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

Abstract

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.

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

“…Previously, we demonstrated that our E. coli strain SBDG419, which has been engineered to have an oxidizing cytoplasm, could express a variety of LCs that could be incorporated into antibodies produced in CFPS reactions. 26 The high titer and productivity observed in the SBDG419 strain suggested that it could be a promising platform for producing antibody fragments and full-length antibodies potentially containing nnAAs. Therefore, an expression screen of several therapeutically relevant proteins without nnAAs was performed in this strain to find potential scaffolds for nnAA incorporation, including a scFv, 29 an anti-CD3 antibody Fab, 30 and a full IgG complex.…”

“… 14 Due to these challenges with periplasmic expression, we focused on the synthesis of mAbs and mAb fragments in the oxidizing cytoplasm of an E. coli strain that was redox optimized for high-level expression of antibody light chain (LC) lacking nnAAs in the cytoplasm. 26 Production of this LC is relatively straightforward because it requires overexpression of only a single protein in the proper redox environment. Notably, it was also shown that the same LC will express poorly in a strain background exhibiting a wild type, reducing cytoplasm.…”

“… 13 This prompted us to test whether this newly engineered E. coli strain could achieve high level soluble expression of other antibody therapeutic scaffolds beyond LCs. 26 …”

“…In this report, we demonstrate soluble expression of a single-chain variable fragment (scFv), an IgG antigen-binding (Fab) fragment, and a full-length IgG mAb complex at high levels in an oxidizing E. coli cytoplasm. Of these scaffold proteins, a LC from our previous report 26 and the full-length IgG were selected for nnAA incorporation with pAMF. 18 Both products were expressed in a bioreactor using high-density fermentation in cells transformed with plasmids bearing the genes for the nnAA tRNA and RS and the product of interest, 27 , 28 resulting in high yields of >1 g/L and pAMF incorporation efficiency > 99% for each protein.…”

State-of-the-Art Approaches to Heterologous Expression of Bispecific Antibodies Targeting Solid Tumors

“…The XpressCF+ platform enables easier incorporation of non-natural amino acids and conjugates, as well as reduces protein heterogenicity and facilitates biosynthesis of structurally complex precisely assembled products. At present, Sutro has four conjugates and bsAbs in its portfolio that have been produced with the XpressCF+™ technology and continues to expand its inf luence by collaborating with other companies [98,99].…”