Frontiers of Medicine

, Volume 11, Issue 4, pp 554–562 | Cite as

CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells

  • Yongping Zhang
  • Xingying Zhang
  • Chen Cheng
  • Wei Mu
  • Xiaojuan Liu
  • Na Li
  • Xiaofei Wei
  • Xiang Liu
  • Changqing Xia
  • Haoyi Wang
Research Article


T cells engineered with chimeric antigen receptor (CAR) have been successfully applied to treat advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of Tcell activity is lymphocyte activation gene-3 (LAG-3). We successfully generated LAG-3 knockout Tand CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.




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We would like to thank Junning Wei and Yi Yang (Beijing Cord Blood Bank) for their help in preparing the cord blood samples. This work was supported by the National Natural Science Foundation of China (No. 31471215), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA01010409), and the National High Technology Research and Development Program of China (863 Program, No. 2015AA020307). Haoyi Wang is supported by the “Young Thousand Talent Project.”

Supplementary material

11684_2017_543_MOESM1_ESM.pdf (147 kb)
Supplementary material, approximately 146 KB.


  1. 1.
    Kakarla S, Gottschalk S. CAR T cells for solid tumors: armed and ready to go? Cancer J 2014; 20(2): 151–155CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, Chung SS, Stefanski J, Borquez-Ojeda O, Olszewska M, Qu J, Wasielewska T, He Q, Fink M, Shinglot H, Youssif M, Satter M, Wang Y, Hosey J, Quintanilla H, Halton E, Bernal Y, Bouhassira DC, Arcila ME, Gonen M, Roboz GJ, Maslak P, Douer D, Frattini MG, Giralt S, Sadelain M, Brentjens R. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 2014; 6(224): 224ra25CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL, June CH, Porter DL, Grupp SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371(16): 1507–1517CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Fry TJ, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 2015; 385(9967): 517–528CrossRefPubMedGoogle Scholar
  5. 5.
    McClanahan F, Riches JC, Miller S, Day WP, Kotsiou E, Neuberg D, Croce CM, Capasso M, Gribben JG. Mechanisms of PD-L1/PD- 1-mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model. Blood 2015; 126(2): 212–221CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Khalil DN, Smith EL, Brentjens RJ, Wolchok JD. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol 2016; 13(5): 273–290CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Triebel F, Jitsukawa S, Baixeras E, Roman-Roman S, Genevee C, Viegas-Pequignot E, Hercend T. LAG-3, a novel lymphocyte activation gene closely related to CD4. J Exp Med 1990; 171(5): 1393–1405CrossRefPubMedGoogle Scholar
  8. 8.
    Huard B, Prigent P, Tournier M, Bruniquel D, Triebel F. CD4/major histocompatibility complex class II interaction analyzed with CD4- and lymphocyte activation gene-3 (LAG-3)-Ig fusion proteins. Eur J Immunol 1995; 25(9): 2718–2721CrossRefPubMedGoogle Scholar
  9. 9.
    Xu F, Liu J, Liu D, Liu B, Wang M, Hu Z, Du X, Tang L, He F. LSECtin expressed on melanoma cells promotes tumor progression by inhibiting antitumor T-cell responses. Cancer Res 2014; 74(13): 3418–3428CrossRefPubMedGoogle Scholar
  10. 10.
    Baixeras E, Huard B, Miossec C, Jitsukawa S, Martin M, Hercend T, Auffray C, Triebel F, Piatier-Tonneau D. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J Exp Med 1992; 176(2): 327–337PubMedGoogle Scholar
  11. 11.
    Huard B, Gaulard P, Faure F, Hercend T, Triebel F. Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand. Immunogenetics 1994; 39(3): 213–217CrossRefPubMedGoogle Scholar
  12. 12.
    Workman CJ, Rice DS, Dugger KJ, Kurschner C, Vignali DA. Phenotypic analysis of the murine CD4-related glycoprotein, CD223 (LAG-3). Eur J Immunol 2002; 32(8): 2255–2263CrossRefPubMedGoogle Scholar
  13. 13.
    Huang CT, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, Hipkiss EL, Ravi S, Kowalski J, Levitsky HI, Powell JD, Pardoll DM, Drake CG, Vignali DA. Role of LAG-3 in regulatory T cells. Immunity 2004; 21(4): 503–513CrossRefPubMedGoogle Scholar
  14. 14.
    Kisielow M, Kisielow J, Capoferri-Sollami G, Karjalainen K. Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells. Eur J Immunol 2005; 35(7): 2081–2088CrossRefPubMedGoogle Scholar
  15. 15.
    Workman CJ, Wang Y, El Kasmi KC, Pardoll DM, Murray PJ, Drake CG, Vignali DA. LAG-3 regulates plasmacytoid dendritic cell homeostasis. J Immunol 2009; 182(4): 1885–1891CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hannier S, Triebel F. The MHC class II ligand lymphocyte activation gene-3 is co-distributed with CD8 and CD3-TCR molecules after their engagement by mAb or peptide-MHC class I complexes. Int Immunol 1999; 11(11): 1745–1752CrossRefPubMedGoogle Scholar
  17. 17.
