SIAAIC Toscana 2014 Nuove opportunità diagnostiche per la gestione clinica delle reazioni avverse a farmaci biologici Alessandra Vultaggio AOU Careggi (Florence, Italy) Department of Biomedicine Immunoallergology Unit [email protected] Adverse Events Adverse Events (AE): Any untoward medical occurrence Infusion Reactions associated with the use of a drug, whether or not drug related Infusion Reactions (IR): Any AE caused by the drug Hyper sensitivity reactions Hypersensitivity Reactions: Antibody- or cellular-mediated IR Biological agents-related hypersensitivity reaction: a SUBMERGED and EMERGENT problem !!! (2014, in press) Clinical management of hypersensitivity reactions Patients with previous reactions Definition of pathogenic mechanisms Patients with no previous reactions Identify patients at risk Prevention of further or first time reactions Infusion reactions to biologicals : extension and timing of onset LOCAL SYSTEMIC IMMEDIATE DELAYED OCCUR DURING OR WITHIN 1 HOUR AFTER INFUSION OCCUR FROM 1 HOUR TO 14 DAYS AFTER INFUSION OCCUR WITHIN A FEW MINUTES AFTER INJECTION OCCUR AT LEAST 1 DAY AFTER INJECTION Pichler WJ, Allergy 2006 Maggi E et al, Exp Rev Clin Immunol 2011 Pathogenic mechanisms of reactions to biological agents (BA) IMMEDIATE systemic REACTIONS • Non antibody-mediated adverse reactions Complement-mediated Cytokine release syndrome (CRS) • Antibody-mediated adverse reactions IgE-mediated Non IgE-mediated Antibody-mediated = Hypersensitivity Vultaggio A et al, Curr Opin Allergy Clin Immunol 2012 BA-induced cytokine release syndrome FcγRI Biological agent n t io a t iv c s A lysi d e t ia med ’ C Target cell Mø MAC Direc ta popto sis AD CC -m ed ia te d ly si s NK cell Vultaggio A, Maggi E, Matucci A Curr Opin Allergy Clin Immunol 2011 C Y T O K I N E R E L E A S E Flu-like reactions Anaphylaxis-like reactions Cytokine storm Overlap between CRS and HYPERSENSITIVITY Antibody-mediated Hypersensitivity CRS Headache Myalgias Diarrhea MOF Fever Angioedema Rash Dizziness Hypotension Bronchospasm Dyspnea Pruritus Tachycardia Nausea Different mechanisms Different approaches to avoid future reactions Which tools are available for the clinician to manage the infusion reactions? – In vitro tests for ADA detection and Isotyping – In vivo tests to • Skin testing for IgEmediated HRs • Non isotype-specific ADA assay • IgE Isotyping T cell assays: their role to be defined Detection of ADAs: current methods Each of these methods has benefits and limitations !! Immunochemical methods Biophysical methods Binding assays (ELISAs, RIA, elettrochemiluminesce) Surface plasmon resonance Bioassays • ELISA bridging format is the most used test Comparison of different tecniques to monitor anti-drug antibdies • 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 ) Immediate systemic reactions to IFX are associated with ADA formation 100 0 17,9 17.6 % of patients 80 61,1 75 60 93,2 100 82,1 82.4 40 20 38,9 25 0 6,8 RESPONDER (n=103) ATI ATI + 96 (93.2%) 7 (6.8%) Vultaggio A, Matucci A et al, Allergy 2010 and unpublished data PARTIALLY NON TOTALLY NON RESPONDER RESPONDER (n=28) (n=36) 21 (75%) 7 (25%) 22 (61.1%) 14(38.9%) REACTIVE (n=34) REACTIVE/NON RESPONDER (n=9) 6 (17.6%) 28 (82.4%) 0 (0%) 9 (100%) IgG ADAs Isotype Antibodies towards biotherapeutics are mainly of the IgG isotype (IgG1 subclass) Monitoring patients treated