Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo

Abstract

The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. We modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

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Figure 1: Mouse CAR-T cells can be co-modified to secrete the mouse PD-1-blocking scFv RMP1-14.
Figure 2: CAR-T cells secreting RMP1-14 scFv have enhanced anti-tumor function in syngeneic mouse tumor models.
Figure 3: Human CAR-T cells can be co-modified to secrete a PD-1-blocking scFv, E27.
Figure 4: Coexpression of CAR and E27 scFv protects proliferative and lytic capacity of T cells in the context of PD-L1+ tumor cells.
Figure 5: CAR-T cells that secrete E27 scFv have enhanced anti-tumor function in vivo.
Figure 6: The PD-1-blocking E27 scFv secreted by CAR-T cells is only detected in the local TME.

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Acknowledgements

We would like to acknowledge Y. Iragashi and A. Rookard for technical assistance with in vivo experiments and the MSKCC Molecular Cytogenetics Core and NIH Cancer Center support grant P30 CA008748 for karyotyping of the ID8 and SKOV3 cells. The authors thank the following for financial support: US National Institutes of Health grants 5 P01 CA190174-03 and 5 P50 CA192937-02 (R.J.B.), The Annual Terry Fox Run for Cancer Research organized by the Canada Club of New York (R.J.B.), Kate's Team (R.J.B.), Carson Family Charitable Trust (R.J.B.), the Leukemia & Lymphoma Society Specialized Center of Research Program (7014) (R.J.B.), William Lawrence and Blanche Hughes Foundation (R.J.B.), the ARD Foundation (R.J.B.), and the Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center (Innovations in the structures, functions and targets of monoclonal antibody-based drugs for cancer) (R.J.B.).

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Authors

Contributions

S.R., O.O.Y., H.J.J. and R.J.B. designed the experiments, interpreted the results and wrote the manuscript. T.J.P., D.G.v.L., D.J.D., M.S., M.M.M., Z.L., P.W., S.Y. J.X. X.M., R.C.H. and C.L. designed, performed and/or analyzed the experiments. V.E.S. performed the statistical analysis.

Corresponding author

Correspondence to Renier J Brentjens.

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Competing interests

R.J.B. is a co-founder and receives royalties from Juno Therapeutics. R.J.B., S.R., H.J.J., O.Y. and C.L. have submitted a patent related to this work.

Integrated supplementary information

Supplementary Figure 1

(a) Entire western blot of analysis shown in Figure 1c on supernatant from equivalent numbers of viral packaging cells transduced to express the secretable scFv with the CAR, detected with anti-myc-tag antibody. Data shown is representative of 3 independent experiments. (b) Representative dot plot examples of flow cytometric data quantifying PD-1 detected on CAR-T cells in Figure 1g. Data shown is representative from 4 independent experiments.

Supplementary Figure 2 Detection of scFv secreted by CAR-T cells in vivo.

Entire western blot from Figure 2b analysis of in vivo secretion of RMP1-14 scFv, detected by harvesting ascites from CAR-T cell treated, tumor-bearing mice after immunoprecipitation with an anti-myc-tag antibody.

Supplementary Figure 3 Endogenous, CAR-T cells extracted from C57BL/6 mice bearing B16-F10 mouse melanoma and treated with PD-1-blocking scFv CAR-T cells have enhanced activation and cytokine levels as compared to mice treated with second generation CAR-T cells.

(a-b) Representative plots demonstrating gating strategy utilized for flow cytometric quantification of CAR-T cells in Figure 2g. Data shown is representative of 2 independent experiments with 3 mice per condition, per experiment.

Supplementary Figure 4

(a) Entire western blot from Figure 3c of analysis on supernatant from equivalent numbers of 293-Glv9 packaging cells transduced to secrete scFvs with the 1928z CAR, stained with anti-HA antibody. Datan show is representative of 2 independent experiments. (b) Entire western blot from Figure 3f on supernatant from CAR-T cells stained with anti-HA mAb, demonstrating a ~30 kDa protein in the 1928z-E27 and 4H1128z-E27 T cells. Data shown is representative of 2 independent experiments. (c) Entire western blot from Figure 3g of 293-Glv9-PD-1+ cells incubated in supernatant (SN) from 1928z and 1928z-E27 T cells, stained with anti-HA mAb, showing a ~30 kDa protein in the PD-1+ cells incubated with supernatant from 1928z-E27. Data shown is representative of 2 independent experiments. (d) Representative dot plot from Figure 3h, showing decreased PD-1 detection by flow cytometry on 1928z-E27 and 4H1128z-E27 T cell, as compared to second-generation CAR-T cells. Data shown is representative of 5 independent donors.

Supplementary Figure 5 Western blot analysis of scFv binding to CAR and CAR+ populations after co-culture.

Entire western blot from Figure 4f showing western blot analysis on flow sorted CAR+ and CAR-T cell populations probed with anti-HA mAb.

Supplementary Figure 6 Quantification utilizing unique peptide sequences by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of serum levels over time of E27 scFv and anti-human PD-1 mAb.

Peptide FGSNLESGIPAR (m/z = 624.3226) from the EH12/2H7 Ab. a) Skyline analysis to identify unique peptides in mAb but absent in the mouse or human proteome. The six y-type ions above or near the doubly charged precursor mass are shown. b) SDS-PAGE gel from EH12/2H7 AB. 6 μg/lane were loaded. Heavy chain and light chain were analyzed by data-dependent LC-MS/MS analysis as described. Light chain is shown labeled at band 2. c) High resolution HCD MS/MS spectra obtained on the (M+2H)2+ ions at 624.3226. Peptide FSGSNSGNTATLTISR (m/z = 806.8999) from the E27 scFv. d) Skyline analysis to identify unique peptides in the scFv but absent in the mouse or human proteome. The six y-type ions above or near the doubly charged precursor mass are shown. e) SDS-PAGE gel from E27 scFv. 6 μg/lane were loaded. ScFv was analyzed by data-dependent LC-MS/MS analysis as described. ScFv is shown labeled at band 2. f) High resolution HCD MS/MS spectra obtained on the(M+2H)2+ ions at 806.8999. Data shown is representative of 5 mice per group.

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Rafiq, S., Yeku, O., Jackson, H. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 36, 847–856 (2018). https://doi.org/10.1038/nbt.4195

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