Sains Malaysiana 51(1)(2022): 271-284

http://doi.org/10.17576/jsm-2022-5101-22

A Safety Evaluation of Intravenous Administration of ex vivo Expanded Human Peripheral Blood-Derived NK Cells: A Preclinical Study

(Penilaian Keselamatan Pentadbiran ex vivo Sel NK Darah Janaan Periferi Manusia yang Dikembangkan secara Intravena: Kajian Praklinikal)

SELLAMUTHU SUBBANNA GOUNDER¹, BASKAR SUBRAMANI¹*, NUR EZZATI IZYAN BINTI MOHD RADZUANB¹, NURHIDAYAH BT MOHAMAD SAIT¹, FARAH DALILA BINTI MOHD ZAIN¹ & BASRI JOHAN JEET ABDULLAH²

¹Nichi-Asia Life Science Sdn Bhd, Kota Damansara PJU 5, 47810 Petaling Jaya, Selangor Darul Ehsan, Malaysia

²Department of Biomedical Imaging, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

Diserahkan: 19 Januari 2021/Diterima: 20 April 2021

ABSTRACT

The use of natural killer (NK) cells in the treatment of various cancers is emerging as a promising approach in adoptive immunotherapy. However, the safety of ex vivo activated and expanded cells in in vivo conditions remain unknown. In this study, the toxicity of NK cells was evaluated at different doses, with 5 × 10⁶, 20 × 10⁶ and 50 × 10⁶ cells injected intravenously into pre-irradiated (30Gy) immunodeficient mice twice a week for three weeks and the mice were followed-up on for 90 days. Throughout the study, no mortality, abnormal clinical signs, or behavioural changes related to the testing material were observed in either the treated or control groups of mice. There were no significant variations in food and water consumption between both genders in the NK cell treated and control groups. However, certain significant changes were observed between the groups in the clinical biochemistry and urine analysis reports. As autopsy showed no significant variations in absolute and relative organ weights between the groups, except for the livers of the treated mice. The histopathological analysis also demonstrated that there were no significant abnormalities in most of the organs of both genders, except for the liver. Some necrotic lesions were observed in the livers of both the treated and control mice, and these lesions may be due to the effects of irradiation or could be common in NOD.SCID mice. The findings of this study indicate that intravenous administration of NK cells is safe and does not cause any adverse effects up to the dose of 50 × 10⁶ cells/mouse.

Keywords: Adoptive immunotherapy; immunotherapy; natural killer cell; NK cell toxicity; pathology

ABSTRAK

Penggunaan sel pemusnah semula jadi (Natural Killer, NK) dalam rawatan pelbagai jenis kanser telah muncul sebagai pendekatan yang berpotensi dalam terapi imuno adoptif. Namun, keselamatan ex vivo teraktif dan sel berkembang dalam keadaan in vivo masih kekal tidak diketahui. Dalam kajian ini, ketoksikan sel NK telah disuntik secara intravena pada dos yang berbeza iaitu 5 × 10⁶, 20 × 10⁶ and 50 × 10⁶ kepada tikus keimunokurangan pre-tersinar (30Gy) sebanyak dua kali seminggu untuk 3 minggu dan tikus tersebut telah melalui pemeriksaan lanjutan untuk 90 hari. Sepanjang kajian ini, tiada kematian, tanda klinikal tidak normal atau perubahan tingkah laku berkait dengan bahan ujian dapat dilihat dalam mana-mana kumpulan tikus terawat atau terkawal. Tiada variasi yang jelas dalam pengambilan makanan dan air antara kedua dua jantina dalam kumpulan sel NK yang terawat dan terkawal. Namun, perbezaan perubahan yang jelas dapat dilihat antara kumpulan biokimia klinikal dan laporan analisis urin. Autopsi mendedahkan tiada perbezaan yang ketara pada variasi dalam berat organ mutlak dan relatif antara kumpulan, melainkan untuk hati tikus yang terawat. Analisis histopatologi juga menunjukkan bahawa tiada perbezaan yang ketara pada keabnormalan dalam kebanyakan organ untuk kedua-dua jantina, melainkan organ hati. Beberapa luka nekrosis dapat dilihat di dalam organ hati untuk kedua-dua tikus yang terawat dan terkawal, dan luka ini mungkin disebabkan oleh kesan penyinaran atau mungkin kesan biasa dalam tikus NOD.SCID. Penemuan kajian ini menunjukkan bahawa pemberian sel secara intravena NK adalah selamat dan tidak mengakibatkan kesan buruk sehingga dos 50 × 10⁶ sel/tikus.

