Disertasi

Pengembangan Panel Antibodi Monoklonal Dari Sel B Manusia Berbasis Protein Spike SARS-Cov-2 Sebagai Prototipe Imunoterapi Pasien COVID-19 = Development of a monoclonal antibody panel from human B cells based on the spike protein of SARS-CoV-2 as a prototype for immunotherapy in COVID-19 patients.

Latar Belakang: Tingginya angka kejadian dan kematian secara global yang disebabkan oleh infeksi SARS-CoV-2 sejak tahun 2020 menyebabkan berbagai pendekatan dilakukan untuk mengatasi wabah tersebut. Pemberian vaksinasi secara global dan pemberian terapi antibodi pada pasien COVID-19 diharapkan dapat mengatasi permasalahan tersebut. Berbagai penelitian terkait pengembangan vaksin SARS-CoV-2 dan terapi COVID-19 masih terus dilakukan. Akan tetapi pengembangan antibodi monoklonal manusia sebagai kandidat terapi antibodi bagi pasien COVID-19 masih belum dilaporkan di Indonesia. Metode: Penelitian ini dilakukan untuk mengembangkan panel antibodi monoklonal yang berasal dari sel B manusia terhadap protein spike SARS-CoV-2 sebaagi prototipe imunoterapi pasien COVID-19. Pada penelitian ini antibodi monoklonal dikembangkan dengan menggabungkan sel limfosit B manusia pada PBMC dengan sel myeloma, K6H6/B5 menggunakan polyethylene glycol. Seleksi hibridoma dilakukan dengan melakukan pengamatan sel secara mikroskopis dan imunologis menggunakan metode in-house indirect ELISA terhadap protein spike dan SARS-CoV-2 inaktif. Antibodi netralisasi dilakukan terhadap klon-klon hibridoma terpilih dengan menggunakan metode ELISA kompetitif. Kami juga melakukan uji suseptibilitas Omicron BA.2 pada sel Vero E6, Huh-7D12, dan HepG2 untuk mengetahui sel yang sesuai untuk propagasi Omicron BA.2 sebagai antigen untuk pengujian secar imunologis. Kami juga mengembangkan metode inhouse indirect ELISA terhadap protein spike SARS-CoV-2. Hasil: Sebanyak 11 klon hibridoma diperoleh dari tiga kumpulan sel hibridoma yang dapat mengenali protein spike dan SARS-CoV-2 inaktif. Klon C.24 dan OM.3 dapat menghasilkan antibodi netralisasi terhadap SARS-CoV-2. Kesimpulan: Penelitian ini memperlihatkan bahwa antibodi monoklonal manusia dapat dikembangkan dengan menggunakan PBMC dan dengan teknologi yang mudah dan sederhana. Penelitian lebih lanjut perlu dilakukan untuk mengetahui karakter dari antibodi monoklonal yang diperoleh sehingga dapat digunakan sebagai terapi ada pasien COVID-19, seperti pengujian antibodi monoklonal terhadap epitop yang dimiliki oleh varian SARS-CoV-2 dan stabilitas antibodi monoklonal.
Kata kunci : SARS-CoV-2, COVID-19, antibodi monoklonal manusia, hibridoma, Omicron, antibodi netralisasi


Background: The high incidence and death rate globally caused by SARS-CoV-2 virus infection since 2020 has led to various approaches being taken to overcome the outbreak. Vaccination globally and the provision of antibody therapy to COVID-19 patients are expected to overcome these problems. Various studies related to the development of SARS-CoV-2 vaccines and COVID-19 therapies are still being carried out. However, the development of human monoclonal antibodies as antibody therapy candidates for COVID-19 patients has not been reported in Indonesia. This research was conducted to develop a monoclonal antibody panel derived from human B cells against the SARS-CoV-2 spike protein as a prototype immunotherapy for COVID-19 patients. Methods: This study developed monoclonal antibodies by combining human B lymphocyte cells in PBMC with myeloma cells, K6H6/B5 using polyethylene glycol. Hybridoma selection was carried out by conducting microscopic and immunological cell observations using the in-house indirect ELISA method against the spike protein and the inactive SARS-CoV-2. Neutralizing antibodies were performed against selected hybridoma clones using competitive ELISA methods. We also performed Omicron BA.2 susceptibility tests on Vero E6, Huh-7D12, and HepG2 cells to determine which cells are suitable for the propagation of the Omicron BA.2 virus as antigens for immunological testing. Additionally, we developed an in-house indirect ELISA method against spike protein of the SARSCoV-2. Results: A total of eleven hybridoma clones could recognize the spike protein and the inactivated virus, and only C.24 and OM.3 could produce neutralizing humanderived monoclonal antibodies. Conclusion: This study shows that human monoclonal antibodies can be developed using PBMCs and with easy and simple technology. Further research needs to be carried out to determine the character of the monoclonal antibodies obtained so that they can be used as therapy for COVID-19 patients, such as testing monoclonal antibodies against epitopes possessed by SARS-CoV-2 variants and the stability of monoclonal antibodies.
Keywords: SARS-CoV-2, COVID-19, human monoclonal antibodies, hybridoma, Omicron, neutralizing monoclonal antibodies