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. 2023 May 9;14(1):2463.
doi: 10.1038/s41467-023-37863-5.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting

Elisa Ruffo  1   2   3   4 , Adam A Butchy  5 , Yaniv Tivon  6 , Victor So  1   2   3   4 , Michael Kvorjak  1   2   3   4 , Avani Parikh  1   2   3   4 , Eric L Adams  1   2   3   4 , Natasa Miskov-Zivanov  5   7   8 , Olivera J Finn  3 , Alexander Deiters  6 , Jason Lohmueller  9   10   11   12
Affiliations

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting

Elisa Ruffo et al. Nat Commun. .

Abstract

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop "universal" receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

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Conflict of interest statement

J.L. and A.D. are inventors on a patent application filed by the University of Pittsburgh on the universal SNAP receptor technology described herein (WO2020072764A1). The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Universal adaptor SNAP-CAR and SNAP-synNotch receptor function.
a A benzylguanine motif (BG) is chemically conjugated to an antibody using a benzylguanine NHS ester. The BG-antibody conjugate then covalently binds to the extracellular SNAPtag enzyme through a self-labeling reaction. b SNAPtag receptors enable the targeting of multiple different antigens using the same receptor by combining SNAP receptor cells with different BG-conjugated antibodies. c The SNAP-synNotch receptor is targeted by a BG-conjugated antibody and upon antigen recognition leads to cleavage of the synNotch receptor, releasing the transcription factor and transcriptional regulation of a target gene or genes. d The SNAP-CAR is targeted by a BG-conjugated antibody to activate T cell signaling and effector functions upon antigen recognition.
Fig. 2
Fig. 2. The SNAP-synNotch receptor can be targeted to desired antigens of interest by benzylguanine-conjugated antibodies.
a Diagram of the SNAP-synNotch receptor. b Lentiviral vector design for SNAP-synNotch receptor expression and the corresponding reporter gene. The SNAP- synNotch receptor contains the Gal4-VP64 transcription factor which upon activation leads to activation of the TagBFP reporter. c Flow cytometry analysis of surface expression and enzymatic functionality of the SNAP-synNotch receptor on transduced vs. mock (non-transduced) Jurkat cells assessed by staining with an anti-mycTag antibody and a SNAP-Surface-AF647 dye (arbitrary units, arb. units). d Flow cytometry analysis of the activation of SNAP-synNotch Jurkat cells co-incubated with the indicated target cell lines and antibody concentrations for TagBFP output gene expression reported as mean fluorescence intensity (MFI) gated on mCherry+ cells and e by ELISA for the production of the IL-7 therapeutic transgene. For d and e, n = 3 biologically-independent experiments ± s.e.m. Source data are available as a Source Data file.
Fig. 3
Fig. 3. The SNAP-CAR can be targeted to desired antigens of interest by benzylguanine-conjugated binding proteins.
a SNAP-CAR design. b SNAP-CAR lentiviral expression construct. c Flow cytometry analysis of the expression and enzymatic functionality of the SNAP-CAR receptor on transduced vs. mock (nontransduced) Jurkat cells, assessed by staining with SNAP-Surface-AF647 dye and recording TagBFP expression (arbitrary units, arb. units). d Flow cytometry analysis of CD25 and CD62L T cell activation markers on Jurkat SNAP-CAR effector cells (gated by TagBFP+ expression) co-incubated with the indicated target cell lines and antibody concentrations reported as mean fluorescence intensity (MFI). CD25 increases while CD62L decreases with activation, n = 3 biologically independent experiments; averages ± s.e.m. Source data are available as a Source Data file.
Fig. 4
Fig. 4. The SNAP-CAR is effective on primary human T cells.
a Flow cytometry analysis of the expression and enzymatic functionality of the SNAP-CAR on transduced vs. mock (un)transduced primary human T cells by staining with SNAP-Surface-AF647 dye and recording TagBFP expression (arbitrary units, arb. units). b ELISA for IFNγ production from primary human SNAP-CAR T effector cells co-incubated with the indicated target cell lines and 1.0 μg/mL of the indicated antibody and c flow cytometry analysis of CD69, CD62L, and CD107a T cell activation markers on the SNAP-CAR (TagBFP + ) population from the co-incubations in b reported as MFI. d Specific lysis of target cell lines by co-incubated primary human SNAP-CAR T cells and 1.0 μg/mL of the indicated BG-conjugated antibodies. e Specific lysis of individual cell lines (left) and mixed cell lines (right) by primary human SNAP-CAR T cells and 1.0 μg/mL of the indicated BG-conjugated antibodies. For b–e two-way ANOVA tests with multiple comparisons were performed. As the data did not have homogeneity of variance (Levene’s test), Tukey’s HSD was used for post-hoc analysis between antibody conditions. “ * ”denotes a significance of p < .0001, n = 3 biologically-independent experiments ± s.e.m. Source data are available as a Source Data file.
