OVERVIEW OF ORAL TOLERANCE THERAPY

Background Information

The immune system is a biological defense mechanism responsible for recognizing and fighting disease. It distinguishes foreign substances (antigens) from the body’s own tissues and eliminates a wide variety of pathogenic agents such as bacteria and viruses. T cells, specialized white cells which circulate throughout the body, are a major component of this system. There are several types of T cells, each of which plays a critical role in recognizing antigens, carrying out the immune response, or regulating the resulting chain of events. These include “helper” T cells, which release factors to amplify the immune response; “killer” T cells, which attack and destroy cells displaying the targeted antigen; and, “regulatory” T cells, which release factors to down-regulate or suppress the immune response and keep it from going out of control. Each of these different types of T cells comes from a common precursor, and it is the conditions under which the precursor is activated that determines the type of T cell produced. Another major component of the immune system, B cells, produce antibodies and perform other functions important in the response to antigenic stimulation.

How an individual responds to a foreign substance is determined by the genetics of their immune system and the method of antigen delivery (route and presence or absence of adjuvants), as well as on parameters related to the antigen itself: its form (soluble or particulate, native or denatured); the amount of antigen given, and the frequency with which it is administered. An individual’s genes determine whether or not they are capable of responding to an antigen, and if so, the potential strength of the response. The method of delivery determines the type of tissue and local milieu in which antigen presentation takes place and thus the conditions of T cell activation. The form of antigen determines how is is processed for presentation, and the amount of antigen determines the nature of the response. The frequency of antigen administration affects the size of the response.

In normal individuals, T cells with specificity for self-antigens are either eliminated during differentiation or “suppressed” by regulatory mechanisms. In cell-mediated autoimmune disease, killer T cells reactive for a self-tissue antigen become active and destroy healthy tissues and organs. The ultimate result is loss of organ functionality. Examples of cell-mediated autoimmune diseases include: Type I diabetes, where the pancreas is attacked with resultant loss of insulin production; multiple sclerosis, where brain or spinal cord tissue is attacked with a resultant loss of central nervous system (CNS) function; and, rheumatoid arthritis, where the cartilage in the joints is attacked and the resultant inflammation leads to joint destruction.

The immune system responds to foreign proteins encountered within most tissues of the body by generating helper T cells which release inflammatory cytokines such as IL-2 and IFN-g. The response to foreign proteins in mucosal tssue, in contrast, stimulates the induction of TGF-ß secreting cells and regulatory T cells which secrete IL-4 and IL-10. This cascade of events results in a suppressive regulatory response as well as induction of B cells that secrete IgA.

The oral tolerance approach for interrupting and suppressing the autoimmune disease process works by stimulating the natural mucosal immune mechanisms in the gut associated lymphoid tissues (GALT) of the small intestine. Autoimmune disease suppression is achieved by orally delivering (in an appropriate amount, form and frequency) protein(s) associated with the tissue under attack in an autoimmune disease. This type of tissue-specific autoimmune disease suppression is called oral tolerance therapy. More generally, proteins can be delivered to any mucosal tissue to induce the same type of immunogolical response (mucosal tolerance therapy).

Mechanisms of Oral Tolerance

Experimental evidence shows that oral tolerance can be induced by three different mechanisms: active suppression, clonal anergy and clonal deletion. Each of these has been shown to be associated with oral tolerance after antigen administration, either as a single mechanism or in combination. Antigen dose is the primary factor determining the form of peripheral tolerance that develops. The generation of tolerance due to regulatory T cells (active suppression) is favored by administration of low doses of antigen, whereas administration of high doses of antigen biases toward development of tolerance due to anergy or deletion. These distinct mechanisms of oral tolerance are not mutually exclusive and may occur concurrently.

Active Suppression
Low doses of antigen presented by gut associated antigen presenting cells preferentially induce regulatory T cells. Regulatory T cells specific for orally administered antigen migrate out of the gut to the lymphoid organs and into the general circulatory system. Upon encountering and recognizing the same or similar antigen in the target (diseased) tissue, the regulatory T cells are stimulated to secrete suppressive cytokines, such as TGF-b, IL-4 and IL-10. These suppressive cytokines, in turn, function to down-regulate the activated, inflammatory Th1 cells. This process is known as active suppression.

The action of the suppressive cytokines is not antigen specific, but stimulation of their secretion by regulatory T cells is dependent on recognition of specific antigenic peptides in the context of antigen presenting cell MHC by the T cell receptor (TcR). Since the tolerizing antigen functions to induce the regulatory T cells and to stimulate them to produce suppressive cytokines at the disease site, the tolerizing antigen need not be identical to the antigen that activates the inflammatory Th1 cells. The tolerizing antigen need only be located in the same vicinity in the target tissue as the antigen responsible for the inflammatory reaction. This phenomenon is called bystander suppression.

Active suppression has been shown to be a primary mechanism of oral tolerance in autoimmune disease models. Regulatory cells have been identified that act via the secretion of antigen-nonspecific, down-regulatory cytokines (TGF-b, IL-4, IL-10) when triggered by an oral antigen found in the diseased tissue. Regulatory cells can be found in Peyer’s patches 24-48 hours after a single feeding of an oral antigen such as myelin basic protein (MBP). The oral antigens do not need to attain access to the circulatory system or organs other than the gut associated lymphoid tissue in order to induce the regulatory cells involved in the suppression of the target autoimmune disease. Circulating blood levels of antigen are not required to induce active suppression.

