Open access peer-reviewed chapter
Abstract
For more than three decades, IFN-α2b has been widely used for the treatment of multiple human viral infections such as chronic hepatitis B and C, and certain types of cancers. However, IFN-α2b can be immunogenic, and these undesired immune responses can lead to a decrease in therapeutic efficacy. In addition, IFN-α therapy has also been associated with the progression of certain autoimmune diseases. For these reasons, the development of new IFN-α2b versions with reduced (or even null) immunogenicity has become the focus of several investigations. The “de-immunization” strategies usually involve several steps starting with T cell epitope identification and mutation of those immunogenic residues using immuno-informatics tools. Then, further experimental validation through in vitro and in vivo experimental platforms is needed to confirm in silico predictions. In this chapter, we will review the main strategies addressed so far to develop more effective and safer IFN-based therapies.
Keywords
- interferon alpha
- antiviral therapy
- immunogenicity
- de-immunization
- biobetters
- in silico epitope prediction
- in vitro assay
- in vivo assay
1. Introduction
Interferons (IFN) were initially discovered in 1957 by Lindenmann and Isaacs while performing infection experiments on chicken cells with the influenza virus [1]. In this study, the investigators observed a factor secreted by the infected cells that induced an antiviral state in the cell itself and in neighboring cells. This and other further studies [2] established the basis for elucidating the precise mechanism of action of IFNs. These secreted factors were later named “interferon” because of their ability to interfere with viral infections.
IFNs constitute a family of species-specific cytokines secreted in an autocrine and paracrine manner by host cells in response to pathogens, mainly viruses, and microbial products such as endotoxins, pyrans, and double-stranded polyribonucleotides, among others. They are composed of polypeptides ranging in molecular weights from 20 to 100 kDa. Like most proteins, they are sensitive to proteolytic enzymes and heat, although relatively stable at low pH values [3]. Essentially, the main function of IFNs is to increase the transcription of hundreds of IFN-stimulated genes (ISGs) whose products play a key role in the innate and adaptive immune response to viral infections, producing profound metabolic changes [4].
Due to the antiviral and immunomodulatory function of IFN-α2b, this protein was developed as a recombinant version (rhIFN-α2b), which was first approved by the Food and Drug Administration (FDA) in June 1986 for the treatment of hairy cell leukemia. Then, in 1988, its use was also approved for the treatment of AIDS-related Kaposi’s Sarcoma and in 1991 and 1992 to treat chronic hepatitis B and C virus infections, respectively.
Since its approval as an antiviral agent more than three decades ago, the use of rhIFN-α2b in the clinic has grown markedly. However, some adverse effects can be observed during rhIFN-α2b-based therapy. Among the most common unwanted effects are flu-like symptoms such as fever, fatigue, myalgia, chills, and headache. Also, subacute and chronic side effects such as myelosuppression, neuropsychiatric effects, and immunogenicity events can occur [5]. Regarding product immunogenicity, the development of antidrug antibodies is the most frequently detected response in patients. As an example, more than 40% of patients with malignant intestinal tumors showed anti-rhIFN-α2b antibody formation [6]. In addition, a similar proportion of patients suffering from renal cell carcinoma showed the presence of neutralizing antibodies after being treated with this biologic [7, 8, 9]. These rhIFN-α2b-based therapy unwanted effects represented a major challenge in the biopharmaceutical industry and prompted the development of new rhIFN-α2b variants with reduced immunogenicity. In the present chapter, we will discuss the main strategies addressed to develop reduced immunogenicity (de-immunized) versions of the cytokine.
Another drawback related to rhIFN-α2b therapy is the protein’s short circulating half-life, which poses the need for repeated high doses to reach the therapeutic window. This can lead to the appearance of treatment adverse effects. In general, the higher the dose of rhIFN-α2b administered and the longer the treatment, the more frequent and severe adverse effects are observed. To circumvent this issue, numerous platforms based on glycoengineering and pegylation have been approached with encouraging results. Here, we will describe the strategies that led to more stable and safer rhIFN-α2b-based products.
