Sains Malaysiana 46(10)(2017): 1831–1838

http://dx.doi.org/10.17576/jsm-2017-4610-21

 

Overcoming the Challenge of Transduction of Human T-cells with Chimeric Antigen Receptor (CAR) Specific for ERBB2 Antigen

(Mengatasi Cabaran Transduksi Sel-T Manusia dengan Reseptor Kimera Antigen (CAR) Khusus kepada Antigen ERBB2)

 

RUSHENI MUNISVARADASS¹, SHIRLEY DING SUET LEE¹, AVIN EE HWAN KOH¹, SURESH KUMAR^3,4, LIM MOON NIAN⁶, SHALINI VELLASAMY⁷, SYAHRIL ABDULLAH^1,4,5, ABDULLAH A. ALARFAJ⁸ & MOK POOI LING^1,2,4*

 

¹Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

²Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Aljouf University, Sakaka, 72442 Aljouf Province, Kingdom of Saudi Arabia

 

³Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

⁴Genetics and Regenerative Medicine Research Centre, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

⁵Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

⁶Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Federal Territory, Malaysia

 

⁷Department of Biomedical Science, Universiti Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

 

⁸Department of Botany and Microbiology, King Saud University, Riyadh 11451, Saudi Arabia

 

Received: 26 November 2016/Accepted: 6 March 2017

 

 

ABSTRACT

Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction protocol using spinoculation on CD³⁺ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene. CD³⁺T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabead-conjugated CD3/CD28 human T-cell activator and interleukin^-2 (IL^-2) before transduction. CD³⁺ T-cells were selected and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells.

 

Keywords: Breast cancer; CD3+ T-cells; chimeric antigen receptor (CAR); immunotherapy

 

 

ABSTRAK

Kanser payudara adalah salah satu kanser yang kerap melanda kaum wanita. Kajian saintifik telah mengaitkan lebihan ekspresi antigen ERBB2 pada barah kanser yang lebih agresif. Untuk menangani masalah ini, teknologi reseptor kimera antigen (CAR) boleh digunakan. Untuk ini, sel T manusia ditransduksi dengan urutan gen pengekodan CAR yang khusus untuk antigen berkaitan-barah (TAA). Sel T yang ditransduksi dengan CAR (CAR-T) secara genetik dapat mensasar kepada antigen kanser tanpa memerlukan pengenalan kompleks kehistoserasian utama (MHC), menjadikan ia pilihan terapi imun berpotensi umum. Walau bagaimanapun, transduksi gen terapeutik ke dalam sel T manusia dan pengembangan sel selanjutnya adalah mencabar. Dalam kajian ini, kami berjaya melaporkan pengoptimuman protokol transduksi menggunakan spinokulasi sel T CD³⁺ dengan kepekatan plasmid lentiviral pengekodan gen CAR yang berbeza. Sel T CD3+ telah diasingkan daripada sel-sel mononuklear darah periferi (PBMCs). Gen CAR yang dibina dimasukkan ke dalam plasmid lentiviral mengandungi protein pendarfluor hijau (GFP) dan zarah lentiviral penuh dihasilkan. Zarah lentiviral digunakan untuk transduksi spinokulasi T-sel yang telah diaktifkan. Sel T diaktifkan menggunakan CD3 berkonjugasi Dynabead/pengaktif manusia sel T CD28 dan interleukin^-2 (IL^-2) sebelum transduksi. Kejayaan transduksi dilaporkan apabila ekspresi GFP diperhatikan di dalam sel T CD³⁺. Kajian masa depan akan memberi tumpuan kepada model in vitro dan in vivo untuk menentukan kecekapan sel CAR-T dalam mensasarkan dan membunuh sel kanser yang mempunyai ekpresi ERBB2 berlebihan.

 

Kata kunci: Kanser payudara; reseptor kimera antigen (CAR); sel CD3+ T; terapi imuno

REFERENCES

Ahmadi, M., King, J., Xue, S., Voisine, C., Holler, A. & Wright, G. 2011. CD3 limits the efficacy of TCR gene therapy in vivo. Blood 118(13): 3528-3537.

Bollard, C., Rooney, C. & Heslop, H. 2012. T-cell therapy in the treatment of post-transplant lymphoproliferative disease. Nature Reviews Clinical Oncology 9(9): 510-519.

Carey, L. 2011. Directed therapy of subtypes of triple-negative breast cancer. The Oncologist 16: 71-78.

Chmielewski, M., Hombach, A. & Abken, H. 2013. Antigen-specific T-cell activation independently of the MHC: Chimeric antigen receptor-redirected T cells. Front Immunology 4: 371.

Cribbs, A., Kennedy, A., Gregory, B. & Brennan, F. 2013. Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T-cells. BMC Biotechnology 13: 98.

De Saint Basile, G., Ménasché, G. & Fischer, A. 2010. Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. Nature Reviews in Immunology 10: 568-579.

