The clinical effect of neutralizing antibodies

against interferon-beta is independent

of the type of interferon-beta used for patients
with relapsing-remitting multiple sclerosis
N Koch-Henriksen
1
, PS Sorensen
2
, K Bendtzen
3
and EM Flachs
4,*
Objective To establish whether the clinical effect of neutralizing antibodies (NAbs) against
interferon-beta (IFN) depends on the type of IFN (1a or 1b) used for treatment of patients with
relapsing-remitting multiple sclerosis (MS).
Introduction NAbs against IFN-1b appear faster and may be more evenly distributed on IgG sub-
classes, whereas NAbs against IFN-1a develop more slowly and may be devoid of IgG3. This might
cause different clinical responses to NAbs.
Design/patients All Danish MS-patients who had started first-time treatment with IFN-1a 22 g s.c
tiw (Rebif22) or IFN-1b 250 g s.c. qod (Betaferon) before January 1st 2003 were included.
Relapses were recorded at bi-annual visit.
Methods We measured NAbs every 12 months using a clinically validated cytopathic effect assay.
A blood sample with a neutralizing capacity of 20% or more was considered as NAb-positive. We
used a mixed logistic regression analysis in which NAb-status (three levels), IFN-preparation, and
time since treatment started were included as explanatory variables, and relapse rate as response
variable.
Results In 1,309 patients, who were observed for 21,958 months, 32.3% were classified as NAb-
positive. The odds-ratio (OR) for relapses in NAb-positive months compared with NAb-negative
months was 1.25; P = 0.02. The risk of relapses was higher with Betaferon than with Rebif22 (OR
1.26; P < 0.01). The effect of NAb-level on relapses was independent of whether the patients were
treated with Betaferon or Rebif22 (P = 0.89) and of time (P = 0.80).
Conclusion NAbs caused by IFN-1a s.c. do not differ from NAbs caused by IFN-1b in their detri-
mental clinical effect. Multiple Sclerosis 2009; 15: 601605. http://msj.sagepub.com
Key words: interferon-beta; interferon-beta antibodies; multiple sclerosis; neutralizing antibodies;
therapy; treatment effect
1
Department of Neurology, Aarhus University Hospital in Aalborg, Denmark and The Danish MS Treatment Register,
Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark
2
Danish Multiple Sclerosis Research Center, Department of Neurology, Copenhagen University Hospital Rigshospitalet,
Copenhagen, Denmark
3
Institute for Inflammation Research, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark
4
National Institute of Public Health, University of Southern Denmark, Copenhagen, Denmark
*
On behalf of the Danish Multiple Sclerosis Study Group: Per Soelberg Sorensen, Rigshospitalet, Copenhagen; Finn
Sellebjerg, Rigshospitalet, Copenhagen; Jette Lautrup Frederiksen, Glostrup University Hospital; Kai Jensen, Hillerod
Hospital; Anne Heltberg, Roskilde Hospital; Ole Kristensen, Holbk Hospital; Sven Deth, Naestved Hospital; Claus
Madsen, Odense University Hospital; Egon Stenager, Vejle, Esbjerg, and Sonderborg Hospital; Thor Petersen, Aarhus
University Hospital; Bjarne Sivertsen, Viborg Hospital; Jesper Torring, Holstebro Hospital; Nils Koch-Henriksen; Aarhus
University Hospital in Aalborg/Danish MS Registry.
Correspondence to: Nils Koch-Henriksen, Aarhus University Hospital in Aalborg/Danish MS Registry.
Email: koch-henriksen@stofanet.dk
Received 6 August 2008; accepted 20 November 2008
RESEARCH PAPER Multiple Sclerosis 2009; 15: 601605
The Author(s), 2009. 10.1177/1352458508101946
Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav
Introduction
A substantial proportion of patients treated with
interferon beta (IFN) for relapsing-remitting multi-
ple sclerosis (MS) develops neutralizing antibodies
(NAbs). Several studies have demonstrated that
presence of NAbs hampers the clinical effect of
IFN-1b [13] as well of IFN-1a [36].
A possible differential clinical importance of
NAbs in patients treated with different types of
IFN has recently been suggested in a study claim-
ing, on indirect evidence, that NAbs against IFN-
1b are not associated with disease worsening [7].
