EB66 cell line, a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity

Abstract

Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of monoclonal antibodies and to potentially reduce the therapeutic cost. We report here that the EB66 cell line derived from duck embryonic stem cells can be efficiently genetically engineered to produce mAbs at yields beyond a 1 g/L, as suspension cells grown in serum-free culture media. EB66 cells display additional attractive grown characteristics such as a very short population doubling time of 12 to 14 hours, a capacity to reach very high cell density (> 30 million cells/mL) and a unique metabolic profile resulting in low ammonium and lactate accumulation and low glutamine consumption, even at high cell densities. Furthermore, mAbs produced on EB66 cells display a naturally reduced fucose content resulting in strongly enhanced ADCC activity. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies.

Figure 1

DNA transfection of duck EB66 cells. An expression vector encoding the red fluorescent protein (DsRed) was transfected into EB66 cells at increasing amounts. (A) 48 h post transfection, viable cells were counted and viability was assessed by a trypan blue exclusion. (B) Efficiency of DNA transfection was determined by flow cytometry analysis of the cells expressing DSRed. High fluorescent cells were gated to specifically determine the percentage of EB66 cells expressing dsRed at high levels. NT: non-transfected cells.

Figure 2

Cell culture growth characteristics and monoclonal antibody production. A stable EB66 clone producing a monoclonal antibody targeting an undisclosed antigen “X” was selected for analysis of its growth properties in 100 mL Erlenmeyer flasks. The population doubling time (PDT) was determined by routine culture of the EB66 producer clone during 30 d (A) and the cell density and antibody production yield was assessed in a fedbatch experiment in which glucose and glutamine concentrations were maintained by addition of concentrated media formulation at 10 g/L and 2 mM, respectively (B). A metabolic analysis (C) was performed by daily analysis of cell culture supernatant samples for the concentration of glucose (dark circles), glutamine (open squares), lactate (dark squares), ammonium (dark triangles) and glutamate (open circles).

Figure 3

Comparative N-linked oligosaccharide analysis of CHO- and EB66-produced antibodies. The N-glycan analysis was performed by time-of-flight mass spectrometry (MALDI-TOF-MS) on the commercial anti-CD20 rituximab antibody produced on CHO cells, as well as on an EB66-produced anti-CD20 antibody with the same sequence than rituximab. A second EB66-produced antibody targeting an undisclosed target “X” was also included in the study. Results are presented for each group of oligosaccharide as the percentage of total glycans. G0, G1 and G2 are non-fucosylated nongalactosylated, non-fucosylated monogalatosylated, non-fucosylated digalactosylated oligosaccharides, respectively. G0F, G1F and G2F are fucosylated nongalactosylated, fucosylated monogalatosylated, fucosylated digalactosylated oligosaccharides, respectively. “Others” represent the hybrid glycans and tri-antennary glycans, with or without fucose.

Figure 4

Stability of the glycosylation profile of EB66-produced monoclonal antibodies. An individual EB66 clone producing the anti-X antibody was selected for its production yields and expanded under fixed culture conditions for 105 generations. Monoclonal antibody batches were purified with EB66 cultures arrested at generation 1, 43 and 105 and were analyzed for their N-linked oligosaccharides composition (A and C). In parallel, an analysis of glycosylation was performed on purified antibody batches prepared from the same EB66 producer clone grown under three fedbatch conditions differing in the feeding strategies and leading to different production yields (B and D).

Figure 5

Comparative analysis of the activation of Natural Killer cells by CHO- and EB66-produced antibodies. Efficiency of activation of NK cells purified from two independent healthy human donors was assessed in parallel for the monoclonal antibody targeting the undisclosed target “X” and produced either on CHO or on EB66 cells. Induction of expression of INFγ (A and B) and of the membrane antigen CD107a (C and D) was determined by flow cytometry with NK cells cultured without Interleukin 2 (IL-2) or with 5 or 100 units of IL-2.

Figure 6

Comparative analysis of the ADCC activity of CHO- and EB66-produced antibodies. The ADCC activity was measured for two independent monoclonal antibodies: the commercial anti-CD20 rituximab and its counterpart anti-CD20 antibody produced on EB66 cells (A), as well as a monoclonal antibody targeting an undisclosed target “Z” produced in parallel on CHO cells and on four individual EB66 clones (B). The anti-hCD20 antibodies were tested using the human Raji cell line as target cells and human peripheral blood mononuclear cells (PBMC) from either a donor homozygous for FcγRIIIa-158V (VV; A, left) or a donor homozygous for FcγRIIIa-158F (FF; A, right). The mean values ± S.D. of triplicates are shown.

Figure 7

Influence of the percentage of fucosylation on the ADCC activity. The monoclonal antibody targeting the undisclosed antigen “X” was produced and purified from three individual EB66 clones specifically selected for the production of IgGs with increasing proportions of total non-fucosylated glycoforms of 29, 48 and 82%, respectively. The three batches of purified antibodies were tested for their ADCC activity as indicated in Materials & Methods, using effector cells from a single human donor. The mean values of specific target cell lysis ±S.D. of triplicates are shown.

Figure 8

In vivo clearance of EB66-produced monoclonal antibodies in mice. BALB/c mice were injected via the retro orbital sinus route with the commercial CHO-produced anti-CD20 rituximab antibody and its counterpart anti-CD20 antibody produced on EB66 cells at 10 mg/kg (A) or 20 mg/kg (B). The antibody concentrations in plasma were monitored using a human IgG-specific ELISA. The serum concentration 5 min after injection is considered as 100%. Data are the mean ± S.D. of four animals per group. The serum half-life of the administrated IgG1 was calculated from the slope of the elimination β-phase.