Chimeric antigen receptor T cell targeting EGFRvIII for metastatic lung cancer therapy

Abstract

Lung cancer is the most common incident cancer and the leading cause of cancer death. In recent years, the development of tumor immunotherapy especially chimeric antigen receptor T (CAR-T) cell has shown a promising future. Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation expressed in various types of tumors and has been detected in non-small cell lung cancer with a mutation rate of 10%. Thus, EGFRvIII is a potential antigen for targeted lung cancer therapy. In this study, CAR vectors were constructed and transfected into virus-packaging cells. Then, activated T cells were infected with retrovirus harvested from stable virus-producing single clone cell lines. CAR expression on the surfaces of the T cells was detected by flow cytometry and Western blot. The function of CAR-T targeting EGFRvIII was then evaluated. The EGFRvIII-CAR vector was successfully constructed and confirmed by DNA sequencing. A stable virus-producing cell line was produced from a single clone by limited dilution. The culture conditions for the cell line, including cell density, temperature, and culture medium were optimized. After infection with retrovirus, CAR was expressed on more than 90% of the T cells. The proliferation of CAR-T cells were induced by cytokine and specific antigen in vitro. More importantly, EGFRvIII-CART specifically and efficiently recognized and killed A549-EGFRvIII cells with an effector/target ratio of 10:1 by expressing and releasing cytokines, including perforin, granzyme B, IFN-g, and TNF-α. The in vivo study indicated that the metastasis of A549-EGFRvIII cells in mice were inhibited by EGFRvIII-CART cells, and the survival of the mice was significantly prolonged with no serious side effects. EGFRvIII-CART showed significantly efficient antitumor activity against lung cancer cells expressing EGFRvIII in vivo and in vitro. Therefore, CAR-T targeting EGFRvIII is a potential therapeutic strategy in preventing recurrence and metastasis of lung cancer after surgery.

Keywords

chimeric antigen receptor T cells / epidermal growth factor receptor / lung cancer / immunotherapy / tumor immunology

References

[1] Chen W, Zheng R, Zhang S, Zhao P, Zeng H, Zou X, He J. Annual report on status of cancer in China, 2010. Chin J Cancer Res 2014; 26(1): 48–58

[2] Chen W, Zheng R, Zeng H, Zhang S, He J. Annual report on status of cancer in China, 2011. Chin J Cancer Res 2015; 27(1): 2–12

[3] Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin 2016; 66(2): 115–132

[4] Reck M, Rabe KF. Precision diagnosis and treatment for advanced non-small-cell lung cancer. N Engl J Med 2017; 377(9): 849–861

[5] Rizvi NA, Peters S. Immunotherapy for unresectable stage III non-small-cell lung cancer. N Engl J Med 2017; 377(20): 1986–1988

[6] Ribas A. Adaptive immune resistance: how cancer protects from immune attack. Cancer Discov 2015; 5(9): 915–919

[7] Rosenberg SA, Lotze MT, Muul LM, Leitman S, Chang AE, Ettinghausen SE, Matory YL, Skibber JM, Shiloni E, Vetto JT, Seipp CA, Simpson C, Reichert CM. Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 1985; 313(23): 1485–1492

[8] Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA 1989; 86(24): 10024–10028

[9] 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): 1507–1517

[10] Park JH, Rivière I, Gonen M, Wang X, Sénéchal B, Curran KJ, Sauter C, Wang Y, Santomasso B, Mead E, Roshal M, Maslak P, Davila M, Brentjens RJ, Sadelain M. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med 2018; 378(5): 449–459

[11] Jindal V, Arora E, Gupta S. Challenges and prospects of chimeric antigen receptor T cell therapy in solid tumors. Med Oncol 2018; 35(6): 87

[12] Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA, Kurpad SN, McLendon RE, Moscatello D, Pegram CN, Reist CJ, Traweek ST, Wong AJ, Zalutsky MR and Bigner DD. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res 1995; 55(14): 3140–3148

[13] Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK, Huang HJ. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 1994; 91(16): 7727–7731

[14] Yu H, Gong X, Luo X, Han W, Hong G, Singh B, Tang CK. Co-expression of EGFRvIII with ErbB-2 enhances tumorigenesis: EGFRvIII mediated constitutively activated and sustained signaling pathways, whereas EGF-induced a transient effect on EGFR-mediated signaling pathways. Cancer Biol Ther 2008; 7(11): 1818–1828

[15] Zeineldin R, Rosenberg M, Ortega D, Buhr C, Chavez MG, Stack MS, Kusewitt DF, Hudson LG. Mesenchymal transformation in epithelial ovarian tumor cells expressing epidermal growth factor receptor variant III. Mol Carcinog 2006; 45(11): 851–860

[16] Garcia de Palazzo IE, Adams GP, Sundareshan P, Wong AJ, Testa JR, Bigner DD, Weiner LM. Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas. Cancer Res 1993; 53(14): 3217–3220

[17] Okamoto I, Kenyon LC, Emlet DR, Mori T, Sasaki J, Hirosako S, Ichikawa Y, Kishi H, Godwin AK, Yoshioka M, Suga M, Matsumoto M, Wong AJ. Expression of constitutively activated EGFRvIII in non-small cell lung cancer. Cancer Sci 2003; 94(1): 50–56

