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VEQ Allergologia
Valutazione risultati VEQ ciclo 2014
Alessandra Vultaggio
SOD Immunoallergologia
Dipartimento di Biomedicina
AOU Careggi
[email protected]
[email protected]
V.E.Q.
ALLERGOLOGIA
Ciclo 2014
- 12 campioni di sieri liofili di origine umana
- Valutazione IgE specifiche per alimenti e inalanti
- Valutazione IgE totali
61
Centri partecipanti
per Regione
11
1
4
1
10
15
-4 laboratori rispetto al 2013
5
129 Laboratori Ciclo 2014
11
63 Laboratori Ciclo 2009
3
2
1
3
22 Allergeni dosati
Bianco d’uovo
Grano
Arachidi
Soia
Pomodoro
Aglio
Mela
Bambagiona
Betulla verrucosa
Olivo
Cipresso medit.
Artemisifolia
Assenzio selvatico
D. pteronyssinus
Epitelio di gatto
Forfora di cavallo
Forfora di cane
Nocciole
Lattice
Parietaria judaica
Gambero
Pesce
N° all. x Camp.
1
x
2
3
x
x
x
x
x
x
4
x
x
x
x
x
x
6
x
x
x
x
x
x
x
x
5
x
x
x
7
x
x
x
x
x
x
x
x
x
x
x
x
x
x
11
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
12
x
x
x
x
10
x
x
x
x
x
x
x
x
9
x
x
x
x
x
x
8
x
x
x
x
10 alimenti + 12 inalanti
x
x
x
x
7
7
8
7
8
7
8
8
8
8
8
8
N° dosaggi
2
5
6
4
4
4
6
5
4
6
5
3
5
5
5
2
3
7
5
2
3
1
92
+ 13 dosaggi (rispetto al 2013)
Metodi IgE Totali (2014)
(6)
(66)
(50)
(2013)
Metodi IgE Specifiche 2014
(9)
(79)
(33)
(2013)
Valori Cut-off utilizzati
F.E.I.A.
Valori Cut-off utilizzati
Chemiluminescenza
20
19
15
12
10
5
6
7
0
0,1
0,35
Nuove prospettive per il laboratorio di
Immunoallergologia:
nuovi farmaci, nuovi tests
Farmaci Biologici
PROTEINE DI
ORIGINE
UMANA
PROTEINE DI
ORIGINE
NON UMANA
ANTICORPI
MONOCLONALI
PROTEINE UMANE
RICOMBINANTI
PROTEINE
DI
FUSIONE
Immunogenicity:
capacity to induce an immunoresponse
Chemical
compounds
•
•
•
•
•
•
•
Biologicals
Large and complex proteins
Different degree of glycosylation
Presence of xenoantigens
Partial or complete human sequence (allotypes)
Neoantigens on junction-sites
Repetitive Idiotype (mAbs)
Cross-reactivity with recall epitopes
All Biological agents
are immunogenic
Anti-drug
antibodies
(ADA)
Why are we interested
to immunogenicity?
Unwanted immunogenicity leads to the
development of anti-drug antibodies (ADA) and
may have two major clinical consequences
No
effects
Adverse events
safety
Immunogenicity
Loss of efficacy
efficacy
Farmaci Biologici:
utilizzo nelle varie specialità mediche
Gastro
enterologia
reumatologia
oncologia
dermatologia
neurologia
Immunoallergologia
Emopatie
congenite
Biologics approved for immuneimmune-mediated diseases
Biologic agent
Infliximab
Action
Anti-TNFα
FDA licence
EMEA licence
NICE approval
RA
RA
RA
AS
AS
PsA
PsA
PsA
CD
CD
CD
AS (appraisal)
UC
UC
UC (submitted)
Paediatric CD
Psoriasis
Psoriasis
Paediatric CD (appraisal)
Etanercept
Adalimumab
Anti-TNFα
Anti-TNFα
RA
RA
RA
JIA
JIA
JIA
AS
AS
PsA
PsA
PsA
Psoriasis
Psoriasis
Psoriasis
AS (appraisal)
RA
RA
RA (appraisal)
PsA
PsA
PsA
AS
AS
AS
Rituximab
Anti-CD20
RA
RA
RA (submitted)
Abatacept
CTLA4Ig
RA
RA (submitted)
RA (submitted)
Efalizumab
Anti‐CD11a
Psoriasis
Psoriasis
Psoriasis
Alefacept
LFA-3/IgG Fc construct
Psoriasis
None
None
Anakinra
Anti‐IL1
RA
RA
None
AS, ankylosing spondylitis; CD, Crohn's disease; CTLA4, cytotoxic T lymphocyte antigen-4; EMEA, European Medicines Agency; FDA, US Food and Drug
Administration; IL, interleukin; JIA, juvenile idiopathic arthritis; NICE, UK National Institute for Health and Clinical Excellence; PsA, psoriatic arthritis; RA,
rheumatoid arthritis; TNFα, tumour necrosis factor-α; UC, ulcerative colitis.
(Kuek A et al. Post Med J. 2007)
Detection of ADAs : current methods

