Current Hematologic Malignancy Reports

, Volume 14, Issue 5, pp 451–459 | Cite as

Multi-Specific CAR Targeting to Prevent Antigen Escape

  • Zachary Walsh
  • Savannah Ross
  • Terry J. FryEmail author
CART and Immunotherapy (M Ruella and P Hanley, Section Editors)
Part of the following topical collections:
  1. Topical Collection on CART and Immunotherapy


Purpose of Review

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable remission induction rates for relapsed/refractory B cell malignancies. However, loss of the CAR-targeted antigen, known as antigen escape, accounts for a substantial percentage of relapses following CAR therapy and is a major barrier to durable remission. Here, we discuss mechanisms for antigen escape and strategies to prevent this pattern of relapse, including the use of multi-specific CARs, which recognize and target multiple tumor-associated antigens simultaneously.

Recent Findings

Preclinical and early clinical trial data indicates that multi-specific CAR therapy for B cell malignancies is both safe and effective. Optimal combinations of target antigens, as well as different multi-specific CAR formats, are currently being evaluated.


Although still in early stages of development, multi-specific CAR therapy represents a promising approach to mitigate antigen loss–related relapses and improve durability of remission in patients with refractory B cell malignancies, and may be applicable to other types of cancer.


CAR T cell Antigen loss Antigen escape Multi-specific Bivalent Lineage switch 


Compliance with Ethical Standards

Conflict of Interest

Zachary Walsh, Savannah Ross, and Terry J. Fry declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

All reported studies/experiments with human or animal subjects performed by the authors have been previously published and complied with all applicable ethical standards (including the Helsinki declaration and its amendments, institutional/national research committee standards, and international/national institutional guidelines).


Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance

  1. 1.
    Ogba N, Arwood NM, Bartlett NL, Bloom M, Brown P, Brown C, et al. Chimeric antigen receptor T-cell therapy. J Natl Compr Cancer Netw. 2018;16(9):1092–106.CrossRefGoogle Scholar
  2. 2.
    Jindal V. Role of chimeric antigen receptor T cell therapy in glioblastoma multiforme. Mol Neurobiol. 2018;55(11):8236–42.CrossRefGoogle Scholar
  3. 3.
    Ruella M, Barrett DM, Kenderian SS, Shestova O, Hofmann TJ, Perazzelli J, et al. Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. J Clin Invest. 2016;126(10):3814–26.CrossRefGoogle Scholar
  4. 4.
    Qin H, Ramakrishna S, Nguyen S, Fountaine TJ, Ponduri A, Stetler-Stevenson M, et al. Preclinical development of bivalent chimeric antigen receptors targeting both CD19 and CD22. Mol Ther - Oncolytics. 2018;11:127–37 Available from: Accessed 11 April 2019.CrossRefGoogle Scholar
  5. 5.
    •• Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S, et al. CD22-targeted CAR T cells induce remission in B-ALL that is or resistant to CD19-targeted CAR immunotherapy. Nat Med. 2018;24(1):20–8. Demonstrated efficacy of CD22-treated CAR for B-ALL and impact of antigen density on CAR functionality. CrossRefGoogle Scholar
  6. 6.
    Ramakrishna S, Highfill SL, Walsh Z, Nguyen SM, Lei H, Shern JF, et al. Modulation of target antigen density improves CAR T cell functionality and persistence. Clin Cancer Res. 2019:clincanres.3784.2018 Available from: Accessed 22 May 2019.
  7. 7.
    Brudno JN, Maric I, Hartman SD, Rose JJ, Wang M, Lam N, et al. T cells genetically modified to express an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of poor-prognosis relapsed multiple myeloma. J Clin Oncol. 2018;36(22):2267–80.CrossRefGoogle Scholar
  8. 8.
    Qin H, Nguyen SM, Ramakrishna S, Tarun S, Yang L, Verdini NP, et al. Novel CD19/CD22 bicistronic chimeric antigen receptors outperform single or bivalent cars in eradicating CD19<sup>+</sup>CD22<sup>+</sup>, CD19<sup>-</sup>, and CD22<sup>-</sup> pre-B leukemia. Blood. 2017;130(Suppl 1):810 LP–810.Google Scholar
  9. 9.
    Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol [Internet]. 2019. Scholar
  10. 10.
    Maude SL, Teachey DT, Porter DL, Grupp SA. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood. 2015;125(26):4017–23.CrossRefGoogle Scholar
  11. 11.
    Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor–modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509–18.CrossRefGoogle Scholar
  12. 12.
    Sotillo E, Barrett DM, Black KL, Bagashev A, Oldridge D, Wu G, et al. Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov. 2015;5(12):1282–95.CrossRefGoogle Scholar
  13. 13.
    Orlando EJ, Han X, Tribouley C, Wood PA, Leary RJ, Riester M, et al. Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia. Nat Med. 2018;24(10):1504–6.CrossRefGoogle Scholar
  14. 14.
    Bagashev A, Sotillo E, Tang C-HA, Black KL, Perazzelli J, Seeholzer SH, et al. CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum. Mol Cell Biol. 2018;38(21).Google Scholar
  15. 15.
    Yates B, Shalabi H, Salem D, Delbrook C, Yuan CM, Stetler-Stevenson M, et al. Sequential CD22 targeting impacts CD22 CAR-T cell response. Blood. 2018;132(Suppl 1):282 LP–282 Available from: Accessed 22 May 2019.CrossRefGoogle Scholar
  16. 16.
    Shalabi H, Kraft IL, Wang H-W, Yuan CM, Yates B, Delbrook C, et al. Sequential loss of tumor surface antigens following chimeric antigen receptor T-cell therapies in diffuse large B-cell lymphoma. Haematologica Italy. 2018;103:e215–8.CrossRefGoogle Scholar
  17. 17.
    Jacoby E, Nguyen SM, Fountaine TJ, Welp K, Gryder B, Qin H, et al. CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat Commun. 2016;7:12320.Google Scholar
  18. 18.
    Evans AG, Rothberg PG, Burack WR, Huntington SF, Porter DL, Friedberg JW, et al. Evolution to plasmablastic lymphoma evades CD19-directed chimeric antigen receptor T cells. Br J Haematol. 2015;171(2):205–9.CrossRefGoogle Scholar
