Chimeric antigen receptor (CAR)-T cells represent a major breakthrough in cancer therapy, wherein a patient’s own T cells are engineered to recognize a tumor antigen, resulting in activation of a local cytotoxic immune response. However, CAR-T cell therapies are currently limited to the treatment of B cell cancers and their effectiveness is hindered by resistance from antigen-negative tumor cells, immunosuppression in the tumor microenvironment, eventual exhaustion of T cell immunologic functions and frequent severe toxicities. To overcome these problems, we have developed a novel class of CAR-T cells engineered to express an enzyme that activates a systemically administered small-molecule prodrug in situ at a tumor site. We show that these synthetic enzyme-armed killer (SEAKER) cells exhibit enhanced anticancer activity with small-molecule prodrugs, both in vitro and in vivo in mouse tumor models. This modular platform enables combined targeting of cellular and small-molecule therapies to treat cancers and potentially a variety of other diseases.
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The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files. Any raw data not provided therein are available from the corresponding authors upon reasonable request.
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We thank E. de Stanchina and C. Hagen (MSK Antitumor Assessment Core Facility) for assistance with mouse toxicology studies, G. Sukenick and R. Wang (MSK Analytical NMR Core Facility) for expert nuclear magnetic resonance and mass spectral support, J. Fraser Glickman and C. Adura Alcaino (Rockefeller High-Throughput and Spectroscopy Resource Center) for assistance with SPE–MS experiments, G. Chiosis and S. Sharma (MSK) for assistance with LC–MS/MS experiments and B. Yoo (MSK) for helpful discussions on the synthesis of APdMG-Glu. Financial support from the NIH (grant nos. P01 CA023766 to D.A.S. and D.S.T., R01 CA55349 and R35 CA241894 to D.A.S., R01 AI118224 to D.S.T. and CCSG P30 CA008748 to C.B. Thompson), the Tudor Fund (to D.A.S.), the Lymphoma Fund (to D.A.S.), the Commonwealth Foundation and MSK Center for Experimental Therapeutics (to D.A.S. and D.S.T.) is gratefully acknowledged.
D.A.S., D.S.T. and R.J.B. are consultants for, have equity in and have sponsored research agreements with CoImmune, which has licensed technology described in this paper from MSK. D.A.S. has equity in or is a consultant for: Actinium Pharmaceuticals, Arvinas, Eureka Therapeutics, Iovance Biotherapeutics, OncoPep, Pfizer, Repretoire and Sellas. D.S.T. has been a consultant and/or paid speaker for Eli Lilly, Elsevier, Emerson Collective, Merck, National Institutes of Health, Venenum Biodesign, the Research Center for Molecular Medicine of the Austrian Academy of Sciences and the Institute for Research in Biomedicine, Barcelona. R.J.B. is a cofounder and receives royalties from Juno Therapeutics/Celgene. MSK has filed for patent protection behalf of T.J.G., J.P.L., D.S.T. and D.A.S. for inventions described in this paper. The remaining authors declare no competing interests.
Peer review information Nature Chemical Biology thanks Yuan Gao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Synthesis of AMS-Glu prodrug (2).
DMA = N,N-dimethylacetamide; DMF = N,N-dimethylformamide; TBAF = tetrabutylammonium fluoride; TBS = t-butyldimethylsilyl.
Extended Data Fig. 2 Synthesis of Ceph-AMS prodrug (3).
(a) Synthesis of protected AMS precursor 16. (b) Synthesis of Ceph-AMS (3). pyr = pyridine; TFA = trifluoroacetic acid; THF = tetrahydrofuran; TMS = trimethylsilyl.
Extended Data Fig. 3 Complete bioluminescent imaging data for in vivo efficacy in mouse intraperitoneal Raji tumor xenografts.
Mice treated with (a) CPG2-19BBz SEAKER cells and AMS-Glu (2) (50 mg/kg, ip, bid, days 2-7 post-CAR engraftment, 12 doses total, gray band) or (b) β-Lac-19BBz SEAKER cells and Ceph-AMS (3) (4 mg/kg, ip, bid, days 2–3 post-CAR engraftment, 3 doses total, gray band). Raw BLI is plotted on log scale; AUC is plotted on split linear scale. (mean ± s.d. of n = 5 mice per group; experiment was repeated with similar results.) Representative images are shown in Fig. 5d and e, respectively, of the manuscript.
Extended Data Fig. 4 Complete bioluminescent imaging data for in vivo efficacy against antigen-negative cells in intraperitoneal heterogeneous tumor xenografts.
(a) Bioluminescent imaging (BLI) and quantification of CD19+ Nalm6 cells expressing mCherry and Gaussia luciferase (Nalm6-mCherry/gLuc [CD19+]) in untreated mice or mice receiving β-Lac-19BBz SEAKER cells plus or minus 3 injections of Ceph-AMS (3: 4 mg/kg, ip, bid). Images taken at day 20 post tumor engraftment; one mouse in the group treated with SEAKER cells and prodrug showed tumor clearance but died after day 14 and is omitted. Complete radiance data is shown in the right two panels, with raw BLI plotted on log scale and AUC plotted on a linear scale. (Mean ± s.d. of n = 4 (untreated, SEAKER + prodrug) or n = 5 (SEAKER alone) mice per group; Student’s two-tailed t-test: ns = not significant; *p < 0.05; experiment was performed once). (b) BLI and quantification of CD19− Nalm6 cells expressing eGFP and firefly luciferase (Nalm6-eGFP/fLuc [CD19−]) in untreated mice or mice receiving β-Lac-19BBz SEAKER cells plus or minus 3 injections of Ceph-AMS (3: 4 mg/kg, ip, bid). Images taken at day 17 post tumor engraftment; one mouse in the group treated with SEAKER cells and prodrug died after day 14 and is omitted. The left two panels also appear in Fig. 5g,h of the manuscript. Complete radiance data is shown in the right two panels, with raw BLI plotted on log scale and AUC plotted on a linear scale. (Mean ± s.d. of n = 4 (untreated, SEAKER + prodrug) or n = 5 (SEAKER alone) mice per group; Student’s two-tailed t-test: ns = not significant; *p < 0.05; experiment was performed once).
