Investigation of Novel Linker Technologies and Payloads for Use in Protein-Drug Conjugates

  • Ivan Paul

Student thesis: Phd


Antibody-drug conjugates (ADCs) are an emerging class of targeted chemotherapies. They consist of an antibody joined to a small molecule payload by a linker. Through the exquisite binding selectivity of the antibody for its specific antigen and subsequent internalisation of the conjugate, this class of therapeutics offers a way to selectively deliver a potent cytotoxin to cancerous cells. Developments in this field have culminated in multiple approved therapeutics that are used to treat a range of cancers. Microbial transglutaminase (MTG) is an enzyme that has been investigated as a means by which to conjugate linker-payloads to antibodies. The native function of MTG is to crosslink proteins by condensing glutamine and lysine residues, which gives rise to one of the drawbacks with this conjugation strategy - under certain conditions MTG can crosslink antibodies, leading to reduced yields. This challenge can be overcome by carrying out a two-step chemoenzymatic conjugation, or simply using the linker-payload in large excess (typically 50-80 equivalents). The extra purification step required for the former adds operational complexity and may reduce overall yield, whereas the latter represents a significant waste of often expensive or synthetically advanced payload. We aim to develop an alternative strategy that allows an efficient one-step conjugation, suppresses antibody crosslinking, and requires only a moderate excess of linker-payload. In order to do this we expanded the known substrate scope of MTG, with the aim of finding a more active substrate than lysine. If a linker-payload uses the improved substrate as a conjugation handle, it may allow the conjugation to outcompete any crosslinking, even when the linker-payload is in moderate excess. During the work we verified some of the few previous results reported in the literature and systematically explored the structure-activity relationship (SAR) by iterative substrate design. Alkyne-containing amines were found to be a promising motif, with some members of this class showing high reactivity. However further work is required to fully develop a general chemical motif that is both highly active and functionalisable. This work also sought to design, synthesise, and test a series of proteolysis-targeting chimeras (PROTACs) targeting the enzyme nicotinamide phosphoribosyltransferase (NAMPT), with a view to develop these compounds as novel ADC payloads. PROTACs are heterobifunctional molecules that induce degradation of a specific protein of interest, which can achieve superb degradation potencies due to their event-driven pharmacology. NAMPT is the rate-limiting enzyme in the NAD salvage pathway in mammals, making it essential for normal metabolism. Cancer cells are particularly sensitive to the suppression of NAMPT function, as they rely more heavily on NAD in their altered metabolic state. PROTACs are an attractive potential payload class for ADCs partly because of the excellent potencies they can achieve, which may allow them to be used in the targeting of low-antigen expressing cells. Their unique mechanism of action also provides an opportunity to diversify the available classes of ADC payloads, expanding the technology's potential to treat a wide range of different conditions. We synthesised a range of PROTACs based on the NAMPT inhibitor FK866 and ligands for both VHL and CRBN E3 ligases. Ultimately the compounds we produce did not induce any observable protein degradation despite engaging with and inhibiting NAMPT, however literature published during the course of this work suggests that screening an expanded collection of linkers and altering the FK866 exit vector may lead to effective PROTACs.
Date of Award31 Dec 2023
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorSam Butterworth (Supervisor) & Michael Greaney (Supervisor)


  • antibody-drug conjugate
  • bioconjugation

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