Skip to main content

DNAM-1-based chimeric antigen receptors enhance T cell effector function and exhibit in vivo efficacy against melanoma

Abstract

Chimeric antigen receptor (CAR) T cell therapies hold great potential for treating cancers, and new CARs that can target multiple tumor types and have the potential to target non-hematological malignancies are needed. In this study, the tumor recognition ability of a natural killer cell-activating receptor, DNAM-1 was harnessed to design CARs that target multiple tumor types. DNAM-1 ligands, PVR and nectin-2, are expressed on primary human leukemia, myeloma, ovarian cancer, melanoma, neuroblastoma, and Ewing sarcoma. DNAM-1 CARs exhibit high tumor cell cytotoxicity but low IFN-γ secretion in vitro. In contrast to other CAR designs, co-stimulatory domains did not improve the expression and function of DNAM-1 CARs. A DNAM-1/CD3zeta CAR reduced tumor burden in a murine melanoma model in vivo. In conclusion, DNAM-1-based CARs may have the potential to treat PVR and nectin-2 expressing hematological and solid tumors.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Abbreviations

B6:

C57BL/6

CAR:

Chimeric antigen receptor

51Cr:

51Chromium

CYP:

Cytoplasmic

DC:

Dendritic cell

DNAM-1:

DNAX accessory molecule-1

EC:

Extracellular

MFI:

Mean fluorescent intensity

NK:

Natural killer

PI-3 kinase:

Phosphoinositide-3 kinase

PIP2:

Phosphatidylinositol-4,5-bisphosphate

scFv:

Single-chain variable fragment

TM:

Transmembrane

TME:

Tumor microenvironment

UnTd:

Untransduced

References

  1. 1.

    Porter DL, Levine BL, Kalos M, Bagg A, June CH (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365(8):725–733

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. 2.

    Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, June CH (2011) T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 3(95):95ra73

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. 3.

    Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, Teachey DT, Chew A, Hauck B, Wright JF, Milone MC, Levine BL, June CH (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368(16):1509–1518

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. 4.

    Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, Bartido S, Stefanski J, Taylor C, Olszewska M, Borquez-Ojeda O, Qu J, Wasielewska T, He Q, Bernal Y, Rijo IV, Hedvat C, Kobos R, Curran K, Steinherz P, Jurcic J, Rosenblat T, Maslak P, Frattini M, Sadelain M (2013) CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 5(177):177

    Article  Google Scholar 

  5. 5.

    Sadelain M, Brentjens R, Riviere I (2013) The basic principles of chimeric antigen receptor design. Cancer Discov 3(4):388–398

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. 6.

    Lanier LL (2005) NK cell recognition. Annu Rev Immunol 23:225–274

    Article  CAS  PubMed  Google Scholar 

  7. 7.

    Kruse PH, Matta J, Ugolini S, Vivier E (2014) Natural cytotoxicity receptors and their ligands. Immunol Cell Biol 92(3):221–229

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Shibuya A, Campbell D, Hannum C, Yssel H, Franz-Bacon K, McClanahan T, Kitamura T, Nicholl J, Sutherland GR, Lanier LL, Phillips JH (1996) DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity 4(6):573–581

    Article  CAS  PubMed  Google Scholar 

  9. 9.

    Tahara-Hanaoka S, Miyamoto A, Hara A, Honda S, Shibuya K, Shibuya A (2005) Identification and characterization of murine DNAM-1 (CD226) and its poliovirus receptor family ligands. Biochem Biophys Res Commun 329(3):996–1000

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Seth S, Georgoudaki AM, Chambers BJ, Qiu Q, Kremmer E, Maier MK, Czeloth N, Ravens I, Foerster R, Bernhardt G (2009) Heterogeneous expression of the adhesion receptor CD226 on murine NK and T cells and its function in NK-mediated killing of immature dendritic cells. J Leukoc Biol 86(1):91–101

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    Dardalhon V, Schubart AS, Reddy J, Meyers JH, Monney L, Sabatos CA, Ahuja R, Nguyen K, Freeman GJ, Greenfield EA, Sobel RA, Kuchroo VK (2005) CD226 is specifically expressed on the surface of Th1 cells and regulates their expansion and effector functions. J Immunol 175(3):1558–1565

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    Bottino C, Castriconi R, Pende D, Rivera P, Nanni M, Carnemolla B, Cantoni C, Grassi J, Marcenaro S, Reymond N, Vitale M, Moretta L, Lopez M, Moretta A (2003) Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J Exp Med 198(4):557–567

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. 13.

