Human <scp>KIT</scp> Antibody Briquilimab Diminishes Anaphylactic Responses in Mice Expressing Chimeric Human‐Mouse <scp>KIT</scp>

Briquilimab (JSP191) is an aglycosylated humanized IgG1 anti-KIT monoclonal antibody (mAb) that blocks SCF binding to KIT, inhibiting SCF/KIT signaling critical for mast cell (MC) survival, and thus a potential therapy for treating MC-mediated diseases [1]. To directly evaluate briquilimab in vivo, we created knock-in mice (JCK) that express chimeric KIT, consisting of mouse signal peptide, human extracellular KIT, and mouse transmembrane/intracellular KIT (mSP-hED-mTM/ID), in lieu of wild-type mouse KIT (Figure 1A). Chimeric KIT responded to mouse SCF, and JCK mice exhibited normal hematopoietic compartments, including MCs and hematopoietic stem and progenitor cells (HSPCs) (Figure S1). Following 25 mg/kg briquilimab (~2 mg/kg human equivalent dose), which cleared by Day 21 (Figure 1B), JCK mice exhibited transient decreases in hemoglobin/reticulocytes and increases in SCF, which recovered to baseline also by Day 21; other peripheral blood counts remained within normal ranges (Figure S2). Briquilimab transiently decreased JCK mast cell progenitors (MCPs), which recovered by 4 weeks (Figure 1C), and JCK bone marrow (BM) hematopoietic stem cells (HSCs) and HSPCs, which recovered by 3 weeks (Figure 1D, Figure S2). Ex vivo differentiated JCK MCs, cultured without IL-3 (24 h), started to undergo apoptosis but were rescued from apoptosis if cultured with SCF (20 h) (Figure 1E), confirming SCF/KIT signaling is essential for MC survival [2, 3]. Briquilimab prevented SCF-mediated JCK MC survival and promoted MC apoptosis in vitro (Figure 1E). In contrast, briquilimab inhibited JCK HSC proliferation in vitro but did not induce significant HSC apoptosis beyond basal levels detected in controls cultured with SCF only (Figure 1F,G), findings which were recapitulated in human HSC experiments showing that briquilimab inhibited proliferation and skewed HSC differentiation without inducing apoptosis (Figure S3). In vivo, 2–4 weeks following 25 mg/kg briquilimab, JCK mice exhibited significant MC reductions (> 80%) across multiple tissues (tongue, back skin, stomach, and lung), with depletion sustainable up to 8–14 weeks (Figure 2A). Compared to BM MCPs (Figure 1C), the rate of tissue-resident MC repopulation to baseline was relatively slower and varied across tissues (Figure 2A), suggesting that MCP differentiation into MCs takes at least several weeks and that the kinetics of MC recovery following briquilimab are not uniform across tissues. Together, these data suggest that briquilimab induces rapid, deep, and durable MC depletion across multiple tissues in JCK mice. Whereas MCs undergo apoptosis, HSCs do not undergo apoptosis upon KIT signal inhibition by briquilimab, and this likely contributes to the differential recovery time of HSPCs versus mature MCs following briquilimab. Anti-KIT mAb ACK2 has been shown to prevent IgE-dependent allergic diarrhea in mice through depleting mucosal/connective tissue MCs [4]. Accordingly, 25 mg/kg briquilimab in JCK mice partially prevented MC-mediated passive systemic anaphylaxis (PSA), measured as severe hypothermia induced by IgE (α-DNP IgE) and allergen/antigen (DNP-HSA) (Figure 2B,C). Clinical anaphylaxis scores (CAS), measured through behavioral changes [5], correlated with the severity of hypothermia (Figure 2D). Despite significant MC number decreases observed in multiple tissues (Figure 2A), only ~50% reduction in the severity of IgE-dependent PSA responses was observed, potentially due to basophils unaffected by briquilimab and/or residual MCs in other tissues. Additionally, briquilimab nearly completely protected JCK mice from IgE-independent anaphylactoid reactions induced by 5 mg/kg compound 48/80 (C48/80), which binds to Mrgprb2 (MRGPRX2 in humans) and has been commonly used to evaluate IgE-independent MC activation (Figure 2E–G) [6]. These data demonstrate that briquilimab ameliorates both IgE-dependent and IgE-independent anaphylactic reactions in JCK mice. JCK mice are a potentially useful animal model to evaluate extracellular KIT-targeting therapeutics, such as briquilimab, in MC-mediated diseases. Since briquilimab induces relatively brief and reversible changes in HSPCs, while inducing relatively profound and longer lasting tissue-resident MC depletion, clinical trials are currently being conducted to evaluate optimal dosing frequencies of briquilimab, which may enable prolonged MC depletion while allowing for recovery of other KIT-expressing cell types between doses to minimize non-MC-related effects of KIT inhibition. H.S.K., S.E.L., M.Y., C.A.C., M. Yu., and W.W.P. contributed to the conception, design, analysis, and interpretation of data. S.E.L., A.S., M.Y., C.A.C., A.W., A.R.R., A.J., S.R., S.K., R.N., K.M., and C.K. contributed to experimental execution and data analysis. H.-S.K., S.E.L., M.Y., and W.W.P. drafted the manuscript. The authors would like to thank Stephen J. Galli, Daniel Adelman, Edwin Tucker, and Judith A. Shizuru for review and insightful guidance, and the Jasper team for critical feedback, operational, and technical support. H.S.K., S.E.L., A.S., M.Y., C.A.C., A.W., A.R.R., A.J., S.R., S.K., R.N., K.M., C.K., M. Yu., W.W.P. are current or former employees of Jasper Therapeutics and may hold Jasper Therapeutics stock or stock options. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1: JCK mice retain relevant KIT function in the hematopoietic lineages, including HSPCs and MCs. Figure S2: The effects of in vivo administration of briquilimab on hematopoiesis in JCK mice. Figure S3: Briquilimab does not induce human HSC apoptosis but suppresses proliferation and skews differentiation of HSC. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.