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Construction and characterization of antibody fragment-based PROTACs engaging EGFR and RNF43

Úlfarsson, Magnús Gauti LU (2026) KIMM01 20261
Department of Immunotechnology
Educational programmes, LTH
Abstract
Conventional monoclonal antibodies that function via occupancy-based inhibition often fall short due to drug resistance and non-enzymatic scaffolding functions that persist despite successful inhibition of the target protein. Antibody-based proteolysis targeting chimeras (AbTACs) utilize a novel event-driven modality allowing for specific degradation of the target protein. This study investigated the construction and characterization of bispecific tandem-single-chain fragment variable (tandem-scFv) AbTACs, designed to bind the oncogenic driver protein epidermal growth factor receptor (EGFR) and the membrane-bound E3 ubiquitin ligase ring finger protein 43 (RNF43). A panel of phage display-derived antibody clones was characterized through... (More)
Conventional monoclonal antibodies that function via occupancy-based inhibition often fall short due to drug resistance and non-enzymatic scaffolding functions that persist despite successful inhibition of the target protein. Antibody-based proteolysis targeting chimeras (AbTACs) utilize a novel event-driven modality allowing for specific degradation of the target protein. This study investigated the construction and characterization of bispecific tandem-single-chain fragment variable (tandem-scFv) AbTACs, designed to bind the oncogenic driver protein epidermal growth factor receptor (EGFR) and the membrane-bound E3 ubiquitin ligase ring finger protein 43 (RNF43). A panel of phage display-derived antibody clones was characterized through parallel ELISA and surface plasmon resonance epitope binning against reference antibodies. Two distinct EGFR-binding Fabs were identified that bind different epitopes on EGFR. These, along with an RNF43-binding Fab were converted to scFv format and also engineered to form bispecific tandem-scFvs. The effect of orientation when constructing tandem-scFvs was investigated in terms of binding affinity and expression yields. Format comparisons between the tandem-scFvs, Fabs, and scFvs were also conducted revealing insightful trends. Ultimately, this work establishes a valuable framework for the design and optimization of compact tandem-scFv degraders. (Less)
Popular Abstract
Cancer remains one of society’s greatest challenges. While traditional chemotherapy has saved countless lives, it acts broadly and destructively, even harming healthy cells along with the cancerous ones. Biologic drugs have the potential to offer results with fewer side effects. One such application is using engineered antibody drugs which act like keys that fit perfectly into a cancer protein “lock”, which could for example drive tumor growth. By plugging into this lock, the drug slows the cancer down, leaving other parts of the body alone. However, this approach has a major flaw. Since most antibody drugs only work as long as they remain jammed inside the lock, the cancer can adapt and simply make more locks to outnumber the medicine.... (More)
Cancer remains one of society’s greatest challenges. While traditional chemotherapy has saved countless lives, it acts broadly and destructively, even harming healthy cells along with the cancerous ones. Biologic drugs have the potential to offer results with fewer side effects. One such application is using engineered antibody drugs which act like keys that fit perfectly into a cancer protein “lock”, which could for example drive tumor growth. By plugging into this lock, the drug slows the cancer down, leaving other parts of the body alone. However, this approach has a major flaw. Since most antibody drugs only work as long as they remain jammed inside the lock, the cancer can adapt and simply make more locks to outnumber the medicine. Also, even though the connection itself is successful, the lock itself remains intact and can sometimes still do damage.
That’s where a new class of drugs called AbTACs come into play. Instead of just occupying a single lock, these act like molecular handcuffs that bind harmful cancer proteins to other cellular proteins that destroy them. Because the drug destroys the target instead of blocking it, it can go on and neutralize multiple cancer proteins one after another.
In this project, a special compact version of AbTACs called tandem-scFvs were constructed from smaller antibody building blocks called Fabs and scFvs. Things such as how strongly the key fits both targets were tested. Additional comparisons were made to see if the recognition was affected when swapping between antibody types or changing their orientation. This project laid the groundwork in learning how to create and optimize AbTACs of this tandem-scFv version. (Less)
Please use this url to cite or link to this publication:
author
Úlfarsson, Magnús Gauti LU
supervisor
organization
course
KIMM01 20261
year
type
H2 - Master's Degree (Two Years)
subject
keywords
AbTAC, Bispecific antibodies, Targeted Protein Degradation, Epitope binning, Epidermal growth factor receptor, RNF43, Tandem antibody fragments, PROTAC
language
English
id
9235866
date added to LUP
2026-06-15 11:46:37
date last changed
2026-06-15 11:46:37
@misc{9235866,
  abstract     = {{Conventional monoclonal antibodies that function via occupancy-based inhibition often fall short due to drug resistance and non-enzymatic scaffolding functions that persist despite successful inhibition of the target protein. Antibody-based proteolysis targeting chimeras (AbTACs) utilize a novel event-driven modality allowing for specific degradation of the target protein. This study investigated the construction and characterization of bispecific tandem-single-chain fragment variable (tandem-scFv) AbTACs, designed to bind the oncogenic driver protein epidermal growth factor receptor (EGFR) and the membrane-bound E3 ubiquitin ligase ring finger protein 43 (RNF43). A panel of phage display-derived antibody clones was characterized through parallel ELISA and surface plasmon resonance epitope binning against reference antibodies. Two distinct EGFR-binding Fabs were identified that bind different epitopes on EGFR. These, along with an RNF43-binding Fab were converted to scFv format and also engineered to form bispecific tandem-scFvs. The effect of orientation when constructing tandem-scFvs was investigated in terms of binding affinity and expression yields. Format comparisons between the tandem-scFvs, Fabs, and scFvs were also conducted revealing insightful trends. Ultimately, this work establishes a valuable framework for the design and optimization of compact tandem-scFv degraders.}},
  author       = {{Úlfarsson, Magnús Gauti}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Construction and characterization of antibody fragment-based PROTACs engaging EGFR and RNF43}},
  year         = {{2026}},
}