2. Antiviral action of IFN-α
IFN-α triggers intracellular signaling cascades that activate multiple immune functions, affecting numerous cellular processes such as proliferation, differentiation, activation, migration, and apoptosis [10].
In the canonical signaling pathway, known as the JAK–STAT pathway (Janus kinase pathway, JAK, and the signal transducer and activator of transcription, STAT), the binding of IFN-α promotes the IFNAR heterodimeric receptor complex dimerization, which enables phosphorylation and activation of the Janus family kinases JAK1 and TYK2. Then, JAK1 and TYK2 recruit and activate STAT1 and STAT2 proteins, which allow their dimerization and consequently the recruitment of IFN regulatory factor nine (IRF9). These three proteins form the ternary ISGF3 complex, capable of translocating to the cell nucleus where they bind to IFN-stimulated response elements (ISREs) in the DNA and mediate the expression of different IFN-stimulated genes (ISGs) involved in the development of antiviral, proinflammatory, and immunomodulatory responses. Additionally, through the noncanonical signaling pathway, IFN-α binding induces the expression of ISGs from the activation of other STAT proteins (such as STAT 3, 5, and 6) or through the mitogen-activated kinases (MAPKs) or phosphatidylinositol 3-kinase (PI3K) pathway [10].
Thus, IFN-α mediates antiviral actions by inhibiting the virion cycle and promoting enzyme synthesis, which interferes with viral particle transport, viral DNA transcription, replication, or RNA translation [11, 12].
Also, IFN-α elicits a powerful immunomodulatory action aiming to promote an effective antiviral immune response. For instance, IFN-α induces the activation of macrophages and NK cells, enhancing their cytotoxic activity, which helps promote the development of adaptive immune responses by increasing the expression of major histocompatibility complex (MHC) class l and ll molecules. As a result, this enhances the cytotoxic T cell-mediated killing efficiency of infected cells. Furthermore, IFN-α favors cell sequestration in lymph nodes, which promotes their activation and acts directly on T and B cells, guiding the progression of the antiviral response [13].
In addition, IFN-α inhibits cell proliferation through the regulation of different genes involved in the cell cycle, such as c-myc oncogenes, cyclin D3, phosphatase CDC25A [14], and cyclin-dependent kinase CDK-2 [15], or through the activation of numerous proapoptotic genes and proteins [16].
3. Immunogenicity of rhIFN-α2b in the clinic
rhIFN-α2b therapy can lead to the formation of antidrug antibodies. These antibodies can have varied consequences on the patient and the therapy efficacy. For example, these antibodies can bind to the biologic without having a significant impact on its biological effect or, on the contrary, neutralize its function or affect its pharmacokinetic properties [17, 18]. Regarding the nature of the antibody response, there are cases where rhIFN-α2b treatment resulted in the appearance of neutralizing IgG1 antibodies [19]. However, there is also evidence demonstrating the emergence of anti-rhIFN-α2b IgM antibodies [20]. Although the persistence and affinity of these antibodies are lower than the IgG antibody-mediated response, the consequences are potentially more severe due to their multivalent nature and the capacity to engage complement molecules and promote Fc-receptor cross-linking [20].
rhIFN-α2b immunogenicity was also evidenced by the appearance or exacerbation of certain autoimmune diseases. This is the case of autoimmune thyroiditis associated with rhIFN-α2b therapy. Initially, the occurrence of this disease was attributed to impurities in the biologic [21, 22]. However, the persistence of this disease after interruption of treatment and replacement with a product of higher purity showed that the observed autoimmunity was a direct consequence of the rhIFN-α2b proinflammatory capacity [23, 24]. The product’s proinflammatory properties could explain the immunogenicity events observed in the clinic. There is growing evidence showing that the presence of T cell epitopes in the molecule can facilitate a proinflammatory context and also contribute to the development of antidrug antibodies [25, 26, 27, 28]. Specifically, three regions enriched in T cell epitopes were identified in the rhIFN-α2b sequence, of which the region located at the c-terminal end of the molecule was found to be the most immunogenic [29].
