razionale per approcci di correlati (razionale per approcci di immunoterapia immunoterapia ““crosscross--reattivareattiva””).).

Personalized Personalized IdiotypicIdiotypic Vaccines: Vaccines: LimitationsLimitations

-- Complex and timeComplex and time--consuming approach;consuming approach;

-- Feasible only in a limited number of highly specialized Feasible only in a limited number of highly specialized Centers;Centers;

-- Drug Industry:Drug Industry:•• Mass produced products for mass marketsMass produced products for mass markets•• High margins between cost of goods and sales priceHigh margins between cost of goods and sales price

-- Regulatory Bodies usually deals with manufacturing Regulatory Bodies usually deals with manufacturing issues and large scale trials;issues and large scale trials;

-- Difficult comparison of the responses induced by Difficult comparison of the responses induced by different Id vaccines in clinical trials.different Id vaccines in clinical trials.

Can we overcome these limitations?Can we overcome these limitations?

TT

TTGerminal centerGerminal center

TT--cellcellhelphelp

TT--cell helpcell help

Viral (HCV)Viral (HCV)or bor bacterialacterialantigen(santigen(s))

ClonalClonalB lymphocytesB lymphocytes

AntigenAntigenpersistencepersistence

DCDC

From antigenFrom antigen--driven Bdriven B--cell cell lymphoproliferationslymphoproliferationsto to ““subsetsubset--specificspecific”” immunotherapyimmunotherapy

SharedSharedAntigenAntigen

SharedSharedIdId

Limited setLimited set ofofrecombinantrecombinantId vaccinesId vaccines

Prevention and treatment of: Prevention and treatment of: -- AntigenAntigen--driven lymphomasdriven lymphomas-- other lymphomas/other lymphomas/leukemiasleukemias

expressing molecularly expressing molecularly correlated Idcorrelated Id

Malignant lymphomaMalignant lymphoma

Ig Ig genesgenes usedused byby B B cellcell NHL in NHL in HCV+HCV+ patientspatients (De Re, (De Re, BloodBlood 2000)2000)

De Re De Re etet al, al, BloodBlood, 2000, 2000

SalivarySalivary glandgland B B cellcell lymphoproliferativelymphoproliferative disordersdisorders in in SjSjöögrengren’’s s syndromesyndrome show a show a restrictedrestricted useuse ofof

Ig gene Ig gene segmentssegments similarsimilar toto thosethose ofof HCVHCV--relatedrelated NHLsNHLs

De Re De Re etet al, EJI, 2002al, EJI, 2002

IdiotypicIdiotypic IgIg of HCVof HCV--related NHL mayrelated NHL mayconstitute an useful target for immunotherapyconstitute an useful target for immunotherapy

•• The highly restricted usage of defined VH and VL segments The highly restricted usage of defined VH and VL segments may allow the use of amay allow the use of a limited numberlimited number of of idiotypicidiotypic IgIg to to prepare vaccines potentially effective in most HCVprepare vaccines potentially effective in most HCV--related related lymphoproliferationslymphoproliferations..

•• Such an approach is largelySuch an approach is largely less complex and timeless complex and time--consumingconsuming than the purification of than the purification of idiotypicidiotypic IgIg from from individual NHL patients previously adopted for HCVindividual NHL patients previously adopted for HCV--unrelated lowunrelated low--grade grade NHLsNHLs..

•• Immunotherapy approaches based on the use of Immunotherapy approaches based on the use of idiotypicidiotypicIgIg are expected to induce onlyare expected to induce only mild side effectsmild side effects since onlysince onlya limited fraction (<5%) of normal B cells use similar gene a limited fraction (<5%) of normal B cells use similar gene segments in assembling the segments in assembling the IgRIgR..

Can Can wewe useuse ““prototypicprototypic”” proteinprotein(s) (s) toto efficientlyefficientlyvaccinate vaccinate mostmost patientspatients withwith HCVHCV--NHL?NHL?

>20 >20 donorsdonors withwith differentdifferentHLA HLA ClassClass I background I background havehave beenbeen testedtested so far.so far.