Kata kunci: Ketoksikan sel NK; patologi; sel pemusnah semula jadi; terapi imuno; terapi imuno adoptif

RUJUKAN

Barkholt, L., Alici, E., Conrad, R., Sutlu, T., Gilljam, M., Stellan, B., Christensson, B., Guven, H., Björkström, N.K., Söderdahl, G., Cederlund, K., Kimby, E., Aschan, J., Ringdén, O., Ljunggren, H.G. & Dilber, M.S. 2009. Safety analysis of ex vivo-expanded NK and NK-like T cells administered to cancer patients: A phase I clinical study. Immunotherapy 1(5): 753-764.

Billiau, A. 1996. Interferon-gamma: Biology and role in pathogenesis. Advances in Immunology 62: 61-130.

Blom, W.M., De Bont, H.J., Meijerman, I., Kuppen, P.J., Mulder, G.J. & Nagelkerke, J.F. 1999. Interleukin-2-activated natural killer cells can induce both apoptosis and necrosis in rat hepatocytes. Hepatology 29(3): 785-792.

Burns, L.J., Weisdorf, D.J., DeFor, T.E., Vesole, D.H., Repka, T.L., Blazar, B.R., Burger, S.R., Panoskaltsis-Mortari, A., Keever-Taylor, C.A., Zhang, M.J. & Miller, J.S. 2003. IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: A phase I/II trial. Bone Marrow Transplant 32(2): 177-186.

Carlens, S., Gilljam, M., Chambers, B.J., Aschan, J., Guven, H.,  Ljunggren, H.G., Christensson, B. & Dilber, M.S.  2001. A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells. Human Immunology 62(10): 1092-1098.

Cho, D. & Campana, D. 2009. Expansion and activation of natural killer cells for cancer immunotherapy. The Korean Journal of Laboratory Medicine 29(2): 89-96.

Denman, C.J., Senyukov, V.V., Somanchi, S.S., Phatarpekar, P.V., Kopp, L.M., Johnson, J.L., Singh, H., Hurton, L., Maiti, S.N., Huls, M.H., Champlin, R.E., Cooper, L.J. & Lee, D.A. 2012. Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PLoS ONE 7(1): e30264.

Diefenbach, A., Jensen, E.R., Jamieson, A.M. & Raulet, D.H. 2001. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413: 165-171.

Doubrovina, E.S., Doubrovin., M.M., Vider, E., Sisson, R.B., O'Reilly, R.J., Dupont, B. & Vyas, Y.M. 2003. Evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma. The Journal of Immunology 171(12): 6891-6899.

Fasbender, F., Widera, A., Hengstler, J.G. & Watzl, C. 2016. Natural killer cells and liver fibrosis. Front Immunol. 7: 19.

Festing, S. & Wilkinson, R. 2007. The ethics of animal research. talking point on the use of animals in scientific research. EMBO Reports 8(6): 526-530.

Gounder, S.S., Abdullah, B.J.J., Radzuanb, N.E.I.B.M., Zain, F.D.B.M., Sait, N.B.M., Chua, C. & Subramani, B. 2018. Effect of aging on NK cell population and their proliferation at ex vivo culture condition. Analytical Cellular Pathology 2: 7871814.

Jung, I.H., Kim, D.H., Yoo, D.K., Baek, S.Y., Jeong, S.H., Jung, D.E., Park, S.W. & Chung, Y.Y. 2018. In vivo study of natural killer (NK) cell cytotoxicity against cholangiocarcinoma in a nude mouse model. In Vivo 32(4): 771-781.

