Modular Protein Expression Toolbox (MoPET), a standardized assembly system for defined expression constructs and expression optimization libraries.

The design and generation of an optimal expression construct is the first and essential step in in the characterization of a protein of interest. Besides evaluation and optimization of process parameters (e.g. selection of the best expression host or cell line and optimal induction conditions and time points), the design of the expression construct itself has a major impact. However, the path to this final expression construct is often not straight forward and includes multiple learning cycles accompanied by design variations and retesting of construct variants, since multiple, functional DNA sequences of the expression vector backbone, either coding or non-coding, can have a major impact on expression yields. To streamline the generation of defined expression constructs of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed. This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules with a minimal cloning burden. Combining these features with a standardized cloning strategy facilitates the identification of optimized DNA expression constructs in shorter time. The MoPET system currently consists of 53 defined DNA modules divided into eight functional classes and can be flexibly expanded. However, already with the initial set of modules, 792,000 different constructs can be rationally designed and assembled. Furthermore, this starting set was used to generate small and mid-sized combinatorial expression optimization libraries. Applying this screening approach, variants with up to 60-fold expression improvement have been identified by MoPET variant library screening

Expression optimization library of an artificial two domain cytokine.

<p>(A) Cytokine expression optimization library design. Listed functional modules were included in two separate reactions. In one reaction domain A is used at the N-TAG position and domain B at the core protein position. In the second reaction domain B is used at the N-TAG position and domain A at the core protein position. The resulting final library has a theoretical complexity of 600. (B) Distribution of the implemented modules in accordance to their functional class of a test set of 88 randomly selected clones. (C) Expression titer of the 88 randomly selected clones as determined by ELISA in two independent experiments (Pearson’s Correlation r = 0.8642, R2 = 0.7468, P (two-tailed) <0.0001). Red diamond represents the starting expression level. All clones are correctly assembled and include all functions essential for expression like promoter and signal peptide. (D) Construct design of the Top 4 expression constructs.</p

Expression test library of hPTK7-ECD1-7.

<p>(A) hPTK7-ECD1-7 expression optimization library design. All listed functional modules were included in a single reaction resulting in a theoretical complexity for the final library of 72. (B) Expression test of 20 unique expression clones in deep well expression (DWP) system (y-axis) or in a tube spin expression system (x-axis). Red diamond represents starting expression level of the original construct. Yellow and green diamonds show the expression level of duplicates of the same construct (yet derived from independent E. coli clones) for 2 cases (Pearson’s Correlation r = 0.7380, R2 = 0.5447, P (two-tailed) <0.0001). (C) Construct design of the Top 5 expression constructs in DWP and Tube spin expression.</p

General overview of the MoPET design and implemented functional parts.

<p>(A) Modular structure of the MoPET system consisting of the eight basic module types Promoter, Signal Peptide, N-TAG (N-terminal tag), N-Linker (N-terminal linker), Core Protein, C-Linker (C-terminal linker), C-TAG (C-terminal tag) and the vector. Boxes show the fusion sites separating the modules and indicating the reading frame. (B) Layout of level 0 storage plasmids for the respective module positions and the destination backbones. The level 0 storage plasmids confer resistance to kanamycin and allow blue white selection for cloning purposes. Cloning into this plasmid set can be performed via BpiI, with a final BsaI based assembly in the ampicillin resistant level 1 backbones. (C) Compilation of the functional modules compatible with the MoPET system. DKTH-hFc-His (human IgG1 Fc sequence starting with DKTH), PKSC-hFc-His (human IgG1 Fc sequence starting with PKSC), HSA (human serum albumin), mFc (murine Fc sequence), Avi-tag [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref024" target="_blank">24</a>]. (D) Overview of the functional features of the three basic backbones. P: OriP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref025" target="_blank">25</a>], pA: poly adenylation site, RBG: rabbit beta-globin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref026" target="_blank">26</a>], SV40 Simian virus 40, bGH: bovine growth hormone [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref027" target="_blank">27</a>], Neo^R: neomycin resistance, Ap^R: ampicillin resistance, Km^R: kanamycin resistance.</p

T cell-mediated elimination of cancer cells by blocking CEACAM6–CEACAM1 interaction

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation