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

 

Diserahkan: 26 November 2016/Diterima: 6 Mac 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

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*Pengarang untuk surat-menyurat; email: rachelmok2005@gmail.com