Abstract
Bispecific antibodies (bsAbs) provide enticing therapeutic opportunities in the area of immunotherapy, especially in the field of immuno-oncology. These constructs can bind two separate antigenic epitopes and thus provide access to unique mechanisms of action (MoAs). A key MoA is unlocked by bispecific T cell engagers (BiTEs), which cause T cells to be cross-linked with a targeted cancer cell, ultimately leading to death of the targeted cell. It has been shown that the combination of a BiTE with checkpoint inhibition, such as blockade of the PD-1/PD-L1 pathway, can lead to a synergistic effect and greater efficacy. Constructs built from a BiTE core (anti-CD3/anti-cancer antigen) with an immunomodulatory protein added, have been dubbed checkpoint-inhibitory T cell-engagers (CiTEs). Both bsAbs and CiTEs have traditionally been generated via protein engineering. However, recently, improved chemical methods for the construction of bsAbs have been reported. This includes a strategy developed by the Chudasama and Baker groups to synthesize homogenous fragment-based bsAbs from antibodies’ fragments antigen binding (Fabs), utilising click-enabled pyridazinediones (PDs) for functional disulfide re-bridging, followed by strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) click chemistry to attach the two Fabs to each other. In this paper, we describe a first-in-class chemical method to generate biotin-functionalized three-protein conjugates, building significantly on the previously described PD-method. The three-protein constructs generated here include two such CiTE molecules, one containing an anti-PD-1 Fab, the other containing an immunomodulatory enzyme Salmonella typhimurium sialidase; FabCD3-FabHER2-FabPD-1-Biotin and FabHER2-FabCD3-Sia-Biotin. These constructs (along with suitable controls) were tested for their biological activity, and each of their protein components were shown to retain their function. Their efficacy was also compared to a simpler BiTE scaffold and was shown to be superior, with the sialidase-containing CiTE especially showing significantly enhanced potency in vitro. The chemical method described here has the potential to enable the rapid generation of a plethora of multi-protein constructs, which we envisage would be especially useful in hit-identification screening but could also potentially be scaled up for drug-development after further optimization.
Supplementary materials
Title
ESI: Chemical generation of checkpoint inhibitory T cell engagers (CiTEs) for the treatment of cancer
Description
Electronic supplementary information with detailed methods and spectral data. Also included are manuscript-style descriptions of alternative methods for three-protein conjugate generation that were explored.
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