The relevance of rhIFN-α2b immunogenicity in the clinic highlights the many efforts made to mitigate the effect of this unwanted drug attribute during treatment. The most promising strategies developed so far will be discussed in detail below.
4. Strategies aimed at reducing rhIFN-α2b immunogenicity
Because of the risk to patient health and therapy efficacy, several strategies are now available to reduce or mitigate the effects of rhIFN-α2b immunogenicity. Some of these involve modification of the protein, whereas others include coadministration of immunosuppressors.
Among the developments involving product modification, changes in the glycosylation pattern and the addition of polyethylene glycol (PEG) residues in the molecule deserve special mention. While the initial goal of these strategies was to increase the plasma half-life through an increase in apparent size and changes in the molecule’s overall charge, these modifications could also exert a masking effect on potentially immunogenic epitopes. More recently, the advent of immuno-informatics and
We will now review these strategies, highlighting their main advantages and limitations.
4.1 Development of rhIFN-α2b variants by glycoengineering
Glycoengineering is a modification strategy that is based on the linkage of glycan residues to the protein structure through covalent bonds [30]. This technology allows optimizing several macromolecular properties, such as increased solubility in aqueous solvents, thermal stability, as well as proteolytic and chemical degradation. In addition, the attachment of glycans can also lead to changes in specificity, affinity, and protein biological activity. Furthermore, there are studies showing that the presence of glycans could reduce the formation of aggregates and improve polypeptide folding and final protein conformation [31, 32, 33, 34].
Protein glycosylation can take place in a variety of ways, on the side chains of the amino acids that constitute the polypeptide chain. However, the main forms of sugar linkage are through binding to the N atoms of the asparagine (Asn) side chain or the oxygen atom of the amino acids serine and threonine. In the first case, glycosylation is called N-glycosylation, while in the second it is known as O-glycosylation.
4.1.1 Development of N and O-glycosylated versions of rhIFN-α2b
rhIFN-α2b is a low molecular mass molecule and, therefore, susceptible to plasma clearance once it reaches the blood circulation. Indeed, the protein is nearly undetectable after 1-day post subcutaneous administration [35]. As a result, multiple doses of the product are required to reach the therapeutic window. For this reason, several approaches have been addressed to increase the apparent molecular size and, consequently, the stability, half-life, and
An interesting study using glycoengineering as a strategy to increase plasma half-life was addressed by Ceaglio et al. [40]. The authors modified the rhIFN-α2b sequence to add N-glycosylation sites to the molecule. These mutations were selected so as not to negatively impact the structure/function of the cytokine. Thus, the new hyperglycosylated version, known as 4 N-IN, evidenced a marked increase in plasma half-life and a significant improvement in systemic clearance in animal experiments. However, the
Similarly, another hyperglycosylated rhIFN-α2b was recently developed and referred to as GMOP-IFN. This new variant consisted of the addition at the N-terminal end of the cytokine of a peptide tag derived from the human granulocyte and macrophage colony-stimulating factor (GM-CSF), which contained additional O-glycosylation sites. As a result, the therapeutic protein was endowed with a higher sialic acid content and larger apparent molecular size. These properties allowed improved pharmacokinetic parameters in rats and increased thermal and plasma stability. In addition, unlike what was observed for 4 N-IFN, GMOP-IFN evidenced an antiviral activity similar to that of the wild-type molecule [41].
However, we further demonstrated using T cell proliferation and cytokine profiling assays that 4 N-IFN and GMOP-IFN were immunogenic. Moreover, through an immuno-informatics toolkit, we not only identified the potentially immunogenic amino acids and regions for both molecules but also the specific mutations that allow for disruption in the binding of IFN-derived peptides to the most prevalent human MHC (HLA, human leukocyte antigen) molecules in the world population [42, 43]. The development of these new variants will be discussed in detail below.