According to these authors, difference, if existing,
might be explained by different antibody responses
to IFN-1b and IFN-1a in terms of NAb levels
[811] and differences in IgG-subclass distribution
of the NAbs [11]. These differences might cause dif-
ferent clinical consequences of NAbs in patients
treated with IFN-1a and IFN-1b.
The aim of this study was, therefore, to compare
the effect of NAbs on relapse rates in patients trea-
ted with IFN-1a 22 g sc tiw (Rebif22) and IFN-1b
250 g s.c. qod (Betaferon).
Patients and methods
As a part of a national protocol for disease modify-
ing treatment of MS, a national MS treatment regis-
ter was established in 1996 [12]. In this register, we
have recorded all Danish patients treated with
IFN. The use of IFN treatment for MS in Denmark
is limitedtodepartments of neurology, andall depart-
ments of neurology send record forms with clinical
information fromall obligatory visits every 6 months
to the database.
We included patients with relapsing-remitting
MS who started first-time treatment with IFN-1a
22 g s.c tiw (Rebif22) or IFN-1b 250 g s.c. qod
(Betaferon) before January 1st 2003. In Denmark,
22 g tiw was the preferred dose of Rebif before Jan-
uary 1st 2004. The reasons for not including
patients treated with IFN-1a 30 g once a week
(Avonex) was that Avonex would be a possible con-
founder because 1) we predominantly use Avonex
for cases with low disease activity and 2) Avonex is
less liable to cause NAbs [13]. This decision was
made a priori.
At the regular visits, relapses were assessed by his-
tory and physical examination and is defined
according to Schumacher [14] as the appearance of
a new symptom or worsening of an old symptom
attributable to MS, accompanied by an appropriate
new neurological abnormality or focal neurological
dysfunction lasting at least 24 h in the absence of
fever and preceded by stability or improvement for
at least 30 days.
Blood samples for measurements of NAbs against
IFN were collected by venipuncture at least 48 h
after an injection of IFN, and the sera were isolated
and stored at 20C until assayed.
We used an antiviral neutralization bioassay
for detection of NAbs, essentially as previously
described [15]. Briefly, MC-5 cells, a subclone of
A549 cells, were seeded in microtrays at a concen-
tration of 15,000 cells per well and incubated for
24 h at 37C in a humidified 5% CO
2
atmosphere.
IFN (using recombinant IFN homologous to the
treatment received by the individual patient) at a
concentration of 10 LU per ml was preincubated
with diluted serum at 5% concentration in a vol-
ume of 240 l. After 1 h, 200 l were transferred to
the A549 cells. The cells were challenged with
encephalo-myocarditis virus at the lowest concen-
tration that by itself caused 100% cell death. After
another 24 h, the antiviral effect of IFN was mea-
sured by use of a 3-(4,5 dimethyl thiazol-2-yl)-
2,5-difenyl tetrazolium bromide assay. To avoid
false-positive and false-negative results, controls
for endogenous antiviral activity and serum toxicity
were included in each assay. The neutralizing capa-
city (NC) of each serum sample was measured;
i.e. the percentage of the added IFN that had
been neutralized by the NAbs. The investigators in
the laboratory had no knowledge of the patients
disease characteristics. We defined a NAb-negative
sample as a sample with NC < 20%, a low-NAb-
positive blood sample as a sample with NC 20
79%, and a medium/high NAb-positive blood
sample as a sample with NC of 80% or above. The
cut-off value for being Nab positive was chosen as
the lowest concentration of NAbs that had signifi-
cant clinical importance as shown in a previous
study [3]. The MS Treatment Register was notified
of the results of all NAb-tests. As clinically relevant
NAbs usually do not appear until 912 months after
initiation of IFN therapy [13], we used the NAb
tests from the 12-, 24-, 36-, 48-, and 60-month visits
in this study.
The ethical committees were not specifically
involved in this study because it was a pure retro-
spective observational study made on patients who
were treated with approved drugs on the basis of
national recommendations. Measuring NAB on reg-
ular basis on all IFN-treated patients is obligatory in
Denmark, a decision that has been approved by the
National Ethics Committee.
Statistical methods
We used the interval analysis or all switches
considered method for defining NAb-positivity
[16,17]. For a period of a half-year before and a
half-year after a NAb-test was taken, each month
602 N Koch-Henriksen et al.
Multiple Sclerosis 2009; 15: 601605 http://msj.sagepub.com
was classified either as NAb-negative or NAb-
positive according to the result of the NAb-test. In
the simple calculations, each individual month of
treatment was regarded as a unit, characterized by
relapse/no relapse, preparation, and NAb-status (+/).