Dieci, M., Griguolo, G., Miglietta, F. & Guarneri, V. 2016. The immune system and hormone-receptor positive breast cancer: Is it really a dead end? Cancer Treatment Reviews 46: 9-19.

Elsahwi, K.S. & Santin, A.D. 2011. ERBB2 overexpression in uterine serous cancer: A molecular target for trastuzumab therapy. Obstetrics and Gynecology International 2011: 128295.

Eroles, P., Bosch, A., Pérez-Fidalgo, J.A. & Lluch, A. 2012. Molecular biology in breast cancer: Intrinsic subtypes and signaling pathways. Cancer Treatment Reviews 38: 698-707.

Govindarajan, S., Sivakumar, J., Garimidi, P., Rangaraj, N., Kumar, J., Rao, N. & Gopal, V. 2012. Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate. Biomaterials 33(8): 2570- 2582.

Han, X., Sun, L., Nishiyama, Y., Feng, B., Michiue, H., Seno, M., Matsui, H. & Tomizawa, K. 2013. Theranostic protein targeting ErbB2 for bioluminescence imaging and therapy for cancer. Plos ONE 8: 9.

Indah Mohd Amin, Roziana Kamaludin, Swee, Keong Yeap Mohamad Rodi Isa, Nik Mohd Mazuan Nik Mohd Rosdy, Rosfaiizah Siran, Siti Hamimah Sheikh Abdul Kadir & Narimah Abdul Hamid Hasani. 2015. Aloe emodin induces apoptosis in ER+-breast cancer cells; MCF-7 through IGF- 1R signalling pathway. Sains Malaysiana 44(4): 1137-1143.

Iqbal, N. & Iqbal, N. 2014. Human epidermal growth factor receptor 2 (HER2) in cancers: Overexpression and therapeutic implications. Molecular Biology International 2014: 1-9.

Jena, B., Dotti, G. & Cooper, L. 2010. Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood 116: 7.

Jensen, M., Popplewell, L., Cooper, L., DiGiusto, D., Kalos, M., Ostberg, J. & Forman, S. 2010. Anti-transgene rejection responses contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans. Biology of Blood and Marrow Transplantation 16(9): 1245-1256.

Maher, J. 2012. Immunotherapy of malignant disease using chimeric antigen receptor engrafted T-cells. ISRN Oncology 2012: 1-23.

Marchini, A., Scott, E. & Rommelaere, J. 2016. overcoming barriers in oncolytic virotherapy with HDAC inhibitors and immune checkpoint blockade. Viruses 8(1): 9.

Porter, D., Levine, B., Kalos, M., Bagg, A. & June, C. 2011. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. New England Journal of Medicine 365(8): 725-733.

Rivkina, A., Holodnuka-Kholodnyuk, I., Murovska, M., Soloveichika, M. & Lejniece, S. 2015. Peripheral blood lymphocyte phenotype of ZAP-70⁺ and ZAP-70⁻ patients with B-cell chronic lymphocytic leukaemia. Experimental Oncology 37: 73-76.

Schroeder, R., Stevens, C. & Sridhar, J. 2014. Small molecule tyrosine kinase inhibitors of ERBB2/HER2/Neu in the treatment of aggressive breast cancer. Molecules 19: 9.

Scott, A.M., Allison, J.P. & Wolchok, J.D. 2012. Monoclonal antibodies in cancer therapy. Cancer Immunity 12: 14.

Shaw, A. & Cornetta, K. 2014. Design and potential of non-integrating lentiviral vectors. Biomedicines 2(1): 14-35.

Sinon, S.H., Rich, A.M., Firth, N.A. & Seymour, G.J. 2013. Qualitative and quantitative assessment of immune cells in oral mucosal lichen planus (OMLP). Sains Malaysiana 42(1): 65-71.

Stemberger, C., Graef, P., Odendahl, M., Albrecht, J., Dossinger, G. & Anderl, F. 2014. Lowest numbers of primary CD⁸⁺ T cells can reconstitute protective immunity upon adoptive immunotherapy. Blood 124(4): 628-637.

Teplinsky, E. & Muggia, F. 2015. EGFR and HER2: Is there a role in ovarian cancer? Translational Cancer Research 4: 1.

Whiteside, T.L. 2010. Immune responses to malignancies. The Journal of Allergy and Clinical Immunology 125(202): 272-283.

Zack, J., Kim, S. & Vatakis, D. 2013. HIV restriction in quiescent CD⁴⁺ T cells. Retrovirology 10(1): 37.

Zhong, X., Matsushita, M., Plotkin, J., Riviere, I. & Sadelain, M. 2009. Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD⁸⁺ T cell-mediated tumor eradication. Molecular Therapy 18(2): 413-420.

 

 

*Corresponding author; email: rachelmok2005@gmail.com