    Tahara-Hanaoka S, Shibuya K, Onoda Y, Zhang H, Yamazaki S, Miyamoto A, Honda S, Lanier LL, Shibuya A (2004) Functional characterization of DNAM-1 (CD226) interaction with its ligands PVR (CD155) and nectin-2 (PRR-2/CD112). Int Immunol 16(4):533–538

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Takai Y, Miyoshi J, Ikeda W, Ogita H (2008) Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation. Nat Rev Mol Cell Biol 9(8):603–615

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Fujito T, Ikeda W, Kakunaga S, Minami Y, Kajita M, Sakamoto Y, Monden M, Takai Y (2005) Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3. J Cell Biol 171(1):165–173

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. 16.

    Verhoeven DH, de Hooge AS, Mooiman EC, Santos SJ, ten Dam MM, Gelderblom H, Melief CJ, Hogendoorn PC, Egeler RM, van Tol MJ, Schilham MW, Lankester AC (2008) NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways. Mol Immunol 45(15):3917–3925

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Cho D, Shook DR, Shimasaki N, Chang YH, Fujisaki H, Campana D (2010) Cytotoxicity of activated natural killer cells against pediatric solid tumors. Clin Cancer Res 16(15):3901–3909

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. 18.

    Castriconi R, Dondero A, Corrias MV, Lanino E, Pende D, Moretta L, Bottino C, Moretta A (2004) Natural killer cell-mediated killing of freshly isolated neuroblastoma cells: critical role of DNAX accessory molecule-1-poliovirus receptor interaction. Cancer Res 64(24):9180–9184

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Pende D, Spaggiari GM, Marcenaro S, Martini S, Rivera P, Capobianco A, Falco M, Lanino E, Pierri I, Zambello R, Bacigalupo A, Mingari MC, Moretta A, Moretta L (2005) Analysis of the receptor-ligand interactions in the natural killer-mediated lysis of freshly isolated myeloid or lymphoblastic leukemias: evidence for the involvement of the Poliovirus receptor (CD155) and Nectin-2 (CD112). Blood 105(5):2066–2073

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    El-Sherbiny YM, Meade JL, Holmes TD, McGonagle D, Mackie SL, Morgan AW, Cook G, Feyler S, Richards SJ, Davies FE, Morgan GJ, Cook GP (2007) The requirement for DNAM-1, NKG2D, and NKp46 in the natural killer cell-mediated killing of myeloma cells. Cancer Res 67(18):8444–8449

    Article  CAS  PubMed  Google Scholar 

  21. 21.

    Carlsten M, Norell H, Bryceson YT, Poschke I, Schedvins K, Ljunggren HG, Kiessling R, Malmberg KJ (2009) Primary human tumor cells expressing CD155 impair tumor targeting by down-regulating DNAM-1 on NK cells. J Immunol 183(8):4921–4930

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Lakshmikanth T, Burke S, Ali TH, Kimpfler S, Ursini F, Ruggeri L, Capanni M, Umansky V, Paschen A, Sucker A, Pende D, Groh V, Biassoni R, Hoglund P, Kato M, Shibuya K, Schadendorf D, Anichini A, Ferrone S, Velardi A, Karre K, Shibuya A, Carbone E, Colucci F (2009) NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo. J Clin Invest 119(5):1251–1263

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. 23.

    Carlsten M, Baumann BC, Simonsson M, Jadersten M, Forsblom AM, Hammarstedt C, Bryceson YT, Ljunggren HG, Hellstrom-Lindberg E, Malmberg KJ (2010) Reduced DNAM-1 expression on bone marrow NK cells associated with impaired killing of CD34 + blasts in myelodysplastic syndrome. Leukemia 24(9):1607–1616

    Article  CAS  PubMed  Google Scholar 

  24. 24.

    Gilfillan S, Chan CJ, Cella M, Haynes NM, Rapaport AS, Boles KS, Andrews DM, Smyth MJ, Colonna M (2008) DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors. J Exp Med 205(13):2965–2973

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. 25.

    Barber A, Zhang T, DeMars LR, Conejo-Garcia J, Roby KF, Sentman CL (2007) Chimeric NKG2D receptor-bearing T cells as immunotherapy for ovarian cancer. Cancer Res 67(10):5003–5008

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Zhang T, Wu MR, Sentman CL (2012) An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo. J Immunol 189(5):2290–2299

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. 27.

    Barth RJ Jr, Mule JJ, Spiess PJ, Rosenberg SA (1991) Interferon gamma and tumor necrosis factor have a role in tumor regressions mediated by murine CD8 + tumor-infiltrating lymphocytes. J Exp Med 173(3):647–658

    Article  CAS  PubMed  Google Scholar 

  28. 28.

