AbstractTargeted lipid nanoparticles (LNPs) offer a nonviral approach for in vivo cell engineering but efficient programming of natural killer (NK) cells remains underexplored. Here we develop NK cell-targeted LNPs (NtLNPs) using engineered biparatopic NK cell ligands to drive receptor-specific uptake and mRNA delivery. Lead NtLNPs achieve up to ~80% NK cell transfection with minimal off-target effects in mice. Systemic delivery of chimeric antigen receptor (CAR) mRNA using NtLNPs generates CAR NK cells in vivo. CD19-targeted CAR NKs completely deplete circulating B cells in healthy mice and CD3-targeted CAR NKs eliminate nearly all autoreactive T cells in an autoimmune hepatitis model, highlighting a potential advantage over CAR T therapies for T cell-driven disease. In solid tumors, NtLNPs targeting the inhibitory checkpoint NKG2A enhance antitumor efficacy by relieving Qa-1b-mediated immunosuppression. Lastly, we optimized NtLNPs for in vivo engineering of human CAR NK cells, enabling regression of two humanized hematologic malignancy models and establishing NtLNPs as a modular immunotherapy system.This is a preview of subscription content, access via your institutionAccess options Access through your institutionAccess Nature and 54 other Nature Portfolio journalsGet Nature+, our best-value online-access subscription27,99 € / 30 dayscancel any timeLearn moreSubscribe to this journalReceive 12 print issues and online access269,00 € per yearonly 22,42 € per issueLearn moreBuy this articlePurchase on SpringerLinkInstant access to the full article PDF.39,95 €Prices may be subject to local taxes which are calculated during checkoutFig. 1: Development and assessment of scFv-conjugated tLNP library.Fig. 2: In vivo CAR NK cell generation and B cell depletion.Fig. 3: In vivo CAR NK cell therapy for T cell-driven AIH treatment.Fig. 4: Targeting NKG2A for tumor immunotherapy.Fig. 5: Antitumor activity of NtLNPs in humanized xenograft models.Data availabilityAll data that support the findings of this study are provided within the paper and its Supplementary Information.Code availabilityAll code and data used for this study can be found on GitHub (https://github.com/elinat98/NK-MD-Analysis) and Zenodo (https://doi.org/10.5281/zenodo.21983910)68.ReferencesIrvine, D. 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Nat. Cancer 6, 786–800 (2025).Article CAS PubMed PubMed Central Google Scholar Download referencesAcknowledgementsWe thank the Koch Institute’s Robert A. Swanson (1969) Biotechnology Center for technical support, specifically the Flow Cytometry Core, the Hope Babette Tang (1983) Histology Core and the Animal Imaging and Preclinical Testing Core.FundingThis work was funded by Sanofi (formerly Translate Bio) (D.G.A.), the Marble Center for Cancer Nanomedicine (to D.G.A. and R.L.) and Koch Institute Support (core) grant 5P30-CA014051 from the National Cancer Institute.Author informationAuthor notesThese authors contributed equally: Yuang Chen, Julia Monkovic, Haseeb Mughal.Authors and AffiliationsDavid H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USAYuang Chen, Julia Monkovic, Haseeb Mughal, Binlong Chen, Elina Thadhani, Patrick McMullen, Ava Self, Yuxuan Chen, Yizong Hu, Samuel Detmer, Min Chen, Anna Lapteva, Ria Kamat, Ashley Vernon, Robert Langer & Daniel G. AndersonDepartment of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USAYuang Chen, Julia Monkovic, Binlong Chen, Patrick McMullen, Ava Self, Yuxuan Chen, Yizong Hu, Min Chen, Robert Langer & Daniel G. AndersonDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USAHaseeb Mughal, Samuel Detmer & Robert LangerHarvard and MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USAElina Thadhani, Robert Langer & Daniel G. AndersonDepartment of Anesthesiology, Critical Care and Pain Medicine, Boston Children’s Hospital, Boston, MA, USAPatrick McMullen, Robert Langer & Daniel G. AndersonDepartment of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USAAnna Lapteva, Ria Kamat & Robert LangerInstitute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USARobert Langer & Daniel G. AndersonAuthorsYuang ChenView author