The analyses were performed with 20% NC as cut-off
value. We also employed a mixed logistic regression
analysis in which the unit was the individual itself
(rather than the individual month) with relapse rate
as the response variable and NAb status (+/), prepa-
ration (Rebif22/Betaferon), and time (period 15,
representing the months 718, 1930, 3142, 4354,
and 5566, counted from treatment start) as the
explanatory variables.
Results
In all, 1,309 patients with clinically definite MS,
398 men and 911 women, fulfilled the criteria of
having started first-time IFN treatment with
Rebif22 (n = 892) or Betaferon (n = 417) before Jan-
uary 1st 2003. Clinical and demographic character-
istics by treatment are shown in Table 1. The
patients were observed for a total of 21,963 months
of which 7,098 (32.3%) were classified as NAb-
positive according to the above given definition.
With Rebif22, 3,927 out of 12,494 months of obser-
vation (31.4%) were NAb-positive, and with Beta-
feron 3,172 out of 9,468 months of observation
(33.0%) were NAb-positive; P = 0.001, (tied observa-
tions). Among the Nab-positive blood tests, 77%
were medium/high positive (NC 80%) with
Rebif22 and 67% with Betaferon (P = 0.004, see
Figure 1). The cumulative risk of being NAb positive
was at 12 months: 0.19 for Rebif22 and 0.35 for
Betaferon, at 24 months: 0.35 for Rebif22 and 0.48
for Betaferon, and at 36 months: 0.55 for Rebif22
and 0.56 for Betaferon. The risk of relapses was
higher with Betaferon than with Rebif22 (odds-
ratio [OR] 1.31, P = 0.0002), which was true for
Nab-positive months (OR 1.43; 95% CI 1.141.78;
P = 0.002) as well as for NAb-negative months (OR
1.22; 95% CI 1.031.46; P = 0.028).
The annualized relapse rate in the NAb-positive
months was 0.54 compared with 0.41 in the NAb-
negative months (OR 1.32, P < 0.0001).
In a mixed logistic regression analysis, we
included the three main effects: period, preparation,
and NAb, and all possible second-order interaction
terms between the three explanatory variables. In
this setting, we partitioned NC of NAbs into three
levels: (negative: <20%; low positive: 2079%;
medium/high positive: 80%).
With this, the OR for relapses in all NAb-positive
months (low positive as well as medium/high posi-
tive) compared with NAb-negative months was 1.25;
P = 0.02. The OR for relapses in medium/high NC
Nab-positive months compared with Nab-negative
months was 1.28 (95% CI: 1.051.55). The OR of
having relapses on treatment with Betaferon com-
pared with Rebif22 was 1.26; (95% CI 1.061.51;
P < 0.01). Period as single factor did influence
relapse rate statistically significantly with an OR of
0.85 per step (95% CI 0.790.90), and this effect was
linear.
We aimed at determining whether preparation
influences the effect of NAbs on relapse frequency.
The null-hypothesis was tested by estimating the
significance of the interaction term NAb*prepara-
tion in the mixed logistic regression equation. The
effect of NAb-status on relapses did not differ
between the two preparations (P = 0.89), meaning,
that NAbs have the same detrimental effect with
Betaferon as with Rebif22. Moreover, there was no
0
200
400
600
800
1000
Rebif 22 Betaferon
N
u
m
b
e
r

o
f

N
A
b
-
t
e
s
t
s
<20%
20-79%
80+%
67.9%
7.3%
24.8%
66.3%
11.2%
22.5%
Figure 1 Absolute and percentage distribution of neutra-
lizing capacities (NC) on negative (NC < 20%), medium
positive (NC 2079%) and highly positive (NC > 80%)
NAb-tests with Rebif22 and Betaferon.
Table 1 Clinical and demographic characteristics at baseline
IFN-1a 22 g
s.c. qod n = 417
IFN-1b 250 g
s.c. tiw n = 892
Female:male ratio 2.37 2.14
Age at treatment start
Mean 37.9 38.2
Minmax 1368 1664
Duration of MS, years
Mean 5.5 7.1
Minmax 044 051
Baseline EDSS
Mean 2.65 2.86
Minmax 06.5 06.0
#Relapses 24 months prior to treatment
Mean 2.56 2.89
Minmax 18 08
IFN, interferon beta; MS, multiple sclerosis; EDSS, expanded
disability status scale.
