Several studies have shown that antibody formation can occur in patients receiving recombinant human proteins for therapy (Wang et al
2008; Kishnani et al
2010; Banugaria et al
2011; Patel et al
2012). Our patients with classic infantile Pompe disease who were treated with alglucosidase alfa showed various immune response patterns. Low and high titers were measured in CRIM-negative and CRIM-positive patients alike. While thorough statistical analysis was prevented by the small sample size and the heterogeneity of our patient population, we noticed that patients who did not learn to walk had a relatively high titer. We also noticed that patients who had started ERT before the age of two months tended to have lower titers than those who had started later. Notably, all CRIM-positive patients were still alive at the time of reporting, whereas all CRIM-negative patients had died.
GAA genotypes and CRIM status
The quality and quantity of acid α-glucosidase in Pompe disease depends on the GAA genotype. Ten different mutations were identified, all causing complete loss of acid α-glucosidase activity.
The commonly used procedure to determine the CRIM status is immunoblotting and normally reveals four distinct acid α-glucosidase species that separately and collectively are called CRIM. In healthy people (Fig.
1, Wt), the long-lived 76 and 70 kD species comprise approximately 80 % of the total CRIM. In patients, however, the synthesis of acid α-glucosidase can be totally lost, which results in a CRIM-negative status. More often, the synthesis is derailed, and CRIM consists mainly of the 110 kD precursor (e.g., Fig.
1, pts. 1 and 3) or a modified derivative thereof (Fig.
1, pts. 2 and 6). As it stands, the CRIM-negative status is well defined, but the CRIM-positive status encompasses a collection of qualitatively and quantitatively abnormal immunoblot profiles.
By and large, a patient’s CRIM status can be predicted on the basis of its genotype (Bali et al
2012). Most non-sense mutations and frame-shift mutations lead to mRNA decay and a CRIM-negative status (e.g., 525delT in pt. 3), truncated protein species are sometimes detectable by immunoblotting (e.g., c.2481 + 102_2646 + 31del in pts. 1, 7, 10, and 11), and thus lead to a CRIM-positive status. Although the effect of missense mutations is hard to predict, they usually lead to a CRIM-positive status.
Information on the effect of missense mutations can be obtained by transient expression (Fig.
2), which is especially informative for the stage at which mutant forms of acid α-glucosidase are degraded. For instance, the c.1460 T > C or c.1799G > A encoded acid a-glucosidase precursors must have traversed the Golgi complex since they are secreted into the medium, whereas the c.1833G > T, c1913G > T and c1115A > T encoded precursors do not appear in the medium (Fig.
2) and are apparently degraded while passing through the ER/Golgi complex. By the current definition of CRIM, these five missense mutations lead to a CRIM-positive status, but obviously do not represent one and the same CRIM-positive condition.
Our application of both methods to determine the CRIM status resulted in the same outcome: eight patients are CRIM-positive and three are CRIM-negative. Notably, the patient homozygous for c.2741delinsCAG was previously designated CRIM-positive (Hermans et al
1998; Banugaria et al
2011).
CRIM status and antibody titer
Antibody formation is a natural response to foreign invading proteins. Thus, ERT is prone to evoke an immune response in CRIM-negative patients, but not necessarily in all CRIM-positive patients. Patients with Pompe disease receiving ERT respond roughly according to these principles. Analysis of the immune response in 34 treated infants showed a strong tendency toward higher and sustained antibody titers in CRIM-negative compared to CRIM-positive infants (Kishnani et al
2010). However, CRIM-positive patients can also develop high titers (Banugaria et al
2011), and CRIM-negative patients low titers (Abbott et al
2011; Al Khallaf et al
2013). Our study in 11 infants has led to similar findings. The highest peak antibody titers were measured in four of the eight CRIM-positive patients and in two of the three CRIM-negative patients. The antibody titer of the third CRIM-negative patient did not exceed 1:6250 and spontaneously regressed to 1:50 after 3 years of ERT. There were two CRIM-positive patients with the same
GAA genotype; one developed a relatively high titer, and the other a relatively low titer.
Although the number of patients in our study is small, we can firmly conclude that the CRIM status alone predicts neither the level nor the duration of the immune response. This may relate to the imprecise definition of CRIM status, which does not describe the amount, the conformation or the location of the endogenous acid α-glucosidase. For instance, secretion of the 110 kD precursor as opposed to intracellular degradation might reduce the immune response and contribute to the relatively low antibody titers in patients 1 and 9. Notably, adult patients with a considerable amount of normally structured and catalytically active acid α-glucosidase can also develop high titers (van der Ploeg et al
2010; de Vries et al
2010; Patel et al
2012; Regnery et al
2012). Altogether, we conclude that genotype and CRIM status are of limited value in predicting the height of the immune response.