with anti-TNFa biopharmaceuticals: assessing serum infliximab and anti-infliximab antibodies Svenson M et al, Rheumatology 2007 ADR towards biologics: a model IgG pathway of anaphylaxis in mice High amount of antibody Maggi E, Vultaggio A, Matucci A Exp Rev Clin Immunol 2011 High amount of antigen IgE ADA isotype IgE-mediated reactions have been described towards several BAs Maggi E, Vultaggio A, Matucci A Exp Rev Clin Immunol 2011 Drug In vivo In vitro Ref Muromonab No Yes Georgitis, 1991 Cetuximab No Yes Chung, 2008 Tocilizumab No Yes Stubenrauch, 2010 Basiliximab No Yes Baudouin , 2003 Omalizumab Yes No Price, 2007 Etanercept Yes No Bavbek, 2011 Adalimumab Yes No Paltiel, 2008 Rituximab Yes No Brennan , 2009 Natalizumab Yes Yes MunozCano, 2010 Infliximab Yes Yes Vultaggio, 2010 Rituximab Yes Yes Vultaggio, 2012 Vultaggio A et al, Allergy 2010 Matucci A et al, Clin Exp Allergy 2013 IgE-mediated IFX-related events: Update of our experience Infliximab-specific IgE ADAs ADR+ patients n=34 ATI+ patients n=28 (82.4%) IgE+ patients n=7 (25%) Vultaggio A et al, Allergy 2010 Matucci A et al, Clin Exp Allergy 2013 And Unpublished data The Relevance of IgE Isotyping: Why not “Just do it”? the analysis of specific IgE anti-drug antibodies is challenging • The level of circulating specific IgE antibodies is usually low (1 ng/ml) • The timeframe in which sIgE is detectable in serum is quite short 1 Non-isotype-specific ADA IgE ADA • Assays are sensitive to: – – Circulating drug Antidrug antibodies of other isotypes OD 0,8 0,6 0,4 0,2 0 BaselineDay of reaction +7 +14 +21 +28 Days post reaction Serum detection of IgE anti-drug antibodies: Type of assay Drug In vitro Ref Muromonab Yes, ELISA Georgitis, 1991 Cetuximab Yes, ImmunoCAP Chung, 2008 Tocilizumab Yes, ImmunoCAP Stubenrauch, 2010 Basiliximab Yes, ELISA Baudouin , 2003 Natalizumab Yes, ImmunoCAP MunozCano, 2010 Infliximab Yes, ImmunoCAP Vultaggio, 2010, Matucci 2013 Rituximab Yes, ImmunoCAP Vultaggio, 2012 Why to measure BA-specific IgE? Highest incidence of anti-drug IgE in severe reactions 1. IgE-mediated reactions have peculiar clinical characteristics 100 p<0.02 Matucci A al, Clin Exp Allergy 2013 % of IgE+ patients 80 60 40 20 0 Hypereactivity Tolerant Severe Hypereacitivy Mariotte D et al, mAbs 2011 Timing of the development of reactions 2. IgE-mediated reactions usually occur earlier and more frequently after a period of interrruption ATI+ IgE+ N° of patients ATI+ IgEATI- (N° of infusions) ATI+ IgE+ ATI+ IgE- 1° Cycle 2/7 (28.6%) 14/16 (87.5%) Re-exposure 5/7 (71.4%) 2/16 (12.5%) Matucci A et al, Clin Exp Allergy 2013 p<0.02 Can diagnostic tools predict BA-induced infusion reactions? A role for ADA assays in the identification of patients at risk (A.Vultaggio, A.Matucci et al Allergy 2010) 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 Days post reaction +28 IgE-mediated reactions at first exposure The Cetuximab case Chung CH et al, NEJM 2008 Sensitization to Gal-1-3Gal M ea t Al lerg IgE G1-3G y First exposure to Cetuximab Cetuximab (SP2) Murine part Gal-1-3-Gal n ctio e j n I G 1-3 l Ga IgE G1-3G Hypersensitivity Reaction al (Matucci A. et al. EAACI 2014 Anti-cetuximab IgE as a valuable screening test 5 out of 6 resulted 6 patients with meat allergy but IFX-naive Delayed anaphylaxis to meat Previous history of tick bites IFX POSITIVE IgE anti-IFX antibodies