4.2 Use of pegylation for the development of rhIFN-α2b versions with enhanced stability
In addition to glycoengineering, another successful strategy to increase the circulating half-life of proteins for human therapeutic use is through the addition of polyethylene glycol (PEG) molecules. One case that exemplifies this is PEGIntron® produced by Schering Corporation. PEGIntron is a pegylated version of rhIFN-α2b which, due to the addition of a small PEG structure (approximately 12 kDa), exhibits increased residual biological activity. However, PEGIntron achieves only a modest improvement in circulating half-life over the original protein [44, 45, 46, 47]. In this product, almost half of the PEG residues added to the molecule are exclusively bound to histidine and show high instability in water. This could explain, at least partially, the limited improvement in the stability of the product with respect to the nonpegylated molecule [44, 46].
To circumvent this drawback, another product based on rhIFN-α2b was developed by Roche and includes the addition of a 40 kDa molecule, called PEGASYS®. This product is more heterogeneous and consists of different isoforms with varying biological potency [47, 48]. As a consequence of the addition of a larger PEG molecule, the plasma half-life is increased. However, this also results in a lower biological activity, which ranges between 1 and 7% with respect to the unmodified cytokine [47].
A remarkable improvement in rhIFN-α2b pegylation technology was addressed by Rosendahl et al. [49]. In this study, the authors demonstrated that the addition of only one cysteine in the rhIFN-α2b sequence followed by pegylation at that residue allowed the development of a homogeneous mono-pegylated product. Among the cysteine residues whose mutation does not lead to a significantly increased immunogenicity, mutein Q5C showed the most encouraging results. In fact, pharmacokinetic studies in rats inoculated subcutaneously with the product showed a plasma half-life 20 to 40 times higher than that of the unmodified protein [49].
In addition to facilitating an increased rhIFN-α2b
4.3 Development of de-immunized therapeutic protein variants
As previously mentioned, although the development of improved versions of rhIFN-α2b with increased plasma half-life proved challenging, the results obtained were encouraging. Moreover, some of the rhIFN-α2b pegylated versions are commercially available on the market. However, the immunogenicity risk in the clinic still prevails [53]. As a result, this raises the need for reduced immunogenicity versions of the product. An interesting strategy is the socalled de-immunization for functional therapeutics (DeFT) [54]. The biotherapeutic de-immunization process involves several stages that will be discussed below. Finally, the de-immunization process of human rhIFN-α2b will be addressed, highlighting the challenges and achievements so far.
4.3.1 In silico analysis
The de-immunization process usually begins with the identification of potentially immunogenic protein regions. To accomplish this task, several
Also, the computational study carries out an iterative process for the identification of changes or mutations that leads to greater disruption in peptide-HLA binding. The selection of which mutations are more appropriate should also be made taking into account the available protein structural information, to avoid significant disruptions in the molecule structure/function. This information can be obtained from a literature search, if available, or through protein molecular modeling.
4.3.2 In vitro studies
Although the results achieved through
For this purpose, peptides defined as potentially immunogenic are synthesized, including the “original” sequences, that is, those derived from the wild-type protein and the “modified” ones, which include the mutations defined from the
Then, the synthesized peptides must be tested using binding assays to specific HLA molecules. These assays allow estimating the binding affinity of the original and modified peptides to multiple HLA molecules and inferring which mutations actually produce a significant disruption in the antigenicity of the tested peptide. One of the most widely used assays consists of a competitive binding assay [57] that allows assaying various selected alleles in order to cover a wide repertoire of HLA pockets [56]. This experiment establishes competition between original and modified untagged peptides at different concentrations and a biotinylated standard peptide for binding to HLA class II molecules. The complexes formed are then captured on multi-well plates using immobilized anti-HLA class II antibodies. Assay development is performed by incubating the immuno-complexes with a streptavidin-enzyme conjugate and subsequent treatment with the enzyme substrate.
