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The Role of Neutralizing Antibodies in MS Treatments
Posted 09/30/2003
Mark S. Freedman, MD, MSc

Both interferon-beta (IFN-beta) and glatiramer acetate have been
established as effective disease-modifying agents for the treatment of
patients with multiple sclerosis (MS). Both of these medications
currently require continued administration by injection in order to
maintain their modest effect at reducing disease activity and slowing
disease progression. As part of the body's normal defense, the immune
system effectively reacts to foreign agents, and particularly to agents
introduced into the body via injection. Thus, in response to the
injection of protein-based products such as IFN-beta or glatiramer
acetate, the immune system typically reacts by producing antibodies.
Binding Abs and Neutralizing Abs

Although IFN-beta is manufactured by gene-recombinant technology that
uses the human gene, the process itself employs either bacterial
(Escherichia coli in the case of IFN-beta 1b) or mammalian (Chinese
hamster ovarian cells in the case of both IFN-beta 1a preparations) cell
lines to manufacture large quantities of drug. Thus, these cells are not
human, and sufficient (if slight) differences remain and can induce the
formation of Ab in some people. Although the protein sequence may be
identical to (in the case of IFN-beta 1a) or only slightly different
from (in the case of IFN-beta 1b) native IFN-beta, the way that sugars
are added (IFN-beta 1a) or not (IFN-beta 1b) in the final processing of
the protein may be important indicators to the immune system that the
agent is indeed "foreign." In the case of glatiramer acetate, the random
number and size of the polymers produced lead to a fairly widespread
immune response that might in fact be part of its mechanism of action.
If the immune system reacts to these injected agents, the response might
well be to generate antibodies.

Antibodies can simply bind to IFN-beta or glatiramer acetate (binding
Ab, or BAb) with no subsequent effect on function, or they can block or
neutralize (neutralizing Ab, or NAb) their biological activity. BAbs can
bind to the agent but do not sterically hinder its biological action; in
some cases BAbs might even extend the half-life of the agent, by
protecting it from elimination. NAbs will partially or completely
abrogate the biological action of an agent by either blocking an
important surface molecule needed for its activity or by interfering
with the binding of the agent to its receptor on a target cell. The type
of Ab and its affinity (stickiness) to either agent are important areas,
but beyond the scope of discussion here.

The development of BAbs or NAbs depends on the dose, frequency, and mode
of injection or route of entry (skin > muscle > intravenous > oral) of
an agent. A low dose given less frequently will induce very few Abs, but
higher doses given more often tend to induce greater Ab formation.
Interestingly, if too little or too much of a given agent is
administered, the production of Ab is actually inhibited -- a phenomenon
known as low- or high-zone tolerance.

The skin is the best site for stimulating Ab formation; hence, vaccines
are often administered subcutaneously. Not surprisingly, both IFN-betas
that are injected subcutaneously (Betaseron, Rebif) induce greater Ab
formation compared with the formulation that is injected into muscle
(Avonex). The big question in the case of MS agents is whether the
formation of NAbs compromises the efficacy of any of the treatments.
Let's look at the information that is available today to help answer
this question.
Antibody Assays

Before discussing the clinical data, several key issues concerning the
detection of Abs should be clarified. The definitions of BAb and NAb
have been derived from in vitro assays. Commonly, BAbs are found with a
capture assay that simply binds the molecule, if it can be found in
serum, and identifies it. The simplest and most widely used method is
the ELISA together with a "capture Ab." Such an assay detects only the
presence of an Ab, and does not provide any information about its
biological activity.

Whether or not the identified Ab is biologically active, which in this
case means "neutralizing," is an area of great controversy: the assays
rely on examining known biological actions of IFN-beta, even though no
single action has ever been clearly linked to its beneficial effect on
MS. One of the 2 most popular assays looks for the induction of a
protein (eg, Mx protein) known to respond stoichiometrically to
IFN-beta; the other, a cytopathic effect (CPE)assay, detects a basic
function of IFN-beta, that of protecting cells from being lysed by a set
amount of introduced virus. These assays are sensitive to even very low
titers of Abs.
In Vivo Effects of Antibodies

Two approaches have been used to elucidate the in vivo effects of Abs:
the first is to assess some of the biological response markers known to
be affected by IFN-beta; the second is to examine the results from
clinical trials. In the first approach, scientists measure certain
biological response markers in the serum of patients receiving an
IFN-beta before and after starting the agent, or compared with a
parallel group that is using only placebo. A normal response would show
elevations in the amount of the biological response marker, which would
remain elevated as long as the agent is being administered. The second
method examines MS-related outcomes in placebo-controlled or comparative
clinical studies. In both cases, it is possible to determine whether the
presence of NAb is at all linked to either smaller increases in levels
of a biological response marker, or a poorer clinical outcome.

