Modulation of Inflammatory Response in Multiple Sclerosis by Altered Peptide Ligand (APL)
Silva Markovic-Plese, MD—Department of Neurology, University of North Carolina at Chapel Hill,
North Carolina School of Medicine, North Carolina
Introduction
The recent expansion in our understanding of the development of the disease in patients with multiple sclerosis (MS) has allowed therapies to be designed with ever-greater specificity. Some of these medications are even able to target interactions between particular molecules. MS is believed to be an autoimmune disease, in which a person’s immune system attacks the individual’s own myelin sheaths within the central nervous system (CNS). A potential new treatment described in this article seeks to directly influence the molecular interactions between immune cells and the myelin protein targets.
In autoimmune diseases, the immune system recognizes a “self antigen,†where an antigen is a molecule that causes an immune response. A set of cells, called T-cells, is critical for the immune attack, because these are the cells that recognize antigens and allow the immune system to “remember†the response to the same antigen in the future.
For a T-cell to recognize and bind to an antigen, the antigen has to be processed by another type of cell, called an antigenpresenting cell (APC). APCs have an important molecule on their surface called the major histocompatibility complex (MHC) molecule, which binds the antigen. T-cells carry on their cell surface “T-cell receptors†(TCR), whose job is to bind antigens on APCs and signal the T-cell to respond appropriately. TCRs will bind an antigen only if the binding site on the TCR matches the complex created by the antigen and the MHC molecule on the APC. TCRs are extremely variable, and each T-cell clone (or copy) carries only one type of TCR, so there is a good chance that at least one T-cell clone in the body will recognize a given antigen through its TCR.
Once the TCR binds the antigen-MHC complex, the TCR will signal the T-cell to respond. If there is a poor match between the TCR and the antigen-MHC complex, the TCR may send a weak signal that induces only a partial immune response. For example, the T-cell might secrete chemicals that influence the immune system, but it might not divide. With a good match, the T-cell can mount a full immune response. The T-cell replicates itself into a clone of cells that recognize the same antigen, and every cell in the clone can secrete powerful immune chemicals that may mobilize other cells to attack. This kind of response becomes a chain reaction that leads to inflammation. Inflammation in MS brain lesions is believed to cause much of the nerve tissue destruction associated with the disease.
One approach to treating autoimmune diseases is to disrupt the binding between the MHC, antigen, and TCR. This could be done by misleading the TCR into binding an altered antigen that closely resembles the native one (see Bielekova and Martin, 2001, for a review). The altered peptide antigen could then affect the T-cell differently from the real native one (Figure 1). For example, it might cause a weak immune response or drive the T-cell into a state called “anergy†where it cannot respond to antigen. Or, it might influence the T-cell to differentiate into a clone that releases chemicals that inhibit inflammation. This final scenario is the most likely to be beneficial to a patient with autoimmune disease. When T-cells mature, they become T helper (TH) cells. One kind of TH cell, TH1, promotes inflammation. Another TH cell, TH2, releases chemicals that down-regulate (inhibit) inflammation. Inducing the clones to become TH2 cells
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is an important approach to therapy of autoimmune diseases. Unfortunately, this result is hard to achieve, because the path taken by the young T-cells depends in large part on the state of the immune system when the altered peptid ligand (APL) is injected. Based on the trials described below, however, it is now known that low doses of the APL tend to induce TH2 cells and high doses TH1 cells. The ideas described above are currently being tested in clinical trials. The altered antigens are referred to as APLs because they are made of a short stretch of amino acids, called a peptide, that has been altered so that it resembles the native antigen but binds the TCR and/or MHC differently. It is called a “ligand†because that word is used to describe any molecule that binds a receptor.
In the case of MS, there is considerable evidence that immune cells recognize and attack proteins that are part of the fatty nerve sheath, myelin, which is destroyed in the disease process. In fact, patients with MS often have a lot of T-cells in their blood that bind myelin proteins, and a particular protein, myelin basic protein (MBP), is common target. Only a small part of the protein is responsible for most of the immune responses, the peptide made from the 83rd to the 99th amino acid in MBP. This peptide is therefore an attractive target for using APLs to alter the immune response.
