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Inhibition of Allergic Reactivity Through Targeting Fcεri-Bound Ige with Humanized Low-Affinity Antibodies

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Inhibition of Allergic Reactivity Through Targeting Fcεri-Bound Ige with Humanized Low-Affinity Antibodies Inhibition of Allergic Reactivity through Targeting Fc εRI-Bound IgE with Humanized Low-Affinity Antibodies This information is current as Ke Zhang, Michael Elias, Hong Zhang, Jeffrey Liu, of September 27, 2021. Christopher Kepley, Yun Bai, Dean D. Metcalfe, Zachary Schiller, Yang Wang and Andrew Saxon J Immunol published online 21 October 2019 http://www.jimmunol.org/content/early/2019/10/19/jimmun ol.1900112 Downloaded from Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 21, 2019, doi:10.4049/jimmunol.1900112 The Journal of Immunology Inhibition of Allergic Reactivity through Targeting Fc«RI-Bound IgE with Humanized Low-Affinity Antibodies Ke Zhang,* Michael Elias,† Hong Zhang,* Jeffrey Liu,* Christopher Kepley,† Yun Bai,‡ Dean D. Metcalfe,‡ Zachary Schiller,x Yang Wang,x and Andrew Saxon* Options for effective prevention and treatment of epidemic allergic diseases remain limited, and particularly so for IgE-mediated 26 28 food allergies. We previously found that mouse low-affinity anti-human IgE mAbs with KD in the 10 –10 M range were capable of blocking allergic reactivity without triggering immediate allergic mediator release. In this study, we humanized three parent low affinity allergic response inhibitor (LARI) mouse anti-human IgE mAbs and characterized their biological and immunological features, refined the lead candidate for further clinical development, examined their safety profiles, determined their therapeutic efficiency, and explored the mechanism of action potentially responsible for their therapeutic effects. LARI profoundly blocked cat- and peanut-allergic IgE-mediated basophil activation, inhibited acute release of both prestored and newly synthesized Downloaded from mediator from human mast cells, suppressed peanut-specific IgE-mediated passive cutaneous anaphylaxis, and attenuated dansyl IgE-mediated systemic anaphylaxis in human Fc«RIa transgenic mice. Safety testing demonstrated that concentrations of LARI well above therapeutic levels failed to trigger immediate release of prestored and newly synthesized allergic mediators, failed to promote robust cytokine/chemokine production from allergic effector cells, and did not elicit allergic reactivity in an animal model of cutaneous and systemic anaphylaxis. Mechanistic studies revealed that LARI downregulated surface Fc«RI receptors and IgE via internalization of the IgE/Fc«RI, promoted a partial mediator depletion pathway leading to slow release of small http://www.jimmunol.org/ amount of mediators, and functioned as a partial antagonist to inhibit Fc«RI signaling phosphorylation of Syk, Akt, Erk, and p38 MAPK. These studies demonstrate that targeting surface-bound IgE with LARI profoundly suppresses human allergic reactivity while displaying an excellent safety profile. The Journal of Immunology, 2019, 203: 000–000. iven their increased prevalence and incidence, IgE- feasible because of the common assumption that anti-IgE Abs mediated allergic disorders have become a major world- would trigger an immediate allergic reaction by cross-linking wide public health concern (1). This epidemic of surface IgE/FcεRI. However, we recently reported that murine G 26 28 IgE-mediated allergic diseases has led to a corresponding interest in prototype low affinity anti-IgE mAbs, with KD in 10 –10 M by guest on September 27, 2021 developing new therapeutic approaches, including novel immune- range, did not trigger anaphylactic reactivity but instead inhibited based biologic therapies (2, 3). In an effort to develop an effec- the reactivity of human allergic effector cells through weak binding tive therapy that would inhibit all IgE mediated reactions and, in to IgE in the FcεRI (4). particular, severe food allergic reactions, we have proposed the To target human surface-bound IgE with low affinity anti-IgE use of specifically designed low affinity anti-IgE Abs to directly mAbs as a therapeutic, we have developed a set of humanized low target IgE bound to its high affinity receptor on allergic effector affinity anti-human IgE mAbs designated as low affinity allergic cells (4). response inhibitors (LARI). We characterized their biological and Previously, direct targeting of surface-bound IgE