As previously suggested (van Gelder et al
2012; Al Khallaf et al
2013), our study indicates that the age at start of ERT might play a role in the immune response since none of the patients who started ERT before 2 months of age developed titers > 1:6250. There are at least two plausible explanations for this: First, the neonatal immune system is immature, and very early administration of ERT might induce tolerance (Brooks
1999; Dierenfeld et al
2010). Second, the ‘danger model’ suggests that the immune system needs alarm signals from injured tissues to be activated. At a less advanced stage of disease these signals will be weaker (Matzinger
2002). Other models may equally apply.
Consequences of antibody formation
Depending on their binding sites antibodies can inhibit catalytic function, block binding to the mannose 6-phosphate receptor and prevent uptake, or otherwise misdirect the protein to macrophages and neutrophils (Brooks et al
2003; Wang et al
2008). Antibodies and antigen can also form immune complexes and trigger a cascade of adverse events (Hunley et al
2004).
In a previous case-report about an adult patient with Pompe disease we have calculated how the concentration of alglucosidase alfa specific antibodies relates to the concentration of alglucosidase alfa during enzyme infusion (de Vries et al
2010). In the present study we have applied the same arithmetic method to infants. For example, patient 11 had an ELISA titer of 1:156,250 at a certain point of treatment. At that time the corresponding titer as measured by immune-precipitation was 40 nmol MUGlc/h.μL (Supplemental Fig.
2) implying that 1 mL of the patient’s serum contained enough antibodies to bind 0.13 mg alglucosidase alfa (de Vries et al
2010). As a child receives 0.24 mg of alglucosidase alfa per mL blood (based on 20 mg/kg and a circulating blood volume of 80 mL/kg), antibodies may bind as much as 54 % of the administered enzyme. By contrast, if the titer is only 4 nmol MUGlc/h.μL, corresponding with an ELISA titer of 1:6250, the circulating antibodies can bind only 5 % of the administered aglucosidase alfa.
Although these estimates are crude they indicate that ELISA titers of 1:6250 and lower probably have no clinical significance, whereas titers of 1:31,250 and higher may counteract ERT at a dose of 20 mg/kg. Accordingly, titers above 1:60,000 are expected to counteract ERT at a dose of 40 mg/kg. Our arithmetic estimates of what should be considered a high titer and what should be considered a low titer remarkably correspond to the cut-off value of 1:51,200 chosen by Banaguria et al (Banugaria et al
2011).
Antibodies can impede the effect of ERT in several ways. In four cases we observed a decrease of catalytic activity suggestive for binding of antibodies to the enzyme’s active site. Uptake was inhibited in three cases. In one case this was due to a combination of inactivation and uptake inhibition possibly by steric hindrance of the ligand-receptor binding. Of note, the effect of antibodies not only depends on the binding sites but also on the stoichiometry of antibodies and alglucosidase alfa in the experimental setting; changing the concentration of one of the two components can tip the balance. Whereas neutralizing antibodies were reported to be more common in CRIM-negative than in CRIM-positive patients (Kishnani et al
2010; Banugaria et al
2011), in our study their presence was not related specifically to the patients’ CRIM status but to the patients’ antibody titer.
It has been shown that high antibody titers in CRIM-negative as well as CRIM-positive patients are associated with shorter ventilator-free survival (Kishnani et al
2010; Banugaria et al
2011). In several adults with Pompe disease, a high titer was also accompanied by poor response to ERT (de Vries et al
2010; Patel et al
2012). Although our study group was too small and too varied to permit statistical analysis, patients with higher titers tended to attain fewer motor milestones. However, with respect to ventilator-free survival, only two of the six patients who developed respiratory insufficiency or died had a titer of ≥ 1:31,250 at the time that these events occurred. Two other patients had low titers, but nevertheless developed respiratory insufficiency at the age of 2. Two patients became ventilator dependent just before or soon after the start of ERT which we ascribe to their advanced disease at the start of ERT.
Conclusions and challenges
The outcome of classic infantile Pompe patients is determined by many factors, among which are the age and disease severity at initiation of ERT, the patient’s genotype, and the height of the antibody response. It is beyond question that high and sustained antibody titers need to be prevented in order to achieve a good response to ERT (Kishnani et al
2010; de Vries et al
2010; Banugaria et al
2011). Our study indicates that the negative effect of antibodies starts at an antibody titer of approximately 1:31,250 when the recommended dose of 20 mg/kg is used. Very early start of ERT (<2 months), achievable by neonatal screening, may help to keep the antibody titers low. Recent publications indicate that immune tolerance can be successfully induced by a combination of rituximab, methotrexate, and intravenous immunoglobulins (Mendelsohn et al
2009; Messinger et al
2012; Banugaria et al
2013b), a regimen likely to be most effective when used prophylactically (Lacaná et al
2012; Banugaria et al
2013a). As the patients’ genotype and related CRIM status alone are obviously not the sole determinants for the patients’ immune response, identification of other triggering factors remains a challenge. Though the number of patients is very small, it occurred to us that the three CRIM-negative patients in our study had an early demise while all CRIM-positive patients survived. If the difference is not made solely by the CRIM-status or antibody titer, might it be that CRIM-positive patients can have a tiny bit of acid α-glucosidase activity in their tissues that we cannot detect with our current methods?