Advanced Bioconjugation Technologies for Site-selective Protein Modification: Antibody Drug Conjugates

Antibody drug conjugates (ADCs) present a targeted cancer chemotherapy, that combine the specificity of an antibody with the toxicity of a small molecule drug. The enormous potential of ADCs, drives the need for the development of novel technologies, allowing for tuning of the properties of ADCs.
Chapter 2 Part I covers the development of a novel site-selective serine conjugation method, inspired by the catalytic triad found in the active site of hydrolytic enzymes. The N-heterocyclic carbene (NHC) organocatalyst IMes was used to introduce O-selectivity and through screening, it was found that short vinyl esters displayed O-selectivity. The subsequent screening of peptides containing a serine, activated by adjacent amino acids, demonstrated the feasibility of this sequence specific serine conjugation strategy. Part II describes the synthesis of a tetra anchor containing four disulfide rebridging head groups able to tether the 4 interchain disulfide bonds of antibodies together. This novel cysteine conjugation strategy, enables access to odd numbered DARs, as well as reforming the covalent bonds responsible for the stability of the antibody.
In Chapter 3, the development of sulfatase cleavable linkers is described. In part I, a novel sulfatase cleavable linker was designed and synthesized for the release of alcohol functionalized drugs. The linker was attached to MMAE and the payload was conjugated to trastuzumab via maleimidecysteine chemistry. Subsequent in vitro testing of the ADC towards HER2 positive breast cancer cells displayed an antigen dependent dose-response curve. To increase the potency of the ADC, a nitro group was introduced in the ortho position to the aryl sulfate of the linker. However, unforeseen acid sensitivity of the o-nitro aryl sulfate moiety was observed during linker synthesis and in vitro assays of the ADC containing the o-nitro aryl sulfate-auristatin E payload are pending. Part II investigates how substitution of the aryl sulfate moiety affects sulfatase mediated hydrolysis rates. Four probes were synthesized and it was observed that introduction of electron withdrawing groups (EWG) enhanced the cleavage rates significantly, highlighting the opportunity to tune the specificity and efficiency of sulfatase cleavable linkers. Part III describes the synthesis of three novel sulfatase cleavable payloads decorated with different EWG. The payloads were conjugated to trastuzumab and in in vitro assays against HER2 positive breast cancer cells, ADC potency varied significantly for the differently substituted aryl sulfates. In conclusion, the work presented in this thesis may provide useful contributions towards the development of safer and more efficacious ADCs to battle cancer.