[18] Duan J, Wang Z, Bai H, An T, Zhuo M, Wu M, Wang Y, Yang L, Wang J. Epidermal growth factor receptor variant III mutation in Chinese patients with squamous cell cancer of the lung. Thorac Cancer 2015; 6(3): 319–326

[19] Onodera M, Yachie A, Nelson DM, Welchlin H, Morgan RA, Blaese RM. A simple and reliable method for screening retroviral producer clones without selectable markers. Hum Gene Ther 1997; 8(10): 1189–1194

[20] Bridgeman JS, Hawkins RE, Bagley S, Blaylock M, Holland M, Gilham DE. The optimal antigen response of chimeric antigen receptors harboring the CD3ζ transmembrane domain is dependent upon incorporation of the receptor into the endogenous TCR/CD3 complex. J Immunol 2010; 184(12): 6938–6949

[21] Heslop HE. Safer CARS. Mol Ther 2010; 18(4): 661–662

[22] Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, Suhoski MM, Varela-Rohena A, Haines KM, Heitjan DF, Albelda SM, Carroll RG, Riley JL, Pastan I, June CH. Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA 2009; 106(9): 3360–3365

[23] 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(10): 2304–2309

[24] 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(2): 123–127

[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(4): 843–851

[26] Hatanpaa KJ, Burma S, Zhao D, Habib AA. Epidermal growth factor receptor in glioma: signal transduction, neuropathology, imaging, and radioresistance. Neoplasia 2010; 12(9): 675–684

[27] Johnson LA, Scholler J, Ohkuri T, Kosaka A, Patel PR, McGettigan SE, Nace AK, Dentchev T, Thekkat P, Loew A, Boesteanu AC, Cogdill AP, Chen T, Fraietta JA, Kloss CC, Posey AD Jr, Engels B, Singh R, Ezell T, Idamakanti N, Ramones MH, Li N, Zhou L, Plesa G, Seykora JT, Okada H, June CH, Brogdon JL, Maus MV. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma. Sci Transl Med 2015; 7(275): 275ra22

[28] Ohno M, Ohkuri T, Kosaka A, Tanahashi K, June CH, Natsume A, Okada H. Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts. J Immunother Cancer 2013; 1(1): 21

[29] Hsu C, Jones SA, Cohen CJ, Zheng Z, Kerstann K, Zhou J, Robbins PF, Peng PD, Shen X, Gomes TJ, Dunbar CE, Munroe DJ, Stewart C, Cornetta K, Wangsa D, Ried T, Rosenberg SA, Morgan RA. Cytokine-independent growth and clonal expansion of a primary human CD8⁺ T-cell clone following retroviral transduction with the IL-15 gene. Blood 2007; 109(12): 5168–5177

[30] Nagaraj S, Ziske C, Schmidt-Wolf IG. Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer. Genet Vaccines Ther 2004; 2(1): 12

[31] Markley JC, Sadelain M. IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell-mediated rejection of systemic lymphoma in immunodeficient mice. Blood 2010; 115(17): 3508–3519

[32] Hsu C, Hughes MS, Zheng Z, Bray RB, Rosenberg SA, Morgan RA. Primary human T lymphocytes engineered with a codon-optimized IL-15 gene resist cytokine withdrawal-induced apoptosis and persist long-term in the absence of exogenous cytokine. J Immunol 2005; 175(11): 7226–7234

[33] Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ, Hwang LN, Yu Z, Wrzesinski C, Heimann DM, Surh CD, Rosenberg SA, Restifo NP. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8⁺ T cells. J Exp Med 2005; 202(7): 907–912

[34] O’Rourke DM, Nasrallah MP, Desai A, Melenhorst JJ, Mansfield K, Morrissette JJD, Martinez-Lage M, Brem S, Maloney E, Shen A, Isaacs R, Mohan S, Plesa G, Lacey SF, Navenot JM, Zheng Z, Levine BL, Okada H, June CH, Brogdon JL, Maus MV. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med 2017; 9(399): eaaa0984

[35] Chen N, Morello A, Tano Z, Adusumilli PS. CAR T-cell intrinsic PD-1 checkpoint blockade: a two-in-one approach for solid tumor immunotherapy. OncoImmunology 2017; 6(2): e1273302

[36] Huang Q, Xia J, Wang L, Wang X, Ma X, Deng Q, Lu Y, Kumar M, Zhou Z, Li L, Zeng Z, Young KH, Yi Q, Zhang M, Li Y. miR-153 suppresses IDO1 expression and enhances CAR T cell immunotherapy. J Hematol Oncol 2018; 11(1): 58

Acknowledgements

This study was supported by National Key Basic Research and Development Plan (No. 2015CB553701).

Compliance with ethics guidelines

Zhao Zhang, Jun Jiang, Xiaodong Wu, Mengyao Zhang, Dan Luo, Renyu Zhang, Shiyou Li, Youwen He, Huijie Bian, and Zhinan Chen declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the appropriate credit is given to the original author(s) and the source, and a link is provided to the Creative Commons license, indicating if changes were made.

RIGHTS & PERMISSIONS

2019 The Author(s) 2019. This article is published with open access at link.springer.com and journal.hep.com.cn