APLs as Therapeutic Agents in Treatment of Animal Models of MS
The first attempts to use APLs to treat an MS-like disease were carried out in rodents with experimental autoimmune encephalomyelitis (EAE), a demyelinating disease that mimics MS in several respects. Although successful in several cases, the animal experiments also illustrate possible difficulties with using APLs to treat autoimmune diseases (Bielekova and Martin, 2001; Kappos, Comi, Panitch, Oger, Antel et al. 2000).
EAE is experimentally induced in rodents, usually by injecting into them a myelin protein or a peptide from a myelin protein.
“Only a small part of the protein is responsible for most of the immune responses, the peptide made from the 83rd to the 99th amino acid in MBP.â€
The animals develop neurological symptoms that worsen with repeated exacerbations. One peptide that causes EAE is part of the myelin basic protein, MBP87-99, which is quite close to the peptide sequence that often causes an immune response in humans with MS. When APLs were used to try to treat EAE, an APL that was based on the MBP87-99 peptide successfully treated EAE caused by injecting MBP87-99, but only in a given rodent strain. The same APL did not improve EAE symptoms in other rodent strains. This effect is explained by the genetic differences among the strains. In a given strain, all the animals are genetically identical. Strains differ genetically, and an important difference, in particular, is in their MHC molecules. Since TCRs recognize antigens only when bound to MHCs, the kind of MHC helps determine whether or not a TCR will recognize a given antigen.
Interestingly, even in the same rat strain, an APL based on MBP87-99 that did treat EAE caused by injecting MBP87-99, did not treat EAE caused by injecting a different part of the same molecule, MBP68-88. Since humans are genetically very diverse, this suggests that a single APL that works in one person or a set of people could be completely ineffective in others.
Animal experiments also suggest that APLs are unlikely to work by simply knocking out given TCR-antigen interactions. When animals are immunized by injecting a given peptide, many different T-cells may respond, because their different TCRs recognize different sets of amino acids in the peptide. An APL with a single amino-acid change might stop the response of one T-cell clone, but it could be very difficult to design APLs that would block all the reactive TCRs.
Finally, once inflammation is under way and tissue is damaged, the immune system is exposed to other self-antigens and can react to them as well, resulting in immune reactions to a number of different proteins. Preventing T-cell responses to a given peptide with an APL would not block the immune responses to a wide number of different proteins.
A better strategy is to influence T-cells to divide into TH2 clones. The chemicals released by these cells would down-regulate (or reduce) inflammation caused by TH1 cells. This effect of one set of immune cells on others is known as the “bystander effect.†For the bystander effect to work, the TCR that responds to the APL by making a clone of TH2 cells must also recognize the native antigen, so that the TH2 cells will migrate to the lesion site and bind the native antigen there, where the inflammation needs to be down-regulated. This approach has been used effectively to treat EAE.
APL Treatment of MS
The APL used in the first (Phase I) trial of APLs in humans with MS was based on MBP83-99 with two amino acid changes as illustrated in Figure 1. (see Bielekova & Martin, 2001; Bielekova et al., 2000). The subjects received 1, 5, 20, or 50 mg APL as weekly subcutaneous injections for 4 weeks. The injections were well tolerated, even at 50 mg, although there was often inflammation at the injection sites. In several patients, T -cells specific for the APL clearly increased in number.
Phase II Trials
Two Phase II trials have now been conducted (Bielekova et al., 2000; Kappos et al., 2000). For the Bielekova study, a 24-subject trial was planned. The patients’ baseline disease activity was established by evaluating the patients by magnetic resonance imaging (MRI), clinical examination, and immunological status assays for 6 months. After the baseline period, the patients were given 50 mg APL weekly by subcutaneous injection for 9 months. Unlike the Phase I trial, the 50 mg dose was poorly tolerated. After reviewing the data for the first seven patients, the safety board recommended a lower dose, and the eighth patient was given 5 mg APL weekly, to be changed to 5 mg monthly after the first 4 weeks. Of the eight patients, only one concluded the 9 months of treatment. Three patients developed unusual exacerbations, and at least two of these exacerbations were probably caused by the APL treatment.
