A Simplified Function-First Method for the Discovery and Optimization of Bispecific Immune Engaging Antibodies

Abstract

Bi-specific T-cell engager antibodies (BITEs) are synthetic soluble molecules derived from antibodies that induce active contact between T-cells and other target cells in the body. BITE therapeutics have shown great promise for the treatment of various forms of cancer; however, the current development process for BITEs is time consuming and costly. BITE development requires empirical testing and characterization of the individual antigen binding domains, followed by extensive engineering and optimization in bi-specific molecular format to generate a molecule with strong biological activity and appropriate characteristics for clinical development. Here, we sought to create a cost efficient high-throughput method for creating and evaluating BITEs using a simplified function first approach to identify bioactive molecules without purification. Using a plasmid with a modular structure to allow high efficiency exchange of either binder arm, we established a simple method to combine many novel tumour-targeting single chain variable (scFv) domains with the well-characterized OKT3 scFv CD3-targeting domain. After generating these novel plasmids, we demonstrate two systems for high throughput functional screening of BITE molecules based on Jurkat T cells (referred to as BITE-J). Using BITE-J we evaluate four EGFRvIII BITEs, identifying two constructs with superior activity. We then confirmed this activity in primary T cells, where novel EGFRvIII-BITEs induced T cell activation and antigen selective tumor killing. We also demonstrate that we can similarly exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BITEs, we were able to identify highly active BITE molecules with desirable properties for downstream development. In summary, BITE-J presents a low cost, high-throughput method for the rapid assessment of novel BITE molecules without the need for purification and quantification.

Mammalian display to secretion switchable libraries for antibody preselection and high throughput functional screening

“…While the setup shares similarities with previously reported mammalian autocrine functional selections, 4 , 18 , 42 the approach applied here includes screening in the final secreted format and in higher throughput than plate-based screens. 43 Although secreting and reporter cell lines were of mammalian origin, identifying applicable cultivation conditions needed assay optimization, highlighting the complexity of such screening setups. GFP reporter cell-based screens yielded enrichment factors comparable to similar recent reports, 42 but background of non-antibody secreting cell (ASC)-containing droplets indicated at least partially leaky reporter systems ( Figure 1e and Figure S1b) as a critical aspect of assay setup potentially leading to lower sorting efficiencies.…”