probably preexist to the treatment Are they specific for glycans? What is their clinical significance? Do 2 different pathway of sensitization toward IFX exist? Serum detection of IgE anti-drug antibodies: Clinical studies Method Pts ELISA CAP Controls 14 78 unreactive 117 HD 30 20 untreated; 15 unreactive; 15 LOR Culprit Sens drugs CTX INF 71.4 26 Spec 82.1 90 PPV 33.3 26 NPV 98.5 89 Author Mariotte D (2011) Matucci A (2013) Preexisting crossreactive ADA New developed non crossreactive ADA ADA-positive but IgE-negative reactive patients: why? TRUE IgE-NEGATIVE Other isotypes of ADAs involved in the induction of hypersensitivity reactions FALSE IgE-NEGATIVE Interference by other antibodies in the assay OR Very low circulating IgE levels (bound on cells) Can skin testing help clinicians in the allergological work-up for IgE-mediated hypersensitivity reactions ? Skin testing for BA In vivo tests are not currently standardized and approved for BA • Limited data in literature • Positive skin testing has been reported mainly in patients with HRs caused by: – antiTNF agents – Rituximab – Trastuzumab • A Few studies (small case series) report results of skin testing confirmed by in vitro assay – Infliximab – Rituximab – Natalizumab What is the appropriate drug concentration? Biological Agent Concentration Dilutions Infliximab 10 mg/ml Prick test 1:100-1:1 IDT (0,02 ml) 1:1000-1:10 Adalimumab 50 mg/ml 1:100-1:1 1:1000-1:10 Etanercept 25 mg/ml 1:100-1:1 1:1000-1:10 Rituximab 10 mg/ml 1:100-1:1 1:1000-1:10 Cetuximab 5 mg/ml 1:100-1:1 1:1000-1:10 Trastuzumab 21 mg/ml 1:100-1:1 1:1000-1:10 Natalizumab 20 mg/ml 1:100-1:1 1:1000-1:10 Skin testing for infliximab: our experience Skin testing Positive Negative Total Patients with HRs (n=23) 7 16 23 Patients with 1 no HRs (n=30) 29 30 Healthy Donors (n=20) 0 20 20 Total 8 65 73 Sensitivity(%) Specificity(%) PPV(%) NPV(%) 30.4 90 Matucci A et al, Clin Exp Allergy 2013 96.6 56 Timing to perfom skin testing for BA REACT ION The time between the reaction and the in vivo evaluation is a crucial point IN VIVO ASSAY • Temporary irresponsiveness due to the massive activation during the reaction • Decrease of specific IgE over time The value of skin testing for BA in the clinical practice Symptom-driven manner • To assess REACTIVE PATIENTS – To define IgE-mediated pathogenesis desensitization Brennan P et al, JACI 2009 Matucci A et al, Clin Exp Allergy 2013 The value of skin testing for BA in the clinical practice Preventive manner ?? • To assess UNREACTIVE PATIENTS – To identify the risk of IgE-mediated events since…… ……..NPV is very high ……..The presence of IgE deeply correlate with the development of (severe) reactions …….skin test are easy and safe …….may be predictable of reactions (at least for some BA) ……BUT not all patients could be submitted to skin testing in a preventive manner (time consuming!!!) …..preventive skin testing could be useful IgE develop early during the treatment IgE develop more frequently after interruption Skin prick test using cetuximab to diagnose meat allergy Conclusions o Biological agents are up-growing cause of drug-related adverse effects, and the majority of these adverse effects are due to antibody formation o In vivo procedures offer a new tool for the diagnosis of at least IgE-mediated HR o IgE and non IgE-mediated mechanisms may be involved o In