Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy

RESEARCH ARTICLE Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Reena Varkey1☯, Qun Du1☯, Jodi L. Karnell2¤a, Xiaodong Xiao1¤b, Kerry A. Casey2, Rob Woods1¤c, Kim Rosenthal1, Susan Wilson1, William F. Dall’Acqua1, Herren Wu1, Ronald Herbst2, Rachel Ettinger2¤a, Melissa Damschroder ID1* 1 Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, Maryland, United States of America, 2 Department of Respiratory, Inflammation, and Autoimmunity, MedImmune LLC, Gaithersburg, Maryland, United States of America ☯ These authors contributed equally to this work. ¤a Current address: Viela Bio, Gaithersburg, Maryland, United States of America ¤b Current address:Jecho Laboratories, Frederick, Maryland, United States of America ¤c Current address: Novavax, Gaithersburg, Maryland, United States of America * OPEN ACCESS Citation: Varkey R, Du Q, Karnell JL, Xiao X, Casey KA, Woods R, et al. (2019) Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy. PLoS ONE 14(1): e0211236. https://doi.org/10.1371/ journal.pone.0211236 Editor: Aftab A. Ansari, Emory University School of Medicine, UNITED STATES Received: September 28, 2018 Accepted: January 9, 2019 Published: January 25, 2019 Copyright: © 2019 Varkey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was funded by MedImmune, the global R&D biologics arm of AstraZeneca. Competing interests: All authors are current or former employees of MedImmune at the time of the research, with stock and/or stock options in AstraZeneca. This does not alter our adherence to PLOS ONE polices on sharing data and materials. Abstract Interleukin-21 (IL-21), a member of the common cytokine receptor γ chain (γc) family, is secreted by CD4+ T cells and natural killer T cells and induces effector function through interactions with the IL-21 receptor (IL-21R)/γc complex expressed on both immune and non-immune cells. Numerous studies suggest that IL-21 plays a significant role in autoimmune disorders. Therapeutic intervention to disrupt the IL-21/IL-21R/γc interaction and inhibit subsequent downstream signal transduction could offer a treatment paradigm for these diseases. Potent neutralizing antibodies reported in the literature were generated after extensive immunizations with human IL-21 alone and in combination with various adjuvants. To circumvent the laborious method of antibody generation while targeting a conserved functional epitope, we designed a novel alternating-antigen immunization strategy utilizing both human and cynomolgus monkey (cyno) IL-21. Despite the high degree of homology between human and cyno IL-21, our alternating-immunization strategy elicited higher antibody titers and more potent neutralizing hybridomas in mice than did the immunization with human IL-21 antigen alone. The lead hybridoma clone was humanized by grafting the murine complementarity-determining regions onto human germline framework templates, using a unique rational design. The final humanized and engineered antibody, MEDI7169, encodes only one murine residue at the variable heavy/light-chain interface, retains the sub-picomolar affinity for IL-21, specifically inhibits IL-21/IL-21R–mediated signaling events and is currently under clinical development as a potential therapeutic agent for autoimmune diseases. This study provides experimental evidence of the immune system’s potential to recognize and respond to shared epitopes of antigens from distinct species, and presents a generally applicable, novel PLOS ONE | https://doi.org/10.1371/journal.pone.0211236 January 25, 2019 1 / 26 Potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy method for the rapid generation of exceptional therapeutic antibodies using the hybridoma platform. Introduction Interleukin-21 (IL-21) belongs to a family of immune modulatory cytokines that includes IL-2, IL-4, IL-7, IL-9, and IL-15 and has a wide range of biologic activities. IL-21 signaling takes place via a receptor complex consisting of its own unique receptor, the IL-21R, and the common gamma receptor chain (γc), leading to the activation of the Janus-activated kinases (JAK) and the signal transducer and activator of transcription (STAT) pathways [1, 2]. IL-21 is mainly produced by activated CD4+ T cells and natural killer (NK) T cells, whereas IL-21R is expressed on a broad array of cell types, including hematopoietic and nonhematopoietic cells [3–5]. IL-21 modulates various aspects of immune function, including differentiation of CD4+ T cells and B cells and upregulation of CD8+ T-cell and NK-cell cytolytic activity. The most profound impact of IL-21 is its ability to shape the humoral immune response. IL-21 has widereaching actions in determining how B cells respond to their environment, as well as the potential to induce robust B-cell activation, class switch recombination, and plasma cell (PC) differentiation in concert with CD40 engagement [6]. Overexpression of IL-21 is a feature of many inflammatory and autoimmune disorders, including Sjögren’s syndrome, systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease [7–14]. The critical role of IL-21 in promoting humoral and cellular immune responses makes it an important focus of potential therapeutic interventions in conditions characterized by both overproduction of IL-21 and pathogenic autoantibodies. Disruption of IL-21/IL-21R–mediated cell signaling has been investigated for disease control through the generation of antibodies directly targeting IL-21 [15], or IL-21R [16, 17] or the use of IL-21R fragment crystallizable (Fc) fusion protein (IL21R-Fc) [18, 19]. The binding affinity of human IL-21 to its receptor is reported to be 70 pM [20] which makes the generation of inhibitory antibodies extremely challenging. Several platforms have been employed to expedite the production of antibodies for research, diagnostic, and therapeutic applications [21]. Although each method has its unique potential, the hybridoma platform continues to be widely used to generate monoclonal antibodies (mAbs) [22, 23]. Of the therapeutic antibodies marketed in 2016 in the United States, only 6 have been derived from the phage display platform, whereas all others trace their origin to the hybridoma platform [24, 25]. One of the major advantages of hybridoma technology is the ability to isola (...truncated)