Protein Immunogenicity

Most traditional pharmaceuticals are relatively low molecular weight substances and generally escape the attention of the immune system. Proteins, on the other hand, are macromolecules and display molecular properties that can potentially trigger a vigorous immune response. During its formation our immune system develops tolerance to self antigens. Such immunological tolerance is generally maintained throughout our lifetime by various regulatory mechanisms that either :-

i) Prevent B- and T-lymphocytes from becoming responsive 
    to self-antigens.

OR

ii) That inactivate such immune effector cells once they 

     encounter self-antigens.

Based on the above principles, it might be assumed that a therapeutic protein obtained by direct extraction from human sources or produced via recombinant expression of a human gene/cDNA sequence would be non-immunogenic in humans whereas ‘foreign’ therapeutic proteins would stimulate a human immune response. This general principle holds in many cases, but not all. 


So why do therapeutic proteins of human amino acid sequences have the potential to trigger an immune response?


Potential reasons can include:


1)      Differences in post-translational modification.

Human therapeutic proteins produced in several recombinant systems (e.g. yeast, plant and insect based systems) can display altered post translational modification detail, particularly in the context of glycosylation. Some sugar residues/motifs characteristic of these systems can be highly immunogenic in humans.

2)      Structural alteration of the protein during processing

         or storage.

Suboptimal product processing or formulation can result in partial degradation, denaturation, aggregation or precipitation of the therapeutic protein. Epitopes normally shielded from immune surveillance may be exposed as a result, triggering an immune response.

3)      Dosage levels and duration of treatment.

High dosage levels (well above normal physiological ranges), in particular if a product is administered on an ongoing and regular basis, may potentially contribute to breaking self-tolerance, particularly if combined with any of the circumstances outlined in the surrounding bulleted points.

4)      Genetic or immunological factors.

Some individuals may display underlining or induced immunological abnormalities, rendering them more susceptible to breakdown of self tolerance. For example, some blood factor and hormone preparations isolated by direct extraction from human serum or tissue stimulated an immunological response in a proportion of human patients receiving them. This may be triggered by some immune deficiency in the patients themselves, although the presence of product impurities or structural altered product forms may also be contributing factors.

Even if a biopharmaceutical triggers an immune response, it does not automatically follow that the response will be clinically significant or undesirable. In some instances, anti-product antibodies have no effect upon safety or efficacy. In other instances, antibody binding may alter the product’s pharmacokinetic properties or directly neutralize the biopharmaceutical’s biological activity. Even more seriously, antibodies raised against the product could potentially cross-react with the endogenous form of the protein, neutralizing it. Eprex provides an example of this latter phenomenon. Antibodies formed against the product cross-reacted with endogenous EPO, causing shutdown of (EPO-stimulated) red blood cell production, triggering antibody-mediated pure red cell aplasia.

A number of approaches may be adopted in an attempt to reduce or eliminate protein immunogenicity. Protein engineering, for example, has been employed to humanize monoclonal antibodies. An alternative approach entails the covalent attachment of polyethylene glycol (PEG) to the protein backbone. This can potentially shield immunogenic epitopes upon the protein from the immune system.

Thrombopoietin (TPO)

A human thrombopoietin (TPO) is a 332 amino acid, 60 kDa glycoprotein, containing six potential N-linked glycosylation sites. These are all localized towards the C terminus of the molecule. The N-terminal half exhibits a high degree of amino acid homology with EPO and represents the biologically active domain of the molecule.

TPO is the haemopoietic growth factor now shown to be the primary physiological regulator of platelet production. This molecule may, therefore, represent an important future therapeutic agent in combating thrombocytopenia, a condition characterized by reduced blood platelet levels. The most likely initial TPO therapeutic target is thrombocytopenia induced by cancer chemo- or radio therapy. This indication generally accounts for up to 80 per cent of all platelet transfusions undertaken. In the USA alone, close to 2 million people receive platelet transfusions annually.