vitro ADA assay in combination with in vivo assay can be suggested to characterize reactive patients and to identify potentially reactive patients Aknowledgement AOU Careggi University of Florence Immunoallergology Unit Andrea Matucci Daniele Cammelli Dept of Internal Medicine Francesca Nencini Sara Pratesi Giulia Petroni Prof. Enrico Maggi External Collaborators Dermatology Unit (Florence) Francesca Prignano Gastroenterology Units Monica Milla (Florence) Marco Bertini (Pisa) G.Luigi De’ Angelis (Parma) Francesca Vincenzi (Parma) Rheumatology Unit (Prato) Fabrizio Cantini Thermofisher (Uppsala) Serum detection of IgE anti-drug antibodies: Clinical studies Method IMMUNO CAP ELISA IMMUNO CAP Patients Controls 25 51 unreactive; 462 HD 14 78 unreactive; 117 HD 30 20 untreated; 15 unreactive; 15 LOR Culprit drugs Cetuximab Cetuximab Infliximab Sensitivity specificity PAPER 92% (68%)° 90% (92%)° Chung CH (2008) 71.4% 82.1% Mariotte D (2011) 26% 90% Matucci A (2013) Preexisting crossreactive ADA New developed non crossreactive ADA Diapo DHM6 berna Aknowledgement AOU Careggi University of Florence Immunoallergology Unit Andrea Matucci Daniele Cammelli Dept of Internal Medicine Francesca Nencini Sara Pratesi Giulia Petroni Prof. Enrico Maggi External Collaborators Dermatology Unit (Florence) Francesca Prignano Gastroenterology Unit (Florence) Monica Milla Gastroenterology Unit (Pisa) Marco Bertini Giampaolo Bresci Gastroenterology Unit (Parma) G.Luigi De’ Angelis Francesca Vincenzi Rheumatology Unit (Prato) Fabrizio Cantini Thermofisher (Phadia) In vitro ADAs assays Stage 1: ADA screening & confirmation ADA screening assay (Bridging ELISA) - No further testing - No further testing + Confirmatory assay (binding inhibition) + Stage 2: ADA characterization Isotype determination Relative ADA concentration Neutralizing activity Skin testing: implications in desensitization • SELECTION of PATIENTS – To define IgE-dependent reactions – To perform risk stratification ?? • Does skin testing positivity correlate with a greater likelood of developing reactions during the subsequent desensitization? • SELECTION of PROCEDURE – Skin test endpoint-titration to choose the starting dose • ASSESSMENT OF DESENSITIZATION OUTCOME – Does skin testing become negative after desensitization? Skin testing for infliximab Skin testing Positive Negative Total Patients with HRs (n=23) 7 16 23 Patients with 1 no HRs (n=30) 29 30 Healthy Donors (n=20) 0 20 20 Total 8 65 73 Sensitivity(%) Specificity(%) PPV(%) NPV(%) 30.4 99 Matucci A et al, Clin Exp Allergy 2013 96.6 55 Still an unsolved question…… • The presence of IgE in a reactive patient does not exclude the presence of IgG ADAs • What is the role for IgG ADAs in IgE+ patients? Khodoun MV et al, PNAS 2011 When do IgE-mediated infusion reactions occur? Hypersensitivity reactions are a clinical concern about biological agents Mild Moderate Severe Maggi E, Vultaggio A, Matucci A Exp Rev Clin Immunol 2011 N° of patients Clinical characteristics of IFX-related Immediate infusion reactions Matucci A et al Clin Exp Allergy 2013, in press % of patients Clinical characteristics of IFX-related Immediate infusion reactions itching urticaria AE Matucci A et al Clin Exp Allergy 2013, in press nausea back throat flushing dyspnea hypo- LOC vomiting pain costriction tension tachy- low O2 cardia RTX-induced infusion