IFN preparation and clinical effect of neutralizing antibodies 603
http://msj.sagepub.com Multiple Sclerosis 2009; 15: 601605
interaction between period and the effect of NAbs
(P = 0.80).
Discussion
We found no indications of less harmful consequ-
ences of NAbs on clinical relapses in NAb-positive
patients treated with Betaferon as hypothesized
by others [7]. Differences in NAb-levels are likely
to impact the clinical consequences of NAbs.
Although moderate and high concentrations of
NAbs reduce or abolish IFN bioactivity, patients
with low NAb concentrations may have retained a
full or partial in-vivo response to IFN. According to
one study, NAb-positive patients treated with IFN-
1b had higher NAb titers compared to NAb-positive
patients on IFN-1a s.c. [11], but two other studies
have shown higher titers with IFN-1a s.c. [10,18],
which also is in accordance with the present
study. This, if true, would hypothetically favor
NAb-positive patients treated with IFN-1b. The dif-
ference between the above-mentioned study [11]
and the present study could be ascribed to differ-
ences in NAb-assay or cut off for NAb positivity.
We did not use a quantitative immunochemical
method for IFN antibody measurements as these
would detect binding anti-IFN antibodies, which
might not neutralize the clinical effects of IFN.
Rather, we used a semi-quantitative functional
assay for ex vivo IFN neutralization to grade blood
samples as NAb-negative, low-level NAb-positive, or
medium/high-level NAb-positive with a ceiling
effect, preventing us from distinguishing very high
levels of NAb from medium/high levels. Further
titration of Nab concentrations is of limited interest
for the risk of having relapses, as all patients with
medium/high Nab levels have lost their in vivo bio-
availability of IFN and thereby lost the therapeutic
benefit of IFN [17].
Another difference between NAbs raised against
IFN-1b and NAbs raised against IFN-1a is the per-
sistence of NAbs. Nab-positive patients treated with
IFN-1b have a higher probability of reverting to
the NAb-negative state within a treatment period
of 35 years [13], and this would favor IFN-1b if
the once positive, always positive method is used
for evaluation of the clinical significance of NAbs
[16]. However, we used the interval analysis or
all switches considered method for defining NAb-
positivity in order to eliminate the error caused by
patients who regain the therapeutic effect of IFN,
as they revert to the NAb-negative state [19].
We found that, even when accounting for NAb-
status, the OR for relapses with Betaferon was 1.26
compared with Rebif22 (P < 0.001). This difference
is probably due to the circumstance that Betaferon
was approved 2 years before Rebif22, and during
this interval, we gradually began to treat patients
with slightly less active disease.
The IgG-subclass distribution in NAb-positive
patients seems to vary with IFN preparation and
time [11]: In Betaferon-treated NAb-positive patients,
all four subclasses of IgG may be induced with IgG1
and IgG3 peaking the first year and IgG4 later on.
In Rebif22-treated NAb-positive patients, the IgG-
subclass profile is dominated by IgG4 emerging
later, and it has been suggested that a distinctive
pattern of IgG-subclasses could be of functional sig-
nificance [11]. We did not perform IgG-subclass
measurements of our blood samples, but our results
do not support the idea that any difference in IgG-
subclass distribution caused by the IFN-preparation
should influence the clinical effect of NAbs, as an
interaction term between preparation and the effect
of NAbs was far from significant and did not even
show a weak trend. Neither did the proposed tempo-
ral differentiation in distribution of IgG-subclasses
result in a time effect on the clinical consequences
of being NAb-positive, as the interaction between
time (period) and clinical NAb-effect was nonexist-
ing (P = 0.90).
In conclusion, we found no indication of less
detrimental effects of NAbs on clinical relapse rate
in NAb-positive patients treated with IFN-1b
compared with NAb-positive patients treated with
IFN-1a s.c.
Acknowledgements
Economic support to the Danish Multiple Sclerosis
Research Center was obtained from the Danish
Multiple Sclerosis Society, the Warwara Larsen
Foundation, the Danish Medical Research Council,
the European Union Sixth Framework Programme:
Life sciences, Genomics and Biotechnology for
health: Contract No. 18926: Neutralizing antibodies
in multiple sclerosis (NABINMS). The MS Treatment
Register is funded by the Copenhagen Hospital
Council and by the Association of Danish Regions.
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