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A. Khalil et al.
serum PEG-ASNase activity levels. Patients with pre-existing antibodies may show mild to moderate signs of hypersensitivity reaction after their first administration PEG-ASNase, which may be successful- ly addressed by re-challenge.
Introduction
Due to its favorable toxicity profile polyethylene glycol (PEG) is widely used in foods, cosmetics, and pharmaceu- ticals.1 Pegylation can improve the therapeutic benefit of protein drugs. It prolongs their elimination by increasing the molecular mass and protecting them from enzymatic cleavage and it decreases their immunogenicity by shield- ing potential antigenic epitopes.2-4 Numerous pegylated drugs are currently marketed in the USA and Europe including pegylated uricase (KrystexxaTM), pegylated inter- feron (PegasysTM, PegIntronTM) and pegylated E. coli asparaginase (PEG-ASNase) (OncasparTM, calaspargase, AsparlasTM).5-8 The asparagine-hydrolyzing enzyme asparaginase (ASNase) is crucial for the successful treat- ment of acute lymphoblastic leukemia (ALL)9,10 and because of its favorable drug characteristics PEG-ASNase is increasingly replacing its unmodified native form in frontline treatment of ALL.11-13
While pegylated proteins, despite their higher molecular mass, tend to be less immunogenic than their non-pegylat- ed forms of protein drugs, antibodies against PEG have been detected in patients treated with pegylated proteins as well as in healthy volunteers.14 The reported prevalence varies widely between studies (0.2-72%) which is partly due to the use of different detection methods and cut- point definitions (Online Supplementary Tables S1 and S2). In animal studies anti-PEG antibodies, especially anti-PEG IgM, were considered responsible for the accelerated blood clearance of pegylated proteins, liposomes, and nanoparticles.15,16 In human studies, the reported effects of anti-PEG antibodies on the therapeutic efficacy of pegylat- ed drugs have been ambiguous; no effects of antibodies against PEG have been observed for pegylated interferons to date,17 whereas in patients with gout anti-PEG IgM and anti-PEG IgG were associated with a faster elimination of PEG-uricase.18,19 Drug authorities now require evaluation of the relevance of anti-PEG antibodies during drug devel- opment and registration processes.20,21
Published data suggest that anti-PEG antibodies may have important effects on the efficacy of PEG-ASNase. Armstrong et al. detected anti-PEG antibodies in 12 of 15 patients with undetectable ASNase activities after PEG- ASNase administration and also in four of 12 patients before their first PEG-ASNase administration.22 Liu et al. recently showed that anti-PEG ASNase antibodies consist- ed mainly of antibodies against PEG rather than E. coli
Table 1. Demographics of the patients in the three cohorts of acute lympho- cytic leukemia cases.
ASNase and were significantly associated with hypersen- sitivity reactions to PEG ASNase.23
Given the increasing use of PEG-ASNase in frontline treatment for ALL, the aims of this study were to: (i) eval- uate the prevalence of anti-PEG antibodies in three cohorts of patients (children and adults with primary ALL and children with relapsed ALL) before and/or immediate- ly after their first dose of PEG-ASNase during induction treatment, and (ii) investigate the effects of pre-existing anti-PEG antibodies on PEG-ASNase activities and hyper- sensitivity reactions.
Methods
Patients
Samples for anti-PEG antibody determination were obtained from children with primary ALL (ALL-cohort 1), children with relapsed ALL (ALL-cohort 2), adults with primary ALL (ALL- cohort 3) and healthy infants, who served as the reference cohort. Patients in ALL-cohort 1 were treated according to the AIEOP- BFM ALL 2009 trial (ClinicalTrials.gov identifier: NCT01117441) and a total of 673 plasma samples were collected from 673 pedi- atric patients (401 males, 272 females) prior to their first adminis- tration of PEG-ASNase. In addition, 646 patients provided one or two more serum samples (1,183 in total) taken within 15 days after the first PEG-ASNase dose on day 12 of induction.
Patients in ALL-cohort 2 were diagnosed with relapsed ALL and treated according to the protocol of the ALL-REZ BFM 2002 (ClinicalTrials.gov identifier: 00114348) or the ALL-REZ BFM Observational Study and Biobank study. Twenty-eight samples were collected from 28 patients (19 males, 9 females) 0 to 2 days after the first dose of PEG-ASNase
Patients in ALL-cohort 3 were treated according to the multi- center GMALL 07/2003 trial (ClinicalTrials.gov identifier: 00198991). A total of 188 samples from 120 males and 68 females were taken on the same day after the first administration of PEG- ASNase (n=16) or the following day (n=172). Further details on the ALL cohorts are provided in Table 1 and in the Online Supplementary Data.
The respective ALL studies were approved by national and local review boards in accordance with the Helsinki Declaration and national laws. The approvals included monitoring antibodies against PEG-ASNase and determination of ASNase activity. Patients and/or their guardians gave their signed informed consent to participate in the monitoring of ASNase activities and antibod- ies against PEG-ASNase.
Serum samples from 58 infants <1 year, who were considered naïve to PEG were used as reference. The Central Laboratory of the University Hospital Muenster provided anonymized remain- ders of routine serum samples from infants. Only age in months was disclosed. Thus, these samples were considered as complete- ly anonymized leftover material.
Determination of antibodies against PEG
For the detection of anti-PEG IgG and anti-PEG IgM the flow cytometry method described by Armstrong et al.22 was transferred to a 96-well format with fluorescent read-out. TentaGel M OCH3 particles (10 mm), to which methoxy-polyethylene glycol chains with a mean molecular weight of 5,000 Da were covalently
ALL-cohort 1 AIEOP-BFM ALL 2009
673 401/272
ALL-cohort 2 ALL-REZ BFM 2002 & ALL-REZ BFM Observ.a
28 19/9
ALL-cohort 3 GMALL 07/2003
188 120/68
Protocol
Number
Sex (M/F)
aObservational Study and Biobank. M: male; F: female.
Age, years Median
5.6
Range 1-18 5-17 18-74
8.5
36
50
haematologica | 2022; 107(1)