Criteri di valutazione della risposta: monitoraggio immunologico … · Da un modello Cell-Oriented...

Attualit e Prospettive in Immuno-Oncologia

Roma, ISS, 4 Novembre 2016

Criteri di valutazione della risposta: monitoraggio

immunologico e nuovi marcatori di efficacia

PAOLA NISTICO M.D.

Laboratorio di ImmunologiaIstituto Nazionale Tumori Regina Elena

Corso ECMAttualit e prospettive in

Immuno-oncologia

4 Novembre 2016

Istituto Superiore di

Sanit

Da un modello Cell-Oriented ad una visione Tumor-Oriented della trasformazione neoplastica

Risposta immunitaria e microbiota

Risposta immunitaria al tumore e microambiente tumorale

Extracellular matrix (ECM)

ECM

ECM

Modified by Adams et al., Nature Rev 2015 and Lee and Mazmanian, Science 2010

4 Novembre 2016


Sanit

Chen and Mellman, Immunity 2013

La generazione dellimmunit anti-tumorale un pro cesso ciclico

The Cancer-Immunity Cycle 4 Novembre 2016


Sanit

Cancer-Immunity Cycle:

Fattori Stimolatori e Inibitori

Chen and Mellman, Immunity 2013

4 Novembre 2016


Sanit

Mahoney et al., Nature Rev, 2015

Lattivazione delle cellule T un processo che coi nvolge segnali attivatori ed inibitori

4 Novembre 2016


Sanit

CTLA-4 e PD-1 operano a differenti stadi nella risp osta immunitaria anti-tumorale

Luke, Oncotarget 2015

Linfonodo Periferia/TumoreCellule T Nave

Cellule TumoraliCellule T inattivate

Cellule del sistema immunitario(macrofagi, cellule dendritiche)

CTLA-4

PD-L1

PD-L2

PD-L1

PD-L1

PD1

PD1

PD1

Cellule T inattivate

4 Novembre 2016


Sanit

I meccanismi inibitori di CTLA -4 e PD-1 sono distinti

Pardoll DM Nat Rev. Imm. 2012

CTLA-4 blocca completamente la co-stimolazione CD28 -mediata

Leffetto inibitorio del PD-1 sul TCR e sul CD28 indiretto, meno completo e pi lento

La nuova era degli immunocheckpoint inibitori

Loncologia incontra limmunologia

Breaking the immune -tolerance 4 Novembre 2016


Sanit

Blocco degli immunocheckpoint per rinvigorire la risposta

linfocitaria T contro il tumore

Sharma and Allison, Science 2015

+

Meccanismi di azione di PD1, PDL1 e PDL2

Nguyen and Ohashi, Nat Rev Immunol 2015

PD-1 PDL1 PDL2

PD-1 PD-L1 PD-L2

Naive T cells

CTLA-4

+

Activated T cells + +

Exhausted T cells +

NK +

T Reg

MDSC

Endothelial cells

B cells

DC

APC

Fibroblast/CAF

Tumor cells

++

+

++

++

+

+

+

-/+ +

+

-/+

Stromal cells +

+

-/+

Macrophages ++

-/+

+ +

Pattern di espressione

-/+ -/+


Immuno-oncologia

4 Novembre 2016

Istituto Superiore di Sanit

To contribute to the success of Immuno-Oncology

we need to focus on the immune assessment

modalities and sites, both systemic and importantly

intratumoral

Refining immune endpoints to better design

clinical trials represents a high priority challenge

Sharma P, Nat Rev. Cancer, 2011

Clinical trial concepts

Progress to identify new and validate current biomarker candidates has been

limited by the use of unstandardized assays that provide limited data and

variable results.

Monitoring

Mechanisms

Standardized monitoring techniques

Understanding immunogenic mechanisms and designing of efficient combined immunotherapy

Immuno-modulatory drugs Molecules


Immuno-oncologia

4 Novembre 2016


Sanit

The development of immune monitoringassays is essential to determine the immuneresponses in clinical trials of novelimmunotherapies and targeted drugs

To discover the immune profiles of disease, leading to new

biomarkers of diagnosis, prognosis, and response to therapy

To transfer these therapies to standard of care in oncology


Immuno-oncologia

4 Novembre 2016


Sanit

1. Standardized immune assays leading to

reproducible results

2. A repository of immune monitoring data

available for mining

Two prerequisites are:


