1. “Clinical pictures of hypersensitivity to biologicals” E. Maggi, MD enrico.maggi@unifi.it Center of Research, “DENOThe”, University of Florence, Italy

2. IMMEDIATE DELAYED OCCUR DURING OR WITHIN 1 HOUR AFTER INFUSION OCCUR FROM 1 HOUR TO 14 DAYS AFTER INFUSION Pichler WJ, Allergy 2006 Maggi E et al, Exp Rev Clin Immunol 2011 OCCUR WITHIN A FEW MINUTES AFTER INJECTION OCCUR AT LEAST 1 DAY AFTER INJECTION LOCAL SYSTEMIC HR to biologicals: Definition All the infusion reactions recognising an antibody- or cellular-mediated mechanism

3. itching urticaria AE nausea vomiting back pain throat costriction flushingdyspneahypo- tension LOCtachy- cardia low O2 % of patients Clinical features of IFX-related Immediate infusion reactions Matucci A et al Clin Exp Allergy 2013, N° of patients

4. Disseminated skin reactions – Maculopapular exanthema – Steven-Johnson’s syndrome – Acneiform eruptions – Psoriasiform eruptions Serum-sickness syndrome Tromboembolism events Skin vasculitis Likely associated with ADA development Likely sustained by T cell response Target-dependent event Delayed reactions to biologicals clinical pictures and pathogenetic mechanisms Torres MJ et al JACI, 2011 T-cell involvement in delayed hypersensitivity reactions to anti-TNFα agents Biopsy of maculopapular exanthema after a drug provocation test with infliximab

5. Unspecific irritative effect – Eccipient (e.g. sorbitol vs citric acid) – Volume of injection (eg. 0,5 ml vs 1 ml) Antibody-mediated reaction – The development of ADA increases the risk of IRS T cell-mediated reaction IMMEDIATE DELAYED T-cell infiltration has been observed in delayed ISR to etanercept (Zelster R, Arch Dermatol 2001) T-cell infiltration has been observed in delayed ISR to etanercept (Zelster R, Arch Dermatol 2001) Local reactions to biologicals

6. Question Can Immunogenicity explain the onset of Hypersentivity Reactions to the drug?

7. V IgG ADAs IL-4; IL-13 The Immune response to Biologicals MHC II TCR Aggregate mAbs PRRs V Fc  R Y BCR IgM ADAs Matucci A, Vultaggio A, et al. (IFIACI 2011) ICC VIRTUALLY ALL BIOLOGICALS ARE IMMUNOGENIC 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 Chimeric Humanized Fully human Fusion protein B B Y B B Th APC IgE ADAs

8. % ATI+ patients 346 infliximab-treated patients: 34,6% ADA+ Tolerant n=200 Non responder n=93 Reactive n=53 346 infliximab-treated patients: 34,6% ADA+ Tolerant n=200 Non responder n=93 Reactive n=53 p<0.005 p<0.0001 (Vultaggio A et al, submitted) ADA were prevalently detected in non-responder or reactive patients The onset of ADA response as well as the HR, develop within the 5-10 infusions The ADA levels in patients with HR are higher than in ADA+ patients who did Not develop HR The detection of ADA were performed with validated commercial kits with parallel evaluation of the drug levels. A confirmatory assay and a speficic inhibitory test was usually run in positive assays (Rup B et al & Abirisk Consortium, Clin Exp Allergy, 2015)

9. IgE-mediated reactions have been described towards several BAs Vultaggio A et al, Allergy 2010 Matucci A et al, Clin Exp Allergy 2013 Drug HR+ patients n=34 ADA+ patients n=28 (82,4%) IgE+ patients n=7 (25%) Infliximab-specific IgE ADAs Maggi E, Vultaggio A, Matucci A Exp Rev Clin Immunol 2011 rugIn vivoIn vitroRef MuromonabNoYes Georgitis, 1991 CetuximabNoYes Chung, 2008 TocilizumabNoYes Stubenrauch, 2010 BasiliximabNoYes Baudouin, 2003 OmalizumabYesNo Price, 2007 EtanerceptYesNo Bavbek, 2011 AdalimumabYesNo Paltiel, 2008 RituximabYesNo Brennan, 2009 NatalizumabYes MunozCano, 2010 InfliximabYes Vultaggio, 2010 p<0.02 ADA of IgE isotype

10. Skin testingPositiveNegativeTotal Patients with HRs (n=23) 71623 Patients with no HRs(n=30) 12930 Healthy Donors(n=20) 020 Total 86573 Sensitivity (%)Specificity (%)PPV(%)NPV(%) 30.496.65599 Matucci A et al, Clin Exp Allergy 2013 ADRLoss of response Treated- controls Healthy donors All ADA pos19114034 ADA neg44112039 Total2315 2073 Skin pos80000 IgE pos70000 P<0.0001 Skin testing for infliximab Sensitivity (%) Specificity (%) PPV(%)NPV(%) 30.496.65690

11. Serum anti-Rituximab IgE Ab increased during the two HRs of a RTX-treated patient RTX-specific IgE (kUA/l) Total IgE (kU/l) 1 2 3 4 5 6 7 8 9 10 Sample # RTX-specific IgE Total IgE (Vultaggio A et al, Int Archs Allergy Clin Immunol, 2012) DilutionPrick testIDT (imm) 1:1000Neg 1:100NegPos 1:10NegPos SFNeg Skin Testing Positivity HR

12. IgG anti-drug antibodies IgE anti-drug antibodies HR towards biologicals: a model

13. Remarks The presence of high affinity ADA of IgG/IgE isotypes and of IgG1/IgG4 subclasses in HR patients indicates that the immune response to IFX is a T-cell dependent phenomenon (Baker MP et al, Slf nonself, 2010)

14. Isotype control Anti-MHC class II (Vultaggio A, Matucci A et al, Int Arch Allergy Immunol 2012) RTX-reactive patient Healthy controls RTX-unexposed patients Rituximab-specific T cells derived from the IgE ADA+ patient produce high type 2 cytokines

15. Local and systemic immediate and delayed HRs to biologicals are due to the immunogenicity of the drug ADA positivity and levels correlate with the clinical outcomes (LOR or HR). IgE ADAs are detected in a proportion of reactive patients and are pathogenetic since associated to positive skin tests and drug-specific type2 response in vitro. Likely both IgE- and non-IgE ADA are responsible for HRs. Anti-IFX T cella are likely induced in the majority of patients during the first infusions, but they are inhibited by drug- driven regulatory mechanisms as IL-10-producing drug- specific T cells. Conclusions

16. “Clinical pictures of Hypersensitivity reactions to biologicals” Internal AOU-Careggi Lab. of Clin. Immunology Unit of Gastroenterology Francesca Nencini V. Annese Giulia Petroni M. Milla Sara PratesiUnit of Rheumatology Francesca Zanieri M. Matucci Cèrinic Alessandra Vultaggio G. Fiore DH Clinical Team Unit of Hematology Andrea Matucci L. Rigacci In/out-Patient Services IMI- EU Project “ABIRISK” Fabio Almerigogna Chaired by M. Pallardy (Paris) Francesco Annunziato Daniele Cammelli Lorenzo Cosmi Francesco Liotta Paola Parronchi Oliviero Rossi