Development of a Mammalian Cell Line for Stable Production of Anti-PD-1

Abstract

Background/Objectives: Immune checkpoint blockade, particularly targeting the programmed cell death 1 (PD-1) receptor, is a promising strategy in cancer immunotherapy. The interaction between PD-1 and its ligands, PD-L1 and PD-L2, is crucial in immune evasion by tumors. Blocking this interaction with monoclonal antibodies like Nivolumab can restore anti-tumor immunity. This study aims to develop a stable expression system for Nivolumab-based anti-PD-1 in the Chinese Hamster Ovary (CHO) DG44 cell line using two different expression vector systems with various signal sequences. Methods: The heavy chain (HC) and light chain (LC) of Nivolumab were cloned into two expression vectors, pOptiVEC and pcDNA3.3. Each vector was engineered with two distinct signal sequences, resulting in the creation of eight recombinant plasmids. These plasmids were co-transfected into CHO DG44 cells in different combinations, allowing for the assessment of stable antibody production. Results: Both pOptiVEC and pcDNA3.3 vectors were successful in stably integrating and expressing the Nivolumab-based anti-PD-1 antibody in CHO DG44 cells. This study found that the choice of signal sequence significantly influenced the quantity of antibodies produced. The optimization of production conditions further enhanced antibody yield, indicating the potential for large-scale production. Conclusions: This study demonstrates that both pOptiVEC and pcDNA3.3 expression systems are effective for the stable production of Nivolumab-based anti-PD-1 in CHO DG44 cells. Signal sequences play a critical role in determining the expression levels, and optimizing production conditions can further increase antibody yield, supporting future applications in cancer immunotherapy.

Keywords: CHO DG44; Nivolumab; anti-PD-1; immune checkpoint molecule; monoclonal antibody production.

Figure 1

Schematic overview of recombinant plasmid construction (BioRender). (A) SP1-LC, SP2-HC; (B) SP3-LC, SP4-HC.

Figure 1

Schematic overview of recombinant plasmid construction (BioRender). (A) SP1-LC, SP2-HC; (B) SP3-LC, SP4-HC.

Figure 2

Schematic representation of the pcDNA3.3N plasmid construction (BioRender).

Figure 3

Separation of digested fragments of the pcDNA3.3N plasmid on agarose gel. Lane M. DNA molecular weight marker (1 kb, Thermo Scientific); Lane 1. undigested pcDNA3.3; Lane 2. undigested pcDNA3.3N; Lanes 3–8. pcDNA3.3N digested with NotI; Lane 9. pcDNA3.3 digested with NotI and PstI; Lanes 10–15. pcDNA3.3N digested with NotI and PstI.

Figure 4

Schematic representation of the recombinant constructs (BioRender). PCMV—cytomegalovirus promoter; TK-PA—thymidine kinase polyA; PSV40—simian virus 40 promoter; geneticin—geneticin resistance gene; IRES—internal ribosome entry site; DHFR—dihydrofolate reductase; Amp—ampicillin resistance gene; pUC ori—bacterial replication origin.

Figure 5

Determination of the molecular weight of Nivolumab with MALDI-TOF. The peak appearing at 146 kDa is Nivolumab. Ionization fragments are visible at 48 kDa and 73 kDa, respectively.

Figure 6

The (A) chromatogram shows fractions of the intact, purified antibody. The (B) chromatogram represents a sample reduced with 1 mM GSH and separated under the same conditions. The first large peak at an RT of 14.729 represents the heavy chain, the next one, at 15.291 is the light chain, and the third small peak represents the non-glycosylated heavy chain.