Kay, H.D., Fagnani, R. & Bonnard, G.D. 1979. Cytotoxicity against the K562 erythroleukemia cell line by human natural killer (NK) cells which do not bear free Fc receptors for IgG. International Journal of Cancer 24(2): 141-150.

Keppel, M.P., Saucier, N., Mah, A.Y., Vogel, T.P. & Cooper, M.A. 2015. Activation-specific metabolic requirements for NK Cell IFN-γ production. The Journal of Immunology 194(4): 1954-1962.

Knorr, D.A., Ni, Z., Hermanson, D., Hexum, M.K., Bendzick, L., Cooper, L.J., Lee, D.A. & Kaufman, D.S. 2013. Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Translational Medicine 2(4): 274-283.

Liedtke, C., Luedde, T., Sauerbruch, T., Scholten, D., Streetz, K., Tacke, F., Tolba, R., Trautwein, C., Trebicka, J. & Weiskirchen, R. 2013. Experimental liver fibrosis research: Update on animal models, legal issues and translational aspects. Fibrogenesis Tissue Repair 6(1): 19.

Motohashi, S., Ishikawa, A., Ishikawa, E., Otsuji, M., Iizasa, T., Hanaoka, H., Shimizu, N., Horiguchi, S., Okamoto, Y., Fujii, S., Taniguchi, M., Fujisawa, T. & Nakayama, T. 2006. A phase I study of in vitro expanded natural killer T cells in patients with advanced and recurrent non-small cell lung cancer. Clinical Cancer Research 12(20): 6079-6086.

Mouiseddine, M., François, S., Souidi, M. & Chapel, A. 2012. Intravenous human mesenchymal stem cells transplantation in NOD/SCID mice preserve liver integrity of irradiation damage. In Methods in Molecular Biology, edited by Walker, J.M. New Jersey: Humana Press. pp. 179-188.

Purdy, A.K. & Campbell, K.S. 2009. Natural killer cells and cancer: Regulation by the killer cell Ig-like receptors (KIR). Cancer Biology & Therapy 8(23): 2211-2220.

Sempoux, C., Horsmans, Y., Geubel, A., Fraikin, J., Van Beers, B.E., Gigot, J.F., Lerut, J. & Rahier, J. 1997. Severe radiation-induced liver disease following localized radiation therapy for biliopancreatic carcinoma: Activation of hepatic stellate cells as an early event. Hepatology 26(1): 128-134.

Shah, N., Martin-Antonio, B., Yang, H., Ku, S., Lee, D.A., Cooper, L.J., Decker, W.K., Li, S., Robinson, S.N., Sekine, T., Parmar, S., Gribben, J., Wang, M., Rezvani, K., Yvon, E., Najjar, A., Burks, J., Kaur, I., Champlin, R.E., Bollard, C.M. & Shpall, E.J. 2013. Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity. PLoS ONE 8: e76781.

Smyth, M.J., Swann, J., Cretney, E., Zerafa, N., Yokoyama, W.M. & Hayakawa, Y. 2005. NKG2D function protects the host from tumor initiation. Journal of Experimental Medicine 202(5): 583-588.

Stern, P., Gidlund, M., Orn, A. & Wigzell, H. 1980. Natural killer cells mediate lysis of embryonal carcinoma cells lacking MHC. Nature 285(5763): 341-342.

Subramani, B., Ratnavelu, K., Pullai, C.R., Krishnan, K., Sugadan, S.D., Deng, X. & Hiroshi, T. 2013 Autologous immune enhancement therapy: A case report of a stage IV colonic cancer. Oncology Letters 5(5): 1611-1614.

Suzuki, Y., Yeung, A.C. & Ikeno, F. 2009. The pre-clinical animal model in the translational research of interventional cardiology. JACC: Cardiovascular Interventions 2(5): 373-383.

Woll, P.S., Grzywacz, B., Tian, X., Marcus, R.K., Knorr, D.A., Verneris, M.R. & Kaufman, D.S. 2009. Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity. Blood 113(24): 6094-6101.

*Pengarang untuk surat-menyurat; email: sudabas23@gmail.com