AbstractDuring the process of engulfment, phosphatidylserine is exposed on the surface of dead cells as an ‘eat-me’ signal and is recognized by Protein S (ProS), a secreted factor that also binds to the Mer tyrosine kinase (MerTK) on phagocytes. Despite its robust activity, this engulfment mechanism has not been exploited for therapeutic purposes. Here we develop a synthetic protein modality called Crunch (connector for removal of unwanted cell habitat) by modifying ProS, inspired by the high engulfment capability of the ProS–MerTK pathway. In Crunch, the phosphatidylserine-binding motif of ProS is replaced with a nanobody or single-chain variable fragment that recognizes the surface proteins of targeted cells. Green fluorescent protein nanobody-conjugated Crunch eliminates green fluorescent protein-expressing melanoma cells in transplantation mouse models. In addition, CD19+B cells are eliminated by anti-CD19 single-chain variable fragment-conjugated Crunch, resulting in a therapeutic effect on systemic lupus erythematosus. Both mouse and human versions of Crunch are effective, establishing this synthetic ligand as a promising tool for the elimination of targeted cells.This is a preview of subscription content, access via your institutionAccess optionsAccess Nature and 54 other Nature Portfolio journalsGet Nature+, our best-value online-access subscription27,99 € / 30 dayscancel any timeLearn moreSubscribe to this journalReceive 12 digital issues and online access to articles118,99 € per yearonly 9,92 € per issueLearn moreBuy this articlePurchase on SpringerLinkInstant access to full article PDFBuy nowPrices may be subject to local taxes which are calculated during checkoutFig. 1: Design for Crunch.Fig. 2: MerTK dimerization and phosphorylation by Crunch.Fig. 3: Engulfment by Crunch in vitro and in vivo.Fig. 4: Suppression of tumour growth and metastasis by Crunch.Fig. 5: Removal of targeted through recognition of an endogenous marker by scFv Crunch.Fig. 6: Removal of targeted cells by scFv Crunch targets for disease treatment.Data availabilityHuman acral melanoma datasets for single-cell analysis were obtained from the GEO database under accession number GSE115978. 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IK cytokine ameliorates the progression of lupus nephritis in MRL/lpr mice. Arthritis Rheumatism 54, 3591–3600 (2006).CAS PubMed Google Scholar Download referencesAcknowledgementsWe thank A. Fujimoto for administrative assistance and A. Wee for proof-reading. This work was supported by JST-CREST (grant no. 1199566), Kusunoki125, Sumitomo Kyoto University Innovation Promotion System (SKIPS) to J. Suzuki. Computation time was provided by the Supercomputer System, Institute for Chemical Research, Kyoto University.Author informationAuthors and AffiliationsInstitute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto, JapanYuki Yamato & Jun SuzukiGraduate School of Biostudies, Kyoto University, Konoe-cho, Yoshida, Sakyoku, Kyoto, JapanYuki Yamato & Jun SuzukiCenter for Integrated Biosystems, Institute of Biomedical Sciences, Academia Sinica, Taipei, TaiwanJun SuzukiCREST, Japan Science and Technology Agency, Kawaguchi, Saitama, JapanJun SuzukiAuthorsYuki YamatoView author publicationsSearch author on:PubMed Google ScholarJun SuzukiView author publicationsSearch author on:PubMed Google ScholarContributionsY.Y. and J.S. designed the overall research and interpreted experimental results. Y.Y. conducted all experiments. Y.Y. and J.S. wrote the paper.Corresponding authorCorrespondence to Jun Suzuki.Ethics declarationsCompeting interestsJ.S. and Y.Y. are inventors on a patent application (2024-191258) of a protein modality to eliminate unwanted cells. This work was supported in part by the Collaborative Research Grant from Sumitomo Pharma Co Ltd.Peer reviewPeer review informationNature Biomedical Engineering thanks Jeanette Leusen, Lun Tsou and Thomas Valerius and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Production and characterization of Crunch.a–b, Generation of high Crunch-secreting CHO cells. CHO cells transduced with