Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli

Figures & data

Figure 1. Expression of therapeutically relevant antibody fragments and full-length IgGs in E. coli strain SBDG419.

(a–c) show SDS-PAGE gels of soluble lysates and purified proteins from each test expression in shake flasks. MW = molecular weight of protein standards in ladder. (a) Trastuzumab scFv (lane 1: soluble lysate (reducing), lane 2: purified (reducing), lane 3: purified (non-reducing). (b) anti-CD3 Fab (lane 1: soluble lysate (non-reducing), lane 2: purified (non-reducing), lane 3: purified (reducing)). (c) anti-CD74 IgG (lane 1: soluble lysate (reducing), lane 2: purified (non-reducing), lane 3: purified (reducing)).

Table 1. Summary of titers and amber suppression efficiency of pAMF incorporation into nnAA-LC and nnAA-IgG produced in E. coli. For titer calculation method descriptions, refer to the materials and methods section within the supplementary information. Amber suppression efficiencies were calculated by dividing the titer (*from bioreactors, **from shake flasks) of nnAA-containing proteins by the titer of the proteins without nnAA incorporation.

Protein	Shake flask titer (g/L)	Bioreactor titer (g/L)	Titer calculation method	Amber suppression efficiency
Trastuzumab scFv	0.04	–	Protein L capture	–
Anti-CD3 Fab	0.03	–	KappaSelect CH1-XL capture	–
Trastuzumab LC	0.72^Citation26	6.2^Citation26	KappaXL HPLC	–
Anti-CD74 IgG	0.23	–	Phytip capture	–
nnAA-LC	0.35	3.9	KappaXL HPLC	63%*
nnAA-IgG	0.12	1.1	Phytip capture	52%**

Figure 2. Expression and activity of nnAA-LC, nnAA-IgG-X and DAR8 ADC-X.

(a) Depiction of two plasmids used for nnAA incorporation in E. coli strain SBDG419 or SBDG175. Left: pJ411 product plasmid bearing the sequence of the protein of interest (POI) behind a T7 promoter (T7 pr.) and followed by a T7 terminator (T7 term.). The product plasmid is high copy (pUC origin of replication) and bears a kanamycin resistance gene (KanR). Right: pJ434 RS plasmid bearing the sequence of the pAMF RS behind a T7 promoter (T7 pr.) and constitutive Pc0 promoter (Pc0 pr.). The pAMF RS sequence is followed by one copy of the AS tRNA and a T7 terminator (T7 term.). The RS plasmid is a medium copy (p15a origin of replication) and bears an ampicillin resistance gene (AmpR). (b) SDS-PAGE analysis of lysates expressing nnAA-LC using different promoter combinations driving pAMF RS and AS tRNA expression. MW = molecular weight of protein standards in ladder. Lane 1: Trastuzumab light chain (Tras. LC) control (no nnAAs incorporated), lane 2: nnAA-LC expression using RS plasmid with a T7pr., no pAMF nnAA added, lane 3: nnAA-LC expression using RS plasmid with a T7pr., pAMF added, lane 4: nnAA-LC expression using RS plasmid with a combined T7pr. and Pc0 promoter, no pAMF added, lane 5: nnAA-LC expression using RS plasmid with a combined T7pr. and Pc0 promoter, pAMF added. The arrow indicates the LC band. (c) Cell killing activity of ADC-X against a cell line expressing its target antigen (left panel, “Antigen (+)”), or with no expression of its target antigen (right panel, “Antigen (-)”). (d) Table describing titer and quality attributes of nnAA-LC and the DAR8 ADC-X. Purity % was assessed by analytical SEC, assembly % was calculated with denaturing Caliper capillary electrophoresis assay, and DAR was calculated by intact LC-MS analysis.

Figure 3. Schematic of cell free protein synthesis-based production of nnAA-IgG-X and ADC-X using nnAA-LC as pre-fabricated light chain.

The nnAA-LC protein, produced in E. coli strain SBDG419, is purified and added as a reagent to a cell free protein synthesis reaction producing the heavy chain of nnAA-IgG-X. The nnAA-IgG-X is purified from the reaction and conjugated with a DBCO-exatecan payload to produce a homogenously-conjugated DAR8 ADC: ADC-X. Modified from reference 26.

Figure 4. Expression, purification, and biological activity of the anti-CD74 nnAA-IgG and its derivative ADC.

(a) Graph showing in-lysate nnAA-IgG concentrations (orange bars, left axis) and nnAA-IgG assembly (black squares, right axis) produced from each expression plasmid construct. In-lysate nnAA-IgG concentrations were quantified by Phytip purification assay and nnAA-IgG assembly was analyzed by a Caliper gel electrophoresis assay. Plasmids are named based on the RBS used for either the heavy chain or light chain, e.g., the H2L1 construct bears heavy chain RBS 2 (H2) and light chain RBS 1 (L1). The RBS sequences are numbered from strongest (RBS #1) to weakest (RBS #4) predicted strength. (b) SDS-PAGE gel of anti-CD74 nnAA-IgG samples. MW = molecular weight of protein standards in ladder. Lane 1: soluble lysate from 5 L fermentation to produce the anti-CD74 nnAA-IgG (reducing), lane 2: purified nnAA-IgG (non-reducing), and lane 3: purified nnAA-IgG (reducing). (c) Cell killing activity of the anti-CD74 ADC (blue squares) compared to the anti-CD74 nnAA-IgG (magenta triangles) observed in cell lines exhibiting CD74 antigen expression (left, “CD74 (+)”) or without CD74 antigen expression (right, “CD74 (-)”). (d) Table summarizing titer, product quality, and biological activity of the anti-CD74 nnAA-IgG and ADC. The % purity was assessed by analytical SEC, % assembly was determined by Caliper electrophoresis assay, DAR was calculated using intact LC-MS, and EC₅₀ cell killing activity was determined by cell viability measurements (see panel C). n.d. = not detected

Hanson J, Groff D, Carlos A, Usman H, Fong K, Yu A, Armstrong S, Dwyer A, Masikat MR, Yuan D. et al. An integrated in vivo/in vitro protein production platform for site-specific antibody drug conjugates. Bioengineering. 2023;10(3):304. doi:10.3390/bioengineering10030304.

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