A Plug-and-Play Platform for the Formation of Trifunctional Cysteine Bioconjugates that also Offers Control over Thiol Cleavability.

Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy in vivo. Cysteine residues have been exploited as a route to site-selectively modify proteins, and many successfully approved therapeutics make use of cysteine directed conjugation reagents. However, commonly used linkers, including maleimide-thiol conjugates, are not stable to the low concentrations of thiol present in blood. Furthermore, only a few cysteine-targeting reagents enable the site-selective attachment of multiple functionalities: a useful tool in the fields of theranostics and therapeutic blood half-life extension. Herein, we demonstrate the application of the pyridazinedione motif to enable site-selective attachment of three functionalities to a protein bearing a single cysteine residue. Extending upon previously documented dual modification work, here we demonstrate that by exploiting a bromide leaving group as an additional reactive point on the pyridazinedione scaffold, a thiol or aniline derivative can be added to a protein, post-conjugation. Thiol cleavability appraisal of the resultant C-S and C-N linked thio-bioconjugates demonstrated C-S functionalized linkers to be cleavable and C-N functionalized linkers to be noncleavable when incubated in an excess of glutathione. The plug-and-play trifunctional platform was exemplified by attaching clinically relevant motifs: biotin, fluorescein, a polyethylene glycol chain, and a model peptide. This platform provides a rare opportunity to combine up to three functionalities on a protein in a site-selective fashion. Furthermore, by selecting the use of a thiol or an amine for functionalization, we provide unique control over linker cleavability toward thiols, allowing this novel linker to be applied in a range of physiological environments.

Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones.

Due to their exquisite cysteine-selectivity, excellent stability, and ability to functionally rebridge disulfide bonds, dibromopyridazinediones are emerging as an exciting new class of bioconjugation reagents, particularly in the field of antibody conjugation. Despite this, relatively little work has been performed on the optimisation of their synthesis and subsequent reaction with immunoglobulins. Herein we present a novel synthetic route towards functionalised dibromopyridazinediones, proceeding via an isolatable dibromopyridazinedione-NHS ester. Reaction of this activated intermediate with a variety of amines produces functional dibromopyridazinediones in good to excellent yields. The disulfide rebridging capacity of these reagents was optimised on the clinically relevant IgG1 trastuzumab, resulting in a general method which allows for the generation of site-selectively modified native trastuzumab with over 90% homogeneity (no disulfide scrambling) without the need for protein engineering or enzymatic conjugation.

Structural basis of human LRG1 recognition by Magacizumab, a humanized monoclonal antibody with therapeutic potential.

The formation of new dysfunctional blood vessels is a crucial stage in the development of various conditions such as macular degeneration, diabetes, cardiovascular disease, neurological disease and inflammatory disorders, as well as during tumor growth, eventually contributing to metastasis. An important factor involved in pathogenic angiogenesis is leucine-rich α-2-glycoprotein 1 (LRG1), the antibody blockade of which has been shown to lead to a reduction in both choroidal neovascularization and tumor growth in mouse models. In this work, the structural interactions between the LRG1 epitope and the Fab fragment of Magacizumab, a humanized function-blocking IgG4 against LRG1, are analysed, determining its specific binding mode and the key residues involved in LRG1 recognition. Based on these structural findings, a series of mutations are suggested that could be introduced into Magacizumab to increase its affinity for LRG1, as well as a model of the entire Fab-LRG1 complex that could enlighten new strategies to enhance affinity, consequently leading towards an even more efficient therapeutic.

Correction: Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation.

Correction for 'Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation' by Calise Bahou et al., Chem. Commun., 2019, 55, 14829-14832, https://doi.org/10.1039/C9CC08362F.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) as a novel ADC target.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) is present abundantly in the microenvironment of many tumours where it contributes to vascular dysfunction, which impedes the delivery of therapeutics. In this work we demonstrate that LRG1 is predominantly a non-internalising protein. We report the development of a novel antibody-drug conjugate (ADC) comprising the anti-LRG1 hinge-stabilised IgG4 monoclonal antibody Magacizumab coupled to the anti-mitotic payload monomethyl auristatin E (MMAE) via a cleavable dipeptide linker using the site-selective disulfide rebridging dibromopyridazinedione (diBrPD) scaffold. It is demonstrated that this ADC retains binding post-modification, is stable in serum and effective in in vitro cell studies. We show that the extracellular LRG1-targeting ADC provides an increase in survival in vivo when compared against antibody alone and similar anti-tumour activity when compared against standard chemotherapy, but without undesired side-effects. LRG1 targeting through this ADC presents a novel and effective proof-of-concept en route to improving the efficacy of cancer therapeutics.

Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation.

Herein we report a retro-Michael deconjugation pathway of thiol-pyridazinedione linked protein bioconjugates to provide a novel cleavable linker technology. We demonstrate that the novel pyridazinedione linker does not suffer from off-target modification with blood thiols (e.g., glutathione, human serum albumin (HSA)), which is in sharp contrast to an analogous maleimide linker.

Advances in targeting the folate receptor in the treatment/imaging of cancers.

The folate receptor (FR) is a recognised biomarker for tumour cells due to its overexpression on a large number of tumours. Consequently, the FR has been exploited by many diagnostic and therapeutic tools to allow targeted delivery to, and imaging of, cancer cells. Herein, we describe the many different approaches by which this has been achieved, including the attachment of folate to potent chemotherapeutic drugs to form FR-targeting small molecule-drug conjugates (SMDCs), FR-targeting antibodies (as antibody alone and as an antibody-drug conjugate), and in the form of complementary nanotechnology-folate platforms; as well as imaging variants thereof. The potential of exploiting the FR for targeted therapy/imaging has the potential to revolutionise the way several cancers are treated. These FR-targeted technologies can also pave the way for inspiring further sophisticated drug conjugates, especially as this receptor is being targeted by use of several complementary technologies: small molecule, nanoparticle and protein-based - thus providing broad and distinct knowledge in the area.