  19. 19.
    Gardner R, Wu D, Cherian S, Fang M, Hanafi L-A, Finney O, et al. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy. Blood. 2016;127(20):2406–10.CrossRefGoogle Scholar
  20. 20.
    Park JH, Geyer MB. Brentjens RJ. CD19-targeted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical outcomes to date. Blood. 2016;127(26):3312 LP–3320 Available from: Accessed 11 April 2019.CrossRefGoogle Scholar
  21. 21.
    Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med. 2018;378(5):439–48. Scholar
  22. 22.
    Majzner RG, Rietberg SP, Labanieh L, Sotillo E, Weber EW, Lynn RC, et al. Low CD19 antigen density diminishes efficacy of CD19 CAR T cells and can be overcome by rational redesign of CAR signaling domains. Blood. 2018. 132(Suppl 1):963 LP – 963963. Available from: Accessed 16 April 2019.
  23. 23.
    Walker AJ, Majzner RG, Zhang L, Wanhainen K, Long AH, Nguyen SM, et al. Tumor antigen and receptor densities regulate efficacy of a chimeric antigen receptor targeting anaplastic lymphoma kinase. Mol Ther. 2017;25(9):2189–201.CrossRefGoogle Scholar
  24. 24.
    Biberacher V, Decker T, Oelsner M, Wagner M, Bogner C, Schmidt B, et al. The cytotoxicity of anti-CD22 immunotoxin is enhanced by bryostatin 1 in B-cell lymphomas through CD22 upregulation and PKC-betaII depletion. Haematologica. 2012;97(5):771–9.CrossRefGoogle Scholar
  25. 25.
    Yoshida T, Mihara K, Takei Y, Yanagihara K, Kubo T, Bhattacharyya J, et al. All-trans retinoic acid enhances cytotoxic effect of T cells with an anti-CD38 chimeric antigen receptor in acute myeloid leukemia. Clin Transl Immunol. 2016;5(12):e116.CrossRefGoogle Scholar
  26. 26.
    Gardner R, Annesley C, Finney O, Summers C, Lamble AJ, Rivers J, et al. Early clinical experience of CD19 x CD22 dual specific CAR T cells for enhanced anti-leukemic targeting of acute lymphoblastic leukemia. Blood. 2018;132(Suppl 1):278 LP–278 Available from: Accessed 11 April 2019.CrossRefGoogle Scholar
  27. 27.
    Grada Z, Hegde M, Byrd T, Shaffer DR, Ghazi A, Brawley VS, et al. TanCAR: a novel bispecific chimeric antigen receptor for cancer immunotherapy. Mol Ther Nucleic Acids. 2013;2:e105.CrossRefGoogle Scholar
  28. 28.
    Hegde M, Mukherjee M, Grada Z, Pignata A, Landi D, Navai SA, et al. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape find the latest version: tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape. J Clin Invest. 2016;126(8):3036–52.CrossRefGoogle Scholar
  29. 29.
    Zah E, Lin M-Y, Silva-Benedict A, Jensen MC, Chen YY. T Cells Expressing CD19/CD20 Bispecific chimeric antigen receptors prevent antigen escape by malignant B cells. Cancer Immunol Res. 2016;4(6):498 LP–508 Available from: Accessed 11 April 2019.CrossRefGoogle Scholar
  30. 30.
    Amrolia PJ, Wynn R, Hough R, Vora A, Bonney D, Veys P, et al. Simultaneous targeting of CD19 and CD22: phase I study of AUTO3, a bicistronic chimeric antigen receptor (CAR) T-cell therapy, in pediatric patients with relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL): Amelia Study. Blood. 2018;132(Suppl 1):279 LP–279 Available from: Accessed 16 April 2019.CrossRefGoogle Scholar
  31. 31.
    • Ardeshna K, Marzolini MAV, Osborne W, Al-Hajj M, Thomas S, Faulkner J, et al. Study of AUTO3, the first bicistronic chimeric antigen receptor (CAR) targeting CD19 and CD22, followed by Anti-PD1 consolidation in patients with relapsed/refractory (r/r) diffuse large B cell lymphoma (DLBCL): Alexander study. Blood. 2018;132(Suppl 1):1679 LP–1679 Available from: Accessed 16 April 2019. Clinical trial demonstrating early efficacy of bicistronic CARs targeting CD19 and CD22 for the treatment of r/r leukemia CrossRefGoogle Scholar
  32. 32.