RecombinantRecombinantVK3VK3--2020

0

20

40

60

80

100

Don 4

134

VK3-

15

Don 4

178

VK3-

15

Don 4

182

VK3-

15

Don 4

193

VK3-

15

Don 7

88 V

K3-2

0

Don 4

134

VK3-

20

Don 4

178

VK3-

20

Don 4

182

VK3-

20

Don 4

193

VK3-

20Don

46

VK3-

20Don

48

VK3-

20

Don 4

810

VK3-

20

Don 4

816

VK3-

20

Sp

eci

fic

kil

lin

g (

%)

VK LCLVK LCL+W6/32CTRL

InducesInduces specificspecific CTL CTL responsesresponses ex vivoex vivo

0

20

40

60

80

100

2 3 5 20 25 26 28 29 32 33 40 42 46 47 48 72 78 86 90 91 95 97

A*0101

A*0201

A*0301

A*1101

A*2402

Peptide Number

% R

elat

ive

Bind

ing

CarryCarry severalseveral HLA HLA ClassClass II--restrictedrestricted CTL CTL epitopesepitopes

In collaboration with:- A. Anichini, INT, Milan,- Areta International, Gerenzano

In In collaborationcollaboration withwith::-- A. A. AnichiniAnichini, INT, Milan,, INT, Milan,-- AretaAreta International, International, GerenzanoGerenzano

RecombinantRecombinantVK3VK3--2020

InducesInduces CTL CTL responsesresponsesagainstagainst differentdifferent VK3VK3--2020

MemoryMemory CD8+ T CD8+ T cellcellresponsesresponses are are detectabledetectable in in

NHL NHL patientspatients

In In collaborationcollaboration withwith::-- A. A. AnichiniAnichini, INT, Milan,, INT, Milan,-- AretaAreta International, International, GerenzanoGerenzano

0

10

20

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50

60

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80

90

100

110

D1380 D1835 D46 D48

% Specific Killing

NHLNHL--22 VK3VK3--20 LCL20 LCLControlControl

NHLNHL--11 VK3VK3--2020--specific specific CTLsCTLs

CTLCTLVK3VK3--2020

VK3-20

Matched target

VK3-15

Mismatched target

CTLCTLVK3VK3--2020

VK3-20

Matched target

VK3-15

Mismatched target

RationaleRationale forfor a a ““crosscross--reactivereactive”” immunotherapyimmunotherapy??

Don 05003656E:T = 2.5:1

0102030405060708090

1

% S

peci

fic L

ysis

Auto LCL vk3-20+

Auto LCL vk3-20+ anti-A2Auto LCL vk3-20-

DG75

DG75 + anti-A2

VK3VK3--20 20 specificspecific CTLsCTLs killskills DG75 DG75 lymphomalymphoma cellscells(HLA(HLA--A2, A2, VK3VK3--20+20+) in HLA) in HLA--A2A2--restrictedrestricted

fashionfashion

A MHC/Peptide A MHC/Peptide BindingBinding AssayAssay thatthat allowsallowsthe the identificationidentification ofof epitopesepitopes::

Peptide Peptide SelectionSelection and and SynthesisSynthesisInitialInitial MHC/Peptide MHC/Peptide BindingBinding StudiesStudiesAffinityAffinity AnalysisAnalysis ofof BindingBinding PeptidesPeptidesOffOff--raterate AnalysisAnalysis ofof BindingBinding PeptidesPeptidesMultiparametricMultiparametric Data Data AnalysisAnalysis

TheThe iTopiaiTopia™™ EpitopeEpitope DiscoveryDiscovery SystemSystem

TheThe iTopiaiTopia™™ EpitopeEpitope DiscoveryDiscovery SystemSystem

T2 T2 stabilizationstabilization assayassayofof VK3VK3--2020--derived derived HLA A*02 HLA A*02 peptidespeptides

IsotypicIsotypiccontrolcontrol

VK3VK3--2020peptidepeptide

ControlControl FluFlupeptidepeptide

PeptidePeptide aaaa sequencesequence

P2P2 IVLIVL…………

P3P3 VLTVLT…………

P20 *P20 * TLSTLS…………

P26P26 SQISQI…………

P33 *P33 * YLAYLA…………

P46 *P46 * RLLRLL…………

P47 *P47 * LLILLI…………

P48 *P48 * LIYLIY…………

P72P72 FTLFTL…………

P78 *P78 * RLERLE…………

P97P97 RTFRTF…………

* HLA* HLA--A02 A02 stabilizingstabilizing peptidespeptides

Preliminary results1. VK3-20-specific cellular responses in HCV+ patients

• Elispot IFNγ

1. Healthy controls matched for age and socio‐economic status

a.CN 24/07;b.CN 31/07;c.CN 07/08.

