STUB1 is an intracellular checkpoint for interferon gamma sensing.

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models-with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer.

Monoclonal antibodies (mAbs) are used as targeted therapies against cancers. These mAbs kill cancer cells via various mechanisms of actions. In this study, human embryonic stem cells (hESCs) was used as the immunogen to generate a panel of antibodies. From this panel of mAbs, A19 was found to bind both hESC and various cancer cell lines. The antigen target of A19 was identified as Erbb-2 and glycan analysis showed that A19 binds to a N-glycan epitope on the antigen. A19 was elucidated to internalize into cancer cells following binding to Erbb-2 and hence developed as an antibody-drug conjugate (ADC). Using ADC as the mechanism of action, A19 was able to kill cancer cells in vitro and delayed the onset of tumour formation in mice xenograft model. When compared to Herceptin, A19 binds to different isoforms of Erbb-2 and does not compete with Herceptin for the same epitope. Hence, A19 has the potential to be developed as an alternative targeted therapeutic agent for cancers expressing Erbb-2.

Proteomic profiling of barley spent grains guides enzymatic solubilization of the remaining proteins.

Within the brewing industry, malted barley is being increasingly replaced by raw barley supplemented with exogenous enzymes to lessen reliance on the time-consuming, high water and energy cost of malting. Regardless of the initial grain of choice, malted or raw, the resultant bulk spent grains are rich in proteins (up to 25% dry weight). Efficient enzymatic solubilization of these proteins requires knowledge of the protein composition within the spent grains. Therefore, a comprehensive proteomic profiling was performed on spent grains derived from (i) malted barley (spent grain A, SGA) and (ii) enzymatically treated raw barley (spent grain B, SGB); data are available via ProteomeXchange with identifier PXD008090. Results from complementary shotgun proteomics and 2D gel electrophoresis showed that the most abundant proteins in both spent grains were storage proteins (hordeins and embryo globulins); these were present at an average of two fold higher in spent grain B. Quantities of other major proteins were generally consistent in both spent grains A and B. Subsequent in silico protein sequence analysis of the predominant proteins facilitated knowledge-based protease selection to enhance spent grain solubilization. Among tested proteases, Alcalase 2.4 L digestion resulted in the highest remaining protein solubilization with 9.2 and 11.7% net dry weight loss in SGA and SGB respectively within 2 h. Thus, Alcalase alone can significantly reduce spent grain side stream, which makes it a possible solution to increase the value of this low-value side stream from the brewing and malt extract beverage manufacturing industry.

Targeting of embryonic annexin A2 expressed on ovarian and breast cancer by the novel monoclonal antibody 2448.

Monoclonal antibodies (mAbs) play an increasingly important role in cancer therapy. To address the wide heterogeneity of the disease, the identification of novel antigen targets and the development of mAbs against them are needed. Our lab previously generated a panel of mAbs against human embryonic stem cells (hESC) using a whole cell immunization approach in mice. These mAbs can potentially target oncofetal antigens and be repurposed for antibody or antibody drug conjugate (ADC) therapy. From this panel, the novel IgG1 2448 was found to bind surface antigens on hESC and multiple cancer cell lines. Here, we show 2448 targets a unique glycan epitope on annexin A2 (ANXA2) and can potentially monitor the Epithelial-Mesenchymal Transition (EMT) in ovarian and breast cancer. To evaluate 2448 as a potential drug, 2448 was engineered and expressed as a chimeric IgG1. Chimeric 2448 (ch2448) demonstrated efficient and specific killing when conjugated to cytotoxic payloads as an ADC. In addition, ch2448 elicited potent antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro and in vivo. Further engineering of ch2448 to remove fucose in the Fc domain enhanced ADCC. Overall, these findings indicate that embryonic ANXA2 is an attractive target and suggest that ch2448 is a promising candidate for further therapeutic development.

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

Molecular portrait of high productivity in recombinant NS0 cells.

Many important therapeutic proteins are produced in recombinant mammalian cells. Upon the introduction of the product gene, the isolated clones typically exhibit a wide range of productivity and high producers are subsequently selected for use in production. Using DNA microarray, two-dimensional gel electrophoresis (2DE), and iTRAQ as global surveying tools, we examined the transcriptome and proteome profiles of 11 lines of NS0 cells producing the same antibody molecule. Genes that are significantly differentially expressed between high and low producer groups statistically fall into a number of functional classes. Their distribution among the functional classes differs somewhat between transcriptomic and proteomic results. Overall, a high degree of consistency between transcriptome and proteome analysis are seen, although some genes exhibiting inconsistent trends between transcript and protein levels were observed as expected. In a novel approach, functional gene networks were retrieved using computational pathway analysis tools and their association with productivity was tested by physiological comprehension of the possible pathways involved in high recombinant protein production. Network analysis indicates that protein synthesis pathways were altered in high producers at both transcriptome and proteome levels, whereas the effect on cell growth/death pathways was more prominent only at the transcript level. The results suggest a common mechanism entailing the alteration of protein synthesis and cell growth control networks leading to high productivity. However, alternate routes with different sets of genes may be invoked to give rise to the same mechanistic outcomes. Such systematic approaches, combining transcriptomic and proteomic tools to examine high and low producers of recombinant mammalian cells will greatly enhance our capability to rationally design high producer cells. This work is a first step towards shedding a new light on the global physiological landscape of hyper productivity of recombinant cells.

Large scale gene expression profiling of metabolic shift of mammalian cells in culture.

The metabolic state of hybridoma cells in continuous culture varies with the cultivation condition from which the culture is initiated. At a metabolically shifted state, cells have markedly reduced glucose and other nutrient consumption and lactate production as compared to cells in batch culture or in continuous culture without a metabolic shift. Taking a combined genomics and proteomics approach, we investigated the molecular mechanism of metabolic shift. Cells from continuous cultures at two different steady states with a glucose consumption to lactate production molar ratio (DeltaL/DeltaG) of 0.08 and 1.4 were studied. Affymetrix GeneChips as well as cDNA microarrays were employed to identify differentially expressed mRNA transcripts, and the differentially expressed proteins were identified using the 2D gel electrophoresis-mass spectrometry approach. The decrease in glucose metabolism upon metabolic shift is accompanied by a decrease in gene expression of a number of genes involved in its metabolism. However, the number of genes differentially expressed and the extent of differential expression upon metabolic shift are relatively moderate. The change in the expression of metabolic genes at the transcriptional level was confirmed by real time PCR. The results suggest that metabolic shift is a combined effect of both biochemical events at reaction level and gene expression at transcription and translation level. This approach of integrating transcriptional profiling, proteomic techniques and biochemical analysis provides a more global view of the metabolism of mammalian cells in culture.