Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Biotechnology volume 36, pages 847–856 (2018)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Jackson, H.J., Rafiq, S. & Brentjens, R.J. Driving CAR T-cells forward. Nat. Rev. Clin. Oncol. 13, 370–383 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Zhang, M. et al. A high M1/M2 ratio of tumor-associated macrophages is associated with extended survival in ovarian cancer patients. J. Ovarian Res. 7, 19 (2014).

    PubMed  PubMed Central  Google Scholar 

  3. Knutson, K.L. et al. Regulatory T cells, inherited variation, and clinical outcome in epithelial ovarian cancer. Cancer Immunol. Immunother. 64, 1495–1504 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Erfani, N. et al. FoxP3+ regulatory T cells in peripheral blood of patients with epithelial ovarian cancer. Iran. J. Immunol. 11, 105–112 (2014).

    PubMed  Google Scholar 

  5. Shevach, E.M. Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 30, 636–645 (2009).

    CAS  PubMed  Google Scholar 

  6. Noy, R. & Pollard, J.W. Tumor-associated macrophages: from mechanisms to therapy. Immunity 41, 49–61 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Abiko, K. et al. IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer. Br. J. Cancer 112, 1501–1509 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yeku, O.O., Purdon, T.J., Koneru, M., Spriggs, D. & Brentjens, R.J. Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment. Sci. Rep. 7, 10541 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. Frey, A.B. Suppression of T cell responses in the tumor microenvironment. Vaccine 33, 7393–7400 (2015).

    CAS  PubMed  Google Scholar 

  10. Kershaw, M.H. et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin. Cancer Res. 12, 6106–6115 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Brahmer, J.R. et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Powles, T. et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515, 558–562 (2014).

    CAS  PubMed  Google Scholar 

  13. Topalian, S.L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443–2454 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Armand, P. Immune checkpoint blockade in hematologic malignancies. Blood 125, 3393–3400 (2015).

    CAS  PubMed  Google Scholar 

  15. Rizvi, N.A. et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348, 124–128 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Tumeh, P.C. et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568–571 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Cha, E. et al. Improved survival with T cell clonotype stability after anti-CTLA-4 treatment in cancer patients. Sci. Transl. Med. 6, 238ra70 (2014).

    PubMed  PubMed Central  Google Scholar 

  18. Gajewski, T.F., Louahed, J. & Brichard, V.G. Gene signature in melanoma associated with clinical activity: a potential clue to unlock cancer immunotherapy. Cancer J. 16, 399–403 (2010).

    CAS  PubMed  Google Scholar 

  19. Ku, G.Y. et al. Single-institution experience with ipilimumab in advanced melanoma patients in the compassionate use setting: lymphocyte count after 2 doses correlates with survival. Cancer 116, 1767–1775 (2010).

    CAS  PubMed  Google Scholar 

  20. Snyder, A. et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N. Engl. J. Med. 371, 2189–2199 (2014).

    PubMed  PubMed Central  Google Scholar 

  21. Curran, K.J. et al. Enhancing antitumor efficacy of chimeric antigen receptor T cells through constitutive CD40L expression. Mol. Ther. 23, 769–778 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Pegram, H.J. et al. Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood 119, 4133–4141 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Koneru, M., Purdon, T.J., Spriggs, D., Koneru, S. & Brentjens, R.J. IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo. OncoImmunology 4, e994446 (2015).

    PubMed  PubMed Central  Google Scholar 

  24. Pegram, H.J. et al. IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia. Leukemia 29, 415–422 (2015).

    CAS  PubMed  Google Scholar 

  25. Rafiq, S. et al. Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen. Leukemia 31, 1788–1797 (2017).

    CAS  PubMed  Google Scholar 

  26. Avanzi, M.P. et al. Engineered tumor-targeted t cells mediate enhanced anti-tumor efficacy both directly and through activation of the endogenous immune system. Cell Reports 23, 2130–2141 (2018).

