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Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency.

ABSTRACT: Antibody drug conjugates (ADCs) have recently been proven to be highly potent anti-tumor drugs, typically exceeding the efficacy of conventional monoclonal antibodies (mAbs). ADCs are currently produced by chemical conjugation of a small-molecule toxin to the mAb through lysine or cysteine side chains. This leads to heterogeneous mixtures of ADCs in which variable numbers of drugs are conjugated to individual antibodies and in which the site of conjugation cannot be defined. Consequently, there is currently significant interest in further development of drug conjugation technologies, with a particular focus on site-specific payload conjugation. Here, we present an enzymatic conjugation platform based on the S. aureus sortase A-mediated transpeptidation reaction, allowing the efficient generation of ADCs with toxins conjugated to pre-defined sites at pre-defined drug-to-antibody ratios. For this, two modifications were introduced: first, immunoglobulin heavy (IgH) and light (IgL) chains were modified at their C-termini by addition of the sortase A recognition motif LPETG, and second, the small molecule tubulin polymerization inhibitors monomethylauristatin E (MMAE) and maytansine were modified by addition of a pentaglycine peptide, thus making them suitable substrates for sortase A-mediated transpeptidation. We demonstrate efficient generation and characterization of the anti-CD30 ADC Ac10-vcPAB-MMAE, an enzymatically conjugated counterpart of brentuximab vedotin (Adcetris), as well as several anti-HER-2 ADCs including trastuzumab-maytansine, the counterpart of trastuzumab emtansine (Kadcyla). ADCs generated in this manner were found to display in vitro cell killing activities indistinguishable from the classic conjugates. Further, when tested in vivo in a HER-2-overexpressing ovarian cancer xenograft mouse model, enzymatically generated trastuzumab-maytansine was found to lead to complete regression of established tumors, similar to Kadcyla.

SUBMITTER: Beerli RR

PROVIDER: S-EPMC4488448 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency.

Beerli Roger R RR Hell Tamara T Merkel Anna S AS Grawunder Ulf U

PloS one 20150701 7

Antibody drug conjugates (ADCs) have recently been proven to be highly potent anti-tumor drugs, typically exceeding the efficacy of conventional monoclonal antibodies (mAbs). ADCs are currently produced by chemical conjugation of a small-molecule toxin to the mAb through lysine or cysteine side chains. This leads to heterogeneous mixtures of ADCs in which variable numbers of drugs are conjugated to individual antibodies and in which the site of conjugation cannot be defined. Consequently, there ...[more]

PMID: 26132162

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Project description: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.

Dataset Information

Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency.

Publications

Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency.

Similar Datasets

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