reactions can be induced by an IgE-mediated mechanism April 2009 RTX (2x1000 mg) 2 weeks February 2010 RTX (2x1000 mg) December 2010 RTX (2x1000 mg) +2 +3 +4 +5 +6 +10 +12 +36 weeks post ADR ADR ADR Sample # 0 1 2 3 4 6 5 7 8 9 10 12 13 RTX-specific IgE (kUA/l) IgE-CAP ADAs Total IgE (kU/l) Anti-RTX non-isotype-specific antibodies (OD) Non isotype-specific ADAs 11 Sample # (Vultaggio A, Matucci A et al, Int Arch Allergy Immunol 2012) Sample # RTX-specific IgE antibodies SKIN TESTING RESULT Prick test 1:1 negative IDT 1:1000 negative IDT 1:100 positive IDT 1:10 positive Saline solution negative % of inhibition Rituximab Mouse IgG Cetuximab Inhibitor added (µg/ml) RTX 10 mg/ml RTX 1 mg/ml RTX 0,1 mg/ml RTX 0,01 mg/ml 1:1 formulation 1:10 1:100 1:1000 The importance of T cells in the development of anti-therapeutic antibodies • The presence of multiple non-IgM ADAs isotypes Vultaggio A et al, Allergy 2010 Bartelds GM et al, Ann Rheum Dis 2010 Ehrenforth S et al, Lancet 1992 • T cell subset polarization in treated haemophilic patients Reding MT et al, Thromb Haemost 2002 • Many therapeutics proteins contain T cell epitope Harding FA et al, Mabs 2010 van Schouwenburg PA et al, Nat Rew Rheumatol 2013 Circulating T-cell response to rituximab is detectable RTX-reactive patient Healthy controls Isotype control Anti-MHC class II RTX-unexposed patients (Vultaggio A, Matucci A et al, Int Arch Allergy Immunol 2012) T cell response for infliximab in treated patients (Vultaggio A, Matucci A et al, in preparation) N° of patients (11) (10) (8) (6) RTX-specific T cell response is more easily detectable N° of patients showing proliferative response to drug by using Patients (N°) _____________________________________________________ PBMNC CD4+T/DC Two-step Atg stim (%) (%) (%) Biologic 8 (38) 14* (66) 7 (33) 0 1 (6) 1 (6) (anti-inflammatory) effects 0 0 0 on transmembrane TNF IFX-treated ADA+ (21) ADA- (15) HC (10) Mitoma et al, 2008 RTX-treated Horiuchi etNDal, 2010 ADA+ ( 1) 1 1 Vos etNDal, 2011 ADA- ( 7) 4 (57) 4 (57) HC ( 10) 0 0 ND ___________________________________________________________ •11 out of 14 patients showed ADR or ADR plus LOR. Only one patient with ADR resulted negative Features of IFX-specific T cells after ADR: a clonal analysis Non specific TCC 188 TCC Clonal Efficiency: Total: 36.6% Specific: 4,7% Non proliferating cytokine-producing TCC N° of clones CLONING IFX-specific TCC High IL-10 production by IFX-specific T cell clones * p<0.002 IL-10-producing TCC * * (Vultaggio A, Matucci A et al, in preparation) Infliximab-specific regulatory T cell clones Annexin V- Treg clones CD73-PE T eff clones + IFX T eff clones + IFX + Treg clones CD39-FITC IFX-specific response (cpm) T eff clones + IFX + no-Treg clones ANNEXIN V-CD39+CD73+ TCC (preliminary unpublished data)) CD39-CD73- TCC Remarks Remarks • Circulating T cells specific to biological agents are detectable in the blood of ADR+ patients and usually parallel the kinetics of serum ADAs • Different subsets of drug-specific Treg cells are detectable in PBMC of ADR+ patients, which can, at least in part, explain the variable T (and B) cell response to the drug • The identification of T cell response to some biologicals (such as IFX) may prove particularly difficult A role for ADA assays in the identification of at risk patients 1 Non-isotype-specific ADA IgE ADA 