Immunochemical methods


Biophysical methods


Binding assays (ELISAs, RIA, elettrochemiluminesce)
Surface plasmon resonance
Bioassays
Each of these methods has
benefits and limitations !!
What are anti-drug antibodies?
* anti-drug antibodies:ADA
• Binding ADA* (BAbs)
– Bind the drug but do not necessarily inhibit its biological
activity
– All detected ADA (anti-drug antibodies) are binding
– They develop early
• Neutralizing ADA (NAbs)
– a subset of Babs that prevent the binding of
biopharmaceutical to target on cell surface
– They develop 4-6 months after the beginning of therapy
(usually within the first two years)
In vitro ADAs assays
Stage 1:
ADA screening
& confirmation
ADA screening assay
-
No further
testing
-
No further
testing
+
Confirmatory assay
+
Stage 2:
ADA
characterization
Isotype
Neutralization
(from Vultaggio A, Nencini F, Pratesi S, Petroni G, Maggi E, Matucci A, J Inter Cyt Research 2013 in press)
ELISA is the preferred format
to measure antibodies
Commercialy available ELISA tests
for ADA detection
• Immunodiagnostick
• Promonitor
• Lisa Tracker
All 3 Bridging ELISA format
“Bridging” assay
(double-antigen sandwich format)
E
BA
BA
E
BA
Low background
High sensitivity
Non-isotype-specific
Not able to detect IgG4 ADA
ADA
Color reaction
Problems associated with
the detection of ADAs

Matrix effects (sample composition may influence
the assay)



Rheumatoid factors
Heterophilic antibodies
Cross-reactive antibodies
Drug interference
 Antigen conformation
 Low affinity ADAs

Comparison of different tecniques to monitor
anti-drug antibodies
• IFX-treated patients
• BID patients (CD)
• ELISA, RIA, EIA, RGA
Performances of assays are comparable.
However, anti-IFX Ab titers show systematic differences,
and in individual patients, only the same assay should be used.
Problems may arise when different assays
are used to manage therapies in the same patient.
(Vande Casteele N et al, Aliment Pharmacol Ther 2012 )
Fattori “confondenti” il test
RISULTATI
RISULTATI
FALSI
POSITIVI
FALSI
NEGATIVI
Fattori “confondenti” il test
RISULTATI
FALSI
POSITIVI
1. Rheumatoid factors
SA
BA
BA
Risultati falsi positivi
SA
BA
RF
Assessment of pre-treatment
serum samples!!
2. Anti-hinge antibodies
Fab
Fc
Anche i soggetti sani (10%) possono avere
anticorpi anti-hinge
Risultati falsi positivi
Fattori “confondenti” il test
RISULTATI
FALSI
NEGATIVI
1. Low affinity ADAs
Several factors may influence the detection of low-affinity ADAs:
1. Washing
2. Buffer constituents
3. Length of incubations
These factors should be considered during assay optimization
Low affinity ADAs may have neutralizing activity
2. Low levels ADAs
27
3. Drug interference
Serum samples with
excess of drug
No signal
ELISA plate with
immobilized drug
Serum TmAb concentration
ADAs detection
28
Approcching drug interference:
1) Appropriate sampling time
Drug infusion
X



Patient
Circulating
drug (IFX)
1
20.3
2
6.5
3
2.33
4
0.05
5
29.4
6
0.001
7
0.001
8
0.001
9
20.3
(Personal data)


I livelli di farmaco circolante
pre-infusione sono piuttosto variabili
da soggetto a soggetto
Time
Approcching drug interference:
2) Acid pretreatment of serum
• Dissociation of soluble IC at acid pH
– Exact conditions are critical
– A change of 0.5 pH Unit can affect both drug
tolerance but also signal loss
– Decrease precision
• Neutralization of the sample is required, but
reformation of IC may occur
Approcching drug interference:
3) Evaluation of drug levels
Risultato interpretabile
ATI
pos
Risultato interpretabile
ATI
IFX
pos
IFX
pos
pos
neg
neg
Cut-off
Cut-off
neg
neg
Risultato interpretabile
ATI
pos
Risultato indeterminato
ATI
IFX
pos
IFX
pos
pos
neg
neg
Cut-off
Cut-off
neg
neg
Immunogenicity testing strategies
When to test the
immunogenicity?
When to monitor immunogenicity?
Symptom-driven
Preventive
manner
manner
Adverse
effects
Loss of
efficacy
?
Preventive role for immunogenicity testing
o Pre-existing ADAs
o Cetuximab case
(Chung, NEJM 2008)
o ADA cross-react with an oligosaccaride of mammalian proteins
o Omalizumab case
(Price, Allergy Asthma Proc 2007)
o ADAs cross-react with polysorbate 80 (additive)
o Infliximab case ????
(Nielsen CH et al Aliment Pharmacol Ther 2013)
o ADAs developed during treatment
o The majority of antibody responses
A role for ADA assays in
the identification of patients at risk
1
Non-isotype-specific ATI
IgE ATI
0,8
Hypersensitivity risk
can be detected prior to
clinical symptoms !!
OD
0,6
0,4
0,2
0
Baseline
Day of
reaction
+7
+14
+21
+28
Days post reaction
(A.Vultaggio, A.Matucci et al Allergy 2010)
Can potentially reactive patients
be identified by clinical features?
BID
patients
AR
patients
SPA
Vasc
p<0.025
(Vultaggio A, Matucci A et al, IJIP 2008)
(Steenholdt C et al, Aliment Pharmacol Ther 2011)
When to monitor immunogenicity?
Symptom-driven
Preventive
manner
manner
Adverse
effects
Loss of
response
At
re-treatment!!
Conclusioni
-Come effettuare il dosaggio?
-Non esiste il test ideale, ma sicuramente la metodologia ELISA
costituisce un buon compromesso di specificità e sensibilità
-Quando effettuare il dosaggio?
-Sicuramente dopo un evento avverso o perdita di efficacia
-In modo predittivo?
-Da tenere presente la questione del tempo di campionamento
-Come interpretare i risultati
nella pratica clinica?
-tenendo presente quando è stato effettuato il prelievo nonchè
i livelli circolanti di farmaco
-Conoscendo i limiti del test che stiamo usando
Azienda Ospedaliero
Ospedaliero--Universitaria Careggi
Azienda Ospedaliero
Ospedaliero-Universitaria Careggi
D.A.I. Biomedicina
D.A.I. Biomedicina
SOD IMMUNOALLERGOLOGIA
Dir. Prof. Fabio Almerigogna
Dr A.Matucci
Dr. O.Rossi
SOD IMMUNOLOGIA
SOD IMMUNOLOGIA
E TERAPIE
CELLULARI
E TERAPIE
CELLULARI
Dir.
Prof. Enrico
Maggi
Dir. Prof.
Sergio
Romagnani
Drssa
Nencini
Francesca
Drssa Pratesi Sara
SOD IMMUNOALLERGOLOGIA
Dir. Prof. Enrico Maggi
Dr. Andrea Matucci
Dr