Extended Data Fig. 5 Persistence of β-Lac enzyme activity and Ceph-AMS prodrug activation in mouse intraperitoneal Raji tumor xenografts after SEAKER cell exhaustion in vivo.
(a) Experimental scheme for rescue of relapsed Raji xenograft by treatment with Ceph-AMS (3: 4 mg/kg, ip, bid, days 4–5, 3 doses total, then days 22–23 (gray bar), 3 doses total) after exhaustion of β-Lac-19BBz SEAKER cells. (b) Quantitation of tumor bioluminescence before and after second dosing period (median with s.d. of n = 4 mice per group; experiment was performed once). (c) Persistence of β-Lac enzyme activity in β-Lac-19BBz SEAKER cells extracted from two of the mice from the experiment in panel a at day 30 (day 28 post administration), in comparison to standard 19BBz CAR-T cell controls (flow cytometry analysis: β-Lac substrate = CCF2-AM).
Extended Data Fig. 6 Construction and characterization of β-Lac-expressing murine SEAKER cells.
(a) SEAKER construct encoding secreted β-Lac and a murine CAR: β-Lac-MUC28z (β-Lac/α-MUC16/CD28/CD3ζ). LTR = long terminal, Ψ = psi packaging element, FLAG = FLAG epitope tag (pink), P2A = 2A self cleaving peptide, α-MUC16 scFv = MUC-16-specific mouse-derived single chain variable fragment, myc = Myc epitope tag (brown), mCD28 = mouse CD28 costimulatory domain (green), mCD3ζ = mouse CD3 zeta chain (red). (b) Flow cytometry analysis of α-MUC16 CAR expression in retrovirally-transduced primary mouse T cells (fluorescently (phycoerythrin, PE) labeled anti-idiotype antibody; representative data from 5 independent experiments). (c) Trans-cytotoxicity of supernatant fluid (sn) from β-Lac-MUC28z SEAKER cells with or without Ceph-AMS (3: 107 nM) against mouse EL4 lymphoma cells, compared to prodrug alone and parent drug AMS (1: 500 nM) (24 h, CellTiter-Glo assay; mean ± s.d. of n = 3 technical replicates; Student’s two-tailed t-test: ns = not significant, ***p < 0.001; experiment was perfomed once).
Extended Data Fig. 7 Assessment of SEAKER cell immunogenicity in an immunocompetent mouse model.
Assessment of SEAKER cell immunogenicity in an immunocompetent mouse model. (a) Experimental scheme to assess immunogenicity of β-Lac-MUC28z SEAKER cells in a syngeneic intraperitoneal ID8 tumor model. Sera were collected on Days 21, 24, 28, and 31 and tested for anti-β-Lac antibodies in panel b. In a separate experiment, ascites were recovered on Day 28 by peritoneal lavage and tested for the presence of SEAKER cells in panels c,d and β-Lac enzyme activity in panel e. (b) Detection anti-β-Lac antibodies in sera over 10 days following SEAKER cell engraftment (Days 21–31) (median with lines representing each individual mouse of n = 12; experiment was performed once). (c,d) Flow cytometry analysis of SEAKER cell (myc+) persistence among T cells (CD3+) and (e) nitrocefin cleavage-based quantitation of β-Lac enzyme activity in ascites recovered 7 days after SEAKER cell engraftment (Day 28) (representative data shown from n = 2 mice per group; on average, 25% of T cells were SEAKER-positive; experiment was performed once). SSC = side scatter. (f) In a third experiment, mice were treated as in panel a, but without cyclophosphamide pretreatment to maximize the antibody response, then sera were recovered 5 days after SEAKER cell engraftment (Day 26) and analyzed for anti-β-Lac antibodies (n = 1 mouse in untreated group; n = 5 mice in treated group; mean ± s.d. of n = 3 technical replicates from each mouse; experiment was performed once). Sera from the 4 mice showing anti-β-Lac antibodies were used for the ex vivo enzyme activity experiment in panel g. (g) Nitrocefin cleavage-based quantitation of enzyme activity of recombinant β-Lac treated with sera from untreated or SEAKER-treated mice from panel f (n = 2 mice in untreated group; mean ± s.d. of n = 4 mice in treated group; experiment was performed once).
Supplementary Tables 1–3, Figs 1–15 and Note (chemistry).
Original western blots from Supplementary Figs. 5, 6 and 8; representative ancestry gate for flow cytometry analysis.
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Gardner, T.J., Lee, J.P., Bourne, C.M. et al. Engineering CAR-T cells to activate small-molecule drugs in situ. Nat Chem Biol 18, 216–225 (2022). https://doi.org/10.1038/s41589-021-00932-1
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