The New Model
In 1982, the first recombinant protein derived from DNA, for human use, was marketed as the human insulin. Since then, many recombinant proteins were introduced among which epoetins and interferons are some of the widely used drug products in the world today. Though, these proteins are developed as close copies human proteins (endogenous), most of these proteins have the ability to induce antibodies, sometimes even in some patients. Further, majority of these drug products are used in patients lack an innate deficiency and are assumed to have immune tolerance to a specific protein.

Initially, it was assumed that production by recombinant technology in downstream processing and non-human host cells modified the drug proteins and the subsequent immunological response is the classical response to a foreign protein. The clinical manifestations these two reactions are entirely different. The vaccine-type response takes place within weeks and often, a single injection is adequate enough to induce an antibody response. Neutralizing antibodies on high levels are induced leading to booster reaction; this indicates a memory response.

In general, the breaking B-cell tolerance usually takes 6 to 12 months of chronic treatment and sometimes leads to binding antibodies production sans biological effect. This response appears to lack memory, because challenging patients whose antibody levels do not induce any response when decreased.

Immunological Response
Currently, therapeutic proteins that are available cover the whole spectrum ranging from human-like interferon α-2a to completely foreign (example, bacterial-derived asparaginase) to everything in the middle. The foreign protein reduces antibodies via the classical pathway that includes cleaving and ingestion of drug proteins into peptides by dendritic cells and macrophages, peptides presentation by the MHC-II system, B-cells activation and boosting, affinity maturation, and isotype switching of B-cells by T-cells (helper).

It is less clear on the how the tolerance of B-cells break. Auto-reactive B-cells are always present and the meeting of B-cell receptor and its epitope does not trigger any activation. B-cells receptor oligomerizes and leads to cell activation and subsequent production of antibodies only when they meet their epitopes in regularly repeated form. This means B-cells are capable of recognizing repeated protein structure (three-dimensional). Naturally occurring proteins (narrowly spaced) are found on viruses and some bacterial surfaces. Further, the B-cell system is known for its potential to react to microbial structures that are system independent discriminating self from non-self.

B-cell activation by aggregates is explained along with how they begin with the production of IgM. Though, it is not clear on how isotype swapping from IgM to IgG occurs, some studies have suggested that the aggregates after reacting with B-cell receptor undergo internalization. Therefore, by internalizing, the B-cells become helper cells producing cytokines that activate other B-cells.

Factors Influencing the Formation of Antibody to Therapeutic Proteins:

Physical Factors
The presence of aggregates and the degree of non-self play the initial triggers of any antibody response to a therapeutic protein. The degree of non-self is important for inducing vaccine-type response and depends on the protein and the divergence site from the endogenous protein's natural sequence. For Insulin, single mutations occur leading to a new epitope and a subsequent antibody response while other mutations may not have any influence. Glycosylation is another key structural factor for immunogenicity of therapeutic proteins. Interferon β produced in E.coli is highly immunogenic when compared to the similar product produced in mammalian cells.

Impurities play a key role in immunogenicity of therapeutic proteins. Certain substances including host cell components, chromatographic resins, or enzymes, and monoclonal antibodies end up in the final product.

Human therapeutic proteins sometimes exhibit high biological activity. Freeze-dried formulations which contain human serum albumin act as a stabilizer. Recently, human serum albumin was replaced by polysorbate 80.

Administration Route
The administration route on the immunogenicity is quite clear. The most immunogenic is the subcutaneous route while the least immunogenic is the intravenous route. However, immunogenicity can be seen after any application route that includes intrapulmonary and mucosal route.

Patient Factors
There are many patient-related factors influencing antibody formation. The product's biological effects can either improve or inhibit formation of antibody as can a patient's concomitant treatment. Sometimes, the concomitant treatment is given to hinder antibody formation (example, methotrexate treatment to hinder antibody formation to etanercept).

Today, the regulatory process for therapeutic proteins includes immunogenicity evaluation. Studies are being conducted on why and how patients generate antibodies to therapeutic proteins.

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