    Zhang T, Barber A, Sentman CL (2007) Chimeric NKG2D modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways. Cancer Res 67(22):11029–11036

    Article  CAS  PubMed  Google Scholar 

  29. 29.

    Faroudi M, Utzny C, Salio M, Cerundolo V, Guiraud M, Muller S, Valitutti S (2003) Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold. Proc Natl Acad Sci USA 100(24):14145–14150

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. 30.

    Hudecek M, Lupo-Stanghellini MT, Kosasih PL, Sommermeyer D, Jensen MC, Rader C, Riddell SR (2013) Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin Cancer Res 19(12):3153–3164

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. 31.

    Shibuya K, Lanier LL, Phillips JH, Ochs HD, Shimizu K, Nakayama E, Nakauchi H, Shibuya A (1999) Physical and functional association of LFA-1 with DNAM-1 adhesion molecule. Immunity 11(5):615–623

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Shibuya K, Shirakawa J, Kameyama T, Honda S, Tahara-Hanaoka S, Miyamoto A, Onodera M, Sumida T, Nakauchi H, Miyoshi H, Shibuya A (2003) CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 costimulatory signal for naive T cell differentiation and proliferation. J Exp Med 198(12):1829–1839

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. 33.

    Cai YC, Cefai D, Schneider H, Raab M, Nabavi N, Rudd CE (1995) Selective CD28pYMNM mutations implicate phosphatidylinositol 3-kinase in CD86-CD28-mediated costimulation. Immunity 3(4):417–426

    Article  CAS  PubMed  Google Scholar 

  34. 34.

    Germain RN (1997) T-cell signaling: the importance of receptor clustering. Curr Biol 7(10):R640–R644

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Choudhuri K, Wiseman D, Brown MH, Gould K, van der Merwe PA (2005) T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand. Nature 436(7050):578–582

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Alarcon B, Swamy M, van Santen HM, Schamel WW (2006) T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity. EMBO Rep 7(5):490–495

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. 37.

    Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18(4):843–851

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. 38.

    Kloss CC, Condomines M, Cartellieri M, Bachmann M, Sadelain M (2013) Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 31(1):71–75

    Article  CAS  PubMed  Google Scholar 

  39. 39.

    Fedorov VD, Themeli M, Sadelain M (2013) PD-1- and CTLA-4-based inhibitory chimeric antigen receptors. Sci Transl Med 5(215):215ra172

    Article  PubMed Central  PubMed  Google Scholar 

  40. 40.

    Pende D, Castriconi R, Romagnani P, Spaggiari GM, Marcenaro S, Dondero A, Lazzeri E, Lasagni L, Martini S, Rivera P, Capobianco A, Moretta L, Moretta A, Bottino C (2006) Expression of the DNAM-1 ligands, Nectin-2 (CD112) and poliovirus receptor (CD155), on dendritic cells: relevance for natural killer-dendritic cell interaction. Blood 107(5):2030–2036

    Article  CAS  PubMed  Google Scholar 

  41. 41.

    Huarte E, Cubillos-Ruiz JR, Nesbeth YC, Scarlett UK, Martinez DG, Buckanovich RJ, Benencia F, Stan RV, Keler T, Sarobe P, Sentman CL, Conejo-Garcia JR (2008) Depletion of dendritic cells delays ovarian cancer progression by boosting antitumor immunity. Cancer Res 68(18):7684–7691

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. 42.

    Reymond N, Imbert AM, Devilard E, Fabre S, Chabannon C, Xerri L, Farnarier C, Cantoni C, Bottino C, Moretta A, Dubreuil P, Lopez M (2004) DNAM-1 and PVR regulate monocyte migration through endothelial junctions. J Exp Med 199(10):1331–1341

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank the National Cancer Institute Biological Resource Branch for providing recombinant human IL-2, Dartmouth College Molecular biology Core for technical support, and the Center for Comparative Medicine and Research for providing animal care. This work was supported by a National Institute of Health grant CA130911.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Charles L. Sentman.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wu, MR., Zhang, T., Alcon, A. et al. DNAM-1-based chimeric antigen receptors enhance T cell effector function and exhibit in vivo efficacy against melanoma. Cancer Immunol Immunother 64, 409–418 (2015). https://doi.org/10.1007/s00262-014-1648-2

Download citation

Keywords

  • CAR
  • Gene therapy
  • Immunotherapy
  • Adoptive T cell therapy
  • Melanoma