Humanization incorporates fully human sequences into mAbs without changing the complementarity-determining regions, but inadequacy of humanization is revealed by unfortunate ADA rates for fully human adalimumab [114, 196, 197]. administered subcutaneously (subcutaneous proteins) and comments on product-related risk factors related to protein structure and stability, dosage form, and aggregation. A two-wave mechanism of antigen presentation in the immune response CDKN2D toward subcutaneous proteins is described, and interaction with dynamic antigen-presenting cells possessing high antigen processing efficiency and migratory activity may drive immunogenicity. Mitigation strategies for immunogenicity UM-164 are discussed, including those in general use UM-164 clinically and those currently in development. Mechanistic insights along with consideration of risk factors involved inspire theoretical strategies to provide antigen-specific, long-lasting effects for maintaining the safety and efficacy of therapeutic proteins. Key Points Immune response toward subcutaneously administered proteins likely entails two waves of antigen presentation by both migratory skin-resident and lymph node-resident dendritic cells, which likely drive immunogenicity.Subcutaneous route of administration as a factor of immunogenicity is intertwined with product-related risk factors including impurities, biophysical characteristics, aggregation, and subvisible particle concentration.Some promising immunogenicity mitigation strategies in the investigative research stage are tolerance induction, T cell engineering, protein de-immunization and tolerization, use of chaperone molecules, and combination approaches. Open in a separate window Introduction Introduction to Immunogenicity of Therapeutic Proteins Immunogenicity is the propensity of a therapeutic protein to induce unwanted immune response toward itself or endogenous proteins [1]. An anti-drug antibody (ADA) response can develop after a single dose and upon repeated administration of a therapeutic protein. ADA with neutralizing or binding capabilities directly or indirectly affect therapeutic protein efficacy, respectively [2]. Neutralizing antibodies targeting active site(s) on the protein can cause direct loss of efficacy. Several important examples underscore the impact of ADA against a therapeutic protein. Hemostatic efficacy of factor VIII (FVIII) is compromised by development of anti-FVIII antibodies with neutralizing activity (termed inhibitors) in approximately 30% of severe hemophilia A (HA) patients [3, 4]. Neutralizing antibody development in mild to moderate HA patients led to spontaneous bleeding episodes due to cross-reaction with endogenous FVIII [5]. Clinical response to Pompe disease treatment is negatively impacted by sustained antibody development toward recombinant human acid-alpha glucosidase (rhGAA), which is more common in infantile-onset patients with negative status for cross-reactive immunological material [6]. Binding ADA can impact pharmacokinetics and pharmacodynamics (PK/PD) of therapeutic proteins by increasing clearance, and anti-adalimumab antibody response is associated with decreased adalimumab serum concentrations and diminished therapeutic response in rheumatoid arthritis patients [7, 8]. Anti-infliximab antibodies increase infliximab clearance, leading to treatment failure and acute hypersensitivity reactions [9]. Although less frequent, immunologically based adverse events have been associated with ADA development during replacement therapy, such as recombinant erythropoietin (EPO), thrombopoietin, interferon (IFN)-, and factor IX [10C16]. Increased relapse rate during recombinant IFN therapy has been observed for multiple sclerosis patients that develop neutralizing anti-IFN ADA, and multiple studies have found neutralizing ADA against recombinant IFN1a and IFN1b are cross-reactive and neutralize endogenous IFN [12, 17C20]. Other well-known examples include pure red-cell aplasia and thrombocytopenia development in patients receiving recombinant EPO or thrombopoietin, respectively, associated with detection of neutralizing ADA that cross-react with endogenous protein [13, 14, 21]. Food and Drug Administration (FDA) Guidance for Industry published in 2014 presents a risk-based approach for evaluation UM-164 and mitigation of immune responses to therapeutic proteins that limit efficacy and negatively impact safety profiles [1]. Efforts to assess risk of immunogenicity have considered the currently known influential factors of immunogenicity, including a multitude of product-, treatment-, and patient-related factors. Examples of patient-related factors are age, immune status, genetic factors such as human leukocyte antigen (HLA) haplotype, and autoimmune condition [22]. Product-related factors include protein structure, stability, and dosage form, and intrinsic features of recombinant proteins can impact immunogenicity, such as sequence variation, post-translational modifications (PTM), UM-164 immunodominant epitopes, and cellular expression system [23, 24]. Treatment-related factors include dose, duration and frequency of treatment, and route of administration [23]. Subcutaneous (SC) administration has unique immunogenicity challenges for some products compared to intravenous (IV) administration that are likely due to differences in immune system exposure and antigen presentation mechanisms [25, 26]. Vaccine development elucidated the capacity of antigens to induce a more efficient and effective host immune response following SC administration compared to IV infusion, likely a consequence of frequent encounter by dynamic skin antigen-presenting cells (APCs) [26C29]. Understanding how route of administration and product-related factors impact immunogenic risk will be critical for mitigating immunogenicity and designing safer biologics for SC delivery. Anatomy of the Subcutaneous Space and Skin-Resident Immune Cells The Epidermis and Langerhans Cells UM-164 Human.