Soggetto FedII-01/08 FedII-01/08 FedII-02/08 FedII-03/08FedII-04/08FedII-05/08FedII-06/08Cot-01/08 CN 24/7 CN 31/7 CN 7/8

PatolHCV+NH+ Crio

HCV+NH+Crio

HCV+NH+Crio

HCV+NH+Crio HCV+ NH HCV+ NH

HCV+ Crio HBV Sani Sani Sani

IFNγ Chemio CHOP CHOPciclofosfamide

Peptide ug/mlVK3-20 31.25 4 26 32 79 23 22

15.62 170 204 37 29 30 50 72 45 32 11 26HCV core Mix 11 21 28 32 80 79 73HCV NS3/4Mix 1 4 36 41 30 61 22Inluenza Mix 20 52 51 400 350 520No stimoloNO 3 5 60 12 30 26 47 51 10 10 23RPMI RPMI 2 0 0 3 4

Folds Vk3-20/ no stim 56.67 40.80 0.62 2.42 1.00 1.92 1.53 0.88 3.20 1.10 1.13

Induzione di IFNgamma 24/06/2008

0

50

100

150

200

1000

.0050

0.00

250.0

012

5.00

62.50

31.25

15.62

no st

imolo

RPMIns

3co

re

IFN

ug/

ml

UO-3 Napoli

2. Monocyte activation induced by recombinant Vk3-20 • Facs analysis of co-stimulatory molecule expression

Control

1.5ug/ml VK3‐20

5ug/ml VK3‐20

15ug/ml VK3‐20

4ug/ml LPS

Healthy individual16’168

13’2970.82x

17’4631.08x

16’5521.02x

19’9391.23x

890

8991.01x

1’0851,22x

9861.10x

1’2651.42x

413

5921.43x

7561.83x

8752,11x

1’4803.58x

1’874

1’1430.61x

1’8280.97x

1’7710.94x

1’7590.94x

HLA‐DR CD83 CD80 CD86

Preliminary results UO-3 Napoli

Control

1.5ug/ml VK3‐20

5ug/ml VK3‐20

15ug/ml VK3‐20

4ug/ml LPS

HCV+cronic active 17’643

27’0371.53x

32’4781.84x

33’6111.91x

1’209

1’8131.49x

1’3641,12x

1’8491.52x

1’6231.34x

322

1’2363.83x

4201.30x

1’0743,33x

1’4834.61x

2’519

2’9321.16x

2’7641.09x

3’0131.19x

4’0511.61x

HLA‐DR CD83 CD80 CD86

26’6731.51x

Preliminary results2. Monocyte activation induced by recombinant Vk3-20

• Facs analysis of co-stimulatory molecule expression

UO-3 Napoli

3. Cytokine activation profiling• Facs analysis of cytokine production

HCVHCV--negativenegativecontrol control

HCV+ HCV+ persistentpersistent

HCV+ active inf.

