As more recombinant human proteins become available on the market, the incidence of immunogenicity problems is rising. The antibodies formed against a therapeutic protein can result in serious clinical effects, such as loss of efficacy and neutralization of the endogenous protein with essential biological functions. Here we review the literature on the relations between the immunogenicity of the therapeutic proteins and their structural properties. The mechanisms by which protein therapeutics can induce antibodies as well as the models used to study immunogenicity are discussed. Examples of how the chemical structure (including amino acid sequence, glycosylation, and pegylation) can influence the incidence and level of antibody formation are given. Moreover, it is shown that physical degradation (especially aggregation) of the proteins as well as chemical decomposition (e.g., oxidation) may enhance the immune response. To what extent the presence of degradation products in protein formulations influences their immunogenicity still needs further investigation. Immunization of transgenic animals, tolerant for the human protein, with well-defined, artificially prepared degradation products of therapeutic proteins may shed more light on the structure-immunogenicity relationships of recombinant human proteins.

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Chapter 6 96 AbstractPurpose. To study the influence of protein structure on the immunogenicity in wildtype and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b).Methods. RhIFNα2b was degraded by metal catalyzed oxidation (M), crosslinking with glutaraldehyde (G), oxidation with hydrogen peroxide (H) and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reversed-phase HPLC, SDS-PAGE, Western blotting and mass spectrometry. The immunogenicity of the products was evaluated in wildtype mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by ELISA or surface plasmon resonance.Results. M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Native (N) rhIFNα2b was immunogenic in the wildtype mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The antirhIFNα2b antibody levels in the wildtype mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ~ N-rhIFNα2b >> B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance.Conclusions. RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size. Structural characterization and immunogenicity of rhIFNα2b97

For largely unknown reasons, biopharmaceuticals evoke potentially harmful antibody formation. Such antibodies can inhibit drug efficacy and, when directed against endogenous proteins, cause life-threatening complications. Insight into the mechanisms by which biopharmaceuticals break tolerance and induce an immune response will contribute to finding solutions to prevent this adverse effect. Using a transgenic mouse model, we here demonstrate that protein misfolding, detected with the use of tissue-type plasminogen activator and thioflavin T, markers of amyloid-like properties, results in breaking of tolerance. In wild-type mice, misfolding enhances protein immunogenicity. Several commercially available biopharmaceutical products were found to contain misfolded proteins. In some cases, the level of misfolded protein was found to increase upon storage under conditions prescribed by the manufacturer. Our results indicate that misfolding of therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity. These findings offer novel possibilities to detect immunogenic protein entities with tPA and reduce immunogenicity of biopharmaceuticals.Over the past decades, the use of therapeutic proteins has become common practice in medicine and as their use is very promising, many more biopharmaceuticals are under development (1, 2). Unfortunately, a major drawback of protein therapeutics is the risk of antibody formation (3-7). These immunogenicity problems are of concern regarding therapeutic efficacy and patient safety (5,8). For example, drug-induced neutralizing antibodies to erythropoietin (EPO) 3 result in pure red cell aplasia (9), whereas drug-induced acquired anti-factor VIII (fVIII) antibodies worsen the pathology associated with hemophilia (10). As more and more recombinant therapeutic proteins become licensed for marketing, the incidence of immunogenicity problems is expected to rise.Initially, when mainly proteins from animal origin were used for therapy, it was thought that their foreign (non-self) nature was the main cause of immunogenicity. Unexpectedly, however, both human plasma derived as well as recombinant human protein therapeutics such as EPO (11) and fVIII (12) also elicit immune responses. This suggests that the molecular characteristic evoking antibody responses is at least more complex than being self or non-self to the human immune system. Several additional factors contributing to immunogenicity have been proposed, including contaminants or impurities, protein aggregation (13), chemical degradation and protein modification, such as differences in glycosylation or oxidation (14,15) to explain the induction of antibodies.Protein misfolding is an intrinsic and problematic property of proteins, which underlies a variety of degenerative diseases, such as Alzheimer disease. These diseases are characterized by the occurrence of fibrillar deposits, classically termed amyloid, containing aggregates of misfolded proteins. Whereas the term amyloid is classically used to classify these fi...

This animal model offers the possibility to study the many factors influencing the immunogenicity of hIFNbeta and test new formulations before going into clinical trials. The model also provides the first evidence that the rhIFNbetas differ in the immunological mechanisms responsible for the development of antibodies.

Eprex/Erypo contains micelle-associated epoetin, which may be a risk factor for the development of antibodies against epoetin.

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We describe a novel strategy to achieve high affinity recognition for the specific, cerebrovascular large neutral amino acid transporter (LAT1) isoform by covalent coupling of small molecules to the amino acid, L-cysteine (L-Cys). L-Cys (as the carrier) was covalently attached via a disulfide bond to either 6-mercaptopurine or 2-methyl-1-propanethiol (IBM) to form the brain-targeted drug delivery systems (BTDS). BTDS were designed for high affinity recognition by LAT1 at the cerebrovasculature. Using an in situ rat brain perfusion technique, competition between BTDS and the radiotracer [14C]L-Leu demonstrated significant inhibition of [14C]L-Leu brain uptake. BTDS possess affinity for cerebrovascular LAT1 in many distinct brain compartments, and the recognition of BTDS by LAT1 is influenced by hydrophobicity of the side-chain in BTDS. Thus, the BTDS strategy may be utilized for rapid shuttling of various neuropharmaceuticals into brain.