Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system.

Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.

High levels of protein expression using different mammalian CMV promoters in several cell lines.

With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

Identification of domain structures in the propeptide of corin essential for the processing of proatrial natriuretic peptide.

Corin is a type II transmembrane serine protease and functions as the proatrial natriuretic peptide (pro-ANP) convertase in the heart. In the extracellular region of corin, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor (LDLR) repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain at the C terminus. To examine the functional importance of the domain structures in the propeptide of corin for pro-ANP processing, we constructed a soluble corin, EKshortCorin, that consists of only the protease domain and contains an enterokinase (EK) recognition sequence at the conserved activation cleavage site. After being activated by EK, EKshortCorin exhibited catalytic activity toward chromogenic substrates but failed to cleave pro-ANP, indicating that certain domain structures in the propeptide are required for pro-ANP processing. We then constructed a series of corin deletion mutants and studied their functions in pro-ANP processing. Compared with that of the full-length corin, a corin mutant lacking frizzled 1 domain exhibited approximately 40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had approximately 49, approximately 12, approximately 53, and approximately 77% activity, respectively. We also made corin mutants with a single mutation at a conserved Asp residue that coordinates Ca(2+)-binding in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y) and showed that these mutants had approximately 25, approximately 11, approximately 16, and approximately 82% pro-ANP processing activity, respectively. Our results indicate that frizzled 1 domain and LDLR repeats 1-4 are important structural elements for corin to recognize its physiological substrate, pro-ANP.