Fig. 5
Fig. 5. Characterizing the activity of SNAP receptors when pre-assembled or with pre-labeled target cells.
a Flow cytometry analysis of SNAP receptor activation for SNAP-synNotch and SNAP-CAR cells co-incubated with target cells that were pre-labeled with the indicated antibodies. b Flow cytometry analysis of SNAP receptor activation for SNAP-synNotch and SNAP-CAR cells that were pre-labeled with the indicated antibodies and co-incubated with target cells. TagBFP output gene expression and CD25 marker expression were evaluated by flow cytometry. c Specific lysis of target cells by co-incubated primary human SNAP-CAR T cells that were pre-incubated with the indicated concentration of adaptor at the indicated cell concentration as compared to SNAP-CAR T cells incubated with 1.0 μg/mL of soluble adaptor or a positive control anti-CD20 CAR. For a and b, two-way ANOVA tests with multiple comparisons were performed. As the data did not have homogeneity of variance (Levene’s test), Tukey’s HSD was used for post-hoc analysis between antibody conditions. “ * ” denotes a significance of p < 0.0001, n = 3 biologically-independent experiments ± s.e.m. For c a one-way ANOVA with Dunnet’s Multiple Comparison tests was performed and all values were significantly different (p < 0.0001) from the no adaptor control (white bar). Source data are available as a Source Data file.
Fig. 6
Fig. 6. In vivo loading of SNAP-CAR T cells with antibody adaptor.
a Diagram of the SNAP-CAR-LNGFR receptor. b Design of the SNAP-CAR gammaretroviral expression construct. c Flow cytometry analysis of SNAP-CAR T cells from the blood of NSG mice injected r.o. 24 h prior with SNAP-CAR T cells with or without Rituximab antibody adaptor i.p. (arbitrary units, arb. units). d Average MFI of anti-human IgG-AF647 on LNGFR + cells. For d an unpaired two-tailed student’s t-test was performed and “ * ” denotes a significance of p = 0.012, n = 3 mice ± s.e.m. Source data are available as a Source Data file.
Fig. 7
Fig. 7. Anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model.
a In vivo experimental design. b IVIS imaging of tumor burden over time. c Quantification of tumor growth via luciferase intensity for mouse images in b. “PR” indicates partial response which is defined by a final tumor size over baseline but <109 at day 33 (relative light units, RLU). d Survival of treated mice over time. For d a Mantel-Cox log-rank test was performed with a Bonferroni correction for multiple comparisons and “ * ” denotes a significance of p < 0.01667 for three comparisons, n = 5 mice. Exact p-values are p = 0.0135 for adaptor only and p = 0.0031 for SNAP-CAR T cells only. Source data are available as a Source Data file.
Fig. 8
Fig. 8. Mathematical model of three-body binding in the context of antibody mediated T cell targeting.
a Schematic of the ODE model for SNAP receptor ternary body formation. b Model simulations using parameters from the literature and from parameter estimation, compared to experimental results for four different antibody and antigen pairs for SNAP-CAR and SNAP-synNotch receptors. c Parameter scans of kf1 (binding rate of T cells to antibody), KD1 (equilibrium dissociation constant between T cell and antibody), and the number of target antigens on the surface of the tumor. For experimental data in c (green), n = 3 biologically-independent experiments ± s.e.m. Source data are available as a Source Data file.
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References

    1. Lim WA, June CH. The principles of engineering immune. Cells Treat. Cancer Cell. 2017;168:724–740. - PMC - PubMed
    1. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc. Natl Acad. Sci. USA. 1989;86:10024–10028. doi: 10.1073/pnas.86.24.10024. - DOI - PMC - PubMed
    1. June CH, Sadelain M. Chimeric antigen receptor therapy. N. Engl. J. Med. 2018;379:64–73. doi: 10.1056/NEJMra1706169. - DOI - PMC - PubMed
    1. Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor design. Cancer Disco. 2013;3:388–398. doi: 10.1158/2159-8290.CD-12-0548. - DOI - PMC - PubMed
    1. Lee DW, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet. 2015;385:517–528. doi: 10.1016/S0140-6736(14)61403-3. - DOI - PMC - PubMed

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