This
4.3.3 Ex vivo assays
Considering the relevance of achieving clinical trials with effective and safe biotherapeutics, it is necessary to rely on preclinical assays to anticipate the risks in the clinic. As a result, in the last decades, much effort has been devoted to the development of experimental platforms based on cell cultures (bioassays). These bioassays engage immune system mediators that could detect the product and consequently trigger an unwanted response in the patient.
One of the most commonly used assays involves the utilization of human peripheral blood mononuclear cells (PBMCs). PBMC samples typically engage monocytes, B cells, T cells, NK cells, and to a lesser extent dendritic cells. Therefore, these cell cultures include antigen-presenting cells (APCs) and T cells, which are the cellular mediators engaged in developing an immune response against immunogenic proteins. Wullner and collaborators [58] developed an experimental strategy based on the utilization of PBMC samples in which the therapeutic product is used to successively “challenge” the cell cultures. Initially, the APCs present in the PBMC sample internalize the product, process it at the lysosomal level, and the resulting peptides are presented in the context of HLA molecules. Thus, if the biotherapeutic contains T cell epitopes, repeated stimulation with the product will lead to activation, proliferation, and differentiation of specific naϊve T cells into a specific T helper (Th) profile. In particular, the most frequently observed effector Th profiles are Th1, Th2, and Th17, by ELISA and/or ELISPot assays.
Due to the HLA allelic diversity in the world population and the potential differences in the elicited immune response, this assay should be performed using PBMC samples from donors expressing different HLA alleles. In particular, it has been reported that HLA-DRB1 alleles are the most frequently involved in antigenic presentation and, therefore, required for T cell-mediated responses [59, 60].
Although this experimental strategy allows predicting with good accuracy the risk of immunogenicity of certain biologics in preclinical stages [27], it has a limitation associated with the direct effect that some biotherapeutics may have on T cells and, consequently, on the results derived from this assay. For example, IFN-α exerts a potent antiproliferative action on naϊve T cells. Therefore, this experimental approach cannot be used to analyze the immunogenicity of rhIFN-α2b-based products.
To circumvent this issue, an interesting approach derived from this bioassay includes an initial step based on the development of immature dendritic cells as antigen-presenting cells [60]. Immature dendritic cells can be obtained
4.3.4 In vivo studies
Initially, toxicity and immunogenicity tests of biological products were carried out using “wild-type” animals (mainly mice). However, these animal models exhibit species-specific characteristics. In particular, the results achieved when studying the immune response developed by these animals may differ considerably from those in humans.
The advent of molecular biology and genetic engineering techniques has allowed the development of animals with particular characteristics of interest for this type of study. Currently, the most commonly used models for immunogenicity analysis are therapeutic protein-tolerant animals and transgenic animals expressing HLA molecules.
4.3.4.1 Mice immune tolerant to human proteins
Human protein-tolerant mice are engineered to express the therapeutic protein of interest. Hence, during cell ontogeny, tolerance processes allow the maintenance of immune homeostasis toward that specific protein.
These mouse strains are useful for evaluating the presence of neo-epitopes as well as subtle changes in product attributes such as formulation.
Several tolerant mouse strains are available. Among them are human tissue plasminogen activator (tPA)-tolerant murine models [62], mice producing human preproinsulin [63], and human IFN-α [64] and IFN-β [65] tolerant animals. Currently, other tolerant murine models that are of great utility for
Although human protein-tolerant mouse strains have proven useful, they have some limitations. These mice express the murine antigenic processing and presentation system components and, consequently, the biologic-derived peptides that are exhibited by APCs and the immune response developed are still mouse-specific. Therefore, this may sometimes lead to results that do not necessarily correlate with those addressed during human clinical trials. In addition, this strategy raises the need to develop and validate a tolerant mouse strain for each therapeutic protein under study.