    Workman CJ, Dugger KJ, Vignali DA. Cutting edge: molecular analysis of the negative regulatory function of lymphocyte activation gene-3. J Immunol 2002; 169(10): 5392–5395CrossRefPubMedGoogle Scholar
  18. 18.
    Sierro S, Romero P, Speiser DE. The CD4-like molecule LAG-3, biology and therapeutic applications. Expert Opin Ther Targets 2011; 15(1): 91–101CrossRefPubMedGoogle Scholar
  19. 19.
    Okamura T, Fujio K, Shibuya M, Sumitomo S, Shoda H, Sakaguchi S, Yamamoto K. CD4+CD25–LAG3+ regulatory T cells controlled by the transcription factor Egr-2. Proc Natl Acad Sci USA 2009; 106(33): 13974–13979CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, Subramaniam S, Blattman JN, Barber DL, Ahmed R. Molecular signature of CD8+T cell exhaustion during chronic viral infection. Immunity 2007; 27(4): 670–684CrossRefPubMedGoogle Scholar
  21. 21.
    Matsuzaki J, Gnjatic S, Mhawech-Fauceglia P, Beck A, Miller A, Tsuji T, Eppolito C, Qian F, Lele S, Shrikant P, Old LJ, Odunsi K. Tumor-infiltrating NY-ESO-1-specific CD8+T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci USA 2010; 107(17): 7875–7880CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Workman CJ, Cauley LS, Kim IJ, Blackman MA, Woodland DL, Vignali DA. Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J Immunol 2004; 172(9): 5450–5455CrossRefPubMedGoogle Scholar
  23. 23.
    Maçon-Lemaître L, Triebel F. The negative regulatory function of the lymphocyte-activation gene-3 co-receptor (CD223) on human T cells. Immunology 2005; 115(2): 170–178CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Blackburn SD, Shin H, Haining WN, Zou T, Workman CJ, Polley A, Betts MR, Freeman GJ, Vignali DA, Wherry EJ. Coregulation of CD8+T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 2009; 10(1): 29–37CrossRefPubMedGoogle Scholar
  25. 25.
    Richter K, Agnellini P, Oxenius A. On the role of the inhibitory receptor LAG-3 in acute and chronic LCMV infection. Int Immunol 2010; 22(1): 13–23CrossRefPubMedGoogle Scholar
  26. 26.
    Butler NS, Moebius J, Pewe LL, Traore B, Doumbo OK, Tygrett LT, Waldschmidt TJ, Crompton PD, Harty JT. Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection. Nat Immunol 2011; 13(2): 188–195CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Woo SR, Turnis ME, Goldberg MV, Bankoti J, Selby M, Nirschl CJ, Bettini ML, Gravano DM, Vogel P, Liu CL, Tangsombatvisit S, Grosso JF, Netto G, Smeltzer MP, Chaux A, Utz PJ, Workman CJ, Pardoll DM, Korman AJ, Drake CG, Vignali DA. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate Tcell function to promote tumoral immune escape. Cancer Res 2012; 72(4): 917–927CrossRefPubMedGoogle Scholar
  28. 28.
    Goding SR, Wilson KA, Xie Y, Harris KM, Baxi A, Akpinarli A, Fulton A, Tamada K, Strome SE, Antony PA. Restoring immune function of tumor-specific CD4+T cells during recurrence of melanoma. J Immunol 2013; 190(9): 4899–4909CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Menger L, Sledzinska A, Bergerhoff K, Vargas FA, Smith J, Poirot L, Pule M, Hererro J, Peggs KS, Quezada SA. TALEN-mediated inactivation of PD-1 in tumor-reactive lymphocytes promotes intratumoral T-cell persistence and rejection of established tumors. Cancer Res 2016; 76(8): 2087–2093CrossRefPubMedGoogle Scholar
  30. 30.
    Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex genome editing to generate universal CAR Tcells resistant to PD1 inhibition. Clin Cancer Res 2017; 23(9): 2255–2266CrossRefPubMedGoogle Scholar
  31. 31.
    Liu X, Zhang Y, Cheng C, Cheng AW, Zhang X, Li N, Xia C, Wei X, Liu X, Wang H. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res 2017; 27(1): 154–157CrossRefPubMedGoogle Scholar
  32. 32.
    Su S, Hu B, Shao J, Shen B, Du J, Du Y, Zhou J, Yu L, Zhang L, Chen F, Sha H, Cheng L, Meng F, Zou Z, Huang X, Liu B. Corrigendum: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci Rep 2017; 7: 40272CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 2014; 42(22): e168CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013; 31(9): 827–832CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Yongping Zhang
    • 1
  • Xingying Zhang
    • 2
    • 3
  • Chen Cheng
    • 2
    • 4
  • Wei Mu
    • 2
    • 3
  • Xiaojuan Liu
    • 2
  • Na Li
    • 2
  • Xiaofei Wei
    • 6
  • Xiang Liu
    • 2
  • Changqing Xia
    • 1
    • 5
  • Haoyi Wang
    • 2
    • 3
  1. 1.Department of Hematology, Xuanwu HospitalCapital Medical UniversityBeijingChina
  2. 2.State Key Laboratory of Stem Cell and Reproductive Biology, Institute of ZoologyChinese Academy of SciencesBeijingChina
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.Graduate SchoolUniversity of Science and Technology of ChinaHefeiChina
  5. 5.Department of Pathology, Immunology and Laboratory MedicineUniversity of FloridaFloridaUSA
  6. 6.Beijing Cord Blood BankBeijingChina

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