Clonal Anergy
Clonal anergy is a condition that may result when high doses of oral antigen induce unresponsiveness in the immunoreactive Th1 cell function. The cells are not deleted, but are rendered intrinsically incapable of responding to a specific antigen in the context of their T cell receptor (TcR) and peptide associated with MHC. The anergy of these cells may be overcome by high concentration of cytokines capable of delivering a secondary stimulatory signal, such as IL-2. Clonal anergy is differentiated from clonal deletion by the presence of antigen-specific TcR clonotypes, and/or release of cells from the unresponsive state by pre-culture in IL-2. These antigen-specific cells are present, they are just incapable of responding. As concentration of antigen increases, Th1 cells are the first to be “turned off” or anergized. This mechanism may help reduce an inflammatory response associated with an autoimmune disease when the specific oral antigen dose is sufficiently high enough to anergize the Th1 cells involved in the initiation or perpetuation of the tissue-distinctive immune response.

Clonal Deletion
The third mechanism known to be associated with oral tolerance is clonal deletion, which results in elimination of antigen responsive cells. Deletion is the normal mechanism responsible for elimination of cells responsive to self proteins. Clonal deletion occurs naturally in the thymus gland during the ontogeny of the immune response and during the maturation cycle of T lymphocytes before they travel to the periphery. In the presence of high concentration of protein, deletion of cells specific for that protein can occur directly within the Peyer’s patch as well as in the thymus. Both Th1 and regulatory T cells may be deleted in response to oral antigen, in a dose-dependent fashion.

Recent work has clarified some of the differences in previously proposed mechanisms of orally induced tolerance. Side by side comparisons, within the same experiments, have demonstrated that high doses of oral antigen induce a state of tolerance characterized by anergy or weak active suppression with increased secretion of IL-4. By contrast, low doses of oral antigen induce a state of tolerance characterized by active suppression effected through soluble mediators, with increased secretion of TGF-b and IL-4, and minimal anergy. These results demonstrate that cytokine secretion profiles may vary with the amount of antigen administered.

In conclusion, oral tolerance is a natural immunological process that can be employed successfully in the treatment of autoimmune diseases. There are three mechanisms of tolerance induction: active suppression, clonal anergy, and clonal deletion. All three can result in suppression of disease, decreased proliferative responses, and decreased inflammatory responses at the site of autoimmune disease. The mechanism induced depends upon antigen dosage and frequency of administration. The figure below diagrammatically summarizes oral tolerance induction evoked at low and high antigen concentrations.

Research studies have shown that oral tolerance may be useful in treating autoimmune disorders by selectively suppressing the specific inflammatory immune responses associated with the disease. These studies have demonstrated that orally administered tissue associated antigens suppress disease in several experimental autoimmune models in a disease-specific fashion. See table below:

Model

• Arthritis (7 different types)
• Multiple sclerosis
• Hives
• Uveitis
• Myasthenia gravis
• Type 1 diabetes
• Transplantation
• Thyroiditis
• Tinnitus
• Colitis

Protein orally delivered

• Type II collagen
• MBP, PLP, MOG
• S-Ag, IRBP
• ArchR
• Insulin, GAD
• Alloantigen, MHC peptide
• Thyroglobulin
• Haptenized colonic proteins

It is not necessary to orally-dose an intact protein to induce the regulatory T cells. In animal models, small peptides have been shown to induce regulatory T cells when the appropriate amino acid sequences (antigenic determinants or epitopes) are present. An epitope capable of inducing tolerance in one species or inbred strain may or may not be capable of inducing tolerance in another strain.

AutoImmune Products

In developing an oral tolerance therapy for a particular disease, AutoImmune uses proteins found in the organs under attack. These proteins are delivered orally and broken down to fragments by the normal digestive processes. Specific fragments of these proteins (peptides) are taken up by antigen-presenting cells on the surface of the gut and processed for presentation to undifferentiated T cells. Regulatory T cells are induced and migrate through the blood and lymph system. Upon encountering antigen in the target organ, these regulatory T cells release cytokines which suppress inflammation, thereby ameliorating the disease. By appropriate selection of the amount and form of orally-delivered protein(s), active suppression is directed toward the specific tissue involved in the patient’s disease.

Because the proteins used in treatment are naturally derived, administered at low dose and subject to normal digestive processes, they are remarkably safe. The processing of the protein material to reach finished dosage form does not concentrate native impurities or processing residues. To date,there have been no serious adverse events attributable to drug product in any of our human clinical studies.

References

1. Weiner HL. Oral Tolerance: Immune Mechanisms and Treatment of Autoimune Diseases. Immunolgy Today 1997; 18:335-343.
2. Weiner, HL and Mayer, LF, editors. Oral Tolerance: Mechanisms and Applications. Annals of the New York Academy of Sciences 1996; 778: 1-453.
3. Weiner, HF, Mayer, LF and Strober, W., editors. Oral Tolerance: New Insights and Prospects for Clinical Applications. Annals of the New York Academy of Sciences 2004; 1029: 1-425.

 

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