Not surprisingly, the clinical trials have yielded some interesting but
controversial results. In the case of IFN-beta, although up to almost
80% of treated patients became BAb positive over time, NAbs were found
in only 2% to 47% of patients. This implies that the majority of Abs to
IFN-beta do not interfere with the biological action of the drug. The
presence of BAbs and particularly NAbs was time dependent, with most
positivity occurring after the first 6 months of treatment and fading
with time. The lower, less frequent dose of intramuscular IFN-beta was
associated with the lowest number of NAb-positive patients. Higher, more
frequent doses of the subcutaneously administered IFN-betas were
associated with a greater proportion of NAb-positive patients; however,
in comparative studies, both higher-dose agents were shown to provide
superior efficacy compared with the lower-dose formulation. In addition,
increasing the dosage of subcutaneous IFN-beta 1a led to a reduction of
NAb positivity, explained perhaps by the phenomenon of "high-zone
tolerance." In studies examining biological response markers, the group
of patients with particularly high NAb titers experienced changes in
biological response markers that were no different from those of the
placebo group, suggesting there was no longer any measurable (by
biological response marker) effect of IFN-beta.

As a group, especially using an interval analysis approach that examines
periods when patients are NAb positive or negative, the highest and most
persistently positive titers of NAb were associated with a greater
number of relapses, more progression, or a higher degree of MRI activity
in patients with MS. However, many individual patients with high NAb
titers had no measurable clinical activity, and others with undetectable
NAbs experienced clinical deterioration. Thus, it was only possible to
determine the effect of NAbs using the group data.

It would appear just as difficult to determine the negative effect of a
NAb as it is to measure the benefit of any single treatment in an
individual patient with MS. A final and curious finding is that in all
studies to date, the individuals who could be identified as those who
would eventually harbor NAbs achieved a greater reduction in relapse
rates during the first year compared with those who would never develop
a NAb titer of significance. Some interesting theories regarding this
latter phenomenon suggest that these patients may actually have a
different and perhaps less biologically potent native IFN-beta than NAb
negative patients. The introduction of a highly potent recombinant
product would therefore generate a greater clinical response when first
injected, but would also be regarded as more "foreign" by the host's
immune system, leading to a greater NAb response (see Opdenakker
review). It is possible that such patients may be naturally predisposed
to greater disease, explaining the association of NAb positivity with
negative clinical outcomes. Such a theory warrants further
investigation.

At this time, it is not possible to determine which patients will
develop the more significant NAbs when they begin therapy, but
scientists are searching for the clues that will identify such
individuals. The incidence of NAbs is greatest (30% to 40%) for
subcutaneous IFN-beta 1b (the most "foreign" of the IFN-betas), second
highest for subcutaneous IFN-beta 1a (12% to 14%), and lowest for
intramuscular IFN-beta 1a (2% to 5%). Although both IFN-beta 1a
molecules are identical, the difference in NAb rates is probably due to
the higher dose and frequency of administration of the subcutaneous
formulation. However, in direct comparative studies, both subcutaneous
formulations demonstrated superior clinical efficacy compared with the
intramuscular formulation, an overall treatment effect that stood out
even if you compared results for the former groups that were NAb
positive with those in the latter group who were NAb negative. In
addition, the majority of patients do not develop persistent NAbs and
the clinical benefits of IFN-beta in these NAb individuals are even
greater than those found for the group as a whole. It is therefore not
at all clear that we should consider sacrificing the greater efficacy of
the subcutaneous formulations in favor of intramuscular IFN-beta in
order to minimize the possible development of NAbs. It would also not
make sense to switch from a subcutaneous to the intramuscular
formulation in case NAbs do develop, because NAbs cross-react and could
just as well negatively affect treatment with the lower-dose
formulation.

Glatiramer acetate treatment has been associated with nearly 100% BAb,
and only recently has been associated with what appear to be NAbs in
some 47% of treated patients. Unlike the studies of IFN-beta, NAbs
associated with glatiramer acetate were determined on the basis of their
ability to "neutralize" some of the known in vitro immunologic effects
of glatiramer acetate that are presumably linked to their proposed
mechanism of action in producing the clinical benefit in patients with
MS.

No consensus has yet been reached regarding which Ab tests are the most
reliable, what titer is most important, when in the course of treatment
measurements should be taken, and most importantly, what, if anything,
should be done for patients who test positive. In addition, because NAbs
are not always associated with a negative clinical outcome, vary in
titer, and more than 50% will disappear completely within a few months
to years with continued IFN-beta treatment, it is not possible to
understand the true significance of being NAb positive. It is therefore
not surprising that experts worldwide have recommended that the response
to IFN-beta or glatiramer acetate treatment be judged clinically, and
that decisions on whether to continue on a given product not be based on
the result of a controversial NAb test.


Mark Freedman, Professor of Medicine (Neurology), University of Ottawa,
and Director, Multiple Sclerosis Research Clinic, The Ottawa Hospital,
Ottawa, Canada
http://www.medscape.com/viewarticle/461270?mpid=19289