These issues could be addressed through the use of transgenic animals expressing HLA molecules, or combinations of these mice with tolerant animals (if available).
4.3.4.2 Mice expressing HLA molecules
In addition to mice tolerant to human therapeutic proteins, genetic engineering strategies have allowed the development of animals that have had the murine antigenic presentation system knocked out and express specific HLA molecules instead. In humans, numerous HLA genes have been identified and classified into three classes. HLA class I comprises the A, B, and C genes; HLA class II contains the DR, DQ, and DP genes; while HLA class III includes complement molecules. Class I and II molecules are involved in the selection processes of T cells in the thymus. Thus, HLA class I is responsible for the negative and positive selection of CD8 T cells, whereas HLA class II is involved in the selection processes of CD4 T cells [68]. Therefore, HLA transgenic mice exhibit an antigenic presentation system analogous to the human one (at least in those specific HLA molecules). Thus, peptides derived from antigenic proteins, including therapeutic proteins, will be identical to those presented by humans expressing those HLA molecules. In addition, due to the selection processes in the thymus mentioned above, these animals possess T cells endowed with TCRs capable of recognizing peptides presented in the context of these HLA molecules. As a result, these animals develop an immune response that mimics the human immune response. The close correlation of T cell-mediated responses observed in patients and in animals expressing HLA molecules represents clear evidence of the utility of this
These transgenic mice strains can be used for multiple purposes, including, in addition to the analysis of the immunogenicity of biological products, autoimmune diseases, vaccine development, certain infectious diseases, and oncology studies. In the immunogenicity analysis of therapeutic proteins, HLA-DR3 and HLA-DR4 mice have proven to be particularly useful [54, 70].
Despite the high correlation that
4.3.5 De-immunization of human rhIFN-α2b
To de-immunize rhIFN-α2b a multi-step strategy was approached [42]. First, the most immunogenic regions of the protein were identified by using the immuno-computational algorithms of EpiVax Inc. Then, mutations leading to a substantial disruption of IFN-derived peptide binding were selected from a group of eight HLA class II archetypes that combined exhibited a coverage of more than 90% of the allelic diversity of the world population. In particular, the DRB1 alleles analyzed in this study were *0101, *0301, *0401, *0701, *0801, *1101, *1301, and *1501.
The results were validated by
However, both muteins exhibited only a residual
5. Conclusion
For more than 30 years, rhIFN-α2b has been used for the treatment of chronic and emerging viral infections. However, rhIFN-α2b is potentially immunogenic, which may compromise the efficacy and safety of rhIFN-α2b-based therapy. In addition, rhIFN-α2b is a low molecular size cytokine, which correlates with its reduced plasma half-life in patients.
For these reasons, several strategies have been addressed to reduce the impact of protein immunogenicity as well as increase the product’s plasma stability.
Glycoengineering and protein pegylation have been proposed as strategies aimed at increasing the apparent macromolecule size and, consequently, reducing the rhIFN-α2b clearance rate. The results obtained so far have been very encouraging, with remarkable pharmacokinetic parameters for these biobetters. In addition, both approaches would allow the addition of glycans or large PEG residues that would generate a shielding effect of protein epitopes. However, neither of these strategies has allowed the development of products that significantly ameliorate the negative impact associated with product immunogenicity.
In contrast, de-immunization for functional therapeutics (DeFT) of rhIFN-α2b has emerged as a promising alternative for markedly reducing product immunogenicity. The success of this strategy is based on the careful selection of the target residues, which will rely on the impact of such a change on the antigenicity/immunogenicity as well as on protein structure/function.
The individual or combined use of these strategies will enable the development of more effective and safer rhIFN-α2b-based antiviral therapies.
Acknowledgments
This work was supported by the Consejo Nacional de Investigaciones Científícas y Técnicas (CONICET, Argentina); Agencia Nacional de Promoción Científica y Tecnológica; and Universidad Nacional del Litoral. EFM and ME are members of CONICET; LCP is a fellow of the same institution.
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