with bivalent immunological features, defined their safety profiles, determined anti-IgE Abs as an allergy therapeutic was thought not to be their therapeutic effect, and further examined their effects related to their therapeutic mechanism of action. In this article, we will *Sixal Inc., Torrance, CA 90502; †Joint School of Nanoscience and Nanoengineering, show that the defined LARI has a profound inhibiting effect on University of North Carolina at Greensboro, Greensboro, NC 27401; ‡Laboratory of allergic reactivity while displaying an excellent safety profile and, Allergic Diseases, National Institute of Allergy and Infectious Diseases, National x in the process, defines a candidate for future clinical development. Institutes of Health, Bethesda, MD 20892; and MassBiologics of the University of Massachusetts Medical School, Boston, MA 02126 ORCIDs: 0000-0002-7781-8260 (D.D.M.); 0000-0002-4185-2416 (Z.S.). Materials and Methods Received for publication January 29, 2019. Accepted for publication September 20, Abs and reagents 2019. The mAb (FITC and/or PE labeled) to CD123 (Clone 6H6), CD63 (clone This work was supported by National Institute of Allergy and Infectious Diseases ε (NIAID), National Institutes of Health Grant AI102279. D.D.M. and Y.B. are sup- H5C6), HLA-Dr (Clone L243), Fc RIa (clone AER-37), CD117 ported through the R Division of Intramural Research, NIAID. (clone 104D2), Mas-related G-protein coupled receptor X2 (MRGPRX2) (clone K125H4), and Igl (clone MHL-38) were from BioLegend. PE- Address correspondence and reprint requests to Dr. Ke Zhang, Sixal Inc., 1124 W and allophycocyanin-labeled Abs to pSyk (y348) (clone moch1ct), pAkt Carson Street, Torrance, CA 90502-2006. E-mail address: [email protected] (S473) (clone SNRNR), and pErk 1/2 (T202/y204) (clone MILAN8R) + Abbreviations used in this article: BAT, basophil activation test; CMC, CD34 he- were from eBioscience, and p-p38MAPK (clone 4NIT4KK) was from matopoietic stem cell–derived mast cell; EBD, Evans blue dye; FcRn, neonatal Fc ε ε Invitrogen. Polyclonal goat anti-human IgE (PAE) was from Abcam receptor; b-hex, b-hexosaminidase; hFc RIa Tg, human Fc RIa transgenic; LARI, (Ab9159). Anti–4-hydroxy-3-nitrophenylacetyl (NP)-IgE (clone JW 8/1) low affinity allergic response inhibitor; LTC4, leukotriene C4; MRGPRX2, Mas- related G-protein coupled receptor X2; NP, 4-hydroxy-3-nitrophenylacetyl; PAE, was from Bio-Rad. Anti-human IgG mAb (ATCC clone HB60) was purified polyclonal goat anti-human IgE; PCA, passive cutaneous anaphylaxis; PO, propylene from mouse ascites. Recombinant mouse IL-3, human IL-3, IL-6, and stem oxide; SCF, stem cell factor; SPR, surface plasmon resonance. cell factor (SCF) were purchased from PeproTech. The cat allergen Fel d1 and peanut allergen Ara h1, Ara h2, and Ara h6 were obtained from Indoor Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 Biotechnologies. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900112 2 LOW-AFFINITY ALLERGIC RESPONSE INHIBITING HUMANIZED ANTI-IgE Humanization of the mouse anti-human IgE mAb corresponding allergen Fel d1 and Ara h126. University of California, Los Angeles institutional review board approved the use of human blood Mouse anti-human IgE mAbs were produced with standard hybridoma from healthy and allergic subjects. technology (5). Humanization service of the parent low affinity mouse anti- human IgE mAbs p6.2, mE17, and F11 was provided by Genscript using Culture of human CD34+ hematopoietic stem cell derived its Ab humanization algorithm. CDR sequences of each VH and VL region mast cells of the parent founder clones were engrafted into the framework regions of + the most homologous human germline sequences. The humanized VH and The human CD34 hematopoietic stem cell–derived mast cells (CMC) 3 9 VL were expressed as human IgG1 (g1, k) to make the low affinity anti- were developed using the protocol of Yin et al. (8). PBMC (2 10 ) were human ε specific mAbs, or LARI. To fine-tune IgE binding affinities, ad- used to isolate the human CD34+ blood hematopoietic stem cells using ditional single mutations were introduced into the humanized VL derived Easysep Human CD34 Positive Selection Kit (Stemcell Technologies) per from the parent founder mE17. Mutated clones without detectable IgE manufacturer’s instructions. CMC were developed over 8 wk in StemPro- binding were discarded, except for clone E4, which was kept to be used as 34 SFM medium (Life Technologies) in the presence
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