Author ORCID Identifier

https://orcid.org/0009-0000-2013-7627

Document Type

Thesis

Date of Award

Fall 12-2025

Keywords

antibody-drug conjugate, immune-stimulating antibody conjugate, immunotherapy, conjugation, TLR7, cancer

Degree Name

Chemistry (BA, BS)

Department

CHEMISTRY

First Advisor

Dr L. Nathan Tumey

Second Advisor

Dr. John Fetse

Third Advisor

Dr. Brian Callahan

Abstract

Recent advances in immunotherapy as a viable cancer treatment modality has led to the development of immune-stimulating antibody conjugates (ISACs). ISACs are analogous to antibody-drug conjugates (ADCs), which utilize the specificity and stability of an antibody to deliver cytotoxic payloads directly to a tumor. ISACs, however, deliver immune stimulating agents instead, activating adaptive and innate immune responses to engage with tumor cells. Among the various immunostimulants currently being explored, Toll-Like Receptor (TLR) agonists have risen as promising inducers of anti-cancer immune responses. Previously, the Tumey lab has explored ISACs employing an imidazole[4,5-c]quinoline TLR7 agonist with drug-antibody ratios (DAR) of ~8. While induction of the NFκB pathway and tumor regression in mouse models were observed, these ISACs face issues of aggregation, poor pharmacokinetics, heterogeneity, and the inability to employ a recently discovered more potent TLR7 agonist due to aggregation. This is a result of the “Hinge” method used to generate these ISACs, which conjugates linker-payloads to free thiols generated by reducing interchain disulfides of the antibody. In response, a site-specific conjugation method developed by the Tumey lab allows for controlled addition of linker-payloads to a thiolated Q295 site of antibodies. Despite producing DAR 2 conjugates, this “Q295” method offers improved aggregation levels, pharmacokinetics, homogeneity, and the potential for conjugation with the previously unconjugatable potent TLR7 agonist. Herein, this work aims to optimize the Q295 method for generating ISACs employing TLR7 agonists across various antibodies and cleaveable linkers. Additionally, Q295 conjugates are characterized and evaluated against their Hinge conjugate counterparts in a variety of co-culture models for NF-κB activation and IL-6 release, as well as lysosomal payload release assays to elucidate possible differences in release mechanisms affecting efficacy.

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