Enzymatic conjugation using branched linkers for constructing homogeneous antibody–drug conjugates with high potency

Abstract

Antibody-drug conjugates (ADCs) are emerging therapeutic agents in the treatment of cancer, and various conjugation strategies and chemical linkers have been developed to efficiently construct ADCs. Despite previous extensive efforts for improving conjugation efficiency and ADC homogeneity, most ADC linkers developed to date load only single payloads. Branched linkers that can load multiple payload molecules have yet to be fully explored. It is logical to envisage that a multi-loading strategy allows for increase in drug-to-antibody ratio (DAR) with less chemical or enzymatic modification to the antibody structure compared to traditional linear linkers, leading to efficient ADC construction, minimal destabilization of the antibody structure, and enhanced ADC efficacy. Herein, we report that the branched linkers we designed can be quantitatively installed on an anti-HER2 monoclonal antibody by microbial transglutaminase (MTGase)-mediated conjugation without impairing its antigen binding affinity, enabling modular installation of payload molecules and construction of homogeneous ADCs with increased DARs (up to 8). An anti-HER2 antibody-monomethyl auristatin F conjugate constructed using our branched linkers showed greater in vitro cytotoxicity against HER2-expressing breast cancer cell lines than that consisting of linear linkers, demonstrating the effectiveness of the branched linker-based payload delivery. Our finding demonstrates that enzymatic ADC construction using branched linkers is a promising strategy, which may lead to innovative cancer therapeutics.

Glutamic acid–valine–citrulline linkers ensure stability and efficacy of antibody–drug conjugates in mice

“…However, the success of such efforts depends on the choice of the linker installation sites, conjugation modality, and payload. Indeed, as we have demonstrated in this and previous reports 29 , our branched linker technology requires both adequate spacers and cleavable mechanisms within the linker scaffold for maximal ADC potency. These components are critical to alleviate the structural congestion and to ensure rapid payload release in an active form from each linker arm.…”

“…3 for synthesis details) to an anti-HER2 mAb at glutamine 295 (Q295) by microbial transglutaminase (MTGase)-mediated conjugation according to the protocol developed by our group with minor modifications (Fig. 2a ) 29 . The anti-HER2 mAb used for linker conjugation contained a mutation of the asparagine 297 into alanine (N297A) 31 .…”

“…These features make the EVCit sequence ideal cleavable ADC linker design for increasing the hydrophilicity under physiological conditions, maximizing the therapeutic potential, and minimizing the risk of systemic toxicity in mouse models caused by premature payload release. Indeed, a homogeneous anti-HER2 ADC constructed using an EVCit–PABC linker along with our branched linker technology 29 exhibited higher hydrophilicity and by far greater long-term in vivo stability than did ADCs equipped with a conventional VCit or SVCit, an analogue of the hydroxy-functionalized tripeptide ADC linker that reportedly exhibited increased stability in mouse plasma 23 . In addition, although treatment with a VCit-based anti-HER2 ADC showed poor therapeutic effect, treatment with the stable EVCit ADC led to complete remission in two xenograft mouse models of HER2-positive breast cancer.…”

“…In addition, this issue significantly limits flexibility in the choice of conjugation sites and linker design. Indeed, ADCs constructed using the multi-loading VCit linker recently developed by our group 29 were shown to have instability in mouse plasma and poor treatment efficacy in mouse breast tumor models (described in detail in the Results section).

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Biocatalysis by Transglutaminases: A Review of Biotechnological Applications

“…Josten and colleagues proposed an original enzymatic biotinylation method useful for the production of low-biotinylated proteins, based on mTG-mediated incorporation of amino-modified derivatives on IgG Gln residues [ 222 ]. More recently, antibody functionalisation by TGs has been applied in radio immunodiagnosis and therapy antibody [ 223 , 224 , 225 ]. Both mTG and, to a lesser extent, human TG2 have shown the ability to perform the selective modification of antibodies heavy chains (IgGs), without interfering with their biological activities, such as antigen affinity and cell internalisation, as tested both in vitro and in vivo [ 223 , 225 ].…”