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Sensitizing solid tumors to CAR-mediated cytotoxicity by lipid nanoparticle delivery of synthetic antigens

Nature Cancer volume 6, pages 1073–1087 (2025)Cite this article

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Abstract

Chimeric antigen receptor (CAR) T cell immunotherapy relies on CAR targeting of tumor-associated antigens; however, heterogenous antigen expression, interpatient variation and off-tumor expression by healthy cells remain barriers. Here we develop synthetic antigens to sensitize solid tumors for recognition and elimination by CAR T cells. Unlike tumor-associated antigens, we design synthetic antigens that are orthogonal to endogenous proteins to eliminate off-tumor targeting and that have a small genetic footprint to facilitate efficient tumor delivery to tumors by lipid nanoparticles. Using a camelid single-___domain antibody (VHH) as a synthetic antigen, we show that adoptive transfer of anti-VHH CAR T cells to female mice bearing VHH-expressing tumors reduced tumor burden in multiple syngeneic and xenograft models of cancer, improved survival, induced epitope spread, protected against tumor rechallenge and mitigated antigen escape in heterogenous tumors. Our work supports the in situ delivery of synthetic antigens to treat antigen-low or antigen-negative tumors with CAR T cells.

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Fig. 1: Design of syntAg constructs.
Fig. 2: mRNA-loaded LNPs deliver syntAgs to the surface of tumor cells.
Fig. 3: Anti-VHH CAR T cells recognize and kill tumor cells expressing their cognate syntAgs in murine and human models.
Fig. 4: Anti-VHH CAR T cells are well tolerated by immunocompetent mice and isolated human PBMCs.
Fig. 5: Adoptive transfer of anti-VHH CAR T cells into mice with VHH-expressing tumors delays tumor growth, enhances infiltration of tumor-reactive T cells and promotes endogenous antitumor immunity.
Fig. 6: LNP-mediated syntAg delivery sensitizes tumors to CAR-mediated cytoxicity in models of TNBC.
Fig. 7: LNP-mediated syntAg treatment augments antitumor immunity in preclinical models of heterogenous tumors and mitigates outgrowth of antigen-negative tumor cells.

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Data availability

All raw IVIS images for Extended Data Fig. 8 are available from the Georgia Institute of Technology Research Repository ([email protected]) upon publication of this paper. The remaining data are available within the article and Supplementary Information or upon reasonable request to the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank D. R. Meyers (Emory) and C. D. Sago for helpful insights. This work was funded by the National Institutes of Health (NIH) Director’s New Innovator Award (DP2-HD091793), NIH Director’s Pioneer Award (DP1-CA280832), National Cancer Institute (R01CA273878), National Institute of Biomedical Imaging and Bioengineering (R01EB032822), National Center for Advancing Translational Sciences (UL1TR000454), Shurl and Kay Curci Foundation and NIH Shared Instrumentation Grant (1S10OD016264-01A1) and was partially performed at the Georgia Tech Institute for Nanotechnology, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (NSF; grant ECCS-1542174). L.G. was supported by the Alfred P. Sloan Foundation and the NIH GT BioMAT Training Grant under award number 5T32EB006343. L.G., A.S. and S.N.D. were supported by the NSF Graduate Research Fellowship under grant number DGE-1451512. A.H.Z. was supported by the NIH Kirschstein National Research Service Award program under award number F31-CA271803. C.A.T. was supported by the NIH Cell and Tissue Engineering training program under grant number 5T32GM145735. F.S. was supported by a postdoctoral fellowship from the Wallace H. Coulter Department of Biomedical Engineering and the College of Engineering at Peking University. This content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author information