    CAS  PubMed  Google Scholar 

  27. John, L.B. et al. Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells. Clin. Cancer Res. 19, 5636–5646 (2013).

    CAS  PubMed  Google Scholar 

  28. Rosewell Shaw, A. et al. Adenovirotherapy delivering cytokine and checkpoint inhibitor augments CAR T cells against metastatic head and neck cancer. Mol. Ther. 25, 2440–2451 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Chekmasova, A.A. et al. Successful eradication of established peritoneal ovarian tumors in SCID-Beige mice following adoptive transfer of T cells genetically targeted to the MUC16 antigen. Clin. Cancer Res. 16, 3594–3606 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Curran, M.A., Montalvo, W., Yagita, H. & Allison, J.P. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc. Natl. Acad. Sci. USA 107, 4275–4280 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Santos, E.B. et al. Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase. Nat. Med. 15, 338–344 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Suarez, E.R. et al. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model. Oncotarget 7, 34341–34355 (2016).

    PubMed  PubMed Central  Google Scholar 

  33. Prosser, M.E., Brown, C.E., Shami, A.F., Forman, S.J. & Jensen, M.C. Tumor PD-L1 co-stimulates primary human CD8+ cytotoxic T cells modified to express a PD1:CD28 chimeric receptor. Mol. Immunol. 51, 263–272 (2012).

    CAS  PubMed  Google Scholar 

  34. Liu, X. et al. A chimeric switch-receptor targeting PD1 augments the efficacy of second-generation CAR T cells in advanced solid tumors. Cancer Res. 76, 1578–1590 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Cherkassky, L. et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J. Clin. Invest. 126, 3130–3144 (2016).

    PubMed  PubMed Central  Google Scholar 

  36. Michot, J.M. et al. Immune-related adverse events with immune checkpoint blockade: a comprehensive review. Eur. J. Cancer 54, 139–148 (2016).

    CAS  PubMed  Google Scholar 

  37. Grosso, J.F. et al. LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems. J. Clin. Invest. 117, 3383–3392 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Workman, C.J. et al. Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J. Immunol. 172, 5450–5455 (2004).

    CAS  PubMed  Google Scholar 

  39. Sabatos, C.A. et al. Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance. Nat. Immunol. 4, 1102–1110 (2003).

    CAS  PubMed  Google Scholar 

  40. Hodi, F.S. et al. Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363, 711–723 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Peterson, A.C., Russell, J.D., Bailey, D.J., Westphall, M.S. & Coon, J.J. Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol. Cell. Proteomics 11, 1475–1488 (2012).

    PubMed  PubMed Central  Google Scholar 

  42. Egertson, J.D., MacLean, B., Johnson, R., Xuan, Y. & MacCoss, M.J. Multiplexed peptide analysis using data-independent acquisition and Skyline. Nat. Protoc. 10, 887–903 (2015).

    PubMed  PubMed Central  Google Scholar 

  43. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    CAS  PubMed  Google Scholar 

  44. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  PubMed  Google Scholar 

  45. Lee, A.Y. et al. Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry. Clin. Chem. 58, 619–627 (2012).

    CAS  PubMed  Google Scholar 

Download references

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.).

Author information

Author notes

  1. Sarwish Rafiq, Oladapo O Yeku and Hollie J Jackson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Sarwish Rafiq, Oladapo O Yeku, Hollie J Jackson, Terence J Purdon, Dayenne G van Leeuwen & Renier J Brentjens

  2. Cellular Therapeutics Center, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Sarwish Rafiq & Renier J Brentjens

  3. Department of Pharmacology, Weill Cornell Graduate School of Medical Sciences, New York, New York, USA

    Dylan J Drakes & Renier J Brentjens

  4. Department of Microbiology and Immunology, Weill Cornell Medicine, New York, New York, USA

    Mei Song & Xiaojing Ma

  5. Proteomics Core Laboratory, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Matthew M Miele, Zhuoning Li & Ronald C Hendrickson

  6. Eureka Therapeutics Inc., Emeryville, California, USA

    Pei Wang, Su Yan, Jingyi Xiang & Cheng Liu

  7. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Venkatraman E Seshan

  8. Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Ronald C Hendrickson

Authors
  1. Sarwish Rafiq
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oladapo O Yeku
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hollie J Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Terence J Purdon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dayenne G van Leeuwen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dylan J Drakes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mei Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Matthew M Miele
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhuoning Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Pei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Su Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jingyi Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Xiaojing Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Venkatraman E Seshan
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Ronald C Hendrickson
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Cheng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Renier J Brentjens
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

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.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.4195

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing