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Reliable and reproducible monitoring of the conformational state of therapeutic protein products remains an unmet technological need. This need is amplified by the increasing number of biosimilars entering the drug development pipeline as many branded biologics are reaching the end of their market exclusivity period. Availability of methods to better characterize protein conformation may improve detection of counterfit and unlicensed therapeutic proteins. In this study, we report the use of a set of modified DNA aptamers with enhanced chemical diversity to probe the conformational state of 12 recombinant human erythropoietin (rHuEPO) therapeutic protein products; one FDA-licensed rHuEPO originator biological product, three rHuEPO products that are approved for marketing in the US or EU as biosimilars, and eight rHuEPO products that are not approved for marketing in the US or EU. We show that several of these modified aptamers are able to distinguish rHuEPO reference products or approved biosimilars from non-licensed rHuEPO products on the basis of differences in binding kinetics and equilibrium affinity constants. These reagents exhibit sensitivity to the conformational integrity of various forms of rHuEPO and as such represent powerful, simple-to-use analytical tools to monitor the conformational integrity of therapeutic-proteins during manufacture and to screen for and identify both substandard and counterfeit products.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Recombinant therapeutic proteins are a broad class of biological products used to replace dysfunctional human proteins in individuals with genetic defects (e.g., factor VIII for hemophilia) or, in the case of monoclonal antibodies, bind to disease targets involved in cancers, autoimmune disorders, or other conditions. Unfortunately, immunogenicity (immune response to the drug) remains a key impediment, potentially affecting the safety and efficacy of these therapeutics. Immunogenicity risk is routinely evaluated during the licensure of therapeutic proteins. However, despite eliciting anti-drug immune responses in at least some patients, most protein drugs are nevertheless licensed as they address unmet medical needs. The pre-licensure immunogenicity assessments of therapeutic proteins are the subject of numerous reviews and white papers. However, observation and clinical management of the immunogenicity of approved therapeutic proteins face additional challenges. We survey the immunogenicity of approved therapeutic proteins, discuss the clinical management of immunogenicity, and identify the challenges to establishing clinically relevant immunogenicity assays for use in routine clinical practice.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog. The reengineered rFVIIa analog variants retained increased intrinsic thrombin generation activity but did not elicit T-cell responses in peripheral blood mononuclear cells isolated from 50 HLA typed subjects representing the human population. Our algorithm, rational immunogenicity determination, offers a broadly applicable deimmunizing strategy for bioengineered proteins.

Fusing the human immunoglobulin G1 (IgG1) constant region (Fc-domain) to therapeutic proteins or peptides increases their circulating plasma half-life via neonatal Fc receptor (FcRn) binding and recycling. However, Fc-mediated interactions with other molecules including complement C1q and Fc gamma receptors (FcγRs) can have immunological consequences and the potential to modulate the immunogenicity of Fc-fusion therapeutics. In a comparative study, we carried out a comprehensive assessment of Fc-mediated interactions for five FDA-approved Fc-fusion therapeutics. C1q binding and complement activation were measured by ELISA, while FcγR binding and signaling were evaluated using BW5147:FcγR-ζ reporter cell lines. We demonstrate that FIX-Fc and FVIII-Fc bound C1q as well as activating and inhibitory FcγRs (I, IIA, IIB, IIIA). These coagulation factor Fc-fusions also signaled via FcγRIIIA, and to a lesser extent via FcγRI and FcγRIIB. TNFR-Fc and CTLA4-Fc bound FcγRI, while TNFR-Fc also bound FcγRIIIA, but these interactions did not result in FcγR signaling. Our comprehensive assessment demonstrates that (i) different Fc-fusion drugs have distinct C1q/FcγR binding and signaling properties, (ii) FcγR binding does not predict signaling, and (iii) the fusion partner (effector molecule) can influence Fc-mediated interactions.

Therapeutic protein drugs are an important class of medicines serving patients most in need of novel therapies. Recently approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancers, autoimmunity/inflammation, exposure to infectious agents, and genetic disorders. The latest advances in protein-engineering technologies have allowed drug developers and manufacturers to fine-tune and exploit desirable functional characteristics of proteins of interest while maintaining (and in some cases enhancing) product safety or efficacy or both. In this review, we highlight the emerging trends and approaches in protein drug development by using examples of therapeutic proteins approved by the U.S. Food and Drug Administration over the previous five years (2011–2016, namely January 1, 2011, through August 31, 2016).