IL- 2

IL- 4

IL- 6

IL-10

TNFα

IFNγ

No Peptide 1.5ug 5ug 15 ug 4.5ug LPS

Preliminary resultsUO-3 Napoli

VK3VK3--2020

IL-10 FOLD

1.5ug

/ml

5ug/

ml

15ug

/ml

LPS 4

ug/m

l0

25

50

75

100

TNF ALPHA FOLD

1.5ug

/ml

5ug/

ml

15ug

/ml

LPS 4

ug/m

l

0

250

500

750

1000

IFN GAMMA FOLD

1.5ug

/ml

5ug/

ml

15ug

/ml

LPS 4

ug/m

l

0

100

200

300

400

500Sero -Sero + ROSero + IA

FoldsIncrease

VK3‐20 VK3‐20 VK3‐20

IL-2 FOLD

1.5ug

/ml

5ug/

ml

15ug

/ml

LPS 4

ug/m

l

0

1

2

IL-4 FOLD

1.5ug

/ml

5ug/

ml

15ug

/ml

LPS 4

ug/m

l0

100

200

300

400

IL-6 FOLD

1.5ug

/ml

5ug/

ml

15ug

/ml

LPS 4

ug/m

l

0

100

200

300

400

500Sero -Sero + ROSero + IA

FoldsIncrease

VK3‐20 VK3‐20VK3‐20

Fold increase of Th1/Th2 cytokines over the control

3. Cytokine activation profiling• Facs analysis of cytokine production

Preliminary resultsUO-3 Napoli

4. Immunogenomic profilingGene expression profiling - microarray

Preliminary resultsUO-3 Napoli

The RNAs of DCs from HCV-infected individuals pulsed withVK3-20 have been collected and cryopreserved for

immunogenomic profiling

The The RNAsRNAs of of DCsDCs fromfrom HCVHCV--infectedinfected individualsindividuals pulsedpulsed withwithVK3VK3--20 20 havehave beenbeen collectedcollected and and cryopreservedcryopreserved forfor

immunogenomicimmunogenomic profilingprofiling

Parallel experience: Genomic

transcriptional profiling of

Dendritic Cells induced by

HIV-VLPA

ParallelParallel experienceexperience: : GenomicGenomic

transcriptionaltranscriptional profilingprofiling of of

DendriticDendritic CellsCells inducedinduced byby

HIVHIV--VLPVLPAA

Differentially expressed324 genes (p<0.005)

DifferentiallyDifferentially expressedexpressed324 genes 324 genes (p<0.005) (p<0.005)

Interim Interim suggestionssuggestionsVK3-20-specific T cell responses are detectable in patients withHCV-related lymphoma.

VK3-20 promotes monocyte maturation in HCV+ patients at lowerdoses when compared to HCV- patients;

VK3-20 induces a mainly Th2-skewed cytokine pattern with a modest Th1 response in patients with chronic active HCV infection.

• HCV infection probably does not hamper the boosting of VK3-20-specific T cell responses for preventive (cryoglobulinemia) and therapeutic (overt NHL) purposes;

• Need of a strong adjuvant to enhance Th1 responsesin NHL patients.

•• HCV HCV infectioninfection probablyprobably doesdoes notnot hamperhamper the the boostingboosting of of VK3VK3--2020--specific T specific T cellcell responsesresponses forfor preventive preventive ((cryoglobulinemiacryoglobulinemia) and ) and therapeutictherapeutic ((overtovert NHL) NHL) purposespurposes;;

•• NeedNeed of a strong of a strong adjuvantadjuvant toto enhanceenhance Th1 Th1 responsesresponsesin NHL in NHL patientspatients..

UO-3 Napoli

ActivationActivation of a of a PhasePhase I/II trial I/II trial basedbased on the VK3on the VK3--20 20 vaccination of vaccination of patientspatients withwith lowlow--gradegrade BB--NHL NHL expressingexpressing molecularlymolecularly relatedrelated IdId proteinsproteins..

Programma 3ACC-4

Programma 3Programma 3ACCACC--44

•• VectorsVectors suitablesuitable forfor GMP production of VK3GMP production of VK3--20 20 havehave beenbeenproducedproduced;;

•• GMP GMP protocolsprotocols forfor VK3VK3--20 production and 20 production and purificationpurification havehavebeenbeen set up;set up;

•• EndotoxinEndotoxin--freefree aliquotsaliquots of VK30 and of VK30 and KLHKLH--conjugatedconjugated VK3VK3--2020 havehave beenbeen distributeddistributed toto the the UnitsUnits involvedinvolved in in in vivoin vivostudiesstudies..