publicationsSearch author on:PubMed Google ScholarJulia MonkovicView author publicationsSearch author on:PubMed Google ScholarHaseeb MughalView author publicationsSearch author on:PubMed Google ScholarBinlong ChenView author publicationsSearch author on:PubMed Google ScholarElina ThadhaniView author publicationsSearch author on:PubMed Google ScholarPatrick McMullenView author publicationsSearch author on:PubMed Google ScholarAva SelfView author publicationsSearch author on:PubMed Google ScholarYuxuan ChenView author publicationsSearch author on:PubMed Google ScholarYizong HuView author publicationsSearch author on:PubMed Google ScholarSamuel DetmerView author publicationsSearch author on:PubMed Google ScholarMin ChenView author publicationsSearch author on:PubMed Google ScholarAnna LaptevaView author publicationsSearch author on:PubMed Google ScholarRia KamatView author publicationsSearch author on:PubMed Google ScholarAshley VernonView author publicationsSearch author on:PubMed Google ScholarRobert LangerView author publicationsSearch author on:PubMed Google ScholarDaniel G. AndersonView author publicationsSearch author on:PubMed Google ScholarContributionsYuang Chen, J.M., H.M., B.C., E.T., P.M., A.S., Yuxuan Chen, Y.H., S.D., M.C., A.L., R.K. and A.V. performed the experiments and analyzed data. Yuang Chen, J.M., H.M. and D.G.A. discussed the results and wrote the paper with input from all authors. R.L. and D.G.A. acquired funding and supervised the project.Corresponding authorCorrespondence to Daniel G. Anderson.Ethics declarationsCompeting interestsD.G.A. discloses potential competing interests because of his advising (past and present) for Alnylam, Sanofi, Translate Bio, CRISPR Tx, oRNA Therapeutics, Soufflé Therapeutics and Combined Therapeutics. R.L. discloses potential competing interests because of his advising (past and present) for Alnylam, Moderna, Soufflé Therapeutics and Combined Therapeutics. A list of entities with which R.L. is involved with, compensated or uncompensated, is available on Dropbox (www.dropbox.com/s/yc3xqb5s8s94v7x/Rev%20Langer%20COI.pdf?dl=0). A patent application covering aspects of this work has been filed by D.G.A., Yuang C., J.M., H.M. and R.L. (application no. 64/059,548) and is currently pending. The other authors declare no competing interests.Peer reviewPeer review informationNature Biotechnology thanks Rafet Basar, Yizhou Dong, Katy Rezvani and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Qa-1b-dependent enhancement of NK cell function by NKG2A targeting.a, Quantification of Qa-1b expression on resting, activated B cell, T cell and B16F10 tumor cell line. b,c, In vitro co-culture of NK cells with B16F10 melanoma cells (Qa-1b high) after pretreatment with unmodified LNP, BiNKG2A-LNP, or BiNKG2D-LNP encapsulating luciferase mRNA. Frequencies of NK cells’ degranulation (CD107a+) and corresponding tumor cell killing are shown. d,e, Co-culture of NK cells with Qa-1b knockout B16F10 cells (B16F10Qa-1b−/−) under the same treatment conditions. f, In vivo growth of parental B16F10 and engineered B16F10-hCD19 tumors after subcutaneous inoculation. g, Quantification and representative flow cytometry plots of CD69 expression on CAR NK cells following treatment of mice bearing parental B16F10 or B16F10-hCD19 tumors. h,i, Individual tumor growth curves of mice under the indicated treatment conditions. Each line represents one mouse. n = 3 for in vitro experiment. Data are mean ± s.d. Statistical significance was assessed by one-way and two-way ANOVA followed by Dunnett’s multiple comparisons test. n = 5 biologically independent mice per group. Data are mean ± s.d. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test.Extended Data Fig. 2 Safety and immunogenicity profile of repeated intravenous NtLNP administration.a, Serum cytokine profiling collected at acute (6 h) time point after repeated intravenous treatment with PBS, unmodified LNP, BiNKG2A-LNP, or BiNKG2D-LNP. b, Longitudinal body weight monitoring during the treatment course. c, Blood chemistry analysis of liver and kidney function collected at short (24 h) and long-term (14 d) time points after repeated treatment. d, Hepatocyte-tumor cell binding was quantified as PB+ APC+ events within a doublet-enriched flow cytometry gate. e, Representative H&E staining of major organs, including heart, liver, spleen, lung, and kidney, collected at short and long-term time points after repeated treatment. f, Anti-scFv antibody titers in serum collected after repeated administration of BiNKG2A-LNP or BiNKG2D-LNP. g,h, Functional evaluation of serum neutralizing activity after repeated NtLNP administration, measured by MFI of scFv binding to primary NK cells. i,j, Functional evaluation of serum dissociated NKG2D scFv on NKG2D-dependent NK cell cytotoxicity after repeated NtLNP administration, measured by CD107a. n = 5 biologically independent mice per group. Data are mean ± s.d. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test or by two-tailed Student’s t-test for comparison.Extended Data Fig. 3 Structural features and cross-species activity of NtLNPs.a, Pairwise human-murine sequence identity, alignment score, and normalized score for the NKG2D homodimer and NKG2A:CD94 heterodimer. b, Conservation maps across receptor domains (dark blue = high, gray = low) show continuous conservation for NKG2D versus fragmented conservation in NKG2A and CD94. c, RAE-1β:NKG2D and BiNKG2D:NKG2D MD structures aligned on murine NKG2D (mNKG2D); side (top), top (bottom) views. d, Residue-level H-bond occupancy across RAE-1β, BiNKG2D, and the NKG2DE2 (single-epitope scFv):NKG2D complexes (avg. frequency, 1 μs trajectory). e, Epitope mapping of RAE-1β (left) vs BiNKG2D (right) on NKG2D. f, Representative H-bond networks at the NKG2D interface; BiNKG2D engages more residues, likely stabilizing binding. g,h, Average H-bond occupancy for NKG2D residues implicated in native binding (g) or unique to engineered ligands (h). n = 3 independent MD simulation replicates per complex, each initiated from the same starting structure with a distinct random seed for initial atomic velocities; mean ± s.d., one-way ANOVA with Dunnett’s test. i, Structural alignment of BiNKG2D with human (purple) and murine (teal) NKG2D. j, Close-up of conserved BiNKG2D interface residues (H174, D173, Y154, G152) through hydrogen bonds, salt bridges, and cation–π interactions. k, MD-structure of the human MICA-NKG2D complex. l, Close-up of the native MICA:hNKG2D interface: salt bridges (A.K197-D66/D164) and hydrogen bonding (A.Y199-D164). m, Residue-level H-bond occupancy heat map for hNKG2D with MICA or BiNKG2D. n,o, Average H-bond occupancy for hNKG2D residues implicated in MICA binding (n) or newly engaged by BiNKG2D (o). n = 3 independent MD simulation replicates per complex, each initiated from the same starting structure with a distinct random seed for initial atomic velocities; mean ± s.d., one-way ANOVA with Dunnett’s test.Extended Data Fig. 4 Extended assessment of tumor recurrence, CAR NK kinetics and T cell depletion in humanized xenograft models.a, Continued bioluminescence imaging of Raji-luciferase-GFP tumor burden from day 36 to day 48 in NSG-IL-15 mice treated with ex vivo hCD19-hCAR NK cells or the indicated LNP formulations. b, Percentages of hCD19-hCAR+ human NK cells in blood at 1, 6, 24, 48, 72 and 96 h after intravenous administration of BiNKG2D-LNP. c, Representative spleen and liver images at day 50. PBS-treated mice inoculated with Raji-luciferase-GFP cells on day 30 served as tumor-bearing positive control. d,e, Flow cytometric analysis of GFP+ Raji tumor cells in spleen, liver, bone marrow and blood at day 50. f, Continued bioluminescence imaging of CCRF-CEM-luciferase-GFP tumor burden from day 36 to day 48 in NSG-IL-15 mice treated with the indicated LNP formulations. g, CAR positivity of infused ex vivo CAR NK cells before each treatment. h,i, Validation of human T cell depletion in the Raji model. Representative flow cytometry plots and quantification of human T cells and NK cells among live hCD45+ peripheral immune cells in mice treated with BiNKG2D-LNP or BiNKG2D-LNP with T cell depletion. n = 5 biologically independent mice per group. 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