Crunch-IRES-RFP lentivirus were RFP-sorted twice (S2), reinfected, and sorted again to yield S3. Cells were seeded, switched to serum-free medium after 16 hr, and supernatant was collected at 48 hr. Crunch expression was analyzed by by immunoblotting with anti-FLAG antibody (a), and binding to GFPm⁺ BDKO cells was assessed by flow cytometry (b). c, Production kinetics. S3 cells were cultured as above, and daily supernatants were analyzed by ELISA (n = 3, independent biological samples). d–e, Crunch purification. Supernatant (540 ml, Day 6) was filtered, pH-adjusted (25 mM HEPES, pH 7.5), and incubated with Ni-NTA beads overnight. Input, flow-through, wash, and elution were assessed by immunoblotting with anti-FLAG (d). Eluates were buffer-exchanged to PBS and analyzed with BSA standards by CBB staining (e). Final concentration: 3.7 mg/ml; yield: ~9.61 mg/L. f, Glycosylation analysis. Crunch was treated with PNGaseF and analyzed by by immunoblotting with anti-FLAG. g, Binding affinity. ELISA using GFP-coated plates compared GFPNb-Crunch and GFP nanobody. Absorbance (450 nm) was used to calculate KD via nonlinear regression (R). h–i, Thermal stability. DSF analysis of 1D3 antibody and Crunch (mean of triplicates shown in h); aggregation onset temperatures (Tm) shown in i (n = 3, triplicate using the same sample). j, Vitamin K effect. GFPNb-Crunch produced with or without vitamin K was PNGaseF-treated and analyzed by by immunoblotting with anti-FLAG. All graphs show mean ± S.D.Source dataExtended Data Fig. 2 MerTK dimerization and phosphorylation.a–b, Split-GFP assay for MerTK-sfGFP. NIH3T3 cells expressing MerTK-sfGFP and tagRFP were co-cultured with BDKO or GFPm⁺ BDKO cells in conditioned medium containing mock or GFPNb-Crunch (from HEK293T) at 37 °C for 2 hr. After removing target cells, RFP⁺ MerTK-sfGFP⁺ NIH3T3 cells were analyzed by flow cytometry. Dot plots show % GFP⁺ cells among RFP⁺ NIH3T3 cells (a); bar plot shows % GFP⁺ cells among MerTK-sfGFP⁺ NIH3T3 cells with BDKO (b) (n = 4, independent biological samples). GFPm⁺ BDKO data in Fig. 2e. Mean ± S.D.; Student’s t test; N.S.P > 0.05 c, MerTK phosphorylation by apoptotic thymocytes and ProS. Mouse thymocytes were treated with PBS (–) or FasL (+) for 3 hr at 37 °C, incubated with 10% FBS medium for ProS loading, washed, and co-cultured with NIH3T3 or MerTK⁺ NIH3T3 cells in serum-free medium. After centrifugation (300 × g, 2 min, 37 °C) and 15 min incubation, NIH3T3 cells were washed, lysed, and analyzed by immunoblotting with anti–phospho-MerTK and anti-MerTK antibodies. d, MerTK phosphorylation by PtdSer-exposed living cells and ProS. Ba/F3 or aXkr4⁺ Ba/F3 cells were incubated with 10% FBS medium, washed, and added to MerTK⁺ NIH3T3 cells. After centrifugation and 15 min incubation, MerTK⁺ cells were lysed and analyzed by immunoblotting as in (c). e, Time-dependent MerTK phosphorylation by GFPNb-Crunch. GFPm⁺ BDKO cells were added to MerTK⁺ NIH3T3 cells with (+) or without (-) 1 µg/ml GFPNb-Crunch in serum-free medium with 5 mg/ml BSA. After centrifugation and incubation (0–30 min, 37 °C), MerTK⁺ cells were lysed and analyzed by immunoblotting as in (c).Source dataExtended Data Fig. 3 Cell survival assay.a, Experimental design. Ba/F3 cells were engineered to express MerTK, aXkr4 (to expose PtdSer), or both. 2.5 × 105 cells were cultured in 500 µl IL-3(–) RPMI1640 with FBS. MerTK activation supports survival and proliferation. b–e, Cell survival by PtdSer and MerTK. Cell number and viability were measured by Trypan blue exclusion every 24 hr. Line graphs show mean cell number (b) and viability (c); bar plots show both parameters at 48 and 72 hr (d, e) (n = 3, independent biological samples). f–i, Cell survival by Crunch. MerTK⁺ Ba/F3 cells were cultured with (+) or without (-) 10 µg/ml GFPNb-Crunch in IL-3(–) medium. Cell number (f) and viability (g) were measured every 24 hr. Bar plots show both at 48 and 72 hr (h, i) (n = 3, independent biological samples). Data are mean ± S.D. Statistical analysis: one-way ANOVA with Tukey-Kramer t-test (d–e, g); Student’s t test (f–i). N.S., P > 0.05; *P