Journal of Clinical Immunology

, Volume 32, Issue 5, pp 1059–1070 | Cite as

Dual Targeting of ErbB2 and MUC1 in Breast Cancer Using Chimeric Antigen Receptors Engineered to Provide Complementary Signaling

  • Scott Wilkie
  • May C. I. van Schalkwyk
  • Steve Hobbs
  • David M. Davies
  • Sjoukje J. C. van der Stegen
  • Ana C. Parente Pereira
  • Sophie E. Burbridge
  • Carol Box
  • Suzanne A. Eccles
  • John MaherEmail author



Chimeric antigen receptor (CAR) engineered T-cells occupy an increasing niche in cancer immunotherapy. In this context, CAR-mediated CD3ζ signaling is sufficient to elicit cytotoxicity and interferon-γ production while the additional provision of CD28-mediated signal 2 promotes T-cell proliferation and interleukin (IL)-2 production. This compartmentalisation of signaling opens the possibility that complementary CARs could be used to focus T-cell activation within the tumor microenvironment.


Here, we have tested this principle by co-expressing an ErbB2- and MUC1-specific CAR that signal using CD3ζ and CD28 respectively. Stoichiometric co-expression of transgenes was achieved using the SFG retroviral vector containing an intervening Thosea asigna peptide.


We found that “dual-targeted” T-cells kill ErbB2+ tumor cells efficiently and proliferate in a manner that requires co-expression of MUC1 and ErbB2 by target cells. Notably, however, IL-2 production was modest when compared to control CAR-engineered T-cells in which signaling is delivered by a fused CD28 + CD3ζ endodomain.


These findings demonstrate the principle that dual targeting may be achieved using genetically targeted T-cells and pave the way for testing of this strategy in vivo.


Adoptive immunotherapy chimeric antigen receptor dual specificity targeting MUC1 ErbB2 



This work was supported by a Breast Cancer Campaign Project Grant (2006NovPR18), the Guy’s and St Thomas’ Charity, Experimental Cancer Medicine Centre (King’s College London) and by Guy’s and the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre (BRC) award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. CB is funded by the Oracle Cancer Trust at the Royal Marsden Hospital. SAE’s research is funded by the Institute of Cancer Research (ICR) and Cancer Research UK grant CA309/A8274 and funding provided via the NIHR specialist BRC award to the Royal Marsden NHS Foundation Trust in partnership with the ICR. We thank colleagues who have generously provided materials that facilitated this work, as indicated in the text.

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

Supplementary material

10875_2012_9689_Fig7_ESM.jpg (41 kb)
Fig S1

Western blotting analysis of ErbB expression. The indicated tumor cell lines were analyzed for expression of ErbB1-4 expression by western blotting under reducing conditions, using rabbit anti-ErbB antiserum. A cross-reactive band in the ErbB4 blot serves as a loading/transfer control. (JPEG 40 kb)

10875_2012_9689_MOESM1_ESM.tif (655 kb)
High resolution image (TIFF 655 kb)