Author notes

  1. These authors contributed equally: Lena Gamboa, Ali H. Zamat.

Authors and Affiliations

  1. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine, Atlanta, GA, USA

    Lena Gamboa, Ali H. Zamat, Chloé A. Thiveaud, Hee Jun Lee, Elif Kulaksizoglu, Zizhen Zha, Noah S. Campbell, Ching Shen Chan, Sydney Fábrega, S. Abbey Oliver, Fang-Yi Su, Hathaichanok Phuengkham, Daryll Vanover, Hannah E. Peck, Anirudh Sivakumar, Shreyas N. Dahotre, Adrian M. Harris, Philip J. Santangelo & Gabriel A. Kwong

  2. Parker H. Petit Institute of Bioengineering and Bioscience, Atlanta, GA, USA

    Gabriel A. Kwong

  3. Institute for Matter and Systems, Georgia Institute of Technology, Atlanta, GA, USA

    Gabriel A. Kwong

  4. The Georgia Immunoengineering Consortium, Emory University and Georgia Tech, Atlanta, GA, USA

    Gabriel A. Kwong

  5. Winship Cancer Institute, Emory University, Atlanta, GA, USA

    Gabriel A. Kwong

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  1. Lena Gamboa
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Contributions

L.G., A.H.Z. and G.A.K. designed the study and wrote the paper. L.G., A.H.Z., C.A.T., H.J.L. and Z.Z. analyzed the data and generated the figures. L.G., A.H.Z., C.A.T., H.J.L., E.K., Z.Z., N.S.C., C.S.C., S.F., S.A.O., A.S., S.N.D., H.P. and A.M.H. performed the experiments. L.G., A.H.Z., P.J.S. and G.A.K. supervised the studies. D.V., H.E.P. and F.S. contributed to the formulation and production of LNPs and provided helpful discussions. All authors reviewed the paper before submission.

Corresponding author

Correspondence to Gabriel A. Kwong.

Ethics declarations

Competing interests

G.A.K. reports equity or consulting roles for Sunbird Bio, Port Therapeutics, Send Biotherapeutics and Ridge Biotechnologies. The terms of this arrangement were reviewed and approved by Georgia Tech in accordance with its conflict-of-interest policies. L.G., A.H.Z., D.V., P.J.S., C.S.C., H.J.L., C.A.T. and G.A.K. are listed as inventors on a patent application related to the results of this paper, titled ‘Synthetic Antigens as Chimeric Antigen Receptor (CAR) Ligands and Uses Thereof’ (US20230390335A1). The patent applicant is the Georgia Tech Research Corporation.

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Nature Cancer thanks Jan Melenhorst and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Kinetics of expression of Kb-SIINFEKL pMHC complex.

Surface expression kinetics of the Kb-SIINFEKL pMHC complex on the surface of MC38 tumor cells following peptide pulsing with the SIINFEKL peptide for one hour at room temperature. Mean of n = 3 technical replicates from a single experiment.

Source data

Extended Data Fig. 2 Kinetics of expression of LNP mediated delivery of VHH synthetic antigen to tumor cells.

VHH expression on MDA-MB-468 (left) and E0771 (right) following VHH-LNP treatment at the indicated doses. VHH mean fluorescent intensity (MFI) quantified by flow cytometry at indicated days after transfection. Mean of n = 4 technical replicates from a single experiment.

Source data

Extended Data Fig. 3 Characterization of murine CAR T cells sourced from either pmel-1 or C57BL6/J mice.

(a) Staining of indicated murine T cell population with activation markers CD25 and CD69 following co-incubation with ST- or VHH-expressing E0771 tumor cells. Data is representative of 3 technical replicates from a single experiment. (b) IFN-γ secretion by Pmel-1 or C57BL/6J derived αVHH CAR T cells quantified following a 24 hr coculture with MC38-VHH tumor cells at a 1:1 E:T ratio. Mean of n = 3 technical replicates from a single experiment. (c) Cytotoxicity of Pmel-1 and C57BL/6 derived αVHH CAR T cells were assessed following a 24 hr coculture with MC38-VHH at a 1:1 E:T ratio. Mean of n = 3 technical replicates from a single experiment.