    • Schultz LM, Davis KL, Baggott C, Chaudry C, Marcy AC, Mavroukakis S, et al. Phase 1 study of CD19/CD22 bispecific chimeric antigen receptor (CAR) therapy in children and young adults with B cell acute lymphoblastic leukemia (ALL). Blood. 2018;132(Suppl 1):898 LP–898 Available from: Accessed 16 April 2019. Clinical trial demonstrating early efficacy of bivalent CARs targeting CD19 and CD22 for the treatment of pediatric and young adult B-ALL.CrossRefGoogle Scholar
  33. 33.
    Hossain N, Sahaf B, Abramian M, Spiegel JY, Kong K, Kim S, et al. Phase I experience with a bi-specific CAR targeting CD19 and CD22 in adults with B-cell malignancies. Blood. 2018;132(Suppl 1):490 LP–490 Available from: Scholar
  34. 34.
    Shah NN, Zhu F, Taylor C, Schneider D, Krueger W, Worden A, et al. A phase 1 study with point-of-care manufacturing of dual targeted, tandem anti-CD19, anti-CD20 chimeric antigen receptor modified T (CAR-T) cells for relapsed, refractory, non-Hodgkin lymphoma. Blood. 2018;132(Suppl 1):4193 LP–4193 Available from: Scholar
  35. 35.
    Li D, Hu Y, Jin Z, Zhai Y, Tan Y, Sun Y, et al. TanCAR T cells targeting CD19 and CD133 efficiently eliminate MLL leukemic cells. Leukemia. 2018;32(9):2012–6.,2018.
  36. 36.
    De Munter S, Ingels J, Goetgeluk G, Bonte S, Pille M, Weening K, et al. Nanobody Based Dual Specific CARs. Int J Mol Sci. 2018;19(2).Google Scholar
  37. 37.
    Lee L, Draper B, Chaplin N, Philip B, Chin M, Galas-Filipowicz D, et al. An APRIL-based chimeric antigen receptor for dual targeting of BCMA and TACI in multiple myeloma. Blood. 2018;131(7):746–58.CrossRefGoogle Scholar
  38. 38.
    Schneider D, Xiong Y, Wu D, Nӧlle V, Schmitz S, Haso W, et al. A tandem CD19/CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines. J Immunother Cancer. 2017;5(1):42. Scholar
  39. 39.
    Qin H, Cho M, Haso W, Zhang L, Tasian SK, Oo HZ, et al. Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein. Blood. 2015;126(5):629–39.Google Scholar
  40. 40.
    Wang Y, Xu Y, Li S, Liu J, Xing Y, Xing H, et al. Targeting FLT3 in acute myeloid leukemia using ligand-based chimeric antigen receptor-engineered T cells. J Hematol Oncol. 2018;11(1):60.Google Scholar
  41. 41.
    Jetani H, Garcia-Cadenas I, Nerreter T, Thomas S, Rydzek J, Meijide JB, et al. CAR T-cells targeting FLT3 have potent activity against FLT3(−)ITD(+) AML and act synergistically with the FLT3-inhibitor crenolanib. Leukemia. 2018;32(5):1168–79.CrossRefGoogle Scholar
  42. 42.
    Fousek K, Watanabe J, George A, An X, Samaha H, Navai SA, et al. Targeting primary pre-B cell acute lymphoblastic leukemia and CD19-negative relapses using trivalent CAR T cells. Blood. 2017;130(Suppl 1):4614 LP–614 Available from: Scholar
  43. 43.
    Bielamowicz K, Fousek K, Byrd TT, Samaha H, Mukherjee M, Aware N, et al. Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma. Neuro-Oncology. 2018;20(4):506–18.Google Scholar
  44. 44.
    Caruso H, Heimberger AB. Comment on “Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma”. Neuro-Oncology. 2018;20(7):1003–4. Scholar
  45. 45.
    Wilkie S, van Schalkwyk MCI, Hobbs S, Davies DM, van der Stegen SJC, Pereira ACP, et al. Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling. J Clin Immunol. 2012;32(5):1059–70.CrossRefGoogle Scholar
  46. 46.
    Kloss CC, Condomines M, Cartellieri M, Bachmann M, Sadelain M. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol. 2013;31(1):71–5.CrossRefGoogle Scholar
  47. 47.
    Lanitis E, Poussin M, Klattenhoff AW, Song D, Sandaltzopoulos R, June CH, et al. Chimeric antigen receptor T cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo. Cancer Immunol Res. 2013;1(1):43–53.CrossRefGoogle Scholar
  48. 48.
    Fedorov VD, Themeli M, Sadelain M. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci Transl Med. 2013;5(215):215ra172.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Center for Cancer and Blood Disorders, Children’s Hospital ColoradoUniversity of Colorado Anschutz Medical CampusAuroraUSA

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