Immuno-oncologia

4 Novembre 2016


Sanit

Assays must have:

Sufficient sensitivity and specificity to be clinically useful

Good intra- and inter-assay precision

Linearity over a biologically useful range

Robust performance across minor lab variables

This will allow:

Ability to detect small changes that may characterize a disease state

Potential for assays to become clinical diagnostics

Standardized immune monitoring


Immuno-oncologia

4 Novembre 2016


Sanit

MIATA

A project for minimal Information about T cell assays

Many assays to evaluate the nature, breadth and

quality of antigen-specific T cell responses are

currently applied in human medicine

Experiments conducted by more than 100 labs have

generated the MIATA guidelines


Immuno-oncologia

4 Novembre 2016


Sanit

Britten et al, Immunity 2012

Minimal Information on the sample: Donors: report all available donor information for interpretation of the data

The Source and Processing: report the source, the conditions of transport and the median time of sample collection

Cryopreservation and storage

Cell Counting: before freezing, after thawing and after overnight resting

Minimal Information on the Assay: Medium/serum

The assay: report details about assay procedure including all reagents used

Controls

Data acquisition: Equipment and Software

Acquisition Strategy and Gating

The (interpretation of) results: Raw data: provide the means, medians and ranges for both background and antigen-

specific reactivity levels

Response determination, statistical tests and empirical rules

The laboratory environment: General Laboratory operation: GMP, GLP, exploratory research etc.

Laboratory procedure standardization

Status of Assay Qualification and validation


Immuno-oncologia

Evaluation of the T-cell response will require the use of spe cific assays toaddress issues related to T-cell frequency, trafficking an d function, aswell as issues related to potential immune-escape mechanis ms that areenlisted by the tumour to evade the T-cell response

Yee et al. Nature Rev Cancer 2002

Monitoring the anti-tumour T-cell responseCorso ECM

Attualit e prospettive in Immuno-oncologia

Flow Cytometry for phenotypic analysis, including leukocyte subset analysis and Intracellular Cytokine staining (ICS)

Peptide/MHC Multimer staining

Enzyme-linked Immunospot Assay (ELISPOT)

Bio-plex Multiplex Immunoassay

TCR V Repertoire Analysis using Flow cytometrystaining, spectratyping and sequencing

Validated assays in immune monitoring


Immuno-oncologia

4 Novembre 2016


Sanit

Multiparameter Flow cytometry assay for phenotypic analysis and intracellular cytokine staining (ICS)


Immuno-oncologia

4 Novembre 2016


La catena pesante della molecola HLAmodificata per contenere un sito dibiotinilazione e la b2-microglobulinavengono espresse in un sistemaprocariotico.

In seguito a purificazione delle proteinericombinanti viene aggiunto il peptide.

Una reazione enzimatica inducebiotinilazione della molecola e laggiuntadi streptavidina, che contiene quattro sitidi legame per la biotina, induce laformazione del complesso tetramerico.

La marcatura della streptavidina con uncomplesso fluorescente permettelidentificazione tramite analisi percitometria a flusso dei linfociti T dotatidi TCR specifico per il complesso HLA-peptide

HLA class I MultimersCorso ECM


Yee et al. Nature Rev Cancer 2002

ex-vivo

Melan-Ain vitro

expansion

PRE T42 T84 T105

1.15%1.04%0.05%0.04%

7.5% 17.0% 44.0% 70.0%

CD8

A2/

Mel

an-A

te

tram

er

0.45% 0.22%

70.0% 90.0%

POST(3 months)

POST(17 months)

Monitoring of a HLA-A2 melanoma patient by A2/Melan-A Multimer staining

HLA-A2/MULTIMER


Immuno-oncologia

4 Novembre 2016


ELISPOT(enzyme-linked immunospot assay) CVIA (computer video image assisted)

Short-term sensitizedIFN- Elispot assay

Pre Post

T2 +Melan-A

T2 empty


Immuno-oncologia

Bio-Plex Multiplex Immunoassay

Cytokines, chemokines and grow factors detection by a bead-based flow cytometric platform


