Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect

Abstract

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer's Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety.

Fig 1

Mass spectrometry MALDI-TOF analysis of protein expression at 48h, 72h, 96h and 120h after induction of EAEA (A) and EAEV (B) variants. Orange arrows indicate the Kex2 cleavage site and green arrows indicate the STE13 one. While the molecular weight of the EAEV variant (26142.0 or 26342.2 Da depending on the protease cleavage site) changed during the induction period, the EAEA variant (26114.0 or 26314.2 Da) maintained a homogenous population. (C) SDS-PAGE of the samples analyzed by mass spectrometry. Secreted proteins rendered a good and similar expression yield.

Fig 2. ScFv-h3D6-Pp purification.

(A) Cation Exchange Chromatography (CEX); (B) Peptide Mass Fingerprinting (PMF) analysis of each peak. PMF analysis of the two differentiated peaks eluted after CEX indicated that the two fractions corresponded to the two possible protein variants depending on the STE13 protease cleavage site. (C) SDS-PAGE of scFv-h3D6-Pp purification. (M) Molecular weight marker; (1) Supernatant after ammonium sulfate precipitation; (2) Pellet after ammonium sulfate precipitation; (3) Sample before Cationic Exchange chromatography (CEX); (4) CEX flow through; (5) CEX elution of the mean peak; (6) PBS-dialyzed scFv-h3D6-Pp.

Fig 3. Large-scale fermentation profile.

Oxygen concentration (expressed as the percentage of air saturation), cell grow (OD₆₀₀) and methanol concentration (mg/mL of culture) during batch and feed-batch phases. Culture was induced 24h after the batch phase started. During the fed-batch phase, methanol was added periodically to reach a maximum concentration of 1% (v/v) (7.9 g/L).

Fig 4. Protein characterization.

(A) Circular Dichroism (CD) spectra at 25°C; (B)Thermal denaturation followed by CD; (C) Thermal denaturation followed by Trp-fluorescence; (D) and (E) TEM micrographs. Black: scFv-h3D6-Ec, Red: scFv-h3D6-Pp. CD analysis showed that the β-conformation characteristic of the immunoglobulin fold is maintained, albeit some differences in the interferences due to the Trp residues in the core of each domain are somehow higher in the scFv-h3D6-Pp spectrum. However, no differences in terms of thermal stability were observed and, therefore, it can be assumed that both molecules are equally folded. As expected, worm-like fibrils, behind the protective effect of scFv-h3D6, are formed upon thermal denaturation in both cases, so that the therapeutic effect should remain.

Fig 5

(A) Sequence of scFv-h3D6-Pp protein. Cysteine residues are indicated in bold red; tryptic peptides containing cysteine residues are highlighted in a blue rectangle. (B) Disulfide bonds present in the scFv-h3D6-Pp. Expected Mw of the tryptic peptides involved in disulfide pairing for each possible disulfide bonding conformation. The peptides detected by MALDI-TOF MS are indicated.

Fig 6. Mass spectrometry analysis of scFv-h3D6-Pp (red) and scFv-h3D6-Ec (black).

Molecular weight analysis determined that no N-glycosylation either O-glycosylation was added to the protein as a posttranslational modification, as the molecular weight corresponded to the amino acid sequence alone (scFv-h3D6-Pp: 26314.2 Da and scFv-h3D6-Ec: 26273.2 Da).

Fig 7. Therapeutic effects of scFv-hD6-Ec and scFv-h3D6-Pp.

(A) Viability assays in SH-SY5Y neuroblastoma cell line. Cells were exposed to Aβ oligomers (10 μM) and different concentrations of scFv-h3D6-Ec or scFv-h3D6-Pp. Black: scFv-h3D6-Ec, Red: scFv-h3D6-Pp. Comparisons of each concentration of scFv-h3D6-Ec or scFv-h3D6-Pp with Aβ alone showed statistical significance for 7.5 and 10 μM in both cases, indicating efficiency of both treatments (unpaired t-test with Welch’s correction, * p<0.05. **p<0.01). (B) Aβ₄₂ ELISA of brain homogenates (Hippocampus and Cortex); Black: scFv-h3D6-Ec, Red: scFv-h3D6-Pp, Grey: 3xTg-AD-Vehicle, Stripped: NTg-vehicle. Both treatments recovered the non-pathological levels of Aβ. *, ** indicate significance compared to the 3xTg-AD-vehicle group (Unpaired t-test with Welch’s correction, *p<0.05, **p<0.01). Arrows indicate comparisons with NTg-vehicle group (n.s. indicates no significance). (C) Aβ Immunohistochemistry of coronal sections. Aβ-immunoreactivity decreased to similar levels as those in non-transgenic animals when 3xTg-AD were treated with scFv-h3D6-Ec or scFv-h3D6-Pp. Bar in panoramic coronal sections (2.5x zoom in) corresponds to 1 mm and in hippocampus and cortex sections (16x zoom in) to 200 μm.