An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates.

The XpressCF+® cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+® reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.

Development of an E. coli strain for cell-free ADC manufacturing.

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody-drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.

Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free Protein Synthesis.

Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvß3, αvß5, and α5ß1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL-Fc-MMAF, showed a modest but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications. Mol Cancer Ther; 15(6); 1291-300. ©2016 AACR.

A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.

Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re-examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell-free system with a 95% reduction in reagent costs (excluding cell-extract, plasmid, and T7 RNA polymerase made in-house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze-thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high-throughput screening, and biotherapeutics.

Factors affecting endotoxin removal from recombinant therapeutic proteins by anion exchange chromatography.

Removal of endotoxins from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of the bacterial expression systems widely used to manufacture therapeutic proteins. In this study we investigated various parameters affecting anion exchange chromatography to selectively remove endotoxins from therapeutic proteins. NY-ESO-1, Melan-A, and SSX-2 are different recombinant proteins used in this study, all of them are cancer antigens currently developed as potential immunotherapeutic agents. We found that by using a commercially available Q XL resin in a flow-through mode, endotoxin could be effectively removed from these proteins while maintaining very acceptable protein yields. The ratio of resin volume to endotoxin load was analyzed to determine the endotoxin binding capacity of the resin. In our hands at least 900,000 endotoxin units (EU) could be loaded per ml of Q XL resin. Solution conductivity could be increased to 20 mS/cm to minimize protein loss by weakening protein-resin attraction, and pH could be increased to enhance endotoxin removal by weakening endotoxin-protein attraction. Endotoxin levels were ultimately decreased to below 0.5 EU per microg of protein, an over 2000-fold reduction in this single step. A successful scale-up of these processes in which column volume was increased 100-fold was performed under cGMP conditions with over 80% protein recovery.

Expression and purification of the cancer antigen SSX2: a potential cancer vaccine.

SSX2 is a cancer testis antigen expressed in a wide variety of cancers, including synovial sarcoma and melanoma. It holds promise as a potential antigen for cancer immunotherapy. A process for the production of recombinant SSX2 was developed by overexpressing a His-tagged fusion protein of SSX2 in Escherichia coli C41 (DE3). A T-7 promoter system was employed and a plasmid was introduced into the strain to compensate for rare codons in the SSX2 sequence. The production of SSX2 was scaled up to a 2-L fermentation that was operated under fed-batch conditions to improve productivity. After 32h cultivation, the wet cell mass reached 260mg/ml, with SSX2 produced mainly as inclusion bodies at a concentration of 1.1g/L. Urea-solubilized SSX2 was purified by nickel affinity, ion exchange and hydrophobic interaction chromatography. The recovery of SSX2 was 20%, and over 87% purity was obtained with an endotoxin level of 0.11EU/microg. The purified recombinant SSX2 was characterized by ELISA and was shown to be recognized by human sera that have been reported to carry anti-SSX2 antibodies.