Immuno-oncologia

4 Novembre 2016


MASS CYTOMETRYBased on the use of monoclonal antibodies labelled with stable

isotopes

ADVANTAGES

Up to 100 independent detection channels

Reproducibility between sample

Absolute quantification of target molecules

Overcomes the compensation step

USES

Analysis of phenotype

Analysis of signaling pathways

Effects of inhibitors on feedback signaling and intercellular comunication


Immuno-oncologia

4 Novembre 2016


To contribute to the success of immuno-

oncology we need to focus on the immune

assessment modalities and sites, both

systemic and importantly intratumoral

The immune contexture

During the past two decades, it has become clear th at the interaction between tumours and

their microenvironment (including stromal, endothel ial and immune cells) is crucial not only

during oncogenesis and tumour progression but also in the context of anticancer therapies

Fridman WH, Nature Rev, Cancer, 2012

TLS conduttori della risposta immunitaria adattativa e sistemica contro il tumore

I tumori sono infiltrati dalle cellule del sistema immunitario

Le strutture linfoidi terziarie (TLS) e linfiltrato linfocitario: fattori prognostici

favorevoli nel Non-Small Cell Lung Cancer

Modified by Dieu-Nosjean et al., Immunological Rev, 2016

4 Novembre 2016


Sanit

Corso ECM

Attualit e prospettive in Immuno-oncologiaTechnologies currently used to assess the potential

immune biomarkers

TUMOR

Type of Assay Tumor-derived Biomarker Immune cell-derived Biomarker

Protein (IHC)

Genomics

Transcriptomics

Function

PD-L1, PD-L2, TAAs

Next-generation sequencing

or Whole exome sequencing

for mutation load,

neoantigens, MMR genes

CD8, CD4, CD3, CTLA-4, PD-1,

LAG-3, CD45RO, CD25, FOXP3,

CD127, CD11B, CD57, CD68,

co-stimulator(s)

T and/or B cell receptor deep

sequencing

Multigene signatures Multigene signatures,

epigenomics

Proliferative marker: Ki67 Ki67, granzyme B, IFN-

by Ma W., J. of Hematology &&&& Oncology 2016

Corso ECM


4 Novembre 2016Istituto Superiore di Sanit

Cell free

genomics or

proteomics

Cell based

ctDNA, ctRNA, miRNA,

exosomes

Circulating tumor cells

miRNA, serological biomarkers

(soluble proteins, autoantibodies,

cytokines or chemokines);

HLA haplotyping

Absolute lymphocyte counts

(ALC), Eosiniphils,

Neutrophil/Leukocyte ratio,

MDSC, Th17,

Tumor-specific Immune cells

BLOOD


Technologies currently used to assess the potential immune biomarkers

Type of Assay Tumor-derived Biomarker Immune cell-derived Biomarker

Corso ECM



Flow and

mass cytometry

Antigen-

specific T cell

monitoring

Phenotypic and functional characterization of different immune

cells:

Activated T cells: CD3, CD4, CD8, Ki67, CTLA-4, PD-1, LAG-3,

TIM-3, ICOS

Regulatory T cells: CD3, CD4, CD25, FOXP3, CD127, Ki67,

CD45RA

MDSC: CD14+HLA-DRlow

Detection and characterization of circulating tumor cells (CTC)

Characterization of tumor-antigen specific T cells:

Sorting, Cloning, Binding avidity, Antigen-specific TCR

Functional assessment of response by tumor antigen-

specific T cells:

HLA-class I and II tetramer, ELISPOT, Polyfunctional ICS,

Natural tumor recognition

BLOOD


Technologies currently used to assess the potential immune biomarkers

TUMOR

Corso ECM



Topalian, Nature Rev Cancer 2016

Multifactorial biomarkers of clinical response to PD-1 pathway blockade

Corso ECM



Topalian Nature Rev 2016

Pitfalls of using PD -L1 immunohistochemistry as a biomarker test for anti-PD -1/PD-L1 therapy

Corso ECM


Mutational load as a potential biomarker for response

A cancer mutation can produce a T cell neoantigenic peptide t hat can confer de

novo peptide binding to MHC molecules.

The antigenicity of a neopeptide is not related to its functi on and a passenger

mutation can be a good tumor antigen.

Tumors with a greater mutational load could possess more neo antigens and thus

the patients may possess a larger repertoire.