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 patients at risk be identified by clinical features? BID patients patients p<0.025 (Vultaggio A, Matucci A et al, IJIP 2008) (Steenholdt C et al, Aliment Pharmacol Ther 2011) Timing of the development of reactions ATI+ IgE+ N° of patients ATI+ IgEATI- (N° of infusions) ATI+ IgE+ ATI+ IgE- 1° Cycle 2/7 (28.6%) 14/16 (87.5%) Re-exposure 5/7 (71.4%) 2/16 (12.5%) Matucci A et al, Clin Exp Allergy 2013, in press p<0.02 When to monitor immunogenicity? Table 1. Patients’ characteristics and monitoring of sensitization to infliximab Patient Prick IDR (age/sex/disease/atopy) Non isotype-specific ATI (OD) IgE ATI A (kUA/l) #1 (33/F/BD/no) 1st course of therapy Baseline Neg Neg 0.142 0.00 3rd infusion (°) Neg Neg 0.112 0.00 T0 Neg Neg 0.136 0.02 T1 Neg Pos (1/100) 2.194 0.19 2nd course of therapy B #2 (42/M/RA/no) 1st course of therapy Baseline Neg Neg 0.126 0.02 5th infusion (°) Neg Neg 0.149 0.01 T0 Neg Neg 0.134 0.01 T1 Neg Pos (1/10) 2.085 0.16 2nd course of therapy IDR: intradermal tests; ATI: anti-infliximab antibodies; OD: optical density; BD: Behçet disease; RA: rheumatoid arthritis. Non isotype-specific (cut-off value: 0.160) and IgE ATI (cut-off value: 0.10) have been measured as reported in the text. (°): serum samples have been collected before the reported infusion. T0: baseline before retreatment; T1: twelve days after the first infusion of the second course. (Vultaggio A, Matucci A, et al. Intern Emerg Medicine 2011) Frequency of ADA formation in different immune-mediated diseases Infliximab-treated patients RA SpA BID Vas ATI + 15 (40.5%) 12 (20.7%) 19 (30.6%) 7 (23.3%) ATI - 22 46 43 23 personal unpublished data % of IgE+ patients Highest incidence of anti-cetuximab IgE in severe reactions Mariotte D et al, mAbs 2011 Do IgE-mediated events have peculiar clinical features? Clinical management of hypersensitivity reactions Patients with previous reactions Definition of pathogenic mechanisms Patients with no previous reactions Identify patients at risk Prevention of further or first time reactions Symptom-driven and preventive manner Da cambiare!!! Symptom-driven and preventive manner Da cambiare!!! Serum detection of IgE anti-drug antibodies: Clinical studies Method IMMUNOCAP ASSAY ELISA IMMUNOCAP ASSAY Patients 13 grade 1-2* 12 grade 3-4* Controls 51 tolerant patients; 462 HD 3 urticaria/angioedema 3 pulmonary/GI/CV symphy toms 6 severe hypotension 2 cardiac/respiratory arrest 78 tolerant patients; 117 HD 10 mild 10 moderate 10 severe 20 untreated, 15 tolerant and 15 non responder patients Confirmed diagnosis 5/8 IgE-neg subjects were rechallenged: 1/5 had HR Histamine and tryptase measurements SPT + IDT Culprit drugs Cetuximab Cetuximab Infliximab Drugs tested Cetuximab Sensitivity Specificity Sensitivity 92% (68%)° Specificity 90% (92%)° Cetuximab Sensitivity 71.4% Specificity 82.1% Infliximab Sensitivity 26% Specificity 90% Comments Retrospective study; Pre-existing IgE that are shown to be specific for Gal1-3Gal Retrospective study; Good potential of developed ELISA for predicting high grade HR at first cetuximab therapy Correlation between skin testing and serum IgE antibodies PAPER Chung CH (2008) Mariotte D (2011) Matucci A (2013) Which tools are available for the clinician to manage the infusion reactions?
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