|Better Cell-Based Assays for Anti-CTLA-4, Anti-PD-1/PD-L1, and Bispecific Immunotherapy Drug Studies|
Richard Somberg, Mei Cong, Pete Stecha, Natasha Karassina, Jim Hartnett, Zhi-Jie Jey Cheng, and Frank Fan
Here we report the development of a panel of robust reporter assays to measure the potencies for biologics in immunotherapy. These assays reflect mode of action and can serve as valuable tools in immunotherapy drug development and discovery.
|Development of a Robust Reporter-based T cell Activation Assay for Therapeutic Biologics in Immunotherapy |
Zhi-jie Jey Cheng, Pete Stecha, Jim Hartnett, Frank Fan, and Mei Cong
Jurkat T-cells stably expressing luciferase reporter driven by IL2 promoter or NFAT-RE, are used as effector cells. Tumor cell lines endogenously expressing cancer antigen are used as antigen presenting cells (APC). By co-cultivating the two cell lines in the presence of CD3 bispecific antibody, TCR/CD3 is activated in Jurkat effector cells. Luciferase activity is up regulated through IL-2 promoter or NFAT-RE activation.
|Reporter Bioassays to Assess Therapeutic Antibodies for Immunotherapy Programs|
Mei Cong, Zhi-Jie Jey Cheng, Pete Stecha, Jun Wang, Jamison Grailer, Natasha Karassina, Jim Hartnett, and Frank Fan
Immunotherapy, also called biologic therapy or biotherapy, stimulates certain parts of the immune system to fight diseases such as cancer. Important drug targets in immunotherapy include: Co-inhibitory receptors, such as PD-1/PD-L1, CTLA-4, LAG3, Tim3; and co-stimulatory receptors, such as GITR, CD40, OX40, 4-1BB.
Current approaches to assaying these targets are cumbersome and variable. Here we offer an improved in vitro bioassay approach.
|Automated in-gel digestion on a commercial autosampler directly coupled to nanoLC-MS/MS|
Achermann François, Bolliger Reto, Buchs Natasha, Doiron Nicholas, Lagache Braga Sophie, Heller Manfred, Boehm Guenter
SDS-PAGE separates protein samples from LC-MS incompatible contaminations, and is frequently used to fractionate proteins of entire proteomes. One disadvantage is that gel lanes have to be cut into many slices, followed by in-gel digestion of proteins and extraction of peptides. The number of these gel slices goes into the hundreds, rendering this process very repetitive and prone to mistakes and errors during sample handling. Automation reduces such risks and improves reproducibility.
|Automated sample preparation workflows for quantitative proteomics applications|
Oliver Popp1, Lucas Luethy2, Tamara Kanashova1, HaAn Nguyen1, Julia Kikuchi1, Guenter Boehm2, Thomas Blenkers3, Andreas Bruchmann3, Gunnar Dittmar1
Mass spectrometry based proteomics requires large scale identification of peptides, and depends upon efficient sample preparation. Recently, we presented two automated protein-digestion setups, in-solution and in-gel digestion. We extended these techniques by implementing dimethyl labelling (DML). Furthermore, we established an automated phospho-peptide (PP) enrichment procedure in a 96-well formate, generating phospho-proteomic data in very short time.
|Characterization of Protein and Protein Aggregates using Temperature-controlled Hollow-Fiber Flow-FFF|
Trevor Havard, Florian Meier, Evelin Moldenhauer, Soheyl Tadjiki, Thorsten Klein
Reproducibilty Improvements in Field Flow Fractionation can be achieved on two fronts. Instrument design and control, the system used in for this poster is does not require flow controllers or switching valves and there for produces the same conditions in every case. Fractionater design, the design of the cartridges used in this poster maintain excellent conditions to maintain constant pressure at the membrane removing unwanted effects of sale relaxation above the membrane s
|Specificity of highly potent miRNA inhibitors|
Barbara Roberston, Andrew Dalby, Yuriy Fedorov, Jon Karpilow, Anastasia Khvorova, Devin Leake, Annaleen Vermeulen
Specificity of highly potent miRNA inhibitors
|HOMOGENEOUS ADCs BEARING TWO DIFFERENT PAYLOADS SHOW STRONG SYNERGY IN TUMOR KILLING|
Lisha Allen, Yi Mi, Muhammad Abbas, Wolfgang Richter, and Sean Hu
We presented a one-pot reaction to prepare site-specific ADCs using available mABs on hand without re-engineering. We have identified a novel non-cleavable linker, which increases not only ADC stability, but also potency. We also demonstrate synergy in cancer cell killing of hybrid ADC loaded with two different toxins
|Toward a Comprehensive Bottom-up and Top-down Analysis of the NIST Reference Monoclonal Antibody|
Chris Becker1, Eric Carlson1, Wilfred Tang1, Yong Kil1, Marshall Bern1, John Schiel2, Lisa Kilpatrick2, Trina Formolo2
Development of ADC and other therapeutic proteins require careful characterization of sequence, post-translational modifications, sequence variants, and degradants. In this study, a NIST mAb interim reference material was analyzed by new bioinformatics tools (Byonic™, Byologic®, and Byomap™) allowing one to efficiently identify and quantify modifications down to trace levels.
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