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Extended Data Fig. 4 Murine αVHH CAR T cells are well tolerated by immunocompetent mice.

(a) Blood serum analysis 7 d post i.v. administration of αVHH CAR T cells, untransduced T cells, or saline into naïve C57BL6/J mice. One-way ANOVA, mean ± s.d., n = 4 mice.; n.s. = not significant. (b) Body weight measurements following i.v. administration of αVHH CAR T cells, wild-type T cells, or saline into naïve C57BL6/J mouse. Two-way ANOVA, mean ± s.d., n = 5 mice per cohort; n.s. = not significant. (c) PBMC analysis before and 3 days after ACT of saline, WT, αVHH CAR, or αCD19 CAR T cells into C57BL6/J mice. One-way ANOVA; mean ± s.e.m., n = 4 mice for αCD19 CAR treated mice, n = 5 mice in all other cohorts; n.s. = not significant ***p = 0.0002. (d) Blood serum analysis 3 d post i.v. administration of saline, WT, αVHH CAR, or αhHER2 CAR T cells into naïve B6-HER2 mice. One-way ANOVA; mean ± s.e.m., n = 3 mice for hHER2 and VHH treatments, n = 4 mice for Saline and WT treatments.; *p = 0.01, **p = 0.0065.

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Extended Data Fig. 5 VHH expression alone does not alter tumor growth.

(a) VHH expression on wildtype or transduced MC38 and E0771 tumor cells. Data is representative of 2 independent experiments. (b) Tumor growth curves of wildtype MC38 (MC38-WT) or MC38 cells transduced to stably express VHH (MC38-VHH) without adoptive cell transfer of αVHH CAR T cells. n = 4 mice inoculated with MC38-WT and n = 3 mice inoculated with MC38-VHH. Two-way ANOVA, mean ± s.e.m. is depicted; n.s. = not significant (c) Tumor growth curves of wildtype E0771 (E0771-WT) or E0771 cells transduced to stably express VHH (E0771-VHH) without adoptive cell transfer of αVHH CAR T cells n = 4 mice inoculated with E0771-WT and n = 6 mice inoculated with E0771-VHH; Two-way ANOVA, mean ± s.e.m. is depicted; n.s. = not significant.

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Extended Data Fig. 6 Adoptive transfer of αVHH CAR T cells to mice bearing wildtype tumors does not alter tumor growth.

Tumor growth curves of mice bearing wiltdype E0771 tumors treated intravenously with either saline, 5 × 106 untransduced (UTD) or αVHH CAR T cells. Two-way ANOVA, mean ± s.e.m., n = 5 mice; n.s. = not significant.

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Extended Data Fig. 7 The antitumor activity αVHH CAR T cells is comparable to αHER2 CAR T cells against VHH + HER2+ tumors.

(a) Representative flow plots of HER2 and VHH expression on either wildtype or 468-HV tumor cells. Data is representative from a single experiment, repeated 2 times with similar results. (b) 468-HV tumor cells were cocultured with either human untransduced (UTD), αHER2 CAR, or αVHH CAR T cells at a 2:1 E:T ratio and assessed for cytotixicity after 24 hrs (Mean of n = 3 technical replicates) and (c) growth curves of 468-HV tumors following treatment with the UTD, αHER2 CAR, or αVHH CAR T cells. (Two-way ANOVA, mean ± s.e.m., n = 6 mice per cohort, αVHH (**p = 0.0080 on day 6, **p = 0.0015 on day 13, ***p = 0.0003 on day 18, ***p = 0.0005 on day 25, ***p = 0.0003 on day 33) and αHER2 (††p = 0.0054 on day 6, ††p = 0.0016 on day 13, †††p = 0.0003 on day 18, †††p = 0.0005 on day 25, †††p = 0.0003 on day 33) CAR T cell treatments are compared to UTD.

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Extended Data Fig. 8 In vivo biodistribution of functional mRNA delivery mediated by CKK-LNPs following intratumoral injection.

(a) In vivo biodistribution following saline or a 2 µg intratumoral injection of nLuc mRNA-loaded LNPs (nLuc-LNPs) as measured by IVIS 24 hrs following injection. Luminesence in organs quantified (left) and representative (of n = 3 organs, each from individual mice) images are displayed on the right. mean ± s.d., multiple unpaired t test, n = 3 organs, each from individual mice; *p = 0.0397, ***p = 0.00047, ****p < 0.0001. (b) In vivo kinetics of nLuc expression following one (light blue) or two (dark blue) intratumoral injections of 5 µg nLuc-LNPs. Transfection kinetics quantified by IVIS. Two-way ANOVA, mean ± s.e.m., n = 4 mice; *p = 0.0196, **p = 0.0080.

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Extended Data Fig. 9 αVHH CAR T cells reduce tumor burden of VHH LNP injected tumors.

(a) Tumor growth curves of wildtype E0771 (E0771-wt) tumors transfected with VHH-LNPs (CKK-E12) by intratumoral injection of 5 µg mRNA-loaded LNPs without adoptive cell transfer of αVHH CAR T cells. Two-way ANOVA, mean ± s.e.m. is depicted; n = 6 saline injected mice and n = 5 for VHH-LNP injected mice.; n.s. = not significant. (b) Tumor growth curves of wildtype E0771 tumor-bearing mice treated with intratumorally with either saline, Fluc-LNP or VHH-LNP and αVHH CAR T cells on days indicated by a dashed line (D0, D7, D14, D21, and D28). Two-way ANOVA, mean ± s.e.m., n = 5 mice. Saline vs. VHH-LNP: *p = 0.0176, ***p = 0.0006, ****p < 0.0001.

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Extended Data Fig. 10 LNP-mediated synthetic antigen treatment augments antitumor immunity in a preclinical model of heterogenous glioblastoma tumors.

(a) EGFRvIII- (vIII-) or EGFRvIII+ (vIII+) SB28 tumor cells were transfected with 100 ng of VHH-LNP and evaluated for VHH expression by flow cytometry after 18 hrs, mean of n = 5 technical replicates from a single experiment (b) Mice bearing a heterogenous tumor mixture comprised of 90% vIII- and 10% vIII+ SB28 tumor cells were treated with saline or VHH-LNP followed by adoptive transfer of indicated CAR T cells (c) 24 hrs following intratumoral injection VHH-LNP (5 µg), SB28 tumors were dissociated and analyzed by flow cytometry for EGFRvIII (left) and VHH (right) expression, one-way unpaired T test, mean ± s.d., n = 3 dissociated tumors, each from individual mice; ***p = 0.0008, n.s.=nonsignficiant. (d) Representative flow plots of VHH expression on SB28 tumors treated with either saline or VHH-LNP. Data is representative of 3 tumors each isolated from one mouse (e) Tumor growth curves of heterogenous SB28 tumor-bearing mice treated with intratumorally with saline or VHH-LNP and systemically with either WT, αVHH, or αEGFRvIII CAR T cells days indicated days by dashed lines (D0, D7, & D14) n = 5 untreated and VHH-LNP + αVHH CAR treated mice, n = 6 mice with αEGFRvIII CAR T cell treatment, n = 7 mice with VHH-LNP + saline treatment. (f) Survival curves from (e) of heterogenous SB28 tumor-bearing mice following synthetic antigen treatment, log-rank (Mantel–Cox) test; **p = 0.0019 comparing EGFRvIII CAR treatment with LNP + αVHH CAR.

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Gamboa, L., Zamat, A.H., Thiveaud, C.A. et al. Sensitizing solid tumors to CAR-mediated cytotoxicity by lipid nanoparticle delivery of synthetic antigens. Nat Cancer 6, 1073–1087 (2025). https://doi.org/10.1038/s43018-025-00968-5

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