  1. 1.
    Altenschmidt U, Kahl R, Moritz D, Schnierle BS, Gerstmayer B, Wels W, et al. Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes. Clin Cancer Res. 1996;2:1001–8.PubMedGoogle Scholar
  2. 2.
    Dotti G, Savoldo B, Brenner M. Fifteen years of gene therapy based on chimeric antigen receptors: "are we nearly there yet?". Hum Gene Ther. 2009;20:1229–39.PubMedCrossRefGoogle Scholar
  3. 3.
    Kahlon KS, Brown C, Cooper LJ, Raubitschek A, Forman SJ, Jensen MC. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res. 2004;64:9160–6.PubMedCrossRefGoogle Scholar
  4. 4.
    Jakobsen MK, Restifo NP, Cohen PA, Marincola FM, Cheshire LB, Linehan WM, et al. Defective major histocompatibility complex class I expression in a sarcomatoid renal cell carcinoma cell line. J Immunother Emphasis Tumor Immunol. 1995;17:222–8.PubMedCrossRefGoogle Scholar
  5. 5.
    Lou Y, Basha G, Seipp RP, Cai B, Chen SS, Moise AR, et al. Combining the antigen processing components TAP and Tapasin elicits enhanced tumor-free survival. Clin Cancer Res. 2008;14:1494–501.PubMedCrossRefGoogle Scholar
  6. 6.
    Singh R, Paterson Y. Immunoediting sculpts tumor epitopes during immunotherapy. Cancer Res. 2007;67:1887–92.PubMedCrossRefGoogle Scholar
  7. 7.
    Ahmed N, Ratnayake M, Savoldo B, Perlaky L, Dotti G, Wels WS, et al. Regression of experimental medulloblastoma following transfer of HER2-specific T cells. Cancer Res. 2007;67:5957–64.PubMedCrossRefGoogle Scholar
  8. 8.
    Cooper LJ, Topp MS, Serrano LM, Gonzalez S, Chang WC, Naranjo A, et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect. Blood. 2003;101:1637–44.PubMedCrossRefGoogle Scholar
  9. 9.
    Hombach A, Heuser C, Sircar R, Tillmann T, Diehl V, Pohl C, et al. Characterization of a chimeric T-cell receptor with specificity for the Hodgkin's lymphoma-associated CD30 antigen. J Immunother. 1999;22:473–80.PubMedCrossRefGoogle Scholar
  10. 10.
    Jensen MC, Cooper LJ, Wu AM, Forman SJ, Raubitschek A. Engineered CD20-specific primary human cytotoxic T lymphocytes for targeting B-cell malignancy. Cytotherapy. 2003;5:131–8.PubMedCrossRefGoogle Scholar
  11. 11.
    Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z, Mavroukakis SA, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res. 2006;12:6106–15.PubMedCrossRefGoogle Scholar
  12. 12.
    Lamers CH, Sleijfer S, Vulto AG, Kruit WH, Kliffen M, Debets R, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol. 2006;24:e20–2.PubMedCrossRefGoogle Scholar
  13. 13.
    Vera J, Savoldo B, Vigouroux S, Biagi E, Pule M, Rossig C, et al. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood. 2006;108:3890–7.PubMedCrossRefGoogle Scholar
  14. 14.
    Park JR, Digiusto DL, Slovak M, Wright C, Naranjo A, Wagner J, et al. Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. Mol Ther. 2007;15:825–33.PubMedGoogle Scholar
  15. 15.
    Finney HM, Lawson AD, Bebbington CR, Weir AN. Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. J Immunol. 1998;161:2791–7.PubMedGoogle Scholar
  16. 16.
    Haynes NM, Trapani JA, Teng MW, Jackson JT, Cerruti L, Jane SM, et al. Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors. Blood. 2002;100:3155–63.PubMedCrossRefGoogle Scholar
  17. 17.
    Maher J, Brentjens RJ, Gunset G, Riviere I, Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor. Nat Biotechnol. 2002;20:70–5.PubMedCrossRefGoogle Scholar
  18. 18.
    Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, Suhoski MM, et al. Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci U S A. 2009;106:3360–5.PubMedCrossRefGoogle Scholar
  19. 19.
    Kowolik CM, Topp MS, Gonzalez S, Pfeiffer T, Olivares S, Gonzalez N, et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res. 2006;66:10995–1004.PubMedCrossRefGoogle Scholar
  20. 20.
    Wilkie S, Picco G, Foster J, Davies DM, Julien S, Cooper L, et al. Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor. J Immunol. 2008;180:4901–9.PubMedGoogle Scholar
  21. 21.
    Brentjens R, Yeh R, Bernal Y, Riviere I, Sadelain M. Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial. Mol Ther. 2010;18:666–8.PubMedCrossRefGoogle Scholar
  22. 22.
    Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum G, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest. 2011;121:1822–6.PubMedCrossRefGoogle Scholar
  23. 23.
    Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced Leukemia. Sci Transl Med. 2011;3:95ra73.Google Scholar
  24. 24.
    Buning H, Uckert W, Cichutek K, Hawkins RE, Abken H. Do CARs need a driver's license? Adoptive cell therapy with chimeric antigen receptor-redirected T cells has caused serious adverse events. Hum Gene Ther. 2010;21:1039–42.PubMedCrossRefGoogle Scholar
  25. 25.
    Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 2010;18:843–51.PubMedCrossRefGoogle Scholar
  26. 26.
    Peinert S, Kershaw MH, Prince HM. Chimeric T cells for adoptive immunotherapy of cancer: using what have we learned to plan for the future. Immunotherapy. 2009;1:905–12.PubMedCrossRefGoogle Scholar
  27. 27.