Despite the poor toleration of the high dose, on average, the disease activity of these patients was not significantly changed as assessed clinically and by MRI. Individually, the disease activity of two patients improved, three stayed the same, and three had worse MS at the end of treatment. Interestingly, both patients who improved had a lower immune response to MBP83-99, and the three who were worse had a stronger immune response to MBP83-99 peptide after treatment than at the baseline of the study.
To examine the response in T-cells, the cells were isolated from the subjects’ blood, and individual T-cells were allowed to divide in culture, where their reactivity to the APL and native antigens could be tested and their pro- or anti-inflammatory differentiation nature could be determined. The APL treatment resulted in a 2- to 1,000-fold increase in T-cells that reacted with the APL in all the patients, and 70% of the T-cells that recognized MBP also recognized the APL, which showed that the cross-reactivity required for the bystander effect had been achieved. Unfortunately, for the bystander effect to work, the cross-reactive T-cells also must be of the TH2 type, and the APL treatment resulted in the APL-reactive T-cells, as well as the MBP-specific T-cells, being mostly of the TH1 type. This was probably caused by a combination of giving the APL subcutaneously, using a high dose and frequent injections, and by the preexisting immune state of the patients with MS, which favored a TH1, that is, proinflammatory, response.
A larger Phase II multi-center study study has also been carried out (Kappos et al., 2000). This study was planned as a multicenter, double blind, placebo-controlled study. One hundred forty-two patients with relapsing-remitting MS (RRMS) were enrolled. They were divided into four groups, one to receive placebo, and the other three to receive 5, 20, or 50 mg APL weekly for 4 months. The treatment phase followed an initial 1-month baseline study.
The subjects were monitored monthly by MRI, and each MRI was compared with the two baseline scans. The number of new gadolinium-enhancing lesions and their volume were monitored. The enhancing lesions indicate active inflammation. There was no significant difference in the frequency or the number of relapses in any of the groups receiving APL compared with those receiving placebo. The trial was stopped because 13 of the 142 subjects showed a “hypersensitivity-type†reaction (itching, nausea, abdominal pain, rash, hives) immediately after an injection. The symptoms did not require treatment and usually resolved in 2 hours. Most of these reactions occurred after the subject had received more than ten injections and a high dose of APL. MRI studies did not show worsening of the MS among the subjects with hypersensitivity reactions. One promising finding was that patients in this study tended to have a TH2 rather than a TH1 response to the APL.
Overall, neither Phase II trial showed significant changes in clinical or MRI measures of MS diseases activity. The analysis of a subgroup of subjects in the multi-center trial suggested there was a lower volume of enhancing lesions in patients who received the lowest 5 mg dose compared with placebo.
A Planned Randomized, DoubleBlind, Placebo-Controlled Study
A new study of APLs is planned to evaluate the safety, tolerability, and efficacy of an MBP83-99 APL in patients with RRMS. It is to take place at 25 study centers in the US and Canada and will evaluate treatment with 5 mg APL injected subcutaneously for 9 months. This study is presently recruiting patients.
The Future of APL
The current studies have shown that T-cells specific for MBP83-99 in at least some patients with MS are a clearly a valid target for modulating the immune system with antigenspecific agents. A better understanding of how T-cells contribute to autoimmune disease and how T-cells and their effects differ in individual patients, however, is required before specific immunotherapies, like APL, can be used effectively and safely. In the future, we will also have to consider alternative routes of administration (intranasal or oral) or use adjuvant (additional) chemicals with the APL injections that will help skew the T-cell response toward TH2. In designing studies, it may also be helpful to consider the similarities between APLs and glatiramer acetate, which also works in part as APL by preventing T-cell responses to MBP and/or eliciting TH2 responses.
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