Platelets (thrombocytes) carry out several functions in the body, all of which relate to the arrest of bleeding. They are disc-shaped structures 1–2 μm in diameter, and are present in the blood of healthy individuals. They are formed by a lineage-specific stem cell differentiation process. The terminal stages of this process entail the maturation of large progenitor cells termed ‘megakaryocytes’. Platelets represent small vesicles that bud off from the megakaryocyte cell surface and enter the circulation.

Leukocytes, their Range & Function

Leukocytes (white blood cells) encompass all blood cells that contain a nucleus, and these cells basically constitute the cells of the immune system. They thus function to protect the body by inactivating and destroying foreign agents. Certain leukocytes are also capable of recognizing and destroying altered body cells, such as cancer cells. Most are not confined exclusively to blood, but can circulate/exchange between blood, lymph and body tissues. This renders them more functionally effective by facilitating migration and congregation at a site of infection.

Leukocytes have been subclassified into three families: mononuclear phagocytes, lymphocytes and granulocytes. These can be differentiated from each other on the basis of their interaction with a dye known as Romanowsky stain.

Mononuclear phagocytes consist of monocytes and macrophages, and execute their defence function primarily by phagocytosis. Like all leukocytes, they are ultimately derived from bone marrow stem cells. Some such stem cells differentiate into monocytes, which enter the bloodstream from the bone marrow. From there, they migrate into most tissues in the body, where they settle and differentiate (mature) to become macrophages (sometimes called histocytes). Macrophages are found in all organs and connective tissue. They are given different names, depending upon in which organ they are located (hepatic macrophages are called Kupffer cells, central nervous system macrophages are called microglia, and lung macrophages are termed alveolar macrophages). All macrophages are effective scavenger cells, engulfing and destroying (by phagocytosis) any foreign substances they encounter. They also play an important role in other aspects of immunity by producing cytokines, and acting as antigen-presenting cells.

MONOCYTES

Lymphocytes are responsible for the specificity of the immune response. They are the only immune cells that recognize and respond to specific antigens, due to the presence on their surface of high-affinity receptors. In addition to blood, lymphocytes are present in high numbers in the spleen and thymus. They may be sub-categorized into antibody-producing B-lymphocytes, T-lymphocytes (which are involved in cell-mediated immunity) and null cells.

T-lymphocytes may be subcategorized on a functional basis into T-helper, T-cytoxic and T-suppressor cells. T-helper cells can produce various cytokines which can stimulate and regulate the immune response. T-cytotoxic cells can induce the lysis of cells exhibiting foreign antigen on their surface. As such, their major target cells are body cells infected by viruses or other intracellular pathogens (e.g. some protozoa). T-suppressor cells function to dampen or suppress an activated immune response, thus functioning as an important ‘off’ switch.

Most T-helper cells express a membrane protein termed CD4 on their surface. Most T-cytotoxic and T-suppressor cells produce a different cell surface protein, termed CD8. Monoclonal antibodies specifically recognizing CD4 or CD8 proteins can thus be used to differentiate between some T cell types.

Null cells are also known as ‘large granular lymphocytes’, but are best known as ‘natural killer’ (NK) cells. These represent a third lymphocyte subgroup. They are capable of directly lysing cancer cells and virally infected cells.

GRANULOCYTES

The third leukocyte cell type is termed granulocytes, due to the presence of large granules in their cytoplasm. Granulocytes, many of which can be activated by cytokines, play a direct role in immunity, and also in inflammation. Granulocytes can be subdivided into three cell types of which neutrophils (also known as polymorphonuclear leukocytes; PMN leukocytes) are the most abundant. Attracted to the site of infection, they mediate acute inflammation and phagocytose opsonized antigen efficiently due to the presence of an IgG Fc receptor on their surface. Eosinophils display a cell surface IgE receptor and, thus, seem to specialize in destroying foreign substances that specifi cally elicit an IgE response (e.g. parasitic worms). These cells also play a direct role in allergic reactions. Basophils also express IgE receptors. Binding of antigen–IgE complex prompts these cells to secrete their granule contents, which mediate hypersensitivity reactions.