Programma 3ACC-4

Programma 3Programma 3ACCACC--44

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1.2

1:50 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400

diluition

Abs

orba

nce

m ouse 1

m ouse 2

m ouse 3

m ouse 4

m ouse 5

m ouse 6

day 0

VK3-20-specific humoral

responses in immunized mice

VK3VK3--2020--specific specific humoralhumoral

responsesresponses in in immunizedimmunized micemice

# 1 # 2 # 3 # 4 # 5 # 6 CTRLMW (kDa)

64

50

22

10

4-6

VK3-20 protein; 1 μg/lane

DGDG--75 cells down75 cells down--regulate HLA class I regulate HLA class I and II and II in vivoin vivo

10 0 10 1 10 2 10 3 0

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n. e

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s

Fluorescence

CtrlCtrlDGDG--7575

CD19CD19 HLAHLA--II HLAHLA--IIII

100% 100% 100%

10 0 10 1 10 2 10 3 0

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40

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100

10 0 10 1 10 2 10 3 0

20

40

60

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100

10 0 10 1 10 2 10 3 0

20

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1% neg100%

in vitroin vitroin vitro

ex vivoex vivoex vivo

10 0 10 1 10 2 10 3 0

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Fluorescence

CD19 HLA-I HLA-II

99% 61% 39%

HLAHLA--A2A2--transfected DGtransfected DG--75 cells retain 75 cells retain HLA class I (and II) expression HLA class I (and II) expression in vivoin vivo

CtrlCtrlDGDG--75 75 ex vivoex vivo

DGDG--75 75 in vivoin vivo

dayday 00 dayday 77 dayday 2121 dayday 2828

The HHD mouseThe HHD mouse

HH--2 class I2 class I--negative, HLAnegative, HLA--A2.1A2.1--transgenictransgenic

Generation of CD8Generation of CD8++ T T lymphocytes restricted for lymphocytes restricted for human MHC class I A2.1human MHC class I A2.1

mouse origin

human origin

Induction of HLA-A2.1-restricted VK3-20-specific CTLs

Induction of Induction of HLAHLA--A2.1A2.1--restricted restricted VK3VK3--2020--specific specific CTLsCTLs

Adoptive cell therapy Id protein injection

HHD mouseHHD mouse

IdId--specific CTLspecific CTL

SCID mouse SCID mouse grafted with grafted with DGDG--75 cells75 cells

Tumor reduction?Tumor reduction?

CTL from donors/patientsCTL from donors/patients

Generation of VK3Generation of VK3--2020--specific specific CTLsCTLs

5151Cr Cr cytotoxicitycytotoxicity testtest

MzMz + Vk3+ Vk3--2020

T T cellcellVk3Vk3--2020--loadedloaded

splenocytesplenocyte

DGDG--7575

γ-irradiatedDG-75

T T cellcell

CytotoxicCytotoxic activity of VK3activity of VK3--2020--specific specific CTLsCTLs obtained from obtained from

immunized HHD miceimmunized HHD mice

Rapporto E:T

25,0 12,0 6,0 3,0 1,5 0,8

% li

si

0

10

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30

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70

DG-75 LCL

Rapporto E:T

25,0 12,0 6,0 3,0 1,5 0,8

% li

si

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30

40

50

60

70

DG-75 LCL

Utilizzo del modello Utilizzo del modello ““huhu--PBLPBL--SCIDSCID”” per valutare e caratterizzare la risposta immune per valutare e caratterizzare la risposta immune umorale e cellulare umana indotta umorale e cellulare umana indotta in vivoin vivo dalldall’’immunizzazione delle xenoimmunizzazione delle xeno--chimere con chimere con IFNIFN--DC DC autologheautologhe pulsate con VK3pulsate con VK3--20 o VK320 o VK3--2020--KLH.KLH.

Hu-PBL

Hu-Monocytes

IFN-DCs

ReconstitutionReconstitution and and immunizationimmunization ofof

huhu--PBLPBL--SCIDSCID micemice

SCID-mouse

VK3VK3--20 20

RecallRecallimmunizationsimmunizations

DayDay 7 7 SacrificeSacrificeand and analysisanalysis ofofhumanhuman immune immune

responseresponse

Day 14 DayDay 2121

ElispotElispotAssayAssay

UO Reparto Immunoterapia Sperimentale, UO Reparto Immunoterapia Sperimentale, DipDip. Biologia Cellulare e Neuroscienze, ISS. Biologia Cellulare e Neuroscienze, ISS(Caterina (Caterina LapentaLapenta, Stefano M. Santini, Simona Donati, Massimo Spada, Maria , Stefano M. Santini, Simona Donati, Massimo Spada, Maria FerrantiniFerrantini))

Generation of VK3Generation of VK3--2020--specific specific humanhuman CD8+ T CD8+ T cellscells in in huhu--PBLPBL--SCIDSCID micemiceimmunizedimmunized withwith VK3VK3--20 or VK320 or VK3--2020--KLHKLH--pulsed DC. pulsed DC.