Lawrence, Nature 2013

The odds of immunotherapy success

Schreiber R, Science 2015

4 Novembre 2016


Sanit


Modified by Chen, Cancer Discov 2016

Immune profiling in early on-treatment biopsies is predictive of response to sequential CTLA-4 and PD-1 blockade

Corso ECM



Optimal time points for tumor- and blood -

based biomarker measurement

To capture the kinetics of the immunologic

phenomena, extensive serial samplings of blood at

every cycle with more frequent collections during the

first cycle may be warranted

We are (fortunately) not alone

Y K Lee, and S K Mazmanian Science 2010

In humans, trillions of bacteria are distributed in complex and site-specific

communities on the skin and at mucosal surfaces, and the larg est community

is found in the distal gut

Round, Nat Rev Immunol 2009

Immunological dysregulation associated with dysbios is of the microbiota

A healthy microbiota contains a balanced composition of man y classes of bacteria

In condition of dysbiosis reduction of symbionts, increase of pathobionts and inflammation

The efficacy of cancer immunotherapy relies on gut

microbiota

Vetizou M, Science Nov 2015

4 Novembre 2016


Sanit

Certain gut microbiota determine the level of effic acy and toxicity during CTLA -4 checkpoint blockade.

Jonathan M. Pitt et al. Cancer Res 2016;76:4602-460 7

4 Novembre 2016


Sanit

Our experience

Peptide (Melan-A/gp100)-based vaccine in combination with chemotherapy (DTIC) in 10

melanoma patients (phase I pilot clinical trial)

Peptide (Melan-A/NY-ESO-1)-based vaccine in combination with chemotherapy (DTIC) in 35

melanoma patients (phase II randomized clinical trial)


Immuno-oncologia

MELANOMA PATIENTS(admitted in the Melanoma Unit)

Sera and Peripheral Blood CollectionNeoplastic Lesions paraffin embedded snap frozen

SerumPeripheral Blood

Lymphocytes immunohistochemical analysis

(tumor phenotype; HLA class I expression; T cell infiltrate

localization and phenotyping)Ex-vivo analysis of

T cell response

Antibody Response

Analysis of in-vitro cultures

T cell cloning and analysis of TCR repertoire

IDENTIFICATION OF PREDICTORS OF

RESPONSE

Tumor immune microenvironment


Immuno-oncologia

IRE Immune Monitoring AssaysFlow cytometry:

Multiparametric immunophenotyping of T (CD4, CD8, ) and B cells

Enumeration of ex-vivo Ag-specific CD8+ T cells by Peptide/MHC Tetramers staining in PBMC, TIL and TILN

Analysis of differentiation phenotype for T and B cells

Analysis of immune checkpoint inhibitory receptors (PD-1, LAG-3, TIM-3) and PD1 ligand (PD-L1) expression

Intracellular staining (ICS) for evaluation of cell ular polyfunctionality

ELISPOT assay CVIA

Ag-specific T-cell lines and clones in vitro expanded

TCR V Repertoire analysis by sequencing Immune cell infiltration

Tumor immune microenvironment

Ag-specific-line -clone

ELISA for antibody response

Ex-vivo T cells

51Cr release cytotoxicity assay

Bio-plex Multiplex Immunoassay for multiple cytokin es detection

BioBanking of tumor tissue, PBMC, serum/plasma, TIL , TILN

Tertiary lymphoid structures CD3 CD20

Exploring the influence of DTIC on the functional activity of CD8+ T cells

Generation of a multitude of Melan-A+ T-cell clones

Characterization of phenotype and TCR repertoire of

T-cell clones

Analysis of the lytic activity

Analysis of co-stimulatory and inhibitory receptor s

4 Novembre 2016


Sanit

Polyfunctionality

Activation of AKT in absence of CD28/C27 co-stimula tion

Immune checkpoint

Searching for biomarkers in patients who benefit from

chemoimmunotherapy treatment

Franzese, Palermo et al. Polyfunctional Melan-A-specific tumor-reactive CD8+ T cells elicited by Dacarbazine treatment before peptide-vaccination depends on AKT activation sustained by ICOS. OncoImmunology 2016

Responder patients

TCR diversity enlargement

Arm2

Proliferative capability

Palermo et al, Cancer Res, 2010

Up-regulation of ICOS co-stimulatory molecule

Limmunoterapia dei tumori personalizzata

Gubin J Clin Invest 2015

Cellule linfocitarie T specifiche per neo-antigeni mutati

Vaccini terapeuticiTrasferimento adottivo di

cellule linfocitarie T

Corso ECM



Criteri di valutazione della risposta: monitoraggio immunologico … · Da un modello Cell-Oriented...

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