    Heslop HE. Safer CARS. Mol Ther. 2010;18:661–2.PubMedCrossRefGoogle Scholar
  28. 28.
    Krause A, Guo HF, Latouche JB, Tan C, Cheung NK, Sadelain M. Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes. J Exp Med. 1998;188:619–26.PubMedCrossRefGoogle Scholar
  29. 29.
    Alvarez-Vallina L, Hawkins RE. Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors. Eur J Immunol. 1996;26:2304–9.PubMedCrossRefGoogle Scholar
  30. 30.
    Burchell J, Durbin H, Taylor-Papadimitriou J. Complexity of expression of antigenic determinants, recognized by monoclonal antibodies HMFG-1 and HMFG-2, in normal and malignant human mammary epithelial cells. J Immunol. 1983;131:508–13.PubMedGoogle Scholar
  31. 31.
    Styles JM, Harrison S, Gusterson BA, Dean CJ. Rat monoclonal antibodies to the external domain of the product of the C-erbB-2 proto-oncogene. Int J Cancer. 1990;45:320–4.PubMedCrossRefGoogle Scholar
  32. 32.
    Gong MC, Latouche JB, Krause A, Heston WD, Bander NH, Sadelain M. Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen. Neoplasia. 1999;1:123–7.PubMedCrossRefGoogle Scholar
  33. 33.
    Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol. 2004;22:589–94.PubMedCrossRefGoogle Scholar
  34. 34.
    Gallardo HF, Tan C, Ory D, Sadelain M. Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes. Blood. 1997;90:952–7.PubMedGoogle Scholar
  35. 35.
    Riviere I, Gallardo HF, Hagani AB, Sadelain M. Retroviral-mediated gene transfer in primary murine and human T-lymphocytes. Mol Biotechnol. 2000;15:133–42.PubMedCrossRefGoogle Scholar
  36. 36.
    Wilkie S, Burbridge SE, Chiapero-Stanke L, Pereira AC, Cleary S, van der Stegen SJ, et al. Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4. J Biol Chem. 2010;285:25538–44.PubMedCrossRefGoogle Scholar
  37. 37.
    Zhang Q, Fan H, Shen J, Hoffman RM, Xing HR. Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo. PLoS One. 2010;5:e9712.PubMedCrossRefGoogle Scholar
  38. 38.
    Hombach A, Sent D, Schneider C, Heuser C, Koch D, Pohl C, et al. T-cell activation by recombinant receptors: CD28 costimulation is required for interleukin 2 secretion and receptor-mediated T-cell proliferation but does not affect receptor-mediated target cell lysis. Cancer Res. 2001;61:1976–82.PubMedGoogle Scholar
  39. 39.
    van de Wiel-van Kemenade E, Ligtenberg MJ, de Boer AJ, Buijs F, Vos HL, Melief CJ, et al. Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction. J Immunol. 1993;151:767–76.PubMedGoogle Scholar
  40. 40.
    Chmielewski M, Hombach AA, Abken H. CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack. Gene Ther. 2011;18:62–72.PubMedCrossRefGoogle Scholar
  41. 41.
    Hombach AA, Abken, H. Costimulation by chimeric antigen receptors revisited: The T cell antitumour response benefits from combined CD28-OX40 signalling. Int J Cancer. 2011: Article first published online: 29 MAR 2011 | doi: 10.1002/ijc.25960.
  42. 42.
    Verwilghen J, Baroja ML, Van Vaeck F, Van Damme J, Ceuppens JL. Differences in the stimulating capacity of immobilized anti-CD3 monoclonal antibodies: variable dependence on interleukin-1 as a helper signal for T-cell activation. Immunology. 1991;72:269–76.PubMedGoogle Scholar
  43. 43.
    Alvarez-Vallina L, Russell SJ. Efficient discrimination between different densities of target antigen by tetracycline-regulatable T bodies. Hum Gene Ther. 1999;10:559–63.PubMedCrossRefGoogle Scholar
  44. 44.
    Weijtens ME, Hart EH, Bolhuis RL. Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production. Gene Ther. 2000;7:35–42.PubMedCrossRefGoogle Scholar
  45. 45.
    Guest RD, Hawkins RE, Kirillova N, Cheadle EJ, Arnold J, O'Neill A, et al. The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J Immunother. 2005;28:203–11.PubMedCrossRefGoogle Scholar
  46. 46.
    Maher J, Wilkie S. CAR mechanics: driving T cells into the MUC of cancer. Cancer Res. 2009;69:4559–62.PubMedCrossRefGoogle Scholar
  47. 47.
    Kofler DM, Chmielewski M, Rappl G, Hombach A, Riet T, Schmidt A, et al. CD28 costimulation Impairs the efficacy of a redirected T-cell antitumor attack in the presence of regulatory T cells which can be overcome by preventing Lck activation. Mol Ther. 2011;19:760–7.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Scott Wilkie
    • 1
  • May C. I. van Schalkwyk
    • 1
  • Steve Hobbs
    • 2
  • David M. Davies
    • 1
  • Sjoukje J. C. van der Stegen
    • 1
  • Ana C. Parente Pereira
    • 1
  • Sophie E. Burbridge
    • 1
  • Carol Box
    • 2
  • Suzanne A. Eccles
    • 2
  • John Maher
    • 1
    • 3
    • 4
    Email author
  1. 1.King’s College London, King’s Health Partners Integrated Cancer Center, Department of Research OncologyGuy’s Hospital CampusLondonUK
  2. 2.Tumour Biology and Metastasis, Cancer Research UK Cancer Therapeutics UnitThe Institute of Cancer ResearchSurreyUK
  3. 3.Department of ImmunologyBarnet and Chase Farm NHS TrustHertfordshireUK
  4. 4.Department of Clinical Immunology and AllergyKing’s College Hospital NHS Foundation TrustLondonUK

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