DC

DC + VK3-20

Stimulators

ELISPOT ELISPOT analysisanalysis ofof antianti--VK3VK3--20 20 CD8+ T CD8+ T cellcell responsesresponses

0

20

40

60

80

IFN-DC IFN-DC+

VK3-20

IFN-DC+

VK3-20-KLH

NoneIFNγ

Spot

form

ing

CD

8+ c

ells

/5x1

04

vaccination

Experimental protocol

Human cells recovered fromperitoneal lavage of three hu-PBL-SCID mice from eachgroup were pooled. The assaywas performed usingautologous VK3-20-pulsed or unpulsed DC as stimulators.

Bars represent the n. of CD8+ T cells secreting IFN-γ in response to VK3-20-pulsed IFN-DC. The CD8+ T cellsrecovered from hu-PBL-SCIDmice immunized with VK3-20-or VK3-20-KLH-pulsed IFN-DC were compared to the basallevel of CD8+ T cells found in non-immunized hu-PBL-SCIDmice or in mice injected withunpulsed autologous DC.

The presence and amount of anti-VK3-20 antibodies and cytokines in peritoneallavages and serum of immunized mice will be also evaluated (CRO-Aviano).

RecombinantRecombinantVK3VK3--2020

RATIONALE FOR “SUBSET-SPECIFIC”IMMUNOTHERAPY

RATIONALE FOR RATIONALE FOR ““SUBSETSUBSET--SPECIFICSPECIFIC””IMMUNOTHERAPYIMMUNOTHERAPY

ImmunologicImmunologiccharacterizationcharacterization

• HCV-related B-NHL;• Autoimmunity-associated B-NHL;• Other VK3-20+ B-NHL.

Distinct sets of stereotyped Igs have been identified in various B cell malignancies, suggesting that a much higher frequency of idiotype sharing exists among

patients than appreciated previously:

ADDITIONAL “SUBSET-SPECIFIC” Id VACCINES MAY BE DEVELOPED

Distinct sets of stereotyped Distinct sets of stereotyped IgsIgs have been identified have been identified in various B cell malignancies, suggesting that a much in various B cell malignancies, suggesting that a much higher frequency of higher frequency of idiotypeidiotype sharing exists among sharing exists among

patients than appreciated previously:patients than appreciated previously:

ADDITIONAL ADDITIONAL ““SUBSETSUBSET--SPECIFICSPECIFIC”” IdId VACCINES VACCINES MAY BE DEVELOPED MAY BE DEVELOPED

OngoingOngoing studiesstudies relative relative totothe the mainmain objectivesobjectives of the project of the project

AssessmentAssessment of the of the efficacyefficacy of VK3of VK3--2020--based based vaccinationvaccination in in animalanimalmodelsmodels..

DefinitionDefinition of the best of the best adjuvantadjuvant formulationformulation, , vaccinationvaccination schedulescheduleand and immunomonitoringimmunomonitoring assaysassays →→ ActivationActivation of the of the PhasePhase I/II trialI/II trial..

AssessmentAssessment of the of the fractionfraction of of patientspatients withwith B B cellcelllymphoproliferationslymphoproliferations whowho maymay benefit benefit fromfrom VK3VK3--2020--based based vaccinationvaccination..

CompletionCompletion of the of the characterizationcharacterization of the of the immunogenicimmunogenic propertiespropertiesofof VH1VH1--69 69 asas anan additionaladditional candidate candidate forfor ““subsetsubset--specificspecific””vaccinationvaccination..

IdentificationIdentification of of furtherfurther IdId proteinsproteins suitablesuitable forfor ““subsetsubset--specificspecific”” immunotherapyimmunotherapy of of BB--cellcell malignanciesmalignancies..